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Sample records for hemocytes

  1. Two Hemocyte Lineages Exist in Silkworm Larval Hematopoietic Organ

    Nakahara, Yuichi; Kanamori, Yasushi; Kiuchi, Makoto; Kamimura, Manabu

    2010-01-01

    BACKGROUND: Insects have multiple hemocyte morphotypes with different functions as do vertebrates, however, their hematopoietic lineages are largely unexplored with the exception of Drosophila melanogaster. METHODOLOGY/PRINCIPAL FINDINGS: To study the hematopoietic lineage of the silkworm, Bombyx mori, we investigated in vivo and in vitro differentiation of hemocyte precursors in the hematopoietic organ (HPO) into the four mature hemocyte subsets, namely, plasmatocytes, granulocytes, oenocyto...

  2. Honey bee hemocyte profiling by flow cytometry.

    Marringa, William J; Krueger, Michael J; Burritt, Nancy L; Burritt, James B

    2014-01-01

    Multiple stress factors in honey bees are causing loss of bee colonies worldwide. Several infectious agents of bees are believed to contribute to this problem. The mechanisms of honey bee immunity are not completely understood, in part due to limited information about the types and abundances of hemocytes that help bees resist disease. Our study utilized flow cytometry and microscopy to examine populations of hemolymph particulates in honey bees. We found bee hemolymph includes permeabilized cells, plasmatocytes, and acellular objects that resemble microparticles, listed in order of increasing abundance. The permeabilized cells and plasmatocytes showed unexpected differences with respect to properties of the plasma membrane and labeling with annexin V. Both permeabilized cells and plasmatocytes failed to show measurable mitochondrial membrane potential by flow cytometry using the JC-1 probe. Our results suggest hemolymph particulate populations are dynamic, revealing significant differences when comparing individual hive members, and when comparing colonies exposed to diverse conditions. Shifts in hemocyte populations in bees likely represent changing conditions or metabolic differences of colony members. A better understanding of hemocyte profiles may provide insight into physiological responses of honey bees to stress factors, some of which may be related to colony failure.

  3. Hemocyte characterization of Nasutitermes coxipoensis (Holmgren) (Isoptera: Termitidae) workers and hemocyte evaluation after parasitism by Metarhizium anisopliae

    Cunha, Franklin M.; Wanderley-Teixeira, Valeria; Albuquerque, Auristela C.; Lima, Elza A.L.A.

    2009-01-01

    We aimed to characterize the morphology and ultrastructure of hemocytes of Nasutitermes coxipoensis (Holmgren) workers and to quantify the cell types 24h, 48h and 72h after inoculation with Metarhizium anisopliae. Six hemocytes types were identified, plasmatocyte, granulocyte, spherulocyte, prohemocyte, adipohemocyte and eonocytoid Hemocytes did not present any morphological alteration at the several observation periods, but they did have a change in their abundance, as observed for spherulocytes, adipohemocytes and eonocytoids at all intervals, and for plasmatocytes and granulocytes at 48h after host inoculation. We argue on the possible reasons and implications of the observed changes. (author)

  4. [Functional morphology of blowfly Calliphora vicina hemocytes].

    Kind, T V

    2012-01-01

    In the hemolymph of Calliphora seven types of hemocytes were revealed. These are prohemocytes, which are the stem cells, stable and unstable hyaline cells, thrombocytoids, spindle cells, juvenile plasmatocytes and plasmatocytes I-IV, which represent sequential stages of one cell line differentiation were registered. The margin between them is completion of the crop emptying and beginning of wandering stage. In the feeding and crop emptying larvae take place rising of hyaline cells, thrombocytoids and hyaline cells amount with parallel growth of their defense function. The second wave of hemogenesis occur in the end of crop emptying period. It is accompanied by burst of plasmatocyte I production with their subsequent differentiation to plasmatocytes II-IV. Production of stable hyaline cells and respectively prothrombocytoids may be regulated not only by hormonal background but also by inorganic or organic particles invaded into the hemocel. Three types of hemocytes are involved in loosing of hemolymph from alien particles, notably thrombocytoids, juvenile plasmatocytes and plasmatocytes I and II. Thrombocytoids are responsible for parasitic eggs encapsulation. In addition they can phagocytize tiny organic and inorganic particles. Juvenile plasmatocytes respond to alien invasion almost as quickly as thrombocytoids at the onset of invasion. Plasmatocytes I and II start phagocytosis more slowly, hours post invasion, frequently accumulating the particles previously catched by thrombocytoids. Plasmatocytes I can absorb foreign particles and group in morules and can also surround filled thrombocytoids forming distinctive capsules. Both morules and capsules are temporary structures and disintegrate some hours lately. It is supposed the existence of three levels of immune defence: the fast response reaction of thrombocytoids and juvenile plasmatocytes and slow cellular reactions of plasmatocytes I. They are prerequisites for more extensive humoral response.

  5. On the origin of the Biomphalaria glabrata hemocytes

    Samaly dos Santos Souza

    2006-10-01

    Full Text Available A histologic, morphometric and ultrastructural study performed on Biomphalaria glabrata submitted to infection with Schistosoma mansoni miracidia failed to provide significant evidences that the so-called amebocyte-producing organ (APO is really the central organ for hemocyte production. In infected snails no general reactive changes appeared in the APO, the mitoses were seen only occasionally, and the possibility of cellular hyperplasia was ruled out by morphometric measurements. Under the electron microscope the APO cells presented an essentially epithelial structure, without features indicative of transition toward hemocytes. On the other hand, the present findings pointed to a multicentric origin for the mollusck hemocytes, as earlier studies had indicated. Dense foci of hemocyte collections appeared sometimes around disintegrating sporocysts and cercariae in several organs and tissues of the infected snails, including a curious accumulation of such cells inside the ventricular cavity of the heart. In the heart and other sites, features suggestive of transformation of vascular space endothelial lining cells into hemocytes were apparent. To some extent, the postulated multicentric origin for B. glabrata hemocytes recapitulates earlier embryologic findings in vertebrates, when mesenchymal vascular spaces generate the circulating and phagocytic blood cells.

  6. Two hemocyte lineages exist in silkworm larval hematopoietic organ.

    Yuichi Nakahara

    Full Text Available BACKGROUND: Insects have multiple hemocyte morphotypes with different functions as do vertebrates, however, their hematopoietic lineages are largely unexplored with the exception of Drosophila melanogaster. METHODOLOGY/PRINCIPAL FINDINGS: To study the hematopoietic lineage of the silkworm, Bombyx mori, we investigated in vivo and in vitro differentiation of hemocyte precursors in the hematopoietic organ (HPO into the four mature hemocyte subsets, namely, plasmatocytes, granulocytes, oenocytoids, and spherulocytes. Five days after implantation of enzymatically-dispersed HPO cells from a GFP-expressing transgenic line into the hemocoel of normal larvae, differentiation into plasmatocytes, granulocytes and oenocytoids, but not spherulocytes, was observed. When the HPO cells were cultured in vitro, plasmatocytes appeared rapidly, and oenocytoids possessing prophenol oxidase activity appeared several days later. HPO cells were also able to differentiate into a small number of granulocytes, but not into spherulocytes. When functionally mature plasmatocytes were cultured in vitro, oenocytoids were observed 10 days later. These results suggest that the hemocyte precursors in HPO first differentiate into plasmatocytes, which further change into oenocytoids. CONCLUSIONS/SIGNIFICANCE: From these results, we propose that B. mori hemocytes can be divided into two major lineages, a granulocyte lineage and a plasmatocyte-oenocytoid lineage. The origins of the spherulocytes could not be determined in this study. We construct a model for the hematopoietic lineages at the larval stage of B. mori.

  7. Two hemocyte lineages exist in silkworm larval hematopoietic organ.

    Nakahara, Yuichi; Kanamori, Yasushi; Kiuchi, Makoto; Kamimura, Manabu

    2010-07-28

    Insects have multiple hemocyte morphotypes with different functions as do vertebrates, however, their hematopoietic lineages are largely unexplored with the exception of Drosophila melanogaster. To study the hematopoietic lineage of the silkworm, Bombyx mori, we investigated in vivo and in vitro differentiation of hemocyte precursors in the hematopoietic organ (HPO) into the four mature hemocyte subsets, namely, plasmatocytes, granulocytes, oenocytoids, and spherulocytes. Five days after implantation of enzymatically-dispersed HPO cells from a GFP-expressing transgenic line into the hemocoel of normal larvae, differentiation into plasmatocytes, granulocytes and oenocytoids, but not spherulocytes, was observed. When the HPO cells were cultured in vitro, plasmatocytes appeared rapidly, and oenocytoids possessing prophenol oxidase activity appeared several days later. HPO cells were also able to differentiate into a small number of granulocytes, but not into spherulocytes. When functionally mature plasmatocytes were cultured in vitro, oenocytoids were observed 10 days later. These results suggest that the hemocyte precursors in HPO first differentiate into plasmatocytes, which further change into oenocytoids. From these results, we propose that B. mori hemocytes can be divided into two major lineages, a granulocyte lineage and a plasmatocyte-oenocytoid lineage. The origins of the spherulocytes could not be determined in this study. We construct a model for the hematopoietic lineages at the larval stage of B. mori.

  8. Hemocyte-mediated phagocytosis differs between honey bee (Apis mellifera worker castes.

    Eva Marit Hystad

    Full Text Available Honey bees as other insects rely on the innate immune system for protection against diseases. The innate immune system includes the circulating hemocytes (immune cells that clear pathogens from hemolymph (blood by phagocytosis, nodulation or encapsulation. Honey bee hemocyte numbers have been linked to hemolymph levels of vitellogenin. Vitellogenin is a multifunctional protein with immune-supportive functions identified in a range of species, including the honey bee. Hemocyte numbers can increase via mitosis, and this recruitment process can be important for immune system function and maintenance. Here, we tested if hemocyte mediated phagocytosis differs among the physiologically different honey bee worker castes (nurses, foragers and winter bees, and study possible interactions with vitellogenin and hemocyte recruitment. To this end, we adapted phagocytosis assays, which-together with confocal microscopy and flow cytometry-allow qualitative and quantitative assessment of hemocyte performance. We found that nurses are more efficient in phagocytic uptake than both foragers and winter bees. We detected vitellogenin within the hemocytes, and found that winter bees have the highest numbers of vitellogenin-positive hemocytes. Connections between phagocytosis, hemocyte-vitellogenin and mitosis were worker caste dependent. Our results demonstrate that the phagocytic performance of immune cells differs significantly between honey bee worker castes, and support increased immune competence in nurses as compared to forager bees. Our data, moreover, provides support for roles of vitellogenin in hemocyte activity.

  9. Hemocyte-mediated phagocytosis differs between honey bee (Apis mellifera) worker castes.

    Hystad, Eva Marit; Salmela, Heli; Amdam, Gro Vang; Münch, Daniel

    2017-01-01

    Honey bees as other insects rely on the innate immune system for protection against diseases. The innate immune system includes the circulating hemocytes (immune cells) that clear pathogens from hemolymph (blood) by phagocytosis, nodulation or encapsulation. Honey bee hemocyte numbers have been linked to hemolymph levels of vitellogenin. Vitellogenin is a multifunctional protein with immune-supportive functions identified in a range of species, including the honey bee. Hemocyte numbers can increase via mitosis, and this recruitment process can be important for immune system function and maintenance. Here, we tested if hemocyte mediated phagocytosis differs among the physiologically different honey bee worker castes (nurses, foragers and winter bees), and study possible interactions with vitellogenin and hemocyte recruitment. To this end, we adapted phagocytosis assays, which-together with confocal microscopy and flow cytometry-allow qualitative and quantitative assessment of hemocyte performance. We found that nurses are more efficient in phagocytic uptake than both foragers and winter bees. We detected vitellogenin within the hemocytes, and found that winter bees have the highest numbers of vitellogenin-positive hemocytes. Connections between phagocytosis, hemocyte-vitellogenin and mitosis were worker caste dependent. Our results demonstrate that the phagocytic performance of immune cells differs significantly between honey bee worker castes, and support increased immune competence in nurses as compared to forager bees. Our data, moreover, provides support for roles of vitellogenin in hemocyte activity.

  10. In vitro cultivation and cryopreservation of Babesia bigemina sporokinetes in hemocytes of Rhipicephalus microplus.

    de Rezende, Jania; Rangel, Charles P; McIntosh, Douglas; Silveira, Júlia A G; Cunha, Nathalie C; Ramos, Carlos A N; Fonseca, Adivaldo H

    2015-09-15

    Cultures of tick hemocytes represent alternative cell lines for the isolation and cultivation of a variety of hemoparasites. The present study reports the development and evaluation of methods for the in vitro culture and maintenance of sporokinetes of Babesia bigemina in association with hemocytes of the tick Rhipicephalus microplus. Hemolymph, from engorged females infected with B. bigemina sporokinetes, was incubated at 28 °C in L15 culture medium supplemented with 40% fetal bovine serum. Adherence of hemocytes to flask surfaces and the development of B. bigemina sporokinetes commenced on the first day of cultivation. The protozoa demonstrated clear motility and the capacity to adhere to hemocyte membranes for up to 25 days, at which time the hemocytes began to show signs of degeneration. Examination of Giemsa stained hemocyte cultures, revealed the presence of pyriformis forms, as well as mature and immature sporokinetes with dark red nuclei, centralized or near the apical extremities. Sporokinetes harvested from culture supernatants were cryopreserved in liquid nitrogen. Inoculation of parasite-free hemocyte cultures with defrosted sporokinetes, demonstrated the viability and interaction of the protozoa with the hemocytes over 21 days. Cultured hemocytes of R. microplus hold potential for development as a tool in the study of host parasite interactions and as a substrate for the in vitro maintenance of B. bigemina sporokinetes. Copyright © 2015 Elsevier B.V. All rights reserved.

  11. Reactive oxygen species in unstimulated hemocytes of the pacific oyster Crassostrea gigas: a mitochondrial involvement.

    Ludovic Donaghy

    Full Text Available The Pacific oyster Crassostrea gigas is a sessile bivalve mollusc whose homeostasis relies, at least partially, upon cells circulating in hemolymph and referred to as hemocytes. Oyster's hemocytes have been reported to produce reactive oxygen species (ROS, even in absence of stimulation. Although ROS production in bivalve molluscs is mostly studied for its defence involvement, ROS may also be involved in cellular and tissue homeostasis. ROS sources have not yet been described in oyster hemocytes. The objective of the present work was to characterize the ROS sources in unstimulated hemocytes. We studied the effects of chemical inhibitors on the ROS production and the mitochondrial membrane potential (Δψ(m of hemocytes. First, this work confirmed the specificity of JC-10 probe to measure Δψ(m in oyster hemocytes, without being affected by ΔpH, as reported in mammalian cells. Second, results show that ROS production in unstimulated hemocytes does not originate from cytoplasmic NADPH-oxidase, nitric oxide synthase or myeloperoxidase, but from mitochondria. In contrast to mammalian cells, incubation of hemocytes with rotenone (complex I inhibitor had no effect on ROS production. Incubation with antimycin A (complex III inhibitor resulted in a dose-dependent ROS production decrease while an over-production is usually reported in vertebrates. In hemocytes of C. gigas, the production of ROS seems similarly dependent on both Δψ(m and ΔpH. These findings point out differences between mammalian models and bivalve cells, which warrant further investigation about the fine characterization of the electron transfer chain and the respective involvement of mitochondrial complexes in ROS production in hemocytes of bivalve molluscs.

  12. Osmoregulated Chloride Currents in Hemocytes from Mytilus galloprovincialis.

    Monica Bregante

    Full Text Available We investigated the biophysical properties of the transport mediated by ion channels in hemocytes from the hemolymph of the bivalve Mytilus galloprovincialis. Besides other transporters, mytilus hemocytes possess a specialized channel sensitive to the osmotic pressure with functional properties similar to those of other transport proteins present in vertebrates. As chloride fluxes may play an important role in the regulation of cell volume in case of modifications of the ionic composition of the external medium, we focused our attention on an inwardly-rectifying voltage-dependent, chloride-selective channel activated by negative membrane potentials and potentiated by the low osmolality of the external solution. The chloride channel was slightly inhibited by micromolar concentrations of zinc chloride in the bath solution, while the antifouling agent zinc pyrithione did not affect the channel conductance at all. This is the first direct electrophysiological characterization of a functional ion channel in ancestral immunocytes of mytilus, which may bring a contribution to the understanding of the response of bivalves to salt and contaminant stresses.

  13. Characterization of myosin light chain in shrimp hemocytic phagocytosis.

    Han, Fang; Wang, Zhiyong; Wang, Xiaoqing

    2010-11-01

    Myosin light chain, a well-known cytoskeleton gene, regulates multiple processes that are involved in material transport, muscle shrink and cell division. However, its function in phagocytosis against invading pathogens in crustacean remains unknown. In this investigation, a myosin light chain gene was obtained from Marsupenaeus japonicus shrimp. The full-length cDNA of this gene was of 766 bp and an open reading frame (ORF) of 462 bp encoding a polypeptide of 153 amino acids. The myosin light chain protein was expressed in Escherichia coli and purified. Subsequently the specific antibody was raised using the purified GST fusion protein. As revealed by immuno-electron microscopy, the myosin light chain protein was only expressed in the dark bands of muscle. In the present study, the myosin light chain gene was up-regulated in the WSSV-resistant shrimp as revealed by real-time PCR and western blot. And the phagocytic percentage and phagocytic index using FITC-labeled Vibrio parahemolyticus were remarkably increased in the WSSV-resistant shrimp, suggesting that the myosin light chain protein was essential in hemocytic phagocytosis. On the other hand, RNAi assays indicated that the phagocytic percentage and phagocytic index were significantly decreased when the myosin light chain gene was silenced by sequence-specific siRNA. These findings suggested that myosin light chain protein was involved in the regulation of hemocytic phagocytosis of shrimp. Copyright 2010 Elsevier Ltd. All rights reserved.

  14. IN VITRO KILLING OF PERKINSUS MARINUS BY HEMOCYTES OF OYSTERS CRASSOSTREA VIRGINICA

    A colorimetric microbicidal assay was adapted, optimized and applied in experiments to characterize the in vitro capacity of eastern oyster (Crassostrea virginica) hemocytes to kill cultured isolates of Perkinsus marinus, a protozoan parasite causing a highly destructive disease...

  15. INFLUENCE OF SEASONAL FACTORS ON OYSTER HEMOCYTE KILLING OF VIBRIO PARAHEMOLYTICUS

    Seasonal variation of cellular defenses of oyster Crassostrea virginica against Vibrio parahaemolyticus were examined from June 1997 to December 1998 using a recently developed bactericidal assay that utilizes a tetrazolium dye. Mean hemocyte numbers, plasma lysozyme, and P. mari...

  16. Concholepas hemocyanin biosynthesis takes place in the hepatopancreas, with hemocytes being involved in its metabolism.

    Manubens, Augusto; Salazar, Fabián; Haussmann, Denise; Figueroa, Jaime; Del Campo, Miguel; Pinto, Jonathan Martínez; Huaquín, Laura; Venegas, Alejandro; Becker, María Inés

    2010-12-01

    Hemocyanins are copper-containing glycoproteins in some molluscs and arthropods, and their best-known function is O(2) transport. We studied the site of their biosynthesis in the gastropod Concholepas concholepas by using immunological and molecular genetic approaches. We performed immunohistochemical staining of various organs, including the mantle, branchia, and hepatopancreas, and detected C. concholepas hemocyanin (CCH) molecules in circulating and tissue-associated hemocytes by electron microscopy. To characterize the hemocytes, we purified them from hemolymph. We identified three types of granular cells. The most abundant type was a phagocyte-like cell with small cytoplasmic granules. The second type contained large electron-dense granules. The third type had vacuoles containing hemocyanin molecules suggesting that synthesis or catabolism occurred inside these cells. Our failure to detect cch-mRNA in hemocytes by reverse transcription with the polymerase chain reaction (RT-PCR) led us to propose that hemocytes instead played a role in CCH metabolism. This hypothesis was supported by colloidal gold staining showing hemocyanin molecules in electron-dense granules inside hemocytes. RT-PCR analysis, complemented by in situ hybridization analyses with single-stranded antisense RNAs as specific probes, demonstrated the presence of cch-mRNA in the hepatopancreas; this was consistent with the specific hybridization signal and confirmed the hepatopancreas as the site of CCH synthesis. Finally, we investigated the possibility that CCH catabolism in hemocytes was involved in the host immune response and in the generation of secondary metabolites such as antimicrobial peptides and phenoloxidase.

  17. Ocean Acidification Affects Hemocyte Physiology in the Tanner Crab (Chionoecetes bairdi)

    Meseck, Shannon L.; Alix, Jennifer H.; Swiney, Katherine M.; Long, W. Christopher; Wikfors, Gary H.; Foy, Robert J.

    2016-01-01

    We used flow cytometry to determine if there would be a difference in hematology, selected immune functions, and hemocyte pH (pHi), under two different, future ocean acidification scenarios (pH = 7.50, 7.80) compared to current conditions (pH = 8.09) for Chionoecetes bairdi, Tanner crab. Hemocytes were analyzed after adult Tanner crabs were held for two years under continuous exposure to acidified ocean water. Total counts of hemocytes did not vary among control and experimental treatments; however, there were significantly greater number of dead, circulating hemocytes in crabs held at the lowest pH treatment. Phagocytosis of fluorescent microbeads by hemocytes was greatest at the lowest pH treatment. These results suggest that hemocytes were dying, likely by apoptosis, at a rate faster than upregulated phagocytosis was able to remove moribund cells from circulation at the lowest pH. Crab hemolymph pH (pHe) averaged 8.09 and did not vary among pH treatments. There was no significant difference in internal pH (pHi) within hyalinocytes among pH treatments and the mean pHi (7.26) was lower than the mean pHe. In contrast, there were significant differences among treatments in pHi of the semi-granular+granular cells. Control crabs had the highest mean semi-granular+granular pHi compared to the lowest pH treatment. As physiological hemocyte functions changed from ambient conditions, interactions with the number of eggs in the second clutch, percentage of viable eggs, and calcium concentration in the adult crab shell was observed. This suggested that the energetic costs of responding to ocean acidification and maintaining defense mechanisms in Tanner crab may divert energy from other physiological processes, such as reproduction. PMID:26859148

  18. The ascidian Styela plicata hemocytes as a potential biomarker of marine pollution: In vitro effects of seawater and organic mercury.

    Parrinello, D; Bellante, A; Parisi, M G; Sanfratello, M A; Indelicato, S; Piazzese, D; Cammarata, M

    2017-02-01

    Toxic metals, such as mercury, contribute substantially to anthropogenic pollution in many estuarine environments. Animals living in those environments, particularly invertebrate filter feeders like tunicates, can be used as bioindicators. In an attempt to identify cellular markers for revealing pollution, this study examined in vitro the effects of different concentrations of methyl mercury on Styela plicata hemocytes. The harvested hemocytes from S. plicata that were exposed to the metal had a significant mortality, cellular count and morphometric alterations. These findings provided evidence of MeHg immunotoxic effects on S. plicata, resulting in hemocyte death and morphological changes induced by cytoskeleton alterations. Thus, a morphometric cellular parameter, such as spreading ability, was used as a complementary method for differentiation between hemocytes treated with a marine solution (as a negative control) and hemocytes incubated with methylmercury and/or Sicilian seawater samples. Copyright © 2016 Elsevier Inc. All rights reserved.

  19. Understanding the Role of Host Hemocytes in a Squid/Vibrio Symbiosis Using Transcriptomics and Proteomics

    Andrew J. Collins

    2012-05-01

    Full Text Available The symbiosis between the squid, Euprymna scolopes, and the bacterium, Vibrio fischeri, serves as a model for understanding interactions between beneficial bacteria and animal hosts. The establishment and maintenance of the association is highly specific and depends on the selection of V. fischeri and exclusion of non-symbiotic bacteria from the environment. Current evidence suggests that the host’s cellular innate immune system, in the form of macrophage-like hemocytes, helps to mediate host tolerance of V. fischeri. To begin to understand the role of hemocytes in this association, we analyzed these cells by high-throughput 454 transcriptomic and liquid chromatography/ tandem mass spectrometry (LC-MS/MS proteomic analyses. 454 high-throughput sequencing produced 650,686 reads totaling 279.9 Mb while LC-MS/MS analyses of circulating hemocytes putatively identified 702 unique proteins. Several receptors involved with the recognition of microbial associated molecular patterns (MAMPs were identified. Among these was a complete open reading frame (ORF to a putative peptidoglycan recognition protein (EsPGRP5 that has conserved residues for amidase activity. Assembly of the hemocyte transcriptome showed EsPGRP5 had high coverage, suggesting it is among the 5% most abundant transcripts in circulating hemocytes. Other transcripts and proteins identified included members of the conserved NFκB signaling pathway, putative members of the complement pathway, the carbohydrate binding protein galectin, and cephalotoxin. Quantitative PCR of complement-related genes, cephalotoxin, EsPGRP5, and a nitric oxide synthase showed differential expression in circulating hemocytes isolated from adult squid with colonized light organs compared to those for which the symbionts were removed. These data suggest that the presence of the symbiont influences gene expression of the cellular innate immune system of the host.

  20. Recognition between symbiotic Vibrio fischeri and the hemocytes of Euprymna scolopes

    Nyholm, Spencer V.; Stewart, Jennifer J.; Ruby, Edward G.; McFall-Ngai, Margaret J.

    2008-01-01

    Summary The light-organ crypts of the squid Euprymna scolopes permit colonization exclusively by the luminous bacterium Vibrio fischeri. Because the crypt interior remains in contact with seawater, the squid must not only foster the specific symbiosis but also continue to exclude other bacteria. Investigation of the role of the innate immune system in these processes revealed that macrophage-like hemocytes isolated from E. scolopes recognized and phagocytosed V. fischeri less than other closely related bacterial species common to the host’s environment. Interestingly, phagocytes isolated from hosts that had been cured of their symbionts bound five-times more V. fischeri cells than those from uncured hosts. No such change in the ability to bind other species of bacteria was observed, suggesting that the host adapts specifically to V. fischeri. Deletion of the gene encoding OmpU, the major outer membrane protein of V. fischeri, increased binding by hemocytes from uncured animals to the level observed for hemocytes from cured animals. Co-incubation with wild-type V. fischeri reduced this binding, suggesting they produce a factor that complements the mutant’s defect. Analyses of the phagocytosis of bound cells by fluorescence-activated cell sorting (FACS) indicated that, once binding to hemocytes had occurred, V. fischeri cells are phagocytosed as effectively as other bacteria. Thus, discrimination by this component of the squid immune system occurs at the level of hemocyte binding, and this response: (i) is modified by previous exposure to the symbiont and, (ii) relies on outer membrane and/or secreted components of the symbionts. These data suggest that regulation of host hemocyte binding by the symbiont may be one of many factors that contribute to specificity in this association. PMID:19196278

  1. Hemocyte characterization of Nasutitermes coxipoensis (Holmgren) (Isoptera: Termitidae) workers and hemocyte evaluation after parasitism by Metarhizium anisopliae; Caracterizacao dos hemocitos de operarios de Nasutitermes coxipoensis (Holmgren) (Isoptera: Termitidae) e avaliacao hemocitaria apos parasitismo por Metarhizium anisopliae

    Cunha, Franklin M.; Wanderley-Teixeira, Valeria; Albuquerque, Auristela C. [Universidade Federal Rural de Pernambuco (UFRPE), Recife, PE (Brazil). Programa de Pos-Graduacao em Entomologia Agricola], e-mail: ukento@yahoo.com.br; Teixeira, Alvaro A.C. [Universidade Federal Rural de Pernambuco (UFRPE), Recife, PE (Brazil). Dept. de Morfologia e Fisiologia Animal], e-mail: valeria@dmfa.ufrpe.br, e-mail: auritermes@yahoo.com.br; Alves, Luiz C. [Universidade Federal de Pernambuco (UFPE), Recife, PE (Brazil). Lab. de Imunopatologia Keizo Asami (LIKA); Lima, Elza A.L.A. [Universidade Federal de Pernambuco (UFPE), Recife, PE (Brazil). Dept. de Micologia. Lab. de Controle Biologico

    2009-03-15

    We aimed to characterize the morphology and ultrastructure of hemocytes of Nasutitermes coxipoensis (Holmgren) workers and to quantify the cell types 24h, 48h and 72h after inoculation with Metarhizium anisopliae. Six hemocytes types were identified, plasmatocyte, granulocyte, spherulocyte, prohemocyte, adipohemocyte and eonocytoid Hemocytes did not present any morphological alteration at the several observation periods, but they did have a change in their abundance, as observed for spherulocytes, adipohemocytes and eonocytoids at all intervals, and for plasmatocytes and granulocytes at 48h after host inoculation. We argue on the possible reasons and implications of the observed changes. (author)

  2. New insights from the oyster Crassostrea rhizophorae on bivalve circulating hemocytes.

    Mauro de Freitas Rebelo

    Full Text Available Hemocytes are the first line of defense of the immune system in invertebrates, but despite their important role and enormous potential for the study of gene-environment relationships, research has been impeded by a lack of consensus on their classification. Here we used flow cytometry combined with histological procedures, histochemical reactions and transmission electron microscopy to characterize the hemocytes from the oyster Crassostrea rhizophorae. Transmission electron microscopy revealed remarkable morphological characteristics, such as the presence of membranous cisternae in all mature cells, regardless of size and granulation. Some granular cells contained many cytoplasmic granules that communicated with each other through a network of channels, a feature never previously described for hemocytes. The positive reactions for esterase and acid phosphatase also indicated the presence of mature cells of all sizes and granule contents. Flow cytometry revealed a clear separation in complexity between agranular and granular populations, which could not be differentiated by size, with cells ranging from 2.5 to 25 µm. Based on this evidence we suggest that, at least in C. rhizophorae, the different subpopulations of hemocytes may in reality be different stages of one type of cell, which accumulates granules and loses complexity (with no reduction in size as it degranulates in the event of an environmental challenge.

  3. Hemocyte-mediated phagocytosis and melanization in the mosquito Armigeres subalbatus following immune challenge by bacteria.

    Hillyer, Julián F; Schmidt, Shelley L; Christensen, Bruce M

    2003-07-01

    Mosquitoes are important vectors of disease. These insects respond to invading organisms with strong cellular and humoral immune responses that share many similarities with vertebrate immune systems. The strength and specificity of these responses are directly correlated to a mosquito's ability to transmit disease. In the current study, we characterized the hemocytes (blood cells) of Armigeres subalbatus by morphology (ultrastructure), lectin binding, enzyme activity, immunocytochemistry, and function. We found four hemocyte types: granulocytes, oenocytoids, adipohemocytes, and thrombocytoids. Granulocytes contained acid phosphatase activity and bound the exogenous lectins Helix pomatia agglutinin, Galanthus nivalis lectin, and wheat germ agglutinin. Following bacteria inoculation, granulocytes mounted a strong phagocytic response as early as 5 min postexposure. Bacteria also elicited a hemocyte-mediated melanization response. Phenoloxidase, the rate-limiting enzyme in the melanization pathway, was present exclusively in oenocytoids and in many of the melanotic capsules enveloping bacteria. The immune responses mounted against different bacteria were not identical; gram(-) Escherichia coli were predominantly phagocytosed and gram(+) Micrococcus luteus were melanized. These studies implicate hemocytes as the primary line of defense against bacteria.

  4. Silkworm (Bombyx mori) hemocytes do not produce reactive oxygen metabolites as a part of defense mechanisms

    Hyršl, P.; Číž, Milan; Kubala, Lukáš; Lojek, Antonín

    2004-01-01

    Roč. 49, č. 3 (2004), s. 315-319 ISSN 0015-5632 R&D Projects: GA AV ČR IBS5004009 Institutional research plan: CEZ:AV0Z5004920 Keywords : hemocytes * Bombyx mori * reactive oxygen species Subject RIV: BO - Biophysics Impact factor: 1.034, year: 2004

  5. Effects of cadmium on aneuploidy and hemocyte parameters in the Pacific oyster, Crassostrea gigas

    Bouilly, Karine; Gagnaire, Beatrice; Bonnard, Marc; Thomas-Guyon, Helene; Renault, Tristan; Miramand, Pierre; Lapegue, Sylvie

    2006-01-01

    Pacific oysters, Crassostrea gigas, are commonly reared in estuaries where they are exposed to anthropogenic pollution. Much research has been made on the toxicity of cadmium to aquatic organisms because the compound recurrently contaminates their environment. Our study examined the influence of cadmium on aneuploidy level (lowered chromosome number in a percentage of somatic cells) and hemocyte parameters in C. gigas at different stages of life. Adults and juveniles were exposed to two different concentrations of cadmium. The first concentration applied was equivalent to a peak value found in Marennes-Oleron bay (Charente-Maritime, France; 50 ng L -1 ) and the second was 10 times higher (500 ng L -1 ). Exposure to 50 ng L -1 cadmium caused a significant decrease in the survival time of C. gigas, but exposure to 500 ng L -1 surprisingly affected the survival time positively. Significant differences in aneuploidy level were observed between the cadmium treatments and the control in adults but not in juveniles or the offspring of the adult groups. The effects of cadmium on hemocyte parameters were analyzed by flow cytometry. Several hemocyte parameters increased significantly after 21 days of cadmium exposure and subsequently decreased. Phenoloxidase-like activity, evaluated by spectrophotometry, varied over the time of the experiment and increased after 66 days of contact with 500 ng L -1 cadmium. Taken together, cadmium at environmentally relevant concentrations seems to have only moderate effects on aneuploidy and hemocyte parameters

  6. Vibrio cholerae interactions with Mytilus galloprovincialis hemocytes mediated by serum components.

    Laura eCanesi

    2013-12-01

    Full Text Available Edible bivalves (e.g., mussels, oysters can accumulate large amount of bacteria in their tissues and act as passive carriers of pathogens to humans. Bacterial persistence inside bivalves depends, at least in part, on hemolymph anti-bacterial activity that is exerted by both serum soluble factors and phagocytic cells (i.e., the hemocytes. It was previously shown that Mytilus galloprovincialis hemolymph serum contains opsonins that mediate D-mannose-sensitive interactions between hemocytes and V. cholerae O1 El Tor bacteria that carry the Mannose–Sensitive Hemagglutinin (MSHA. These opsonins enhance phagocytosis and killing of vibrios by facilitating their binding to hemocytes. Since V. cholerae strains not carrying the MSHA ligand (O1 classical, non O1/O139 are present in coastal water and can be entrapped by mussels, we studied whether in mussel serum, in addition to opsonins directed towards MSHA, other components can mediate opsonization of these bacteria. By comparing interactions of O1 classical and non O1/O139 strains with hemocytes in ASW and serum, it was found that M. galloprovincialis serum contains components that increase by at approximately two fold their adhesion to, association with and killing by hemocytes. Experiments conducted with high and low molecular mass fractions obtained by serum ultrafiltration indicated that these compounds have molecular mass higher than 5000 Da. Serum exposure to high temperature (80°C abolished its opsonizing capability suggesting that the involved serum active components are of protein nature. Further studies are needed to define the chemical properties and specificity of both the involved bacterial ligands and hemolymph opsonins. This information will be central not only to better understand V. cholerae ecology, but also to improve current bivalve depuration practices and properly protect human health.

  7. Immune responses and ultrastructural changes of hemocytes in freshwater crab Sinopotamon henanense exposed to elevated cadmium

    Qin Qin [College of Life Science, Shanxi University, Taiyuan, Shanxi, 030006 (China); Central Laboratory, Shanxi Provincial People' s Hospital, Taiyuan, Shanxi, 030012 (China); Qin Shengjuan [College of Life Science, Shanxi University, Taiyuan, Shanxi, 030006 (China); Wang Lan, E-mail: lanwang@sxu.edu.cn [College of Life Science, Shanxi University, Taiyuan, Shanxi, 030006 (China); Lei Wenwen [College of Life Science, Shanxi University, Taiyuan, Shanxi, 030006 (China)

    2012-01-15

    Cadmium (Cd) is one of the most toxic heavy metals that can impact immunological parameters in aquatic animals. To investigate the immunotoxicity and ultrastructural changes of hemocytes, specimens of Sinopotamon henanense were exposed to different concentrations of cadmium and the differences in immunologic parameters between Cd exposure groups and control groups were investigated. Total hemocyte count (THC) in Cd-exposure groups were decreased significantly when compared with the control groups, especially in the groups treated with higher Cd concentrations and longer exposure time, while no significant differences were observed in the proportions of the three types of hemocytes. Phenoloxidase (PO) activities were significantly higher in Cd-exposure groups than the control groups. Superoxide dismutase (SOD) activities gradually increased in 7.25 and 14.5 mg L{sup -1} Cd groups, but in other higher Cd groups, they showed first increase and following decrease with the exposure time prolonged. Acid phosphatase (ACP) activities were induced at 48 h, and then decreased, while alkaline phosphatase (AKP) activities increased gradually until 96 h. Electron microscopic results showed that nucleus, mitochondria and rough endoplasm recutulum (rER) of three types of hemocytes were sensitive to acute Cd toxicity. In Cd-exposed groups, chromatin condensation, nucleus deformation and nucleus envelope rupture were noted. Additionally, mitochondrial dilation and rER degranulation were observed in Cd-treated crabs. These results suggested that immune response and organelles of hemocyte of S. henanense were impacted by Cd exposure, and the changes of these immunologic parameters reflect changes in crab immune response capability consequent to Cd exposure.

  8. Immune responses and ultrastructural changes of hemocytes in freshwater crab Sinopotamon henanense exposed to elevated cadmium

    Qin Qin; Qin Shengjuan; Wang Lan; Lei Wenwen

    2012-01-01

    Cadmium (Cd) is one of the most toxic heavy metals that can impact immunological parameters in aquatic animals. To investigate the immunotoxicity and ultrastructural changes of hemocytes, specimens of Sinopotamon henanense were exposed to different concentrations of cadmium and the differences in immunologic parameters between Cd exposure groups and control groups were investigated. Total hemocyte count (THC) in Cd-exposure groups were decreased significantly when compared with the control groups, especially in the groups treated with higher Cd concentrations and longer exposure time, while no significant differences were observed in the proportions of the three types of hemocytes. Phenoloxidase (PO) activities were significantly higher in Cd-exposure groups than the control groups. Superoxide dismutase (SOD) activities gradually increased in 7.25 and 14.5 mg L −1 Cd groups, but in other higher Cd groups, they showed first increase and following decrease with the exposure time prolonged. Acid phosphatase (ACP) activities were induced at 48 h, and then decreased, while alkaline phosphatase (AKP) activities increased gradually until 96 h. Electron microscopic results showed that nucleus, mitochondria and rough endoplasm recutulum (rER) of three types of hemocytes were sensitive to acute Cd toxicity. In Cd-exposed groups, chromatin condensation, nucleus deformation and nucleus envelope rupture were noted. Additionally, mitochondrial dilation and rER degranulation were observed in Cd-treated crabs. These results suggested that immune response and organelles of hemocyte of S. henanense were impacted by Cd exposure, and the changes of these immunologic parameters reflect changes in crab immune response capability consequent to Cd exposure.

  9. Rac1 mediates cytokine-stimulated hemocyte spreading via prostaglandin biosynthesis in the beet armyworm, Spodoptera exigua

    Cell spreading is an integral component of insect hemocytic immune reactions to infections and invasions. Cell spreading is accomplished by cytoskeleton rearrangement, which is activated by three major immune mediators, biogenic monoamines, plasmatocyte-spreading peptide (PSP), and eicosanoids, part...

  10. Ultrastructural and functional characterization of circulating hemocytes from Galleria mellonella larva: Cell types and their role in the innate immunity.

    Wu, Gongqing; Liu, Yi; Ding, Ying; Yi, Yunhong

    2016-08-01

    Galleria mellonella larvae have been widely used as a model to study the virulence of various human pathogens. Hemocytes play important roles in the innate immune response of G. mellonella. In this study, the hemocytes of G. mellonella larvae were analyzed by transmission electron microscope, light microscope, and cytochemistry. The cytological and morphological analyses revealed four types of hemocytes; Plasmatocytes, granular cells, spherule cells and oenocytoids. Differential hemocyte counts showed that under our conditions plasmatocytes and granular cells were the most abundant circulating cell types in the hemolymph. We also investigated the role of different types of hemocytes in the cellular and humoral immune defenses. The in-vivo experiment showed that plasmatocytes, granular cells and oenocytoids phagocytized FITC-labelled Escherichia coli bacteria in larvae of G. mellonella, whereas the granular cells exhibited the strongest phagocytic ability against these microbial cells. After incubation with L-DOPA, plasmatocytes, granular cells and oenocytoids are stained brown, indicating the presence of phenoloxidase activity. These results shed new light on our understanding of the immune function of G. mellonella hemocytes. Copyright © 2016 Elsevier Ltd. All rights reserved.

  11. Use of Biomphalaria glabrata hemocytes as a biomarker of exposure to ionizing radiation

    Silva, Hianna A.M.F.; Lima, Maira V.; Sa, Jose L.F.; Siqueira, Williams N.; Luna Filho, Ricardo L.C.; Melo, Larissa S.A.; Morais, Vinicius H.T.; Melo, Ana M.M.A., E-mail: hiannaamfs@gmail.com, E-mail: mairavasconceloslima@gmail.com, E-mail: luismuma6@gmail.com, E-mail: williams.wns@gmail.com, E-mail: ricardolclf@hotmail.com, E-mail: larissamelo.pe@gmail.com, E-mail: viniciushtmorais@hotmail.com, E-mail: amdemelo@hotmail.com [Universidade Federal de Pernambuco (UFPE), Recife, PE (Brazil). Departamento de Biofisica e Radiobiologia

    2017-11-01

    The increase of the applications of ionizing radiation in several areas and sectors of modern society has given rise to a greater probability of occurrence of accidents. These accidental occurrences have revealed the need for methods that provide quantitative data on the radiation doses absorbed by biological systems. The mollusk Biomphalaria glabrata presents as a good bioindicator in several works referenced in the literature. In this way, the objective of this work was to evaluate the morphological and quantitative alterations of hemocytes of the Biomphalaria glabrata exposed to ionizing radiation. For the experiments, adult mollusks of the species B. glabrata pigmented were used. The selected mollusks were divided into six groups: five submitted to doses of 10, 20, 30, 40, 50 Gy of {sup 60}Co gamma radiation and the control group. After 48 h, the slides were prepared and then read in a microscope. Quantitative analysis showed a decrease in the total number of hemocytes after irradiation. In the cell classification, a higher number of hyalinocytes were observed in relation to the number of granulocytes, except for the animals exposed to a dose of 30 Gy. The presence of micronuclei and binucleations were observed at all doses used. Apoptosis was observed at doses starting at 30 Gy. Therefore, it is possible to conclude that the morphological and quantitative analysis of B. glabrata hemocytes provided significant data for the identification of biological damage caused by ionizing radiation, allowing the beginning of standardization of the morphological alteration counting technique in B. glabrata hemocytes as An environmental biomarker for the action of physical agents. (author)

  12. Use of Biomphalaria glabrata hemocytes as a biomarker of exposure to ionizing radiation

    Silva, Hianna A.M.F.; Lima, Maira V.; Sa, Jose L.F.; Siqueira, Williams N.; Luna Filho, Ricardo L.C.; Melo, Larissa S.A.; Morais, Vinicius H.T.; Melo, Ana M.M.A.

    2017-01-01

    The increase of the applications of ionizing radiation in several areas and sectors of modern society has given rise to a greater probability of occurrence of accidents. These accidental occurrences have revealed the need for methods that provide quantitative data on the radiation doses absorbed by biological systems. The mollusk Biomphalaria glabrata presents as a good bioindicator in several works referenced in the literature. In this way, the objective of this work was to evaluate the morphological and quantitative alterations of hemocytes of the Biomphalaria glabrata exposed to ionizing radiation. For the experiments, adult mollusks of the species B. glabrata pigmented were used. The selected mollusks were divided into six groups: five submitted to doses of 10, 20, 30, 40, 50 Gy of "6"0Co gamma radiation and the control group. After 48 h, the slides were prepared and then read in a microscope. Quantitative analysis showed a decrease in the total number of hemocytes after irradiation. In the cell classification, a higher number of hyalinocytes were observed in relation to the number of granulocytes, except for the animals exposed to a dose of 30 Gy. The presence of micronuclei and binucleations were observed at all doses used. Apoptosis was observed at doses starting at 30 Gy. Therefore, it is possible to conclude that the morphological and quantitative analysis of B. glabrata hemocytes provided significant data for the identification of biological damage caused by ionizing radiation, allowing the beginning of standardization of the morphological alteration counting technique in B. glabrata hemocytes as An environmental biomarker for the action of physical agents. (author)

  13. Effects of Copper on Hemocyte Apoptosis, ROS Production, and Gene Expression in White Shrimp Litopenaeus vannamei.

    Guo, Hui; Li, Kexu; Wang, Wei; Wang, Chenggui; Shen, Yuchun

    2017-10-01

    Copper, a common chemical contaminant in aquatic environment, is known to be toxic to aquatic life at high concentrations. In the present study, we evaluated the apoptotic cell ratio and ROS production in hemocytes of the white shrimp Litopenaeus vannamei exposed to 1 or 5 mg L -1 Cu for 0, 3, 6, 12, 24, and 48 h. The expression changes of antioxidant biomarker genes, i.e., copper-zinc superoxide dismutase (Cu-Zn SOD) and catalase (CAT), apoptosis-related genes, i.e., caspase-3 and inhibitor of apoptosis protein (IAP), and a specific biomarker gene of heavy metal pollution, i.e., metallothionein (MT), were also determined in hemocytes. Significant increases in ROS production were observed in both treatment groups at each time points. The apoptotic cell ratios were significantly increased at 6-48 h among shrimp exposed to 1 mg L -1 Cu and at each time points in 5 mg L -1 Cu group. These results indicated that Cu would induce oxidative stress and apoptosis in the hemocyte of L. vannamei. Quantitative real-time PCR analysis revealed that the relative expression levels of Cu-Zn SOD, CAT, caspase-3, IAP, and MT were upregulated in a dose-dependent and time-dependent manner, suggesting the involvement of these genes in stress response against Cu exposure.

  14. Effect of gamma irradiation on the hemocyte-mediated immune response of Aedes aegypti against microfilariae

    Christensen, B.M.; Huff, B.M.; Li, J.

    1990-01-01

    The effect of gamma irradiation on the melanotic encapsulation response of Aedes aegypti black eye Liverpool strain against inoculated Dirofilaria immitis microfilariae (mff) was assessed at 1, 2, 3, and 6 days postinoculation (PI). Mosquitoes received 6000 rad from a 137Cs source (Shepard Mark I irradiator) at 3 days postemergence and were inoculated with 15-20 mff 24 hr later. These mosquitoes were compared to nonirradiated controls that also were inoculated with 15-20 mff at 3 days postemergence. The immune response was significantly reduced in irradiated mosquitoes as compared with controls at all days PI. Although the response was significantly inhibited compared with controls, irradiated mosquitoes were still capable of eliciting a response against 69% of recovered mff at 6 days PI. External gamma irradiation did not significantly affect the proliferation of hemocytes associated with the melanotic encapsulation response of A. aegypti. The number of circulating hemocytes increased in irradiated mosquitoes in response to inoculated mff in a manner similar to nonirradiated, inoculated controls. Hemocyte monophenol oxidase activity, however, was significantly reduced in gamma-irradiated mosquitoes at 12 hr PI as compared with controls. The reduced immunological capacity of irradiated mosquitoes might be related to an interference with gene activity required for the synthesis or activation of enzymes that are directly or indirectly involved in the biochemical processes associated with the production of melanotic substances that sequester mff

  15. Morphologic, cytometric and functional characterization of the common octopus (Octopus vulgaris) hemocytes.

    Castellanos-Martínez, S; Prado-Alvarez, M; Lobo-da-Cunha, A; Azevedo, C; Gestal, C

    2014-05-01

    The hemocytes of Octopus vulgaris were morphologically and functionally characterized. Light and electron microscopy (TEM and SEM), and flow cytometry analyses revealed the existence of two hemocyte populations. Large granulocytes showed U-shaped nucleus, a mean of 11.6 μm±1.2 in diameter with basophilic granules, polysaccharide and lysosomic deposits in the cytoplasm. Small granulocytes measured a mean of 8.1 μm±0.7 in diameter, and have a round nucleus occupying almost the entire cell and few or not granules in the cytoplasm. Flow cytometry analysis showed that large granulocytes are the principal cells that develop phagocytosis of latex beads (rising up to 56%) and ROS after zymosan stimulation. Zymosan induced the highest production of both ROS and NO. This study is the first tread towards understanding the O. vulgaris immune system by applying new tools to provide a most comprehensive morpho-functional study of their hemocytes. Copyright © 2013 Elsevier Ltd. All rights reserved.

  16. The immune response of hemocytes of the insect Oncopeltus fasciatus against the flagellate Phytomonas serpens.

    Thiago L Alves e Silva

    Full Text Available The genus Phytomonas includes parasites that are etiological agents of important plant diseases, especially in Central and South America. These parasites are transmitted to plants via the bite of an infected phytophagous hemipteran. Despite the economic impact of these parasites, many basic questions regarding the genus Phytomonas remain unanswered, such as the mechanism by which the parasites cope with the immune response of the insect vector. In this report, using a model of systemic infection, we describe the function of Oncopeltus fasciatus hemocytes in the immune response towards the tomato parasite Phytomonas serpens. Hemocytes respond to infection by trapping parasites in nodular structures and phagocytizing the parasites. In electron microscopy of hemocytes, parasites were located inside vacuoles, which appear fused with lysosomes. The parasites reached the O. fasciatus salivary glands at least six hours post-infection. After 72 hours post-infection, many parasites were attached to the salivary gland outer surface. Thus, the cellular responses did not kill all the parasites.

  17. Endogenous growth factor stimulation of hemocyte proliferation induces resistance to Schistosoma mansoni challenge in the snail host.

    Pila, Emmanuel A; Gordy, Michelle A; Phillips, Valerie K; Kabore, Alethe L; Rudko, Sydney P; Hanington, Patrick C

    2016-05-10

    Digenean trematodes are a large, complex group of parasitic flatworms that infect an incredible diversity of organisms, including humans. Larval development of most digeneans takes place within a snail (Gastropoda). Compatibility between snails and digeneans is often very specific, such that suitable snail hosts define the geographical ranges of diseases caused by these worms. The immune cells (hemocytes) of a snail are sentinels that act as a crucial barrier to infection by larval digeneans. Hemocytes coordinate a robust and specific immunological response, participating directly in parasite killing by encapsulating and clearing the infection. Hemocyte proliferation and differentiation are influenced by unknown digenean-specific exogenous factors. However, we know nothing about the endogenous control of hemocyte development in any gastropod model. Here, we identify and functionally characterize a progranulin [Biomphalaria glabrata granulin (BgGRN)] from the snail B. glabrata, a natural host for the human blood fluke Schistosoma mansoni Granulins are growth factors that drive proliferation of immune cells in organisms, spanning the animal kingdom. We demonstrate that BgGRN induces proliferation of B. glabrata hemocytes, and specifically drives the production of an adherent hemocyte subset that participates centrally in the anti-digenean defense response. Additionally, we demonstrate that susceptible B. glabrata snails can be made resistant to infection with S. mansoni by first inducing hemocyte proliferation with BgGRN. This marks the functional characterization of an endogenous growth factor of a gastropod mollusc, and provides direct evidence of gain of resistance in a snail-digenean infection model using a defined factor to induce snail resistance to infection.

  18. Hemiuroid trematode sporocysts are undetected by hemocytes of their intermediate host, the ark cockle Anadara trapezia: potential role of surface carbohydrates in successful parasitism.

    Kawasaki, Minami; Delamare-Deboutteville, Jerome; Dang, Cecile; Barnes, Andrew C

    2013-12-01

    In order to establish a successful relationship with their hosts, parasites must subvert or evade immune defences. Cockle Anadara trapezia and Sydney Rock oyster (SRO) Saccostrea glomerata live in the same location but only ark cockles are infected by sporocysts of hemiuroid trematode. This provides an opportunity to explore differing interactions between the parasite and the immune system of susceptible and refractive hosts. Rapid migration and encapsulation of sporocysts was observed by SRO hemocytes but not by cockle hemocytes. This migration/encapsulation was inhibited by N-acetylglucosamine or N-acetylgalactosamine but not by the other sugars, implicating specific surface carbohydrates in immune detection. Effector responses of hemocytes were investigated in vitro in terms of production of reactive oxygen production (ROS). Hemocytes of both species strongly reacted to Zymosan, but only SRO hemocytes responded to live sporocysts. Neither species' hemocytes produced ROS in the presence of dead/fixed sporocysts, and there was no suppression of Zymosan-induced respiratory burst by sporocysts. This suggests that immune escape is mediated by avoiding encapsulation, perhaps through molecular mimicry. Membrane-shaving with proteases indicated that sporocyst surface proteins are not a key factors in hemocytic detection. Surface carbohydrates of SRO and cockle hemocytes and of sporocysts were profiled with a panel of biotinylated lectins. This revealed substantial differences between cockle and SRO hemocytes, but greater similarity between cockle hemocytes and sporocysts. Results suggest that surface carbohydrates play an integral role in hemocyte immunorecognition and that surface carbohydrate molecular mimicry is a potential strategy for immune evasion in cockles by hemiuroid trematode sporocysts. Crown Copyright © 2013. Published by Elsevier Ltd. All rights reserved.

  19. Pacific oyster (Crassostrea gigas) hemocyte are not affected by a mixture of pesticides in short-term in vitro assays.

    Moreau, Pierrick; Burgeot, Thierry; Renault, Tristan

    2014-04-01

    Pesticides are frequently detected in estuaries among the pollutants found in estuarine and coastal areas and may have major ecological consequences. They could endanger organism growth, reproduction, or survival. In the context of high-mortality outbreaks affecting Pacific oysters, Crassostrea gigas, in France since 2008, it appears of importance to determine the putative effects of pesticides on oyster susceptibility to infectious agents. Massive mortality outbreaks reported in this species, mainly in spring and summer, may suggest an important role played by the seasonal use of pesticides and freshwater input in estuarine areas where oyster farms are frequently located. To understand the impact of some pesticides detected in French waters, their effects on Pacific oyster hemocytes were studied through short-term in vitro experiments. Bivalve immunity is mainly supported by hemocytes eliminating pathogens by phagocytosis and producing compounds including lysosomal enzymes and antimicrobial molecules. In this study, oyster hemocytes were incubated with a mixture of 14 pesticides and metaldehyde alone, a molecule used to eliminate land mollusks. Hemocyte parameters including dead/alive cells, nonspecific esterase activities, intracytoplasmic calcium, lysosome number and activity, and phagocytosis were monitored by flow cytometry. No significant effect of pesticides tested at different concentrations was reported on oyster hemocytes maintained in vitro for short-term periods in the present study. It could be assumed that these oyster cells were resistant to pesticide exposure in tested conditions and developing in vivo assays appears as necessary to better understand the effects of pollutants on immune system in mollusks.

  20. A hemocyte gene expression signature correlated with predictive capacity of oysters to survive Vibrio infections

    Rosa Rafael

    2012-06-01

    Full Text Available Abstract Background The complex balance between environmental and host factors is an important determinant of susceptibility to infection. Disturbances of this equilibrium may result in multifactorial diseases as illustrated by the summer mortality syndrome, a worldwide and complex phenomenon that affects the oysters, Crassostrea gigas. The summer mortality syndrome reveals a physiological intolerance making this oyster species susceptible to diseases. Exploration of genetic basis governing the oyster resistance or susceptibility to infections is thus a major goal for understanding field mortality events. In this context, we used high-throughput genomic approaches to identify genetic traits that may characterize inherent survival capacities in C. gigas. Results Using digital gene expression (DGE, we analyzed the transcriptomes of hemocytes (immunocompetent cells of oysters able or not able to survive infections by Vibrio species shown to be involved in summer mortalities. Hemocytes were nonlethally collected from oysters before Vibrio experimental infection, and two DGE libraries were generated from individuals that survived or did not survive. Exploration of DGE data and microfluidic qPCR analyses at individual level showed an extraordinary polymorphism in gene expressions, but also a set of hemocyte-expressed genes whose basal mRNA levels discriminate oyster capacity to survive infections by the pathogenic V. splendidus LGP32. Finally, we identified a signature of 14 genes that predicted oyster survival capacity. Their expressions are likely driven by distinct transcriptional regulation processes associated or not associated to gene copy number variation (CNV. Conclusions We provide here for the first time in oyster a gene expression survival signature that represents a useful tool for understanding mortality events and for assessing genetic traits of interest for disease resistance selection programs.

  1. Nematocytes: Discovery and characterization of a novel anculeate hemocyte in Drosophila falleni and Drosophila phalerata.

    Julianna Bozler

    Full Text Available Immune challenges, such as parasitism, can be so pervasive and deleterious that they constitute an existential threat to a species' survival. In response to these ecological pressures, organisms have developed a wide array of novel behavioral, cellular, and molecular adaptations. Research into these immune defenses in model systems has resulted in a revolutionary understanding of evolution and functional biology. As the field has expanded beyond the limited number of model organisms our appreciation of evolutionary innovation and unique biology has widened as well. With this in mind, we have surveyed the hemolymph of several non-model species of Drosophila. Here we identify and describe a novel hemocyte, type-II nematocytes, found in larval stages of numerous Drosophila species. Examined in detail in Drosophila falleni and Drosophila phalerata, we find that these remarkable cells are distinct from previously described hemocytes due to their anucleate state (lacking a nucleus and unusual morphology. Type-II nematocytes are long, narrow cells with spindle-like projections extending from a cell body with high densities of mitochondria and microtubules, and exhibit the ability to synthesize proteins. These properties are unexpected for enucleated cells, and together with our additional characterization, we demonstrate that these type-II nematocytes represent a biological novelty. Surprisingly, despite the absence of a nucleus, we observe through live cell imaging that these cells remain motile with a highly dynamic cellular shape. Furthermore, these cells demonstrate the ability to form multicellular structures, which we suggest may be a component of the innate immune response to macro-parasites. In addition, live cell imaging points to a large nucleated hemocyte, type-I nematocyte, as the progenitor cell, leading to enucleation through a budding or asymmetrical division process rather than nuclear ejection: This study is the first to report such a

  2. Jaburetox-induced toxic effects on the hemocytes of Rhodnius prolixus (Hemiptera: Reduviidae).

    Moyetta, Natalia R; Broll, Valquiria; Perin, Ana Paula A; Uberti, Augusto F; Coste Grahl, Matheus V; Staniscuaski, Fernanda; Carlini, Celia R; Fruttero, Leonardo L

    2017-10-01

    Jaburetox is a recombinant peptide derived from a Canavalia ensiformis urease that presents toxic effects upon several species of insects, phytopathogenic fungi and yeasts of medical importance. So far, no toxicity of Jaburetox to mammals has been shown. Previous reports have identified biochemical targets of this toxic peptide in insect models, although its mechanism of action is not completely understood. In this work, we aimed to characterize the effects of Jaburetox in hemolymphatic insect cells. For this purpose, the model insect and Chagas' disease vector Rhodnius prolixus was used. In vivo and in vitro experiments indicated that Jaburetox interacts with a subset of hemocytes and it can be found in various subcellular compartments. In insects injected with Jaburetox there was an increase in the gene expression of the enzymes UDP-N-acetylglucosamine pyrophosphorylase (UAP), chitin synthase and nitric oxide synthase (NOS). Nevertheless, the expression of NOS protein, the enzyme activities of UAP and acid phosphatase (a possible link between UAP and NOS) as well as the phosphorylation state of proteins remained unchanged upon the in vivo Jaburetox treatment. Nitric oxide (NO) imaging using fluorescent probes showed that Jaburetox augmented NO production in the hemocyte aggregates when compared to controls. Even though Jaburetox activated the hemocytes, as demonstrated by wheat germ agglutinin binding assays, the peptide did not lead to an increase of their phagocytic behavior. Taken together, these findings contribute to our understanding of toxic effects of Jaburetox, a peptide with biotechnological applications and a prospective tool for rational insect control. Copyright © 2017 Elsevier Inc. All rights reserved.

  3. Virus-like particles in venom of Meteorus pulchricornis induce host hemocyte apoptosis.

    Suzuki, M; Tanaka, T

    2006-06-01

    Ultrastructural studies on the reproductive tract and venom apparatus of a female braconid, Meteorus pulchricornis, revealed that the parasitoid lacks the calyx region in its oviduct, but possesses a venom gland with two venom gland filaments and a venom reservoir filled with white and cloudy fluid. Its venom gland cell is concaved and has a lumen filled with numerous granules. Transmisson electron microscopic (TEM) observation revealed that virus-like particles (VLPs) were produced in venom gland cells. The virus-like particle observed in M. pulchricornis (MpVLP) is composed of membranous envelopes with two different parts: a high-density core and a whitish low-density part. The VLPs of M. pulchricornis is also found assembling ultimately in the lumen of venom gland cell. Microvilli were found thrusting into the lumen of the venom gland cell and seem to aid in driving the matured MpVLPs to the common duct of the venom gland filament. Injection of MpVLPs into non-parasitized Pseudaletia separata hosts induced apoptosis in hemocytes, particularly granulocytes (GRs). Rate of apoptosis induced in GRs peaked 48h after VLP injection. While a large part of the GR population collapsed due to apoptosis caused by MpVLPs, the plasmatocyte population was minimally affected. The capacity of MpVLPs to cause apoptosis in host's hemocytes was further demonstrated by a decrease ( approximately 10-fold) in ability of host hemocytes to encapsulate fluorescent latex beads when MpVLPs were present. Apparently, the reduced encapsulation ability was due to a decrease in the GR population resulting from MpVLP-induced apoptosis.

  4. Hemocyte quantitative changes in Anticarsia gemmatalis (Lepidoptera: Noctuidae larvae infected by AgMNPV

    Fábio Goulart de Andrade

    2010-04-01

    Full Text Available The initial effects of the infection by AgMNPV in the total and differential counts of the hemocytes in Anticarsia gemmatalis (Lepidoptera: Noctuidae larvae were studied. The total number of the hemocytes did not decrease in infected larvae, as it occurred in non infected larvae. In infected larvae, the hemocyte types showed the following frequencies: plasmatocytes - 47.8%, esferulocytes - 25.9%, granulocytes - 15.8%, oenocytoids - 7.2%, prohemocytes - 2.8%, vermicytes - 0,5%. Only the percentage of the granulocytes was different among infected and non infected larvae, indicating that these cells responded quickly to the initial viral infection. These results showed the effective role of the hemocytes in the response of the A. gemmatalis to the infection by AgMNPV. The comprehension of the immunological mechanisms of this insect is an important tool to understand its biological control.Os efeitos iniciais da infecção por AgMNPV nas contagens total e diferencial dos hemócitos em Anticarsia gemmatalis (Lepidoptera: Noctuidae foram estudados. O número total de hemócitos não diminuiu nas larvas infectadas, como ocorreu nas larvas não infectadas. Nas larvas infectadas, os tipos de hemócitos apresentaram as seguintes freqüências: plasmatócitos - 47,8%, esferulócitos - 25,9%, granulócitos - 15,8%, oenocitóides - 7,2%, prohemócitos - 2,8%, vermiformes - 0,5%. Apenas a porcentagem de granulócitos foi diferente entre larvas infectadas e não infectadas, indicando que estas células responderam rapidamente à infecção viral inicial. Estes resultados mostraram o papel efetivo que dos hemócitos na resposta de A. gemmatalis à infecção por AgMNPV. A compreensão dos mecanismos imunológicos deste inseto é uma ferramenta importante para compreender seu controle biológico.

  5. Immune Defenses of the Invasive Apple Snail Pomacea canaliculata (Caenogastropoda, Ampullariidae: Phagocytic Hemocytes in the Circulation and the Kidney.

    Juan A Cueto

    Full Text Available Hemocytes in the circulation and kidney islets, as well as their phagocytic responses to microorganisms and fluorescent beads, have been studied in Pomacea canaliculata, using flow cytometry, light microscopy (including confocal laser scanning microscopy and transmission electron microscopy (TEM. Three circulating hemocyte types (hyalinocytes, agranulocytes and granulocytes were distinguished by phase contrast microscopy of living cells and after light and electron microscopy of fixed material. Also, three different populations of circulating hemocytes were separated by flow cytometry, which corresponded to the three hemocyte types. Hyalinocytes showed a low nucleus/cytoplasm ratio, and no apparent granules in stained material, but showed granules of moderate electron density under TEM (L granules and at least some L granules appear acidic when labeled with LysoTracker Red. Both phagocytic and non-phagocytic hyalinocytes lose most (if not all L granules when exposed to microorganisms in vitro. The phagosomes formed differed whether hyalinocytes were exposed to yeasts or to Gram positive or Gram negative bacteria. Agranulocytes showed a large nucleus/cytoplasm ratio and few or no granules. Granulocytes showed a low nucleus/cytoplasm ratio and numerous eosinophilic granules after staining. These granules are electron dense and rod-shaped under TEM (R granules. Granulocytes may show merging of R granules into gigantic ones, particularly when exposed to microorganisms. Fluorescent bead exposure of sorted hemocytes showed phagocytic activity in hyalinocytes, agranulocytes and granulocytes, but the phagocytic index was significantly higher in hyalinocytes. Extensive hemocyte aggregates ('islets' occupy most renal hemocoelic spaces and hyalinocyte-like cells are the most frequent component in them. Presumptive glycogen deposits were observed in most hyalinocytes in renal islets (they also occur in the circulation but less frequently and may mean that

  6. Evaluation of radiosensitivity hemocytes of Biomphalaria glabrata exposed to gamma radiation

    Silva, L.R.S.; Amaral, A.J.; Silva, E.B.; Amancio, F.F.; Melo, A.M.M.A.

    2013-01-01

    The mollusc Biomphalaria glabrata have characteristics that allow them to be identified as an animal model ideal for monitoring areas exposed to chemical agents and physical. This study evaluated the effect of ionizing radiation from Cobalt-60 in haemocytes present in the hemolymph of Biomphalaria glabrata, with the goal of using these cells as indicators of the presence of radiation in aquatic environments. The mollusks were divided into five groups: one control and four subjected doses of 25, 35, 45 and 55 Gy of gamma radiation. After 48 hours of irradiation, the clam hemolymph was collected and slides were prepared and stained with Giemsa for analyses under a light microscope. Statistical analysis was performed using ANOVA and Tukey's test, p <0.05. The results showed that the total number of cells after irradiation reduced compared to control except at a dose of 55 Gy. During data analysis, morphological changes were observed in haemocytes of mollusks subjected to doses of 35, 45 and 55 Gy. These modifications consisted of nucleus bilobulated and nucleo plasmatic bridges. Another change was exclusively observed in the cellular exposure of 55 Gy, where hemocytes showed misshapen nuclei and cytoplasm vacuolisation, suggestive of apoptosis. It is concluded that hemocytes are sensitive to radiation and can be used as indicators of the presence of high doses of ionizing radiation in aquatic environments. (author)

  7. Coronatin-2 from the entomopathogenic fungus Conidiobolus coronatus kills Galleria mellonella larvae and incapacitates hemocytes.

    Boguś, M I; Wieloch, W; Ligęza-Żuber, M

    2017-02-01

    Coronatin-2, a 14.5 kDa protein, was isolated from culture filtrates of the entomopathogenic fungus Conidiobolus coronatus (Costantin) Batko (Entomophthoramycota: Entomophthorales). After LC-MS/MS (liquid chromatography tandem mass spectrometry) analysis of the tryptic peptide digest of coronatin-2 and a mass spectra database search no orthologs of this protein could be found in fungi. The highest homology was observed to the partial translation elongation factor 1a from Sphaerosporium equinum (protein sequence coverage, 21%), with only one peptide sequence, suggesting that coronatin-2 is a novel fungal protein that has not yet been described. In contrast to coronatin-1, an insecticidal 36 kDa protein, which shows both elastolytic and chitinolytic activity, coronatin-2 showed no enzymatic activity. Addition of coronatin-2 into cultures of hemocytes taken from larvae of Galleria mellonella Linnaeus (Lepidoptera: Pyralidae), resulted in progressive disintegration of nets formed by granulocytes and plasmatocytes due to rapid degranulation of granulocytes, extensive vacuolization of plasmatocytes accompanied by cytoplasm expulsion, and cell disintegration. Spherulocytes remained intact, while oenocytes rapidly disintegrated. Coronatin-2 produced 80% mortality when injected into G. mellonella at 5 µg larva-1. Further study is warranted to determine the relevance of the acute toxicity of coronatin-2 and its effects on hemocytes in vitro to virulence of C. coronatus against its hosts.

  8. TAT improves in vitro transportation of fortilin through midgut and into hemocytes of white shrimp Litopenaeus vannamei

    Zhou, Yi; Zhang, Wenbing; Mai, Kangsen; Xu, Wei; Zhang, Yanjiao; Ai, Qinghui; Wang, Xiaojie

    2012-06-01

    Fortilin is a multifunctional protein implicated in many important cellular processes. Since injection of Pm-fortilin reduces shrimp mortality caused by white spot syndrome virus (WSSV), there is potential application of fortilin in shrimp culture. In the present study, in order to improve trans-membrane transportation efficiency, the protein transduction domain of the transactivator of transcription (TAT) peptide was fused to fortilin. The Pichia pastoris yeast expression system, which is widely accepted in animal feeds, was used for production of recombinant fusion protein. Green fluorescence protein (GFP) was selected as a reporter because of its intrinsic visible fluorescence. The fortilin, TAT and GFP fusion protein were constructed. Their trans-membrane transportation efficiency and effects on immune response of shrimp were analyzed in vitro. Results showed that TAT peptide improved in vitro uptake of fortilin into the hemocytes and midgut of Litopenaeus vannamei. The phenoloxidase (PO) activity of hemocytes incubated with GFP-Fortilin or GFP-Fortilin-TAT was significantly increased compared with that in the control without expressed fortilin. The PO activity of hemocytes incubated with 200 μg mL-1 GFP-Fortilin-TAT was significantly higher than that in the group with the same concentration of GFP-Fortilin. Hemocytes incubated with GFP-Fortilin-TAT at all concentrations showed significantly higher nitric oxide synthase (NOS) activity than those in the control or in the GFP-Fortilin treatment. The present in vitro study indicated that TAT fusion protein improved the immune effect of fortilin.

  9. Phagocytic activities of hemocytes from the deep-sea symbiotic mussels Bathymodiolus japonicus, B. platifrons, and B. septemdierum.

    Tame, Akihiro; Yoshida, Takao; Ohishi, Kazue; Maruyama, Tadashi

    2015-07-01

    Deep-sea mytilid mussels harbor symbiotic bacteria in their gill epithelial cells that are horizontally or environmentally transmitted to the next generation of hosts. To understand the immune defense system in deep-sea symbiotic mussels, we examined the hemocyte populations of the symbiotic Bathymodiolus mussel species Bathymodiolus japonicus, Bathymodiolus platifrons, and Bathymodiolus septemdierum, and characterized three types of hemocytes: agranulocytes (AGs), basophilic granulocytes (BGs), and eosinophilic granulocytes (EGs). Of these, the EG cells were the largest (diameter, 8.4-10.0 μm) and had eosinophilic cytoplasm with numerous eosinophilic granules (diameter, 0.8-1.2 μm). Meanwhile, the BGs were of medium size (diameter, 6.7-8.0 μm) and contained small basophilic granules (diameter, 0.3-0.4 μm) in basophilic cytoplasm, and the AGs, the smallest of the hemocytes (diameter, 4.8-6.0 μm), had basophilic cytoplasm lacking granules. A lectin binding assay revealed that concanavalin A bound to all three hemocyte types, while wheat germ agglutinin bound exclusively to EGs and BGs. The total hemocyte population densities within the hemolymph of all three Bathymodiolus mussel species were similar (8.4-13.3 × 10(5) cells/mL), and the percentages of circulating AGs, BGs, and EGs in the hemolymph of these organisms were 44.7-48.5%, 14.3-17.6%, and 34.3-41.0%, respectively. To analyze the functional differences between these hemocytes, the phagocytic activity and post-phagocytic phagosome-lysosome fusion events were analyzed in each cell type using a fluorescent Alexa Fluor(®) 488-conjugated Escherichia coli bioparticle and a LysoTracker(®) lysosomal marker, respectively. While the AGs exhibited no phagocytic activity, both types of granulocytes were phagocytic. Of the three hemocyte types, the EGs exhibited the highest level of phagocytic activity as well as rapid phagosome-lysosome fusion, which occurred within 2 h of incubation. Meanwhile, the BGs showed

  10. Morphological and functional characterization of hemocytes from two deep-sea vesicomyid clams Phreagena okutanii and Abyssogena phaseoliformis.

    Tame, Akihiro; Ozawa, Genki; Maruyama, Tadashi; Yoshida, Takao

    2018-03-01

    Deep-sea vesicomyid clams harboring intracellular symbiotic sulfur-oxidizing bacteria are often dominant in chemosynthetic animal communities. Although they are known to have erythrocytes, little is known about other hemocytes. To investigate the types and roles of various hemocytes in vesicomyid clams, we performed morphological, histochemical and functional characterization of the hemocytes in two species, Phreagena okutanii, collected from 873 to 978 m depth, and Abyssogena phaseoliformis, from 5199 to 5355 m. Both were found to have three types of hemocytes: erythrocytes (ERCs), eosinophilic granulocytes (EGs), and basophilic granulocytes (BGs). The ERCs contain hemoglobin in the cytoplasm, with basophilic vacuoles containing acid polysaccharide, neutral lipids, and peroxidase. The EGs were found to contain acid polysaccharides and eosinophilic granules containing lysosomal enzymes, acid and alkaline phosphatases, chloroacetate esterase, and peroxidase. Although BGs had some basophilic granules with alkaline phosphatase, they lacked acid phosphatase and acid polysaccharides. The EGs and BGs were shown to have phagocytic ability, while the ERCs exhibited no phagocytosis. The EGs showed higher phagocytic activity as well as a higher phagosome-lysosome fusion rate than BGs. The hemocytes of the two vesicomyid species differed in the intracellular structures. In A. phaseoliformis, ERCs additionally contained neutral polysaccharides in vacuoles and had vesicles with acinus-like acidic mucus in the cytoplasm, neither of which were observed in P. okutanii. The eosinophilic granules in the EGs had heteromorphically-elongated shapes containing homogeneously electron-dense material in P. okutanii, but were more spherical and composed of fibrous structures in A. phaseoliformis. The difference in hemocytes between the two clams seems to be reflective of phylogenetically differentiated lineages adapting to differing conditions in their respective deep-sea environments

  11. Immunomodulation by different types of N-oxides in the hemocytes of the marine bivalve Mytilus galloprovincialis.

    Caterina Ciacci

    Full Text Available The potential toxicity of engineered nanoparticles (NPs for humans and the environment represents an emerging issue. Since the aquatic environment represents the ultimate sink for NP deposition, the development of suitable assays is needed to evaluate the potential impact of NPs on aquatic biota. The immune system is a sensitive target for NPs, and conservation of innate immunity represents an useful basis for studying common biological responses to NPs. Suspension-feeding invertebrates, such as bivalves, are particularly at risk to NP exposure, since they have extremely developed systems for uptake of nano and microscale particles integral to intracellular digestion and cellular immunity. Evaluation of the effects of NPs on functional parameters of bivalve immunocytes, the hemocytes, may help understanding the major toxic mechanisms and modes of actions that could be relevant for different NP types in aquatic organisms.In this work, a battery of assays was applied to the hemocytes of the marine bivalve Mytilus galloprovincialis to compare the in vitro effects of different n-oxides (n-TiO(2, n-SiO(2, n-ZnO, n-CeO(2 chosen on the basis of their commercial and environmental relevance. Physico-chemical characterization of both primary particles and NP suspensions in artificial sea water-ASW was performed. Hemocyte lysosomal and mitochondrial parameters, oxyradical and nitric oxide production, phagocytic activity, as well as NP uptake, were evaluated. The results show that different n-oxides rapidly elicited differential responses hemocytes in relation to their chemical properties, concentration, behavior in sea water, and interactions with subcellular compartments. These represent the most extensive data so far available on the effects of NPs in the cells of aquatic organisms. The results indicate that Mytilus hemocytes can be utilized as a suitable model for screening the potential effects of NPs in the cells of aquatic invertebrates, and may

  12. Gene expression profile of Bombyx mori hemocyte under the stress of destruxin A.

    Liang Gong

    Full Text Available Destruxin A (DA is a cyclo-peptidic mycotoxin from the entomopathogenic fungus Metarhizium anisopliae. To uncover potential genes associated with its molecular mechanisms, a digital gene expression (DGE profiling analysis was used to compare differentially expressed genes in the hemocytes of silkworm larvae treated with DA. Ten DGE libraries were constructed, sequenced, and assembled, and the unigenes with least 2.0-fold difference were further analyzed. The numbers of up-regulated genes were 10, 20, 18, 74 and 8, as well as the numbers of down-regulated genes were 0, 1, 8, 13 and 3 at 1, 4, 8, 12 and 24 h post treatment, respectively. Totally, the expression of 132 genes were significantly changed, among them, 1, 3 and 12 genes were continually up-regulated at 4, 3 and 2 different time points, respectively, while 1 gene was either up or down-regulated continually at 2 different time points. Furthermore, 68 genes were assigned to one or multiple gene ontology (GO terms and 89 genes were assigned to specific Kyoto Encyclopedia of Genes and Genomes (KEGG Orthology. In-depth analysis identified that these genes putatively involved in insecticide resistance, cell apoptosis, and innate immune defense. Finally, twenty differentially expressed genes were randomly chosen and validated by quantitative real-time PCR (qRT-PCR. Our studies provide insights into the toxic effect of this microbial insecticide on silkworm's hemocytes, and are helpful to better understanding of the molecular mechanisms of DA as a biological insecticide.

  13. The virus-like particles of a braconid endoparasitoid wasp, Meteorus pulchricornis, inhibit hemocyte spreading in its noctuid host, Pseudaletia separata.

    Suzuki, M; Miura, K; Tanaka, T

    2008-06-01

    We previously reported that the virus-like particles of Meteorus pulchricornis (MpVLPs) are capable of inducing apoptosis by around 6h in the hemocytes of the host, Pseudaletia separata [Suzuki, M., Tanaka, T., 2006. Virus-like particles in venom of Meteorus pulchricornis induce host hemocyte apoptosis. Journal of Insect Physiology 52, 602-611], thereby protecting the oviposited egg. In the present study, we focused on analyses of the earlier events caused by the MpVLPs upon the host immune response, namely their effects on hemocyte spreading. After recognition and attachment on foreign substance, the granulocytes and plasmatocytes assemble focal complexes and focal adhesions and spread by protruding filopodia/lamellipodia. The well-spread, cultured hemocytes were subjected to MpVLPs exposure, and the morphological changes were observed. The granulocytes lost the focal complexes/adhesions visualized as phosphotyrosine clusters and retracted the filopodia/lamellipodia within 30min after exposure, while the plasmatocytes exhibited similar but distinct responses. The two hemocyte species prepared from either parasitized or MpVLP-injected hosts lost the ability to form both filopodia/lamellipodia and phosphotyrosine clusters. A caspase inhibitor, Z-VAD-FMK, did not affect these MpVLP-induced morphological changes, indicating that these earlier changes found in the hemocytes precede apoptosis. The present study together with our previous data has established that the attenuation of host immune defense by the MpVLPs comprises at least two temporally distinguishable phases: immediate and early inhibition of hemocyte spreading and the eventual induction of hemocyte apoptosis.

  14. In vivo effects of metaldehyde on Pacific oyster, Crassostrea gigas: comparing hemocyte parameters in two oyster families.

    Moreau, Pierrick; Burgeot, Thierry; Renault, Tristan

    2015-06-01

    Pollutants via run-off into the ocean represent a potential threat to marine organisms, especially bivalves such as oysters living in coastal environments. These organisms filter large volumes of seawater and may accumulate contaminants within their tissues. Pesticide contamination in water could have a direct or indirect toxic action on tissues or cells and could induce alteration of immune system. Bivalve immunity is mainly supported by hemocytes and participates directly by phagocytosis to eliminate pathogens. Some studies have shown that pesticides can reduce immune defences and/or modify genomes in vertebrates and invertebrates. Metaldehyde is used to kill slugs, snails and other terrestrial gastropods. Although metaldehyde has been detected in surface waters, its effects on marine bivalves including the Pacific oyster, Crassostrea gigas, have never been studied. Given the mode of action of this molecule and its targets (molluscs), it could be potentially more toxic to oysters than other pesticides (herbicides, fungicides, insecticides, etc.). Effects of metaldehyde on oyster hemocyte parameters were thus monitored through in vivo experiments based on a short-term exposure. In this work, metaldehyde at 0.1 μg/L, which corresponds to an average concentration detected in the environment, modulated hemocyte activities of Pacific oysters after an in vivo short-term contact. Individuals belonging to two families showed different behaviours for some hemocyte activities after contamination by metaldehyde. These results suggested that effects of pollutants on oysters may differ from an individual to another in relation to genetic diversity. Finally, it appears essential to take an interest in the effects of metaldehyde on a wide variety of aquatic invertebrates including those that have a significant economic impact.

  15. Genotoxic and immunotoxic potential effects of selected psychotropic drugs and antibiotics on blue mussel (Mytilus edulis) hemocytes

    Lacaze, Emilie; Pédelucq, Julie; Fortier, Marlène; Brousseau, Pauline; Auffret, Michel; Budzinski, Hélène; Fournier, Michel

    2015-01-01

    The potential toxicity of pharmaceuticals towards aquatic invertebrates is still poorly understood and sometimes controversial. This study aims to document the in vitro genotoxicity and immunotoxicity of psychotropic drugs and antibiotics on Mytilus edulis. Mussel hemocytes were exposed to fluoxetine, paroxetine, venlafaxine, carbamazepine, sulfamethoxazole, trimethoprim and erythromycin, at concentrations ranging from μg/L to mg/L. Paroxetine at 1.5 μg/L led to DNA damage while the same concentration of venlafaxine caused immunomodulation. Fluoxetine exposure resulted in genotoxicity, immunotoxicity and cytotoxicity. In the case of antibiotics, trimethoprim was genotoxic at 200 μg/L and immunotoxic at 20 mg/L whereas erythromycin elicited same detrimental effects at higher concentrations. DNA metabolism seems to be a highly sensitive target for psychotropic drugs and antibiotics. Furthermore, these compounds affect the immune system of bivalves, with varying intensity. This attests the relevance of these endpoints to assess the toxic mode of action of pharmaceuticals in the aquatic environment. - Highlights: • Psychotropic drugs and antibiotics affect the immune system of Mytilus edulis. • Genotoxic and immunotoxic endpoints were relevant to assess pharmaceuticals toxicity. • DNA metabolism is a highly sensitive target for pharmaceuticals. • Fluoxetine and paroxetine were the most toxic compounds on mussel hemocytes. - Psychotropic drugs and antibiotics have the potential to cause immune toxicity and genotoxicity on Mytilus edulis hemocytes

  16. Multixenobiotic resistance in Mytilus edulis: Molecular and functional characterization of an ABCG2- type transporter in hemocytes and gills.

    Ben Cheikh, Yosra; Xuereb, Benoit; Boulangé-Lecomte, Céline; Le Foll, Frank

    2018-02-01

    Among the cellular protection arsenal, ABC transporters play an important role in xenobiotic efflux in marine organisms. Two pumps belonging to B and C subfamily has been identified in Mytilus edulis. In this study, we investigated the presence of the third major subtype ABCG2/BCRP protein in mussel tissues. Transcript was expressed in hemocytes and with higher level in gills. Molecular characterization revealed that mussel ABCG2 transporter shares the sequence and organizational structure with mammalian and molluscan orthologs. Overall identity of the predicted amino acid sequence with corresponding homologs from other organisms was between 49% and 98%. Moreover, protein efflux activity was demonstrated using a combination of fluorescent allocrites and specific inhibitors. The accumulation of bodipy prazosin and pheophorbide A was heterogeneous in gills and hemocytes. Most of the used blockers enhanced probe accumulation at different levels, most significantly for bodipy prazosin. Moreover, Mrp classical blocker MK571 showed a polyspecificity. In conclusion, our data demonstrate that several ABC transporters contribute to MXR phenotype in the blue mussel including ABCG2 that forms an active pump in hemocytes and gills. Efforts are needed to distinguish between the different members and to explore their single function and specificity towards allocrites and chemosensitizers. Copyright © 2017 Elsevier B.V. All rights reserved.

  17. Apoptosis of hemocytes from lions-paw scallop Nodipecten subnodosus induced with paralyzing shellfish poison from Gymnodinium catenatum.

    Estrada, Norma; Ascencio, Felipe; Shoshani, Liora; Contreras, Rubén G

    2014-12-01

    The toxic dinoflagellate Gymnodinium catenatum produces paralyzing shellfish poisons (PSPs) that are consumed and accumulated by bivalves. Previously, we recorded a decrease in hemocytes 24h after injection of PSPs (gonyautoxin 2/3 epimers, GTX2/3) in the adductor muscle in the lions-paw scallop Nodipecten subnodosus. In this work, qualitative and quantitative analyses, in in vivo and in vitro experiments, revealed that the lower count of hemocytes results from cells undergoing typical apoptosis when exposed to GTX 2/3 epimers. This includes visible morphological alterations of the cytoplasmic membrane, damage to the nuclear membrane, condensation of chromatin, DNA fragmentation, and release of DNA fragments into the cytoplasm. Induction of apoptosis was accompanied by phosphatidylserine exposure to the outer cell membrane and activation of cysteine-aspartic proteases, caspase 3 and caspase 8. Addition of an inhibitor of caspase to the medium suppressed activation in hemocytes exposed to the toxins, suggesting that cell death was induced by a caspase-dependent apoptotic pathway. The results are important for future investigation of the scallop's immune system and should provide new insights into apoptotic processes in immune cells of scallops exposed to PSPs. Copyright © 2014 Elsevier GmbH. All rights reserved.

  18. Effect of dopamine injection on the hemocyte count and prophenoloxidase system of the white shrimp Litopenaeus vannamei

    Pan, Luqing; Hu, Fawen; Zheng, Debin

    2011-09-01

    Effects of dopamine injection on the hemocyte count, phenoloxidase activity, serine proteinase activity, proteinase inhibitor activity and α2-macroglobulin-like activity in L. vannamei were studied. Results showed that dopamine injection resulted in a significant effect on the parameters measured ( P < 0.05), while no significant difference was observed in the control group (0.85% NaCl). In the experimental groups, the hemocyte count reached the minimum in 3 h; granular and semi-granular cells became stable after 12 h and hyaline cells and the total hemocyte count became stable after 18 h. Phenoloxidase activity reached the minimum in 6 h, and then became stable after 9 h. Serine protease activity and proteinase inhibitor activity reached the minimum in 3 h, and α2-macroglobulin-like activity reached the maximum in 3 h, and all the three parameters became stable after 12 h. The results suggest that the activating mechanisms of the proPO system triggered by dopamine are different from those triggered by invasive agents or spontaneously activated under a normal physical condition.

  19. Hemocytes of zebra mussels (Dreissena polymorpha are relevant cells for the monitoring of environmental genotoxicity by the comet assay.

    Marc Bonnard

    2015-06-01

    Full Text Available The measure of DNA integrity by the single cell gel electrophoresis (SCGE or comet assay is especially recommended for its sensitivity and its capacity for detecting different types of damages. Therefore, it has been applied in environmental genotoxicity in a variety of organisms. It appears today necessary to define both reference and threshold levels of DNA damage, for their application in in situ biomonitoring. However, little is known about the influence of both biological (sex, reproduction status or external (temperature… confounding factors on the measure of DNA damage by the comet assay. These variables need to be taken into account if the robustness of the assay is to be established (Jha, 2008. In the zebra mussel Dreissena polymorpha (recommended as a sentinel species in the evaluation of freshwater quality the measure of DNA damage by the comet assay is mainly performed on hemocytes, which are circulating cells involved in key physiological functions such as immunity, homeostasis, detoxication…. This communication will present and discuss results from an innovative study about the variability of the baseline level of DNA damage in hemocytes of mussels encaged for one year in the canal de l’Aisne à la Marne (Reims, according to their sex and their reproductive status. The sensitivity and the suitability of hemocytes in the evaluation of environmental genotoxicity will also be discussed, referring to observations during a 6 month-exposure of mussels in mesocosms to environmentally realistic concentrations of carbamazepine.

  20. RNA-Seq Study of Microbially Induced Hemocyte Transcripts from Larval Heliothis virescens (Lepidoptera: Noctuidae

    Kent S. Shelby

    2012-08-01

    Full Text Available Larvae of the tobacco budworm are major polyphagous pests throughout the Americas. Development of effective microbial biopesticides for this and related noctuid pests has been stymied by the natural resistance mediated innate immune response. Hemocytes play an early and central role in activating and coordinating immune responses to entomopathogens. To approach this problem we completed RNA-seq expression profiling of hemocytes collected from larvae following an in vivo challenge with bacterial and fungal cell wall components to elicit an immune response. A de novo exome assembly was constructed by combination of sequence tags from all treatments. Sequence tags from each treatment were aligned separately with the assembly to measure expression. The resulting table of differential expression had > 22,000 assemblies each with a distinct combination of annotation and expression. Within these assemblies > 1,400 were upregulated and > 1,500 downregulated by immune activation with bacteria or fungi. Orthologs to innate immune components of other insects were identified including pattern recognition, signal transduction pathways, antimicrobial peptides and enzymes, melanization and coagulation. Additionally orthologs of components regulating hemocytic functions such as autophagy, apoptosis, phagocytosis and nodulation were identified. Associated cellular oxidative defenses and detoxification responses were identified providing a comprehensive snapshot of the early response to elicitation.

  1. The hemocytes of Panstrogyllus Megistus (Hemiptera: Reduviidae Os hemócitos de Panstrongylus megistus (Hemiptera: Reduviidae

    Margherita Anna Barracco

    1987-09-01

    Full Text Available Five hemocyte types were identified in the hemolymph of Panstrongylus megistus by phase contrast and common light microscopy using some histochemical methods. These are: Prohemocytes, small cells presenting a great nucleus/cytoplasm ratio; Plasmatocytes, the most numerous hemocytes, are polymorphic cells mainly characterized by a large amount of lysosomes; Granulocytes, hemocytes very similar to plasmatocytes which contain cytoplasmic granules and are especially rich in polysaccharides; Oenocytoids, cells presenting a small nucleus and a thick cytoplasm; they show many small round vacuoles when observed in Giemsa smears and many cytoplasmic granules under phase microscopy; Adipohemocytes, very large hemocytes, presenting many fat droplet inclusions which could correspond to free fat bodies which entered the hemolymph. Only prohemocytes and plasmatocytes can be clearly classified; all the other hemocyte types have a more ambiguous classification.Cinco tipos de hemócitos foram identificados na hemolinfa de Panstrongylus megistus através da microscopia de constraste de fase e de luz, usando alguns testes histoquímicos: Pró-hemócitos-células pequenas que mostram uma grande relação núcleocitoplasmática; Plasmotócitos-células polimporficas, que se caracterizam principalmente pela sua grande abundância em lisossomos - são os hemócitos mais numerosos; Granulócitos-células muito semelhantes aos plasmatócitos que contêm grânulos citoplasmáticos particularmente ricos em polissacarídeos; Enocitóides-hemócitos que apresentam um núcleo pequeno e cujo citoplasma basofílico revela-se muito denso e homogênio - mostram uma série de pequenos vacúolos esféricos quando observados nos esfregaços corados pelo Giemsa, mas a microscopia de fase revela uma grande quantidade de pequenos grânulo ao invés de vacúolos; Adipo-hemócitos-hemócitos muito grandes que contêm uma grande quantidade de inclusões lípicas - poderiam corresponder a

  2. Sepsis and Hemocyte Loss in Honey Bees (Apis mellifera) Infected with Serratia marcescens Strain Sicaria.

    Burritt, Nancy L; Foss, Nicole J; Neeno-Eckwall, Eric C; Church, James O; Hilger, Anna M; Hildebrand, Jacob A; Warshauer, David M; Perna, Nicole T; Burritt, James B

    2016-01-01

    Global loss of honey bee colonies is threatening the human food supply. Diverse pathogens reduce honey bee hardiness needed to sustain colonies, especially in winter. We isolated a free-living Gram negative bacillus from hemolymph of worker honey bees (Apis mellifera) found separated from winter clusters. In some hives, greater than 90% of the dying bees detached from the winter cluster were found to contain this bacterium in their hemolymph. Throughout the year, the same organism was rarely found in bees engaged in normal hive activities, but was detected in about half of Varroa destructor mites obtained from colonies that housed the septic bees. Flow cytometry of hemolymph from septic bees showed a significant reduction of plasmatocytes and other types of hemocytes. Interpretation of the16S rRNA sequence of the bacterium indicated that it belongs to the Serratia genus of Gram-negative Gammaproteobacteria, which has not previously been implicated as a pathogen of adult honey bees. Complete genome sequence analysis of the bacterium supported its classification as a novel strain of Serratia marcescens, which was designated as S. marcescens strain sicaria (Ss1). When compared with other strains of S. marcescens, Ss1 demonstrated several phenotypic and genetic differences, including 65 genes not previously found in other Serratia genomes. Some of the unique genes we identified in Ss1 were related to those from bacterial insect pathogens and commensals. Recovery of this organism extends a complex pathosphere of agents which may contribute to failure of honey bee colonies.

  3. Sepsis and Hemocyte Loss in Honey Bees (Apis mellifera) Infected with Serratia marcescens Strain Sicaria

    Burritt, Nancy L.; Foss, Nicole J.; Neeno-Eckwall, Eric C.; Church, James O.; Hildebrand, Jacob A.; Warshauer, David M.; Perna, Nicole T.; Burritt, James B.

    2016-01-01

    Global loss of honey bee colonies is threatening the human food supply. Diverse pathogens reduce honey bee hardiness needed to sustain colonies, especially in winter. We isolated a free-living Gram negative bacillus from hemolymph of worker honey bees (Apis mellifera) found separated from winter clusters. In some hives, greater than 90% of the dying bees detached from the winter cluster were found to contain this bacterium in their hemolymph. Throughout the year, the same organism was rarely found in bees engaged in normal hive activities, but was detected in about half of Varroa destructor mites obtained from colonies that housed the septic bees. Flow cytometry of hemolymph from septic bees showed a significant reduction of plasmatocytes and other types of hemocytes. Interpretation of the16S rRNA sequence of the bacterium indicated that it belongs to the Serratia genus of Gram-negative Gammaproteobacteria, which has not previously been implicated as a pathogen of adult honey bees. Complete genome sequence analysis of the bacterium supported its classification as a novel strain of Serratia marcescens, which was designated as S. marcescens strain sicaria (Ss1). When compared with other strains of S. marcescens, Ss1 demonstrated several phenotypic and genetic differences, including 65 genes not previously found in other Serratia genomes. Some of the unique genes we identified in Ss1 were related to those from bacterial insect pathogens and commensals. Recovery of this organism extends a complex pathosphere of agents which may contribute to failure of honey bee colonies. PMID:28002470

  4. Bombyx mori and Aedes aegypti form multi-functional immune complexes that integrate pattern recognition, melanization, coagulants, and hemocyte recruitment.

    Phillips, Dennis R; Clark, Kevin D

    2017-01-01

    The innate immune system of insects responds to wounding and pathogens by mobilizing multiple pathways that provide both systemic and localized protection. Key localized responses in hemolymph include melanization, coagulation, and hemocyte encapsulation, which synergistically seal wounds and envelop and destroy pathogens. To be effective, these pathways require a targeted deposition of their components to provide protection without compromising the host. Extensive research has identified a large number of the effectors that comprise these responses, but questions remain regarding their post-translational processing, function, and targeting. Here, we used mass spectrometry to demonstrate the integration of pathogen recognition proteins, coagulants, and melanization components into stable, high-mass, multi-functional Immune Complexes (ICs) in Bombyx mori and Aedes aegypti. Essential proteins common to both include phenoloxidases, apolipophorins, serine protease homologs, and a serine protease that promotes hemocyte recruitment through cytokine activation. Pattern recognition proteins included C-type Lectins in B. mori, while A. aegypti contained a protein homologous to Plasmodium-resistant LRIM1 from Anopheles gambiae. We also found that the B. mori IC is stabilized by extensive transglutaminase-catalyzed cross-linking of multiple components. The melanization inhibitor Egf1.0, from the parasitoid wasp Microplitis demolitor, blocked inclusion of specific components into the IC and also inhibited transglutaminase activity. Our results show how coagulants, melanization components, and hemocytes can be recruited to a wound surface or pathogen, provide insight into the mechanism by which a parasitoid evades this immune response, and suggest that insects as diverse as Lepidoptera and Diptera utilize similar defensive mechanisms.

  5. Hemocyte responses of Dreissena polymorpha following a short-term in vivo exposure to titanium dioxide nanoparticles: Preliminary investigations

    Couleau, Nicolas; Techer, Didier [Universite de Lorraine, Laboratoire des Interactions Ecotoxicologie, Biodiversite, Ecosystemes (LIEBE), CNRS UMR 7146, IUT Thionville-Yutz, Espace Cormontaigne, Yutz, F-57970 (France); Pagnout, Christophe [Universite de Lorraine, Laboratoire des Interactions Ecotoxicologie, Biodiversite, Ecosystemes (LIEBE), UMR 7146, Campus Bridoux, rue du General Delestraint, Metz, F-57070 (France); International Consortium for the Environmental Implications of Nanotechnology, iCEINT, http://www.i-ceint.org (France); Jomini, Stephane [Universite de Lorraine, Laboratoire des Interactions Ecotoxicologie, Biodiversite, Ecosystemes (LIEBE), UMR 7146, Campus Bridoux, rue du General Delestraint, Metz, F-57070 (France); Foucaud, Laurent; Laval-Gilly, Philippe; Falla, Jairo [Universite de Lorraine, Laboratoire des Interactions Ecotoxicologie, Biodiversite, Ecosystemes (LIEBE), CNRS UMR 7146, IUT Thionville-Yutz, Espace Cormontaigne, Yutz, F-57970 (France); Bennasroune, Amar, E-mail: amar.bennasroune@univ-metz.fr [Universite de Lorraine, Laboratoire des Interactions Ecotoxicologie, Biodiversite, Ecosystemes (LIEBE), CNRS UMR 7146, IUT Thionville-Yutz, Espace Cormontaigne, Yutz, F-57970 (France)

    2012-11-01

    The widespread use of titanium-based nanoparticles and their environmental release may pose a significant risk to aquatic organisms within freshwater ecosystems. Suspension-feeder invertebrates like bivalve molluscs represent a unique target group for nanoparticle toxicology. The aim of this work was to investigate the short-term responses of Dreissena polymorpha hemocytes after in vivo exposure to titanium dioxide nanoparticles (TiO{sub 2} NP). For this purpose, freshwater mussels were exposed to P25 TiO{sub 2} NP at the concentrations of 0.1, 1, 5 and 25 mg/L during 24 h. Viability, phagocytosis activity and mitogen activated protein kinase (MAPK) phosphorylation level of ERK 1/2 and p38 in hemocytes extracted from exposed mussels were compared to those from control specimens. Results demonstrated an inhibition of the phagocytosis activity after exposure to TiO{sub 2} NP at 0.1 and 1 mg/L. Similar trends, albeit less pronounced, were reported for higher concentrations of NP. Transmission electron microscopy showed for the first time the internalization of TiO{sub 2} NP into Dreissena polymorpha hemocytes. Besides, exposure to NP increased the ERK 1/2 phosphorylation levels in all treatments. Concerning the phosphorylation level of p38, only exposures to 5 and 25 mg/L of NP induced significant p38 activation in comparison to that of the control. Finally, these short-term effects observed at environmentally relevant concentrations highlighted the need for further studies concerning ecotoxicological evaluation of nanoparticle release into an aquatic environment. -- Highlights: Black-Right-Pointing-Pointer Phagocytosis inhibition at TiO{sub 2} NP exposure concentrations of 0.1 and 1 mg/L. Black-Right-Pointing-Pointer Internalization of TiO{sub 2} NP in freshwater mussel hemocytes. Black-Right-Pointing-Pointer Increased phosphorylation level of p38 and ERK 1/2 after in vivo exposure to TiO{sub 2} NP.

  6. Oxidative stress and cytotoxicity elicited lipid peroxidation in hemocytes of Bombyx mori larva infested with dipteran parasitoid, Exorista bombycis.

    Pooja, Makwana; Pradeep, Appukuttan Nair R; Hungund, Shambhavi P; Sagar, Chandrashekhar; Ponnuvel, Kangayam M; Awasthi, Arvind K; Trivedy, Kanika

    2017-12-20

    Parasitization of silkworm, Bombyx mori by invasive larva of dipteran parasitoid Exorista bombycis caused upto 20% revenue loss in sericulture. The parasitism was successful by suppressing host immune system however mechanism of immune suppression induced by E. bombycis is unknown which is unravelled here. The infestation induced cytotoxic symptoms in host hemocytes, such as vacuolated cytoplasm, porous plasma membrane, indented nuclei with condensed chromatin and dilated RER. One of the markers of necrosis is cell permeabilization, which can be measured as released lactate dehydrogenase (LDH). LDH level showed significantly (Pmori.

  7. Nitric oxide production by hemocytes of larva and pharate prepupa of Galleria mellonella in response to bacterial lipopolysaccharide: cytoprotective or cytotoxic?

    Krishnan, Natraj; Hyršl, P.; Šimek, V.

    2006-01-01

    Roč. 142, 1-2 (2006), s. 103-110 ISSN 1532-0456 Institutional research plan: CEZ:AV0Z50070508 Keywords : nitric oxide * hemocytes * lipopolysaccharide Subject RIV: ED - Physiology Impact factor: 1.991, year: 2006

  8. Hemocyte parameters of the Pacific oyster Crassostrea gigas a year after the Hebei Spirit oil spill off the west coast of Korea

    Donaghy, Ludovic; Hong, Hyun-Ki; Lee, Hee-Jung; Jun, Je-Cheon; Park, Young-Je; Choi, Kwang-Sik

    2010-12-01

    In marine bivalves, hemocytes support various physiological functions, including immune defense, nutrient transport, shell repair, and homeostatic maintenance. Although the effects of marine contaminants on the immunological functions of bivalves have been extensively investigated, the impacts of oil spills are not well understood. Therefore, we investigated hemocyte parameters in the Pacific oyster Crassostrea gigas 13 months after the Hebei Spirit oil spill (December 2007) off the west coast of Korea. The parameters studied included hemocyte concentration and mortality, relative proportion of hemocyte populations, and immunological functions such as phagocytosis and oxidative activity using flow cytometry. These immune-related parameters in oysters damaged by the oil spill were also compared to control oysters that were collected from an area unaffected by the spill. The flow cytometry study indicated that granulocyte population, phagocytic capacity, and reactive oxygen species production in oysters exposed to crude oil 13 months prior were depressed compared to the unexposed control oysters. Our data suggest that immunocompetence in oysters affected by the oil spill had not fully recovered 1 year after the accident, although more detailed studies on the physiology and disease resistance should be performed.

  9. Cytotoxicity and cellular mechanisms involved in the toxicity of CdS quantum dots in hemocytes and gill cells of the mussel Mytilus galloprovincialis

    Katsumiti, A. [CBET Research Group, Dept. Zoology and Animal Cell Biology, Faculty of Science and Technology and Research Centre for Experimental Marine Biology and Biotechnology PIE, University of the Basque Country UPV/EHU, Basque Country (Spain); Gilliland, D. [EU Commission–Joint Research Centre, Institute of Health and Consumer Protection, NSB Unit, Ispra (Italy); Arostegui, I. [Department of Applied Mathematics, Statistics and Operations Research, Faculty of Science and Technology, University of the Basque Country UPV/EHU, Leioa (Spain); Cajaraville, M.P., E-mail: mirenp.cajaraville@ehu.es [CBET Research Group, Dept. Zoology and Animal Cell Biology, Faculty of Science and Technology and Research Centre for Experimental Marine Biology and Biotechnology PIE, University of the Basque Country UPV/EHU, Basque Country (Spain)

    2014-08-15

    Highlights: • CdS QDs were cytotoxic for mussel hemocytes and gill cells in vitro. • Ionic Cd was the most toxic form, followed by CdS QDs and bulk CdS. • CdS QDs altered oxidative balance and caused DNA damage in mussel cells. • CdS QDs caused a particle-specific immunostimulation on phagocytosis of hemocytes. • Conceptual models for cellular handling and toxicity of CdS QDs are proposed. - Abstract: CdS quantum dots (QDs) show a great promise for treatment and diagnosis of cancer and for targeted drug delivery, due to their size-tunable fluorescence and ease of functionalization for tissue targeting. In spite of their advantages it is important to determine if CdS QDs can exert toxicity on biological systems. In the present work, cytotoxicity of CdS QDs (5 nm) at a wide range of concentrations (0.001–100 mg Cd/L) was screened using neutral red (NR) and thiazolyl blue tetrazolium bromide (MTT) assays in isolated hemocytes and gill cells of mussels (Mytilus galloprovincialis). The mechanisms of action of CdS QDs were assessed at sublethal concentrations (0.31–5 mg Cd/L) in the same cell types through a series of functional in vitro assays: production of reactive oxygen species (ROS), catalase (CAT) activity, DNA damage, lysosomal acid phosphatase (AcP) activity, multixenobiotic resistance (MXR) transport activity, Na-K-ATPase activity (only in gill cells) and phagocytic activity and damage to actin cytoskeleton (only in hemocytes). Exposures to CdS QDs lasted for 24 h and were performed in parallel with exposures to bulk CdS and ionic Cd. Ionic Cd was the most toxic form to both cell types, followed by CdS QDs and bulk CdS. ROS production, DNA damage, AcP activity and MXR transport were significantly increased in both cell types exposed to the 3 forms of Cd. CAT activity increased in hemocytes exposed to the three forms of Cd while in gill cells only in those exposed to ionic Cd. No effects were found on hemocytes cytoskeleton integrity. Effects on

  10. Immune responses of mussel hemocyte subpopulations are differentially regulated by enzymes of the PI 3-K, PKC, and ERK kinase families.

    García-García, Erick; Prado-Alvarez, Maria; Novoa, Beatriz; Figueras, Antonio; Rosales, Carlos

    2008-01-01

    Various hemocyte cell types have been described in invertebrates, but for most species a functional characterization of different hemocyte cell types is still lacking. In order to characterize some immunological properties of mussel (Mytilus galloprovincialis) hemocytes, cells were separated by flow cytometry and their capacity for phagocytosis, production of reactive oxygen species (ROS), and production of nitric oxide (NO), was examined. Phosphatidylinositol 3-kinase (PI 3-K), protein kinase C (PKC), and extracellular signal-regulated kinase (ERK) inhibitors were also used to biochemically characterize these cell responses. Four morphologically distinct subpopulations, designated R1-R4, were detected. R1, R2, and R3 cells presented different levels of phagocytosis towards zymosan, latex beads, and two bacteria species. Similarly, R1 to R3, but not R4, cells produced ROS, while all subpopulations produced NO, in response to zymosan. Internalization of all phagocytic targets was blocked by PI 3-K inhibition. In addition, internalization of latex particles, but not of bacteria, was partially blocked by PKC or ERK inhibition. Interestingly, phagocytosis of zymosan was impaired by PKC, or ERK inhibitors, only in R2 cells. Zymosan-induced ROS production was blocked by PI 3-K inhibition, but not by PKC, or ERK inhibition. In addition, zymosan-stimulated NO production was affected by PI 3-K inhibition in R1 and R2, but not in R3 or R4 cells. NO production in all cell types was unaffected by PKC inhibition, but ERK inhibition blocked it in R2 cells. These data reveal the existence of profound functional and biochemical differences in mussel hemocytes and indicate that M. galloprovincialis hemocytes are specialized cells fulfilling specific tasks in the context of host defense.

  11. Comparative transcriptomic analysis of shrimp hemocytes in response to acute hepatopancreas necrosis disease (AHPND) causing Vibrio parahemolyticus infection.

    Zheng, Zhihong; Wang, Fan; Aweya, Jude Juventus; Li, Ruiwei; Yao, Defu; Zhong, Mingqi; Li, Shengkang; Zhang, Yueling

    2018-03-01

    The recent emergence of acute hepatopancreas necrosis disease (AHPND) in shrimps has posed a major challenge in the shrimp aquaculture industry. The Pir toxin proteins carried by some strains of Vibrio parahaemolyticus are believed to play essential roles in the pathogenesis of AHPND. However, few studies have so far explored how the host immune system responds to these bacteria. In this study, AHPND V. parahaemolyticus (with Pir) and non-AHPND V. parahaemolyticus (without Pir) were injected into two groups of shrimps, and the hemocytes collected for comparative transcriptomic analyses. A total of 1064 differentially expressed genes (DEGs) were identified, of which 910 were up-regulated and 154 were down-regulated. Gene Ontology (GO) annotation and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis showed that many DEGs were involved in a number of biological processes such as cellular process, metabolic process and single-organism process in the AHPND V. parahaemolyticus injected group than the non-AHPND V. parahaemolyticus injected group. Among these, major metabolic processes such as carbohydrate metabolism, lipid metabolism and amino acid metabolism were further identified as the major responsive gene groups. We observed that genes involved in cell growth and anti-apoptosis including src, iap2, cas2, cytochrome P450, gst and cytochromecoxidase were strongly activated in the AHPND V. parahaemolyticus group than in the non-AHPND V. parahaemolyticus group. Collectively, our results unveiled that shrimp hemocytes respond to AHPND related strain of Vibrio parahaemolyticus infection at the transcriptional level, which is useful in furthering our understanding of AHPND. Copyright © 2017 Elsevier Ltd. All rights reserved.

  12. The effects of ocean acidification on hemocyte of crab species in Alaska from laboratory experiment studies from 2011-07-01 to 2013-07-06 (NODC Accession 0123400)

    National Oceanic and Atmospheric Administration, Department of Commerce — This archival package contains laboratory experiment data that were collected to examine the effects of ocean acidification on hemocyte of crab species in Alaska....

  13. Ultrastructural studies of the hemocytes of Panstrongylus megistus (Hemiptera: Reduvidae Ultra-estrutura dos hemócitos de Panstrongylus megistus (Hemiptera: Reduviidae

    Margherita A. Barracco

    1989-06-01

    Full Text Available Ultrastructural analyses revealed the presence of six hemocyte types in the hemolymph of Panstrogylus megistus, partially confirming our previous results obtained through light microscopy. Prohemocytes: small, round hemocytes with a thin cytoplasm layer, espcieally rich in free ribosomes and poor in membranous systems. Plasmatocytes: polymorphic cells, whose cytoplasm contains many lysosomes and a well developed rough endoplasmic reticulum (RER.They are extremely phagocytic. Sometimes, they show a large vacuolation. Granulocytes: granular hemocytes whose granules show different degrees of electrondensity. Most of them, have an internal structuration. Coagulocytes: oval or elongated hemocytes, which show pronounced perinuclear cisternae as normally observed in coagulocytes. The cytoplasm is usually electrondense, poor in membranous systems and contains many labile granules. Oenocytoids: large and very stable hemocytes, whose homogeneous cytoplasme is rich in loose ribosomes and poor in membranous systems. Adipohemocytes: large cells, containing several characteristic lipid droplets. The cytoplasm is also rich in glycogen, RER and large mitochondria. The total and differential hemocyte count (THC and DHC were also calculated for this reduviid. THC increases from 2,900 hemocytes/cubic millimeter of hemolymph in the 4th intar to 4,350 in the 5th and then, decreases to 1,950 in the adults. Plasmatocytes and coagulocytes are the predominant hemocyte types.Estudos ao microscópio eletrônico de transmissão revelaram a presença de seis tipos de hemócitos na hemolinfa de Panstrongylus megistus. Estes resultados confirmam parcialmente os obtidos anteriormente através da microscopia de luz. Pró-hemócitos: células pequenas e arredondadas, cuja delgada faixa citoplasmática é especialmente rica em ribossomos livres e pobre em sistemas membranosos. Plasmatócitos: células polimórficas, cujo citoplasma caracteriza-se por um retículo endoplasm

  14. Comparative venom toxicity between Pteromalus puparum and Nasonia vitripennis (Hymenoptera: Pteromalidae) toward the hemocytes of their natural hosts, non-target insects and cultured insect cells.

    Zhang, Zhong; Ye, Gong-Yin; Cai, Jun; Hu, Cui

    2005-09-01

    Crude venoms from two parasitoid species, Pteromalus puparum and Nasonia vitripennis (Hymenoptera: Pteromalidae) were assayed for biological activities toward hemocytes from two species of their natural hosts and eight species of their non-natural hosts as well as two lines of cultured Lepidoptera cells, respectively. By inhibiting the spreading and viability of insect hemocytes, the venom from P. puparum displayed significantly higher activities toward plasmatocytes and granular cells from both larvae and pupae of two natural hosts, Pieris rapae and Papilio xuthus, and lower activity toward those from Spodoptera litura, Musca domestica and Sarcophaga peregrina. However, no effect was found towards any type of hemocytes from other five insects tested, namely, Ectropis oblique, Galleria mellonella, Sesamia inferens, Bombyx mori and Parnara guttata. In contrast, the venom from N. vitripennis showed a narrower range of targeted insects. It appeared to have highly adverse effects on the spreading and viability of plasmatocytes and granular cells only from the natural hosts, M. domestica and S. peregrina, little toxicity to cells from P. rapae and P. xuthus, and no effect on any of the other insects tested. Pteromalus puparum venom also apparently presented a high ability to block the spreading of Tn-5B1-4 cells derived from Trichoplusia ni, and high cytotoxicity to the cells and Ha cells derived from Helicoverpa armigera. Nasonia vitripennis venom, however, only had a marked lethal effect to Ha cells. In addition, the possibility that the host range of a defined parasitoid could be assessed using our method of treating hemocytes from candidate insects with venom in vitro, and the potential of our venoms tested in the development of bio-insecticides, insect-resistant transgenic plants, are discussed.

  15. De novo transcriptome sequencing of the Octopus vulgaris hemocytes using Illumina RNA-Seq technology: response to the infection by the gastrointestinal parasite Aggregata octopiana.

    Castellanos-Martínez, Sheila; Arteta, David; Catarino, Susana; Gestal, Camino

    2014-01-01

    Octopus vulgaris is a highly valuable species of great commercial interest and excellent candidate for aquaculture diversification; however, the octopus' well-being is impaired by pathogens, of which the gastrointestinal coccidian parasite Aggregata octopiana is one of the most important. The knowledge of the molecular mechanisms of the immune response in cephalopods, especially in octopus is scarce. The transcriptome of the hemocytes of O. vulgaris was de novo sequenced using the high-throughput paired-end Illumina technology to identify genes involved in immune defense and to understand the molecular basis of octopus tolerance/resistance to coccidiosis. A bi-directional mRNA library was constructed from hemocytes of two groups of octopus according to the infection by A. octopiana, sick octopus, suffering coccidiosis, and healthy octopus, and reads were de novo assembled together. The differential expression of transcripts was analysed using the general assembly as a reference for mapping the reads from each condition. After sequencing, a total of 75,571,280 high quality reads were obtained from the sick octopus group and 74,731,646 from the healthy group. The general transcriptome of the O. vulgaris hemocytes was assembled in 254,506 contigs. A total of 48,225 contigs were successfully identified, and 538 transcripts exhibited differential expression between groups of infection. The general transcriptome revealed genes involved in pathways like NF-kB, TLR and Complement. Differential expression of TLR-2, PGRP, C1q and PRDX genes due to infection was validated using RT-qPCR. In sick octopuses, only TLR-2 was up-regulated in hemocytes, but all of them were up-regulated in caecum and gills. The transcriptome reported here de novo establishes the first molecular clues to understand how the octopus immune system works and interacts with a highly pathogenic coccidian. The data provided here will contribute to identification of biomarkers for octopus resistance against

  16. The Silencing of a 14-3-3ɛ Homolog in Tenebrio molitor Leads to Increased Antimicrobial Activity in Hemocyte and Reduces Larval Survivability.

    Seo, Gi Won; Jo, Yong Hun; Seong, Jeong Hwan; Park, Ki Beom; Patnaik, Bharat Bhusan; Tindwa, Hamisi; Kim, Sun-Am; Lee, Yong Seok; Kim, Yu Jung; Han, Yeon Soo

    2016-08-20

    The 14-3-3 family of phosphorylated serine-binding proteins acts as signaling molecules in biological processes such as metabolism, division, differentiation, autophagy, and apoptosis. Herein, we report the requirement of 14-3-3ɛ isoform from Tenebrio molitor (Tm14-3-3ɛ) in the hemocyte antimicrobial activity. The Tm14-3-3ɛ transcript is 771 nucleotides in length and encodes a polypeptide of 256 amino acid residues. The protein has the typical 14-3-3 domain, the nuclear export signal (NES) sequence, and the peptide binding residues. The Tm14-3-3ɛ transcript shows a significant three-fold expression in the hemocyte of T. molitor larvae when infected with Escherichia coli Tm14-3-3ɛ silenced larvae show significantly lower survival rates when infected with E. coli. Under Tm14-3-3ɛ silenced condition, a strong antimicrobial activity is elicited in the hemocyte of the host inoculated with E. coli. This suggests impaired secretion of antimicrobial peptides (AMP) into the hemolymph. Furthermore, a reduction in AMP secretion under Tm14-3-3ɛ silenced condition would be responsible for loss in the capacity to kill bacteria and might explain the reduced survivability of the larvae upon E. coli challenge. This shows that Tm14-3-3ɛ is required to maintain innate immunity in T. molitor by enabling antimicrobial secretion into the hemolymph and explains the functional specialization of the isoform.

  17. The Silencing of a 14-3-3ɛ Homolog in Tenebrio molitor Leads to Increased Antimicrobial Activity in Hemocyte and Reduces Larval Survivability

    Gi Won Seo

    2016-08-01

    Full Text Available The 14-3-3 family of phosphorylated serine-binding proteins acts as signaling molecules in biological processes such as metabolism, division, differentiation, autophagy, and apoptosis. Herein, we report the requirement of 14-3-3ɛ isoform from Tenebrio molitor (Tm14-3-3ɛ in the hemocyte antimicrobial activity. The Tm14-3-3ɛ transcript is 771 nucleotides in length and encodes a polypeptide of 256 amino acid residues. The protein has the typical 14-3-3 domain, the nuclear export signal (NES sequence, and the peptide binding residues. The Tm14-3-3ɛ transcript shows a significant three-fold expression in the hemocyte of T. molitor larvae when infected with Escherichia coli Tm14-3-3ɛ silenced larvae show significantly lower survival rates when infected with E. coli. Under Tm14-3-3ɛ silenced condition, a strong antimicrobial activity is elicited in the hemocyte of the host inoculated with E. coli. This suggests impaired secretion of antimicrobial peptides (AMP into the hemolymph. Furthermore, a reduction in AMP secretion under Tm14-3-3ɛ silenced condition would be responsible for loss in the capacity to kill bacteria and might explain the reduced survivability of the larvae upon E. coli challenge. This shows that Tm14-3-3ɛ is required to maintain innate immunity in T. molitor by enabling antimicrobial secretion into the hemolymph and explains the functional specialization of the isoform.

  18. Interactions of cationic polystyrene nanoparticles with marine bivalve hemocytes in a physiological environment: Role of soluble hemolymph proteins

    Canesi, Laura, E-mail: Laura.Canesi@unige.it [Dept. of Earth, Environmental and Life Sciences – DISTAV, University of Genoa (Italy); Ciacci, Caterina [Dept. of Biomolecular Sciences – DIBS, University of Urbino (Italy); Fabbri, Rita; Balbi, Teresa [Dept. of Earth, Environmental and Life Sciences – DISTAV, University of Genoa (Italy); Salis, Annalisa; Damonte, Gianluca [Centre of Excellence for Biomedical Research – CEBR, University of Genoa (Italy); Cortese, Katia [Department of Experimental Medicine – DIMES, University of Genoa (Italy); Caratto, Valentina [Dept. of Earth, Environmental and Life Sciences – DISTAV, University of Genoa (Italy); Monopoli, Marco P. [Centre for BioNanoInteractions, School of Chemistry and Chemical Biology, University College Dublin (Ireland); Department of Pharmaceutical and Medical Chemistry, Royal College of Surgeons, 123 St. Stephen Green, Dublin (Ireland); Dawson, Kenneth [Centre for BioNanoInteractions, School of Chemistry and Chemical Biology, University College Dublin (Ireland); Bergami, Elisa; Corsi, Ilaria [Dept. of Physical, Earth and Environmental Sciences, University of Siena (Italy)

    2016-10-15

    The bivalve Mytilus galloprovincialis has proven as a suitable model invertebrate for evaluating the potential impact of nanoparticles (NPs) in the marine environment. In particular, in mussels, the immune system represents a sensitive target for different types of NPs. In environmental conditions, both NP intrinsic properties and those of the receiving medium will affect particle behavior and consequent bioavailability/uptake/toxicity. However, the evaluation of the biological effects of NPs requires additional understanding of how, once within the organism, NPs interact at the molecular level with cells in a physiological environment. In mammalian systems, different NPs associate with serum soluble components, organized into a “protein corona”, which affects particle interactions with target cells. However, no information is available so far on the interactions of NPs with biological fluids of aquatic organisms. In this work, the influence of hemolymph serum (HS) on the in vitro effects of amino modified polystyrene NPs (PS-NH{sub 2}) on Mytilus hemocytes was investigated. Hemocytes were incubated with PS-NH{sub 2} suspensions in HS (1, 5 and 50 µg/mL) and the results were compared with those obtained in ASW medium. Cell functional parameters (lysosomal membrane stability, oxyradical production, phagocytosis) were evaluated, and morphological changes were investigated by TEM. The activation state of the signalling components involved in Mytilus immune response (p38 MAPK and PKC) was determined. The results show that in the presence of HS, PS-NH{sub 2} increased cellular damage and ROS production with respect to ASW medium. The effects were apparently mediated by disregulation of p38 MAPK signalling. The formation of a PS-NH{sub 2}-protein corona in HS was investigated by centrifugation, and 1D- gel electrophoresis and nano-HPLC-ESI-MS/MS. The results identified the Putative C1q domain containing protein (MgC1q6) as the only component of the PS-NH{sub 2} hard

  19. Interactions of cationic polystyrene nanoparticles with marine bivalve hemocytes in a physiological environment: Role of soluble hemolymph proteins

    Canesi, Laura; Ciacci, Caterina; Fabbri, Rita; Balbi, Teresa; Salis, Annalisa; Damonte, Gianluca; Cortese, Katia; Caratto, Valentina; Monopoli, Marco P.; Dawson, Kenneth; Bergami, Elisa; Corsi, Ilaria

    2016-01-01

    The bivalve Mytilus galloprovincialis has proven as a suitable model invertebrate for evaluating the potential impact of nanoparticles (NPs) in the marine environment. In particular, in mussels, the immune system represents a sensitive target for different types of NPs. In environmental conditions, both NP intrinsic properties and those of the receiving medium will affect particle behavior and consequent bioavailability/uptake/toxicity. However, the evaluation of the biological effects of NPs requires additional understanding of how, once within the organism, NPs interact at the molecular level with cells in a physiological environment. In mammalian systems, different NPs associate with serum soluble components, organized into a “protein corona”, which affects particle interactions with target cells. However, no information is available so far on the interactions of NPs with biological fluids of aquatic organisms. In this work, the influence of hemolymph serum (HS) on the in vitro effects of amino modified polystyrene NPs (PS-NH 2 ) on Mytilus hemocytes was investigated. Hemocytes were incubated with PS-NH 2 suspensions in HS (1, 5 and 50 µg/mL) and the results were compared with those obtained in ASW medium. Cell functional parameters (lysosomal membrane stability, oxyradical production, phagocytosis) were evaluated, and morphological changes were investigated by TEM. The activation state of the signalling components involved in Mytilus immune response (p38 MAPK and PKC) was determined. The results show that in the presence of HS, PS-NH 2 increased cellular damage and ROS production with respect to ASW medium. The effects were apparently mediated by disregulation of p38 MAPK signalling. The formation of a PS-NH 2 -protein corona in HS was investigated by centrifugation, and 1D- gel electrophoresis and nano-HPLC-ESI-MS/MS. The results identified the Putative C1q domain containing protein (MgC1q6) as the only component of the PS-NH 2 hard protein corona in

  20. Interactions of cationic polystyrene nanoparticles with marine bivalve hemocytes in a physiological environment: Role of soluble hemolymph proteins.

    Canesi, Laura; Ciacci, Caterina; Fabbri, Rita; Balbi, Teresa; Salis, Annalisa; Damonte, Gianluca; Cortese, Katia; Caratto, Valentina; Monopoli, Marco P; Dawson, Kenneth; Bergami, Elisa; Corsi, Ilaria

    2016-10-01

    The bivalve Mytilus galloprovincialis has proven as a suitable model invertebrate for evaluating the potential impact of nanoparticles (NPs) in the marine environment. In particular, in mussels, the immune system represents a sensitive target for different types of NPs. In environmental conditions, both NP intrinsic properties and those of the receiving medium will affect particle behavior and consequent bioavailability/uptake/toxicity. However, the evaluation of the biological effects of NPs requires additional understanding of how, once within the organism, NPs interact at the molecular level with cells in a physiological environment. In mammalian systems, different NPs associate with serum soluble components, organized into a "protein corona", which affects particle interactions with target cells. However, no information is available so far on the interactions of NPs with biological fluids of aquatic organisms. In this work, the influence of hemolymph serum (HS) on the in vitro effects of amino modified polystyrene NPs (PS-NH2) on Mytilus hemocytes was investigated. Hemocytes were incubated with PS-NH2 suspensions in HS (1, 5 and 50µg/mL) and the results were compared with those obtained in ASW medium. Cell functional parameters (lysosomal membrane stability, oxyradical production, phagocytosis) were evaluated, and morphological changes were investigated by TEM. The activation state of the signalling components involved in Mytilus immune response (p38 MAPK and PKC) was determined. The results show that in the presence of HS, PS-NH2 increased cellular damage and ROS production with respect to ASW medium. The effects were apparently mediated by disregulation of p38 MAPK signalling. The formation of a PS-NH2-protein corona in HS was investigated by centrifugation, and 1D- gel electrophoresis and nano-HPLC-ESI-MS/MS. The results identified the Putative C1q domain containing protein (MgC1q6) as the only component of the PS-NH2 hard protein corona in Mytilus

  1. TOTAL HEMOCYTE COUNT AND HEMOLYMPH GLUCOSE CONCENRATION RESPONSE OF SPINY LOBSTER Panulirus homarus ON RATIO OF SHELTER

    Suhaiba Djai

    2017-11-01

    Full Text Available This research was conducted to assess the physiological response of the lobster Panulirus homarus for the ratio of the shelters. The method used completely randomized design with two replicates of each treatments with shelter ratio (A 1 : 5, (B 3 : 5, (C 4 : 5, (D 5 : 5. Weight average for 184 lobsters with the stocking density of 23 lobsters for each treatment was 32.64 ± 0.58 g. The experiment was conducted for 60 days. The lobster was fed with trash fish and acclimatized for 7 days before the experiment. Observations on the physiologycal of every 10 days. The physiological responses that observed were total hemocyte count (THC and hemolymph glucose concentration. The results showed that 4:5 was the best lobster shelter ratio because it could reduce stress levels. This is indicated by the stable values of THC and hemolymph glucose level during the experiment and supported by the growth of 57.28 ± 0.15 g and survival rate of 91.31 ± 2.60%. Keywords: lobster, Panulirus homarus, ratio, shelter, THC, glucose

  2. Ocean Acidification Affects the Cytoskeleton, Lysozymes, and Nitric Oxide of Hemocytes: A Possible Explanation for the Hampered Phagocytosis in Blood Clams, Tegillarca granosa.

    Su, Wenhao; Rong, Jiahuan; Zha, Shanjie; Yan, Maocang; Fang, Jun; Liu, Guangxu

    2018-01-01

    An enormous amount of anthropogenic carbon dioxide (CO 2 ) has been dissolved into the ocean, leading to a lower pH and changes in the chemical properties of seawater, which has been termed ocean acidification (OA). The impacts of p CO 2 -driven acidification on immunity have been revealed recently in various marine organisms. However, the mechanism causing the reduction in phagocytosis still remains unclear. Therefore, the impacts of p CO 2 -driven OA at present and near-future levels (pH values of 8.1, 7.8, and 7.4) on the rate of phagocytosis, the abundance of cytoskeleton components, the levels of nitric oxide (NO), and the concentration and activity of lysozymes (LZM) of hemocytes were investigated in a commercial bivalve species, the blood clam ( Tegillarca granosa ). In addition, the effects of OA on the expression of genes regulating actin skeleton and nitric oxide synthesis 2 ( NOS2 ) were also analyzed. The results obtained showed that the phagocytic rate, cytoskeleton component abundance, concentration and activity of LZM of hemocytes were all significantly reduced after a 2-week exposure to the future OA scenario of a pH of 7.4. On the contrary, a remarkable increase in the concentration of NO compared to that of the control was detected in clams exposed to OA. Furthermore, the expression of genes regulating the actin cytoskeleton and NOS were significantly up-regulated after OA exposure. Though the mechanism causing phagocytosis seemed to be complicated based on the results obtained in the present study and those reported previously, our results suggested that OA may reduce the phagocytosis of hemocytes by (1) decreasing the abundance of cytoskeleton components and therefore hampering the cytoskeleton-mediated process of engulfment, (2) reducing the concentration and activity of LZM and therefore constraining the degradation of the engulfed pathogen through an oxygen-independent pathway, and (3) inducing the production of NO, which may negatively

  3. Functional Conservation of the Glide/Gcm Regulatory Network Controlling Glia, Hemocyte, and Tendon Cell Differentiation in Drosophila

    Cattenoz, Pierre B.; Popkova, Anna; Southall, Tony D.; Aiello, Giuseppe; Brand, Andrea H.; Giangrande, Angela

    2016-01-01

    High-throughput screens allow us to understand how transcription factors trigger developmental processes, including cell specification. A major challenge is identification of their binding sites because feedback loops and homeostatic interactions may mask the direct impact of those factors in transcriptome analyses. Moreover, this approach dissects the downstream signaling cascades and facilitates identification of conserved transcriptional programs. Here we show the results and the validation of a DNA adenine methyltransferase identification (DamID) genome-wide screen that identifies the direct targets of Glide/Gcm, a potent transcription factor that controls glia, hemocyte, and tendon cell differentiation in Drosophila. The screen identifies many genes that had not been previously associated with Glide/Gcm and highlights three major signaling pathways interacting with Glide/Gcm: Notch, Hedgehog, and JAK/STAT, which all involve feedback loops. Furthermore, the screen identifies effector molecules that are necessary for cell-cell interactions during late developmental processes and/or in ontogeny. Typically, immunoglobulin (Ig) domain–containing proteins control cell adhesion and axonal navigation. This shows that early and transiently expressed fate determinants not only control other transcription factors that, in turn, implement a specific developmental program but also directly affect late developmental events and cell function. Finally, while the mammalian genome contains two orthologous Gcm genes, their function has been demonstrated in vertebrate-specific tissues, placenta, and parathyroid glands, begging questions on the evolutionary conservation of the Gcm cascade in higher organisms. Here we provide the first evidence for the conservation of Gcm direct targets in humans. In sum, this work uncovers novel aspects of cell specification and sets the basis for further understanding of the role of conserved Gcm gene regulatory cascades. PMID:26567182

  4. White spot syndrome virus induces metabolic changes resembling the warburg effect in shrimp hemocytes in the early stage of infection.

    Chen, I-Tung; Aoki, Takashi; Huang, Yun-Tzu; Hirono, Ikuo; Chen, Tsan-Chi; Huang, Jiun-Yan; Chang, Geen-Dong; Lo, Chu-Fang; Wang, Han-Ching

    2011-12-01

    The Warburg effect is an abnormal glycolysis response that is associated with cancer cells. Here we present evidence that metabolic changes resembling the Warburg effect are induced by a nonmammalian virus. When shrimp were infected with white spot syndrome virus (WSSV), changes were induced in several metabolic pathways related to the mitochondria. At the viral genome replication stage (12 h postinfection [hpi]), glucose consumption and plasma lactate concentration were both increased in WSSV-infected shrimp, and the key enzyme of the pentose phosphate pathway, glucose-6-phosphate dehydrogenase (G6PDH), showed increased activity. We also found that at 12 hpi there was no alteration in the ADP/ATP ratio and that oxidative stress was lower than that in uninfected controls. All of these results are characteristic of the Warburg effect as it is present in mammals. There was also a significant decrease in triglyceride concentration starting at 12 hpi. At the late stage of the infection cycle (24 hpi), hemocytes of WSSV-infected shrimp showed several changes associated with cell death. These included the induction of mitochondrial membrane permeabilization (MMP), increased oxidative stress, decreased glucose consumption, and disrupted energy production. A previous study showed that WSSV infection led to upregulation of the voltage-dependent anion channel (VDAC), which is known to be involved in both the Warburg effect and MMP. Here we show that double-stranded RNA (dsRNA) silencing of the VDAC reduces WSSV-induced mortality and virion copy number. For these results, we hypothesize a model depicting the metabolic changes in host cells at the early and late stages of WSSV infection.

  5. Oxidative stress parameters induced by exposure to either cadmium or 17β-estradiol on Mytilus galloprovincialis hemocytes. The role of signaling molecules

    Koutsogiannaki, Sophia; Franzellitti, Silvia; Fabbri, Elena; Kaloyianni, Martha

    2014-01-01

    Highlights: •Oxidative parameters in Mytilus galloprovincialis hemocytes were measured. •Comparison between cadmium and 17β-estradiol cytotoxicity is discussed. •NHE, PKC, PI3-K, NADPH oxidase, NO synthase, JNK involvement was observed. •Protective role of cAMP is suggested. •Signaling molecules studied could constitute novel biomarkers. -- Abstract: The aim of the present study was to determine and compare the possible effects of exposure to an estrogen, 17β-estradiol and to a metal, cadmium on oxidative parameters of Mytilus galloprovincialis hemocytes and to elucidate the signaling pathways that probably mediate the studied effects exerted by these two chemicals. In addition, it was of interest to investigate if the studied parameters could constitute biomarkers for aquatic pollution monitoring. Our results suggest that micromolar concentrations of either cadmium or 17β-estradiol affected the redox status of mussels by modulating oxidative parameters and antioxidant enzymes gene expression in mussel M. galloprovincialis hemocytes. In particular, our results showed that treatment of hemocytes with either 5 μM of cadmium chloride or with 25 nM of 17β-estradiol for 30 min caused significant increased ROS production; this led to oxidative damage exemplified by significant increased DNA damage, protein carbonylation and lipid peroxidation, as well as increased mRNA levels of the antioxidant enzymes catalase (CAT), superoxide dismoutase (SOD) and glutathione S-transferase (GST). Furthermore, our results suggest that either cadmium or 17β-estradiol signal is mediated either through one of the already known pathways initiated by photatidyl-inositol 3-kinase (PI3 K) and reaching Na + /H + exchanger (NHE) probably through protein kinase C (PKC) or a kinase-mediated signaling pathway that involves in most of the cases NHE, PKC, Ca 2+ -dependent PKC isoforms, PI3-K, NADPH oxidase, nitric oxide (NO) synthase, c-Jun N-terminal kinase (JNK) and cyclic adenosine

  6. Oxidative stress parameters induced by exposure to either cadmium or 17β-estradiol on Mytilus galloprovincialis hemocytes. The role of signaling molecules

    Koutsogiannaki, Sophia [Laboratory of Animal Physiology, Zoology Department, School of Biology, Faculty of Science, Aristotle University of Thessaloniki, 54124 Thessaloniki (Greece); Franzellitti, Silvia [University of Bologna, Interdepartment Centre for Environmental Science Research, via S. Alberto 163, 48123 Ravenna (Italy); Fabbri, Elena [University of Bologna, Interdepartment Centre for Environmental Science Research, via S. Alberto 163, 48123 Ravenna (Italy); University of Bologna, Department of Biological, Geological, and Environmental Sciences, via Selmi 3, 40100 Bologna (Italy); Kaloyianni, Martha, E-mail: kaloyian@bio.auth.gr [Laboratory of Animal Physiology, Zoology Department, School of Biology, Faculty of Science, Aristotle University of Thessaloniki, 54124 Thessaloniki (Greece)

    2014-01-15

    Highlights: •Oxidative parameters in Mytilus galloprovincialis hemocytes were measured. •Comparison between cadmium and 17β-estradiol cytotoxicity is discussed. •NHE, PKC, PI3-K, NADPH oxidase, NO synthase, JNK involvement was observed. •Protective role of cAMP is suggested. •Signaling molecules studied could constitute novel biomarkers. -- Abstract: The aim of the present study was to determine and compare the possible effects of exposure to an estrogen, 17β-estradiol and to a metal, cadmium on oxidative parameters of Mytilus galloprovincialis hemocytes and to elucidate the signaling pathways that probably mediate the studied effects exerted by these two chemicals. In addition, it was of interest to investigate if the studied parameters could constitute biomarkers for aquatic pollution monitoring. Our results suggest that micromolar concentrations of either cadmium or 17β-estradiol affected the redox status of mussels by modulating oxidative parameters and antioxidant enzymes gene expression in mussel M. galloprovincialis hemocytes. In particular, our results showed that treatment of hemocytes with either 5 μM of cadmium chloride or with 25 nM of 17β-estradiol for 30 min caused significant increased ROS production; this led to oxidative damage exemplified by significant increased DNA damage, protein carbonylation and lipid peroxidation, as well as increased mRNA levels of the antioxidant enzymes catalase (CAT), superoxide dismoutase (SOD) and glutathione S-transferase (GST). Furthermore, our results suggest that either cadmium or 17β-estradiol signal is mediated either through one of the already known pathways initiated by photatidyl-inositol 3-kinase (PI3 K) and reaching Na{sup +}/H{sup +} exchanger (NHE) probably through protein kinase C (PKC) or a kinase-mediated signaling pathway that involves in most of the cases NHE, PKC, Ca{sup 2+}-dependent PKC isoforms, PI3-K, NADPH oxidase, nitric oxide (NO) synthase, c-Jun N-terminal kinase (JNK) and

  7. Circulating Hemocytes from Larvae of the Japanese Rhinoceros Beetle Allomyrina dichotoma (Linnaeus) (Coleoptera: Scarabaeidae) and the Cellular Immune Response to Microorganisms.

    Hwang, Sejung; Bang, Kyeongrin; Lee, Jiae; Cho, Saeyoull

    2015-01-01

    Hemocytes of the last larva of the Japanese rhinoceros beetle A. dichotoma (Linnaeus) (Coleoptera: Scarabaeidae) were classified as granulocytes, plasmatocytes, oenocytoids, spherulocytes, prohemocytes, and adipohemocytes. Among these cell types, only the granulocytes became immunologically activated with obvious morphological changes, displaying large amoeba-like, lobopodia-like, and fan-like structures. In addition, their cytoplasmic granules became larger and greatly increased in number. To explore whether these granules could be immunologically generated as phagosomes, total hemocytes were stained with LysoTracker. Greater than 90% of the granulocytes retained the LysoTracker dye at 4 h post-bacterial infection. In flow cytometry analysis, the red fluorescent signal was highly increased at 4 h post-bacterial infection (60.36%) compared to controls (5.08%), as was confirmed by fluorescent microscopy. After 12 h post-infection, these signals returned to basal levels. The uptake of pathogens by granulocytes rapidly triggered the translocation of the microtubule-associated protein 1 light chain 3 alpha (LC3) to the phagosome, which may result in enhanced pathogen killing.

  8. Circulating Hemocytes from Larvae of the Japanese Rhinoceros Beetle Allomyrina dichotoma (Linnaeus (Coleoptera: Scarabaeidae and the Cellular Immune Response to Microorganisms.

    Sejung Hwang

    Full Text Available Hemocytes of the last larva of the Japanese rhinoceros beetle A. dichotoma (Linnaeus (Coleoptera: Scarabaeidae were classified as granulocytes, plasmatocytes, oenocytoids, spherulocytes, prohemocytes, and adipohemocytes. Among these cell types, only the granulocytes became immunologically activated with obvious morphological changes, displaying large amoeba-like, lobopodia-like, and fan-like structures. In addition, their cytoplasmic granules became larger and greatly increased in number. To explore whether these granules could be immunologically generated as phagosomes, total hemocytes were stained with LysoTracker. Greater than 90% of the granulocytes retained the LysoTracker dye at 4 h post-bacterial infection. In flow cytometry analysis, the red fluorescent signal was highly increased at 4 h post-bacterial infection (60.36% compared to controls (5.08%, as was confirmed by fluorescent microscopy. After 12 h post-infection, these signals returned to basal levels. The uptake of pathogens by granulocytes rapidly triggered the translocation of the microtubule-associated protein 1 light chain 3 alpha (LC3 to the phagosome, which may result in enhanced pathogen killing.

  9. Shrimp hemocyte homeostasis-associated protein (PmHHAP) interacts with WSSV134 to control apoptosis in white spot syndrome virus infection.

    Apitanyasai, Kantamas; Amparyup, Piti; Charoensapsri, Walaiporn; Sangsuriya, Pakkakul; Tassanakajon, Anchalee

    2018-05-01

    Hemocyte homeostasis-associated protein (PmHHAP) was first identified as a viral-responsive gene, due to a high upregulation in transcription following white spot syndrome virus (WSSV) infection. Functional studies using RNA interference have suggested that PmHHAP is involved in hemocyte homeostasis by controlling apoptosis during WSSV infection. In this study, the role of PmHHAP in host-viral interactions was further investigated. Yeast two-hybrid assay and co-immunoprecipitation revealed that PmHHAP binds to an anti-apoptosis protein, WSSV134. The viral protein WSSV134 is a late protein of WSSV, expressed 24 h post infection (hpi). Gene silencing of WSSV134 in WSSV-infected shrimp resulted in a reduction of the expression level of the viral replication marker genes VP28, wsv477, and ie-1, which suggests that WSSV134 is likely involved in viral propagation. However, co-silencing of PmHHAP and WSSV134 counteracted the effects on WSSV infection, which implies the importance of the host-pathogen interaction between PmHHAP and WSSV134 in WSSV infection. In addition, caspase 3/7 activity was noticeably induced in the PmHHAP and WSSV134 co-silenced shrimp upon WSSV infection. Moreover, PmHHAP and WSSV134 inhibited caspase-induced activation of PmCasp in vitro in a non-competitive manner. Taken together, these results suggest that PmHHAP and WSSV134 play a role in the host-pathogen interaction and work concordantly to control apoptosis in WSSV infection. Copyright © 2018 Elsevier Ltd. All rights reserved.

  10. HSP70 gene expression in Mytilus galloprovincialis hemocytes is triggered by moderate heat shock and Vibrio anguillarum, but not by V. splendidus or Micrococcus lysodeikticus.

    Cellura, Cinzia; Toubiana, Mylène; Parrinello, Nicolo; Roch, Philippe

    2006-01-01

    Complete sequence of HSP70 cDNA from the mussel, Mytilus galloprovincialis was established before quantifying its expression following moderate heat shock or injection of heat-killed bacteria. HSP70 cDNA is comprised of 2378 bp including one ORF of 654 aa, with a predicted 70 bp 5'-UTR and a 343 bp 3'-UTR (GenBank, 18 Jan 05, AY861684). Alignment identity ranged from 89% for Crassostrea ariakensis to 72% for C. virginica. Curiously, HSP70 gene and cDNA sequences from M. galloprovincialis, deposited later (03 and 27 May), show only 73% identity with the present sequence. Meanwhile, characteristic motifs of the HSP70 family were located in conserved positions. Expression of HSP70 gene was quantified on circulating hemocyte mRNA using Q-PCR after RT using random hexaprimers. Housekeeping gene was 28S rRNA. Four stresses were applied: heat shock that consisted of immersing mussels for 90 min at 30 degrees C and returning them to 20 degrees C sea water, one injection of heat-killed Gram-negative bacteria, Vibrio splendidus LGP32, one injection of heat-killed Gram-negative bacteria Vibrio anguillarum, one injection of heat-killed Gram-positive bacteria Micrococcus lysodeikticus. We found no significant modification of 28S rRNA gene expression. Significant increase of 5.2 +/- 0.4 fold the ratio HSP70/28S rRNA was observed 6 h after heat shock and was maximum at 15 h (6.1 +/- 1.1), and still significant after 24 h (1.7 +/- 0.03). Similarly, injecting V. anguillarum resulted in a significant increase of 2.7 +/- 0.1 after 12 h. Expression was maximum after 48 h (5.2 +/- 0.05) and returned to baseline after 72 h. In contrast, injecting V. splendidus or M. lysodeikticus failed to significantly modulate HSP70 gene expression at least during the first 3 days post-injection. Consequently, mussel hemocytes appeared to discriminate between pathogenic and non-pathogenic Vibrios, as well as between Gram-negative and Gram-positive bacteria.

  11. Molecular cloning and characterization of autophagy-related gene TmATG8 in Listeria-invaded hemocytes of Tenebrio molitor.

    Tindwa, Hamisi; Jo, Yong Hun; Patnaik, Bharat Bhusan; Lee, Yong Seok; Kang, Sang Sun; Han, Yeon Soo

    2015-07-01

    Macroautophagy (hereinafter called autophagy) is a highly regulated process used by eukaryotic cells to digest portions of the cytoplasm that remodels and recycles nutrients and disposes of unwanted cytoplasmic constituents. Currently 36 autophagy-related genes (ATG) and their homologs have been characterized in yeast and higher eukaryotes, including insects. In the present study, we identified and functionally characterized the immune function of an ATG8 homolog in a coleopteran insect, Tenebrio molitor (TmATG8). The cDNA of TmATG8 comprises of an ORF of 363 bp that encodes a protein of 120 amino acid residues. TmATG8 transcripts are detected in all the developmental stages analyzed. TmAtg8 protein contains a highly conserved C-terminal glycine residue (Gly116) and shows high amino acid sequence identity (98%) to its Tribolium castaneum homolog, TcAtg8. Loss of function of TmATG8 by RNAi led to a significant increase in the mortality rates of T. molitor larvae against Listeria monocytogenes. Unlike dsEGFP-treated control larvae, TmATG8-silenced larvae failed to turn-on autophagy in hemocytes after injection with L. monocytogenes. These data suggest that TmATG8 play a role in mediating autophagy-based clearance of Listeria in T. molitor. Copyright © 2015 Elsevier Ltd. All rights reserved.

  12. New polymorphic microsatellite markers derived from hemocyte cDNA library of Manila clam Ruditapes philippinarum challenged by the protozoan parasite Perkinsus olseni

    Kang, Hyun-Sil; Hong, Hyun-Ki; Park, Kyung-Il; Cho, Moonjae; Youn, Seok-Hyun; Choi, Kwang-Sik

    2017-03-01

    Manila clam Ruditapes philippinarum is one of the most important benthic animals in the coastal north Pacific region, where clam populations have been mixed genetically through trade and aquaculture activities. Accordingly, identification of the genetically different clam populations has become one of the most important issues to manage interbreeding of the local and introduced clam populations. To identify genetically different populations of clam populations, we developed 11 expressed sequence tag (EST)-microsatellite loci (i.e., simple sequence repeat, SSR) from 1,128 clam hemocyte cDNA clones challenged by the protozoan parasite Perkinsus olseni. Genotype analysis using the markers developed in this study demonstrated that clams from a tidal flat on the west coast contained 6 to 19 alleles per locus, and a population from Jeju Island had 4 to 20 alleles per locus. The expected heterozygosity of the 2 clam populations ranged from 0.472 to 0.919 for clams from the west coast, and 0.494 to 0.919 for clams from Jeju Island, respectively. Among the 11 loci discovered in this study, 7 loci significantly deviated from the Hardy-Weinberg equilibrium after Bonferroni correction. The 5 loci developed in this study also successfully amplified the SSRs of R. variegatus, a clam species taxonomically very close to R. philippinarum, from Hong Kong and Jeju Island. We believe that the 11 novel polymorphic SSR developed in this study can be utilized successfully in Manila clam genetic diversity analysis, as well as in genetic discrimination of different clam populations.

  13. Effects of tributyltin and benzo[a]pyrene on the immune-associated activities of hemocytes and recovery responses in the gastropod abalone, Haliotis diversicolor.

    Gopalakrishnan, Singaram; Huang, Wei-Bin; Wang, Qiang-Wei; Wu, Man-Li; Liu, Jie; Wang, Ke-Jian

    2011-08-01

    Our previous study reports that short-term exposure to sublethal concentrations of benzo[a]pyrene (BaP) induces immunomodulation in the gastropod abalone, Haliotis diversicolor. In the present study, it was further observed that long-term chronic exposure to sublethal concentrations of BaP modulated the immunocompetence of abalones in terms of the change in activity of the antioxidant and immune associated parameters tested. In addition, the effect of tributyltin (TBT), another important genotoxicant in the aquatic environment, was investigated. Exposure of abalones to sublethal concentrations of TBT and BaP for 21 days resulted in significant decrease of total hemocyte count, phagocytosis, membrane stability and lysozyme activity. Conversely induction of extra and intra cellular superoxide generation, nitric oxide, nitric oxide synthase and myeloperoxidase activity was present when the abalones were exposed to TBT and BaP. Most of the immune associated parameters tested showed clear time dependent response to both toxicants. Within 14 days after the 21 day exposure to BaP, recovery was observed as evidenced by most of the parameters returning to their normal level. However, no recovery was observed within 14 days after the 21 day exposure to TBT as evidenced by continued elevation of intra cellular superoxide and nitrite production and decrease in THC, membrane stability and lysozyme activity. This suggested a prolonged TBT-induced impact on the immune reaction and possibly more damage than that caused by BaP. Overall the results suggest that chronic exposure to sublethal concentrations of TBT or BaP causes modulations in the immunocompetence of abalones with most of the immune associated parameters tested being stimulated, and this might be harmful to the host. Copyright © 2011 Elsevier Inc. All rights reserved.

  14. New functions of arthropod bursicon: inducing deposition and thickening of new cuticle and hemocyte granulation in the blue crab, Callinectes sapidus.

    J Sook Chung

    Full Text Available Arthropod growth requires molt-associated changes in softness and stiffness of the cuticle that protects from desiccation, infection and injury. Cuticle hardening in insects depends on the blood-borne hormone, bursicon (Burs, although it has never been determined in hemolymph. Whilst also having Burs, decapod crustaceans reiterate molting many more times during their longer life span and are encased in a calcified exoskeleton, which after molting undergoes similar initial cuticle hardening processes as in insects. We investigated the role of homologous crustacean Burs in cuticular changes and growth in the blue crab, Callinectes sapidus. We found dramatic increases in size and number of Burs cells during development in paired thoracic ganglion complex (TGC neurons with pericardial organs (POs as neurohemal release sites. A skewed expression of Burs β/Burs α mRNA in TGC corresponds to protein contents of identified Burs β homodimer and Burs heterodimer in POs. In hemolymph, Burs is consistently present at ∼21 pM throughout the molt cycle, showing a peak of ∼89 pM at ecdysis. Since initial cuticle hardness determines the degree of molt-associated somatic increment (MSI, we applied recombinant Burs in vitro to cuticle explants of late premolt or early ecdysis. Burs stimulates cuticle thickening and granulation of hemocytes. These findings demonstrate novel cuticle-associated functions of Burs during molting, while the unambiguous and constant presence of Burs in cells and hemolymph throughout the molt cycle and life stages may implicate further functions of its homo- and heterodimer hormone isoforms in immunoprotective defense systems of arthropods.

  15. Mediated effect of ultrasound treated Diclofenac on mussel hemocytes: First evidence for the involvement of respiratory burst enzymes in the induction of DCF-mediated unspecific mode of action.

    Toufexi, Eirini; Dailianis, Stefanos; Vlastos, Dimitris; Manariotis, Ioannis D

    2016-06-01

    The present study investigates the toxic behavior of diclofenac (DCF) before and after its ultrasound (US) treatment, as well as the involvement of intracellular target molecules, such as NADPH oxidase and NO synthase, in the DCF-induced adverse effects on hemocytes of mussel Mytilus galloprovincialis. In this context, appropriate volumes (350 and 500mL) of DCF solutions (at concentrations of 2, 2.5, 5 and 10mgL(-1)) were treated under different ultrasound operating conditions (frequency at 582 and 862kHz, electric power density at 133 and 167W) for assessing US method efficiency. In parallel, DCF and US DCF-mediated cytotoxic (in terms of cell viability measured with the use of neutral red uptake/NRU method), oxidative (in terms of superoxide anions/(.)O2(-), nitric oxides such as NO2(-) and lipid peroxidation products, such as malondialdehyde/MDA content) and genotoxic (DNA damage measured by the use of Comet assay method) effects were investigated in hemocytes exposed for 1h to 5, 10 and 100ngL(-1) and 1, 10 and 20μgL(-1) of DCF. The involvement of NADPH oxidase and NO synthase to the DCF-induced toxicity was further investigated by the use of 10μΜ L-NAME, a NO synthase inhibitor and 10μΜ DPI, a NADPH oxidase inhibitor. According to the results, 350mL of 2mgL(-1) DCF showed higher degradation (>50%) under 167W electric power density and frequency at 862kHz for 120min, compared to degradation in all other cases, followed by a significant elimination of its toxicity. Specifically, US DCF-treated hemocytes showed a significant attenuation of DCF-mediated cytotoxic, oxidative and genotoxic effects, which appeared to be caused by NADPH oxidase and NO synthase activation, since their inhibition was followed by a significant elimination of (.)O2(-) and NO2(-) generation and the concomitant oxidative damage within cells. The results of the present study showed for the first time that unspecific mode of action of DCF, associated with the induction of NADPH oxidase

  16. Big defensins, a diverse family of antimicrobial peptides that follows different patterns of expression in hemocytes of the oyster Crassostrea gigas.

    Rosa, Rafael D; Santini, Adrien; Fievet, Julie; Bulet, Philippe; Destoumieux-Garzón, Delphine; Bachère, Evelyne

    2011-01-01

    Big defensin is an antimicrobial peptide composed of a highly hydrophobic N-terminal region and a cationic C-terminal region containing six cysteine residues involved in three internal disulfide bridges. While big defensin sequences have been reported in various mollusk species, few studies have been devoted to their sequence diversity, gene organization and their expression in response to microbial infections. Using the high-throughput Digital Gene Expression approach, we have identified in Crassostrea gigas oysters several sequences coding for big defensins induced in response to a Vibrio infection. We showed that the oyster big defensin family is composed of three members (named Cg-BigDef1, Cg-BigDef2 and Cg-BigDef3) that are encoded by distinct genomic sequences. All Cg-BigDefs contain a hydrophobic N-terminal domain and a cationic C-terminal domain that resembles vertebrate β-defensins. Both domains are encoded by separate exons. We found that big defensins form a group predominantly present in mollusks and closer to vertebrate defensins than to invertebrate and fungi CSαβ-containing defensins. Moreover, we showed that Cg-BigDefs are expressed in oyster hemocytes only and follow different patterns of gene expression. While Cg-BigDef3 is non-regulated, both Cg-BigDef1 and Cg-BigDef2 transcripts are strongly induced in response to bacterial challenge. Induction was dependent on pathogen associated molecular patterns but not damage-dependent. The inducibility of Cg-BigDef1 was confirmed by HPLC and mass spectrometry, since ions with a molecular mass compatible with mature Cg-BigDef1 (10.7 kDa) were present in immune-challenged oysters only. From our biochemical data, native Cg-BigDef1 would result from the elimination of a prepropeptide sequence and the cyclization of the resulting N-terminal glutamine residue into a pyroglutamic acid. We provide here the first report showing that big defensins form a family of antimicrobial peptides diverse not only in terms

  17. Big Defensins, a Diverse Family of Antimicrobial Peptides That Follows Different Patterns of Expression in Hemocytes of the Oyster Crassostrea gigas

    Rosa, Rafael D.; Santini, Adrien; Fievet, Julie; Bulet, Philippe; Destoumieux-Garzón, Delphine; Bachère, Evelyne

    2011-01-01

    Background Big defensin is an antimicrobial peptide composed of a highly hydrophobic N-terminal region and a cationic C-terminal region containing six cysteine residues involved in three internal disulfide bridges. While big defensin sequences have been reported in various mollusk species, few studies have been devoted to their sequence diversity, gene organization and their expression in response to microbial infections. Findings Using the high-throughput Digital Gene Expression approach, we have identified in Crassostrea gigas oysters several sequences coding for big defensins induced in response to a Vibrio infection. We showed that the oyster big defensin family is composed of three members (named Cg-BigDef1, Cg-BigDef2 and Cg-BigDef3) that are encoded by distinct genomic sequences. All Cg-BigDefs contain a hydrophobic N-terminal domain and a cationic C-terminal domain that resembles vertebrate β-defensins. Both domains are encoded by separate exons. We found that big defensins form a group predominantly present in mollusks and closer to vertebrate defensins than to invertebrate and fungi CSαβ-containing defensins. Moreover, we showed that Cg-BigDefs are expressed in oyster hemocytes only and follow different patterns of gene expression. While Cg-BigDef3 is non-regulated, both Cg-BigDef1 and Cg-BigDef2 transcripts are strongly induced in response to bacterial challenge. Induction was dependent on pathogen associated molecular patterns but not damage-dependent. The inducibility of Cg-BigDef1 was confirmed by HPLC and mass spectrometry, since ions with a molecular mass compatible with mature Cg-BigDef1 (10.7 kDa) were present in immune-challenged oysters only. From our biochemical data, native Cg-BigDef1 would result from the elimination of a prepropeptide sequence and the cyclization of the resulting N-terminal glutamine residue into a pyroglutamic acid. Conclusions We provide here the first report showing that big defensins form a family of antimicrobial

  18. Big defensins, a diverse family of antimicrobial peptides that follows different patterns of expression in hemocytes of the oyster Crassostrea gigas.

    Rafael D Rosa

    Full Text Available BACKGROUND: Big defensin is an antimicrobial peptide composed of a highly hydrophobic N-terminal region and a cationic C-terminal region containing six cysteine residues involved in three internal disulfide bridges. While big defensin sequences have been reported in various mollusk species, few studies have been devoted to their sequence diversity, gene organization and their expression in response to microbial infections. FINDINGS: Using the high-throughput Digital Gene Expression approach, we have identified in Crassostrea gigas oysters several sequences coding for big defensins induced in response to a Vibrio infection. We showed that the oyster big defensin family is composed of three members (named Cg-BigDef1, Cg-BigDef2 and Cg-BigDef3 that are encoded by distinct genomic sequences. All Cg-BigDefs contain a hydrophobic N-terminal domain and a cationic C-terminal domain that resembles vertebrate β-defensins. Both domains are encoded by separate exons. We found that big defensins form a group predominantly present in mollusks and closer to vertebrate defensins than to invertebrate and fungi CSαβ-containing defensins. Moreover, we showed that Cg-BigDefs are expressed in oyster hemocytes only and follow different patterns of gene expression. While Cg-BigDef3 is non-regulated, both Cg-BigDef1 and Cg-BigDef2 transcripts are strongly induced in response to bacterial challenge. Induction was dependent on pathogen associated molecular patterns but not damage-dependent. The inducibility of Cg-BigDef1 was confirmed by HPLC and mass spectrometry, since ions with a molecular mass compatible with mature Cg-BigDef1 (10.7 kDa were present in immune-challenged oysters only. From our biochemical data, native Cg-BigDef1 would result from the elimination of a prepropeptide sequence and the cyclization of the resulting N-terminal glutamine residue into a pyroglutamic acid. CONCLUSIONS: We provide here the first report showing that big defensins form a family

  19. Identification of expressed genes in cDNA library of hemocytes from the RLO-challenged oyster, Crassostrea ariakensis Gould with special functional implication of three complement-related fragments (CaC1q1, CaC1q2 and CaC3).

    Xu, Ting; Xie, Jiasong; Li, Jianming; Luo, Ming; Ye, Shigen; Wu, Xinzhong

    2012-06-01

    A SMARTer™ cDNA library of hemocyte from Rickettsia-like organism (RLO) challenged oyster, Crassostrea ariakensis Gould was constructed. Random clones (400) were selected and single-pass sequenced, resulted in 200 unique sequences containing 96 known genes and 104 unknown genes. The 96 known genes were categorized into 11 groups based on their biological process. Furthermore, we identified and characterized three complement-related fragments (CaC1q1, CaC1q2 and CaC3). Tissue distribution analysis revealed that all of three fragments were ubiquitously expressed in all tissues studied including hemocyte, gills, mantle, digestive glands, gonads and adductor muscle, while the highest level was seen in the hemocyte. Temporal expression profile in the hemocyte monolayers reveled that the mRNA expression levels of three fragments presented huge increase after the RLO incubation at 3 h and 6 h in post-challenge, respectively. And the maximal expression levels at 3 h in post-challenge are about 256, 104 and 64 times higher than the values detected in the control of CaC1q1, CaC1q2 and CaC3, respectively. Copyright © 2012 Elsevier Ltd. All rights reserved.

  20. Shellfish Hemocyte Data from Flow Cytometers

    National Oceanic and Atmospheric Administration, Department of Commerce — Various immune function tests using shellfish hemolymph have been developed to determine shellfish health. These tests including viability, phagocytosis, adhesion,...

  1. Aspectos ultraestruturais de hemócitos de Biomphalaria glabrata Say (1818 (Gastropoda: Planorbidae analisados sob microscopia eletrônica de transmissão Ultrastructural aspects of hemocytes from Biomphalaria glabrata Say (1818 (Gastropoda: Planorbidae analysed with transmission eletronic microscopy

    Marco Antonio Vasconcelos Santos

    2009-09-01

    Full Text Available Os hemócitos do caramujo Biomphalaria glabrata, um importante transmissor do trematódeo Schistosoma mansoni no Brasil, foram coletados de especimens na região Bragantina, localizada a oeste do estado do Pará. Os hemócitos foram examinados por meio de microscopia eletrônica de transmissão. As células foram fixadas pelo método de rotina com o uso do tampão PHEM (PIPES, HEPES, EGTA e Magnésio. Foram descritos os aspectos ultra-estruturais celulares como inclusões citoplasmáticas limitadas por membranas, mitocôndrias, retículos endoplasmáticos e outros. As observações mostram que esse tampão possui a propriedade de preservação do citoesqueleto celular, apresentando bons resultados na preservação das estruturas dos hemócitos e suas organelas.The blood cells of the pulmonate snail Biomphalaria glabrata from the region of Bragantina in the state of Pará, an important vector of the trematode Schistosoma mansoni in Brazil, were collected and fixed by routine method with PHEM buffer and examined with transmission electron microscopy. Ultrastructural cellular aspects like cytoplasmic inclusions, mitochondrion, vesicles and others organelles are described. The buffer used resulted in good preservations of the hemocytes and their organelles.

  2. Rapid extraction of virus-contaminated hemocytes from oysters

    Rapid viral detection methods are necessary to employ diagnostic testing for viral contamination in shellfish to prevent and control foodborne illness. Current shellfish viral RNA extraction methods, which are time-consuming and not applicable for routine monitoring, require the testing of whole or ...

  3. Hemocytes are sites of persistence for virus-contaminated oysters

    Like fecal bacteria, waterborne enteric viruses are readily bioconcentrated by bivalve shellfish. However while many bacteria decline rapidly when bivalves are placed in uncontaminated water, viruses tend to be retained within shellfish. In this study, we offer evidence that phagocytic blood cells...

  4. Hemocytes from Pediculus humanus humanus are hosts for human bacterial pathogens.

    Eric eGhigo

    2015-01-01

    Full Text Available Pediculus humanus humanus is an human ectoparasite which represents a serious public health threat because it is vector for pathogenic bacteria. It is important to understand and identify where bacteria reside in human body lice to define new strategies to counterstroke the capacity of vectorization of the bacterial pathogens by body lice. It is known that phagocytes from vertebrates can be hosts or reservoirs for several microbes. Therefore, we wondered if Pediculus humanus humanus phagocytes could hide pathogens. In this study, we characterized the phagocytes from Pediculus humanus humanus and evaluated their contribution as hosts for human pathogens such as Rickettsia prowazekii, Bartonella quintana and Acinetobacter baumannii.

  5. Purification and characterization of hemocyte phenoloxidases in Chilo suppressalis walker (Lepidoptera: Crambidae

    Mirhaghparast Seyyedeh Kimia

    2015-01-01

    Full Text Available In the current study, two phenoloxidases (POs from the larvae of Chilo suppressalis Walker were extracted and purified by column chromatography using Sepharyl G-100 and DEAE-Cellulose fast flow column. Two proteins possessing PO activity, named as POI and POII, were extracted by purification, 5.08- and 5.62-fold, respectively, with 8.94% and 7.31% recoveries, respectively. Also, the specific activities of POI and POII were 0.478 and 0.529 U/mg protein, respectively. Finally, the molecular weights of POI and POII were calculated as 94.6 and 95.7 kDa, respectively. Kinetic parameters of the purified phenoloxidases by Lineweaver-Burk analysis were Vmax of 2.27 and 1.11 U/mg protein and Km of 15.51 and 17.31 mM for POI and POII, respectively. Mg2+ and Cu2+ significantly increased the PO activities. Ca2+ decreased the activity of POI and showed no statistical effects on POII activity. EDTA and DTC significantly inhibited the activities of the purified enzymes, while triethylenetetramine hexaacetic acid (TTHA and RGTA showed no significant effects on enzymatic activities.

  6. The cell-mediated immunity of Drosophila melanogaster: hemocyte lineages, immune compartments, microanatomy and regulation.

    Honti, Viktor; Csordás, Gábor; Kurucz, Éva; Márkus, Róbert; Andó, István

    2014-01-01

    In the animal kingdom, innate immunity is the first line of defense against invading pathogens. The dangers of microbial and parasitic attacks are countered by similar mechanisms, involving the prototypes of the cell-mediated immune responses, the phagocytosis and encapsulation. Work on Drosophila has played an important role in promoting an understanding of the basic mechanisms of phylogenetically conserved modules of innate immunity. The aim of this review is to survey the developments in the identification and functional definition of immune cell types and the immunological compartments of Drosophila melanogaster. We focus on the molecular and developmental aspects of the blood cell types and compartments, as well as the dynamics of blood cell development and the immune response. Further advances in the characterization of the innate immune mechanisms in Drosophila will provide basic clues to the understanding of the importance of the evolutionary conserved mechanisms of innate immune defenses in the animal kingdom. Copyright © 2013 Elsevier Ltd. All rights reserved.

  7. Bacterial Infection of Fly Ovaries Reduces Egg Production and Induces Local Hemocyte Activation

    Brandt, Stephanie M.; Schneider, David S.

    2009-01-01

    Summary Morbidity, the state of being diseased, is an important aspect of pathogenesis that has gone relatively unstudied in fruit flies. Our interest is in characterizing how bacterial pathogenesis affects various physiologies of the fly. We chose to examine the fly ovary because we found bacterial infection had a striking effect on fly reproduction. We observed decreased egg laying after bacterial infection that correlated with increased bacterial virulence. We also found that bacteria colonized the ovary in a previously undescribed manner; bacteria were found in the posterior of the ovary, adjacent to the lateral oviduct. This local infection in the ovary resulted in melanization and activation of the cellular immune response at the site of infection. PMID:17400292

  8. Bacterial Infection of Fly Ovaries Reduces Egg Production and Induces Local Hemocyte Activation

    Brandt, Stephanie M.; Schneider, David S.

    2007-01-01

    Morbidity, the state of being diseased, is an important aspect of pathogenesis that has gone relatively unstudied in fruit flies. Our interest is in characterizing how bacterial pathogenesis affects various physiologies of the fly. We chose to examine the fly ovary because we found bacterial infection had a striking effect on fly reproduction. We observed decreased egg laying after bacterial infection that correlated with increased bacterial virulence. We also found that bacteria colonized th...

  9. Induction of micronuclei in hemocytes of Mytilus edulis and statistical analysis

    Wrisberg, M. N.; Bilbo, Carl M.; Spliid, Henrik

    1992-01-01

    biological variation, emphasizing the importance of application of a correct statistical method. A systematic approach to the statistical evaluation of the mussel MN test is outlined. The statistical model includes three different situations: (a) estimation of parameters of a single sample, (b) estimation...

  10. Notch co-operates with Lozenge/Runx to lock hemocytes into a differentiation programme

    Terriente-Felix, A.; Li, J.; Collins, S.; Mulligan, A.; Reekie, I.; Bernard, F.; Krejčí, Alena; Bray, S.

    2013-01-01

    Roč. 140, č. 4 (2013), s. 926-937 ISSN 0950-1991 R&D Projects: GA ČR(CZ) GAP305/11/0126 Grant - others:MRC programme(GB) G0800034 Institutional support: RVO:60077344 Keywords : Lozenge/Runx * Notch * chromatin immunoprecipitation Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 6.273, year: 2013

  11. Rapid detection of hepatitis A virus and murine norovirus in hemocytes of contaminated oysters

    The human enteric pathogens, hepatitis A virus and human norovirus, have been shown to contaminate molluscan shellfish and cause foodborne disease in consumers. Rapid viral extraction methods are needed to replace current time consuming methods, which use whole oysters or dissected tissues. In our ...

  12. GREATER HEMOCYTE BACTERICIDAL ACTIVITY IN OYSTERS (CRASSOSTREA VIRGINICA) FROM A RELATIVELY CONTAMINATED SITE IN PENSACOLA BAY, FLORIDA.

    Bivalve mollusks such as Crassostrea virginica inhabiting polluted estuaries and coastal areas may bioaccumulate high concentrations of contaminants without apparent ill effects. However, changes in putative internal defense activities have been associated with contaminant accumu...

  13. Encapsulation and Hemocyte Numbers in Crocidolomia pavonana and Spodoptera litura Fabricius (Lepidoptera Attacked by Parasitoid Eriborus argenteopilosus Cameron (Hymenoptera

    DAMAYANTI BUCHORI

    2009-12-01

    Full Text Available Eriborus argenteopilosus is the most important parasitoid attacking cabbage pest Crocidolomia pavonana in Indonesia. Previous studies proved that parasitoid encapsulation was found to be an important factor limiting the effectiveness of the parasitoid in controlling pest population in the field. Since 1998, we have conducted series studies to investigate encapsulation mechanism developed by hosts against parasitoid, responses of parasitoid toward encapsulation ability and to determine factors that may help parasitoid avoid encapsulation. Parasitoid responses were examined on two different hosts C. pavonana and Spodoptera litura. Our findings showed that parasitization level was found to be high both on C. pavonana and S. litura. Encapsulation occurred to be high in all larva stages of C. pavonana, in contrast encapsulation was recorded very low in all larvae stages of S. litura. We recorded that encapsulation in the larval body of C. pavonana was completed in 72 hours and mostly occurred in higher larval stage. Melanization was only recorded in encapsulated parasitoid inside larva body of C. pavonana, not in S. litura. We recorded that encapsulation increased blood cell number of both larvae C. pavonana and S. litura. Encapsulation may affect development of immature parasitoid. Weight of S. litura's pupae containing encapsulated parasitoid was found to be lower in S. litura, but not in C. pavonana. Our investigation also proved that superparasitism may help parasitoid avoid encapsulation.

  14. Dopamine modulates hemocyte phagocytosis via a D1-like receptor in the rice stem borer, Chilo suppressalis

    Dopamine (DA) is a signal moiety bridging the nervous and immune systems. DA dysregulation is linked to serious human diseases, including addiction, schizophrenia, and Parkinson's disease. However, DA actions in the immune system remain incompletely understood. In this study, we found that DA modula...

  15. Development of rapid hemocyte-based extraction methods for detection of hepatitis A virus and murine norovirus in contaminated oysters

    The human enteric pathogens, hepatitis A virus and human norovirus, have been shown to contaminate molluscan shellfish and cause foodborne disease in consumers. Rapid viral extraction methods are needed to replace current time consuming methods, which use whole oysters or dissected tissues. In our ...

  16. The entomopathogenic fungus Nomuraea rileyi impairs cellular immunity of its host Helicoverpa armigera.

    Zhong, Ke; Liu, Zhan-Chi; Wang, Jia-Lin; Liu, Xu-Sheng

    2017-09-01

    In this study, we investigated the effect of the entomopathogenic fungus Nomuraea rileyi on Helicoverpa armigera cellular immune responses. Nomuraea rileyi infection had no effect on total hemocyte count (THC), but impaired hemocyte-mediated phagocytosis, nodulation, and encapsulation responses. Nomuraea rileyi infection led to a significant reduction in hemocyte spreading. An in vitro assay revealed that plasma from N. rileyi infected H. armigera larvae suppressed the spreading ability of hemocytes from naïve larvae. We infer that N. rileyi suppresses the cellular immune response of its host, possibly by secreting exogenous, cytotoxic compounds into the host's hemolymph. © 2017 Wiley Periodicals, Inc.

  17. Effects of heavy-ion radiosurgery on the hemopoietic function of the silkworm Bombyx mori

    Tu, Zhen-Li; Kobayashi, Yasuhiko; Watanabe, Hiroshi; Yamamoto, Kazuo [Japan Atomic Energy Research Inst., Takasaki, Gunma (Japan). Takasaki Radiation Chemistry Research Establishment; Kiguchi, Kenji [SHINSHU Univ., Ueda, Nagano (Japan). Faculty of Textile Science and Technology

    2002-09-01

    To study the effects of heavy-ion radiosurgery on the hemopoietic function of a silkworm, hemopoietic organs of larvae were locally irradiated with carbon-ion beams, and the changes in the hemocyte density and in the hemocyte function were investigated. When the larvae were irradiated by 50 Gy to 300 Gy carbon ions on the 3rd day of the 4th instar, the hemocyte densities did not change for a while, though they gradually increased at a later stage, but were finally still significantly lower than those of unirradiated controls. The hemocyte densities of the larvae irradiated at different developmental stages showed suppressed increments, and carbon-ion irradiation given to larvae at early stages compared to the later stages had a significant suppressive effect on the hemocyte densities. On unilateral irradiated larvae a hemocyte intermediate increment between those of bilateral irradiated larvae and unirradiated controls was observed. The percentage of dead hemocytes was obviously higher for irradiated larvae than unirradiated controls during the later 5th instar. Thus, it is evident that carbon-ion radiosurgery on hemopoietic organs of silkworm induced not only a quantitative change, but also a qualitative change in the hemocytes. (author)

  18. Transient expression of protein tyrosine phosphatases encoded in Cotesia plutellae bracovirus inhibits insect cellular immune responses

    Ibrahim, Ahmed M. A.; Kim, Yonggyun

    2008-01-01

    Several immunosuppressive factors are associated with parasitism of an endoparasitoid wasp, Cotesia plutellae, on the diamondback moth, Plutella xylostella. C. plutellae bracovirus (CpBV) encodes a large number of putative protein tyrosine phosphatases (PTPs), which may play a role in inhibiting host cellular immunity. To address this inhibitory hypothesis of CpBV-PTPs, we performed transient expression of individual CpBV-PTPs in hemocytes of the beet armyworm, Spodoptera exigua, and analyzed their cellular immune responses. Two different forms of CpBV-PTPs were chosen and cloned into a eukaryotic expression vector under the control of the p10 promoter of baculovirus: one with the normal cysteine active site (CpBV-PTP1) and the other with a mutated active site (CpBV-PTP5). The hemocytes transfected with CpBV-PTP1 significantly increased in PTP activity compared to control hemocytes, but those with CpBV-PTP5 exhibited a significant decrease in the PTP activity. All transfected hemocytes exhibited a significant reduction in both cell spreading and encapsulation activities compared to control hemocytes. Co-transfection of CpBV-PTP1 together with its double-stranded RNA reduced the messenger RNA (mRNA) level of CpBV-PTP1 and resulted in recovery of both hemocyte behaviors. This is the first report demonstrating that the polydnaviral PTPs can manipulate PTP activity of the hemocytes to interrupt cellular immune responses.

  19. Hematological changes in white spot syndrome virus-infected shrimp, Fenneropenaeus chinensis (Osbeck)

    Feng, Shouming; Zhan, Wenbin; Xing, Jing; Li, Jun; Yang, Kai; Wang, Jing

    2008-08-01

    The pathological changes of hemocytes in the haemolymph and hepatopancreas were examined in experimentally and naturally WSSV (white spot syndrome virus) infected Fenneropenaeus chinensis. The results showed that the pathological manifestations of hemocytes were similar among moribund shrimps infected via injection, feeding and by nature. Firstly, the total hemocyte counts (THCs) in WSSV-infected shrimp were significantly lower than those in healthy shrimp. Secondly, necrotic, broken and disintegrated cells were often observed, and a typical hematolysis was present in the haemolymph smear of WSSV-infected shrimp. Thirdly, necrosis and typical apoptosis of hemocytes were detected with TEM in the peripheral haemolymph of WSSV-infected shrimp. Hyalinocytes and semi-granulocytes with masses of WSSVs in their nuclei often appeared, whereas no granular hemocytes with WSSV were found in the hepatopancreas of moribund infected shrimps. All our results supported that hemocytes were the main target cells of WSSV, and hyalinocytes and semigranular hemocytes seemed to be more favorable for WSSV infection in F. chinensis.

  20. Antimicrobial lipids from the hemolymph of brachyuran crabs

    Ravichandran, S.; Wahidullah, S.; DeSouza, L.; Rameshkumar, G.

    The potential of marine crabs as a source of biologically active products is largely unexplored. In the present study, antimicrobial activity of the hemolymph (plasma) and hemocytes (plasma cells) of six brachyuran crabs was investigated against 16...

  1. Serratia marcescens Suppresses Host Cellular Immunity via the Production of an Adhesion-inhibitory Factor against Immunosurveillance Cells*

    Ishii, Kenichi; Adachi, Tatsuo; Hamamoto, Hiroshi; Sekimizu, Kazuhisa

    2014-01-01

    Injection of a culture supernatant of Serratia marcescens into the bloodstream of the silkworm Bombyx mori increased the number of freely circulating immunosurveillance cells (hemocytes). Using a bioassay with live silkworms, serralysin metalloprotease was purified from the culture supernatant and identified as the factor responsible for this activity. Serralysin inhibited the in vitro attachment of both silkworm hemocytes and murine peritoneal macrophages. Incubation of silkworm hemocytes or murine macrophages with serralysin resulted in degradation of the cellular immune factor BmSPH-1 or calreticulin, respectively. Furthermore, serralysin suppressed in vitro phagocytosis of bacteria by hemocytes and in vivo bacterial clearance in silkworms. Disruption of the ser gene in S. marcescens attenuated its host killing ability in silkworms and mice. These findings suggest that serralysin metalloprotease secreted by S. marcescens suppresses cellular immunity by decreasing the adhesive properties of immunosurveillance cells, thereby contributing to bacterial pathogenesis. PMID:24398686

  2. Serratia marcescens suppresses host cellular immunity via the production of an adhesion-inhibitory factor against immunosurveillance cells.

    Ishii, Kenichi; Adachi, Tatsuo; Hamamoto, Hiroshi; Sekimizu, Kazuhisa

    2014-02-28

    Injection of a culture supernatant of Serratia marcescens into the bloodstream of the silkworm Bombyx mori increased the number of freely circulating immunosurveillance cells (hemocytes). Using a bioassay with live silkworms, serralysin metalloprotease was purified from the culture supernatant and identified as the factor responsible for this activity. Serralysin inhibited the in vitro attachment of both silkworm hemocytes and murine peritoneal macrophages. Incubation of silkworm hemocytes or murine macrophages with serralysin resulted in degradation of the cellular immune factor BmSPH-1 or calreticulin, respectively. Furthermore, serralysin suppressed in vitro phagocytosis of bacteria by hemocytes and in vivo bacterial clearance in silkworms. Disruption of the ser gene in S. marcescens attenuated its host killing ability in silkworms and mice. These findings suggest that serralysin metalloprotease secreted by S. marcescens suppresses cellular immunity by decreasing the adhesive properties of immunosurveillance cells, thereby contributing to bacterial pathogenesis.

  3. Prostaglandin-mediated recovery from bacteriosemia delays larval development in fall armyworms, Spodoptera frugiperda

    Insect immunity includes a surveillance system that detects and signals infections, coupled with hemocytic and humoral immune functions. These functions are signaled and coordinated by several biochemicals, including biogenic amines, insect cytokines, peptides and prostaglandins (PGs). The actions o...

  4. DIFFERENTIAL EFFECTS OF OYSTER (CRASSOSTREA VIRGINICA) DEFENSES ON CLINICAL AND ENVIRONMENTAL ISOLATES OF VIBRIO PARAHEMOLYTICUS

    Three clinical (2030, 2062, and 2107) and three environmental (1094, 1163, and ATCC 17802) isolates of Vibrio parahaemolyticus were exposed to hemocytes and plasma collected from oysters (Crassostrea virginica) to determine their susceptibility to putative oyster defenses. Clinic...

  5. Nitric oxide mediates insect cellular immunity via phospholipase A2 activation

    After infection or invasion is recognized, biochemical mediators act in signaling insect immune functions. These include biogenic amines, insect cytokines, eicosanoids and nitric oxide (NO). Treating insects or isolated hemocyte populations with different mediators often leads to similar results. Se...

  6. IS COPPER REQUIRED FOR EASTERN OYSTER SETTING AND METAMORPHOSIS?

    Recent field research with eastern oysters demonstrated higher defense activities, including hemocyte numbers, locomotion and bactericidal ability, associated with locations exhibiting relatively high contamination. Copper and zinc, found in high concentrations in tissues of oyst...

  7. The Roles of Heat Shock Proteins 70 and 90 in Exopalaemon carinicauda After WSSV and Vibrio anguillarum Challenges

    Li, Jitao; Li, Jian; Duan, Yafei; Chen, Ping; Liu, Ping

    2018-04-01

    Heat shock proteins (HSPs), such as HSP70 and HSP90, are a suite of highly conserved proteins produced in all cellular organisms when they are exposed to stresses. In aquatic animals, they have been proved to play important roles in response to environmental pollutants and particularly in the non-specific immune responses to pathogen infections. In the present study, the expression profiles of HSP70 and HSP90 genes in hemocytes and hepatopancreas from the ridgetail white prawn Exopalaemon carinicauda infected with WSSV and Vibrio anguillarum were detected using reverse transcription polymerase chain reaction (RT-PCR). After WSSV challenge, the expression level of HSP 70 gene transcripts in the hemocytes and hepatopancreas increased to peak level at 6 h and 48 h, respectively. HSP90 gene transcripts in hemocytes and hepatopancreas were up-regulated significantly at 12 h and 6 h, respectively. During V. anguillarum challenge, the mRNA content of HSP70 gene in hemocytes and hepatopancreas increased significantly at 12 h and 6 h post-infection, respectively. The expression level of HSP90 gene both in hemocytes and hepatopancreas were up-regulated in the first 3 h. The expression patterns of HSP70 and HSP90 genes in hemocytes and hepatopancreas showed temporal and spatial differences after challenged with WSSV and V. anguillarum. The results suggested that HSPs might be involved in immune responses to pathogens challenge in E. carinicauda.

  8. Genotoxic and Cytotoxic Effects on the Immune Cells of the Freshwater Bivalve Dreissena polymorpha Exposed to the Environmental Neurotoxin BMAA.

    Lepoutre, Alexandra; Milliote, Nadia; Bonnard, Marc; Palos-Ladeiro, Mélissa; Rioult, Damien; Bonnard, Isabelle; Bastien, Fanny; Faassen, Elisabeth; Geffard, Alain; Lance, Emilie

    2018-03-01

    The environmental neurotoxin β- N -Methylamino-l-alanine (BMAA) has been pointed out to be involved in human neurodegenerative diseases. This molecule is known to be bioaccumulated by bivalves. However, little data about its toxic effects on freshwater mussels is available, particularly on the hemolymphatic compartment and its hemocyte cells involved in various physiological processes such as immune defenses, digestion and excretion, tissue repair, and shell production. Here we exposed Dreissena polymorpha to dissolved BMAA, at the environmental concentration of 7.5 µg of /mussel/3 days, during 21 days followed by 14 days of depuration in clear water, with the objective of assessing the BMAA presence in the hemolymphatic compartment, as well as the impact of the hemocyte cells in terms of potential cytotoxicity, immunotoxicity, and genotoxiciy. Data showed that hemocytes were in contact with BMAA. The presence of BMAA in hemolymph did not induce significant effect on hemocytes phagocytosis activity. However, significant DNA damage on hemocytes occurred during the first week (days 3 and 8) of BMAA exposure, followed by an increase of hemocyte mortality after 2 weeks of exposure. Those effects might be an indirect consequence of the BMAA-induced oxidative stress in cells. However, DNA strand breaks and mortality did not persist during the entire exposure, despite the BMAA persistence in the hemolymph, suggesting potential induction of some DNA-repair mechanisms.

  9. The effect of Beauveria bassiana infection on cell mediated and humoral immune response in house fly, Musca domestica L.

    Mishra, Sapna; Kumar, Peeyush; Malik, Anushree

    2015-10-01

    Entomopathogenic fungi that manifest infections by overcoming insect's immune response could be a successful control agent for the house fly, Musca domestica L. which is a major domestic, medical, and veterinary pest. In this study, the immune response of house fly to Beauveria bassiana infection was investigated to reveal fundamental aspects of house fly hemocyte biology, such as hemocyte numbers and size, which is poorly understood. The total hemocyte counts (THCs) in B. bassiana-infected house fly showed an initial increase (from 6 to 9 h), followed by subsequent decrease (9 to 12 h) with increase in time of infection. The THCs was slightly greater in infected flies than the non-infected ones. Insight into relative hemocyte counts depicted a significant increase in prohemocyte (PR) and decrease in granulocyte (GR) in infected house flies compared to non-infected ones. The relative cell area of hemocyte cells showed a noticeable increase in PR and intermediate cells (ICs), while a considerable reduction was observed for plasmatocyte (PL) and GR. The considerable variation in relative cell number and cell area in the B. bassiana-infected house flies indicated stress development during infection. The present study highlights changes occurring during B. bassiana invasion to house fly leading to establishment of infection along with facilitation in understanding of basic hemocyte biology. The results of the study is expected to help in better understanding of house fly immune response during fungal infection, so as to assist production of more efficient mycoinsecticides for house fly control using B. bassiana.

  10. Genotoxic and Cytotoxic Effects on the Immune Cells of the Freshwater Bivalve Dreissena polymorpha Exposed to the Environmental Neurotoxin BMAA

    Alexandra Lepoutre

    2018-03-01

    Full Text Available The environmental neurotoxin β-N-Methylamino-l-alanine (BMAA has been pointed out to be involved in human neurodegenerative diseases. This molecule is known to be bioaccumulated by bivalves. However, little data about its toxic effects on freshwater mussels is available, particularly on the hemolymphatic compartment and its hemocyte cells involved in various physiological processes such as immune defenses, digestion and excretion, tissue repair, and shell production. Here we exposed Dreissena polymorpha to dissolved BMAA, at the environmental concentration of 7.5 µg of /mussel/3 days, during 21 days followed by 14 days of depuration in clear water, with the objective of assessing the BMAA presence in the hemolymphatic compartment, as well as the impact of the hemocyte cells in terms of potential cytotoxicity, immunotoxicity, and genotoxiciy. Data showed that hemocytes were in contact with BMAA. The presence of BMAA in hemolymph did not induce significant effect on hemocytes phagocytosis activity. However, significant DNA damage on hemocytes occurred during the first week (days 3 and 8 of BMAA exposure, followed by an increase of hemocyte mortality after 2 weeks of exposure. Those effects might be an indirect consequence of the BMAA-induced oxidative stress in cells. However, DNA strand breaks and mortality did not persist during the entire exposure, despite the BMAA persistence in the hemolymph, suggesting potential induction of some DNA-repair mechanisms.

  11. Morula-like cells in photo-symbiotic clams harboring zooxanthellae.

    Nakayama, K; Nishijima, M; Maruyama, T

    1998-06-01

    Symbiosis is observed between zooxanthellae, symbiotic dinoflagellates, and giant clams and related clams which belong to the families Tridacnidae and Cardiidae. We have previously shown that a photo-symbiotic clam Tridacna crocea has three types of hemocytes, the eosinophilic granular hemocyte with phagocytic activity, the agranular cell with electron lucent granules, and the morula-like cell with large (ca. 2 mum in diameter) colorless granules. The function of the morula-like cell is not clear, but it has not been reported in any other bivalves except photo-symbiotic clams T. crocea and Tridacna maxima. In order to clarify whether it is specific to photo-symbiotic clams or not, we studied hemocytes in the photo-symbiotic clams Tridacna derasa (Tridacnidae), Hippopus hippopus (Tridacnidae) and Corculum cardissa (Cardiidae), and a closely related non-symbiotic clam Fulvia mutica (Cardiidae). The eosinophilic granular hemocytes and the agranular cells were found in all of the clams examined. However, the morula-like cells which were packed with many large electron dense granules (ca. 2 mum in diameter), were observed only in the photo-symbiotic clams. In F. mutica, a closely related non-symbiotic clam, this type of hemocyte was not found. Instead a hemocyte with vacuoles and a few large granules containing peroxidase activity was observed. The large granules of F. mutica varied in size from ca. 1-9 mum in diameter. Present data suggests that the presence of morula-like cells is restricted to photo-symbiotic clams and that the hemocytes associated with the morula-like cells may have some functional relationship to symbiosis with zooxanthellae.

  12. Imidacloprid intensifies its impact on honeybee and bumblebee cellular immune response when challenged with LPS (lippopolysacharide) of Escherichia coli.

    Walderdorff, Louise; Laval-Gilly, Philippe; Bonnefoy, Antoine; Falla-Angel, Jaïro

    2018-05-16

    Insect hemocytes play an important role in insects' defense against environmental stressors as they are entirely dependent on their innate immune system for pathogen defense. In recent years a dramatic decline of pollinators has been reported in many countries. The drivers of this declines appear to be associated with pathogen infections like viruses, bacteria or fungi in combination with pesticide exposure. The aim of this study was thus to investigate the impact of imidacloprid, a neonicotinoid insecticide, on the cellular immune response of two pollinators (Apis mellifera and Bombus terrestris) during simultaneous immune activation with LPS (lipopolysaccharide) of Escherichia coli. For this purpose the phagocytosis capacity as well as the production of H 2 O 2 and NO of larval hemocytes, exposed to five different imidacloprid concentrations in vitro, was measured. All used pesticide concentrations showed a weakening effect on phagocytosis with but also without LPS activation. Imidacloprid decreased H 2 O 2 and increased NO production in honeybees. Immune activation by LPS clearly reinforced the effect of imidacloprid on the immune response of hemocytes in all three immune parameters tested. Bumblebee hemocytes appeared more sensitive to imidacloprid during phagocytosis assays while imidacloprid showed a greater impact on honeybee hemocytes during H 2 O 2 and NO production. Copyright © 2018 Elsevier Ltd. All rights reserved.

  13. Hematopoiesis and hematopoietic organs in arthropods.

    Grigorian, Melina; Hartenstein, Volker

    2013-03-01

    Hemocytes (blood cells) are motile cells that move throughout the extracellular space and that exist in all clades of the animal kingdom. Hemocytes play an important role in shaping the extracellular environment and in the immune response. Developmentally, hemocytes are closely related to the epithelial cells lining the vascular system (endothelia) and the body cavity (mesothelia). In vertebrates and insects, common progenitors, called hemangioblasts, give rise to the endothelia and blood cells. In the adult animal, many differentiated hemocytes seem to retain the ability to proliferate; however, in most cases investigated closely, the bulk of hemocyte proliferation takes place in specialized hematopoietic organs. Hematopoietic organs provide an environment where undifferentiated blood stem cells are able to self-renew, and at the same time generate offspring that differentiate into different blood cell types. Hematopoiesis in vertebrates, taking place in the bone marrow, has been subject to intensive research by immunologists and stem cell biologists. Much less is known about blood cell formation in invertebrate animals. In this review, we will survey structural and functional properties of invertebrate hematopoietic organs, with a main focus on insects and other arthropod taxa. We will then discuss similarities, at the molecular and structural level, that are apparent when comparing the development of blood cells in hematopoietic organs of vertebrates and arthropods. Our comparative review is intended to elucidate aspects of the biology of blood stem cells that are more easily missed when focusing on one or a few model species.

  14. Antimicrobial histones and DNA traps in invertebrate immunity: evidences in Crassostrea gigas.

    Poirier, Aurore C; Schmitt, Paulina; Rosa, Rafael D; Vanhove, Audrey S; Kieffer-Jaquinod, Sylvie; Rubio, Tristan P; Charrière, Guillaume M; Destoumieux-Garzón, Delphine

    2014-09-05

    Although antimicrobial histones have been isolated from multiple metazoan species, their role in host defense has long remained unanswered. We found here that the hemocytes of the oyster Crassostrea gigas release antimicrobial H1-like and H5-like histones in response to tissue damage and infection. These antimicrobial histones were shown to be associated with extracellular DNA networks released by hemocytes, the circulating immune cells of invertebrates, in response to immune challenge. The hemocyte-released DNA was found to surround and entangle vibrios. This defense mechanism is reminiscent of the neutrophil extracellular traps (ETs) recently described in vertebrates. Importantly, oyster ETs were evidenced in vivo in hemocyte-infiltrated interstitial tissues surrounding wounds, whereas they were absent from tissues of unchallenged oysters. Consistently, antimicrobial histones were found to accumulate in oyster tissues following injury or infection with vibrios. Finally, oyster ET formation was highly dependent on the production of reactive oxygen species by hemocytes. This shows that ET formation relies on common cellular and molecular mechanisms from vertebrates to invertebrates. Altogether, our data reveal that ET formation is a defense mechanism triggered by infection and tissue damage, which is shared by relatively distant species suggesting either evolutionary conservation or convergent evolution within Bilateria. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

  15. Wash functions downstream of Rho1 GTPase in a subset of Drosophila immune cell developmental migrations

    Verboon, Jeffrey M.; Rahe, Travis K.; Rodriguez-Mesa, Evelyn; Parkhurst, Susan M.

    2015-01-01

    Drosophila immune cells, the hemocytes, undergo four stereotypical developmental migrations to populate the embryo, where they provide immune reconnoitering, as well as a number of non–immune-related functions necessary for proper embryogenesis. Here, we describe a role for Rho1 in one of these developmental migrations in which posteriorly located hemocytes migrate toward the head. This migration requires the interaction of Rho1 with its downstream effector Wash, a Wiskott–Aldrich syndrome family protein. Both Wash knockdown and a Rho1 transgene harboring a mutation that prevents Wash binding exhibit the same developmental migratory defect as Rho1 knockdown. Wash activates the Arp2/3 complex, whose activity is needed for this migration, whereas members of the WASH regulatory complex (SWIP, Strumpellin, and CCDC53) are not. Our results suggest a WASH complex–independent signaling pathway to regulate the cytoskeleton during a subset of hemocyte developmental migrations. PMID:25739458

  16. Relevant principal factors affecting the reproducibility of insect primary culture.

    Ogata, Norichika; Iwabuchi, Kikuo

    2017-06-01

    The primary culture of insect cells often suffers from problems with poor reproducibility in the quality of the final cell preparations. The cellular composition of the explants (cell number and cell types), surgical methods (surgical duration and surgical isolation), and physiological and genetic differences between donors may be critical factors affecting the reproducibility of culture. However, little is known about where biological variation (interindividual differences between donors) ends and technical variation (variance in replication of culture conditions) begins. In this study, we cultured larval fat bodies from the Japanese rhinoceros beetle, Allomyrina dichotoma, and evaluated, using linear mixed models, the effect of interindividual variation between donors on the reproducibility of the culture. We also performed transcriptome analysis of the hemocyte-like cells mainly seen in the cultures using RNA sequencing and ultrastructural analyses of hemocytes using a transmission electron microscope, revealing that the cultured cells have many characteristics of insect hemocytes.

  17. A Drosophila Model to Image Phagosome Maturation

    Douglas A. Brooks

    2013-03-01

    Full Text Available Phagocytosis involves the internalization of extracellular material by invagination of the plasma membrane to form intracellular vesicles called phagosomes, which have functions that include pathogen degradation. The degradative properties of phagosomes are thought to be conferred by sequential fusion with endosomes and lysosomes; however, this maturation process has not been studied in vivo. We employed Drosophila hemocytes, which are similar to mammalian professional macrophages, to establish a model of phagosome maturation. Adult Drosophila females, carrying transgenic Rab7-GFP endosome and Lamp1-GFP lysosome markers, were injected with E. coli DH5α and the hemocytes were collected at 15, 30, 45 and 60 minutes after infection. In wild-type females, E. coli were detected within enlarged Rab7-GFP positive phagosomes at 15 to 45 minutes after infection; and were also observed in enlarged Lamp1-GFP positive phagolysosomes at 45 minutes. Two-photon imaging of hemocytes in vivo confirmed this vesicle morphology, including enlargement of Rab7-GFP and Lamp1-GFP structures that often appeared to protrude from hemocytes. The interaction of endosomes and lysosomes with E. coli phagosomes observed in Drosophila hemocytes was consistent with that previously described for phagosome maturation in human ex vivo macrophages. We also tested our model as a tool for genetic analysis using 14-3-3e mutants, and demonstrated altered phagosome maturation with delayed E. coli internalization, trafficking and/or degradation. These findings demonstrate that Drosophila hemocytes provide an appropriate, genetically amenable, model for analyzing phagosome maturation ex vivo and in vivo.

  18. Uranium concentration by Crustacea: a structural, ultrastructural and microanalytical study by secondary ion emission and electron probe X ray microanalysis

    Chassard-Bouchaud, Colette

    1982-01-01

    Experimental intoxications were performed on the Crayfish Pontastacus leptodactylus using hydrosoluble uranium nitrate. Investigations demonstrate that Crustacea are able to concentrate both uranium main radioactive isotopes 238 U and 235 U within the cuticle, gill epithelium, midgut gland (=hepatopancreas) and macrophagic hemocytes. The storage occurs within nucleus and lysosomal system where uranium is precipitated in the form of an unsoluble phosphate. The proposed hypothesis for the metal extrusion is the following: residual bodies containing the uranium precipitates are extruded into the extracellular space where they are absorbed by phagocytosis, by the macrophagic hemocytes [fr

  19. Evaluation of the immune responses of the brown mussel Perna perna as indicators of fecal pollution.

    Silva Dos Santos, Fernanda; Neves, Raquel Almeida Ferrando; Carvalho, Wanderson Fernandes de; Krepsky, Natascha; Crapez, Mirian Araújo Carlos

    2018-06-01

    The mussel Perna perna is an intertidal bivalve that is widely distributed, cultivated and consumed in South Africa, Brazil and Venezuela. Among marine resources, bivalve mollusks are one of the most impacted by anthropogenic pollution, as they can accumulate pathogenic bacteria and water pollutants. Hemocytes are molluscan defense cells, and their abundance and functions can be affected in response to contaminants, such as bacterial load. However, no previous study has investigated the immune response of P. perna hemocytes. The aim of this study was to evaluate several immune parameters in P. perna as indicators of fecal pollution in mussel hemolymph and in seawater. We collected mussels and adjacent seawater from beaches with different levels of fecal contamination in Rio de Janeiro state (Brazil): Vermelha Beach (VB); Icaraí Beach (IB); Urca Beach (UB); and Jurujuba Beach (JB). Hemocyte parameters (density, morphology, phagocytic activity and production of Reactive Oxygen Species - ROS) were evaluated using flow cytometry. We quantified Fecal Indicator Bacteria (FIB) in seawater by the multiple tubes technique for each beach and for hemolymph by the spread-plate technique. In agreement with historical evaluation of fecal contamination levels, UB presented the highest FIB abundance in seawater (thermotolerant coliforms, TEC = 1600 NMP 100 mL -1 ), whereas VB exhibited the lowest (TEC = 17 NMP 100 mL -1 ). UB mussels had six and eight times higher hemocyte density and phagocytic activity, respectively, than mussels from VB. Mussels from VB and IB presented a significantly lower number of total coliforms in hemolymph and a significantly higher relative internal complexity of hemocytes than those from UB and JB (p ≤ 0.01, PERMANOVA). ROS production by hemocytes was significantly lower in mussels from VB compared to those from JB (p = 0.04, ANOVA). Our results indicate a significant relationship between the level of fecal contamination in

  20. Reexamination of the role of hematopoietic organs on the ...

    STORAGESEVER

    2009-08-04

    Aug 4, 2009 ... Sciences, 300 Fenglin Rd. Shanghai 200032, P.R. China. ... Key words: Hematopoietic organ, wing disc, hemocytes, surgical operation, silkworm, ... They were reared on artificial diet at 25°C under a 16 h ..... The image was.

  1. Clearance of bacteria and differential involvement of mussel hyalinocytes, small and large granulocytes in antibacterial immunity

    Jouvet, Lionel

    2008-01-01

    injected into the circulation of the mussel, the number of living intra-hemocyte bacteria dramatically increased in less than an hour, suggesting intense phagocytosis, then gradually decreased, with no viable bacteria remaining 12h post-injection for Micrococcus lysodeikticus, 24h for Vibrio splendidus...

  2. Persistence of enteric viruses within oysters (Crassostrea virginica)

    It is well known that water-borne enteric viruses are concentrated by bivalves. Why these viruses are selectively retained and remain infectious within shellfish tissues for extended periods is unknown. Our current hypothesis is that phagocytic hemocytes (blood cells) are a site of virus persiste...

  3. Serratia marcescens Induces Apoptotic Cell Death in Host Immune Cells via a Lipopolysaccharide- and Flagella-dependent Mechanism*

    Ishii, Kenichi; Adachi, Tatsuo; Imamura, Katsutoshi; Takano, Shinya; Usui, Kimihito; Suzuki, Kazushi; Hamamoto, Hiroshi; Watanabe, Takeshi; Sekimizu, Kazuhisa

    2012-01-01

    Injection of Serratia marcescens into the blood (hemolymph) of the silkworm, Bombyx mori, induced the activation of c-Jun NH2-terminal kinase (JNK), followed by caspase activation and apoptosis of blood cells (hemocytes). This process impaired the innate immune response in which pathogen cell wall components, such as glucan, stimulate hemocytes, leading to the activation of insect cytokine paralytic peptide. S. marcescens induced apoptotic cell death of silkworm hemocytes and mouse peritoneal macrophages in vitro. We searched for S. marcescens transposon mutants with attenuated ability to induce apoptosis of silkworm hemocytes. Among the genes identified, disruption mutants of wecA (a gene involved in lipopolysaccharide O-antigen synthesis), and flhD and fliR (essential genes in flagella synthesis) showed reduced motility and impaired induction of mouse macrophage cell death. These findings suggest that S. marcescens induces apoptosis of host immune cells via lipopolysaccharide- and flagella-dependent motility, leading to the suppression of host innate immunity. PMID:22859304

  4. Environmental and human risk assessment of landfill leachate: An integrated approach with the use of cytotoxic and genotoxic stress indices in mussel and human cells

    Toufexi, Eirini; Tsarpali, Vasiliki [Section of Animal Biology, Department of Biology, School of Natural Sciences, University of Patras, GR 26500 Patras (Greece); Efthimiou, Ioanna; Vidali, Maria-Sophia; Vlastos, Dimitris [Department of Environmental and Natural Resources Management, University of Patras, 2 Seferi Str., GR 30100 Agrinio (Greece); Dailianis, Stefanos, E-mail: sdailianis@upatras.gr [Section of Animal Biology, Department of Biology, School of Natural Sciences, University of Patras, GR 26500 Patras (Greece)

    2013-09-15

    Highlights: • Landfill leachate poses a threat for aquatic biota and humans. • Leachate induces cytotoxic and oxidative effects on mussel hemocytes. • Increased levels of DNA damage were observed both in vivo and in vitro in hemocytes. • Leachate low doses enhance MN formation in human lymphocyte cultures. • Potential leachate aneugenic activity was detected in human lymphocytes. -- Abstract: The present study investigates leachate hazardous effects on marine biota and human cells, with the use of a battery of assays, both under in vivo and in vitro conditions. According to the results, mussels exposed for 4 days to 0.01 and 0.1% (v/v) of leachate showed increased levels of DNA damage and micronuclei (MN) frequencies in their hemocytes. Similarly, enhanced levels of DNA damage were also observed in hemocytes treated in vitro with relevant concentrations of leachate, followed by a significant enhancement of both superoxide anions (·O{sub 2}{sup −}) and lipid peroxidation products (malondialdehyde/MDA). On the other hand, human lymphocyte cultures treated with such a low concentrations of leachate (0.1, 0.2 and 1%, v/v), showed increased frequencies of MN formation and large MN size ratio, as well as decreased cell proliferation, as indicated by the use of the cytokinesis block micronucleus (CBMN) assay and Cytokinesis Block Proliferation Index (CBPI) respectively. These findings showed the clear-cut genotoxic and cytotoxic effects of leachate on both cellular types, as well as its potential aneugenic activity in human lymphocytes.

  5. Genome-wide transcriptional analysis of Drosophila larvae infected by entomopathogenic nematodes shows involvement of complement, recognition and extracellular matrix proteins

    Arefin, B.; Kučerová, Lucie; Dobeš, P.; Markus, R.; Strnad, H.; Wang, Z.; Hyršl, P.; Žurovec, Michal; Theopold, U.

    2014-01-01

    Roč. 6, č. 2 (2014), s. 192-204 ISSN 1662-811X EU Projects: European Commission(XE) 316304 - MODBIOLIN Institutional support: RVO:60077344 Keywords : basement membrane * coagulation * hemocyte Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 4.352, year: 2014

  6. Environmental and human risk assessment of landfill leachate: An integrated approach with the use of cytotoxic and genotoxic stress indices in mussel and human cells

    Toufexi, Eirini; Tsarpali, Vasiliki; Efthimiou, Ioanna; Vidali, Maria-Sophia; Vlastos, Dimitris; Dailianis, Stefanos

    2013-01-01

    Highlights: • Landfill leachate poses a threat for aquatic biota and humans. • Leachate induces cytotoxic and oxidative effects on mussel hemocytes. • Increased levels of DNA damage were observed both in vivo and in vitro in hemocytes. • Leachate low doses enhance MN formation in human lymphocyte cultures. • Potential leachate aneugenic activity was detected in human lymphocytes. -- Abstract: The present study investigates leachate hazardous effects on marine biota and human cells, with the use of a battery of assays, both under in vivo and in vitro conditions. According to the results, mussels exposed for 4 days to 0.01 and 0.1% (v/v) of leachate showed increased levels of DNA damage and micronuclei (MN) frequencies in their hemocytes. Similarly, enhanced levels of DNA damage were also observed in hemocytes treated in vitro with relevant concentrations of leachate, followed by a significant enhancement of both superoxide anions (·O 2 − ) and lipid peroxidation products (malondialdehyde/MDA). On the other hand, human lymphocyte cultures treated with such a low concentrations of leachate (0.1, 0.2 and 1%, v/v), showed increased frequencies of MN formation and large MN size ratio, as well as decreased cell proliferation, as indicated by the use of the cytokinesis block micronucleus (CBMN) assay and Cytokinesis Block Proliferation Index (CBPI) respectively. These findings showed the clear-cut genotoxic and cytotoxic effects of leachate on both cellular types, as well as its potential aneugenic activity in human lymphocytes

  7. Avian Schistosomes and Outbreaks of Cercarial Dermatitis

    Horák, P.; Mikeš, L.; Lichtenbergová, L.; Skála, V.; Soldánová, Miroslava; Brant, S. V.

    2015-01-01

    Roč. 28, č. 1 (2015), s. 165-190 ISSN 0893-8512 Institutional support: RVO:60077344 Keywords : fresh water snails * Lymnae stagnalis hemocytes * Trichobilharzia regenti schistosomatidae Subject RIV: FN - Epidemiology, Contagious Diseases ; Clinical Immunology Impact factor: 16.187, year: 2015

  8. MicroRNA-100 is involved in shrimp immune response to white spot syndrome virus (WSSV) and Vibrio alginolyticus infection.

    Wang, Zhi; Zhu, Fei

    2017-02-09

    In this study, we discovered that shrimp miR-100 was up-regulated at 24 h after WSSV or Vibrio alginolyticus infection, confirming its participation in the innate immune system of shrimp. The anti-miRNA oligonucleotide (AMO-miR-100) was applied to inhibit the expression of miR-100. After AMO-miR-100 treatment, the shrimp was challenged with WSSV or V. alginolyticus. The knockdown of miR-100 expression decreased the mortality of WSSV-infected shrimp from 24 h to 72 h post-infection and enhanced the mortality of V. alginolyticus-infected shrimp significantly. The knockdown of miR-100 affected phenoloxidase (PO) activity, superoxide dismutase (SOD) activity and total hemocyte count (THC) after the infection with WSSV or V. alginolyticus, indicating a regulative role of miR-100 in the immune potential of shrimp in the response to WSSV or V. alginolyticus infection. The knockdown of miR-100 induced the apoptosis of shrimp hemocytes, and V. alginolyticus + AMO-miR-100 treatment caused more hemocyte apoptosis than V. alginolyticus treatment. The miR-100 influenced also the morphology of shrimp hemocytes and regulated the phagocytosis of WSSV or V. alginolyticus. Thus, we concluded that miR-100 may promote the anti-Vibrio immune response of shrimp through regulating apoptosis, phagocytosis and PO activity and affects the progression of WSSV infection at a certain level.

  9. Immunotoxicity in green mussels under perfluoroalkyl substance (PFAS) exposure: Reversible response and response model development.

    Liu, Changhui; Gin, Karina Yew-Hoong

    2018-04-01

    The immunotoxicity of 4 commonly detected perfluoroalkyl substances (PFASs), namely, perfluorooctanesulfonate (PFOS), perfluorooctanoic acid (PFOA), perfluorononanoic acid (PFNA), and perfluorodecanoic acid (PFDA) was investigated by measuring biomarkers of the immune profile of green mussels, Perna viridis. The biomarkers included neutral red retention, phagocytosis, and spontaneous cytotoxicity, all of which were tested on mussel hemocytes. Hemocytes are an important component of the invertebrate immune system. We found that exposure to PFASs could lead to reduced hemocyte cell viability and suppress immune function by up to 50% of normal performance within the experimental exposure range. The results indicate that PFASs have an immunotoxic potential and thus could pose severe health risks to aquatic organisms. The reported immunotoxicity is likely to result from the compounds' direct and indirect interactions with the hemocyte membrane, and therefore likely to affect the functionality of these cells. The immunotoxic response was found to be related to the organism's burden of PFASs, and was reversible when the compounds were removed from the test organisms. Based on this relationship, models using an organism's PFAS concentration and bioaccumulation factor (BAF) as the independent variables were established to quantify PFAS-induced immunotoxicity. The models help us to gain a better understanding of the toxic mechanism of PFASs, and provide a tool to evaluate adverse effects for the whole group of compounds with one mathematical equation. Environ Toxicol Chem 2018;37:1138-1145. © 2018 SETAC. © 2018 SETAC.

  10. Investigation of microgravity effects on basic imune functions on the cellular level - The TRIPLELUX-B experiment

    Unruh, Eckehardt; Hansen, Peter-Diedrich

    Hemocytes are the primary defence of the Blue Mussel against invading microorganisms and foreign particles. The hemocytes of mussels as part of the immune system of invertebrates has not been studied so far in space. The choice of the phagocytes from invertebrates is justified by the claim to study the universal validity of innate immune responses. The hemocytes of mussels have a lot in common with macrophages of higher organisms. They are able to detect the presence of microorganisms and kill these microorganisms by phagocytosis. The phagocy-tosis related production of ROS will be stimulated with opsonised zymosan. The hemocytes will be stored frozen and reconstituted in-flight for the experiment. The signals of the im-muno cellular responses are translated into luminescence as a rapid optical reporter system. The primary aim of Triplelux B is to investigate under space flight conditions the effect of microgravity on the ability of isolated Blue Mussel hemocytes to perform phagocytosis. As a secondery objectiv, the results expected will allow to conclude whether the observed responses are caused by microgravity and/or radiation (change in permeability, endpoints in genotoxicity: DNA unwinding). The TRIPLELUX-B Experiment contributes to risk assessment concerning immunotoxicity under space flight conditions. The components of the fully automated AEC (Advanced Experimental Containment) will be demonstrated. The AEC of the TRIPLELUX-B experiment will contribute to a real time operational monitoring for immunotoxicity testing for earth. Blue mussels have been used repeatedly for monitoring imunotoxicity and genotoxicity in coastal waters. Based on the AEC an automatet measuring device will allow "real time monitoring" providing observations of immunotoxicity in coastal and inland waters.

  11. A shell-formation related carbonic anhydrase in Crassostrea gigas modulates intracellular calcium against CO2 exposure: Implication for impacts of ocean acidification on mollusk calcification.

    Wang, Xiudan; Wang, Mengqiang; Jia, Zhihao; Song, Xiaorui; Wang, Lingling; Song, Linsheng

    2017-08-01

    Ocean acidification (OA) could decrease the shells and skeletons formation of mollusk by reducing the availability of carbonate ions at calcification sites. Carbonic anhydrases (CAs) convert CO 2 to HCO 3 - and play important roles in biomineralization process from invertebrate to vertebrate. In the present study, a CA (designated as CgCA) was identified and characterized in Pacific oyster C. gigas. The cDNA of CgCA was of 927bp encoding a predicted polypeptide of 308 amino acids with a signal peptide and a CA catalytic function domain. The mRNA transcripts of CgCA were constitutively expressed in all tested tissues with the highest levels in mantle and hemocytes. During the early development period, the mRNA transcripts of CgCA could be detected in all the stages with the highest level in D-veliger larvae. Elevated CO 2 increased the mRNA transcripts of CgCA in muscle, mantle, hepatopancreas, gill and hemocytes significantly (p<0.05) and induced the translocation of CgCA in hemocytes and mantle. Moreover, elevated CO 2 also caused the decrease of intracellular Ca 2+ in hemocytes (p<0.05). The inhibition of CA by acetazolamide and suppression of CgCA gene via RNA interference could increase the intracellular Ca 2+ in hemocytes (p<0.05). Besides, the decrease of intracellular Ca 2+ content caused by Ca 2+ reagent ionomycin could affect localization of CgCA in mantle tissue. The results indicated CgCA played essential roles in calcification and elevated CO 2 accelerated the mutual modulation between calcium and CgCA, implying reduced calcification rate and dissolved shells under OA. Copyright © 2017 Elsevier B.V. All rights reserved.

  12. Differential Protein Expression in the Hemolymph of Bithynia siamensis goniomphalos Infected with Opisthorchis viverrini.

    Kulwadee Suwannatrai

    2016-11-01

    Full Text Available Bithynia siamensis goniomphalos is a freshwater snail that serves as the first intermediate host of the human liver fluke Opisthorchis viverrini. This parasite is a major public health problem in different countries throughout the Greater Mekong sub-region (Thailand, southern Vietnam, Lao PDR and Cambodia. Chronic O. viverrini infection also results in a gradual increase of fibrotic tissues in the biliary tract that are associated with hepatobiliary diseases and contribute to cholangiocarcinoma (a fatal type of bile duct cancer. Infectivity of the parasite in the snail host is strongly correlated with destruction of helminths by the snail's innate immune system, composed of cellular (hemocyte and humoral (plasma defense factors. To better understand this important host-parasite interface we applied sequential window acquisition of all theoretical spectra mass spectrometry (SWATH-MS to identify and quantify the proteins from the hemolymph of B. siamensis goniomphalos experimentally infected with O. viverrini and compare them to non-infected snails (control group. A total of 362 and 242 proteins were identified in the hemocytes and plasma, respectively. Of these, 145 and 117 proteins exhibited significant differences in expression upon fluke infection in hemocytes and plasma, respectively. Among the proteins with significantly different expression patterns, we found proteins related to immune response (up-regulated in both hemocyte and plasma of infected snails and proteins belonging to the structural and motor group (mostly down-regulated in hemocytes but up-regulated in plasma of infected snails. The proteins identified and quantified in this work will provide important information for the understanding of the factors involved in snail defense against O. viverrini and might facilitate the development of new strategies to control O. viverrini infection in endemic areas.

  13. Transcriptional analysis of disk abalone (Haliotis discus discus) antioxidant enzymes against marine bacteria and virus challenge.

    De Zoysa, Mahanama; Whang, Ilson; Nikapitiya, Chamilani; Oh, Chulhong; Choi, Cheol Young; Lee, Jehee

    2011-07-01

    Diverse antioxidant enzymes are essential for marine organisms to overcome oxidative stress as well as for the fine-tuning of immune reactions through activating different signal transduction pathways. This study describes the transcriptional analysis of antioxidant enzymes of disk abalone by challenging with bacteria (Vibrio alginolyticus, Vibrio parahemolyticus, and Listeria monocytogenes) and viral hemorrhagic septicemia virus (VHSV). Upon bacteria and VHSV challenge, Manganese superoxide dismutase (MnSOD), Copper, Zinc superoxide dismutase (CuZnSOD), catalase, thioredoxin peroxidase (TPx), Selenium-dependent glutathione peroxidase (SeGPx), and thioredoxin-2 (TRx-2) expression levels were altered in gills, and hemocytes at different magnitudes. In gills, only MnSOD, catalase, and SeGPx genes were completely upregulated by post-challenge of bacterial and VHSV. Among them, SeGPx demonstrated strong upregulation by 16-fold (bacteria) and 2-fold (VHSV) in gills, and 5-fold (bacteria) and 3.0-fold (VHSV) in hemocytes. None of the genes examined were downregulated (in gills and hemocytes) by bacteria challenge even though CuZnSOD and TPx showed downregulation (completely) in hemocytes by VHSV. In general, abalone hemocytes had lower potential to induce antioxidant enzyme transcripts upon bacteria and VHSV challenge than gills. Based upon these results, we suggest that abalones induce oxidative stress in tissues during the bacteria and VHSV challenge, and the identified response of antioxidant enzymes could be supported for maintaining a low-level of reactive oxygen species (ROS) that may serve as a signal for activating immune reactions against pathogenic conditions. Copyright © 2011 Elsevier Ltd. All rights reserved.

  14. The crustin-like peptide plays opposite role in shrimp immune response to Vibrio alginolyticus and white spot syndrome virus (WSSV) infection.

    Sun, Baozhen; Wang, Zhi; Zhu, Fei

    2017-07-01

    Crustin is an antimicrobial peptide (AMP) that plays a key role in innate immunity of crustaceans. In this study, we cloned the entire 660 bp crustin-like sequence with a 507 bp open reading frame encoding a 168 amino acid from Marsupenaeus japonicus. The crustin-like gene was primarily expressed in gills and over-expressed in shrimp hemocytes after challenge with WSSV or Vibrio alginolyticus. After knockdown crustin-like gene using specific double-stranded RNA (CRU-dsRNA), IMD, Rab7, L-lectin, mitogen-activated protein kinase, p53, prophenoloxidase and Rho were down-regulated and nitric oxide synthase, myosin and tumor necrosis factor-α were up-regulated in hemocytes at 24 h post dsRNA treatment. After WSSV challenge, The mortality, WSSV copy number and expressions of WSSV immediate early genes (IE1, IE2, DNA polymerase, VP28) were both decreased but the apoptosis rate was increased in CRU-dsRNA-treated shrimps, indicating that WSSV may take advantage of crustin-like to benefit its replication. After silenced the crustin-like, the results of phagocytosis showed that the phagocytic rate of shrimp hemocytes on WSSV decreased significantly. In contrast, the absence of crustin-like in shrimps increased the mortality following V. alginolyticus challenge, indicating that crustin-like may play a positive role in the antibacterial process. The phagocytosis experiment showed there was a higher phagocytosis rate of hemocytes after CRU-dsRNA treatment. The result indicated that V. alginolyticus may be able to use crustin-like to avoid phagocytosis of shrimp hemocytes. These results further added to our understanding of the function of crustin-like peptide and also provided its potential role in innate immunity in shrimp. Copyright © 2017 Elsevier Ltd. All rights reserved.

  15. HMGB in mollusk Crassostrea ariakensis Gould: structure, pro-inflammatory cytokine function characterization and anti-infection role of its antibody.

    Ting Xu

    Full Text Available BACKGROUND: Crassostrea ariakensis Gould is a representative bivalve species and an economically important oyster in China, but suffers severe mortalities in recent years that are caused by rickettsia-like organism (RLO. Prevention and control of this disease is a priority for the development of oyster aquaculture. It has been proven that mammalian HMGB (high mobility group box can be released extracellularly and acts as an important pro-inflammatory cytokine and late mediator of inflammatory reactions. In vertebrates, HMGB's antibody (anti-HMGB has been shown to confer significant protection against certain local and systemic inflammatory diseases. Therefore, we investigated the functions of Ca-HMGB (oyster HMGB and anti-CaHMGB (Ca-HMGB's antibody in oyster RLO/LPS (RLO or LPS-induced disease or inflammation. METHODOLOGY/PRINCIPAL FINDINGS: Sequencing analysis revealed Ca-HMGB shares conserved structures with mammalians. Tissue-specific expression indicates that Ca-HMGB has higher relative expression in hemocytes. Significant continuous up-regulation of Ca-HMGB was detected when the hemocytes were stimulated with RLO/LPS. Recombinant Ca-HMGB protein significantly up-regulated the expression levels of some cytokines. Indirect immunofluorescence study revealed that Ca-HMGB localized both in the hemocyte nucleus and cytoplasm before RLO challenge, but mainly in the cytoplasm 12 h after challenge. Western blot analysis demonstrated Ca-HMGB was released extracellularly 4-12 h after RLO challenge. Anti-CaHMGB was added to the RLO/LPS-challenged hemocyte monolayer and real-time RT-PCR showed that administration of anti-CaHMGB dramatically reduced the rate of RLO/LPS-induced up-regulation of LITAF at 4-12 h after treatment. Flow cytometry analysis indicated that administration of anti-CaHMGB reduced RLO/LPS-induced hemocyte apoptosis and necrosis rates. CONCLUSIONS/SIGNIFICANCE: Ca-HMGB can be released extracellularly and its subcellular localization

  16. Caracterização dos tipos celulares presentes na hemolinfa de larvas e ninfas de Rhipicephalus sanguineus (Latreille (Ixodoidea, Ixodidae em diferentes estados nutricionais Characterization of the celular types present in the Haemolymph of larvae and nimphs of Rhipicephalus sanguineus (Latreille (Ixodoidea, Ixodidae in different nutritional stages

    Maria Elisa Carneiro

    1996-01-01

    Full Text Available With the purpose of characterize morphologically the hemocytes of larvae and nymphs of Rhipicephalus sanguineus on different nutritional phases, it was obtained samples of haemolymph were obtained by sectioning the forelegs and collecting the drop there formed. After dried, the samples were fixed by methanol and stained by Giemsa. Microscopical observation resulted in the characterization of five basic cellular types: prohemocytes, plasmatocytes, granulocytes, spherulocytes and oenocytoids. Moreover, undefined cell types, whose morphological patterns didn't have correlation with the characteristics cited for hemocytes, were found in low frequency. The change in the relative composition of the haemolimph was characterized by decrease of the number of granulocytes and greater variabilily of the cell types present in the hemolymph as the tick evolved. This fact may be linked to the alterations that these cells suffer along the development of the tick.

  17. Effect of inactive yeast cell wall on growth performance, survival rate and immune parameters in Pacific White Shrimp (Litopenaeus vannamei

    Rutchanee Chotikachinda

    2008-10-01

    Full Text Available Effects of dietary inactive yeast cell wall on growth performance, survival rate, and immune parameters in pacific white shrimp (Litopenaeus vannamei was investigated. Three dosages of inactive yeast cell wall (0, 1, and 2 g kg-1 were tested in three replicate groups of juvenile shrimps with an average initial weight of 7.15±0.05 g for four weeks. There was no significant difference in final weight, survival rate, specific growth rate, feed conversion ratio, feed intake, protein efficiency ratio, and apparent net protein utilization of each treatments. However, different levels of inactive yeast cell wall showed an effect on certain immune parameters (p<0.05. Total hemocyte counts, granular hemocyte count, and bacterial clearance were better in shrimp fed diets supplemented with 1 and 2 g kg-1 inactive yeast cell wall as compared with thecontrol group.

  18. Immunosuppression in Honeybee Queens by the Neonicotinoids Thiacloprid and Clothianidin.

    Brandt, Annely; Grikscheit, Katharina; Siede, Reinhold; Grosse, Robert; Meixner, Marina Doris; Büchler, Ralph

    2017-07-05

    Queen health is crucial to colony survival of honeybees, since reproduction and colony growth rely solely on the queen. Queen failure is considered a relevant cause of colony losses, yet few data exist concerning effects of environmental stressors on queens. Here we demonstrate for the first time that exposure to field-realistic concentrations of neonicotinoid pesticides can severely affect the immunocompetence of queens of western honeybees (Apis mellifera L.). In young queens exposed to thiacloprid (200 µg/l or 2000 µg/l) or clothianidin (10 µg/l or 50 µg/l), the total hemocyte number and the proportion of active, differentiated hemocytes was significantly reduced. Moreover, functional aspects of the immune defence namely the wound healing/melanisation response, as well as the antimicrobial activity of the hemolymph were impaired. Our results demonstrate that neonicotinoid insecticides can negatively affect the immunocompetence of queens, possibly leading to an impaired disease resistance capacity.

  19. Cellular Immune Reactions of the Sunn Pest, Eurygaster integriceps, to the Entomopathogenic Fungus, Beauveria bassiana and Its Secondary Metabolites

    Zibaee, Arash; Bandani, Ali Reza; Talaei-Hassanlouei, Reza; Malagoli, Davide

    2011-01-01

    In this study, five morphological types of circulating hemocytes were recognized in the hemolymph of the adult sunn pest, Eurygaster integriceps Puton (Hemiptera: Scutelleridae), namely prohemocytes, plasmatocytes, granulocytes, adipohemocytes, and oenocytoids. The effects of the secondary metabolites of the entomopathogenic fungus Beauveria bassiana on cellular immune defenses of Eurygaster integriceps were investigated. The results showed that the fungal secondary metabolites inhibited phagocytic activity of E. integriceps hemocytes and hampered nodule formation. A reduction of phenoloxidase activity was also observed. The data suggest that B. bassiana produce secondary metabolites that disable several immune mechanisms allowing the fungus to overcome and then kill its host. This characteristic makes B. bassiana a promising model for biological control of insect pests such as E. integriceps. PMID:22233481

  20. The dipteran parasitoid Exorista bombycis induces pro- and anti-oxidative reactions in the silkworm Bombyx mori: Enzymatic and genetic analysis.

    Makwana, Pooja; Pradeep, Appukuttan Nair R; Hungund, Shambhavi P; Ponnuvel, Kangayam M; Trivedy, Kanika

    2017-02-01

    Hymenopteran parasitoids inject various factors including polydnaviruses along with their eggs into their host insects that suppress host immunity reactions to the eggs and larvae. Less is known about the mechanisms evolved in dipteran parasitoids that suppress host immunity. Here we report that the dipteran, Exorista bombycis, parasitization leads to pro-oxidative reactions and activation of anti-oxidative enzymes in the silkworm Bombyx mori larva. We recorded increased activity of oxidase, superoxide dismutase, thioredoxin peroxidase, catalase, glutathione-S-transferase (GST), and peroxidases in the hemolymph plasma, hemocytes, and fat body collected from B. mori after E. bombycis parasitization. Microarray and qPCR showed differential expression of genes encoding pro- and anti-oxidant enzymes in the hemocytes. The significance of this work lies in increased understanding of dipteran parasitoid biology. © 2017 Wiley Periodicals, Inc.

  1. Isolation and risk assessment of Geotrichum spp. in the white shrimp (Litopenaeus vannamei Boone, 1931 from culture ponds

    José Luis Ochoa

    2015-09-01

    Full Text Available The present study was done in order to identify the fungus invading some of the supralittoral ponds used for shrimp aquaculture in the CIBNOR facilities in La Paz, Baja California Sur (BCS, México during the summer season. From the walls and bottoms of the ponds, two strains of Geotrichum spp. were isolated and morphologically identified. Fungal adhesion towards hemocytes and primary cultures of various white shrimp (Litopeneaus vannamei tissues (gill, tegument, and gut was analyzed to determine infectivity. Extracellular protease, lipase, and amylase activity were evaluated as virulence factors. Survival of shrimp post-larvae (PL8 exposed to fungal culture supernatant or to their filaments was also investigated. The results showed that shrimp tegument cells and hemocytes were very susceptible to Geotrichum spp. invasion, and that this fungus provokes great mortality of post-larvae. Hence, Geotrichum spp. could be considered an opportunistic pathogen that might represent a serious health risk to shrimp in culture.

  2. Molecular cloning of Kazal-type proteinase inhibitor of the shrimp Fenneropenaeus chinensis.

    Kong, Hee Jeong; Cho, Hyun Kook; Park, Eun-Mi; Hong, Gyeong-Eun; Kim, Young-Ok; Nam, Bo-Hye; Kim, Woo-Jin; Lee, Sang-Jun; Han, Hyon Sob; Jang, In-Kwon; Lee, Chang Hoon; Cheong, Jaehun; Choi, Tae-Jin

    2009-01-01

    Proteinase inhibitors play important roles in host defence systems involving blood coagulation and pathogen digestion. We isolated and characterized a cDNA clone for a Kazal-type proteinase inhibitor (KPI) from a hemocyte cDNA library of the oriental white shrimp Fenneropenaeus chinensis. The KPI gene consists of three exons and two introns. KPI cDNA contains an open reading frame of 396 bp, a polyadenylation signal sequence AATAAA, and a poly (A) tail. KPI cDNA encodes a polypeptide of 131 amino acids with a putative signal peptide of 21 amino acids. The deduced amino acid sequence of KPI contains two homologous Kazal domains, each with six conserved cysteine residues. The mRNA of KPI is expressed in the hemocytes of healthy shrimp, and the higher expression of KPI transcript is observed in shrimp infected with the white spot syndrome virus (WSSV), suggesting a potential role for KPI in host defence mechanisms.

  3. Cellular and biochemical responses of the oyster Crassostrea gigas to controlled exposures to metals and Alexandrium minutum

    Haberkorn, Hansy; Lambert, Christophe; Le Goïc, Nelly [Laboratoire des Sciences de l‘Environnement Marin, UMR 6539, Institut Universitaire Européen de la Mer, Université de Bretagne Occidentale, Place Copernic, Technopôle Brest-Iroise, 29280 Plouzané (France); Quéré, Claudie [IFREMER Centre de Brest, Laboratoire de Physiologie des Invertébrés, Unité Physiologie Fonctionnelle des Organismes Marins, BP 70, 29280 Plouzané (France); Bruneau, Audrey; Riso, Ricardo; Auffret, Michel [Laboratoire des Sciences de l‘Environnement Marin, UMR 6539, Institut Universitaire Européen de la Mer, Université de Bretagne Occidentale, Place Copernic, Technopôle Brest-Iroise, 29280 Plouzané (France); Soudant, Philippe, E-mail: Philippe.Soudant@univ-brest.fr [Laboratoire des Sciences de l‘Environnement Marin, UMR 6539, Institut Universitaire Européen de la Mer, Université de Bretagne Occidentale, Place Copernic, Technopôle Brest-Iroise, 29280 Plouzané (France)

    2014-02-15

    Highlights: •Oysters, C. gigas, were exposed to both metals and PST-producer A. minutum. •Oysters exposed to metals accumulated about thirty-six times less PSTs. •Exposure to both metals and A. minutum induced antagonistic or synergetic effects. -- Abstract: Effects of simultaneous exposure of Pacific oyster, Crassostrea gigas, to both a harmful dinoflagellate that produces Paralytic Shellfish Toxins (PST), Alexandrium minutum, and cadmium (Cd) and copper (Cu), were assessed. Oysters were exposed to a mix of Cd–Cu with two different diets (i.e. A. minutum or Tisochrysis lutea) and compared to control oysters fed A. minutum or T. lutea, respectively, without metal addition. Metals and PST accumulations, digestive gland lipid composition, and cellular and biochemical hemolymph variables were measured after 4 days of exposure. Oysters exposed to Cd–Cu accumulated about thirty-six times less PSTs than oysters exposed to A. minutum alone. Exposure to Cd–Cu induced significant changes in neutral lipids (increase in diacylglycerol – DAG – and decrease in sterols) and phospholipids (decreases in phosphatidylcholine, phosphatidylethanolamine, cardiolipin and ceramide aminoethylphosphonate) of digestive gland suggesting that lipid metabolism disruptions and/or lipid peroxidation have occurred. Simultaneously, concentrations, percentages of dead cells and phenoloxidase activity of hemocytes increased in oysters exposed to metals while reactive oxygen species production of hemocytes decreased. Feeding on the harmful dinoflagellate A. minutum resulted in significant decreases in monoacylglycerol (MAG) and DAG and ether glycerides (EG), as well as significant increases in hemocyte concentration and phagocytic activity as compared to oysters fed T. lutea. Finally, the present study revealed that short-term, simultaneous exposure to Cd–Cu and A. minutum may induce antagonistic (i.e. hemocyte concentration and phagocytosis) or synergic (i.e. DAG content in

  4. Insect immunology and hematopoiesis

    Hillyer, Julián F.

    2015-01-01

    Insects combat infection by mounting powerful immune responses that are mediated by hemocytes, the fat body, the midgut, the salivary glands and other tissues. Foreign organisms that have entered the body of an insect are recognized by the immune system when pathogen-associated molecular patterns bind host-derived pattern recognition receptors. This, in turn, activates immune signaling pathways that amplify the immune response, induce the production of factors with antimicrobial activity, and...

  5. deep-orange and carnation define distinct stages in late endosomal biogenesis in Drosophila melanogaster

    Sriram, V.; Krishnan, K.S.; Mayor, Satyajit

    2003-01-01

    Endosomal degradation is severely impaired in primary hemocytes from larvae of eye color mutants of Drosophila. Using high resolution imaging and immunofluorescence microscopy in these cells, products of eye color genes, deep-orange (dor) and carnation (car), are localized to large multivesicular Rab7-positive late endosomes containing Golgi-derived enzymes. These structures mature into small sized Dor-negative, Car-positive structures, which subsequently fuse to form tubular lysosomes. Defec...

  6. Molecular characterization of shrimp harbinger transposase derived 1 (HARBI1)-like and its role in white spot syndrome virus and Vibrio alginolyticus infection.

    Sun, Baozhen; Qian, Xiyi; Zhu, Fei

    2018-07-01

    The role of the nuclease, HARBI1-like protein (mjHARBI1-like) in the innate immunity of Marsupenaeus japonicus was explored in this study. The 1361 bp cDNA sequence of mjHARBI1-like was cloned from M. japonicus using RACE. RT-qPCR analysis results showed that the gills and hepatopancreas of M. japonicus were the main tissues where mjHARBI1-like is expressed. In addition, it was also found that white spot syndrome virus (WSSV) or Vibrio alginolyticus challenge could stimulate mjHARBI1-like expression. After mjHARBI1-likewas inhibited, expression of immune genes such as toll, p53, myosin, and proPO were significantly downregulated (P shrimp hemocytes, hemocyanin and tumor necrosis factor-α (TNF-α) were up-regulated significantly (P shrimp was significantly advanced by double-strand RNA interference (dsRNAi) of mjHARBI1-like. Apoptosis studies indicated that mjHARBI1-dsRNA treatment caused a reduction in hemocyte apoptosis in bacterial and viral groups. In addition, phagocytosis experiments illustrated that mjHARBI1-dsRNA treatment led to a lower phagocytosis rate in hemocytes of V. alginolyticus-challenged shrimp. It was also found that knockdown of mjHARBI1-like inhibited shrimp phenoloxidase (PO) activity, superoxide dismutase (SOD) activity, and total hemocyte count (THC) after WSSV or V. alginolyticus infection. These data indicate a regulative role of mjHARBI1-likein the immunity of shrimp in response to pathogen infection. Resultantly, it was concluded that mjHARBI1-like might have a positive effect on the anti-WSSV immune response of shrimp by regulating apoptosis, THC, PO activity, and SOD activity. Additionally, mjHARBI1-like might promote anti-V. alginolyticus infection by participating in regulating phagocytosis, apoptosis, SOD activity, PO activity, and THC. Copyright © 2018 Elsevier Ltd. All rights reserved.

  7. Scavenger Receptor C Mediates Phagocytosis of White Spot Syndrome Virus and Restricts Virus Proliferation in Shrimp

    Yang, Ming-Chong; Shi, Xiu-Zhen; Yang, Hui-Ting; Sun, Jie-Jie; Xu, Ling; Wang, Xian-Wei; Zhao, Xiao-Fan

    2016-01-01

    Scavenger receptors are an important class of pattern recognition receptors that play several important roles in host defense against pathogens. The class C scavenger receptors (SRCs) have only been identified in a few invertebrates, and their role in the immune response against viruses is seldom studied. In this study, we firstly identified an SRC from kuruma shrimp, Marsupenaeus japonicus, designated MjSRC, which was significantly upregulated after white spot syndrome virus (WSSV) challenge at the mRNA and protein levels in hemocytes. The quantity of WSSV increased in shrimp after knockdown of MjSRC, compared with the controls. Furthermore, overexpression of MjSRC led to enhanced WSSV elimination via phagocytosis by hemocytes. Pull-down and co-immunoprecipitation assays demonstrated the interaction between MjSRC and the WSSV envelope protein. Electron microscopy observation indicated that the colloidal gold-labeled extracellular domain of MjSRC was located on the outer surface of WSSV. MjSRC formed a trimer and was internalized into the cytoplasm after WSSV challenge, and the internalization was strongly inhibited after knockdown of Mjβ-arrestin2. Further studies found that Mjβ-arrestin2 interacted with the intracellular domain of MjSRC and induced the internalization of WSSV in a clathrin-dependent manner. WSSV were co-localized with lysosomes in hemocytes and the WSSV quantity in shrimp increased after injection of lysosome inhibitor, chloroquine. Collectively, this study demonstrated that MjSRC recognized WSSV via its extracellular domain and invoked hemocyte phagocytosis to restrict WSSV systemic infection. This is the first study to report an SRC as a pattern recognition receptor promoting phagocytosis of a virus. PMID:28027319

  8. Consequences of Food Restriction for Immune Defense, Parasite Infection, and Fitness in Monarch Butterflies.

    McKay, Alexa Fritzsche; Ezenwa, Vanessa O; Altizer, Sonia

    2016-01-01

    Organisms have a finite pool of resources to allocate toward multiple competing needs, such as development, reproduction, and enemy defense. Abundant resources can support investment in multiple traits simultaneously, but limited resources might promote trade-offs between fitness-related traits and immune defenses. We asked how food restriction at both larval and adult life stages of the monarch butterfly (Danaus plexippus) affected measures of immunity, fitness, and immune-fitness interactions. We experimentally infected a subset of monarchs with a specialist protozoan parasite to determine whether parasitism further affected these relationships and whether food restriction influenced the outcome of infection. Larval food restriction reduced monarch fitness measures both within the same life stage (e.g., pupal mass) as well as later in life (e.g., adult lifespan); adult food restriction further reduced adult lifespan. Larval food restriction lowered both hemocyte concentration and phenoloxidase activity at the larval stage, and the effects of larval food restriction on phenoloxidase activity persisted when immunity was sampled at the adult stage. Adult food restriction reduced only adult phenoloxidase activity but not hemocyte concentration. Parasite spore load decreased with one measure of larval immunity, but food restriction did not increase the probability of parasite infection. Across monarchs, we found a negative relationship between larval hemocyte concentration and pupal mass, and a trade-off between adult hemocyte concentration and adult life span was evident in parasitized female monarchs. Adult life span increased with phenoloxidase activity in some subsets of monarchs. Our results emphasize that food restriction can alter fitness and immunity across multiple life stages. Understanding the consequences of resource limitation for immune defense is therefore important for predicting how increasing constraints on wildlife resources will affect fitness and

  9. Hematological Analysis of the Ascidian Botrylloides leachii (Savigny, 1816) During Whole-Body Regeneration.

    Blanchoud, Simon; Zondag, Lisa; Lamare, Miles D; Wilson, Megan J

    2017-06-01

    Whole-body regeneration (WBR)-the formation of an entire adult from only a small fragment of its own tissue-is extremely rare among chordates. Exceptionally, in the colonial ascidian Botrylloides leachii (Savigny, 1816) a fully functional adult is formed from their common vascular system after ablation of all adults from the colony in just 10 d, thanks to their high blastogenetic potential. While previous studies have identified key genetic markers and morphological changes, no study has yet focused on the hematological aspects of regeneration despite the major involvement of the remaining vascular system and the contained hemocytes in this process. To dissect this process, we analyzed colony blood flow patterns using time-lapse microscopy to obtain a quantitative description of the velocity, reversal pattern, and average distance traveled by hemocytes. We also observed that flows present during regeneration are powered by temporally and spatially synchronized contractions of the terminal ampullae. In addition, we revised previous studies of B. leachii hematology as well as asexual development using histological sectioning and compared the role played by hemocytes during WBR. We found that regeneration starts with a rapid healing response characterized by hemocyte aggregation and infiltration of immunocytes, followed by increased activity of hemoblasts, recruitment of macrophage-like cells for clearing the tissues of debris, and their subsequent disappearance from the circulation concomitant with the maturation of a single regenerated adult. Overall, we provide a detailed account of the hematological properties of regenerating B. leachii colonies, providing novel lines of inquiry toward the decipherment of regeneration in chordates.

  10. Ecotoxicity of CdTe quantum dots to freshwater mussels: Impacts on immune system, oxidative stress and genotoxicity

    Gagne, F.; Auclair, J.; Turcotte, P.; Fournier, M.; Gagnon, C.; Sauve, S.; Blaise, C.

    2008-01-01

    The purpose of this study was to examine the toxic effects of cadmium-telluride (CdTe) quantum dots on freshwater mussels. Elliption complanata mussels were exposed to increasing concentrations of CdTe (0, 1.6, 4 and 8 mg/L) and cadmium sulfate (CdSO 4 , 0.5 mg/L) for 24 h at 15 o C. After the exposure period, they were removed for assessments of immunocompetence, oxidative stress (lipid peroxidation) and genotoxicity (DNA strand breaks). Preliminary experiments revealed that CdTe dissolved in aquarium water tended to aggregate in the particulate phase (85%) while 15% of CdTe was found in the dissolved phase. Immunotoxicity was characterized by a significant decrease in the number of hemocytes capable of ingesting fluorescent beads, and hemocyte viability. The cytotoxic capacity of hemocytes to lyse mammalian K-562 cells was significantly increased, but the number of circulating hemocytes remained unchanged. Lipid peroxidation was significantly increased at a threshold concentration of 5.6 mg/L in gills and significantly reduced in digestive glands at a threshold concentration <1.6 mg/L CdTe. The levels of DNA strand breaks were significantly reduced in gills at <1.6 mg/L CdTe. In digestive glands, a transient but marginal increase in DNA strand breaks occurred at the lowest concentration and dropped significantly at the higher concentrations. A multivariate analysis revealed that the various response patterns differed based on the concentration of CdTe, thus permitting the identification of biomarkers associated with the form (colloidal vs. molecular) of cadmium

  11. Ecotoxicity of CdTe quantum dots to freshwater mussels: Impacts on immune system, oxidative stress and genotoxicity

    Gagne, F. [Fluvial Ecosystem Research, Environment Canada, 105 McGill Street, Montreal, Quebec, H2Y 2E7 (Canada)], E-mail: francois.gagne@ec.gc.ca; Auclair, J.; Turcotte, P. [Fluvial Ecosystem Research, Environment Canada, 105 McGill Street, Montreal, Quebec, H2Y 2E7 (Canada); Fournier, M. [INRS-Institut Armand-Frappier, 245 Hymus, Pointe-Claire, Quebec, H9R 3G6 (Canada); Gagnon, C. [Fluvial Ecosystem Research, Environment Canada, 105 McGill Street, Montreal, Quebec, H2Y 2E7 (Canada); Sauve, S. [Departement de Chimie, Universite de Montreal, C.P. 6128, Succursale Centre-ville, Montreal, Quebec, H3C 3J7 (Canada); Blaise, C. [Fluvial Ecosystem Research, Environment Canada, 105 McGill Street, Montreal, Quebec, H2Y 2E7 (Canada)

    2008-02-18

    The purpose of this study was to examine the toxic effects of cadmium-telluride (CdTe) quantum dots on freshwater mussels. Elliption complanata mussels were exposed to increasing concentrations of CdTe (0, 1.6, 4 and 8 mg/L) and cadmium sulfate (CdSO{sub 4}, 0.5 mg/L) for 24 h at 15 {sup o}C. After the exposure period, they were removed for assessments of immunocompetence, oxidative stress (lipid peroxidation) and genotoxicity (DNA strand breaks). Preliminary experiments revealed that CdTe dissolved in aquarium water tended to aggregate in the particulate phase (85%) while 15% of CdTe was found in the dissolved phase. Immunotoxicity was characterized by a significant decrease in the number of hemocytes capable of ingesting fluorescent beads, and hemocyte viability. The cytotoxic capacity of hemocytes to lyse mammalian K-562 cells was significantly increased, but the number of circulating hemocytes remained unchanged. Lipid peroxidation was significantly increased at a threshold concentration of 5.6 mg/L in gills and significantly reduced in digestive glands at a threshold concentration <1.6 mg/L CdTe. The levels of DNA strand breaks were significantly reduced in gills at <1.6 mg/L CdTe. In digestive glands, a transient but marginal increase in DNA strand breaks occurred at the lowest concentration and dropped significantly at the higher concentrations. A multivariate analysis revealed that the various response patterns differed based on the concentration of CdTe, thus permitting the identification of biomarkers associated with the form (colloidal vs. molecular) of cadmium.

  12. Effects of ergothioneine from mushrooms (Flammulina velutipes) on melanosis and lipid oxidation of kuruma shrimp (Marsupenaeus japonicus).

    Encarnacion, Angel B; Fagutao, Fernand; Hirono, Ikuo; Ushio, Hideki; Ohshima, Toshiaki

    2010-02-24

    The antimelanosic and antioxidative properties of a hot water extract prepared from the fruiting body of the edible mushroom (Flammulina velutipes) were evaluated by dietary supplementation in Kuruma shrimp (Marsupenaeus japonicus) for possible aquaculture application. The extract contained ergothioneine (ERT) at a level of 2.05 mg/mL. A commercial standard of l-ergothioneine (l-ERT) and the mushroom extract showed inhibitory activity against mushroom polyphenoloxidase (PPO). Feeding of the extract had no adverse effects on the immune systems of the shrimp under the present experimental conditions. Supplementation of the extract in the diet significantly suppressed PPO activities in the hemolymphs of the shrimp. Expression of the prophenoloxidase (proPO) gene decreased in the hemocyte of the Kuruma shrimp fed with the mushroom extract. Consequently, development of melanosis was significantly suppressed in the supplement fed shrimp during ice storage. Lipid oxidation was also effectively controlled in the supplement fed group throughout the storage period. In vitro experiments showed that l-ERT effectively inhibited the activation of proPO in the hemocyte lysate supernatant (HLS). The transcript of the proPO gene in the hemocyte showed lower expression in the l-ERT-treated HLS. It was concluded that dietary supplementation of the mushroom extract in shrimp could be a promising approach to control post mortem development of melanosis and lipid oxidation in shrimp muscles.

  13. Lineage tracing of lamellocytes demonstrates Drosophila macrophage plasticity.

    Martin Stofanko

    2010-11-01

    Full Text Available Leukocyte-like cells called hemocytes have key functions in Drosophila innate immunity. Three hemocyte types occur: plasmatocytes, crystal cells, and lamellocytes. In the absence of qimmune challenge, plasmatocytes are the predominant hemocyte type detected, while crystal cells and lamellocytes are rare. However, upon infestation by parasitic wasps, or in melanotic mutant strains, large numbers of lamellocytes differentiate and encapsulate material recognized as "non-self". Current models speculate that lamellocytes, plasmatocytes and crystal cells are distinct lineages that arise from a common prohemocyte progenitor. We show here that over-expression of the CoREST-interacting transcription factor Chn in plasmatocytes induces lamellocyte differentiation, both in circulation and in lymph glands. Lamellocyte increases are accompanied by the extinction of plasmatocyte markers suggesting that plasmatocytes are transformed into lamellocytes. Consistent with this, timed induction of Chn over-expression induces rapid lamellocyte differentiation within 18 hours. We detect double-positive intermediates between plasmatocytes and lamellocytes, and show that isolated plasmatocytes can be triggered to differentiate into lamellocytes in vitro, either in response to Chn over-expression, or following activation of the JAK/STAT pathway. Finally, we have marked plasmatocytes and show by lineage tracing that these differentiate into lamellocytes in response to the Drosophila parasite model Leptopilina boulardi. Taken together, our data suggest that lamellocytes arise from plasmatocytes and that plasmatocytes may be inherently plastic, possessing the ability to differentiate further into lamellocytes upon appropriate challenge.

  14. Condition-Dependent Trade-Off Between Weapon Size and Immunity in Males of the European Earwig.

    Körner, Maximilian; Vogelweith, Fanny; Foitzik, Susanne; Meunier, Joël

    2017-08-11

    Investigating the expression of trade-offs between key life-history functions is central to our understanding of how these functions evolved and are maintained. However, detecting trade-offs can be challenging due to variation in resource availability, which masks trade-offs at the population level. Here, we investigated in the European earwig Forficula auricularia whether (1) weapon size trades off with three key immune parameters - hemocyte concentration, phenoloxidase and prophenoloxidase activity - and whether (2) expression and strength of these trade-offs depend on male body condition (body size) and/or change after an immune challenge. Our results partially confirmed condition dependent trade-offs between weapon size and immunity in male earwigs. Specifically, we found that after an immune challenge, weapon size trades off with hemocyte concentrations in low-condition, but not in good-condition males. Contrastingly, weapon size was independent of pre-challenge hemocyte concentration. We also found no trade-off between weapon size and phenoloxidase activity, independent of body condition and immune challenge. Overall, our study reveals that trade-offs with sexual traits may weaken or disappear in good-condition individuals. Given the importance of weapon size for male reproductive success, our results highlight how low-condition individuals may employ alternative life-history investment strategies to cope with resource limitation.

  15. dOCRL maintains immune cell quiescence by regulating endosomal traffic.

    Steven J Del Signore

    2017-10-01

    Full Text Available Lowe Syndrome is a developmental disorder characterized by eye, kidney, and neurological pathologies, and is caused by mutations in the phosphatidylinositol-5-phosphatase OCRL. OCRL plays diverse roles in endocytic and endolysosomal trafficking, cytokinesis, and ciliogenesis, but it is unclear which of these cellular functions underlie specific patient symptoms. Here, we show that mutation of Drosophila OCRL causes cell-autonomous activation of hemocytes, which are macrophage-like cells of the innate immune system. Among many cell biological defects that we identified in docrl mutant hemocytes, we pinpointed the cause of innate immune cell activation to reduced Rab11-dependent recycling traffic and concomitantly increased Rab7-dependent late endosome traffic. Loss of docrl amplifies multiple immune-relevant signals, including Toll, Jun kinase, and STAT, and leads to Rab11-sensitive mis-sorting and excessive secretion of the Toll ligand Spåtzle. Thus, docrl regulation of endosomal traffic maintains hemocytes in a poised, but quiescent state, suggesting mechanisms by which endosomal misregulation of signaling may contribute to symptoms of Lowe syndrome.

  16. Effect of temperature on immunocompetence of the blue mussel (Mytilus edulis

    Alexandre Beaudry

    2016-08-01

    Full Text Available The blue mussel is a filter-feeding bivalve commonly used in ecotoxicological monitoring as a sentinel species. Due to climate change and the increase of temperature expected in marine environment, it is important to anticipate potential impacts on this species. The aim of this study was to investigate the immunocompetence of blue mussels acclimated to different temperatures and on the effects of increasing temperatures (5, 10 and 20°C. Different indices and gonad maturation stages were also determined throughout the experiments. Cell viability, phagocytosis, serum lysozyme activity and cyclooxygenase (COX activity were evaluated as immune parameters. The cellular immunity was also evaluated after hemocytes exposure to various cadmium concentrations in vitro. The results obtained demonstrate modulation of hemocyte viability and the ability of these cells to phagocytize in absence of contaminants. After the exposure to cadmium, hemocytes showed greater viability at 5°C while maintaining a higher phagocytic competence. In addition, the lysozyme activity stayed stable at all tested temperatures, contrary to that of COX, which increased when the mussels were maintained at 20°C. The evaluation of indices demonstrated no reduction of general conditions during all the experiment despite the increase of temperature and the reduction of the digestive gland weight. Moreover, the lack of food does not affect gonad maturation and the spawning process.

  17. Assessing potential harmful effects of CdSe quantum dots by using Drosophila melanogaster as in vivo model

    Alaraby, Mohamed; Demir, Esref; Hernández, Alba; Marcos, Ricard

    2015-01-01

    Since CdSe QDs are increasingly used in medical and pharmaceutical sciences careful and systematic studies to determine their biosafety are needed. Since in vivo studies produce relevant information complementing in vitro data, we promote the use of Drosophila melanogaster as a suitable in vivo model to detect toxic and genotoxic effects associated with CdSe QD exposure. Taking into account the potential release of cadmium ions, QD effects were compared with those obtained with CdCl 2 . Results showed that CdSe QDs penetrate the intestinal barrier of the larvae reaching the hemolymph, interacting with hemocytes, and inducing dose/time dependent significant genotoxic effects, as determined by the comet assay. Elevated ROS production, QD biodegradation, and significant disturbance in the conserved Hsps, antioxidant and p53 genes were also observed. Overall, QD effects were milder than those induced by CdCl 2 suggesting the role of Cd released ions in the observed harmful effects of Cd based QDs. To reduce the observed side-effects of Cd based QDs biocompatible coats would be required to avoid cadmium's undesirable effects. - Highlights: • CdSe QDs were able to cross the intestinal barrier of Drosophila. • Elevated ROS induction was detected in larval hemocytes. • Changes in the expression of Hsps and p53 genes were observed. • Primary DNA damage was induced by CdSe QDs in hemocytes. • Overall, CdSe QD effects were milder than those induced by CdCl 2

  18. Characteristic and functional analysis of toll-like receptors (TLRs in the lophotrocozoan, Crassostrea gigas, reveals ancient origin of TLR-mediated innate immunity.

    Yang Zhang

    Full Text Available The evolution of TLR-mediated innate immunity is a fundamental question in immunology. Here, we report the characterization and functional analysis of four TLR members in the lophotrochozoans Crassostreagigas (CgTLRs. All CgTLRs bear a conserved domain organization and have a close relationship with TLRs in ancient non-vertebrate chordates. In HEK293 cells, every CgTLR could constitutively activate NF-κB responsive reporter, but none of the PAMPs tested could stimulate CgTLR-activated NF-κB induction. Subcellular localization showed that CgTLR members have similar and dual distribution on late endosomes and plasma membranes. Moreover, CgTLRs and CgMyD88 mRNA show a consistent response to multiple PAMP challenges in oyster hemocytes. As CgTLR-mediated NF-κB activation is dependent on CgMyD88, we designed a blocking peptide for CgTLR signaling that would inhibit CgTLR-CgMyD88 dependent NF-κB activation. This was used to demonstrate that a Vibrio parahaemolyticus infection-induced enhancement of degranulation and increase of cytokines TNF mRNA in hemocytes, could be inhibited by blocking CgTLR signaling. In summary, our study characterized the primitive TLRs in the lophotrocozoan C. gigas and demonstrated a fundamental role of TLR signaling in infection-induced hemocyte activation. This provides further evidence for an ancient origin of TLR-mediated innate immunity.

  19. Immunity of an alternative host can be overcome by higher densities of its parasitoids Palmistichus elaeisis and Trichospilus diatraeae.

    Gilberto Santos Andrade

    Full Text Available Interactions of the parasitoids Palmistichus elaeisis Delvare & LaSalle and Trichospilus diatraeae Cherian & Margabandhu (Hymenoptera: Eulophidae with its alternative host Anticarsia gemmatalis (Hübner (Lepidoptera: Noctuidae affect the success or failure of the mass production of these parasitoids for use in integrated pest management programs. The aim of this study was to evaluate changes in the cellular defense and encapsulation ability of A. gemmatalis pupae against P. elaeisis or T. diatraeae in adult parasitoid densities of 1, 3, 5, 7, 9, 11 or 13 parasitoids/pupae. We evaluated the total quantity of circulating hemocytes and the encapsulation rate versus density. Increasing parasitoid density reduced the total number of hemocytes in the hemolymph and the encapsulation rate by parasitized pupae. Furthermore, densities of P. elaeisis above 5 parasitoids/pupae caused higher reduction in total hemocyte numbers. The encapsulation rate fell with increasing parasitoid density. However, parasitic invasion by both species induced generally similar responses. The reduction in defensive capacity of A. gemmatalis is related to the adjustment of the density of these parasitoids to their development in this host. Thus, the role of the density of P. elaeisis or T. diatraeae by pupa is induced suppression of cellular defense and encapsulation of the host, even without them possesses a co-evolutionary history. Furthermore, these findings can predict the success of P. elaeisis and T. diatraeae in the control of insect pests through the use of immunology as a tool for evaluation of natural enemies.

  20. Azadirachtin-induced effects on various life history traits and cellular immune reactions of Galleria mellonella (Lepidoptera: Pyralidae

    Er Aylin

    2017-01-01

    Full Text Available The effects of the botanical insecticide azadirachtin were examined on the life history traits, fecundity and immune parameters of Galleria mellonella L. (Lepidoptera: Pyralidae. We determined that for the topical application of azadirachtin, the LC50 was 16.564 ppm; at 100 ppm the adult emergence time was prolonged, however the longevity of adults remained unchanged above sublethal concentrations. The mean number of healthy eggs and the fecundity of adults decreased, whereas the number of defective eggs increased with azadirachtin treatment. At concentrations >50 ppm female G. mellonella adults laid no eggs. Azadirachtin reduced total hemocyte counts at 24 and 48 h posttreatment, however the alterations in differential hemocyte counts were only significant at 100 ppm. Laminarin-induced nodulation response and the spreading ability of hemocytes were also suppressed with azadirachtin treatment. Our results suggest that azadirachtin, as a good candidate for integrated pest control, has the capability to affect the biological parameters and cellular immunity of the model insect G. mellonella.

  1. Health status and bioremediation capacity of wild freshwater mussels (Diplodon chilensis) exposed to sewage water pollution in a glacial Patagonian lake.

    Bianchi, Virginia A; Castro, Juan M; Rocchetta, Iara; Bieczynski, Flavia; Luquet, Carlos M

    2014-04-01

    Deleterious effects on health and fitness are expected in mussels chronically exposed to sewage water pollution. Diplodon chilensis inhabiting SMA, an area affected by untreated and treated sewage water, shows increased hemocyte number and phagocytic activity, while bacteriolytic and phenoloxidase activities in plasma and reactive oxygen species production in hemocytes are lower compared to mussels from an unpolluted area (Yuco). There are not differences in cell viability, lysosomal membrane stability, lipid peroxidation and total oxygen scavenging capacity between SMA and Yuco mussels' hemocytes. Energetic reserves and digestive gland mass do not show differences between groups; although the condition factor is higher in SMA than in Yuco mussels. Gills of SMA mussels show an increase in mass and micronuclei frequency compared to those of Yuco. Mussels from both sites reduce bacterial loads in polluted water and sediments, improving their quality with similar feeding performance. These findings suggest that mussels exposed to sewage pollution modulate physiological responses by long-term exposure; although, gills are sensitive to these conditions and suffer chronic damage. Bioremediation potential found in D. chilensis widens the field of work for remediation of sewage bacterial pollution in water and sediments by filtering bivalves. Copyright © 2014 Elsevier Ltd. All rights reserved.

  2. Field biomonitoring using the zebra mussel Dreissena polymorpha and the quagga mussel Dreissena bugensis following immunotoxic reponses. Is there a need to separate the two species?

    Evariste, Lauris; David, Elise; Cloutier, Pierre-Luc; Brousseau, Pauline; Auffret, Michel; Desrosiers, Mélanie; Groleau, Paule Emilie; Fournier, Michel; Betoulle, Stéphane

    2018-04-02

    The zebra mussel, Dreissena polymorpha constitutes an extensively used sentinel species for biomonitoring in European and North American freshwater systems. However, this invasive species is gradually replaced in freshwater ecosystem by Dreissena bugensis, a closely related dreissenid species that shares common morphological characteristics but possess some physiological differences. However, few are known about differences on more integrated physiological processes that are generally used as biomarkers in biological monitoring studies. Declining of zebra mussel populations raises the question of the sustainability of using one or both species indifferently to maintain the quality of environmental pollution monitoring data. In our study, we performed a field comparative study measuring immune-related markers and bioaccumulation of PCBs, PAHs and PBDEs in sympatrically occurring mussel populations from three sites of the St. Lawrence River. For tested organisms, species were identified using RFLP analysis. Measurement of bioaccumulated organic compounds indicated a higher accumulation of PCBs and PBDEs in D. bugensis soft tissues compared to D. polymorpha while no differences were noticed for PAHs. Results of hemocytic parameters highlighted that differences of hemocyte distributions were associated to modulations of phagocytic activities. Moreover, marked differences occurred in measurement of hemocytic oxidative activity, indicating divergences between the two species for ROS regulation strategies. This physiological characteristic may deeply influence species responses facing environmental or pollution related stress and induce bias if the two species are not differentiated in further biomarker or bioaccumulation measurement-based studies. Copyright © 2018 Elsevier Ltd. All rights reserved.

  3. Experimental infection of octopus vulgaris (Cuvier, 1797) with Photobacterium damsela subsp. piscicida. Immunohistochemical tracking of antigen and tissue responses.

    Bakopoulos, Vasileios; White, Daniella; Valsamidis, Michail-Aggelos; Vasilaki, Feli

    2017-03-01

    Adult common octopus individuals were intramuscularly infected with Photobacterium damsela subsp. piscicida in order to investigate if this species is sensitive to this common and important fish pathogen. The fate of the bacterial antigens and the tissue responses of Octopus vulgaris were studied employing immunohistochemical techniques. Strong reaction at the site of injection was evident from day 2 post-infection that continued until day 14. Great numbers of hemocytes that were attracted at the site of infection were involved in phagocytosis of bacteria. Very early in the infection, a transition of cells to fibroblasts and an effort to isolate the infection was observed. During the course of the study, very large necrotic cells were seen at the site of infection, whereas during the later stages hemocytes with phagocytosed bacteria were observed in well-defined pockets inside the muscle tissue. None of the internal organs tested for the presence of the bacterium were positive with the exception of the digestive gland where antigen staining was observed which was not associated with hemocyte infiltration. The high doses of bacterial cells used in this experimental infection and the lack of disease signs from Octopus vulgaris suggest that, under normal conditions, octopus is resistant to Photobacterium damsela subsp. piscicida. Copyright © 2017 Elsevier Inc. All rights reserved.

  4. A novel immune-related gene HDD1 of silkworm Bombyx mori is involved in bacterial response.

    Zhang, Kui; Pan, Guangzhao; Zhao, Yuzu; Hao, Xiangwei; Li, Chongyang; Shen, Li; Zhang, Rui; Su, Jingjing; Cui, Hongjuan

    2017-08-01

    Insects have evolved an effective immune system to respond to various challenges. In this study, a novel immune-related gene, called BmHDD1, was first charactered in silkworm, Bombyx mori. BmHDD1 contained an ORF of 837bp and encoding a deduced protein of 278 amino acids. BmHDD1 was specifically expressed in hemocytes, and highly expressed at the molting and metamorphosis stages under normal physiological conditions. Our results suggested that BmHDD1 was mainly generated by hemocytes and secreted into hemolymph. Our results also showed that the expression level of BmHDD1 was significantly increased after 20E injection, which indicated that BmHDD1 might be regulated by ecdysone. More importantly, BmHDD1 was dramatically induced after injected with different types of PAMPs or bacteria, either in hemocytes or fat body. Those results suggested that BmHDD1 plays a role in developing and immunity system in silkworm, Bombyx mori. Copyright © 2017. Published by Elsevier Ltd.

  5. A Carbonic Anhydrase Serves as an Important Acid-Base Regulator in Pacific Oyster Crassostrea gigas Exposed to Elevated CO2: Implication for Physiological Responses of Mollusk to Ocean Acidification.

    Wang, Xiudan; Wang, Mengqiang; Jia, Zhihao; Qiu, Limei; Wang, Lingling; Zhang, Anguo; Song, Linsheng

    2017-02-01

    Carbonic anhydrases (CAs) have been demonstrated to play an important role in acid-base regulation in vertebrates. However, the classification and modulatory function of CAs in marine invertebrates, especially their responses to ocean acidification remain largely unknown. Here, a cytosolic α-CA (designated as CgCAII-1) was characterized from Pacific oyster Crassostrea gigas and its molecular activities against CO 2 exposure were investigated. CgCAII-1 possessed a conserved CA catalytic domain, with high similarity to invertebrate cytoplasmic or mitochondrial α-CAs. Recombinant CgCAII-1 could convert CO 2 to HCO 3 - with calculated activity as 0.54 × 10 3  U/mg, which could be inhibited by acetazolamide (AZ). The mRNA transcripts of CgCAII-1 in muscle, mantle, hepatopancreas, gill, and hemocytes increased significantly after exposure to elevated CO 2 . CgCAII-1 could interact with the hemocyte membrane proteins and the distribution of CgCAII-1 protein became more concentrated and dense in gill and mantle under CO 2 exposure. The intracellular pH (pHi) of hemocytes under CO 2 exposure increased significantly (p ocean acidification and participate in acid-base regulation. Such cytoplasmic CA-based physiological regulation mechanism might explain other physiological responses of marine organisms to OA.

  6. Molecular cloning and tissue expression of the fatty acid-binding protein (Es-FABP gene in female Chinese mitten crab (Eriocheir sinensis

    He Lin

    2010-09-01

    Full Text Available Abstract Background Fatty acid-binding proteins (FABPs, small cytosolic proteins that function in the uptake and utilization of fatty acids, have been extensively studied in higher vertebrates while invertebrates have received little attention despite similar nutritional requirements during periods of reproductive activity. Results Therefore, a cDNA encoding Eriocheir sinensis FABP (Es-FABP was cloned based upon EST analysis of a hepatopancreas cDNA library. The full length cDNA was 750 bp and encoded a 131 aa polypeptide that was highly homologous to related genes reported in shrimp. The 9108 bp Es-FABP gene contained four exons that were interrupted by three introns, a genomic organization common among FABP multigene family members in vertebrates. Gene expression analysis, as determined by RT-PCR, revealed the presence of Es-FABP transcripts in hepatopancreas, hemocytes, ovary, gills, muscle, thoracic ganglia, heart, and intestine, but not stomach or eyestalk. Real-time quantitative RT-PCR analysis revealed that Es-FABP expression in ovary, hemocytes, and hepatopancreas was dependent on the status of ovarian development, with peak expression observed in January. Conclusions Evidence provided in the present report supports a role of Es-FABP in lipid transport during the period of rapid ovarian growth in E. sinensis, and indirectly confirms the participation of the hepatopancreas, ovary, and hemocytes in lipid nutrient absorption and utilization processes.

  7. Culture conditions improvement of Crassostrea gigas using a potential probiotic Bacillus sp strain.

    Fdhila, Kais; Haddaji, Najla; Chakroun, Ibtissem; Dhiaf, Amel; Macherki, Mohammed Ezz Edine; Khouildi, Bochra; Lamari, Faouzi; Chaieb, Kamel; Abid, Nabil; Marzougui, Hajer; Khouadja, Sadok; Missaoui, Hechmi

    2017-09-01

    It is well demonstrated that some probiotics improve rearing water quality and thereby have beneficial effects on reared organisms. We conducted this study to determine the effect of Bacillus consortium on Crassostrea gigas reared in contemned seawater with indigo dye priory treated with Bacillus or no treated. This effect was studied by assessing hemocytes death using flow cytometry analysis. We found that the percentage of decolorization of indigo dye in polluted seawater in presence of C. gigas increased from 41% to 90% when using Bacillus consortium. In these conditions, the hemocytes mortality of reared C. gigas decreased from 87% to 56%. We have demonstrated also that seawater contemned with priory treated indigo with Bacillus consortium is less toxic than seawater contemned with the no treated indigo. The percentage of hemocytes death is 81% for the contemned seawater with indigo and 56% for no contemned seawater. This consortium shows a protector effect of C. gigas against Vibrio harveyi contemning reared seawater. Copyright © 2017 Elsevier Ltd. All rights reserved.

  8. PmVRP15, a Novel Viral Responsive Protein from the Black Tiger Shrimp, Penaeus monodon, Promoted White Spot Syndrome Virus Replication

    Vatanavicharn, Tipachai; Prapavorarat, Adisak; Jaree, Phattarunda; Somboonwiwat, Kunlaya; Tassanakajon, Anchalee

    2014-01-01

    Suppression subtractive hybridization of Penaeus monodon hemocytes challenged with white spot syndrome virus (WSSV) has identified the viral responsive gene, PmVRP15, as the highest up-regulated gene ever reported in shrimps. Expression analysis by quantitative real time RT-PCR revealed 9410–fold up-regulated level at 48 h post WSSV injection. Tissue distribution analysis showed that PmVRP15 transcript was mainly expressed in the hemocytes of shrimp. The full-length cDNA of PmVRP15 transcript was obtained and showed no significant similarity to any known gene in the GenBank database. The predicted open reading frame of PmVRP15 encodes for a deduced 137 amino acid protein containing a putative transmembrane helix. Immunofluorescent localization of the PmVRP15 protein revealed it accumulated around the nuclear membrane in all three types of shrimp hemocytes and that the protein was highly up-regulated in WSSV-infected shrimps. Double-stranded RNA interference-mediated gene silencing of PmVRP15 in P. monodon significantly decreased WSSV propagation compared to the control shrimps (injected with GFP dsRNA). The significant decrease in cumulative mortality rate of WSSV-infected shrimp following PmVRP15 knockdown was observed. These results suggest that PmVRP15 is likely to be a nuclear membrane protein and that it acts as a part of WSSV propagation pathway. PMID:24637711

  9. Selectively enhanced expression of prophenoloxidase activating enzyme 1 (PPAE1 at a bacteria clearance site in the white shrimp, Litopenaeus vannamei

    Jang In-Kwon

    2011-12-01

    Full Text Available Abstract Background The prophenoloxidase-activating (PO activating system plays an important role in the crustacean innate immunity, particularly in wound healing and pathogen defense. A key member of this system is prophenoloxidase-activating enzyme (PPAE, which is the direct activator of prophenoloxidase (proPO. Despite their importance in crustacean PO activating system, the studies on them remain limited. Results Here we report on a PPAE of white shrimp, Litopenaeus vannamei (lvPPAE1, which showed 94% similarity to PPAE1 of Penaeus monodon. We found that lvPPAE1 in fluid hemocytes was down regulated after challenge by Vibrio harveyi but was enhanced when shrimps were exposed to a bacteria-rich environment for long-term. In vivo gene silence of lvPPAE1 by RNAi can significantly reduce the phenoloxidase activity (PO and increase the susceptibility of shrimps to V. harveyi. Although lvPPAE1 was down-regulated in fluid hemocytes by Vibrio challenge, its expression increased significantly in gill after bacteria injection, which is the primary bacteria-clearance tissue. Conclusion Suppressed expression in fluid hemocytes and enhanced expression in gill indicates selectively enhanced expression at the bacterial clearance site. This is a novel feature for PPAE expression. The results will contribute to our understanding of the PO activating system in crustaceans.

  10. Immune-physiological aspects of synergy between avermectins and the entomopathogenic fungus Metarhizium robertsii in Colorado potato beetle larvae.

    Tomilova, Oksana G; Kryukov, Vadim Yu; Duisembekov, Bahytzhan A; Yaroslavtseva, Olga N; Tyurin, Maksim V; Kryukova, Natalia A; Skorokhod, Valery; Dubovskiy, Ivan M; Glupov, Viktor V

    2016-10-01

    The interaction between the entomopathogenic fungus Metarhizium robertsii and natural avermectin metabolites of the actinomycete Streptomyces avermitilis were investigated on Colorado potato beetle larvae. A synergy in the mortality of larvae was detected after simultaneous treatment with half-lethal doses of avermectins (commercial name actarophit) 0.005% and fungus (5×10 5 conidia/ml). The treatment with avermectins led to rapid fungal colonization of the hemolymph. The defense strategies of insects infected by fungus and treated with avermectins and untreated insects were compared to investigate the mechanisms of this synergy. We have shown an increase in hemocytes, especially immunocompetent cells - plasmatocytes and granular cells in the initial stages of mycosis (third day post inoculation). In contrast, avermectins suppressed cellular immunity in hemolymph. Specifically, avermectins dramatically decreased the count of granular cells in larvae infected and uninfected with fungus. Apoptosis inducement and hemocyte necrosis under the influence of avermectins has been shown in vitro as one of the possible reasons for hemocyte mortality. In addition, avermectins enhanced the activity of phenoloxidases in integuments and hemolymph and increased the activity of glutathione-S-transferases activity in the fat body and hemolymph of infected and uninfected larvae, thereby intensifying the development of fungal infection by M. robertsii in Colorado potato beetle larvae. The combination of fungal infection and avermectins constitutes a new perspective for developing multicomponent bioinsecticides. Copyright © 2016 Elsevier Inc. All rights reserved.

  11. GENOTOXICITY OF SHALLOW WATERS NEAR THE BRAZILIAN ANTARCTIC STATION "COMANDANTE FERRAZ" (EACF, ADMIRALTY BAY, KING GEORGE ISLAND, ANTARCTICA

    Arthur José da Silva Rocha

    2015-03-01

    Full Text Available Series of biomonitoring surveys were undertaken weekly in February 2012 to investigate the genotoxicity of the shallow waters around the Brazilian Antarctic Station "Comandante Ferraz" (EACF. The comet assay was applied to assess the damage to the DNA of hemocytes of the crustacean amphipods Gondogeneia antarctica collected from shallow waters near the Fuel Tanks (FT and Sewage Treatment Outflow (STO of the research station, and compare it to the DNA damage of animals from Punta Plaza (PPL and Yellow Point (YP, natural sites far from the EACF defined as experimental controls. The damage to the DNA of hemocytes of G. antarctica was not significantly different between sites in the biomonitoring surveys I and II. In survey III, the damage to the DNA of animals captured in shallow waters near the Fuel Tanks (FT and Sewage Treatment Outflow (STO was significantly higher than that of the control site of Punta Plaza (PPL. In biomonitoring survey IV, a significant difference was detected only between the FT and PPL sites. Results demonstrated that the shallow waters in front of the station may be genotoxic and that the comet assay and hemocytes of G. antarctica are useful tools for assessing genotoxicity in biomonitoring studies of Antarctic marine coastal habitats.

  12. Immunological responses of the mangrove oysters Crassostrea gasar naturally infected by Perkinsus sp. in the Mamanguape Estuary, Paraíba state (Northeastern, Brazil).

    Queiroga, Fernando Ramos; Marques-Santos, Luis Fernando; Hégaret, Hélène; Soudant, Philippe; Farias, Natanael Dantas; Schlindwein, Aline Daiane; Mirella da Silva, Patricia

    2013-08-01

    Perkinsus genus includes protozoan parasites of marine mollusks, especially bivalves. In the last four years, this parasite has been detected in mangrove oysters Crassostrea rhizophorae and Crassostrea gasar from the Northeastern region of Brazil. Hemocytes are the key cells of the oyster immune system, being responsible for a variety of cellular and humoral reactions, such as phagocytosis, encapsulation and the release of several effector molecules that control the invasion and proliferation of microorganisms. In Brazil, there is little information on perkinsosis and none on the immune responses of native oysters' species against Perkinsus spp. The objective of this study was to determine the effects of natural infection by Perkinsus sp. on the immunological parameters of mangrove oysters C. gasar cultured in the Mamanguape River Estuary (Paraíba, Brazil). Adults oysters (N = 40/month) were sampled in December 2011, March, May, August and October 2012. Gills were removed and used to determine the presence and intensity of the Perkinsus sp. infection, according to a scale of four levels (1-4), using the Ray's fluid thioglycollate medium assay. Immunological parameters were measured in hemolymph samples by flow cytometry, including: total hemocyte count (THC), differential hemocyte count (DHC), cell mortality, phagocytic capacity, and production of Reactive Oxygen Species (ROS). The plasma was used to determine the hemagglutination activity. The results showed the occurrence of Perkinsus sp. with the highest mean prevalence (93.3%) seen so far in oyster populations in Brazil. Despite that, no oyster mortality was associated. In contrast, we observed an increase in hemocyte mortality and a suppression of two of the main defense mechanisms, phagocytosis and ROS production in infected oysters. The increase in the percentage of blast-like cells on the hemolymph, and the increase in THC in oysters heavily infected (at the maximum intensity, 4) suggest an induction of

  13. C-type lectin B (SpCTL-B) regulates the expression of antimicrobial peptides and promotes phagocytosis in mud crab Scylla paramamosain.

    Wei, Xiaoyuan; Wang, Limin; Sun, Wanwei; Zhang, Ming; Ma, Hongyu; Zhang, Yueling; Zhang, Xinxu; Li, Shengkang

    2018-07-01

    As pattern recognition receptors, C-type lectins (CTLs) play important roles in immune system of crustaceans through identifying and binding to the conservative pathogen-associated molecular patterns (PAMPs) on pathogen surfaces. In this study, a new CTL, SpCTL-B, was identified from the hemocytes of mud crab Scylla paramamosain. The full-length of SpCTL-B cDNA was 1278 bp with an open reading frame (ORF) of 348 bp. The predicted SpCTL-B protein contains a single carbohydrate-recognition domain (CRD). SpCTL-B transcripts were distributed in all examined tissues with the highest levels in hepatopancreas. After challenged with Vibrio parahaemolyticus, LPS, polyI:C and white spot syndrome virus (WSSV), the mRNA levels of SpCTL-B in hemocytes and hepatopancreas were up-regulated. The recombinant SpCTL-B (rSpCTL-B) purified by Ni-affinity chromatography showed stronger binding activities with Staphylococcus aureus, β-hemolytic Streptococcus, Escherichia coli, Aeromonas hydrophila, Vibrio alginolyticus than those with V. parahaemolyticus and Saccharomyces cerevisiae. rSpCTL-B exhibited a broad spectrum of microorganism-agglutination activities against Gram-positive bacteria (S. aureus, β-hemolytic Streptococcus) and Gram-negative bacteria (E. coli, V. parahaemolyticus, A. hydrophila, V. alginolyticus) in a Ca 2+ -dependent manner. The agglutination activities of rSpCTL-B could be inhibited by D-mannose and LPS, but not by d-fructose and galactose. The antimicrobial assay showed that rSpCTL-B exhibited the growth inhibition against all examined gram-positive bacteria and gram-negative bacteria. When SpCTL-B was silenced by RNAi, the bacterial clearance ability in mud crab was decreased and the transcript levels of five antimicrobial peptides (AMPs) (SpCrustin, SpHistin, SpALF4 (anti-lipopolysaccharide factor), SpALF5 and SpALF6) were significantly decreased in hemocytes. In our study, knockdown of SpCTL-B could down-regulate the expression of SpSTAT at m

  14. L-3,4-Dihydroxyphenylalanine (l-DOPA) induces neuroendocrinological, physiological, and immunological regulation in white shrimp, Litopenaeus vannamei.

    Mapanao, Ratchaneegorn; Kuo, Hsin-Wei; Chang, Chin-Chuan; Liu, Kuan-Fu; Cheng, Winton

    2018-03-01

    L-3,4-Dihydroxyphenylalanine (l-DOPA) is a precursor for dopamine (DA) synthesis. Assessments were conducted to analyze the effects of l-DOPA on mediating regulation of neuroendocrinological, immunological, and physiological parameters in the shrimp, Litopenaeus vannamei when they were individually injected with 0.01 N HCl or l-DOPA at 0.5 or 1.0 μmol shrimp -1 for 60, 120, and 240 min. For catecholamine synthesis evaluation, tyrosine hydroxylase (TH) and DA beta hydroxylase (DBH) activities, l-DOPA, DA, and norepinephrine (NE) levels in hemolymph were determined. The total hemocyte count (THC), differential hemocyte count (DHC), phenoloxidase (PO) activity, respiratory bursts (RBs), superoxide dismutase (SOD) activity, phagocytic activity, and clearance efficiency in response to the pathogen, Vibrio alginolyticus were assessed for immune responses, and plasma glucose and lactate levels were for physiological response. Results showed that the TH activity, THC, hyaline cells (HCs), and semigranular cells (SGCs) at 120 min, DA levels at 60-240 min, PO activity in hemocytes per 50 μL of hemolymph at 60-120 min, and PO activity per granulocyte (granular cells (GCs) + SGCs) at 60 min significantly increased, but TH activity, l-DOPA levels, GCs, SGCs, and respiratory bursts in hemocytes per 10 μL of hemolymph at 60 min, respiratory bursts per hemocyte and SOD activity at 120 min, phagocytic activity at 60-240 min, and the clearance efficiency at 60-120 min significantly decreased in shrimp injected with l-DOPA at 1.0 μmol shrimp -1 . In another experiment, 60 min after shrimp had received l-DOPA at 0.5 or 1.0 μmol shrimp -1 , they were challenged with an injection of V. alginolyticus at 2 × 10 5  colony-forming units (cfu) shrimp -1 . The injection of l-DOPA at 1.0 μmol shrimp -1 also significantly increased the cumulative mortality of shrimp by 16.7%, compared to the HCl-challenged control after 120 h. These results suggest

  15. Assessing potential harmful effects of CdSe quantum dots by using Drosophila melanogaster as in vivo model

    Alaraby, Mohamed [Grup de Mutagènesi, Departament de Genètica i de Microbiologia, Facultat de Biociències, Universitat Autònoma de Barcelona, Campus de Bellaterra, 08193 Cerdanyola del Vallès (Spain); Sohag University, Faculty of Sciences, Zoology Department, 82524-Campus, Sohag (Egypt); Demir, Esref [Akdeniz University, Faculty of Sciences, Department of Biology, 07058-Campus, Antalya (Turkey); Hernández, Alba [Grup de Mutagènesi, Departament de Genètica i de Microbiologia, Facultat de Biociències, Universitat Autònoma de Barcelona, Campus de Bellaterra, 08193 Cerdanyola del Vallès (Spain); CIBER Epidemiología y Salud Pública, ISCIII, Madrid (Spain); Marcos, Ricard, E-mail: ricard.marcos@uab.es [Grup de Mutagènesi, Departament de Genètica i de Microbiologia, Facultat de Biociències, Universitat Autònoma de Barcelona, Campus de Bellaterra, 08193 Cerdanyola del Vallès (Spain); CIBER Epidemiología y Salud Pública, ISCIII, Madrid (Spain)

    2015-10-15

    Since CdSe QDs are increasingly used in medical and pharmaceutical sciences careful and systematic studies to determine their biosafety are needed. Since in vivo studies produce relevant information complementing in vitro data, we promote the use of Drosophila melanogaster as a suitable in vivo model to detect toxic and genotoxic effects associated with CdSe QD exposure. Taking into account the potential release of cadmium ions, QD effects were compared with those obtained with CdCl{sub 2}. Results showed that CdSe QDs penetrate the intestinal barrier of the larvae reaching the hemolymph, interacting with hemocytes, and inducing dose/time dependent significant genotoxic effects, as determined by the comet assay. Elevated ROS production, QD biodegradation, and significant disturbance in the conserved Hsps, antioxidant and p53 genes were also observed. Overall, QD effects were milder than those induced by CdCl{sub 2} suggesting the role of Cd released ions in the observed harmful effects of Cd based QDs. To reduce the observed side-effects of Cd based QDs biocompatible coats would be required to avoid cadmium's undesirable effects. - Highlights: • CdSe QDs were able to cross the intestinal barrier of Drosophila. • Elevated ROS induction was detected in larval hemocytes. • Changes in the expression of Hsps and p53 genes were observed. • Primary DNA damage was induced by CdSe QDs in hemocytes. • Overall, CdSe QD effects were milder than those induced by CdCl{sub 2}.

  16. Bombyx mori cecropin A has a high antifungal activity to entomopathogenic fungus Beauveria bassiana.

    Lu, Dingding; Geng, Tao; Hou, Chengxiang; Huang, Yuxia; Qin, Guangxing; Guo, Xijie

    2016-05-25

    A cDNA encoding cecropin A (CecA) was cloned from the larvae of silkworm, Bombyx mori, using RT-PCR. It encodes a protein of 63 amino acids, containing a 22 amino acid signal peptide and a 37 amino acid mat peptide of functional domain. The CecA secondary structure contains two typical amphiphilic α-helices. Real-time qPCR analysis revealed that CecA was expressed in all the tissues tested, including cuticle, fat body, hemocytes, Malpighian tubule, midgut and silk gland in the silkworm larvae with the highest expression in the fat body and hemocytes. The gene expression of B. mori CecA was rapidly induced by Beauveria bassiana challenge and reached maximum levels at 36h after inoculation in third instar larvae. In the fifth instar larvae infected with B. bassiana, the relative expression level of CecA was upregulated in fat body and hemocytes, but not in cuticle, Malpighian tubule, midgut and silk gland. The cDNA segment of the CecA was inserted into the expression plasmid pET-30a(+) to construct a recombinant expression plasmid. Western blot results revealed that his-tagged fusion protein was successfully expressed and purified. Then the mat peptide of CecA was chemically synthesized with C-terminus amidation for in vivo antifungal assay and purity achieved 93.7%. Mass spectrometry and SDS-PAGE showed its molecular weight to be 4046.95Da. Antifungal assays indicated that the B. mori CecA had a high antifungal activity to entomopathogenic fungus B. bassiana both in vitro and in vivo in the silkworm larvae. This is the first report that the CecA is effective to inhibit B. bassiana inside the body of silkworm. Copyright © 2016 Elsevier B.V. All rights reserved.

  17. Immunity in Protochordates: The Tunicate Perspective

    Nicola Franchi

    2017-06-01

    Full Text Available Tunicates are the closest relatives of vertebrates, and their peculiar phylogenetic position explains the increasing interest toward tunicate immunobiology. They are filter-feeding organisms, and this greatly influences their defense strategies. The majority of the studies on tunicate immunity were carried out in ascidians. The tunic acts as a first barrier against pathogens and parasites. In addition, the oral siphon and the pharynx represent two major, highly vascularized, immune organs, where circulating hemocytes can sense non-self material and trigger immune responses that, usually, lead to inflammation and phagocytosis. Inflammation involves the recruitment of circulating cytotoxic, phenoloxidase (PO-containing cells in the infected area, where they degranulate as a consequence of non-self recognition and release cytokines, complement factors, and the enzyme PO. The latter, acting on polyphenol substrata, produces cytotoxic quinones, which polymerize to melanin, and reactive oxygen species, which induce oxidative stress. Both the alternative and the lectin pathways of complement activation converge to activate C3: C3a and C3b are involved in the recruitment of hemocytes and in the opsonization of foreign materials, respectively. The interaction of circulating professional phagocytes with potentially pathogenic foreign material can be direct or mediated by opsonins, either complement dependent or complement independent. Together with cytotoxic cells, phagocytes are active in the encapsulation of large materials. Cells involved in immune responses, collectively called immunocytes, represent a large fraction of hemocytes, and the presence of a cross talk between cytotoxic cells and phagocytes, mediated by secreted humoral factors, was reported. Lectins play a pivotal role as pattern-recognition receptors and opsonizing agents. In addition, variable region-containing chitin-binding proteins, identified in the solitary ascidian Ciona

  18. Lactobacillus paracasei modulates the immune system of Galleria mellonella and protects against Candida albicans infection.

    Rossoni, Rodnei Dennis; Fuchs, Beth Burgwyn; de Barros, Patrícia Pimentel; Velloso, Marisol Dos Santos; Jorge, Antonio Olavo Cardoso; Junqueira, Juliana Campos; Mylonakis, Eleftherios

    2017-01-01

    Probiotics have been described as a potential strategy to control opportunistic infections due to their ability to stimulate the immune system. Using the non-vertebrate model host Galleria mellonella, we evaluated whether clinical isolates of Lactobacillus spp. are able to provide protection against Candida albicans infection. Among different strains of Lactobacillus paracasei, Lactobacillus rhamnosus and Lactobacillus fermentum, we verified that L. paracasei 28.4 strain had the greatest ability to prolong the survival of larvae infected with a lethal dose of C. albicans. We found that the injection of 107 cells/larvae of L. paracasei into G. mellonella larvae infected by C. albicans increased the survival of these insects compared to the control group (P = 0.0001). After that, we investigated the immune mechanisms involved in the protection against C. albicans infection, evaluating the number of hemocytes and the gene expression of antifungal peptides. We found that L. paracasei increased the hemocyte quantity (2.38 x 106 cells/mL) in relation to the control group (1.29 x 106 cells/mL), indicating that this strain is capable of raising the number of circulating hemocytes into the G. mellonella hemolymph. Further, we found that L. paracasei 28.4 upregulated genes that encode the antifungal peptides galiomicin and gallerymicin. In relation to the control group, L. paracasei 28.4 increased gene expression of galiomicin by 6.67-fold and 17.29-fold for gallerymicin. Finally, we verified that the prophylactic provision of probiotic led to a significant reduction of the number of fungal cells in G. mellonella hemolymph. In conclusion, L. paracasei 28.4 can modulate the immune system of G. mellonella and protect against candidiasis.

  19. Age, pathogen exposure, but not maternal care shape offspring immunity in an insect with facultative family life.

    Vogelweith, Fanny; Körner, Maximilian; Foitzik, Susanne; Meunier, Joël

    2017-03-07

    To optimize their resistance against pathogen infection, individuals are expected to find the right balance between investing into the immune system and other life history traits. In vertebrates, several factors were shown to critically affect the direction of this balance, such as the developmental stage of an individual, its current risk of infection and/or its access to external help such as parental care. However, the independent and/or interactive effects of these factors on immunity remain poorly studied in insects. Here, we manipulated maternal presence and pathogen exposure in families of the European earwig Forficula auricularia to measure whether and how the survival rate and investment into two key immune parameters changed during offspring development. The pathogen was the entomopathogenic fungus Metarhizium brunneum and the immune parameters were hemocyte concentration and phenol/pro-phenoloxidase enzyme activity (total-PO). Our results surprisingly showed that maternal presence had no effect on offspring immunity, but reduced offspring survival. Pathogen exposure also lowered the survival of offspring during their early development. The concentration of hemocytes and the total-PO activity increased during development, to be eventually higher in adult females compared to adult males. Finally, pathogen exposure overall increased the concentration of hemocytes-but not the total-PO activity-in adults, while it had no effect on these measures in offspring. Our results show that, independent of their infection risk and developmental stage, maternal presence does not shape immune defense in young earwigs. This reveals that pathogen pressure is not a universal evolutionary driver of the emergence and maintenance of post-hatching maternal care in insects.

  20. Massively parallel amplicon sequencing reveals isotype-specific variability of antimicrobial peptide transcripts in Mytilus galloprovincialis.

    Umberto Rosani

    Full Text Available BACKGROUND: Effective innate responses against potential pathogens are essential in the living world and possibly contributed to the evolutionary success of invertebrates. Taken together, antimicrobial peptide (AMP precursors of defensin, mytilin, myticin and mytimycin can represent about 40% of the hemocyte transcriptome in mussels injected with viral-like and bacterial preparations, and unique profiles of myticin C variants are expressed in single mussels. Based on amplicon pyrosequencing, we have ascertained and compared the natural and Vibrio-induced diversity of AMP transcripts in mussel hemocytes from three European regions. METHODOLOGY/PRINCIPAL FINDINGS: Hemolymph was collected from mussels farmed in the coastal regions of Palavas (France, Vigo (Spain and Venice (Italy. To represent the AMP families known in M. galloprovincialis, nine transcript sequences have been selected, amplified from hemocyte RNA and subjected to pyrosequencing. Hemolymph from farmed (offshore and wild (lagoon Venice mussels, both injected with 10(7 Vibrio cells, were similarly processed. Amplicon pyrosequencing emphasized the AMP transcript diversity, with Single Nucleotide Changes (SNC minimal for mytilin B/C and maximal for arthropod-like defensin and myticin C. Ratio of non-synonymous vs. synonymous changes also greatly differed between AMP isotypes. Overall, each amplicon revealed similar levels of nucleotidic variation across geographical regions, with two main sequence patterns confirmed for mytimycin and no substantial changes after immunostimulation. CONCLUSIONS/SIGNIFICANCE: Barcoding and bidirectional pyrosequencing allowed us to map and compare the transcript diversity of known mussel AMPs. Though most of the genuine cds variation was common to the analyzed samples we could estimate from 9 to 106 peptide variants in hemolymph pools representing 100 mussels, depending on the AMP isoform and sampling site. In this study, no prevailing SNC patterns related

  1. Protection from Oxidative Stress in Immunocytes of the Colonial Ascidian Botryllus schlosseri: Transcript Characterization and Expression Studies.

    Franchi, Nicola; Ballin, Francesca; Ballarin, Loriano

    2017-02-01

    Botryllus schlosseri is a cosmopolitan colonial ascidian that undergoes cyclical generation changes, or take-overs, during which adult zooids are resorbed and replaced by their buds. At take-over, adult tissues undergo diffuse apoptosis and effete cells are massively ingested by circulating phagocytes, with a consequent increase in oxygen consumption and in production of reactive oxygen species (ROS). The latter are responsible for the death of phagocytes involved in the clearance of apoptotic cells and corpses by phagocytosis-induced apoptosis. However, the majority of phagocytes and hemocytes do not die, even if they experience oxidative stress. This fact suggests the presence of detoxification mechanisms assuring their protection. To test this assumption, we searched for transcripts of genes involved in detoxification in the transcriptome of B. schlosseri. We identified and characterized transcripts for Cu/Zn superoxide dismutase (SOD), γ-glutamyl-cysteine ligase modulatory subunit (GCLM), glutathione synthase (GS), and two glutathione peroxidases (i.e., GPx3 and GPx5), all involved in protection from ROS. We also carried out a phylogenetic analysis of the putative amino acid sequences, confirming their similarity to their vertebrate counterparts, and studied the location of their mRNAs by in situ hybridization on hemocyte monolayers. We also analyzed gene transcription during the colonial blastogenetic cycle, which is the interval of time between one take-over and the next, by qRT-PCR. In addition, we investigated the effects of cadmium (Cd), an inducer of oxidative stress, on gene transcription. Our results indicated that i) antioxidant gene expression is modulated in the course of the blastogenetic cycle and upon exposure to Cd, and ii) hemocytes synthesize both enzymatic and nonenzymatic antioxidants, in line with the idea that they represent a major detoxification system for ascidians.

  2. Lactobacillus paracasei modulates the immune system of Galleria mellonella and protects against Candida albicans infection

    Rossoni, Rodnei Dennis; Fuchs, Beth Burgwyn; de Barros, Patrícia Pimentel; Velloso, Marisol dos Santos; Jorge, Antonio Olavo Cardoso; Junqueira, Juliana Campos; Mylonakis, Eleftherios

    2017-01-01

    Probiotics have been described as a potential strategy to control opportunistic infections due to their ability to stimulate the immune system. Using the non-vertebrate model host Galleria mellonella, we evaluated whether clinical isolates of Lactobacillus spp. are able to provide protection against Candida albicans infection. Among different strains of Lactobacillus paracasei, Lactobacillus rhamnosus and Lactobacillus fermentum, we verified that L. paracasei 28.4 strain had the greatest ability to prolong the survival of larvae infected with a lethal dose of C. albicans. We found that the injection of 107 cells/larvae of L. paracasei into G. mellonella larvae infected by C. albicans increased the survival of these insects compared to the control group (P = 0.0001). After that, we investigated the immune mechanisms involved in the protection against C. albicans infection, evaluating the number of hemocytes and the gene expression of antifungal peptides. We found that L. paracasei increased the hemocyte quantity (2.38 x 106 cells/mL) in relation to the control group (1.29 x 106 cells/mL), indicating that this strain is capable of raising the number of circulating hemocytes into the G. mellonella hemolymph. Further, we found that L. paracasei 28.4 upregulated genes that encode the antifungal peptides galiomicin and gallerymicin. In relation to the control group, L. paracasei 28.4 increased gene expression of galiomicin by 6.67-fold and 17.29-fold for gallerymicin. Finally, we verified that the prophylactic provision of probiotic led to a significant reduction of the number of fungal cells in G. mellonella hemolymph. In conclusion, L. paracasei 28.4 can modulate the immune system of G. mellonella and protect against candidiasis. PMID:28267809

  3. Characterization of a novel anti-lipopolysaccharide factor isoform (SpALF5) in mud crab, Scylla paramamosain.

    Sun, Wanwei; Wan, Weisong; Zhu, Shuo; Wang, Shasha; Wang, Shuqi; Wen, Xiaobo; Zheng, Huaiping; Zhang, Yueling; Li, Shengkang

    2015-04-01

    Anti-lipopolysaccharide factors (ALFs), the potential antimicrobial peptides that bind and neutralize lipopolysaccharide (LPS), are common effectors of innate immunity in crustaceans. In this study, a novel isoform of ALFs (SpALF5) was isolated from the hemocytes of mud crab Scylla paramamosain. The full-length 975bp SpALF5 contains a 375bp open reading frame (ORF) encoding 125 amino acids. Although SpALF5 exhibits a low degree of nucleotide homology with other reported ALFs, it contains the conserved amino acid sequence with a signal peptide and a LPS-binding domain including two conservative cysteine residues. The genomic organization of SpALF5 consists of four exons and three introns, with each intron containing one or more tandem repeats. Unlike most of ALFs mainly distributed in crab hemocytes, SpALF5 transcript was predominantly observed in the brain, muscle and skin, while barely detected in the hemocytes in our study. In situ hybridization assay also showed that SpALF5 mRNA was localized in brain, muscle and skin tissues of mud crab. Further, SpALF5 transcript was significantly up-regulated after challenge with LPS, polyinosinic polycytidylic acid (PolyI:C) (with the except of that in brain), Vibrio parahemolyticus or white spot syndrome virus (WSSV). The recombinant SpALF5 protein showed a varying degree of binding activity towards bacteria and fungus. Moreover, in vitro, the recombinant SpALF5 revealed a strong antimicrobial activity against Gram-negative bacteria (V. parahemolyticus, Vibrio alginolyticus, Escherichia coli, Aeromonas hydrophila) and fungus (Sacchromyces cerevisiae), but could only inhibited the growth of some Gram-positive bacteria like Staphylococcus aureus. The results suggest that SpALF5 is a potent immune protector and plays an important role in immune defense against invading pathogens in S. paramamosain. Copyright © 2014 Elsevier Ltd. All rights reserved.

  4. Cloning and characterisation of the SpToll gene from green mud crab, Scylla paramamosain.

    Lin, Zhongyang; Qiao, Jie; Zhang, Yueling; Guo, Lingling; Huang, He; Yan, Fang; Li, Yuanyou; Wang, Xiuying

    2012-05-01

    Toll/Toll-like receptors (TLRs), one of the most important pattern recognition receptors (PRRs), play a crucial role in innate immune responses in both invertebrates and vertebrates. In this study, we cloned and characterised a Toll gene from Scylla paramamosain (SpToll). Bioinformatic analysis predicted that SpToll contained one open reading frame of 3018bp and encoded a single-pass transmembrane domain protein of 1005 amino acids. Further, SpToll could be clustered into one branch along with other arthropod Tolls in a phylogenetic tree. SpToll transcripts could be detected by RT-PCR from all tissues examined including the heart, gill, hepatopancreas, stomach, intestine, muscle, eyestalk and hemocytes. Infection by Vibrio parahemolyticus up-regulated SpToll mRNA expression in hemocytes after 48h. The profile of single nucleotide polymorphisms (SNPs) in the leucine-rich repeats (LRRs) domain of SpToll in three healthy crabs was then evaluated. Two hundred and twenty SNPs with a frequency of about 1.0-4.0% were identified in hemocyte DNA/cDNA. Surprisingly, the adenine to guanine transition at position 1372 (c.1372A>G) had a frequency of about 50%. Finally, the results showed that challenge with V. parahemolyticus stimulated the appearance of two sets of SNPs in crabs. More importantly, the c.1372A>G mutation could contribute to a low mortality after V. parahemolyticus infection and introduce variation of charge and secondary structure into the SpToll polypeptide. In summary, these studies suggested a novel Toll homologue in crab and identified a SNP with potential pathogen-resistant activities. The result will be important for the investigation of crab immune defense mechanisms. Copyright © 2011 Elsevier Ltd. All rights reserved.

  5. Molecular cloning of Kuruma shrimp Marsupenaeus japonicus endonuclease-reverse transcriptase and its positive role in white spot syndrome virus and Vibrio alginolyticus infection.

    Ma, Xiongchao; Sun, Baozhen; Zhu, Fei

    2018-02-01

    This study investigated the function of endonuclease-reverse transcriptase (mjERT) in Marsupenaeus japonicus. The 1129 bp cDNA sequence of mjERT was cloned from M. japonicus using rapid amplification of cDNA ends (RACE) PCR, and RT-qPCR analysis indicated that mjERT was highly expressed in the gills and hepatopancreas of M. japonicus. We also found that white spot syndrome virus (WSSV) or Vibrio alginolyticus challenge could enhance the expression of mjERT. When mjERT was inhibited, immune genes such as toll, p53, hemocyanin and tumor necrosis factor-α (TNF-α) were significantly down-regulated (P shrimp, while myosin was significantly up-regulated (P shrimps was significantly increased following mjERT RNA interfere (RNAi). Apoptosis data provided information to suggest that mjERT-dsRNA challenge caused less apoptosis in hemocytes in both the disease-free and viral group. We also revealed that mjERT-dsRNA treatment resulted in a lower phagocytosis rate in the hemocytes of V. alginolyticus-challenged shrimp. Finally, we found that the absence of mjERT had an significantly negative impact upon shrimp phenoloxidase (PO) activity, superoxide dismutase (SOD) activity and total hemocyte count (THC) following WSSV or V. alginolyticus infection, indicating a regulative role for mjERT in the innate immunity of shrimp in response to pathogenic infection. In summary, we concluded that mjERT might promote the anti-WSSV immune response of shrimp by regulating apoptosis, PO activity, THC and SOD activity, and also exert a positive role in the immune response against V. alginolyticus by regulating phagocytosis, SOD activity, PO activity and THC. Copyright © 2017 Elsevier Ltd. All rights reserved.

  6. Tsetse immune system maturation requires the presence of obligate symbionts in larvae.

    Brian L Weiss

    2011-05-01

    Full Text Available Beneficial microbial symbionts serve important functions within their hosts, including dietary supplementation and maintenance of immune system homeostasis. Little is known about the mechanisms that enable these bacteria to induce specific host phenotypes during development and into adulthood. Here we used the tsetse fly, Glossina morsitans, and its obligate mutualist, Wigglesworthia glossinidia, to investigate the co-evolutionary adaptations that influence the development of host physiological processes. Wigglesworthia is maternally transmitted to tsetse's intrauterine larvae through milk gland secretions. We can produce flies that lack Wigglesworthia (Gmm(Wgm- yet retain their other symbiotic microbes. Such offspring give rise to adults that exhibit a largely normal phenotype, with the exception being that they are reproductively sterile. Our results indicate that when reared under normal environmental conditions Gmm(Wgm- adults are also immuno-compromised and highly susceptible to hemocoelic E. coli infections while age-matched wild-type individuals are refractory. Adults that lack Wigglesworthia during larval development exhibit exceptionally compromised cellular and humoral immune responses following microbial challenge, including reduced expression of genes that encode antimicrobial peptides (cecropin and attacin, hemocyte-mediated processes (thioester-containing proteins 2 and 4 and prophenoloxidase, and signal-mediating molecules (inducible nitric oxide synthase. Furthermore, Gmm(Wgm- adults harbor a reduced population of sessile and circulating hemocytes, a phenomenon that likely results from a significant decrease in larval expression of serpent and lozenge, both of which are associated with the process of early hemocyte differentiation. Our results demonstrate that Wigglesworthia must be present during the development of immature progeny in order for the immune system to function properly in adult tsetse. This phenomenon provides

  7. Pathogen-Specific Binding Soluble Down Syndrome Cell Adhesion Molecule (Dscam Regulates Phagocytosis via Membrane-Bound Dscam in Crab

    Xue-Jie Li

    2018-04-01

    Full Text Available The Down syndrome cell adhesion molecule (Dscam gene is an extraordinary example of diversity that can produce thousands of isoforms and has so far been found only in insects and crustaceans. Cumulative evidence indicates that Dscam may contribute to the mechanistic foundations of specific immune responses in insects. However, the mechanism and functions of Dscam in relation to pathogens and immunity remain largely unknown. In this study, we identified the genome organization and alternative Dscam exons from Chinese mitten crab, Eriocheir sinensis. These variants, designated EsDscam, potentially produce 30,600 isoforms due to three alternatively spliced immunoglobulin (Ig domains and a transmembrane domain. EsDscam was significantly upregulated after bacterial challenge at both mRNA and protein levels. Moreover, bacterial specific EsDscam isoforms were found to bind specifically with the original bacteria to facilitate efficient clearance. Furthermore, bacteria-specific binding of soluble EsDscam via the complete Ig1–Ig4 domain significantly enhanced elimination of the original bacteria via phagocytosis by hemocytes; this function was abolished by partial Ig1–Ig4 domain truncation. Further studies showed that knockdown of membrane-bound EsDscam inhibited the ability of EsDscam with the same extracellular region to promote bacterial phagocytosis. Immunocytochemistry indicated colocalization of the soluble and membrane-bound forms of EsDscam at the hemocyte surface. Far-Western and coimmunoprecipitation assays demonstrated homotypic interactions between EsDscam isoforms. This study provides insights into a mechanism by which soluble Dscam regulates hemocyte phagocytosis via bacteria-specific binding and specific interactions with membrane-bound Dscam as a phagocytic receptor.

  8. Kazal-type proteinase inhibitor from disk abalone (Haliotis discus discus): molecular characterization and transcriptional response upon immune stimulation.

    Wickramaarachchi, W D Niroshana; De Zoysa, Mahanama; Whang, Ilson; Wan, Qiang; Lee, Jehee

    2013-09-01

    Proteinases and proteinase inhibitors are involved in several biological and physiological processes in all multicellular organisms. Proteinase inhibitors play a key role in regulating the activity of the respective proteinases. Among serine proteinase inhibitors, kazal-type proteinase inhibitors (KPIs) are widely found in mammals, avians, and a variety of invertebrates. In this study, we describe the identification of a kazal-type serine proteinase inhibitor (Ab-KPI) from the disk abalone, Haliotis discus discus, which is presumably involved in innate immunity. The full-length cDNA of Ab-KPI includes 600 bp nucleotides with an open reading frame (ORF) encoding a polypeptide of 143 amino acids. The deduced amino acid sequence of Ab-KPI contains a putative 17-amino acid signal peptide and two tandem kazal domains with high similarity to other kazal-type SPIs. Each kazal domain consists of reactive site (P1) residue containing a leucine (L), and a threonine (T) located in the second amino acid position after the second conserved cysteine of each domain. Temporal expression of Ab-KPI was assessed by real time quantitative PCR in hemocytes and mantle tissue following bacterial and viral hemorrhagic septicemia virus (VHSV) challenge, and tissue injury. At 6 h post-bacterial and -VHSV challenge, Ab-KPI expression in hemocytes was increased 14-fold and 4-fold, respectively, compared to control samples. The highest up-regulations upon tissue injury were shown at 9 h and 12 h in hemocytes and mantle, respectively. The transcriptional modulation of Ab-KPI following bacterial and viral challenges and tissue injury indicates that it might be involved in immune defense as well as wound healing process in abalone. Copyright © 2013 Elsevier Ltd. All rights reserved.

  9. Developing a Drosophila Model of Schwannomatosis

    2013-02-01

    processed for ChIP as described above. Cell culture and dsRNA S2 cells were cultured at 25°C in Schneider’s insect medium (Sigma; 10% fetal bovine serum...destroy pathogens. In Drosophila, circulating blood cells called hemocytes phagocytose bacteria, fungi, and parasitic wasp eggs [28]. RBF1 and dCAP-D3...hTERT-RPE-1 cells were grown in Dulbecco’sModified Essential Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin

  10. Respiratory and Metabolic Impacts of Crustacean Immunity: Are there Implications for the Insects?

    Burnett, Karen G; Burnett, Louis E

    2015-11-01

    Extensive similarities in the molecular architecture of the crustacean immune system to that of insects give credence to the current view that the Hexapoda, including Insecta, arose within the clade Pancrustacea. The crustacean immune system is mediated largely by hemocytes, relying on suites of pattern recognition receptors, effector functions, and signaling pathways that parallel those of insects. In crustaceans, as in insects, the cardiovascular system facilitates movement of hemocytes and delivery of soluble immune factors, thereby supporting immune surveillance and defense along with other physiological functions such as transport of nutrients, wastes, and hormones. Crustaceans also rely heavily on their cardiovascular systems to mediate gas exchange; insects are less reliant on internal circulation for this function. Among the largest crustaceans, the decapods have developed a condensed heart and a highly arteriolized cardiovascular system that supports the metabolic demands of their often large body size. However, recent studies indicate that mounting an immune response can impair gas exchange and metabolism in their highly developed vascular system. When circulating hemocytes detect the presence of potential pathogens, they aggregate rapidly with each other and with the pathogen. These growing aggregates can become trapped in the microvasculature of the gill where they are melanized and may be eliminated at the next molt. Prior to molting, trapped aggregates of hemocytes also can impair hemolymph flow and oxygenation at the gill. Small shifts to anaerobic metabolism only partially compensate for this decrease in oxygen uptake. The resulting metabolic depression is likely to impact other energy-expensive cellular processes and whole-animal performance. For crustaceans that often live in microbially-rich, but oxygen-poor aquatic environments, there appear to be distinct tradeoffs, based on the gill's multiple roles in respiration and immunity. Insects have

  11. Immunotoxicity and genotoxicity testing for in-flight experiments under microgravity

    Hansen, Peter-Diedrich; Hansen, Peter-Diedrich; Unruh, Eckehardt

    Life Sciences as Related to Space (F) Influence of Spaceflight Environment on Biological Systems (F44) Immunotoxicity and genotoxicity testing for In-flight experiments under microgravity Sensing approaches for ecosystem and human health Author: Peter D. Hansen Technische Universit¨t Berlin, Faculty VI - Planen, Bauen, Umwelt, a Institute for Ecological Research and Technology, Department for Ecotoxicology, Berlin, Germany Peter-diedrich.hansen@tu-berlin.de Eckehardt Unruh Technische Universit¨t Berlin, Faculty VI - Planen, Bauen, Umwelt, Institute a for Ecological Research and Technology, Department for Ecotoxicology, Berlin, Germany An immune response by mussel hemocytes is the selective reaction to particles which are identified as foreign by its immune system shown by phagocytosis. Phagocytotic activity is based on the chemotaxis and adhesion, ingestion and phagosome formation. The attachment at the surface of the hemocytes and consequently the uptake of the particles or bacteria can be directly quantified in the format of a fluorescent assay. Another relevant endpoint of phagocytosis is oxidative burst measured by luminescence. Phagocytosis-related production of ROS will be stimulated with opsonised zymosan. The hemocytes will be stored frozen at -80oC and reconstituted in-flight for the experiment. The assay system of the TRIPLELUX-B Experiment has been performed with a well-defined quantification and evaluation of the immune function phagocytosis. The indicator cells are the hemocytes of blue mussels (Mytilus edulis). The signals of the immuno cellular responses are translated into luminescence as a rapid optical reporter system. The results expected will determine whether the observed responses are caused by microgravity and/or radiation (change in permeability, endpoints in genotoxicity: DNA unwinding). The samples for genotoxicity will be processed after returning to earth. The immune system of invertebrates has not been studied so far in space. The

  12. BgTEP: An Antiprotease Involved in Innate Immune Sensing in Biomphalaria glabrata

    Anaïs Portet

    2018-05-01

    Full Text Available Insect thioester-containing protein (iTEP is the most recently defined group among the thioester-containing protein (TEP superfamily. TEPs are key components of the immune system, and iTEPs from flies and mosquitoes were shown to be major immune weapons. Initially characterized from insects, TEP genes homologous to iTEP were further described from several other invertebrates including arthropods, cniderians, and mollusks albeit with few functional characterizations. In the freshwater snail Biomphalaria glabrata, a vector of the schistosomiasis disease, the presence of a TEP protein (BgTEP was previously described in a well-defined immune complex involving snail lectins (fibrinogen-related proteins and schistosome parasite mucins (SmPoMuc. To investigate the potential role of BgTEP in the immune response of the snail, we first characterized its genomic organization and its predicted protein structure. A phylogenetic analysis clustered BgTEP in a well-conserved subgroup of mollusk TEP. We then investigated the BgTEP expression profile in different snail tissues and followed immune challenges using different kinds of intruders during infection kinetics. Results revealed that BgTEP is particularly expressed in hemocytes, the immune-specialized cells in invertebrates, and is secreted into the hemolymph. Transcriptomic results further evidenced an intruder-dependent differential expression pattern of BgTEP, while interactome experiments showed that BgTEP is capable of binding to the surface of different microbes and parasite either in its full length form or in processed forms. An immunolocalization approach during snail infection by the Schistosoma mansoni parasite revealed that BgTEP is solely expressed by a subtype of hemocytes, the blast-like cells. This hemocyte subtype is present in the hemocytic capsule surrounding the parasite, suggesting a potential role in the parasite clearance by encapsulation. Through this work, we report the first

  13. Modulating effects of orally supplied Euglena gracilis on the physiological responses of the freshwater mussel Diplodon chilensis, exposed to sewage water pollution in a Patagonian river (Argentina).

    Bianchi, Virginia A; Castro, Juan M; Rocchetta, Iara; Conforti, Visitación; Pascual, Mariano; Luquet, Carlos M

    2016-04-01

    In order to test if orally supplied Euglena sp. cells modulate the physiological status of bivalves during bioremediation procedures, we evaluated the effect of Euglena gracilis diet on the immune response, oxidative balance and metabolic condition of Diplodon chilensis exposed to sewage water pollution. Mussels were fed for 90 days with E. gracilis (EG) or Scenedesmus vacuolatus (SV, control diet), and then exposed for 10 days at three sites along the Pocahullo river basin: 1) an unpolluted site, upstream of the city (control, C); 2) upstream (UpS) and 3) downstream (DoS) from the main tertiary-treated sewage discharge, in the city of San Martín de los Andes, Northwest Patagonia, Argentina. Our results show that the total hemocyte number decreases while pollution load increases along the river course for both, EG and SV mussels. Phagocytic activity is higher in EG mussels than in SV ones under all conditions. Reactive oxygen species (ROS) production in hemocytes increases with the increase in the pollution load, being significantly higher for EG mussels than for SV ones at DoS; no changes are observed for total oxyradical scavenging capacity (TOSC). Hemocytes' viability is increased for E. gracilis diet at C and remains unchanged in this group of mussels when exposed at the polluted sites. Lysosomal membrane stability is higher in EG mussels than in SV ones for all conditions, although it is decreased at polluted sites compared with that at C. Antioxidant (catalase) and detoxifying (gluthatione S-transferase) defenses are generally lower in gills and digestive gland of EG mussels than in SV ones. Lipid peroxidation (TBARS) is evident in gills of EG mussels at C, and in digestive gland of the same group, at all the sites. Gill mass factor (GF) is affected by the E. gracilis diet; it is increased at C and decreased at polluted sites when compared with that of SV ones. Digestive gland mass factor (DGF) is higher in EG mussels than in SV ones. In D

  14. Infection of silkworm larvae by the entomopathogenic fungus Metarhizium anisopliae

    Lucineia de Fátima Chasko Ribeiro

    Full Text Available ABSTRACT: The isolate E9 of Metarhizium anisopliae was used in commercial hybrids of Bombyx mori larvae to evaluate its biological effect. Symptomatological analyses showed typical signs of fungal infection. Histopathology revealed the presence of large numbers of hemocytes in the hemocoel, and on the sixth dpi the bodies of the insects appeared to be colonised by the fungus. The isolate E9 is pathogenic to larvae B. mori and; therefore, death of the insects was caused by the colonization of fungus in the epidermal and mesodermal tissues.

  15. Molecular and functional analyses of novel anti-lipopolysaccharide factors in giant river prawn (Macrobrachium rosenbergii, De Man) and their expression responses under pathogen and temperature exposure.

    Srisapoome, Prapansak; Klongklaew, Nawanith; Areechon, Nontawith; Wongpanya, Ratree

    2018-06-15

    Anti-lipopolysaccharide factor (ALF) is an immune-related protein that is crucially involved in immune defense mechanisms against invading pathogens in crustaceans. In the current study, three different ALFs of giant river prawn (Mr-ALF3, Mr-ALF8 and Mr-ALF9) were discovered. Based on sequence analysis, Mr-ALF3 and Mr-ALF9 were identified as new members of ALFs in crustaceans (groups F and G, respectively). Structurally, each newly identified Mr-ALF contained three α-helices packed against a four-stranded β-sheet bearing the LPS-binding motif, which usually binds to the cell wall components of bacteria. Tissue expression analysis using quantitative real-time RT-PCR (qRT-PCR) demonstrated that Mr-ALF3 was expressed in most tissues, and the highest expression was in the heart and hemocytes. The Mr-ALF8 gene was highly expressed in the heart, hemocytes, midgut, hepatopacreas and hindgut, respectively, while the Mr-ALF9 gene was modestly expressed in the heart and hemocytes, respectively. The transcriptional responses of the Mr-ALFs to Aeromonas hydrophila and hot/cold temperatures were investigated by qRT-PCR in the gills, hepatopancreas and hemocytes. We found that all Mr-ALFs were clearly suppressed in all tested tissues when the experimental prawns were exposed to extreme temperatures (25 and 35 °C). Moreover, the expression levels of these genes were significantly induced in all examined tissues by 2 different concentrations of A. hydrophila (1 × 10 6 and 1 × 10 9  CFU/ml), particularly 12 and 96 h after the injection. Finally, binding activity analysis of LPS-motif peptides of each Mr-ALF revealed that the LPS peptide of Mr-ALF3 exhibited the strongest adhesion to two pathogenic Gram-negative bacteria, A. hydrophila and Vibrio harveyi, and the non-pathogenic Gram-positive Bacillus megaterium. The results also showed that the Mr-ALF8 and Mr-ALF9 peptides had mild antimicrobial effects against similar tested bacteria. Based on information

  16. viking: identification and characterization of a second type IV collagen in Drosophila.

    Yasothornsrikul, S; Davis, W J; Cramer, G; Kimbrell, D A; Dearolf, C R

    1997-10-01

    We have taken an enhancer trap approach to identify genes that are expressed in hematopoietic cells and tissues of Drosophila. We conducted a molecular analysis of two P-element insertion strains that have reporter gene expression in embryonic hemocytes, strain 197 and vikingICO. This analysis has determined that viking encodes a collagen type IV gene, alpha2(IV). The viking locus is located adjacent to the previously described DCg1, which encodes collagen alpha1(IV), and in the opposite orientation. The alpha2(IV) and alpha1(IV) collagens are structurally very similar to one another, and to vertebrate type IV collagens. In early development, viking and DCg1 are transcribed in the same tissue-specific pattern, primarily in the hemocytes and fat body cells. Our results suggest that both the alpha1 and alpha2 collagen IV chains may contribute to basement membranes in Drosophila. This work also provides the foundation for a more complete genetic dissection of collagen type IV molecules and their developmental function in Drosophila.

  17. Reaction of the mussel Mytilus galloprovincialis (Bivalvia) to Eugymnanthea inquilina (Cnidaria) and Urastoma cyprinae (Turbellaria) concurrent infestation.

    Mladineo, Ivona; Petrić, Mirela; Hrabar, Jerko; Bočina, Ivana; Peharda, Melita

    2012-05-01

    In total 480 individuals of Mytilus galloprovincialis were sampled monthly from October 2009 to September 2010, at the shellfish farm in the Mali Ston Bay, south Adriatic Sea (Croatia) in order to assess the extent of pathology imposed by two parasites, Eugymnanthea inquilina (Cnidaria) and Urastoma cyprinae (Turbellaria). Although a deteriorating impact on host reproduction or condition index was lacking, we evidenced ultrastructural and functional alteration in host cells at the attachment site. Ultrastructural changes included hemocytic encapsulation of the turbellarian and cell desquamation in medusoid infestation. Caspase positive reaction inferred by immunohistochemistry (IHC) was triggered in cases of turbellarian infestation, in contrast with hydroids, suggesting that the former exhibits more complex host-parasite interaction, reflected in the persistent attempts of the parasite to survive bivalve reaction. We have evidenced that both organisms trigger specific host reaction that although not costly in terms of host reproductive cycle or growth, results in mild tissue destruction and hemocyte activation. A lower degree of tissue reaction was observed in cases of hydroid infestation, compared to turbellarian. Copyright © 2012 Elsevier Inc. All rights reserved.

  18. The neonicotinoids thiacloprid, imidacloprid, and clothianidin affect the immunocompetence of honey bees (Apis mellifera L.).

    Brandt, Annely; Gorenflo, Anna; Siede, Reinhold; Meixner, Marina; Büchler, Ralph

    2016-03-01

    A strong immune defense is vital for honey bee health and colony survival. This defense can be weakened by environmental factors that may render honey bees more vulnerable to parasites and pathogens. Honey bees are frequently exposed to neonicotinoid pesticides, which are being discussed as one of the stress factors that may lead to colony failure. We investigated the sublethal effects of the neonicotinoids thiacloprid, imidacloprid, and clothianidin on individual immunity, by studying three major aspects of immunocompetence in worker bees: total hemocyte number, encapsulation response, and antimicrobial activity of the hemolymph. In laboratory experiments, we found a strong impact of all three neonicotinoids. Thiacloprid (24h oral exposure, 200 μg/l or 2000 μg/l) and imidacloprid (1 μg/l or 10 μg/l) reduced hemocyte density, encapsulation response, and antimicrobial activity even at field realistic concentrations. Clothianidin had an effect on these immune parameters only at higher than field realistic concentrations (50-200 μg/l). These results suggest that neonicotinoids affect the individual immunocompetence of honey bees, possibly leading to an impaired disease resistance capacity. Copyright © 2016 Elsevier Ltd. All rights reserved.

  19. Physiological and immunological characterization of Caribbean spiny lobsters Panulirus argus naturally infected with Panulirus argus Virus 1 (PaV1).

    Pascual Jiménez, Cristina; Huchin-Mian, Juan Pablo; Simões, Nuno; Briones-Fourzán, Patricia; Lozano-Álvarez, Enrique; Sánchez Arteaga, Ariadna; Pérez-Vega, Juan Antonio; Simá-Álvarez, Raúl; Rosas Vazquez, Carlos; Rodríguez-Canul, Rossanna

    2012-08-27

    The present study compares 13 physiological and immunological variables between a group of healthy Panulirus argus lobsters and a group of lobsters naturally infected with Panulirus argus Virus 1 (PaV1). Viral infection was determined through histopathology and PCR. Ten of the 13 variables differed significantly between the 2 groups. Using these variables, a principal component analysis yielded 2 separate clusters: one corresponding to the healthy group and the other corresponding to the infected group. In particular, infected lobsters exhibited significantly lower levels of osmotic pressure, total hemocyte counts, plasmatic proteins, and total phenoloxidase (PO) activity in plasma, as well as significantly higher levels of cholesterol and acylglycerides. These features are consistent with metabolic wasting, hyperlipidemia, and presumed immune suppression. Infection with PaV1 appears to increase the susceptibility of lobsters to some other opportunistic pathogens, as 61.1% of infected lobsters presented infestations of ciliate epibionts (Epystilis and Zoothamniun) in the gill chamber compared with 11.5% lobsters in the healthy group. Infected lobsters also showed significantly higher levels of total PO activity in degranulated hemocytes and trypsin inhibitor activity, potentially indicating activation of immune response by the PO system during the systemic infection with PaV1.

  20. Identification and Characterization of Pathogen-Response Genes (repat) in Spodoptera frugiperda (Lepidoptera: Noctuidae).

    Machado, Vilmar; Serrano, Jose; Galián, Jose

    2016-01-01

    The fall armyworm (Spodoptera frugiperda, Noctuidae, Lepidoptera) is one of the most important crop pests in the Americas, causing significant damage to maize, rice and sorghum. The mechanisms that determine its defences against pathogens are particularly relevant for the development of management and control strategies. We used an in silico approach to identify and characterize pathogen response genes (repat) present in different tissue libraries of S. fugiperda. The analyses revealed complete cDNA for nine repat genes; of these, repat15 and repat39 were found in libraries from a specific tissue--the midgut of larvae fed with xenobiotic substances. High expression levels of some genes were found in different libraries: 39 hits in repat30 in challenged hemocytes, 16 hits in repat31 in fat body, 10 hits in repat32 in fat body and 10 in challenged hemocytes, and 10 hits in repat38 in midgut of non-treated larvae and midgut of larvae fed with natural and xenobiotic substances. The genes corresponded to two ontology categories, stress response and immune response, and their phylogenetic relationships, nucleotide similarity, number of amino acid residues and molecular weights agree with what has been described for repat genes. It is noteworthy that proteins encoded by the repat genes of S. frugiperda have important defence functions in other tissues beyond midgut and that their functional categories are likely diverse, as they are related to cell envelope structure, energy metabolism, transport and binding.

  1. The Ciona intestinalis immune-related galectin genes (CiLgals-a and CiLgals-b) are expressed by the gastric epithelium.

    Parrinello, Daniela; Sanfratello, Maria Antonietta; Vizzini, Aiti; Testasecca, Lelia; Parrinello, Nicolò; Cammarata, Matteo

    2017-03-01

    The transcription of two Ciona intestinalis galectin genes (CiLgals-a and CiLgals-b) is uparegulated by LPS in the pharynxis (hemocytes, vessel epithelium, endostilar zones) which is retained the main organ of the immunity. In this ascidian, for the first time we show, by immunohistochemistry and in situ hybridization methods, that these two immune-related genes are expressed in the gastric epithelium of naïve ascidians, whereas the galectins appear to be only contained in the intestine columnar epithelium. In addition, according to previous results on the pharynx, the genes are also expressed and galectins produced by hemocytes scattered in the connective tissue surrounding the gut. The genes expression and galectin localization in several tissues, including the previous findings on the transcription upregulation, the constitutive expression of these genes by endostylar zones and by the gastric epithelium suggest a potential multifunctional role of these galectins. In this respect, it is of interest to define where the CiLgals are normally found as related to the tissue functions. Such an approach should be a starting point for further investigations. Copyright © 2017 Elsevier Ltd. All rights reserved.

  2. Lysosomal membrane stability of the mussel, Mytilus galloprovincialis (L.), as a biomarker of tributyltin exposure.

    Okoro, Hussein K; Snyman, Reinette G; Fatoki, Olalekan S; Adekola, Folahan A; Ximba, Bhekumusa J; Slabber, Michelle Y

    2015-05-01

    The effect of tributyltin (TBT) on the stability of hemocytic lysosome membranes of the mussel, Mytilus galloprovincialis, and the use thereof as a biomarker of TBT-induced stress, was investigated. Mussels were exposed to 0.1 and 1.0 µg/L tributyltin respectively for 4 weeks. Lysosomal membrane stability of hemocytes was tested weekly by means of the neutral red retention time (NRRT) assay, after which the mussel samples were analyzed for TBT content. The two exposed groups exhibited significantly increased (p < 0.05) whole body TBT concentrations with concomitant significant decreases (p < 0.05) in NRRT (R(2) values of 0.85 and 0.971 for lower and higher exposure groups, respectively). The higher exposure group showed a typical dose-response curve. For the control, no TBT was detected and NRRT remained stable. It was concluded that the NRRT assay could be considered as a useful technique, and lysosomal membrane destabilization a useful early warning and cellular biomarker of stress due to TBT exposure in M. galloprovincialis.

  3. Short-term heat shock affects the course of immune response in Galleria mellonella naturally infected with the entomopathogenic fungus Beauveria bassiana.

    Vertyporokh, Lidiia; Taszłow, Paulina; Samorek-Pieróg, Małgorzata; Wojda, Iwona

    2015-09-01

    We aimed to investigate how exposition of infected insects to short-term heat shock affects the biochemical and molecular aspects of their immune response. Galleria mellonella larvae were exposed to 43°C for 15min, at the seventy second hour after natural infection with entomopathogenic fungus Beauveria bassiana. As a result, both qualitative and quantitative changes in hemolymph protein profiles, and among them infection-induced changes in the amount of apolipophorin III (apoLp-III), were observed. Heat shock differently affects the expression of the tested immune-related genes. It transiently inhibits expression of antifungal peptides gallerimycin and galiomicin in both the fat body and hemocytes of infected larvae. The same, although to a lesser extent, concerned apoLp-III gene expression and was observed directly after heat shock. Nevertheless, in larvae that had recovered from heat shock, apoLp-III expression was higher in comparison to unshocked larvae in the fat body but not in hemocytes, which was consistent with the higher amount of this protein detected in the hemolymph of the infected, shocked larvae. Furthermore, lysozyme-type activity was higher directly after heat shock, while antifungal activity was significantly higher also in larvae that had recovered from heat shock, in comparison to the respective values in their non-shocked, infected counterparts. These results show how changes in the external temperature modulate the immune response of G. mellonella suffering from infection with its natural pathogen B. bassiana. Copyright © 2015 Elsevier Inc. All rights reserved.

  4. Effect of Propolis Oral Intake on Physiological Condition of Young Worker Honey Bees, Apis Mellifera L.

    Damiani Natalia

    2017-12-01

    Full Text Available Honey bees collect resin from various plant species and transform it into propolis that is incorporated into the nest. The role of resins in the bee health field is poorly understood. The aim was to evaluate the effects of forced consumption of propolis on the physiological condition and short-term survival of Apis mellifera worker bees. It was tested if the number of circulating hemocytes in hemolymph, the abdominal fat bodies and the hypopharyngeal glands development were affected by the feeding with propolis extracts in laboratory conditions during the warm and the cold seasons. Propolis added to sugar candy was consumed by workers for fourteen days without affecting the bee survival. The number of circulating hemocytes in hemolymph remained constant despite the differential diet during the experiment. However, the development of fat bodies and hypopharyngeal glands was altered by propolis ingestion. The abdominal fat body development in winter bees diminished after fourteen days of propolis consumption, while it increased in summer bees. The hypopharyngeal gland development decreased for the assayed period in workers from both seasons. Our results encourage us to continue exploring this research field and learn how long-term forced ingestion of a plant-derived compound, a non-nutritive substance, can modify physiological bee parameters. A broader understanding of the multiple roles of propolis in the health of the honey bee colonies could be obtained by studying the ways in which it is processed and metabolized and the effect that generates in another physiological responses.

  5. Cecropins from Plutella xylostella and Their Interaction with Metarhizium anisopliae.

    Lina Ouyang

    Full Text Available Cecropins are the most potent induced peptides to resist invading microorganisms. In the present study, two full length cDNA encoding cecropin2 (Px-cec2 and cecropin3 (Px-cec3 were obtained from P. xylostella by integrated analysis of genome and transcriptome data. qRT-PCR analysis revealed the high levels of transcripts of Px-cecs (Px-cec1, Px-cec2 and Px-cec3 in epidermis, fat body and hemocytes after 24, 30 and 36 h induction of Metarhizium anisopliae, respectively. Silencing of Spätzle and Dorsal separately caused the low expression of cecropins in the fat body, epidermis and hemocytes, and made the P.xylostella larvae more susceptible to M. anisopliae. Antimicrobial assays demonstrated that the purified recombinant cecropins, i.e., Px-cec1, Px-cec2 and Px-cec3, exerted a broad spectrum of antimicrobial activity against fungi, as well as Gram-positive and Gram-negative bacteria. Especially, Px-cecs showed higher activity against M. anisopliae than another selected fungi isolates. Scanning electron microscopy (SEM and transmission electron microscopy (TEM revealed that cecropins exerted the vital morphological alterations to the spores of M. anisopliae. Based on our results, cecropins played an imperative role in resisting infection of M. anisopliae, which will provide the foundation of biological control of insect pests by using cecorpins as a target in the future.

  6. Characterization of Ixodes ricinus Fibrinogen-Related Proteins (Ixoderins Discloses Their Function in the Tick Innate Immunity

    Helena Honig Mondekova

    2017-12-01

    Full Text Available Ticks are important vectors of serious human and animal disease-causing organisms, but their innate immunity can fight invading pathogens and may have the ability to reduce or block transmission to mammalian hosts. Lectins, sugar-binding proteins, can distinguish between self and non-self-oligosaccharide motifs on pathogen surfaces. Although tick hemolymph possesses strong lectin activity, and several lectins have already been isolated and characterized, little is known about the implementation of these molecules in tick immunity. Here, we have described and functionally characterized fibrinogen-related protein (FReP lectins in Ixodes ticks. We have shown that the FReP family contains at least 27 genes (ixoderins, ixo that could, based on phylogenetic and expression analyses, be divided into three groups with differing degrees of expansion. By using RNA interference-mediated gene silencing (RNAi we demonstrated that IXO-A was the main lectin in tick hemolymph. Further, we found that ixoderins were important for phagocytosis of Gram-negative bacteria and yeasts by tick hemocytes and that their expression was upregulated upon injection of microbes, wounding, or after blood feeding. However, although the tick hemocytes could swiftly phagocytose Borrelia afzelii spirochetes, their transmission and burst of infection in mice was not altered. Our results demonstrate that tick ixoderins are crucial immune proteins that work as opsonins in the tick hemolymph, targeting microbes for phagocytosis or lysis.

  7. Insect immunology and hematopoiesis.

    Hillyer, Julián F

    2016-05-01

    Insects combat infection by mounting powerful immune responses that are mediated by hemocytes, the fat body, the midgut, the salivary glands and other tissues. Foreign organisms that have entered the body of an insect are recognized by the immune system when pathogen-associated molecular patterns bind host-derived pattern recognition receptors. This, in turn, activates immune signaling pathways that amplify the immune response, induce the production of factors with antimicrobial activity, and activate effector pathways. Among the immune signaling pathways are the Toll, Imd, Jak/Stat, JNK, and insulin pathways. Activation of these and other pathways leads to pathogen killing via phagocytosis, melanization, cellular encapsulation, nodulation, lysis, RNAi-mediated virus destruction, autophagy and apoptosis. This review details these and other aspects of immunity in insects, and discusses how the immune and circulatory systems have co-adapted to combat infection, how hemocyte replication and differentiation takes place (hematopoiesis), how an infection prepares an insect for a subsequent infection (immune priming), how environmental factors such as temperature and the age of the insect impact the immune response, and how social immunity protects entire groups. Finally, this review highlights some underexplored areas in the field of insect immunobiology. Copyright © 2015 Elsevier Ltd. All rights reserved.

  8. Origins and activation of prophenoloxidases in the digestive tract of the cricket, Gryllus bimaculatus.

    Joseph, Woodring

    2014-10-01

    The function of Phenoloxidases (POs) in sclerotization and defense in insects is well understood, but little is known concerning their occurrence, origins, and function in the digestive tract. In Gyrllus bimaculatus gut all of the PO activity is found in the lumen of the digestive tract, and no detectible activity is found in homogenates of the gut epithelium or secretions from incubated epithelial tissues. Prophenoloxidases (PPOs) are synthesized in the hemocytes of  Bombyx mori and are transported into the cuticle. It is suggested that the PPOs in the caecal lumen of G. bimaculatus likewise are synthesized in hemocytes and are transported by unknown means into the caecal lumen, where they are activated to POs by trypsin. Peristalsis transports the POs both forward into the crop and posterior within the peritrophic membrane into the hind gut. The PPOs in the hemolymph consist of a trimer (270-280 kDa) and a tetramer (340-370 kDa). The active POs in the gut lumen consist of a monomer (85-95 kDa) in addition to an activated trimer and tetramer. © 2014 Wiley Periodicals, Inc.

  9. RNA interference silences Microplitis demolitor bracovirus genes and implicates glc1.8 in disruption of adhesion in infected host cells

    Beck, Markus; Strand, Michael R.

    2003-01-01

    The family Polydnaviridae consists of ds-DNA viruses that are symbiotically associated with certain parasitoid wasps. PDVs are transmitted vertically but also are injected by wasps into hosts where they cause several physiological alterations including immunosuppression. The PDV genes responsible for mediating immunosuppression and other host alterations remain poorly characterized in large measure because viral mutants cannot be produced to study gene function. Here we report the use of RNA interference (RNAi) to specifically silence the glc1.8 and egf1.0 genes from Microplitis demolitor bracovirus (MdBV) in High Five cells derived from the lepidopteran Trichoplusia ni. Dose-response studies indicated that MdBV infects High Five cells and blocks the ability of these cells to adhere to culture plates. This response was very similar to what occurs in two classes of hemocytes, granular cells, and plasmatocytes, after infection by MdBV. Screening of monoclonal antibody (mAb) markers that distinguish different classes of lepidopteran hemocytes indicated that High Five cells cross-react with three mAbs that recognize granular cells from T. ni. Double-stranded RNA (dsRNA) complementary to glc1.8 specifically silenced glc1.8 expression and rescued the adhesive phenotype of High Five cells. Reciprocally, dsRNA complementary to egf1.0 silenced egf1.0 expression but had no effect on adhesion. The simplicity and potency of RNAi could be extremely useful for analysis of other PDV genes

  10. Repair and regeneration of heavy ion beam locally irradiated embryonic hemopoietic organs of silkworm, Bombyx mori

    Xu Shiqing; Tu Zhenli

    2003-01-01

    Eggs of silkworm, Bombyx mori were irradiated by 12 C 5+ ions, at the embryonic wings, where the hemopoietic organs attached. Changes in the development of the silkworm larvae and pupae, especially in the repair and regeneration of the hemopoietic organs represented by their morphological and functional characteristics, were observed. The results showed that the silkworm larvae and pupae developed from the irradiated eggs grew with the physiological obstruction due to exsanguinations, such as difficulty in their metamorphosis and their marked lower survival rates. After 100 Gy irradiation, some of the larvae and pupae died during the periods of larval molting stages, pupation stage and eclosion stage. With increased doses, the deficiencies in the silkworm development became more significant. After 200 Gy irradiation, the average hemocyte density for the 5 th larvae decreased markedly, but the hemocyte density for some individuals were comparable to the control, and regenerated hemopoietic organs was found in these larvae. The results suggested that the silkworm larvae might be capable of self-repair and self-regeneration of the hemopoietic organs after the heave ion irradiation

  11. Bombyx mori Serpin6 regulates prophenoloxidase activity and the expression of antimicrobial proteins.

    Li, Bing; Yu, Hai-Zhong; Ye, Chong-Jun; Ma, Yan; Li, Xing; Fan, Tao; Chen, Fu-Sheng; Xu, Jia-Ping

    2017-04-30

    Serpins are a family of serine protease inhibitors that are found widely in insects. They play an important role in insect physiological responses, such as innate immunity and development. In this study, we obtained the Bombyx mori serpin6 (BmSerpin6) sequence from National Center for Biotechnology Information (NCBI) and the silkworm genome database (SilkDB). Reverse transcription PCR (RT-PCR) results showed that BmSerpin6 was expressed highly in hemocytes, the midgut, and the fat body. After challenging with Micrococcus luteus (Mi) and Serratia marcescens (Sm), the BmSerpin6 expression level was induced significantly. Transcript levels of gloverin2 and prophenoloxidase (PPO) activity were reduced significantly in the fat body and hemocytes after injecting the recombinant BmSerpin6 protein into silkworm larvae. A BmSerpin6 recombinant plasmid (BmSerpin6-pAC 5.1) was constructed successfully and transfected into Drosophila S2 cells, which resulted in significantly reduced expression of the drosomycin protein. These results indicated that BmSerpin6 might regulate silkworm immune responses. Copyright © 2017 Elsevier B.V. All rights reserved.

  12. Cationic antimicrobial peptides in penaeid shrimp.

    Tassanakajon, Anchalee; Amparyup, Piti; Somboonwiwat, Kunlaya; Supungul, Premruethai

    2011-08-01

    Penaeid shrimp aquaculture has been consistently affected worldwide by devastating diseases that cause a severe loss in production. To fight a variety of harmful microbes in the surrounding environment, particularly at high densities (of which intensive farming represents an extreme example), shrimps have evolved and use a diverse array of antimicrobial peptides (AMPs) as part of an important first-line response of the host defense system. Cationic AMPs in penaeid shrimps composed of penaeidins, crustins, and anti-lipopolysaccharide factors are comprised of multiple classes or isoforms and possess antibacterial and antifungal activities against different strains of bacteria and fungi. Shrimp AMPs are primarily expressed in circulating hemocytes, which is the main site of the immune response, and hemocytes expressing AMPs probably migrate to infection sites to fight against pathogen invasion. Indeed, most AMPs are produced as early as the nauplii developmental stage to protect shrimp larvae from infections. In this review, we discuss the sequence diversity, expression, gene structure, and antimicrobial activities of cationic AMPs in penaeid shrimps. The information available on antimicrobial activities indicates that these shrimp AMPs have potential therapeutic applications in the control of disease problems in aquaculture.

  13. Identification of an entomopathogenic bacterium, Serratia sp. ANU101, and its hemolytic activity.

    Kim, Yonggyun; Kim, Keunseob; Seo, Jiae; Shrestha, Sony; Kim, Hosanna H; Nalini, Madanagopal; Yi, Youngkeun

    2009-03-01

    Four different bacterial colonies were isolated from an old stock of an entomopathogenic nematode, Steinernema monticolum. They all showed entomopathogenicity to final instar larvae of beet armyworm, Spodoptera exigua, by hemocoelic injection. However, they varied in colony form, susceptibility to antibiotics, and postmortem change of the infected host insects. Biolog microbial identification and 16S rDNA sequence analyses indicate that these are four different species classified into different bacterial genera. owing to high entomopathogenicity and a cadaver color of infected insect host, Serratia sp. was selected as a main symbiotic bacterial species and analyzed for its pathogenicity. Although no virulence of Serratia sp. was detected at oral administration, the bacteria gave significant synergistic pathogenicity to fifth instar S. exigua when it was treated along with a spore-forming entomopathogenic bacterium, Bacillus thuringiensis. The synergistic effect was explained by an immunosuppressive effect of Serratia sp. by its high cytotoxic effect on hemocytes of S. exigua, because Serratia sp. caused septicemia of S. exigua when the bacterial cells were injected into S. exigua hemocoel. The cytotoxic factor(s) was present in the culture medium because the sterilized culture broth possessed high potency in the cytotoxicity, which was specific to granular cells and plasmatocytes, two main immune-associated hemocytes in insects.

  14. Three STATs are involved in the regulation of the expression of antimicrobial peptides in the triangle sail mussel, Hyriopsis cumingii.

    Dai, Yun-Jia; Hui, Kai-Min; Zhang, Ying-Hao; Liu, Yan; Wang, Yu-Qing; Zhao, Li-Juan; Lin, Li; Chai, Lian-Qin; Wei, Shun; Lan, Jiang-Feng

    2017-04-01

    Janus kinase (Jak) and signal transducers and activators of transcription (STAT) signaling pathway is associated in antiviral and antibacterial immune response. Previous studies primarily investigated the function of STATs in mammals. For most invertebrates, only one STAT was found in each species, such as STAT92E was found in Drosophila melanogaster. The studies, which focus on the functional difference between various STATs in the same species of invertebrate, are limited. In the present study, three STATs (HcSTAT1, HcSTAT2 and HcSTAT3) were identified in triangle shell pearl mussel, Hyriopsis cumingii. Phylogenetic analysis showed that HcSTAT1 and HcSTAT3 were clustered with Homo sapiens STAT5, and HcSTAT2 was clustered with Pinctada fucata STAT and Crassostea gigas STAT6. All three STATs could be detected in all tested tissues (hemocytes, hepatopancreas, gill, mantle and foot), and were induced expression when challenged with Staphylococcus aureus or Aeromonas hydrophilia in hemocytes and hepatopancreas. HcSTAT1 regulated the expression of HcDef, HcWAP, HcThe and HcTNF. The expression of HcWAP and HcTNF was down-regulated in HcSTAT2-RNAi mussel. And HcSTAT3 affected the expression of HcTNF. The study is the first report of different functions in antibacterial immune responses between STATs in mollusks. Copyright © 2017 Elsevier Ltd. All rights reserved.

  15. Somatic and Germline Diversification of a Putative Immunoreceptor within One Phylum: Dscam in Arthropods.

    Brites, Daniela; Du Pasquier, Louis

    2015-01-01

    Arthropod Dscam, the homologue of the human Down Syndrome cell adhesion molecule, is a receptor used by the nervous and immune systems. Unlike in vertebrates, evolutionary pressure has selected and maintained a vast Dscam diversity of isoforms, known to specifying neuronal identity during the nervous system differentiation. This chapter examines the different modes of Dscam diversification in the context of arthropods' evolution and that of their immune system, where its role is controversial. In the single Dscam gene of insects and crustaceans, mutually exclusive alternative splicing affects three clusters of duplicated exons encoding the variable parts of the receptor. The Dscam gene produces over 10,000 isoforms. In the more basal arthropods such as centipedes, Dscam diversity results from a combination of many germline genes (over 80) with, in about half of those, the possibility of alternative splicing affecting only one exon cluster. In the even more basal arthropods, such as chelicerates, no splicing possibility is detected, but there exist dozens of germline Dscam genes. Compared to controlling the expression of multiple germline genes, the somatic mutually alternative splicing within a single gene may offer a simplified way of expressing a large Dscam repertoire. Expressed by hemocytes, Dscam is considered a phagocytic receptor but is also encountered in solution. More information is necessary about its binding to pathogens, its role in phagocytosis, its possible role in specifying hemocyte identity, its kinetics of expression, and the regulation of its RNA splicing to understand how its diversity is linked to immunity.

  16. BRICHOS domain-containing leukocyte cell-derived chemotaxin 1-like cDNA from disk abalone Haliotis discus discus.

    Kim, Yucheol; De Zoysa, Mahanama; Lee, Youngdeuk; Whang, Ilson; Lee, Jehee

    2010-11-01

    A BRICHOS domain-containing leukocyte cell-derived chemotaxin 1-like cDNA was cloned from the disk abalone (Haliotis discus discus) and designated as AbLECT-1. A full-length (705 bp) of AbLECT-1 cDNA was composed of a 576 bp open reading frame that translates into a putative peptide of 192 amino acids. Deduced amino acid sequence of AbLECT-1 had 15.5- and 27.8% identity and similarity to human LECT-1, respectively. Quantitative real-time PCR analysis results showed that the mRNA of AbLECT-1 was constitutively expressed in abalone hemocytes, gills, mantle, muscle, digestive tract and hepatopancreas in a tissue-specific manner. Moreover, the AbLECT-1 transcription level was induced in hemocytes after challenge with Vibrio alginolyticus, Vibrio parahemolyticus, and Listeria monocytogenes suggesting that it may be involved in immune response reactions in abalone. Copyright 2010 Elsevier Ltd. All rights reserved.

  17. Identification of a serine proteinase homolog (Sp-SPH) involved in immune defense in the mud crab Scylla paramamosain.

    Zhang, Qiu-xia; Liu, Hai-peng; Chen, Rong-yuan; Shen, Kai-li; Wang, Ke-jian

    2013-01-01

    Clip domain serine proteinase homologs are involved in many biological processes including immune response. To identify the immune function of a serine proteinase homolog (Sp-SPH), originally isolated from hemocytes of the mud crab, Scylla paramamosain, the Sp-SPH was expressed recombinantly and purified for further studies. It was found that the Sp-SPH protein could bind to a number of bacteria (including Aeromonas hydrophila, Escherichia coli, Staphylococcus aureus, Vibrio fluvialis, Vibrio harveyi and Vibrio parahemolyticus), bacterial cell wall components such as lipopolysaccharide or peptidoglycan (PGN), and β-1, 3-glucan of fungus. But no direct antibacterial activity of Sp-SPH protein was shown by using minimum inhibitory concentration or minimum bactericidal concentration assays. Nevertheless, the Sp-SPH protein was found to significantly enhance the crab hemocyte adhesion activity (paired t-test, Pparahemolyticus which were both recognized by Sp-SPH protein, if pre-incubated with Sp-SPH protein, respectively. Whereas, the crabs died much faster when challenged with Vibrio alginolyiicus, a pathogenic bacterium not recognized by Sp-SPH protein, compared to those of crabs challenged with A. hydrophila or V. parahemolyticus when pre-coated with Sp-SPH protein. Taken together, these data suggested that Sp-SPH molecule might play an important role in immune defense against bacterial infection in the mud crab S. paramamosain.

  18. Structural characterization and expression analysis of a beta-thymosin homologue (Tβ) in disk abalone, Haliotis discus discus.

    Kasthuri, Saranya Revathy; Premachandra, H K A; Umasuthan, Navaneethaiyer; Whang, Ilson; Lee, Jehee

    2013-09-15

    Repertoires of proteins and small peptides play numerous physiological roles as hormones, antimicrobial peptides, and cellular signaling factors. The beta-thymosins are a group of small acidic peptides involved in processes such as actin sequestration, neuronal development, wound healing, tissue repair, and angiogenesis. Recent characterization of the beta thymosins as immunological regulators in invertebrates led to our identification and characterization of a beta-thymosin homologue (Tβ) from Haliotis discus discus. The cDNA possessed an ORF of 132 bp encoding a protein of 44 amino acids with a molecular mass of 4977 Da. The amino acid sequence shows high identity with another molluskan beta-thymosin and has a characteristic actin binding motif (LKKTET) and glutamyl donors. Phylogenetic analysis showed a close relationship with molluskan homologues, as well as its distinct identity and common ancestral origin. Genomic analysis revealed a 3 exon-2 intron structure similar to the other homologues. In silico promoter analysis also revealed significant transcription factor binding sites, providing evidence for the expression of this gene under different cellular conditions, including stress or pathogenic attack. Tissue distribution profiling revealed a ubiquitous presence in all the examined tissues, but with the highest expression in mantle and hemocyte. Immune challenge with lipopolysaccharide, poly I:C and Vibrio parahemolyticus induced beta-thymosin expression in gill and hemocytes, affirming an immune-related role in invertebrates. Copyright © 2013 Elsevier B.V. All rights reserved.

  19. Identification of a Serine Proteinase Homolog (Sp-SPH) Involved in Immune Defense in the Mud Crab Scylla paramamosain

    Zhang, Qiu-xia; Liu, Hai-peng; Chen, Rong-yuan; Shen, Kai-li; Wang, Ke-jian

    2013-01-01

    Clip domain serine proteinase homologs are involved in many biological processes including immune response. To identify the immune function of a serine proteinase homolog (Sp-SPH), originally isolated from hemocytes of the mud crab, Scylla paramamosain, the Sp-SPH was expressed recombinantly and purified for further studies. It was found that the Sp-SPH protein could bind to a number of bacteria (including Aeromonas hydrophila, Escherichia coli, Staphylococcus aureus, Vibrio fluvialis, Vibrio harveyi and Vibrio parahemolyticus), bacterial cell wall components such as lipopolysaccharide or peptidoglycan (PGN), and β-1, 3-glucan of fungus. But no direct antibacterial activity of Sp-SPH protein was shown by using minimum inhibitory concentration or minimum bactericidal concentration assays. Nevertheless, the Sp-SPH protein was found to significantly enhance the crab hemocyte adhesion activity (paired t-test, Pparahemolyticus which were both recognized by Sp-SPH protein, if pre-incubated with Sp-SPH protein, respectively. Whereas, the crabs died much faster when challenged with Vibrio alginolyiicus, a pathogenic bacterium not recognized by Sp-SPH protein, compared to those of crabs challenged with A. hydrophila or V. parahemolyticus when pre-coated with Sp-SPH protein. Taken together, these data suggested that Sp-SPH molecule might play an important role in immune defense against bacterial infection in the mud crab S. paramamosain. PMID:23724001

  20. Encapsulated Synbiotic Dietary Supplementation at Different Dosages to Prevent Vibriosis in White Shrimp, Litopenaeus vannamei

    Anis Zubaidah

    2015-10-01

    Full Text Available The aim of this study was to evaluate the effect of encapsulated synbiotic (Bacillus sp. NP5 and oligosaccharide dietary at different dosages on growth performance, survival rate, feed conversion ratio, and immune responses of Litopenaeus vannamei against Vibrio infection. The shrimps of the main treatments were fed by the diet that contained three different dosages of encapsulated synbiotic [0.5% (A, 1% (B, and 2% (C (w/w] with feeding rate of 5% of shrimp biomass (4 times a day. The shrimps of two control treatments (negative control and positive control were fed only by commercial feed without supplementation of encapsulated synbiotic. The growth, feed conversion ratio, and survival rate were observed after 30 days of encapsulated synbiotic dietary. The shrimps were then challenged by injection of Vibrio harveyi (6 log colony forming units/mL 0.1 mL/shrimp, excluded the negative control treatment. Afterward, the survival and immune responses were observed for 9 days after experimental infection. The shrimps treated with 2% encapsulated synbiotic (treatment C in the diet showed the highest growth performance (2.98 ± 0.42%, feed conversion ratio (1.26 ± 0.19, and better immune responses i.e. total hemocyte counts, differential hemocyte count, phenoloxidase, and intestine bacteria observation compared to those of positive control treatment.

  1. Transcriptional profiling of immune-related genes in Pacific white shrimp (Litopenaeus vannamei) during ontogenesis.

    Quispe, Ruth L; Justino, Emily B; Vieira, Felipe N; Jaramillo, Michael L; Rosa, Rafael D; Perazzolo, Luciane M

    2016-11-01

    We have performed here a gene expression analysis to determine the developmental stage at the main genes involved in crustacean immune response begin to be expressed and their changes in mRNA abundance during shrimp development. By using a quantitative PCR-based approach, we have measured the mRNA abundance of 24 immune-related genes from different functional categories in twelve developmental stages ranging from fertilized eggs to larval and postlarval stages and also in juveniles. We showed for the first time that the main genes from the RNAi-based post-transcriptional pathway involved in shrimp antiviral immunity are transcribed in all developmental stages, but exhibit a diverse pattern of gene expression during shrimp ontogenesis. On the other hand, hemocyte-expressed genes mainly involved in antimicrobial defenses appeared to be transcribed in larval stages, indicating that hematopoiesis initiates early in development. Moreover, transcript levels of some genes were early detected in fertilized eggs at 0-4 h post-spawning, suggesting a maternal contribution of immune-related transcripts to shrimp progeny. Altogether, our results provide important clues regarding the ontogenesis of hemocytes as well the establishment of antiviral and antimicrobial defenses in shrimp. Copyright © 2016 Elsevier Ltd. All rights reserved.

  2. Immunomodulatory effects and anti-Candida activity of lactobacilli in macrophages and in invertebrate model of Galleria mellonella.

    de Oliveira, Felipe Eduardo; Rossoni, Rodnei Dennis; de Barros, Patricia Pimentel; Begnini, Barbara Evelyn; Junqueira, Juliana Campos; Jorge, Antonio Olavo Cardoso; Leão, Mariella Vieira Pereira; de Oliveira, Luciane Dias

    2017-09-01

    Due to the growing number of multi-resistant Candida spp., adjuvant treatments that may help combat these fungal pathogens are relevant and useful. This study evaluated the immunomodulation and anti-Candida activity of Lactobacillus rhamnosus (LR), Lactobacillus acidophilus and Lactobacillus paracasei suspensions, either single- or multiple-strain, in mouse macrophages (RAW 264.7) and Galleria mellonella (GM). Mouse macrophages were activated by different lactobacilli suspensions and challenged with C. albicans (CA). Tumor necrosis factor (TNF)-α, interleukin IL-1β, IL-6 and IL-17 production and cell viability were investigated. LR was the best suspension for stimulating all evaluated cytokines and thus was used in subsequent in vivo assays. Two C. albicans clinical strains, CA21 and CA60, were then added to the GM assays to further confirm the results. LR suspension was injected into the larvae 24 h before challenging with CA. Survival curve, CFU per larva and hemocytes were counted. In the GM, the LR suspension increased the survival rate and hemocyte counts and decreased the CFU per larva counts for all groups. Lactobacilli suspensions presented strain-dependent immunomodulation; however, single suspensions showed better results. Anti-Candida activity was demonstrated by decreased Candida counts in the GM with the use of LR. Copyright © 2017 Elsevier Ltd. All rights reserved.

  3. Isolation, cDNA cloning and gene expression of an antibacterial protein from larvae of the coconut rhinoceros beetle, Oryctes rhinoceros.

    Yang, J; Yamamoto, M; Ishibashi, J; Taniai, K; Yamakawa, M

    1998-08-01

    An antibacterial protein, designated rhinocerosin, was purified to homogeneity from larvae of the coconut rhinoceros beetle, Oryctes rhinoceros immunized with Escherichia coli. Based on the amino acid sequence of the N-terminal region, a degenerate primer was synthesized and reverse-transcriptase PCR was performed to clone rhinocerosin cDNA. As a result, a 279-bp fragment was obtained. The complete nucleotide sequence was determined by sequencing the extended rhinocerosin cDNA clone by 5' rapid amplification of cDNA ends. The deduced amino acid sequence of the mature portion of rhinocerosin was composed of 72 amino acids without cystein residues and was shown to be rich in glycine (11.1%) and proline (11.1%) residues. Comparison of the deduced amino acid sequence of rhinocerosin with those of other antibacterial proteins indicated that it has 77.8% and 44.6% identity with holotricin 2 and coleoptrecin, respectively. Rhinocerosin had strong antibacterial activity against E. coli, Streptococcus pyogenes, Staphylococcus aureus but not against Pseudomonas aeruginosa. Results of reverse-transcriptase PCR analysis of gene expression in different tissues indicated that the rhinocerosin gene is strongly expressed in the fat body and the Malpighian tubule, and weakly expressed in hemocytes and midgut. In addition, gene expression was inducible by bacteria in the fat body, the Malpighian tubule and hemocyte but constitutive expression was observed in the midgut.

  4. Parallel and costly changes to cellular immunity underlie the evolution of parasitoid resistance in three Drosophila species.

    John E McGonigle

    2017-10-01

    Full Text Available A priority for biomedical research is to understand the causes of variation in susceptibility to infection. To investigate genetic variation in a model system, we used flies collected from single populations of three different species of Drosophila and artificially selected them for resistance to the parasitoid wasp Leptopilina boulardi, and found that survival rates increased 3 to 30 fold within 6 generations. Resistance in all three species involves a large increase in the number of the circulating hemocytes that kill parasitoids. However, the different species achieve this in different ways, with D. melanogaster moving sessile hemocytes into circulation while the other species simply produce more cells. Therefore, the convergent evolution of the immune phenotype has different developmental bases. These changes are costly, as resistant populations of all three species had greatly reduced larval survival. In all three species resistance is only costly when food is in short supply, and resistance was rapidly lost from D. melanogaster populations when food is restricted. Furthermore, evolving resistance to L. boulardi resulted in cross-resistance against other parasitoids. Therefore, whether a population evolves resistance will depend on ecological conditions including food availability and the presence of different parasite species.

  5. Chinese mitten crab (Eriocheir sinensis) iron-sulphur cluster assembly protein 2 (EsIscA2) is differentially regulated after immune and oxidative stress challenges.

    Zhang, Peng; Liu, Yu; Wang, Min; Dong, Miren; Liu, Zhaoqun; Jia, Zhihao; Wang, Weilin; Zhang, Anguo; Wang, Lingling; Song, Linsheng

    2018-07-01

    Iron-sulphur clusters (ISCs), one of the oldest and most versatile cofactors of proteins, are involved in catalysis reactions, electron transport reactions, regulation processes as well as sensing of ambient conditions. Iron-sulphur cluster assembly protein (IscA) is a scaffold protein member of ISC formation system, which plays a significant role in the assembly and maturation process of ISC proteins. In the present study, the cDNA sequence of iron-sulphur cluster assembly protein 2 (designated as EsIscA2) was cloned from Eriocheir sinensis. The open reading frame (ORF) of EsIscA2 was of 507 bp, encoding a peptide of 168 amino acids with a typically conserved Fe-S domain. A tetrameric form was predicated by the SWISS-MODEL prediction algorithm, and three conserved cysteine residues (Cys-93, Cys-158, Cys-160) from each IscA monomer were predicted to form a 'cysteine pocket'. The deduced amino acid sequence of EsIscA2 shared over 50% similarity with that of other IscAs. EsIscA2 was clustered with IscA2 proteins from invertebrates and vertebrates, indicating that the protein was highly conservative in the evolution. rEsIscA2 exhibited a high iron binding affinity in the concentration ranging from 2 to 200 μM. EsIscA2 transcripts were detected in all the tested tissues including gonad, hemocytes, gill, muscle, heart, hepatopancreas and eyestalk, and EsIscA2 protein was detected in the mitochondria of hemocytes. The highest mRNA expression level of EsIscA2 was detected in muscle and hepatopancreas, which was about 34.66-fold (p < 0.05) and 27.07-fold (p < 0.05) of that in hemocytes, respectively. After Aeromonas hydrophila and lipopolysaccharide (LPS) stimulations, the mRNA expression of EsIscA2 in hemocytes was down-regulated and reached the lowest level at 24 h (0.31-fold, p < 0.05) and 48 h (0.29-fold, p < 0.05) compared to control group, respectively. And the expression of EsIscA2 mRNA in hepatopancreas was repressed from 6 h to 48 h post

  6. Oxidative stress induced by chlorine dioxide as an insecticidal factor to the Indian meal moth, Plodia interpunctella.

    Kumar, Sunil; Park, Jiyeong; Kim, Eunseong; Na, Jahyun; Chun, Yong Shik; Kwon, Hyeok; Kim, Wook; Kim, Yonggyun

    2015-10-01

    A novel fumigant, chlorine dioxide (ClO2) is a commercial bleaching and disinfection agent. Recent study indicates its insecticidal activity. However, its mode of action to kill insects is yet to be understood. This study set up a hypothesis that an oxidative stress induced by ClO2 is a main factor to kill insects. The Indian meal moth, Plodia interpunctella, is a lepidopteran insect pest infesting various stored grains. Larvae of P. interpunctella were highly susceptible to ClO2 gas, which exhibited an acute toxicity. Physiological damages by ClO2 were observed in hemocytes. At high doses, the larvae of P. interpunctella suffered significant reduction of total hemocytes. At low doses, ClO2 impaired hemocyte behaviors. The cytotoxicity of ClO2 was further analyzed using two insect cell lines, where Sf9 cells were more susceptible to ClO2 than High Five cells. The cells treated with ClO2 produced reactive oxygen species (ROS). The produced ROS amounts increased with an increase of the treated ClO2 amount. However, the addition of an antioxidant, vitamin E, significantly attenuated the cytotoxicity of ClO2 in a dose-dependent manner. To support the oxidative stress induced by ClO2, two antioxidant genes (superoxide dismutase (SOD) and thioredoxin-peroxidase (Tpx)) were identified from P. interpunctella EST library using ortholog sequences of Bombyx mori. Both SOD and Tpx were expressed in larvae of P. interpunctella especially under oxidative stress induced by bacterial challenge. Exposure to ClO2 gas significantly induced the gene expression of both SOD and Tpx. RNA interference of SOD or Tpx using specific double stranded RNAs significantly enhanced the lethality of P. interpunctella to ClO2 gas treatment as well as to the bacterial challenge. These results suggest that ClO2 induces the production of insecticidal ROS, which results in a fatal oxidative stress in P. interpunctella. Copyright © 2015 Elsevier Inc. All rights reserved.

  7. Expression of immune-response genes in lepidopteran host is suppressed by venom from an endoparasitoid, Pteromalus puparum

    Fang Qi

    2010-09-01

    Full Text Available Abstract Background The relationships between parasitoids and their insect hosts have attracted attention at two levels. First, the basic biology of host-parasitoid interactions is of fundamental interest. Second, parasitoids are widely used as biological control agents in sustainable agricultural programs. Females of the gregarious endoparasitoid Pteromalus puparum (Hymenoptera: Pteromalidae inject venom along with eggs into their hosts. P. puparum does not inject polydnaviruses during oviposition. For this reason, P. puparum and its pupal host, the small white butterfly Pieris rapae (Lepidoptera: Pieridae, comprise an excellent model system for studying the influence of an endoparasitoid venom on the biology of the pupal host. P. puparum venom suppresses the immunity of its host, although the suppressive mechanisms are not fully understood. In this study, we tested our hypothesis that P. puparum venom influences host gene expression in the two main immunity-conferring tissues, hemocytes and fat body. Results At 1 h post-venom injection, we recorded significant decreases in transcript levels of 217 EST clones (revealing 113 genes identified in silico, including 62 unknown contigs derived from forward subtractive libraries of host hemocytes and in transcript levels of 288 EST clones (221 genes identified in silico, including 123 unknown contigs from libraries of host fat body. These genes are related to insect immune response, cytoskeleton, cell cycle and apoptosis, metabolism, transport, stress response and transcriptional and translational regulation. We verified the reliability of the suppression subtractive hybridization (SSH data with semi-quantitative RT-PCR analysis of a set of randomly selected genes. This analysis showed that most of the selected genes were down-regulated after venom injection. Conclusions Our findings support our hypothesis that P. puparum venom influences gene expression in host hemocytes and fat body. Specifically

  8. Efficient gene transfer into silkworm larval tissues by a combination of sonoporation and lipofection.

    Lee, Jae Man; Takahashi, Masateru; Mon, Hiroaki; Koga, Katsumi; Kawaguchi, Yutaka; Kusakabe, Takahiro

    2005-11-01

    Sonoporation (ultrasound treatment) provides a new and attractive nonviral way of in vivo gene transfer. To access the applicability of this method to the silkworm, Bombyx mori, we have compared the efficiencies of gene transfer by means of lipofection (using an appropriate agent, PDD111), sonoporation (ditto, FluoroGene), and lipofection followed by sonoporation. By these methods, a luciferase expression plasmid was found to be markedly transferred into the haemocoel of newly ecdysed fifth instar silkworm larvae, and also into other tissues although with lower rates compared with the haemocoel. In terms of luciferase activity, the efficiencies of transgene by lipofection plus sonoporation were approximately 6 (hemocytes), 20 (silk glands), 8 (mid-gut), 38 (fat body), 10 (Malpighian tubules), 33 (ovaries), and 16 (testes) times as high as those by lipofection or sonoporation alone. These results demonstrated that the present method is useful to introduce the exogenous DNA into insect organs in vivo.

  9. Characterization of A Baculovirus of Spodoptera litura (Lepidoptera : Noctuidae Isolated from Yogyakarta

    Arman Wijonarko

    2007-07-01

    Full Text Available A Baculovirus has been isolated from cadaver of larvae of Spodoptera litura, a Noctuidae of agricultural pest, importance due to its wide-range hosts and the damage to their respective host. Phase contrast light microscopy observation from infected larvae showed that the fat body, hemocyte cells, and cells surrounding the trachea or tracheolus were the most tissue invaded by polyhedra. Transmission electron microscopy analysis of the occlusion body purified from diseased larva showed that the baculovirus envelope containing multiple nucleocapsid. Digestion of viral DNA with three restriction enzymes showed that the genome pattern of baculovirus isolated from Bantul were close to SpliNPV isolated from Japan and those of Spodoptera littoralis and quite distinct from those isolated from Southeast Asia region. Bioassay test performed on first to fifth instar larvae showed that the virus effectively control the young larvae, but showed some level of resistance against older larvae of Spodoptera litura.

  10. Effects of an environmentally relevant concentration of diuron on oyster genitors during gametogenesis: responses of early molecular and cellular markers and physiological impacts.

    Akcha, F; Barranger, A; Bachère, E; Berthelin, C Heude; Piquemal, D; Alonso, P; Sallan, R Rondon; Dimastrogiovanni, G; Porte, C; Menard, D; Szczybelski, A; Benabdelmouna, A; Auffret, M; Rouxel, J; Burgeot, T

    2016-04-01

    Genitors of the Pacific oyster Crassostrea gigas were submitted during gametogenesis to a short pulse exposure to the herbicide diuron at a realistic environmental concentration. Histological analysis showed no effect of diuron on gametogenesis course, sex ratio and reproductive effort. A non-significant increase in testosterone and progesterone levels was observed in genitors exposed to the herbicide. At cell level, diuron exposure was shown to modulate the phagocytic activity of circulating hemocytes. The results of a transcriptional analysis showed that diuron affected the expression of genes belonging to functions known to play a major role during oyster gametogenesis such as gene transcription regulation, DNA replication and repair, DNA methylation and cytokinesis. Taking into account the results we previously obtained on the same genitors, this study showed a negative effect of diuron on oyster reproduction by inducing both structural and functional modifications of the DNA.

  11. Sublethal pesticide doses negatively affect survival and the cellular responses in American foulbrood-infected honeybee larvae

    López, Javier Hernández; Krainer, Sophie; Engert, Antonia; Schuehly, Wolfgang; Riessberger-Gallé, Ulrike; Crailsheim, Karl

    2017-02-01

    Disclosing interactions between pesticides and bee infections is of most interest to understand challenges that pollinators are facing and to which extent bee health is compromised. Here, we address the individual and combined effect that three different pesticides (dimethoate, clothianidin and fluvalinate) and an American foulbrood (AFB) infection have on mortality and the cellular immune response of honeybee larvae. We demonstrate for the first time a synergistic interaction when larvae are exposed to sublethal doses of dimethoate or clothianidin in combination with Paenibacillus larvae, the causative agent of AFB. A significantly higher mortality than the expected sum of the effects of each individual stressor was observed in co-exposed larvae, which was in parallel with a drastic reduction of the total and differential hemocyte counts. Our results underline that characterizing the cellular response of larvae to individual and combined stressors allows unmasking previously undetected sublethal effects of pesticides in colony health.

  12. Dicty_cDB: Contig-U02054-1 [Dicty_cDB

    Full Text Available 0.080 1 ( DT656898 ) pgr1n.UA001.227 Normalized chicken reproductive t... 50 0.080 1 ( AJ454996 ) Gallus gallus EST, clone library...90810 XtSt10-30 Xenopus (Silurana) t... 36 0.17 2 ( DV037739 ) BRS3230 storage root cDNA library Ipomoea bat..... 44 4.9 1 ( BM959958 ) cihA1L9S Ascidian hemocytes cDNA library Ciona in... 44 4.9 1 ( BM230365 ) K0294C12-3 NIA Mouse Unferti...... 36 0.010 3 ( EY189411 ) LLAE1039S Spider Loxosceles laeta cDNA library Lo... ... riken1, clone 4e... 50 0.080 1 ( AJ453299 ) Gallus gallus EST, clone library riken1,

  13. Genotoxic effects induced by the exposure to an environmental mixture of illicit drugs to the zebra mussel.

    Parolini, Marco; Magni, Stefano; Castiglioni, Sara; Binelli, Andrea

    2016-10-01

    Despite the growing interest on the presence of illicit drugs in freshwater ecosystems, just recently the attention has been focused on their potential toxicity towards non-target aquatic species. However, these studies largely neglected the effects induced by exposure to complex mixtures of illicit drugs, which could be different compared to those caused by single psychoactive molecules. This study was aimed at investigating the genetic damage induced by a 14-day exposure to a realistic mixture of the most common illicit drugs found in surface waters worldwide (cocaine, benzoylecgonine, amphetamine, morphine and 3,4-methylenedioxymethamphetamine) on the zebra mussel (Dreissena polymorpha). The mixture caused a significant increase of DNA fragmentation and triggered the apoptotic process and micronuclei formation in zebra mussel hemocytes, pointing out its potential genotoxicity towards this bivalve species. Copyright © 2016 Elsevier Inc. All rights reserved.

  14. Effects of chronic exposure to ionising radiation in the Pacific oyster Crassostrea gigas

    Fievet, B.; Devos, A.; Voiseux, C.; Leconte-Pradines, C. [Institut de Radioprotection et de Surete' Nucleaire (France); Dallas, L.; Jha, A. [University of Plymouth (United Kingdom)

    2014-07-01

    The Cotentin peninsula (Normandy, France) hosts nuclear industry facilities which operate with controlled discharges of radionuclides in the marine environment. Compared to natural radioactivity, the increase by artificial radionuclides is small but constant. As a consequence, marine species are chronically exposed to low additional doses of ionizing radiation (IR). The effects of chronic exposure to radionuclides were investigated in early stages of development of the Japanese oyster Crassostrea gigas. On the basis of literature, mollusks are expected to be particularly resistant to acute IR (UNSCEAR, 1996. Sources and effects of ionizing radiation. Report to the General Assembly, with Scientific Annex. 86 p). Two different chronic exposure conditions consisted in external ({sup 137}Cs) and internal ({sup 241}Am) irradiation for two weeks. Biological endpoints were analyzed in parallel at both the integrated (growth) and molecular (target stress gene expression) levels. To identify potential biological targets of IR, oysters were first exposed to very high dose rates and radionuclide activities with the perspective to reduce the levels and to derive dose-response curves. Although the initial exposure levels ({sup 137}Cs 30 000 μGy.h{sup -1}; {sup 241}Am 57 000 Bq.L{sup -1}) were many orders of magnitude higher than those encountered in the natural environment, no significant change in the measured parameters was observed. This result was surprising because data from the literature showed that exposure of mussel Mytilus edulis to {sup 3}H at lower doses rates (10-100 μGy.h{sup -1}) induced DNA damage in hemocytes (Jha et al., 2005. Impact of low doses of tritium on the marine mussel, Mytilus edulis: Genotoxic effects and tissue-specific bioconcentration. Mutation Research/Genetic Toxicology and Environmental Mutagenesis 586, 47-57). To understand this apparent discrepancy between those two filtering bivalves, a new experiment was performed to compare the response

  15. Humoral and Haemocytic Responses of Litopenaeus vannamei to Cd Exposure

    Juan C. Bautista-Covarrubias

    2014-01-01

    Full Text Available White shrimp, Litopenaeus vannamei, subadults were exposed to four dilutions of the 96 h cadmium LC50 reported for postlarvae (PL12 of this species, and the effects were evaluated after 5, 48, and 96 h of exposure. While treatments did not affect survival and hemolymph clotting time increased with time, but not as a response to Cd exposure, the intensity of other responses was related to concentration, to time of exposure, and to their interaction. Hemocyanin decreased with time in all metal concentrations but increased in the control treatment, and an almost similar trend was observed with hemocyte numbers. As an initial response, phenoloxidase activity decreased with all metal concentrations, but it increased later to values similar or higher than the control treatment.

  16. Parasitic castration in Fissurella crassa (Archaeogastropoda due to an adult Digenea, Proctoeces lintoni (Fellodistomidae

    Marcelo E. Oliva

    1992-03-01

    Full Text Available Specimens of Fissurella crassa (Archaeogastropoda from Ilo, southern Perú, are infected with the adult stage of the digenetic trematode Proctoeces lintoni (Fellodistomidae. The histopatological analysis of the male and female gonads show a strong effect of the parasite on the structure and function of these organs. P. lintoni live unencysted in the gonads, and the main mechanical damage is originated by the action of a well developed acetabulum. Chemical actions of parasitic secretions may also be involved. The infected gonads show altered structure and the gametogenic processes is aborted. There is no evidence of hemocytic response, but leucocite infiltration is evident at least in male infected gonads. An increased content of polysaccarides is evident in infected gonads.

  17. Viral gametocytic hypertrophy caused by a papova-like virus infection in the Pacific oyster Crassostrea gigas in Korea.

    Choi, Dong Lim; Lee, Nam-Sil; Choi, Hee Jung; Park, Myoung-Ae; McGladdery, Sharon E; Park, Mi Seon

    2004-06-11

    During a routine survey of the Pacific oyster Crassostrea gigas in Tongyoung (previously Chungmu) on the southern coast of Korea, basophilic inclusions were observed in the gonadal tissues. They were detected from March to May at a prevalence rate of 3.3 to 7.1%. The inclusion bodies were Feulgen-positive and stained orange-red with phloxine tartrazine. Electron microscopic observation revealed non-enveloped, icosahedral particles 40 to 45 nm in diameter. These morphological characteristics resemble those of papova virus-like inclusions previously described from Pacific and eastern (American) oysters C. virginica in North America. Although many mitochondrial bodies and intact sperm cells were observed around the inclusion body, no host reaction, such as hemocytic infiltration, was detected.

  18. Physiological markers in insects indicating treatment with ionizing radiation

    Nation, J.L.; Smittle, B.J.; Milne, K.R.

    1999-01-01

    Seven markers or tests that can be applied to 3rd instars of the Caribbean fruit fly as indicators of exposure to ionizing radiation are described, including (1) whole body melanization, (2) phenoloxidase spot test, (3) quantitative phenoloxidase measurement, (4) measurement of the ratio between size of the supraesophageal ganglion and the proventriculus, (5) development of imaginal discs, (6) number of hemocytes in one μl of hemolymph, and (7) larval weight. The markers work best and are most definitive when larvae are exposed to at least 50 Gy no later than the first day of the 3rd instar. All of the tests are sensitive enough to be applied to a single 3rd instar larva. Combinations of some of the tests could be used on a single larva. Tests (1) and (2) are easiest to use and require no specific technical training, and seem to have the most potential for practical use in quarantine. (author)

  19. Parasites of the hard clam Meretrix meretrix Linnaeus from Western Johor Straits, Malaysian

    Azmi, Nur Fauzana; Ghaffar, Mazlan Abd.; Cob, Zaidi Che

    2014-09-01

    This study describes the apicomplexa as well as other parasites infecting organs/tissues of the hard clam Meretrix meretrix Linnaeus, from Merambong Shoal, Western Johor Straits, Malaysia. Samples were collected randomly by hand picking, in November and December 2013. Histological techniques were performed, stained using Masson's Trichrome protocol and observed under light microscope. The results showed that gonad and gill were the most infected organs followed by digestive gland, intestine and adductor muscle. No pathology condition was observed in the mantle. Histophatological examination showed that the gregarine, Nematopsis, unidentified coccidian and Perkinsus were found in the gill and gonad, and also in the numerous hemocytes. Other pathological conditions such as bacteria-like inclusion and intracellular bacteria were also observed in the same organs. Further investigations are needed particularly on other molluscs present at the study area. Understanding the morphology and pathology of parasites infecting mollusks are very important for management of the resources.

  20. Low-cost motility tracking system (LOCOMOTIS for time-lapse microscopy applications and cell visualisation.

    Adam E Lynch

    Full Text Available Direct visualisation of cells for the purpose of studying their motility has typically required expensive microscopy equipment. However, recent advances in digital sensors mean that it is now possible to image cells for a fraction of the price of a standard microscope. Along with low-cost imaging there has also been a large increase in the availability of high quality, open-source analysis programs. In this study we describe the development and performance of an expandable cell motility system employing inexpensive, commercially available digital USB microscopes to image various cell types using time-lapse and perform tracking assays in proof-of-concept experiments. With this system we were able to measure and record three separate assays simultaneously on one personal computer using identical microscopes, and obtained tracking results comparable in quality to those from other studies that used standard, more expensive, equipment. The microscopes used in our system were capable of a maximum magnification of 413.6×. Although resolution was lower than that of a standard inverted microscope we found this difference to be indistinguishable at the magnification chosen for cell tracking experiments (206.8×. In preliminary cell culture experiments using our system, velocities (mean µm/min ± SE of 0.81 ± 0.01 (Biomphalaria glabrata hemocytes on uncoated plates, 1.17 ± 0.004 (MDA-MB-231 breast cancer cells, 1.24 ± 0.006 (SC5 mouse Sertoli cells and 2.21 ± 0.01 (B. glabrata hemocytes on Poly-L-Lysine coated plates, were measured and are consistent with previous reports. We believe that this system, coupled with open-source analysis software, demonstrates that higher throughput time-lapse imaging of cells for the purpose of studying motility can be an affordable option for all researchers.

  1. Low-cost motility tracking system (LOCOMOTIS) for time-lapse microscopy applications and cell visualisation.

    Lynch, Adam E; Triajianto, Junian; Routledge, Edwin

    2014-01-01

    Direct visualisation of cells for the purpose of studying their motility has typically required expensive microscopy equipment. However, recent advances in digital sensors mean that it is now possible to image cells for a fraction of the price of a standard microscope. Along with low-cost imaging there has also been a large increase in the availability of high quality, open-source analysis programs. In this study we describe the development and performance of an expandable cell motility system employing inexpensive, commercially available digital USB microscopes to image various cell types using time-lapse and perform tracking assays in proof-of-concept experiments. With this system we were able to measure and record three separate assays simultaneously on one personal computer using identical microscopes, and obtained tracking results comparable in quality to those from other studies that used standard, more expensive, equipment. The microscopes used in our system were capable of a maximum magnification of 413.6×. Although resolution was lower than that of a standard inverted microscope we found this difference to be indistinguishable at the magnification chosen for cell tracking experiments (206.8×). In preliminary cell culture experiments using our system, velocities (mean µm/min ± SE) of 0.81 ± 0.01 (Biomphalaria glabrata hemocytes on uncoated plates), 1.17 ± 0.004 (MDA-MB-231 breast cancer cells), 1.24 ± 0.006 (SC5 mouse Sertoli cells) and 2.21 ± 0.01 (B. glabrata hemocytes on Poly-L-Lysine coated plates), were measured and are consistent with previous reports. We believe that this system, coupled with open-source analysis software, demonstrates that higher throughput time-lapse imaging of cells for the purpose of studying motility can be an affordable option for all researchers.

  2. Low-Cost Motility Tracking System (LOCOMOTIS) for Time-Lapse Microscopy Applications and Cell Visualisation

    Lynch, Adam E.; Triajianto, Junian; Routledge, Edwin

    2014-01-01

    Direct visualisation of cells for the purpose of studying their motility has typically required expensive microscopy equipment. However, recent advances in digital sensors mean that it is now possible to image cells for a fraction of the price of a standard microscope. Along with low-cost imaging there has also been a large increase in the availability of high quality, open-source analysis programs. In this study we describe the development and performance of an expandable cell motility system employing inexpensive, commercially available digital USB microscopes to image various cell types using time-lapse and perform tracking assays in proof-of-concept experiments. With this system we were able to measure and record three separate assays simultaneously on one personal computer using identical microscopes, and obtained tracking results comparable in quality to those from other studies that used standard, more expensive, equipment. The microscopes used in our system were capable of a maximum magnification of 413.6×. Although resolution was lower than that of a standard inverted microscope we found this difference to be indistinguishable at the magnification chosen for cell tracking experiments (206.8×). In preliminary cell culture experiments using our system, velocities (mean µm/min ± SE) of 0.81±0.01 (Biomphalaria glabrata hemocytes on uncoated plates), 1.17±0.004 (MDA-MB-231 breast cancer cells), 1.24±0.006 (SC5 mouse Sertoli cells) and 2.21±0.01 (B. glabrata hemocytes on Poly-L-Lysine coated plates), were measured and are consistent with previous reports. We believe that this system, coupled with open-source analysis software, demonstrates that higher throughput time-lapse imaging of cells for the purpose of studying motility can be an affordable option for all researchers. PMID:25121722

  3. Identification and function of myeloid differentiation factor 88 (MyD88 in Litopenaeus vannamei.

    Shuang Zhang

    Full Text Available Myeloid differentiation factor 88 (MyD88 is a universal and essential signaling protein in Toll-like receptor/interleukin-1 receptor-induced activation of nuclear factor-kappa B. In this study, two MyD88 protein variants (LvMyD88 and LvMyD88-1 were identified in Litopenaeus vannamei. The LvMyD88 cDNA is 1,848 bp in length and contains an open reading frame (ORF of 1,428 bp, whereas the LvMyD88-1 cDNA is 1,719 bp in length and has an ORF of 1,299 bp. Both variants encode proteins with death and Toll/interleukin-1 receptor domains and share 91% sequence identity. In healthy L. vannamei, the LvMyD88 genes were highly expressed in hemocytes but at a low level in the hepatopancreas. The LvMyD88s expression was induced in hemocytes after challenge with lipopolysaccharide, CpG-ODN2006, Vibrio parahaemolyticus, Staphyloccocus aureus, and white spot syndrome virus, but not by poly I∶C. Overexpression of LvMyD88 and LvMyD88-1 in Drosophila Schneider 2 cells led to activation of antimicrobial peptide genes and wsv069 (ie1, wsv303, and wsv371. These results suggested that LvMyD88 may play a role in antibacterial and antiviral response in L. vannamei. To our knowledge, this is the first report on MyD88 in shrimp and a variant of MyD88 gene in invertebrates.

  4. Phenotypic Plasticity, Epigenetic or Genetic Modifications in Relation to the Duration of Cd-Exposure within a Microevolution Time Range in the Beet Armyworm.

    Maria Augustyniak

    Full Text Available In the case of the pests inhabiting metal polluted or fields where the use of pesticides is common, a natural selection of resistant individuals can occur. This may pose serious problems for humans, agriculture, as well as the economies of many countries. In this study, the hypothesis that multigenerational (120 generations exposure to cadmium of a beet armyworm population could be a selecting factor toward a more efficient DNA protection was verified. The hemocytes of individuals from two culture strains (control and Cd-exposed were treated with H2O2 (a DNA-damaging agent or PBS (reference. The level of DNA damage was assessed using the Comet assay immediately and 5, 15 and 30 min. after the treatment. The immediate result of the contact with H2O2 was that the level of DNA damage in the hemocytes of the insects from both strains increased significantly. However, in the cells of the Cd-exposed individuals, the level of DNA damage decreased over time, while in the cells from the control insects it remained at the same level with no evidence of repair. These results suggest that efficient defense mechanisms may exist in the cells of insects that have prolonged contact with cadmium. Some evolutionary and trade-off aspects of the phenomenon are discussed. In a wider context, comparing the results obtained in the laboratory with field studies may be beneficial for understanding basic mechanisms of the resistance of an organism. To summarize, the high potential for the repair of DNA damage that was observed in the insects from the cadmium strain may confirm the hypothesis that multigenerational exposure to that metal may possibly contribute to the selection of insects that have a wider tolerance to oxidative stress. However, our investigations of polymorphism using AFLP did not reveal differences between the two main insect strains.

  5. New insights into the apoptotic process in mollusks: characterization of caspase genes in Mytilus galloprovincialis.

    Alejandro Romero

    2011-02-01

    Full Text Available Apoptosis is an essential biological process in the development and maintenance of immune system homeostasis. Caspase proteins constitute the core of the apoptotic machinery and can be categorized as either initiators or effectors of apoptosis. Although the genes encoding caspase proteins have been described in vertebrates and in almost all invertebrate phyla, there are few reports describing the initiator and executioner caspases or the modulation of their expression by different stimuli in different apoptotic pathways in bivalves. In the present work, we characterized two initiator and four executioner caspases in the mussel Mytilus galloprovincialis. Both initiators and executioners showed structural features that make them different from other caspase proteins already described. Evaluation of the genes' tissue expression patterns revealed extremely high expression levels within the gland and gills, where the apoptotic process is highly active due to the clearance of damaged cells. Hemocytes also showed high expression values, probably due to of the role of apoptosis in the defense against pathogens. To understand the mechanisms of caspase gene regulation, hemocytes were treated with UV-light, environmental pollutants and pathogen-associated molecular patterns (PAMPs and apoptosis was evaluated by microscopy, flow cytometry and qPCR techniques. Our results suggest that the apoptotic process could be tightly regulated in bivalve mollusks by overexpression/suppression of caspase genes; additionally, there is evidence of caspase-specific responses to pathogens and pollutants. The apoptotic process in mollusks has a similar complexity to that of vertebrates, but presents unique features that may be related to recurrent exposure to environmental changes, pollutants and pathogens imposed by their sedentary nature.

  6. The genomic features of parasitism, Polyembryony and immune evasion in the endoparasitic wasp Macrocentrus cingulum.

    Yin, Chuanlin; Li, Meizhen; Hu, Jian; Lang, Kun; Chen, Qiming; Liu, Jinding; Guo, Dianhao; He, Kang; Dong, Yipei; Luo, Jiapeng; Song, Zhenkun; Walters, James R; Zhang, Wenqing; Li, Fei; Chen, Xuexin

    2018-05-30

    Parasitoid wasps are well-known natural enemies of major agricultural pests and arthropod borne diseases. The parasitoid wasp Macrocentrus cingulum (Hymenoptera: Braconidae) has been widely used to control the notorious insect pests Ostrinia furnacalis (Asian Corn Borer) and O. nubilalis (European corn borer). One striking phenomenon exhibited by M. cingulum is polyembryony, the formation of multiple genetically identical offspring from a single zygote. Moreover, M. cingulum employs a passive parasitic strategy by preventing the host's immune system from recognizing the embryo as a foreign body. Thus, the embryos evade the host's immune system and are not encapsulated by host hemocytes. Unfortunately, the mechanism of both polyembryony and immune evasion remains largely unknown. We report the genome of the parasitoid wasp M. cingulum. Comparative genomics analysis of M. cingulum and other 11 insects were conducted, finding some gene families with apparent expansion or contraction which might be linked to the parasitic behaviors or polyembryony of M. cingulum. Moreover, we present the evidence that the microRNA miR-14b regulates the polyembryonic development of M. cingulum by targeting the c-Myc Promoter-binding Protein 1 (MBP-1), histone-lysine N-methyltransferase 2E (KMT2E) and segmentation protein Runt. In addition, Hemomucin, an O-glycosylated transmembrane protein, protects the endoparasitoid wasp larvae from being encapsulated by host hemocytes. Motif and domain analysis showed that only the hemomucin in two endoparasitoids, M. cingulum and Venturia canescens, possessing the ability of passive immune evasion has intact mucin domain and similar O-glycosylation patterns, indicating that the hemomucin is a key factor modulating the immune evasion. The microRNA miR-14b participates in the regulation of polyembryonic development, and the O-glycosylation of the mucin domain in the hemomucin confers the passive immune evasion in this wasp. These key findings provide

  7. Galleria mellonella larvae are capable of sensing the extent of priming agent and mounting proportionatal cellular and humoral immune responses.

    Wu, Gongqing; Xu, Li; Yi, Yunhong

    2016-06-01

    Larvae of Galleria mellonella are useful models for studying the innate immunity of invertebrates or for evaluating the virulence of microbial pathogens. In this work, we demonstrated that prior exposure of G. mellonella larvae to high doses (1×10(4), 1×10(5) or 1×10(6) cells/larva) of heat-killed Photorhabdus luminescens TT01 increases the resistance of larvae to a lethal dose (50 cells/larva) of viable P. luminescens TT01 infection administered 48h later. We also found that the changes in immune protection level were highly correlated to the changes in levels of cellular and humoral immune parameters when priming the larvae with different doses of heat-killed P. luminescens TT01. Priming the larvae with high doses of heat-killed P. luminescens TT01 resulted in significant increases in the hemocytes activities of phagocytosis and encapsulation. High doses of heat-killed P. luminescens TT01 also induced an increase in total hemocyte count and a reduction in bacterial density within the larval hemocoel. Quantitative real-time PCR analysis showed that genes coding for cecropin and gallerimycin and galiomycin increased in expression after priming G. mellonella with heat-killed P. luminescens TT01. All the immune parameters changed in a dose-dependent manner. These results indicate that the insect immune system is capable of sensing the extent of priming agent and mounting a proportionate immune response. Copyright © 2016 European Federation of Immunological Societies. Published by Elsevier B.V. All rights reserved.

  8. Ulcerative enteritis in Homarus americanus: case report and molecular characterization of intestinal aerobic bacteria of apparently healthy lobsters in live storage.

    Battison, Andrea L; Després, Béatrice M; Greenwood, Spencer J

    2008-10-01

    An intermoult male American lobster, Homarus americanus, with severe intestinal lesions was encountered while collecting samples of aerobic intestinal bacteria from lobsters held in an artificial sea-water recirculation aquarium system. Grossly, the intestine was firm, thickened, and white. Histologic examination revealed a severe, diffuse, ulcerative enteritis which spared the chitin-lined colon, somewhat similar to hemocytic enteritis of shrimp. The bacterial isolates from this lobster were compared to 11 other lobsters lacking gross intestinal lesions. Two organisms, one identified as Vibrio sp. and another most similar to an uncultured proteobacterium (98.9%), clustering with Rhanella and Serratia species using 16S rDNA PCR, were isolated from the intestines of the 11, grossly normal, lobsters and the affected lobster. An additional two intestinal isolates were cultured only from the lobster with ulcerative enteritis. One, a Flavobacterium, similar to Lutibacter litoralis (99.3%), possibly represented a previously described commensal of the distal intestine. The second, a Vibrio sp., was unique to the affected animal. While the etiology of the ulcerative enteritis remains undetermined, this report represents the first description of gross and histologic findings in H. americanus of a condition which has morphologic similarities to hemocytic enteritis of shrimp. An additional observation was a decrease in the number of intestinal isolates recovered from the 11 apparently healthy lobsters compared to that previously reported for recently harvested lobster. More comprehensive studies of the relationship between the health of lobsters, gut microbial flora and the husbandry and environment maintained within holding units are warranted.

  9. Responses to a warming world: Integrating life history, immune investment, and pathogen resistance in a model insect species.

    Laughton, Alice M; O'Connor, Cian O; Knell, Robert J

    2017-11-01

    Environmental temperature has important effects on the physiology and life history of ectothermic animals, including investment in the immune system and the infectious capacity of pathogens. Numerous studies have examined individual components of these complex systems, but little is known about how they integrate when animals are exposed to different temperatures. Here, we use the Indian meal moth ( Plodia interpunctella ) to understand how immune investment and disease resistance react and potentially trade-off with other life-history traits. We recorded life-history (development time, survival, fecundity, and body size) and immunity (hemocyte counts, phenoloxidase activity) measures and tested resistance to bacterial ( E. coli ) and viral ( Plodia interpunctella granulosis virus) infection at five temperatures (20-30°C). While development time, lifespan, and size decreased with temperature as expected, moths exhibited different reproductive strategies in response to small changes in temperature. At cooler temperatures, oviposition rates were low but tended to increase toward the end of life, whereas warmer temperatures promoted initially high oviposition rates that rapidly declined after the first few days of adult life. Although warmer temperatures were associated with strong investment in early reproduction, there was no evidence of an associated trade-off with immune investment. Phenoloxidase activity increased most at cooler temperatures before plateauing, while hemocyte counts increased linearly with temperature. Resistance to bacterial challenge displayed a complex pattern, whereas survival after a viral challenge increased with rearing temperature. These results demonstrate that different immune system components and different pathogens can respond in distinct ways to changes in temperature. Overall, these data highlight the scope for significant changes in immunity, disease resistance, and host-parasite population dynamics to arise from small

  10. Potensi sinbiotik lokal terhadap respon imun non spesifik udang vaname (Litopenaeus vannamei

    Indri Saputri Ramadhani

    2017-09-01

    Full Text Available The aims of the study were to examine the exact percentage of prebiotics in sinbiotics, administered through feed, so as to enhance nonspecific immune responses in white shrimp. The sweet potato extract was used as prebiotic, combined with local isolated Bacillus sp. D2.2 as a probiotic applied simultaneously as a synbiotic. The feed was treated with 0% sinbiotic treatment (treatment A / control, 0% prebiotic and 6% probiotics (B treatment, 2% prebiotic and 6% probiotics (treatment C, 4% prebiotics and 6% probiotics (treatment D. Examination of nonspecific immune responses to shrimp included total hemocyte count (THC, phagocytosis activity (AP, phagocytosis index (IP, phenoloxidase activity (PO, and superoxide dismutase activity (SOD. Observation of the non-specific immune response of vaname shrimp after treatment showed that treatment C was the best prebiotic  to increase non-specific immune response in vaname shrimp. Penelitian ini bertujuan untuk mempelajari persentase prebiotik yang tepat dalam sinbiotik, yang diberikan melalui pakan, sehingga dapat meningkatkan respon imun nonspesifik pada udang vaname. Dalam penelitian ini digunakan ekstrak tepung ubi jalar sebagai prebiotik, dikombinasikan dengan isolat lokal Bacillus sp. D2.2 sebagai probiotik yang diaplikasikan secara bersamaan sebagai sinbiotik. Pakan yang diberikan adalah pakan dengan perlakuan 0% sinbiotik (perlakuan A/kontrol, 0% prebiotik dan 6% probiotik (perlakuan B, 2% prebiotik dan 6% probiotik (perlakuan C, 4% prebiotik dan 6% probiotik (perlakuan D. Pemeriksaan respon imun nonspesifik pada udang meliputi total hemocyte count (THC, aktivitas fagositosis (AF, indeks fagositosis (IF, aktivitas phenoloxidase (PO, dan aktivitas superoxide dismutase (SOD. Pengamatan pada respon imun non spesifik udang vaname setelah diberi perlakuan menunjukan bahwa perlakuan C merupakan persentase prebiotik terbaik untuk meningkatkan respon imun non spesifik pada udang vaname.

  11. Grasshopper Lazarillo, a GPI-anchored Lipocalin, increases Drosophila longevity and stress resistance, and functionally replaces its secreted homolog NLaz.

    Ruiz, Mario; Wicker-Thomas, Claude; Sanchez, Diego; Ganfornina, Maria D

    2012-10-01

    Lazarillo (Laz) is a glycosyl-phosphatidylinositol (GPI)-linked glycoprotein first characterized in the developing nervous system of the grasshopper Schistocerca americana. It belongs to the Lipocalins, a functionally diverse family of mostly secreted proteins. In this work we test whether the protective capacity known for Laz homologs in flies and vertebrates (NLaz, GLaz and ApoD) is evolutionarily conserved in grasshopper Laz, and can be exerted from the plasma membrane in a cell-autonomous manner. First we demonstrate that extracellular forms of Laz have autocrine and paracrine protecting effects for oxidative stress-challenged Drosophila S2 cells. Then we assay the effects of overexpressing GPI-linked Laz in adult Drosophila and whether it rescues both known and novel phenotypes of NLaz null mutants. Local effects of GPI-linked Laz inside and outside the nervous system promote survival upon different stress forms, and extend lifespan and healthspan of the flies in a cell-type dependent manner. Outside the nervous system, expression in fat body cells but not in hemocytes results in protection. Within the nervous system, glial cell expression is more effective than neuronal expression. Laz actions are sexually dimorphic in some expression domains. Fat storage promotion and not modifications in hydrocarbon profiles or quantities explain the starvation-desiccation resistance caused by Laz overexpression. This effect is exerted when Laz is expressed ubiquitously or in dopaminergic cells, but not in hemocytes. Grasshopper Laz functionally restores the loss of NLaz, rescuing stress-sensitivity as well as premature accumulation of aging-related damage, monitored by advanced glycation end products (AGEs). However Laz does not rescue NLaz courtship behavioral defects. Finally, the presence of two new Lipocalins with predicted GPI-anchors in mosquitoes shows that the functional advantages of GPI-linkage have been commonly exploited by Lipocalins in the arthropodan lineage

  12. Antioxidant deficit in gills of Pacific oyster (Crassostrea gigas) exposed to chlorodinitrobenzene increases menadione toxicity

    Trevisan, Rafael; Arl, Miriam; Sacchet, Cássia Lopes; Engel, Cristiano Severino; Danielli, Naissa Maria; Mello, Danielle Ferraz; Brocardo, Caroline; Maris, Angelica Francesca; Dafre, Alcir Luiz

    2012-01-01

    Disturbances in antioxidant defenses decrease cellular protection against oxidative stress and jeopardize cellular homeostasis. To knock down the antioxidant defenses of Pacific oyster Crassostrea gigas, animals were pre-treated with 1-chloro-2,4-dinitrobenzene (CDNB) and further challenged with pro-oxidant menadione (MEN). CDNB pre-treatment (10 μM for 18 h) was able to consume cellular thiols in gills, decreasing GSH (53%) and decrease protein thiols (25%). CDNB pre-treatment also disrupted glutathione reductase and thioredoxin reductase activity in the gills, but likewise strongly induced glutathione S-transferase activity (270% increase). Surprisingly, hemocyte viability was greatly affected 24 h after CDNB removal, indicating a possible vulnerability of the oyster immune system to electrophilic attack. New in vivo approaches were established, allowing the identification of higher rates of GSH–CDNB conjugate export to the seawater and enabling the measurement of the organic peroxide consumption rate. CDNB-induced impairment in antioxidant defenses decreased the peroxide removal rate from seawater. After showing that CDNB decreased gill antioxidant defenses and increased DNA damage in hemocytes, oysters were further challenged with 1 mM MEN over 24 h. MEN treatment did not affect thiol homeostasis in gills, while CDNB pre-treated animals recovered GSH and PSH to the control level after 24 h of depuration. Interestingly, MEN intensified GSH and PSH loss and mortality in CDNB-pre-treated animals, showing a clear synergistic effect. The superoxide-generating one-electron reduction of MEN was predominant in gills and may have contributed to MEN toxicity. These results support the idea that antioxidant-depleted animals are more susceptible to oxidative attack, which can compromise survival. Data also corroborate the idea that gills are an important detoxifying organ, able to dispose of organic peroxides, induce phase II enzymes, and efficiently export GSH

  13. Antioxidant deficit in gills of Pacific oyster (Crassostrea gigas) exposed to chlorodinitrobenzene increases menadione toxicity

    Trevisan, Rafael; Arl, Miriam [Departamento de Bioquimica, Universidade Federal de Santa Catarina, 88040-900 Florianopolis, SC (Brazil); Sacchet, Cassia Lopes [Universidade do Oeste do Estado de Santa Catarina, 89600-000 Joacaba, SC (Brazil); Engel, Cristiano Severino; Danielli, Naissa Maria; Mello, Danielle Ferraz [Departamento de Bioquimica, Universidade Federal de Santa Catarina, 88040-900 Florianopolis, SC (Brazil); Brocardo, Caroline [Universidade do Oeste do Estado de Santa Catarina, 89600-000 Joacaba, SC (Brazil); Maris, Angelica Francesca [Departamento de Biologia Celular, Embriologia e Genetica, Universidade Federal de Santa Catarina, 88040-900 Florianopolis, SC (Brazil); Dafre, Alcir Luiz, E-mail: alcir@ccb.ufsc.br [Departamento de Bioquimica, Universidade Federal de Santa Catarina, 88040-900 Florianopolis, SC (Brazil)

    2012-02-15

    Disturbances in antioxidant defenses decrease cellular protection against oxidative stress and jeopardize cellular homeostasis. To knock down the antioxidant defenses of Pacific oyster Crassostrea gigas, animals were pre-treated with 1-chloro-2,4-dinitrobenzene (CDNB) and further challenged with pro-oxidant menadione (MEN). CDNB pre-treatment (10 {mu}M for 18 h) was able to consume cellular thiols in gills, decreasing GSH (53%) and decrease protein thiols (25%). CDNB pre-treatment also disrupted glutathione reductase and thioredoxin reductase activity in the gills, but likewise strongly induced glutathione S-transferase activity (270% increase). Surprisingly, hemocyte viability was greatly affected 24 h after CDNB removal, indicating a possible vulnerability of the oyster immune system to electrophilic attack. New in vivo approaches were established, allowing the identification of higher rates of GSH-CDNB conjugate export to the seawater and enabling the measurement of the organic peroxide consumption rate. CDNB-induced impairment in antioxidant defenses decreased the peroxide removal rate from seawater. After showing that CDNB decreased gill antioxidant defenses and increased DNA damage in hemocytes, oysters were further challenged with 1 mM MEN over 24 h. MEN treatment did not affect thiol homeostasis in gills, while CDNB pre-treated animals recovered GSH and PSH to the control level after 24 h of depuration. Interestingly, MEN intensified GSH and PSH loss and mortality in CDNB-pre-treated animals, showing a clear synergistic effect. The superoxide-generating one-electron reduction of MEN was predominant in gills and may have contributed to MEN toxicity. These results support the idea that antioxidant-depleted animals are more susceptible to oxidative attack, which can compromise survival. Data also corroborate the idea that gills are an important detoxifying organ, able to dispose of organic peroxides, induce phase II enzymes, and efficiently export GSH

  14. Morphological and histological studies on freshwater prawn Macrobrachium rosenbergii (de man) irradiated with (60)Co gamma radiation.

    Stalin, A; Broos, K V; Sadiq Bukhari, A; Syed Mohamed, H E; Singhal, R K; Venu-Babu, P

    2013-11-15

    This study was framed to investigate the (60)Co gamma radiation induced morphological and histological variations in freshwater prawn Macrobrachium rosenbergii. The LD50 value of (60)Co gamma irradiated M. rosenbergii observed (by probit analysis) at 30 Gy. Prawns were irradiated to four different dose levels (3 mGy, 30 mGy, 300 mGy and 3,000 mGy) using Theratron Phoenix TeleCobalt Unit [P-33] and one control group (without irradiation) maintained separately. Irradiated groups exhibited several morphological variations such as discoloration; damaged rostrum; opaque coloration in cephalothorax; black bands and dot formation in abdomen; deformed uropods and telson in tail regions when compared with control group. The Hepato Somatic Index reflected the severity of radiation on hepatopancreas. Histological variations in gills, hepatopancreas and muscles of irradiated groups were observed. In gills, structural changes such as swollen and fused lamellae, abnormal gill tips, hyperplasic, necrotic and clavate-globate lamellae were observed in gamma irradiated prawns. Accumulation of hemocytes in hemocoelic space, interstitial sinuses filled with abnormal infiltrated hemocytes, the tubular epithelium with ruptured basal laminae, abnormal and coagulated lumen, necrotic tubules, thickened basal laminae, tissue debris, necrotic hepatocytes were observed in irradiated prawn hepatopancreas. In muscle, shrinkage of muscular fiber and necrotic musculature were observed in irradiated prawns. These structural alterations of the organs it is felt could affect the vital physiological functions such as respiration, osmotic and ionic regulation in gills and muscles; absorption, storage and secretion of the hepatopancreas which in turn could adversely affect the growth and survival of freshwater prawn M. rosenbergii. Copyright © 2013 Elsevier B.V. All rights reserved.

  15. Characterization and transcription studies of a phytochelatin synthase gene from the solitary tunicate Ciona intestinalis exposed to cadmium

    Franchi, Nicola; Piccinni, Ester; Ferro, Diana; Basso, Giuseppe; Spolaore, Barbara; Santovito, Gianfranco; Ballarin, Loriano

    2014-01-01

    Highlights: • Ciona intestinalis have a functional phytochelatin synthase (PCS) gene (cipcs). • CiPCS amino acid sequence is phylogentically related to other metazoan PCSs. • CiPCS catalyze the synthesis of PC2. • cipcs are mostly transcribed in circulating hemocytes, in both tunic and blood lacunae. • Cadmium exposure results in a significant increase of cipcs and cipcna transcription. - Abstract: The major thiol-containing molecules involved in controlling the level of intracellular ROS in eukaryotes, acting as a nonenzymatic detoxification system, are metallothioneins (MTs), glutathione (GSH) and phytochelatins (PCs). Both MTs and GSH are well-known in the animal kingdom. PC was considered a prerogative of the plant kingdom but, in 2001, a phytochelatin synthase (PCS) gene was described in the nematode Caenorhabditis elegans; additional genes encoding this enzyme were later described in the earthworm Eisenia fetida and in the parasitic nematode Schistosoma mansoni but scanty data are available, up to now, for Deuterostomes. Here, we describe the molecular characteristics and transcription pattern, in the presence of Cd, of a PCS gene from the invertebrate chordate Ciona intestinalis, a ubiquitous solitary tunicate and demonstrate the presence of PCs in tissue extracts. We also studied mRNA localization by in situ hybridization. In addition, we analyzed the behavior of hemocytes and tunic cells consequent to Cd exposure as well as the transcription pattern of the Ciona orthologous for proliferating cell nuclear antigen (PCNA), usually considered a proliferation marker, and observed that cell proliferation occurs after 96 h of Cd treatment. This matches the hypothesis of Cd-induced cell proliferation, as already suggested by previous data on the expression of a metallothionein gene in the same animal

  16. A semiquantitative PCR assay for assessing Perkinsus marinus infections in the eastern oyster, Crassostrea virginica.

    Marsh, A G; Gauthier, J D; Vasta, G R

    1995-08-01

    A 3.2-kb fragment of Perkinsus marinus DNA was cloned and sequenced. A noncoding domain was identified and targeted for the development of a semiquantitative polymerase chain reaction (PCR) assay for the presence of P. marinus in eastern oyster tissues. The assay involves extracting total DNA from oyster hemolymph and using 1 microgram of that DNA as template in a stringent PCR amplification with oligonucleotide primers that are specific for the P. marinus 3.2-kb fragment. With this assay, we can detect 10 pg of total P. marinus DNA per 1 microgram of oyster hemocyte DNA with ethidium bromide (EtBr) staining of agarose gels, 100 fg total P. marinus DNA with Southern blot autoradiography, and 10 fg of total P. marinus DNA with dot-blot hybridizations. We have used the sensitivity of the PCR assay to develop a method for estimating the level of P. marinus DNA in oyster hemolymph and have successfully applied this technique to gill tissues. Our semiquantitative assay uses a dilution series to essentially titrate the point at which a P. marinus DNA target is no longer amplified in a sample. We refer to this technique as "dilution endpoint" PCR. Using hemocytes obtained by withdrawing a 1-ml sample of hemolymph, this assay provides a nondestructive methodology for rapidly screening large numbers of adult oysters for the presence and quantification of P. marinus infection levels. This technique is applicable to other tissues (gills) and could potentially be applied to DNA extracts of whole larvae or spat.

  17. Identification of clam plasma proteins that bind its pathogen Quahog Parasite Unknown.

    Hartman, Rachel; Pales Espinosa, Emmanuelle; Allam, Bassem

    2018-06-01

    The hard clam (Mercenaria mercenaria) is among the most economically-important marine species along the east coast of the United States, representing the first marine resource in several Northeastern states. The species is rather resilient to infections and the only important disease of hard clams results from an infection caused by Quahog Parasite Unknown (QPX), a protistan parasite that can lead to significant mortality events in wild and aquacultured clam stocks. Though the presence of QPX disease has been documented since the 1960s, little information is available on cellular and molecular interactions between the parasite and the host. This study examined the interactions between the clam immune system and QPX cells. First, the effect of clam plasma on the binding of hemocytes to parasite cells was evaluated. Second, clam plasma proteins that bind QPX cells were identified through proteomic (LC-MS/MS) analyses. Finally, the effect of prior clam exposure to QPX on the abundance of QPX-reactive proteins in the plasma was evaluated. Results showed that plasma factors enhance the attachment of hemocytes to QPX. Among the proteins that specifically bind to QPX cells, several lectins were identified, as well as complement component proteins and proteolytic enzymes. Furthermore, results showed that some of these lectins and complement-related proteins are inducible as their abundance significantly increased following QPX challenge. These results shed light on plasma proteins involved in the recognition and binding of parasite cells and provide molecular targets for future investigations of factors involved in clam resistance to the disease, and ultimately for the selection of resistant clam stocks. Copyright © 2018 Elsevier Ltd. All rights reserved.

  18. Characterization of a novel gene encoding ankyrin repeat domain from Cotesia vestalis polydnavirus (CvBV)

    Shi Min; Chen Yafeng; Huang Fang; Liu Pengcheng; Zhou Xueping; Chen Xuexin

    2008-01-01

    Cotesia vestalis (Haliday) is an endoparasitoid of Plutella xylostella (L.) larvae and injects a polydnavirus (CvBV) into its host during oviposition. In this report we describe the characterization of a gene (CvBV805) and its products. CvBV805 is located on the segment S8 of CvBV genome; it has a size of 909 bp and encodes a predicted protein of 125 amino acids. This protein contains an ankyrin repeat domain with a high degree of similarity with IκB-like genes. Gene transcripts were detected in extracts of the host as early as 2 h post-parasitization (p.p.) and continued to be detected through 24 h. Tissue-specific expression patterns showed that CvBV805 might be involved in early host immunosuppression. CvBV805 was detected in parasitized hosts at 12 h p.p. and in rBac-eGFP-CvBV805-infected Tn-5B1-4 cells at 72 h.p.i. by using western blots analysis. The size of the protein expressed in the host hemocytes and infected Tn-5B1-4 cells was 17 kDa and 56 kDa (including eGFP), respectively, which nearly corresponded with the predicted molecular weight (14.31 kDa) of CvBV805, suggesting that the protein did not undergo extensive post-translational modification. The protein was confirmed to be present within the nuclear region in hemocytes of the parasitized P. xylostella larvae at 48 h p.p. using confocal laser scanning microscopy

  19. Essential roles of Cdc42 and MAPK in cadmium-induced apoptosis in Litopenaeus vannamei

    Peng, Ting; Wang, Wei-Na; Gu, Mei-Mei; Xie, Chen-Ying; Xiao, Yu-Chao; Liu, Yuan; Wang, Lei

    2015-01-01

    Highlights: • Cd 2+ induces Cdc42 and MAPKs pathway related gene of Litopenaeus vannamei up-regulation. • Reduction of THC, increase of ROS production and apoptotic cell rate were observed when the shrimps exposure to Cd 2+ . • DsRNA-suppression of LvCdc42 and MAPKs during Cd 2+ stress reduces the ROS production and apoptosis. • We conclude that LvCdc42 and MAPKs play key roles in Cd 2+ stress responses of shrimps. - Abstract: Cadmium, one of the most toxic heavy metals in aquatic environments, has severe effects on marine invertebrates and fishes. The MAPK signaling pathway plays a vital role in stress responses of animals. The mitogen-activated protein kinase (MAPK) signaling pathway plays a vital role in animals’ stress responses, including mediation of apoptosis induced by the Rho GTPase Cdc42. However, there is limited knowledge about its function in shrimps, although disorders exacerbated by environmental stresses (including heavy metal pollution) have caused serious mortality in commercially cultured shrimps. Thus, we probed roles of Cdc42 in Litopenaeus vannamei shrimps (LvCdc42) during cadmium exposure by inhibiting its expression using dsRNA-mediated RNA interference. The treatment successfully reduced expression levels of MAPKs (including p38, JNK, and ERK). Cadmium exposure induced significant increases in expression levels of LvCdc42 and MAPKs, accompanied by reductions in total hemocyte counts (THC) and increases in apoptotic hemocyte ratios and ROS production. However, all of these responses were much weaker in LvCdc42-suppressed shrimps, in which mortality rates were higher than in controls. Our results suggest that the MAPK pathway plays a vital role in shrimps’ responses to Cd 2+ . They also indicate that LvCdc42 in shrimps participates in its regulation, and thus plays key roles in ROS production, regulation of apoptosis and associated stress responses

  20. Real-time in vivo imaging of butterfly wing development: revealing the cellular dynamics of the pupal wing tissue.

    Masaki Iwata

    Full Text Available Butterfly wings are covered with regularly arranged single-colored scales that are formed at the pupal stage. Understanding pupal wing development is therefore crucial to understand wing color pattern formation. Here, we successfully employed real-time in vivo imaging techniques to observe pupal hindwing development over time in the blue pansy butterfly, Junonia orithya. A transparent sheet of epithelial cells that were not yet regularly arranged was observed immediately after pupation. Bright-field imaging and autofluorescent imaging revealed free-moving hemocytes and tracheal branches of a crinoid-like structure underneath the epithelium. The wing tissue gradually became gray-white, epithelial cells were arranged regularly, and hemocytes disappeared, except in the bordering lacuna, after which scales grew. The dynamics of the epithelial cells and scale growth were also confirmed by fluorescent imaging. Fluorescent in vivo staining further revealed that these cells harbored many mitochondria at the surface of the epithelium. Organizing centers for the border symmetry system were apparent immediately after pupation, exhibiting a relatively dark optical character following treatment with fluorescent dyes, as well as in autofluorescent images. The wing tissue exhibited slow and low-frequency contraction pulses with a cycle of approximately 10 to 20 minutes, mainly occurring at 2 to 3 days postpupation. The pulses gradually became slower and weaker and eventually stopped. The wing tissue area became larger after contraction, which also coincided with an increase in the autofluorescence intensity that might have been caused by scale growth. Examination of the pattern of color development revealed that the black pigment was first deposited in patches in the central areas of an eyespot black ring and a parafocal element. These results of live in vivo imaging that covered wide wing area for a long time can serve as a foundation for studying the

  1. Real-time in vivo imaging of butterfly wing development: revealing the cellular dynamics of the pupal wing tissue.

    Iwata, Masaki; Ohno, Yoshikazu; Otaki, Joji M

    2014-01-01

    Butterfly wings are covered with regularly arranged single-colored scales that are formed at the pupal stage. Understanding pupal wing development is therefore crucial to understand wing color pattern formation. Here, we successfully employed real-time in vivo imaging techniques to observe pupal hindwing development over time in the blue pansy butterfly, Junonia orithya. A transparent sheet of epithelial cells that were not yet regularly arranged was observed immediately after pupation. Bright-field imaging and autofluorescent imaging revealed free-moving hemocytes and tracheal branches of a crinoid-like structure underneath the epithelium. The wing tissue gradually became gray-white, epithelial cells were arranged regularly, and hemocytes disappeared, except in the bordering lacuna, after which scales grew. The dynamics of the epithelial cells and scale growth were also confirmed by fluorescent imaging. Fluorescent in vivo staining further revealed that these cells harbored many mitochondria at the surface of the epithelium. Organizing centers for the border symmetry system were apparent immediately after pupation, exhibiting a relatively dark optical character following treatment with fluorescent dyes, as well as in autofluorescent images. The wing tissue exhibited slow and low-frequency contraction pulses with a cycle of approximately 10 to 20 minutes, mainly occurring at 2 to 3 days postpupation. The pulses gradually became slower and weaker and eventually stopped. The wing tissue area became larger after contraction, which also coincided with an increase in the autofluorescence intensity that might have been caused by scale growth. Examination of the pattern of color development revealed that the black pigment was first deposited in patches in the central areas of an eyespot black ring and a parafocal element. These results of live in vivo imaging that covered wide wing area for a long time can serve as a foundation for studying the cellular dynamics of living

  2. Two's a crowd: phenotypic adjustments and prophylaxis in Anticarsia gemmatalis larvae are triggered by the presence of conspecifics.

    Farley W S Silva

    Full Text Available Defence from parasites and pathogens involves a cost. Thus, it is expected that organisms use this only at high population densities, where the risk of pathogen transmission may be high, as proposed by the "density-dependent prophylaxis" (DDP hypothesis. These predictions have been tested in a wide range of insects, both in comparative and experimental studies. We think it pertinent to consider a continuum between solitarious and gregarious living insects, wherein: (1 solitarious insects are those that are constitutively solitary and do not express any phenotypic plasticity, (2 the middle of the continuum is represented by insects that are subject to fluctuations in local density and show a range of facultative and plastic changes; and (3 constitutively gregarious forms live gregariously and show the gregarious phenotype even in the absence of crowding stimuli. We aimed to chart some of the intermediary continuum with an insect that presents solitarious aspects, but that is subject to fluctuations in density. Thus, Anticarsia gemmatalis (Lepidoptera: Noctuidae larvae reared at higher densities showed changes in coloration, a greater degree of encapsulation, had higher hemocyte densities and were more resistant to Baculovirus anticarsia, but not to Bacillus thuringiensis. Meanwhile, with increased rearing density there was reduced capsule melanization. Hemocyte density was the only variable that did not vary according to larval phenotype. The observed responses were not a continuous function of larval density, but an all-or-nothing response to the presence of a conspecific. As A. gemmatalis is not known for gregarious living, yet shows these density-dependent changes, it thus seems that this plastic phenotypic adjustment may be a broader phenomenon than previously thought.

  3. The glucuronyltransferase GlcAT-P is required for stretch growth of peripheral nerves in Drosophila.

    Rahul Pandey

    Full Text Available During development, the growth of the animal body is accompanied by a concomitant elongation of the peripheral nerves, which requires the elongation of integrated nerve fibers and the axons projecting therein. Although this process is of fundamental importance to almost all organisms of the animal kingdom, very little is known about the mechanisms regulating this process. Here, we describe the identification and characterization of novel mutant alleles of GlcAT-P, the Drosophila ortholog of the mammalian glucuronyltransferase b3gat1. GlcAT-P mutants reveal shorter larval peripheral nerves and an elongated ventral nerve cord (VNC. We show that GlcAT-P is expressed in a subset of neurons in the central brain hemispheres, in some motoneurons of the ventral nerve cord as well as in central and peripheral nerve glia. We demonstrate that in GlcAT-P mutants the VNC is under tension of shorter peripheral nerves suggesting that the VNC elongates as a consequence of tension imparted by retarded peripheral nerve growth during larval development. We also provide evidence that for growth of peripheral nerve fibers GlcAT-P is critically required in hemocytes; however, glial cells are also important in this process. The glial specific repo gene acts as a modifier of GlcAT-P and loss or reduction of repo function in a GlcAT-P mutant background enhances VNC elongation. We propose a model in which hemocytes are required for aspects of glial cell biology which in turn affects the elongation of peripheral nerves during larval development. Our data also identifies GlcAT-P as a first candidate gene involved in growth of integrated peripheral nerves and therefore establishes Drosophila as an amenable in-vivo model system to study this process at the cellular and molecular level in more detail.

  4. Essential roles of Cdc42 and MAPK in cadmium-induced apoptosis in Litopenaeus vannamei

    Peng, Ting; Wang, Wei-Na, E-mail: weina63@aliyun.com; Gu, Mei-Mei; Xie, Chen-Ying; Xiao, Yu-Chao; Liu, Yuan; Wang, Lei

    2015-06-15

    Highlights: • Cd{sup 2+} induces Cdc42 and MAPKs pathway related gene of Litopenaeus vannamei up-regulation. • Reduction of THC, increase of ROS production and apoptotic cell rate were observed when the shrimps exposure to Cd{sup 2+}. • DsRNA-suppression of LvCdc42 and MAPKs during Cd{sup 2+} stress reduces the ROS production and apoptosis. • We conclude that LvCdc42 and MAPKs play key roles in Cd{sup 2+} stress responses of shrimps. - Abstract: Cadmium, one of the most toxic heavy metals in aquatic environments, has severe effects on marine invertebrates and fishes. The MAPK signaling pathway plays a vital role in stress responses of animals. The mitogen-activated protein kinase (MAPK) signaling pathway plays a vital role in animals’ stress responses, including mediation of apoptosis induced by the Rho GTPase Cdc42. However, there is limited knowledge about its function in shrimps, although disorders exacerbated by environmental stresses (including heavy metal pollution) have caused serious mortality in commercially cultured shrimps. Thus, we probed roles of Cdc42 in Litopenaeus vannamei shrimps (LvCdc42) during cadmium exposure by inhibiting its expression using dsRNA-mediated RNA interference. The treatment successfully reduced expression levels of MAPKs (including p38, JNK, and ERK). Cadmium exposure induced significant increases in expression levels of LvCdc42 and MAPKs, accompanied by reductions in total hemocyte counts (THC) and increases in apoptotic hemocyte ratios and ROS production. However, all of these responses were much weaker in LvCdc42-suppressed shrimps, in which mortality rates were higher than in controls. Our results suggest that the MAPK pathway plays a vital role in shrimps’ responses to Cd{sup 2+}. They also indicate that LvCdc42 in shrimps participates in its regulation, and thus plays key roles in ROS production, regulation of apoptosis and associated stress responses.

  5. The glucuronyltransferase GlcAT-P is required for stretch growth of peripheral nerves in Drosophila.

    Pandey, Rahul; Blanco, Jorge; Udolph, Gerald

    2011-01-01

    During development, the growth of the animal body is accompanied by a concomitant elongation of the peripheral nerves, which requires the elongation of integrated nerve fibers and the axons projecting therein. Although this process is of fundamental importance to almost all organisms of the animal kingdom, very little is known about the mechanisms regulating this process. Here, we describe the identification and characterization of novel mutant alleles of GlcAT-P, the Drosophila ortholog of the mammalian glucuronyltransferase b3gat1. GlcAT-P mutants reveal shorter larval peripheral nerves and an elongated ventral nerve cord (VNC). We show that GlcAT-P is expressed in a subset of neurons in the central brain hemispheres, in some motoneurons of the ventral nerve cord as well as in central and peripheral nerve glia. We demonstrate that in GlcAT-P mutants the VNC is under tension of shorter peripheral nerves suggesting that the VNC elongates as a consequence of tension imparted by retarded peripheral nerve growth during larval development. We also provide evidence that for growth of peripheral nerve fibers GlcAT-P is critically required in hemocytes; however, glial cells are also important in this process. The glial specific repo gene acts as a modifier of GlcAT-P and loss or reduction of repo function in a GlcAT-P mutant background enhances VNC elongation. We propose a model in which hemocytes are required for aspects of glial cell biology which in turn affects the elongation of peripheral nerves during larval development. Our data also identifies GlcAT-P as a first candidate gene involved in growth of integrated peripheral nerves and therefore establishes Drosophila as an amenable in-vivo model system to study this process at the cellular and molecular level in more detail.

  6. Characterization and transcription studies of a phytochelatin synthase gene from the solitary tunicate Ciona intestinalis exposed to cadmium

    Franchi, Nicola [Department of Biology, University of Padova, Padova (Italy); Department of Biological, Chemical, Pharmaceutical Science and Technology, University of Palermo, Palermo (Italy); Piccinni, Ester [Department of Biology, University of Padova, Padova (Italy); Ferro, Diana [Department of Biology, University of Padova, Padova (Italy); Institute for Evolution and Biodiversity, Westfälische Wilhelms-Universität, Münster (Germany); Basso, Giuseppe [Department of Woman and Child Health, University of Padova, Padova (Italy); Spolaore, Barbara [CRIBI Biotechnology Centre, University of Padova, Padova (Italy); Department of Pharmaceutical and Pharmacological Sciences, University of Padova, Padova (Italy); Santovito, Gianfranco, E-mail: gianfranco.santovito@unipd.it [Department of Biology, University of Padova, Padova (Italy); Ballarin, Loriano [Department of Biology, University of Padova, Padova (Italy)

    2014-07-01

    Highlights: • Ciona intestinalis have a functional phytochelatin synthase (PCS) gene (cipcs). • CiPCS amino acid sequence is phylogentically related to other metazoan PCSs. • CiPCS catalyze the synthesis of PC2. • cipcs are mostly transcribed in circulating hemocytes, in both tunic and blood lacunae. • Cadmium exposure results in a significant increase of cipcs and cipcna transcription. - Abstract: The major thiol-containing molecules involved in controlling the level of intracellular ROS in eukaryotes, acting as a nonenzymatic detoxification system, are metallothioneins (MTs), glutathione (GSH) and phytochelatins (PCs). Both MTs and GSH are well-known in the animal kingdom. PC was considered a prerogative of the plant kingdom but, in 2001, a phytochelatin synthase (PCS) gene was described in the nematode Caenorhabditis elegans; additional genes encoding this enzyme were later described in the earthworm Eisenia fetida and in the parasitic nematode Schistosoma mansoni but scanty data are available, up to now, for Deuterostomes. Here, we describe the molecular characteristics and transcription pattern, in the presence of Cd, of a PCS gene from the invertebrate chordate Ciona intestinalis, a ubiquitous solitary tunicate and demonstrate the presence of PCs in tissue extracts. We also studied mRNA localization by in situ hybridization. In addition, we analyzed the behavior of hemocytes and tunic cells consequent to Cd exposure as well as the transcription pattern of the Ciona orthologous for proliferating cell nuclear antigen (PCNA), usually considered a proliferation marker, and observed that cell proliferation occurs after 96 h of Cd treatment. This matches the hypothesis of Cd-induced cell proliferation, as already suggested by previous data on the expression of a metallothionein gene in the same animal.

  7. Relative roles of the cellular and humoral responses in the Drosophila host defense against three gram-positive bacterial infections.

    Nadine T Nehme

    2011-03-01

    Full Text Available Two NF-kappaB signaling pathways, Toll and immune deficiency (imd, are required for survival to bacterial infections in Drosophila. In response to septic injury, these pathways mediate rapid transcriptional activation of distinct sets of effector molecules, including antimicrobial peptides, which are important components of a humoral defense response. However, it is less clear to what extent macrophage-like hemocytes contribute to host defense.In order to dissect the relative importance of humoral and cellular defenses after septic injury with three different gram-positive bacteria (Micrococcus luteus, Enterococcus faecalis, Staphylococcus aureus, we used latex bead pre-injection to ablate macrophage function in flies wildtype or mutant for various Toll and imd pathway components. We found that in all three infection models a compromised phagocytic system impaired fly survival--independently of concomitant Toll or imd pathway activation. Our data failed to confirm a role of the PGRP-SA and GNBP1 Pattern Recognition Receptors for phagocytosis of S. aureus. The Drosophila scavenger receptor Eater mediates the phagocytosis by hemocytes or S2 cells of E. faecalis and S. aureus, but not of M. luteus. In the case of M. luteus and E. faecalis, but not S. aureus, decreased survival due to defective phagocytosis could be compensated for by genetically enhancing the humoral immune response.Our results underscore the fundamental importance of both cellular and humoral mechanisms in Drosophila immunity and shed light on the balance between these two arms of host defense depending on the invading pathogen.

  8. Effects of heavy metals on the oyster (Crassostrea virginica at Mandinga Lagoon, Veracruz, Mexico

    X Guzmán-García

    2009-12-01

    Full Text Available The Mandinga Lagoon in the Mexican State of Veracruz is an important ecological zone that produces 32% of the oyster output in the state of Veracruz, the main oyster producer in Mexico. Samples of water, sediment, and oysters were collected in 2003 and 2004 to study heavy metal pollution. Metal concentrations were determined in water, soil, and oyster tissues from fresh and detoxified Crassostrea virginica, and histology samples were analyzed. Metal (Cr, Cd, and Pb concentrations in water were within the Mexican legal limits. The recorded values in sediments corresponded to those not producing biological effects (ERL. In the tissues, the highest concentrations corresponded to Pb, above 5.84 μgg-1 dry weight (d.w.; Cd was of 2.23 μgg-1 d.w., and Cr above 6 μgg-1 d.w. The metal levels detected in oysters exceeded the maximum permissible limits (MPL for Cd and Pb, and oysters were unable to eliminate the concentrations of the bioaccumulated metals during the detoxification stage. The histopathological analysis revealed lesions in the digestive gland, edema, atrophy of epithelia in the digestive tubules, the presence of brown vesicles, hemocytic reaction, and necrosis. During detoxification, a higher number of epithelia were observed in the tubules, as well as an increase in brown vesicles and hemocytic reaction. Forty seven percent of oysters presented histopathological lesions related to metal concentrations. It is important to monitor metal concentrations, to detect the source of pollution, andto evaluate the effects on organisms to establish culture areas and adequate criteria for the exploitation of such an important fishery resource. Rev. Biol. Trop. 57 (4: 955-962. Epub 2009 December 01.

  9. Type I and type II crustins from Penaeus monodon, genetic variation and antimicrobial activity of the most abundant crustinPm4.

    Donpudsa, Suchao; Visetnan, Suwattana; Supungul, Premruethai; Tang, Sureerat; Tassanakajon, Anchalee; Rimphanitchayakit, Vichien

    2014-11-01

    An antimicrobial protein, crustin, is involved in the innate immunity of crustacean by defending the host directly against the microbial pathogens. By data mining the Penaeus monodon EST database, two type I crustins, carcininPm1 and 2, and ten type II crustins, crustinPm1-10, were identified. The abundant crustins were crustinPm1, 4 and 7, each with variation in the length of Gly-rich repeat among its members. A few crustinPm1, 4 and 7 with deletion in the Cys-rich region were also observed. Furthermore, the crustinPm4 with the longest N-terminal Gly-rich region was characterized. The crustinPm4 allelic genes were expressed mainly from the hemocytes. Its expression was up-regulated readily by WSSV infection and gradually decreased to normal level afterwards. The recombinant crustinPm4-1 (rcrustinPm4-1) isoform was produced using the Escherichia coli expression system and tested for its antimicrobial activity. The rcrustinPm4-1 was able to inhibit the growth of a Gram-positive bacterium, Bacillus megaterium but not Bacillus subtilis, Micrococcus luteus and Staphylococcus aureus. It also inhibited the growth of two Gram-negative bacteria, E. coli 363 and Vibrio harveyi 639 at lower potency. The rcrustinPm4-1 affected the WSSV infection because the expression of an intermediate early gene ie1 in WSSV-infected hemocyte cell culture was reduced. It was shown further that the rcrustinPm4-1 could delay by about one and a half days the manifestation of disease by WSSV. Copyright © 2014 Elsevier Ltd. All rights reserved.

  10. Tumor necrosis factor receptor-associated factor 6 (TRAF6) participates in anti-lipopolysaccharide factors (ALFs) gene expression in mud crab.

    Sun, Wan-Wei; Zhang, Xin-Xu; Wan, Wei-Song; Wang, Shu-Qi; Wen, Xiao-Bo; Zheng, Huai-Ping; Zhang, Yue-Ling; Li, Sheng-Kang

    2017-02-01

    Tumor necrosis factor receptor-associated factor 6 (TRAF6) is a key cytoplasm signal adaptor that mediates signals activated by tumor necrosis factor receptor (TNFR) superfamily and the Interleukin-1 receptor/Toll-like receptor (IL-1/TLR) superfamily. The full-length 2492 bp TRAF6 (Sp-TRAF6) from Scylla paramamosain contains 1800 bp of open reading frame (ORF) encoding 598 amino acids, including an N-terminal RING-type zinc finger, two TRAF-type zinc fingers and a conserved C-terminal meprin and TRAF homology (MATH) domain. Multiple alignment analysis shows that the putative amino acid sequence of Sp-TRAf6 has highest identity of 88% with Pt-TRAF6 from Portunus trituberculatus, while the similarity of Sp-TRAF6 with other crustacean sequences was 54-55%. RT-PCR analysis indicated that Sp-TRAF6 transcripts were predominantly expressed in the hepatopancreas and stomach, whereas it was barely detected in the heart and hemocytes in our study. Moreover, Sp-TRAF6 transcripts were significantly up-regulated after Vibrio parahemolyticus and LPS challenges. RNA interference assay was carried out used by siRNA to investigate the genes expression patterns regulated by Sp-TRAF6. The qRT-PCR results showed that silencing Sp-TRAF6 gene could inhibit SpALF1, SpALF2, SpALF5 and SpALF6 expression in hemocytes, while inhibit SpALF1, SpALF3, SpALF4, SpALF5 and SpALF6 expression in hepatopancreas. Taken together, the acute-phase response to immune challenges and the inhibition of SpALFs gene expression indicate that Sp-TRAF6 plays an important role in host defense against pathogen invasions via regulation of ALF gene expression in S. paramamosain. Copyright © 2016. Published by Elsevier Ltd.

  11. Identification of a serine proteinase homolog (Sp-SPH involved in immune defense in the mud crab Scylla paramamosain.

    Qiu-xia Zhang

    Full Text Available Clip domain serine proteinase homologs are involved in many biological processes including immune response. To identify the immune function of a serine proteinase homolog (Sp-SPH, originally isolated from hemocytes of the mud crab, Scylla paramamosain, the Sp-SPH was expressed recombinantly and purified for further studies. It was found that the Sp-SPH protein could bind to a number of bacteria (including Aeromonas hydrophila, Escherichia coli, Staphylococcus aureus, Vibrio fluvialis, Vibrio harveyi and Vibrio parahemolyticus, bacterial cell wall components such as lipopolysaccharide or peptidoglycan (PGN, and β-1, 3-glucan of fungus. But no direct antibacterial activity of Sp-SPH protein was shown by using minimum inhibitory concentration or minimum bactericidal concentration assays. Nevertheless, the Sp-SPH protein was found to significantly enhance the crab hemocyte adhesion activity (paired t-test, P<0.05, and increase phenoloxidase activity if triggered by PGN in vitro (paired t-test, P<0.05. Importantly, the Sp-SPH protein was demonstrated to promote the survival rate of the animals after challenge with A. hydrophila or V. parahemolyticus which were both recognized by Sp-SPH protein, if pre-incubated with Sp-SPH protein, respectively. Whereas, the crabs died much faster when challenged with Vibrio alginolyiicus, a pathogenic bacterium not recognized by Sp-SPH protein, compared to those of crabs challenged with A. hydrophila or V. parahemolyticus when pre-coated with Sp-SPH protein. Taken together, these data suggested that Sp-SPH molecule might play an important role in immune defense against bacterial infection in the mud crab S. paramamosain.

  12. Identification and characterization of the related immune-enhancing proteins in crab Scylla paramamosain stimulated with rhubarb polysaccharides.

    Cao, Jingsong; Wang, Zehuan; Zhang, Yueling; Qu, Fengliang; Guo, Lingling; Zhong, Mingqi; Li, Shengkang; Zou, Haiying; Chen, Jiehui; Wang, Xiuying

    2014-02-01

    Recently, considerable interest has been focused on immunostimulants to reduce diseases in crab aquaculture. However, information regarding to the related immune-enhancing proteins in crabs is not available yet. In this study, rhubarb polysaccharides were tested for enhancement of the immune activity in crab Scylla paramamosain. Compared with those in the control group, values of, phenoloxidase (PO), alkaline phosphatase (AKP) and alkaline phosphatasein (ACP) activity in the, experimental group were improved significantly 4 d after the treatment. Furthermore, 15 and 17 altered proteins from haemocytes and hepatopancreas, respectively, were found in rhubarb polysaccharide-treated crabs using 2-DE approach. Of these, hemocyanin, chymotrypsin, cryptocyanin, C-type lectin receptor, and ferritin protein were identified by mass spectrometry. In addition, RT-PCR, analysis showed that the mRNA levels of hemocyanin and chymotrypsin increased about 2.4- and 1.4-fold in the experiment group. Moreover, the hemocyanin gene in S. paramamosain (SpHMC) was, cloned and characterized. SpHMC contains one open reading frame of 2022 bp and encodes a polypeptide of 673 amino acids. It is clustered into one branch along with crab hemocyanin in a phylogenetic tree. The mRNA transcripts of SpHMC were detected mainly in the tissues of, hepatopancreas, hemocyte and intestines, and its levels were up-regulated significantly in hemocytes, of S. paramamosain treated with Vibrio parahemolyticus, Beta streptococcus or poly I:C for 6-48 h. Taken together, these studies found 5 related immune-enhancing proteins and a novel heomcyanin homologue with potential pathogen-resistant activities in crab. Copyright © 2013 Elsevier Ltd. All rights reserved.

  13. Analysis on the expression and function of syndecan in the Pacific white shrimp Litopenaeus vannamei.

    Yang, Hui; Li, Shihao; Li, Fuhua; Wen, Rong; Xiang, Jianhai

    2015-08-01

    Syndecan is considered to be a multifunctional protein which functions as a cell surface receptor involved in cell adhesion, migration, cytoskeleton organization and differentiation. Previous bioinformatic analysis has revealed that syndecan in shrimp might interact with white spot syndrome virus (WSSV). In the present study, we experimentally studied the function of syndecan in shrimp immunity. The syndecan from Litopenaeus vannamei (LvSDC) was cloned and analyzed. The full-length cDNA of LvSDC was 1005 bp, consisting of 59 bp 5'-UTR, 253 bp 3'-UTR, and 693 bp open reading frame encoding 230 amino acids. LvSDC consisted of an extracellular domain (ED), a transmembrane domain (TM) and a cytoplasmic domain (CD). TM and CD shared high similarities with those of syndecan proteins from other species. LvSDC was ubiquitously expressed in all tested tissues, with the highest level in Oka. After WSSV challenge, the transcription level of LvSDC in Oka was apparently up-regulated. Recombinant LvSDC protein and its rabbit polyclonal antibody were prepared for detecting the location of LvSDC in hemocytes using immunocytochemistry approach. Data showed that LvSDC mainly located at the cell membrane and the cytoplasm of hemocytes. After silencing of LvSDC with siRNA, the WSSV copy numbers and mortality of shrimp after WSSV infection were both significantly decreased. These data provide useful information for understanding the immune mechanism of shrimp to WSSV infection. Copyright © 2015 Elsevier Ltd. All rights reserved.

  14. The novel white spot syndrome virus-induced gene, PmERP15, encodes an ER stress-responsive protein in black tiger shrimp, Penaeus monodon.

    Leu, Jiann-Horng; Liu, Kuan-Fu; Chen, Kuan-Yu; Chen, Shu-Hwa; Wang, Yu-Bin; Lin, Chung-Yen; Lo, Chu-Fang

    2015-04-01

    By microarray screening, we identified a white spot syndrome virus (WSSV)-strongly induced novel gene in gills of Penaeus monodon. The gene, PmERP15, encodes a putative transmembrane protein of 15 kDa, which only showed some degree of similarity (54-59%) to several unknown insect proteins, but had no hits to shrimp proteins. RT-PCR showed that PmERP15 was highly expressed in the hemocytes, heart and lymphoid organs, and that WSSV-induced strong expression of PmERP15 was evident in all tissues examined. Western blot analysis likewise showed that WSSV strongly up-regulated PmERP15 protein levels. In WSSV-infected hemocytes, immunofluorescence staining showed that PmERP15 protein was colocalized with an ER enzyme, protein disulfide isomerase, and in Sf9 insect cells, PmERP15-EGFP fusion protein colocalized with ER -Tracker™ Red dye as well. GRP78, an ER stress marker, was found to be up-regulated in WSSV-infected P. monodon, and both PmERP15 and GRP78 were up-regulated in shrimp injected with ER stress inducers tunicamycin and dithiothreitol. Silencing experiments showed that although PmERP15 dsRNA-injected shrimp succumbed to WSSV infection more rapidly, the WSSV copy number had no significant changes. These results suggest that PmERP15 is an ER stress-induced, ER resident protein, and its induction in WSSV-infected shrimp is caused by the ER stress triggered by WSSV infection. Furthermore, although PmERP15 has no role in WSSV multiplication, its presence is essential for the survival of WSSV-infected shrimp. Copyright © 2014 Elsevier Ltd. All rights reserved.

  15. A Novel Vascular Endothelial Growth Factor Receptor Participates in White Spot Syndrome Virus Infection in Litopenaeus vannamei

    Shihao Li

    2017-11-01

    Full Text Available Vascular endothelial growth factor (VEGF signaling pathway is known to play key roles in endothelial cell proliferation, migration, angiogenesis, vascular permeability, inhibition of apoptosis, and virus infection. In the present study, a novel VEGFR gene (LvVEGFR2 was identified and characterized from Litopenaeus vannamei. The deduced amino acid sequence of LvVEGFR2 possessed typical features of VEGFRs reported in other species, including six IG-like domains, a transmembrane motif, a protein kinase (PK domain, and one tyrosine-PK active site. The transcripts of LvVEGFR2 were mainly detected in hemocytes and lymphoid organ (Oka. Subcellular localization analysis showed that LvVEGFR2 was a membrane protein. Its expression level was obviously upregulated in hemocytes and Oka of the shrimp after white spot syndrome virus (WSSV infection. Knockdown of LvVEGFR2 gene expression by double-strand RNA mediated interference could lead to a decrease of virus copy number in WSSV-infected shrimp. The interaction between LvVEGFR2 and different LvVEGFs (LvVEGF1, LvVEGF2, and LvVEGF3 in shrimp was analyzed at the transcription level and protein level, respectively. Knockdown of LvVEGF2 or LvVEGF3 could downregulate the expression level of LvVEGFR2, and injection of the recombinant LvVEGF2 or LvVEGF3 could upregulate the expression level of LvVEGFR2. Yeast two-hybrid analysis showed that LvVEGFR2 could interact with LvVEGF2 and LvVEGF3 directly. The study improved our understanding on the VEGF signaling pathway of shrimp and its role during WSSV infection.

  16. Immunoevasive protein (IEP)-containing surface layer covering polydnavirus particles is essential for viral infection.

    Furihata, Shunsuke; Tanaka, Kohjiro; Ryuda, Masasuke; Ochiai, Masanori; Matsumoto, Hitoshi; Csikos, Gyorge; Hayakawa, Yoichi

    2014-01-01

    Polydnaviruses (PDVs) are unique symbiotic viruses associated with parasitoid wasps: PDV particles are injected into lepidopteran hosts along with the wasp eggs and express genes that interfere with aspects of host physiology such as immune defenses and development. Recent comparative genomic studies of PDVs have significantly improved our understanding of their origin as well as the genome organization. However, the structural features of functional PDV particles remain ambiguous. To clear up the structure of Cotesia kariyai PDV (CkPDV) particles, we focused on immunoevasive protein (IEP), which is a mediator of immunoevasion by the wasp from the encapsulation reaction of the host insect's hemocytes, because it has been demonstrated to be present on the surface of the virus particle. We discovered that IEP tends to polymerize and constitutes a previously unidentified thin surface layer covering CkPDV particles. This outermost surface layer looked fragile and was easily removed from CkPVD particles by mechanical stressors such as shaking, which prevented CkPDV from expressing the encoded genes in the host target tissues such as fat body or hemocytes. Furthermore, we detected IEP homologue gene expression in the wasp's venom reservoirs, implying IEP has another unknown biological function in the wasp or parasitized hosts. Taken together, the present results demonstrated that female C. kariyai wasps produce the fragile thin layer partly composed of IEP to cover the outer surfaces of CkPDV particles; otherwise, they cannot function as infectious agents in the wasp's host. The fact that IEP family proteins are expressed in both venom reservoirs and oviducts suggests an intimate relationship between both tissues in the development of the parasitism strategy of the wasp. Copyright © 2013 Elsevier Inc. All rights reserved.

  17. Expression of matrix metalloproteinase genes during basement membrane degradation in the metamorphosis of Bombyx mori.

    Kawasaki, Hideki; Manickam, Asaithambi; Shahin, Rima; Ote, Manabu; Iwanaga, Masashi

    2018-01-05

    The present study was conducted to clarify the involvement of the basement membrane (BM) in insect metamorphosis through analysis of the expression profile of two types of metalloproteinase (MMP and ADAMTS) genes in several organs, their ecdysone involvement, and the histological change of BM. BM was observed around wing sac and in the wing cavity and around fat bodies at the W0 stage but disappeared after the W3 stage, and wing discs evaginated and fat body cells scattered after the W3 stage. The disappearance of the BM of midgut and silk glands was not observed after the W3 stage, but degenerated epithelium cells in the midgut and shrunken cells in the silk gland were observed after the W3 stage. BmMMP1 showed a peak at P0 in the wing discs, fat bodies, midgut, and silk gland. BmMMP2 showed a broad peak around pupation in the wing discs, fat bodies, midgut, and silk gland. BmADAMTS-1 showed enhanced expression at W2 in the wing discs, fat bodies, midgut, and hemocyte, while BmADAMTS-L showed enhanced expression at W3 in the fat bodies, midgut, silk gland, and hemocyte. After pupation, they showed a different expression in different organs. All of four genes were induced by 20-hydroxyecdysone in wing discs in vitro. The present results suggested the involvement of MMPs and ADAMTS in the BM digestion and the morphogenesis of organs during Bombyx metamorphosis. Copyright © 2017 Elsevier B.V. All rights reserved.

  18. Structural and functional characterization of a novel molluskan ortholog of TRAF and TNF receptor-associated protein from disk abalone (Haliotis discus discus).

    Lee, Youngdeuk; Elvitigala, Don Anushka Sandaruwan; Whang, Ilson; Lee, Sukkyoung; Kim, Hyowon; Zoysa, Mahanama De; Oh, Chulhong; Kang, Do-Hyung; Lee, Jehee

    2014-09-01

    Immune signaling cascades have an indispensable role in the host defense of almost all the organisms. Tumor necrosis factor (TNF) signaling is considered as a prominent signaling pathway in vertebrate as well as invertebrate species. Within the signaling cascade, TNF receptor-associated factor (TRAF) and TNF receptor-associated protein (TTRAP) has been shown to have a crucial role in the modulation of immune signaling in animals. Here, we attempted to characterize a novel molluskan ortholog of TTRAP (AbTTRAP) from disk abalone (Haliotis discus discus) and analyzed its expression levels under pathogenic stress. The complete coding sequence of AbTTRAP consisted of 1071 nucleotides, coding for a 357 amino acid peptide, with a predicted molecular mass of 40 kDa. According to our in-silico analysis, AbTTRAP resembled the typical TTRAP domain architecture, including a 5'-tyrosyl DNA phosphodiesterase domain. Moreover, phylogenetic analysis revealed its common ancestral invertebrate origin, where AbTTRAP was clustered with molluskan counterparts. Quantitative real time PCR showed universally distributed expression of AbTTRAP in selected tissues of abalone, from which more prominent expression was detected in hemocytes. Upon stimulation with two pathogen-derived mitogens, lipopolysaccharide (LPS) and polyinosinic:polycytidylic acid (poly I:C), transcript levels of AbTTRAP in hemocytes and gill tissues were differentially modulated with time. In addition, the recombinant protein of AbTTRAP exhibited prominent endonuclease activity against abalone genomic DNA, which was enhanced by the presence of Mg(2+) in the medium. Collectively, these results reinforce the existence of the TNF signaling cascade in mollusks like disk abalone, further implicating the putative regulatory behavior of TTRAP in invertebrate host pathology. Copyright © 2014 Elsevier Ltd. All rights reserved.

  19. Proteomic characterization of the hemolymph of Octopus vulgaris infected by the protozoan parasite Aggregata octopiana.

    Castellanos-Martínez, Sheila; Diz, Angel P; Álvarez-Chaver, Paula; Gestal, Camino

    2014-06-13

    The immune system of cephalopods is poorly known to date. The lack of genomic information makes difficult to understand vital processes like immune defense mechanisms and their interaction with pathogens at molecular level. The common octopus Octopus vulgaris has a high economic relevance and potential for aquaculture. However, disease outbreaks provoke serious reductions in production with potentially severe economic losses. In this study, a proteomic approach is used to analyze the immune response of O. vulgaris against the coccidia Aggregata octopiana, a gastrointestinal parasite which impairs the cephalopod nutritional status. The hemocytes and plasma proteomes were compared by 2-DE between sick and healthy octopus. The identities of 12 differentially expressed spots and other 27 spots without significant alteration from hemocytes, and 5 spots from plasma, were determined by mass spectrometry analysis aided by a six reading-frame translation of an octopus hemocyte RNA-seq database and also public databases. Principal component analysis pointed to 7 proteins from hemocytes as the major contributors to the overall difference between levels of infection and so could be considered as potential biomarkers. Particularly, filamin, fascin and peroxiredoxin are highlighted because of their implication in octopus immune defense activity. From the octopus plasma, hemocyanin was identified. This work represents a first step forward in order to characterize the protein profile of O. vulgaris hemolymph, providing important information for subsequent studies of the octopus immune system at molecular level and also to the understanding of the basis of octopus tolerance-resistance to A. octopiana. The immune system of cephalopods is poorly known to date. The lack of genomic information makes difficult to understand vital processes like immune defense mechanisms and their interaction with pathogens at molecular level. The study herein presented is focused to the comprehension of

  20. TmSR-C, scavenger receptor class C, plays a pivotal role in antifungal and antibacterial immunity in the coleopteran insect Tenebrio molitor.

    Kim, Soo Gon; Jo, Yong Hun; Seong, Jeong Hwan; Park, Ki Beom; Noh, Mi Young; Cho, Jun Ho; Ko, Hye Jin; Kim, Chang Eun; Tindwa, Hamisi; Patnaik, Bharat Bhusan; Bang, In Seok; Lee, Yong Seok; Han, Yeon Soo

    2017-10-01

    Scavenger receptors (SRs) constitute a family of membrane-bound receptors that bind to multiple ligands. The SR family of proteins is involved in removing cellular debris, oxidized low-density lipoproteins, and pathogens. Specifically, class C scavenger receptors (SR-C) have also been reported to be involved in phagocytosis of gram-positive and -negative bacteria in Drosophila and viruses in shrimp. However, reports are unavailable regarding the role of SR-C in antifungal immune mechanisms in insects. In this study, a full-length Tenebrio molitor SR-C (TmSR-C) sequence was obtained by 5'- and 3'-Rapid amplification of cDNA ends-polymerase chain reaction (RACE-PCR). The TmSR-C full-length cDNA comprised 1671 bp with 5'- and 3'-untranslated regions of 23- and 107-bp, respectively. TmSR-C encodes a putative protein of 556 amino acid residues that is constitutively expressed in all tissues of late instar larvae and 2-day-old adults, with the highest transcript levels observed in hemocytes of larvae and adults. TmSR-C mRNA showed a 2.5-fold and 3-fold increase at 24 and 6 h after infection with Candida albicans and β-glucan, respectively. Immunoassay with TmSR-C polyclonal antibody showed induction of the putative protein in the cytosols of hemocytes at 3 h after inoculation of C. albicans. RNA interference (RNAi)-based gene silencing and phagocytosis assays were used to understand the role of TmSR-C in antifungal immunity. Silencing of TmSR-C transcripts reduced the survivability of late instar larvae at 2 days post-inoculation of C. albicans, Escherichia coli, or Staphylococcus aureus. Furthermore, in TmSR-C-silenced larvae, there was a decline in the rate of microorganism phagocytosis. Taken together, results of this study suggest that TmSR-C plays a pivotal role in phagocytosing not only fungi but also gram-negative and -positive bacteria in T. molitor. Copyright © 2017. Published by Elsevier Ltd.

  1. The effects of stress hormones on immune function may be vital for the adaptive reconfiguration of the immune system during fight-or-flight behavior.

    Adamo, Shelley A

    2014-09-01

    Intense, short-term stress (i.e., robust activation of the fight-or-flight response) typically produces a transient decline in resistance to disease in animals across phyla. Chemical mediators of the stress response (e.g., stress hormones) help induce this decline, suggesting that this transient immunosuppression is an evolved response. However, determining the function of stress hormones on immune function is difficult because of their complexity. Nevertheless, evidence suggests that stress hormones help maintain maximal resistance to disease during the physiological changes needed to optimize the body for intense physical activity. Work on insects demonstrates that stress hormones both shunt resources away from the immune system during fight-or-flight responses as well as reconfigure the immune system. Reconfiguring the immune system minimizes the impact of the loss of these resources and reduces the increased costs of some immune functions due to the physiological changes demanded by the fight-or-flight response. For example, during the stress response of the cricket Gryllus texensis, some molecular resources are shunted away from the immune system and toward lipid transport, resulting in a reduction in resistance to disease. However, insects' immune cells (hemocytes) have receptors for octopamine (the insect stress neurohormone). Octopamine increases many hemocyte functions, such as phagocytosis, and these changes would tend to mitigate the decline in immunity due to the loss of molecular resources. Moreover, because the stress response generates oxidative stress, some immune responses are probably more costly when activated during a stress response (e.g., those that produce reactive molecules). Some of these immune responses are depressed during stress in crickets, while others, whose costs are probably not increased during a stress response, are enhanced. Some effects of stress hormones on immune systems may be better understood as examples of reconfiguration

  2. Bacillus NP5 Improves Growth Performance and Resistance Against Infectious Myonecrosis Virus in White Shrimp (Litopenaeus vannamei (Bacillus NP5 Meningkatkan Pertumbuhan dan Ketahanan Terhadap Infeksi Virus Myonecrosis pada Udang Putih (L. vannamei

    Widanarni Widanarni

    2015-07-01

    Full Text Available Infectious Myonecrosis (IMN merupakan salah satu penyakit yang sering menyerang udang vaname. Probiotik banyak digunakan pada budidaya udang karena terbukti mampu mengurangi serangan penyakit pada udang. Penelitian ini bertujuan untuk menguji pengaruh pemberian probiotik Bacillus NP5 melalui pakan terhadap kinerja pertumbuhan, respons imun, dan resistensi udang vaname terhadap infeksi Infectious Myonecrosis Virus (IMNV. Udang vaname Litopenaeus vannamei (2.41±0.07 g ekor-1 diberi pakan yang disuplementasi probiotik Bacillus NP5 dengan dosis yang berbeda, 102 CFU.g-1 (A, 104 CFU.g-1 (B, 106 CFU.g-1 (C, dan kontrol tanpa suplementasi probiotik (kontrol negatif, KN; kontrol positif, KP selama 30 hari dan dengan tiga ulangan untuk masing-masing dosis, kemudian KP, perlakuan A, B, dan C diuji tantang secara intramuskular dengan IMNV (100 µl.ekor-1. Hasil penelitian ini menunjukkan bahwa udang vaname yang diberi pakan dengan suplementasi probiotik mempunyai laju pertumbuhan harian (LPH, rasio konversi pakan (RKP, dan respons imun yang lebih tinggi. Udang tersebut juga mempunyai total hemocyte count (THC dan resistensi terhadap IMNV yang lebih tinggi dibandingkan kontrol positif. Konsentrasi probiotik 106 CFU.g-1 memberikan hasil terbaik dalam meningkatkan pertumbuhan, respon imun, dan resistensi udang vaname terhadap infeksi IMNV. Kata kunci: probiotik, Bacillus NP5, Litopenaeus vannamei, pertumbuhan, IMNV Infectious Myonecrosis (IMN is one of the most prevalent white shrimp diseases. Probiotics are widely used in shrimp cultivation because they have been proven to reduce shrimp disease outbreak. This study aimed to observe the effect of oraly administered probiotic Bacillus NP5 on the white shrimp's growth performance, immune response, and resistance to Infectious Myonecrosis Virus (IMNV infection. White shrimp Litopenaeus vannamei (2.41±0.07 g individual-1 were fed with a feed supplemented with different doses of the probiotic Bacillus NP5, i

  3. Characterization and metal-induced gene transcription of two new copper zinc superoxide dismutases in the solitary ascidian Ciona intestinalis

    Ferro, Diana; Franchi, Nicola; Mangano, Valentina; Bakiu, Rigers; Cammarata, Matteo; Parrinello, Nicolò; Santovito, Gianfranco; Ballarin, Loriano

    2013-01-01

    Highlights: •Ciona intestinalis express two copper-zinc superoxide dismutases (Cu,Zn SODs), one extracellular (Ci-SODa) and one intracellular isoform (Ci-SODb). •Promoters contain consensus sequences similar to mammalian MRE. •Metal exposure results in a significant increase of gene transcription: ci-soda is induced especially by copper and zinc, the increase of ci-sodb transcription is more evident after cadmium exposure. •Genes are mostly transcribed in circulating hemocytes and in ovarian follicular cells. -- Abstract: Antioxidant enzymes are known to protect living organisms against the oxidative stress risk, also induced by metals. In the present study, we describe the purification and molecular characterization of two Cu,Zn superoxide dismutases (SODs), referred to as Ci-SODa and Ci-SODb, from Ciona intestinalis, a basal chordate widely distributed in temperate shallow seawater. The putative amino acid sequences were compared with Cu,Zn SODs from other metazoans and phylogenetic analyses indicate that the two putative Ci-SODs are more related to invertebrate SODs than vertebrate ones. Both phylogenetic and preliminary homology modeling analyses suggest that Ci-SODa and Ci-SODb are extracellular and intracellular isoform, respectively. The mRNA of the two Cu,Zn SODs was localized in hemocytes and in ovarian follicular cells, as revealed by in situ hybridization. The time course of SOD mRNA levels in the presence of three different metals showed upregulation of ci-soda and inhibition of ci-sodb. Spectrophotometric analysis confirms the presence of SOD activity in Ciona tissues. Our in silico analyses of the ci-soda promoter region revealed putative consensus sequences similar to mammalian metal-responsive elements (MRE), suggesting that the transcription of these genes directly depends on metals. These data emphasize the importance of complex metal regulation of ci-soda and ci-sodb transcription, as components of an efficient detoxification pathway

  4. Morphological and histological studies on freshwater prawn Macrobrachium rosenbergii (de man) irradiated with {sup 60}Co gamma radiation

    Stalin, A.; Broos, K.V. [P.G. and Research Department of Zoology, Jamal Mohamed College (Autonomous), Tiruchirappalli 620020, Tamil Nadu (India); Sadiq Bukhari, A., E-mail: abjmc@yahoo.in [P.G. and Research Department of Zoology, Jamal Mohamed College (Autonomous), Tiruchirappalli 620020, Tamil Nadu (India); Syed Mohamed, H.E. [P.G. and Research Department of Zoology, Jamal Mohamed College (Autonomous), Tiruchirappalli 620020, Tamil Nadu (India); Singhal, R.K. [Analytical Spectroscopy Section, Analytical Chemistry Division, Bhabha Atomic Research Center, Trombay, Mumbai 4000085 (India); Venu-Babu, P. [Nuclear Agriculture and Biotechnology Division, Bhabha Atomic Research Center, Trombay, Mumbai 4000085 (India)

    2013-11-15

    Graphical abstract: -- Highlights: •Analyzing the impact of {sup 60}Co gamma radiation (low doses) in M. rosenbergii. •LD{sub 50} identified in 30 Gy; Irradiated exhibits morphological changes than control. •Significantly decreased HSI and histological changes in irradiated groups. •Aberration severities increased as increased of dose level. •Biomonitoring tool in determining low dose radiation pollution in aquatic ecosystem. -- Abstract: This study was framed to investigate the {sup 60}Co gamma radiation induced morphological and histological variations in freshwater prawn Macrobrachium rosenbergii. The LD{sub 50} value of {sup 60}Co gamma irradiated M. rosenbergii observed (by probit analysis) at 30 Gy. Prawns were irradiated to four different dose levels (3 mGy, 30 mGy, 300 mGy and 3000 mGy) using Theratron Phoenix TeleCobalt Unit [P-33] and one control group (without irradiation) maintained separately. Irradiated groups exhibited several morphological variations such as discoloration; damaged rostrum; opaque coloration in cephalothorax; black bands and dot formation in abdomen; deformed uropods and telson in tail regions when compared with control group. The Hepato Somatic Index reflected the severity of radiation on hepatopancreas. Histological variations in gills, hepatopancreas and muscles of irradiated groups were observed. In gills, structural changes such as swollen and fused lamellae, abnormal gill tips, hyperplasic, necrotic and clavate-globate lamellae were observed in gamma irradiated prawns. Accumulation of hemocytes in hemocoelic space, interstitial sinuses filled with abnormal infiltrated hemocytes, the tubular epithelium with ruptured basal laminae, abnormal and coagulated lumen, necrotic tubules, thickened basal laminae, tissue debris, necrotic hepatocytes were observed in irradiated prawn hepatopancreas. In muscle, shrinkage of muscular fiber and necrotic musculature were observed in irradiated prawns. These structural alterations of

  5. Translational Control of Host Gene Expression by a Cys-Motif Protein Encoded in a Bracovirus.

    Eunseong Kim

    Full Text Available Translational control is a strategy that various viruses use to manipulate their hosts to suppress acute antiviral response. Polydnaviruses, a group of insect double-stranded DNA viruses symbiotic to some endoparasitoid wasps, are divided into two genera: ichnovirus (IV and bracovirus (BV. In IV, some Cys-motif genes are known as host translation-inhibitory factors (HTIF. The genome of endoparasitoid wasp Cotesia plutellae contains a Cys-motif gene (Cp-TSP13 homologous to an HTIF known as teratocyte-secretory protein 14 (TSP14 of Microplitis croceipes. Cp-TSP13 consists of 129 amino acid residues with a predicted molecular weight of 13.987 kDa and pI value of 7.928. Genomic DNA region encoding its open reading frame has three introns. Cp-TSP13 possesses six conserved cysteine residues as other Cys-motif genes functioning as HTIF. Cp-TSP13 was expressed in Plutella xylostella larvae parasitized by C. plutellae. C. plutellae bracovirus (CpBV was purified and injected into non-parasitized P. xylostella that expressed Cp-TSP13. Cp-TSP13 was cloned into a eukaryotic expression vector and used to infect Sf9 cells to transiently express Cp-TSP13. The synthesized Cp-TSP13 protein was detected in culture broth. An overlaying experiment showed that the purified Cp-TSP13 entered hemocytes. It was localized in the cytosol. Recombinant Cp-TSP13 significantly inhibited protein synthesis of secretory proteins when it was added to in vitro cultured fat body. In addition, the recombinant Cp-TSP13 directly inhibited the translation of fat body mRNAs in in vitro translation assay using rabbit reticulocyte lysate. Moreover, the recombinant Cp-TSP13 significantly suppressed cellular immune responses by inhibiting hemocyte-spreading behavior. It also exhibited significant insecticidal activities by both injection and feeding routes. These results indicate that Cp-TSP13 is a viral HTIF.

  6. Characterization and metal-induced gene transcription of two new copper zinc superoxide dismutases in the solitary ascidian Ciona intestinalis

    Ferro, Diana [Department of Biology, University of Padova, Padova (Italy); Institute for Evolution and Biodiversity, Westfälische Wilhelms-Universität, Münster (Germany); Franchi, Nicola [Department of Biology, University of Padova, Padova (Italy); Department of Biological, Chemical, Pharmaceutical Science and Technology, University of Palermo, Palermo (Italy); Mangano, Valentina [Department of Biological, Chemical, Pharmaceutical Science and Technology, University of Palermo, Palermo (Italy); Bakiu, Rigers [Department of Crop Production, Agricultural University of Tirana, Tirana (Albania); Cammarata, Matteo; Parrinello, Nicolò [Department of Biological, Chemical, Pharmaceutical Science and Technology, University of Palermo, Palermo (Italy); Santovito, Gianfranco, E-mail: gianfranco.santovito@unipd.it [Department of Biology, University of Padova, Padova (Italy); Ballarin, Loriano [Department of Biology, University of Padova, Padova (Italy)

    2013-09-15

    Highlights: •Ciona intestinalis express two copper-zinc superoxide dismutases (Cu,Zn SODs), one extracellular (Ci-SODa) and one intracellular isoform (Ci-SODb). •Promoters contain consensus sequences similar to mammalian MRE. •Metal exposure results in a significant increase of gene transcription: ci-soda is induced especially by copper and zinc, the increase of ci-sodb transcription is more evident after cadmium exposure. •Genes are mostly transcribed in circulating hemocytes and in ovarian follicular cells. -- Abstract: Antioxidant enzymes are known to protect living organisms against the oxidative stress risk, also induced by metals. In the present study, we describe the purification and molecular characterization of two Cu,Zn superoxide dismutases (SODs), referred to as Ci-SODa and Ci-SODb, from Ciona intestinalis, a basal chordate widely distributed in temperate shallow seawater. The putative amino acid sequences were compared with Cu,Zn SODs from other metazoans and phylogenetic analyses indicate that the two putative Ci-SODs are more related to invertebrate SODs than vertebrate ones. Both phylogenetic and preliminary homology modeling analyses suggest that Ci-SODa and Ci-SODb are extracellular and intracellular isoform, respectively. The mRNA of the two Cu,Zn SODs was localized in hemocytes and in ovarian follicular cells, as revealed by in situ hybridization. The time course of SOD mRNA levels in the presence of three different metals showed upregulation of ci-soda and inhibition of ci-sodb. Spectrophotometric analysis confirms the presence of SOD activity in Ciona tissues. Our in silico analyses of the ci-soda promoter region revealed putative consensus sequences similar to mammalian metal-responsive elements (MRE), suggesting that the transcription of these genes directly depends on metals. These data emphasize the importance of complex metal regulation of ci-soda and ci-sodb transcription, as components of an efficient detoxification pathway

  7. Habitat pollution and thermal regime modify molecular stress responses to elevated temperature in freshwater mussels (Anodonta anatina: Unionidae)

    Falfushynska, H.; Gnatyshyna, L.; Yurchak, I.; Ivanina, A.; Stoliar, O.; Sokolova, I.

    2014-01-01

    Elevated temperature and pollution are common stressors in freshwater ecosystems. We study cellular stress response to acute warming in Anodonta anatina (Unionidae) from sites with different thermal regimes and pollution levels: a pristine area and an agriculturally polluted site with normal temperature regimes (F and A, respectively) and a polluted site with elevated temperature (N) from the cooling pond of an electrical power plant. Animals were exposed to different temperatures for 14 days and stress response markers were measured in gills, digestive gland and hemocytes. Mussels from site N and A had elevated background levels of lactate dehydrogenase activity indicating higher reliance on anaerobic metabolism for ATP production and/or redox maintenance. Exposure to 25 °C and 30 °C induced oxidative stress (indicated by elevated levels of lipid peroxidation products) in digestive gland and gills of mussels from A and F sites, while in mussels from N sites elevated oxidative stress was only apparent at 30 °C. Temperature-induced changes in levels of antioxidants (superoxide dismutase, metallothioneins and glutathione) were tissue- and population-specific. Acute warming led to destabilization of lysosomal membranes and increased frequencies of nuclear lesions in mussels from F and A sites but not in their counterparts from N site. Elevated temperature led to an increase in the frequency of micronuclei in hemocytes in mussels from F and A sites at 25 °C and 30 °C and in mussels from N site at 30 °C. The mussels from N site also demonstrated better survival at elevated temperature (30 °C) than their counterparts from the F and A sites. Taken together, these data indicate that long-term acclimation and/or adaptation of A. anatina to elevated temperatures result in increased thermotolerance and alleviate stress response to moderate temperature rise. In contrast, extreme warming (30 °C) is harmful to mussels from all populations indicating limit to this induced

  8. Studies on the immunomodulatory effect of polysaccharide gel extracted from Durio zibethinus in Penaeus monodon shrimp against Vibrio harveyi and WSSV.

    Pholdaeng, Komsil; Pongsamart, Sunanta

    2010-04-01

    Oral administration of polysaccharide gel (PG) in shrimp diets revealed immunostimulating potential and disease resistance in Penaeus monodon (black tiger shrimp). PG from the fruit-rind of Durio zibethinus has been characterized to be a pectic polysaccharide with immunomodulating and antibacterial activities. PG inhibited growth of the shrimp bacterial pathogen, Vibrio harveyi 1526, by agar diffusion and broth microdilution tests. Clear inhibition zones on agar plates were observed at the lowest PG concentration of 3.1 mg/ml, where minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) values for PG were 6.3 and 12.5 mg/ml, respectively. Each group of juvenile shrimps, initial mean body weight 0.29 +/- 0.04 g, was housed in a closed-recirculating treated water system and was fed with PG-supplemented diets containing 1, 2 and 3% PG or shrimp basal diet in the control group for 8 and 12 weeks. PG-supplemented diets did not contribute to the overall growth of black tiger shrimp. The immune response was evaluated by analysis of prophenoloxidase activity and total hemocyte count in the shrimp fed PG-supplemented diets for 12 weeks. Prophenoloxidase activity in shrimp fed the 1, 2 and 3% PG-supplemented diet and total hemocyte count in shrimp fed the 1 and 2% PG-supplemented diet were higher (P < 0.05) than those of the control group. The percent survival was higher in groups fed the 1-3% PG-supplemented diets in challenge tests with either white spot syndrome virus (WSSV) or the bacterium V. harveyi 1526 than that of the control group. Relative percent survival (RPS) values in groups fed the 2% PG-supplemented diet showed the highest RPS value for disease resistance of 100% (at Day 6) and 36% (at Day 4) in treated shrimp against viral and bacterial infection, respectively. Mortality of PG-supplemented diets in treated shrimps against WSSV infection was also found to be much lower (P < 0.05) than that of the control group. Copyright 2009

  9. PcToll2 positively regulates the expression of antimicrobial peptides by promoting PcATF4 translocation into the nucleus.

    Lan, Jiang-Feng; Zhao, Li-Juan; Wei, Shun; Wang, Yuan; Lin, Li; Li, Xin-Cang

    2016-11-01

    Drosophila Toll and mammalian Toll-like receptors (TLRs) are a family of evolutionarily conserved immune receptors that play a crucial role in the first-line defense against intruded pathogens. Activating transcription factor 4 (ATF4), a member of the ATF/CREB transcription factor family, is an important factor that participates in TLR signaling and other physiological processes. However, in crustaceans, whether ATF4 homologs were involved in TLR signaling remains unclear. In the current study, we identified a Toll homolog PcToll2 and a novel ATF4 homolog PcATF4 from Procambarus clarkii, and analyzed the likely regulatory activity of PcATF4 in PcToll2 signaling. The complete cDNA sequence of PcToll2 was 4175 bp long containing an open reading frame of 2820 bp encoding a 939-amino acid protein, and the cDNA sequence of PcATF4 was 2027 bp long with an open reading frame of 1296 bp encoding a 431-amino acid protein. PcToll2 and human TLR4 shared the high identity and they were grouped into a cluster. Furthermore, PcToll2 had a close relationship with other shrimp TLRs that possessed potential antibacterial activity. PcToll2 was highly expressed in the hemocytes, heart and gills, while PcATF4 mainly distributed in gills. Upon challenge with Vibrio parahemolyticus, PcToll2 and PcATF4 together with the antimicrobial peptides of ALF1 and ALF2 were significantly up-regulated in the hemocytes, and the PcATF4 was translocated into the nucleus. After PcToll2 silencing and challenge with Vibrio, the translocation of PcATF4 into the nucleus was inhibited and the expression of ALF1 and ALF2 was reduced, but the expression of PcDorsal and PcSTAT was not affected. Furthermore, after PcATF4 knockdown and challenge with or without Vibrio, the expression of ALF1 and ALF2 was also decreased while the expression of PcToll2 was upregulated. These results suggested that PcToll2 might regulate the expression of ALF1 and ALF2 by promoting the import of PcATF4, instead of the routine

  10. Genomic characterization and expression profiles upon bacterial infection of a novel cystatin B homologue from disk abalone (Haliotis discus discus).

    Premachandra, H K A; Wan, Qiang; Elvitigala, Don Anushka Sandaruwan; De Zoysa, Mahanama; Choi, Cheol Young; Whang, Ilson; Lee, Jehee

    2012-12-01

    Cystatins are a large family of cysteine proteinase inhibitors which are involved in diverse biological and pathological processes. In the present study, we identified a gene related to cystatin superfamily, AbCyt B, from disk abalone Haliotis discus discus by expressed sequence tag (EST) analysis and BAC library screening. The complete cDNA sequence of AbCyt B is comprised of 1967 nucleotides with a 306 bp open reading frame (ORF) encoding for 101 amino acids. The amino acid sequence consists of a single cystatin-like domain, which has a cysteine proteinase inhibitor signature, a conserved Gly in N-terminal region, QVVAG motif and a variant of PW motif. No signal peptide, disulfide bonds or carbohydrate side chains were identified. Analysis of deduced amino acid sequence revealed that AbCyt B shares up to 44.7% identity and 65.7% similarity with the cystatin B genes from other organisms. The genomic sequence of AbCyt B is approximately 8.4 Kb, consisting of three exons and two introns. Phylogenetic tree analysis showed that AbCyt B was closely related to the cystatin B from pacific oyster (Crassostrea gigas) under the family 1.Functional analysis of recombinant AbCyt B protein exhibited inhibitory activity against the papain, with almost 84% inhibition at a concentration of 3.5 μmol/L. In tissue expression analysis, AbCyt B transcripts were expressed abundantly in the hemocyte, gill, mantle, and digestive tract, while weakly in muscle, testis, and hepatopancreas. After the immune challenge with Vibrio parahemolyticus, the AbCyt B showed significant (P<0.05) up-regulation of relative mRNA expression in gill and hemocytes at 24 and 6 h of post infection, respectively. These results collectively suggest that AbCyst B is a potent inhibitor of cysteine proteinases and is also potentially involved in immune responses against invading bacterial pathogens in abalone. Copyright © 2012 Elsevier Ltd. All rights reserved.

  11. Vaccination with multimeric recombinant VP28 induces high protection against white spot syndrome virus in shrimp.

    Taengchaiyaphum, Suparat; Nakayama, Hideki; Srisala, Jiraporn; Khiev, Ratny; Aldama-Cano, Diva January; Thitamadee, Siripong; Sritunyalucksana, Kallaya

    2017-11-01

    To improve the efficacy of WSSV protection, multimeric (tetrameric) recombinant VP28 (4XrVP28) was produced and tested in comparison with those of monomeric VP28 (1XrVP28). In vitro binding of either 1XrVP28 or 4XrVP28 to shrimp hemocyte surface was evident as early as 10 min after protein inoculation. Similar results were obtained in vivo when shrimp were injected with recombinant proteins that the proteins bound to the hemocyte surface could be detected since 5 min after injection. Comparison of the WSSV protection efficiencies of 1XrVP28 or 4XrVP28 were performed by injection the purified 1XrVP28 or 4XrVP28 (22.5 μg/shrimp) and WSSV inoculum (1000 copies/shrimp) into shrimp. At 10 dpi, while shrimp injected with WSSV inoculum reached 100% mortality, shrimp injected with 1XrVP28 + WSSV or 4XrVP28 + WSSV showed relative percent survival (RPS) of 67% and 81%, respectively. PCR quantification revealed high number of WSSV in the moribund shrimp of WSSV- and 1XrVP28+WSSV-injected group. In contrast, lower number of WSSV copies were found in the survivors both from 1XrVP28+WSSV- or 4XrVP28+WSSV- injected groups. Histopathological analysis demonstrated the WSSV infected lesions found in the moribund from WSSV-infected group and 1XrVP28+WSSV-injected group, but less or none in the survivors. ELISA demonstrated that 4XrVP28 exhibited higher affinity binding to rPmRab7, a WSSV binding protein essential for WSSV entry to the cell than 1XrVP28. Taken together, the protection against WSSV in shrimp could be improved by application of multimeric rVP28. Copyright © 2017 Elsevier Ltd. All rights reserved.

  12. Habitat pollution and thermal regime modify molecular stress responses to elevated temperature in freshwater mussels (Anodonta anatina: Unionidae)

    Falfushynska, H.; Gnatyshyna, L.; Yurchak, I. [Research Laboratory of Comparative Biochemistry and Molecular Biology, Ternopil National Pedagogical University, Kryvonosa Str 2, 46027 Ternopil (Ukraine); Ivanina, A. [Department of Biological Sciences, University of North Carolina at Charlotte, 9201 University City Blvd., Charlotte, NC 28223 (United States); Stoliar, O. [Research Laboratory of Comparative Biochemistry and Molecular Biology, Ternopil National Pedagogical University, Kryvonosa Str 2, 46027 Ternopil (Ukraine); Sokolova, I., E-mail: isokolov@uncc.edu [Department of Biological Sciences, University of North Carolina at Charlotte, 9201 University City Blvd., Charlotte, NC 28223 (United States)

    2014-12-01

    Elevated temperature and pollution are common stressors in freshwater ecosystems. We study cellular stress response to acute warming in Anodonta anatina (Unionidae) from sites with different thermal regimes and pollution levels: a pristine area and an agriculturally polluted site with normal temperature regimes (F and A, respectively) and a polluted site with elevated temperature (N) from the cooling pond of an electrical power plant. Animals were exposed to different temperatures for 14 days and stress response markers were measured in gills, digestive gland and hemocytes. Mussels from site N and A had elevated background levels of lactate dehydrogenase activity indicating higher reliance on anaerobic metabolism for ATP production and/or redox maintenance. Exposure to 25 °C and 30 °C induced oxidative stress (indicated by elevated levels of lipid peroxidation products) in digestive gland and gills of mussels from A and F sites, while in mussels from N sites elevated oxidative stress was only apparent at 30 °C. Temperature-induced changes in levels of antioxidants (superoxide dismutase, metallothioneins and glutathione) were tissue- and population-specific. Acute warming led to destabilization of lysosomal membranes and increased frequencies of nuclear lesions in mussels from F and A sites but not in their counterparts from N site. Elevated temperature led to an increase in the frequency of micronuclei in hemocytes in mussels from F and A sites at 25 °C and 30 °C and in mussels from N site at 30 °C. The mussels from N site also demonstrated better survival at elevated temperature (30 °C) than their counterparts from the F and A sites. Taken together, these data indicate that long-term acclimation and/or adaptation of A. anatina to elevated temperatures result in increased thermotolerance and alleviate stress response to moderate temperature rise. In contrast, extreme warming (30 °C) is harmful to mussels from all populations indicating limit to this induced

  13. Knockdown of SCF(Skp2 function causes double-parked accumulation in the nucleus and DNA re-replication in Drosophila plasmatocytes.

    Paul T Kroeger

    Full Text Available In Drosophila, circulating hemocytes are derived from the cephalic mesoderm during the embryonic wave of hematopoiesis. These cells are contributed to the larva and persist through metamorphosis into the adult. To analyze this population of hemocytes, we considered data from a previously published RNAi screen in the hematopoietic niche, which suggested several members of the SCF complex play a role in lymph gland development. eater-Gal4;UAS-GFP flies were crossed to UAS-RNAi lines to knockdown the function of all known SCF complex members in a plasmatocyte-specific fashion, in order to identify which members are novel regulators of plasmatocytes. This specific SCF complex contains five core members: Lin-19-like, SkpA, Skp2, Roc1a and complex activator Nedd8. The complex was identified by its very distinctive large cell phenotype. Furthermore, these large cells stained for anti-P1, a plasmatocyte-specific antibody. It was also noted that the DNA in these cells appeared to be over-replicated. Gamma-tubulin and DAPI staining suggest the cells are undergoing re-replication as they had multiple centrioles and excessive DNA content. Further experimentation determined enlarged cells were BrdU-positive indicating they have progressed through S-phase. To determine how these cells become enlarged and undergo re-replication, cell cycle proteins were analyzed by immunofluorescence. This analysis identified three proteins that had altered subcellular localization in these enlarged cells: Cyclin E, Geminin and Double-parked. Previous research has shown that Double-parked must be degraded to exit S-phase, otherwise the DNA will undergo re-replication. When Double-parked was titrated from the nucleus by an excess of its inhibitor, geminin, the enlarged cells and aberrant protein localization phenotypes were partially rescued. The data in this report suggests that the SCF(Skp2 complex is necessary to ubiquitinate Double-parked during plasmatocyte cell division

  14. Molecular identification of disk abalone (Haliotis discus discus) tetraspanin 33 and CD63: Insights into potent players in the disk abalone host defense system.

    Priyathilaka, Thanthrige Thiunuwan; Bathige, S D N K; Herath, H M L P B; Lee, Sukkyoung; Lee, Jehee

    2017-10-01

    Tetraspanins are a superfamily of transmembrane proteins involved in a diverse range of physiological processes including differentiation, adhesion, signal transduction, cell motility, and immune responses. In the present study, two tetraspanins, CD63 and tetraspanin 33 (TSPAN33) from disk abalone (AbCD63 and AbTSPAN33), were identified and characterized at the molecular level. The coding sequences for AbCD63 and AbTSPAN33 encoded polypeptides of 234 and 290 amino acids (aa) with predicted molecular mass of 25.3 and 32.5 kDa, respectively. The deduced AbCD63 and AbTSPAN33 protein sequences were also predicted to have a typical tetraspanin domain architecture, including four transmembrane domains (TM), short N- and C- terminal regions, a short intracellular loop, as well as a large and small extracellular loop. A characteristic CCG motif and cysteine residues, which are highly conserved across CD63 and TSPAN33 proteins of different species, were present in the large extracellular loop of both abalone tetraspanins. Phylogenetic analysis revealed that the AbCD63 and AbTSPAN33 clustered in the invertebrate subclade of tetraspanins, thus exhibiting a close relationship with tetraspanins of other mollusks. The AbCD63 and AbTSPAN33 mRNA transcripts were detected at early embryonic development stages of disk abalone with significantly higher amounts at the trochophore stage, suggesting the involvement of these proteins in embryonic development. Both AbCD63 and AbTSPAN33 were ubiquitously expressed in all the tissues of unchallenged abalones analyzed, with the highest expression levels found in hemocytes. Moreover, significant induction of AbCD63 and AbTSPAN33 mRNA expression was observed in immunologically important tissues, such as hemocytes and gills, upon stimulation with live bacteria (Vibrio parahaemolyticus and Listeria monocytogenes), virus (viral hemorrhagic septicemia virus), and two potent immune stimulators [polyinosinic:polycytidylic acid (poly I:C) and

  15. A Tendon Cell Specific RNAi Screen Reveals Novel Candidates Essential for Muscle Tendon Interaction.

    Prabhat Tiwari

    Full Text Available Tendons are fibrous connective tissue which connect muscles to the skeletal elements thus acting as passive transmitters of force during locomotion and provide appropriate body posture. Tendon-derived cues, albeit poorly understood, are necessary for proper muscle guidance and attachment during development. In the present study, we used dorsal longitudinal muscles of Drosophila and their tendon attachment sites to unravel the molecular nature of interactions between muscles and tendons. We performed a genetic screen using RNAi-mediated knockdown in tendon cells to find out molecular players involved in the formation and maintenance of myotendinous junction and found 21 candidates out of 2507 RNAi lines screened. Of these, 19 were novel molecules in context of myotendinous system. Integrin-βPS and Talin, picked as candidates in this screen, are known to play important role in the cell-cell interaction and myotendinous junction formation validating our screen. We have found candidates with enzymatic function, transcription activity, cell adhesion, protein folding and intracellular transport function. Tango1, an ER exit protein involved in collagen secretion was identified as a candidate molecule involved in the formation of myotendinous junction. Tango1 knockdown was found to affect development of muscle attachment sites and formation of myotendinous junction. Tango1 was also found to be involved in secretion of Viking (Collagen type IV and BM-40 from hemocytes and fat cells.

  16. Soft shell clams Mya arenaria with disseminated neoplasia demonstrate reverse transcriptase activity

    House, M.L.; Kim, C.H.; Reno, P.W.

    1998-01-01

    Disseminated neoplasia (DN), a proliferative cell disorder of the circulatory system of bivalves, was first reported in oysters in 1969. Since that time, the disease has been determined to be transmissible through water-borne exposure, but the etiological agent has not been unequivocally identified. In order to determine if a viral agent, possibly a retrovirus, could be the causative agent of DN, transmission experiments were performed, using both a cell-free filtrate and a sucrose gradient-purified preparation of a cell-free filtrate of DN positive materials. Additionally, a PCR-enhanced reverse transcriptase assay was used to determine if reverse transcriptase was present in tissues or hemolymph from DN positive soft shell clams Mya arenaria. DN was transmitted to healthy clams by injection with whole DN cells, but not with cell-free flitrates prepared from either tissues from DN positive clams, or DN cells. The cell-free preparations from DN-positive tissues and hemolymph having high levels of DN cells in circulation exhibited positive reactions in the PCR-enhanced reverse transcriptase assay. Cell-free preparations of hemolymph from clams having low levels of DN (<0.1% of cells abnormal), hemocytes from normal soft shell clams, and normal soft shell clam tissues did not produce a positive reaction in the PCR enhanced reverse transcriptase assay.

  17. Current applications, selection, and possible mechanisms of actions of synbiotics in improving the growth and health status in aquaculture: A review.

    Huynh, Truong-Giang; Shiu, Ya-Li; Nguyen, Thanh-Phuong; Truong, Quoc-Phu; Chen, Jiann-Chu; Liu, Chun-Hung

    2017-05-01

    Synbiotics, a conjunction between prebiotics and probiotics, have been used in aquaculture for over 10 years. However, the mechanisms of how synbiotics work as growth and immunity promoters are far from being unraveled. Here, we show that a prebiotic as part of a synbiotic is hydrolyzed to mono- or disaccharides as the sole carbon source with diverse mechanisms, thereby increasing biomass and colonization that is established by specific crosstalk between probiotic bacteria and the surface of intestinal epithelial cells of the host. Synbiotics may indirectly and directly promote the growth of aquatic animals through releasing extracellular bacterial enzymes and bioactive products from synbiotic metabolic processes. These compounds may activate precursors of digestive enzymes of the host and augment the nutritional absorptive ability that contributes to the efficacy of food utilization. In fish immune systems, synbiotics cause intestinal epithelial cells to secrete cytokines which modulate immune functional cells as of dendritic cells, T cells, and B cells, and induce the ability of lipopolysaccharides to trigger tumor necrosis factor-α and Toll-like receptor 2 gene transcription leading to increased respiratory burst activity, phagocytosis, and nitric oxide production. In shellfish, synbiotics stimulate the proliferation and degranulation of hemocytes of shrimp due to the presence of bacterial cell walls. Pathogen-associated molecular patterns are subsequently recognized and bound by specific pattern-recognition proteins, triggering melanization and phagocytosis processes. Copyright © 2017 Elsevier Ltd. All rights reserved.

  18. Optimization of Non-Thermal Plasma Treatment in an In Vivo Model Organism.

    Amanda Lee

    Full Text Available Non-thermal plasma is increasingly being recognized for a wide range of medical and biological applications. However, the effect of non-thermal plasma on physiological functions is not well characterized in in vivo model systems. Here we use a genetically amenable, widely used model system, Drosophila melanogaster, to develop an in vivo system, and investigate the role of non-thermal plasma in blood cell differentiation. Although the blood system in Drosophila is primitive, it is an efficient system with three types of hemocytes, functioning during different developmental stages and environmental stimuli. Blood cell differentiation in Drosophila plays an essential role in tissue modeling during embryogenesis, morphogenesis and also in innate immunity. In this study, we optimized distance and frequency for a direct non-thermal plasma application, and standardized doses to treat larvae and adult flies so that there is no effect on the viability, fertility or locomotion of the organism. We discovered that at optimal distance, time and frequency, application of plasma induced blood cell differentiation in the Drosophila larval lymph gland. We articulate that the augmented differentiation could be due to an increase in the levels of reactive oxygen species (ROS upon non-thermal plasma application. Our studies open avenues to use Drosophila as a model system in plasma medicine to study various genetic disorders and biological processes where non-thermal plasma has a possible therapeutic application.

  19. The role of iron in the proliferation of Drosophila l(2)mbn cells

    Metzendorf, Christoph [Department of Comparative Physiology, Uppsala University, Norbyvaegen 18A, SE-752 36 Uppsala (Sweden); Lind, Maria I., E-mail: maria.lind@ebc.uu.se [Department of Comparative Physiology, Uppsala University, Norbyvaegen 18A, SE-752 36 Uppsala (Sweden)

    2010-09-24

    Research highlights: {yields} Establishment of a model system to study the role of iron during proliferation. {yields} Iron deprivation of insect tumorous cell line inhibits cell proliferation. {yields} Iron deprivation causes a reversible cell cycle arrest in G1/S-phase. {yields} Iron deprivation promotes decreased gene expression of cycE. -- Abstract: Iron is essential for life and is needed for cell proliferation and cell cycle progression. Iron deprivation results first in cell cycle arrest and then in apoptosis. The Drosophila tumorous larval hemocyte cell line l(2)mbn was used to study the sensitivity and cellular response to iron deprivation through the chelator desferrioxamine (DFO). At a concentration of 10 {mu}M DFO or more the proliferation was inhibited reversibly, while the amount of dead cells did not increase. FACS analysis showed that the cell cycle was arrested in G1/S-phase and the transcript level of cycE was decreased to less than 50% of control cells. These results show that iron chelation in this insect tumorous cell line causes a specific and coordinated cell cycle arrest.

  20. Tissue-specific tagging of endogenous loci in Drosophila melanogaster

    Kate Koles

    2016-01-01

    Full Text Available Fluorescent protein tags have revolutionized cell and developmental biology, and in combination with binary expression systems they enable diverse tissue-specific studies of protein function. However these binary expression systems often do not recapitulate endogenous protein expression levels, localization, binding partners and/or developmental windows of gene expression. To address these limitations, we have developed a method called T-STEP (tissue-specific tagging of endogenous proteins that allows endogenous loci to be tagged in a tissue specific manner. T-STEP uses a combination of efficient CRISPR/Cas9-enhanced gene targeting and tissue-specific recombinase-mediated tag swapping to temporally and spatially label endogenous proteins. We have employed this method to GFP tag OCRL (a phosphoinositide-5-phosphatase in the endocytic pathway and Vps35 (a Parkinson's disease-implicated component of the endosomal retromer complex in diverse Drosophila tissues including neurons, glia, muscles and hemocytes. Selective tagging of endogenous proteins allows, for the first time, cell type-specific live imaging and proteomics in complex tissues.

  1. Parasitization by Scleroderma guani influences expression of superoxide dismutase genes in Tenebrio molitor.

    Zhu, Jia-Ying; Ze, Sang-Zi; Stanley, David W; Yang, Bin

    2014-09-01

    Superoxide dismutase (SOD) is an antioxidant enzyme involved in detoxifying reactive oxygen species. In this study, we identified genes encoding the extracellular and intracellular copper-zinc SODs (ecCuZnSOD and icCuZnSOD) and a manganese SOD (MnSOD) in the yellow mealworm beetle, Tenebrio molitor. The cDNAs for ecCuZnSOD, icCuZnSOD, and MnSOD, respectively, encode 24.55, 15.81, and 23.14 kDa polypeptides, which possess structural features typical of other insect SODs. They showed 20-94% identity to other known SOD sequences from Bombyx mori, Musca domestica, Nasonia vitripennis, Pediculus humanus corporis, and Tribolium castaneum. Expression of these genes was analyzed in selected tissues and developmental stages, and following exposure to Escherichia coli and parasitization by Scleroderma guani. We recorded expression of all three SODs in cuticle, fat body, and hemocytes and in the major developmental stages. Relatively higher expressions were detected in late-instar larvae and pupae, compared to other developmental stages. Transcriptional levels were upregulated following bacterial infection. Analysis of pupae parasitized by S. guani revealed that expression of T. molitor SOD genes was significantly induced following parasitization. We infer that these genes act in immune response and in host-parasitoid interactions. © 2014 Wiley Periodicals, Inc.

  2. The scavenger endothelial cell: a new player in homeostasis and immunity.

    Sørensen, Karen Kristine; McCourt, Peter; Berg, Trond; Crossley, Clive; Le Couteur, David; Wake, Kenjiro; Smedsrød, Bård

    2012-12-15

    To maintain homeostasis, the animal body is equipped with a powerful system to remove circulating waste. This review presents evidence that the scavenger endothelial cell (SEC) is responsible for the clearance of blood-borne waste macromolecules in vertebrates. SECs express pattern-recognition endocytosis receptors (mannose and scavenger receptors), and in mammals, the endocytic Fc gamma-receptor IIb2. This cell type has an endocytic machinery capable of super-efficient uptake and degradation of physiological and foreign waste material, including all major classes of biological macromolecules. In terrestrial vertebrates, most SECs line the wall of the liver sinusoid. In phylogenetically older vertebrates, SECs reside instead in heart, kidney, or gills. SECs, thus, by virtue of their efficient nonphagocytic elimination of physiological and microbial substances, play a critical role in the innate immunity of vertebrates. In major invertebrate phyla, including insects, the same function is carried out by nephrocytes. The concept of a dual-cell principle of waste clearance is introduced to emphasize that professional phagocytes (macrophages in vertebrates; hemocytes in invertebrates) eliminate larger particles (>0.5 μm) by phagocytosis, whereas soluble macromolecules and smaller particles are eliminated efficiently and preferentially by clathrin-mediated endocytosis in nonphagocytic SECs in vertebrates or nephrocytes in invertebrates. Including these cells as important players in immunology and physiology provides an additional basis for understanding host defense and tissue homeostasis.

  3. Linking δ15N and histopathological effects in molluscs exposed in situ to effluents from land-based marine fish farms

    Carballeira, C.; Espinosa, J.; Carballeira, A.

    2011-01-01

    Highlights: ► Land-based marine aquaculture effluents induce branchial exfoliation and phagocytic haemocytosis in exposed molluscs. ► Transplanted clams are more sensitive to aquaculture discharges than native mussels. ► δ 15 N in organisms is a cost-effective means of biomonitoring exposure to contamination from aquaculture. ► δ 15 N analysis may facilitate the evaluation of potential effects at the tissue level. - Abstract: Histopathological alterations can indicate time-integrated impacts on organisms stemming from alterations at lower biological organisation levels. Long-term (native mussels) and short-term (transplanted clams) changes in the tissues of molluscs exposed to the effluents from two land-based marine fish farms (LBMFFs) were determined. Histological alterations were related to the δ 15 N isotopic signal measured in mussels and macroalgae. Effluents from LBMFFs were found to cause severe and moderate gill filament exfoliation in clams and mussels, respectively. Some transplanted clams showed severe degrees of hemocytic phagocytosis in gonads and connective tissue. In an attempt to semi-quantitatively summarize the observed histopathological alterations, a weighted index of damage (WID) was calculated for each type of alteration, species and sampling site. The WID was clearly related to the δ 15 N descriptor of exposure. Further studies aimed at standardizing this relationship may establish critical thresholds of the descriptor for its implementation within environmental monitoring plans for LBMFFs.

  4. Interactive effects of metal contamination and pathogenic organisms on the introduced marine bivalve Ruditapes philippinarum in European populations

    Paul-Pont, Ika; Montaudouin, Xavier de; Gonzalez, Patrice; Jude, Florence; Raymond, Natalie; Paillard, Christine; Baudrimont, Magalie

    2010-01-01

    In natural environment, marine organisms are concomitantly exposed to pollutants and multiple disease agents resulting in detrimental interactions. The present study evaluated interactive effects of metal contamination (cadmium) and pathogenic organisms (trematode parasites Himasthla elongata and pathogenic bacteria Vibrio tapetis) singularly and in combination on the bivalve Ruditapes philippinarum, an introduced species to Europe, under laboratory controlled conditions. After 7 days, metal bioaccumulation and pathogen load were analyzed as well as metallothionein (MT) response and hemocyte concentrations and activities. Results showed that infection by opportunistic pathogens affects metal accumulation, leading to maximal Cd accumulation in co-infected clams. Among stressors only V. tapetis induced significant effects on immune parameters whereas a particular interaction 'trematode-bacteria' was shown on MT responses. Despite low trematode infection in agreement with the resistant status of R. philippinarum to these macroparasites, significant interaction with bacteria and metal occurred. Such results highlight the necessity of taking pathogens into account in ecotoxicological studies. - Co-infection by opportunistic pathogens affects metal accumulation and some defense-related activities in the Manila clam Ruditapes philippinarum.

  5. Interactive effects of metal contamination and pathogenic organisms on the introduced marine bivalve Ruditapes philippinarum in European populations

    Paul-Pont, Ika, E-mail: i.paulpont@epoc.u-bordeaux1.f [Universite Bordeaux 1, UMR 5805 CNRS, Station Marine d' Arcachon, place du Dr. Peyneau, Arcachon 33120 (France); Montaudouin, Xavier de; Gonzalez, Patrice; Jude, Florence; Raymond, Natalie [Universite Bordeaux 1, UMR 5805 CNRS, Station Marine d' Arcachon, place du Dr. Peyneau, Arcachon 33120 (France); Paillard, Christine [Universite de Bretagne Occidentale-IUEM, LEMAR UMR 6539 CNRS, Place Nicolas Copernic, Technopole Brest Iroise, 29280 Plouzane (France); Baudrimont, Magalie [Universite Bordeaux 1, UMR 5805 CNRS, Station Marine d' Arcachon, place du Dr. Peyneau, Arcachon 33120 (France)

    2010-11-15

    In natural environment, marine organisms are concomitantly exposed to pollutants and multiple disease agents resulting in detrimental interactions. The present study evaluated interactive effects of metal contamination (cadmium) and pathogenic organisms (trematode parasites Himasthla elongata and pathogenic bacteria Vibrio tapetis) singularly and in combination on the bivalve Ruditapes philippinarum, an introduced species to Europe, under laboratory controlled conditions. After 7 days, metal bioaccumulation and pathogen load were analyzed as well as metallothionein (MT) response and hemocyte concentrations and activities. Results showed that infection by opportunistic pathogens affects metal accumulation, leading to maximal Cd accumulation in co-infected clams. Among stressors only V. tapetis induced significant effects on immune parameters whereas a particular interaction 'trematode-bacteria' was shown on MT responses. Despite low trematode infection in agreement with the resistant status of R. philippinarum to these macroparasites, significant interaction with bacteria and metal occurred. Such results highlight the necessity of taking pathogens into account in ecotoxicological studies. - Co-infection by opportunistic pathogens affects metal accumulation and some defense-related activities in the Manila clam Ruditapes philippinarum.

  6. Perna perna (LINNAEUS, 1758) mussels irradiated by 60CO gamma rays cytotoxicity evaluation

    Martini, Gisela A.; Pusceddu, Fabio H.; Rogero, Sizue O.; Rogero, Jose Roberto

    2013-01-01

    The aim of the present work was the study of ionizing radiation effects on aquatic biota regarding the location of nuclear facilities nearby coastal areas assuming the risk of leaks and nuclear accidents. Bivalve mollusks have been widely used in the monitoring of aquatic environment studies mainly for their sessile habit and pollutants bioconcentration ability. So marine mussel Perna perna (Bivalvia: Mytilidae) was used as organism test in this study. The study of radioactive toxicity was performed by cytotoxicity test exposing the organisms to 11Gy gamma radiation dose. After radiation the neutral red retention assay evaluated the lysosomal membrane integrity in the mussel hemocytes. 50% lethal dose assay (LD50) of gamma radiation on Perna perna mussels was carried out by exposure the organisms to 60 Co gamma rays at doses ranging from 0 to 3000 Gy. The result of gamma radiation LD50 for these mussels was 1068 Gy and the neutral red retention time of irradiated organisms was about 47% lower than the control, non irradiated organisms. With the obtained results is expected to contribute in the study to identify the range of ionizing radiation doses which can cause toxic effects in marine invertebrates. (author)

  7. Prostaglandin-mediated recovery from bacteremia delays larval development in fall armyworm, Spodoptera frugiperda.

    Zhang, Lei; Ringbauer, Joseph A; Goodman, Cynthia L; Reall, Tamra; Jiang, Xing-Fu; Stanley, David

    2018-04-01

    Insect immunity includes a surveillance system that detects and signals infections, coupled with hemocytic and humoral immune functions. These functions are signaled and coordinated by several biochemicals, including biogenic amines, insect cytokines, peptides, and prostaglandins (PGs). The actions of these mediators are coordinated within cells by various forms of cross-talk among the signaling systems and they result in effective reactions to infection. While this is well understood, we lack information on how immune-mediated recovery influences subsequent juvenile development in surviving insects. We investigated this point by posing the hypothesis that PG signaling is necessary for larval recovery, although the recovery imposes biological costs, registered in developmental delays and failures in surviving individuals. Here, we report that nodulation responses to infections by the bacterium, Serratia marcescens, increased over time up to 5 h postinfection, with no further nodulation; it increased in a linear manner with increasing bacterial dosages. Larval survivorship decreased with increasing bacterial doses. Treating larvae with the PG-biosynthesis inhibitor, indomethacin, led to sharply decreased nodulation reactions to infection, which were rescued in larvae cotreated with indomethacin and the PG-precursor, arachidonic acid. Although nodulation was fully rescued, all bacterial challenged larvae suffered reduced survivorship compared to controls. Bacterial infection led to reduced developmental rates in larvae, but not pupae. Adult emergence from pupae that developed from experimental larvae was also decreased. Taken together, our data potently bolster our hypothesis. © 2018 Wiley Periodicals, Inc.

  8. Isolation of a novel LPS-induced component of the ML superfamily in Ciona intestinalis.

    Vizzini, Aiti; Bonura, Angela; Longo, Valeria; Sanfratello, Maria Antonietta; Parrinello, Daniela; Cammarata, Matteo; Colombo, Paolo

    2015-11-01

    ML superfamily represents a group of proteins playing important roles in lipid metabolism and innate immune response. In this study, we report the identification of the first component of the ML superfamily in the invertebrate Ciona intestinalis by means of a subtractive hybridization strategy. Sequence homology and phylogenetic analysis showed that this protein forms a specific clade with vertebrate components of the Niemann-Pick type C2 protein and, for this reason, it has been named Ci-NPC2. The putative Ci-NPC2 is a 150 amino acids long protein with a short signal peptide, seven cysteine residues, three putative lipid binding site and a three-dimensional model showing a characteristic β-strand structure. Gene expression analysis demonstrated that the Ci-NPC2 protein is positively upregulated after LPS inoculum with a peak of expression 1 h after challenge. Finally, in-situ hybridization demonstrated that the Ci-NPC2 protein is preferentially expressed in hemocytes inside the vessel lumen. Copyright © 2015 Elsevier Ltd. All rights reserved.

  9. Transforming growth factor β (CiTGF-β) gene expression is induced in the inflammatory reaction of Ciona intestinalis.

    Vizzini, Aiti; Di Falco, Felicia; Parrinello, Daniela; Sanfratello, Maria Antonietta; Cammarata, Matteo

    2016-02-01

    Transforming growth factor (TGF-β) is a well-known component of a regulatory cytokines superfamily that has pleiotropic functions in a broad range of cell types and is involved, in vertebrates, in numerous physiological and pathological processes. In the current study, we report on Ciona intestinalis molecular characterisation and expression of a transforming growth factor β homologue (CiTGF-β). The gene organisation, phylogenetic tree and modelling supported the close relationship with the mammalian TGF suggesting that the C. intestinalis TGF-β gene shares a common ancestor in the chordate lineages. Functionally, real-time PCR analysis showed that CiTGF-β was transcriptionally upregulated in the inflammatory process induced by LPS inoculation, suggesting that is involved in the first phase and significant in the secondary phase of the inflammatory response in which cell differentiation occurs. In situ hybridisation assays revealed that the genes transcription was upregulated in the pharynx, the main organ of the ascidian immune system, and expressed by cluster of hemocytes inside the pharynx vessels. These data supported the view that CiTGF-β is a potential molecule in immune defence systems against bacterial infection. Copyright © 2015 Elsevier Ltd. All rights reserved.

  10. LPS challenge regulates gene expression and tissue localization of a Ciona intestinalis gene through an alternative polyadenylation mechanism.

    Aiti Vizzini

    Full Text Available A subtractive hybridization strategy for the identification of differentially expressed genes was performed between LPS-challenged and naive Ciona intestinalis. This strategy allowed the characterization of two transcripts (Ci8short and Ci8long generated by the use of two Alternative Polyadenylation sites. The Ci8long transcript contains a protein domain with relevant homology to several components of the Receptor Transporting Protein (RTP family not present in the Ci8short mRNA. By means of Real Time PCR and Northern Blot, the Ci8short and Ci8long transcripts showed a different pattern of gene expression with the Ci8short mRNA being strongly activated after LPS injection in the pharynx. In situ hybridization analysis demonstrated that the activation of the APA site also influenced the tissue localization of the Ci8short transcript. This analysis showed that the Ci8long mRNA was expressed in hemocytes meanwhile the Ci8short mRNA was highly transcribed also in vessel endothelial cells and in the epithelium of pharynx. These findings demonstrated that regulation of gene expression based on different polyadenylation sites is an ancestral powerful strategy influencing both the level of expression and tissue distribution of alternative transcripts.

  11. A neurotropic route for Maize mosaic virus (Rhabdoviridae) in its planthopper vector Peregrinus maidis.

    Ammar, El-Desouky; Hogenhout, Saskia A

    2008-01-01

    To investigate the dissemination route of Maize mosaic virus (MMV, Rhabdoviridae) in its planthopper vector Peregrinus maidis (Delphacidae, Hemiptera), temporal and spatial distribution of MMV was studied by immunofluorescence confocal laser scanning microscopy following 1-week acquisition feeding of planthoppers on infected plants. MMV was detected 1-week post first access to diseased plants (padp) in the midgut and anterior diverticulum, 2-week padp in the esophagus, nerves, nerve ganglia and visceral muscles, and 3-week padp in hemocytes, tracheae, salivary glands and other tissues. MMV is neurotropic in P. maidis; infection was more extensive in the nervous system compared to other tissues. A significantly higher proportion of planthoppers had infected midguts (28.1%) compared to those with infected salivary glands (20.4%) or to those that transmitted MMV (15.7%), suggesting the occurrence of midgut and salivary gland barriers to MMV transmission in P. maidis. In this planthopper, the esophagus and anterior diverticulum are located between the compound ganglionic mass and the salivary glands. We postulate that MMV may overcome transmission barriers in P. maidis by proceeding from the midgut to the anterior diverticulum and esophagus, and from these to the salivary glands via the nervous system: a neurotropic route similar to that of some vertebrate-infecting rhabdoviruses.

  12. Investigations of Salmonella enterica serovar newport infections of oysters by using immunohistochemistry and knockout mutagenesis.

    Morrison, Christopher M; Dial, Sharon M; Day, William A; Joens, Lynn A

    2012-04-01

    The consumption of raw oysters is an important risk factor in the acquisition of food-borne disease, with Salmonella being one of a number of pathogens that have been found in market oysters. Previous work by our lab found that Salmonella was capable of surviving in oysters for over 2 months under laboratory conditions, and this study sought to further investigate Salmonella's tissue affinity and mechanism of persistence within the oysters. Immunohistochemistry was used to show that Salmonella was capable of breaching the epithelial barriers, infecting the deeper connective tissues of the oysters, and evading destruction by the oysters' phagocytic hemocytes. To further investigate the mechanism of these infections, genes vital to the function of Salmonella's two main type III secretion systems were disrupted and the survivability of these knockout mutants within oysters was assayed. When the Salmonella pathogenicity island 1 and 2 mutant strains were exposed to oysters, there were no detectable deficiencies in their abilities to survive, suggesting that Salmonella's long-term infection of oysters does not rely upon these two important pathogenicity islands and must be due to some other, currently unknown, mechanism.

  13. White Shrimp Litopenaeus vannamei That Have Received Gracilaria tenuistipitata Extract Show Early Recovery of Immune Parameters after Ammonia Stressing

    Yu-Yuan Chen

    2015-06-01

    Full Text Available White shrimp Litopenaeus vannamei immersed in seawater (35‰ containing Gracilaria tenuistipitata extract (GTE at 0 (control, 400, and 600 mg/L for 3 h were exposed to 5 mg/L ammonia-N (ammonia as nitrogen, and immune parameters including hyaline cells (HCs, granular cells (GCs, including semi-granular cells, total hemocyte count (THC, phenoloxidase (PO activity, respiratory bursts (RBs, superoxide dismutase (SOD activity, lysozyme activity, and hemolymph protein level were examined 24~120 h post-stress. The immune parameters of shrimp immersed in 600 mg/L GTE returned to original values earlier, at 96~120 h post-stress, whereas in control shrimp they did not. In another experiment, shrimp were immersed in seawater containing GTE at 0 and 600 mg/L for 3 h and examined for transcript levels of immune-related genes at 24 h post-stress. Transcript levels of lipopolysaccharide and β-1,3-glucan binding protein (LGBP, peroxinectin (PX, cytMnSOD, mtMnSOD, and HSP70 were up-regulated at 24 h post-stress in GTE receiving shrimp. We concluded that white shrimp immersed in seawater containing GTE exhibited a capability for maintaining homeostasis by regulating cellular and humoral immunity against ammonia stress as evidenced by up-regulated gene expression and earlier recovery of immune parameters.

  14. Sensitivity of Larvae and Adult and the Immunologic Characteristics of Litopenaeus vannamei under the Acute Hypoxia

    Hailong Zhou

    2014-01-01

    Full Text Available Litopenaeus vannamei is one of the most commercially important species of shrimp in the world. In this study, we performed acute hypoxia tests with Litopenaeus vannamei to estimate 12 h median lethal concentration (LC50 values at different life stages. The results indicated that the 12 h LC50 values were significantly different in different life stages of shrimp (P<0.05. The maximum value of 12 h LC50 was 2.113 mg L−1 for mysis III, and the minimum value was 0.535 mg L−1 for adult shrimp with an average total length of 6 cm. The study also determined the hemocyanin concentration (HC and the total hemocyte counts (THC in the conditions of hypoxia and reoxygenation. These results showed that the THC decreased and the HC increased under hypoxia, and the THC increased and the HC decreased in the condition of reoxygenation. These results can provide fundamental information for shrimp farming and seedling and also can guide the breeding selection, as well as being very helpful to better understand the hypoxia stress mechanism of shrimp.

  15. Accumulation, Biotransformation, Histopathology and Paralysis in the Pacific Calico Scallop Argopecten ventricosus by the Paralyzing Toxins of the Dinoflagellate Gymnodinium catenatum

    Rosalba Alonso-Rodriguez

    2012-05-01

    Full Text Available The dinoflagellate Gymnodinium catenatum produces paralyzing shellfish poisons that are consumed and accumulated by bivalves. We performed short-term feeding experiments to examine ingestion, accumulation, biotransformation, histopathology, and paralysis in the juvenile Pacific calico scallop Argopecten ventricosus that consume this dinoflagellate. Depletion of algal cells was measured in closed systems. Histopathological preparations were microscopically analyzed. Paralysis was observed and the time of recovery recorded. Accumulation and possible biotransformation of toxins were measured by HPLC analysis. Feeding activity in treated scallops showed that scallops produced pseudofeces, ingestion rates decreased at 8 h; approximately 60% of the scallops were paralyzed and melanin production and hemocyte aggregation were observed in several tissues at 15 h. HPLC analysis showed that the only toxins present in the dinoflagellates and scallops were the N-sulfo-carbamoyl toxins (C1, C2; after hydrolysis, the carbamate toxins (epimers GTX2/3 were present. C1 and C2 toxins were most common in the mantle, followed by the digestive gland and stomach-complex, adductor muscle, kidney and rectum group, and finally, gills. Toxin profiles in scallop tissue were similar to the dinoflagellate; biotransformations were not present in the scallops in this short-term feeding experiment.

  16. Accumulation, biotransformation, histopathology and paralysis in the Pacific calico scallop Argopecten ventricosus by the paralyzing toxins of the dinoflagellate Gymnodinium catenatum.

    Escobedo-Lozano, Amada Y; Estrada, Norma; Ascencio, Felipe; Contreras, Gerardo; Alonso-Rodriguez, Rosalba

    2012-05-01

    The dinoflagellate Gymnodinium catenatum produces paralyzing shellfish poisons that are consumed and accumulated by bivalves. We performed short-term feeding experiments to examine ingestion, accumulation, biotransformation, histopathology, and paralysis in the juvenile Pacific calico scallop Argopecten ventricosus that consume this dinoflagellate. Depletion of algal cells was measured in closed systems. Histopathological preparations were microscopically analyzed. Paralysis was observed and the time of recovery recorded. Accumulation and possible biotransformation of toxins were measured by HPLC analysis. Feeding activity in treated scallops showed that scallops produced pseudofeces, ingestion rates decreased at 8 h; approximately 60% of the scallops were paralyzed and melanin production and hemocyte aggregation were observed in several tissues at 15 h. HPLC analysis showed that the only toxins present in the dinoflagellates and scallops were the N-sulfo-carbamoyl toxins (C1, C2); after hydrolysis, the carbamate toxins (epimers GTX2/3) were present. C1 and C2 toxins were most common in the mantle, followed by the digestive gland and stomach-complex, adductor muscle, kidney and rectum group, and finally, gills. Toxin profiles in scallop tissue were similar to the dinoflagellate; biotransformations were not present in the scallops in this short-term feeding experiment.

  17. Effects of fluorine-containing usnic acid and fungus Beauveria bassiana on the survival and immune-physiological reactions of Colorado potato beetle larvae.

    Kryukov, Vadim Yu; Tomilova, Oksana G; Luzina, Olga A; Yaroslavtseva, Olga N; Akhanaev, Yuriy B; Tyurin, Maksim V; Duisembekov, Bahytzhan A; Salakhutdinov, Nariman F; Glupov, Viktor V

    2018-03-01

    The search for compounds that interact synergistically with entomopathogenic fungi is aimed at enhancing the efficacy and stability of biological products against pest insects, for example, against the Colorado potato beetle (CPB). We hypothesized that fluorine-containing derivatives of usnic acid (FUA) might be candidates for the development of multicomponent bio-insecticides. The aim of this study was to analyze the co-influence of FUA and Beauveria bassiana on the survival and immune-physiological reactions of CPB larvae. Synergy between FUA and B. bassiana was observed after treatment of second, third and fourth larvae instars under laboratory conditions. Furthermore, synergy was observed in field trials in continental climate conditions in southeastern Kazakhstan. In a field experiment, the median lethal time was shortened three-fold, and cumulative mortality for 15 days increased by 36% in the combined treatment compared with a fungal infection alone. FUA treatment delayed larval development, decreased the total hemocyte count, and increased both the phenoloxidase activity in integuments and the detoxification enzyme rate in hemolymph. A combined treatment with fungus and FUA led to increases in the aforementioned changes. Toxicosis caused by FUA provides a stable synergistic effect between FUA and B. bassiana. The combination can be promising for the development of highly efficient products against CPB. © 2017 Society of Chemical Industry. © 2017 Society of Chemical Industry.

  18. Perkinsus beihaiensis (Perkinsozoa in oysters of Bahia State, Brazil

    M. S. A. Luz

    2017-08-01

    Full Text Available Abstract This study reports the pathogen Perkinsus beihaiensis in oysters of the genus Crassostrea on the coast of the State of Bahia (Brazil, its prevalence, infection intensity and correlation with salinity. Oysters (n = 240 were collected between October and December 2014 at eight sampling stations between latitudes 13°55'S and 15°42'S. The laboratory procedures included macroscopic analysis, histology, culture in Ray's fluid thioglycollate medium (RFTM, Polymerase Chain Reaction (PCR and DNA sequencing. PCR and sequencing have been used for the genetic identification of oysters as well. Two species of oysters have been identified: Crassostrea rhizophorae and C. brasiliana. In both oyster species P. beihaiensis was the only Perkinsus species detected. In C. rhizophorae, the average prevalence was 82.8% by histology and 65.2% by RFTM. In C. brasiliana, the prevalences were 70.5% and 35.7%, respectively. The higher prevalence of P. beihaiensis in C. rhizophorae was probably influenced by salinity, with which was positively correlated (r> 0.8. In both oysters, P. beihaiensis was located mainly in the gastric epithelium. The infection was generally mild or moderate, without apparent harm to the hosts, but in cases of severe infection, there was hemocytical reaction and tissue disorganization. The generally high prevalence in the region suggests that oysters should be monitored with respect to this pathogen, especially in growing areas.

  19. An anionic defensin from Plutella xylostella with potential activity against Bacillus thuringiensis.

    Xu, X-X; Zhang, Y-Q; Freed, S; Yu, J; Gao, Y-F; Wang, S; Ouyang, L-N; Ju, W-Y; Jin, F-L

    2016-12-01

    Insect defensins, are cationic peptides that play an important role in immunity against microbial infection. In the present study, an anionic defensin from Plutella xylostella, (designated as PxDef) was first cloned and characterized. Amino acid sequence analysis showed that the mature peptide owned characteristic six-cysteine motifs with predicted isoelectric point of 5.57, indicating an anionic defensin. Quantitative real-time polymerase chain reaction analysis showed that PxDef was significantly induced in epidermis, fat body, midgut and hemocytes after injection of heat-inactivated Bacillus thuringiensis, while such an induction was delayed by the injection of live B. thuringiensis in the 4th instar larvae of P. xylostella. Knocking down the expression of nuclear transcription factor Dorsal in P. xylostella by RNA interference significantly decreased the mRNA level of PxDef, and increased the sensitivity of P. xylostella larvae to the infection by live B. thuringiensis. The purified recombinant mature peptide (PxDef) showed higher activity against Gram-positive bacteria, with the minimum inhibition concentrations of 1.6 and 2.6 µM against B. thuringiensis and Bacillus subtilis, respectively. To our knowledge, this is the first report about an anionic PxDef, which may play an important role in the immune system of P. xylostella against B. thuringiensis.

  20. IDENTIFICATION AND MOLECULAR CHARACTERIZATION OF TWO SERINE PROTEASES AND THEIR POTENTIAL INVOLVEMENT IN PROPHENOLOXIDASE ACTIVATION IN Plutella xylostella.

    Gao, Gang; Xu, Xiao-Xia; Yu, Jing; Li, Lin-Miao; Ju, Wen-Yan; Jin, Feng-Liang; Freed, Shoaib

    2016-09-01

    The proteolytic activation of prophenoloxidase (proPO) is a humoral defense mechanism in insects and crustaceans. Phenoloxidase (PO) is produced as an inactive precursor namely, proPO and is activated via specific proteolytic cleavage by proPO-activating proteinase. The current research reports two novel serine proteinase genes (PxSP1-768 bp and PxSP2-816 bp) from Plutella xylostella, encoding 255 and 271 amino acid residues, respectively. Tissue distribution analyses by semiquantitative reverse transcription-PCR (RT-PCR) revealed the resultant genes to be primarily expressed in the hemocytes, while quantitative-RT-PCR (qRT-PCR) assay showed that transcription level of PxSP1 and PxSP2 increased significantly after injection of the fungal pathogen Beauveria bassiana. Purified recombinant fusion proteins of PxSP2 and PxSP1 were injected to New Zealand white rabbits and polyclonal antibodies were generated with the titers of 1:12,800. After silencing the expression of PxSP2 by RNAi, the PO activity decreased significantly. The results show that PxSP2 is involved in prophenoloxidase activation in P. xylostella. © 2016 Wiley Periodicals, Inc.

  1. Cloning, Expression, and Characterization of Prophenoloxidases from Asian Corn Borer, Ostrinia furnacalis (Gunée

    Shasha Zhang

    2016-01-01

    Full Text Available Insect phenoloxidase (PO belongs to the type 3 copper protein family and possesses oxidoreductase activities. PO is typically synthesized as a zymogen called prophenoloxidase (PPO and requires the proteolytic activation to function. We here cloned full-length cDNA for 3 previously unidentified PPOs, which we named OfPPO1a, OfPPO1b, and OfPPO3, from Asian corn borer, Ostrinia furnacalis (Gunée, in addition to the previously known OfPPO2. These conceptual PPOs and OfPPO2 all contain two common copper-binding regions, two potential proteolytic activation sites, a plausible thiol-ester site, and a conserved C-terminal region but lack a secretion signal peptide sequence at the N-terminus. O. furnacalis PPOs were highly similar to other insect PPOs (42% to 79% identity and clustered well with other lepidopteran PPOs. RT-PCR assay showed the transcripts of the 4 OfPPOs were all detected at the highest level in hemocytes and at the increased amounts after exposure to infection by bacteria and fungi. Additionally, we established an Escherichia coli (E. coli expression system to produce recombinant O. furnacalis PPO proteins for future use in investigating their functions. These insights could provide valuable information for better understanding the activation and functioning mechanisms of O. furnacalis PPOs.

  2. Effects of azadirachtin on Rhodnius prolixus: immunity and trypanosoma interaction

    Patricia de Azambuja

    1992-01-01

    Full Text Available The effects of azadirachtin, a tetranortriterpenoid from the neem tree Aradirachta indica J. on both immunity and Trypanosoma cruzi interaction within Rhodniusprolixus and other triatomines, were presented Given through a blood meal, azadirachtin affected the immune reactivity as shown by a significant reduction in numbers of hemocytes and consequently nodule formation follwing challenge with Enterobacter cloacae ß12, reduction in ability to produce antibacterial activities in the hemolymph when injected with bacteria, and decreased ability to destroy the infection caused by inoculation of E. cloacae cells. A single dose of azadirachtin was able to block the development of T. cruzi in R. prolixus if given through the meal at different intervals, together with, before or after parasite infection. Similary, these results were observed with different triatomine species and different strains of T. cruzi. Azadirachtin induced a permanent resistance of the vector against reinfection with T. cruzi. The significance of these data is discussed in relation to the general mode of azadirachtin action in insects.

  3. Fusarium pathogenesis investigated using Galleria mellonella as a heterologous host

    Coleman, Jeffrey J.; Muhammed, Maged; Kasperkovitz, Pia V.; Vyas, Jatin M.; Mylonakis, Eleftherios

    2011-01-01

    Members of the fungal genus Fusarium are capable of manifesting in a multitude of clinical infections, most commonly in immunocompromised patients. In order to better understand the interaction between the fungus and host, we have developed the larvae of the greater wax moth, Galleria mellonella, as a heterologous host for fusaria. When conidia are injected into the hemocoel of this Lepidopteran system, both clinical and environmental isolates of the fungus are able to kill the larvae at 37°C, although killing occurs more rapidly when incubated at 30°C. This killing was dependent on several other factors besides temperature, including the Fusarium strain, the number of conidia injected, and the conidia morphology, where macroconidia are more virulent than their microconidia counterpart. There was a correlation in the killing rate of Fusarium spp. when evaluated in G. mellonella and a murine model. In vivo studies indicated G. mellonella hemocytes were capable of initially phagocytosing both conidial morphologies. The G. mellonella system was also used to evaluate antifungal agents, and amphotericin B was able to confer a significant increase in survival to Fusarium infected-larvae. The G. mellonella-Fusarium pathogenicity system revealed that virulence of Fusarium spp. is similar, regardless of the origin of the isolate, and that mammalian endothermy is a major deterrent for Fusarium infection and therefore provides a suitable alternative to mammalian models to investigate the interaction between the host and this increasingly important fungal pathogen. PMID:22115447

  4. Cerebral Innate Immunity in Drosophila Melanogaster

    Brian P. Leung

    2015-03-01

    Full Text Available Modeling innate immunity in Drosophila melanogaster has a rich history that includes ground-breaking discoveries in pathogen detection and signaling. These studies revealed the evolutionary conservation of innate immune pathways and mechanisms of pathogen detection, resulting in an explosion of findings in the innate immunity field. In D. melanogaster, studies have focused primarily on responses driven by the larval fat body and hemocytes, analogs to vertebrate liver and macrophages, respectively. Aside from pathogen detection, many recent mammalian studies associate innate immune pathways with development and disease pathogenesis. Importantly, these studies stress that the innate immune response is integral to maintain central nervous system (CNS health. Microglia, which are the vertebrate CNS mononuclear phagocytes, drive vertebrate cerebral innate immunity. The invertebrate CNS contains microglial-like cells-ensheathing glia and reticular glia-that could be used to answer basic questions regarding the evolutionarily conserved innate immune processes in CNS development and health. A deeper understanding of the relationship between D. melanogaster phagocytic microglial-like cells and vertebrate microglia will be key to answering basic and translational questions related to cerebral innate immunity.

  5. In vitro immunotoxicity of environmentally representative antibiotics to the freshwater mussel Elliptio complanata

    Gust, M.; Gélinas, M.; Fortier, M.; Fournier, M.; Gagné, F.

    2012-01-01

    The separate and combined in vitro toxic effects of antibiotics (ciprofloxacin, erythromycin, novobiocin, oxytetracycline, sulfamethazole and trimethoprim) commonly found in urban wastewater effluents were assessed on the immune parameters of Elliptio complanata at environmentally relevant concentrations. The observed responses were then compared to those produced by the physicochemical-treated wastewater effluent of a major city before and after the removal of microorganisms. Most of the selected antibiotics, separately and as mixture, induced changes in immune responses. The removal of microorganisms and fine particles from the effluent increased or decreased the resulting immunotoxic effects, depending of the observed parameter. The immunotoxic effects of erythromycin, sulfamethoxazole and trimethoprim were closely associated to the antibiotic mixture and the filtered effluent. In conclusion, the data revealed that the removal of fine particles and microorganisms from municipal effluents can alter the toxic nature of the effluent that is closely associated with the cumulative effects of antibiotics. - Highlights: ► Environmental concentrations of antibiotics induce the immune response in mussels. ► Antibiotic mixture also modulate the immune response. ► Urban effluent, both filtered and unfiltered also increase the immune response. ► Filtered effluent, antibiotic mixture and 3 antibiotics induced similar responses. - Environmentally relevant concentration of antibiotics and filtered urban effluent similarly modulate the in vitro immune response of mussels’ hemocytes.

  6. In vitro immunotoxicity of environmentally representative antibiotics to the freshwater mussel Elliptio complanata

    Gust, M [Fluvial Ecosystem Research, Environment Canada, 105 McGill St., Montreal, Quebec H2Y 2E7 (Canada); IRSTEA, UR MAEP, Laboratoire d' ecotoxicologie, 3 bis quai Chauveau, 69009 Lyon (France); Gelinas, M [Fluvial Ecosystem Research, Environment Canada, 105 McGill St., Montreal, Quebec H2Y 2E7 (Canada); Fortier, M; Fournier, M [INRS - Institut Armand-Frappier, 531 Blvd. des Prairies, Laval, Quebec H7V 1B7 (Canada); Gagne, F [Fluvial Ecosystem Research, Environment Canada, 105 McGill St., Montreal, Quebec H2Y 2E7 (Canada); INRS - Institut Armand-Frappier, 531 Blvd. des Prairies, Laval, Quebec H7V 1B7 (Canada)

    2012-10-15

    The separate and combined in vitro toxic effects of antibiotics (ciprofloxacin, erythromycin, novobiocin, oxytetracycline, sulfamethazole and trimethoprim) commonly found in urban wastewater effluents were assessed on the immune parameters of Elliptio complanata at environmentally relevant concentrations. The observed responses were then compared to those produced by the physicochemical-treated wastewater effluent of a major city before and after the removal of microorganisms. Most of the selected antibiotics, separately and as mixture, induced changes in immune responses. The removal of microorganisms and fine particles from the effluent increased or decreased the resulting immunotoxic effects, depending of the observed parameter. The immunotoxic effects of erythromycin, sulfamethoxazole and trimethoprim were closely associated to the antibiotic mixture and the filtered effluent. In conclusion, the data revealed that the removal of fine particles and microorganisms from municipal effluents can alter the toxic nature of the effluent that is closely associated with the cumulative effects of antibiotics. - Highlights: Black-Right-Pointing-Pointer Environmental concentrations of antibiotics induce the immune response in mussels. Black-Right-Pointing-Pointer Antibiotic mixture also modulate the immune response. Black-Right-Pointing-Pointer Urban effluent, both filtered and unfiltered also increase the immune response. Black-Right-Pointing-Pointer Filtered effluent, antibiotic mixture and 3 antibiotics induced similar responses. - Environmentally relevant concentration of antibiotics and filtered urban effluent similarly modulate the in vitro immune response of mussels' hemocytes.

  7. Effects of the toxic dinoflagellate, Gymnodinium catenatum on hydrolytic and antioxidant enzymes, in tissues of the giant lions-paw scallop Nodipecten subnodosus.

    Estrada, Norma; de Jesús Romero, Maria; Campa-Córdova, Angel; Luna, Antonio; Ascencio, Felipe

    2007-11-01

    This study documents effects of the toxic dinoflagellate Gymnodinium catenatum, a producer of paralytic shellfish poison, on juvenile farmed (5.9+/-0.39 cm) giant lions-paw scallop Nodipecten subnodosus. Scallops were fed bloom concentrations of toxic dinoflagellate G. catenatum for 7 h. The effect of the toxic dinoflagellate in different tissues was determined by analysis of antioxidant enzymes (catalase, superoxide dismutase, gluthathione peroxidase), thiobarbituric acid reactive substances (lipid peroxidation), and hydrolytic enzymes (proteases, glycosidases, phosphatases, lipases, and esterases). Histopathological photos record the effects of the toxic dinoflagellate in various tissues. The results show that juvenile lions-paw scallops produce pseudo-feces, partially close their shell, increase melanization, and aggregate hemocytes. Several enzymes were affected and could serve as biological markers. In general, the adductor muscle was not affected. In the digestive gland, some enzymes could be the result of defensive and digestive processes. Gills and mantle tissue were markedly affected because these sites respond first to toxic dinoflagellates, leading to the idea that proteolytic cascades could be involved.

  8. Transcriptome analysis of the white body of the squid Euprymna tasmanica with emphasis on immune and hematopoietic gene discovery.

    Karla A Salazar

    Full Text Available In the mutualistic relationship between the squid Euprymna tasmanica and the bioluminescent bacterium Vibrio fischeri, several host factors, including immune-related proteins, are known to interact and respond specifically and exclusively to the presence of the symbiont. In squid and octopus, the white body is considered to be an immune organ mainly due to the fact that blood cells, or hemocytes, are known to be present in high numbers and in different developmental stages. Hence, the white body has been described as the site of hematopoiesis in cephalopods. However, to our knowledge, there are no studies showing any molecular evidence of such functions. In this study, we performed a transcriptomic analysis of white body tissue of the Southern dumpling squid, E. tasmanica. Our primary goal was to gain insights into the functions of this tissue and to test for the presence of gene transcripts associated with hematopoietic and immune processes. Several hematopoiesis genes including CPSF1, GATA 2, TFIID, and FGFR2 were found to be expressed in the white body. In addition, transcripts associated with immune-related signal transduction pathways, such as the toll-like receptor/NF-κβ, and MAPK pathways were also found, as well as other immune genes previously identified in E. tasmanica's sister species, E. scolopes. This study is the first to analyze an immune organ within cephalopods, and to provide gene expression data supporting the white body as a hematopoietic tissue.

  9. Immunohistochemical localization of cardio-active neuropeptides in the heart of a living fossil, Nautilus pompilius L. (Cephalopoda, Tetrabranchiata).

    Springer, J; Ruth, P; Beuerlein, K; Westermann, B; Schipp, R

    2004-01-01

    Neuropeptides play an important role in modulating the effects of neurotransmitters such as acetylcholine and noradrenaline in the heart and the vascular system of vertebrates and invertebrates. Various neuropeptides, including substance P (SP), vasoactive intestinal polypeptide (VIP) and FMRFamide, have been localized in the brain in cephalopods and the neurosecretory system of the vena cava. Previous studies involving cephalopods have mainly focussed on the modern, coleoid cephalopods, whereas little attention was paid to the living fossil Nautilus. In this study, the distributions of the peptides related to tachykinins (TKs) and the high affinity receptor for the best characterized TK substance P (tachykinin NK-1), VIP, as well as FMRFamide were investigated in the heart of Nautilus pompilius L. by immunohistochemistry. TK-like immunoreactivity (TK-LI) was seen associated to a sub-population of hemocytes, VIP-LI glial cells in larger nerves entering the heart, whereas FMRFamide immunoreactivity was distributed throughout the entire heart, including the semilunar atrioventricular valves. The pattern of FMRFamide immunoreactivity matched that of Bodian silver staining for nervous tissue. The NK-1-LI receptor was located on endothelial cells, which were also positive for endothelial nitric oxide synthase-LI (eNOS). The results indicate that neuropeptides may be involved in the regulation of the Nautilus heart via different mechanisms, (1) by direct interaction with myocardial receptors (FMRFamide), (2) by interacting with the nervus cardiacus (VIP-related peptides) and (3) indirectly by stimulating eNOS in the endothelium throughout the heart (TK-related peptides).

  10. deep-orange and carnation define distinct stages in late endosomal biogenesis in Drosophila melanogaster.

    Sriram, V; Krishnan, K S; Mayor, Satyajit

    2003-05-12

    Endosomal degradation is severely impaired in primary hemocytes from larvae of eye color mutants of Drosophila. Using high resolution imaging and immunofluorescence microscopy in these cells, products of eye color genes, deep-orange (dor) and carnation (car), are localized to large multivesicular Rab7-positive late endosomes containing Golgi-derived enzymes. These structures mature into small sized Dor-negative, Car-positive structures, which subsequently fuse to form tubular lysosomes. Defective endosomal degradation in mutant alleles of dor results from a failure of Golgi-derived vesicles to fuse with morphologically arrested Rab7-positive large sized endosomes, which are, however, normally acidified and mature with wild-type kinetics. This locates the site of Dor function to fusion of Golgi-derived vesicles with the large Rab7-positive endocytic compartments. In contrast, endosomal degradation is not considerably affected in car1 mutant; fusion of Golgi-derived vesicles and maturation of large sized endosomes is normal. However, removal of Dor from small sized Car-positive endosomes is slowed, and subsequent fusion with tubular lysosomes is abolished. Overexpression of Dor in car1 mutant aggravates this defect, implicating Car in the removal of Dor from endosomes. This suggests that, in addition to an independent role in fusion with tubular lysosomes, the Sec1p homologue, Car, regulates Dor function.

  11. Antioxidant response of ridgetail white prawn Exopalaemon carinicauda to harmful dinoflagellate Prorocentrum minimum exposure and its histological change

    Mu, Cuimin; Ren, Xianyun; Ge, Qianqian; Wang, Jiajia; Li, Jian

    2017-04-01

    The dinoflagellate Prorocentrum minimum, one of the most widespread red tide causing species, affects marine aquaculture and ecosystems worldwide. In this study, ridgetail white prawn Exopalaemon carinicauda were exposed to P. minimum cells (5 × 104 cells mL-1) to investigate its harmful effects on the shrimp. Antioxidant activities and histological changes were used as indicators of health status of the shrimp. In 72 hours, the mortality of E. carinicauda was not affected, but its antioxidant response and histology were statistically different from those of control. Elevated superoxide dismutase (SOD) and glutathione peroxidase (GPX) activities and depressed catalase (CAT) activity were observed in gill; while increased SOD, glutathione S-transferase (GST), CAT activities and modulated GPX activity were observed in hepatopancreas. Thus, antioxidant activities in gill and hepatopancreas seem to respond differentially to harmful alga exposure. Increased malondialdehyde (MDA) content in early a few hours indicates the damage of the antioxidant defense system. Although MDA content recovered to a low level thereafter, a series of histological abnormalities including accumulation or infiltration of hemocytes, tissue lesions and necrosis were discovered in gill and hepatopancreas. Exposure to P. minimum induced sublethal effects on E. carinicauda, including temporary oxidative damage and histological injury.

  12. First characterization of three cyclophilin family proteins in the oyster, Crassostrea ariakensis Gould.

    Xu, Ting; Xie, Jiasong; Yang, Shoubao; Ye, Shigen; Luo, Ming; Wu, Xinzhong

    2016-08-01

    Cyclophilins (CyPs) are a family of proteins that bind the immunosuppressive agent cyclosporin A (CsA) with high-affinity and belong to one of the three superfamilies of peptidyl-prolyl cis-trans isomerases (PPIase). In this report, three cyclophilin genes (Ca-CyPs), including Ca-CyPA, Ca-CyPB and Ca-PPIL3, were identified from oyster, Crassostrea ariakensis Gould in which Ca-CyPA encodes a protein with 165 amino acid sequences, Ca-CyPB encodes a protein with 217 amino acid sequences and Ca-PPIL3 encodes a protein with 162 amino acid sequences. All of the three Ca-CyPs genes contain a typical CyP-PPIase domain with its signature sequences and Ca-CyPB contains an N-signal peptide sequences. Tissue distribution study revealed that Ca-CyPs were ubiquitously expressed in all examined tissues and the highest levels were observed in hemocytes. RLO incubation upregulated the mRNA expression levels of Ca-CyPs, indicating that three Ca-CyPs might be involved in oyster immune response against RLO infection. Copyright © 2016 Elsevier Ltd. All rights reserved.

  13. Multiple roles for apoptosis facilitating condensation of the Drosophila ventral nerve cord.

    Page, Damon T; Olofsson, Birgitta

    2008-02-01

    At the end of embryogenesis, the ventral nerve cord (VNC) of Drosophila undergoes a shape change, termed condensation. During condensation the length of the VNC shortens by 25%, a process dependent on extracellular matrix deposited by hemocytes, an intact cytoskeleton of glia and neurons and neural activity. Here we show that cell death contributes to nerve cord shortening. Firstly, apoptosis occurs at the interface of the epidermis and the nerve cord where it plays a role in the separation of these two tissues. Separation precedes condensation and in conditions where separation is prevented, condensation fails. Secondly, many cells undergo apoptosis within VNC during condensation. This cell death is localized mainly to the posterior part of the nerve cord where more than half of all cell death occurs. Preventing apoptosis either in neurons or glia partially inhibits VNC shortening during condensation. Despite the importance of midline glia in axon tract development, preventing midline glia cell death results in normal hatching and adult formation. We find that undead midline glia are eliminated from the midline and become mispositioned or expelled from the nervous system. We suggest that this represent a form of pattern repair that operates to reduce the impact of the additional cells. (c) 2008 Wiley-Liss, Inc.

  14. Arthropod Innate Immune Systems and Vector-Borne Diseases.

    Baxter, Richard H G; Contet, Alicia; Krueger, Kathryn

    2017-02-21

    Arthropods, especially ticks and mosquitoes, are the vectors for a number of parasitic and viral human diseases, including malaria, sleeping sickness, Dengue, and Zika, yet arthropods show tremendous individual variation in their capacity to transmit disease. A key factor in this capacity is the group of genetically encoded immune factors that counteract infection by the pathogen. Arthropod-specific pattern recognition receptors and protease cascades detect and respond to infection. Proteins such as antimicrobial peptides, thioester-containing proteins, and transglutaminases effect responses such as lysis, phagocytosis, melanization, and agglutination. Effector responses are initiated by damage signals such as reactive oxygen species signaling from epithelial cells and recognized by cell surface receptors on hemocytes. Antiviral immunity is primarily mediated by siRNA pathways but coupled with interferon-like signaling, antimicrobial peptides, and thioester-containing proteins. Molecular mechanisms of immunity are closely linked to related traits of longevity and fertility, and arthropods have the capacity for innate immunological memory. Advances in understanding vector immunity can be leveraged to develop novel control strategies for reducing the rate of transmission of both ancient and emerging threats to global health.

  15. Preparation and evaluation of squid ink polysaccharide-chitosan as a wound-healing sponge.

    Huang, Na; Lin, Jiali; Li, Sidong; Deng, Yifeng; Kong, Songzhi; Hong, Pengzhi; Yang, Ping; Liao, Mingneng; Hu, Zhang

    2018-01-01

    A new type of wound healing agent was developed using two marine biomaterials (squid ink polysaccharide and chitosan) as carriers and calcium chloride as an initiator for coagulation. Based on central composite design-response surface methodology, comprehensive evaluation of appearance quality for composite sponges and water absorbency were used as evaluation indices to identify the optimized preparation conditions and further evaluate the performance of the squid ink polysaccharide-chitosan sponge (SIP-CS). The optimized formulation of SIP-CS was as follows: chitosan concentration, 2.29%; squid ink polysaccharide concentration, 0.55%; and calcium chloride concentration, 2.82%, at a volume ratio of 15:5:2. SIP-CS was conducive to sticking on the wound, characterized by the spongy property, strong absorptivity, and tackiness. Rabbit ear arterial, hepatic, and femoral artery hemorrhage experiments indicated that, compared with chitosan dressings and absorbable gelatin, the hemostatic times were shorter and the bleeding volume was smaller. Furthermore, SIP-CS absorbed a large amount of hemocytes, leading to rapid hemostasis. The healing areas and wound pathological sections in scalded New Zealand rabbits indicated that SIP-CS promoted wound healing more rapidly than chitosan and better than commercially available burn cream. Thus, SIP-CS is a good wound healing agent for rapid hemostasis, promoting burn/scalded skin healing, and protecting from wound infection. Copyright © 2017 Elsevier B.V. All rights reserved.

  16. Female-specific wing degeneration caused by ecdysteroid in the Tussock Moth, Orgyia recens: Hormonal and developmental regulation of sexual dimorphism

    Saori Lobbia

    2003-04-01

    Full Text Available Females of the tussock moth Orgyia recens have vestigial wings, whereas the males have normal wings. During early pupal development, female wings degenerate drastically compared with those of males. To examine whether ecdysteroid is involved in this sex-specific wing development, we cultured pupal wings just after pupation with ecdysteroid (20-hydroxyecdysone, 20E. In the presence of 20E, the female wings degenerated to about one-fifth their original size. In contrast, the male wings cultured with 20E showed only peripheral degeneration just outside the bordering lacuna, as in other butterflies and moths. TUNEL analysis showed that apoptotic signals were induced by 20E over the entire region of female wings, but only in the peripheral region of male wings. Semi-thin sections of the wings cultured with ecdysteroid showed that phagocytotic hemocytes were observed abundantly throughout the female wings, but in only peripheral regions of male wings. These observations indicate that both apoptotic events and phagocytotic activation are triggered by ecdysteroid, in sex-specific and region-specific manners.

  17. Biological activity of Penaeus monodon GILT in shrimp pathogen protection

    Aekkaraj Nualla-ong

    2017-04-01

    Full Text Available Gamma-interferon-inducible lysosomal thiol reductase (GILT contains a CXXC active site motif that possesses thiol reductase activity by catalyzing the disulfide bond reduction of exogenous antigens. Mutating the active site of human GILT to change the cysteine residues to serine residues eliminates this property. Our previous study reported that Penaeus monodon GILT (PmGILT contained a CXXS active site motif. Therefore, we assessed the enzymatic activity of PmGILT and demonstrated that it displayed identical thiol reductase activity at an acidic pH. In addition, the biological activity of PmGILT against shrimp pathogens, including white spot syndrome virus (WSSV and Gram-negative bacteria, was investigated. The neutralization of WSSV with PmGILT indicated the inhibition of WSSV invasion into shrimp hemocyte cells. Moreover, the relative percentage survival of shrimp injected with PmGILT-treated virus solution was 75%. Finally, the antimicrobial activity of PmGILT was confirmed by the growth inhibition of Vibrio harveyi. These results establish the role of PmGILT in the inhibition of the virulence of two major shrimp pathogens.

  18. Growth, nonspecific immune characteristics, and survival upon challenge with Vibrio harveyi in Pacific white shrimp (Litopenaeus vannamei) raised on diets containing algal meal.

    Nonwachai, Thasanee; Purivirojkul, Watchariya; Limsuwan, Chalor; Chuchird, Niti; Velasco, Mario; Dhar, Arun K

    2010-08-01

    A 70-day growth trial was conducted with postlarvae 12 (PL12) Pacific white shrimp (Litopenaeus vannamei) to study the suitability of soybean meal and oil originating from a single-celled microorganism (thraustochytrid) as fishmeal and fish oil substitutes in practical diets for L. vannamei. The growth, survival rate and immune characteristics were evaluated. Seven experimental diets were designed with soybean meal used as the primary protein source; each formulation contained 33% crude protein and 8% lipid. Fish oil was completely substituted with 3% soybean oil and meals originating from single-celled heterotrophs rich in docosahexaenoic acid (DHA) and arachidonic acid (ARA) were added at different concentrations. A commercial shrimp feed was used as the control diet. The final weights and survival rates of the shrimp were not significantly different among all treatments. However, shrimp raised on diets supplemented with marine algal meals rich in DHA and ARA showed significant improvement in immune parameters, such as total hemocyte count, phenoloxidase activity, superoxide dismutase activity, and bactericidal activity. Additionally, the survival rate after challenge with Vibrio harveyi was increased. These findings demonstrated that substitution of thraustochytrid-derived meals as an alternative to fish-based ingredients in shrimp diets provided similar growth rates while increasing the immune parameters and providing vibriosis resistance. Copyright 2010 Elsevier Ltd. All rights reserved.

  19. Editing Transgenic DNA Components by Inducible Gene Replacement in Drosophila melanogaster

    Lin, Chun-Chieh; Potter, Christopher J.

    2016-01-01

    Gene conversions occur when genomic double-strand DNA breaks (DSBs) trigger unidirectional transfer of genetic material from a homologous template sequence. Exogenous or mutated sequence can be introduced through this homology-directed repair (HDR). We leveraged gene conversion to develop a method for genomic editing of existing transgenic insertions in Drosophila melanogaster. The clustered regularly-interspaced palindromic repeats (CRISPR)/Cas9 system is used in the homology assisted CRISPR knock-in (HACK) method to induce DSBs in a GAL4 transgene, which is repaired by a single-genomic transgenic construct containing GAL4 homologous sequences flanking a T2A-QF2 cassette. With two crosses, this technique converts existing GAL4 lines, including enhancer traps, into functional QF2 expressing lines. We used HACK to convert the most commonly-used GAL4 lines (labeling tissues such as neurons, fat, glia, muscle, and hemocytes) to QF2 lines. We also identified regions of the genome that exhibited differential efficiencies of HDR. The HACK technique is robust and readily adaptable for targeting and replacement of other genomic sequences, and could be a useful approach to repurpose existing transgenes as new genetic reagents become available. PMID:27334272

  20. Complement C3 gene: Expression characterization and innate immune response in razor clam Sinonovacula constricta.

    Peng, Maoxiao; Niu, Donghong; Wang, Fei; Chen, Zhiyi; Li, Jiale

    2016-08-01

    Complement component 3 (C3) is central to the complement system, playing an important role in immune defense, immune regulation and immune pathology. Several C3 genes have been characterized in invertebrates but very few in shellfish. The C3 gene was identified from the razor clam Sinonovacula constricta, referred to here as Sc-C3. It was found to be highly homologous with the C3 gene of Ruditapes decussatus. All eight model motifs of the C3 gene were found to be included in the thiolester bond and the C345C region. Sc-C3 was widely expressed in all healthy tissues with expression being highest in hemolymph. A significant difference in expression was revealed at the umbo larvae development stage. The expression of Sc-C3 was highly regulated in the hemolymph and liver, with a distinct response pattern being noted after a challenge with Micrococcus lysodeikticus and Vibrio parahemolyticus. It is therefore suggested that a complicated and unique response pathway may be present in S. constricta. Further, serum of S. constricta containing Sc-C3 was extracted. This was activated by LPS or bacterium for verification for function. The more obvious immune function of Sc-C3 was described as an effective membrane rupture in hemocyte cells of rabbit, V. parahemolyticus and Vibrio anguillarum. Thus, Sc-C3 plays an essential role in the immune defense of S. constricta. Copyright © 2016 Elsevier Ltd. All rights reserved.

  1. Proteomic identification of the related immune-enhancing proteins in shrimp Litopenaeus vannamei stimulated with vitamin C and Chinese herbs.

    Qiao, Jie; Du, Zhiheng; Zhang, Yueling; Du, Hong; Guo, Lingling; Zhong, Mingqi; Cao, Jingsong; Wang, Xiuying

    2011-12-01

    Recently, strong interest has been focused on immunostimulants to reducing the diseases in shrimp aquaculture. However, information regarding to the related immune-enhancing proteins in shrimps is not available yet. In this study, vitamin C (Vc), Chinese herbs (CH), and the mixture of vitamin C and Chinese herbs (Mix) were tested for their enhancement on shrimp's immune activity. Compared with those in the control group, values of phenoloxidase (PO), superoxide dismutase (SOD) and antibacterial (Ua) activity in the Mix-treated group were improved significantly 12 or 24 days after the treatment. The cumulative mortality was also lower in the Mix-treated group after infection with Vibrio parahemolyticus. Furthermore, comparative proteomic approach was used to assess the protein expression profile in shrimps. Approximately 220-290 and 300-400 protein spots were observed in the 2-DE gels. Among them, 29 and 28 altered proteins from hemocytes and hepatopancreas, respectively, were subjected to matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF/MS) analysis. The results revealed that the main altered proteins showed high homologies with Litopenaeus vannamei hemocyanin, hemolymph clottable protein, hemoglobin beta, cytosolic MnSOD, trypsin, cathepsin I(L) and zinc proteinase Mpc1. Together, these studies found Vc and CH were suitable immunostimulants to shrimp L. vannamei, and 7 altered proteins could be involved in the enhanced immune activities. Copyright © 2011 Elsevier Ltd. All rights reserved.

  2. Transcriptome Analysis Revealed Changes of Multiple Genes Involved in Haliotis discus hannai Innate Immunity during Vibrio parahemolyticus Infection.

    Nam, Bo-Hye; Jung, Myunghee; Subramaniyam, Sathiyamoorthy; Yoo, Seung-il; Markkandan, Kesavan; Moon, Ji-Young; Kim, Young-Ok; Kim, Dong-Gyun; An, Cheul Min; Shin, Younhee; Jung, Ho-jin; Park, Jun-hyung

    2016-01-01

    Abalone (Haliotis discus hannai) is one of the most valuable marine aquatic species in Korea, Japan and China. Tremendous exposure to bacterial infection is common in aquaculture environment, especially by Vibrio sp. infections. It's therefore necessary and urgent to understand the mechanism of H. discus hannai host defense against Vibrio parahemolyticus infection. However studies on its immune system are hindered by the lack of genomic resources. In the present study, we sequenced the transcriptome of control and bacterial challenged H. discus hannai tissues. Totally, 138 MB of reference transcriptome were obtained from de novo assembly of 34 GB clean bases from ten different libraries and annotated with the biological terms (GO and KEGG). A total of 10,575 transcripts exhibiting the differentially expression at least one pair of comparison and the functional annotations highlight genes related to immune response, cell adhesion, immune regulators, redox molecules and mitochondrial coding genes. Mostly, these groups of genes were dominated in hemocytes compared to other tissues. This work is a prerequisite for the identification of those physiological traits controlling H. discus hannai ability to survive against Vibrio infection.

  3. Transcriptome Analysis Revealed Changes of Multiple Genes Involved in Haliotis discus hannai Innate Immunity during Vibrio parahemolyticus Infection

    Nam, Bo-Hye; Jung, Myunghee; Subramaniyam, Sathiyamoorthy; Yoo, Seung-il; Markkandan, Kesavan; Moon, Ji-Young; Kim, Young-Ok; Kim, Dong-Gyun; An, Cheul Min; Shin, Younhee; Jung, Ho-jin; Park, Jun-hyung

    2016-01-01

    Abalone (Haliotis discus hannai) is one of the most valuable marine aquatic species in Korea, Japan and China. Tremendous exposure to bacterial infection is common in aquaculture environment, especially by Vibrio sp. infections. It’s therefore necessary and urgent to understand the mechanism of H. discus hannai host defense against Vibrio parahemolyticus infection. However studies on its immune system are hindered by the lack of genomic resources. In the present study, we sequenced the transcriptome of control and bacterial challenged H. discus hannai tissues. Totally, 138 MB of reference transcriptome were obtained from de novo assembly of 34 GB clean bases from ten different libraries and annotated with the biological terms (GO and KEGG). A total of 10,575 transcripts exhibiting the differentially expression at least one pair of comparison and the functional annotations highlight genes related to immune response, cell adhesion, immune regulators, redox molecules and mitochondrial coding genes. Mostly, these groups of genes were dominated in hemocytes compared to other tissues. This work is a prerequisite for the identification of those physiological traits controlling H. discus hannai ability to survive against Vibrio infection. PMID:27088873

  4. Transcriptome Analysis Revealed Changes of Multiple Genes Involved in Haliotis discus hannai Innate Immunity during Vibrio parahemolyticus Infection.

    Bo-Hye Nam

    Full Text Available Abalone (Haliotis discus hannai is one of the most valuable marine aquatic species in Korea, Japan and China. Tremendous exposure to bacterial infection is common in aquaculture environment, especially by Vibrio sp. infections. It's therefore necessary and urgent to understand the mechanism of H. discus hannai host defense against Vibrio parahemolyticus infection. However studies on its immune system are hindered by the lack of genomic resources. In the present study, we sequenced the transcriptome of control and bacterial challenged H. discus hannai tissues. Totally, 138 MB of reference transcriptome were obtained from de novo assembly of 34 GB clean bases from ten different libraries and annotated with the biological terms (GO and KEGG. A total of 10,575 transcripts exhibiting the differentially expression at least one pair of comparison and the functional annotations highlight genes related to immune response, cell adhesion, immune regulators, redox molecules and mitochondrial coding genes. Mostly, these groups of genes were dominated in hemocytes compared to other tissues. This work is a prerequisite for the identification of those physiological traits controlling H. discus hannai ability to survive against Vibrio infection.

  5. Involvement of clip-domain serine protease in the anti-Vibrio immune response of abalone (Haliotis discus hannai)-Molecular cloning, characterization and functional analysis.

    Hu, Jian-Jian; Chen, Yu-Lei; Duan, Xue-Kun; Jin, Teng-Chuan; Li, Yue; Zhang, Ling-Jing; Liu, Guang-Ming; Cao, Min-Jie

    2018-01-01

    Vibrio parahemolyticus (V. parahemolyticus) is a major pathogen for abalone, an important economical shellfish in coastal area of China. There is little known about the abalone innate immune system against pathogen infection. Clip-domain serine proteases (cSPs) are increasingly recognized to play important roles in host immune defense in invertebrates. In this study, we cloned a cSP (Hdh-cSP) from abalone (Haliotis discus hannai). We found out that Hdh-cSP was widely expressed in multiple tissues of abalone, with highest level in the immune-like organ, hepatopancreas. V. parahemolyticus infection induced significantly elevated expression of Hdh-cSP in addition to better-characterized innate immune component genes including Rel/NF-κB, allograft inflammatory factor (ALInFa), macrophage expressed protein (MEP) and caspase-8. Importantly, the silencing of Hdh-cSP reduced the expression of these genes, suggesting that Hdh-cSP was an upstream regulatory factor in V. parahemolyticus infection. Further analysis showed that apoptosis of hemocytes was inhibited when the transcription of Hdh-cSP was knocked down, suggesting that Hdh-cSP participated in cell apoptosis by regulation of caspase 8 expression in V. parahemolyticus infection. Therefore, our study established an important role of cSP in the innate immunity against V. parahemolyticus infection in abalone. Copyright © 2017 Elsevier Ltd. All rights reserved.

  6. Identification and function of leucine-rich repeat flightless-I-interacting protein 2 (LRRFIP2 in Litopenaeus vannamei.

    Shuang Zhang

    Full Text Available Leucine-rich repeat flightless-I-interacting protein 2 (LRRFIP2 is a myeloid differentiation factor 88-interacting protein with a positive regulatory function in toll-like receptor signaling. In this study, seven LRRFIP2 protein variants (LvLRRFIP2A-G were identified in Litopenaeus vannamei. All the seven LvLRRFIP2 protein variants encode proteins with a DUF2051 domain. LvLRRFIP2s were upregulated in hemocytes after challenged with lipopolysaccharide, poly I:C, CpG-ODN2006, Vibrio parahaemolyticus, Staphylococcus aureus, and white spot syndrome virus (WSSV. Dual-luciferase reporter assays in Drosophila Schneider 2 cells revealed that LvLRRFIP2 activates the promoters of Drosophila and shrimp AMP genes. The knockdown of LvLRRFIP2 by RNA interference resulted in higher cumulative mortality of L. vannamei upon V. parahaemolyticus but not S. aureus and WSSV infections. The expression of L. vannamei AMP genes were reduced by dsLvLRRFIP2 interference. These results indicate that LvLRRFIP2 has an important function in antibacterials via the regulation of AMP gene expression.

  7. A chitinase from pacific white shrimp Litopenaeus vannamei involved in immune regulation.

    Niu, Shengwen; Yang, Linwei; Zuo, Hongliang; Zheng, Jiefu; Weng, Shaoping; He, Jianguo; Xu, Xiaopeng

    2018-08-01

    Chitinases are a group of hydrolytic enzymes that hydrolyze chitin and widely exist in organisms. Studies in mammals have demonstrated that chitinases play important roles in regulation of humoral and cellular immune responses. In arthropods, although it is well known that chitinases are involved in growth, molting and development, the current knowledge on the role of chitinases in immunity, especially in immune regulation, remains largely unknown. In this study, a chitinase (LvChi5) from Litopenaeus vannamei was representatively selected for studying its immune function. The start codon of LvChi5 was corrected by 5'RACE analysis and its protein sequence was reanalyzed. LvChi5 contains a catalytic domain and a chitin binding domain and shows no inhibitory effect on growth of bacteria in vitro. However, in vivo experiments demonstrated that silencing of LvChi5 increased the mortality of shrimp infected with white spot syndrome virus (WSSV) and Vibro parahaemolyticus and significantly upregulated the load of pathogens in tissues. The expression of various immune related genes, including transcription factors, antimicrobial peptides and other functional proteins with antibacterial and antiviral activities, was widely changed in LvChi5 silencing shrimp. Moreover, the recombinant LvChi5 protein could enhance the phagocytic activity of hemocytes against bacteria. These suggested that shrimp chitinase could play a role in regulation of both humoral and cellular immune responses in shrimp. Copyright © 2018 Elsevier Ltd. All rights reserved.

  8. A single WAP domain (SWD)-containing protein with antiviral activity from Pacific white shrimp Litopenaeus vannamei.

    Yang, Linwei; Niu, Shengwen; Gao, Jiefeng; Zuo, Hongliang; Yuan, Jia; Weng, Shaoping; He, Jianguo; Xu, Xiaopeng

    2018-02-01

    The single whey acidic protein (WAP) domain (SWD)-containing proteins, also called type III crustins, are a group of antimicrobial peptides (AMPs) in crustaceans. At present, a number of SWDs have been identified in shrimp, which showed essential antibacterial activities. However, the roles of SWDs in antiviral immune responses have not been reported up to now. In this study, a novel SWD (LvSWD3) was identified from Pacific white shrimp, Litopenaeus vannamei, which contained a typical single WAP domain homologous to those of other crustacean SWDs. Although lacking the pro and arg-rich region between the signal peptide and the WAP domain, LvSWD3 was closely clustered with other shrimp SWDs in the phylogenetic tree. Similar to many shrimp SWDs, the highest expression of LvSWD3 was detected in hemocytes. The LvSWD3 expression exhibited only limited changes after challenges with Vibrio parahaemolyticus, Poly (I:C) and lipopolysaccharide, but was significantly up-regulated after white spot syndrome virus (WSSV) infection. Silencing of LvSWDs significantly accelerated the death of the WSSV-infected but not the V. parahaemolyticus-infected shrimp. The recombinant LvSWD3 protein did not show proteinase inhibitory and antibacterial activities but could significantly postpone the death of WSSV-infected shrimp and reduce the viral load in tissues. These suggested that LvSWD3 was a novel SWD with antiviral activity. Copyright © 2017 Elsevier Ltd. All rights reserved.

  9. Actin genes and their expression in pacific white shrimp, Litopenaeus vannamei.

    Zhang, Xiaoxi; Zhang, Xiaojun; Yuan, Jianbo; Du, Jiangli; Li, Fuhua; Xiang, Jianhai

    2018-04-01

    Actin is a multi-functional gene family that can be divided into muscle-type actins and non-muscle-type actins. In this study, 37 unigenes encoding actins were identified from RNA-Seq data of Pacific white shrimp, Litopenaeus vannamei. According to phylogenetic analysis, four and three cDNAs belong to cytoplasmic- and heart-type actins and were named LvActinCT and LvActinHT, respectively. 10 cDNAs belong to the slow-type skeletal muscle actins, and 18 belong to the fast-type skeletal muscle actins; they were designated LvActinSSK and LvActinFSK, respectively. Some muscle actin genes formed gene clusters in the genome. Multiple alternative transcription starts sites (ATSSs) were found for LvActinCT1. Based on the early developmental expression profile, almost all LvActins were highly expressed between the early limb bud and post-larval stages. Using LvActinSSK5 as probes, slow-type muscle was localized in pleopod muscle and superficial ventral muscle. We also found three actin genes that were down-regulated in the hemocytes of white spot syndrome virus (WSSV)- and Vibrio parahaemolyticus-infected L. vannamei. This study provides valuable information on the actin gene structure of shrimp, furthers our understanding of the shrimp muscle system and helps us develop strategies for disease control and sustainable shrimp farming.

  10. Epigallocatechin-3-gallate protects Kuruma shrimp Marsupeneaus japonicus from white spot syndrome virus and Vibrio alginolyticus.

    Wang, Zhi; Sun, Baozhen; Zhu, Fei

    2018-07-01

    Epigallocatechin-3-gallate (EGCG) is the most abundant catechin in green tea and exhibits potential antibacterial and anticancer activities. In this study, EGCG was used in pathogen-challenge experiments in shrimp to discover its effect on the innate immune system of an invertebrate. Kuruma shrimp Marsupeneaus japonicus was used as an experimental model and challenged with white spot syndrome virus (WSSV) and the Gram-negative bacterium Vibrio alginolyticus. Pathogen-challenge experiments showed that EGCG pretreatment significantly delayed and reduced mortality upon WSSV and V. alginolyticus infection, with VP-28 copies of WSSV also reduced. Quantitative reverse transcription polymerase chain reaction revealed the positive influence of EGCG on several innate immune-related genes, including IMD, proPO, QM, myosin, Rho, Rab7, p53, TNF-alpha, MAPK, and NOS, and we observed positive influences on three immune parameters, including total hemocyte count and phenoloxidase and superoxide dismutase activities, by EGCG treatment. Additionally, results showed that EGCG treatment significantly reduced apoptosis upon V. alginolyticus challenge. These results indicated the positive role of EGCG in the shrimp innate immune system as an enhancer of immune parameters and an inhibitor of apoptosis, thereby delaying and reducing mortality upon pathogen challenge. Our findings provide insight into potential therapeutic or preventive functions associated with EGCG to enhance shrimp immunity and protect shrimp from pathogen infection. Copyright © 2018 Elsevier Ltd. All rights reserved.

  11. White Shrimp Litopenaeus vannamei That Have Received Gracilaria tenuistipitata Extract Show Early Recovery of Immune Parameters after Ammonia Stressing.

    Chen, Yu-Yuan; Chen, Jiann-Chu; Lin, Yong-Chin; Yeh, Su-Tuen; Huang, Chien-Lun

    2015-06-05

    White shrimp Litopenaeus vannamei immersed in seawater (35‰) containing Gracilaria tenuistipitata extract (GTE) at 0 (control), 400, and 600 mg/L for 3 h were exposed to 5 mg/L ammonia-N (ammonia as nitrogen), and immune parameters including hyaline cells (HCs), granular cells (GCs, including semi-granular cells), total hemocyte count (THC), phenoloxidase (PO) activity, respiratory bursts (RBs), superoxide dismutase (SOD) activity, lysozyme activity, and hemolymph protein level were examined 24~120 h post-stress. The immune parameters of shrimp immersed in 600 mg/L GTE returned to original values earlier, at 96~120 h post-stress, whereas in control shrimp they did not. In another experiment, shrimp were immersed in seawater containing GTE at 0 and 600 mg/L for 3 h and examined for transcript levels of immune-related genes at 24 h post-stress. Transcript levels of lipopolysaccharide and β-1,3-glucan binding protein (LGBP), peroxinectin (PX), cytMnSOD, mtMnSOD, and HSP70 were up-regulated at 24 h post-stress in GTE receiving shrimp. We concluded that white shrimp immersed in seawater containing GTE exhibited a capability for maintaining homeostasis by regulating cellular and humoral immunity against ammonia stress as evidenced by up-regulated gene expression and earlier recovery of immune parameters.

  12. Hypoxia increases susceptibility of Pacific white shrimp to whitespot syndrome virus (WSSV

    M. Lehmann

    2016-04-01

    Full Text Available The present study aimed to evaluate the mortality, reactive oxygen species production (ROS and total hemocyte counts (THC of the marine shrimp Litopenaeus vannamei infected with the white spot syndrome virus (WSSV at three levels of oxygen saturation. For this, 360 shrimp (20±2g were distributed in 24 tanks (60L, divided in two groups (infected and non-infected, which were subjected to 30, 60 and 100% of dissolved oxygen saturation (in quadruplicate. During 96 hours after infection, daily hemolymph samples were collected for hemato-immunological parameter evaluation (THC and ROS and dead animals were removed and computed to assess cumulative mortality rates. In the infected group, animals subjected to 100% saturation showed higher ROS production (P<0.05 after 48 hours, while THC was significantly reduced (P<0.05, regardless of oxygen saturation. The hypoxia resulted in high mortality when compared to 100% saturation condition. In the uninfected group, no significant differences were observed in all evaluated parameters. Thus, the hypoxia condition increased the susceptibility of shrimp to the infection of WSSV, which may be partly related to the low ROS production showed by the animals subjected to 30% oxygen saturation.

  13. Specific Molecular Signatures for Type II Crustins in Penaeid Shrimp Uncovered by the Identification of Crustin-Like Antimicrobial Peptides in Litopenaeus vannamei

    Barreto, Cairé; Coelho, Jaqueline da Rosa; Yuan, Jianbo; Xiang, Jianhai; Perazzolo, Luciane Maria

    2018-01-01

    Crustins form a large family of antimicrobial peptides (AMPs) in crustaceans composed of four sub-groups (Types I-IV). Type II crustins (Type IIa or “Crustins” and Type IIb or “Crustin-like”) possess a typical hydrophobic N-terminal region and are by far the most representative sub-group found in penaeid shrimp. To gain insight into the molecular diversity of Type II crustins in penaeids, we identified and characterized a Type IIb crustin in Litopenaeus vannamei (Crustin-like Lv) and compared Type II crustins at both molecular and transcriptional levels. Although L. vannamei Type II crustins (Crustin Lv and Crustin-like Lv) are encoded by separate genes, they showed a similar tissue distribution (hemocytes and gills) and transcriptional response to the shrimp pathogens Vibrio harveyi and White spot syndrome virus (WSSV). As Crustin Lv, Crustin-like Lv transcripts were found to be present early in development, suggesting a maternal contribution to shrimp progeny. Altogether, our in silico and transcriptional data allowed to conclude that (1) each sub-type displays a specific amino acid signature at the C-terminal end holding both the cysteine-rich region and the whey acidic protein (WAP) domain, and that (2) shrimp Type II crustins evolved from a common ancestral gene that conserved a similar pattern of transcriptional regulation. PMID:29337853

  14. Interaction of the tick immune system with transmitted pathogens

    Ondrej eHajdusek

    2013-07-01

    Full Text Available Ticks are hematophagous arachnids transmitting a wide variety of pathogens including viruses, bacteria, and protozoans to their vertebrate hosts. The tick vector competence has to be intimately linked to the ability of transmitted pathogens to evade tick defense mechanisms encountered on their route through the tick body comprising midgut, hemolymph, salivary glands or ovaries. Tick innate immunity is, like in other invertebrates, based on an orchestrated action of humoral and cellular immune responses. The direct antimicrobial defense in ticks is accomplished by a variety of small molecules such as defensins, lysozymes or by tick-specific antimicrobial compounds such as microplusin/hebraein or 5.3-kDa family proteins. Phagocytosis of the invading microbes by tick hemocytes seems to be mediated by the primordial complement-like system composed of thioester-containing proteins, fibrinogen-related lectins and convertase-like factors. Moreover, an important role in survival of the ingested microbes seems to be played by host proteins and redox balance maintenance in the tick midgut. Here, we summarize recent knowledge about the major components of tick immune system and focus on their interaction with the relevant tick-transmitted pathogens, represented by spirochetes (Borrelia, rickettsiae (Anaplasma, and protozoans (Babesia. Availability of the tick genomic database and feasibility of functional genomics based on RNA interference greatly contribute to the understanding of molecular and cellular interplay at the tick-pathogen interface and may provide new targets for blocking the transmission of tick pathogens.

  15. Ophryoglena hemophaga n. sp. (Ciliophora: Ophryoglenidae): a parasite of the digestive gland of zebra mussels Dreissena polymorpha.

    Molloy, Daniel P; Lynn, Denis H; Giamberini, Laure

    2005-07-18

    Ophryoglena hemophaga n. sp. is described from a freshwater Dreissena polymorpha population in the Rhine delta of the Netherlands. This is the first ophryoglenine species (order Hymenostomatida, suborder Ophryoglenina) recorded as a molluscan parasite. As is typical of ciliates in the suborder Ophryoglenina, O. hemophaga exhibits a polymorphic life history with cystment and reproduction by palintomy. Trophonts were observed within digestive gland lumina, and zebra mussel hemocytes were present in some of their digestive vacuoles. The presence of a single, longitudinal tract of multiple contractile vacuoles represents its most unique feature and distinguishes it from all other described Ophryoglena spp. The number of somatic kineties of O. hemophaga (range 100 to 124) is also distinguishing as it is one of the lowest for [corrected] an Ophryoglena sp. Other characteristics of this species include: ovoid to elongate trophonts 96 to 288 microm in length, with an elongate macronucleus 41 to 65 microm in length; tomonts 50 to 150 microm in diameter producing a clear mucous cyst envelope, whose thickness is approximately half of the tomont diameter; elongated theronts 96 to 131 microm in length which emerge after 1 to 3 cell divisions taking 36 to 48 h at 20 +/- 3 degrees C. Protomonts and theronts are, respectively, negatively and positively phototactic--characteristics that likely aid in maintenance of infection in zebra mussel populations.

  16. Involvement of apoptosis in host-parasite interactions in the zebra mussel.

    Laëtitia Minguez

    Full Text Available The question of whether cell death by apoptosis plays a biological function during infection is key to understanding host-parasite interactions. We investigated the involvement of apoptosis in several host-parasite systems, using zebra mussels Dreissena polymorpha as test organisms and their micro- and macroparasites. As a stress response associated with parasitism, heat shock proteins (Hsp can be induced. In this protein family, Hsp70 are known to be apoptosis inhibitors. Mussels were diagnosed for their respective infections by standard histological methods; apoptosis was detected using the TUNEL methods on paraffin sections and Hsp70 by immunohistochemistry on cryosections. Circulating hemocytes were the main cells observed in apoptosis whereas infected tissues displayed no or few apoptotic cells. Parasitism by intracellular bacteria Rickettsiales-like and the trematode Bucephalus polymorphus were associated with the inhibition of apoptosis whereas ciliates Ophryoglena spp. or the trematode Phyllodistomum folium did not involve significant differences in apoptosis. Even if some parasites were able to modulate apoptosis in zebra mussels, we did not see evidence of any involvement of Hsp70 on this mechanism.

  17. Effects of Freshwater Pollution on the Genetics of Zebra Mussels (Dreissena polymorpha at the Molecular and Population Level

    Emilia G. Thomas

    2014-01-01

    Full Text Available Revealing long-term effects of contaminants on the genetic structure of organisms inhabiting polluted environments should encompass analyses at the population, molecular, and cellular level. Following this concept, we studied the genetic constitution of zebra mussel populations from a polluted (Dp and reference sites (Cl at the river Drava, Croatia, and applied microsatellite and DNA damage analyses (Comet assay, micronucleus test (MNT. Additionally, mussels from both populations were exposed to polluted wastewater in the laboratory for three days, and DNA damage was analyzed to evaluate acclimatization and genetic adaptation of the investigated populations to the polluted environment. The two populations differed in their genetic constitution. Microsatellite analysis suggested that Dp had undergone a genetic bottleneck. Comet assay did not indicate any difference in DNA damage between the two populations, but MNT revealed that Dp had an increased percentage of micronuclei in hemocytes in comparison to Cl. The laboratory experiment revealed that Dp had a lower percentage of tail DNA and a higher percentage of micronuclei than Cl. These differences between populations were possibly caused by an overall decreased fitness of Dp due to genetic drift and by an enhanced DNA repair mechanism due to acclimatization to pollution in the source habitat.

  18. Effects of freshwater pollution on the genetics of zebra mussels (Dreissena polymorpha) at the molecular and population level.

    Thomas, Emilia G; Srut, Maja; Stambuk, Anamaria; Klobučar, Göran I V; Seitz, Alfred; Griebeler, Eva Maria

    2014-01-01

    Revealing long-term effects of contaminants on the genetic structure of organisms inhabiting polluted environments should encompass analyses at the population, molecular, and cellular level. Following this concept, we studied the genetic constitution of zebra mussel populations from a polluted (Dp) and reference sites (Cl) at the river Drava, Croatia, and applied microsatellite and DNA damage analyses (Comet assay, micronucleus test (MNT)). Additionally, mussels from both populations were exposed to polluted wastewater in the laboratory for three days, and DNA damage was analyzed to evaluate acclimatization and genetic adaptation of the investigated populations to the polluted environment. The two populations differed in their genetic constitution. Microsatellite analysis suggested that Dp had undergone a genetic bottleneck. Comet assay did not indicate any difference in DNA damage between the two populations, but MNT revealed that Dp had an increased percentage of micronuclei in hemocytes in comparison to Cl. The laboratory experiment revealed that Dp had a lower percentage of tail DNA and a higher percentage of micronuclei than Cl. These differences between populations were possibly caused by an overall decreased fitness of Dp due to genetic drift and by an enhanced DNA repair mechanism due to acclimatization to pollution in the source habitat.

  19. Involvement of Apoptosis in Host-Parasite Interactions in the Zebra Mussel

    Minguez, Laëtitia; Brulé, Nelly; Sohm, Bénédicte; Devin, Simon; Giambérini, Laure

    2013-01-01

    The question of whether cell death by apoptosis plays a biological function during infection is key to understanding host-parasite interactions. We investigated the involvement of apoptosis in several host-parasite systems, using zebra mussels Dreissena polymorpha as test organisms and their micro- and macroparasites. As a stress response associated with parasitism, heat shock proteins (Hsp) can be induced. In this protein family, Hsp70 are known to be apoptosis inhibitors. Mussels were diagnosed for their respective infections by standard histological methods; apoptosis was detected using the TUNEL methods on paraffin sections and Hsp70 by immunohistochemistry on cryosections. Circulating hemocytes were the main cells observed in apoptosis whereas infected tissues displayed no or few apoptotic cells. Parasitism by intracellular bacteria Rickettsiales-like and the trematode Bucephalus polymorphus were associated with the inhibition of apoptosis whereas ciliates Ophryoglena spp. or the trematode Phyllodistomum folium did not involve significant differences in apoptosis. Even if some parasites were able to modulate apoptosis in zebra mussels, we did not see evidence of any involvement of Hsp70 on this mechanism. PMID:23785455

  20. Gut immunity in Lepidopteran insects.

    Wu, Kai; Yang, Bing; Huang, Wuren; Dobens, Leonard; Song, Hongsheng; Ling, Erjun

    2016-11-01

    Lepidopteran insects constitute one of the largest fractions of animals on earth, but are considered pests in their relationship with man. Key to the success of this order of insects is its ability to digest food and absorb nutrition, which takes place in the midgut. Because environmental microorganisms can easily enter Lepidopteran guts during feeding, the innate immune response guards against pathogenic bacteria, virus and microsporidia that can be devoured with food. Gut immune responses are complicated by both resident gut microbiota and the surrounding peritrophic membrane and are distinct from immune responses in the body cavity, which depend on the function of the fat body and hemocytes. Due to their relevance to agricultural production, studies of Lepidopteran insect midgut and immunity are receiving more attention, and here we summarize gut structures and functions, and discuss how these confer immunity against different microorganisms. It is expected that increased knowledge of Lepidopteran gut immunity may be utilized for pest biological control in the future. Copyright © 2016 Elsevier Ltd. All rights reserved.

  1. Characterizing the host and symbiont proteomes in the association between the Bobtail squid, Euprymna scolopes, and the bacterium, Vibrio fischeri.

    Tyler R Schleicher

    Full Text Available The beneficial symbiosis between the Hawaiian bobtail squid, Euprymna scolopes, and the bioluminescent bacterium, Vibrio fischeri, provides a unique opportunity to study host/microbe interactions within a natural microenvironment. Colonization of the squid light organ by V. fischeri begins a lifelong association with a regulated daily rhythm. Each morning the host expels an exudate from the light organ consisting of 95% of the symbiont population in addition to host hemocytes and shed epithelial cells. We analyzed the host and symbiont proteomes of adult squid exudate and surrounding light organ epithelial tissue using 1D- and 2D-polyacrylamide gel electrophoresis and multidimensional protein identification technology (MudPIT in an effort to understand the contribution of both partners to the maintenance of this association. These proteomic analyses putatively identified 1581 unique proteins, 870 proteins originating from the symbiont and 711 from the host. Identified host proteins indicate a role of the innate immune system and reactive oxygen species (ROS in regulating the symbiosis. Symbiont proteins detected enhance our understanding of the role of quorum sensing, two-component signaling, motility, and detoxification of ROS and reactive nitrogen species (RNS inside the light organ. This study offers the first proteomic analysis of the symbiotic microenvironment of the adult light organ and provides the identification of proteins important to the regulation of this beneficial association.

  2. Squid-derived chitin oligosaccharides are a chemotactic signal during colonization by Vibrio fischeri.

    Mandel, Mark J; Schaefer, Amy L; Brennan, Caitlin A; Heath-Heckman, Elizabeth A C; Deloney-Marino, Cindy R; McFall-Ngai, Margaret J; Ruby, Edward G

    2012-07-01

    Chitin, a polymer of N-acetylglucosamine (GlcNAc), is noted as the second most abundant biopolymer in nature. Chitin serves many functions for marine bacteria in the family Vibrionaceae ("vibrios"), in some instances providing a physical attachment site, inducing natural genetic competence, and serving as an attractant for chemotaxis. The marine luminous bacterium Vibrio fischeri is the specific symbiont in the light-emitting organ of the Hawaiian bobtail squid, Euprymna scolopes. The bacterium provides the squid with luminescence that the animal uses in an antipredatory defense, while the squid supports the symbiont's nutritional requirements. V. fischeri cells are harvested from seawater during each host generation, and V. fischeri is the only species that can complete this process in nature. Furthermore, chitin is located in squid hemocytes and plays a nutritional role in the symbiosis. We demonstrate here that chitin oligosaccharides produced by the squid host serve as a chemotactic signal for colonizing bacteria. V. fischeri uses the gradient of host chitin to enter the squid light organ duct and colonize the animal. We provide evidence that chitin serves a novel function in an animal-bacterial mutualism, as an animal-produced bacterium-attracting synomone.

  3. Sampling the light-organ microenvironment of Euprymna scolopes: description of a population of host cells in association with the bacterial symbiont Vibrio fischeri.

    Nyholm, S V; McFall-Ngai, M J

    1998-10-01

    The symbiosis between the squid Euprymna scolopes and the luminous bacterium Vibrio fischeri has a pronounced diel rhythm, one component of which is the venting of the contents of the light organ into the surrounding seawater each day at dawn. In this study, we explored the use of this behavior to sample the microenvironment of the light-organ crypts. Intact crypt contents, which emerge from the lateral pores of the organ as a thick paste-like exudate, were collected from anesthetized host animals that had been exposed to a light cue. Microscopy revealed that the expelled material is composed of a conspicuous population of host cells in association with the bacterial symbionts, all of which are embedded in a dense acellular matrix that strongly resembles the bacteria-based biofilms described in other systems. Assays of the viability of expelled crypt cells revealed no dead bacterial symbionts and a mixture of live and dead host cells. Analyses of the ultrastructure, biochemistry, and phagocytic activity of a subset of the host cell population suggested that some of these cells are macrophage-like molluscan hemocytes.

  4. Litopenaeus vannamei sterile-alpha and armadillo motif containing protein (LvSARM is involved in regulation of Penaeidins and antilipopolysaccharide factors.

    Pei-Hui Wang

    Full Text Available The Toll-like receptor (TLR-mediated NF-κB pathway is tightly controlled because overactivation may result in severe damage to the host, such as in the case of chronic inflammatory diseases and cancer. In mammals, sterile-alpha and armadillo motif-containing protein (SARM plays an important role in negatively regulating this pathway. While Caenorhabditis elegans SARM is crucial for an efficient immune response against bacterial and fungal infections, it is still unknown whether Drosophila SARM participates in immune responses. Here, Litopenaeus vannamei SARM (LvSARM was cloned and functionally characterized. LvSARM shared signature domains with and exhibited significant similarities to mammalian SARM. Real-time quantitative PCR analysis indicated that the expression of LvSARM was responsive to Vibrio alginolyticus and white spot syndrome virus (WSSV infections in the hemocyte, gill, hepatopancreas and intestine. In Drosophila S2 cells, LvSARM was widely distributed in the cytoplasm and could significantly inhibit the promoters of the NF-κB pathway-controlled antimicrobial peptide genes (AMPs. Silencing of LvSARM using dsRNA-mediated RNA interference increased the expression levels of Penaeidins and antilipopolysaccharide factors, which are L.vannamei AMPs, and increased the mortality rate after V. alginolyticus infection. Taken together, our results reveal that LvSARM may be a novel component of the shrimp Toll pathway that negatively regulates shrimp AMPs, particularly Penaeidins and antilipopolysaccharide factors.

  5. CO2 sequestration using principles of shell formation

    Lee, Seung-Woo; Jang, Young-Nam [CO2 Sequestration Research Department, Korea Institute of Geoscience and Mineral Resources (Korea, Republic of); Lee, Si-Hyun; Lim, Kyoung-Soo; Jeong, Soon-Kwan [Energy Conservation Research Department of Clean Energy System Research Center, Korea Institute of Energy Research (Korea, Republic of)

    2011-06-15

    The biomimetic sequestration of carbon dioxide to reduce the CO2 emitted into the atmosphere is introduced in this paper. Bivalve shells are used as a good model of CO2 sequestration in this paper, because the shell is derived from the calcium ions and CO2 in seawater. Carbonic anhydrase, hemocyte from diseased shell (HDS) and extrapallial fluid (EFP) are involved in shell formation. This paper compares the soluble protein extracted from Crassostrea gigas with bovine carbonic anhydrase II in terms of their ability to promote CO2 hydration and the production of calcium precipitates. The result demonstrates that HDS has more functional groups to bind calcium ions in aqueous systems, and a different process of calcium precipitation, than does bovine carbonic anhydrase II. To understand molecular weight and secondary protein structure, mass-spectroscopic analysis (MALDI-TOF) and circular dichroism (CD) analysis were used. With regard to EPF, EPF related to shell formation is composed of several fractions and plays a role in sequestration of CO2.

  6. Entomopathogenic Fungi: New Insights into Host-Pathogen Interactions.

    Butt, T M; Coates, C J; Dubovskiy, I M; Ratcliffe, N A

    2016-01-01

    Although many insects successfully live in dangerous environments exposed to diverse communities of microbes, they are often exploited and killed by specialist pathogens. Studies of host-pathogen interactions (HPI) provide valuable insights into the dynamics of the highly aggressive coevolutionary arms race between entomopathogenic fungi (EPF) and their arthropod hosts. The host defenses are designed to exclude the pathogen or mitigate the damage inflicted while the pathogen responds with immune evasion and utilization of host resources. EPF neutralize their immediate surroundings on the insect integument and benefit from the physiochemical properties of the cuticle and its compounds that exclude competing microbes. EPF also exhibit adaptations aimed at minimizing trauma that can be deleterious to both host and pathogen (eg, melanization of hemolymph), form narrow penetration pegs that alleviate host dehydration and produce blastospores that lack immunogenic sugars/enzymes but facilitate rapid assimilation of hemolymph nutrients. In response, insects deploy an extensive armory of hemocytes and macromolecules, such as lectins and phenoloxidase, that repel, immobilize, and kill EPF. New evidence suggests that immune bioactives work synergistically (eg, lysozyme with antimicrobial peptides) to combat infections. Some proteins, including transferrin and apolipophorin III, also demonstrate multifunctional properties, participating in metabolism, homeostasis, and pathogen recognition. This review discusses the molecular intricacies of these HPI, highlighting the interplay between immunity, stress management, and metabolism. Increased knowledge in this area could enhance the efficacy of EPF, ensuring their future in integrated pest management programs. Copyright © 2016 Elsevier Inc. All rights reserved.

  7. Perna perna (LINNAEUS, 1758) mussels irradiated by {sup 60}CO gamma rays cytotoxicity evaluation

    Martini, Gisela A.; Pusceddu, Fabio H.; Rogero, Sizue O.; Rogero, Jose Roberto, E-mail: gisela.martini@usp.br [Instituto de Pesquisas Energeticas e Nucleares (IPEN/CNEN-SP), Sao Paulo, SP (Brazil)

    2013-07-01

    The aim of the present work was the study of ionizing radiation effects on aquatic biota regarding the location of nuclear facilities nearby coastal areas assuming the risk of leaks and nuclear accidents. Bivalve mollusks have been widely used in the monitoring of aquatic environment studies mainly for their sessile habit and pollutants bioconcentration ability. So marine mussel Perna perna (Bivalvia: Mytilidae) was used as organism test in this study. The study of radioactive toxicity was performed by cytotoxicity test exposing the organisms to 11Gy gamma radiation dose. After radiation the neutral red retention assay evaluated the lysosomal membrane integrity in the mussel hemocytes. 50% lethal dose assay (LD50) of gamma radiation on Perna perna mussels was carried out by exposure the organisms to {sup 60}Co gamma rays at doses ranging from 0 to 3000 Gy. The result of gamma radiation LD50 for these mussels was 1068 Gy and the neutral red retention time of irradiated organisms was about 47% lower than the control, non irradiated organisms. With the obtained results is expected to contribute in the study to identify the range of ionizing radiation doses which can cause toxic effects in marine invertebrates. (author)

  8. Analysis of Thioester-Containing Proteins during the Innate Immune Response of Drosophila melanogaster

    Bou Aoun, Richard; Hetru, Charles; Troxler, Laurent; Doucet, Daniel; Ferrandon, Dominique; Matt, Nicolas

    2010-01-01

    Thioester-containing proteins (TEPs) are conserved proteins among insects that are thought to be involved in innate immunity. In Drosophila, the Tep family is composed of 6 genes named Tep1–Tep6. In this study, we investigated the phylogeny, expression pattern and roles of these genes in the host defense of Drosophila. Protostomian Tep genes are clustered in 3 distinct branches, 1 of which is specific to mosquitoes. Most D. melanogaster Tep genes are expressed in hemocytes, can be induced in the fat body, and are expressed in specific regions of the hypodermis. This expression pattern is consistent with a role in innate immunity. However, we find that TEP1, TEP2, and TEP4 are not strictly required in the body cavity to fight several bacterial and fungal infections. One possibility is that Drosophila TEPs act redundantly or that their absence can be compensated by other components of the immune response. TEPs may thus provide a subtle selective advantage during evolution. Alternatively, they may be required in host defense against specific as yet unidentified natural pathogens of Drosophila. PMID:21063077

  9. Quorum-sensing regulator RhlR but not its autoinducer RhlI enables Pseudomonas to evade opsonization.

    Haller, Samantha; Franchet, Adrien; Hakkim, Abdul; Chen, Jing; Drenkard, Eliana; Yu, Shen; Schirmeier, Stefanie; Li, Zi; Martins, Nelson; Ausubel, Frederick M; Liégeois, Samuel; Ferrandon, Dominique

    2018-03-09

    When Drosophila melanogaster feeds on Pseudomonas aeruginosa , some bacteria cross the intestinal barrier and eventually proliferate in the hemocoel. This process is limited by hemocytes through phagocytosis. P. aeruginosa requires the quorum-sensing regulator RhlR to elude the cellular immune response of the fly. RhlI synthesizes the autoinducer signal that activates RhlR. Here, we show that rhlI mutants are unexpectedly more virulent than rhlR mutants, both in fly and in nematode intestinal infection models, suggesting that RhlR has RhlI-independent functions. We also report that RhlR protects P. aeruginosa from opsonization mediated by the Drosophila thioester-containing protein 4 (Tep4). RhlR mutant bacteria show higher levels of Tep4- mediated opsonization, as compared to rhlI mutants, which prevents lethal bacteremia in the Drosophila hemocoel. In contrast, in a septic model of infection, in which bacteria are introduced directly into the hemocoel, Tep4 mutant flies are more resistant to wild-type P. aeruginosa, but not to the rhlR mutant. Thus, depending on the infection route, the Tep4 opsonin can either be protective or detrimental to host defense. © 2018 The Authors.

  10. Effects of waterborne nickel on the physiological and immunological parameters of the Pacific abalone Haliotis discus hannai during thermal stress.

    Min, Eun Young; Cha, Yong-Joo; Kang, Ju-Chan

    2015-09-01

    In this study, the 96-h LC50 at 22 and 26 °C values was 28.591 and 11.761 mg/L, respectively, for NiCl2 exposure in the abalone. The alteration of physiological and immune-toxicological parameters such as the total hemocyte count (THC), lysozyme, phenoloxidase (PO), and phagocytosis activity was measured in the abalone exposed to nickel (200 and 400 μg/L) under thermal stress for 96 h. In this study, Mg and THC decreased, while Ca, lysozyme, PO, and phagocytosis activity increased in the hemolymph of Pacific abalone exposed to NiCl2 when compared to a control at both 22 and 26 °C. However, these parameters were not affected by a rise in temperature from 22 to 26 °C in non-exposed groups. Our results showed that NiCl2 below 400 μg/L was able to stimulate immune responses in abalone. However, complex stressors, thermal changes, or NiCl2 can modify the immunological response and lead to changes in the physiology of host-pollutant interactions in the abalone.

  11. The early stages of the immune response of the European abalone Haliotis tuberculata to a Vibrio harveyi infection.

    Cardinaud, Marion; Dheilly, Nolwenn M; Huchette, Sylvain; Moraga, Dario; Paillard, Christine

    2015-08-01

    Vibrio harveyi is a marine bacterial pathogen responsible for episodic abalone mortalities in France, Japan and Australia. In the European abalone, V. harveyi invades the circulatory system in a few hours after exposure and is lethal after 2 days of infection. In this study, we investigated the responses of European abalone immune cells over the first 24 h of infection. Results revealed an initial induction of immune gene expression including Rel/NF-kB, Mpeg and Clathrin. It is rapidly followed by a significant immuno-suppression characterized by reduced cellular hemocyte parameters, immune response gene expressions and enzymatic activities. Interestingly, Ferritin was overexpressed after 24 h of infection suggesting that abalone attempt to counter V. harveyi infection using soluble effectors. Immune function alteration was positively correlated with V. harveyi concentration. This study provides the evidence that V. harveyi has a hemolytic activity and an immuno-suppressive effect in the European abalone. Copyright © 2015 Elsevier Ltd. All rights reserved.

  12. Gills are an initial target of zinc oxide nanoparticles in oysters Crassostrea gigas, leading to mitochondrial disruption and oxidative stress

    Trevisan, Rafael; Delapedra, Gabriel; Mello, Danielle F.; Arl, Miriam; Schmidt, Éder C.; Meder, Fabian; Monopoli, Marco; Cargnin-Ferreira, Eduardo; Bouzon, Zenilda L.; Fisher, Andrew S.; Sheehan, David; Dafre, Alcir L.

    2014-01-01

    Graphical abstract: - Highlights: • ZnONP exposure causes an initial accumulation of zinc in gills and later in digestive gland. • Zinc burden occurs by ZnONP endocytosis or uptake of ionic zinc after dissociation. • ZnONP exposure disrupts mitochondrial ultrastructure in both tissues. • Mitochondrial damage and oxidative stress are major features of ZnONP acute toxicity. - Abstract: The increasing industrial use of nanomaterials during the last decades poses a potential threat to the environment and in particular to organisms living in the aquatic environment. In the present study, the toxicity of zinc oxide nanoparticles (ZnONP) was investigated in Pacific oysters Crassostrea gigas. The nanoscale of ZnONP, in vehicle or ultrapure water, was confirmed, presenting an average size ranging from 28 to 88 nm. In seawater, aggregation was detected by TEM and DLS analysis, with an increased average size ranging from 1 to 2 μm. Soluble or nanoparticulated zinc presented similar toxicity, displaying a LC 50 (96 h) around 30 mg/L. High zinc dissociation from ZnONP, releasing ionic zinc in seawater, is a potential route for zinc assimilation and ZnONP toxicity. To investigate mechanisms of toxicity, oysters were treated with 4 mg/L ZnONP for 6, 24 or 48 h. ZnONP accumulated in gills (24 and 48 h) and digestive glands (48 h). Ultrastructural analysis of gills revealed electron-dense vesicles near the cell membrane and loss of mitochondrial cristae (6 h). Swollen mitochondria and a more conspicuous loss of mitochondrial cristae were observed after 24 h. Mitochondria with disrupted membranes and an increased number of cytosolic vesicles displaying electron-dense material were observed 48 h post exposure. Digestive gland showed similar changes, but these were delayed relative to gills. ZnONP exposure did not greatly affect thiol homeostasis (reduced and oxidized glutathione) or immunological parameters (phagocytosis, hemocyte viability and activation and total hemocyte

  13. Investigation of toxic effects of imidazolium ionic liquids, [bmim][BF{sub 4}] and [omim][BF{sub 4}], on marine mussel Mytilus galloprovincialis with or without the presence of conventional solvents, such as acetone

    Tsarpali, Vasiliki; Belavgeni, Alexia; Dailianis, Stefanos, E-mail: sdailianis@upatras.gr

    2015-07-15

    Highlights: • The toxic effects of [bmim][BF{sub 4}] and [omim][BF{sub 4}] on mussels were investigated. • Both ILs could induce lethal and nonlethal effects on Mytilus galloprovincialis. • Different extent of IL-mediated adverse effects was observed in mussel hemocytes. • The alkyl chain length and lipophilicity of ILs are crucial for their toxicity. • Acetone influences the oxidative and genotoxic effects of [omim][BF{sub 4}]. - Abstract: This study investigated the cytotoxic, oxidative and genotoxic effects of two commonly used imidazolium ionic liquids (ILs), [bmim][BF{sub 4}] (1-butyl-3-methylimidazolium) and [omim][BF{sub 4}] (1-methyl-3-octylimidazolium tetrafluoroborate), on the marine mussel Mytilus galloprovincialis, as well as whether acetone could mediate their toxic profile. In this context, mussels were firstly exposed to different concentrations of [bmim][BF{sub 4}] or [omim][BF{sub 4}], with or without the presence of acetone (at a final concentration of 0.06% v/v), for a period of 96 h, in order to determine the concentration that causes 50% mussel mortality (LC{sub 50} values) in each case. Thereafter, mussels were exposed to sub- and non-lethal concentrations of ILs for investigating their ability to cause lysosomal membrane impairment (with the use of neutral red retention assay/NRRT), superoxide anion and lipid peroxidation byproduct (malondialdehyde/MDA) formation, as well as DNA damage and formation of nuclear abnormalities in hemocytes. The results showed that [omim][BF{sub 4}] was more toxic than [bmim][BF{sub 4}] in all cases, while the presence of acetone resulted in a slight attenuation of its toxicity. The different toxic behavior of ILs was further revealed by the significantly lower levels of NRRT values observed in [omim][BF{sub 4}]-treated mussels, compared to those occurring in [bmim][BF{sub 4}] in all cases. Similarly, [bmim][BF{sub 4}]-mediated oxidative and genotoxic effects were observed only in the highest

  14. Gills are an initial target of zinc oxide nanoparticles in oysters Crassostrea gigas, leading to mitochondrial disruption and oxidative stress

    Trevisan, Rafael; Delapedra, Gabriel; Mello, Danielle F.; Arl, Miriam [Department of Biochemistry, Federal University of Santa Catarina, 88040-900 Florianópolis, SC (Brazil); Schmidt, Éder C. [Department of Cell Biology, Embryology and Genetic, Federal University of Santa Catarina, 88049-900 Florianópolis, SC (Brazil); Meder, Fabian; Monopoli, Marco [Centre for Bionano Interactions, University College Dublin, Dublin (Ireland); Cargnin-Ferreira, Eduardo [Federal Institute of Santa Catarina, Campus Garopaba, Laboratory of Histological Markers, 88495-000 Garopaba, SC (Brazil); Bouzon, Zenilda L. [Department of Cell Biology, Embryology and Genetic, Federal University of Santa Catarina, 88049-900 Florianópolis, SC (Brazil); Fisher, Andrew S. [School of Geography, Earth and Environmental Sciences, University of Plymouth, PL4 8AA Plymouth (United Kingdom); Sheehan, David [Department of Biochemistry, University College Cork, Cork (Ireland); Dafre, Alcir L., E-mail: alcir.dafre@ufsc.br [Department of Biochemistry, Federal University of Santa Catarina, 88040-900 Florianópolis, SC (Brazil)

    2014-08-15

    Graphical abstract: - Highlights: • ZnONP exposure causes an initial accumulation of zinc in gills and later in digestive gland. • Zinc burden occurs by ZnONP endocytosis or uptake of ionic zinc after dissociation. • ZnONP exposure disrupts mitochondrial ultrastructure in both tissues. • Mitochondrial damage and oxidative stress are major features of ZnONP acute toxicity. - Abstract: The increasing industrial use of nanomaterials during the last decades poses a potential threat to the environment and in particular to organisms living in the aquatic environment. In the present study, the toxicity of zinc oxide nanoparticles (ZnONP) was investigated in Pacific oysters Crassostrea gigas. The nanoscale of ZnONP, in vehicle or ultrapure water, was confirmed, presenting an average size ranging from 28 to 88 nm. In seawater, aggregation was detected by TEM and DLS analysis, with an increased average size ranging from 1 to 2 μm. Soluble or nanoparticulated zinc presented similar toxicity, displaying a LC{sub 50} (96 h) around 30 mg/L. High zinc dissociation from ZnONP, releasing ionic zinc in seawater, is a potential route for zinc assimilation and ZnONP toxicity. To investigate mechanisms of toxicity, oysters were treated with 4 mg/L ZnONP for 6, 24 or 48 h. ZnONP accumulated in gills (24 and 48 h) and digestive glands (48 h). Ultrastructural analysis of gills revealed electron-dense vesicles near the cell membrane and loss of mitochondrial cristae (6 h). Swollen mitochondria and a more conspicuous loss of mitochondrial cristae were observed after 24 h. Mitochondria with disrupted membranes and an increased number of cytosolic vesicles displaying electron-dense material were observed 48 h post exposure. Digestive gland showed similar changes, but these were delayed relative to gills. ZnONP exposure did not greatly affect thiol homeostasis (reduced and oxidized glutathione) or immunological parameters (phagocytosis, hemocyte viability and activation and total

  15. Molecular identification and functional analysis of two variants of myeloid differentiation factor 88 (MyD88) from disk abalone (Haliotis discus discus).

    Priyathilaka, Thanthrige Thiunuwan; Bathige, S D N K; Lee, Seongdo; Lee, Jehee

    2018-02-01

    Myeloid differentiation factor 88 (MyD88) is a crucial adaptor protein of the Toll-like receptor (TLR)- and interleukin 1 receptor-mediated signaling pathways and is involved in a diverse array of inflammatory responses via NF-κB activation. In the present study, two MyD88 variants were identified from disk abalone (Haliotis discus discus) and designated AbMyD88-2 and AbMyD88-X. The deduced AbMyD88-2 and AbMyD88-X comprised 433 and 354 amino acids with predicted molecular masses of 48.85 kDa and 40.17 kDa, respectively. AbMyD88-2 and AbMyD88-X possessed typical MyD88 domain structural features including an N-terminal death domain (DD) and C-terminal toll interleukin 1 receptor (TIR) domain similar to those in mammals. Expression analysis of AbMyD88-2 and AbMyD88-X mRNA at different early embryonic developmental stages of abalone by qPCR revealed that their constitutive expression at all developmental stages analyzed with the considerably higher values at the 16-cell (AbMyD88-2) and morula stages (AbMyD88-X). In unchallenged disk abalones, AbMyD88-2 was highly expressed in muscles, while AbMyD88-X mRNA was predominantly transcribed in hemocytes. Moreover, AbMyD88-2 and AbMyD88-X mRNA were differentially modulated in abalone hemocytes after a challenge with live bacteria (Vibrio parahaemolyticus, Listeria monocytogenes), virus (viral hemorrhagic septicemia virus), and pathogen-associated molecular patterns (lipopolysaccharides and Poly I:C). Overexpression of AbMyD88-2 and AbMyD88-X in HEK293T cells induced the activation of the NF-κB promoter. AbMyD88-2 and AbMyD88-X involvement in inflammatory responses was characterized by their overexpression in RAW264.7 murine macrophage cells. These results revealed comparatively higher NO (Nitric oxide) production, induction of inflammatory mediator genes (iNOS and COX2), and proinflammatory genes (IL1β, IL6 and TNFα) expression in abalone MyD88s-overexpressing cells than in mock control in the presence or absence of LPS

  16. Sea urchin coelomocytes are resistant to a variety of DNA damaging agents

    Loram, Jeannette; Raudonis, Renee; Chapman, Jecar; Lortie, Mae [Bermuda Institute of Ocean Sciences, St. George' s, Bermuda, GE 01 (Bermuda); Bodnar, Andrea, E-mail: andrea.bodnar@bios.edu [Bermuda Institute of Ocean Sciences, St. George' s, Bermuda, GE 01 (Bermuda)

    2012-11-15

    Increasing anthropogenic activities are creating environmental pressures that threaten marine ecosystems. Effective environmental health assessment requires the development of rapid, sensitive, and cost-effective tools to predict negative impacts at the individual and ecosystem levels. To this end, a number of biological assays using a variety of cells and organisms measuring different end points have been developed for biomonitoring programs. The sea urchin fertilization/development test has been useful for evaluating environmental toxicology and it has been proposed that sea urchin coelomocytes represent a novel cellular biosensor of environmental stress. In this study we investigated the sensitivity of coelomocytes from the sea urchin Lytechinus variegatus to a variety of DNA-damaging agents including ultraviolet (UV) radiation, hydrogen peroxide (H{sub 2}O{sub 2}), methylmethane sulfonate (MMS) and benzo[a]pyrene (BaP). LD{sub 50} values determined for coelomocytes after 24 h of exposure to these DNA damaging agents indicated a high level of resistance to all treatments. Significant increases in the formation of apurinic/apyrimidinic (AP or abasic) sites in DNA were only detected using high doses of H{sub 2}O{sub 2}, MMS and UV radiation. Comparison of sea urchin coelomocytes with hemocytes from the gastropod mollusk Aplysia dactylomela and the decapod crustacean Panulirus argus indicated that sensitivity to different DNA damaging agents varies between species. The high level of resistance to genotoxic agents suggests that DNA damage may not be an informative end point for environmental health assessment using sea urchin coelomocytes however, natural resistance to DNA damaging agents may have implications for the occurrence of neoplastic disease in these animals.

  17. Insecticidal activity of the metalloprotease AprA occurs through suppression of host cellular and humoral immunity.

    Lee, Seung Ah; Jang, Seong Han; Kim, Byung Hyun; Shibata, Toshio; Yoo, Jinwook; Jung, Yunjin; Kawabata, Shun-Ichiro; Lee, Bok Luel

    2018-04-01

    The biochemical characterization of virulence factors from entomopathogenic bacteria is important to understand entomopathogen-insect molecular interactions. Pseudomonas entomophila is a typical entomopathogenic bacterium that harbors virulence factors against several insects. However, the molecular actions of these factors against host innate immune responses are not clearly elucidated. In this study, we observed that bean bugs (Riptortus pedestris) that were injected with P. entomophila were highly susceptible to this bacterium. To determine how P. entomophila counteracts the host innate immunity to survive within the insect, we purified a highly enriched protein with potential host insect-killing activity from the culture supernatant of P. entomophila. Then, a 45-kDa protein was purified to homogeneity and identified as AprA which is an alkaline zinc metalloprotease of the genus Pseudomonas by liquid chromatography mass spectrometry (LC-MS). Purified AprA showed a pronounced killing effect against host insects and suppressed both host cellular and humoral innate immunity. Furthermore, to show that AprA is an important insecticidal protein of P. entomophila, we used an aprA-deficient P. entomophila mutant strain (ΔaprA). When ΔaprA mutant cells were injected to host insects, this mutant exhibited extremely attenuated virulence. In addition, the cytotoxicity against host hemocytes and the antimicrobial peptide-degrading ability of the ΔaprA mutant were greatly decreased. These findings suggest that AprA functions as an important insecticidal protein of P. entomophila via suppression of host cellular and humoral innate immune responses. Copyright © 2017 Elsevier Ltd. All rights reserved.

  18. Hydroxyapatite growth induced by native extracellular matrix deposition on solid surfaces

    Pramatarova L.

    2005-02-01

    Full Text Available Biological systems have a remarkable capability to produce perfect fine structures such as seashells, pearls, bones, teeth and corals. These structures are composites of interacting inorganic (calcium phosphate or carbonate minerals and organic counterparts. It is difficult to say with certainty which part has the primary role. For example, the growth of molluscan shell crystals is thought to be initiated from a solution by the extracellular organic matrix (ECM. According to this theory, the matrix induces nucleation of calcium containing crystals. Recently, an alternative theory has been put forward, stating that a class of granulocytic hemocytes would be directly involved in shell crystal production in oysters. In the work presented here the surface of AISI 316 stainless steel was modified by deposition of ECM proteins. The ability of the modified substrates to induce nucleation and growth of hydroxyapatite (HA from simulated body fluid (SBF was examined by a kinetic study using two methods: (1 a simple soaking process in SBF and (2 a laser-liquid-solid interaction (LLSI process which allows interaction between a scanning laser beam and a solid substrate immersed in SBF. The deposited HA layers were investigated by Fourier transform infrared spectroscopy (FTIR and scanning electron microscopy (SEM. It was found that a coating of stainless steel surface with native ECM proteins induced nucleation and growth of HA and facilitated its crystallization. By the process of simple soaking of the samples, irrespective of their horizontal or vertical position in the solution, HA layers were grown due to the reactive ECM-coated stainless steel surface. It was shown that the process occurring in the first stages of the growth was not only a result of the force of gravity. The application of the LLSI process strongly influenced HA formation on the ECM-modified substrates by promoting and enhancing the HA nucleation and growth through a synergistic effect

  19. bantam miRNA is important for Drosophila blood cell homeostasis and a regulator of proliferation in the hematopoietic progenitor niche

    Lam, Victoria; Tokusumi, Tsuyoshi; Tokusumi, Yumiko; Schulz, Robert A., E-mail: rschulz@nd.edu

    2014-10-24

    Highlights: • bantam miRNA is endogenously expressed in the hematopoietic progenitor niche. • bantam is necessary and sufficient to induce cellular proliferation in the PSC. • bantam is upstream of the Insulin Receptor signaling pathway. • A model for positive regulation of hematopoietic niche growth is proposed. - Abstract: The Drosophila hematopoietic system is utilized in this study to gain novel insights into the process of growth control of the hematopoietic progenitor niche in blood development. The niche microenvironment is an essential component controlling the balance between progenitor populations and differentiated, mature blood cells and has been shown to lead to hematopoietic malignancies in humans when misregulated. MicroRNAs are one class of regulators associated with blood malignancies; however, there remains a relative paucity of information about the role of miRNAs in the niche. Here we demonstrate that bantam miRNA is endogenously active in the Drosophila hematopoietic progenitor niche, the posterior signaling center (PSC), and functions in the primary hematopoietic organ, the lymph gland, as a positive regulator of growth. Loss of bantam leads to a significant reduction in the PSC and overall lymph gland size, as well as a loss of the progenitor population and correlative premature differentiation of mature hemocytes. Interestingly, in addition to being essential for proper lymph gland development, we have determined bantam to be a novel upstream component of the insulin signaling cascade in the PSC and have unveiled dMyc as one factor central to bantam activity. These important findings identify bantam as a new hematopoietic regulator, place it in an evolutionarily conserved signaling pathway, present one way in which it is regulated, and provide a mechanism through which it facilitates cellular proliferation in the hematopoietic niche.

  20. Sea urchin coelomocytes are resistant to a variety of DNA damaging agents

    Loram, Jeannette; Raudonis, Renee; Chapman, Jecar; Lortie, Mae; Bodnar, Andrea

    2012-01-01

    Increasing anthropogenic activities are creating environmental pressures that threaten marine ecosystems. Effective environmental health assessment requires the development of rapid, sensitive, and cost-effective tools to predict negative impacts at the individual and ecosystem levels. To this end, a number of biological assays using a variety of cells and organisms measuring different end points have been developed for biomonitoring programs. The sea urchin fertilization/development test has been useful for evaluating environmental toxicology and it has been proposed that sea urchin coelomocytes represent a novel cellular biosensor of environmental stress. In this study we investigated the sensitivity of coelomocytes from the sea urchin Lytechinus variegatus to a variety of DNA-damaging agents including ultraviolet (UV) radiation, hydrogen peroxide (H 2 O 2 ), methylmethane sulfonate (MMS) and benzo[a]pyrene (BaP). LD 50 values determined for coelomocytes after 24 h of exposure to these DNA damaging agents indicated a high level of resistance to all treatments. Significant increases in the formation of apurinic/apyrimidinic (AP or abasic) sites in DNA were only detected using high doses of H 2 O 2 , MMS and UV radiation. Comparison of sea urchin coelomocytes with hemocytes from the gastropod mollusk Aplysia dactylomela and the decapod crustacean Panulirus argus indicated that sensitivity to different DNA damaging agents varies between species. The high level of resistance to genotoxic agents suggests that DNA damage may not be an informative end point for environmental health assessment using sea urchin coelomocytes however, natural resistance to DNA damaging agents may have implications for the occurrence of neoplastic disease in these animals.

  1. Cytotoxic effects of gamma radiation and DEET on Perna perna mussels

    Martini, Gisela A.; Rogero, Sizue O.; Silva, Marina V.; Rogero, Jose Roberto, E-mail: gisela.martini@usp.br [Instituto de Pesquisas Energeticas e Nucleares (IPEN/CNEN-SP), Sao Paulo, SP (Brazil)

    2015-07-01

    Aiming at the protection of aquatic biota becomes desirable progress in identifying and quantifying the presence of chemical contaminants to an evaluation of its bioavailability and harmful effects to the aquatic ecosystem. Diethyltoluamide (DEET) is one of the most commonly used active ingredients in insect repellents and is considered an emergent contaminant. In addition the environment in regions near nuclear facilities (mostly coastal) and in regions with higher level of background radiation, many aquatic organisms could be exposed to radiation, becoming more susceptible to other contaminants. Therefore, the purpose of this study was the investigation of the biological effects of ionizing radiation in combination with the exposure of Perna perna mussels to DEET. Previously it was determined the doses of {sup 60}Co gamma radiation (3 and 107 Gy) and the DEET concentrations (0.02 and 0.001mg L-1) which causes cytotoxicity in lysosomes of Perna perna mussels hemocytes. The effects were evaluated by the neutral red retention time assay in irradiated and non-irradiated organisms. The assays were performed in 72 hours, with readings on each 24 hours. The endpoint was the lysosome membrane disruption causing the decrease of the neutral red retention time observed . The results showed that was no statistic difference of irradiated organisms with 3 Gy and exposed to different concentrations of DEET in comparison with non-irradiated organisms, the mean neutral red retention time in assays was around 40 minutes. The organisms that were exposed to DEET after 107 Gy irradiation dose showed cell lysis and neutral red retention time less than 30 minutes, this suggests that synergistic effect can be observed depending on the irradiated dose and the DEET concentration. (author)

  2. Do mollusks use vertebrate sex steroids as reproductive hormones? II. Critical review of the evidence that steroids have biological effects.

    Scott, Alexander P

    2013-02-01

    In assessing the evidence as to whether vertebrate sex steroids (e.g. testosterone, estradiol, progesterone) have hormonal actions in mollusks, ca. 85% of research papers report at least one biological effect; and 18 out of 21 review papers (published between 1970 and 2012) express a positive view. However, just under half of the research studies can be rejected on the grounds that they did not actually test steroids, but compounds or mixtures that were only presumed to behave as steroids (or modulators of steroids) on the basis of their effects in vertebrates (e.g. Bisphenol-A, nonylphenol and sewage treatment effluents). Of the remaining 55 papers, some can be criticized for having no statistical analysis; some for using only a single dose of steroid; others for having irregular dose-response curves; 40 out of the 55 for not replicating the treatments; and 50 out of 55 for having no within-study repetition. Furthermore, most studies had very low effect sizes in comparison to fish-based bioassays for steroids (i.e. they had a very weak 'signal-to-noise' ratio). When these facts are combined with the fact that none of the studies were conducted with rigorous randomization or 'blinding' procedures (implying the possibility of 'operator bias') one must conclude that there is no indisputable bioassay evidence that vertebrate sex steroids have endocrinological or reproductive roles in mollusks. The only observation that has been independently validated is the ability of estradiol to trigger rapid (1-5 min) lysosomal membrane breakdown in hemocytes of Mytilus spp. This is a typical 'inflammatory' response, however, and is not proof that estradiol is a hormone - especially when taken in conjunction with the evidence (discussed in a previous review) that mollusks have neither the enzymes necessary to synthesize vertebrate steroids nor nuclear receptors with which to respond to them. Crown Copyright © 2012. Published by Elsevier Inc. All rights reserved.

  3. Depletion of autophagy-related genes ATG3 and ATG5 in Tenebrio molitor leads to decreased survivability against an intracellular pathogen, Listeria monocytogenes.

    Tindwa, Hamisi; Jo, Yong Hun; Patnaik, Bharat Bhusan; Noh, Mi Young; Kim, Dong Hyun; Kim, Iksoo; Han, Yeon Soo; Lee, Yong Seok; Lee, Bok Luel; Kim, Nam Jung

    2015-01-01

    Macroautophagy (autophagy) is an evolutionarily conserved catabolic process involved in physiological and developmental processes including cell survival, death, and innate immunity. Homologues of most of 36 originally discovered autophagy-related (ATG) genes in yeast have been characterized in higher eukaryotes including insects. In this study, the homologues of ATG3 (TmATG3) and ATG5 (TmATG5) were isolated from the coleopteran beetle, Tenebrio molitor by expressed sequence tag and RNAseq approaches. The cDNA of TmATG3 and TmATG5 comprise open-reading frame sizes of 963 and 792 bp encoding polypeptides of 320 and 263 amino acid residues, respectively. TmATG3 and TmATG5 mRNA are expressed in all developmental stages, and mainly in fat body and hemocytes of larvae. TmATG3 and TmATG5 showed an overall sequence identity of 58-95% to other insect Atg proteins. There exist clear one-to-one orthologs of TmATG3 and TmATG5 in Tribolium and that they clustered together in the gene tree. Depletion of TmATG3 and TmATG5 by RNA interference led to a significant reduction in survival ability of T. molitor larvae against an intracellular pathogen, Listeria monocytogenes. Six days post-Listeria challenge, the survival rate in the dsEGFP-injected (where EGFP is enhanced green fluorescent protein) control larvae was significantly higher (55%) compared to 4 and 3% for TmATG3 and TmATG5 double-stranded RNA injected larvae, respectively. These data suggested that TmATG3 and TmATG5 may play putative role in mediating autophagy-based clearance of Listeria in T. molitor model. © 2014 Wiley Periodicals, Inc.

  4. Characterization of the secretomes of two vibrios pathogenic to mollusks.

    Stéphanie Madec

    Full Text Available Vibrio tapetis causes the brown ring disease in the Japanese clam Ruditapes philippinarum while Vibrio aestuarianus is associated with massive oyster mortalities. As extracellular proteins are often associated with the virulence of pathogenic bacteria, we undertook a proteomic approach to characterize the secretomes of both vibrios. The extracellular proteins (ECPs of both species were fractionated by SEC-FPLC and in vitro assays were performed to measure the effects of each fraction on hemocyte cellular parameters (phagocytosis and adhesion. Fractions showing a significant effect were subjected to SDS-PAGE, and proteins were identified by nano LC-MS/MS. 45 proteins were identified for V. aestuarianus and 87 for V. tapetis. Most of them belonged to outer membrane or were periplasmic, including porins or adhesins that were already described as virulence factors in other bacterial species. Others were transporter components, flagella proteins, or proteins of unknown function (14 and 15 respectively. Interestingly, for V. aestuarianus, we noted the secretion of 3 extracellular enzymes including the Vam metalloprotease and two other enzymes (one putative lipase and one protease. For V. tapetis, we identified five extracellular enymes, i.e. two different endochitinases, one protease, one lipase and an adhesin. A comparison of both secretomes also showed that only the putative extracellular lipase was common to both secretomes, underscoring the difference in pathogenicity mechanisms between these two species. Overall, these results characterize for the first time the secretomes of these two marine pathogenic vibrios and constitute a useful working basis to further analyze the contribution of specific proteins in the virulence mechanisms of these species.

  5. Cytotoxic effects of gamma radiation and DEET on Perna perna mussels

    Martini, Gisela A.; Rogero, Sizue O.; Silva, Marina V.; Rogero, Jose Roberto

    2015-01-01

    Aiming at the protection of aquatic biota becomes desirable progress in identifying and quantifying the presence of chemical contaminants to an evaluation of its bioavailability and harmful effects to the aquatic ecosystem. Diethyltoluamide (DEET) is one of the most commonly used active ingredients in insect repellents and is considered an emergent contaminant. In addition the environment in regions near nuclear facilities (mostly coastal) and in regions with higher level of background radiation, many aquatic organisms could be exposed to radiation, becoming more susceptible to other contaminants. Therefore, the purpose of this study was the investigation of the biological effects of ionizing radiation in combination with the exposure of Perna perna mussels to DEET. Previously it was determined the doses of 60 Co gamma radiation (3 and 107 Gy) and the DEET concentrations (0.02 and 0.001mg L-1) which causes cytotoxicity in lysosomes of Perna perna mussels hemocytes. The effects were evaluated by the neutral red retention time assay in irradiated and non-irradiated organisms. The assays were performed in 72 hours, with readings on each 24 hours. The endpoint was the lysosome membrane disruption causing the decrease of the neutral red retention time observed . The results showed that was no statistic difference of irradiated organisms with 3 Gy and exposed to different concentrations of DEET in comparison with non-irradiated organisms, the mean neutral red retention time in assays was around 40 minutes. The organisms that were exposed to DEET after 107 Gy irradiation dose showed cell lysis and neutral red retention time less than 30 minutes, this suggests that synergistic effect can be observed depending on the irradiated dose and the DEET concentration. (author)

  6. An evaluation of replacing fish meal with fermented soybean meal in the diet of Macrobrachium nipponense: Growth, nonspecific immunity, and resistance to Aeromonas hydrophila.

    Ding, Zhili; Zhang, Yixiang; Ye, Jinyun; Du, Zhenyu; Kong, Youqin

    2015-05-01

    Partial or complete replacement of fish meal (FM) with fermented soybean meal (FSM) was examined in Macrobrachium nipponense over an 8-week growth trial. Growth and immune characteristics were evaluated. Fermented soybean meal replaced 0 (FM, control), 25% (R25), 50% (R50), 75% (R75), or 100% of the FM (R100) in five isocaloric and isonitrogenous diets. Each diet was fed to juvenile prawns (mean weight, 0.103 ± 0.0009 g) twice daily to apparent satiation in five replicates. Weight gain and specific growth rate of M. nipponense were significantly higher in prawns fed the R25 diet than that of prawns fed the FM diet. No significant differences were observed among the other treatments. Total hemocyte count and hemolymph phagocytic activity decreased as the proportion of FSM increased. Total antioxidant activity competence and malondialdehyde level in the hepatopancreas were highest in prawns fed the R100 diet. mRNA levels of the antioxidant genes Cu-Zn superoxide dismutase and catalase, heat shock cognate protein 70, and heat shock protein 90 were significantly differentially regulated in the prawn hepatopancreas. In addition, percent mortality increased after challenge with live Aeromonas hydrophila. Percent mortality of prawns fed the R100 diet was significantly higher than that of prawns fed the FM and R25 diets. These findings demonstrate that (1) M. nipponense growth performance was not affected by including a high proportion of FSM in the diet, and the best growth performance was obtained when 25% of the FM was replaced with FSM; (2) nonspecific immunity was impaired when all of the FM was replaced with FSM. Copyright © 2015 Elsevier Ltd. All rights reserved.

  7. Toxicity assessment and comparison between two types of iron oxide nanoparticles in Mytilus galloprovincialis

    Taze, Chrysa; Panetas, Ioannis [Laboratory of Animal Physiology, Department of Zoology, School of Biology, Aristotle University of Thessaloniki, 54124 Thessaloniki (Greece); Kalogiannis, Stavros [Alexander Technological Educational Institution of Thessaloniki, Department of Nutrition and Dietetics, Thessaloniki (Greece); Feidantsis, Konstantinos [Laboratory of Animal Physiology, Department of Zoology, School of Biology, Aristotle University of Thessaloniki, 54124 Thessaloniki (Greece); Gallios, George P. [Laboratory of General & Inorganic Chemical Technology, School of Chemistry, Aristotle University, GR-54124 Thessaloniki (Greece); Kastrinaki, Georgia [Aerosol & Particle Technology Laboratory, CERTH/CPERI, P.O. Box 60361, 57001 Thessaloniki (Greece); Konstandopoulos, Athanasios G. [Aerosol & Particle Technology Laboratory, CERTH/CPERI, P.O. Box 60361, 57001 Thessaloniki (Greece); Department of Chemical Engineering, Aristotle University, PO. Box 1517, 54006 Thessaloniki (Greece); Václavíková, Miroslava; Ivanicova, Lucia [Institute of Geotechnics, Slovak Academy of Sciences, Watsonova 45, SK-04001 Kosice (Slovakia); Kaloyianni, Martha, E-mail: kaloyian@bio.auth.gr [Laboratory of Animal Physiology, Department of Zoology, School of Biology, Aristotle University of Thessaloniki, 54124 Thessaloniki (Greece)

    2016-03-15

    Highlights: • The impact of two types of iron oxide nanoparticles on the physiological status of mussels was studied. • Oxidative parameters significantly changed after 1, 3, 7 days of exposure. • The nanoparticles induced oxidative stress to the animals. • All the parameters measured could be applied in biomonitoring studies. - Abstract: Nanoparticles (NPs), due to their increased application and production, are being released into the environment with unpredictable impact on the physiology of marine organisms, as well as on entire ecosystems and upcoming effects on human health. The aim of the present study was to evaluate and compare the oxidative responses of the mussel Mytilus galloprovincialis after exposure to iron oxide NPs and to iron oxide NPs incorporated into zeolite for 1, 3 and 7 days. Our results showed that both effectors induced changes on animal physiology by causing oxidative stress in hemocytes of exposed mussels compared to control animals. This was shown by the significant increase in reactive oxygen species (ROS) production, protein carbonylation, lipid peroxidation, ubiquitin conjugates and DNA damage. In addition an increase in prooxidant levels as measured by the prooxidant-antioxidant balance (PAB) assay was observed in exposed mussels’ hemolymph. The results show that ROS, DNA damage, protein and lipid oxidation, ubiquitin conjugates and PAB could constitute, after further investigation, reliable biomarkers for the evaluation of pollution or other environmental stressors. In addition, more studies are needed in order to ensure the safety of these NPs on various biomedical applications, since it is critical to design NPs that they meet the demands of application without causing cellular toxicity.

  8. A novel C-type lysozyme from Mytilus galloprovincialis: insight into innate immunity and molecular evolution of invertebrate C-type lysozymes.

    Qing Wang

    Full Text Available A c-type lysozyme (named as MgCLYZ gene was cloned from the mussel Mytilus galloprovincialis. Blast analysis indicated that MgCLYZ was a salivary c-type lysozyme which was mainly found in insects. The nucleotide sequence of MgCLYZ was predicted to encode a polypeptide of 154 amino acid residues with the signal peptide comprising the first 24 residues. The deduced mature peptide of MgCLYZ was of a calculated molecular weight of 14.4 kD and a theoretical isoelectric point (pI of 8.08. Evolution analysis suggested that bivalve branch of the invertebrate c-type lysozymes phylogeny tree underwent positive selection during evolution. By quantitative real-time RT-PCR (qRT-PCR analysis, MgCLYZ transcript was widely detected in all examined tissues and responded sensitively to bacterial challenge in hemocytes and hepatopancreas. The optimal temperature and pH of recombinant MgCLYZ (rMgCLYZ were 20°C and 4, respectively. The rMgCLYZ displayed lytic activities against Gram-positive bacteria including Micrococcus luteus and Staphyloccocus aureus, and Gram-negative bacteria including Vibrio anguillarum, Enterobacter cloacae, Pseudomonas putida, Proteus mirabilis and Bacillus aquimaris. These results suggest that MgCLYZ perhaps play an important role in innate immunity of M. galloprovincialis, and invertebrate c-type lysozymes might be under positive selection in a species-specific manner during evolution for undergoing adaptation to different environment and diverse pathogens.

  9. Next-generation sequencing-based transcriptome analysis of Helicoverpa armigera Larvae immune-primed with Photorhabdus luminescens TT01.

    Zengyang Zhao

    Full Text Available Although invertebrates are incapable of adaptive immunity, immunal reactions which are functionally similar to the adaptive immunity of vertebrates have been described in many studies of invertebrates including insects. The phenomenon was termed immune priming. In order to understand the molecular mechanism of immune priming, we employed Illumina/Solexa platform to investigate the transcriptional changes of the hemocytes and fat body of Helicoverpa armigera larvae immune-primed with the pathogenic bacteria Photorhabdus luminescens TT01. A total of 43.6 and 65.1 million clean reads with 4.4 and 6.5 gigabase sequence data were obtained from the TT01 (the immune-primed and PBS (non-primed cDNA libraries and assembled into 35,707 all-unigenes (non-redundant transcripts, which has a length varied from 201 to 16,947 bp and a N50 length of 1,997 bp. For 35,707 all-unigenes, 20,438 were functionally annotated and 2,494 were differentially expressed after immune priming. The differentially expressed genes (DEGs are mainly related to immunity, detoxification, development and metabolism of the host insect. Analysis on the annotated immune related DEGs supported a hypothesis that we proposed previously: the immune priming phenomenon observed in H. armigera larvae was achieved by regulation of key innate immune elements. The transcriptome profiling data sets (especially the sequences of 1,022 unannotated DEGs and the clues (such as those on immune-related signal and regulatory pathways obtained from this study will facilitate immune-related novel gene discovery and provide valuable information for further exploring the molecular mechanism of immune priming of invertebrates. All these will increase our understanding of invertebrate immunity which may provide new approaches to control insect pests or prevent epidemic of infectious diseases in economic invertebrates in the future.

  10. Feminizing Wolbachia: a transcriptomics approach with insights on the immune response genes in Armadillidium vulgare

    Chevalier Frédéric

    2012-01-01

    Full Text Available Abstract Background Wolbachia are vertically transmitted bacteria known to be the most widespread endosymbiont in arthropods. They induce various alterations of the reproduction of their host, including feminization of genetic males in isopod crustaceans. In the pill bug Armadillidium vulgare, the presence of Wolbachia is also associated with detrimental effects on host fertility and lifespan. Deleterious effects have been demonstrated on hemocyte density, phenoloxidase activity, and natural hemolymph septicemia, suggesting that infected individuals could have defective immune capacities. Since nothing is known about the molecular mechanisms involved in Wolbachia-A. vulgare interactions and its secondary immunocompetence modulation, we developed a transcriptomics strategy and compared A. vulgare gene expression between Wolbachia-infected animals (i.e., “symbiotic” animals and uninfected ones (i.e., “asymbiotic” animals as well as between animals challenged or not challenged by a pathogenic bacteria. Results Since very little genetic data is available on A. vulgare, we produced several EST libraries and generated a total of 28 606 ESTs. Analyses of these ESTs revealed that immune processes were over-represented in most experimental conditions (responses to a symbiont and to a pathogen. Considering canonical crustacean immune pathways, these genes encode antimicrobial peptides or are involved in pathogen recognition, detoxification, and autophagy. By RT-qPCR, we demonstrated a general trend towards gene under-expression in symbiotic whole animals and ovaries whereas the same gene set tends to be over-expressed in symbiotic immune tissues. Conclusion This study allowed us to generate the first reference transcriptome ever obtained in the Isopoda group and to identify genes involved in the major known crustacean immune pathways encompassing cellular and humoral responses. Expression of immune-related genes revealed a modulation of host

  11. Melatonin Promotes Cheliped Regeneration, Digestive Enzyme Function, and Immunity Following Autotomy in the Chinese Mitten Crab, Eriocheir sinensis

    Cong Zhang

    2018-03-01

    Full Text Available In the pond culture of juvenile Eriocheir sinensis, a high limb-impairment rate seriously affects the culture success. Therefore, it is particularly important to artificially promote limb regeneration. This study evaluated the effects of melatonin on cheliped regeneration, digestive ability, and immunity, as well as its relationship with the eyestalk. It was found that the injection of melatonin significantly increased the limb regeneration rate compared with the saline group (P < 0.05. The qRT-PCR results of growth-related genes showed that the level of EcR-mRNA (ecdysteroid receptor and Chi-mRNA (chitinase expression was significantly increased following the melatonin injection, while the expression of MIH-mRNA (molt-inhibiting hormone was significantly decreased (P < 0.05. Melatonin significantly increased lipase activity (P < 0.05. We observed that the survival rates of limb-impaired and unilateral eyestalk-ablated crabs were substantially improved following melatonin treatment, whereas the survival of the unilateral eyestalk-ablated crabs was significantly decreased compared with the control group (P < 0.05. Furthermore, the results of serum immune and antioxidant capacity revealed that melatonin significantly increased the total hemocyte counts (THC, hemocyanin content, total antioxidant capacity (T-AOC, acid phosphatase (ACP, and glutathione peroxidase activity (GSH-Px, whereas the immune-related parameters were significantly decreased in eyestalk-ablated crabs (P < 0.05. Therefore, these findings indicate that melatonin exerts a protective effect on organism injury, which could promote limb regeneration by up-regulating the expression of growth-related genes, improve digestive enzyme activity, and strengthen the immune response, particularly antioxidant capacity.

  12. Hepato-Nephrocitic System: A Novel Model of Biomarkers for Analysis of the Ecology of Stress in Environmental Biomonitoring.

    Fábio Camargo Abdalla

    Full Text Available Bombus presents a serious global decline of populations and even loss of species. This phenomenon is complex and multifactorial: environmental degradation due to increasing cultivation and grazing areas, indiscriminate use of agrochemicals, and a plethora of xenobiotics daily discharged in the environment. We proposed that bees have an integrated cell system, which ensures protection against chemical stressors up to a certain limit. Therefore, this hypothesis was tested, exposing workers of Bombus morio to cadmium, a harmful trace metal nowadays widespread in our society. The workers were kept in BOD (26°C, RH 70%, in the dark, fed ad libitum, and divided into a control group (n = 20 and an experimental group (n = 20. For the first group, we offered 2 mL of distilled water; for the experimental groups, 2 mL of cadmium at 1 ppb. In relation to the control group, exposed bees showed that their fat body and hemocytes responded in synchronization with pericardial cells in a topographical and temporal cascade of events, where the fat body is the first barrier against xenobiotics, followed by pericardial cells. The immune cells participate throughout the process. To this system, we proposed the name of hepato-nephrocitic system (HNS, which may explain many phenomena that remain unclear in similar research with Apis mellifera and other species of bees, as shown in this paper. The bee's HNS is a system of highly responsive cells to toxicants, considered a novel parameter for the study of the ecology of stress applied in environmental management.

  13. Histopathological effect and stress response of mantle proteome following TBT exposure in the Hooded oyster Saccostrea cucullata.

    Khondee, Phattirapa; Srisomsap, Chantragan; Chokchaichamnankit, Daranee; Svasti, Jisnuson; Simpson, Richard J; Kingtong, Sutin

    2016-11-01

    Tributyltin (TBT), an environmental pollutant in marine ecosystems, is toxic to organisms. Although contamination by and bioaccumulation and toxicity of this compound have been widely reported, its underlying molecular mechanisms remain unclear. In the present study, we exposed the Hooded oyster Saccostrea cucullata to TBT to investigate histopathological effects and proteome stress response. Animals were exposed to three TBT sub-lethal concentrations, 10, 50 and 150 μg/l for 48 h. TBT produced stress leading to histopathological changes in oyster tissues including mantle, gill, stomach and digestive diverticula. TBT induced mucocyte production in epithelia and hemocyte aggregation in connective tissue. Cell necrosis occurred when exposure dosages were high. Comparative proteome analyses of mantle protein of oysters exposed to 10 μg/l and control animals were analyzed by a 2-DE based proteomic approach. In total, 32 protein spots were found to differ (p TBT induced the expression of proteins involved in defensive mechanisms (HSP-78, HSP-70, aldehyde dehydrogenase and catalase), calcium homeostasis (VDAC-3), cytoskeleton and cytoskeleton-associated proteins, energy metabolism and amino acid metabolism. Our study revealed that TBT disturbs calcium homeostasis via VDAC-3 protein in mantle and this probably is the key molecular mechanism of TBT acting to distort shell calcification. Moreover, proteins involved in cell structure (tubulin-alpha and tubulin-beta) and protein synthesis were reduced after TBT exposure. Additionally, differential proteins obtained from this work will be useful as potential TBT biomarkers. Copyright © 2016 Elsevier Ltd. All rights reserved.

  14. Chemical modulators of the innate immune response alter gypsy moth larval susceptibility to Bacillus thuringiensis

    Broderick Nichole A

    2010-04-01

    Full Text Available Abstract Background The gut comprises an essential barrier that protects both invertebrate and vertebrate animals from invasion by microorganisms. Disruption of the balanced relationship between indigenous gut microbiota and their host can result in gut bacteria eliciting host responses similar to those caused by invasive pathogens. For example, ingestion of Bacillus thuringiensis by larvae of some species of susceptible Lepidoptera can result in normally benign enteric bacteria exerting pathogenic effects. Results We explored the potential role of the insect immune response in mortality caused by B. thuringiensis in conjunction with gut bacteria. Two lines of evidence support such a role. First, ingestion of B. thuringiensis by gypsy moth larvae led to the depletion of their hemocytes. Second, pharmacological agents that are known to modulate innate immune responses of invertebrates and vertebrates altered larval mortality induced by B. thuringiensis. Specifically, Gram-negative peptidoglycan pre-treated with lysozyme accelerated B. thuringiensis-induced killing of larvae previously made less susceptible due to treatment with antibiotics. Conversely, several inhibitors of the innate immune response (eicosanoid inhibitors and antioxidants increased the host's survival time following ingestion of B. thuringiensis. Conclusions This study demonstrates that B. thuringiensis infection provokes changes in the cellular immune response of gypsy moth larvae. The effects of chemicals known to modulate the innate immune response of many invertebrates and vertebrates, including Lepidoptera, also indicate a role of this response in B. thuringiensis killing. Interactions among B. thuringiensis toxin, enteric bacteria, and aspects of the gypsy moth immune response may provide a novel model to decipher mechanisms of sepsis associated with bacteria of gut origin.

  15. The Mosquito Melanization Response Is Implicated in Defense against the Entomopathogenic Fungus Beauveria bassiana

    Osta, Mike A.

    2012-01-01

    Mosquito immunity studies have focused mainly on characterizing immune effector mechanisms elicited against parasites, bacteria and more recently, viruses. However, those elicited against entomopathogenic fungi remain poorly understood, despite the ubiquitous nature of these microorganisms and their unique invasion route that bypasses the midgut epithelium, an important immune tissue and physical barrier. Here, we used the malaria vector Anopheles gambiae as a model to investigate the role of melanization, a potent immune effector mechanism of arthropods, in mosquito defense against the entomopathogenic fungus Beauveria bassiana, using in vivo functional genetic analysis and confocal microscopy. The temporal monitoring of fungal growth in mosquitoes injected with B. bassiana conidia showed that melanin eventually formed on all stages, including conidia, germ tubes and hyphae, except the single cell hyphal bodies. Nevertheless, melanin rarely aborted the growth of any of these stages and the mycelium continued growing despite being melanized. Silencing TEP1 and CLIPA8, key positive regulators of Plasmodium and bacterial melanization in A. gambiae, abolished completely melanin formation on hyphae but not on germinating conidia or germ tubes. The detection of a layer of hemocytes surrounding germinating conidia but not hyphae suggested that melanization of early fungal stages is cell-mediated while that of late stages is a humoral response dependent on TEP1 and CLIPA8. Microscopic analysis revealed specific association of TEP1 with surfaces of hyphae and the requirement of both, TEP1 and CLIPA8, for recruiting phenoloxidase to these surfaces. Finally, fungal proliferation was more rapid in TEP1 and CLIPA8 knockdown mosquitoes which exhibited increased sensitivity to natural B. bassiana infections than controls. In sum, the mosquito melanization response retards significantly B. bassiana growth and dissemination, a finding that may be exploited to design transgenic

  16. Antibacterial activity of hemocyanin from red swamp crayfish (Procambarus clarkii).

    Qin, Zhendong; Babu, V Sarath; Wan, Quanyuan; Muhammad, Asim; Li, Jun; Lan, Jiangfeng; Lin, Li

    2018-04-01

    Hemocyanins (HMC): the copper-containing respiratory proteins present in invertebrate hemolymph, which plays many essential roles in the immune system. Currently, little is known about the HMC domains of Procambarus clarkii (P. clarkii) and their function in antimicrobial immune response. In this present study, we comparatively studied the expression pattern of native PcHMC with the three recombinant proteins of variable domains of crayfish hemocyanin (PcHMC-N, N-terminal domain of hemocyanin; PcHMC-T, tyrosinase domain of hemocyanin; PcHMC-C, C-terminal domain of hemocyanin). The results showed that three purified recombinant proteins had a strong binding to various bacteria and lipopolysaccharides that further highly agglutinated. The HMCs recombinant proteins showed strong antibacterial activity against V. parahaemolyticus and S. aureus by bacterial growth inhibition, phenoloxidase (PO) and phagocytosis assays. Specifically, rPcHMC1-T and rPcHMC1-C inhibited both the bacteria efficiently, rPcHMC1-T was highly upregulated the PO activity than the other recombinant proteins. Whereas, recombinant proteins pretreated crayfish hemocytes participated in phagocytosis activity, rPcHMC1-N and rPcHMC1-C proteins had a profound effect than the rPcHMC1-T on S. aureus and V. parahaemolyticus phagocytosis. The crayfish hemocyanin domains clearly exhibited antibacterial and phagocytic activities against both the bacteria, suggesting that its variable domains of hemocyanin have the different function on specific pathogen during the assault of pathogens. Copyright © 2018 Elsevier Ltd. All rights reserved.

  17. A silencing suppressor protein (NSs) of a tospovirus enhances baculovirus replication in permissive and semipermissive insect cell lines.

    Oliveira, Virgínia Carla; Bartasson, Lorrainy; de Castro, Maria Elita Batista; Corrêa, José Raimundo; Ribeiro, Bergmann Morais; Resende, Renato Oliveira

    2011-01-01

    The nonstructural protein (NSs) of the Tomato spotted wilt virus (TSWV) has been identified as an RNAi suppressor in plant cells. A recombinant Autographa californica multiple nucleopolyhedrovirus (AcMNPV) designated vAcNSs, containing the NSs gene under the control of the viral polyhedrin (polh) gene promoter, was constructed and the effects of NSs in permissive, semipermissive and nonpermissive insect cells to vAcNSs infection were evaluated. vAcNSs produced more budded virus when compared to wild type in semipermissive cells. Co-infection of vAcNSs with wild type baculoviruses clearly enhanced polyhedra production in all host cells. Confocal microscopy analysis showed that NSs accumulated in abundance in the cytoplasm of permissive and semipermissive cells. In contrast, high amounts of NSs were detected in the nuclei of nonpermissive cells. Co-infection of vAcNSs with a recombinant AcMNPV containing the enhanced green fluorescent protein (egfp) gene, significantly increased EGFP expression in semipermissive cells and in Anticarsia gemmatalis-hemocytes. Absence of small RNA molecules of egfp transcripts in this cell line and in a permissive cell line indicates the suppression of gene silencing activity. On the other hand, vAcNSs was not able to suppress RNAi in a nonpermissive cell line. Our data showed that NSs protein of TSWV facilitates baculovirus replication in different lepidopteran cell lines, and these results indicate that NSs could play a similar role during TSWV-infection in its thrips vector. Copyright © 2010 Elsevier B.V. All rights reserved.

  18. Effect of silver nanoparticles on the metabolic rate, hematological response, and survival of juvenile white shrimp Litopenaeus vannamei.

    Juarez-Moreno, Karla; Mejía-Ruiz, Claudio Humberto; Díaz, Fernando; Reyna-Verdugo, Horacio; Re, Ana Denisse; Vazquez-Felix, Edgar F; Sánchez-Castrejón, Edna; Mota-Morales, Josué D; Pestryakov, Alexey; Bogdanchikova, Nina

    2017-02-01

    White spot syndrome virus (WSSV) is highly lethal and contagious in shrimps; its outbreaks causes an economic crisis for aquaculture. Several attempts have been made to treat this disease; however, to date, there is no effective cure. Because of their antimicrobial activities, silver nanoparticles (AgNPs) are the most studied nanomaterial. Although the antiviral properties of AgNPs have been studied, their antiviral effect against viral infection in aquaculture has not been reported. The AgNPs tested herein are coated with polyvinylpyrrolidone (PVP) and possess multiple international certifications for their use in veterinary and human applications. The aim of this work was to evaluate the survival rate of juvenile white shrimps (Litopenaeus vannamei) after the intramuscular administration of AgNPs. For this, different concentrations of metallic AgNPs and PVP alone were injected into the organisms. After 96 h of administration, shrimp survival was more than 90% for all treatments. The oxygen consumption routine rate and total hemocyte count remained unaltered after AgNP injection, reflecting no stress caused. We evaluated whether AgNPs had an antiviral effect in shrimps infected with WSSV. The results revealed that the survival rate of WSSV-infected shrimps after AgNP administration was 80%, whereas the survival rate of untreated organisms was only 10% 96 h after infection. These results open up the possibility to explore the potential use of AgNPs as antiviral agents for the treatment of diseases in aquaculture organisms, particularly the WSSV in shrimp culture. Copyright © 2016 Elsevier Ltd. All rights reserved.

  19. Genome-wide investigation of transcription factors provides insights into transcriptional regulation in Plutella xylostella.

    Zhao, Qian; Ma, Dongna; Huang, Yuping; He, Weiyi; Li, Yiying; Vasseur, Liette; You, Minsheng

    2018-04-01

    Transcription factors (TFs), which play a vital role in regulating gene expression, are prevalent in all organisms and characterization of them may provide important clues for understanding regulation in vivo. The present study reports a genome-wide investigation of TFs in the diamondback moth, Plutella xylostella (L.), a worldwide pest of crucifers. A total of 940 TFs distributed among 133 families were identified. Phylogenetic analysis of insect species showed that some of these families were found to have expanded during the evolution of P. xylostella or Lepidoptera. RNA-seq analysis showed that some of the TF families, such as zinc fingers, homeobox, bZIP, bHLH, and MADF_DNA_bdg genes, were highly expressed in certain tissues including midgut, salivary glands, fat body, and hemocytes, with an obvious sex-biased expression pattern. In addition, a number of TFs showed significant differences in expression between insecticide susceptible and resistant strains, suggesting that these TFs play a role in regulating genes related to insecticide resistance. Finally, we identified an expansion of the HOX cluster in Lepidoptera, which might be related to Lepidoptera-specific evolution. Knockout of this cluster using CRISPR/Cas9 showed that the egg cannot hatch, indicating that this cluster may be related to egg development and maturation. This is the first comprehensive study on identifying and characterizing TFs in P. xylostella. Our results suggest that some TF families are expanded in the P. xylostella genome, and these TFs may have important biological roles in growth, development, sexual dimorphism, and resistance to insecticides. The present work provides a solid foundation for understanding regulation via TFs in P. xylostella and insights into the evolution of the P. xylostella genome.

  20. Pesticide residues, heavy metals, and DNA damage in sentinel oysters Crassostrea gigas from Sinaloa and Sonora, Mexico.

    Vázquez-Boucard, Celia; Anguiano-Vega, Gerardo; Mercier, Laurence; Rojas del Castillo, Emilio

    2014-01-01

    Pesticides and heavy metals were analyzed in sentinel Crassostrea gigas oysters placed in six aquaculture sites close to a contaminated agricultural region. Each site was sampled twice. Tests revealed the presence of organochlorine (OC) pesticides in the oysters at concentrations varying from 31.8 to 72.5 μg/kg for gamma-hexachlorocyclohexane (γ-HCH); from 1.2 to 3.1 μg/kg for dichlorodiphenyldichloroethylene (4,4-DDE); from 1.6 to 2.3 μg/kg for endosulfan I; and from 1.4 to 41.2 μg/kg for endosulfan II, as well as heavy metals in concentrations that exceeded Mexican tolerance levels (405.5 to 987.8 μg/g for zinc; 4.2 to 7.3 μg/g for cadmium; and 7.2 to 9.9 μg/g for lead). Significant levels of DNA damage in oyster hemocytes were also detected. There was a significant, positive correlation between genotoxic damage and concentration of nickel or the presence of endosulfan II. Cellular viability evaluated by cytotoxic analyses was found to be high at 80%. Marked inhibition in activity of acetylcholinesterase (AChE ) and induction of glutathione S-transferase (GST) activity was noted. Data demonstrated a significant relation between AChE activity inhibition and presence of endosulfan II, γ-HCH, copper, lead, and 4,4-DDE, as well as between AChE and GST activity at different sites.

  1. Exposure to bacterial signals does not alter pea aphids' survival upon a second challenge or investment in production of winged offspring.

    Bas ter Braak

    Full Text Available Pea aphids have an obligate nutritional symbiosis with the bacteria Buchneraaphidicola and frequently also harbor one or more facultative symbionts. Aphids are also susceptible to bacterial pathogen infections, and it has been suggested that aphids have a limited immune response towards such pathogen infections compared to other, more well-studied insects. However, aphids do possess at least some of the genes known to be involved in bacterial immune responses in other insects, and immune-competent hemocytes. One possibility is that immune priming with microbial elicitors could stimulate immune protection against subsequent bacterial infections, as has been observed in several other insect systems. To address this hypothesis we challenged aphids with bacterial immune elicitors twenty-four hours prior to live bacterial pathogen infections and then compared their survival rates to aphids that were not pre-exposed to bacterial signals. Using two aphid genotypes, we found no evidence for immune protection conferred by immune priming during infections with either Serratia marcescens or with Escherichia coli. Immune priming was not altered by the presence of facultative, beneficial symbionts in the aphids. In the absence of inducible immune protection, aphids may allocate energy towards other defense traits, including production of offspring with wings that could escape deteriorating conditions. To test this, we monitored the ratio of winged to unwinged offspring produced by adult mothers of a single clone that had been exposed to bacterial immune elicitors, to live E. coli infections or to no challenge. We found no correlation between immune challenge and winged offspring production, suggesting that this mechanism of defense, which functions upon exposure to fungal pathogens, is not central to aphid responses to bacterial infections.

  2. Factors affecting post-capture survivability of lobster Homarus americanus.

    Basti, David; Bricknell, Ian; Hoyt, Ken; Chang, Ernest S; Halteman, William; Bouchard, Deborah

    2010-06-11

    Technological advances in gear and fishing practices have driven the global expansion of the American lobster live seafood market. These changes have had a positive effect on the lobster industry by increasing capture efficiency. However, it is unknown what effect these improved methods will have on the post-capture fitness and survival of lobsters. This project utilized a repeated measures design to compare the physiological changes that occur in lobsters over time as the result of differences in depth, hauling rate, and storage methodology. The results indicate that lobsters destined for long distance transport or temporary storage in pounds undergo physiological disturbance as part of the capture process. These changes are significant over time for total hemocyte counts, crustacean hyperglycemic hormone, L-lactate, ammonia, and glucose. Repeated measures multivariate analysis of variance (MANOVA) for glucose indicates a significant interaction between depth and storage methodology over time for non-survivors. A Gram-negative bacterium, Photobacterium indicum, was identified in pure culture from hemolymph samples of 100% of weak lobsters. Histopathology revealed the presence of Gram-negative bacteria throughout the tissues with evidence of antemortem edema and necrosis suggestive of septicemia. On the basis of these findings, we recommend to the lobster industry that if a reduction in depth and hauling rate is not economically feasible, fishermen should take particular care in handling lobsters and provide them with a recovery period in recirculating seawater prior to land transport. The ecological role of P. indicum is not fully defined at this time. However, it may be an emerging opportunistic pathogen of stressed lobsters. Judicious preemptive antibiotic therapy may be necessary to reduce mortality in susceptible lobsters destined for high-density holding facilities.

  3. Antimicrobial activity of a novel hypervariable immunoglobulin domain-containing receptor Dscam in Cherax quadricarinatus.

    Li, Dan; Yu, Ai-Qing; Li, Xue-Jie; Zhu, You-Ting; Jin, Xing-Kun; Li, Wei-Wei; Wang, Qun

    2015-12-01

    Down syndrome cell adhesion molecule (Dscam) mediates innate immunity against pathogens in arthropods. Here, a novel Dscam from red claw crayfish Cherax quadricarinatus (CqDscam) was isolated. The CqDscam protein contains one signal peptide, ten immunoglobulin domains, six fibronectin type III domains, one transmembrane domain and cytoplasmic tail. CqDscam phylogenetically clustered with other invertebrate Dscams. Variable regions of CqDscam in N-terminal halves of Ig2 and Ig3 domains, complete Ig7 domain and TM domain can be reshuffled after transcription to produce a deluge of >37,620 potential alternative splice forms. CqDscam was detected in all tissues tested and abundantly expressed in immune system and nerve system. Upon lipopolysaccharides (LPS) and b-1, 3-glucans (Glu) challenged, the expression of CqDscam was up-regulated, while no response in expression occurred after injection with peptidoglycans (PG). Membrane-bound and secreted types of CqDscam were separated on the protein level, and were both extensively induced post LPS challenge. Membrane-bound CqDscam protein was not detected in the serum, but localized to the hemocyte surface by immuno-localization assay. In the antimicrobial assays, the recombinant LPS-induced isoform of CqDscam protein displayed bacterial binding and growth inhibitory activities, especially with Escherichia coli. These results suggested that CqDscam, as one of pattern-recognition receptors (PRRs), involved in innate immune recognition and defense mechanisms in C. quadricarinatus, possibly through alternative splicing. Copyright © 2015 Elsevier Ltd. All rights reserved.

  4. Isolation and characterization of potential probiotic bacteria from pustulose ark (Anadara tuberculosa suitable for shrimp farming

    Ana Claudia Sánchez-Ortiz

    2015-03-01

    Full Text Available In aquaculture, probiotics have been tested for enhancing the immune system and promoting growth and survival rate of many marine species like shrimp and mollusks. In order to isolate bacteria with a high probiotic potential for marine shellfish aquaculture, homogenates of the gastrointestinal tract from adult mangrove cockle, Anadara tuberculosa, were obtained to perform in vitro and in vivo assays. Isolates were tested in vitro for hemolytic activity, hydrophobicity, tolerance to ammonia nitrogen, salinity and pH as well as for growth kinetics, extracellular enzymatic activity, autoaggregation, coaggregation and molecular identification. Three bacteria with high degree of hydrophobicity (>60% adherence to p-xylene and four bacteria with medium hydrophobicity, which showed different patterns of attachment to monopolar solvents (chloroform and ethyl acetate and a high tolerance to ammonia nitrogen (200 mg L-1, were selected. Six different treatments: T1 (without addition of cultured bacteria; T2 (MAt29, Enterococcus casseliflavus; T3 (MAt35, Citrobacter koseri; T4 (GAtBl, Bacillus subtilis subtilis; T5 (GAt7, Staphylococcus sp.; and T6 (1:1:1:1 mix of strains T2, T3, T4 and T5, were used to evaluate the specific growth rate, and cellular immune response of the shrimp Litopenaeus vannamei. The best specific growth rate was observed for T6 and T4 treatments related to Bacillus subtilis subtilis. A significant difference in total hemocytes count (P < 0.05 was found for T4 treatment with respect to control group. Strains isolated from A. tuberculosa had a beneficial effect on the growth and immune response of L. vannamei, so they have potential use as probiotics in aquaculture of marine shellfish.

  5. Molecular characterization and expression profiles of nicotinic acetylcholine receptors in the rice striped stem borer, Chilo suppressalis (Lepidoptera: Crambidae).

    Xu, Gang; Wu, Shun-Fan; Teng, Zi-Wen; Yao, Hong-Wei; Fang, Qi; Huang, Jia; Ye, Gong-Yin

    2017-06-01

    Nicotinic acetylcholine receptors (nAChRs) are members of the cys-loop ligand-gated ion channel (cysLGIC) superfamily, mediating fast synaptic cholinergic transmission in the central nervous system in insects. Insect nAChRs are the molecular targets of economically important insecticides, such as neonicotinoids and spinosad. Identification and characterization of the nAChR gene family in the rice striped stem borer, Chilo suppressalis, could provide beneficial information about this important receptor gene family and contribute to the investigation of the molecular modes of insecticide action and resistance for current and future chemical control strategies. We searched our C. suppressalis transcriptome database using Bombyx mori nAChR sequences in local BLAST searches and obtained the putative nAChR subunit complementary DNAs (cDNAs) via reverse transcription polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends methods. Similar to B. mori, C. suppressalis possesses 12 nAChR subunits, including nine α-type and three β-type subunits. Quantitative RT-PCR analysis revealed the expression profiles of the nAChR subunits in various tissues, including the brain, subesophageal ganglion, thoracic ganglion, abdominal ganglion, hemocytes, fat body, foregut, midgut, hindgut and Malpighian tubules. Developmental expression analyses showed clear differential expression of nAChR subunits throughout the C. suppressalis life cycle. The identification of nAChR subunits in this study will provide a foundation for investigating the diverse roles played by nAChRs in C. suppressalis and for exploring specific target sites for chemicals that control agricultural pests while sparing beneficial species. ©2016 The Authors Insect Science published by John Wiley & Sons Australia, Ltd on behalf of Institute of Zoology, Chinese Academy of Sciences.

  6. Involvement of a Serpin serine protease inhibitor (OoSerpin) from mollusc Octopus ocellatus in antibacterial response.

    Wei, Xiumei; Xu, Jie; Yang, Jianmin; Liu, Xiangquan; Zhang, Ranran; Wang, Weijun; Yang, Jialong

    2015-01-01

    Serpin is an important member of serine protease inhibitors (SPIs), which is capable of regulating proteolytic events and involving in a variety of physiological processes. In present study, a Serpin homolog was identified from Octopus ocellatus (designated as OoSerpin). Full-length cDNA of OoSerpin was of 1735 bp, containing a 5' untranslated region of 214 bp, a 3' UTR of 282 bp, and an open reading frame of 1239 bp. The open reading frame encoded a polypeptide of 412 amino acids which has a predicted molecular weight of 46.5 kDa and an isoelectric point of 8.52. The OoSerpin protein shares 37% sequence identity with other Serpins from Mus musculus (NP_941373) and Ixodes scapularis (XP_002407493). The existence of a conserved SERPIN domain strongly suggested that OoSerpin was a member of the Serpin subfamily. Expression patterns of OoSerpin, both in tissues and towards bacterial stimulation, were then characterized. The mRNA of OoSerpin was constitutively expressed at different levels in all tested tissues of untreated O. ocellatus, including mantle (lowest), muscle, renal sac, gill, hemocyte, gonad, systemic heart, and hepatopancreas (highest). The transcriptional level of OoSerpin was significantly up-regulated (P<0.01) in O. ocellatus upon bacterial challenges with Vibrio anguillarum and Micrococcus luteus, indicating its involvement in the antibacterial immune response. Furthermore, rOoSerpin, the recombinant protein of OoSerpin, exhibited strong abilities to inhibit proteinase activities of trypsin and chymotrypsin as well as the growth of Escherichia coli. Our results demonstrate that OoSerpin is a potential antibacterial factor involved in the immune response of O. ocellatus against bacterial infection. Copyright © 2014 Elsevier Ltd. All rights reserved.

  7. Circadian transitions in radiation dose-dependent augmentation of mRNA levels for DNA damage-induced genes elicited by accurate real-time RT-PCR quantification

    Ishihara, Hiroshi; Tanaka, Izumi; Yakumaru, Haruko

    2010-01-01

    Molecular mechanisms of intracellular response after DNA-damage by exposure to ionizing radiation have been studied. In the case of cells isolated from living body of human and experimental animals, alteration of the responsiveness by physiological oscillation such as circadian rhythm must be considered. To examine the circadian variation in the response of p53-responsible genes p21, mdm2, bax, and puma, we established a method to quantitate their mRNA levels with high reproducibility and accuracy based on real-time reverse transcription polymerase chain reaction (RT-PCR) and compared the levels of responsiveness in mouse hemocytes after diurnal irradiation to that after nocturnal irradiation. Augmentations of p21 and mdm2 mRNA levels with growth-arrest and of puma mRNA before apoptosis were confirmed by time-course experiment in RAW264.7, and dose-dependent increases in the peak levels of all the RNA were shown. Similarly, the relative RNA levels of p21, mdm2, bax, and puma per glyceraldehyde-3-phosphate dehydrogenase (GAPDH) also increased dose-dependently in peripheral blood and bone marrow cells isolated from whole-body-irradiated mice. Induction levels of all messages reduced by half after nighttime irradiation as compared with daytime irradiation in blood cells. In marrow cells, nighttime irradiation enhanced the p21 and mdm2 mRNA levels than daytime irradiation. No significant difference in bax or puma mRNA levels was observed between nighttime and daytime irradiation in marrow cells. This suggests that early-stage cellular responsiveness in DNA damage-induced genes is modulated between diurnal and nocturnal irradiation. (author)

  8. Cytotoxicity evaluation of Diethyltoluamide (DEET) in Perna perna (Linnaeus, 1758) mussels non-irradiated and irradiated with 60Co gamma radiation

    Martini, Gisela de Assis

    2013-01-01

    Recent studies have identified the presence of several emerging pollutants in aquatic environments. The occurrence in different environmental matrices has been continuously reported, highlighting the need for toxicity studies. The DEET (N,N-diethyl-meta-toluamide) is the active ingredient used in most insect repellents, and is present in many commercially available formulations. Apart from chemical pollutants, aquatic organisms are subject to exposure of ionizing radiation from natural sources or in the vicinity of nuclear power plants. The present study evaluated the toxicity of DEET in organisms irradiated and non-irradiated with 60 Co gamma radiation, and the effects that radiation causes in lysosomes of hemocytes of Perna perna mussel. For this purpose, assays were performed to identify the acute toxicity of DEET concentration and the dose of gamma radiation able to cause mortality. Subsequently, cytotoxicity assays were carried out to assess the stability of the lysosomal membrane in organisms exposed to ionizing radiation and DEET. According to the results obtained in acute toxicity tests, the concentration of DEET that causes mortality of 50% exposed organisms (LC50) is 114,27 mg L -1 , and the radiation dose that causes mortality (LD50) is 1068 Gy. In the cytotoxicity assays, the concentration of the non-observed effect (NOEC) for irradiated and non-irradiated organisms 0.0001 mg L-1 and observed effect concentration (LOEC) at concentrations above this. The IC25 (72h) for non-irradiated organisms was 0.0003 mg L-1 and IC50 (72h) was 0.0008 mg L -1 for irradiated and non-irradiated organisms. Despite of the concentrations of effect found in this study were higher than in the environment, both measurements are in the same order of magnitude and should be also take into account the possible synergistic effects of DEET with other contaminants in the aquatic environment. (author)

  9. Genome-wide identification, characterization of sugar transporter genes in the silkworm Bombyx mori and role in Bombyx mori nucleopolyhedrovirus (BmNPV) infection.

    Govindaraj, Lekha; Gupta, Tania; Esvaran, Vijaya Gowri; Awasthi, Arvind Kumar; Ponnuvel, Kangayam M

    2016-04-01

    Sugar transporters play an essential role in controlling carbohydrate transport and are responsible for mediating the movement of sugars into cells. These genes exist as large multigene families within the insect genome. In insects, sugar transporters not only have a role in sugar transport, but may also act as receptors for virus entry. Genome-wide annotation of silkworm Bombyx mori (B. mori) revealed 100 putative sugar transporter (BmST) genes exists as a large multigene family and were classified into 11 sub families, through phylogenetic analysis. Chromosomes 27, 26 and 20 were found to possess the highest number of BmST paralogous genes, harboring 22, 7 and 6 genes, respectively. These genes occurred in clusters exhibiting the phenomenon of tandem gene duplication. The ovary, silk gland, hemocytes, midgut and malphigian tubules were the different tissues/cells enriched with BmST gene expression. The BmST gene BGIBMGA001498 had maximum EST transcripts of 134 and expressed exclusively in the malphigian tubule. The expression of EST transcripts of the BmST clustered genes on chromosome 27 was distributed in various tissues like testis, ovary, silk gland, malphigian tubule, maxillary galea, prothoracic gland, epidermis, fat body and midgut. Three sugar transporter genes (BmST) were constitutively expressed in the susceptible race and were down regulated upon BmNPV infection at 12h post infection (hpi). The expression pattern of these three genes was validated through real-time PCR in the midgut tissues at different time intervals from 0 to 30hpi. In the susceptible B. mori race, expression of sugar transporter genes was constitutively expressed making the host succumb to viral infection. Copyright © 2015 Elsevier B.V. All rights reserved.

  10. Norepinephrine and dopamine increase motility, biofilm formation and virulence of Vibrio harveyi

    Qian eYang

    2014-11-01

    Full Text Available Vibrio harveyi is one of the major pathogens of aquatic organisms, affecting both vertebrates and invertebrates, and causes important losses in the aquaculture industry. In order to develop novel methods to control disease caused by this pathogen, we need to obtain a better understanding of pathogenicity mechanisms. Sensing of catecholamines increases both growth and production of virulence-related factors in pathogens of terrestrial animals and humans. However, at this moment, knowledge on the impact of catecholamines on the virulence of pathogens of aquatic organisms is lacking. In the present study, we report that in V. harveyi, norepinephrine and dopamine increased growth in serum-supplemented medium, siderophore production, swimming motility and expression of genes involved in flagellar motility, biofilm formation, and exopolysaccharide production. Consistent with this, pretreatment of V. harveyi with catecholamines prior to inoculation into the rearing water resulted in significantly decreased survival of gnotobiotic brine shrimp larvae, when compared to larvae challenged with untreated V. harveyi. Further, norepinephrine-induced effects could be neutralized by α-adrenergic antagonists or by the bacterial catecholamine receptor antagonist LED209, but not by β-adrenergic or dopaminergic antagonists. Dopamine-induced effects could be neutralized by dopaminergic antagonists or LED209, but not by adrenergic antagonists. Together, our results indicate that catecholamine sensing increases the success of transmission of V. harveyi and that interfering with catecholamine sensing might be an interesting strategy to control vibriosis in aquaculture. We hypothesise that upon tissue and/or hemocyte damage during infection, pathogens come into contact with elevated catecholamine levels, and that this stimulates the expression of virulence factors that are required to colonize a new host.

  11. Identification and expression analysis of a novel stylicin antimicrobial peptide from Kuruma shrimp (Marsupenaeus japonicus).

    Liu, Hong-tao; Wang, Jun; Mao, Yong; Liu, Min; Niu, Su-fang; Qiao, Ying; Su, Yong-quan; Wang, Chun-zhong; Zheng, Zhi-peng

    2015-12-01

    Antimicrobial peptides (AMPs) are important components of the innate immune system and function as the first line of defense against invading pathogens. In current study we identified, cloned and characterized a novel stylicin AMP from Kuruma shrimp Marsupenaeus japonicus (Mj-sty). The full-length cDNA of Mj-sty was 428 bp with an open reading frame of 315 bp that encoded 104 amino acids. The theoretical molecular mass of mature Mj-sty was 8.693 kDa with an isoelectric point (pI) of 4.79. A proline-rich N-terminal region and a C-terminal region contained 13 cysteine residues were identified. Genomic sequence analysis with respect to its cDNA showed that Mj-sty was organized into two exons interrupted by one intron. Tissue-specific expression revealed that Mj-sty was mainly transcribed in gills and hemocytes. Expression of Mj-sty in early developmental stages demonstrated that Mj-sty mRNA were present from fertilized eggs to post-larvae of 17 days (PL17), and the expression levels showed a significant variation in different developmental stages. After challenge of white spot syndrome virus (WSSV), the time-dependent expression pattern of Mj-sty in both gills and hepatopancrease showed down-regulation at the early hours of infection, subsequently up-regulation and down-regulation, and then up-regulation at the end hours to almost the half of the controls. The results indicate that Mj-sty is potentially involved in the ontogenesis and immune responses against WSSV. Copyright © 2015 Elsevier Ltd. All rights reserved.

  12. Histopathologic and biochemical responses in Arctic marine bivalve molluscs exposed to experimentally spilled oil

    Neff, J.M.; Hillman, R.E.; Carr, R.S.; Buhl, R.L.; Lahey, J.I.

    1987-01-01

    Following two experimental spills of chemically dispersed and undispersed crude oil in shallow bays on the northwest coast of Baffin Island bivalve molluscs accumulated significant amounts of petroleum hydrocarbons in bay receiving dispersed oil and in those receiving crude oil alone. Specimens of Mya truncata and Macoma calcarea for histopathologic examination were collected immediately before, immediately after and one year after the experimental oil spills. Immediately after there was increased gill and digestive tract necrosis in Mya from the chemically dispersed oil. After one year a few clams had granulocytomas throughout the tissues. Three clams receiving oil alone collected one year after the spill had invasive neoplasias. There were few lesions in Macoma immediatelly after or one year after the spill; animals had a high incidence of vacuolization of the digestive tubule epithelium. The incidence of parasitism and hemocytic infiltration also was higher in Maccoma. Clams Mya truncata were collected for biochemical analysis before, after and two weeks after the simulated oil spills. Concentations in the clam tissues of glucose, glycogen, trehalose, total lipid, and free amino acids were measured; free amino acids in adductor muscles were the most useful indices of pollutant stress. The results of the biochemical analyses indicate that Mya were not severely stressed by either dispersed oil or oil alone. After two weeks, clams from the dispersed oil bays were nearly normal, while those from the bay receiving oil alone appeared stressed. These results seem to corroborate results that the acute effects of dispersed oil are greater than those of undispersed oil, but effects of undispersed oil on infaunal molluscs develop more slowly and persist longer than those from dispersed oil. 43 refs., 2 figs., 6 tabs.

  13. Lipid reserves and immune defense in healthy and diseased migrating monarchs Danaus plexippus

    Dara A. SATTERFIELD, Amy E. WRIGHT, Sonia ALTIZER

    2013-06-01

    Full Text Available Recent studies suggest that the energetic demands of long-distance migration might lower the pool of resources available for costly immune defenses. Moreover, migration could amplify the costs of parasitism if animals suffering from parasite-induced damage or depleted energy reserves are less able to migrate long distances. We investigated relationships between long-distance migration, infection, and immunity in wild fall-migrating monarch butterflies Danaus plexippus. Monarchs migrate annually from eastern North America to central Mexico, accumulating lipids essential for migration and winter survival as they travel southward. Monarchs are commonly infected by the debilitating protozoan parasite Ophryocystis elektroscirrha (OE. We collected data on lipid reserves, parasite loads, and two immune measures (hemocyte concentration and phenoloxidase activity from wild monarchs migrating through north GA (USA to ask whether (1 parasite infection negatively affects lipid reserves, and (2 greater investment in lipid reserves is associated with lower immune measures. Results showed that monarchs sampled later in the fall migration had lower but not significantly different immune measures and significantly higher lipid reserves than those sampled earlier. Lipid measures correlated negatively but only nearly significantly with one measure of immune defense (phenoloxidase activity in both healthy and infected monarchs, but did not depend on monarch infection status or parasite load. These results provide weak support for a trade-off between energy reserves and immune defense in migrants, and suggest that previously-demonstrated costs of OE infection for monarch migration are not caused by depleted lipid reserves [Current Zoology 59 (3: 393–402, 2013].

  14. Changes in blood aggregation with differences in molecular weight and degree of deacetylation of chitosan

    Hattori, Hidemi; Ishihara, Masayuki

    2015-01-01

    Because the molecular weight (Mw) and degree of deacetylation (DDA) of chitosan can vary depending on the purification method, the identification of appropriate chitosan structures is important for developing more effective hemostatic agents. In this study, the influence of varying Mw and DDA of chitosan on blood aggregation was characterized by 10 types of chitosan with different Mw and DDA, including oligomers. The highest aggregation of whole blood, washed erythrocytes and platelets in platelet-rich plasma (PRP) were observed in chitosan with Mw of 8.6 kDa or more and with DDA of 75 to 88%. However, chitosan with too high Mw (247 kDa) inhibited the aggregation of whole blood, washed erythrocytes and PRP at high chitosan concentration. At certain concentrations, chitosan with 75–85% DDA and 50–190 kDa and chitosan with 87.6% DDA and 247 kDa both aggregated proteins in PRP. Chitosan oligomer did not affect blood aggregation. The results suggested that the aggregation by chitosan depended on the interaction of positively charged chitosan with negatively charged erythrocytes, platelets and plasma protein. It seemed that a suitable balance of positive charge in chitosan and negative charge in hemocytes and some kinds of proteins was important. To develop a hemostatic with effective blood aggregation, the chitosan should not be limited to a single Mw and a single DDA but may be a mixed chitosan with wide range of Mw (8.6–247 kDa) and DDA of 75 to 88%. (paper)

  15. Norepinephrine and dopamine increase motility, biofilm formation, and virulence of Vibrio harveyi.

    Yang, Qian; Anh, Nguyen D Q; Bossier, Peter; Defoirdt, Tom

    2014-01-01

    Vibrio harveyi is one of the major pathogens of aquatic organisms, affecting both vertebrates and invertebrates, and causes important losses in the aquaculture industry. In order to develop novel methods to control disease caused by this pathogen, we need to obtain a better understanding of pathogenicity mechanisms. Sensing of catecholamines increases both growth and production of virulence-related factors in pathogens of terrestrial animals and humans. However, at this moment, knowledge on the impact of catecholamines on the virulence of pathogens of aquatic organisms is lacking. In the present study, we report that in V. harveyi, norepinephrine (NE) and dopamine (Dopa) increased growth in serum-supplemented medium, siderophore production, swimming motility, and expression of genes involved in flagellar motility, biofilm formation, and exopolysaccharide production. Consistent with this, pretreatment of V. harveyi with catecholamines prior to inoculation into the rearing water resulted in significantly decreased survival of gnotobiotic brine shrimp larvae, when compared to larvae challenged with untreated V. harveyi. Further, NE-induced effects could be neutralized by α-adrenergic antagonists or by the bacterial catecholamine receptor antagonist LED209, but not by β-adrenergic or dopaminergic antagonists. Dopa-induced effects could be neutralized by dopaminergic antagonists or LED209, but not by adrenergic antagonists. Together, our results indicate that catecholamine sensing increases the success of transmission of V. harveyi and that interfering with catecholamine sensing might be an interesting strategy to control vibriosis in aquaculture. We hypothesize that upon tissue and/or hemocyte damage during infection, pathogens come into contact with elevated catecholamine levels, and that this stimulates the expression of virulence factors that are required to colonize a new host.

  16. Toxicity assessment and comparison between two types of iron oxide nanoparticles in Mytilus galloprovincialis

    Taze, Chrysa; Panetas, Ioannis; Kalogiannis, Stavros; Feidantsis, Konstantinos; Gallios, George P.; Kastrinaki, Georgia; Konstandopoulos, Athanasios G.; Václavíková, Miroslava; Ivanicova, Lucia; Kaloyianni, Martha

    2016-01-01

    Highlights: • The impact of two types of iron oxide nanoparticles on the physiological status of mussels was studied. • Oxidative parameters significantly changed after 1, 3, 7 days of exposure. • The nanoparticles induced oxidative stress to the animals. • All the parameters measured could be applied in biomonitoring studies. - Abstract: Nanoparticles (NPs), due to their increased application and production, are being released into the environment with unpredictable impact on the physiology of marine organisms, as well as on entire ecosystems and upcoming effects on human health. The aim of the present study was to evaluate and compare the oxidative responses of the mussel Mytilus galloprovincialis after exposure to iron oxide NPs and to iron oxide NPs incorporated into zeolite for 1, 3 and 7 days. Our results showed that both effectors induced changes on animal physiology by causing oxidative stress in hemocytes of exposed mussels compared to control animals. This was shown by the significant increase in reactive oxygen species (ROS) production, protein carbonylation, lipid peroxidation, ubiquitin conjugates and DNA damage. In addition an increase in prooxidant levels as measured by the prooxidant-antioxidant balance (PAB) assay was observed in exposed mussels’ hemolymph. The results show that ROS, DNA damage, protein and lipid oxidation, ubiquitin conjugates and PAB could constitute, after further investigation, reliable biomarkers for the evaluation of pollution or other environmental stressors. In addition, more studies are needed in order to ensure the safety of these NPs on various biomedical applications, since it is critical to design NPs that they meet the demands of application without causing cellular toxicity.

  17. A novel toll-like receptor from Mytilus coruscus is induced in response to stress.

    Xu, Mengshan; Wu, Jiong; Ge, Delong; Wu, Changwen; Changfeng Chi; Lv, Zhenming; Liao, Zhi; Liu, Huihui

    2018-07-01

    Toll-like receptor (TLR) is considered to be an evolutionarily conserved transmembrane protein which promotes the Toll signal pathway to active the expression of transcription factors in the innate immunity of the organism. In this study, a full length of TLR homologue of 2525bp in Mytilus coruscus (named as McTLR-a, GenBank accession no: KY940571) was characterized. Its ORF was 1815 bp with a 5'untranslated region (UTR) of 128 bp and a 3'UTR of 582 bp, encoding 602 amino acid residues with a calculated molecular weight of 70.870 kDa (pI = 6.10). BLASTn analysis and phylogenetic relationship strongly suggested that this cDNA sequence was a member of TLR family. Quantitative real time RT-PCR showed that constitutive expression of McTLR-a was occurred, with increasing order in hemocyte, gonad, mantle, adducter, gill and hepatopancreas. Bacterial infection and heavy metals stimulation up-regulated the expression of McTLR-a mRNA in hepatopancreas with time-dependent manners. The maximum expression appeared at 12 h after pathogenic bacteria injection, with approximately 22-fold in Aeromonas hydrophila and 17-fold in Vibrio parahemolyticus higher than that of the blank group. In heavy metals stress group, they all reached peaks at 3d, while the diverse concentration caused the maximum expression were different. The highest expression reached approximately 7-fold higher than the blank in low concentration of Pb 2+ exposure. In Cu 2+ treated group, it reached the peak (approximately 12-fold higher than the blank)in middle concentration. These results indicated that McTLR-a might be involved in the defense response and had a significant role in mediating the environmental stress. Copyright © 2018 Elsevier Ltd. All rights reserved.

  18. PcToll3 was involved in anti-Vibrio response by regulating the expression of antimicrobial peptides in red swamp crayfish, Procambarus clarkii.

    Lan, Jiang-Feng; Wei, Shun; Wang, Yu-Qing; Dai, Yun-Jia; Tu, Jia-Gang; Zhao, Li-Juan; Li, Xin-Cang; Qin, Qi-Wei; Chen, Nan; Lin, Li

    2016-10-01

    Tolls and Toll-like receptors (TLRs) play an important role in host immune defenses by regulating the expression of antimicrobial peptides (AMPs) and cytokines, but the functional differences of crustacean Tolls from Drosophila Tolls or Mammal TLRs are largely unknown. A novel Toll receptor, named PcToll3, was identified from red swamp crayfish, Procambarus clarkii. It was widely expressed in all detected tissues, and its transcript in hemocytes was up-regulated at 12 h after Vibrio parahemolyticus (Vibrio) injection or at 24 h post white spot syndrome virus (WSSV) challenge. After knockdown of PcToll3, the activity of bacterial clearance was inhibited, and the expression levels of AMPs including Crustin1 (Cru1), Anti-lippopolysaccharide factor 1 (ALF1), and Lysozymes1 (Lys1), which could be up-regulated by Vibrio, were all affected. Meanwhile, PcToll3 silencing influenced the expression of myeloid differentiation factor 88 (PcMyd88), tumor necrosis factor-associated factor 6 (PcTRAF6), and PcDorsal, which were the counterparts of Drosophila Toll signaling pathway. Interestingly, PcToll3 silencing inhibited translocation of PcDorsal from cytoplasm to nucleus. Furthermore, the knockdown of PcDorsal also impaired the expression of AMPs after Vibrio challenge. Hence, we concluded that, besides participating in antiviral immunity, PcToll3 might also regulate the expression of Cru1 and Lys1 to participate in anti-Vibrio immune responses by promoting PcDorsal translocation into nucleus. Copyright © 2016 Elsevier Ltd. All rights reserved.

  19. Identification and functional characterization of the TAB2 gene from Litopenaeus vannamei.

    Wang, Sheng; Li, Haoyang; Qian, Zhe; Song, Xuan; Zhang, Zijian; Zuo, Hongliang; Xu, Xiaopeng; Weng, Shaoping; He, Jianguo; Li, Chaozheng

    2015-10-01

    In Drosophila, TAB2, an important intermediate in the IMD signaling pathway, plays critical roles in the innate immune response in response to bacterial and viral infection. However, the role of TAB-related proteins in the immune response of shrimp has not yet been established. Here, we reported the identification of a TAB2-like gene in Litopenaeus vannamei designated as LvTAB2. The full-length cDNA of LvTAB2 was 2160 bp with an open reading frame of 1827 bp, which encoded a putative protein of 608 amino acids including a ubiquitin binding domain (CUE) at the N-terminal and a Zinc Finger domain (ZnF) at the C-terminus. Real-time RT-PCR analysis showed that LvTAB2 was expressed in all tested tissues and the expression levels of LvTAB2 in gills and hemocytes were positively induced in response to LPS, Vibrio parahemolyticus and White Spot Syndrome Virus (WSSV) challenges. Dual luciferase reporter assays demonstrated that LvTAB2 was able to induce the expression of antimicrobial peptide (AMP) genes, including Drosophila Attacin A and shrimp Penaeidins. Interestingly, over-expression of LvTAB2 could up-regulate the promoter activities of L. vannamei Vago1, Vago3 and Vago4 genes in S2 cells. To our knowledge, it was the first report that TAB2 participated in innate immune signaling to regulate the expression of Vago genes in invertebrates. Moreover, RNAi-mediated knockdown of LvTAB2 enhanced sensitivity of L. vannamei to Vibrio parahaemolyticus infection and caused elevated virus loads after WSSV infection. We suggested that the LvTAB2 may play important roles in the shrimp innate immunity. Copyright © 2015 Elsevier Ltd. All rights reserved.

  20. Dm5-HT2B: Pharmacological Characterization of the Fifth Serotonin Receptor Subtype of Drosophila melanogaster

    Wolfgang Blenau

    2017-05-01

    Full Text Available Serotonin (5-hydroxytryptamine, 5-HT is an important regulator of physiological and behavioral processes in both protostomes (e.g., insects and deuterostomes (e.g., mammals. In insects, serotonin has been found to modulate the heart rate and to control secretory processes, development, circadian rhythms, aggressive behavior, as well as to contribute to learning and memory. Serotonin exerts its activity by binding to and activating specific membrane receptors. The clear majority of these receptors belong to the superfamily of G-protein-coupled receptors. In Drosophila melanogaster, a total of five genes have been identified coding for 5-HT receptors. From this family of proteins, four have been pharmacologically examined in greater detail, so far. While Dm5-HT1A, Dm5-HT1B, and Dm5-HT7 couple to cAMP signaling cascades, the Dm5-HT2A receptor leads to Ca2+ signaling in an inositol-1,4,5-trisphosphate-dependent manner. Based on sequence similarity to homologous genes in other insects, a fifth D. melanogaster gene was uncovered coding for a Dm5-HT2B receptor. Knowledge about this receptor’s pharmacological properties is very limited. This is quite surprising because Dm5-HT2B has been attributed to distinct physiological functions based on genetic interference with its gene expression. Mutations were described reducing the response of the larval heart to 5-HT, and specific knockdown of Dm5-HT2B mRNA in hemocytes resulted in a higher susceptibility of the flies to bacterial infection. To gain deeper understanding of Dm5-HT2B’s pharmacology, we evaluated the receptor’s response to a series of established 5-HT receptor agonists and antagonists in a functional cell-based assay. Metoclopramide and mianserin were identified as two potent antagonists that may allow pharmacological interference with Dm5-HT2B signaling in vitro and in vivo.

  1. Dm5-HT2B: Pharmacological Characterization of the Fifth Serotonin Receptor Subtype of Drosophila melanogaster.

    Blenau, Wolfgang; Daniel, Stöppler; Balfanz, Sabine; Thamm, Markus; Baumann, Arnd

    2017-01-01

    Serotonin (5-hydroxytryptamine, 5-HT) is an important regulator of physiological and behavioral processes in both protostomes (e.g., insects) and deuterostomes (e.g., mammals). In insects, serotonin has been found to modulate the heart rate and to control secretory processes, development, circadian rhythms, aggressive behavior, as well as to contribute to learning and memory. Serotonin exerts its activity by binding to and activating specific membrane receptors. The clear majority of these receptors belong to the superfamily of G-protein-coupled receptors. In Drosophila melanogaster , a total of five genes have been identified coding for 5-HT receptors. From this family of proteins, four have been pharmacologically examined in greater detail, so far. While Dm5-HT 1A , Dm5-HT 1B , and Dm5-HT 7 couple to cAMP signaling cascades, the Dm5-HT 2A receptor leads to Ca 2+ signaling in an inositol-1,4,5-trisphosphate-dependent manner. Based on sequence similarity to homologous genes in other insects, a fifth D. melanogaster gene was uncovered coding for a Dm5-HT 2B receptor. Knowledge about this receptor's pharmacological properties is very limited. This is quite surprising because Dm5-HT 2B has been attributed to distinct physiological functions based on genetic interference with its gene expression. Mutations were described reducing the response of the larval heart to 5-HT, and specific knockdown of Dm5-HT 2B mRNA in hemocytes resulted in a higher susceptibility of the flies to bacterial infection. To gain deeper understanding of Dm5-HT 2B 's pharmacology, we evaluated the receptor's response to a series of established 5-HT receptor agonists and antagonists in a functional cell-based assay. Metoclopramide and mianserin were identified as two potent antagonists that may allow pharmacological interference with Dm5-HT 2B signaling in vitro and in vivo .

  2. Shrimp miRNAs regulate innate immune response against white spot syndrome virus infection.

    Kaewkascholkul, Napol; Somboonviwat, Kulwadee; Asakawa, Shuichi; Hirono, Ikuo; Tassanakajon, Anchalee; Somboonwiwat, Kunlaya

    2016-07-01

    MicroRNAs are short noncoding RNAs of RNA interference pathways that regulate gene expression through partial complementary base-pairing to target mRNAs. In this study, miRNAs that are expressed in white spot syndrome virus (WSSV)-infected Penaeus monodon, were identified using next generation sequencing. Forty-six miRNA homologs were identified from WSSV-infected shrimp hemocyte. Stem-loop real-time RT-PCR analysis showed that 11 out of 16 selected miRNAs were differentially expressed upon WSSV infection. Of those, pmo-miR-315 and pmo-miR-750 were highly responsive miRNAs. miRNA target prediction revealed that the miRNAs were targeted at 5'UTR, ORF, and 3'UTR of several immune-related genes such as genes encoding antimicrobial peptides, signaling transduction proteins, heat shock proteins, oxidative stress proteins, proteinases or proteinase inhibitors, proteins in blood clotting system, apoptosis-related proteins, proteins in prophenoloxidase system, pattern recognition proteins and other immune molecules. The highly conserved miRNA homolog, pmo-bantam, was characterized for its function in shrimp. The pmo-bantam was predicted to target the 3'UTR of Kunitz-type serine protease inhibitor (KuSPI). Binding of pmo-bantam to the target sequence of KuSPI gene was analyzed by luciferase reporter assay. Correlation of pmo-bantam and KuSPI expression was observed in lymphoid organ of WSSV-infected shrimp. These results implied that miRNAs might play roles as immune gene regulators in shrimp antiviral response. Copyright © 2016. Published by Elsevier Ltd.

  3. Effects of carotenoid sources on growth performance, blood parameters, disease resistance and stress tolerance in black tiger shrimp (Penaeus monodon Fabricius

    Supamattaya, K.

    2005-02-01

    Full Text Available Two feeding trial were conducted to determine the effects of various sources of carotenoid on growth performance, disease resistance, blood parameters, stress tolerance and pigmentation in juvenile black tiger shrimp (Penaeus monodon. Trial I was performed in small shrimp (1 g average body weight. The shrimp were fed with control diet without carotenoid (diet 1 while diets 2 to 6 contained 50 mg/kg astaxanthin (Lucanthin Pink®, 125 mg/kg β-carotene (Lucarotin®, 200 mg/kg β-carotene (Lucarotin®, 125 mg/kg Betatene® extracted from Dunaliella and 3% dried Spirulina respectively. There was an improvement in color in all groups of shrimp fed caroteniod supplemented diets, but no significant differences in weight gain or survival among the shrimps fed each test diet (p>0.05. Resistance to white spot syndrome virus (WSSV infection and stress tolerance (salinity stress, were not significantly different among treatments. Trial II was performed in juvenile shrimp (10 g average body weight fed test diets containing 100 ppm astaxanthin (Lucanthin pink®, 125 mg/kg β-carotene (Lucarotin®, 250 mg/kg β-carotene (Lucarotin®, 250 mg/kg Betatene® and 3% dried Spirulina compared with those fed control diet without carotenoid. At the end of 6 weeks feeding period, shrimp fed control diet as well as astaxanthin and dried Spirulina supplemented diets had higher levels of total hemocyte counts than those of all β-carotene supplemented diets feeding group. However, phenoloxidase activity and clearance of pathogenic vibrio from the hemolymphwere not significantly different among the treatments (p>0.05. Astaxanthin levels were highest in the shrimp fed all carotenoid-supplemented diets. In conclusion, a natural carotenoid i.e. dried Spirulina and carotenoid extracted from Dunaliella which have a lower production cost than analytical carotenoid showed beneficial effects on shrimp feed supplement.

  4. Effect of various Na/K ratios in low-salinity well water on growth performance and physiological response of Pacific white shrimp Litopenaeus vannamei

    Liu, Hongyu; Tan, Beiping; Yang, Jinfang; Lin, Yingbo; Chi, Shuyan; Dong, Xiaohui; Yang, Qihui

    2014-09-01

    To investigate the influence of sodium to potassium (Na/K) ratios on the growth performance and physiological response of the Pacific white shrimp ( Litopenaeus vananmei), various concentrations of KCl were added to low-salinity well water (salinity 4) in an 8-week culture trial. Six treatments with Na/K ratios of 60:1, 42:1, 33:1, 23:1, 17:1, and 14:1 were replicated in triplicate. The highest weight-gain rate (3 506±48)% and survival rate (89.38±0.88)% was observed in well water with Na/K ratios of 23:1 and 42:1, respectively, while the feed conversion ratio (1.02±0.01), oxygen consumption, and ammonia-N excretion rate was the lowest in the medium with a Na/K ratio of 23:1. Gill Na+-K+-ATPase activity, as an indicator of osmoregulation, peaked in the treatment where the Na/K ratio was 17:1. The total hemocyte count, respiratory burst, and immune-related enzyme activities (ALP, LSZ, PO, and SOD) of L. vananmei were affected significantly by Na/K ratios ( Pshrimp reared in a Na/K ratio of 23:1 (30±14.14)% was significantly lower than the control (75±7.07)%. In conclusion, the addition of K+ to low-salinity well water in L. vannamei cultures is feasible. Na/K ratios ranging from 23:1 to 33:1 might improve survival and growth. Immunity and disease resistance are also closely related to the Na/K ratio of the low-salinity well water. The findings may contribute to the development of more efficient K + remediation strategies for L. vananmei culture in low-salinity well water.

  5. Injected phage-displayed-VP28 vaccine reduces shrimp Litopenaeus vannamei mortality by white spot syndrome virus infection.

    Solís-Lucero, G; Manoutcharian, K; Hernández-López, J; Ascencio, F

    2016-08-01

    White spot syndrome virus (WSSV) is the most important viral pathogen for the global shrimp industry causing mass mortalities with huge economic losses. Recombinant phages are capable of expressing foreign peptides on viral coat surface and act as antigenic peptide carriers bearing a phage-displayed vaccine. In this study, the full-length VP28 protein of WSSV, widely known as potential vaccine against infection in shrimp, was successfully cloned and expressed on M13 filamentous phage. The functionality and efficacy of this vaccine immunogen was demonstrated through immunoassay and in vivo challenge studies. In ELISA assay phage-displayed VP28 was bind to Litopenaeus vannamei immobilized hemocyte in contrast to wild-type M13 phage. Shrimps were injected with 2 × 10(10) cfu animal(-1) single dose of VP28-M13 and M13 once and 48 h later intramuscularly challenged with WSSV to test the efficacy of the vaccine against the infection. All dead challenged shrimps were PCR WSSV-positive. The accumulative mortality of the vaccinated and challenged shrimp groups was significantly lower (36.67%) than the unvaccinated group (66.67%). Individual phenoloxidase and superoxide dismutase activity was assayed on 8 and 48 h post-vaccination. No significant difference was found in those immunological parameters among groups at any sampled time evaluated. For the first time, phage display technology was used to express a recombinant vaccine for shrimp. The highest percentage of relative survival in vaccinated shrimp (RPS = 44.99%) suggest that the recombinant phage can be used successfully to display and deliver VP28 for farmed marine crustaceans. Copyright © 2016 Elsevier Ltd. All rights reserved.

  6. Replication of the Shrimp Virus WSSV Depends on Glutamate-Driven Anaplerosis.

    Chun-Yuan Li

    Full Text Available Infection with the white spot syndrome virus (WSSV induces a metabolic shift in shrimp that resembles the "Warburg effect" in mammalian cells. This effect is triggered via activation of the PI3K-Akt-mTOR pathway, and it is usually accompanied by the activation of other metabolic pathways that provide energy and direct the flow of carbon and nitrogen. Here we show that unlike the glutamine metabolism (glutaminolysis seen in most cancer cells to double deaminate glutamine to produce glutamate and the TCA cycle intermediate α-ketoglutarate (α-KG, at the WSSV genome replication stage (12 hpi, although glutaminase (GLS expression was upregulated, only glutamate was taken up by the hemocytes of WSSV-infected shrimp. At the same time, we observed an increase in the activity of the two enzymes that convert glutamate to α-KG, glutamate dehydrogenase (GDH and aspartate aminotransferase (ASAT. α-ketoglutarate concentration was also increased. A series of inhibition experiments suggested that the up-regulation of GDH is regulated by mTORC2, and that the PI3K-mTORC1 pathway is not involved. Suppression of GDH and ASAT by dsRNA silencing showed that both of these enzymes are important for WSSV replication. In GDH-silenced shrimp, direct replenishment of α-KG rescued both ATP production and WSSV replication. From these results, we propose a model of glutamate-driven anaplerosis that fuels the TCA cycle via α-KG and ultimately supports WSSV replication.

  7. Transgenic Expression of the Anti-parasitic Factor TEP1 in the Malaria Mosquito Anopheles gambiae.

    Gloria Volohonsky

    2017-01-01

    Full Text Available Mosquitoes genetically engineered to be resistant to Plasmodium parasites represent a promising novel approach in the fight against malaria. The insect immune system itself is a source of anti-parasitic genes potentially exploitable for transgenic designs. The Anopheles gambiae thioester containing protein 1 (TEP1 is a potent anti-parasitic protein. TEP1 is secreted and circulates in the mosquito hemolymph, where its activated cleaved form binds and eliminates malaria parasites. Here we investigated whether TEP1 can be used to create malaria resistant mosquitoes. Using a GFP reporter transgene, we determined that the fat body is the main site of TEP1 expression. We generated transgenic mosquitoes that express TEP1r, a potent refractory allele of TEP1, in the fat body and examined the activity of the transgenic protein in wild-type or TEP1 mutant genetic backgrounds. Transgenic TEP1r rescued loss-of-function mutations, but did not increase parasite resistance in the presence of a wild-type susceptible allele. Consistent with previous reports, TEP1 protein expressed from the transgene in the fat body was taken up by hemocytes upon a challenge with injected bacteria. Furthermore, although maturation of transgenic TEP1 into the cleaved form was impaired in one of the TEP1 mutant lines, it was still sufficient to reduce parasite numbers and induce parasite melanization. We also report here the first use of Transcription Activator Like Effectors (TALEs in Anopheles gambiae to stimulate expression of endogenous TEP1. We found that artificial elevation of TEP1 expression remains moderate in vivo and that enhancement of endogenous TEP1 expression did not result in increased resistance to Plasmodium. Taken together, our results reveal the difficulty of artificially influencing TEP1-mediated Plasmodium resistance, and contribute to further our understanding of the molecular mechanisms underlying mosquito resistance to Plasmodium parasites.

  8. bantam miRNA is important for Drosophila blood cell homeostasis and a regulator of proliferation in the hematopoietic progenitor niche

    Lam, Victoria; Tokusumi, Tsuyoshi; Tokusumi, Yumiko; Schulz, Robert A.

    2014-01-01

    Highlights: • bantam miRNA is endogenously expressed in the hematopoietic progenitor niche. • bantam is necessary and sufficient to induce cellular proliferation in the PSC. • bantam is upstream of the Insulin Receptor signaling pathway. • A model for positive regulation of hematopoietic niche growth is proposed. - Abstract: The Drosophila hematopoietic system is utilized in this study to gain novel insights into the process of growth control of the hematopoietic progenitor niche in blood development. The niche microenvironment is an essential component controlling the balance between progenitor populations and differentiated, mature blood cells and has been shown to lead to hematopoietic malignancies in humans when misregulated. MicroRNAs are one class of regulators associated with blood malignancies; however, there remains a relative paucity of information about the role of miRNAs in the niche. Here we demonstrate that bantam miRNA is endogenously active in the Drosophila hematopoietic progenitor niche, the posterior signaling center (PSC), and functions in the primary hematopoietic organ, the lymph gland, as a positive regulator of growth. Loss of bantam leads to a significant reduction in the PSC and overall lymph gland size, as well as a loss of the progenitor population and correlative premature differentiation of mature hemocytes. Interestingly, in addition to being essential for proper lymph gland development, we have determined bantam to be a novel upstream component of the insulin signaling cascade in the PSC and have unveiled dMyc as one factor central to bantam activity. These important findings identify bantam as a new hematopoietic regulator, place it in an evolutionarily conserved signaling pathway, present one way in which it is regulated, and provide a mechanism through which it facilitates cellular proliferation in the hematopoietic niche

  9. Presence of gonadotropin-releasing hormone-like peptide in the central nervous system and reproductive organs of the male blue swimming crab, Portunus pelagicus, and its effect on spermatogenesis.

    Senarai, Thanyaporn; Saetan, Jirawat; Tamtin, Montakan; Weerachatyanukul, Wattana; Sobhon, Prasert; Sretarugsa, Prepee

    2016-08-01

    Our previous studies have demonstrated that lamprey gonadotropin-releasing hormone-III (lGnRH-III)-like peptide occurs in the central nervous system (CNS) of decapod crustaceans (Macrobrachium rosenbergii, Penaeus monodon, Portunus pelagicus), and that lGnRH-III is the most potent in stimulating ovarian maturation compared with other GnRH isoforms. In this study, we examined the localization of lGnRH-III-like peptide in the CNS and male reproductive organs of the blue swimming crab by using anti-lGnRH-III as a probe. In the brain, lGnRH-III immunoreactivity (-ir) was detected in neurons of clusters 6, 10, 11, 14/15, 16, and 17 and in many neuropils. In the subesophageal ganglion, lGnRH-III-ir was present in neurons of the dorso-lateral and ventro-medial clusters. In the thoracic ganglia, lGnRH-III-ir was observed in the large-sized neurons between the thoracic neuropils and in the ventromedial cluster of the abdominal ganglia. In the testis, lGnRH-III-ir was detected in nurse cells, hemocytes, spermatids 2, and the outer and inner zones of the acrosomes of spermatozoa. Bioassay showed that lGnRH-III significantly increased the testis-somatic index, the percentage of late stages of seminiferous tubules (stages VII-IX), the diameter of the seminiferous tubules, and the number of BrdU-labeled early germ cells compared with the control groups. Thus, lGnRH-III-like peptide exists in the male crab and possibly enhances germ cell proliferation and maturation in the testes, leading to increased sperm production.

  10. Ultrastructure of the salivary glands in Lithobius forficatus (Myriapoda, Chilopoda, Lithobiidae) according to seasonal and circadian rhythms.

    Kamińska, K; Włodarczyk, A; Sonakowska, L; Ostróżka, A; Marchewka, A; Rost-Roszkowska, M

    2016-11-01

    The salivary glands (mandibular epidermal glands) of adult males and females of Lithobius forficatus (Myriapoda, Chilopoda) were isolated during spring, summer and autumn. In addition, the organs were isolated at different times of the day - at about 12:00 (noon) and about 00:00 (midnight). The ultrastructure of these organs depending on seasonal and circadian rhythms was analyzed using transmission and scanning electron microscopy and histochemical methods. The paired salivary glands of L. forficatus are situated in the vicinity of the foregut and they are formed by numerous acini that are surrounded by the fat body, hemocytes and tracheolae. The salivary glands are composed of a terminal acinar component and a system of tubular ducts that are lined with a cuticle. The glandular part is composed of secretory epithelial cells that are at various stages of their secretory activity. The saliva that is produced by the secretory cells of the acini is secreted into the salivary ducts, which are lined with a simple epithelium that is based on the non-cellular basal lamina. The ultrastructural variations suggest that salivary glands function differently depending on seasonal rhythms and prepare the animal for overwintering. Therefore, the salivary glands of the centipedes that were analyzed participate in the accumulation of proteins, lipids and polysaccharides during the spring, summer and autumn. Subtle differences in the ultrastructure of the secretory cells of the salivary glands during the circadian cycle must be related to the physiological reactions of the organism. The salivary ducts showed no differences in the specimens that were analyzed during the day/night cycle or during the seasonal cycle. Copyright © 2016 Elsevier Ltd. All rights reserved.

  11. Development of Meteorus pulchricornis and regulation of its noctuid host, Pseudaletia separata.

    Suzuki, M; Tanaka, T

    2007-10-01

    The solitary endoparasitoid Meteorus pulchricornis can parasitize many lepidopteran host species successfully. In the case of parasitization of Pseudaletia separata, developmental duration of M. pulchricornis was 8-9 days from egg to larval emergence and 6 days from prepupa to adult emergence. Successful parasitism by M. pulchricornis decreased with host age. Following parasitization of day-0 4th host instar, the parasitoid embryo, whilst still enclosed in serosal cell membrane, hatched out of the egg chorion 2 days after oviposition. Subsequently, the 1st instar parasitoid emerged from the surrounding serosal cell membrane. Serosal cells dissociated and developed as teratocytes 3.5 days after oviposition. One embryo of M. pulchricornis gave rise to approximately 1200 teratocytes, a number that remained constant until 6 days after parasitization, but decreased drastically to 200 at 7 days post-oviposition. The teratocytes of M. pulchricornis were round- or oval-shaped and grew from 65 microm at 4 days to 200 microm in the long axis at 6 days post-parasitization. At 4 days post-parasitization, many cells or cell clusters with lipid particles were observed in the hemocoels of parasitized hosts. In addition, paraffin sections of parasitized hosts revealed that many teratocytes were attached to the host's fat body and contributed to disrupting the fat body tissue. Further, examination of the total hemocyte count (THC) during parasitization revealed that THC was maintained at low levels. Surprisingly, a temporal decrease followed by restoration of THC was observed in hosts injected with virus-like particles of M. pulchricornis (MpVLPs) plus venom, which contrasts with the constant THC suppression seen in parasitized hosts. This indicates that MpVLP function is temporal and is involved in regulation of the host during early parasitism. Therefore, teratocytes, a host regulation factor in late parasitism, could be involved in keeping THC at a low level.

  12. [Treatment of Chemotherapy Related Leukocytopenia by Oral Administration of Multiple Leucogenic Drugs Combined with G-CSF: an Experimental Study].

    Zhang, Xi-ping; Zhang, Xiang; Yang, Hong-jian; Zou, De-hong; He, Xiang-ming; Yu, Xing-fei; Li, Yong-feng

    2015-07-01

    To evaluate efficacies of three commonly used oral drugs including Berbamine Hydrochloride Tablet (B), Qijiao Shengbai Capsule (Q), and Leucogen Tablet (L) (by single drug, two drugs or three drugs) combined with granulocyte colony-stimulating factor (G-CSF) for treat ment of chemotherapy related leukocytopenia in mice. Totally 156 Kunming male mice were divided into the normal control group (A, n=24), the model group (B, n=24), the G-CSF group (C, n =24), the G-CSF+Q group (D, n=12), G-CSF+ B (E, n=12), the G-CSF+L group (F, n=12), the G-CSF + Q + B group (G, n=12), the G-CSF + Q + L group (H, n=12), the G-CSF + L + B group (I, n=12), and the G-CSF + L + Q + B (J, n=12). Mouse models of chemotherapy related leukocytopenia were established by intraperitoneal injection of cyclophosphamide (CTX). A G-CSF group was set up as a positive control. Mice were treated by a single oral drug, a single oral drug combined with G-CSF, and two or three drugs combined with G-CSF respectively, and the death rate calculated. Hemocytes [such as white blood cells (WBC) and its classification, red blood cells (RBC), platelet (PLT), hemoglobin (Hb)] were calculated by hematology analyzer. Mice were anatomized and important organs weighed. Organ indices were calculated. There was no statistical difference in the mortality rate among all groups (P > 0.05). Compared with Group B, WBC was elevated in all other groups (P drug B and L (P chemotherapy related leukopenia or decreased three blood series was to administrate three commonly oral drugs combined with G-CSF. Authors speculated that G-CSF and Q might have a certain effect on CTX induced immune inhibition.

  13. A novel biomarker for marine environmental pollution of pi-class glutathione S-transferase from Mytilus coruscus.

    Liu, Huihui; He, Jianyu; Zhao, Rongtao; Chi, Changfeng; Bao, Yongbo

    2015-08-01

    Glutathione S-transferases (GSTs) are the superfamily of phase II detoxification enzymes that play crucial roles in innate immunity. In this study, a pi-class GST homolog was identified from Mytilus coruscus (named as McGST1, KC525103). The full-length cDNA sequence of McGST1 was 621bp with a 5' untranslated region (UTR) of 70bp and a 3'-UTR of 201bp. The deduced amino acid sequence was 206 residues in length with theoretical pI/MW of 5.60/23.72kDa, containing the conserved G-site and diversiform H-site. BLASTn analysis and phylogenetic relationship strongly suggested that this cDNA sequence was a member of pi class GST family. The prediction of secondary structure displayed a preserved N-terminal and a C-terminal comprised with α-helixes. Quantitative real time RT-PCR showed that constitutive expression of McGST1 was occurred, with increasing order in mantle, muscle, gill, hemocyte, gonad and hepatopanc