Hemocyte Gene Expression Research Articles

Differential responses of selectively bred mussels (Perna canaliculus) to heat stress-survival, immunology, gene expression and microbiome diversity.

New Zealand's green-lipped mussel (Perna canaliculus) is an ecologically and economically important species. Marine heatwaves are increasing in frequency around NZ's coastline, and these events are correlated with increased stress and mortality of some aquaculture species. This study aimed to identify general biomarkers of heat stress in P. canaliculus and to assess whether responses differed between genetically distinct selectively bred mussels. We exposed three families of selectively bred mussels (families A, B and C) to three seawater temperature regimes in the laboratory: 1) a "control" treatment (ambient 12°C), 2) a 26°C heat challenge with a subsequent recovery period, and 3) a sustained 26°C heat challenge with no recovery. We investigated whether the survival, immune response (hemocyte concentration and viability, oxidative stress and total antioxidant capacity), hemocyte gene expression and gill microbiome differed between the families during the temperature challenges. In the sustained heat-stress treatment, family A had the highest survival rate (42% compared with 25% and 5% for families C and B, respectively). Gene expression levels significantly shifted during thermal stress and differed between families, with family A more dissimilar than families B and C. Family C had substantially more genes impacted by temperature treatment and timepoint than the other families, while family B had very little genes/pathways that responded to thermal stress. Genes related to heat shock proteins and immune responses (e.g., AIF1, CTSC, TOLL8, CASP9, FNTA, AHCY, CRYAB, PPIF) were upregulated in all families during heat stress. Microbiome species-richness differed between families before and during heat-stress, with family A having a distinctly different microbiome flora than the other families. Microbial diversity changed similarly in all families exposed to prolonged heat-stress, with species of Vibrio and Campylobacter increasing in these mussels. Our study highlights the use of non-lethal sampling of hemocytes as a diagnostic tool to explore the immune response and gene expression of selectively bred mussels, to predict their response to ocean warming. This approach can identify potential thermotolerant candidates for further selective breeding, which may increase the resilience of the mussel aquaculture industry in a warming ocean.

Effects of copper pyrithione (CuPT) on apoptosis, ROS production, and gene expression in hemocytes of white shrimp Litopenaeus vannamei

Copper pyrithione (CuPT) is used globally to prevent biofouling. However, it poses certain risks to aquatic ecosystems. To understand the effects of CuPT on Litopenaeus vannamei after exposure to different concentrations of CuPT (0, 64, and 128 μg/L), the apoptotic cell ratio, production of reactive oxygen species (ROS), and gene expression in the hemocytes were studied at 0, 3, 12, 24, and 48 h. The results revealed that ROS production was induced significantly at 3–48 h only in the 128 μg/L groups. The apoptotic cell ratio was increased significantly at 12 and 24 h in the 64 μg/L groups, and at 3–48 h in the 128 μg/L groups. Meanwhile, CuPT exposure changed gene expression in hemocytes at different levels. In the 64 μg/L groups, the expression of Mn-superoxide dismutase (MnSOD) was induced at 12 h, glutathione peroxidase (GPx) was induced at 24 and 48 h, caspase-3 induced at 24 h, metallothionein (MT) and HSP70 were increased at 3 h. In the 128 μg/L groups, MnSOD was increased at 3 h and then decreased at 12–48 h, GPx was up-regulated at 3, 24 h and then decreased at 48 h, caspase-3 was increased at 24 h, MT was increased at 3–48 h, HSP60 and HSP70 were up-regulated at 3–12 h. These results indicated that CuPT induced ROS production and the expression of caspase-3 in hemocytes, then caused hemocyte apoptosis. Expression levels of MnSOD, GPx, MT, HSP60, and HSP70 were up-regulated to protect the hemocyte against CuPT stress.

Dual VP28 and VP37 dsRNA encapsulation in IHHNV virus-like particles enhances shrimp protection against white spot syndrome virus

Accumulative evidence of using double stranded (ds) RNA encapsulated into virus like particle (VLP) nanocarrier has open feasibility to fight against shrimp viral infection in aquaculture field. In this study, we co-encapsulated VP37 and VP28 dsRNA into hypodermal and hematopoietic necrosis virus (IHHNV) like particle and investigated its protection against white spot syndrome virus (WSSV). Five micrograms of each dsRNA were used as starting materials to load into VLP, while the loading efficiency was slightly different, i.e, VP37 dsRNA had somewhat a better load into VLP's cavity. It was apparent that co-encapsulation of dual dsRNA showed a superior WSSV silencing ability than the single dsRNA counterpart as evidence by the lower WSSV gene expression and its copy number in the gill tissues. Besides, we also demonstrated that co-encapsulated dual dsRNA into IHHNV-VLP stimulated the increased number of hemocytes and the corresponding PO activity as well as up-regulated proPO gene expression in hemocytes to resist viral invasion after an acute stage of WSSV infection. This synergistic action of dual dsRNA encapsulated into IHHNV-VLPs could thus act to delay time of shrimp death and reduced shrimp cumulative mortality greater than the single, naked dsRNA treatment and positive control groups. The obtaining results would encourage the feasibility to use it as a new weapon to fight WSSV infection in shrimp aquaculture.

Effects of Eicosanoid Biosynthesis Inhibitors on the Prophenoloxidase Transcript Level inBombyxmoriL

Abstract The silkworm, Bombyxmori L., has developed various defense mechanisms that reducethe negative effects of adverse environmental conditions. However, the mechanisms by which many physiological and ecological aspects work, including the functioning of the prophenoloxidase (pro-PO) system in Bombyxmori (BmPPO), are not completely understood. In our previous study, we found that eicosanoids mediated the innate immunity of the Chinese oak silkworm, Antheraeapernyi. To investigate the role of the pro-PO system in the silk-producing insect, Bombyxmori, the transcripts and activity of BmPPO were studied. We found that lipopolysaccharides (LPS) could induce the transcripts of BmPPO1, while eicosanoids could mediatethe induction of BmPPO1 gene expression in hemocytes. Most interestingly, the expression of BmPPO2 had no response to LPS challenge and treatment with eicosanoids. These results support the hypothesis that the B.mori pro-POsystem is associated with the eicosanoids signaling pathway.

Molecular Profiling of Phagocytic Immune Cells in Anopheles gambiae Reveals Integral Roles for Hemocytes in Mosquito Innate Immunity

The innate immune response is highly conserved across all eukaryotes and has been studied in great detail in several model organisms. Hemocytes, the primary immune cell population in mosquitoes, are important components of the mosquito innate immune response, yet critical aspects of their biology have remained uncharacterized. Using a novel method of enrichment, we isolated phagocytic granulocytes and quantified their proteomes by mass spectrometry. The data demonstrate that phagocytosis, blood-feeding, and Plasmodium falciparum infection promote dramatic shifts in the proteomic profiles of An. gambiae granulocyte populations. Of interest, large numbers of immune proteins were induced in response to blood feeding alone, suggesting that granulocytes have an integral role in priming the mosquito immune system for pathogen challenge. In addition, we identify several granulocyte proteins with putative roles as membrane receptors, cell signaling, or immune components that when silenced, have either positive or negative effects on malaria parasite survival. Integrating existing hemocyte transcriptional profiles, we also compare differences in hemocyte transcript and protein expression to provide new insight into hemocyte gene regulation and discuss the potential that post-transcriptional regulation may be an important component of hemocyte gene expression. These data represent a significant advancement in mosquito hemocyte biology, providing the first comprehensive proteomic profiling of mosquito phagocytic granulocytes during homeostasis blood-feeding, and pathogen challenge. Together, these findings extend current knowledge to further illustrate the importance of hemocytes in shaping mosquito innate immunity and their principal role in defining malaria parasite survival in the mosquito host.

Survival and immune-related gene expression in Litopenaeus vannamei co-infected with WSSV and Vibrio parahaemolyticus

In the present study, survival and immune-related gene expression were investigated in Litopenaeus vannamei co-infected with white spot syndrome virus (WSSV) and Vibrio parahaemolyticus by ingestion and immersion, respectively. The expression of five immune-related genes (translationally controlled tumor protein [TCTP], toll receptor [LvToll], penaeidin4, crustin, and lysozyme) in hemocytes of experimental shrimp was measured by real-time quantitative PCR. Shrimp infected first with V. parahaemolyticus and then with WSSV showed a mortality of 46.66±0.88%. Shrimp infected first with WSSV and then with V. parahaemolyticus showed a mortality of 45.00±1.53%. Shrimp infected with WSSV and V. parahaemolyticus at the same time showed a mortality of 55.00±2.08%. Gene expression of LvToll, crustin, and lysozyme was upregulated whereas gene expression of TCTP and penaeidin4 was downregulated. Under the present experimental conditions and infection routes, V. parahaemolyticus was the main responsible for the mortality of cultured white shrimp. Regarding immune response, significant changes were found in gene expression in hemocytes of L. vannamei during the time-course of a successful immune response to pathogens V. parahaemolyticus and WSSV. Statement of relevanceBacterial and viral co-infection in white shrimp culture.

Immune response and gene expression in hemocytes of Portunus trituberculatus inoculated with the parasitic dinoflagellate Hematodinium

The parasitic dinoflagellates in the genus of Hematodinium infect broad range of crustaceans around the world, causing fatal diseases in multiple species of wild and cultured crabs and subsequent economic loss. In order to explore this host–parasite interaction in the early development stage of infection, mRNA transcript levels of eight key immune-related genes, including LGBP, proPO, PPAF, serpin, α2m, and three PTcSPs were quantitatively assessed in Portunus trituberculatus artificially inoculated with the Hematodinium parasites. The fluctuation of proPO gene expression indicated that the host proPO system was disturbed overtly due to the intrusion of the parasites. And as manifested by the suppressed expression of LGBP and PPAF, an immunosuppressive mechanism was likely induced by the parasites against being entrapped or killed by the host proPO system. Furthermore, the significant variations of the transcript levels of serpin, α2m, and the three PTcSPs suggested that the parasites affected the proteinase cascade reactions associated with the immune response by destroying the balance between serine proteinases and the proteinase inhibitors. The hemocytes counts and PO activity varied accordingly over the time course of infection, showing that hemocytes were actively involved in the immune response against the parasitic invasion. This study primarily highlighted the anti-parasitic immune response of crab hosts, and presented the first report of the immune response of P. trituberculatus to the parasitic dinoflagellate Hematodinium.

Molecular cloning and expression of IRAK-4, IL-17 and I-κB genes in Haliotis rufescens challenged with Vibrio anguillarum

The candidate genes interleukin-1 receptor associated kinase 4 (IRAK-4), Interleukin 17 (IL-17) and Inhibitor of NF-κB (I-κB) were cloned and evaluated in Californian abalone (Haliotis rufescens) hemocytes in response to Vibrio anguillarum. Molecular characterization evidenced that HrI-κB has a full cDNA sequence of 3027 bp with an encoding region of 401 amino acids (aa), HrIRAK-4 comprised 1969 bp that encoded for 516 aa, and Hr-IL17 had a full sequence of 806 bp encoding for 165 aa. qPCR analysis showed the higher constitutive expression level of Hr-IL17 in hemocytes; meanwhile Hr-IκB and Hr-IRAK4 gene expression levels were higher in gills and mantle. The assessment of gene expression in hemocytes after infection with V. anguillarum evidences the immune responses of Hr-IκB, Hr-IRAK4, and Hr-IL17 and their relationships through the NF-κB signaling pathway.

Molecular signatures at imminent death: Hemocyte gene expression profiling of shrimp succumbing to viral and fungal infections

Infectious diseases represent the most serious threat to shrimp farming worldwide. Understanding the molecular mechanisms driving shrimp-pathogen interactions is necessary for developing strategies to control disease outbreaks in shrimp production systems. In the current study, we experimentally reproduced mortality events using standardized infections to characterize the hemocyte transcriptome response of the shrimp Litopenaeus vannamei succumbing to infectious diseases. By using a high-throughput microfluidic RT-qPCR approach, we identified molecular signatures in shrimp during lethal infections caused by the White Spot Syndrome Virus (WSSV) or the filamentous fungus Fusarium solani. We successfully identified gene expression signatures shared by both infections but also pathogen-specific gene responses. Interestingly, whereas lethal WSSV infection induced the expression of antiviral-related genes, the transcript abundance of many antimicrobial effectors was reduced by lethal F. solani infection. To our knowledge, this is the first report of the immune-gene repertoire of infected shrimp at the brink of death.

Accumulation of Enterococcus faecalis and variation of SOD and ACP activities in scallop Pactinopecten yessoensis tissues

Enterococcus faecalis is a common bacterium found in aquatic environment and intestines of warm-blooded animals and human.In summer,it can reach high level in sewage-polluted seawater and be accumulated by scallops.Large numbers of bacteria may constitute significant health risk to cultured animals at high temperature or in other specific conditions.However,the retention of E.faecalis in scallop remains unclear,and information about hosts' growth performance,innate immunity and disease outbreak after a long-term exposure to non-pathogenic fecal bacteria is limited.The present study aims to investigate the relationship between retention of an opportunistic bacterium and variation in immune enzyme activity in the scallop,Pactinopecten yessoensis.The findings of this study will be useful for developing aquaculture probiotics and will provide scientific basis for sustainable aquaculture of bivalves. By bath exposure,the accumulation and distribution of E.faecalis in scallop tissues were analyzed,and the variation in activities of two immunity-related enzymes in haemolymph were also detected.After exposure to seawater inoculum of 5 lg(CFU/mL) E.faecalis for two weeks,the uptake of bacteria at day 1,3,5,7 and 14 and their distribution in scallop tissues were detected by plate counting method respectively.The activity of acid phosphatase(ACP) and superoxide dismutase(SOD) were also determined both in haemocytes and in serum of P.yessoensis.The uptake results showed that at 7 d top level of E.faecalis[5.20 lg(CFU/g)]were tested in scallop tissues,but at 14 d it decreased to the level of 3 d after treatment.However,the rate of bacterial accumulation(RA) was the highest at 1 d,and then significantly decreased(P0.05).It indicated that the bacterial accumulation rate in scallops decreased with exposure time.The top level of E.faecalis,over ten times higher than the average density of total tissues,was observed in digestive tract,mantle lobes and gills,but the lowest was detected in haemolymph,over 100 times lower than the average.SOD and ACP activities in cell-free haemolymph significantly increased to the maximum at 7 d,and then decreased.It can be deduced that there was a positive correlation between enzyme activities in serum and the bacterial accumulation in scallop(P0.05).Soluble factors in serum were more sensitive to bacterial accumulation than those in haemocytes,therefore,the serum may be a chief part of innate immune system of bivalves to digest and kill invading bacteria.Bivalves have an open circulatory and innate immune system.Haemocytes and soluble factors operate in a coordinated way to provide protection from invading microorganisms,and different types of haemocytes play various roles in innate immunity.Understanding the immune response in bivalves to invasive infections at cellular and molecular levels offers a theoretical base for exploring molluscan probiotics.Further study on variations in activities and gene expression in haemocytes and the roles of different scallop tissues in immune system is required.

Cloning and tissue expression of hemocyanin gene in Cherax quadricarinatus during white spot syndrome virus infection

The role of hemocyanin in terms of immune defense was explored in Cherax quadricarinatus infected by WSSV. The cDNA of a hemocyanin gene (CqHC) was cloned from the hepatopancreas of C. quadricarinatus (crayfish) by the Rapid Amplification Complementary DNA Ends (RACE) method. The full-length cDNA of CqHC consisted of 2167bp with a 2043bp open reading frame encoding a 680 amino acid protein with a predicted signal peptide of 19 amino acids. The deduced CqHC protein sequence was found to contain two conserved copper-binding sites and to share 74%, 73%, 72%, 71% and 71% similarity with Penaeus monodon, Marsupenaeus japonicus, Fenneropenaeus chinensis, Metacarcinus magister and Litopenaeus vannamei, respectively. Phylogenetic analysis revealed that CqHC and HC from F. chinensis, M. japonicus and P. monodon clustered in the same phylogenetic branch. Real-time PCR analysis showed that CqHC was widely distributed, with the highest level in hepatopancreas, at lower amounts in hemocytes, gills and antennal gland. HC levels in hepatopancreas, hemocytes and gills from C. quadricarinatus were also compared after white spot syndrome virus (WSSV) infection. After 24h of infection, CqHC levels rose significantly higher than that of the controls (P<0.05) but then sharply declined over the infection period. The HC gene expression in hemocytes and gills (but not hepatopancreas) of crayfish injected with polysaccharides before infection were higher than that of the non-polysaccharide injected infected groups, showing an immunoprotective rate of 51.86% at 7days after exposure, indicating that polysaccharide can act as immunopotentiator and could improve the antiviral ability of C. quadricarinatus against WSSV.

VENOM OF THE ECTOPARASITOID, NASONIA VITRIPENNIS, INFLUENCES GENE EXPRESSION IN Musca domestica HEMOCYTES

Insect hosts have evolved potent innate immunity against invasion by parasitoid wasps. Host/parasitoids live in co-evolutionary relationships. Nasonia vitripennis females inject venom into their dipteran hosts just prior to laying eggs on the host's outer integument. The parasitoid larvae are ectoparasitoids because they feed on their hosts within the puparium, but do not enter the host body. We investigated the influence of N. vitripennis venom on the gene expression profile of hemocytes of their hosts, pupae of the housefly, Musca domestica. We prepared venom by isolating venom glands and treated experimental host pupae with venom. We used suppression subtractive hybridization (SSH) to determine the influence of venom on hemocyte gene expression. At 1 h post treatment, we recorded decreases in transcript levels of 133 EST clones derived from forward a subtractive library of host hemocytes and upregulation in transcript levels of 111 EST clones from the reverse library. These genes are related to immune and stress response, cytoskeleton, cell cycle and apoptosis, metabolism, transport, and transcription/translation regulation. We verified the reliability of our data with reverse transcription quantitative real-time PCR analysis of randomly selected genes, and with assays of enzyme activities. These analyses showed that the expression level of all selected genes were downregulated after venom treatment. Outcomes of our experiments support the hypothesis that N. vitripennis venom influences the gene expression in host hemocytes. We conclude that the actions of venom on host gene expression influence host biology in ways that benefit the development and emergence of the next generation of parasitoids.

Early differential gene expression in haemocytes from resistant and susceptible Biomphalaria glabrata strains in response to Schistosoma mansoni.

The outcome of infection in the host snail Biomphalaria glabrata with the digenean parasite Schistosoma mansoni is determined by the initial molecular interplay occurring between them. The mechanisms by which schistosomes evade snail immune recognition to ensure survival are not fully understood, but one possibility is that the snail internal defence system is manipulated by the schistosome enabling the parasite to establish infection. This study provides novel insights into the nature of schistosome resistance and susceptibility in B. glabrata at the transcriptomic level by simultaneously comparing gene expression in haemocytes from parasite-exposed and control groups of both schistosome-resistant and schistosome-susceptible strains, 2 h post exposure to S. mansoni miracidia, using an novel 5K cDNA microarray. Differences in gene expression, including those for immune/stress response, signal transduction and matrix/adhesion genes were identified between the two snail strains and tests for asymmetric distributions of gene function also identified immune-related gene expression in resistant snails, but not in susceptible. Gene set enrichment analysis revealed that genes involved in mitochondrial electron transport, ubiquinone biosynthesis and electron carrier activity were consistently up-regulated in resistant snails but down-regulated in susceptible. This supports the hypothesis that schistosome-resistant snails recognize schistosomes and mount an appropriate defence response, while in schistosome-susceptible snails the parasite suppresses this defence response, early in infection.

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

BackgroundThe 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.ResultsUsing 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).ConclusionsWe 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.

Biochemical intervention of ergothioneine-rich edible mushroom ( Flammulina velutipes) extract inhibits melanosis in crab ( Chionoecetes japonicus)

We investigated the effects of decreasing phenoloxidase (PO) activity and prophenoloxidase ( proPO) gene expression on the inhibition of postharvest melanosis formation in the red queen crab, Chionoecetes japonicus. The cDNA of proPO from hemocytes of C. japonicus was partially cloned and sequenced. Immersion of live crabs in a 1.0% ergothioneine (ESH)-rich mushroom extract ( Flammulina velutipes; ME) solution resulted in significant inhibition of haemolymph PO activity and a reduction of the proPO gene expression in hemocytes that consequently controlled melanosis in the crabs during ice storage. Treatments with a 0.05% w/v sodium sulphite solution or a 0.05% w/v 4-hexyl-1,3-benzenediol solution had similar positive effects as the treatment with a 1.0% ME solution in vivo. In vitro experiments showed that authentic l-(+)-ESH inhibited PO activity and decreased proPO gene expression in crab hemocytes. Thus, the application of ESH-rich ME can be a novel alternative to synthetic melanosis-inhibiting agents to control postharvest melanosis in crabs.

Cloning and expression pattern analysis of a lipopolysaccharide -and β-1,3-glucan-binding protein in Portunus trituberculatus

The lipopolysaccharide -and beta-1,3-glucan-binding protein (LGBP) is a pattern recognition receptor, which is fundamental for the innate immune response of crustaceans. A LGBP gene was cloned from the haemocytes of Portunus trituberculatus using SMART RACE methods. The full-length LGBP cDNA (1 378 bp) had a 1 095 bp open reading frame encoding a protein of 365 amino acid residues including a 16 amino acid residues signal peptide, a 138 bp 5' untranslated region (UTR) and a 144 bp untranslated region in the 3' UTR with a 29 bp polyA tail. The calculated molecular mass of the mature protein (349 amino acid residues) is 39,825.24 with an estimated pI of 4.49. The gene sequence and secondary structure of LGBP were analyzed by bio-informatics. Additionally, a Glyco hydro 16 domain was identified. The expression of P. trituberculatus in various tissues were detected through RT-PCR methods. The results showed that the LGBP gene expressed in all the tissues detected, including haemocytes, hepatopancreas, heart, gills and muscle. In response to the challenge of Staphyloccocus aureus and Vibrio alginolyticus, the LGBP gene expression in haemocytes of the group challenged with mixed bacteria were higher than the control group within 48 h. It suggested that the LGBP gene plays an active role in immunologic process against bacterial infection.

Differential gene expression in White Spot Syndrome Virus (WSSV)-infected naïve and previously challenged Pacific white shrimp Penaeus ( Litopenaeus) vannamei

White Spot Syndrome Virus (WSSV) is the most devastating viral disease affecting cultivated shrimp around the world. The CENIACUA breeding programme included host resistance as strategy to control WSSV. Since 1999, all families were subjected to WSSV challenge tests and up to 5% of the animals survived. We suspect that a unique genetic profile could be responsible for survival. By Subtractive Suppression Hybridization improved with Mirror Orientation Selection we compared gene expression in haemocytes of WSSV-infected shrimp; seven were previously challenged (pre-challenged) and 10 naïve. Out of 1167 clones, 993 (85%) were obtained from the WSSV-infected pre-challenged library, while 174 (15%) were from the WSSV-infected naïve library. Differential expression was confirmed by hybridization using PCR DIG-labelled probes from unsubtracted cDNA. The 216 (18.5%) clones differentially expressed in the WSSV-infected pre-challenged library and 23 (2%) in the WSSV-infected naïve animals were sequenced. Relative expression of relevant gene products from both libraries was confirmed by reverse transcription real time PCR. Genes related to shrimp immune response, such as the antimicrobial peptide penaeidin, crustin, C-type lectin, protease inhibitor and chitin binding domain-containing protein were found in the library of pre-challenged shrimp. On the other hand, genes from the naïve library coded for viral structural and functional proteins. To our knowledge, this is the first report demonstrating that penaeidin-3 isoforms and crustin are over expressed in haemocytes of WSSV-infected pre-challenged P. vannamei.

Biomphalaria glabrata transcriptome: cDNA microarray profiling identifies resistant- and susceptible-specific gene expression in haemocytes from snail strains exposed to Schistosoma mansoni.

BackgroundBiomphalaria glabrata is an intermediate snail host for Schistosoma mansoni, one of the important schistosomes infecting man. B. glabrata/S. mansoni provides a useful model system for investigating the intimate interactions between host and parasite. Examining differential gene expression between S. mansoni-exposed schistosome-resistant and susceptible snail lines will identify genes and pathways that may be involved in snail defences.ResultsWe have developed a 2053 element cDNA microarray for B. glabrata containing clones from ORESTES (Open Reading frame ESTs) libraries, suppression subtractive hybridization (SSH) libraries and clones identified in previous expression studies. Snail haemocyte RNA, extracted from parasite-challenged resistant and susceptible snails, 2 to 24 h post-exposure to S. mansoni, was hybridized to the custom made cDNA microarray and 98 differentially expressed genes or gene clusters were identified, 94 resistant-associated and 4 susceptible-associated. Quantitative PCR analysis verified the cDNA microarray results for representative transcripts. Differentially expressed genes were annotated and clustered using gene ontology (GO) terminology and Kyoto Encyclopaedia of Genes and Genomes (KEGG) pathway analysis. 61% of the identified differentially expressed genes have no known function including the 4 susceptible strain-specific transcripts. Resistant strain-specific expression of genes implicated in innate immunity of invertebrates was identified, including hydrolytic enzymes such as cathepsin L, a cysteine proteinase involved in lysis of phagocytosed particles; metabolic enzymes such as ornithine decarboxylase, the rate-limiting enzyme in the production of polyamines, important in inflammation and infection processes, as well as scavenging damaging free radicals produced during production of reactive oxygen species; stress response genes such as HSP70; proteins involved in signalling, such as importin 7 and copine 1, cytoplasmic intermediate filament (IF) protein and transcription enzymes such as elongation factor 1α and EF-2.ConclusionProduction of the first cDNA microarray for profiling gene expression in B. glabrata provides a foundation for expanding our understanding of pathways and genes involved in the snail internal defence system (IDS). We demonstrate resistant strain-specific expression of genes potentially associated with the snail IDS, ranging from signalling and inflammation responses through to lysis of proteinacous products (encapsulated sporocysts or phagocytosed parasite components) and processing/degradation of these targeted products by ubiquitination.

Differentially enhanced gene expression in hemocytes from Macrobrachium rosenbergii challenged in vivo with lipopolysaccharide

In order to better understand the immune response in prawns after treatment with the immunostimulant lipopolysaccharide (LPS), in this study, the differential gene expression of the hemocytes from LPS-injected versus non-injected prawns ( Macrobrachium rosenbergii) were isolated and identified using suppression subtractive hybridization (SSH). The hemocytes were extracted after treatment for 1, 6, and 12 h. The upregulated genes (i.e., where gene expression was elevated) were identified and could be divided into four classes on the basis of physiological function: genes concerning defense-related molecules, genes involved in energy-production (respiration), genes related to protein synthesis and folding, and genes with unknown function. The time-course for gene expression indicated that, except for expression of the gene anti-microbial peptide ( amp), which was increased at 12 h after LPS treatment, the expression of the other two immune-related genes was much earlier (at 1 h), including alpha-2-macroglobulin ( α2-M) and Mas-like protein ( mas). These results suggest that in the early phase of LPS stimulation some immune reactions regulated by α-2M and Mas may be induced, such as the activation of prophenoloxidase activating system, opsonization, and anti-microbial activity. In addition, six unigenes with unproven function were particularly interesting and worthy of further study because their expression in LPS-treated hemocytes was clearly enhanced.

Combination of a pesticide exposure and a bacterial challenge: In vivo effects on immune response of Pacific oyster, Crassostrea gigas (Thunberg)

To assess the impact of pollution induced by pesticides on Pacific oyster, Crassostrea gigas, health in France, in vivo effects of combined pesticide exposure and bacterial challenge on cell activities and gene expression in hemocytes were tested using flow cytometry and real-time PCR. As a first step, an in vivo model of experimental contamination was developed. Pacific oysters were exposed to a mixture of eight pesticides (atrazine, glyphosate, alachlor, metolachlor, fosetyl-alumimium, terbuthylazine, diuron and carbaryl) at environmentally relevant concentrations over a 7-day period. Hemocyte parameters (cell mortality, enzyme activities and phagocytosis) were monitored using flow cytometry and gene expression was evaluated by real-time PCR (RT-PCR). The expression of 19 genes involved in C. gigas hemocyte functions was characterized using RT-PCR. After 7 days of exposure, phagocytosis was significantly reduced and the 19 selected genes were down-regulated in treated animals. As a second step, the experimental contamination method previously developed was used to study interactions between pesticide exposure and bacterial challenge by intramuscular injection of two Vibrio splendidus-related pathogenic strains. Oyster mortality and expression of 10 of the 19 selected genes were followed 4 and 24 h post-injection. Oyster mortality was higher in pesticide-treated oysters compared to untreated oysters after the bacterial challenge. Gene expression was up-regulated in pesticide-treated oysters compared to untreated oysters after the bacterial challenge. We hypothesize that gene over-expression due to an interaction between pesticides and bacteria could lead to an injury of host tissues, resulting in higher mortality rates. In conclusion, this study is the first to show effects of pesticides at environmentally relevant concentrations on C. gigas hemocytes and to hypothesize that pesticides modulate the immune response to a bacterial challenge in oysters.