Transcriptional changes in innate immunity genes in head kidneys from Aeromonas salmonicida-challenged rainbow trout fed a mixture of polycyclic aromatic hydrocarbons.

We previously observed that exposure to a complex mixture of high molecular weight polycyclic aromatic hydrocarbons (PAHs) increased sensitivity of rainbow trout (Oncorhynchus mykiss) to subsequent challenge with Aeromonas salmonicida, the causative agent of furunculosis. In this study, we evaluate potential mechanisms associated with disease susceptibility from combined environmental factors of dietary PAH exposure and pathogen challenge. Rainbow trout were fed a mixture of ten high molecular weight PAHs at an environmentally relevant concentration (7.82µg PAH mixture/g fish/day) or control diet for 50 days. After 50 days of PAH exposure, fish were challenged with either Aeromonas salmonicida at a lethal concentration 30 (LC30) or growth media without the pathogen (mock challenge). Head kidneys were collected 2, 4, 10 and 20 days after challenge and gene expression (q<0.05) was evaluated among treatments. In animals fed the PAH contaminated diet, we observed down-regulation of expression for innate immune system genes in pathways (p<0.05) for the terminal steps of the complement cascade (complement component C6) and other bacteriolytic processes (lysozyme type II) potentially underlying increased disease susceptibility after pathogen challenge. Increased expression of genes associated with hemorrhage/tissue remodeling/inflammation pathways (p<0.05) was likely related to more severe head kidney damage due to infection in PAH-fed compared to control-fed fish. This study is the first to evaluate transcriptional signatures associated with the impact of chronic exposure to an environmentally relevant mixture of PAHs in disease susceptibility and immunity.

Successful parasitism of vector snail Biomphalaria glabrata by the human blood fluke (trematode) Schistosoma mansoni: a 2009 assessment.

Schistosomiasis, caused by infections by human blood flukes (Trematoda), continues to disrupt the lives of over 200,000,000 people in over 70 countries, inflicting misery and precluding the individuals' otherwise reasonable expectations of productive lives. Infection requires contact with freshwater in which infected snails (the intermediate hosts of schistosomes) have released cercariae larvae. Habitats suitable for the host snails continue to expand as a consequence of water resource development. No vaccine is available, and resistance has emerged towards the single licensed schistosomicide drug. Since human infections would cease if parasite infections in snails were prevented, efforts are being made to discover requirements of intra-molluscan development of these parasites. Wherever blood flukes occur, naturally resistant conspecific snails are present. To understand the mechanisms used by parasites to ensure their survival in immunocompetent hosts, one must comprehend the interior defense mechanisms that are available to the host. For one intermediate host snail (Biomphalaria glabrata) and trematodes for which it serves as vector, molecular genetic and proteomic surveys for genes and proteins influencing the outcomes on infections are yielding lists of candidates. A comparative approach drawing on data from studies in divergent species provides a robust basis for hypothesis generation to drive decisions as to which candidates merit detailed further investigation. For example, reactive oxygen and nitrogen species are known mediators or effectors in battles between infectious agents and their hosts. An approach targeting genes involved in relevant pathways has been fruitful in the Schistosoma mansoni -- B. glabrata parasitism, leading to discovery of a functionally relevant gene set (encoding enzymes responsible for the leukocyte respiratory burst) that associates significantly with host resistance phenotype. This review summarizes advances in the understanding of strategies used by both this trematode parasite and its molluscan host to ensure their survival.

Clearance of schistosome parasites by resistant genotypes at a single genomic region in Biomphalaria glabrata snails involves cellular components of the hemolymph.

Schistosomiasis is one of the most detrimental neglected tropical diseases. Controlling the spread of this parasitic illness requires effective sanitation, access to chemotherapeutic drugs, and control over populations of the freshwater snails, such as Biomphalaria glabrata, that are essential intermediate hosts for schistosomes. Effectively controlling this disease, while minimising ecological implications of such control, will require an extensive understanding of the immunological interactions between schistosomes and their molluscan intermediate hosts. Here we histologically characterise the clearance of schistosome larvae by snails that exhibit allelic variation at a single genomic region, the Guadeloupe resistance complex. We show that snails with a resistant Guadeloupe resistance complex genotype clear schistosomes within the first 24-48 h, and that this resistance can be transferred to susceptible snails via whole hemolymph but not cell-free plasma. These findings imply that Guadeloupe resistance complex-coded proteins help to coordinate hemocyte-mediated immune responses to schistosome infections in Guadeloupean snails.

Variation in expression of Biomphalaria glabrata SOD1: a potential controlling factor in susceptibility/resistance to Schistosoma mansoni.

The snail Biomphalaria glabrata kills the blood fluke Schistosoma mansoni by a mechanism involving production of hydrogen peroxide, the enzymatic product of cytosolic Cu/Zn superoxide dismutase (SOD1). This enzyme exhibits higher activity in blood cells (hemocytes) from a predominantly resistant strain of B. glabrata than in hemocytes from a susceptible strain. Additionally, B. glabrata SOD1 polymorphisms have been associated with susceptibility/resistance to the parasite. To address the hypothesis that SOD1 transcription levels differ in accordance with variation at the SOD1 locus, quantitative PCR was performed using hemocyte-derived cDNA prepared from SOD1-genotyped snails. Here we report that individuals possessing the allele previously associated with resistance to S. mansoni express significantly higher levels of hemocyte SOD1 transcripts than individuals lacking this allele. A causal relationship between SOD1 expression and susceptibility/resistance to S. mansoni is supported by the correlation of transcript quantity with data (from a previous study) on the probability of infection.

Schistosome infectivity in the snail, Biomphalaria glabrata, is partially dependent on the expression of Grctm6, a Guadeloupe Resistance Complex protein.

Schistosomiasis is one of the most important neglected tropical diseases. Despite effective chemotherapeutic treatments, this disease continues to afflict hundreds of millions of people. Understanding the natural intermediate snail hosts of schistosome parasites is vital to the suppression of this disease. A recently identified genomic region in Caribbean Biomphalaria glabrata snails strongly influences their resistance to infection by Schistosoma mansoni. This region contains novel genes having structural similarity to known pathogen recognition proteins. Here we elaborate on the probable structure and role of one of these genes, grctm6. We characterised the expression of Grctm6 in a population of Caribbean snails, and performed a siRNA knockdown of Grctm6. We show that this protein is not only expressed in B. glabrata hemolymph, but that it also has a role in modulating the number of S. mansoni cercariae released by infected snails, making it a possible target for the biological control of schistosomiasis.

Corrigendum: Whole genome analysis of a schistosomiasis-transmitting freshwater snail.

This corrects the article DOI: 10.1038/ncomms15451.

Whole genome analysis of a schistosomiasis-transmitting freshwater snail.

Biomphalaria snails are instrumental in transmission of the human blood fluke Schistosoma mansoni. With the World Health Organization's goal to eliminate schistosomiasis as a global health problem by 2025, there is now renewed emphasis on snail control. Here, we characterize the genome of Biomphalaria glabrata, a lophotrochozoan protostome, and provide timely and important information on snail biology. We describe aspects of phero-perception, stress responses, immune function and regulation of gene expression that support the persistence of B. glabrata in the field and may define this species as a suitable snail host for S. mansoni. We identify several potential targets for developing novel control measures aimed at reducing snail-mediated transmission of schistosomiasis.

Biomphalaria glabrata cytosolic copper/zinc superoxide dismutase (SOD1) gene: association of SOD1 alleles with resistance/susceptibility to Schistosoma mansoni.

Variation in susceptibility of the snail Biomphalaria glabrata to infection by the parasite Schistosoma mansoni is, at least in part, genetically determined. Functional studies have demonstrated that hemocyte-mediated killing of the parasite involves hydrogen peroxide, the enzymatic product of superoxide dismutase (SOD). The present study identifies alleles of the gene coding for cytosolic copper/zinc SOD (SOD1). The resistance/susceptibility phenotypes and SOD1 genotypes were determined for 354 snails of the predominantly resistant 13-16-R1 strain of B. glabrata. Resistance to the parasite was found to be significantly associated with one allele of the SOD1 gene. Conversely, a separate SOD1 allele was significantly associated with susceptibility.

Use of a rainbow trout oligonucleotide microarray to determine transcriptional patterns in aflatoxin B1-induced hepatocellular carcinoma compared to adjacent liver.

Hepatocellular carcinoma (HCC) is one of the most common malignant tumors worldwide, and its occurrence is associated with a number of environmental factors including ingestion of the dietary contaminant aflatoxin B(1) (AFB(1)). Research over the last 40 years has revealed rainbow trout (Oncorhynchus mykiss) to be an excellent research model for study of AFB(1)-induced hepatocarcinogenesis; however, little is known about changes at the molecular level in trout tumors. We have developed a rainbow trout oligonucleotide array containing 1672 elements representing over 1400 genes of known or probable relevance to toxicology, comparative immunology, carcinogenesis, endocrinology, and stress physiology. In this study, we applied microarray technology to examine gene expression of AFB(1)-induced HCC in the rainbow trout tumor model. Carcinogenesis was initiated in trout embryos with 50 ppb AFB(1), and after 13 months control livers, tumors, and tumor-adjacent liver tissues were isolated from juvenile fish. Global gene expression was determined in histologically confirmed HCCs compared to noncancerous adjacent tissue and sham-initiated control liver. We observed distinct gene regulation patterns in HCC compared to noncancerous tissue including upregulation of genes important for cell cycle control, transcription, cytoskeletal formation, and the acute phase response and downregulation of genes involved in drug metabolism, lipid metabolism, and retinol metabolism. Interestingly, the expression profiles observed in trout HCC are similar to the transcriptional signatures found in human and rodent HCC, further supporting the validity of the model. Overall, these findings contribute to a better understanding of the mechanism of AFB(1)-induced hepatocarcinogenesis in trout and identify conserved genes important for carcinogenesis in species separated evolutionarily by more than 400 million years.

Respiratory burst of Biomphalaria glabrata hemocytes: Schistosoma mansoni-resistant snails produce more extracellular H2O2 than susceptible snails.

The production of reactive oxygen species by hemocytes from the gastropod Biomphalaria glabrata has been linked to their ability to kill the trematode parasite Schistosoma mansoni. For 2 laboratory strains of the snail, 1 resistant (13-16-R1) and 1 susceptible (MO) to the PR1 strain of S. mansoni, we compared hemocyte production of extracellular hydrogen peroxide when stimulated with the protein kinase C agonist phorbol myristate acetate (PMA). The time course of the PMA-induced response is similar in both strains with respect to onset, peak production, and termination of the respiratory burst. However, the magnitude of the response differs between strains, in that hemocytes from resistant snails generate significantly more hydrogen peroxide. These findings suggest that the capacity to produce hydrogen peroxide could be critical in determining susceptibility or resistance to S. mansoni.

Genome-Wide Scan and Test of Candidate Genes in the Snail Biomphalaria glabrata Reveal New Locus Influencing Resistance to Schistosoma mansoni.

BACKGROUND: New strategies to combat the global scourge of schistosomiasis may be revealed by increased understanding of the mechanisms by which the obligate snail host can resist the schistosome parasite. However, few molecular markers linked to resistance have been identified and characterized in snails. METHODOLOGY/PRINCIPAL FINDINGS: Here we test six independent genetic loci for their influence on resistance to Schistosoma mansoni strain PR1 in the 13-16-R1 strain of the snail Biomphalaria glabrata. We first identify a genomic region, RADres, showing the highest differentiation between susceptible and resistant inbred lines among 1611 informative restriction-site associated DNA (RAD) markers, and show that it significantly influences resistance in an independent set of 439 outbred snails. The additive effect of each RADres resistance allele is 2-fold, similar to that of the previously identified resistance gene sod1. The data fit a model in which both loci contribute independently and additively to resistance, such that the odds of infection in homozygotes for the resistance alleles at both loci (13% infected) is 16-fold lower than the odds of infection in snails without any resistance alleles (70% infected). Genome-wide linkage disequilibrium is high, with both sod1 and RADres residing on haplotype blocks >2 Mb, and with other markers in each block also showing significant effects on resistance; thus the causal genes within these blocks remain to be demonstrated. Other candidate loci had no effect on resistance, including the Guadeloupe Resistance Complex and three genes (aif, infPhox, and prx1) with immunological roles and expression patterns tied to resistance, which must therefore be trans-regulated. CONCLUSIONS/SIGNIFICANCE: The loci RADres and sod1 both have strong effects on resistance to S. mansoni. Future approaches to control schistosomiasis may benefit from further efforts to characterize and harness this natural genetic variation.

Constitutive differences in Cu/Zn superoxide dismutase mRNA levels and activity in hemocytes of Biomphalaria glabrata (Mollusca) that are either susceptible or resistant to Schistosoma mansoni (Trematoda).

Genetic strains of the snail Biomphalaria glabrata vary in their resistance to the parasite Schistosoma mansoni. Phagocytic cells (hemocytes) circulating in the hemolymph of B. glabrata play an essential role in the snail's innate immune response. Hemocytes of resistant B. glabrata kill S. mansoni in vitro via a mechanism which involves a respiratory burst. Reactive oxygen species (ROS), which are products of the respiratory burst, can act as mediators of both oxidative damage and of immune-related intracellular signaling pathways. One specific ROS, hydrogen peroxide (H2O2), has been shown to be involved in hemocyte-mediated sporocyst killing. We tested the hypothesis that Cu/Zn superoxide dismutase (SOD), a cytosolic enzyme that catalyzes the conversion of superoxide anion to H2O2, is somehow different between resistant and susceptible snail strains. We report a hemocyte transcript with all the features of a typical cytosolic Cu/Zn SOD (GenBank accession numbers AY505496 and AY505497). The amount of Cu/Zn SOD mRNA in hemocytes from resistant snails was double that of hemocytes from susceptible snails, and this correlated directly with an increased Cu/Zn SOD enzymatic activity in resistant hemocytes. Additional experiments determined that in vitro interaction/encapsulation of sporocysts did not influence Cu/Zn SOD mRNA levels in hemocytes from either snail strain. Thus, resistance in this host-parasite system does not appear to depend on a transcriptional response of hemocyte Cu/Zn SOD, but may be due, at least in part, to a constitutively elevated enzymatic level of Cu/Zn SOD.

The symbiont Capsaspora owczarzaki, nov. gen. nov. sp., isolated from three strains of the pulmonate snail Biomphalaria glabrata is related to members of the Mesomycetozoea.

While investigating the resistance of some strains of Biomphalaria glabrata to infection with Schistosoma mansoni, a unicellular eukaryotic symbiont was noted in the snail haemolymph. It was similar in appearance to Nuclearia sp. reported from B. glabrata. Sequences comprising the 18S, ITS1, 5.8S, ITS2 and the beginning of the 28S rDNA gene regions were obtained from symbionts isolated from three strains of B. glabrata, and compared with the same sequences obtained from a culture of Nuclearia sp. 18S rDNA sequences were identical for all four isolates. 18S rDNA sequences were used in a phylogenetic analysis to produce minimum evolution, maximum parsimony, maximum likelihood and Bayesian trees. All four analyses indicated that the B. glabrata symbiont is not closely related to Nuclearia but instead to the Mesomycetozoea, a recently recognised clade of symbiotic eukaryotes. Based on phylogenetic analysis, life history and morphological differences, the symbiont is described as a new genus and species, Capsaspora owczarzaki. Distinguishing characters are the presence of life cycle stage(s) that occur within snail haemolymph; ability to kill and ingest digenetic trematode larvae; ability to undergo asexual fission to produce daughter cells; absence of flagella, a mucous sheath and membranes containing chitin, elastin, or collagen; and presence of long unbranching pseudopodia and a penetration process. Using both polymerase chain reaction (PCR) and culturing techniques, the S. mansoni-resistant Salvador and 13-16-R1 strains were found to be significantly more likely to harbour the symbiont than the susceptible M line strain. Small but consistent sequence differences were noted among symbiont isolates from different snail strains, raising the possibility that the symbiont has diverged in different snail lineages. This suggests further that the symbiont is not restricted to albino lab-reared snails. A role, if any, of the symbiont in resistance awaits further study.

Schistosoma mansoni sporocysts in culture: host plasma hemoglobin contributes to in vitro oxidative stress.

The initiation and promotion of sporocyst propagation and subsequent production of cercariae by intramolluscan larval stages of digenic trematodes are thought to depend on mollusc-derived factors. The ability to investigate this using in vitro cultures of Schistosoma mansoni sporocysts has been impeded by the fact that plasma from the host, Biomphalaria glabrata, becomes toxic to the parasite in long-term cultures. The present study identifies hemoglobin as the plasma component responsible for this toxicity. The addition of the enzyme catalase to sporocyst cultures neutralized the toxic effects of both purified hemoglobin and whole plasma, suggesting that the generation of H2O2 as a consequence of hemoglobin oxidation is the mechanism of plasma toxicity. Furthermore, cultures incubated in unconditioned schistosome medium with plasma plus catalase yielded significantly higher numbers of daughter sporocysts than cultures with media or plasma alone, but not higher than cultures with catalase alone. These latter results suggest that the oxidative environment and the antioxidant capacity of the media are critical factors for in vitro propagation of S. mansoni sporocysts.

Resistance of Biomphalaria glabrata 13-16-R1 snails to Schistosoma mansoni PR1 is a function of haemocyte abundance and constitutive levels of specific transcripts in haemocytes.

Continuing transmission of human intestinal schistosomiasis depends on the parasite's access to susceptible snail intermediate hosts (often Biomphalaria glabrata). Transmission fails when parasite larvae enter resistant individuals in wild snail populations. The genetic basis for differences in snail susceptibility/resistance is being intensively investigated as a means to devise novel control strategies based on resistance genes. Reactive oxygen species produced by the snail's defence cells (haemocytes) are effectors of resistance. We hypothesised that genes relevant to production and consumption of reactive oxygen species would be expressed differentially in the haemocytes of snail hosts with different susceptibility/resistance phenotypes. By restricting the genetic diversity of snails, we sought to facilitate identification of resistance genes. By inbreeding, we procured from a 13-16-R1 snail population with both susceptible and resistant individuals 52 lines of B. glabrata (expected homozygosity ~87.5%), and determined the phenotype of each in regard to susceptibility/resistance to Schistosoma mansoni. The inbred lines were found to have line-specific differences in numbers of spreading haemocytes; these were enumerated in both juvenile and adult snails. Lines with high cell numbers were invariably resistant to S. mansoni, whereas lines with lower cell numbers could be resistant or susceptible. Transcript levels in haemocytes were quantified for 18 potentially defence-related genes. Among snails with low cell numbers, the different susceptibility/resistance phenotypes correlated with differences in transcript levels for two redox-relevant genes: an inferred phagocyte oxidase component and a peroxiredoxin. Allograft inflammatory factor (potentially a regulator of leucocyte activation) was expressed at higher levels in resistant snails regardless of spread cell number. Having abundant spreading haemocytes is inferred to enable a snail to kill parasite sporocysts. In contrast, snails with fewer spreading haemocytes seem to achieve resistance only if specific genes are expressed constitutively at levels that are high for the species.

Aggregation of sea urchin phagocytes is augmented in vitro by lipopolysaccharide.

Development of protocols and media for culturing immune cells from marine invertebrates has not kept pace with advancements in mammalian immune cell culture, the latter having been driven by the need to understand the causes of and develop therapies for human and animal diseases. However, expansion of the aquaculture industry and the diseases that threaten these systems creates the need to develop cell and tissue culture methods for marine invertebrates. Such methods will enable us to better understand the causes of disease outbreaks and to develop means to avoid and remedy epidemics. We report a method for the short-term culture of phagocytes from the purple sea urchin, Strongylocentrotus purpuratus, by modifying an approach previously used to culture cells from another sea urchin species. The viability of cultured phagocytes from the purple sea urchin decreases from 91.6% to 57% over six days and phagocyte morphology changes from single cells to aggregates leading to the formation of syncytia-like structures. This process is accelerated in the presence of lipopolysaccharide suggesting that phagocytes are capable of detecting this molecular pattern in culture conditions. Sea urchin immune response proteins, called Sp185/333, are expressed on the surface of a subset of phagocytes and have been associated with syncytia-like structures. We evaluated their expression in cultured phagocytes to determine their possible role in cell aggregation and in the formation of syncytia-like structures. Between 0 and 3 hr, syncytia-like structures were observed in cultures when only ~10% of the cells were positive for Sp185/333 proteins. At 24 hr, ~90% of the nuclei were Sp185/333-positive when all of the phagocytes had aggregated into syncytia-like structures. Consequently, we conclude that the Sp185/333 proteins do not have a major role in initiating the aggregation of cultured phagocytes, however the Sp185/333 proteins are associated with the clustered nuclei within the syncytia-like structures.

Three genes involved in the oxidative burst are closely linked in the genome of the snail, Biomphalaria glabrata.

Allelic variation at the Cu-Zn superoxide dismutase (SOD1) locus has been shown to be associated with resistance of the snail, Biomphalaria glabrata, to infection by the trematode parasite, Schistosoma mansoni. SOD1 catalyses the production of hydrogen peroxide, a known cytotoxic component of the oxidative burst used in defence against pathogens. In our laboratory population of B. glabrata, the most resistant allele at SOD1 is over-expressed relative to the other two alleles. Because hydrogen peroxide also causes oxidative stress on host tissues, we hypothesised that over-expression of SOD1 might be compensated by epistatic interactions with other loci involved in oxidation-reduction (redox) pathways. Catalase, peroxiredoxins and glutathione peroxidases all degrade hydrogen peroxide. We tested whether alleles at each of these loci were in linkage disequilibrium with SOD1 in our population, as might be expected given strong epistatic selection. We found that SOD1, catalase (CAT) and a peroxiredoxin locus (PRX4) are in strong linkage disequilibrium in our population. We also found that these loci are tightly linked, within 1-2cM of each other, which explains the high linkage disequilibrium. This result raises the possibility that there is a linked cluster of redox genes, and perhaps other defence-relevant genes, in the B. glabrata genome. Whether epistatic interactions for fitness actually exist among these loci still needs to be tested. However the close physical linkage among SOD1, PRX4 and CAT, and subsequent high disequilibrium, makes such interactions a plausible hypothesis.

Effects of Cu/Zn superoxide dismutase (sod1) genotype and genetic background on growth, reproduction and defense in Biomphalaria glabrata.

Resistance of the snail Biomphalaria glabrata to the trematode Schistosoma mansoni is correlated with allelic variation at copper-zinc superoxide dismutase (sod1). We tested whether there is a fitness cost associated with carrying the most resistant allele in three outbred laboratory populations of snails. These three populations were derived from the same base population, but differed in average resistance. Under controlled laboratory conditions we found no cost of carrying the most resistant allele in terms of fecundity, and a possible advantage in terms of growth and mortality. These results suggest that it might be possible to drive resistant alleles of sod1 into natural populations of the snail vector for the purpose of controlling transmission of S. mansoni. However, we did observe a strong effect of genetic background on the association between sod1 genotype and resistance. sod1 genotype explained substantial variance in resistance among individuals in the most resistant genetic background, but had little effect in the least resistant genetic background. Thus, epistatic interactions with other loci may be as important a consideration as costs of resistance in the use of sod1 for vector manipulation.

Analysis and functional annotation of expressed sequence tags from in vitro cell lines of elasmobranchs: Spiny dogfish shark (Squalus acanthias) and little skate (Leucoraja erinacea).

Elasmobranchs are the most commonly used experimental models among the jawed, cartilaginous fish (Chondrichthyes). Previously we developed cell lines from embryos of two elasmobranchs, Squalus acanthias the spiny dogfish shark (SAE line), and Leucoraja erinacea the little skate (LEE-1 line). From these lines cDNA libraries were derived and expressed sequence tags (ESTs) generated. From the SAE cell line 4303 unique transcripts were identified, with 1848 of these representing unknown sequences (showing no BLASTX identification). From the LEE-1 cell line, 3660 unique transcripts were identified, and unknown, unique sequences totaled 1333. Gene Ontology (GO) annotation showed that GO assignments for the two cell lines were in general similar. These results suggest that the procedures used to derive the cell lines led to isolation of cell types of the same general embryonic origin from both species. The LEE-1 transcripts included GO categories "envelope" and "oxidoreductase activity" but the SAE transcripts did not. GO analysis of SAE transcripts identified the category "anatomical structure formation" that was not present in LEE-1 cells. Increased organelle compartments may exist within LEE-1 cells compared to SAE cells, and the higher oxidoreductase activity in LEE-1 cells may indicate a role for these cells in responses associated with innate immunity or in steroidogenesis. These EST libraries from elasmobranch cell lines provide information for assembly of genomic sequences and are useful in revealing gene diversity, new genes and molecular markers, as well as in providing means for elucidation of full-length cDNAs and probes for gene array analyses. This is the first study of this type with members of the Chondrichthyes.

Transcriptome profiling of selectively bred Pacific oyster Crassostrea gigas families that differ in tolerance of heat shock.

Sessile inhabitants of marine intertidal environments commonly face heat stress, an important component of summer mortality syndrome in the Pacific oyster Crassostrea gigas. Marker-aided selection programs would be useful for developing oyster strains that resist summer mortality; however, there is currently a need to identify candidate genes associated with stress tolerance and to develop molecular markers associated with those genes. To identify candidate genes for further study, we used cDNA microarrays to test the hypothesis that oyster families that had high (>64%) or low (<29%) survival of heat shock (43 degrees C, 1 h) differ in their transcriptional responses to stress. Based upon data generated by the microarray and by real-time quantitative PCR, we found that transcription after heat shock increased for genes putatively encoding heat shock proteins and genes for proteins that synthesize lipids, protect against bacterial infection, and regulate spawning, whereas transcription decreased for genes for proteins that mobilize lipids and detoxify reactive oxygen species. RNAs putatively identified as heat shock protein 27, collagen, peroxinectin, S-crystallin, and two genes with no match in Genbank had higher transcript concentrations in low-surviving families than in high-surviving families, whereas concentration of putative cystatin B mRNA was greater in high-surviving families. These ESTs should be studied further for use in marker-aided selection programs. Low survival of heat shock could result from a complex interaction of cell damage, opportunistic infection, and metabolic exhaustion.