ROS-induced moderate autophagy of haemocytes confers resistance of Mercenaria mercenaria to air exposure stress.

Air exposure (AE) is a significant environmental stressor that can lead to desiccation, hypoxia, starvation, and disruption of cellular homeostasis in marine bivalves. Autophagy is a highly conserved catabolic pathway that facilitates the degradation of damaged macromolecules and organelles, thereby supporting cellular stress responses. To date, autophagy-mediated resistance mechanisms to AE stress remain largely elusive in bivalves. In this study, we performed a multi-tool approach to investigate the autophagy-related physiological regulation in hard clams (Mercenaria mercenaria) under different duration of AE (T = 0, 1, 5, 10, 20, 30 days). We observed that autophagy of haemocytes was significantly activated on day 5. However, autophagy activity began to significantly decline from day 10 to day 30. Autophagy was significantly inhibited after antioxidant treatment, indicating that reactive oxygen species (ROS) was an endogenous inducer of autophagy. A significant decline in the survival rate of hard clams was observed after injection of ammonium chloride or carbamazepine during AE stress, suggesting that moderate autophagy was conducive for clam survival under AE stress. We also observed DNA breaks and high levels of apoptosis in haemocytes on day 10. Activation of apoptosis lagged behind autophagy, and the relationship between autophagy and apoptosis might shift from antagonism to synergy with the duration of stress. This study provides novel insights into the stress resistance mechanisms in marine bivalves.

Molecular Features Associated with Resilience to Ocean Acidification in the Northern Quahog, Mercenaria mercenaria.

The increasing concentration of CO2 in the atmosphere and resulting flux into the oceans will further exacerbate acidification already threatening coastal marine ecosystems. The subsequent alterations in carbonate chemistry can have deleterious impacts on many economically and ecologically important species including the northern quahog (Mercenaria mercenaria). The accelerated pace of these changes requires an understanding of how or if species and populations will be able to acclimate or adapt to such swift environmental alterations. Thus far, studies have primarily focused on the physiological effects of ocean acidification (OA) on M. mercenaria, including reductions in growth and survival. However, the molecular mechanisms of resilience to OA in this species remains unclear. Clam gametes were fertilized under normal pCO2 and reared under acidified (pH ~ 7.5, pCO2 ~ 1200 ppm) or control (pH ~ 7.9, pCO2 ~ 600 ppm) conditions before sampled at 2 days (larvae), 32 days (postsets), 5 and 10 months (juveniles) and submitted to RNA and DNA sequencing to evaluate alterations in gene expression and genetic variations. Results showed significant shift in gene expression profiles among clams reared in acidified conditions as compared to their respective controls. At 10 months of exposure, significant shifts in allele frequency of single nucleotide polymorphisms (SNPs) were identified. Both approaches highlighted genes coding for proteins related to shell formation, bicarbonate transport, cytoskeleton, immunity/stress, and metabolism, illustrating the role these pathways play in resilience to OA.

Massive expansion of P-selectin genes in two Venerida species, Sinonovacula constricta and Mercenaria mercenaria: evidence from comparative genomics of Bivalvia.

BACKGROUND: P-selectin is a molecule participating in the inflammatory response through mediating cellular adhesion and essential for wound repair. However, studies regarding P-selectin in Bivalvia are rare. This study identified 90 P-selectin genes among nine bivalve genomes and classified them into 4 subfamilies according to phylogenetic analysis. RESULTS: Notable P-selectin gene expansion was observed in two Venerida species, Sinonovacula constricta and Mercenaria mercenaria. The synteny analysis revealed that P-selectin gene expansion was mostly caused by tandem duplication. In addition, the expression profiles of P-selectin genes in S. constricta showed that many P-selectins were specifically highly expressed in the gills, and the P-selectin expression patterns changed dramatically under low salt stress and ammonia nitrogen stress. CONCLUSIONS: The massive expansion of P-selectins may facilitate the tolerance to environmental stresses. This study sheds light on the characterizations and expression profiles of P-selectin genes in Bivalvia and provides an integrated framework for further investigation of the role of P-selectins in the environmental tolerance of bivalves.

Transcriptomic, Proteomic, and Functional Assays Underline the Dual Role of Extrapallial Hemocytes in Immunity and Biomineralization in the Hard Clam Mercenaria mercenaria.

Circulating hemocytes in the hemolymph represent the backbone of innate immunity in bivalves. Hemocytes are also found in the extrapallial fluid (EPF), the space delimited between the shell and the mantle, which is the site of shell biomineralization. This study investigated the transcriptome, proteome, and function of EPF and hemolymph in the hard clam Mercenaria mercenaria. Total and differential hemocyte counts were similar between EPF and hemolymph. Overexpressed genes in the EPF were found to have domains previously identified as being part of the "biomineralization toolkit" and involved in bivalve shell formation. Biomineralization related genes included chitin-metabolism genes, carbonic anhydrase, perlucin, and insoluble shell matrix protein genes. Overexpressed genes in the EPF encoded proteins present at higher abundances in the EPF proteome, specifically those related to shell formation such as carbonic anhydrase and insoluble shell matrix proteins. Genes coding for bicarbonate and ion transporters were also overexpressed, suggesting that EPF hemocytes are involved in regulating the availability of ions critical for biomineralization. Functional assays also showed that Ca2+ content of hemocytes in the EPF were significantly higher than those in hemolymph, supporting the idea that hemocytes serve as a source of Ca2+ during biomineralization. Overexpressed genes and proteins also contained domains such as C1q that have dual functions in biomineralization and immune response. The percent of phagocytic granulocytes was not significantly different between EPF and hemolymph. Together, these findings suggest that hemocytes in EPF play a central role in both biomineralization and immunity.

Comparative analysis of the Mercenaria mercenaria genome provides insights into the diversity of transposable elements and immune molecules in bivalve mollusks.

BACKGROUND: The hard clam Mercenaria mercenaria is a major marine resource along the Atlantic coasts of North America and has been introduced to other continents for resource restoration or aquaculture activities. Significant mortality events have been reported in the species throughout its native range as a result of diseases (microbial infections, leukemia) and acute environmental stress. In this context, the characterization of the hard clam genome can provide highly needed resources to enable basic (e.g., oncogenesis and cancer transmission, adaptation biology) and applied (clam stock enhancement, genomic selection) sciences. RESULTS: Using a combination of long and short-read sequencing technologies, a 1.86 Gb chromosome-level assembly of the clam genome was generated. The assembly was scaffolded into 19 chromosomes, with an N50 of 83 Mb. Genome annotation yielded 34,728 predicted protein-coding genes, markedly more than the few other members of the Venerida sequenced so far, with coding regions representing only 2% of the assembly. Indeed, more than half of the genome is composed of repeated elements, including transposable elements. Major chromosome rearrangements were detected between this assembly and another recent assembly derived from a genetically segregated clam stock. Comparative analysis of the clam genome allowed the identification of a marked diversification in immune-related proteins, particularly extensive tandem duplications and expansions in tumor necrosis factors (TNFs) and C1q domain-containing proteins, some of which were previously shown to play a role in clam interactions with infectious microbes. The study also generated a comparative repertoire highlighting the diversity and, in some instances, the specificity of LTR-retrotransposons elements, particularly Steamer elements in bivalves. CONCLUSIONS: The diversity of immune molecules in M. mercenaria may allow this species to cope with varying and complex microbial and environmental landscapes. The repertoire of transposable elements identified in this study, particularly Steamer elements, should be a prime target for the investigation of cancer cell development and transmission among bivalve mollusks.

Comparative Transcriptomics of the Northern Quahog Mercenaria mercenaria and Southern Quahog Mercenaria campechiensis in Response to Chronic Heat Stress.

The northern quahog (Mercenaria mercenaria) supports lucrative aquaculture industries in the USA. In the southeastern USA, aquacultured M. mercenaria faces increasing risks of summer die-offs from prolonged heat waves. We used a comparative transcriptomic approach to investigate the molecular responses of M. mercenaria and its southern congener, Mercenaria campechiensis, to controlled incremental heat stress over a 4-week period. Mercenaria were exposed to temperatures from 24 to 34 °C with 2.5 °C/week, after which, gill transcriptomes were de novo assembled and annotated. During the 4 weeks of chronic heat exposure, both species had the same survival rate (96%); M. mercenaria experienced body weight gain/loss depending on the originated hatcheries while M. campechiensis experienced an average net weight loss. The upregulated genes in both species included those in chaperone-mediated protein folding and regulation of cell death pathways, while the downregulated genes in both species involved in mRNA processing and splicing pathways. Compared to M. mercenaria, M. campechiensis appears to be more sensitive to prolonged heat stress as indicated by upregulating significantly more genes in coping with oxidative stress and in the protein degradation pathways, while downregulating some inhibitors of apoptosis. We discussed this finding within their ecological and evolutionary context. Our findings highlighted the potential vulnerability of the two quahogs, especially the southern quahog, to continued ocean warming.

RNA-Seq analysis and WGCNA reveal dynamic molecular responses to air exposure in the hard clam Mercenaria mercenaria.

Intertidal bivalves are constantly exposed to air due to daily and seasonal tidal cycles. The hard clam Mercenaria mercenaria is an economically important bivalve species and often subjected to air exposure for more than 10 days during long-distance transportation. Hard clam exhibits remarkable tolerance to air exposure. In this study, we performed RNA sequencing on hemocytes of M. mercenaria exposed to air for 0, 1, 5, 10, 20 and 30 days. The overall and dynamic molecular responses of hard clams to air exposure were revealed by different transcriptomic analysis strategies. As a result, most cytochrome P450 1A and 3A, and monocarboxylate transporter family members were up-regulated during air exposure. Additionally, the dominant molecular process in response to 5-d, 10-d, 20-d and 30-d air exposure was refolding of misfolded proteins in endoplasmic reticulum, lysosome-mediated degradation of phospholipids, protein metabolism and reorganization of cytoskeleton, and activation of anti-apoptotic process, respectively. Our results facilitated comprehensive understanding of the tolerance mechanisms of intertidal bivalves to air exposure.

Single-molecule long-read (SMRT) transcriptome sequencing of Mercenaria mercenaria reveals a powerful anti-apoptotic system critical for air exposure endurance.

Mercenaria mercenaria is an economically important clam species and exhibits an outstanding resistance to multiple environmental stressors. However, our understanding of their stress adaptability is limited due to a lack of genomic information, such as transcriptome resources. In this study, single-molecule long-read (SMRT) mRNA sequencing was performed to obtain the full-length gill transcriptome reference sequences of M. mercenaria under air exposure stress. In all, 14.5 G subreads were obtained and assembled into 64,603 unigenes, among which 50,613 were successfully annotated. Additionally, 56,295 SSRs, 1457 transcription factors, and 5924 lncRNAs were identified in M. mercenaria transcriptome. Furthermore, numerous apoptosis-related transcripts were identified according to Swiss-Prot annotation and their numbers were counted. We also found that most apoptosis-related transcripts exhibited typical domains of a certain protein family through conserved domain prediction. Additionally, eight typical sequences related to apoptosis pathway were detected by RT-PCR, with the aim to show the sequential variation of gene expression levels under air exposure. These results implied that the complicated apoptosis system, especially the powerful anti-apoptotic system was critical for M. mercenaria to endure air exposure.

Identification of variants associated with hard clam, Mercenaria mercenaria, resistance to Quahog Parasite Unknown disease.

Severe losses in aquacultured and wild hard clam (Mercenaria mercenaria) stocks have been previously reported in the northeastern United States due to a protistan parasite called QPX (Quahog Parasite Unknown). Previous work demonstrated that clam resistance to QPX is under genetic control. This study identifies single nucleotide polymorphism (SNP) associated with clam survivorship from two geographically segregated populations, both deployed in an enzootic site. The analysis contrasted samples collected before and after undergoing QPX-related mortalities and relied on a robust draft clam genome assembly. ~200 genes displayed significant variant enrichment at each sampling point in both populations, including 18 genes shared between both populations. Markers from both populations were identified in genes related to apoptosis pathways, protein-protein interaction, receptors, and signaling. This research begins to identify genetic markers associated with clam resistance to QPX disease, leading the way for the development of resistant clam stocks through marker-assisted selection.

Effects of seawater salinity and pH on cellular metabolism and enzyme activities in biomineralizing tissues of marine bivalves.

Molluscan shell formation is a complex energy demanding process sensitive to the shifts in seawater CaCO3 saturation due to changes in salinity and pH. We studied the effects of salinity and pH on energy demand and enzyme activities of biomineralizing cells of the Pacific oyster (Crassostrea gigas) and the hard-shell clam (Mercenaria mercenaria). Adult animals were exposed for 14 days to high (30), intermediate (18), or low (10) salinity at either high (8.0-8.2) or low (7.8) pH. Basal metabolic cost as well as the energy cost of the biomineralization-related cellular processes were determined in isolated mantle edge cells and hemocytes. The total metabolic rates were similar in the hemocytes of the two studied species, but considerably higher in the mantle cells of C. gigas compared with those of M. mercenaria. Cellular respiration was unaffected by salinity in the clams' cells, while in oysters' cells the highest respiration rate was observed at intermediate salinity (18). In both studied species, low pH suppressed cellular respiration. Low pH led to an upregulation of Na+/K+ ATPase activity in biomineralizing cells of oysters and clams. Activities of Ca2+ ATPase and H+ ATPase, as well as the cellular energy costs of Ca2+ and H+ transport in the biomineralizing cells were insensitive to the variation in salinity and pH in the two studied species. Variability in cellular response to low salinity and pH indicates that the disturbance of shell formation under these conditions has different underlying mechanisms in the two studied species.

De novo assembly transcriptome analysis reveals the preliminary molecular mechanism of pigmentation in juveniles of the hard clam Mercenaria mercenaria.

Color plays a vital function in camouflage, sexual selection, immunity, and evolution. Mollusca possess vivid shell colors and pigmentation starts at the juvenile stage. The hard clam Mercenaria mercenaria is a widely cultivated bivalve of high economic value. To explore the molecular mechanism of pigmentation in juvenile clams, here, we performed RNA-Seq analysis on non-pigmented, white, and red M. mercenaria specimens. Clean reads were assembled into 358,285 transcripts and 149,234 unigenes, whose N50 lengths were 2107 bp and 1567 bp, respectively. Differentially expressed genes were identified and analyzed for KEGG enrichment. "Melanoma/Melanogenesis", "ABC transporters", and "Porphyrin and chlorophyll metabolism" pathways appeared to be associated with pigmentation. Pathways related to carotenoid metabolism seemed to also play a vital role in pigmentation in juveniles. Our results provide new insights into the formation of shell color in juvenile hard clams.

Linking paternally inherited mtDNA variants and sperm performance.

Providing robust links between mitochondrial genotype and phenotype is of major importance given that mitochondrial DNA (mtDNA) variants can affect reproductive success. Because of the strict maternal inheritance (SMI) of mitochondria in animals, haplotypes that negatively affect male fertility can become fixed in populations. This phenomenon is known as 'mother's curse'. Doubly uniparental inheritance (DUI) of mitochondria is a stable exception in bivalves, which entails two mtDNA lineages that evolve independently and are transmitted separately through oocytes and sperm. This makes the DUI mitochondrial lineages subject to different sex-specific selective sieves during mtDNA evolution, thus DUI is a unique model to evaluate how direct selection on sperm mitochondria could contribute to male reproductive fitness. In this study, we tested the impact of mtDNA variants on sperm performance and bioenergetics in DUI and SMI species. Analyses also involved measures of sperm performance following inhibition of main energy pathways and sperm response to oocyte presence. Compared to SMI, DUI sperm exhibited (i) low speed and linearity, (ii) a strict OXPHOS-dependent strategy of energy production, and (iii) a partial metabolic shift towards fermentation following egg detection. Discussion embraces the adaptive value of mtDNA variation and suggests a link between male-energetic adaptation, fertilization success and paternal mitochondria preservation. This article is part of the theme issue 'Linking the mitochondrial genotype to phenotype: a complex endeavour'.

Transcriptome analysis of shell color-related genes in the hard clam Mercenaria mercenaria.

The shell color of marine bivalves shows great diversity and is considered as an economic trait. In China, the hard clam, Mercenaria mercenaria, commonly has three shell colors in the wild: red, white, and mottled. The genetic mechanisms controlling color segregation are not fully understood. In this study, RNA-seq was performed to exploit the related shell color genes and determine the genetic basis of the different shell colors. Nine sequence libraries with those three shell colors of hard clam were constructed; 406,377 transcripts and 248,251 unigenes were obtained with N50 values of 1365 and 1682 base pairs, respectively. Cluster analysis identified 363, 392, and 220 genes exclusively highly expressed in red, white, and mottled clams, respectively. We further classified differentially expressed genes (DEGs), the genes involved in lipid binding and transport, signal transduction, ATP synthesis, and other processes in the red vs white comparison were found, which may participate in red shell formation. DEGs related to signal transduction, particularly G protein-coupled receptor activity, were found in the red vs mottled comparison, suggesting that these genes might be important in mottled shell formation. In the white vs mottled comparison, DEGs involved in zinc ion binding were found. Our results provide new insights into shell color formation mechanisms in molluscs. This information could be used in selective breeding and marker-assisted breeding of this economically important clam species.

Isolation and characterization of novel microsatellite markers in Mercenaria mercenaria.

Mercenaria mercenaria, also known as the hard clam, is widely distributed in the coastal waters of temperate and tropical areas in the Asian Pacific region. This species is widely popular in the international market, especially in the United States, Europe, and other Western countries, because of its high protein value, taste, and simple farming requirements. In this study, 17 novel microsatellite loci from the M. mercenaria genome were developed using the fast isolation by amplified fragment length polymorphism of sequences containing repeats protocol. Thirty-two wild individuals were used to evaluate the degree of polymorphism of these markers. Results indicated that there were 11 polymorphic loci and six monomorphic loci, and the number of alleles per locus and the polymorphism information content ranged from two to six and from 0.059 to 0.498, respectively. The observed and expected heterozygosity varied from 0.0625 to 0.5333 and 0.0615 to 0.4977, respectively. The Y1-4 locus deviated significantly from Hardy-Weinberg equilibrium (HWE) after Bonferroni correction was applied, while the other loci were in HWE. These loci will provide useful information for M. mercenaria population genetic studies.

A Distinct Mitochondrial Genome with DUI-Like Inheritance in the Ocean Quahog Arctica islandica.

Mitochondrial DNA (mtDNA) is strictly maternally inherited in metazoans. The major exception to this rule has been found in many bivalve species which allow the presence of different sex-linked mtDNA molecules. This mechanism, named doubly uniparental inheritance (DUI), is characterized by the presence of two mtDNAs: The female mtDNA is found in somatic tissue and female gonads, whereas the male mtDNA is usually found in male gonads and sperm. In this study we highlight the existence of two divergent mitochondrial haplotypes with a low genetic difference around 6-8% in Arctica islandica, a long-lived clam belonging to the Arcticidae, a sister group to the Veneridae in which DUI has been found. Phylogenetic analysis on cytochrome b and 16S sequences from somatic and gonadic tissues of clams belonging to different populations reveals the presence of the "divergent" type in male gonads only and the "normal" type in somatic tissues and female gonads. This peculiar segregation of divergent mtDNA types speaks for the occurrence of the DUI mechanism in A. islandica. This example also highlights the difficulties to assess the presence of such particular mitochondrial inheritance system and underlines the possible misinterpretations in phylogeographic and phylogenetic studies of bivalve species linked to the presence of two poorly differentiated mitochondrial genomes.

Alterations of the immune transcriptome in resistant and susceptible hard clams (Mercenaria mercenaria) in response to Quahog Parasite Unknown (QPX) and temperature.

Quahog Parasite Unknown (QPX) is a fatal protistan parasite that causes severe losses in the hard clam (Mercenaria mercenaria) fisheries along the northeastern coast of the US. Field and laboratory studies of QPX disease have demonstrated a major role for water temperature and M. mercenaria genetic origin in disease development. Infections are more likely to occur at cold temperatures, with clam stocks originating from southern states being more susceptible than clams from northern origin where disease is enzootic. Even though the influence of temperature on QPX infection have been examined in susceptible and resistant M. mercenaria at physiological and cellular scales, the underlying molecular mechanisms associated with host-pathogen interactions remain largely unknown. This study was carried out to explore the molecular changes in M. mercenaria in response to temperature and QPX infection on the transcriptomic level, and also to compare molecular responses between susceptible and resistant clam stocks. A M. mercenaria oligoarray (15 K Agilent) platform was produced based on our previously generated transcriptomic data and was used to compare gene expression profiles in naive and QPX-infected susceptible (Florida stock) and resistant (Massachusetts) clams maintained at temperatures favoring disease development (13 °C) or clam healing (21 °C). In addition, transcriptomic changes reflecting focal (the site of infection, mantle) and systemic (circulating hemocytes) responses were also assessed using the oligoarray platform. Results revealed significant regulation of multiple biological pathways by temperature and QPX infection, mainly associated with immune recognition, microbial killing, protein synthesis, oxidative protection and metabolism. Alterations were widely systemic with most changes in gene expression revealed in hemocytes, highlighting the role of circulating hemocytes as the first line of defense against pathogenic stress. A large number of complement-related recognition molecules with fibrinogen or C1q domains were shown to be specially induced following QPX challenge, and the expression of these molecules was significantly higher in resistant clams as compared to susceptible ones. These highly variable immune proteins may be potent candidate molecular markers for future study of M. mercenaria resistance against QPX. Beyond the specific case of clam response to QPX, this study also provides insights into the primitive complement-like system in the hard clam.

Development of genomic resources for a thraustochytrid pathogen and investigation of temperature influences on gene expression.

Understanding how environmental changes influence the pathogenicity and virulence of infectious agents is critical for predicting epidemiological patterns of disease. Thraustochytrids, part of the larger taxonomic class Labyrinthulomycetes, contain several highly pathogenic species, including the hard clam pathogen quahog parasite unknown (QPX). QPX has been associated with large-scale mortality events along the northeastern coast of North America. Growth and physiology of QPX is temperature-dependent, and changes in local temperature profiles influence pathogenicity. In this study we characterize the partial genome of QPX and examine the influence of temperature on gene expression. Genes involved in several biological processes are differentially expressed upon temperature change, including those associated with altered growth and metabolism and virulence. The genomic and transcriptomic resources developed in this study provide a foundation for better understanding virulence, pathogenicity and life history of thraustochytrid pathogens.

Differential immune response in the hard clam (mercenaria mercenaria) against bacteria and the protistan pathogen QPX (quahog parasite unknown).

The immune response of the hard clam (quahog) Mercenaria mercenaria following challenge with live bacteria (Vibrio alginolyticus) and the protist QPX (Quahog Parasite Unknown) was investigated. The study also compared immune responses following QPX challenge in two different hard clam broodstocks exhibiting different degrees of susceptibility toward this parasite. Different immune and stress-related cellular and humoral factors were assessed including general hemocyte parameters (total and differential hemocyte counts, percentage of dead cells, reactive oxygen production, phagocytosis), parameters geared toward QPX (anti-QPX activity in plasma and hemocyte resistance to the cytotoxicity of QPX extracellular products). Two genes (ferritin and metallothionein) previously shown to be modulated following QPX exposure were molecularly characterized by rapid amplification of cDNA ends (RACE) and their transcription levels were determined in resistant and susceptible clams in response to QPX and bacterial challenge. Results indicated that both V. alginolyticus and QPX challenge triggered significant immune responses in clams with similar trends for most measured parameters. However, specific responses were observed for anti-QPX activity in plasma and hemocyte resistance to QPX products as well as ferritin and metallothionein expression according to each inoculum. Similarly, different response patterns were detected following QPX challenge in susceptible and resistant clam stocks. Resistant clams were able to elicit effective response against the parasite leading to the elimination of QPX and the restoration of constitutive immune status whereas QPX-susceptible clams triggered a strong immune modulation characterized by an acute phase response and associated acute phase protein but appeared to be less active in eliminating the parasite. These results suggest that different signaling pathways are triggered during V. alginolyticus and QPX challenge. Moreover, differences in the immune response toward QPX might be linked to the susceptibility or resistance of different clam stocks to the infection by this parasite.

Identification and expression of differentially expressed genes in the hard clam, Mercenaria mercenaria, in response to quahog parasite unknown (QPX).

BACKGROUND: The hard clam, Mercenaria mercenaria, has been affected by severe mortality episodes associated with the protistan parasite QPX (Quahog Parasite Unknown) for several years. Despite the commercial importance of hard clams in the United States, molecular bases of defense mechanisms in M. mercenaria, especially during QPX infection, remain unknown. RESULTS: Our study used suppression subtractive hybridization (SSH), as well as the construction of cDNA libraries from hemocytes to identify genes related to the defense of the hard clam against its parasite. Hard clams were experimentally infected with QPX and SSH was performed on mRNA samples extracted from mantle and gill tissues at different times post-challenge. A total of 298 clones from SSH libraries and 1352 clones from cDNA libraries were sequenced. Among these sequences, homologies with genes involved in different physiological processes related to signal transduction, stress response, immunity and protein synthesis were identified. Quantitative PCR revealed significant changes in the expression of several of these genes in response to QPX challenge and demonstrated significant correlations in terms of levels of gene expression between intermediates of signalling pathways and humoral defense factors, such as big defensin and lysozyme. CONCLUSION: Results of this study allowed the detection of modifications caused by QPX at the transcriptional level providing insight into clam immune response to the infection. These investigations permitted the identification of candidate genes and pathways for further analyses of biological bases of clam resistance to QPX allowing for a better understanding of bivalve immunity in general.

Molecular identification of glutathione S-transferase gene and cDNAs of two isotypes from northern quahog (Mercenaria mercenaria).

Glutathione S-transferases (GSTs) are universally xenobiotic detoxifying enzymes which have been shown to play a unique detoxifying role in northern quahogs. GST consists of two distinct domains: N-terminal domain which contributes residues to form G-site (GSH-binding site) and C-terminal domain which provides residues to form a hydrophobic H-site (second substrate-binding site). In this study, glutathione S-transferases (GSTs) gene and cDNAs of two isotypes from the northern quahog (Mercenaria mercenaria) were cloned and characterized for the first time. Two full-length GST cDNAs were obtained and named as GST Pi-1 and Pi-2, respectively. Both cDNAs have an open reading frame of 624 bp, which encodes a 207-amino acid protein. Multiple sequences alignment analysis shows that the deduced amino acid sequences of GST Pi-1 and Pi-2 have high homology with other Pi class GSTs. The evolutionary relationship assessment indicates that the two deduced amino acid sequences are closely related to Pi class GSTs. The GST Pi-1 gene is an intronless gene so that it has the same sequence as the transcript. The digested peptide fragments of the purified northern quahog GSTs were analyzed by tandem mass spectrometry and the results confirm the existence of the translation products of the GST Pi-1 and Pi-2 transcripts. The predicted three-dimensional structures of GST Pi-1 and Pi-2 showed that both proteins have two domains, N-terminal domain and C-terminal domain. The conserved phenylalanine-48 serving as the ball in ball and socket style interface in all reported Pi class GSTs was found in both proteins. These results suggest that GST Pi-1 and Pi-2 from the northern quahog belong to the Pi class GSTs which may be involved in polychlorinated biphenyl (PCB) dechlorination.