Development and validation of a 66K SNP array for the hard clam (Mercenaria mercenaria).

BACKGROUND: The hard clam (Mercenaria mercenaria), a marine bivalve distributed along the U.S. eastern seaboard, supports a significant shellfish industry. Overharvest in the 1970s and 1980s led to a reduction in landings. While the transition of industry from wild harvest to aquaculture since that time has enhanced production, it has also exacerbated challenges such as disease outbreaks. In this study, we developed and validated a 66K SNP array designed to advance genetic studies and improve breeding programs in the hard clam, focusing particularly on the development of markers that could be useful in understanding disease resistance and environmental adaptability. RESULTS: Whole-genome resequencing of 84 individual clam samples and 277 pooled clam libraries yielded over 305 million SNPs, which were filtered down to a set of 370,456 SNPs that were used as input for the design of a 66K SNP array. This medium-density array features 66,543 probes targeting coding and non-coding regions, including 70 mitochondrial SNPs, to capture the extensive genetic diversity within the species. The SNPs were distributed evenly throughout the clam genome, with an average interval of 25,641 bp between SNPs. The array incorporates markers for detecting the clam pathogen Mucochytrium quahogii (formerly QPX), enhancing its utility in disease management. Performance evaluation on 1,904 samples demonstrated a 72.7% pass rate with stringent quality control. Concordance testing affirmed the array's repeatability, with an average agreement of allele calls of 99.64% across multiple tissue types, highlighting its reliability. The tissue-specific analysis demonstrated that some tissue types yield better genotyping results than others. Importantly, the array, including its embedded mitochondrial markers, effectively elucidated complex genetic relationships across different clam groups, both wild populations and aquacultured stocks, showcasing its utility for detailed population genetics studies. CONCLUSIONS: The 66K SNP array is a powerful and robust genotyping tool that offers unprecedented insights into the species' genomic architecture and population dynamics and that can greatly facilitate hard clam selective breeding. It represents an important resource that has the potential to transform clam aquaculture, thereby promoting industry sustainability and ecological and economic resilience.

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.

Disseminated neoplasia in cultured hard clams (Mercenaria mercenaria).

Marine bivalves are commonly affected by disseminated neoplasia of presumed hemocytic origin (i.e., hemic neoplasia and hemocytic neoplasia). Histopathology of 520 cultured hard clams (Mercenaria mercenaria) from Florida was performed for health surveillance over a consecutive 13-month period. Disseminated neoplasia was identified in 9 of 520 animals (1.7%). The neoplasia was characterized by the presence of large, round to oval, anaplastic cells within hemolymphatic vessels and sinusoids with variable infiltration into adjacent connective tissues of the visceral mass, mantle, foot, and/or adductor muscles. Frequent involvement and/or infiltration of the gill was also identified (5/9). Disseminated neoplasia in other species of clams, mussels, and cockles is considered a transmissible disease. At this time, it is unknown if these hard clams represent de novo development of the disease or potential transmission; however, this report expands both the geographic and host range for this condition.

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.

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.

Keeping up with advances in qPCR pathogen detection: an example for QPX disease in hard clams.

With marine diseases on the rise and increased reliance on molecular tools for disease surveillance, validated pathogen detection capabilities are important for effective management, mitigation, and response to disease outbreaks. At the same time, in an era of continual evolution and advancement of molecular tools for pathogen detection, it is critical to regularly reassess previously established assays to incorporate improvements of common practices and procedures, such as the minimum information for publication of quantitative real-time PCR experiments (MIQE) guidelines. Here, we reassessed, re-optimized, and improved the quantitative PCR (qPCR) assay routinely used for Quahog Parasite Unknown (QPX) disease monitoring. We made 19 significant changes to the qPCR assay, including improvements to PCR amplification efficiency, DNA extraction efficiency, inhibition testing, incorporation of linearized standards for absolute quantification, an inter-plate calibration technique, and improved conversion from copy number to number of cells. These changes made the assay a more effective and efficient tool for disease monitoring and pathogen detection, with an improved linear relationship with histopathology compared to the previous version of the assay. To support the wide adoption of validated qPCR assays for marine pathogens, we provide a simple workflow that can be applied to the development of new assays, re-optimization of old or suboptimal assays, or assay validation after changes to the protocol and a MIQE-compliant checklist that should accompany any published qPCR diagnostic assay to increase experimental transparency and reproducibility amongst laboratories.

The hard clam genome reveals massive expansion and diversification of inhibitors of apoptosis in Bivalvia.

BACKGROUND: Inhibitors of apoptosis (IAPs) are critical regulators of programmed cell death that are essential for development, oncogenesis, and immune and stress responses. However, available knowledge regarding IAP is largely biased toward humans and model species, while the distribution, function, and evolutionary novelties of this gene family remain poorly understood in many taxa, including Mollusca, the second most speciose phylum of Metazoa. RESULTS: Here, we present a chromosome-level genome assembly of an economically significant bivalve, the hard clam Mercenaria mercenaria, which reveals an unexpected and dramatic expansion of the IAP gene family to 159 members, the largest IAP gene repertoire observed in any metazoan. Comparative genome analysis reveals that this massive expansion is characteristic of bivalves more generally. Reconstruction of the evolutionary history of molluscan IAP genes indicates that most originated in early metazoans and greatly expanded in Bivalvia through both lineage-specific tandem duplication and retroposition, with 37.1% of hard clam IAPs located on a single chromosome. The expanded IAPs have been subjected to frequent domain shuffling, which has in turn shaped their architectural diversity. Further, we observed that extant IAPs exhibit dynamic and orchestrated expression patterns among tissues and in response to different environmental stressors. CONCLUSIONS: Our results suggest that sophisticated regulation of apoptosis enabled by the massive expansion and diversification of IAPs has been crucial for the evolutionary success of hard clam and other molluscan lineages, allowing them to cope with local environmental stresses. This study broadens our understanding of IAP proteins and expression diversity and provides novel resources for studying molluscan biology and IAP function and evolution.

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.

Effect of Activated Carbon in Thin Sand Caps Challenged with Ongoing PCB Inputs from Sediment Deposition: PCB Uptake in Clams (Mercenaria mercenaria) and Passive Samplers.

Ongoing inputs, in the form of sediment deposition along with associated dissolved contaminants, have challenged the assessment of cap performance at contaminated sediment sites. To address this issue, thin 2-3 cm layer sand caps amended with activated carbon (AC) were investigated for the remediation of polychlorinated biphenyl (PCB) contaminated marine sediments using 90-day mesocosms. All treatments were challenged with (1) ongoing clean or marker-PCB-spiked sediment inputs and (2) bioturbation. Bioaccumulation in hard clams (filter feeding near the cap-water interface) was evaluated to best understand cap effectiveness, relative to sheepshead minnows (confined to the surface water) and sandworms (which burrowed through the caps). All caps (sand and AC amended sand) provided isolation of native bedded PCBs (i.e., PCBs sourced from the bed), reducing uptake in organisms. Total PCB bioaccumulation in clams indicated that AC addition to the cap provided no benefit with spiked influx, or some benefit (56% reduction) with clean influx. Spiked input PCBs, when added to the depositional input sediment, were consistently detected in clams and passive samplers, with and without AC in the cap. PCB uptake by passive samplers located in the caps did not reflect the performance of the remedy, as defined by clam bioaccumulation. However, PCB uptake by passive samplers in the overlying water reasonably represented clam bioaccumulation results.

Proteomic and Transcriptomic Responses Enable Clams to Correct the pH of Calcifying Fluids and Sustain Biomineralization in Acidified Environments.

Seawater pH and carbonate saturation are predicted to decrease dramatically by the end of the century. This process, designated ocean acidification (OA), threatens economically and ecologically important marine calcifiers, including the northern quahog (Mercenaria mercenaria). While many studies have demonstrated the adverse impacts of OA on bivalves, much less is known about mechanisms of resilience and adaptive strategies. Here, we examined clam responses to OA by evaluating cellular (hemocyte activities) and molecular (high-throughput proteomics, RNASeq) changes in hemolymph and extrapallial fluid (EPF-the site of biomineralization located between the mantle and the shell) in M. mercenaria continuously exposed to acidified (pH ~7.3; pCO2 ~2700 ppm) and normal conditions (pH ~8.1; pCO2 ~600 ppm) for one year. The extracellular pH of EPF and hemolymph (~7.5) was significantly higher than that of the external acidified seawater (~7.3). Under OA conditions, granulocytes (a sub-population of hemocytes important for biomineralization) were able to increase intracellular pH (by 54% in EPF and 79% in hemolymph) and calcium content (by 56% in hemolymph). The increased pH of EPF and hemolymph from clams exposed to high pCO2 was associated with the overexpression of genes (at both the mRNA and protein levels) related to biomineralization, acid-base balance, and calcium homeostasis, suggesting that clams can use corrective mechanisms to mitigate the negative impact of OA.

Mitochondrial capacity and reactive oxygen species production during hypoxia and reoxygenation in the ocean quahog, Arctica islandica.

Oxygen fluctuations are common in marine waters, and hypoxia-reoxygenation (H-R) stress can negatively affect mitochondrial metabolism. The long-lived ocean quahog, Arctica islandica, is known for its hypoxia tolerance associated with metabolic rate depression, yet the mechanisms that sustain mitochondrial function during oxygen fluctuations are not well understood. We used top-down metabolic control analysis (MCA) to determine aerobic capacity and control over oxygen flux in the mitochondria of quahogs exposed to short-term hypoxia (24 h <0.01% O2) and subsequent reoxygenation (1.5 h 21% O2) compared with normoxic control animals (21% O2). We demonstrated that flux capacity of the substrate oxidation and proton leak subsystems were not affected by hypoxia, while the capacity of the phosphorylation subsystem was enhanced during hypoxia associated with a depolarization of the mitochondrial membrane. Reoxygenation decreased the oxygen flux capacity of all three mitochondrial subsystems. Control over oxidative phosphorylation (OXPHOS) respiration was mostly exerted by substrate oxidation regardless of H-R stress, whereas control by the proton leak subsystem of LEAK respiration increased during hypoxia and returned to normoxic levels during reoxygenation. During hypoxia, reactive oxygen species (ROS) efflux was elevated in the LEAK state, whereas it was suppressed in the OXPHOS state. Mitochondrial ROS efflux returned to normoxic control levels during reoxygenation. Thus, mitochondria of A. islandica appear robust to hypoxia by maintaining stable substrate oxidation and upregulating phosphorylation capacity, but remain sensitive to reoxygenation. This mitochondrial phenotype might reflect adaptation of A. islandica to environments with unpredictable oxygen fluctuations and its behavioural preference for low oxygen levels.

Immunological assays of hemocytes in the Northern Quahog Mercenaria mercenaria.

The northern quahog Mercenaria mercenaria (commonly named hard clam) is an important aquaculture and fishery species along the Atlantic west coast. Environmental stresses, such as heat shock, fluctuating salinity, and harmful algal blooms are major challenges for clam aquaculture. In response to environmental stresses, hemocytes would change dynamically for defense and immunity. The goal of this study was to characterize basic immunological assays of hemocytes in the northern quahog by use of flow cytometry. The objectives were to: 1) develop a non-lethal method for hemolymph collection and dilution; 2) verify the capability of flow cytometry for hemocyte count and type identification through comparison with microscopic observation; 3) validate hemocyte viability assay based on plasma membrane integrity, and 4) develop hemocyte phagocytosis assay by use of fluorescein labeled microbeads. A non-lethal hemocyte collection method was developed using needle insertion through the ligament. Osmolality measurement of serum was the same as that of culture seawater. The pH measurement of serum (7.2) was significantly different from that of culture seawater (8.4). By microscopic observation, three types of hemocytes were identified with granulocytes, the dominant cell type (70 ± 16%), agranulocyte (14 ± 4%), and blast-like cell (16 ± 4%), and no differences were found from the measurements by flow cytometer on FSC/SSC plot (cell size/granularity). The viability of hemocytes based on plasma membrane integrity was 88 ± 6% ranging from 70 to 97% (n = 60, three populations), and viability protocol was further validated with the pre-set expected viability (p ≥ 0.424). Phagocytosis assay of hemocytes with fluorescence beads showed a mean capacity of 10 ± 5% (n = 60, three populations). Incubation time (up to 6 h) or bead concentrations (2:1 or 5:1 to hemocytes) did not affect the phagocytosis measurement. Overall, this study reported the basic characteristics of hemolymph (serum and hemocytes) of northern quahogs. It is expected that the assay methodologies will be applied to evaluation of hemocyte responses to environmental stresses for clam aquaculture.

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.

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.

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.

Bioreactivity and Microbiome of Biodeposits from Filter-Feeding Bivalves.

Bivalves serve an important ecosystem function in delivering organic matter from pelagic to benthic zones and are important in mediating eutrophication. However, the fate of this organic matter (i.e., biodeposits) is an important consideration when assessing the ecological roles of these organisms in coastal ecosystems. In addition to environmental conditions, the processing of biodeposits is dependent on its composition and the metabolic capacity of the associated microbial community. The objectives of this study were to compare the biological reactivity, potential denitrification rates, and microbial communities of biodeposits sourced from different bivalve species: hard clam (Mercenaria mercenaria), eastern oyster (Crassostrea virginica), and ribbed mussel (Geukensia demissa). To our knowledge, this is the first study to investigate and compare the microbiome of bivalve biodeposits using high-throughput sequencing and provide important insight into the mechanisms by which bivalves may alter sediment microbial communities and benthic biogeochemical cycles. We show that clam biodeposits had significantly higher bioreactivity compared to mussel and oyster biodeposits, as reflected in higher dissolved inorganic carbon and ammonium production rates in controlled incubations. Potential denitrification rates were also significantly higher for clam biodeposits compared to oyster and mussel biodeposits. The microbial communities associated with the biodeposits were significantly different across bivalve species, with significantly greater abundances of Alteromonadales, Chitinophagales, Rhodobacterales, and Thiotrichales associated with the clam biodeposits. These bioreactivity and microbial differences across bivalve species are likely due to differences in bivalve physiology and feeding behavior and should be considered when evaluating the effects of bivalves on water quality and ecosystem function.

Modeling the odor-landscape resulting from the pumping behavior of bivalve clams in the presence of predators.

Motivated by experimental findings, a computational fluid dynamics (CFD) model was used to investigate whether the clam Mercenaria mercenaria may alter its cue downstream variability by an exhalant random pumping behavior. This behavior was hypothesized to occur in the presence of predator chemical signals in order to prevent successful tracking by the predator. Simulated downstream flow and mixing conditions derived from the random nature of the clam exhalant jet in a crossflow were analyzed by computing an intermittency factor, determining the field of finite-time Lyapunov exponents (FTLEs) and identifying the resulting Lagrangian coherent structures (LCSs). Numerical simulations illustrate that the effectiveness of a fluctuating exhalant jet to prevent downstream tracking by a crab, depends on the ratio of the exhalant jet to the crossflow. Specifically, the clam could effectively enhance the downstream dispersion to prevent tracking, but only in the range of parameters where LCSs are generated (jet-to-crossflow ratio ≥ 1). Then, the probability of detection is reduced with respect to the case of a less fluctuating exhalant jet.

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

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

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.

Differential Gene Expression in Five Isolates of the Clam Pathogen, Quahog Parasite Unknown (QPX).

Quahog parasite unknown (QPX) is a thraustochytrid protist that infects the hard clam, Mercenaria mercenaria, causing significant economic losses along the northeastern coast of North America. Previous investigations noted differences in growth dynamics and virulence in QPX cells from different geographic locations. In order to probe the molecular determinants for these variations, we investigated the transcriptomic profiles of five geographically distinct QPX isolates using custom 15k 60-mer oligonucleotide arrays. A total of 1,263 transcripts were differentially expressed (DE) among the five QPX isolates. The hierarchical clustering of gene expression profiles showed that the QPX isolates from Raritan Bay (RB, NY) and from Provincetown Harbor (MA) were more similar to each other and diverged from QPX isolates from Peconic Bay (PB, NY) and Old Plantation Creek (VA), which had more similar gene expression profiles. The most prominent difference was based on 78 transcripts coding for heat shock proteins DE between the five QPX isolates. The study generated contrasting transcriptomic profiles for QPX isolated from northern (MA) and deeper (RB, NY) locations as compared to southern (VA) and shallower (PB, NY) areas, suggesting the adaptation of the parasite to local environmental, in particular temperature, conditions.