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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; p CO 2 ~2700 ppm) and normal conditions (pH ~8.1; p CO 2 ~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 p CO 2 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.

Bivalve mollusks thrive in environments rich in microorganisms, such as estuarine and coastal waters, and they tend to accumulate various particles, including viruses. However, the current knowledge on mollusk viruses is mainly centered on few pathogenic viruses, whereas a general view of bivalve-associated viromes is lacking. This study was designed to explore the viral abundance and diversity in bivalve mollusks using transcriptomic datasets. From analyzing RNA-seq data of 58 bivalve species, we have reconstructed 26 nearly complete and over 413 partial RNA virus genomes. Although 96.4% of the predicted viral proteins refer to new viruses, some sequences belong to viruses associated with bivalve species or other marine invertebrates. We considered short non-coding RNAs (sncRNA) and post-transcriptional modifications occurring specifically on viral RNAs as tools for virus host-assignment. We could not identify virus-derived small RNAs in sncRNA reads obtained from the oyster sample richest in viral reads. Single Nucleotide Polymorphism (SNP) analysis revealed 938 A-to-G substitutions occurring on the 26 identified RNA viruses, preferentially impacting the AA di-nucleotide motif. Under-representation analysis revealed that the AA motif is under-represented in these bivalve-associated viruses. These findings improve our understanding of bivalve viromes, and set the stage for targeted investigations on the specificity and dynamics of identified viruses.

Background: The hard clam Mercenaria mercenaria is an important seafood species widely exploited along the eastern coasts of the United States and play a crucial role in coastal ecology and economy. Severe hard clam mortalities have been associated with the protistan parasite QPX (Quahog Parasite Unknown). QPX infection establishes in pallial organs with the lesions typically characterized as nodules, which represent inflammatory masses formed by hemocyte infiltration and encapsulation of parasites. QPX infection is known to induce host changes on both the whole-organism level and at specific lesion areas, which imply systemic and focal defense responses, respectively. However, little is known about the molecular mechanisms underlying these alterations., Results: RNA-seq was performed using Illumina Hiseq 2000 (641 Million 100 bp reads) to characterize M. mercenaria focal and systemic immune responses to QPX. Transcripts were assembled and the expression levels were compared between nodule and healthy tissues from infected clams, and between these and tissues from healthy clams. De novo assembly reconstructed a consensus transcriptome of 62,980 sequences that was functionally-annotated. A total of 3,131 transcripts were identified as differentially expressed in different tissues. Results allowed the identification of host immune factors implicated in the systemic and focal responses against QPX and unraveled the pathways involved in parasite neutralization. Among transcripts significantly modulated upon host-pathogen interactions, those involved in non-self recognition, signal transduction and defense response were over-represented. Alterations in pathways regulating hemocyte focal adhesion, migration and apoptosis were also demonstrated., Conclusions: Our study is the first attempt to thoroughly characterize M. mercenaria transcriptome and identify molecular features associated with QPX infection. It is also one of the first studies contrasting focal and systemic responses to infections in invertebrates using high-throughput sequencing. Results identified the molecular signatures of clam systemic and focal defense responses, to collectively mediate immune processes such as hemocyte recruitment and local inflammation. These investigations improve our understanding of bivalve immunity and provide molecular targets for probing the biological bases of clam resistance towards QPX.

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., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Brown Ring Disease (BRD) is a bacterial infection affecting the economically-important clam Ruditapes philippinarum. The disease is caused by a bacterium, Vibrio tapetis, that colonizes the edge of the mantle, altering the biomineralization process and normal shell growth. Altered organic shell matrices accumulate on the inner face of the shell leading to the formation of the typical brown ring in the extrapallial space (between the mantle and the shell). Even though structural and functional changes have been described in solid (mantle) and fluid (hemolymph and extrapallial fluids) tissues from infected clams, the underlying molecular alterations and responses remain largely unknown. This study was designed to gather information on clam molecular responses to the disease and to compare focal responses at the site of the infection (mantle and extrapallial fluid) with systemic (hemolymph) responses. To do so, we designed and produced a Manila clam expression oligoarray (15K Agilent) using transcriptomic data available in public databases and used this platform to comparatively assess transcriptomic changes in mantle, hemolymph and extrapallial fluid of infected clams. Results showed significant regulation in diseased clams of molecules involved in pathogen recognition (e.g. lectins, C1q domain-containing proteins) and killing (defensin), apoptosis regulation (death-associated protein, bcl-2) and in biomineralization (shell matrix proteins, perlucin, galaxin, chitin- and calcium-binding proteins). While most changes in response to the disease were tissue-specific, systemic alterations included co-regulation in all 3 tested tissues of molecules involved in microbe recognition and killing (complement-related factors, defensin). These results provide a first glance at molecular alterations and responses caused by BRD and identify targets for future functional investigations., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Competing Interests: The authors have declared that no competing interests exist.

Background: The hard clam or northern quahog, Mercenaria mercenaria, is one of the most valuable seafood products in the United States representing the first marine resource in some Northeastern states. Severe episodes of hard clam mortality have been consistently associated with infections caused by a thraustochytrid parasite called Quahog Parasite Unknown (QPX). QPX is considered as a cold/temperate water organism since the disease occurs only in the coastal waters of the northwestern Atlantic Ocean from Maritime Canada to Virginia. High disease development at cold temperatures was also confirmed in laboratory studies and is thought to be caused predominantly by immunosuppression of the clam host even though the effect of temperature on QPX virulence has not been fully investigated. In this study, the QPX transcriptome was sequenced using Roche 454 technology to better characterize this microbe and initiate research on the molecular basis of QPX virulence towards hard clams., Results: Close to 18,000 transcriptomic sequences were generated and functionally annotated. Results revealed a wide array of QPX putative virulence factors including a variety of peptidases, antioxidant enzymes, and proteins involved in extracellular mucus production and other secretory proteins potentially involved in interactions with the clam host. Furthermore, a 15 K oligonucleotide array was constructed and used to investigate the effect of temperature on QPX fitness and virulence factors. Results identified a set of QPX molecular chaperones that could explain its adaptation to cold temperatures. Finally, several virulence-related factors were up-regulated at low temperature providing molecular targets for further investigations of increased QPX pathogenicity in cold water conditions., Conclusions: This is one of the first studies to characterize the transcriptome of a parasitic labyrinthulid, offering new insights into the molecular bases of the pathogenicity of members of this group. Results from the oligoarray study demonstrated the ability of QPX to cope with a wide range of environmental temperatures, including those considered to be suboptimal for clam immunity (low temperature) providing a mechanistic scenario for disease distribution in the field and for high disease prevalence and intensity at low temperature. These results will serve as basis for studies aimed at a better characterization of specific putative virulence factors.