Since 2019, outbreaks of an apicomplexan parasite caused catastrophic mortalities of adult northern bay scallops (Argopecten irradians irradians) in New York, USA. Anecdotal observations suggested different levels of mortality between different scallop stocks and mortality outbreaks appeared to occur in years displaying positive temperature anomalies. In this study, field and laboratory experiments were designed to evaluate the effect of environmental conditions and host (scallop) background on disease susceptibility and mortality. Wild and aquacultured scallops originally sourced from the same area were deployed in lantern nets (suspended in the water column) or oyster culture bags (on bottom) at two different enzootic sites showing contrasting environmental conditions. Subsets of wild and aquacultured stocks were also used in laboratory experiments exposing scallops to different temperatures and dissolved oxygen levels, mimicking different environmental scenarios. Results showed higher mortality in aquacultured scallops compared to wild scallops, especially in the more stressful conditions. Laboratory experiments showed that the trajectories of disease development and mortality were significantly affected by initial disease levels and environmental factors. For instance, high temperature and low dissolved oxygen were shown to favor disease development when initial disease levels were low (wild scallops) but also appear to lead to an overall reduction in disease via culling of the most heavily infected scallops when initial disease levels and mortality during the experiment were high (aquacultured scallops). Altogether, these findings underline the impact of environmental conditions, in particular temperature, on host-parasite interactions and suggest the potential existence of more resilient scallop stocks.

Eastern oyster aquaculture has increased steadily over the last three decades, but the industry has not achieved its full potential. In the Northeast USA, production is limited in part by a lack of high performing stocks suited to geographically broad and heterogenous growing environments. Hatchery lines from Maine, New York, and New Jersey and their crosses were evaluated at oyster farms in Rhode Island and New Jersey. Replicate bags of each line were maintained according to standard husbandry practices, and survival, growth and final yield were monitored for 16 months. During the second summer of the evaluation period, subsamples from each line were tested for parasites Perkinsus marinus (Dermo) and Haplosporidium nelsoni (MSX) to assess disease acquisition at both sites. Significant site, line, and line by site (GxE) interaction effects were detected for growth and mortality. Differences in growth between sites reflect food availability while differences in mortality coincide with disease and potential heat stress. Trait differences observed among lines within sites are consistent with selection history and local adaptation. Top performing lines were not consistent across sites, suggesting advantages of long-term selection/local adaptation may not transfer when lines are grown outside their site of origin. Our results highlight the role of GxE interactions in the expression of commercially important traits and can contribute to the development of future breeding strategies and more informed on-farm seed deployments that will augment eastern oyster aquaculture production in the Northeast USA.

As the microbiome becomes increasingly recognized for its role in driving animal health and disease, accurate characterization of microbial community composition is essential. With this research comes the critical need for standardized sampling methods along with an understanding of the biases inherent to these methods. In oysters, hemolymph is a valuable system for microbiome research due to its diverse physiological functions, role as an indicator of health, and ease of collection. Unfortunately, much of the current literature regarding oyster hemolymph microbiome composition is opaque in its description of tissue collection methods. When these methods are clearly described, investigators often collect hemolymph via a needle inserted into the adductor muscle through a notch cut in the shell. However, due to the needle’s movement through soft tissues and mucous membranes exposed to the surrounding seawater, this notch method carries significant risk of sample contamination. An alternative hemolymph sampling method, hereafter referred to as the hole method, minimizes this potential contamination as the needle is inserted through a hole drilled in the shell directly above the adductor muscle, circumventing other soft tissues. Here, we tested whether notch and hole hemolymph collection methods produce different 16S rRNA bacterial community profiles in C. virginica. To compare these two sampling methods and evaluate the potential method-specific biases, particularly biases that may result from tissue contamination, oysters had hemolymph collected through both a notch and hole while alternating method order. Following taxonomic classification and differential abundance analysis of the resultant 16S rRNA sequences, the presence of disproportionate Vibrio spp. enrichment in notch-associated samples was evident. Notch-collected samples yielded significantly higher 16S rRNA gene copy numbers (p = 0.037) and a 16-fold higher relative abundance of Vibrio spp. compared to hole-collected samples (52.6% vs. 3.2%, padj = 0.006) despite no significant change in absolute counts of the dominant genus Poseidonibacter. Overall, this analysis demonstrates both the potential of the hole sampling method and the need for microbiome researchers to account for biases in bacterial community composition associated with their chosen sampling method.

Substantial progress has been made in understanding hemocytes within the hemolymph (herein referred to as circulatory hemocytes), which are essential for immune defense and various physiological functions in bivalve mollusks. Yet, our knowledge of peripheral immunity, particularly the role of hemocytes associated with mucosal surfaces covering pallial (gills, mantle) organs (herein referred to as mucosal hemocytes), remains limited. While mucosal and circulatory hemocytes share similar morphologies, they exhibit distinct functional profiles and cell surface epitopes. The molecular mechanisms underlying these functional differences remain poorly understood. To address this knowledge gap, we characterized gene expression profiles (using RNA sequencing) in granulocytes and agranulocytes isolated via flow cytometry from hemolymph and mucus covering pallial tissues of the eastern oyster, Crassostrea virginica. Mucosal hemocytes showed overexpression of genes related to cell motility, cytokine activity, signaling, and cell adhesion, supporting the hypothesis that they may have sentinel functions. Despite a consistent dichotomy in gene expression between granulocytes and agranulocytes across both body fluids, it was more pronounced in circulatory hemocytes. Circulatory granulocytes showed functions linked to phagocytosis and pathogen killing, whereas circulatory agranulocytes overexpressed genes associated with mitosis and early inflammation compared to their mucosal counterparts. To our knowledge, this is the first study combining flow cytometry sorting and transcriptomic methods to characterize hemocytes from different body fluids in a marine invertebrate. Results underline the potential role of mucosal hemocytes as immune sentinels, although more studies, possibly using single-cell transcriptomic methods and functional assays associated with pathogen challenge experiments, are needed to probe their specific functions.

Ocean warming (OW) and ocean acidification (OA) are expected to have wide-ranging consequences for populations in marine systems caused by organism-level responses at a range of life history stages, including early larval stages. The Atlantic sea scallop, Placopecten magellanicus, is an economically important bivalve species that may be vulnerable to ocean change, but larval responses remain unclear. While the effects of OW and OA are known to impact adult sea scallops, little is known about the interactive effects of these two factors on early life stages. Larval dispersal is key to population-level patterns (e.g. abundance and distribution), but is contingent on growth rates and vertical swimming behaviors, which are both influenced by environmental conditions. Here, we evaluate the combined effects of OW and OA on larval development and behavior in a factorial lab experiment. We maintained larval sea scallop cultures up to metamorphosis under a range of physico-chemical conditions (17℃ and 14℃, and pH 7.3, 7.6, 7.9). Sea scallop larval development time was temperature dependent (1 vs. 1.5 months). There was an interactive effect of OW and OA on growth, where OA limited gains in growth experienced in warmer conditions. OA limited a decline in distribution late in development, while OW dominated individual vertical swimming behavior in intermediate development. Finally, ΩAr growth sensitivity was intermediate compared to that of other bivalve taxa. These results contribute to an improved understanding of developmental and behavioral responses to OW and OA, and provide information necessary for improved larval dispersal models under ocean change.

Margolisiella madlensis, previously known as bay scallop Marosporida (BSM), is an apicomplexan parasite implicated in recurrent summer mortality of the bay scallop Argopecten irradians in the Northeastern United States. It is a member of the class Marosporida, a major apicomplexan lineage that has so far lacked nuclear genomic resources. Here we report the draft nuclear genome of M. madlensis 23004-MA1, assembled from PacBio HiFi and Illumina sequencing data. The assembly comprises 41.0 Mb in 6,697 scaffolds. Interspersed repeats accounted for 40.3% of the assembly, while repetitive and low-complexity sequences together accounted for 45.8%. To our knowledge, this is the first nuclear genome available for the class Marosporida. Therefore, this genome provides an essential reference for comparative studies of apicomplexan evolution and establishes a basis for future work on parasite biology, molecular detection, and host-parasite interactions in marine invertebrates.

BACKGROUND:Understanding the genetic basis of resilience in marine organisms is critical for conservation and management, particularly in the face of escalating environmental stress and disease outbreaks. The bay scallop Argopecten irradians is a commercially and recreationally important shellfish species found in estuarine and coastal environments of the United States from New England to the Gulf of Mexico. In New York, adult bay scallop populations have been decimated every summer since 2019 leading to the collapse of their fishery. These mortality events were associated with annual outbreaks of an undescribed apicomplexan parasite recently named Bay Scallop Marosporida (BSM) that disrupts scallop kidneys. RESULTS:This study investigates host-pathogen interactions and assesses changes in population structure during BSM-associated mortality events. The research compared wild and aquacultured scallops used for stock enhancement in New York, revealing significant change in population structures throughout the mortality outbreak. The results underscore the selective pressures exerted by BSM infection and environmental stressors, as evidenced by shifts in genetic divergence and allele frequencies particularly in genes associated with kidney function, stress and infection response. Through a detailed genomic and population genetic approach, this research represents a unique case study highlighting the impact of disease on marine biodiversity and advances our understanding of the impact of summer mortality events on the scallop population in NY. CONCLUSIONS:This study highlights changes in the genomic structure of bay scallops during a BSM-associated mortality event. Identified mutations (such as the one in the nephrocystin-3-like gene) represent prime candidates for specific targeted investigations to link genotypes to phenotypes. By integrating genomic and epidemiological data, the research provides a basis for understanding the impact of disease on scallop biodiversity. These findings may help guide conservation strategies for sustainable fisheries in the face of environmental change and disease outbreaks.

The northern quahog Mercenaria mercenaria is a major aquaculture species on the US East Coast, and heat resistance is the most sought trait for aquaculture. This study aimed to establish a genome-wide association for heat tolerance using a 66K SNP array for M. mercenaria. Quahogs from three farms were combined for a heat challenge at 1 °C per day from 24 °C to 35 °C and stay for two days (Phase I), decreasing to 27 °C in 24 h, to 24 °C in another 24 h, and maintaining at 24 °C (Phase II) until no one dead within 48 h at 24 °C (Phase III). Dead and live quahogs were sampled for genotyping using the SNP array. During the heat challenge, different mortalities among the quahogs from the three farms were identified at 38, 46, and 55

Over the last two decades, oyster aquaculture has largely shifted from the use of diploids to the near-exclusive use of triploids in many regions, with some hatcheries now producing >90 % triploid oyster spat. The increased demand for triploids is primarily driven by substantial growth advantages and more consistent meat quality associated with sterile triploid animals, with most data suggesting triploids and diploids display comparable adult survivorship. Although adult performance appears similar, anecdotal reports from farmers have suggested that triploids may exhibit greater frailty during earlier age classes, particularly in response to bacterial infections. Due to the extent of triploid production now taking place, the possibility of early ontogenetic frailty in triploids could pose a significant risk to oyster aquaculture production. To evaluate these claims, two cohorts of halfsibling diploid and triploid eastern oyster lines were generated in 2020 and 2021. A subset of larvae and juveniles from these lines were then exposed to a cocktail of bacterial (Vibrio) pathogens and monitored for viability. The remaining oysters were then allowed to grow for another 2 months before being deployed in either Peconic Bay, New York (2020), or Patuxent River, Maryland (2021), where their survivorship was followed for another year. Results showed that triploids were at significantly greater risk of mortality during the larvae and juvenile stages, though differential mortality decreased with age. These trends were consistent across the two spawning events, and the extent of early ontogenetic triploid frailty was observed to vary between the lines tested. This work provides valuable data for hatchery managers and farmers alike and suggests areas where specific attention and further work are required.

Mucochytrium quahogii, also known as QPX or Quahog Parasite Unknown, is the causative agent of QPX disease in the hard clam (Mercenaria mercenaria). Host–pathogen–environment interactions between M. quahogii, the hard clam, and temperature were explored in a microcosm experiment. Hard clams were housed in individual tanks with sterile seawater under two temperature regimes: low (13 °C) temperature, which is thought to be optimal for QPX disease development, and high (20 °C) temperature, which has been shown to promote “healing” of QPX-infected clams. Hard clam tissue, pallial fluid, seawater, and shell biofilms were collected and assayed for M. quahogii. The release of M. quahogii from naturally infected live hard clams into seawater was detected only in the low temperature treatment, suggesting that temperature influences the release of potentially infectious cells. M. quahogii was commonly found in hard clam pallial fluid, even after 9 weeks in the lab, suggesting pallial fluid is a stable reservoir of M. quahogii within its primary host and that M. quahogii is not a transient component of the hard clam microbiota. Overall, results support a host-specific relationship and that M. quahogii is a commensal member of the hard clam microbiota, supporting its classification as an opportunistic pathogen.

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. 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

Suspension-feeding bivalves, including the oyster Crassostrea virginica, use mucosal lectins to capture food particles. For instance, oysters can increase the transcription of these molecules to enhance food uptake. However, the regulatory processes influencing food uptake remain unclear although likely involve neuropeptides. Information on the neuropeptidome of C. virginica is limited, hindering the comprehension of its physiology, including energy homeostasis. This study explored the genome of C. virginica to identify neuropeptide precursors in silico and compared these with orthologs from other mollusks. A special focus was given to genes with potential implication in feeding processes. qPCR was used to determine the main organs of transcription of feeding-related genes. To further probe the function of target neuropeptides, visceral ganglia extracts and synthetic NPF were injected into oysters to evaluate their impact on genes associated with feeding and energy homeostasis. A total of eighty-five neuropeptides genes were identified in C. virginica genome. About 50 % of these are suggested to play a role in feeding processes. qPCR analyses showed that visceral ganglia and digestive system are the main organs for the synthesis of feeding-related neuropeptides. Further, results showed that the transcription of several neuropeptide genes in the visceral ganglia, including NPF and insulin-like peptide, increased after starvation. Finally, the injection of visceral ganglia extracts and synthetic NPF increased the transcription of a mucosal lectin and a glycogen synthase, known to be involved in food capture and glucose storage. Overall, this study identifies key genes regulating oyster physiology, enhancing the understanding of the control of basic physiological mechanisms in C. virginica.

The reduction in pH from atmospheric inputs of CO2 (ocean acidification, OA) threatens marine calcifiers, including the eastern oyster (Crassostrea virginica), that precipitate biogenic CaCO3 for shell formation. Recent investigations have demonstrated that alterations in gene expression enable bivalves to respond to episodic low pH. Evidence generated from several studies highlighted the importance of upregulating genes related to biomineralization, ion transport, and acid-base balance such as carbonic anhydrase (CA) genes. Two experiments were designed to evaluate the effect of acidification on calcification processes and to probe the specific role of CA in oyster resilience to low pH. First, adult oysters were exposed to eight months of chronic acidification stress (pH similar to 7.3, pCO(2) similar to 3300 ppm) or control conditions (pH similar to 7.9, pCO(2) similar to 500 ppm) before shells were artificially damaged and shell repair monitored. Results showed a dramatic decrease in shell regeneration after chronic high pCO(2) exposure (only 30% of oysters regrew any shell) suggesting that mechanisms that promote calcification under high pCO(2) conditions may not be sustainable for extended periods of time. To further explore these mechanisms, a second experiment was designed by focusing on the role of CA in mitigating acidification stress. Here, adult oysters received an injection of acetazolamide in dimethyl sulfoxide (DMSO) to inhibit CA or DMSO (control) before rearing in control (pH similar to 8.1, pCO(2) similar to 340 ppm) or acidified (pH similar to 7.3, pCO(2) similar to 3300 ppm) conditions. After three weeks, oyster shells were damaged and shell repair monitored. Oysters incubated at low pH seawater with CA inhibition had the least amount of shell regeneration at the end of 21-day regrowth period. Interestingly, oysters were able to increase intracellular pH (pH(i)) of hemocytes under low pH conditions; however, this ability was significantly diminished with CA inhibition. Results highlight the role of CA in maintaining calcification under low pH conditions by establishing an intracellular environment favorable to calcium carbonate precipitation.

Temperature and food availability play large roles in bivalve energetics. Understanding bivalve responses to variability in temperature and food availability (i.e., phytoplankton), is important as climate change leads to ocean warming and changes in phytoplankton production. However, few studies address how changes in seasonal temperature regimes, such as an elevated fall or accelerated spring temperature regime affect bivalves via carryover effects, whereby response signals are detected months following temperature regime exposure. Few studies also address how bivalve feeding preferences may respond to variability in temperature and food availability. Here, controlled laboratory experiments simulated climate changed-induced fall and spring temperature regimes for Atlantic surfclams, Spisula solidissima solidissima. A variety of physiological responses were measured, including scope for growth, gonad development and feeding behavior, plus preferences for different phytoplankton groups. Carryover effects were observed where surfclams that experienced an elevated (+ 3.0 C) fall temperature regime yielded enhanced gonad development the following spring (i.e., > 6 months later). An accelerated spring temperature regime (a more rapid temperature increase to 17 C from 7 C) also impacted surfclam scope for growth. Temperature was the primary driver of surfclam clearance rates, but food concentration was the primary driver of surfclam feeding preferences. Surfclams displayed preferential selection of diatoms and chlorophytes over cryptophytes and cyanobacteria, but increased food availability led to decreased selection of diatoms and a relative increase in the uptake of cyanobacteria. These results suggest that climate change induced alterations in food availability and seasonal temperature regimes may affect surfclam metabolism, reproduction and feeding preferences.

Ocean acidification (OA) is recognized as a major stressor for a broad range of marine organisms, particularly shell-building invertebrates. OA can cause alterations in various physiological processes such as growth and metabolism, although its effect on host-pathogen interactions remains largely unexplored. In this study, we used transcriptomics, proteomics, and physiological assays to evaluate changes in immunity of the eastern oyster Crassostrea virginica exposed to OA conditions (pH = 7.5 vs pH = 7.9) at various life stages. The susceptibility of oyster larvae to Vibrio infection increased significantly (131 % increase in mortality) under OA conditions, and was associated with significant changes in their transcriptomes. The significantly higher mortality of larvae exposed to pathogens and acidification stress could be the outcome of an increased metabolic demand to cope with acidification stress (as seen by upregulation of metabolic genes) at the cost of immune function (downregulation of immune genes). While larvae were particularly vulnerable, juveniles appeared more robust to the stressors and there were no differences in mortality after pathogen (Aliiroseovarius crassostrea and Vibrio spp.) exposure. Proteomic investigations in adult oysters revealed that acidification stress resulted in a significant downregulation of mucosal immune proteins including those involved in pathogen recognition and microbe neutralization, suggesting weakened mucosal immunity. Hemocyte function in adults was also impaired by high pCO2, with a marked reduction in phagocytosis (67 % decrease in phagocytosis) in OA conditions. Together, results suggest that OA impairs immune function in the eastern oyster making them more susceptible to pathogen-induced mortality outbreaks. Understanding the effect of multiple stressors such as OA and disease is important for accurate predictions of how oysters will respond to future climate regimes.

The bay scallop, Argopecten irradians, is a species of major commercial, cultural, and ecological importance. It is endemic to the eastern coast of the United States, but has also been introduced to China, where it supports a significant aquaculture industry. Here, we provide an annotated chromosome-level reference genome assembly for the bay scallop, assembled using PacBio and Hi-C data. The total genome size is 845.9 Mb, distributed over 1,503 scaffolds with a scaffold N50 of 44.3 Mb. The majority (92.9%) of the assembled genome is contained within the 16 largest scaffolds, corresponding to the 16 chromosomes confirmed by Hi-C analysis. The assembly also includes the complete mitochondrial genome. Approximately 36.2% of the genome consists of repetitive elements. The BUSCO analysis showed a completeness of 96.2%. We identified 33,772 protein-coding genes. This genome assembly will be a valuable resource for future research on evolutionary dynamics, adaptive mechanisms, and will support genome-assisted breeding, contributing to the conservation and management of this iconic species in the face of environmental and pathogenic challenges.

Atlantic menhaden are a highly migratory marine species in the Eastern United States that suffer from seasonal chronic mortality. Affected fish show neurologic signs referred to as spinning disease, including circling at the surface and erratic corkscrew swimming before death. We investigated three similar menhaden mortality events consistent with spinning disease in coastal New Jersey and New York between 2020 and 2021 to understand the cause. A unique strain of Vibrio (Listonella) anguillarum (serogroup O3) was detected regularly in high loads, particularly in the brains of moribund fish, by both metagenomics and bacterial isolation. The most common histopathological changes in moribund fish were hemorrhagic meningitis, encephalitis, pyknosis, and karyorrhexis of hematopoietic tissues in the kidney and spleen. Whole genome sequencing of isolates from moribund fish representing a wide spatial and temporal range showed that they were nearly identical clones, suggesting it to be a pathogenic strain circulating in the population. Though V. anguillarum is believed to be the main pathogen associated with spinning disease and mortality, Yersinia ruckeri (serotype O1) was isolated from smaller numbers of fish. Considering the highly migratory nature of Atlantic menhaden throughout the eastern United States and their use as bait for other fisheries, these findings identify potential biosecurity challenges that should be considered in Atlantic salmon aquaculture, fisheries, and emerging marine aquaculture in the region.