Mesocosm study of PAC-modified clay effects on Karenia brevis cells and toxins, chemical dynamics, and benthic invertebrate physiology.

Modified clay compounds are used globally as a method of controlling harmful algal blooms, and their use is currently under consideration to control Karenia brevis blooms in Florida, USA. In 1400 L mesocosm tanks, chemical dynamics and lethal and sublethal impacts of MC II, a polyaluminum chloride (PAC)-modified kaolinite clay, were evaluated over 72 h on a benthic community representative of Sarasota Bay, which included blue crab (Callinectes sapidus), sea urchin (Lytechinus variegatus), and hard clam (Mercenaria campechiensis). In this experiment, MC II was dosed at 0.2 g L-1 to treat bloom-level densities of K. brevis at 1 × 106 cells L-1. Cell removal in MC II-treated tanks was 57% after 8 h and 95% after 48 h. In the water column, brevetoxin analogs BTx-1 and BTx-2 were found to be significantly higher in untreated tanks at 24 and 48 h, while in MC II-treated tanks, BTx-3 was found to be higher at 48 h and BTx-B5 was found to be higher at 24 and 48 h. In MC II floc, we found no significant differences in BTx-1 or BTx-2 between treatments for any time point, while BTx-3 was found to be significantly higher in the MC II-treated tanks at 48 and 72 h, and BTx-B5 was higher in MC II-treated tanks at 24 and 72 h. Among various chemical dynamics observed, it was notable that dissolved phosphorus was consistently significantly lower in MC II tanks after 2 h, and that turbidity in MC II tanks returned to control levels 48 h after treatment. Dissolved inorganic carbon and total seawater alkalinity were significantly reduced in MC II tanks, and partial pressure of CO2 (pCO2) was significantly higher in the MC II-only treatment after 2 h. In MC II floc, particulate phosphorus was found to be significantly higher in MC II tanks after 24 h. In animals, lethal and sublethal responses to MC II-treated K. brevis did not differ from untreated K. brevis for either of our three species at any time point, suggesting MC II treatment at this dosage has negligible impacts to these species within 72 h of exposure. These results appear promising in terms of the environmental safety of MC II as a potential bloom control option, and we recommend scaling up MC II experiments to field trials in order to gain deeper understanding of MC II performance and dynamics in natural waters.

Predator-prey interactions in a coastal setting: Linking crab feeding rates to small scale distribution of clams.

A coastal predator-prey system, juvenile green crabs (Carcinus maenas) preying upon juvenile hard clams (Mercenaria mercenaria), was used to explore the link between crab predation rates and clam density and small-scale distribution patterns. The channel working area of a racetrack flume was adapted to form a sedimentary arena in a flowing seawater system (5 cm s-1) to assess crab predation rates in relation to clam density and distribution patterns (clams clustered in one patch vs two nearby vs two farther apart). The trials detected significant differences in relation to clam initial density and distribution with strong (∼50%) declines in clam mortality levels among spatial arrangements (one patch > two nearby > two farther apart). Feeding of clams was associated with the time taken by crabs to handle the first clam (first patch), and the frequency of three distinct types of crab behavior (eating, resting, and searching). Altogether these results suggest that small-scale changes in number and distribution of juvenile clams matter and may have unexpectedly strong effects on the outcome of predator-prey interactions.

A new member of the Nudiviridae from the Florida stone crab (Menippe mercenaria).

Menippe mercenaria, the Florida stone crab, supports an unconventional fishery across the southern USA and Caribbean that involves claw-removal and the return of de-clawed animals to the sea. We provide pathological, ultrastructural, and genomic detail for a novel hepatopancreatic, nucleus-specific virus - Menippe mercenaria nudivirus (MmNV) - isolated from M. mercenaria, captured during fisheries-independent monitoring. The virus has a genome of 99,336 bp and encodes 84 predicted protein coding genes and shows greatest similarity to Aratus pisonii nudivirus (ApNV) (<60% protein similarity and 31 shared genes of greatest similarity), collected from the Florida Keys, USA. MmNV is a member of the Gammanudivirus genus (Naldaviricetes: Lefavirales: Nudiviridae). Comparisons of virus genome size, preferred host environment, and gene number revealed no clear associations between the viral traits and phylogenetic position. Evolution of the virus alongside the diversification of host taxa, with the potential for host-switching, remain more likely evolutionary pathways.

Autophagy contributes to increase the content of intracellular free amino acids in hard clam (Mercenaria mercenaria) during prolonged exposure to hypersaline environments.

Marine bivalves in intertidal zones and land-based seawater ponds are constantly subjected to a wide range of salinity fluctuations due to heavy rainfall, intense drought, and human activities. As osmoconformers, bivalves rely primarily on rapid release or accumulation of free amino acids (FAAs) for osmoregulation. Euryhaline bivalves are capable of withstanding hyposaline and hypersaline environments through regulation of physiology, metabolism, and gene expression. However, current understanding of the molecular mechanisms underlying osmoregulation and salinity adaptation in euryhaline bivalves remains largely limited. In this study, RNA-seq, WGCNA and flow cytometric analysis were performed to investigate the physiological responses of hard clams (Mercenaria mercenaria) to acute short-term hyposalinity (AL) and hypersalinity (AH), and chronic long-term hyposalinity (CL) and hypersalinity (CH) stress. We found that amino acids biosynthesis was significantly inhibited and aminoacyl-tRNA biosynthesis was augmented to decrease intracellular osmolarity during hyposaline exposure. Under CH, numerous autophagy-related genes (ATGs) were highly expressed, and the autophagy activity of gill cells were significantly up-regulated. A significant decrease in total FAAs content was observed in gills after NH4Cl treatment, indicating that autophagy was crucial for osmoregulation in hard clams during prolonged exposure to hypersaline environments. To prevent premature or unnecessary apoptosis, the expression of cathepsin L was inhibited under AL and AH, and inhibitors of apoptosis was augmented under CL and CH. Additionally, neuroendocrine regulation was involved in salinity adaption in hard clams. This study provides novel insights into the physiological responses of euryhaline marine bivalves to hyposaline and hypersaline environments.

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.

Comparison of the flavor qualities between two varieties of Mercenaria mercenaria.

The saltwater hard clam Mercenaria mercenaria (M. mercenaria) as a representative of low-value shellfish, enhancing its flavor quality, is the key to enter the high-end market. Nevertheless, there has not been reported research on the flavor quality of M. mercenaria. This study compared the flavor quality of selective and non-selective saltwater hard clams of M. mercenaria by using various indicators: proximate component, free amino acids, nucleotides, and metabolomic analysis. The results indicated that selective breeding contributed to the significant improvement contents of crude protein, flavor-associated free amino acids (glutamic acid, aspartic acid, proline, etc.), and nucleotides (AMP) (P < 0.05). Then, the metabolome was utilized to assess the metabolite changes in the pre/post-selective breeding of M. mercenaria and further understand the flavor characteristics and metabolic status. In the metabolomics assay, among the 3143 quantified metabolites, a total of 102 peaks were identified as significantly different metabolites (SDMs) between the selective and non-selective varieties of M. mercenaria (VIP > 1 and P < 0.05). These results can provide new insights for future research on improving the quality of saltwater bivalves through selective breeding.

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.

Sublethal effects on the hard clam Mercenaria mercenaria after exposure to Aureococcus anophagefferens Chinese strain isolates.

Brown tides caused by Aureococcus anophagefferens occur frequently worldwide and have contributed to the collapse of Mercenaria mercenaria farming in the United States. This economically valuable hard clam has been used in China for more than 20 years. To date, it has remained unknown whether A. anophagefferens Chinese strain has an impact on hard clam cultivation in the coastal areas of China or other sea areas worldwide if it enters through ship ballast water and other ways. In this study, a Chinese strain of A. anophagefferens isolated from the brown tide waters of Bohai Bay, China, was selected to explore its influence on the feedback of hard clams. After being fed with A. anophagefferens, hard clams showed characteristics similar to starvation. The reduced feeding efficiency of hard clams leads to reduced energy intake. However, the immune response and oxidative stress, result in increased energy consumption. An imbalance in the energy budget may be an important reason for hard clam starvation. This study has described the response characteristics of the A. anophagefferens Chinese strain to M. mercenaria, explored the reasons for the negative impact of A. anophagefferens on hard clams, and provides ideas for reducing shellfish aquaculture caused by brown tides.

Baseline assessment of microplastics in commercially important marine bivalves from New York, U.S.A.

Microplastic (MP) contamination in bivalve mollusks has become a significant concern over the last few years. These ecologically and economically valuable species are popular seafood items for human consumption. As filter feeders, bivalves may ingest MPs in their bodies, possibly impacting their physiology and fitness. Additionally, a considerable amount of the seafood that humans consume comes from coastal areas where MP concentrations tend to be the highest. This research provides the first examination of MPs in eastern oysters (Crassostrea virginica) and hard clams (Mercenaria mercenaria) that were grown locally in coastal areas of New York, contributing to a baseline for the northeast and mid-Atlantic regions of the U.S. A total of 48 eastern oysters (n = 12 per site, at four sites) and hard clams (n = 24 per site, at two sites) were sampled in summer 2021. While MP fibers and fragments (i.e. polyethylene terephthalate, polystyrene, and polypropylene) were found in some oysters, other contaminants (e.g. indigo dye, phthalocyanine, dye 823, etc.) were found in both bivalve species. Particle composition was verified using Raman microspectroscopy. Although mean MP concentrations were low in eastern oysters (i.e. 0.008 MPs g-1 of soft tissue wet weight; 0.125 MPs ind-1) and not found in hard clams, more research is needed to assess the magnitude of contamination in these edible bivalves.

Effect of heat and hypoxia stress on mitochondrion and energy metabolism in the gill of hard clam.

Aquatic animals suffer from heat and hypoxia stress more frequently due to global climate change and other anthropogenic activities. Heat and hypoxia stress can significantly affect mitochondrial function and energy metabolism. Here, the response and adaptation characteristics of mitochondria and energy metabolism in the gill of the hard clam Mercenaria mercenaria under heat (35 °C), hypoxia (0.2 mg/L), and heat plus hypoxia stress (35 °C, 0.2 mg/L) after 48 h exposure were investigated. Mitochondrial membrane potentials were depolarized under environmental stress. Mitochondrial fusion, fission and mitophagy played a key role in maintain mitochondrion function. The AMPK subunits showed different expression under environmental stress. Acceleration of enzyme activities (phosphofructokinase, pyruvate kinase and lactic dehydrogenase) and accumulation of anaerobic metabolites in glycolysis and TCA cycle implied that the anaerobic metabolism might play a key role in providing energy. Accumulation of amino acids might help to increase tolerance under heat and heat combined hypoxia stress. In addition, urea cycle played a key role in amino acid metabolism to prevent ammonia/nitrogen toxicity. This study improved our understanding of the mitochondrial and energy metabolism responses of marine bivalves exposed to environmental stress.

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.

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.

The invasive red seaweed, Dasysiphonia japonica, forms harmful algal blooms: Mortality in early life stage fish and bivalves and identification of putative toxins.

In recent decades, the rate of introduction of non-indigenous macroalgae has increased. While invasive seaweeds often outcompete native species for substrata, their direct effects on marine life are rarely described. Here, we describe 'red water' events caused by the decay of blooms of the invasive red seaweed, Dasysiphonia japonica, in Great South Bay, NY, USA, and the ability of water from such events to induce rapid and significant mortality in larval and juvenile fish (Menidia beryllina, Menidia menidia, and Cyprinodon variegatus) and larval bivalves (Mercenaria mercenaria and Crassostrea virginica). All species studied experienced significant (p<0.05) reductions in survival when exposed to macroalgae in a state of decay, seawater in which the alga was previously decayed, or both. Both bivalve species experienced 50-60% increases in mortality when exposed to decaying D. japonica for ∼ one week, despite normoxic conditions. Among fish, significant increases (40-80%) in mortality were observed after 24 h exposure to decayed D. japonica and one-week exposures caused, on average, 90% mortality in larval M. beryllina, 50% mortality in juvenile (∼3 cm) M. menidia, and 50% mortality in larval C. variegatus. All fish and bivalve mortality occurred under normoxic conditions (dissolved oxygen (DO) >7 mg L-1) and low ammonium levels (< 20 µM), with the exception of C. variegatus, which expired under conditions of decayed D. japonica coupled with reduced DO caused by the alga. Screening of water with decayed D. japonica using liquid chromatography-mass spectrometry revealed compounds with mass-to-charge ratios matching caulerpin, a known algal toxin that causes fish and shellfish mortality, and several other putative toxicants at elevated levels. Collectively, the high levels of mortality (50-90%) of larval and juvenile fish and bivalves exposed to decaying D. japonica under normoxic conditions coupled with the observation of 'red water' events in estuaries collectively indicate the red seaweed, D. japonica, can create harmful algal blooms (HABs).

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 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.

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.

Metabolic response of Mercenaria mercenaria under heat and hypoxia stress by widely targeted metabolomic approach.

In the context of global climatic changes, marine organisms have been exposed to environmental stressors including heat and hypoxia. This calls for the design of multi-stressors to uncover the impact of oceanic factors on aquatic organisms. So far, little is known about the metabolic response of marine organisms, especially bivalves, to the combined effects of heat and hypoxia. In this study, we employed widely targeted metabolomic analysis to study the metabolic response of gills in hard clam, a heat- and hypoxia-tolerant bivalve. A total of 810 metabolites were identified. Results showed that the heat group (HT) and heat plus hypoxia group (HL) had a higher number of differential metabolites than the hypoxia group (LO). Glycolysis was affected by the heat and heat plus hypoxia stress. Moreover, anaerobic metabolic biomarkers were accumulated marking the onset of anaerobic metabolism. Environmental stresses may affect Tricarboxylic acid (TCA) cycle. Accumulation of carnitine and glycerophospholipid may promote fatty acid ß oxidation and maintain cell membrane stability, respectively. The high content of oxidized lipids (i.e., Leukotriene) in HL and HT groups implied that the organisms were under ROS stress. The significantly differential metabolites of organic osmolytes and vitamins might relieve ROS stress. Moreover, accumulation of thermoprotective osmolytes (monosaccharide, Trimethylamine N-oxide (TMAO)) accumulation was helpful to maintain protein homeostasis. This investigation provided new insights into the adaptation mechanisms of hard clam to heat, hypoxia and combined stress at the metabolite level and highlighted the roles of molecules and protectants.

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.