Survival and detection of bivalve transmissible neoplasia from the soft-shell clam Mya arenaria (MarBTN) in seawater

Many pathogens can cause cancer, but cancer itself does not normally act as an infectious agent. However, transmissible cancers have been found in a few cases in nature: in Tasmanian devils, dogs, and several bivalve species. The transmissible cancers in dogs and devils are known to spread through direct physical contact, but the exact route of transmission of bivalve transmissible neoplasia (BTN) has not yet been confirmed. It has been hypothesized that cancer cells could be released by diseased animals and spread through the water column to infect/engraft into other animals. To test the feasibility of this proposed mechanism of transmission, we tested the ability of BTN cells from the soft-shell clam (Mya arenaria BTN, or MarBTN) to survive in artificial seawater. We found that BTN cells are highly sensitive to salinity, with acute toxicity at salinity levels lower than those found in their environment. BTN cells also survive longer at lower temperatures, with >48% of cells surviving a week in seawater at temperatures from 4°C to 16°C, and 49% surviving for more than two weeks at 4°C. With one clam donor, living cells were observed for more than eight weeks at 4°C. We also used qPCR of environmental DNA (eDNA) to detect the presence of BTN-specific DNA in the environment. We observed release of BTN-specific DNA into the water of aquaria from tanks with highly BTN-positive clams, and we detected BTN-specific DNA in seawater samples collected from BTN-endemic areas, although the level detected was much lower. Overall, these data show that BTN cells can survive well in seawater, and they are released into the water by diseased animals, supporting the hypothesis that BTN is spread from animal-to-animal by cells through seawater.

The truncate soft-shell clam, Mya truncata, as a biomonitor of municipal wastewater exposure and historical anthropogenic impacts in the Canadian Arctic.

Municipal wastewater is a large source of pollution to Canadian waters, yet its effects on Arctic marine ecosystems remains relatively unknown. We characterized the impacts of municipal wastewater from a growing northern community, Iqaluit, Nunavut on the Arctic truncate soft-shell clam,Mya truncata. Clams were sampled from six locations that varied in proximity to the wastewater treatment plant and shell biogeochemical analysis revealed that clams nearest the wastewater treatment plant had slower growth rates, lower carbon and oxygen stable isotope ratios, and elevated concentrations of copper and lead. A parallel analysis on mRNA expression profiles characterized M. truncata’s physiological response to wastewater effluent. Clams nearest the wastewater treatment plant had significantly lower mRNA expression of genes associated with metabolism, antioxidants, molecular chaperones, and phase I and II detoxification, but had heightened mRNA expression in genes coding for enzymes that bind and remove contaminants. These results demonstrated a biological response to Iqaluit’s wastewater effluent and highlight M. truncata’s potential to act as a biomonitor of municipal wastewater along Arctic coastlines in Canada.

The truncate soft-shell clam, Mya truncata, as a biomonitor of municipal wastewater exposure and historical anthropogenic impacts in the Canadian Arctic.

Municipal wastewater is a large source of pollution to Canadian waters, yet its effects on Arctic marine ecosystems remains relatively unknown. We characterized the impacts of municipal wastewater from a growing northern community, Iqaluit, Nunavut on the Arctic truncate soft-shell clam, Mya truncata. Clams were sampled from six locations that varied in proximity to the wastewater treatment plant and shell biogeochemical analysis revealed that clams nearest the wastewater treatment plant had slower growth rates, lower carbon and oxygen stable isotope ratios, and elevated concentrations of copper and lead. A parallel analysis on mRNA expression profiles characterized M. truncata's physiological response to wastewater effluent. Clams nearest the wastewater treatment plant had significantly lower mRNA expression of genes associated with metabolism, antioxidants, molecular chaperones, and phase I and II detoxification, but had heightened mRNA expression in genes coding for enzymes that bind and remove contaminants. These results demonstrated a biological response to Iqaluit's wastewater effluent and highlight M. truncata's potential to act as a biomonitor of municipal wastewater along Canadian Arctic coastlines.

The influence of different oxygen regimes on metabolism and behavior of a soft shell clam Mya arenaria

<p>Hypoxia is widely distributed in coastal benthic habitats and is driven by warming, nutrient pollution and the diurnal cycles of photosynthesis and respiration. Benthic sessile species, such as the soft shell clam <em>Mya arenaria</em>, are commonly exposed to oxygen fluctuations in their habitats which might negatively impact the performance and metabolism of clams. To determine the effects of different oxygen regime on metabolism and behavior of <em>M. arenaria</em>, we exposed the clams for 21 days to chronic (constant) hypoxia at 20% of air saturation, fluctuating (cyclic) hypoxia (~10-50% of air saturation) and normoxia (100% of air saturation). To mimic conditions occurring in coastal hypoxic zones, CO2 and pH levels varied with the oxygen. We assessed the digging performance, bioirrigation capacity and bioenergetics of the clams. Acclimation to constant or cyclic hypoxia did not affect the oxygen consumption of the clams, but the oxygen consumption rates declined at low ambient oxygen concentrations regardless of the acclimation to different oxygen regimes. Clams acclimated to constant hypoxia mainly used lipids, whereas clams acclimated to cyclic hypoxia used carbohydrates as energy fuel. Clams acclimated to constant or cyclic hypoxia dug slower compared to the clams acclimated to normoxia. Furthermore, bioirrigation capacity decreased in clams acclimated to constant hypoxia. Our results indicate that constant and cyclic hypoxia impair bioturbation and bioirrigation capacity of clams which has implications for their ecological function as ecosystem engineers in benthic soft bottom habitats. </p>

Extracellular Vesicles and Post-Translational Protein Deimination Signatures in Mollusca—The Blue Mussel (Mytilus edulis), Soft Shell Clam (Mya arenaria), Eastern Oyster (Crassostrea virginica) and Atlantic Jacknife Clam (Ensis leei)

Oysters and clams are important for food security and of commercial value worldwide. They are affected by anthropogenic changes and opportunistic pathogens and can be indicators of changes in ocean environments. Therefore, studies into biomarker discovery are of considerable value. This study aimed at assessing extracellular vesicle (EV) signatures and post-translational protein deimination profiles of hemolymph from four commercially valuable Mollusca species, the blue mussel (Mytilus edulis), soft shell clam (Mya arenaria), Eastern oyster (Crassostrea virginica), and Atlantic jacknife clam (Ensis leei). EVs form part of cellular communication by transporting protein and genetic cargo and play roles in immunity and host–pathogen interactions. Protein deimination is a post-translational modification caused by peptidylarginine deiminases (PADs), and can facilitate protein moonlighting in health and disease. The current study identified hemolymph-EV profiles in the four Mollusca species, revealing some species differences. Deiminated protein candidates differed in hemolymph between the species, with some common targets between all four species (e.g., histone H3 and H4, actin, and GAPDH), while other hits were species-specific; in blue mussel these included heavy metal binding protein, heat shock proteins 60 and 90, 2-phospho-D-glycerate hydrolyase, GTP cyclohydrolase feedback regulatory protein, sodium/potassium-transporting ATPase, and fibrinogen domain containing protein. In soft shell clam specific deimination hits included dynein, MCM3-associated protein, and SCRN. In Eastern oyster specific deimination hits included muscle LIM protein, beta-1,3-glucan-binding protein, myosin heavy chain, thaumatin-like protein, vWFA domain-containing protein, BTB domain-containing protein, amylase, and beta-catenin. Deiminated proteins specific to Atlantic jackknife clam included nacre c1q domain-containing protein and PDZ domain-containing protein In addition, some proteins were common as deiminated targets between two or three of the Bivalvia species under study (e.g., EP protein, C1q domain containing protein, histone H2B, tubulin, elongation factor 1-alpha, dominin, extracellular superoxide dismutase). Protein interaction network analysis for the deiminated protein hits revealed major pathways relevant for immunity and metabolism, providing novel insights into post-translational regulation via deimination. The study contributes to EV characterization in diverse taxa and understanding of roles for PAD-mediated regulation of immune and metabolic pathways throughout phylogeny.

Population Genetic Structure Is Unrelated to Shell Shape, Thickness and Organic Content in European Populations of the Soft-Shell Clam Mya Arenaria

The soft-shell clam Mya arenaria is one of the most ancient invaders of European coasts and is present in many coastal ecosystems, yet little is known about its genetic structure in Europe. We collected 266 samples spanning a latitudinal cline from the Mediterranean to the North Sea and genotyped them at 12 microsatellite loci. In parallel, geometric morphometric analysis of shell outlines was used to test for associations between shell shape, latitude and genotype, and for a selection of shells we measured the thickness and organic content of the granular prismatic (PR), the crossed-lamellar (CL) and the complex crossed-lamellar (CCL) layers. Strong population structure was detected, with Bayesian cluster analysis identifying four groups located in the Mediterranean, Celtic Sea, along the continental coast of the North Sea and in Scotland. Multivariate analysis of shell shape uncovered a significant effect of collection site but no associations with any other variables. Shell thickness did not vary significantly with either latitude or genotype, although PR thickness and calcification were positively associated with latitude, while CCL thickness showed a negative association. Our study provides new insights into the population structure of this species and sheds light on factors influencing shell shape, thickness and microstructure.