Biogeography of resistance to paralytic shellfish toxins in softshell clam, Mya arenaria (L.), populations along the Atlantic coast of North America.

Blooms of Alexandrium spp., the causative agent of paralytic shellfish poisoning (PSP), recur with varying frequency and intensity on the Northwest Atlantic coast of North America, from New York, USA, to northern Canadian waters. Along this latitudinal range blooms co-occur with abundant, intertidal populations of softshell clams, Mya arenaria. Prior work identified a naturally-occurring genetic mutation in Domain II α-subunit of the clams' voltage-gated sodium channels (NaV), which significantly reduces the binding affinity of the paralytic shellfish toxin, saxitoxin (STX). This mutation provides clams with resistance to the deleterious effects of STX, allowing them to continue feeding during Alexandrium spp. blooms and attain very high tissue toxicities. This study used genetic sequencing of the NaV mutation locus in clams from four coastal regions of the Bay of Fundy-Gulf of Maine and the mid-Atlantic to determine the percentage of clams in each region that possess the resistant NaV mutation. The genotype composition was related to the occurrence and magnitude of PSP outbreaks based on shellfish toxicity, primarily that of mussels, Mytilus edulis, used as a proxy for the prevalence and severity of Alexandrium blooms in each region. As hypothesized, the proportion of clams bearing the resistant mutation generally matched up well with the historical incidence and intensity of Alexandrium spp. blooms. The highest percentage of homozygote resistant clams (RR = 70.0%), and the lowest percentage of sensitive clams (SS = 4.5%) were found in eastern Gulf of Maine populations. Exceptions at a few sites where anomalously high numbers of M. arenaria with the resistant mutation were found despite the absence of blooms, may be attributable to larval gene flow. There was no evidence that Alexandrium blooms occurring in Northport Harbor, Long Island, have resulted in a shift in genotypic composition of the local clam population, presumably due to their low cell toxicity. Seasonal mismatch of highly vulnerable M. arenaria postset with toxic blooms at this latitude may also partly explain this result. This study provides strong supporting evidence that Alexandrium blooms can select for resistance to PSP-toxins in M. arenaria populations and proposes a mechanism for the persistence of the sensitive allele throughout the region. Implications for clam aquaculture (seeding) efforts, as well as for shellfish toxicity monitoring are discussed.

Shell matrix proteins of the clam, Mya truncata: Roles beyond shell formation through proteomic study.

Mya truncata, a soft shell clam, is presented as a new model to study biomineralization through a proteomics approach. In this study, the shell and mantle tissue were analysed in order to retrieve knowledge about the secretion of shell matrix proteins (SMPs). Out of 67 and 127 shell and mantle proteins respectively, 16 were found in both shell and mantle. Bioinformatic analysis of SMP sequences for domain prediction revealed the presence of several new domains such as fucolectin tachylectin-4 pentraxin-1 (FTP), scavenger receptor, alpha-2-macroglobulin (α2 M), lipocalin and myosin tail along with previously reported SMP domains such as chitinase, carbonic anhydrase, tyrosinase, sushi, and chitin binding. Interestingly, these newly predicted domains are attributed with molecular functions other than biomineralization. These findings suggest that shells may not only act as protective armour from predatory action, but could also actively be related to other functions such as immunity. In this context, the roles of SMPs in biomineralization need to be looked in a new perspective.

Mining the transcriptomes of four commercially important shellfish species for single nucleotide polymorphisms within biomineralization genes.

Transcriptional profiling not only provides insights into patterns of gene expression, but also generates sequences that can be mined for molecular markers, which in turn can be used for population genetic studies. As part of a large-scale effort to better understand how commercially important European shellfish species may respond to ocean acidification, we therefore mined the transcriptomes of four species (the Pacific oyster Crassostrea gigas, the blue mussel Mytilus edulis, the great scallop Pecten maximus and the blunt gaper Mya truncata) for single nucleotide polymorphisms (SNPs). Illumina data for C. gigas, M. edulis and P. maximus and 454 data for M. truncata were interrogated using GATK and SWAP454 respectively to identify between 8267 and 47,159 high quality SNPs per species (total=121,053 SNPs residing within 34,716 different contigs). We then annotated the transcripts containing SNPs to reveal homology to diverse genes. Finally, as oceanic pH affects the ability of organisms to incorporate calcium carbonate, we honed in on genes implicated in the biomineralization process to identify a total of 1899 SNPs in 157 genes. These provide good candidates for biomarkers with which to study patterns of selection in natural or experimental populations.

Characterisation of the mantle transcriptome and biomineralisation genes in the blunt-gaper clam, Mya truncata.

Members of the Myidae family are ecologically and economically important, but there is currently very little molecular data on these species. The present study sequenced and assembled the mantle transcriptome of Mya truncata from the North West coast of Scotland and identified candidate biomineralisation genes. RNA-Seq reads were assembled to create 20,106 contigs in a de novo transciptome, 18.81% of which were assigned putative functions using BLAST sequence similarity searching (cuttoff E-value 1E-10). The most highly expressed genes were compared to the Antarctic clam (Laternula elliptica) and showed that many of the dominant biological functions (muscle contraction, energy production, biomineralisation) in the mantle were conserved. There were however, differences in the constitutive expression of heat shock proteins, which were possibly due to the M. truncata sampling location being at a relatively low latitude, and hence relatively warm, in terms of the global distribution of the species. Phylogenetic analyses of the Tyrosinase proteins from M. truncata showed a gene expansion which was absent in L. elliptica. The tissue distribution expression patterns of putative biomineralisation genes were investigated using quantitative PCR, all genes showed a mantle specific expression pattern supporting their hypothesised role in shell secretion. The present study provides some preliminary insights into how clams from different environments - temperate versus polar - build their shells. In addition, the transcriptome data provides a valuable resource for future comparative studies investigating biomineralisation.

Impacts of stage-specific acute pesticide exposure on predicted population structure of the soft-shell clam, Mya arenaria.

A combined laboratory and modeling approach was used to assess the impact of selected pesticides on early life stages of the soft-shell clam, Mya arenaria. Clams were exposed for 24h as veligers or pediveligers to the broad-spectrum herbicide hexazinone [3-cyclohexyl-6-(dimethylamino)-1-methyl-1,3,5-triazine-2,4(1h,3h)-dione; Velpar], the phenoxyacetic acid herbicide, 2,4-D (2,4-dichlorophenoxyacetic acid; Agway Super BK 32), or phosmet (Imidan). In addition, juvenile clams were exposed for 24h to 2,4-D and their growth monitored for 21 months. Laboratory experiments indicated veligers were more sensitive to acute pesticide exposure than pediveligers, with 2,4-D exposed veligers exhibiting the lowest survival among all treatments. Relative to controls, juvenile clams exposed to 0.5 ppm 2,4-D had enhanced survival following the initial 3 months of grow out. Juveniles exposed to 0.5, 5 and 10 ppm 2,4-D showed an initial growth delay relative to control clams, but at 21 months post-exposure these clams were significantly larger than control clams. Data from the larval and juvenile exposures were used to generate a stage-specific matrix model to predict the effect of pesticide exposure on clam populations. Impacts on simulated clam populations varied with the pesticide and stage exposed. For example, 2,4-D exposure of veligers and pediveligers significantly reduced predicted recruitment as well as population growth rate compared to controls, but juvenile exposure to 2,4-D did not significantly reduce population growth rate. With the exception of veligers exposed to 10 ppm, hexazinone exposure at the both veliger and pediveliger stages significantly reduced predicted recruitment success compared to 0 ppm controls. Hexazinone exposure also reduced modeled population growth rates, but these reductions were only slight in the pediveliger exposure simulations. Veliger and pediveliger exposure to phosmet reduced modeled population growth rate in a dose-dependent fashion. Changes in modeled population stable stage distributions were also observed when veligers were exposed to any pesticide. These results suggest that both the stage of exposure and the specific toxicant are important in predicting effects of pesticide exposure on soft-shell clam populations, with earlier life stages showing greater sensitivity to the pesticides tested.

Impacts of pollution in feral Mya arenaria populations: the effects of clam bed distance from the shore.

This study examined the relationships between population characteristics and the expression of physiological biomarkers of stress in an intertidal clam population under pollution at sites differing in thermal history and coastline distance. The clam population metrics were age distribution, growth, condition factor, distance of the clam beds from the shore, and gonad development. Physiological biomarkers comprised biomarkers of defence such as superoxide dismutase, labile IIb metals in tissues, redox status of metallothioneins and glutathione S-transferase, of tissue damage such as lipid peroxidation and DNA strand breaks, of reproduction as determined by vitellogenin-like proteins and gonadosomatic index and immunocompetence such as phagocytosis and hemocyte viability. Age-related pigments were also examined to compare the physiological age of the clams with their chronological age. The results showed that all the above biomarkers were significantly affected at one of the two polluted sites at least. Distance from the shore was significantly correlated with most (81%) of the biomarkers examined. Clams collected at one polluted site were physiologically older than clams from the corresponding reference site. Canonical and adaptive regression (artificial neural networks) analyses found that the biomarkers measured in this study were able to predict the ecologically relevant endpoints. Biomarkers implicated in defense mechanisms, tissue damage and age-related pigments were most closely related to the clam population characteristics. Sensitivity analysis of the learning algorithm found that the following physiological and biochemical markers were the most predictive, in decreasing order, of clam population characteristics: glutathione S-transferase, phagocytosis, age pigments, lipid peroxidation in the gills, labile IIb metals and total MT levels. These biomarkers were affected by the distance of the clam beds from the shore, site quality (pollution) and reproduction activity.

Iron and radical content in Mya arenaria. Possible sources of NO generation.

The objective of this work was to analyze oxidative metabolism in Mya arenaria. Total Fe content in M. arenaria collected in the German Wadden Sea was 1.9+/-0.7, 0.7+/-0.1 and 0.17+/-0.01 nmol/mg fresh weight (FW), in digestive glands (DG), mantle and gills, respectively. Labile Fe pool, assessed by electronic paramagnetic resonance (EPR), was 146+/-10 pmol/mg FW, and by the fluorescence method employing calcein it was 118+/-9 pmol/mg FW. The lipid radical content in the DG, assessed by EPR, was 27+/-7 pmol/mg FW, and the thiobarbituric reactive substances content amounted to 57+/-8 pmol/mg FW. Ascorbyl radical (A) content, assessed by quantification of EPR signals, was 0.04+/-0.01 pmol/mg FW, and the ascorbate content (AH(-)) was 478+/-12 pmol/mg FW. The ratio A/AH(-) was (8+/-1)x10(-5)AU, suggesting a minimum oxidative stress even under physiological conditions, presumably depending on basal metabolic functions. The content of nitric oxide (NO), assessed by EPR, was 99+/-3 pmol/mg FW. The generation rate of NO by nitric oxide synthase-like activity (NOS-like) was assayed as NO production detected by EPR in the presence of l-arginine and NADPH, and was 3.16+/-0.06 pmol/(mg FW min). The data presented here document the detectable presence of highly reactive species in M. arenaria.

Disparate distributions of chemolithotrophs containing form IA or IC large subunit genes for ribulose-1,5-bisphosphate carboxylase/oxygenase in intertidal marine and littoral lake sediments.

The distributions of bacterial form IA and form IC ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) were investigated using Lowes Cove intertidal mudflat and Damariscotta Lake littoral sediments by PCR amplification of 492-495 bp fragments of the large subunit RuBisCO gene, cbbL. Genomic extracts for amplification were obtained from lake surface (upper 2 mm), mudflat surface (upper 2 mm), subsurface (5-7 cm), and soft-shell clam (Mya arenaria) burrow-wall sediments, as well as from a sulfide-oxidizing mat. Phylogenetic analyses of cbbL clone libraries revealed that Lowes Cove sediments were dominated by form IA cbbL-containing sequences most closely related to cbbL genes of sulfur-oxidizing bacteria or sulfide-oxidizing mats. In contrast, Damariscotta Lake cbbL clones contained primarily form IC cbbL sequences, which typify aerobic CO- and hydrogen-oxidizing facultative chemolithotrophs. Statistical analyses supported clear differentiation of intertidal and lake chemolithotroph communities, and provided evidence for some differentiation among intertidal communities. amova and libshuff analyses of Lowes Cove libraries suggested that M. arenaria burrow-wall sediments did not harbour distinct communities compared with surface and subsurface sediments, but that surface and subsurface libraries displayed moderate differences. The results collectively support a conceptual model in which the relative distribution of form IA- and IC-containing bacterial chemolithotrophs depends on sulfide availability, which could reflect the role of sulfate reduction in sediment organic matter metabolism, or the presence of geothermal sulfide sources.

Estradiol-17beta and testosterone concentrations in male and female Mya arenaria (Mollusca bivalvia) during the reproductive cycle.

Perturbation of the reproductive cycle as well as vitellin-like protein synthesis have already been reported in Mya arenaria sampled in contaminated areas of the St. Lawrence maritime estuary (Québec, Canada). To assess the potential role of endocrine disruptors in modulating the reproductive cycle in clams, the role of sex steroids has to be clarified. We determined the levels of estradiol-17beta and testosterone during the reproductive cycle in female and male M. arenaria. Both steroids were measured by ELISA in the gonads and no differences in steroid profiles appeared between sexes. Estrogen levels varied between 150 and 400 pg g(-1) wet weight over gametogenesis and were near 10 times higher than testosterone levels. Results showed transient increases of both steroids at the onset of vitellogenesis in females and during the spawning stage in both sexes. These findings indicate that these hormones could have a role as endogenous modulators of gametogenesis. Further studies are, however, needed to describe the pathway of steroid synthesis in clam gonad and elucidate steroid involvement in controlling gametogenesis and as well as their relationship with neurohormones since these latter are required to promote sexual maturation.