Evidence of polystyrene nanoplastic contamination and potential impacts in Mya arenaria clams in the Saint-Lawrence estuary (Canada).

Plastic materials found in the environment are expected to degrade into smaller plastic nanoparticles (NPs) posing a greater toxic risk because they sorb contaminants and pass physiological barriers. Moreover the presence and effects of NPs is difficult to tease out from the contamination background at polluted sites. The purpose of this study was to examine for the presence of polystyrene NPs in feral Mya arenaria clam population near anthropogenic sources of pollution and potential toxic effects. Polystyrene NPs were determined by a newly developed fluorescence-based and size exclusion chromatography methodologies. Clam health status was determined by following changes in air survival time, condition factor, growth, alcohol/aldehyde dehydrogenase (AADH), protein aggregation and lactate dehydrogenase (LDH). In addition, multi-elemental analysis in tissues was also determined. The results revealed that clams collected at 2 polluted sites contained elevated amounts of polystyrene-like NPs between 10 and 110 nm in size based on size exclusion chromatography. Elevated levels of AADH suggest the presence of hydroxylated products and were correlated with plastic NPs in tissues. Moreover, principal component analysis revealed that As, Ca, Cu, Sn and V were closely related to either polystyrene-like NPs in tissues or AADH activity. Although we cannot rule out other pollutants, clams contaminated by polystyrene-like NPs had lower condition, growth rate, air survival time and LDH activity. Increased metal/element contamination reported to sorb onto plastic polymers were also related to NPs in tissues. In conclusion, clams populations close to anthropogenic sources of pollution show evidence of polystyrene-like NPs contamination and could contribute to decreased clam health status.

Effects of different oxygen regimes on ecological performance and bioenergetics of a coastal marine bioturbator, the soft shell clam Mya arenaria.

Benthic species are exposed to oxygen fluctuations that can affect their performance and survival. Physiological effects and ecological consequences of fluctuating oxygen are not well understood in marine bioturbators such as the soft-shell clam Mya arenaria. We explored the effects of different oxygen regimes (21 days of exposure to constant hypoxia (~4.1 kPa PO2), cyclic hypoxia (~2.1-~10.4 kPa PO2) or normoxia (~21 kPa PO2)) on energy metabolism, oxidative stress and ecological behaviors (bioirrigation and bioturbation) of M. arenaria. Constant hypoxia and post-hypoxic recovery in cyclic hypoxia led to oxidative injury of proteins and lipids, respectively. Clams acclimated to constant hypoxia maintained aerobic capacity similar to the normoxic clams. In contrast, clams acclimated to cyclic hypoxia suppressed aerobic metabolism and activated anaerobiosis during hypoxia, and strongly upregulated aerobic metabolism during recovery. Constant hypoxia led to decreased lipid content, whereas in cyclic hypoxia proteins and glycogen accumulated during recovery and were broken down during the hypoxic phase. Digging of clams was impaired by constant and cyclic hypoxia, and bioirrigation was also suppressed under constant hypoxia. Overall, cyclic hypoxia appears less stressful for M. arenaria due to the metabolic flexibility that ensures recovery during reoxygenation and mitigates the negative effects of hypoxia, whereas constant hypoxia leads to depletion of energy reserves and impairs ecological functions of M. arenaria potentially leading to negative ecological consequences in benthic ecosystems.

Effects of prolonged food limitation on energy metabolism and burrowing activity of an infaunal marine bivalve, Mya arenaria.

Benthic organisms are subject to prolonged seasonal food limitation in the temperate shallow coastal waters that can cause energetic stress and affect their performance. Sediment-dwelling marine bivalves cope with prolonged food limitation by adjusting different physiological processes that might cause trade-offs between maintenance and other fitness-related functions. We investigated the effects of prolonged (42 days) food deprivation on bioenergetics, burrowing performance and amino acid profiles in a common marine bivalve, Mya arenaria collected in winter and spring. Food limitation of >15 days decreased respiration of the clams by 80%. Total tissue energy content was higher in spring-collected clams (reflecting higher lipid content) than in their winter counterparts. Prolonged food deprivation decreased the tissue energy content of clams, especially in winter. The levels of free amino acids transiently increased during the early phase of food deprivation possibly reflecting suppression of the protein synthesis or enhanced protein degradation. The levels of amino acids considered essential for bivalves were more tightly conserved than those of non-essential amino acids during starvation. The burrowing capacity of clams was negatively affected by food deprivation so that the time required for a burial cycle increased by 35-50% after 22-42 days of starvation. During the early phase of starvation, clams preferentially used lipids as fuel for burrowing, whereas carbohydrates were used at the later phase. These findings suggest that although M. arenaria can withstand prolonged food deprivation by lowering their basal maintenance costs and switching their fuel usage, their ecological functions (e.g. bioturbation and the energy transferable to the next trophic level) could be negatively impacted by starvation.

Cellular stress responses to chronic heat shock and shell damage in temperate Mya truncata.

Acclimation, via phenotypic flexibility, is a potential means for a fast response to climate change. Understanding the molecular mechanisms underpinning phenotypic flexibility can provide a fine-scale cellular understanding of how organisms acclimate. In the last 30 years, Mya truncata populations around the UK have faced an average increase in sea surface temperature of 0.7 °C and further warming of between 1.5 and 4 °C, in all marine regions adjacent to the UK, is predicted by the end of the century. Hence, data are required on the ability of M. truncata to acclimate to physiological stresses, and most notably, chronic increases in temperature. Animals in the present study were exposed to chronic heat-stress for 2 months prior to shell damage and subsequently, only 3, out of 20 damaged individuals, were able to repair their shells within 2 weeks. Differentially expressed genes (between control and damaged animals) were functionally enriched with processes relating to cellular stress, the immune response and biomineralisation. Comparative transcriptomics highlighted genes, and more broadly molecular mechanisms, that are likely to be pivotal in this lack of acclimation. This study demonstrates that discovery-led transcriptomic profiling of animals during stress-response experiments can shed light on the complexity of biological processes and changes within organisms that can be more difficult to detect at higher levels of biological organisation.

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.

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.

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.

Location is everything: evaluating the effects of terrestrial and marine resource subsidies on an estuarine bivalve.

Estuaries are amongst the world's most productive ecosystems, lying at the intersection between terrestrial and marine environments. They receive substantial inputs from adjacent landscapes but the importance of resource subsidies is not well understood. Here, we test hypotheses for the effects of both terrestrial- and salmon-derived resource subsidies on the diet (inferred from stable isotopes of muscle tissue), size and percent nitrogen of the soft-shell clam (Mya arenaria), a sedentary estuarine consumer. We examine how these relationships shift across natural gradients among 14 estuaries that vary in upstream watershed size and salmon density on the central coast of British Columbia, Canada. We also test how assimilation and response to subsidies vary at smaller spatial scales within estuaries. The depletion and enrichment of stable isotope ratios in soft-shell clam muscle tissue correlated with increasing upstream watershed size and salmon density, respectively. The effects of terrestrial- and salmon-derived subsidies were also strongest at locations near stream outlets. When we controlled for age of individual clams, there were larger individuals with higher percent nitrogen content in estuaries below larger watersheds, though this effect was limited to the depositional zones below river mouths. Pink salmon exhibited a stronger effect on isotope ratios of clams than chum salmon, which could reflect increased habitat overlap as spawning pink salmon concentrate in lower stream reaches, closer to intertidal clam beds. However, there were smaller clams in estuaries that had higher upstream pink salmon densities, possibly due to differences in habitat requirements. Our study highlights the importance of upstream resource subsidies to this bivalve species, but that individual responses to subsidies can vary at smaller scales within estuaries.

Predicting bioaccumulation of polycyclic aromatic hydrocarbons in soft-shelled clams (Mya arenaria) using field deployments of polyethylene passive samplers.

Biota-sediment accumulation factors (BSAF), frequently used to predict tissue concentrations of organisms living within and above sediments contaminated with hydrophobic organic chemicals, often produce inaccurate estimates. Hence, freely dissolved porewater concentrations, CW , have also been investigated as predictors of organism tissue concentrations, but they are more difficult to measure than bulk sediment concentrations (used with BSAF). In situ passive sampling methods, however, make it possible to deduce CW with less effort than required to measure the value directly and make it possible to relate CW with tissue concentrations of undisturbed, native organisms. In the present study, polyethylene passive samplers containing performance reference compounds (d10-phenanthrene, d10-pyrene, and d12-chrysene) were deployed in diverse sediment beds near Boston, Massachusetts, USA, for a 1-wk period. Clams (Mya arenaria) and sediments were then collected from the deployed sediment beds. Concentrations of 3 polycyclic aromatic hydrocarbons (PAHs; phenanthrene, pyrene, and chrysene) were measured in the porewaters, in clam tissues, and in the bulk sediment. Biota-sediment accumulation factors and polyethylene-deduced CW were used to predict organism tissue concentrations. Ratios of predicted-to-measured values showed that the BSAF method over-predicted tissue concentrations in M. arenaria by up to 2 orders of magnitude. The polyethylene-deduced CW method resulted in average ratios closer to 1 (0.43 ± 0.26, 3.7 ± 2.5, and 1.1 ± 1.2 for phenanthrene, pyrene, and chrysene, respectively, N = 26, uncertainty = ± 1σ).

Activation of transcription and retrotransposition of a novel retroelement, Steamer, in neoplastic hemocytes of the mollusk Mya arenaria.

Bivalve mollusks of the North Atlantic, most prominently the soft shell clam Mya arenaria, are afflicted with an epidemic transmissible disease of the circulatory system closely resembling leukemia. The disease is characterized by a dramatic expansion of blast-like cells in the hemolymph with high mitotic index. Examination of hemolymph of diseased clams revealed high levels of reverse transcriptase activity, the hallmark of retroviruses and retroelements. By deep sequencing of RNAs from hemolymph, we identified transcripts of a novel retroelement, here named Steamer. The DNA of the element is marked by long terminal repeats and encodes a single large protein with similarity to mammalian retroviral Gag-Pol proteins. Steamer mRNA levels were specifically elevated in diseased hemocytes, and high expression was correlated with disease status. DNA copy number per genome was present at enormously high levels in diseased hemocytes, indicative of extensive reverse transcription and retrotransposition. Steamer activation in M. arenaria is an example of a catastrophic induction of genetic instability that may initiate or advance the course of leukemia.

Disseminated neoplasia in the soft-shell clam Mya arenaria: membrane lipid composition and functional parameters of circulating cells.

In a previous study we compared lipid composition and functional parameters of circulating cells from Cerastoderma edule affected or not by disseminated neoplasia (neoplastic cells vs hemocytes) (Le Grand et al. Chem Phys Lipids 167:9-20 2013). Neoplastic cells presented morpho-functional modifications concomitant to striking membrane lipid alterations: the proportion of particular plasmalogen molecular species was drastically decreased. We wanted to test whether this pattern was representative of bivalve neoplastic cells. For the purpose, a similar study was conducted on another bivalve species affected by disseminated neoplasia, the soft-shell clam (Mya arenaria). Although total reactive oxygen species production was unaffected, M. arenaria neoplastic cells presented some functional alterations: phagocytosis activity was reduced by 33 %. However, lipid compositions were not drastically altered. Particularly, sterol and plasmalogen levels did not differ between both cell types (about 43 % of membrane lipids and 35 % of phospholipids, respectively in hemocytes and neoplastic cells). This could be related to the fact that disseminated neoplasia was not related to hemolymph cell proliferation in M. arenaria (0.9 ± 0.2 10(6)cell mL(-1), considering both healthy and neoplastic clams, n = 6). Nevertheless this study highlighted minor but specific alterations of membrane lipid composition in M. arenaria neoplastic cells. The only phospholipid subclass in which the fatty acid profile strongly differed between both cell types was serine plasmalogen (PlsSer), with neoplastic cells presenting lower specific enrichment of 20:1n-11 in PlsSer. Such specific alteration of membrane lipid composition strengthened the assumption of an implication of key plasmalogen molecular species in this leukemia-like disease in bivalves.

A mu class glutathione S-transferase from Manila clam Ruditapes philippinarum (RpGSTµ): cloning, mRNA expression, and conjugation assays.

Glutathione S-transferases (GSTs) are enzymes that catalyze xenobiotic metabolism in the phase II detoxification process. GSTs have a potential for use as indicators or biomarkers to assess the presence of organic and inorganic contaminants in aquatic environments. In this study, a full-length cDNA of a mu (µ) class GST (RpGSTµ) was identified from Manila clam (Ruditapes philippinarum) and biochemically characterized. The 1356 bp of the cDNA included an open reading frame of 651 bp encoding a polypeptide of 217 amino acid residues with a molecular mass of 25.04 kDa and an estimated pI of 6.34. Sequence analysis revealed that the RpGSTµ possessed several characteristic features of µ class GSTs, such as a thioredoxin-like N-terminal domain containing binding sites for glutathione (GSH), a C-terminal domain containing substrate binding sites, and a µ loop. The recombinant RpGSTµ (rRpGSTµ) protein exhibited GSH-conjugating catalytic activity towards several substrates, and significantly strong activity was detected against 4-nitrophenethyl bromide (5.77 ± 0.55) and 1-chloro-2,4-dinitrobenzene (CDNB, 3.19 ± 0.05). Kinetic analysis as a function of GSH and CDNB concentrations revealed relatively low Km values of 1.03 ± 0.46 mM and 0.56 ± 0.20 mM, respectively, thereby indicating a GSH-conjugation attributed with high rates. The optimum pH and temperature for the catalytic activity of the rRpGSTµ protein were 7.7 and 37°C, respectively. The effect of two inhibitors, Cibacron blue and hematin, on the activity of rRpGSTµ was evaluated and the IC50 values of 0.65 µM and 9 µM, respectively, were obtained. While RpGSTµ transcripts were highly expressed in gills and hemocytes, a significant elevation in mRNA levels was detected in these tissues after lipopolysaccharide (LPS), polyinosinic-polycytidylic acid (poly I:C) and live bacterial (Vibrio tapetis) challenges. These findings collectively suggest that RpGSTµ functions as a potent detoxifier of xenobiotic toxicants present in the aquatic environment, and that its mRNA expression could be modulated by pathogenic stress signal(s).

Low hydrogen peroxide production in mitochondria of the long-lived Arctica islandica: underlying mechanisms for slow aging.

The observation of an inverse relationship between lifespan and mitochondrial H2O2 production rate would represent strong evidence for the disputed oxidative stress theory of aging. Studies on this subject using invertebrates are surprisingly lacking, despite their significance in both taxonomic richness and biomass. Bivalve mollusks represent an interesting taxonomic group to challenge this relationship. They are exposed to environmental constraints such as microbial H2S, anoxia/reoxygenation, and temperature variations known to elicit oxidative stress. Their mitochondrial electron transport system is also connected to an alternative oxidase that might improve their ability to modulate reactive oxygen species (ROS) yield. Here, we compared H2O2 production rates in isolated mantle mitochondria between the longest-living metazoan--the bivalve Arctica islandica--and two taxonomically related species of comparable size. In an attempt to test mechanisms previously proposed to account for a reduction of ROS production in long-lived species, we compared oxygen consumption of isolated mitochondria and enzymatic activity of different complexes of the electron transport system in the two species with the greatest difference in longevity. We found that A. islandica mitochondria produced significantly less H2O2 than those of the two short-lived species in nearly all conditions of mitochondrial respiration tested, including forward, reverse, and convergent electron flow. Alternative oxidase activity does not seem to explain these differences. However, our data suggest that reduced complex I and III activity can contribute to the lower ROS production of A. islandica mitochondria, in accordance with previous studies. We further propose that a lower complex II activity could also be involved.

A kinetic approach to assess oxidative metabolism related features in the bivalve Mya arenaria.

Electron paramagnetic resonance uses the resonant microwave radiation absorption of paramagnetic substances to detect highly reactive and, therefore, short-lived oxygen and nitrogen centered radicals. Previously, steady state concentrations of nitric oxide, ascorbyl radical (A·) and the labile iron pool (LIP) were determined in digestive gland of freshly collected animals from the North Sea bivalve Mya arenaria. The application of a simple kinetic analysis of these data based on elemental reactions allowed us to estimate the steady state concentrations of superoxide anion, the rate of A· disappearance and the content of unsaturated lipids. This analysis applied to a marine invertebrate opens the possibility of a mechanistic understanding of the complexity of free radical and LIP interactions in a metabolically slow, cold water organism under unstressed conditions. This data can be further used as a basis to assess the cellular response to stress in a simple system as the bivalve M. arenaria that can then be compared to cells of higher organisms.

Haemocytic leukemia in Prince Edward Island (PEI) soft shell clam (Mya arenaria): spatial distribution in agriculturally impacted estuaries.

Intensive farming of potatoes in Prince Edward Island (PEI) relies on the repeated and widespread application of fertilizers and pesticides. In PEI the main potato farming areas are in close proximity and drain directly to estuaries. Runoff from high agricultural activity watersheds could impact benthic organism health in the depositional zone of downstream estuaries. The estuarine filter feeder Mya arenaria (soft-shell clam) could be particularly vulnerable to both particle-adsorbed and water soluble contaminants. M. arenaria is susceptible to haemocytic leukemia. In May 2009, we established that heavily proliferated leukemia (HPL) prevalence was generally higher in PEI estuaries located downstream of high intensity potato farming (Dunk and Wilmot estuaries) watersheds than in estuaries downstream of lower intensity areas. Using Mab-1E10 based immunocytochemistry we observed that leukemic haemocytes from the Dunk and Wilmot estuaries were 1E10 negative whereas those from the Ox/Sheep estuary (low potato farming intensity) were 1E10 positive. The expression of genes in the p53 tumour suppressor pathway enabled us to differentiate groups of leukemic and normal M. arenaria, validating our diagnoses. In October 2009, we confirmed that HPL prevalence was elevated in the Dunk and Wilmot estuaries compared to reference (Souris River). Moreover, leukemia prevalence declined with distance from the river mouths along transects through the Dunk and Wilmot estuaries. The pesticides ß-endosulfan and α-endosulfan were detected in surface sediments from the Dunk and Wilmot estuaries, but not in sediments from either the Souris River or several other lower intensity potato farming watersheds. Our study provides evidence of an association between intensity of potato farming and prevalence of clam leukemia at downstream estuaries in PEI.

Biota: sediment partitioning of aluminium smelter related PAHs and pulp mill related diterpenes by intertidal clams at Kitimat, British Columbia.

The question of polycyclic aromatic hydrocarbon (PAH) bioavailability and its relationship to specific PAH sources with different PAH binding characteristics is an important one, because bioavailability drives PAH accumulation in biota and ultimately the biochemical responses to the PAH contaminants. The industrial harbour at Kitimat (British Columbia, Canada) provides an ideal location to study the bioavailability and bioaccumulation of sediment hydrocarbons to low trophic level biota. Samples of soft shell clams (Mya arenaria) and intertidal sediment collected from multiple sites over six years at various distances from an aluminium smelter and a pulp and paper mill were analysed for 106 PAHs, plant diterpenes and other aromatic fraction hydrocarbons. Interpretation using PAH source ratios and multivariate data analysis reveals six principal hydrocarbon sources: PAHs in coke, pitch and emissions from anode combustion from the aluminium smelter, vascular plant terpenes and aromatised terpenes from the pulp and paper mill, petroleum PAHs from shipping and other anthropogenic activities and PAHs from natural plant detritus. Harbour sediments predominantly contain either pitch or pyrogenic PAHs from the smelter, while clams predominantly contain plant derived PAHs and diterpenes from the adjacent pulp mill. PAHs from the smelter have low bioavailability to clams (Biota-Sediment Accumulation Factors; BSAFs <1 for pitch and coke; <10 for anode combustion, decreasing to ∼0.1 for the mass 300 and 302 PAHs), possibly due to binding to pitch or soot carbon matrices. Decreases in PAH isomer ratios between sediments and clams likely reflect a combination of variation in uptake kinetics of petroleum PAHs and compound specific metabolism, with the importance of petroleum PAHs decreasing with increasing molecular weight. Plant derived compounds exhibit little natural bioaccumulation at reference sites, but unsaturated and aromatised diterpenes released from resins by industrial pulping processes are readily accumulated by the clams (BSAFs >500). Thus while most of the smelter associated PAHs in sediments may not be bioavailable to benthic organisms, the plant terpenes (including retene, totarol, ferruginol, manool, dehydroabietane and other plant terpenes that form the chemical defence mechanism of conifers) released by pulp mills are bioavailable and possess demonstrated toxic properties. The large scale release of plant terpenes by some of the many pulp mills located in British Columbia and elsewhere represents a largely undocumented risk to aquatic biota.

Induction of transposase and polyprotein RNA levels in disseminated neoplastic hemocytes of soft-shell clams: Mya arenaria.

In Prince Edward Island, a high mortality of soft-shell clams Mya arenaria was found to be related to the disease known as disseminated neoplasia (DN). However, the molecular mechanisms by which hemocytes of clams are transformed in the course of DN remain by far unknown. This study aims at identifying the transcripts involved in the development of the disease. Four subtractive cDNA sequence libraries were generated and more than 200,000 reads were obtained. Following similarity searches in genome databases, the transcripts were assigned to cellular functions including mitochondrial respiration, structural proteins, cytoskeleton, nucleic acid regulation, general metabolism, signal transduction, apoptosis, cell cycle regulation, as well as virus transcripts. The expression levels of transposase and polyprotein genes were evaluated in clams with various percentages of tetraploid hemocytes. Data have shown that expression levels were significantly higher in clams with a high percentage of tetraploid hemocytes. These results reinforce the hypothesis of endogenous retrotransposon involvement in the etiology of the disease. Further investigations are needed, however, to elucidate the role of transposase and polyprotein in the disease development.

Physiological effects of temperature and a herbicide mixture on the soft-shell clam Mya arenaria (Mollusca, Bivalvia).

The aim of the current study was to investigate effects of temperature and a mixture of herbicides on the physiological status of the bivalve Mya arenaria. Bivalves acclimated to two temperatures (7 and 18°C) were exposed for 28 d to 0.01 mg/L of a pesticide formulation containing dichlorophenoxyacetic acid (2,4-D), 2-(2-methyl-4-chlorophenoxy) propionic acid (mecoprop), and 3,6-dichloro-2-methoxybenzoic acid (dicamba). At days 7, 14, and 28, mortality, immune parameters (hemocyte number, phagocytic activity, and efficiency), biomarkers of oxidative stress (catalase [CAT] and superoxide dismutase [SOD] activities and malondialdehyde [MDA] content), the metabolic enzyme cytochrome C oxidase (CCO), a biomarker of pesticide exposure (acetylcholinesterase [AChE]), and the activity of an enzyme related to gametogenesis (aspartate transcarbamylase [ATCase]) were monitored in clam tissues. Gonadosomatic index (GSI), condition factor (CF), and sex were also assessed. In clams acclimated to 7°C, exposure to pesticide enhanced CCO activity and CF and decreased MDA content, hemocyte number, CAT, and SOD activities. In clams kept at 18°C, pesticide effects appeared minor compared with samples kept at 7°C. In bivalves acclimated to 18°C, CCO, SOD, and ATCase activity and MDA content were enhanced, and hemocyte number, CAT, and AchE activities and phagocytosis were suppressed. In samples exposed to pesticides, increased temperature enhanced MDA content and CCO and SOD activity and suppressed hemocyte number and CAT and AchE activity. A gradual sexual maturation was observed in both sexes through experimental time, but females had a higher sensitivity to temperature and pesticides compared to males. Increased temperature altered the ability of the sentinel species Mya arenaria to respond to pesticide exposures. Further work is needed to understand the impacts of increasing temperature on the whole St. Lawrence estuary ecosystem.

Exposure to excess dissolved iron in vivo affects oxidative status in the bivalve Mya arenaria.

The effect of in vivo Fe exposure on the oxidative metabolism of the bivalve Myaarenaria was studied. Fe was supplemented in natural seawater and resulted in a significant increase in the total Fe content in the bivalve digestive gland (DG) between 9 to 17days of exposure. Mortality of treated animals increased drastically after day 18. Oxidative stress conditions were characterized in DG through assessment of the generation of reactive oxygen species (ROS) and ascorbyl radical (A) content. Both parameters were affected following a biphasic profile showing significant increases by days 2 and 9 of Fe exposure. The content of 2-thiobarbituric acid reactive substances (TBARS) was significantly increased over control values by days 2, 9 and 17 of treatment. The labile Fe pool (LIP) in isolated DG was elevated over control values by day 7, and maintained this increase until day 17 of Fe exposure. The content of NO, assessed by EPR spin trapping, was 60% lower in DG of animals exposed for 2days to Fe than in control values, with no further changes. The biphasic profile of oxidative stress response to Fe exposure in DG suggests that at early stages of Fe supplementation the cellular control mechanisms, such as CAT activity, were operative to limit oxidative damage, but further Fe exposure overwhelmed these abilities. Moreover, the second phase could be understood as the consequence of the exhaustion of cellular protective systems that could also involve NO.

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.