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.

Distribution of haemic neoplasia of soft-shelled clams in Prince Edward Island: an examination of anthropogenic factors and effects of experimental fungicide exposure.

Haemic neoplasia was first considered a disease of concern for soft-shell clams in Prince Edward Island (PEI) when it was diagnosed as the cause of mass mortalities in 1999. The aetiology of the disease remains elusive, but has been associated with environmental degradation. In this study, a 2-year (2001-2002) geographic and seasonal survey was conducted for haemic neoplasia, using histology, in soft-shell clams from PEI. In addition, using geographic information system, the association between anthropogenic factors in the watersheds at sites affected by haemic neoplasia and the prevalence of the disease was investigated. Finally, histopathological changes were assessed in soft-shell clams experimentally exposed to four concentrations of chlorothalonil for 27 days. Haemic neoplasia could not be induced at any concentration of chlorothalonil. Clams exposed to a concentration of 1000 µg L(-1) of the fungicide, however, exhibited an LC50 of 17 days. Although this information provides additional toxicity information (LC50) for soft-shell clams, further experiments are required to assess longer term exposure to the fungicide. The highest prevalences of haemic neoplasia in PEI were found in North River and Miscouche (28.3-50.9% and 33.0-77.8%, respectively). No clear seasonal patterns were found. There was a correlation between haemic neoplasia prevalence and watersheds with a high percentage of potato acreage and forest coverage (P = 0.026 and P = 0.045, respectively), suggesting a link between anthropogenic activity and the prevalence of the disease.

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.

Effects of pesticide compounds (chlorothalonil and mancozeb) and benzo[a]pyrene mixture on aryl hydrocarbon receptor, p53 and ubiquitin gene expression levels in haemocytes of soft-shell clams (Mya arenaria).

The aim of this study is to investigate the effects of the pesticides/polycyclic aromatic hydrocarbon mixture on aryl hydrocarbon receptor (AhR), p53 and ubiquitin mRNA level in haemocytes of Mya arenaria exposed to a mixture of chlorothalonil, mancozeb and benzo[a]pyrene (BaP) for 48 and 72 h. AhR, p53 and ubiquitin gene expression levels were quantified using quantitative Real-time PCR. For robust and accurate quantification of transcripts, suitable housekeeping genes were selected from four sets of ribosomal and elongation factors transcripts previously sequenced from Mya arenaria using geNorm open source software. Quantitative Real-time PCR data exhibited a significantly high expression of AhR after 72 h of exposure (P ≤ 0.05). p53 gene expression seems to be up-regulated by the mixture after 48 h, however not significantly; but the level of p53 mRNA is down-regulated by the xenobiotics between 48 and 72 h after exposure. This study postulates that AhR mRNA levels could be used as an indicator of the exposure of clams' haemocytes to a mixture of xenobiotics such as chlorothalonil, mancozeb and BaP. However, further studies have to be pursued in order to unravel the molecular mechanisms involved in the p53 signaling pathway.

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.

A critical review: 2,3,7,8 -tetrachlorodibenzo-p-dioxin (2,3,7,8-TCDD) effects on gonad development in bivalve mollusks.

Bivalve mollusks are equally sensitive to 2,3,7,8-tetrachlorodibenzo-p-dioxin's (2,3,7,8-TCDD) effect on gonad development, embryonic development, and epithelial lesion occurrence as higher vertebrates. 2,3,7,8-TCDD alters normal development of reproductive organs and early development in bivalve mollusks at 2 to 20 pg/g wet weight. In both Crassostria virginica and Mya arenaria, 2,3,7,8-TCDD preferentially accumulates into the gonads. The sensitivity of gonad maturation is likely due to disruption of cross-talk between highly conserved steroid, insulin, and metabolic pathways involved in gonad differentiation. The altered gonad development and decreased veliger larval survival can partially explain the lack of self-sustaining bivalve populations in 2,3,7,8-TCDD contaminated estuaries.

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.

Comparison of bioaccumulation of metals and induction of metallothioneins in two marine bivalves (Mytilus edulis and Mya arenaria).

The St. Lawrence maritime estuary (Quebec, Canada) is subjected to mixed inputs of pollutants and the study of the induction of metallothionein in species of economic and ecologic importance such as Mytilus edulis and Mya arenaria was pertinent to assess the consequences of pollution in this northern estuary. Bivalves from an area devoid of anthropogenic influences but characterized by background metal contamination (Franquelin) were actively transplanted within this location and in a site contaminated by urban, industrial and endogenous pollutants, Baie-Comeau (Baie-des-Anglais). Spatial differences in metal concentrations were shown between sites. Cu and Zn concentrations were higher in mussels from Baie-des-Anglais at the beginning of the transfer and after 1 and 2 months. In clams, Zn concentrations were significantly higher in gills and digestive gland tissues for organisms transplanted in Baie-des-Anglais thus showing that spatio-temporal variations of metal concentrations were different between the two species studied. Mussels and clams partitioning of metals were shown to be different depending of the species, metal and/or tissue studied. In mussels, Cd and Cu concentrations decreased in both organs and both groups after the 3-month transfer in the polluted site. In mussels, total metal and metallothionein (MT) concentrations were positively correlated in digestive gland while in clams a positive correlation was only observed in gills.

Potential link between exposure to fungicides chlorothalonil and mancozeb and haemic neoplasia development in the soft-shell clam Mya arenaria: a laboratory experiment.

The aetiology of haemic neoplasia (HN) is unknown, so far but many causative factors are suggested such as viral, pollution and genetics. The aim of this study was to determine if, under chronic exposure, two major pesticides (chlorothalonil and mancozeb) which are used in potato production could induce HN in soft-shell clams (Mya arenaria). Short-term experiments with acute exposure were also performed. Clams were collected from an epizootic site (North River, PEI) and from a site free of the disease (Magdalen Islands, Quebec). The tetraploid level of haemocytes was assessed by flow cytometry for each clam to determine the HN status. The bioaccumulation of pesticides in tissues was quantified by gas chromatography/mass spectrometry (GC/MS) for chlorothalonil while mancozeb and manganese were quantified by inductively coupled plasma-mass spectrometer (ICP/MS). Long term exposure to fungicide Bravo 500((R)) did not induce high tetraploid levels on negative calm from North River and the analysis of the digestive gland and the mantle did not reveal any detectable level of chlorothalonil. In the Manzate 200 DF((R)), some clams revealed high level of tetraploid cells but no difference were observed between the treatments and the control. The analysis of the digestive gland and the mantle for manganese did not highlight any significant difference in tissue concentration (p=0.05). For the acute exposure, chlorothalonil analysis showed that the active ingredient is distributed between four chlorinated compounds: 99.5% for chlorothalonil isomers, 0.4% for pentachlorothalonil and 0.1% for trichlorothalonil isomers. For a 72 h experiment, the accumulation was within 4h; the higher tissue concentration of chlorothalonil was 59.2 microg g(-1) in the mantle after 48 h, following by a decrease to an undetectable level at the end. For the manganese, the accumulation was detected after 4h; the higher tissue concentration was 48.8 microg g(-1) in the mantle after 24h and, over the following 48 h, the accumulation decreased until the end of the trial. Based on the data, the accumulation of these fungicides seems to be transitory. Chlorothalonil and mancozeb are both oxidative-stress promoters and could have induced cell dysfunction while in the tissue. Study on the effect of these fungicides on the p53 protein system is an example of strategy that would provide information on cellular events promoting neoplasia.

Genotoxic stress-induced expression of p53 and apoptosis in leukemic clam hemocytes with cytoplasmically sequestered p53.

In nature, the soft shell clam, Mya arenaria, develops a fatal blood cancer in which a highly conserved homologue for wild-type human p53 protein is rendered nonfunctional by cytoplasmic sequestration. In untreated leukemic clam hemocytes, p53 is complexed throughout the cytoplasm with overexpressed variants for both clam homologues (full-length variant, 1,200-fold and truncated variant, 620-fold above normal clam hemocytes) of human mortalin, an Hsp70 family protein. In vitro treatment with etoposide only and in vivo treatment with either etoposide or mitoxantrone induces DNA damage, elevates expression (600-fold) and promotes nuclear translocation of p53, and results in apoptosis of leukemic clam hemocytes. Pretreatment with wheat germ agglutinin followed by etoposide treatment induces DNA damage and elevates p53 expression (893-fold) but does not overcome cytoplasmic sequestration of p53 or induce apoptosis. We show that leukemic clam hemocytes have an intact p53 pathway, and that maintenance of this tumor phenotype requires nuclear absence of p53, resulting from its localization in the cytoplasm of leukemic clam hemocytes. The effects of these topoisomerase II poisons may result as mortalin-based cytoplasmic tethering is overwhelmed by de novo expression of p53 protein after DNA damage induced by genotoxic stress. Soft shell clam leukemia provides excellent in vivo and in vitro models for developing genotoxic and nongenotoxic cancer therapies for reactivating p53 transcription in human and other animal cancers displaying mortalin-based cytoplasmic sequestration of the p53 tumor suppressor, such as colorectal cancers and primary and secondary glioblastomas, though not apparently leukemias or lymphomas.

Receptor activated C kinase is down-regulated in the male gonad of the marine bivalve mollusc Mya arenaria exposed to tributyltin (TBT).

This study was aimed at investigating the molecular mechanisms by which tributyltin (TBT) impairs the reproductive processes in the marine bivalve Mya arenaria. The suppression polymerase chain reaction subtractive hybridization (SSH) method was used to identify differentially expressed transcripts in the gonads of adult M. arenaria 72 h after a single injection of 160 ng TBT in the adductor muscle. Subtractive cDNA libraries comprising 322 clones were obtained. These clones were sequenced and corresponded to 55 female and 26 single male non-redundant cDNAs. Following similarity searches in genome databases, some of the transcripts could be assigned to cellular functions including mitochondrial respiration, structural proteins, structure of cytoskeleton, nucleic acid regulation, general metabolism and signal transduction. Among the potentially differentially regulated transcripts, Receptor for activated C kinase 1 (RACK1) represented 6% of the total down-regulated clones in males and the corresponding protein exhibited a high degree of similarity (80%) with the human polypeptide. The RACK1 cDNA from M. arenaria consists of 1085 bp, encoding a 318 deduced polypeptide which contains five internal tryptophan-aspartate (WD) repeats, six putative PKC phosphorylation sites, one tyrosine kinase site, four putative N-myristoylation sites as well as a transmembrane segment spanning amino acid 228-251. A significant down-regulation (by approximately 30% (p<0.05)) of RACK1 expression in male gonads exposed to TBT was confirmed by quantitative real-time RT-PCR. Transcript levels of RACK1 were higher in the female gonads than in the mantle, gills and male gonads. Gene expression as detected by in situ hybridization was strong in mature oocytes comparatively to primary germ cells. RACK1 may be a useful biomarker for TBT exposure in the reproductive system of bivalve molluscs.

Physiological effects of polycyclic aromatic hydrocarbons on soft-shell clam Mya arenaria.

The aim of this study was to investigate the effects of polycyclic aromatic hydrocarbons (PAHs) on the physiological status of the bivalve Mya arenaria. Specimens were exposed to four different sources of PAHs: aluminium smelter soot, sediment from an industrial discharge pound, charcoal fine particles and dietary PAHs assessed by feeding clams with phytoplankton freshly impregnated with dissolved PAHs. The exposure period lasted 30 days and bivalves were let to recover for an additional 20 days. At days 8, 15, 30 and 50, immune parameters (phagocytic activity and efficiency) were monitored in haemocytes. Oxidative stress measures such as catalase and lipid peroxidation were quantified in digestive gland as well as concentrations of bioaccumulated PAHs. In a second experiment, clams were exposed to [(14)C]-pyrene via the phytoplankton, and the tissue distribution of radiolabelled compound was studied. Glycogen levels in gonad and digestive gland were also measured and gametogenesis stages were investigated. Results showed a high bioaccumulation in clams exposed to dietary PAHs and contaminated sediments. Tissue distribution of [(14)C]-pyrene revealed that the radiolabelled compound persisted mainly in the gonad during 14 days. A decrease of phagocytosis was observed in contaminated male clams. The lipid peroxidation (malondialdehyde) was found to increase in the digestive gland tissues of clams exposed to dietary PAHs, smelter soot and discharge, but no differences were observed in the catalase activity. A delay in gametogenesis occurred in all exposed males and in females contaminated with coke dust and dietary PAHs. Males were more sensitive than females to PAH exposure. A dysfunction in steroid synthesis is suspected to occur due to the exposure to all sources of PAHs.

Neuroendocrine disruption in Mya arenaria clams during gametogenesis at sites under pollution stress.

This study examined the neuroendocrine status of clams on intertidal mud flats in the St. Lawrence Estuary and Saguenay Fjord areas during late gametogenesis. The impact of pollution was determined by a test battery of early stress markers (metallothioneins, heme levels, glutathione S-transferase activity), tissue damage (lipid peroxidation and DNA damage) and morphologic characteristics (age, soft-tissue weight ratio and growth index). Neuroendocrinal status was examined by tracking serotonin and dopamine metabolism, monoamine adenylate cyclase activity in synaptosomes, monoamine oxidase and arachidonate cyclooxygenase activities in relation to gametogenetic activity: pyrimidine synthesis, (aspartate transcarbamoylase activity or ATC), vitellogenin-like proteins and gonado-somatic index. The results show that clam soft tissue weights were reduced at sites close to harbours and higher at sites near domestic wastewater outfalls. The age-to-length ratio of clams was generally higher at impacted sites, suggesting reduced growth. The biomarkers of stress or damage confirmed that oxidative stress, DNA damage, metallothioneins and glutathione S-transferase activity were significantly increased at varying degrees, at the polluted sites. Vitellogenin-like proteins and gametogenetic activity were significantly increased at sites influenced by domestic wastewaters. Furthermore, the clams were still in active gametogenesis and not ready for spawning, as indicated by the concordance of the serotonin/dopamine ratio with vitellogenin-like proteins and pyrimidine synthesis. However, gonadal cyclooxygenase activity was increased at polluted sites and significantly correlated with most of the stress biomarkers, suggesting that the clams were in a state of inflammation rather than at the spawning stage. Finally, a multivariate analysis revealed that the sites were readily discriminated with high efficiency (>71%) and that both neuroendocrine physiological markers and stress responses were identified as the major components, thus explaining the global physiological response of the clams. We conclude that the effects of pollution compromise the clams' health status and that the initiation of gametogenesis in environments contaminated by municipal wastewaters or harbours contributes to the toxic effects of pollution.

Implication of site quality on mitochondrial electron transport activity and its interaction with temperature in feral Mya arenaria clams from the Saguenay Fjord.

The advent of global warming has given rise to questions about the impact of temperature/pollution interactions on the integrity of certain benthic organisms like bivalves. This interaction was examined in intertidal Mya arenaria clams from the Saguenay Fjord using the concepts of cellular energy allocation and temperature-dependent mitochondrial electron transport (METT) activity. Clams were collected at low tide from six sites (two clean, four polluted) for determinations of condition factor (weight/shell length), growth index (age-to-length ratio), gonadal lipids and maturation index, gonad MET at various habitat temperatures, METT, gill xanthine oxidase and gill DNA damage. Condition factor was generally lower at the four polluted sites, with growth index being severely affected at two of them. Gonadal maturation was also significantly dampened at two of the four pollution-impacted sites. Gill xanthine oxidase (purine bases salvage pathway) and DNA strand breaks were significantly increased at most of the polluted sites, confirming pollution-mediated damage in clams. Moreover, MET at 20 degrees C, METT and gonad lipids were significantly induced at the polluted sites. Clam condition factor was negatively correlated with most of the biomarkers for cellular energy allocation (gonadal lipids, MET and METT), but not with gonadal maturation. DNA damage and xanthine oxidase were positively correlated with MET at 20 degrees C and METT. This is the first report of electron transport in mitochondria being more sensitive to incremental temperature increases in clams under pollution stress. The gradual warming of clam habitats would likely worsen the impacts of pollution in feral clam populations.

Health status of Mya arenaria bivalves collected from contaminated sites in Canada (Saguenay Fjord) and Denmark (Odense Fjord) during their reproductive period.

The purpose of this study was to examine the health status and gametogenetic activity in Mya arenaria clams collected at various sites in the St. Lawrence Estuary (Quebec, Canada) and in the Odense Fjord (Denmark). Clam soft tissues were analyzed for metals/metalloids and organotin compounds to confirm their exposure to these contaminants. Their health status was assessed by a test battery of biomarkers designed to measure the early biological effects of contaminants, which include expression of defence mechanisms such as xenobiotic conjugation (glutathione S-transferase), expression of stress proteins (i.e., heme oxygenase and metallothioneins), changes in gametogenetic activity, and individual morphometric characteristics. Clam tissues were also examined for the presence of oxidative damage to lipids, formation of DNA strand breaks, and alterations in heme metabolism. The results showed that clams sampled from sites with either ferry activity or intensive boat traffic in marinas were contaminated by metals/metalloids such as Ag, Al, As, Co, Cr, Cu, Fe, Hg, Mn, Mo, Ni, Pb, Sn, V, and Zn. The clams also contained relatively high amounts of tributyltin (TBT) in their tissues (in the ng TBT/g range for both areas), with digestive glands containing more organotins than did gonadal tissues. Moreover, clams collected from TBT-contaminated sites had higher amounts of tin-heme adducts and lower total heme in their digestive glands. Condition factor, age distribution, and sex ratio were significantly altered in clams from impacted sites in the Saguenay Fjord and accompanied by an increased male/female sex ratio. Gametogenetic activity was also negatively affected, as revealed by reductions in gonadosomatic index, maturation index, aspartate transcarbamylase activity, and vitellogenin-like proteins. The Saguenay Fjord clams displayed a complex pattern of stress responses and damage such as increased heme oxygenase activity, phase 2 conjugation enzyme activity, lipid peroxidation, and altered DNA strand breaks. The integration of biomarker response data into a biomarker index at the whole-individual level (morphometric characteristics) and for various organs (gill, digestive gland, and gonad) revealed that, relative to the control site, morphological characteristics and gonadal activity were most affected at the most contaminated site, while the effects were more pronounced in the digestive gland and gill at moderately impacted sites. We conclude that the health status of M. arenaria clams at these contaminated sites is compromised, with obvious disruption of reproductive activity.