Protein polymorphisms in the hard clams Mercenaria mercenaria and Mercenaria campechiensis.
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In vivo metallothionein and glutathione status in an acute response to cadmium in Mercenaria mercenaria brown cells.
Brown cells that are found in the red glands of Mercenaria mercenaria accumulate, detoxify and excrete cadmium. Brown cell involvement in metal detoxification was due in part to endogenous glutathione (GSH) and protein sulfhydryl. Metallothionein (MT) and GSH have been shown to play an important role in metal detoxification in bivalve molluscs. This study showed that the protein sulfhydryl in brown cells of Mercenaria was in fact MT, that brown cell GSH functioned in acute protection against Cd2+ toxicity, that GSH provided the initial defense against Cd2+ toxicity prior to MT induction and that MT variants were unequal in response to Cd2+. During treatment of Mercenaria with 0.5 and 1.0 ppm Cd2+, brown cells were analyzed for MT by capillary electrophoresis and GSH colorimetrically after 0.25, 1, 2, 3, and 4 days. The data indicated that the cadmium-binding protein was MT with an apparent molecular weight of 9 kDa determined by gel filtration or 6 kDa as indicated by capillary electrophoresis. Glutathione appeared to prevail in the brown cell acute response to 0.5 ppm Cd2+, whereas MT appeared to prevail in the acute response to 1.0 ppm Cd2+. Capillary electrophoresis can be used to monitor and quantify MT and its variants in brown cells without need for prior separation of cytosolic components by chromatography. The change in MT-II was greater relative to the change in MT-I in the brown cell acute response to 0.5 ppm Cd2+, whereas the change in MT-1 was greater relative to the change in MT-II in the acute response to 1.0 ppm Cd2+. The variants of brown cell MT appeared to respond differentially to Cd2+ depending upon the Cd2+ treatment concentration.
Algorithms↗
Molecular diagnostics, field validation, and phylogenetic analysis of Quahog Parasite Unknown (QPX), a pathogen of the hard clam Mercenaria mercenaria.
Quahog Parasite Unknown (QPX) is a protistan parasite that causes disease and mortality in the hard clam Mercenaria mercenaria. PCR primers and DNA oligonucleotide probes were designed and evaluated for sensitivity and specificity for the QPX organism specifically and for the phylum Labyrinthulomycota in general. The best performing QPX-specific primer pair amplified a 665 bp region of the QPX small-subunit ribosomal DNA (SSU rDNA) and detected as little as 1 fg cloned QPX SSU rDNA and 20 fg QPX genomic DNA. The primers did not amplify DNA of uninfected hard clams M. mercenaria or of the thraustochytrids Schizochytrium aggregatum, Thraustochytrium aureum, and T. striatum. The general labyrinthulomycete primers, which were designed to offer broader specificity than the QPX primers, amplified a 435 bp region of SSU rDNA from QPX, and a 436 to 437 bp region of SSU rDNA from S. aggregatum, T. aureum, and T. striatum, but did not amplify that of the clam M. mercenaria. Field validation of the QPX-specific primer pair, through comparative sampling of 224 clams collected over a 16 mo period from a QPX endemic site in Virginia, USA, indicated that the PCR assay is equivalent to histological diagnosis if initially negative PCR products are reamplified. Oligonucleotide DNA probes specific for QPX and the phylum Labyrinthulomycota were evaluated for in situ hybridization assays of cell smears or paraffin-embedded tissues. Two DNA probes for QPX offered limited sensitivity when used independently; however, when used together as a probe cocktail, sensitivity was greatly enhanced. The probe cocktail hybridized to putative QPX organisms in tissues of hard clams collected from Virginia, New Jersey, Massachusetts and Canada, suggesting that the QPX organisms in these areas are either very closely related or the same species. The QPX probe cocktail did not hybridize with clam tissue or with the thraustochytrids S. aggregatum, T. aureum, and T. striatum. The labyrinthulomycete DNA probe hybridized with QPX and the 3 thraustochytrids, with no background hybridization to clam tissue. SSU rDNA sequences were obtained for the putative QPX organisms from geographically distinct sites. Phylogenetic analyses based on the QPX and Labyrinthulomycota sequences confirmed earlier reports that QPX is a member of this phylum, but could not definitively demonstrate that all of the QPX organisms were the same species.
Animals↗
Toxicity of atrazine to the juvenile hard clam, Mercenaria mercenaria.
The herbicide atrazine is one of the most heavily used pesticides in the United States. The effects of atrazine on the clam Mercenaria mercenaria were evaluated in aqueous and sediment laboratory assays. Juvenile clams of approximately 1mm in size were used for all experiments. An acute aqueous bio-assay was used to determine the 96-h LC(50) for the juvenile clams. A chronic aqueous bioassay was conducted at lower atrazine concentrations over a 10-day exposure period to examine both lethal and sublethal (dry mass, shell size, and condition index) endpoints. A chronic sediment bioassay examined mortality and sublethal endpoints in a 10-day exposure. The acute 96-h LC(50) was 5608 microg/L with 95% confidence intervals ranging from 5003 to 6287 microg/L. Results of the chronic aqueous assay indicated both lethal and sublethal (reduced shell size) effects at high atrazine concentrations. In the 10-day chronic aqueous assay, the no observable effect concentration was 500 microg/L, the lowest observable effect concentration was 1000 microg/L, and the maximum allowable toxicant concentration (MATC) was 707 microg/L. There were no significant effects of atrazine in the chronic sediment exposure. Safe concentrations for the aqueous experiments were estimated by applying an uncertainty factor of 10 to the calculated MATC values. While there were adverse effects of atrazine at high concentrations, these results suggest that atrazine is not directly toxic to M. mercenaria at environmentally relevant concentrations.
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Influence of alpha- and beta-adrenergic antagonists on dopamine-induced responses in the isolated heart of Mercenaria mercenaria.
Agents with predominantly adrenergic antagonistic properties (the alpha-blockers, phentolamine and tolazoline, and the beta-blocker propranolol), and those with suspected alpha-blocking capabilities [hydralazine and 3,3'-(4,4'-biphenylene)bis(2,5-diphenyl-2H-tetrazolium chloride) (neotetrazolium chloride); I] were added individually to a bath containing an isolated Mercenaria mercenaria heart. Two and one-half minutes later, dopamine was added to the bath as the second drug and cardiac responses were noted. Pretreatment with saline controls, followed by dopamine 2.5 min later, produced results that were identical with those which occurred after the administration of dopamine alone, i.e., marked stimulation and cardiac arrest. Pretreatment with phentolamine and I were the only procedures that prevented dopamine-induced cardiac arrest. Phentolamine, tolazoline, and hydralazine generally produced positive inotropic responses when initially added to the bath, whereas propranolol mimicked the effects caused by the addition of dopamine alone or after saline pretreatment. The M. mercenaria heart appears to possess an adrenergic receptor of an alpha-configuration.
Adrenergic alpha-Antagonists↗
Identification of two binding proteins for halogenated aromatic hydrocarbons in the hard-shell clam, Mercenaria mercenaria.
Earlier studies have reported an unusually high incidence of gonadal tumors in marine bivalves in areas of potentially high exposure to herbicides including 2,4-dichlorophenoxyacetic acid and 2,4,5-trichlorophe-noxyacetic acid. Some herbicides can be contaminated by halogenated aromatic compounds including 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Most of the effects of planar halogenated aromatic compounds, including carcinogenic effects in vertebrates, appear to be mediated through binding to the aryl hydrocarbon receptor. The current study investigated whether halogenated aromatic hydrocarbon-binding proteins are present in the marine bivalve, Mercenaria mercenaria. We used the TCDD photoaffinity analog 2-azido-3-[125I]-iodo-7, 8-dibromodibenzo-p-dioxin to detect the presence of two proteins (28 and 39 kDa) in cytosols prepared from M. mercenaria that specifically bound this ligand. Expression of these proteins is tissue-dependent with the highest concentrations being observed in gill and gonadal tissue. Gonadal tissue also exhibited gender-specific expression with female clams exhibiting higher levels of the 39-kDa protein. Gender-and tissue-specific expression are consistent with the hypothesis that these proteins might be involved in the carcinogenic response observed in clams exposed to herbicides.
Affinity Labels↗
Glutathione and metallothionein status in an acute response by Mercenaria mercenaria brown cells to copper in vivo.
Red glands of Mercenaria mercenaria comprise brown cells that accumulate, detoxify, and excrete copper. Brown cell involvement in metal detoxification is due in part to endogenous glutathione (GSH) and metallothionein (MT). The intent of this study was to test the hypotheses that brown cell GSH functions in protection against Cu2+ toxicity, that brown cell GSH provides the initial defense against Cu2+ prior to brown cell MT induction, and that MT variants (MTI, MTII), if present are unequal in response to Cu2+. Brown cells were analyzed for GSH and MT after 0.25, 1, 2, 3, and 4 days of treatment of Mercenaria with 0.01 and 0.05 ppm Cu2+. Glutathione initiated the brown cell acute response (within the first day of treatment) to both the 0.01 and 0.05 ppm Cu2+ treatments. Metallothionein in brown cells increased to Day 4 during treatment with 0.01 ppm Cu2+, whereas MT concentration was greatest at Day 2 after which it decreased to Day 4 with treatment of 0.05 ppm Cu2+. The change in MTII relative to its control was greater than that of MTI in the brown cell acute response to 0.01 ppm Cu2+ and also for Days 0.25 to 2 in response to the 0.05 ppm Cu2+ treatment. At Days 3 and 4 with the 0.05 ppm Cu2+ the change in MTI/MTII ratio was due to a greater change in MTI than MTII relative to their respective controls. The variants of brown cell MT appear to respond differently to Cu2+ depending on the Cu2+ treatment concentration.
Animals↗
Anticoagulantly active heparin from clam (Mercenaria mercenaria).
Heparin was isolated from Mercenaria mercenaria by ion-exchange chromatography and was fractionated into two distinct populations with immobilized antithrombin. The high-affinity glycosaminoglycan accelerated dramatically the inhibition of purified human factors IIa and Xa via purified human antithrombin. Specific anti-factor IIa and anti-factor Xa activities were 363 and 348 U.S.P. units/mg, respectively. The highly active clam heparin exhibited a molecular weight of approximately 18,000 and contained approximately 2.5 sulfate groups per disaccharide. The intrinsic fluorescence of purified human antithrombin was enhanced in the presence of the high-affinity invertebrate glycosaminoglycan to an extent comparable to the level induced by vertebrate heparin. In addition, the critical tetrasaccharides containing 3-O-sulfated glucosamine residues, which constitute part of the unique antithrombin-binding domain of mammalian heparin, were also detected in high-affinity Mercenaria heparin.
Animals↗
Phagocytosis by hemocytes of the hard clam, Mercenaria mercenaria.
Large granular hemocytes of Mercenaria mercenaria avidly phagocytose a variety of biological particles (red blood cells of six species, yeast, and gram-positive and gram-negative bacteria) as well as polystyrene spheres. Clam hemolymph is not necessary for phagocytosis but may have some opsonic effect in certain circumstances (e.g., low temperature and low particle density). Formaldehyde treatment of red blood cells enhances susceptibility to phagocytosis. Phagocytosis by Mercenaria hemocytes in vitro appears to be a nonspecific process.
Animals↗
Agglutinins in the hemolymph of the hard clam, Mercenaria mercenaria.
Lectins in the serum of the clam Mercenaria mercenaria agglutinate some red blood cells, bacteria, and yeast. The interaction of these substances with particles is affected by sugars, ions, temperature, and alteration of particle surfaces. Lectins are not needed for phagocytosis of foreign particles in vitro. In M. mercenaria these recognition molecules do not enhance defense mechanisms.
Agglutinins↗
Glutathione concentrations in the hard clam, Mercenaria mercenaria, following laboratory exposure to lead (a potential model system for evaluating exposure to carcinogens and toxins).
This study determined whether M. mercenaria retain Pb from sea water and whether endogenous GSH acts as an important primary response modulator of heavy metal detoxification. Lead accumulation in M. mercenaria may be related to the rate of endogenous formation of GSH. Glutathione concentrations decrease with increasing early exposure to Pb and increase after continued acute exposure. M. mercenaria do not accumulate Pb but appear to reach an equilibrium with their environment. GSH formation may protect the hard clam from accumulating excess Pb by forming insoluble sulfide adducts with Pb and excreting these complexes.
Animals↗
Oxidative burst in hard clam (Mercenaria mercenaria) haemocytes.
Haemocytes of bivalve molluscs are known to be responsible for many immunological functions, including recognition, phagocytosis, and killing or elimination of invading microorganisms, such as potentially infective bacteria and parasites. In many bivalves, killing of microorganisms engulfed by haemocytes is accomplished by a sudden release of reactive oxygen species (ROS) within the haemocytes; this response is referred to as an oxidative burst. Previous studies have failed to detect oxidative burst in haemocytes of the hard clam (northern quahog), Mercenaria mercenaria. In the present study, we applied a widely used chemical probe for ROS detection in haemocytes, dichlorofluorescin-diacetate (DCFH-DA), to haemocytes from this clam species and used flow cytometry to quantify fluorescence in individual haemocytes. Oxidation of DCFH-DA to the fluorescent product, DCF, within unstimulated haemocytes indicated that ROS were clearly produced in these cells. Two activators of oxidative burst, zymosan and bacterial extracellular products, which have been applied successfully to haemocytes in other species, stimulated large increases in ROS production in hard clam haemocytes. Furthermore, two inhibitors of ROS production, W-13 and diphenylene iodinium (DPI), significantly suppressed ROS production by haemocytes. Nitric oxide synthase inhibitors, NMMA and L-NIO, did not suppress ROS production, indicating that the observed oxidation of DCFH-DA is not mediated by nitric oxide. These results show unequivocally that haemocyte oxidative burst is active in M. mercenaria and, therefore, is a likely mechanism in host response to pathogens and parasites.
Analysis of Variance↗
The role of various dissolved organic matter forms on chlorpyrifos bioavailability to the estuarine bivalve Mercenaria mercenaria.
Dissolved organic matter (DOM) is comprised of a myriad of macromolecules with specific physical and chemical properties that may influence the bioavailability of hydrophobic pesticides to animals. This study was conducted to assess the role of various forms of DOM on the uptake and bioconcentration of the organophosphate insecticide chlorpyrifos (CHPY) to the bivalve Mercenaria mercenaria. Bivalves were exposed to DOM-free seawater (30 per thousand) or to seawater containing a single form of DOM. DOM forms included two filtrate fractions of natural salt-marsh sediment DOM (DOM-(<0.45 mum) and DOM-(<3 kDa)); natural purified humic (HA) and fulvic (FA) acids; and water soluble cyclic oligosaccharides alpha and beta cyclodextrins (CD-alpha and CD-beta). In (14)C-CHPY uptake and elimination experiments, juvenile bivalves were exposed to uniformly-labeled (14)C-CHPY and collected at time intervals during 48 h. The remaining bivalves were transferred to (14)C-CHPY-free elimination chambers with bivalve collection at time intervals over 144 h. Total uptake of (14)C-CHPY by bivalves in DOM-free seawater was >40% greater than in bivalves exposed to (14)C-CHPY in the presence of most DOM forms. These results are consistent with much faster (14)C-CHPY uptake rates estimated using a simple two parameter model. After the elimination period, bivalves exposed to DOM-free seawater had (14)C-CHPY body residue concentrations between 25% and 86% greater than bivalves in the presence of DOM forms. Experiments with larger bivalves showed that pulse-chase exposures with a 1.5 h exposure period to (14)C-CHPY was not long enough to detect differences in (14)C-CHPY tissue accumulation efficiencies across treatments. Our findings suggest that natural forms of DOM, at environmentally realistic organic carbon concentrations, reduced pesticide uptake and bioconcentration, consistent with much lower uptake rates relative to bivalves exposed to (14)C-CHPY in the absence of DOM. Interestingly, at the tested organic carbon concentrations CD-alpha and CD-beta did not reduce (14)C-CHPY bioconcentration in M. mercenaria.
Analysis of Variance↗
Photoreceptor spike responses in the hardshell clam, Mercenaria mercenaria.
Spikes were recorded from single axons in the siphonal nerve of the hardshell clam Mercenaria mercenaria which respond to dimming of light. No axons were found to respond to the onset, or increase, of illumination. In a dark-adapted state there is little or no ongoing spike activity. The responsive area of a single axon is a circle of approximately 85 microm diameter on the inner siphon wall. The number of spikes elicited at the off of constant-duration flashes grows as approximately the 0.4 power of flash intensity. For constant intensity and constant light-time fraction, the off-response increases with increasing duration at least up to 500 s duration. For long durations, the response grows as the logarithm of stimulus duration. Subthreshold light can suppress the off-response from preceding illumination. In a light-adapted state, the off-response is greater and its latency shorter than in the dark-adapted state. The fine structure of groups of cell processes thought to comprise the photoreceptor in Mercenaria is described. On the basis of morphological and physiological findings it is suggested that phototransduction occurs in the fine distal processes of the axons from which we have recorded. Axonal processes were found to contain well organized pentalaminar whorls which may be the site of photo-pigment concentration. The action spectrum obtained from the integrated responses of nerve bundles appears to be that of a single Dartnall pigment having maximal absorption at about 510 nm.
Animals↗
Bioavailability of the organophosphorous insecticide chlorpyrifos to the suspension-feeding bivalve, Mercenaria mercenaria, following exposure to dissolved and particulate matter.
Under estuarine conditions, hydrophobic pesticides undergo physical/chemical partitioning into dissolved and particulate fractions of sediments. Our experimental study using 14C-chlorpyrifos (14C-CHPY) showed that sorptive associations of this common insecticide with particulate (>0.2 microm) and dissolved fractions (<0.2 microm) of sediments strongly influenced uptake, tissue absorption, and elimination in the estuarine bivalve Mercenaria mercenaria. Bivalves were fed algae mixed with particulate and dissolved fractions of 14C-CHPY-contaminated estuarine sediment particles, components of estuarine sediments (silica, humic acids), and a naturally occurring food source (phytoplankton) to quantify relative uptake, tissue absorption efficiencies, and elimination of 14C-CHPY. Measurements of 14C-CHPY tissue absorption efficiencies (AE%) indicated that particle source qualities influenced much of the insecticide bioavailability. Mean tissue AE% of 14C-CHPY associated with different particulate fractions ranged from 23 to 31%, while uptake from the dissolved/colloidal forms resulted in low (7-17%) AE%. Our data indicated that 14C-CHPY is more likely to bioaccumulate through ingestion of contaminated particulate material than through filtration/ingestion of dissolved/colloidal material. The high selectivity and digestibility of algae particles by M. mercenaria, a potentially ingested food source, may play an important role in the bioavailability of chlorpyrifos in this animal.
Animals↗
Tetraploid induction by inhibiting mitosis I with heat shock, cold shock, and nocodazole in the hard clam Mercenaria mercenaria (Linnaeus, 1758).
Tetraploid induction by inhibiting mitosis I with heat shock (32, 35, and 38 degrees C), cold shock (1, 4, and 7 degrees C), and nocodazole (0.02 to 1.6 mg/L) was investigated in the hard clam Mercenaria mercenaria. All treatments were applied to fertilized eggs about 5 min before the first cell division at 22 to 23 degrees C, and lasted for 10, 15, and 20 min. Three replicates were produced for each treatment with different parents. The ploidy of resultant larvae and juveniles was determined with flow cytometry. Heat shock of 35 and 38 degrees C was effective in inhibiting mitosis I, producing 54% to 89% tetraploid larvae. Heat shock of 32 degrees C accelerated embryonic development without inhibiting mitosis or producing tetraploids. In all heat-shock groups, the survival to D-stage larvae was lower than in controls, suggesting that heat-shock treatments and tetraploidy were detrimental to larval development. At the juvenile stage, survivors from heat-shock groups contained no tetraploids. Cold shocks suspended the first cell division during the treatment, but produced no tetraploids in the 4 degrees C and 7 degrees C treatment groups. Cold shock of 1 degrees C produced 31% tetraploid larvae in one replicate, with none surviving to juvenile stage. Nocodazole inhibited mitosis I at concentrations of 0.04 mg/L or higher, but did not produce tetraploids. This study indicates that heat shock is most effective in inducing tetraploids through mitosis I inhibition, although none of the induced tetraploids survived to juvenile stage.
Animals↗
Inter- and intra-annual variations of Pb/Ca ratios in clam shells (Mercenaria mercenaria): a record of anthropogenic lead pollution?
In this study, we re-assess the use of bivalve shells as a proxy of lead pollution. Previous studies have stressed that shells display little variability compared to soft tissues and thus are better for pollution biomonitoring. However, in this manuscript we illustrate that there is large inter- and intra-annual Pb variability between shells of the clam Mercenaria mercenaria collected in North Carolina, USA. Therefore, year to year, as well as intra-annual variations in Pb/Ca ratios should be interpreted with caution. Despite this variability, we were able to obtain an annual Pb chronology from 1949 to 2002 using 11 shells collected at different times which clearly exhibited the late 1970's peak in Pb from leaded gasoline use. This indicates that when enough specimens are pooled together, bivalve shells can be used to reconstruct large, long term changes in environmental Pb concentrations. Our data compare well with other studies of aragonite clams from sites with low regional lead pollution. From this we conclude that the Cape Lookout region of North Carolina has not received extensive pollution over the 1949-2002 period. The Pb concentration in shells growing in the 1949-1976 period was not significantly different from those growing in the 1982-2002 period, although other proxies suggest that the 1949-1976 period should be considerably higher. Therefore, our data suggest that there is still a modern low-level source of Pb in the coastal North Carolina environment.
Animals↗
Mitochondria from the hepatopancreas of the marine clam Mercenaria mercenaria: substrate preferences and salt and pH effects on the oxidation of palmitoyl-L-carnitine and succinate.
A method is presented for the isolation of mitochondria with good respiratory control from the hepatopancreas of the marine clam Mercenaria mercenaria. Palmitoyl-L-carnitine is the preferred substrate of the mitochondria of the hepatopancreas based on state 3 rates of oxidation (in the presence of ADP). Rates of oxidation of pyruvate and glutamate were about one-half that of the lipid substrate in state 3. alpha-Glycerophosphate was oxidized at a rate about one-third that of palmitoyl-L-carnitine. All Krebs cycle intermediates were oxidized to some extent. Proline was not oxidized at detectable levels. The optimal range of KCl concentrations for the oxidation of palmitoyl-L-carnitine is between 250 and 500 mM whereas the optimal range of KCl concentration for the oxidation of succinate is between 200 and 350 mM. The optimal range of pH for the oxidation of succinate and for the oxidation of palmitoyl-L-carnitine lies between pH 6.5 and 7.5 based on the respiratory control ratio.
Animals↗