Mesocosm study of PAC-modified clay effects on Karenia brevis cells and toxins, chemical dynamics, and benthic invertebrate physiology.

Modified clay compounds are used globally as a method of controlling harmful algal blooms, and their use is currently under consideration to control Karenia brevis blooms in Florida, USA. In 1400 L mesocosm tanks, chemical dynamics and lethal and sublethal impacts of MC II, a polyaluminum chloride (PAC)-modified kaolinite clay, were evaluated over 72 h on a benthic community representative of Sarasota Bay, which included blue crab (Callinectes sapidus), sea urchin (Lytechinus variegatus), and hard clam (Mercenaria campechiensis). In this experiment, MC II was dosed at 0.2 g L-1 to treat bloom-level densities of K. brevis at 1 × 106 cells L-1. Cell removal in MC II-treated tanks was 57% after 8 h and 95% after 48 h. In the water column, brevetoxin analogs BTx-1 and BTx-2 were found to be significantly higher in untreated tanks at 24 and 48 h, while in MC II-treated tanks, BTx-3 was found to be higher at 48 h and BTx-B5 was found to be higher at 24 and 48 h. In MC II floc, we found no significant differences in BTx-1 or BTx-2 between treatments for any time point, while BTx-3 was found to be significantly higher in the MC II-treated tanks at 48 and 72 h, and BTx-B5 was higher in MC II-treated tanks at 24 and 72 h. Among various chemical dynamics observed, it was notable that dissolved phosphorus was consistently significantly lower in MC II tanks after 2 h, and that turbidity in MC II tanks returned to control levels 48 h after treatment. Dissolved inorganic carbon and total seawater alkalinity were significantly reduced in MC II tanks, and partial pressure of CO2 (pCO2) was significantly higher in the MC II-only treatment after 2 h. In MC II floc, particulate phosphorus was found to be significantly higher in MC II tanks after 24 h. In animals, lethal and sublethal responses to MC II-treated K. brevis did not differ from untreated K. brevis for either of our three species at any time point, suggesting MC II treatment at this dosage has negligible impacts to these species within 72 h of exposure. These results appear promising in terms of the environmental safety of MC II as a potential bloom control option, and we recommend scaling up MC II experiments to field trials in order to gain deeper understanding of MC II performance and dynamics in natural waters.

Impacts of exposure to the toxic dinoflagellate Karenia brevis on reproduction of the northern quahog, Mercenaria mercenaria.

The Gulf of Mexico, including the southwest Florida coast, USA, experience recurrent blooms of the brevetoxin (PbTx)-producing dinoflagellate, Karenia brevis. Northern quahogs (hard clams) Mercenaria mercenaria, are an important commercial species in this region. This study examined the effects of field and laboratory exposure of adult clams to K. brevis during their reproductive period, and effects on their subsequently produced offspring. Ripe adult clams were collected from a site which had been exposed to an eight-month natural bloom of K. brevis and an unaffected reference site. Ripe adult clams were also exposed to bloom concentrations of K. brevis for 10 days in the laboratory. Clams exposed to K. brevis accumulated PbTx at concentrations of 1508 (field exposure), 1444 (1000 cells mL-1 laboratory treatment) and 5229 ng g-1 PbTx-3 eq (5000 cells mL-1 laboratory treatment). Field-exposed clams showed histopathological effects: a significantly higher prevalence of mucus in the stomach/ intestine (23.3%), edema in gill tissues (30%) and presence of the cestode parasite, Tylocephalum spp. in whole tissue (40%), compared to non-exposed clams (0, 3.3 and 6.7% respectively). These clams also showed reduced gonadal allocation (23% gonadal area) and a higher prevalence of clams of undetermined sex (20%) compared to those sampled from the non-exposed site (43% and 0%, respectively). It is hypothesized that less energy may be channeled into reproduction as more is allocated for homeostasis or tissue repair. The fertilization success of gametes obtained from both field and laboratory-exposed adults was significantly lower in clams that had been exposed to K. brevis and development of these offspring was negatively affected at Days 1 and 4 post-fertilization (in field- and laboratory-exposed clams at the higher K. brevis concentration and in laboratory-exposed clams at the higher K. brevis concentration, respectively). Negative effects may be due to toxin accumulation in the gametes of field-exposed clams (244 ± 50 ng PbTx g-1 and 470 ± 82 ng g-1 wet weight in oocytes and sperm, respectively). Adverse effects in M. mercenaria are compared to those previously reported in oysters, Crassostrea virginica, under similar conditions of exposure. This study provides further evidence of the impacts of K. brevis and its associated toxins on the adults and offspring of exposed shellfish. Site-selection for the collection of broodstock and aquaculture grow-out efforts should therefore consider the local occurrence of K. brevis blooms.

Effects of elevated CO2 levels on subcellular distribution of trace metals (Cd and Cu) in marine bivalves.

Hypercapnia (elevated CO2 levels) and pollution with trace metals such as Cu and Cd are common stressors in estuarine habitats that can negatively affect physiology and health of marine organisms. Hypercapnia can modulate toxicity of trace metals including Cu and Cd; however, the physiological and cellular mechanisms of the metal-CO2 interactions are not well understood. We investigated the effects of elevated PCO2 (∼800 and 2000µatm) and metal exposure (50µgl-1 of Cu or Cd) on subcellular distribution of metals in two common species of marine bivalves, Eastern oysters Crassostrea virginica and hard shell clams Mercenaria mercenaria. Oysters accumulated higher burdens of Cu and Cd in the gill tissues compared to clams. In both studied species, Cu was predominantly associated with the metabolically active cell compartments (mitochondria, lysosomes, microsomes and cytosolic enzymes), with a modest fraction sequestered by metallothioneins (∼30%) and the insoluble metal-containing granules (MCG) (∼15-20%). Unlike Cu, Cd was largely sequestered by metallothioneins (∼60-70%), with a relatively small fraction associated with the organelles and the cytosolic enzymes. Mitochondria were the main intracellular target for trace metals accumulating higher concentrations of Cd (and in the case of oysters - of Cu) than other organelles or cytosolic enzymes. Cu accumulation in the metabolically active cellular compartments was independent of the CO2 levels, while Cd content of the organelles and cytosolic enzymes increased at elevated PCO2 in both studied species indicating that hypercapnia may enhance cellular toxicity of Cd in bivalves. Hypercapnia suppressed the sequestration capacity of metallothioneins for Cu and Cd in oysters but increased Cu and Cd load in clam metallothioneins. Thus, metal-induced metabolic injury in oysters may be exaggerated by hypercapnia which enhances metal accumulation in the potentially sensitive intracellular fractions and suppresses the metal detoxification capacity. In contrast, clams appear to be more resistant to the combined effects of hypercapnia and metal exposure reflecting more efficient and robust detoxification mechanisms of this species.

Intermittent hypoxia leads to functional reorganization of mitochondria and affects cellular bioenergetics in marine molluscs.

Fluctuations in oxygen (O2) concentrations represent a major challenge to aerobic organisms and can be extremely damaging to their mitochondria. Marine intertidal molluscs are well-adapted to frequent O2 fluctuations, yet it remains unknown how their mitochondrial functions are regulated to sustain energy metabolism and prevent cellular damage during hypoxia and reoxygenation (H/R). We used metabolic control analysis to investigate the mechanisms of mitochondrial responses to H/R stress (18 h at <0.1% O2 followed by 1 h of reoxygenation) using hypoxia-tolerant intertidal clams Mercenaria mercenaria and hypoxia-sensitive subtidal scallops Argopecten irradians as models. We also assessed H/R-induced changes in cellular energy balance, oxidative damage and unfolded protein response to determine the potential links between mitochondrial dysfunction and cellular injury. Mitochondrial responses to H/R in scallops strongly resembled those in other hypoxia-sensitive organisms. Exposure to hypoxia followed by reoxygenation led to a strong decrease in the substrate oxidation (SOX) and phosphorylation (PHOS) capacities as well as partial depolarization of mitochondria of scallops. Elevated mRNA expression of a reactive oxygen species-sensitive enzyme aconitase and Lon protease (responsible for degradation of oxidized mitochondrial proteins) during H/R stress was consistent with elevated levels of oxidative stress in mitochondria of scallops. In hypoxia-tolerant clams, mitochondrial SOX capacity was enhanced during hypoxia and continued rising during the first hour of reoxygenation. In both species, the mitochondrial PHOS capacity was suppressed during hypoxia, likely to prevent ATP wastage by the reverse action of FO,F1-ATPase. The PHOS capacity recovered after 1 h of reoxygenation in clams but not in scallops. Compared with scallops, clams showed a greater suppression of energy-consuming processes (such as protein turnover and ion transport) during hypoxia, indicated by inactivation of the translation initiation factor EIF-2α, suppression of 26S proteasome activity and a dramatic decrease in the activity of Na(+)/K(+)-ATPase. The steady-state levels of adenylates were preserved during H/R exposure and AMP-dependent protein kinase was not activated in either species, indicating that the H/R exposure did not lead to severe energy deficiency. Taken together, our findings suggest that mitochondrial reorganizations sustaining high oxidative phosphorylation flux during recovery, combined with the ability to suppress ATP-demanding cellular functions during hypoxia, may contribute to high resilience of clams to H/R stress and help maintain energy homeostasis during frequent H/R cycles in the intertidal zone.

Effects of pH and bicarbonate on mitochondrial functions of marine bivalves.

Estuarine organisms including mollusks are exposed to periodic oxygen deficiency (hypoxia) that leads to a decrease in intracellular pH and accumulation of bicarbonate (HCO3(-)). These changes can affect cellular bioenergetics; however, their effects on mitochondria of estuarine mollusks are not well understood. We determined the interactive effects of bicarbonate (0-10mM) and pH (7.2 and 6.5) on mitochondrial oxygen consumption (MO2), membrane potential (Δψ) and production of reactive oxygen species (ROS) in two common estuarine bivalves - hard clams Mercenaria mercenaria, and bay scallops Argopecten irradians. In both species, elevated HCO3(-) levels suppressed ADP-stimulated (state 3) MO2 but had little effect on the resting (state 4) respiration. These effects were not mediated by the soluble adenylyl cyclase or cyclic AMP. Effects of the low pH (6.5) on mitochondrial traits were species-specific and depended on the substrate oxidized by the mitochondria. Mild acidosis (pH6.5) had minimal effects on MO2 and Δψ of the bivalve mitochondria oxidizing pyruvate but led to increased rates of ROS production in clams (ROS production could not be measured in scallops). In succinate-respiring mitochondria of clams, mild acidosis suppressed MO2 and increased mitochondrial coupling, while in scallop mitochondria the effects of low pH were opposite. Suppression of mitochondrial oxidative phosphorylation by bicarbonate and/or acidosis may contribute to the metabolic rate depression during shell closure or environmental hypoxia/hypercapnia. These findings have implications for understanding the physiological mechanisms involved in regulation of mitochondrial bioenergetics during hypoxia exposure in estuarine bivalves.

Interactive effects of copper exposure and environmental hypercapnia on immune functions of marine bivalves Crassostrea virginica and Mercenaria mercenaria.

Estuarine organisms such as bivalves are commonly exposed to trace metals such as copper (Cu) and hypercapnia (elevated CO2 levels) in their habitats, which may affect their physiology and immune function. This study investigated the combined effects of elevated CO2 levels (∼800-2000 µatm PCO2, such as predicted by the near-future scenarios of global climate change) and Cu (50 µg l(-1)) on immune functions of the sediment dwelling hard clams Mercenaria mercenaria and an epifaunal bivalve, the eastern oyster Crassostrea virginica. Clams and oysters were exposed for 4 weeks to different CO2 and Cu levels, and tissue Cu burdens and immune parameters were assessed to test the hypothesis that hypercapnia will enhance Cu uptake due to the higher bioavailability of free Cu(2+) and increase the immunomodulatory effects of Cu. Exposure to Cu stimulated key immune parameters of clams and oysters leading to increased number of circulating hemocytes, higher phagocytosis and adhesion ability of hemocytes, as well as enhanced antiparasitic and antibacterial properties of the hemolymph reflected in higher activities of lysozyme and inhibitors of cysteine proteases. Lysozyme activation by Cu exposure was most prominent in normocapnia (∼400 µatm PCO2) and an increase in the levels of the protease inhibitors was strongest in hypercapnia (∼800-2000 µatm PCO2), but other immunostimulatory effects of Cu were evident in all PCO2 exposures. Metabolic activity of hemocytes of clams and oysters (measured as routine and mitochondrial oxygen consumption rates) was suppressed by Cu exposure likely reflecting lower rates of ATP synthesis and/or turnover. However, this metabolic suppression had no negative effects of the studied immune functions of hemocytes such as phagocytosis or adhesion capacity. Hypercapnia (∼800-2000 µatm PCO2) slightly but significantly enhanced accumulation of Cu in hemocytes, consistent with higher Cu(2+) bioavailability in CO2-acidified water, but had little effect on cellular and humoral immune traits of clams and oysters. These findings indicate that low levels of Cu contamination may enhance immunity of estuarine bivalves while moderate hypercapnia (such as predicted by the near future scenarios of the global climate change) does not strongly affect their immune parameters.

Effects of environmental hypercapnia and metal (Cd and Cu) exposure on acid-base and metal homeostasis of marine bivalves.

Elevated CO2 levels reduce seawater pH and may affect bioavailability of trace metals in estuaries. We studied the interactive effects of common metal pollutants (50 µg l(-1) Cd or Cu) and PCO2 (~395, 800 and 2000 µatm) on metal levels, intracellular pH, expression of metal binding proteins and stress biomarkers in estuarine bivalves Crassostrea virginica (oysters) and Mercenaria mercenaria (hard clams). Cd (but not Cu or hypercapnia) exposure affected the acid-base balance of hemocytes resulting in elevated intracellular pH. Cd and Cu exposure led to the increase in the tissue metal burdens, and metal accumulation was reduced by elevated PCO2 in the mantle but not hemocytes. No change was found in the intracellular free Cd(2+), Cu(2+) or Fe(2+) during Cu or Cd exposure indicating that these metals are bound to intracellular ligands. Free Zn(2+) content in oyster hemocytes was suppressed by Cd and Cu exposure and below the detection limits in clam hemocytes, which went hand-in-hand with the elevated mRNA expression of metallothioneins and ferritin in Cd- and Cu-exposed bivalves, enhanced by hypercapnia. The metal-binding and antioxidant mechanisms of oysters and clams were sufficient to effectively maintain intracellular redox status, even though metal exposure combined with moderate hypercapnia (~800 µatm PCO2) led to the elevated production of reactive oxygen species in hemocytes. Overall, while hypercapnia modulates metal accumulation, binding capacity and oxidative stress in estuarine bivalves, the physiological effects of elevated CO2 are mild compared to the effects of other common stressors.

Effects of the red tide dinoflagellate, Karenia brevis, on early development of the eastern oyster Crassostrea virginica and northern quahog Mercenaria mercenaria.

The brevetoxin-producing dinoflagellate, Karenia brevis, adversely affects many shellfish species including the commercially and ecologically important bivalve molluscs, the northern quahog (=hard clam) Mercenaria mercenaria and eastern oyster Crassostrea virginica, in the Gulf of Mexico, USA. This study assessed the effects of exposure of these bivalves to K. brevis during their early development. In separate experiments, embryos of 2-4 cell stage of M. mercenaria and C. virginica were exposed to both whole and lysed K. brevis cells isolated from Manasota Key, Florida. Low bloom concentrations of 500 to 3000 cells mL(-1) were simulated for 96 h. Shell length, percent abnormality (and normality), and percent mortality of resulting larvae were measured. Percentages were recorded after 6, 24, and 96 h of exposure; larval shell length was measured at 24 and 96 h. For both quahogs and oysters, the effects of exposing embryos to K. brevis on all larval responses were generally dose- and time-dependent. Percent mortalities and abnormalities of both clam and oyster embryos increased significantly after only 6h of exposure to whole cells of K. brevis. For clams, these parameters were significantly higher in whole and lysed treatments (at 3000 cells mL(-1)) than in controls. Percent mortalities of oysters were significantly higher in the whole-cell treatment (3000 cells mL(-1)) than under control conditions. After 24h of exposure, mean larval shell length of both bivalve species was significantly reduced relative to controls. This was evident for clam larvae in both the lysed treatment at 1500 cells mL(-1) and in whole and lysed treatments at 3000 cells mL(-1), and for oyster larvae in the lysed treatment at 3000 cells mL(-1). After 96 h, both species exposed to the lysed cell treatment at 3000 cells mL(-1) had significantly smaller larvae compared to those in the control. Overall, lysed cells of K. brevis had a more pronounced effect on shell length, percent abnormality, and mortality in both clams and oysters than did whole cells. Given the fact that blooms of K. brevis overlap with the spawning periods of these two bivalves, and that cells of this naked dinoflagellate are readily lysed by wave action, these results suggest that exposure to K. brevis during the early life history stages of clams and oysters could adversely affect their population recruitment. Further, the presence of whole or lysed cells of K. brevis in hatcheries could have a major negative impact on production.

Effects of contaminated St. Lucie River saltwater sediments on an amphipod (Ampelisca abdita) and a hard-shell clam (Mercenaria mercenaria).

The St. Lucie estuary (SLE) ecosystem in South Florida has been shown to be contaminated with metals and pesticides. Our earlier studies also showed that aquatic organisms, especially benthic species in the SLE ecosystem, might be potentially at high risk from copper (Cu) exposure. The objectives of this study were to conduct studies with separate groups of organisms exposed to seven field-collected sediment samples from the St. Lucie River according to standard procedures to evaluate toxicity and tissue concentrations of Cu and zinc (Zn). Short term and longer term whole sediment acute toxicity studies were performed with Ampelisca abdita and Mercenaria mercenaria. Analysis of sediment chemical characteristics showed that Cu and Zn are of most concern because their concentrations in 86 % of the sediments were higher than the threshold effect concentrations for Florida sediment quality criteria and the National Oceanic and Atmospheric Administration Screening Quick Reference Tables (SQuiRTs) sediment values. There was no significant effect on survival of the tested organisms. However, increased Cu and Zn concentrations in the test organisms were found. Dry weight of the tested organisms was also inversely related to Cu and Zn concentrations in sediments and organisms. The effects on organism weight and Cu and Zn uptake raise concerns about the organism population dynamics of the ecosystem because benthic organisms are primary food sources in the SLE system and are continuously exposed to Cu- and Zn-contaminated sediments throughout their life cycle. The results of the present study also indicate that Cu and Zn exposures by way of sediment ingestion are important routes of exposure.

Immunomodulation by the interactive effects of cadmium and hypercapnia in marine bivalves Crassostrea virginica and Mercenaria mercenaria.

Estuarine organisms are exposed to multiple stressors including large fluctuations in partial pressure of carbon dioxide (P2CO) and concentrations of trace metals such as cadmium (Cd) that can affect their survival and fitness. Ocean acidification due to the increasing atmospheric (P2CO) leads to a decrease in pH and shifts in the carbonate chemistry of seawater which can change bioavailability and toxicity of metals. We studied the interactive effects of (P2CO) and Cd exposure on metal levels, metabolism and immune-related functions in hemocytes of two ecologically and economically important bivalve species, Mercenaria mercenaria (hard shell clam) and Crassostrea virginica (Eastern oyster). Clams and oysters were exposed to combinations of three (P2CO) levels (∼400, 800 and 2000 µatm (P2CO), corresponding to the present day conditions and the projections for the years 2100 and 2250, respectively) and two Cd concentrations (0 and 50 µg l(-1)) in seawater. Following four weeks of exposure to Cd, hemolymph of both species contained similar Cd levels (50-70 µg l(-1)), whereas hemocytes accumulated intracellular Cd burdens up to 15-42 mg l(-1), regardless of the exposure P2CO. Clam hemocytes had considerably lower Cd burdens than those of oysters (0.7-1 ng 10(-6) cells vs. 4-6 ng 10(-6) cells, respectively). Cd exposure suppressed hemocyte metabolism and increased the rates of mitochondrial proton leak in normocapnia indicating partial mitochondrial uncoupling. This Cd-induced mitochondrial uncoupling was alleviated in hypercapnia. Cd exposure suppressed immune-related functions in hemocytes of clams and oysters, and these effects were exacerbated at elevated (P2CO). Thus, elevated (P2CO) combined with Cd exposure resulted in decrease in phagocytic activity and adhesion capacity as well as lower expression of mRNA for lectin and heat shock protein (HSP70) in clam and oyster hemocytes. In oysters, combined exposure to elevated (P2CO) and Cd also led to reduced activity of lysozyme in hemocytes and hemolymph. Overall, our study shows that moderately elevated (P2CO) (∼800-2000 µatm P2CO) potentiates the negative effects of Cd on immunity and thus may sensitize clams and oysters to pathogens and diseases during seasonal hypercapnia and/or ocean acidification in polluted estuaries.

Interactive effects of CO2 and trace metals on the proteasome activity and cellular stress response of marine bivalves Crassostrea virginica and Mercenaria mercenaria.

Increased anthropogenic emission of CO2 changes the carbonate chemistry and decreases the pH of the ocean. This can affect the speciation and the bioavailability of metals in polluted habitats such as estuaries. However, the effects of acidification on metal accumulation and stress response in estuarine organisms including bivalves are poorly understood. We studied the interactive effects of CO2 and two common metal pollutants, copper (Cu) and cadmium (Cd), on metal accumulation, intracellular ATP/ubiquitin-dependent protein degradation, stress response and energy metabolism in two common estuarine bivalves-Crassostrea virginica (eastern oyster) and Mercenaria mercenaria (hard shell clam). Bivalves were exposed for 4-5 weeks to clean seawater (control) and to either 50 µg L(-1) Cu or 50 µg L(-1) Cd at one of three partial pressures of CO2 ( [Formula: see text] ∼ 395, ∼ 800 and ∼ 1500 µatm) representative of the present-day conditions and projections of the Intergovernmental Panel for Climate Change (IPCC) for the years 2100 and 2250, respectively. Clams accumulated lower metal burdens than oysters, and elevated [Formula: see text] enhanced the Cd and Cu accumulation in mantle tissues in both species. Higher Cd and Cu burdens were associated with elevated mRNA expression of metal binding proteins metallothionein and ferritin. In the absence of added metals, proteasome activities of clams and oysters were robust to elevated [Formula: see text] , but [Formula: see text] modulated the proteasome response to metals. Cd exposure stimulated the chymotrypsin-like activity of the oyster proteasome at all CO2 levels. In contrast, trypsin- and caspase-like activities of the oyster proteasome were slightly inhibited by Cd exposure in normocapnia but this inhibition was reversed at elevated [Formula: see text] . Cu exposure inhibited the chymotrypsin-like activity of the oyster proteasome regardless of the exposure [Formula: see text] . The effects of metal exposure on the proteasome activity were less pronounced in clams, likely due to the lower metal accumulation. However, the general trends (i.e. an increase during Cd exposure, inhibition during exposure to Cu, and overall stimulatory effects of elevated [Formula: see text] ) were similar to those found in oysters. Levels of mRNA for ubiquitin and tumor suppressor p53 were suppressed by metal exposures in normocapnia in both species but this effect was alleviated or reversed at elevated [Formula: see text] . Cellular energy status of oysters was maintained at all metal and CO2 exposures, while in clams the simultaneous exposure to Cu and moderate hypercapnia (∼ 800 µatm [Formula: see text] ) led to a decline in glycogen, ATP and ADP levels and an increase in AMP indicating energy deficiency. These data suggest that environmental CO2 levels can modulate accumulation and physiological effects of metals in bivalves in a species-specific manner which can affect their fitness and survival during the global change in estuaries.

Interactive effects of pH and metals on mitochondrial functions of intertidal bivalves Crassostrea virginica and Mercenaria mercenaria.

Intertidal bivalves experience broad fluctuations of environmental temperature, pH and oxygen content which could change their intracellular pH. They are also exposed to trace metals such as cadmium (Cd) and copper (Cu) that accumulate in their tissues and may negatively affect mitochondrial functions and bioenergetics. We determined the interactive effects of pH and trace metals (25 µM Cd or Cu) on mitochondrial functions (including respiration and membrane potentials in both ADP-stimulated (state 3) and resting (state 4) states) of two common marine bivalves, the hard clams (Mercenaria mercenaria) and eastern oysters (Crassostrea virginica). In the absence of the trace metals, mitochondrial functions of C. virginica and M. mercenaria were insensitive to pH in a broad physiologically relevant range (6.6-7.8). Mitochondrial respiration was generally suppressed by 25 µM Cd or Cu (with the stronger effects observed for ADP-stimulated compared to the resting respiration) while the mitochondrial membrane potential was unaffected. pH modulated the effects of Cu and Cd on mitochondrial respiration of the bivalves. In oysters, Cu suppressed ADP-stimulated mitochondrial respiration at high and low pH values (6.6 and 7.8, respectively), but had no effect in the intermediate pH range (7.0-7.4). In clams, the negative effect of Cu on ADP-stimulated respiration was only observed at extremely high pH (7.8). A decrease in pH was also protective against Cd in mitochondria of clams and oysters. In clams, 25 µM Cd suppressed ADP-stimulated respiration at all pH; however, at low pH (6.6-7.0) this suppression was paralleled by a decrease in the rates of proton leak thereby effectively restoring mitochondrial coupling. In oysters, the inhibitory effects of Cd on ADP-stimulated respiration were fully abolished at low pH (6.6-7.0). This indicates that moderate acidosis (such as occurs during exposure to air, extreme salinities or elevated CO2 levels in the intertidal zone) may have a beneficial side-effect of protecting mitochondria of clams and oysters against the toxic effects of trace metals in polluted estuaries.

Short-term acute hypercapnia affects cellular responses to trace metals in the hard clams Mercenaria mercenaria.

Estuarine and coastal habitats experience large fluctuations of environmental factors such as temperature, salinity, partial pressure of CO2 ( [Formula: see text] ) and pH; they also serve as the natural sinks for trace metals. Benthic filter-feeding organisms such as bivalves are exposed to the elevated concentrations of metals in estuarine water and sediments that can strongly affect their physiology. The effects of metals on estuarine organisms may be exacerbated by other environmental factors. Thus, a decrease in pH caused by high [Formula: see text] (hypercapnia) can modulate the effects of trace metals by affecting metal bioavailability, accumulation or binding. To better understand the cellular mechanisms of interactions between [Formula: see text] and trace metals in marine bivalves, we exposed isolated mantle cells of the hard clams (Mercenaria mercenaria) to different levels of [Formula: see text] (0.05, 1.52 and 3.01 kPa) and two major trace metal pollutants - cadmium (Cd) and copper (Cu). Elevated [Formula: see text] resulted in a decrease in intracellular pH (pHi) of the isolated mantle cells from 7.8 to 7.4. Elevated [Formula: see text] significantly but differently affected the trace metal accumulation by the cells. Cd uptake was suppressed at elevated [Formula: see text] levels while Cu accumulation has greatly accelerated under hypercapnic conditions. Interestingly, at higher extracellular Cd levels, labile intracellular Cd(2+) concentration remained the same, while intracellular levels of free Zn(2+) increased suggesting that Cd(2+) substitutes bound Zn(2+) in these cells. In contrast, Cu exposure did not affect intracellular Zn(2+) but led to a profound increase in the intracellular levels of labile Cu(2+) and Fe(2+). An increase in the extracellular concentrations of Cd and Cu led to the elevated production of reactive oxygen species under the normocapnic conditions (0.05 kPa [Formula: see text] ); surprisingly, this effect was mitigated in hypercapnia (1.52 and 3.01 kPa). Overall, our data reveal complex and metal-specific interactions between the cellular effects of trace metals and [Formula: see text] in clams and indicate that variations in environmental [Formula: see text] may modulate the biological effects of trace metals in marine organisms.

Interactive effects of elevated temperature and CO2 levels on energy metabolism and biomineralization of marine bivalves Crassostrea virginica and Mercenaria mercenaria.

The continuing increase of carbon dioxide (CO2) levels in the atmosphere leads to increases in global temperatures and partial pressure of CO2 (PCO2) in surface waters, causing ocean acidification. These changes are especially pronounced in shallow coastal and estuarine waters and are expected to significantly affect marine calcifiers including bivalves that are ecosystem engineers in estuarine and coastal communities. To elucidate potential effects of higher temperatures and PCO2 on physiology and biomineralization of marine bivalves, we exposed two bivalve species, the eastern oysters Crassostrea virginica and the hard clams Mercenaria mercenaria to different combinations of PCO2 (~400 and 800µatm) and temperatures (22 and 27°C) for 15weeks. Survival, bioenergetic traits (tissue levels of lipids, glycogen, glucose and high energy phosphates) and biomineralization parameters (mechanical properties of the shells and activity of carbonic anhydrase, CA) were determined in clams and oysters under different temperature and PCO2 regimes. Our analysis showed major inter-species differences in shell mechanical traits and bioenergetics parameters. Elevated temperature led to the depletion of tissue energy reserves indicating energy deficiency in both species and resulted in higher mortality in oysters. Interestingly, while elevated PCO2 had a small effect on the physiology and metabolism of both species, it improved survival in oysters. At the same time, a combination of high temperature and elevated PCO2 lead to a significant decrease in shell hardness in both species, suggesting major changes in their biomineralization processes. Overall, these studies show that global climate change and ocean acidification might have complex interactive effects on physiology, metabolism and biomineralization in coastal and estuarine marine bivalves.

Interactive effects of elevated temperature and CO(2) levels on metabolism and oxidative stress in two common marine bivalves (Crassostrea virginica and Mercenaria mercenaria).

Marine bivalves such as the hard shell clams Mercenaria mercenaria and eastern oysters Crassostrea virginica are affected by multiple stressors, including fluctuations in temperature and CO2 levels in estuaries, and these stresses are expected to be exacerbated by ongoing global climate change. Hypercapnia (elevated CO2 levels) and temperature stress can affect survival, growth and development of marine bivalves, but the cellular mechanisms of these effects are not yet fully understood. In this study, we investigated whether oxidative stress is implicated in cellular responses to elevated temperature and CO2 levels in marine bivalves. We measured the whole-organism standard metabolic rate (SMR), total antioxidant capacity (TAOC), and levels of oxidative stress biomarkers in the muscle tissues of clams and oysters exposed to different temperatures (22 and 27°C) and CO2 levels (the present day conditions of ~400ppm CO2 and 800ppm CO2 predicted by a consensus business-as-usual IPCC emission scenario for the year 2100). SMR was significantly higher and the antioxidant capacity was lower in oysters than in clams. Aerobic metabolism was largely temperature-independent in these two species in the studied temperature range (22-27°C). However, the combined exposure to elevated temperature and hypercapnia led to elevated SMR in clams indicating elevated costs of basal maintenance. No persistent oxidative stress signal (measured by the levels of protein carbonyls, and protein conjugates with malondialdehyde and 4-hydroxynonenal) was observed during the long-term exposure to moderate warming (+5°C) and hypercapnia (~800ppm CO2). This indicates that long-term exposure to moderately elevated CO2 and temperature minimally affects the cellular redox status in these bivalve species and that the earlier observed negative physiological effects of elevated CO2 and temperature must be explained by other cellular mechanisms.

Extreme longevity is associated with increased resistance to oxidative stress in Arctica islandica, the longest-living non-colonial animal.

We assess whether reactive oxygen species production and resistance to oxidative stress might be causally involved in the exceptional longevity exhibited by the ocean quahog Arctica islandica. We tested this hypothesis by comparing reactive oxygen species production, resistance to oxidative stress, antioxidant defenses, and protein damage elimination processes in long-lived A islandica with the shorter-lived hard clam, Mercenaria mercenaria. We compared baseline biochemical profiles, age-related changes, and responses to exposure to the oxidative stressor tert-butyl hydroperoxide (TBHP). Our data support the premise that extreme longevity in A islandica is associated with an attenuated cellular reactive oxygen species production. The observation of reduced protein carbonyl concentration in A islandica gill tissue compared with M mercenaria suggests that reduced reactive oxygen species production in long-living bivalves is associated with lower levels of accumulated macromolecular damage, suggesting cellular redox homeostasis may determine life span. Resistance to aging at the organismal level is often reflected in resistance to oxidative stressors at the cellular level. Following TBHP exposure, we observed not only an association between longevity and resistance to oxidative stress-induced mortality but also marked resistance to oxidative stress-induced cell death in the longer-living bivalves. Contrary to some expectations from the oxidative stress hypothesis, we observed that A islandica exhibited neither greater antioxidant capacities nor specific activities than in M mercenaria nor a more pronounced homeostatic antioxidant response following TBHP exposure. The study also failed to provide support for the exceptional longevity of A islandica being associated with enhanced protein recycling. Our findings demonstrate an association between longevity and resistance to oxidative stress-induced cell death in A islandica, consistent with the oxidative stress hypothesis of aging and provide justification for detailed evaluation of pathways involving repair of free radical-mediated macromolecular damage and regulation of apoptosis in the world's longest-living non-colonial animal.

Assessment of cellular and functional biomarkers in bivalves exposed to ecologically relevant abiotic stressors.

An understanding of the complex effects of the environment on biomarkers of bivalve health is essential for aquaculturists to successfully select field culture sites and monitor bivalve health in these sites and in hatcheries. We tested several whole-organism (functional) and cellular-level biomarkers as indicators of health of the cultured, stress-tolerant northern quahog (hard clam) Mercenaria mercenaria. We performed single- and dual-stressor experiments that were consistent with available water quality data from a clam culture area on the Gulf coast of Florida. Clams from the culture area were exposed over a 14-d period to low O2 (hypoxia), elevated temperature, hyposalinity, and a combination of elevated temperature and hyposalinity. There was no clear relationship between the functional and cellular-level biomarkers, with most of the treatment effects being detected at the whole-organism level but not the cellular level. Survival and burial ability were significantly affected by elevated temperature and by the combination of elevated temperature and hyposalinity. Glycogen content decreased over the experiment duration and did not differ significantly among treatments. There were no significant changes in expression patterns of eight stress proteins or in the levels of oxidatively damaged RNA. The results highlight the importance of investigating the effects of multiple stressors in short-term, controlled laboratory conditions and suggest that such cellular-level biomarker assays should be paired with functional biomarkers to better understand the responses of highly stress-tolerant species.

Do toxicity identification and evaluation laboratory-based methods reflect causes of field impairment?

Sediment toxicity identification and evaluation (TIE) methods are relatively simple laboratory methods designed to identify specific toxicants or classes of toxicants in sediments; however, the question of whether the same toxicant identified in the laboratory is causing effects in the field remains unanswered. The objective of our study was to determine if laboratory TIE methods accurately reflect field effects. A TIE performed on sediments collected from the Elizabeth River (ER) in Virginia identified polycyclic aromatic hydrocarbons (PAHs) as the major toxicants. Several lines of evidence indicated PAHs were the major toxic agents in the field, including elevated PAH concentrations in ER sediments, comet assay results from in situ caged Merceneria merceneria, and chemical analyses of exposed M. merceneria, which indicated high PAH concentrations in the bivalve tissue. Our final evidence was the response from test organisms exposed to ER sediment extracts and then ultraviolet (UV) radiation. UV radiation caused a toxic diagnostic response unique to PAHs. The aggregation of these various lines of evidence supports the conclusion that PAHs were the likely cause of effects in laboratory- and field-exposed organisms, and that laboratory-based TIE findings reflect causes of field impairment

Use of the juvenile clam, Mercenaria mercenaria, as a sensitive indicator of aqueous and sediment toxicity.

The hard clam is an economically important bivalve and is abundant along the East Coast of the US. The goal of this research was to evaluate the sensitivity of this test species as compared to that of other benthic and epibenthic organisms. Toxic effects of cadmium (inorganic metal), DDT (organochlorine pesticide), and fluoranthene (polycyclic aromatic hydrocarbon) exposure in sediments (10-day) and seawater (24-h) on juvenile (212-350-microm) hard clams Mercenaria mercenaria were determined. The aqueous 24-h LC(50) values were 0.42 mg/L cadmium (95% CL=0.35-0.45 mg/L), 0.61 mg/L DDT (95% CL=0.40-0.95 mg/L), and 0.65 mg/L fluoranthene (95% CL=0.44-1.23 mg/L). Results of sediment toxicity tests indicated that the 10-day LC(50) values were 1.66 mg/kg cadmium (95% CL=1.21-2.28 mg/kg), 5.8 mg/kg DDT (95% CL=4.8-8.3mg/kg), and 1.75 mg/kg fluoranthene (95% CL=1.38-2.09 mg/kg). Based on comparisons to toxicity data for other marine species, these findings suggest that the juvenile clam is one of the more sensitive species to a variety of contaminants and may be a valuable indicator for potential sediment toxicity.

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.