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.

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 field and laboratory exposure to the toxic dinoflagellate Karenia brevis on the reproduction of the eastern oyster, Crassostrea virginica, and subsequent development of offspring.

Blooms of the brevetoxin-producing dinoflagellate, Karenia brevis, are a recurrent and sometimes devastating phenomenon in the Gulf of Mexico. The eastern oyster, Crassostrea virginica, is exposed regularly to these blooms, yet little is known about the impacts of K. brevis upon this important species. The present study considered the effects of exposure to both a natural bloom and cultured K. brevis on the reproductive development of C. virginica. Oysters had been exposed to a bloom of K. brevis that occurred in Lee County, Florida, from September 2012 through May 2013, during a period of gametogenesis and gamete ripening. Ripe adult oysters were collected from this bloom-exposed site and from a site 200 miles north which was not exposed to any bloom. In addition, responses to two 10-day laboratory exposures of either unripe or ripe adult oysters to whole cells of K. brevis at high bloom concentrations (1000 and 5000cellsmL-1) were determined. Both field- and laboratory-exposed adult oysters accumulated PbTx (attaining ∼22×103ngg-1 and 922ngg-1 PbTx-3 equivalents in the laboratory and the field, respectively), and significant mucal, edematous, and inflammatory features, indicative of a defense response, were recorded in adult tissues in direct contact with K. brevis cells. Laboratory-exposed oysters also showed an increase in the total number of circulating hemocytes suggesting that: (1) new hemocytes may be moving to sites of tissue inflammation, or, (2) hemocytes are released into the circulatory system from inflamed tissues where they may be produced. The area of oyster tissue occupied by gonad (representative of reproductive effort) and reactive oxygen species production in the spermatozoa of oysters exposed to the natural bloom of K. brevis were significantly lower compared to oysters that were not exposed to K. brevis. Additionally, following 10-day exposure of ripe oysters, a significant, 46% reduction in the prevalence of individuals with ripe gametes was obtained in the 5000cellsmL-1K. brevis treatment. Brevetoxin (PbTx) was recorded within the spermatozoa and oocytes of naturally exposed oysters and was estimated to be 18 and 26% of the adult PbTx load, respectively. Larvae derived from gametes containing PbTx showed significantly higher mortalities and attained a smaller larval size for the first 6 days post-fertilization. These negative effects on larval development may be due to the presence of PbTx in the lipid droplets of the oocytes, which is mobilized by the larvae during embryonic and lecithotrophic larval development. Provision of a non-contaminated food source to larvae however, appeared to mitigate the early negative effects of this neonatal PbTx exposure. Results herein show that adult eastern oysters and their offspring are susceptible to exposure to K. brevis. Caution should therefore be exercised when identifying oyster reef restoration areas and in efforts to establish aquaculture in areas prone to red tides.

Susceptibility of gametes and embryos of the eastern oyster, Crassostrea virginica, to Karenia brevis and its toxins.

The bivalve mollusc, Crassostrea virginica, is frequently exposed to blooms of Karenia brevis along the west coast of Florida during periods of spawning and early larval development. A continuous 4-day exposure of gametes and 2-4 cell stage embryos of C. virginica to whole-cell and culture filtrate of K. brevis at 500 and 5000 cells mL(-1), was followed by a 4-day 'recovery' period. Larval growth, percent of normal, abnormal and dead larvae, and the presence of food in the larval gut were measured throughout the exposure period. Results suggest that negative effects mainly occur during embryogenesis and early development. Damage to feeding apparatus/gut may occur during embryonic development or exposure to toxins may act as a feeding deterrent on non-toxic algae. Following 2-h in vitro exposure of gametes, differences in oocyte and sperm cell parameters were investigated using flow cytometry. The reduced sperm viability in the whole-cell 5000 cells mL(-1) treatment suggests the involvement of extracellular brevetoxins (PbTx) and perhaps other harmful, uncharacterized compounds associated with the K. brevis cell membrane. The cumulative effects of reduced sperm viability, fertilization success, embryonic and larval survival, and the near-annual exposure to blooms of K. brevis could cause significant bottlenecks on oyster recruitment.

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.

In vivo effects of brown tide on the feeding function of the gill of the northern quahog Mercenaria mercenaria (Bivalvia: Veneridae).

The in vivo response of adult northern quahogs, Mercenaria mercenaria, to Aureococcus anophagefferens (brown tide) at the level of the gill was determined using video-endoscopy. Feeding activity, particle-approach velocities, and ventral-groove-transport velocities were documented after the quahogs were exposed to Isochryis galbana (baseline observations) supplemented with either toxic or nontoxic A. anophagefferens at two bloom concentrations (8 x 10(5) or 2 x 10(6) cells ml(-1)). Externally, there was no evidence of adverse effects of brown tide on feeding, as siphons remained extended and dilated. Toxic brown tide at both concentrations elicited gill muscular contractions, intermittent cessation of water flow, and decreased particle loading within the pallial cavity. The 8 x 10(5) cell ml(-1) toxic treatment had no significant effect on approach velocities or ventral-groove-transport velocities after 2 h, although time-averaging showed significant reduction of the latter during the last 30 min of exposure. The higher concentration of toxic brown tide caused a significant decrease in these velocities after only 1 h. Nontoxic brown tide produced none of these effects. Thus, A. anophagefferens compromised quahog feeding by stimulating contractions of the branchial musculature and interfering with lateral and ventral groove ciliary beating. These effects were both time- and concentration-dependent and could be caused by either a dopaminergic or a serotonergic toxic factor.

Domoic acid uptake and elimination kinetics in oysters and mussels in relation to body size and anatomical distribution of toxin.

Toxin accumulation by suspension-feeding qualifier depends on a balance between processes regulating toxin uptake (i.e. ingestion and absorption of toxic cells) and elimination (i.e. egestion, exchange among tissues, excretion, degradation and/or biotransformation) during exposure to toxic blooms. This laboratory study compares the size-specific uptake and elimination kinetics of domoic acid (DA) from Pseudo-nitzschia multiseries in two co-occurring bivalves, the oyster Crassostrea virginica and the mussel Mytilus edulis. Domoic acid concentrations were measured in visceral and non-visceral tissues of different-sized oysters and mussels during simultaneous long-term exposure to toxic P. multiseries cells in the laboratory, followed by depuration on a non-toxic algal diet. Mussels attained 7-17-fold higher DA concentrations than oysters, depending on the body size and exposure time, and also detoxified DA at higher rates (1.4-1.6 d(-1)) than oysters (0.25-0.88 d(-1)) of a comparable size. Small oysters attained markedly higher weight-specific DA concentrations (maximum=78.6 µg g(-1)) than large, market-sized individuals (≤ 13 µg g(-1)), but no clear relationship was found between body size and DA concentration in mussels (maximum=460 µg g(-1)). Therefore, differential DA accumulation by the two species was, on average, approximately 3-fold more pronounced for large bivalves. An inverse relationship between DA elimination rate and body size was established for oysters but not mussels. Elimination of DA was faster in viscera than in other tissues of both bivalves; DA exchange rate from the former to the latter was higher in oysters. The contribution of viscera to the total DA burden of mussels was consistently greater than that of other tissues during both uptake (>80%) and depuration (>65%) phases, whereas it rapidly decreased from 70-80% to 30-40% in oysters, and this occurred faster in smaller individuals. Residual DA concentrations (≤ 0.25 µg g(-1)) were detected at later depuration stages (up to 14 d), mainly in viscera of oysters and non-visceral tissues of mussels, suggesting that a second, slower-detoxifying toxin compartment exists in both species. However, a simple exponential decay model was found to adequately describe DA elimination kinetics in these bivalves. The lower capacity for DA accumulation in oysters compared to mussels can thus only be explained by the former's comparatively low toxin intake rather than faster toxin elimination.

Feeding mechanics as the basis for differential uptake of the neurotoxin domoic acid by oysters, Crassostrea virginica, and mussels, Mytilus edulis.

The neurotoxin domoic acid (DA), produced by diatoms Pseudo-nitzschia spp., is transferred to humans via consumption of contaminated bivalves. This study examines feeding mechanisms, namely reduced filtration, pre-ingestive rejection and poor absorption, that might explain the comparatively low DA levels commonly found in oysters during toxic Pseudo-nitzschia blooms. Clearance rate (CR), absorption efficiency (AE) of organic matter and selective rejection in pseudofeces of oysters (Crassostrea virginica) and mussels (Mytilus edulis) were investigated in relation to the DA levels accumulated during 2-wk, simultaneous exposure to toxic Pseudo-nitzschia multiseries. Effects of temperature and P. multiseries cell size were also tested to identify conditions, if any, under which oysters can accumulate unsafe DA levels. Oysters accumulated 3.0-7.5x less DA than mussels from a short-celled P. multiseries clone (length=24microm) at 12 degrees C. This was related to the 7.4-8.5x lower CRs determined for oysters relative to mussels at this temperature. Exposure to a longer-celled P. multiseries clone (81microm) resulted in up to 70x lower toxin levels in oysters compared to mussels, which was attributed to differential feeding selectivity. Mussels were unable to discriminate between long- and short-celled P. multiseries clones from a mixed suspension, whereas oysters were previously shown to preferentially reject long cells (>70microm) in pseudofeces. Both bivalves selectively rejected P. multiseries cells from mixed suspensions containing a flagellate but not another diatom. AE of organics from P. multiseries cells by oysters and mussels was comparably low (42 and 39%, respectively) and thus unlikely to explain their differential DA accumulation. CR and DA uptake by oysters were negligible at

Sodium channel mutation leading to saxitoxin resistance in clams increases risk of PSP.

Bivalve molluscs, the primary vectors of paralytic shellfish poisoning (PSP) in humans, show marked inter-species variation in their capacity to accumulate PSP toxins (PSTs) which has a neural basis. PSTs cause human fatalities by blocking sodium conductance in nerve fibres. Here we identify a molecular basis for inter-population variation in PSP resistance within a species, consistent with genetic adaptation to PSTs. Softshell clams (Mya arenaria) from areas exposed to 'red tides' are more resistant to PSTs, as demonstrated by whole-nerve assays, and accumulate toxins at greater rates than sensitive clams from unexposed areas. PSTs lead to selective mortality of sensitive clams. Resistance is caused by natural mutation of a single amino acid residue, which causes a 1,000-fold decrease in affinity at the saxitoxin-binding site in the sodium channel pore of resistant, but not sensitive, clams. Thus PSTs might act as potent natural selection agents, leading to greater toxin resistance in clam populations and increased risk of PSP in humans. Furthermore, global expansion of PSP to previously unaffected coastal areas might result in long-term changes to communities and ecosystems.

Metabolic depression and whole-body response to enforced starvation by Crassostrea gigas postlarvae.

Physiological and biochemical measurements were performed on six oyster (Crassostrea gigas) cohorts, in order to: (a) investigate the whole-body response (growth, energy content, metabolic and excretion rates) of 2-week-old postlarvae (spat) to enforced (0-8 days) starvation, and (b) test the potential use of three aerobic enzyme systems as indices of physiological condition. Starvation resulted in exponential reduction of postlarval metabolic and excretion rates, as well as a linear decrease in enzyme activity. These response mechanisms effectively limited the loss of endogenous reserves after 2 days of starvation and maintained the oyster's functional integrity over prolonged (8 days) starvation. Proteins appeared to be selectively conserved during short-term (2 days) starvation, as suggested by a decrease in total protein content, while maintaining constant weight-specific enzyme activity. Postlarvae starved for 2 days exhibited relatively higher lipid losses, lower mortality and lower metabolism than metamorphosing stages, thus suggesting a greater buffering capacity to starvation in the former. The activity of the electron transport system may be a useful indicator of long-term stress or developmental condition of oyster postlarvae, while citrate synthase and cytochrome oxidase could be used as indicators of growth rate. None of these enzyme systems is recommended as an index of aerobic metabolism during short-term starvation.