Testing for resistance of pelagic marine copepods to a toxic dinoflagellate

With few exceptions, the evolutionary consequences of harmful algae to grazers in aquatic systems remain unexplored. To examine both the ecological and evolutionary consequences of harmful algae on marine zooplankton, we used a two-fold approach. In the first approach, we examined the life history responses of two geographically separate Acartia hudsonica (Copepoda Calanoida) populations reared on diets containing the toxic dinoflagellate Alexandrium fundyense . One copepod population was from a region, Casco Bay, Maine, USA, that has experienced recurrent blooms of highly toxic Alexandrium spp. for decades; whereas the other population from Great Bay, New Jersey, USA, has never been exposed to toxic Alexandrium blooms. The life history experiment demonstrated that when the copepod population from New Jersey was reared on a diet containing toxic A. fundyense it exhibited lower somatic growth, size at maturity, egg production and survival than the same population reared on a diet without toxic A. fundyense . In contrast, toxic A. fundyense did not affect the life-history traits of the Maine population. Fitness, finite population growth rate (), was significantly reduced in the New Jersey population, but not in the Maine population. These results are consistent with the hypothesis of local adaptation (resistance) of the historically exposed copepod population to the toxic dinoflagellate. In the second approach, we further tested the resistance hypothesis with a laboratory genetic selection experiment with the naïve New Jersey copepod population exposed to a diet containing toxic A. fundyense. This experiment demonstrated that the ingestion and egg production of adult females of naïve copepods fed A. fundyense improved after three generations of being reared on a diet containing the toxic dinoflagellate. The results of the present study have important implications for understanding how grazer populations may respond to the introduction of toxic algae to their environment, and suggest that grazer resistance may be a feedback mechanism that may lead to bloom control.Co-ordinating editor: Hurst     

Differential responses of populations of the copepod Acartia hudsonica to toxic and nutritionally insufficient food algae

Nutritional insufficiency and toxicity are deleterious effects of phytoplankton on grazers. We hypothesize that toxic food is likely to have stronger evolutionary selective effects on grazers than nutritionally insufficient food. We explore this hypothesis in comparative studies of egg production and egg hatching of the copepod Acartia hudsonica challenged with both a toxic and a nutritionally insufficient alga. Experiments lasting 6 days, in which mixtures of different proportions of the suspect and a control alga were offered as food to female copepods, showed that the dinoflagellate Alexandrium fundyense, which bears paralytic shellfish toxins, was toxic to A. hudsonica. In contrast, the diatom Phaeodactylum tricornutum was nutritionally insufficient to A. hudsonica. In another set of experiments, the effects of A. fundyense and P. tricornutum, respectively, as sole foods on egg production and egg hatching success of two geographically separated populations (Maine and Connecticut) of the copepod A. hudsonica were examined in common-environment experiments, after being raised under identical conditions for two generations. The location in Maine regularly experiences toxic blooms of Alexandrium sp. whereas the location in Connecticut does not. During a 6-day period, A. fundyense reduced the egg production rates of the Connecticut copepod population, but not of the Maine population. In contrast, the diatom P. tricornutum reduced the egg production of both populations. These results of this study are consistent with the hypothesis of local adaptation to toxic food, but not to nutritionally insufficient food.     

Ingestion of the dinoflagellate, Pfiesteria piscicida, by the calanoid copepod, Acartia tonsa

The dinoflagellate, Pfiesteria piscicida, can form harmful algal blooms in estuarine environments. The dominant copepod species usually found in these waters is Acartia tonsa. We tested the ability of A. tonsa to graze the non-toxic zoospore stage of P. piscicida and thus serve as a potential biological control of blooms of this algal species. A. tonsa grazed the non-toxic zoospore stages of both a non-inducible P. piscicida strain (FDEPMDR23) and a potentially toxic strain (Tox-B101156) at approximately equal rates. Ingestion of P. piscicida increased with cell concentration and exhibited a saturated feeding response. Both the maximum number of cells ingested (I_max) and the slope of the ingestion curve (α) of A. tonsa feeding on P. piscicida were comparable to these ingestion parameters for A. tonsa fed similar-sized phytoplankton and protozoan species. When these laboratory ingestion rates were combined with abundance estimates of A. tonsa from the Pocomoke Estuary and Chesapeake Bay, we found that significant grazing control of the non-toxic zoospore stage of P. piscicida by A. tonsa would only occur at high copepod abundances (>10 copepods L⁻¹). We conclude that under most in situ conditions the potential biological control of blooms of P. piscicida is exerted by microzooplankton grazers. However, in the less saline portions of estuaries where maximum concentrations of copepods often occur with low abundances of microzooplankton, copepod grazing coefficients can be similar to the growth rates of P. piscicida.     

Comparison of the functional and numerical responses of resistant versus non-resistant populations of the copepod Acartia hudsonica fed the toxic dinoflagellate Alexandrium tamarense

The functional and numerical responses of grazers are key pieces of information in predicting and modeling predator–prey interactions. It has been demonstrated that exposure to toxic algae can lead to evolved resistance in grazer populations. However, the influence of resistance on the functional and numerical response of grazers has not been studied to date. Here, we compared the functional and numerical responses of populations of the copepod Acartia hudsonica that vary in their degree of resistance to the toxic dinoflagellate Alexandrium tamarense. In common environment experiments carried out after populations had been grown under identical conditions for several generations, female copepods were offered solutions containing different concentrations of either toxic A. tamarense or the non-toxic green flagellate Tetraselmis sp. ranging from 25 to 500 μgC L⁻¹, and ingestion and egg production rates were measured. Throughout most of the range of concentrations of the toxic diet, copepod populations that had been historically exposed to toxic blooms of Alexandrium exhibited significantly higher ingestion and egg production rates than populations that had little or no exposure to these blooms. In contrast, there were no significant differences between populations in ingestion or egg production for the non-toxic diet. Hence, the between population differences in functional and numerical response to A. tamarense were indeed related to resistance. We suggest that the effect of grazer toxin resistance should be incorporated in models of predator and toxic prey interactions. The potential effects of grazer toxin resistance in the development and control of Alexandrium blooms are illustrated here with a simple simulation exercise.     

Experimental study on the impact of dinoflagellate Alexandrium species on populations of the rotifer Brachionus plicatilis

To investigate harmful effects of the dinoflagellate Alexandrium species on microzooplankton, the rotifer Brachionus plicatilis was chosen as an assay species, and tested with 10 strains of Alexandrium including one known non-PSP-producer (Alexandrium tamarense, AT-6). HPLC analysis confirmed the PSP-content of the various strains: Alexandrium lusitanicum, Alexandrium minutum and Alexandrium tamarense (ATHK, AT5-1, AT5-3, ATCI02, ATCI03) used in the experiment were PSP-producers. No PSP toxins were detected in the strains Alexandrium sp1, Alexandrium sp2.Exposing rotifer populations to the densities of 2000 cells ml⁻¹ of each of these 10 Alexandrium strains revealed that the (non-PSP) A. tamarense (AT-6) and two other PSP-producing algae: A. lusitanicum, A. minutum, did not appear to adversely impact rotifer populations. Rotifers exposed to these three strains were able to maintain their population numbers, and in some cases, increase them. Although some increases in rotifer population growth following exposures to these three algal species were noted, the rate was less than for the non-exposed control rotifer groups.In contrast, the remaining seven algal strains (A. tamarense ATHK, AT5-1, AT5-3, ATCI02, ATCI03; also Alexandrium sp1 and Alexandrium sp2) all have adverse effects on the rotifers. Dosing rotifers with respective algal cell densities of 2000 cells ml⁻¹ each, for Alexandrium sp1, Alexandrium sp2, and A. tamarense strains ATHK and ATCI03 showed mean lethal time (LT₅₀) on rotifer populations of 21, 28, 29, and 36h, respectively. The remaining three species (A. tamarense strains AT5-1, AT5-3, ATCI02) caused respective mean rotifer LT₅₀s of 56, 56, and 71 h, compared to 160 h for the unexposed “starved control” rotifers. Experiments to determine ingestion rates for the rotifers, based on changes in their Chlorophyll a content, showed that the rotifers could feed on A. lusitanicum, A. minutum and A. tamarense strain AT-6, but could graze to little or no extent upon algal cells of the other seven strains. The effects on rotifers exposed to different cell densities, fractions, and growth phases of A. tamarense algal culture were respectively compared. It was found that only the whole algal cells had lethal effects, with strongest impact being shown by the early exponential growth phase of A. tamarense. The results indicate that some toxic mechanism(s), other than PSP and present in whole algal cells, might be responsible for the adverse effects on the exposed rotifers.     

Effects of Protoceratium reticulatum yessotoxin on feeding rates of Acartia hudsonica: A bioassay using artificial particles coated with purified toxin

Paralytic shellfish toxins produced by dinoflagellates are known to deter copepod grazing. Dinoflagellate species, including Protoceratium reticulatum, also produce disulfated polyether yessotoxins that were previously referred to as diarrheic shellfish toxins. However, the role of yessotoxins in predator–prey relationships is not yet clear. In the present study, the effects of purified yessotoxin (YTX) on feeding activities of Acartia hudsonica (Copepoda, Calanoida) were experimentally investigated. Polystyrene fluorescent microspheres (10 μm in diameter) colored bright blue or yellow-green were coated with cell extracts of P. reticulatum that do not produce yessotoxins. The bright blue microspheres were further coated with YTX, and the yellow-green microspheres were used as the reference. The microspheres were then given to the copepods separately or in combination to measure clearance rates and feeding selectivity. A. hudsonica was found to feed on the yellow-green microspheres without YTX at twice the rate of the bright blue microspheres with YTX. We also confirmed that microsphere color per se did not affect the feeding rates. The bright blue microspheres adsorbed 1.8–43.3 pg of YTX per microsphere, which is similar to the cell-specific yessotoxin content of toxic P. reticulatum found in natural environments. These results suggest that production of yessotoxin is advantageous for P. reticulatum by deterring predation by copepods.     

No evidence for induction or selection of mutant sodium channel expression in the copepod Acartia husdsonica challenged with the toxic dinoflagellate Alexandrium fundyense

Some species in the dinoflagellate genus Alexandrium spp. produce a suite of neurotoxins that block sodium channels, known as paralytic shellfish toxins (PST), which have deleterious effects on grazers. Populations of the ubiquitous copepod grazer Acartia hudsonica that have co‐occurred with toxic Alexandrium spp. are better adapted than naïve populations. The mechanism of adaptation is currently unknown. We hypothesized that a mutation in the sodium channel could account for the grazer adaptation. We tested two hypotheses: (1) Expression of the mutant sodium channel could be induced by exposure to toxic Alexandrium fundyense; (2) in the absence of induction, selection exerted by toxic A. fundyense would favor copepods that predominantly express the mutant isoform. In the copepod A. hudsonica, both isoforms are expressed in all individuals in varying proportions. Thus, in addition to comparing expression ratios of wild‐type to mutant isoforms for individual copepods, we also partitioned copepods into three groups: those that predominantly express the mutant (PMI) isoform, the wild‐type (PWI) isoform, or both isoforms approximately equally (EI). There were no differences in isoform expression between individuals that were fed toxic and nontoxic food after three and 6 days; induction of mutant isoform expression did not occur. Furthermore, the hypothesis that mutant isoform expression responds to toxic food was also rejected. That is, no consistent evidence showed that the wild‐type to mutant isoform ratios decreased, or that the relative proportion of PMI individuals increased, due to the consumption of toxic food over four generations. However, in the selected line that was continuously exposed to toxic food sources, egg production rate increased, which suggested that adaptation occurred but was unrelated to sodium channel isoform expression.     

Selective grazing of Temora longicornis in different stages of a Phaeocystis globosa bloom—a mesocosm study

Selective grazing of a calanoid copepod Temora longicornis was measured during different stages of a Phaeocystis globosa bloom, in order to reveal (1) if T. longicornis feeds on single cells and/or colonies of P. globosa in the presence of alternative food sources, (2) if copepod food selection changes during the initiation, maintenance, collapse and decay of a P. globosa bloom and (3) if P. globosa dominated food assemblage provides a good diet for copepod egg production. Our results show low but constant feeding on small colonies of P. globosa, irrespective of the type or concentration of alternative food sources. In contrast, feeding on single cells was never significant, and the total contribution of P. globosa to carbon ingestion of T. longicornis was minor. T. longicornis fed most actively on the decaying colonies, whereas during the peak of the bloom copepods selected against P. globosa. Mostly, T. longicornis fed unselectively on different food particles: before the bloom, the major part of the diet consisted of diatoms, whereas during and after the bloom copepod diet was dominated by dinoflagellates and ciliates. Egg production was highest during the decay of the bloom, coinciding with highest proportional ingestion of heterotrophic organisms, but was not seriously reduced even during the peak of the bloom. We conclude that P. globosa blooms should not threaten survival of copepod populations, but the population recruitment may depend on the type (and concentration) of the dominant heterotrophs present during the blooms. Due to relatively unselective grazing, the impact of T. longicornis to the initiation of a Phaeocystis bloom is considered small, although grazing on decaying colonies may contribute to the faster termination of a bloom.     

Copepod feeding response to varying Alexandrium spp. cellular toxicity and cell concentration among natural plankton samples

We challenged four species of copepod grazers (Acartia hudsonica, Centropages hamatus, Eurytemora herdmani, Calanus finmarchicus) with natural water samples containing non-toxic algae mixed with one of three clones of Alexandrium spp.—A. tamarense GTCN16 (non-toxic), A. fundyense GTCA28 (moderate toxicity), and A. fundyense BC1 (higher toxicity), each at relatively high (10⁵ cells L⁻¹) and low (10⁴ cells L⁻¹) concentrations. Within any one copepod species, significant differences existed in copepod clearance rates and total food ingested between high and low Alexandrium cell concentrations, and between levels of toxicity, but feeding response did not follow a predictable relationship proportional to toxin levels—rather, the presence or absence of toxin was more important than the level of toxicity. C. finmarchicus behaved differently from the smaller coastal copepods, showing less selectivity and greater concentration dependence. In low Alexandrium concentration treatments, copepod clearance rates on Alexandrium were usually higher, and electivity indices for Alexandrium less negative (indicating less avoidance), compared to high concentration treatments. In high toxicity (BC1) treatments of all copepod species (except C. finmarchicus), total food consumption was consistently less at high Alexandrium concentrations compared to low concentrations, suggesting that high toxicity and concentration suppress overall feeding, while in non-toxic (GTCN 16) treatments total consumption was always higher at high Alexandrium concentrations. Copepod grazers do not follow predictable feeding rules throughout a continuum of conditions, but become more predictable at extremes of concentration and toxicity of prey, consistent with the conclusion that both factors are important. Results support the hypothesis that grazer deterrence imparted by toxicity is only effective at high cell concentrations, but even then will not protect against all grazers.     

Food selectivity and grazing impact on toxic Dinophysis spp. by copepods feeding on natural plankton assemblages

Food selectivity and grazing impact by Acartia bifilosa, Temora longicornis and Centropages typicus on Dinophysis spp. plankton assemblages were experimentally investigated in the Baltic Sea. Toxin analyses were carried out on phyto- and zooplankton-dominated size fractions from field-collected samples to assess if toxins produced by Dinophysis spp. would end up in the zooplankton. All copepod species fed actively on toxic Dinophysis spp. (Dinophysis acuta and Dinophysis norvegica). Despite the non-selective feeding behaviour by T. longicornis and C. typicus, selectivity coefficients on D. acuta progressively decreased as food availability increased. Similar response was not observed for A. bifilosa, which displayed an even less selective behaviour. A. bifilosa had no significant negative effect on the net growth of D. norvegica at the lowest food concentration offered, whereas T. longicornis and C. typicus had significant negative effects on the net growth of D. acuta at low concentrations, similar to those observed in situ. Both species could potentially contribute as a substantial loss factor for Dinophysis spp. provided they are abundant at the onset of the blooms. The estimated grazing impact by the copepod populations was only considerable when C. typicus abundance was high and D. acuta population in sharp decline. Our results suggest that when high abundance of grazers and poor growth condition of prey populations prevail, grazing impact by copepods can contribute considerably to prevent Dinophysis spp. populations to grow or to cause the populations to decline. Okadaic acid was detected in the zooplankton size fraction at one occasion, but the concentration was far lower than the one expected from the ingested toxins. Thus, even if copepods may act as vectors of DSP-toxins to higher trophic levels, the amount of these toxins transported in the food web by copepods seems limited.     

The interplay between host toxins and parasitism by Amoebophrya

The parasitic dinoflagellate Amoebophrya sp. ex Karlodinium veneficum was used to test two hypotheses: (1) infection of cells decreases with increasing host toxicity and (2) parasitism causes the catabolism of host toxin. To test the first hypothesis, host strains differing in toxin content were inoculated with dinospores of Amoebophrya sp. derived from infected cultures of toxic and non-toxic K. veneficum, with resulting infections assessed following 24-h incubations. Contrary to expectations, infection of K. veneficum by Amoebophrya sp. was positively correlated with host toxicity. To examine the second hypothesis, synchronous infection with >80% of cells being parasitized was induced using a toxic strain of K. veneficum, and total toxin concentration (intracellular plus extracellular levels of KmTX1) was followed over the 3-day infection cycle. Toxin content ml⁻¹ increased with growth of K. veneficum in uninfected control cultures, but declined in infected cultures as the parasite completed its life cycle. On a cellular basis, toxin content of infected and uninfected cultures differed little during the experiment, suggesting that the parasite does not actively catabolise host toxin. Rather, infection appears to promote degradation of toxins via death of host cells and subsequent bacterial activity. Results indicate that Amoebophrya sp. ex K. veneficum has greater potential to impact toxic strains relative to non-toxic host strains in natural systems. Thus, Amoebophrya sp. ex. K. veneficum may limit the occurrence of toxic K. veneficum blooms in marine and estuarine environments, while simultaneously functioning as a pathway for dissipation of host toxin.     

Karlotoxin mediates grazing by Oxyrrhis marina on strains of Karlodinium veneficum

Karlodinium veneficum is a common member of temperate, coastal phytoplankton assemblages that occasionally forms blooms associated with fish kills. Here, we tested the hypothesis that the cytotoxic and ichthyotoxic compounds produced by K. veneficum, karlotoxins, can have anti-grazing properties against the heterotrophic dinoflagellate, Oxyrrhis marina. The sterol composition of O. marina (>80% cholesterol) renders it sensitive to karlotoxin, and does not vary substantially when fed different algal diets even for prey that are resistant to karlotoxin. At in situ bloom concentrations (10⁴–10⁵ K. veneficum ml⁻¹), grazing rates (cells ingested per Oxyrrhis h⁻¹) on toxic K. veneficum strain CCMP 2064 were 55% that observed on the non-toxic K. veneficum strain MD5. At lower prey concentrations typical of in situ non-bloom levels (<10³ cells ml⁻¹), grazing rates (cells ingested per Oxyrrhis h⁻¹) on toxic K. veneficum strain CCMP 2064 were 70–80% of rates on non-toxic strain MD5. Growth of O. marina was significantly suppressed when fed the toxic strain of K. veneficum. Experiments with mixed prey cultures, where non-toxic strain MD5 was fluorescently stained, showed that the presence of toxic strain CCMP 2064 inhibited grazing of O. marina on the co-occurring non-toxic strain MD5. Exogenous addition of a sub-lethal dose (100 ng ml⁻¹) of purified karlotoxin inhibited grazing of O. marina by approximately 50% on the non-toxic K. veneficum strain MD5 or the cryptophyte S. major. These results identify karlotoxin as an anti-grazing compound for those grazers with appropriate sterol composition (i.e., desmethyl sterols). This strategy is likely to be an important mechanism whereby growth of K. veneficum is uncoupled from losses due to grazing, allowing it to form ichthyotoxic blooms in situ.     

Morphogenetic diversity and biotoxin composition of Alexandrium (Dinophyceae) in Irish coastal waters

The diversity of Alexandrium spp. in Irish coastal waters was investigated through the morphological examination of resting cysts and vegetative cells, the determination of PSP toxin and spirolide profiles and the sequence analysis of rDNA genes. Six morphospecies were characterised: A. tamarense, A. minutum, A. ostenfeldii, A. peruvianum, A. tamutum and A. andersoni. Both PSP toxin producing and non-toxic strains of A. tamarense and A. minutum were observed. The average toxicities of toxic strains for both cultured species were respectively 11.3 (8.6 S.D.) and 2.3 (0.5 S.D.) pg STX equiv. cell⁻¹. Alexandrium ostenfeldii and A. peruvianum did not synthesise PSP toxins but HPLC–MS analysis of two strains showed distinct spirolide profiles. A cyst-derived culture of A. peruvianum from Lough Swilly mainly produced spirolides 13 desmethyl-C and 13 desmethyl-D whereas one of A. ostenfeldii, from Bantry Bay, produced spirolides C and D. Species identification was confirmed through the analyses of SSU, ITS1-5.8S-ITS2 and LSU rDNA genes. Some nucleotide variability was observed among clones of toxic strains of A. tamarense, which all clustered within the North American clade. However, rDNA sequencing did not allow discrimination between the toxic and non-toxic forms of A. minutum. Phylogenetic analysis also permitted the differentiation of A. ostenfeldii from A. peruvianum. Resting cysts of PSP toxin producing Alexandrium species were found in Cork Harbour and Belfast Lough, locations where shellfish contamination events have occurred in the past, highlighting the potential for the initiation of harmful blooms from cyst beds. The finding of supposedly non-toxic and biotoxin-producing Alexandrium species near aquaculture production sites will necessitate the use of reliable discriminative methods in phytoplankton monitoring.     

Feeding,egg production,and egg hatching success of the copepods Acartia tonsa and Temora longicornis on diets of the toxic diatom Pseudo-nitzschia multiseries and the non-toxic diatom Pseudo-nitzschia pungens

In 1987, there was an episode of shellfish poisoning in Canada with human fatalities caused by the diatom Pseudo-nitzschia multiseries, which produced the toxin domoic acid. In order to examine whether domoic acid in this diatom serves as a grazing deterrent for copepods, we compared feeding rates, egg production rates, egg hatching success and mortality of the calanoid copepods Acartia tonsa and Temora longicornis feeding on unialgal diets of the toxic diatom P. multiseries and the similarly-sized non-toxic diatom Pseudo-nitzschia pungens. Copepods were collected in summers of 1994, 1995 and 1996 from Shediac Bay, New Brunswick, Canada, near Prince Edward Island, the site of the 1987 episode of domoic acid shellfish poisoning. Rates of ingestion of the toxic versus the non-toxic diatom by A. tonsa and T. longicornis were similar, with only one significantly different pair of values obtained in 1994, for which A. tonsa had a higher mean rate of ingestion of the toxic than the non-toxic diatom. Thus, domoic acid did not appear to retard grazing. Analyses of copepods with high performance liquid chromatography (HPLC) revealed that copepods accumulated domoic acid when feeding on P. multiseries. Egg production rates of copepods when feeding on P. multiseries and P. pungens were very low, ranging from 0 to 2.79 eggs female^–1 d^–1. There did not appear to be differential egg production or egg hatching success on diets of the toxic and non-toxic diatoms. Mortality of females on the toxic diet was low, ranging from 0 to 20%, with a mean of 13%, and there was no apparent difference between mortality of copepods feeding on toxic versus non-toxic diatoms. Egg hatching success on both diets, although based on few eggs, ranged between 22% and 76%, with a mean percentage hatching of 45%. Diets of the non-toxic diatom plus natural seawater assemblages supplemented with dissolved domoic acid, revealed similar rates and percentages when compared to previous experiments. In summary, none of the variables measured indicated adverse effects on copepods feeding on the toxic compared to the non-toxic diatom.     

Development of a qualitative PCR method for the Alexandrium spp. (Dinophyceae) detection in contaminated mussels (Mytilus galloprovincialis)

Paralytic shellfish poisoning (PSP) is a syndrome caused by the consumption of shellfish contaminated with neurotoxins produced by organisms of the marine dinoflagellate genus Alexandrium. A. minutum is the most widespread species responsible for PSP in the Western Mediterranean basin. The standard monitoring of shellfish farms for the presence of harmful algae and related toxins usually requires the microscopic examination of phytoplankton populations, bioassays and toxin determination by HPLC. These procedures are time-consuming and require remarkable experience, thus limiting the number of specimens that can be analyzed by a single laboratory unit. Molecular biology techniques may be helpful in the detection of target microorganisms in field samples. In this study, we developed a qualitative PCR assay for the rapid detection of all potentially toxic species belonging to the Alexandrium genus and specifically A. minutum, in contaminated mussels. Alexandrium genus-specific primers were designed to target the 5.8S rDNA region, while an A. minutum species-specific primer was designed to bind in the ITS1 region. The assay was validated using several fixed seawater samples from the Mediterranean basin, which were analyzed using PCR along with standard microscopy procedures. The assay provided a rapid method for monitoring the presence of Alexandrium spp. in mussel tissues, as well as in seawater samples. The results showed that PCR is a valid, rapid alternative procedure for the detection of target phytoplankton species either in seawater or directly in mussels, where microalgae can accumulate.     

Experimental exposure of the blue mussel (Mytilus edulis, L.) to the toxic dinoflagellate Alexandrium fundyense: Histopathology, immune responses, and recovery

Mussels (Mytilus edulis) were exposed to cultures of the toxic dinoflagellate Alexandrium fundyense or the non-toxic alga Rhodomonas sp. to evaluate the effects of the harmful alga on the mussels and to study recovery after discontinuation of the A. fundyense exposure. Mussels were exposed for 9 days to the different algae and then all were fed Rhodomonas sp. for 6 more days. Samples of hemolymph for hemocyte analyses and tissues for histology were collected before the exposure and periodically during exposure and recovery periods.Mussels filtered and ingested both microalgal cultures, producing fecal pellets containing degraded, partially degraded, and intact cells of both algae. Mussels exposed to A. fundyense had an inflammatory response consisting of degranulation and diapedesis of hemocytes into the alimentary canal and, as the exposure continued, hemocyte migration into the connective tissue between the gonadal follicles. Evidence of lipid peroxidation, similar to the detoxification pathway described for various xenobiotics, was found; insoluble lipofuchsin granules formed (ceroidosis), and hemocytes carried the granules to the alimentary canal, thus eliminating putative dinoflagellate toxins in feces. As the number of circulating hemocytes in A. fundyense-exposed mussels became depleted, mussels were immunocompromised, and pathological changes followed, i.e., increased prevalences of ceroidosis and trematodes after 9 days of exposure. Moreover, the total number of pathological changes increased from the beginning of the exposure until the last day (day 9). After 6 days of the exposure, mussels in one of the three tanks exposed to A. fundyense mass spawned; these mussels showed more severe effects of the toxic algae than non-spawning mussels exposed to A. fundyense.No significant differences were found between the two treatments during the recovery period, indicating rapid homeostatic processes in tissues and circulating hemocytes.     

Determining the Advantages,Costs, and Trade-Offs of a Novel Sodium Channel Mutation in the Copepod Acartia hudsonica to Paralytic Shellfish Toxins (PST)

The marine copepod Acartia hudsonica was shown to be adapted to dinoflagellate prey, Alexandrium fundyense, which produce paralytic shellfish toxins (PST). Adaptation to PSTs in other organisms is caused by a mutation in the sodium channel. Recently, a mutation in the sodium channel in A. hudsonica was found. In this study, we rigorously tested for advantages, costs, and trade-offs associated with the mutant isoform of A. hudsonica under toxic and non-toxic conditions. We combined fitness with wild-type: mutant isoform ratio measurements on the same individual copepod to test our hypotheses. All A. hudsonica copepods express both the wild-type and mutant sodium channel isoforms, but in different proportions; some individuals express predominantly mutant (PMI) or wild-type isoforms (PWI), while most individuals express relatively equal amounts of each (EI). There was no consistent pattern of improved performance as a function of toxin dose for egg production rate (EPR), ingestion rate (I), and gross growth efficiency (GGE) for individuals in the PMI group relative to individuals in the PWI expression group. Neither was there any evidence to indicate a fitness benefit to the mutant isoform at intermediate toxin doses. No clear advantage under toxic conditions was associated with the mutation. Using a mixed-diet approach, there was also no observed relationship between individual wild-type: mutant isoform ratios and among expression groups, on both toxic and non-toxic diets, for eggs produced over three days. Lastly, expression of the mutant isoform did not mitigate the negative effects of the toxin. That is, the reductions in EPR from a toxic to non-toxic diet for copepods were independent of expression groups. Overall, the results did not support our hypotheses; the mutant sodium channel isoform does not appear to be related to adaptation to PST in A. hudsonica. Other potential mechanisms responsible for the adaptation are discussed.     

Detection of potential microcystin-producing cyanobacteria in Brazilian reservoirs with a mcyB molecular marker

One of the most serious problems related to water eutrophication is the occurrence of increasingly frequent blooms of toxic cyanobacteria in freshwater ecosystems. Microcystin (MCYST) molecular markers may be used for the detection of toxic cyanobacteria, both cultivated strains and environmental samples, independently of their taxonomic category and production of the toxin at the moment of analysis. Sixty Microcystis spp. strains from 15 water reservoirs of south, southeastern and northeastern Brazil were analyzed by polymerase chain reaction (PCR) with oligonucleotide primers for mcyB gene of the operon that encodes a microcystin synthetase. It was found out that the presence of a unique amplified product of approximately 780 bp in 18 strains, indicated the presence of the microcystin-producing genotype. There was correspondence between the presence of the mcyB gene and microcystin determined by ELISA. Eight reservoirs contained toxic strains, two of these reservoirs being used mainly for public water supply. The coexistence of a mixture of toxic and non-toxic genotypes in populations of several reservoirs was found. Thus, it is evident that Microcystis populations present in blooms compose a mosaic, with genetically different individuals within the same population, each one, possibly, with its own tolerance to environmental factors and with distinct toxicity potential.     

Feeding interactions of the copepods Eurytemora affinis and Acartia bifilosa with the cyanobacteria Nodularia sp.

We measured ingestion and clearance rates of two Baltic Seacalanoid copepods, Eurytemora affinis and Acartia bifilosa,on toxic and non-toxic cyanobacteria Nodularia sp. using theisotope technique. Eurytemora affinis fed actively on the non-toxicstrain and moderately actively on the toxic strain, whereasA.bifilosa totally avoided feeding on both strains. This suggeststhat A.bifilosa rejected cyanobacterial filaments due to theirnutritional inadequacy or difficult manageability. The differentresponse of E.affinis to the non-toxic and toxic strains, inturn, shows that this copepod species was able to sense thepresence of the toxin in cyanobacterial filaments and thereforefed less on the toxic strain. The interaction between A.bifilosaand Nodularia sp. was further examined (with the particle countingmethod) by measuring the clearance rates of A.bifilosa on ediblegreen flagellates in the presence of cyanobacteria. The presenceor concentration of toxic Nodularia sp. did not affect grazingrates of A.bifilosa on Brachiomonas submarina. Since earlierstudies have shown that ingestion of Nodularia sp. decreasesegg production and increases mortality in E.affinis, we suggestthat the occurrence of Nodularia sp. blooms in the Baltic Seamay favour individuals of copepod species capable of selectivefeeding, such as A.bifilosa.     

Fitness consequences for copepods feeding on a red tide dinoflagellate: deciphering the effects of nutritional value, toxicity, and feeding behavior

Phytoplankton exhibit a diversity of morphologies, nutritional values, and potential chemical defenses that could affect the feeding and fitness of zooplankton consumers. However, how phytoplankton traits shape plant–herbivore interactions in the marine plankton is not as well understood as for terrestrial or marine macrophytes and their grazers. The occurrence of blooms of marine dinoflagellates such as Karenia brevis suggests that, for uncertain reasons, grazers are unable to capitalize on, or control, this phytoplankton growth—making these systems appealing for testing mechanisms of grazing deterrence. Using the sympatric copepod Acartia tonsa, we conducted a mixed diet feeding experiment to test whether K. brevis is beneficial, toxic, nutritionally inadequate, or behaviorally rejected as food relative to the palatable and nutritionally adequate phytoplankter Rhodomonas lens. On diets rich in K. brevis, copepods experienced decreased survivorship and decreased egg production per female, but the percentage of eggs that hatched was unaffected. Although copepods showed a 6–17% preference for R. lens over K. brevis on some mixed diets, overall high ingestion rates eliminated the possibility that reduced copepod fitness was caused by copepods avoiding K. brevis, leaving nutritional inadequacy and toxicity as remaining hypotheses. Because egg production was dependent on the amount of R. lens consumed regardless of the amount of K. brevis eaten, there was no evidence that fitness costs were caused by K. brevis toxicity. Copepods limited to K. brevis ate 480% as much as those fed only R. lens, suggesting that copepods attempted to compensate for low food quality with increased quantity ingested. Our results indicate that K. brevis is a poor food for A. tonsa, probably due to nutritional inadequacy rather than toxicity, which could affect bloom dynamics in the Gulf of Mexico where these species co-occur.