Dynamics of Alexandrium fundyense blooms and shellfish toxicity in the Nauset Marsh System of Cape Cod (Massachusetts,USA)

Paralytic Shellfish Poisoning (PSP) toxins are annually recurrent along the Massachusetts coastline (USA), which includes many small embayments and salt ponds. Among these is the Nauset Marsh System (NMS), which has a long history of PSP toxicity. Little is known, however, about the bloom dynamics of the causative organism Alexandrium fundyense within that economically and socially important system. The overall goal of this work was to characterize the distribution and dynamics of A. fundyense blooms within the NMS and adjacent coastal waters by documenting the distribution and abundance of resting cysts and vegetative cells. Cysts were found predominantly in three drowned kettle holes or salt ponds at the distal ends of the NMS – Salt Pond, Mill Pond, and Town Cove. The central region of the NMS had a much lower concentration of cysts. Two types of A. fundyense blooms were observed. One originated entirely within the estuary, seeded by cysts in the three seedbeds. These blooms developed independently of each other and of the A. fundyense population observed in adjacent coastal waters outside the NMS. The temporal development of the blooms was different in the three salt ponds, with initiation differing by as much as 30 days. These differences do not appear to reflect the initial cyst abundances in these locations, and may simply result from higher cell retention and higher nutrient concentrations in Mill Pond, the first site to bloom. Germination of cysts accounted for a small percentage of the peak cell densities in the ponds, so population size was influenced more by the factors affecting growth than by cyst abundance. Subsurface cell aggregation (surface avoidance) limited advection of the vegetative A. fundyense cells out of the salt ponds through the shallow inlet channels. Thus, the upper reaches of the NMS are at the greatest risk for PSP since the highest cyst abundances and cell concentrations were found there. After these localized blooms in the salt ponds peaked and declined, a second, late season bloom occurred within the central portions of the NMS. The timing of this second bloom relative to those within the salt ponds and the coastal circulation patterns at that time strongly suggest that those cells originated from a regional A. fundyense bloom in the Gulf of Maine, delivered to the central marsh from coastal waters outside the NMS through Nauset Inlet. These results will guide policy decisions about water quality as well as shellfish monitoring and utilization within the NMS and highlight the potential for “surgical” closures of shellfish during PSP events, leaving some areas open for harvesting while others are closed.     

Extensive genetic diversity and rapid population differentiation during blooms of Alexandrium fundyense (Dinophyceae) in an isolated salt pond on Cape Cod,MA, USA

In Massachusetts, paralytic shellfish poisoning (PSP) is annually recurrent along the coastline, including within several small embayments on Cape Cod. One such system, the Nauset Marsh System (NMS), supports extensive marshes and a thriving shellfishing industry. Over the last decade, PSP in the NMS has grown significantly worse; however, the origins and dynamics of the toxic Alexandrium fundyense (Balech) populations that bloom within the NMS are not well known. This study examined a collection of 412 strains isolated from the NMS and the Gulf of Maine (GOM) in 2006–2007 to investigate the genetic characteristics of localized blooms and assess connectivity with coastal populations. Comparisons of genetic differentiation showed that A. fundyense blooms in the NMS exhibited extensive clonal diversity and were genetically distinct from populations in the GOM. In both project years, genetic differentiation was observed among temporal samples collected from the NMS, sometimes occurring on the order of approximately 7 days. The underlying reasons for temporal differentiation are unknown, but may be due, in part, to life‐cycle characteristics unique to the populations in shallow embayments, or possibly driven by selection from parasitism and zooplankton grazing; these results highlight the need to investigate the role of selective forces in the genetic dynamics of bloom populations. The small geographic scale and limited connectivity of NMS salt ponds provide a novel system for investigating regulators of blooms, as well as the influence of selective forces on population structure, all of which are otherwise difficult or impossible to study in the adjacent open‐coastal waters or within larger estuaries.     

Toxin production in migrating dinoflagellates: a modelling study of PSP producing Alexandrium

A mechanistic model of dinoflagellate physiology, previously developed and parameterised to simulate paralytic shellfish poison (PSP) content and cell growth for Alexandrium fundyense in response to N and P nutrition, was operated within a vertical water structure in which the organism migrated. Simulations showed the expected development of vertical migration behaviour in response to light and mineral nutrient interactions. Growth in a N-limited water column resulted in a continual, though low level, PSP production with a large population biomass. A sequence of P-stress and nutrient re-feeding during vertical migration stimulated an enhancement of PSP content even with only moderately elevated supply of N:P ratios. This was exacerbated by low absolute P concentrations below the nutricline as well as by the N:P ratio. Although the final biomass was lower in these P-limited simulations, the total toxin production was much higher. The simulations suggest that vertical migration in stratified waters in even moderately high N:P waters could result in the formation of highly toxic populations of Alexandrium. One may expect a similar enhancement of toxicity in other harmful algal species that are engaged in vertical migration, where nutrient supply ratios affect toxin production.     

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.     

Intercalibration of classical and molecular techniques for identification of Alexandrium fundyense (Dinophyceae) and estimation of cell densities

A workshop with the aim to compare classical and molecular techniques for phytoplankton enumeration took place at Kristineberg Marine Research Station, Sweden, in August 2005. Seventeen different techniques – nine classical microscopic-based and eight molecular methods – were compared. Alexandrium fundyense was the target organism in four experiments. Experiment 1 was designed to determine the range of cell densities over which the methods were applicable. Experiment 2 tested the species specificity of the methods by adding Alexandrium ostenfeldii, to samples containing A. fundyense. Experiments 3 and 4 tested the ability of the methods to detect the target organism within a natural phytoplankton community. Most of the methods could detect cells at the lowest concentration tested, 100 cells L⁻¹, but the variance was high for methods using small volumes, such as counting chambers and slides. In general, the precision and reproducibility of the investigated methods increased with increased target cell concentration. Particularly molecular methods were exceptions in that their relative standard deviation did not vary with target cell concentration. Only two of the microscopic methods and three of the molecular methods had a significant linear relationship between their cell count estimates and the A. fundyense concentration in experiment 2, where the objective was to discriminate that species from a morphologically similar and genetically closely related species. None of the investigated methods were affected by the addition of a natural plankton community background matrix in experiment 3. The results of this study are discussed in the context of previous intercomparisons and the difficulties in defining the absolute, true target cell concentration.     

Does uptake of Alexandrium fundyense cysts contribute to the levels of PSP toxin found in the sea scallop, Placopecten magellanicus?

Atlantic sea scallops, Placopecten magellanicus, in most areas of the Bay of Fundy, New Brunswick, Canada, have year-round concentrations of paralytic shellfish posioning (PSP) toxins greater than the regulatory concentration of 80 μg STX eq. 100 g⁻¹ wet weight. Scallops (mean shell height of 10.7 cm, age 3–5 years) were collected by SCUBA and individually tagged near Parker Island, Bay of Fundy. Half were hung 2 m below the low tide water level and the remainder were placed on the bottom (11 m depth at low tide) under the scallops held at 2 m. Scallop, water and sediment samples were collected monthly for determination of concentrations of PSP toxins and Alexandrium fundyense.In October, 1993, mean concentrations of PSP toxins in digestive gland, and mantle were 3205 and 1018 μg STX eq. 100 g⁻¹ wet weight, respectively. Eight months later (June 1994), PSP concentrations in digestive glands from the surface and bottom had declined to 504 and 682 μg STX eq. 100 g⁻¹ wet weight, respectively, whereas those in the mantle had declined to 802 and 681 μg STX eq. 100 g⁻¹ wet weight. During July 1994, A. fundyense concentrations observed at Parker Island and offshore were 320 cells l⁻¹ and 14,200 cells l⁻¹, respectively. Subsequently, toxin concentrations in surface and bottom scallop digestive glands increased to 12,720 and 11,408 μg STX eq. 100 g⁻¹ wet weight, whereas concentrations in mantles increased to 2126 and 1748 μg STX eq. 100 g⁻¹ wet weight, respectively. Concentrations of PSP toxins in these tissues in October 1994 were similar to those measured in October 1993. Concentrations of PSP toxin were less than the regulatory concentration in the gonads and non-detectable in adductor muscles of all scallops sampled.There were no statistically significant differences in profiles for uptake and depuration of PSP toxins in scallops held at the surface compared to those from bottom, suggesting that A. fundyense cysts at the concentrations found in the sediment (45 cysts cm⁻³) did not contribute significantly to the year-round presence of PSP toxins within scallop tissues. The year-round occurrence of PSP toxin is probably due to accumulation during summer blooms followed by a very slow rate of depuration.     

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     

Alexandrium (Dinophyceae) species in Malaysian waters

A study was carried out to determine the presence of paralytic shellfish poisoning (PSP) toxin-producing dinoflagellates in the coastal waters of Peninsula Malaysia. This followed first ever occurrences of PSP in the Straits of Malacca and the northeast coast of the peninsula. The toxic tropical dinoflagellate Pyrodinium bahamense var. compressum was never encountered in any of the plankton samples. On the other hand, five species of Alexandrium were found. They were Alexandrium affine, Alexandrium leei, Alexandrium minutum, Alexandrium tamarense and Alexandrium tamiyavanichii. Not all species were present at all sites. A. tamiyavanichii was present only in the central to southern parts of the Straits of Malacca. A. tamarense was found in the northern part of the straits, while A. minutum was only found in samples from the northeast coast of the peninsula. A. leei and A. affine were found in both the north and south of the straits. Cultured isolates of A. minutum and A. tamiyavanichii were proven toxic by the receptor binding assay for PSP toxins but A. tamarense clones were not toxic. Mean toxin content for the A. tamiyavanichii and A. minutum clones were 26 and 15 fmol per cell STX equivalent, respectively. This study has provided evidence on the presence of PSP toxin-producing Alexandrium species in Malaysian waters which suggests that PSP could increase in importance in the future.     

The role of dissolved organic matter (DOM) quality in the growth enhancement of Alexandrium fundyense (Dinophyceae) in laboratory culture1

Several algal species responsible for harmful algal blooms (HABs), such as Alexandrium fundyense, are mixotrophic under certain environmental conditions. The ability to switch between photosynthetic and heterotrophic modes of growth may play a role in the development of HABs in coastal regions. We examined the influence of humic dissolved organic matter (HDOM) derived from terrestrial (plant/soil) and microbial sources on the growth of A. fundyense. We found that a terrestrially derived HDOM, Suwannee River humic acid (SRHA), did enhance A. fundyense growth; however, a microbially derived HDOM, Pony Lake fulvic acid (PLFA) did not enhance growth. A. fundyense grows in association with bacteria in culture and we observed that bacterial cell densities were much lower in A. fundyense cultures than in bacteria‐only cultures, consistent with bacterial grazing by A. fundyense in culture. In bacteria‐only cultures with added algal exudates (EX), the addition of PLFA and SRHA resulted in a slight increase in bacterial cell density compared to cultures without HDOM added. Changes over time in the chemical quality of the HDOM in the A. fundyense cultures reflected contributions of microbially derived material with similar characteristics as the PLFA. Overall, these results suggest that the chemical differences between SRHA and PLFA are responsible for the greater effect of SRHA on A. fundyense growth, and that the differential effect is not a result of an effect on the growth of associated bacteria.     

Application of real-time PCR assay for detection and quantification of Alexandrium tamarense and Alexandrium catenella cysts from marine sediments

The dinoflagellates Alexandrium tamarense (Lebor) Balech and Alexandrium catenella (Whedon and Kofoid) Balech (Dinophyceae) are believed to be the main species responsible for paralytic shellfish poisoning (PSP) all over the world. It is necessary to identify A. tamarense and A. catenella cysts and to monitor their distribution in sediment in order to minimize the damages caused by PSP to the economy and food quality because cysts are the seed population for blooms caused by motile vegetative cells. In this study, we developed an efficient DNA extraction method from the natural cysts present in marine sediments after they were size fractionated with a plankton net (mesh size of 20–150 μm). The 10–3000 cysts were added to the sediments collected from the Ariake Sea, and for which the primuline-staining method did not reveal any cysts. DNA was then extracted from each sample, and linear standard curves for A. tamarense and A. catenella cysts were obtained from the correlation between the Ct values by real-time PCR and the log of the initial densities of cysts. We monitored the A. tamarense and A. catenella cyst densities in the environmental samples. This assay was demonstrated to be a powerful tool for the identification, detection, and quantification of the cysts of the toxic dinoflagellates.     

Paralytic shellfish toxins in zooplankton, mussels, lobsters and caged Atlantic salmon, Salmo salar, during a bloom of Alexandrium fundyense off Grand Manan Island, in the Bay of Fundy

Substantial mortalities of Atlantic salmon (Salmo salar) at two aquaculture sites in Long Island Sound, off Grand Manan Island, Bay of Fundy (BoF) (New Brunswick, Canada) in September 2003, were associated with a bloom of Alexandrium fundyense (>3 × 10⁵ cells L⁻¹), a dinoflagellate alga that produces toxins which cause paralytic shellfish poisoning (PSP). Cells of A. fundyense collected from surface waters while fish were dying had total paralytic shellfish (PS) toxin concentrations of 70.6 pg STX equiv. (saxitoxin equivalents) cell⁻¹ and PS toxin profiles rich in carbamate toxins (78.2%). The zooplankton sampled contained PS toxins (63.1 pg STX equiv. g⁻¹ wet wt) and the toxin profile matched that of A. fundyense cells.Mean PS toxin levels were low (<4 μg STX equiv. 100 g⁻¹ wet wt) in stomach, gill and muscle tissues of moribund salmon, suggesting that PS toxins are very lethal to salmon.The PS toxin concentrations in blue mussels (Mytilus edulis) growing on the salmon cages (37; 526 μg STX equiv. 100 g⁻¹ wet wt) were the highest recorded to date from this region. Their PS toxin profiles showed enhanced carbamate contents (85.5%) compared with that found in A. fundyense. Blue mussels collected from an adjacent Canadian Food Inspection Agency (CFIA) monitoring site in Grand Manan had PS toxin concentrations of 4214 and 150 μg STX equiv. 100 g⁻¹ wet wt in late September and December, respectively, well above the regulatory limit (RL), and horse mussels (Modiolus modiolus) collected in late September had PS toxin concentrations of 2357 μg STX equiv. 100 g⁻¹ wet wt. Detoxification under laboratory conditions suggested that blue mussels may require up to 19 weeks for elimination below RL when they accumulate these high concentrations of PS toxins. This depuration period may be shorter in the field.PS toxin levels above RL were detected in hepatopancreatic tissues of lobster (Homarus americanus), with lower levels (<16 μg STX equiv. 100 g⁻¹ wet wt) in tail muscle and gills.These results illustrate the movement of PS toxins through the marine food chain following an A. fundyense bloom in the BoF, and support earlier studies suggesting that kills from the region of zooplanktivorous fish, such as herring (Clupea harengus harengus), can be attributed to blooms of A. fundyense. This is the first reported incident of PSP associated with mortalities of caged Atlantic salmon in the BoF. Analyses of muscle tissues and viscera from the affected salmon indicated that any portion would not be a health hazard if consumed.     

In vitro transformation of PSP toxins by different shellfish tissues

Many in vivo shellfish feeding experiments have been carried out in order to investigate the fate of PSP toxins in the marine food chain. A focal point of these studies concerns the species- and tissue-specific differences in toxin metabolism. However, tissue specific effects are often overlapped by selective toxin retention as well as transfer between individual compartiments. In in vitro experiments presented here digestive tissue and adductor homogenates of 10 shellfish species (bivalvia: Mytilus edulis, Crassostrea gigas, Cardium edule, Arctica islandica, Ensis ensis, Modiolus modiolus, Mactra stultorum, Pecten maximus as well as two snails: Littorina littorea and Buccinum undatum) were incubated with an extract of the toxic strain Alexandrium fundyense CCMP 1719. After incubation, changes in the toxin pattern could be observed in all samples with significant differences occuring between both the species and tissues. The greatest metabolic activity was found in digestive tissue samples. Among the organisms, the species with a non-filtering lifestyle, L. littorea and B. undatum, showed the highest conversion rates. Interestingly, the high metabolic transformation rate of the PSP toxins was accompanied with a fast reduction (up to 73%) of toxicity in the homogenates.     

Phycotoxin accumulation in zooplankton feeding on Alexandrium fundyense--vector or sink?

Zooplankton can consume toxic Alexandrium spp. dinoflagellatesin the Gulf of Maine and retain paralytic shellfish poisoning(PSP) toxins, potentially acting as toxin vectors. We performedexperiments to determine toxin budgets for common species ofcopepods (Acartia hudsonica, Eurytemora herdmani, Centropageshamatus) feeding on toxic Alexandrium fundyense, offered asmonocultures or in mixtures of algal prey, by comparing calculatedtoxin ingestion rates and toxin content of copepod body tissueand fecal pellets. When fed monocultures, both copepod tissueand fecal pellet fractions accounted for 5% each of the calculatedingested toxin, and thus by difference 90% was lost as a dissolvedfraction into the seawater medium. The presence of alternativefood did not significantly alter the efficiency of toxin retention.Sloppy feeding or regurgitation are probable mechanisms forrelease of toxin to sea water. Experiments using varying concentrationsof A. fundyense and alternativenon-toxic species did not showsignificant effects of cell concentration on toxin retentionefficiency. Total toxin retained and efficiency of retentionvaried among copepod species. Toxin profiles (% molar composition)of dinoflagellates, copepod tissues and fecal pellets differedslightly, suggesting some metabolic transformation. Becauseof their low retention efficiency, copepod grazers can effectivelydisperse PSP toxins produced by Alexandrium spp. into the environment,where they are much less likely to be harmful—zooplanktonact as a sink for PSP toxins. Nevertheless, sufficient toxinbody burdens are attained to contribute to propagation of PSPtoxins to other trophic levels.     

First report of paralytic shellfish poisoning (PSP) caused by Alexandrium tamiyavanichii in Kuantan Port,Pahang, East Coast of Malaysia

Harmful algal bloom (HAB) is a proliferation of algae, which naturally produce biotoxins and cause harmful effects to humans, the environment and organisms associated with it. Paralytic shellfish poisoning (PSP) was reported for the first time in Kuantan Port, Pahang, Malaysia, in November 2013, followed by a second episode in August 2014. The toxicity level reported during the second event was as high as 3500 μg of STX equiv./100 g shellfish. Ten people were hospitalized with PSP symptoms after consuming contaminated shellfish. This study was conducted at Kuantan Port to identify the organisms responsible for these events. Water samples were collected monthly for a period of 12 months beginning in September 2014. HAB species were identified based on their morphology using light and fluorescence microscopes, and their classification was supported by molecular evidence based on internal transcribed spacer (ITS) sequences. Monthly cell abundance of Alexandrium tamiyavanichii was measured at four sampling stations. Toxin production by three strains isolated from the area was determined using HPLC. Our results revealed the presence of several HAB species, including the PSP‐producing species A . tamiyavanichii . The highest cell density of A . tamiyavanichii was 840 cells L^?1. The presence of GTX components was detected in these strains. However, other toxin components could not be determined. This study reported, for the first time, the presence of PSP‐producing A . tamiyavanichii on the Pahang coast of east Peninsular Malaysia and confirmed that the PSP events in Kuantan Port were attributable to this species. The presence of this species further indicates that several safety measures need to be considered to safeguard public health, particularly in Pahang coastal waters.     

Dinoflagellate cyst production in one-liter containers

Methods for the production of dinoflagellate cysts in two types of 1 L containers have been developed. Using these methods, dinoflagellate cysts can be produced in amounts large enough for shellfish grazing experiments or whenever large amounts of cysts are needed. The species used were Scrippsiella lachrymosa (B-10) and toxic Alexandrium fundyense (CB501 and GTM25). Cultures of S. lachrymosa yielded 628 ± 74 cysts mL^–1 and A. fundyense cultures yielded 350 ± 98 cysts mL^–1. Findings suggest that aspects of the boundary layer between the media and the wall of the container are important for gamete mating; especially, the slope of the container wall appears to be relevant, which offers some explanation of previous observations that the shape of the container is important in the formation of dinoflagellate resting cysts. These observations may support the theory that physical interfaces in nature facilitate dinoflagellate encystment.     

Effects of increasing temperature and acidification on the growth and competitive success of Alexandrium catenella from the Gulf of Maine

Climate driven increases in ocean temperature and pCO₂ have the potential to alter the growth and prevalence of future Harmful Algal Blooms (HABs), but systematic studies on how climate drivers influence toxic algal species relative to non-toxic phytoplankton are lacking. In particular, little is known about how future climate scenarios will affect the growth of the toxic dinoflagellate Alexandrium catenella, which is responsible for the paralytic shellfish poisoning (PSP) events that threaten the health and economy of coastal communities in the Gulf of Maine and elsewhere. The growth responses of A. catenella and two other naturally co-occurring dinoflagellates in the Gulf of Maine—Scrippsiella sp., and Amphidinium carterae—were studied in mono and mixed species cultures. Experimental treatments tested the effects of elevated temperature (20 °C), lower pH (7.8), and the combination of elevated temperature and lower pH on growth rates relative to those in near-current conditions (15 °C; pH 8.1). Growth rates of A. catenella decreased under elevated temperature and lower pH conditions, a response that was largely attributable to the effect of temperature. In contrast, growth rates of Scrippsiella sp. and A. carterae increased under elevated temperature and lower pH conditions, with temperature also being the primary driver of the response. These trends did not change substantially when these species were grown in mixed cultures (A. catenella + Scrippsiella sp., and A. catenella + A. carterae), indicating that allelopathic or competitive interactions did not affect the experimental outcome under the conditions tested. These findings suggest that A. catenella blooms may become less prevalent in the southern regions of the Gulf of Maine, but potentially more prevalent in the northeastern regions of the Gulf of Maine with continued climate change.     

Toxin profile of Alexandrium catenella from the Chilean coast as determined by liquid chromatography with fluorescence detection and liquid chromatography coupled with tandem mass spectrometry

The profile of tetrahydropurine neurotoxins associated with paralytic shellfish poisoning (PSP) was determined from a Chilean strain of the marine dinoflagellate Alexandrium catenella. The toxin composition was compared with that of toxic shellfish, presumably contaminated by natural blooms of A. catenella from the same region in southern Chile. Ion pair-liquid chromatography with post-column derivatization and fluorescence detection (LC-FD) was employed for relative quantitative analysis of the toxin components, whereas unambiguous identification of the toxins was confirmed by tandem mass spectrometry (LC–MS/MS). In the dinoflagellate strain from Chile, the N-sulfocarbamoyl derivatives (C1/C2, B1) and the carbamoyl gonyautoxins GTX1/GTX4 comprise >90% of the total PSP toxin content on a molar basis. This toxin composition is consistent with that determined for A. catenella populations from the Pacific coast in the northern hemisphere. The characteristic toxin profile is also reflected in the shellfish, but with evidence of epimerization and metabolic transformations of C1 and C2 to GTX2 and GTX3, respectively. This work represents the first unequivocal identification and confirmation of such PSP toxin components from the Chilean coast.     

A novel approach to identifying PST tolerant copepods: An individual ingestion assay

Phenotypic tolerance of individual copepods to paralytic shellfish toxins (PSTs), as determined by fecundity, has recently been reported. In the present study, we tested whether similar phenotypic tolerance related to ingestion also exists. In short-term feeding assays, ingestion rates of individual females of the copepod Acartia hudsonica were measured on a sole diet of toxic Alexandrium fundyense as well as on a sole diet of nontoxic Alexandrium tamarense. When copepods fed on A. fundyense, the frequency distribution of ingestion rates was polymodal, consistent with the hypothesis of levels of phenotypic tolerance associated with toxin ingestion. Four distinct groups of ingestion rates on toxic algae were observed. Mean rates ranged from near zero to more than 100 cells copepod⁻¹ h⁻¹. In contrast, when the same individuals fed on the nontoxic A. tamarense diet, the polymodal distribution of ingestion rates was not apparent. Furthermore, group-specific mean ingestion rates, for two ingestion-defined groups, were always significantly greater on the toxic diet than they were on the nontoxic diet. These results support the hypothesis that discrete groups of grazer ingestion of toxic A. fundyense are related to PST tolerance.     

Quantification methods for Alexandrium catenella, a toxic dinoflagellate: Comparison of competitive enzyme-linked immunosorbent assay and sandwich hybridization integrated with a nuclease protection assay

Alexandrium catenella (Whedon et Kofoid) Balech, a toxic dinoflagellate, is a bloom-forming planktonic species in cold water coastal regions. It produces strong paralytic shellfish poisoning (PSP) toxins which are transmitted via tainted shellfish. These toxins can affect humans, other mammals, fish and birds. In this study, polyclonal antiserum against A. catenella was produced, and a competitive enzyme-linked immunosorbent assay (cELISA) was developed to detect A. catenella. The antiserum against A. catenella showed good specificity, the linear detection range was relatively large, between 38 and 600,000 cells. In addition, specific probes were designed to target the small subunit ribosomal RNA (SSU rRNA) of A. catenella, and quantitative sandwich hybridization integrated with a nuclease protection assay (NPA-SH) was established in order to identify and quantify A. catenella. The NPA-SH assay did not show good specificity as well as cELISA, by which A. catenella and A. tamarense could not be distinguished. Samples in different cell growth phases were analyzed with cELISA and NPA-SH. The results showed that the cell concentration calculated by cELISA was very similar with microscopy, while that of NPA-SH was sometimes higher than that of microscopy, especially in log phase. Comparing the two methods, both assays allow rapid identification of A. catenella without time-consuming microscopy when multiple sites need to be considered in routine monitoring. Meanwhile, cELISA was more specific and accurate in detection of A. catenella than NPA-SH.