Effects of nutrient limitation on toxin production and composition in the marine dinoflagellate Protogonyaulax tamarensis

Toxin production was measured by high pressure liquid chromatography (HPLC) when the marine dinoflagellate Protogonyaulax tamarensis (NEPCC 255) was grown under nitrogen or phosphorus limitation. The major toxins found in P. tamarensis (255) consisted of (N21-SO ₃ ^- )STX (11%), (N21-SO ₃ ^- )NeoSTX (44%), and [(N21-SO ₃ ^- )GTX₂ plus (N21-SO ₃ ^- )GTX₃] (20%). Total toxin content on a per cell basis was high for cultures in log phase (30 to 40 fmol cell^-1) and then decreased to ca 20 fmol cell^-1 as the cultures entered stationary phase. There was a gradual decrease in the toxin content per cell during nitrogen-limited stationary phase to ca 3 fmol cell^-1 or less. Phosphorus-limited cultures showed a markedly different response than nitrogen-limited cultures. Toxin content in P-limited cells dramatically increased at the start of stationary phase, reaching levels 3 to 4 times that observed in control and nitrogen-limited cultures. These results cannot be explained by changes in the average cell volume. Eventhough dramatic effects on the total toxin concentration were observed in response to nutrient limitation (N or P), the toxin composition (on a percent basis) remained constant. This suggests that the individual toxin composition of a given isolate is a fixed genetic trait and not a transient response to changing environmental factors.     

Toxin accumulation and feeding behaviour of the planktonic copepod Calanus finmarchicus exposed to the red-tide dinoflagellate Alexandrium excavatum

The planktonic copepod Calanus finmarchicus is a dominant member of the zooplankton community in the lower St. Lawrence Estuary in eastern Canada. Blooms of the toxic marine dinoflagellate Alexandrium excavatum which produces high cellular levels of paralytic shellfish poisoning (PSP) toxins, occur during the period of high C. finmarchicus production in summer in this region. To study the feeding behaviour of C. finmarchicus in the presence of Alexandrium spp., experiments were conducted in which female adult copepods collected from the St. Lawrence Estuary between May and September 1991 were exposed under controlled conditions to two toxic isolates of A. excavatum (Pr18b and Pr11f) from the estuary and to a non-toxic control (PLY 173) of a closely related species, A. tamarense isolated from the Tamar Estuary, Plymouth, U.K. Clearance rates on non-toxic A. tamarense cells averaged 5.5 ml ind^-1 h^-1 but were nearzero with either toxic isolate. When presented with a mixture of A. excavatum and the non-toxic diatom Thalassiosira weissflogii in varying proportions, C. finmarchicus fed upon the diatom but avoided the toxic dinoflagellate. Although feeding rates on A. excavatum were very low, toxin analysis by high-performance liquid chromatography with fluorescence detection (HPLC-FD) revealed that the PSP toxins were accumulated in copepods exposed to toxigenic dinoflagellates.The toxin composition in copepods was similar to that of the toxic dinoflagellate, but not necessarily identical, particularly after short-term (2-h) exposure, when relatively elevated levels of N-sulfocarbamoyl toxins were detected. The evidence suggests that C. finmarchicus ingests toxic dinoflagellate cells, either mistakenly or during exploratory bouts of feeding, and accumulates PSP toxins in its gut system and perhaps in other tissues.     

<Emphasis Type="Italic">Alexandrium catenella</Emphasis> (Dinophyceae), a toxic ribotype expanding in the NW Mediterranean Sea

The presence of the paralytic shellfish poisoning (PSP) dinoflagellate Alexandrium catenella in the north western (NW) Mediterranean Sea has been known since 1983. From this date on, the species has spread along the Spanish and Italian coastlines. Information concerning A. catenella isolates in the NW Mediterranean Sea was gained through phylogenetic studies. Twenty established toxic cultures of A. catenella taken from various NW Mediterranean Sea locations were analysed by nucleotide sequencing of the 5.8S rDNA and internal transcribed spacer regions. These rDNA ribosomal markers resulted useful in delineating the phylogenetic position of this species in the genus Alexandrium as well as in determining relationships between A. catenella isolates from different geographic areas. The phylogenetic position of the Mediterranean A. catenella ribotype, when compared to the “Alexandrium tamarense/catenella/fundyense species complex”, fits this species complex well. All the Mediterranean A. catenella isolates were constituted by only one genetic ribotype. By comparing the isolate sequences with those of other geographic areas, it revealed that the Mediterranean A. catenella ribotype was closely related to the A. catenella from Japan, Western Pacific Ocean. It was also evident that in temperate Japanese waters, a genetic variability was detected within A. catenella isolates; in fact, all strains resulted divergent showing as many as 15 mutational steps. The possibility that A. catenella has been recently introduced into the Mediterranean basin from temperate Asian areas is discussed.     

An integrative response by Mytilus chilensis to the toxic dinoflagellate Alexandrium catenella

Physiological responses of Mytilus chilensis exposed to the toxic dinoflagellate Alexandrium catenella were measured over 21 days in the laboratory and were compared with control mussels not exposed to the dinoflagellate. Mussels were collected from culturing ropes at Yaldad Bay, southern Chile (43o08′S 73o44′W), in August 2004 and acclimated to laboratory conditions for one week prior to the experiment. After 8 days, the paralytic shellfish poisoning (PSP) toxins (i.e. saxitoxin) in the tissues of exposed mussels exceeded safe levels for human consumption. Clearance rates, ingestion of organic matter, and absorption efficiency of exposed mussels were significantly lower than those of controls on day 0, but this was followed by an increase on day 3. The exposed mussels also increased their excretion rate over time, and this increase was significantly correlated with the accumulation of PSP toxins in their tissues. Oxygen consumption was not affected by the PSP toxins. The scope for growth (SFG) on day 0 was negative in exposed mussels, but it increased during the experiment. Although feeding activity and absorption efficiency were adversely affected during the first few days of exposure to PSP toxins from A. catenella in the laboratory, the M. chilensis cultured in Yaldad Bay may have evolved mechanisms that allow them to exploit the toxic dinoflagellate as a food source.     

Accumulation of paralytic shellfish poisoning toxins in planktonic copepods during a bloom of the toxic dinoflagellate<Emphasis Type="Italic"> Alexandrium tamarense</Emphasis> in Hiroshima Bay,western Japan

Concentrations of paralytic shellfish poisoning (PSP) toxins in toxic dinoflagellate cells and in marine planktonic copepods were monitored during the bloom of Alexandrium tamarense in Hiroshima Bay, western Japan. Concentration of the toxins retained by copepods was a function of the ambient toxin concentration, i.e. the product of A. tamarense cell density and cellular toxicity. The toxin concentration in copepods increased with the increase of toxicants in the seawater then leveled off, but decreased significantly at higher concentrations. In the field, the maximum toxin concentration was 1.2 pmol ind^-1, whereas in the laboratory, the copepod Acartia omorii accumulated a much higher concentration of PSP toxins (24 pmol ind^-1). Feeding avoidance against Alexandrium tamarense and a shift to alternative food sources such as diatoms in the field might keep their toxin levels lower than their potentially maximum level. The copepod toxin levels in the field were not so high as to cause an instantaneous lethal effect on their predator fishes but may reach possibly lethal levels after a few days' continuous feeding. Overall toxin retention by copepods after 12 h feeding and 2 h starvation was only 2.5% of total ingested toxins, which suggested that a significant amount of toxins was released into the seawater. Measurements of toxin reduction and gut evacuation suggested that the toxins were removed through both fecal evacuation and metabolism (e.g. excretion, decomposition and transformation). The results, as a whole, imply that copepods can be a link for PSP toxin flux in both pelagic and benthic food webs and can also be a sink for toxins by metabolizing and removing them from the environment.Communicated by T. Ikeda, Hakodate     

Toxic dinoflagellates (Alexandrium fundyense and A. catenella) have minimal apparent effects on oyster hemocytes

The possible effect of Alexandrium spp. containing paralytic shellfish poisoning (PSP) toxins on the hemocytes of oysters was tested experimentally. In one trial, eastern oysters, Crassostrea virginica Gmelin, were exposed to bloom concentrations of the sympatric dinoflagellate, Alexandrium fundyense Balech, alone and in a mixture with a non-toxic diatom, Thalassiosira weissflogii (Grun) Fryxell et Hasle. Subsequently, another experiment exposed Pacific oysters, Crassostrea gigas Thunberg, to a mixed suspension of the sympatric, toxic species Alexandrium catenella (Whedon et Kofoid) Balech, with T. weissflogii. Measurements of numbers of oyster hemocytes, percentages of different cell types, and functions (phagocytosis, reactive oxygen species (ROS) production, and mortality) were made using flow-cytometry. During and after exposure, almost no significant effects of Alexandrium spp. upon hemocyte numbers, morphology, or functions were detected, despite observations of adductor-muscle paralysis in C. virginica and measured toxin accumulation in C. gigas. The only significant correlation found was between toxin accumulation at one temperature and higher numbers of circulating live and dead hemocytes in C. gigas. The PSP toxins are known to interfere specifically with sodium-channel function; therefore, the finding that the toxins had no effect on measured hemocyte functions suggests that sodium-channel physiology is not important in these hemocyte functions. Finally, because oysters were exposed to the living algae, not purified toxins, there was no evidence of bioactive compounds other than PSP toxins affecting hemocytes in the two species of Alexandrium studied.     

Paralytic shellfish poison in Spisula solidissima: Anatomical location and ozone detoxification

The surf clam Spisula solidissima, when exposed to a northern bloom of the toxic dinoflagellate Gonyaulax tamarensis, concentrates paralytic shellfish poison (PSP) and retains it for periods of over 1 year. The purpose of this investigation was to identify those tissues in which S. solidissima concentrates PSP and to examine the efficacy of ozone gas in PSP detoxification. Various levels of the toxin were found in every untreated tissue examined: the mantle and gill containing high concentrations (>1600 g/100 g tissue); the visceral mass, siphon, and foot showing less toxicity (1100 to 200 g/100 g tissue); and the adductor muscle yielding a level of toxin considered safe for human consumption (<60 g/100 g tissue). Toxic clams exposed to ozonized seawater for 2 weeks exhibited rapid detoxification in all tissues examined.This work was supported, in part, by a grant from the Massachusetts Science and Technology Foundation, Wakefield, Massachusetts 01880, USA.     

Paralytic shellfish poison in the “hiogi” scallop Chlamys nobilis

Attempts were made to analyze the toxin composition of the toxic hiogi scallop Chlamys nobilis. The toxins were partially purified from the digestive glands by column chromatography using Bio-Gel P-2 and Bio-Rex 70 (H⁺ form), resulting in separation into protogonyautoxin (PX), gonyautoxin (GTX) and saxitoxin (STX) fractions. Their total potencies were scored to be <100 mouse units (MU), 3200 MU and 3700 MU, respectively. A 5-min hydrolysis with 0.1 N HCl enhanced the potencies of PX and GTX fractions to 450 MU and 12000 MU respectively, whereas no enhancement occurred in the STX fraction at all. Electrophoretic, thin-layer chromatographic and high performance liquid chromatographic analyses demonstrated that the PX fraction consisted mainly of GTX₈ and its epimer, the GTX fraction of GTX₅, GTX₆, along with two unidentified toxins, and the STX fraction exclusively of two unidentified toxins. This rather unique composition suggested a complex metabolism of PSP in this species.     

Dissolved saxitoxin causes transient inhibition of sensorimotor function in larval Pacific herring (<Emphasis Type="Italic">Clupea harengus pallasi</Emphasis>)

Herring (Clupea harengus pallasi) spawning sites in Puget Sound, Washington overlap spatially and temporally with blooms of Alexandrium catenella, a toxic dinoflagellate species responsible for paralytic shellfish poisoning. Consequently, newly hatched herring larvae may be regularly exposed to the suite of dissolved paralytic shellfish toxins that are released into the water column from toxic cells during blooms. To date, virtually nothing is known about the impacts of these neurotoxins on early developmental stages of marine fish. In the present study, herring larvae at three ages, 0 days post hatch (dph), 4 dph, and 11 dph, were exposed to dissolved saxitoxin (STX) in 24-h and multi-day exposures. All larvae were examined for sensorimotor function (i.e. spontaneous swimming behavior and touch response). Significant reductions in spontaneous and touch-activated swimming behavior occurred within 1 h of exposure. EC₅₀s at 1 h of exposure were 1,500, 840, and 700 μg STX equiv. l⁻¹ for larvae introduced to STX at 0, 4, and 11 dph, respectively. This progressive age-specific increase in STX-induced paralysis suggests that older larvae were more sensitive to the toxin than younger larvae. Interestingly, herring larvae at all ages exhibited a significant degree of neurobehavioral recovery within 4–24 h of continuous exposure relative to the 1-h time point. This recovery of normal motor behaviors was not observed in previous studies with freshwater zebrafish (Danio rerio) larvae under the same continuous exposure conditions, suggesting that an adaptive detoxification or toxin sequestration mechanism may have evolved in some species of marine fish larvae. Our data reveal that (1) dissolved STX is bioavailable to marine finfish larvae, (2) the toxin is a paralytic agent with potencies that differ between developmental stages, and (3) STX-induced sensorimotor inhibition occurs rapidly but is transient in marine larvae. Collectively, these results suggest that dissolved algal toxins may have important sublethal effects on marine fish populations.     

Effects of the toxic dinoflagellate <Emphasis Type="Italic">Gymnodinium catenatum</Emphasis> on uptake and fate of paralytic shellfish poisons in the Pacific giant lions-paw scallop <Emphasis Type="Italic">Nodipecten subnodosus</Emphasis>

Juvenile Pacific giant lions-paw scallops Nodipecten subnodosus were fed the toxic dinoflagellate Gymnodinium catenatum, a producer of paralytic shellfish poison (PSP), supplied with Isochrysis galbana (a nontoxic microalgae). Short-term (<24 h) experiments were performed to determine clearance and ingestion rates of G. catenatum. Kinetics of PSP was examined in longer-term experiments (>2 days). At high food concentrations, juvenile scallops showed production of pseudofeces, partial shell valve closure, and reduction in feeding. According to HPLC analysis, the only toxins present in the dinoflagellate G. catenatum and in the scallops were the gonyautoxins (GTXs), except in the labial palps and digestive gland, where trace amounts of saxitoxin (STX) were present in scallops. These tissues could play an important role in toxin biotransformation. The ranking of toxin concentration in tissues was: digestive gland > labial palps > intestine > gills > mantle > adductor muscle, where the total contribution of viscera was more than 80% of the total toxin body burden. Juvenile scallops exhibited no apparent detrimental physiological responses during the long-term feeding experiment. The dinoflagellate may contribute nutrients to the scallop, in addition to the microalgae I. galbana. The dinoflagellate may enhance cell uptake and byssus production. Once PSP accumulated during the first 12 days, it was slowly eliminated. The limited capacity for accumulating toxins in the adductor muscle favors domestic marketing of scallops.     

Dynamics and physiology of saxitoxin production by the dinoflagellatesAlexandrium spp.

Toxin content (fmol cell^–1) and a suite of elemental and macromolecular variables were measured in batch cultures of the dinoflagellatesAlexandrium fundyense, A. tamarense andAlexandrium sp. from the southern New England region, USA. A different perspective was provided by semicontinuous cultures which revealed sustained, steady-state physiological adaptations by cells to N and P limitation. Two types of variability were investigated. In batch culture, changes in nutrient availability with time caused growth stage variability in toxin content, which often peaked in mid-exponential growth. A second type of variability that could be superimposed on growth stage differences is best exemplified by the high toxin content of cells grown at suboptimal temperatures. Calculations of the net rate of toxin production (R _tox ; fmol cell^–1 d^–1) for these different culture treatments and modes made it possible to separate the dynamics of toxin production from cell division. Over a wide range of growth rates, cells produced toxin at rates approximating those needed to replace losses to daughter cells during division. The exception to this direct proportionality was with P limitation, which was associated with a dramatic increase in the rate of toxin production as cells stopped dividing due to nutrient limitation in batch culture. Growth stage variability in batch culture thus reflects small imbalances (generally within a factor of two) between the specific rates of toxin production and cell division. N limitation and CO₂ depletion both affect pathways involved in toxin synthesis before those needed for cell division; P limitation does the opposite. The patterns of toxin accumulation were the same as for major cellular metabolites or elemental pools. The highest rates of toxin production appear to result from an increased availability of arginine (Arg) within the cell, due to either a lack of competition for this amino acid from pathways involved in cell division or to increased de novo synthesis. There were no significant changes in toxin content with either acclimated growth at elevated salinity, or with short term increases or decreases of salinity. These results demonstrate that toxin production is a complex process which, under some conditions, is closely coupled to growth rate; under other conditions, these processes are completely uncoupled. Explanations for the observed variability probably relate to pool sizes of important metabolites and to the differential response of key biochemical reactions to these pool sizes and to environmental conditions.     

Presence of Alexandrium catenella and paralytic shellfish toxins in finfish, shellfish and rock crabs in Monterey Bay, California, USA

The central California coast is a highly productive, biodiverse region that is frequently affected by the toxin-producing dinoflagellate Alexandrium catenella. Despite the consistent presence of A. catenella along our coast, very little is known about the movement of its toxins through local marine food webs. In the present study, we investigated 13 species of commercial finfish and rock crabs harvested in Monterey Bay, California for the presence of paralytic shellfish toxins (PSTs) and compared them to the presence of A. catenella and PSTs in sentinel shellfish over a 3-year period. Between 2003 and 2005, A. catenella was noted in 55% of surface water samples (n = 307) and reached a maximum concentration of 17,387 cells L⁻¹ at our nearshore site in Monterey Bay. Peak cell densities occurred in the month of July and were associated with elevated shellfish toxicity in the summers of 2004 and 2005. When A. catenella was present, particulate PSTs were detected 71% of the time and reached a maximum concentration of 962 ng STXeq L⁻¹. Of the 13 species tested, we frequently detected PSTs in Pacific sardines (Sardinops sagax; maximum 250 μg STXeq 100 g⁻¹), northern anchovies (Engraulis mordax; maximum 23.2 μg STXeq 100 g⁻¹), brown rock crabs (Cancer antennarius; maximum 49.3 μg STXeq 100 g⁻¹) and red rock crabs (C. productus; 23.8 μg STXeq 100 g⁻¹). PSTs were also present in one sample of Pacific herring (Clupea pallas; 13.3 μg STXeq 100 g⁻¹) and one sample of English sole (Pleuronectes vetulus; 4.5 μg STXeq 100 g⁻¹), and not detected in seven other species of flatfish tested. The presence of PSTs in several of these organisms reveals that toxins produced by A. catenella are more prevalent in California food webs than previously thought and also indicates potential routes of toxin transfer to higher trophic levels. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Cell cycle and toxin production in the benthic dinoflagellate Prorocentrum lima

Profiles of diarrhetic shellfish poisoning (DSP) toxins produced throughout the growth cycle and the cell cycle of the toxigenic marine dinoflagellate Prorocentrum lima were studied in triplicate unialgal batch cultures. Cells were pre-conditioned at 18 ± 1 °C, under a photon flux density (PFD) of 90 ± 5 μmol m⁻² s⁻¹ on a 14 h light:10 h dark photoperiod. In exponential growth phase, cultures were synchronized in darkness for 17 d. After dark synchronization, cultures were transferred back to the original photoperiod regime. Cells were harvested for DSP toxin analysis by LC-MS (liquid chromatography with mass spectrometry), and double-stranded (nuclear) DNA was quantified by flow cytometry. The cell populations became asynchronous within approximately 3 d after transition from darkness to the 14 h light:10 h dark photoperiod. This may be due to the prolonged division cycle (5 to 7 d) that is not tightly phased by the photoperiod. Unlike other planktonic Prorocentrum spp., cytokinesis in P. lima occurred early in the dark and ceased by “midnight”. Cellular levels of the four principal DSP toxins, okadaic acid (OA), OA C8-diol-ester (OA-D8), dinophysistoxin-1 (DTX1) and dinophysistoxin-4 (DTX4), ranged from 0.37 to 6.6, 0.02 to 1.5, 0.04 to 2.6, and 1.8 to 7.8 fmol cell⁻¹, respectively. No toxin production was evident during the extended period of dark synchronization nor during the initial period when NH₄ was consumed as the major nitrogen source. Soon after the cells were returned to the 14 h light:10 h dark cycle and they began to take up NO₃, cellular levels of all four toxins gradually increased. This increase in DSP toxins usually occurred in the light, marked by a rise in DTX4 levels that preceded an increase in the cellular concentration of OA and DTX1 (delayed by 3 to 6 h). Thus, DTX4 synthesis is initiated in the G1 phase of the cell cycle and persists into S phase (“morning” of the photoperiod), whereas OA and DTX1 production occurs later during S and G2 phases (“afternoon”). No toxin production was measured during cytokinesis, which happened early in the dark. The evidence indicates that toxin synthesis is restricted to the light period and is coupled to cell cycle events. Received: 3 September 1998 / Accepted: 30 March 1999     

Deleterious effects of a nonPST bioactive compound(s) from <Emphasis Type="Italic">Alexandrium tamarense</Emphasis> on bivalve hemocytes

The known negative effects of shellfish toxin-producing dinoflagellates on feeding, burrowing and survival of some bivalve mollusks has prompted questions concerning whether they might also impair the internal defense system of affected bivalves and make them more susceptible to disease agents. The primary components of the cellular defense system are hemocytes. Many toxic dinoflagellates are too large to be ingested whole by hemocytes and would most likely be exposed to intracellular toxins only after the algae are consumed, broken down, and the water-soluble toxins, released. Therefore, we conducted a series of experiments in which hemocytes from two suspension-feeding bivalves—the Manila clam, Ruditapes philippinarum, and the softshell clam, Mya arenaria—were exposed in vitro to filtered extracts of one highly toxic paralytic shellfish toxin (PST)-producing and one nonPST-producing strain of Alexandrium tamarense (isolates PR18b, 76 ± 6 STXeq cell⁻¹ and CCMP115, with undetectable PST, respectively). We measured adherence and phagocytosis, two hemocyte attributes known to be inhibited by bacterial pathogens and other stressors. We found no measurable effect of a cell-free extract from a highly concentrated suspension of the PST-producing strain on hemocytes of either bivalve species. Instead, extract from the nonPST-producing strain had a consistent negative effect on both clams, resulting in significantly lower adherence and phagocytosis compared to strain PR18b and filtered seawater controls. The bioactive compound produced by strain CCMP115, which has yet to be characterized, may be similar to the PST-independent allelopathic compounds described for Alexandrium spp., which act on other plankters. These compounds and those produced by other harmful algae are known to cause immobilization, cellular deformation and lysis of co-occurring target organisms. Thus, nonPST producing Alexandrium spp., which do not cause paralysis and burrowing incapacitation of clams, may still produce a compound(s) that has negative effects not only on hemocytes, but on other molluscan cell types and their functions, as well.     

Field depuration and biotransformation of paralytic shellfish toxins in scallop<Emphasis Type="Italic"> Chlamys nobilis</Emphasis> and green-lipped mussel<Emphasis Type="Italic"> Perna viridis</Emphasis>

Under laboratory conditions, the scallop Chlamys nobilis and the mussel Perna viridis were exposed to N-sulfocarbamoyl toxins (C2 toxin), a paralytic shellfish toxin (PST), by feeding a local toxic strain of the dinoflagellate Alexandrium tamarense (ATDP) that produced C2 toxin exclusively. The bivalves were subsequently depurated in the field, and their depuration kinetics, biotransformation and toxin distribution were quantified. Depuration was characterized by a rapid loss within the first day, followed by a secondary slower loss of toxins. In the fast depuration phase, scallops detoxified PSTs more quickly than the mussels (depuration rate constants for scallops and mussels were 1.16 day^–1 and 0.87 day^–1, respectively). In contrast, the mussels detoxified PSTs more quickly than the scallops in the slow depuration phase, and the calculated depuration rate constants (mean+SE) from day 2 to day 13 were 0.063+0.009 day^–1 and 0.040+0.019 day^–1 for mussels and scallops, respectively. The differences in the appearances of gonyautoxins, GTX2 and GTX3, and their decarbamoyl derivatives, dcGTX2, dcGTX3 and GTX5, which are all derivatives of C2 toxin, indicated active and species-specific biotransformation of the algal toxins in the two bivalves. In both species of bivalves, the non-viscera tissue contained fewer toxins and lower concentrations than the viscera-containing tissue compartment. In scallops, very little toxin was distributed in the adductor muscle. In mussels, most of the PSTs were found in the digestive gland with significant transport of toxins into the digestive gland from other tissues during the course of depuration. The toxin profiles of scallops and mussels differed from each other and from that of the toxic algae fed. A significant fraction of GTX5 was detected in the mussels but not in the scallops. Our study demonstrates a species specificity in the depuration kinetics, biotransformation and tissue distribution of PSTs among different bivalves.Communicated by T. Ikeda, Hakodate     

Biogeography of toxic dinoflagellates in the genusAlexandrium from the northeastern United States and Canada

Twenty-eight strains of toxic dinoflagellates in the genusAlexandrium from the northeastern United States and Canada were characterized on the basis of morphology, bioluminescence capacity, mating compatibility, and toxin composition. The distributions of these characters were evaluated in the context of regional patterns of paralytic shellfish poisoning (PSP) and coastal hydrography. Two morphospecies were identified-A. tamarense Lebour andA. fundyense Balech. The two are interspersed geographically though there are areas, such as the Gulf of Maine, where apparently onlyA. fundyense occurs. Southern waters (Cape Cod, Connecticut, and Long Island) have especially diverse populations. The two species are sexually compatible. Virtually all northern isolates are bioluminescent, whereas southern isolates include bioluminescent and non-bioluminescent strains. Cluster analyses, based on high performance liquid chromatography (HPLC) determinations of the suite of toxins produced by each isolate, revealed two and perhaps three distinct groups. One is comprised almost exclusively of northern strains, and the other of southern strains. A Cape Cod cluster may be separable from the southern group. These analyses explain a previously reported north-to-south trend of decreasing toxicity, as the northern isolates produce greater proportions of the more potent toxins than do southern forms. The overall perspective is that the biogeography of toxicAlexandrium spp. in the study region is not that of a single, widespread, homogeneous population, but rather is comprised of several sub-populations, each with its own physiological characteristics and history. Two scenarios are considered with respect to this regional biogeography. The first invokes recent and continuing dispersal of isolates to the south from a center of origin in the north, followed by recombination and strong selection. The second holds that the northern and southern populations diverged from a common ancestor (vicariance), but now represent localized populations with little mixing of genotypes. Neither hypothesis can be completely refuted by the data presented here, though the weight of the evidence favors the latter. The correct scenario may be a combination of both, with recent and continuous speading occuring within the Gulf of Maine and perphaps the Gulf of St. Laerence, but with endemic localized populations persisting without genetic exchange in most southern locations. These data also indicate that although morphological criteria separate toxicAlexandrim isolates from the study region into two morphospecies, these assignments do not coincide with clusterings based on toxin composition or allozyme electrophoresis, and they are further violated by mating results. A revision of taxon designations to the varietal level could be justified.     

Light-shade adaptation by the oceanic dinoflagellates Pyrocystis noctiluca and P. fusiformis

Species-specific rates of photosynthetic carbon uptake (P), chlorophyll a content and P versus irradiance (P-I), have been measured for cells of Pyrocystis noctiluca and P. fusiformis isolated from natural populations collected in the euphotic zone within and below the surface mixed layer in the Sargasso Sea. These same measurements and the assay for ribulose bis-phosphate carboxylase (RuBP-Case), have been made for cultures of P. noctiluca in a 12 h L: 12 h D photoperiod at 9 different constant or at changing light intensities. In nature chl a cell^-1 was constant throughout the euphotic zone. The photosynthetic capacity (P_max), of cells captured below the surface mixed layer was lower by a factor of 10 compared with cells collected from the surface mixed layer. The P_max for P. noctiluca collected and incubated within the surface mixed layer was the same as for cell cultures grown under high light, non nutrient-limiting conditions, suggesting that photosynthesis in the natural system was not nutrient limited. In laboratory cultures under constant low light intensities, chl a cell^-1 increased by a factor of 5 while both P_max and RuBPCase activity decreased by a factor of ca 4 compared with high light intensities. In changing light intensities both P_max and RuBPCase activities were decreased by factors of 4 during low light intervals while chl a cell^-1 approached a constant intermediate value. The change in chl a cell^-1 in response to prolonged exposure to constant low light intensities was first order with a rate constant of 0.33 d^-1. For all irradiance conditions in culture, the P-I dependence could be described by the simple Michaelis-Menten formula. The ratio of P_max to K_I, (the light intensity where P=P_max/2) was a constant with a Coefficient of Variation of 12%: The constancy of this ratio, the parallel changes in RuBPCase activity with P_max and the constant chl a cell^-1 in the Sargasso Sea imply that for P. noctiluca and presumably P. fusiformis in nature, a dark enzymatic step rather than changes in photosynthetic pigment concentrations may regulate the photosynthetic capacity in the changing photic environment.Contribution no. 1141 from McCollum-Pratt Institute and Department of Biology, The Johns Hopkins University. Supported by DOE contract no. EY 76S20 3278, NSF no. OCE 76-02571 and ONR no. N300014-81-C-0062     

Ichthyotoxicity found in cultured media of Protogonyaulax spp.

Ichthyotoxicity was observed in the cultured media from Protogonyaulax catenella and P. tamarensis. No paralytic shellfish poison was detected in the cultured media. Test fish in the cultured media died showing signs of hypoxia. Histological observations of the gills of intoxicated fish indicated that the epithelial cells were swollen and exfoliated from the pillar cell. The cultured media had hemolytic activity against erythrocytes of various animals. The media also decreased the elasticity of eggs of rainbow trout Salmo gairdneri. From these results, it is concluded that the cultured media of both species of Protogonyaulax contain a cytotoxin which is lethal to fish.     

Feeding and absorption of the toxic dinoflagellate Alexandrium tamarense by two marine bivalves from the South China Sea

Paralytic shellfish poisoning (PSP) toxins can be accumulated by bivalves through the feeding process; therefore, knowledge on feeding and the assimilation of PSP-toxin-containing algae is critical to understand the kinetics of PSP toxins in these bivalves. In the South China Sea, it has been documented that the scallop Chlamys nobilis has a much higher PSP toxin burden than the clam Ruditapes philippinarum. Experiments were therefore carried out to assess whether the difference in toxin burden between these two species of bivalves was due to differences in feeding and absorption. In a mixed diet of Alexandrium tamarense (a PSP-toxin-producing dinoflagellate) and Thalassiosira pseudonana (a non-toxic diatom), the maximum clearance and filtration rates were about two times higher in the scallop C. nobilis than in the clam R. philippinarum. Furthermore, the clams produced pseudofeces at a lower cell density than the scallops. However, we found that the clams were unable to selectively exclude the toxic dinoflagellates by pseudofeces production. The scallop C. nobilis also possessed a greater ability to assimilate A. tamarense with a comparable carbon absorption efficiency to the diatom T. pseudonana. In contrast, the carbon absorption in the clam R. philippinarum was lower when feeding on A. tamarense than on the diatom. In general, the absorption efficiency decreased with increasing concentration of A. tamarense. Thus, it is likely that the higher PSP toxin levels in the scallops compared with clams can be partly explained by differences in their feeding and absorption behavior. Other processes, especially the biotransformation and biokinetics of PSP toxins, may also play a significant role in defining the inter-species differences in PSP body burden in marine bivalves.     

The chelation of metal ions by the acylpolyamine toxins from the web-spider Nephilengys cruentata: effects in the intoxication/detoxification of preys

Summary. Orb-web-spiders present a series of different strategies for prey capture, involving the use of different types of silk for web building, the use of adhesive traps in the webs, the secretion of toxic compounds to the spider’s preys in the adhesive coating of the capture web and the biosynthesis of a wide range of structurally related acylpolyamine toxins in their venoms. The polyamine toxins usually block neuromuscular junctions and/or the central nervous system (CNS) of Arthropods, targeting specially the ionotropic glutamate receptors; this way these toxins are used are as chemical weapons to kill / paralyze the spider’s prey. Polyamine toxins contain many azamethylene groups involved with the chelation of metal ions, which in turn can interact with the glutamate receptors, affecting the toxicity of these toxins. It was demonstrated that the chelation of Ni⁺², Fe⁺², Pb⁺², Ca⁺² and Mg⁺² ions by the desalted crude venom of Nephilengys cruentata and by the synthetic toxin JSTX-3, did not cause any significant change in the toxicity of the acylpolyamine toxins to the model-prey insect (honeybees). However, it was also reported that the chelation of Zn⁺² ions by the acylpolyamines potentiated the lethal / paralytic action of these toxins to the honeybees, while the chelation of Cu⁺² ions caused the inverse effect. Atomic absorption spectrometry and Plasma-ICP analysis both of N. cruentata venom and honeybee’s hemolymph revealed that the spider’s venom concentrates Zn⁺² ions, while the honeybee’s hemolymph concentrates Cu⁺² ions. These results are suggesting that the natural accumulation of Zn⁺² ions in N. cruentata venom favors the prey catching and/or its maintenance in the web, while the natural accumulation of Cu⁺² ions in prey’s hemolymph minimizes the efficiency of the acylpolyamine toxins as killing/paralyzing tool.