Variation in paralytic shellfish toxin composition within the Protogonyaulax tamaronsis/catenella species complex; red tide dinoflagellates

Unialgal isolates of the Protogonyaulax (—Gonyaulax) tamarensis/catenella species complex, a group of dinoflagellates which causes paralytic shellfish poisoning (PSP), were subjected to toxin analysis by HPLC. Protogonyaulax isolates from widely separated geographical locations were compared, including the northeastern Pacific (British Columbia and Washington State), eastern Canada, Portugal, the United Kingdom and New Zealand. Two distantly related gonyaulacoid species were also analyzed, but the presence of PSP toxins was not detected. Although Protogonyaulax isolates varied markedly in total toxin concentration and toxicity, even through the culture cycle, the toxin ratios of individual isolates were distinctive and relatively constant. No toxins were detected in the Plymouth (U.K.) isolate of P. tamarensis, from the species type locality. Two isolates from Vancouver Island (British Columbia), which were previously considered to be non-toxic according to the mouse bioassay, revealed weak toxin spectra by HPLC. Within populations from English Bay (British Columbia) the toxin profiles of tamarensoid isolates tended to be conservative. However, this was not the case for the catenelloid forms from Washington State, which displayed a greater degree of toxin heterogeneity. Significantly, there was no identifiable relationship between toxicity or toxin profiles and the morphological characteristics conventionally used to separate the two dominant morphotypes into species within this species complex.     

Characterization of Satellite DNA from Three Marine Dinoflagellates (Dinophyceae): Glenodinium sp. and Two Members of the Toxic Genus, Protogonyaulax

Using CsCl-Hoechst dye or CsCl-ethidium bromide gradients, satellite and nuclear DNAs were separated and characterized in three marine dinoflagellates: Glenodinium sp., and two toxic dinoflagellates, Protogonyaulax tamarensis and Protogonyaulax catenella. In all three dinoflagellates, the lowest density fraction, satellite DNA₁, hybridized to chloroplast genes derived from terrestrial plants and/or other algae. Dinoflagellate chloroplast DNAs exhibited molecular sizes of 114 to 125 kilobase pairs, which is consistent with plastid sizes determined for other chromophytic algae (120-150 kilobase pairs). Mitochondrial DNA was not resolved from nuclear DNA in this system. Two additional satellite DNAs, satellite DNA₂ and satellite DNA₃, recovered from P. tamarensis and P. catenella were similar to one another, both within and between species, when characterized by restriction enzyme analysis. These satellites were 85 to 95 kilobase pairs in size, and exhibited restriction fragments that hybridized to yeast nuclear ribosomal RNA genes. Restriction enzyme analyses and DNA hybridization studies of cpDNA document that the two Protogonyaulax isolates are not evolutionarily identical.     

Electrophoretic variability within the Protogonyaulax tamarensis/catenella species complex: Pyridine linked dehydrogenases

Extracts from 20 isolates of the Protogonyaulax (= Gonyaulax) tamarensis/catenella species complex from diverse geographical locations, including ten contemporaneous isolates from the same geographical population, were subjected to enzyme electrophoresis. Analysis of isozyme banding patterns produced by eight pyridine-linked dehydrogenases revealed a high degree of genetic polymorphism within and between morphotypes and geographical populations. Since there was little apparent correlation between electromorphs and variants assigned provisionally to the catenelloid or tamarensoid morphotype, and because morphological intermediates exist, the presently used morphological characters used to discriminate unequivocally between P. catenella and P. tamarensis appear to be inadequate. Nevertheless, within a given morphotype, isolates from the same location were more similar than to those from elsewhere. Isozyme electrophoresis offers a means of biochemically discriminating between isolates of this species complex, for which the plate patterns are substantially the same.     

Molecular phylogeny and toxin profiles of Alexandrium tamarense (Lebour) Balech (Dinophyceae) from the west coast of Greenland

Detection of paralytic shellfish poisoning (PSP) toxins in scallops from the west coast of Greenland exceeding the 800 μg toxin/kg shellfish limit led to an investigation with the aim of finding the responsible organism(s). Three strains of Alexandrium Halim were established from single cell isolations. Morphological identification of the strains and determination of their position within the genus by LSU rDNA sequences was carried out. Light microscopy revealed that the three strains was of the Alexandrium tamarense morphotype, and bayesian and neighbor-joining analyses of the LSU rDNA sequences placed them within Group I of the A. tamarense species complex. The toxicity and toxin profiles of the strains were measured by liquid chromatography fluorescence detection (LC-FD) and their identity was confirmed by liquid chromatography coupled with tandem mass spectrometry (LC–MS/MS). The three strains all turned out to be toxic and all produced large proportions (>60% total mol) of gonyautoxins 1 and 4 (GTX1/GTX4). This is the first record of saxitoxin producers from western Greenland. The toxin profiles were atypical for A. tamarense in their absence of N-sulfocarbanoyl C1/C2 or B1/B2 toxins. Rather the high molar percentage of GTX1/GTX4, the lesser amounts of only carbamoyl toxins and the absence of decarbamoyl derivatives are more characteristic features of A. minutum strains. This may indicate that the genetically determined toxin profiles in Alexandrium species are more complex than previously appreciated.     

Alkaline phosphatase activity in Protogonyaulax tamarensis

A non-toxic strain of the marine dinoflagellate Protogonyaulaxtamarensis (= Gonyaulax tamarensis has been isolated from abloom in the Adriatic Sea, off the Emilia-Romagna coast. Culturesof the cells were grown in the laboratory in enriched seawaterat various initial ambient orthophosphate (P_i concentrations,ranging from 0.3 to 40.5 µM. The growth rate varied from0.3 to 0.8 divisions day^–1 depending on the P_i concentration.Alkaline phosphatase activity was inversely proportional toambient P levels. From measurements of kinetic parameters, thebinding of the artificial substrate p-nitrophenylphosphate tothe P.tamarensis alkaline phosphatase was quite strong (K_m=50µM). Maximal activity was observed at pH 8.4, althoughthe pH-activity curve was broad, in contrast to that of otheralkaline phosphatases. Protogonyaulax tamarensis alkaline phosphatase,measured over a 24h period, exhibited an apparent diurnal fluctuationin activity, in common with the enzyme from other dinoflagellates.     

Physiological effects of Protogonyaulax tamarensis on cardiac activity in bivalve molluscs

1. In Spisula solidissima, Mercenaria mercenaria, Artica islandica, and Placopecten magellanicus, there was no effect on cardiac activity after exposure to Protogonyaulax tamarensis (GT429).2. In Mya arenaria, there was transient cardiac inhibition in 40% of the individuals tested after exposure to GT429.3. In Ostrea edulis there was long term decrease in heart rate in 22% of the animals after exposure to GT429.4. In Geukensia demissa, there was transient cardiac inhibition in 10% of the animals, transient excitation in 40%, and long term inhibition in 10% after exposure to GT429.5. In Mytilus edulis, there was transient inhibition in 25% of the animals, long term inhibition in 21% and long term excitation in 11%, after exposure to GT429.6. There was no difference in the distribution of responses in between Mytilus that had prior exposure to GT429 and Mytilus that had no prior exposure.     

Standardized extraction method for paralytic shellfish toxins in phytoplankton

The optimal conditions were established for extraction of paralytic shellfish toxins from a Danish clone of Alexandrium tamarense using extraction with acetic acid and HCl in the concentration range 0.01–1.0 N. Physical destruction of the cells was investigated microscopically to select the most efficient extraction procedure.The toxin content was quantitated by an automized isocratic reversed-phase high-performance liquid chromatography (HPLC) method. The best results as judged from the total amount of toxins and the toxin profile were obtained using 0.05–1.0 N acetic acid and 0.01–0.02 N HCl. Hydrochloric acid in the concentration range 0.03–1.0 N caused the amount of C1 and C2 toxins to decrease sharply and concomitant increase of gonyautoxins 2 and 3.The phytoplankton extracts with 0.1 to 0.5 N acetic acid or 0.01 N HCl were stable during 6 months at –20 °C, but the extracts with HCl 0.02 N underwent a change in toxin profile, although the total amount of toxins was constant.     

Production and excretion of okadaic acid,pectenotoxin-2 and a novel dinophysistoxin from the DSP-causing marine dinoflagellate Dinophysis acuta – Effects of light,food availability and growth phase

Diarrhetic shellfish poisoning (DSP) toxins constitute a severe economic threat to shellfish industries and a major food safety issue for shellfish consumers. The prime producers of the DSP toxins that end up in filter feeding shellfish are species of the marine mixotrophic dinoflagellate genus Dinophysis. Intraspecific toxin contents of Dinophysis spp. vary a lot, but the regulating factors of toxin content are still poorly understood. Dinophysis spp. have been shown to sequester and use chloroplasts from their ciliate prey, and with this rare mode of nutrition, irradiance and food availability could play a key role in the regulation of toxins contents and production. We investigated toxin contents, production and excretion of a Dinophysis acuta culture under different irradiances, food availabilities and growth phases. The newly isolated strain of D. acuta contained okadaic acid (OA), pectenotoxins-2 (PTX-2) and a novel dinophysistoxin (DTX) that we tentatively describe as DTX-1b isomer. We found that all three toxins were excreted to the surrounding seawater, and for OA and DTX-1b as much as 90% could be found in extracellular toxin pools. For PTX-2 somewhat less was excreted, but often >50% was found extracellularly. This was the case both in steady-state exponential growth and in food limited, stationary growth, and we emphasize the need to include extracellular toxins in future studies of DSP toxins. Cellular toxin contents were largely unaffected by irradiance, but toxins accumulated both intra- and extracellularly when starvation reduced growth rates of D. acuta. Toxin production rates were highest during exponential growth, but continued at decreased rates when cell division ceased, indicating that toxin production is not directly associated with ingestion of prey. Finally, we explore the potential of these new discoveries to shed light on the ecological role of DSP toxins.     

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.     

EFFECT OF CULTURE AND BLOOM DEVELOPMENT AND OF SAMPLE STORAGE ON PARALYTIC SHELLFISH POISONS IN THE CYANOBACTERIUM ANABAENA CIRCINALIS

Paralytic shellfish poisons (PSPs) were detected in 24 of 31 bloom samples dominated by the cyanobacterium Anabaena circinalis Rabenhorst, collected from across Austraia. The ability to produce PSPs has been maintained in everal non-axenic strains of A. circinalis kept in culture, whereas strains that were non-toxin-producing when isolated have remained as such. PSPs were detected and quantified by high-performance liquid chromatography (HPLC), and the structures were confirmed by electrospray mass spectroscopy. The concentration of toxins in PSP positive samples ranged from 50 to 3400 μg.g^-1 dry weight. Toxin profiles were always dominated by the N-sulfocarbamoyl-11-hydroxysulfate C toxins, C1 and C2 (44–85 mol%), with the remainder consisting of gonyautoxins-2, −3, and −5, decarbamoylgonyautoxins-2 and −3, saxitoxin, and decarbamoylraxitoxin. N₁-_-hydroxy PSPs, commonly found in marine dinoflagellates, were absent, suggesting that A. circinalis lacks the enzyme responsible for N₁-_-hydroxylation. On a dry weight basis, the amount of toxin in cultured Anabaena circinalis (strain ACMB06)rose significantly (P < 0.05)over time from 570 to 3400 μg.g.^-1 cells in late stationary phase. However, there was no significant trend in cellular toxin quota (toxin per cell) over the life of the culture; this may be explained by variation in cell mass. On average, batch cultures of Anabaena circinalis contained 19% extracellular toxin, which increased slightly over the growth cycle and had a composition similar to that of the intracellular toxins. As cultures aged, the formation of decarbamoyl toxins and increases in theα-/β-epimer ratios of C toxins and gonyautoxins were observed. The variation in these components during stationary phase in culture was sufficient to explain the variation in relative PSP composition observed among natural bloom samples. Because decarbamoylgonyautoxins are much more toxic than C toxins on a molar basis, these transformations also lead to an increase in toxicity of the sample or bloom over time. The transformations of PSPs, which occur during aging and sample storage, render the comparison of PSPs by HPLC unreliable for phenotyping Anabaena circinalis, unless strains are cultured, harvested, and analyzed under standard conditions.     

INFLUENCE OF PHOSPHORUS LIMITATION ON TOXICITY AND PHOTOSYNTHESIS OF ALEXANDRIUM MINUTUM (DINOPHYCEAE) MONITORED BY IN‐LINE DETECTION OF VARIABLE CHLOROPHYLL FLUORESCENCE1

The effects of phosphorus (P) limitation on growth, toxicity, and variable chl fluorescence of Alexandrium minutum were examined in batch culture experiments. Cell division was greatly impaired in P‐limited cultures, but P spiking of these cultures after 9 days stimulated high levels of cell division equivalent to P‐replete cultures. The cellular concentration of paralytic shellfish toxins was consistent over the growth cycle of control cultures from lag phase into logarithmic growth phase, with toxins repeatedly lost to daughter cells during division. The low level of cell division in P‐limited cultures resulted in a 10‐fold increase of cellular toxin compared with controls, but this dropped upon P spiking due to increased rates of cell division. The history of phosphorus supply had an important effect on toxin concentration, with the P‐limited and the P‐spiked cultures showing values 2‐fold higher than the P‐replete cultures. Toxin profiles of the A. minutum strain used in these experiments were dominated by the N₁‐hydroxy toxins, gonyautoxins (GTX) GTX1 and GTX4, which were approximately 40 times more abundant than their analogues, GTX2 and GTX3, in P‐limited cultures. The dominance of the N₁‐hydroxy toxins increased significantly in control cultures as they advanced through logarithmic growth. In‐line measurements of the variable chl fluorescence of light‐adapted cells indicated consistent photochemical efficiency under P‐replete conditions. P limitation induced a drop in fluorescence‐based photochemical efficiency that was reversible by P spiking. There was an inverse linear relationship between in‐line fluorescence and cell toxin quota (r = ?0.88). Monitoring fluorescence in‐line may be valuable in managing efficient biotechnological production of toxins.     

Diagnostic deficiencies of C. difficile infection among patients in a tertiary hospital in Saudi Arabia: A laboratory-based case series

Clostridioides difficile infection (CDI) has become a threatening public health problem in the developed world. In the kingdom of Saudi Arabia, prevalence of CDI is still unknown due to limited surveillance protocols and diagnostic resources. We used a two-step procedure to study and confirm C. difficile cases. We also studied toxin profiles of these isolates.Stool samples were collected from symptomatic patients and clinically suspected of CDI for almost 12 months. Isolates were confirmed by culture method followed by 16S rRNA sequencing. Multiplex PCR was performed for the identification of toxin A, toxin B and binary toxin genes and compared to Gene Expert results.Out of the 47 collected samples, 27 were successfully grown on culture media. 18 samples were confirmed as C. difficile by both culture and 16S rRNA sequencing. Interestingly, the rest of the isolates (9 species) belonged to different genera. Our results showed 95% of samples were positive for both toxin A and B (tcdA, tcdB) and all samples exhibited the toxin gene regulator tcdC. All samples were confirmed negative for the binary toxin gene ctdB and 11% of the isolates were positive for ctdA gene. Interestingly, one isolate harbored the binary toxin gene (cdtA⁺) and tested negative for both toxins A and B.We believe that combining the standard culture method with molecular techniques can make the detection of C. difficile more accurate.     

Production of dinophysistoxin-1 and pectenotoxin-2 by a culture of Dinophysis acuminata (Dinophyceae)

The production of diarrhetic shellfish poisoning toxins (okadaic acid analogues and other lipophilic toxins) by a culture of Dinophysis acuminata, fed with the autotrophic ciliate Myrionecta rubra, was confirmed by LC–MS analysis, and the toxin profile compared with that in the field assemblage of the same species. The growth response of D. acuminata to the density of the food organism was also examined in laboratory experiments. In semi-continuous culture experiments, the growth rates of D. acuminata increased with increasing density of M. rubra and a maximum growth rate of 0.67 per day was calculated. In batch culture experiments; the cellular content of PTX2 and DTX1 were 14.7–14.8 and 2.5–4.8 pg cell^?1, respectively. Okadaic acid, dinophysistoxin-3, pectenotoxin-1, pectenotoxin-6, yessotoxin (YTX) and 45-OHYTX were not detected. PTX2 was detected (cellular toxin content: 22 pg cell^?1), but DTX1 was not detected, in an extract of D. acuminata collected from natural seawater at the same location where the cultured D. acuminata specimens were isolated. These results strongly suggest that D. acuminata produces these toxins during cell growth and that environmental factors influence variations in the toxin composition and specific cellular toxicity.     

Warm temperature acclimation impacts metabolism of paralytic shellfish toxins from Alexandrium minutum in commercial oysters

Species of Alexandrium produce potent neurotoxins termed paralytic shellfish toxins and are expanding their ranges worldwide, concurrent with increases in sea surface temperature. The metabolism of molluscs is temperature dependent, and increases in ocean temperature may influence both the abundance and distribution of Alexandrium and the dynamics of toxin uptake and depuration in shellfish. Here, we conducted a large‐scale study of the effect of temperature on the uptake and depuration of paralytic shellfish toxins in three commercial oysters (Saccostrea glomerata and diploid and triploid Crassostrea gigas, n = 252 per species/ploidy level). Oysters were acclimated to two constant temperatures, reflecting current and predicted climate scenarios (22 and 27 °C), and fed a diet including the paralytic shellfish toxin‐producing species Alexandrium minutum. While the oysters fed on A. minutum in similar quantities, concentrations of the toxin analogue GTX1,4 were significantly lower in warm‐acclimated S. glomerata and diploid C. gigas after 12 days. Following exposure to A. minutum, toxicity of triploid C. gigas was not affected by temperature. Generally, detoxification rates were reduced in warm‐acclimated oysters. The routine metabolism of the oysters was not affected by the toxins, but a significant effect was found at a cellular level in diploid C. gigas. The increasing incidences of Alexandrium blooms worldwide are a challenge for shellfish food safety regulation. Our findings indicate that rising ocean temperatures may reduce paralytic shellfish toxin accumulation in two of the three oyster types; however, they may persist for longer periods in oyster tissue.     

Occurrence ofAlexandrium cohorticula in Japanese coastal water

Two clones ofAlexandrium cohorticula were isolated at Aburatsubo, Sagami Bay, Japan. Cultured cells of both contained high amounts of paralytic shellfish toxins. The toxicity of these isolates was comparable with that of highly toxic Thai clones. No significant difference in toxin components or their proportions was observed between Japanese and Thai strains. The optimum growth temperature of both strains was around 25 °C. Japanese strains survived at 15 °C, whereas Thai strains did not; the latter grew faster than the former at 30 °C.     

The Toxins of Helminthosporium maydis (Race T) : A Colorimetric Determination of the Toxins, Their Appearance in Culture and in Infected Plants

Host-specific toxins produced by Helminthosporium maydis, race T, are measured quantitatively by a chemical assay procedure involving reaction of the toxins with a sulfuric acidacetic anhydride reagent and measurement of the absorbance of the product at 330 nm. The assay was shown to measure total toxin concentrations after only limited fractionation of the culture medium. Using the assay it was possible to show that the highest amount of toxin per gram of fungus mycelium occurs early in the growth cycle of H. maydis. Toxins I, II, and V are the predominant toxins at these early times both in culture and in infected corn and wheat varieties. Some chromatographic and spectral properties of toxin V, a previously unreported toxin, are described. Since toxin V appears in culture prior to toxins I, II, III and IV, a precursor-product relationship can be suggested.     

Changes in toxins, intracellular and dissolved free amino acids of the toxic dinoflagellate Gymnodinium catenatum in response to changes in inorganic nutrients and salinity

The paralytic shellfish poison prducing dinoflagellate Gymnodiniuncatemrum was subjected to changes in salinity, phosphate, ammoniumand nitrate using continuous culture and batch culture methods.In contrast with other algae, this species showed very slowchanges in the concentration of intracellular amino acids, inthe Gln:Glu ratio, and, in contrast with Alrsandnum spp., onlyslow changes in toxin content, during such events as N-feedingof Ndeprived cells or during nutrient deprivation. This organismwas found to be very susceptible to disturbance; maximum growthrates around 0.25–0.3 day^–1 with a minimum C:N massratio of 5.5, were attained when cultures were only disturbedby sampling once a day. P-deprived cells were larger (twicethe usual C content of 4 ng C cell^–1 and volume of 20pl). The content of free amino acids was always low (5% of cell-N),with low contributions made by arginine (the precursor for paralyticshellfish toxins). Cells growing using ammonium had the lowestC:N ratios and the highest proportion of intracellular aminoacids as arginine. The toxin profile (equal mole ratios of dcSTX,GTX₅, dcGT_2/3 C₁ and C₂, and half those values for C₃ and C₄)was stable and the toxin concentration varied between 0.2 and1 mM STX equivalents (highest when ammonium was not limiting,lowest in P-deprived cells, though as the latter were largertoxin per cell was not so variable). Decreased salinity didnot result in increases in toxin content. Significant amountsof amino acids (mainly serine and glycine, with a total oftenexceeding 4 µM) accumulated in the growth medium duringbatch growth even though the cultures were not bacteria free. ⁴Present address: Instituto Español de Oceanografia,Apdo 1552, 36280, Vigo, Spain     

Enzymatic transformation of PSP toxins in the littleneck clam (Protothacastaminea)

Stemming from investigations into the relationship between toxins produced by Gonyaulax sp. and accumulated in shellfish, we wish to report enzymatic transformations of the PSP toxins to decarbamoyl derivatives in the littleneck clam (Protothaca staminea). No toxin transformations were observed in either mussels (Mytilus edulis) or in butter clams (Saxidomus giganteus). In addition, littleneck clam samples from the natural environment contained predominantly the decarbamoyl derivatives, while other shellfish species collected from the same vicinity contained the previously reported PSP toxins.     

Possible mechanisms underlying copepod grazing responses to levels of toxicity in red tide dinoflagellates

A previous study indicated that rates of ingestion exhibited by adult female copepods of Acartia hudsonica (Pinhey) and Pseudocalanus spp. were lowered by increasing levels of toxicity in clonal cultures of the bloom-causing dinoflagellate Protogonyaulax tamarensis (Taylor). In the present study, three types of laboratory grazing experiments were performed to determine which of two contending hypotheses —behavioural rejection or physiological incapacitation - could explain the observed relationship best. The experimental results consistently supported the postulated mechanism of physiological incapacitation, and not the mechanism of behavioural rejection, as the reason for the lowered rates of ingestion on the more toxic dinoflagellate clones.     

Differentiation and distribution of three types of exfoliative toxin produced by Staphylococcus hyicus from pigs with exudative epidermitis

Exfoliative toxins of approximately 30 kDa produced by Staphylococcus hyicus strains NCTC 10350, 1289D-88 and 842A-88 were purified and specific polyclonal antisera were raised against each of the toxins. It was shown by immunoblot analysis and ELISA that three exfoliative toxins from S. hyicus were antigenically distinct. The three toxins were designated ExhA, ExhB and ExhC. From 60 diseased pigs, each representing an outbreak of exudative epidermitis, a total of 584 isolates of S. hyicus were phage typed and tested for production of exfoliative toxin. ExhA-, ExhB- and ExhC-producing S. hyicus isolates were found in 12 (20%), 20 (33%) and 11 (18%), respectively, of the 60 pig herds investigated. Production of the different types of exfoliative toxin was predominantly associated with certain phage groups. However, toxin production was found in all of the six phage groups defined by the phage typing system. Some changes in the distribution of isolates between phage groups were observed when the results of this study were compared to previous investigations. In this study two new antigenically distinct exfoliative toxins were isolated and tools for in vitro detection of toxin producing S. hyicus isolates and for further studies on the exfoliative toxins from S. hyicus have been provided.