Paralytic shellfish toxin profiles and toxin variability of the genus Alexandrium (Dinophyceae) isolated from the Southeast China Sea.

Paralytic shellfish toxin (PST) profiles of 16 Alexandrium isolates from the Southeast China Sea were analyzed by high-pressure liquid chromatography. Toxin content and composition of three A. tamarense isolates, ATDH01, ATGX02 and ATMJ02, were also investigated at different growth phases and under various culture conditions. Our results showed that six strains of A. affine were non-toxic, while 10 strains of A. tamarense and A. catenella were toxic. These toxic isolates grown in the same culture conditions consistently produced an unusually high proportion of the N-sulfocarbamoyl toxin C1/2 (around 60-80% of total toxins) and medium amounts of gonyautoxin GTX5 (around 15-30% of total) with only trace quantities (<5% of total) of other saxitoxin derivatives (i.e. GTX1, GTX3, GTX4 and neoSTX). The toxin composition of three A. tamarense isolates did not vary with the growth phases, although higher toxin contents (Qt, fmolcell(-1)) were found in the exponential phase. Variations in temperature, salinity and nutrient levels affected toxin content of three A. tamarense isolates but they did not have pronounced effects on the toxin composition (mole %). These results indicate that toxin composition remained relatively constant under various culture conditions, suggesting that toxin composition could be used as a stable biomarker for the Alexandrium species in this region. However, comparison of toxin profiles between isolates from different localities require special caution since isolates even from the same region can have distinct toxin profiles.     

Variability in toxicity of the dinoflagellate Alexandrium tamarense in response to different nitrogen sources and concentrations.

Nitrogen (N) supply in pulses was simulated by exposing Alexandrium tamarense which was acclimatized at low N concentration (6 microM-N) to sudden increase in concentrations of nitrate, ammonium and urea, and the variability in toxicity due to nutrient status of A. tamarense was examined. The toxin composition did not vary dramatically among the three N sources, however, ammonium induced the highest concentration of intracellular toxin, followed by urea and then nitrate. Therefore, populations utilizing high ammonium concentration could be more toxic than those growing on nitrate or urea. The toxin content was dependent on the cellular N status of nitrate grown cells only, suggesting that the competition for N in toxin production with other metabolic pathways such as growth may be different among N sources. The relationship between toxin and nutrient status is a complex interaction and it involves the redistribution of cellular N within the cells. Understanding the toxin dynamics of natural populations in relation to nutrient is essential for the mitigation of harmful dinoflagellates in a given coastal ecosystem.     

Influence of inorganic nutrition on growth and PSP toxin production of Alexandrium minutum (Dinophyceae) from Cork Harbour, Ireland.

The physiological response of the PSP toxin producing dinoflagellate Alexandrium minutum isolated from the Irish coast was assessed after modulating the initial concentrations of nitrate and phosphate in batch cultures. The cell growth in cultures of strain CK.A02 was primarily controlled by nitrate availability. In all experiments, only gonyautoxins 2 and 3 (GTX2 and 3) were synthesized along the different growth phases, with GTX3 dominating ( approximately 80%) at all stages, making the GTX2-3 toxin profile a possible population marker of A. minutum in Cork Harbour. The cellular toxin quotas remained low and relatively stable at around 2pgcell(-1), except when high N:P ratios were initially used for culture inoculations; in these conditions PSP toxins accumulated up to 14pgcell(-1). Due to the composition of the toxin profile, the toxicity of strain CK.A02 was generally relatively low (from 1.1 to 1.7pg STX eqcell(-1)) in comparison with strains from other geographic areas except when phosphate limiting culture conditions were applied (maximum of 12.5pg STX eqcell(-1)). Results showed that sufficient soluble protein quotas were necessary to observe the intra-cellular accumulation of PSP toxins in phosphate limiting conditions, highlighting also the requirement of adequate nitrogen supplies. The possible existence of localized toxicity hot spots in the field, linked to the accumulation of PSP toxins within A. minutum cells as a metabolic response to adverse environmental conditions, could potentially increase risks for shellfish farming operations.     

Growth response and toxin concentration of cultured Pyrodinium bahamense var. compressum to varying salinity and temperature conditions.

The growth and toxin production of a Philippine Pyrodinium bahamense isolate in nutrient replete batch cultures were investigated under conditions affected by varying salinity, temperature and combined effects of salinity and temperature. Early exponential growth stage was reached after 7 days with a cell division rate of 0.26 div day(-1). The toxin content reached a peak of 298 fmol cell-1 at mid exponential phase and rapidly declined to 54 fmol cell-1 as it approached the death phase. Only three sets of toxins composed of STX, dcSTX and B1 were detected in which STX made up to 85-98 mol%toxincell-1. P. bahamense was able to grow in salinities and temperatures ranging from 26 per thousand to 36 per thousand and 23 to 36 degrees C, respectively. The optimum growth under varying salinity and temperature conditions was observed at 36 per thousand and 25 degrees C. Toxin content reached a peak of 376 fmol cell-1 at 25 degrees C and was lower (80-116 fmol cell-1) at higher temperatures (32-35 degrees C). Combined effects of salinity and temperature showed that P. bahamense was not able to grow at low salinity and temperature (i.e. below 26 per thousand-28 degrees C). Optimum growth was observed in higher salinities at all temperature conditions.     

Variety of PSP toxins in four culture strains of Alexandrium minutum collected from southern Taiwan.

Paralytic shellfish poisoning (PSP) toxin profiles were compared among four culture strains of Alexandrium minutum. GTX-1, 2, 3 and 4 are the PSP toxins that occur in A. minutum, and other PSP toxins were not detected. When comparing the toxin profile of four A. minutum strains, GTX1 and 4 were the major toxins in Amtk1, Amtk2, and Amtk4, but in Amtk7, GTX3 and 2 were the major toxins. The results indicate that strains with various toxin profiles exist in southern Taiwan, and suggest that the comparison of the toxin profiles between strains at different localities is difficult. Additionally, the toxin profiles of A. minutum strains cultured in the same environment were different, suggesting that it was owing to the intrinsic nature of toxic algae.     

Influence of environmental and nutritional factors on growth, toxicity, and toxin profile of dinoflagellate Alexandrium minutum.

The toxic dinoflagellate Alexandrium minutum T1 was isolated from southern Taiwan in February 1996, grown under various environmental and nutritional conditions, and then examined for growth, toxicity, and toxin profile. It was found that the optimal environmental conditions for cell growth and toxin production of A. minutum T1 was as follows: temperature 25 degrees C, pH 7.5, light strength 120 microEm(-2) s(-1), and salinity 15 ppt. The optimal level of nutrients supplemented in the 50% natural seawater medium was as follows: phosphate 0.002%, nitrate 0.01%, cupric ion 5.0 ppb, ferric ion 270 ppb and humic acid free. Both cell toxicity and total toxicity reached the maximum level at the post-stationary growth phase and decreased quickly. The toxic components of A. minutum T1 were assayed by high performance liquid chromatography, and found to be gonyautoxin (GTX) 1-4 only. Among these four toxin components, toxins GTX 1 and 4 were the predominant components throughout the growth curve when the cells were grown in the optimal environmental and nutritional conditions. But toxins GTX 2 and 3 increased when the cells were cultured in high salinity medium.     

Paralytic shellfish poisoning toxin profile of mussels Perna perna from southern Atlantic coasts of Morocco

During the monitoring programme of harmful algal blooms established along the south Atlantic coast of Morocco, a bimonthly determination of harmful algae and phycotoxins analysis in Perna perna was carried out from May 2003 to December 2004. Results of mouse bioassay (in organs and whole flesh) showed a seasonal evolution of paralytic shellfish poisoning (PSP) toxin. The mussel's contamination was associated with the occurrence in water of Alexandrium minutum.The PSP toxin profile obtained with high-performance liquid chromatography (HPLC/FD) revealed the dominance of gonyautoxins GTX2 and GTX3 and a minority of GTX1, GTX4 and saxitoxin (STX). This profile explains that the toxicity was mainly associated with A. minutum.     

Nitrogen:Phosphorus supply ratio may control the protein and total toxin of dinoflagellate Alexandrium tamarense.

The protein and total toxin of dinoflagellate Alexandrium tamarense at the exponential growth phase were studied at four N:P supply ratios=8, 16, 24 and 48 by maintaining the N concentration at 880 microM with variable P concentrations without nutrient limitation. Because A. tamarense grew well at all N:P supply ratios, they might synthesize RNA which contains high phosphorus and consequently low N:P atomic ratio of cells might be retained during exponential growth phase. Cellular protein:C ratio and toxin:C ratio depended on N:P supply ratio, suggesting that intracellular biochemical composition of A. tamarense might vary due to N:P supply conditions. These biochemical changes could not be detected by only investigations of cellular N:C atomic ratio which was independent on N:P supply ratio. Total cellular toxin contents of A. tamarense increased with increasing N:P supply ratio, indicated that total cellular toxin contents of A. tamarense might be stimulated by relative P-deficiency. In situ P concentration of the Seto Inland Sea of Japan has been reduced since 1980s the environmental regulation issued by Japanese Government, and therefore N:P supply ratio of input water from adjacent rivers has became higher than the Redfield ratio. The present study may suggest that the reduction of P supply into ambient sea water might cause A. tamarense to accumulate more toxin within the cell, in result, noxious A. tamarense would be more influential to marine organisms in coastal ecosystem.     

The loss of PSP toxin production in a formerly toxic Alexandrium lusitanicum clone.

Toxin production has always been considered a constitutive characteristic of dinoflagellates in the genus Alexandrium. Here we demonstrate that saxitoxin production can be lost by an Alexandrium species during routine culture maintenance. This is the first report of any marine saxitoxin-producing alga ever to have completely lost the ability to produce toxins. A clonal toxic isolate of Alexandrium lusitanicum from Portugal has been maintained in culture since 1962. In 1992, a subculture was established and sent to a different laboratory. Recent comparisons of the parental strain and the subculture revealed that the former had lost its toxicity, whereas the latter still produces saxitoxins. This loss of toxicity was confirmed by three independent toxin detection methods: mouse bioassay, mouse neuroblastoma assay and HPLC. Sequence analyses of different rRNA domains demonstrated that the toxic and non-toxic cultures are genetically identical for those markers. Morphological analysis showed that both cultures have the same plate tabulation and are A. lusitanicum. These results strongly argue that the loss of toxicity is not a result of a culturing artifact or mistake, such as mislabeling or contamination. The clonal cultures also show a significant difference in growth. Possible explanations for the change include genetic mutations or the effects of prolonged treatment of the non-toxic culture with antibiotics.     

Estimating the accumulation and transfer of Nodularia spumigena toxins by the blue mussel Mytilus edulis: an appraisal from culture and mesocosm experiments.

Accumulation of Nodularia spumigena toxins by Mytilus edulis was studied during laboratory and mesocosm experiments in order to investigate the possible pathways of nodularin in mussels and calculate toxin budgets. Mussels were exposed to 0.2-15.6 microg nodularin l(-1), fed for up to 5 days with Nodularia cells from culture, or blooming in different nutrient-treated seawater. Toxin concentration was monitored with LC-ESI-MS. During different exposures, the amount of nodularin detected in mussels increased linearly with increasing toxin concentration in food and attained 0.28-13.8 microg of nodularin g dw(-1) of the mussel whole body tissue after 12 h. The digestive gland was found to be the tissue with the highest toxin concentration. Nodularin concentration in faeces was not proportional to faeces production or to toxin concentration in food; however, it seemed to be mostly related to food quality as well as to food availability. The percentage of nodularin taken up by the mussels, relative to the amount contained in the offered food, varied from 10% to 20%, depending on food quality. During a 5-day toxin accumulation experiment, the acute reduction of the toxin in mussel tissues the second day and the following stabilization, showed that probably mussels maintain low toxin levels via efficient elimination and/or toxin metabolism. After a 72 h depuration period, mussels showed 75% reduction in their toxin content.     

Comparison of paralytic shellfish poisoning toxin between carnivorous crabs (Telmessus acutidens and Charybdis japonica) and their prey mussel (Mytilus galloprovincialis) in an inshore food chain.

Paralytic shellfish poisoning toxin in two shore crab species, Telmessus acutidens and Charybdis japonica, were compared with the toxin in the prey mussel Mytilus galloprovincialis and causative dinoflagellates Alexandrium tamarense, all having been collected at Onahama, Fukushima Prefecture, in the northern part of Japan. When the toxicities were detected in mussels by mouse bioassays, 73.7% of the sampled T. acutidens were toxic in the hepatopancreas. T. acutidens has been found to become toxic for three years, therefore, it can be concluded that the crab commonly and repeatedly accumulate the toxins via the food chain at Onahama. C. japonica was also expected to be a possible vector species, because small quantities of the toxins were detected in eight specimens of the crab by HPLC analysis. By the comparison of the toxin profiles in the dinoflagellates, mussels and the crab T. acutidens, reductive conversions of GTX1 and GTX4 were observed when the toxins passed through the three species in the food chain. But increases of STX and neoSTX by further reductive process were not observed in the crab. The absence of the STX group toxins in the crab suggests that the crab eliminates the toxin before such reductive process occur.     

Remarkable difference in accumulation of paralytic shellfish poisoning toxins among bivalve species exposed to Pyrodinium bahamense var. compressum bloom in Masinloc Bay, Philippines.

Seasonal variation of bivalve toxicity was monitored in association with the abundance of the toxic dinoflagellate Pyrodinium bahamense var. compressum in Masinloc Bay, Luzon Island. Among 7 species of bivalve, 6 species became toxic during a bloom of the dinoflagellate. However, remarkable difference in the toxicity was observed among the species. The toxicity of thorny oyster Spondylus squamosus was the highest among the species, showing more than 30 times that of safety consumption level after the peak bloom of the dinoflagellate, while other bivalve species showed much lower toxicity. The toxicity of thorny oyster decreased under absence of the dinoflagellate, but this species maintained a considerably high toxicity throughout a year. Similar trend was observed in penshell Atrina vexillum in a small scale, indicating that these species in the bay are not safe for human consumption almost throughout a year. The toxicity of green mussel Perna viridis increased to significant level during a bloom, but it decreased rapidly when the dinoflagellate disappeared. Toxin analysis of cultured and natural cells showed typical toxin profile of the dinoflagellate. Toxin profile of all the bivalve species reflected the characteristic toxin profile of the dinoflagellate.     

Dynamics of C2 toxin and chlorophyll-a formation in the dinoflagellate Alexandrium tamarense during large scale cultivation.

The production of paralytic shellfish toxins (PSTs) by the dinoflagellate Alexandrium tamarense ATCI01, a toxigenic strain isolated from South China coastal waters, was studied in batch cultures in relatively large volumes (20l). Under nutrient-replete conditions, this strain produced C2 toxin (C2T) as a predominant PST. In a 15-day production culture, phosphate was depleted by day 4, the stationary phase began at day 6, and the toxin productivity peaked at day 10, in which the cell content of C2T reached 76 fmol per cell. Much of the toxin was produced after the depletion of phosphate in the medium suggesting that C2T is a secondary metabolite. Aeration with small bubbles was useful in increasing cell mass and toxin yield. Chlorophyll-a (Chl-a) was formed in algal cells until the culture entered the stationary phase, after which Chl-a began to disappear rapidly from the culture while the C2T content continued to rise. These results suggest a metabolic relationship between Chl-a and C2T.     

从两株微小亚历山大藻中分离纯化膝沟藻毒素

目的从两株微小亚历山大藻(Alexandrium minutum Halim)中分离纯化膝沟藻毒素(Gonyautoxins).方法用胶滤层析、离子交换层析等方法从人工培养的两株微小亚历山大藻酸酒精萃取物中分离纯化膝沟藻毒素,再用HPLC法对毒素进行定性定量测定.结果从两株藻株培养液中均分离得到膝沟藻毒素GTX-4,GTX-1,GTX-3,GTX-2,其中Amtk4藻株的毒素含量为Amtk2的近10倍.结论微小亚历山大藻Amtk4藻株适于制备膝沟藻毒素.     

Growth and toxin production in the ciguatera-causing dinoflagellate Gambierdiscus polynesiensis (Dinophyceae) in culture

The growth and toxin production in a clonal strain of Gambierdiscus polynesiensis, TB-92, was examined in batch culture conditions. The mean growth rate at exponential phase was (0.13 ± 0.03) division day⁻¹. Regardless of the age of cultures, all mice injected with dichloromethanolic and methanolic extracts showed symptoms specific to ciguatoxin (CTX) and maitotoxin (MTX) bioactivity, respectively. The highest total toxicity assessed in TB-92 cultures was 10.4 × 10⁻⁴ mouse unit cell⁻¹. The toxin production pattern reveals an enhanced cellular toxin content with the age of the culture. CTX- and MTX-like compounds each accounted for approx. 50% of the total toxicity of TB-92 cultures, except in aged cells where CTXs were dominant. The high ciguatoxic activity of TB-92 was further confirmed in dichloromethanolic extracts by means of the receptor-binding assay. The highest CTX level monitored at late stationary phase was (11.9 ± 0.4) pg P-CTX-3C equiv cell⁻¹. Further HPLC and LC-MS analysis revealed the presence of five CTXs congeners in lipid-soluble extracts, i.e. CTX-3C, -3B, -4A, -4B and M-seco-CTX-3C, and of new CTX congeners. Toxin composition comparison between two G. polynesiensis strains suggests that the toxin profile is a stable characteristic in this species. G. polynesiensis clones also proved inherently more toxic than other Gambierdiscus species isolated from other geographical areas.     

Effects of toxic dinoflagellates and toxin biotransformation in bivalves

Attempts were made to elucidate the different responses of shellfish to paralytic shellfish poison (PSP) and the PSP donor Alexandrium minutum T1. Five species of edible bivalves (Crassostrea gigas, Meretrix lusoria, Mytilus edulis, Ruditapes philippinarum, and Soletellina diphos) were collected and examined for susceptibility to PSP and PSP donor. It was determined that all five bivalves had low susceptibility to PSP following an intramuscular injection (> 300 MU/20 g). The abnormal effects on bivalves were species-specific and varied with the concentration of A. minutum T1. Judging from the LC50 data (medium lethal concentration), the resistance of bivalves to the toxic dinoflagellate was as follows (least to most resistant): C. gigas < R. philippinarum < M. lusoria < M. edulis, S. diphos. With the exception of S. diphos, the bivalves accumulated very little toxin (< 2 MU/g edible tissue) when they were exposed to 10(7) cells/L of A. minutum for four days. The toxin levels in S. diphos increased with exposure time to the toxic dinoflagellates and accumulated primarily in the digestive gland (88-100%), followed by the gill (0-10%), and other organs (0-8%). Although the concentrations of toxin components in the digestive gland were found to be variable during the exposure period, the toxin profile in the digestive gland of S. diphos during the early exposure period was similar to that of A. minutum. Moreover, toxin components in the gills and in other organs were retained at near constant concentrations during the exposure period.     

Variation in toxin compositions of two harmful raphidophytes, Chattonella antiqua and Chattonella marina, at different salinities

Toxin compositions of the two species of raphidophytes, Chattonella antiqua (Hada) Ono and Chattonella marina (Subrahmanyan) Hara et Chihara, were investigated at different salinities under laboratory conditions. C. antiqua contained toxin components CaTx-I, CaTx-II, CaTx-III, and CaTx-IV, which corresponded to brevetoxin components PbTx-1, PbTx-2, PbTx-3, and oxidized PbTx-2. Similarly, C. marina included CmTx-I, CmTx-II, CmTx-III, and CmTx-IV corresponding to PbTx-2, PbTx-9, PbTx-3, and oxidized PbTx-2. Toxin yields in both species varied markedly with a change in salinity concentration. In C. antiqua CaTx-I, CaTx-II, and CaTx-III peaked at 25 P.P.t. with yields of 0.99, 0.42, and 2.90 pg/cell, but the highest yield (2.35 pg/cell) of CaTx-IV was attained at 30 P.P.t. The yields of all CaTx components decreased sharply at salinities exceeding 30 P.P.t. On the other hand, C. marina yielded higher proportions of CmTx-I (0.55 pg/cell) and CmTx-III (2.50 pg/cell) at 25 P.P.t. However, CmTx-IV was present in its highest amount (1.65 pg/cell) at 30 P.P.t., as seen in C. antiqua. A small amount of CmTx-II was also detected at 20 P.P.t.-35 P.P.t. Both species showed the highest ichthyotoxicities at 25 P.P.t., at which the maximum cell division rate was obtained.     

Accumulation and depuration profiles of PSP toxins in the short-necked clam Tapes japonica fed with the toxic dinoflagellate Alexandrium catenella.

A toxic dinoflagellate responsible for paralytic shellfish poisoning (PSP), Alexandrium catenella (Ac) was fed to the short-necked clam Tapes japonica, and the accumulation and depuration profiles of PSP toxins were investigated by means of high-performance liquid chromatography with postcolumn fluorescence derivatization (HPLC-FLD). The short-necked clams ingested more than 99% of the Ac cells (4 x 10(7)cells) supplied once at the beginning of experiment, and accumulated a maximal amount of toxin (185 nmol/10 clams) after 12h. The rate of toxin accumulation at that time was 23%, which rapidly decreased thereafter. Composition of the PSP toxin accumulated in the clams obviously different from that of Ac even 0.5h after the cell supply, the proportion of C1+2 being much higher than in Ac, although the reason remains to be elucidated. In contrast, a higher ratio of gonyautoxin (GTX)1+4 than in Ac was detected in the toxin profiles of clam excrements. The variation in toxin composition derived presumably from the transformation of toxin analogues in clams was observed from 0.5h, such as reversal of the ratio of C1 to C2, and appearance of carbamate (saxitoxin (STX), neoSTX and GTX2, 3) and decarbamoyl (dc) derivatives (dcSTX and dcGTX2, 3), which were undetectable in Ac cells. The total amount of toxin distributed over Ac cells, clams and their excrements gradually declined, and only 1% of supplied toxin was detected at the end of experiment.     

Identification of pectenotoxins in plankton, filter feeders, and isolated cells of a Dinophysis acuminata with an atypical toxin profile, from Chile.

A bloom of Dinophysis acuminata produced, in autumn of 2005, a closure of the scallop harvesting in Bahía Inglesa, in the Chilean III region. Isolated cells of this Dinophysis species were shown to contain 180 pg cell(-1) of pectenotoxin 2 but neither okadaic acid nor any of its analogs or derivatives (at least at a detectable level). Examination of plankton and filter-feeder samples covering an area of ca. 350 km, from the location where the toxicity was recorded to Bahía Tongoy, showed that the unique toxin profile found in the first bloom was widespread over that part of Chile and persisted for months. The analysis were carried out by HPLC-ESI-MS using positive ionization mode, with a detection limit below 2 ng of OA mL(-1) of methanolic extract. This is the first report of the presence of pectenotoxins in the plankton of the Pacific coast of America and in the studied filter feeders. This is also the first report of a Dinophysis species containing pectenotoxins and not any toxin of the okadaic acid group.