Toxin composition variations in cultures of Alexandrium species isolated from the coastal waters of southern China

The composition of the paralytic shellfish toxins (PSTs) of five Alexandrium tamarense strains isolated from the coastal waters of southern China and one Alexandrium minutum strain from Taiwan Island were investigated. A. tamarense CI01 and A. tamarense Dapeng predominantly produced C2 toxin (over 90%) with trace amounts of C1 toxin (C1), gonyautoxin-2 (GTX2) and GTX3; two strains of A. tamarense HK9301 maintained in different locations produced C1-4 toxins and GTX1, 4, 5 and 6; no PSTs were found in A. tamarense NEW, while A. minutum TW produced only GTX1-4. The toxin compositions of cultured A. tamarense strains did not vary as much during different growth phases as did the toxin composition of A. minutum TW. The toxin compositions of A. tamarense HK9301-1 did not change significantly under different salinity, light intensity, and nitrate and phosphate levels in the culture medium, although the toxin productivity varied expectably. Another strain HK9301-2 maintained in a different location produced much less toxins with a considerably different toxin composition. Under similar culture maintenance conditions for 3 years, the toxin profiles of A. tamarense HK9301-1 did not change as much as did A. tamarense CI01. Our results indicate that toxin compositions of the dinoflagellate strains are strain-specific and are subject to influence by nutritional and environmental conditions but not as much by the growth phase. Use of toxin composition in identifying a toxigenic strain requires special caution.     

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.     

The combined effect of salinity and temperature on the growth and toxin content of four Chilean strains of Alexandrium catenella (Whedon and Kofoid) Balech 1985 (Dinophyceae) isolated from an outbreak occurring in southern Chile in 2009

The toxic dinoflagellate Alexandrium catenella has been detected in the southern Chile since 1972, causing severe negative impacts on public health and aquaculture activities. Several environmental factors have been determined to affect growth and toxin production in Alexandrium strains. The aim of this study was to determine the effect of four combined conditions of two temperatures (10 and 15 °C) and two salinities (15 and 35 psu) on the growth and the Paralytic Shellfish Poisoning (PSP) toxin content and composition in four Chilean strains of A. catenella (PFB41, PFB42, PFB37 and PFB38), isolated during a summer outbreak occurred in southern Chile in 2009. The growth curves showed a higher effect of the salinity in strains PFB41 and PFB42 than in strains PFB37 and PFB38. The values of growth rates and maximum cell densities ranged from 0.25 to 0.73 div day⁻¹ and 1.1 × 10⁴ to 5.2 × 10⁴ cells mL⁻¹, respectively. All of the strains showed the highest values for both growth parameters at 15 °C and 35 psu. In general, growth parameters were higher at 35 psu independently of the temperature. On the other hand, the total PSP toxin content ranged widely from 3.99 to 239 fmol cell⁻¹. The highest values of PSP toxin content were attained at 10 °C and 35 psu for all of the strains, at both stages of growth. All of the strains displayed different toxin compositions, with neoSTX, GTX4-1, GTX3-2 and GTX5 being the main toxins detected. The results showed significant differences in the absolute values of growth and toxin production parameters among the strains grown under the same culture conditions, and for each strain grown under different combined conditions of temperature and salinity. These findings demonstrate that abiotic factors can differentially affect the population dynamics of the A. catenella toxic genotypes, thus making it extremely difficult to predict the ecological behavior of this species in the field in terms of the intensity of a potential outbreak.     

Effect of Associated Bacteria on the Growth and Toxicity of Alexandrium catenella

Saprophytic bacteria in cultures of the marine dinoflagellate Alexandrium catenella were removed to assess their effect on growth and paralytic shellfish poisoning toxin production of this dinoflagellate. The actual axenic status was demonstrated by the lack of observable bacteria both immediately after treatment and following extended incubation in the absence of antibiotics. Bacteria were measured by counting CFU and also by epifluorescence microscopy and PCR amplification of bacterial 16S-23S spacer ribosomal DNA to detect noncultivable bacteria. Removal of bacteria did not have any effect on the growth of the dinoflagellate except for the inhibition of A. catenella disintegration after reaching the stationary phase. Toxicity was determined in dinoflagellate cell extracts by different methods: high-performance liquid chromatography (HPLC); an electrophysiological test called the Electrotest, which measures the inhibition of saxitoxin-sensitive Na⁺ channels expressed in a cell line; and a mouse bioassay, which measures the toxic effect on the whole mammal neuromuscular system. A lower toxicity of the dinoflagellates in axenic culture was observed by these three methods, though the difference was significant only by the mouse bioassay and HPLC methods. Altogether the results indicate that axenic cultures of A. catenella are able to produce toxin, though the total toxicity is probably diminished to about one-fifth of that in nonaxenic cultures.     

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

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

Inheritance of chromosome length polymorphisms inCoprinus cinereus

The sexually compatible strains ofCoprinus cinereus 5302 and Dd 13 revealed chromosome length polymorphisms in their electrophoretic karyotypes. The dikaryon derived from two monokaryons contained a mixture of the two electrophoretic patterns. F₁ progenies were isolated by crossingC. cinereus 5302 and Dd 13 strains and it showed unique karyotypes. Chromosome length polymorphisms of both parental strains were inherited at random in the F₁ progenies. As a result, several novel electrophoretic karyotypes which had not been observed in either parental strains were found in the F₁ progeny. The rDNA probe hybridized with one chromosome in both parental strains, with two chromosomes in the hybridization pattern of both parental strains in the dikaryon, and with one to two chromosomes in the F₁ progenies. The relation between mating type and hybridization pattern has thus not been made clear in the case of F₁ progeny.     

The growth,toxicity and genetic characterization of seven strains of Alexandrium catenella (Whedon and Kofoid) Balech 1985 (Dinophyceae) isolated during the 2009 summer outbreak in southern Chile

The dinoflagellate Alexandrium catenella causes recurrent harmful algal blooms in southern Chile. This species belongs to the “Alexandrium tamarense/catenella/fundyense species complex” (the “tamarensis complex”), defined by morphological attributes. Ribosomal sequences serve to differentiate five evolutionary lineages (clades) in this species complex. These distinctions reflect the geographic distribution and toxicity of the populations rather than their morphological designations. Despite the social and economic impact that harmful blooms produce in Chile, few strains of A. catenella have been isolated. Moreover, physiological and/or genetic studies of the group are scarce. The aim of this work was to examine possible physiological and genetic variability among populations of A. catenella having different geographical origins but isolated from the same toxic event. Seven strains of A. catenella were isolated and established from phytoplankton samples collected in the Aysén and Los Lagos regions of southern Chile during a recent outbreak (February–March 2009). Growth, toxicity, and ITS sequences were compared among these strains. All the strains included in this study were grouped with strains belonging to the previously described “North America” clade. The genetic diversity detected among Chilean strains was 3%, a much higher value than those reported for comparisons among strains from other parts of the world. In addition, a remarkable variability of growth parameters and toxicity was detected among strains. Strain PFB45 showed the highest PSP toxin content, whereas strain PFB41 had the lowest value of this parameter but had the highest maximum cell density. In strains PFB38, PFB42, and PFB37, more than 98% of the total PSP toxin content occurred in the form of gonyautoxins (primarily GTX-4,1 and GTX-3,2). In strains PFB39, PFB36, and PFB45, neoSTX, and STX toxins were detected. These results demonstrated remarkable variability at the genetic and physiological level among strains of A. catenella isolated from the same outbreak. No correlations were found between the phenotypic traits (growth and toxicity) and the genetic affiliation of the strains studied.     

Species diversity and abundance of dinoflagellate resting cysts seven months after a bloom of Alexandrium catenella in two contrasting coastal systems of the Chilean Inland Sea

In Chile, 90% of the fish farms and major natural shellfish beds are located in the region surrounding the Inland Sea, where over the last few decades harmful phytoplankton blooms have often been observed. The onset and recurrence of bloom events are often related to the resuspension and germination of resting cysts that have accumulated in the sediments. The degree of cyst settling, accumulation and germination is highly variable between areas and depends on physical and environmental factors. To learn how differences in oceanographic exposure, amount of river runoff and bathymetry affect dinoflagellate cyst deposition, we examined the diversity and abundance of dinoflagellate resting cysts from two hydrographically contrasting coastal areas (oceanic Guaitecas Archipelago and estuarine Pitipalena Fjord) of the Chilean Inland Sea in September 2006, seven months after a bloom of Alexandrium catenella, a producer of paralytic shellfish toxin. Cyst species diversity consisted of 18 taxa, including A. catenella and the noxious species Protoceratium reticulatum, both of which have caused blooms in the study area. Our results revealed significant differences between the two study sites in terms of the abundance and diversity of resting cysts, suggesting that in the specific case of A. catenella, only Guaitecas stations have potential for cyst accumulation and successful growth of cells. However, there was no evidence of long-term resting cyst beds of A. catenella at either study site.     

Biological interactions in enclosed plankton communities including Alexandrium catenella and copepods: Role of phosphorus

A microcosm approach was used to test whether: a) growth under unbalanced nutrient conditions (varying N:P ratios) affected the susceptibility of a phytoplankton community including the dinoflagellate Alexandrium catenella (a paralytic shellfish toxin producer) to mesozooplankton grazing, and b) the potential effects of unbalanced nutrient conditions were mediated by changes in toxicity of A. catenella or by other mechanisms. The experimental setup consisted of fifteen 30 l microcosms, filled with water from the Barcelona Harbour and subjected to treatments combining nutrient inputs at three different N:P ratios (Redfield N:P ratio or nutrient-balanced, high N:P and low N:P), addition or omission of A. catenella (an estimated initial concentration of 38 A. catenella cells ml^− 1, a value typical for blooms in harbours of the Catalan coast), and selective addition of a cultured population of Acartia grani. P sufficiency had a strong positive effect on the growth of A. grani, both with or without A. catenella addition, presumably due to enhanced food quality of the prey community. The presence of this copepod resulted in lower concentrations of ciliates, A. catenella, and other dinoflagellates, suggesting active grazing by the copepods. No noxious effects of A. catenella on the copepods were detected at the relatively low cell concentrations of that dinoflagellate used in the experiment.     

Growth and toxin production in batch cultures of a marine dinoflagellate Alexandrium tamarense HK9301 isolated from the South China Sea

Nutritional and environmental conditions were characterized for a batch culture of the marine dinoflagellate Alexandrium tamarense HK9301 isolated from the South China Sea for its growth (cells ml⁻¹), cellular toxin content (Qt in fmol cell⁻¹) and toxin composition (mol%). Under a nutrient replete condition, Qt increased with cell growth and peaked at the late stationary phase. Toxin content increased with the nitrate concentration in the culture while it reached a maximum at 5 μM phosphate. When nitrate was replaced with ammonia, Qt decreased by 4.5-fold. Salinity and light intensity were important factors affecting Qt. The latter increased two-fold over the range of salinity from 15 to 30‰, while decreased 38% as light intensity increased from 80 to 220 μE m⁻² s⁻¹. Toxin composition varied with growth phase and culture conditions. In nutrient replete cultures, toxin composition varied greatly in the early growth phase (first 3 days) and then C1/C2, C3/C4 and GTX1 remained relatively constant while GTX4 increased from 32 to 46% and GTX5 decreased from 28 to 15%. In general, the composition of GTXs was affected in a much greater extent than C toxins by changes in nutrient conditions, salinity and light intensity. This is especially true with GTX4 and GTX5. These data indicate that the cellular toxin content and toxin composition of A. tamarense HK9301 are not constant, but that they vary with growth phase and culture conditions. Use of toxin composition to identify a toxigenic marine dinoflagellate is not always valid. The data also reveal that high salinity and low light intensity, together with high nitrate and low phosphate concentrations, would favor toxin production by this species.     

Paralytic shellfish toxin profiles of different geographic populations of Gymnodinium catenatum (Dinophyceae) in Korean coastal waters

Paralytic shellfish poisoning toxin profiles of dinoflagellate cultures of Gymnodinium catenatum Graham from the Yellow and South Seas in Korea were investigated by high performance liquid chromatography fluorometric detection. Strains from the Yellow Sea had predominantly carbamate toxins, while strains from Sujeongri and Chindong in the South Sea contained the N‐sulfocarbamoyl toxins, Cl,2, as major components including the presence of GTX5 and dcSTX in some strains. Toxin profiles from St. Deukryang Bay strains (South Sea) showed both characteristics of those in the South Sea and those in the Yellow Sea. Thirty strains could be divided into three groups based on cluster analysis of toxin compositions. Group I (Yellow Sea strains) was distinguished from Group II (Sujeongri and Chindong strains) by the absence of GTX5 and dcSTX. Group III comprised Deukryang Bay strains. In conclusion, the Yellow Sea and the South Sea were found to have different dinoflagellate populations with different toxin compositions.     

Grazing on a toxic Alexandrium catenella bloom by the lobster krill Munida gregaria (Decapoda: Galatheoidea: Munididae)

Specimens of Munida gregaria were collected within and in the vicinity of a bloom of the toxic dinoflagellate Alexandrium catenella in Queen Charlotte Sound, New Zealand. The crustacean contained paralytic shellfish toxin (PST) with an analogue profile dominated by N-sulfocarbamoyl analogues (C1,2 and GTX5) and carbamate gonyautoxins (GTX1,4), similar to that of the dinoflagellate. A feeding experiment showed that M. gregaria is capable of actively grazing on A. catenella and it may play a role in controlling population growth of the dinoflagellate. This is the first account of the accumulation of PST by M. gregaria. When it is periodically abundant, M. gregaria is an important food item for fish, birds and other marine fauna and they are a vector by which PST may be transferred to higher trophic levels.     

INHIBITORY EFFECTS OF CONCANAVALIN A AND TUNICAMYCIN ON SEXUAL ATTACHMENT OF ALEXANDRIUM CATENELLA (DINOPHYCEAE)1

The effect of concanavalin A (a lectin) and tunicamycin (an inhibitor of protein glycosylation) on sexual attachment of gamete pairs of the dinoflagellate Alexandrium catenella Whedon & Kofoid was studied. Concanavalin A inhibited sexual attachment at a concentration of 0.005%, and this inhibition was released by the addition of glucose or mannose at 10 mM. Mating type plus cells of A. catenella treated with tunicamycin for 12 h were not capable of sexual attachment. These results are the first to suggest the existence of a cell–cell recognition system in sexual attachment in A. catenella.     

Relative importance of nitrogen sources,algal alarm cues and grazer exposure to toxin production of the marine dinoflagellate Alexandrium catenella

Dinoflagellate paralytic shellfish toxin (PST) production is mediated by several abiotic and biotic factors. This study compared the relative importance of nitrogen source and concentration, prey alarm cues and grazer presence on toxin production of the marine dinoflagellate Alexandrium catenella (Group I, strain BF-5). In separate assays run under either nutrient-replete (F/2 medium) or nutrient-depleted (filtered seawater) conditions, PST production of A. catenella was measured as a function of varying concentrations of added nitrogen sources (ammonium and urea), alarm cues from lysed conspecific (A. catenella Group I strains) and interspecific (the diatom, Thalassiosira weissflogii, and the green flagellate, Tetraselmis sp.) algae, and the presence of a grazer (the copepod Acartia hudsonica). Results showed that addition of ammonium or urea did not increase PST production. Unexpectedly, interspecific alarm cues increased toxin production but conspecific ones did not. Grazer presence dramatically induced PST production in A. catenella, irrespective of nutrient conditions, and this effect was an order of magnitude greater than any of the other variables tested. These results corroborate previous studies on grazer-induced PST production, and support the hypothesis that grazer-induced toxin production is not an experimental artifact, but rather a prey defense mechanism.     

Characterization of protoplasts prepared from the edible fungus, Stropharia rugoso-annulata

Protoplast preparation and regeneration conditions of the edible fungus, Stropharia rugoso-annulata Farlow apud Murrill were studied, and the regenerated progenies were characterized in this study. The optimal condition for protoplast preparation was incubation of young mycelia with gentle shaking in 1.5%(w/v) Lywallzyme at 30 °C for 3 h. PGPM (potato/glucose/peptone/mannitol) was the most suitable regeneration medium. Served as osmotic stabilizer, sugars (mannitol and sucrose) were better than inorganic salts (MgSO₄) for clone development and growth. Pre-incubation of protoplasts in liquid regeneration medium resulted in a significantly decreased regeneration rate. Both dikaryotic isolates and monokaryotic isolates could be identified from protoplast-regenerated progenies, with a much higher frequency of monokaryotic isolates identified from the early-developed and fast-growing regenerated clones. Two parental mating types were also identified from protoplasted monokaryotic isolates, but not segregated by 1:1. The mycelial growth rate of protoplasted monokaryotic isolates showed a mating type-dependent model when cultured at different incubation temperatures and pH values, with A2B2 mating type monokaryotic isolates growing faster than those of A1B1 mating type monokaryotic isolates.     

Large subunit rDNA sequences from Alexandrium catenella strains isolated during harmful algal blooms in Hong Kong

The partial sequences of the large subunit (24S) ribosomal RNA gene(LsurRNA) of three Alexandrium catenella strains isolatedduring the 1998 and 1989 red tide incidents in Hong Kong were compared,togetherwith the sequences of other Asian A. catenella speciescomplex. The sequences of two A. catenella strainsisolatedin 1998 were identical, despite being isolated at different time (March andApril) and different locations (eastern and southern Hong Kong). In cladisticanalysis, the two A. catenella HK98A & B strains, theA. catenella HK1989 strain, the Chinese strain clade andtheKorean/Japanese A. catenella form a clade distinct fromother A. catenella strains. Interestingly, the twoA. catenella HK98A & B strains only form a clade withtheA. catenella HK1989 strain/ChineseA. catenella (CCMP1493) with low bootstrap values (49%).     

Feeding and intoxication–detoxification dynamics in two populations of the mussel Mytilus chilensis (Hupé, 1854) with different histories of exposure to paralytic shellfish poisoning (PSP)

Individuals of Mytilus chilensis with different histories of exposure to paralytic shellfish poisoning (PSP) were exposed to a diet containing the dinoflagellate Alexandrium catenella. Feeding and intoxication–detoxification dynamics were evaluated over a period of 12–16?days. Feeding activity was reduced during the first days of exposure, followed by a period of recovery from day 5 to the end of the experiment. Mussels from Corral population (no history of PSP exposure) exceeded the concentration of 80?μg STX eq. 100?g^?1 tissue, although filtration activity was significantly lower compared with individuals from Melinka (frequent PSP exposure). The higher feeding activity and the lower degree of toxin accumulation in the Melinka population appear be associated with frequent exposure to PSP in the natural environment. The use of A. catenella as food resource and the capacity of a rapid intoxication of both populations showed that M. chilensis is an adequate indicator for early detection of PSP.     

Use of PCR and partial sequencing of the large-subunit rRNA gene to identify Alexandrium catenella (Dinophyceae) from the South of Chile

A fragment of the large-subunit ribosomal DNA gene (LSU rDNA) from Chilean Alexandrium catenella clones isolated from two different geographic regions (XI and XII) was amplified by PCR and the products cloned and sequenced. Based on the analysis of the PCR products it is possible to distinguish two strains of A. catenella, denominated strain type 1 (a single PCR product band) and strain type 2 (two PCR product bands). These two strains proliferate in both, the XI and XII regions. Only in the XI region, there is evidence that they bloom simultaneously. The LSU rDNA sequence analysis indicate that the Chilean A. catenella isolated clones are more related to the North American ribotype-Western subribotype.     

Genetic analysis ofCochliobolus heterostrophus polyoxin-resistant mutants

Nine polyoxin-resistant mutants ofCochliobolus heterostrophus were isolated after ethyl methanesulphonate mutagenesis. All were highly resistant to polyoxin (MIC≥1,600 ppm). Crosses between the mutants and a wild-type strain revealed that the resistance trait was inherited to the offsprings in different fashions. Four of the mutant strains inherited polyoxin resistance in a 1∶1 segregation ratio, indicating that the phenotypes in these strains were due to alteration at a single locus. Allelism tests revealed four new loci,Pol1, Pol2, Pol3 andPol4, for polyoxin resistance in these mutant strains. The genes responsible for the phenotypes of the other five mutant strains were not determined, because of extremely slow growth of progenies in one cross, sterility in another cross, and inexplicable responses to polyoxin of the progenies in the other crosses. No linkage was detected between the genes for polyoxin resistance and mating type.