Saxitoxin is an alkaloid neurotoxin originally isolated from the clam Saxidomus giganteus in 1957. This group of neurotoxins is produced by several species of freshwater cyanobacteria and marine dinoflagellates. The saxitoxin biosynthesis pathway was described for the first time in the 1980s and, since then, it was studied in more than seven cyanobacterial genera, comprising 26 genes that form a cluster ranging from 25.7 kb to 35 kb in sequence length. Due to the complexity of the genomic landscape, saxitoxin biosynthesis in dinoflagellates remains unknown. In order to reveal and understand the dynamics of the activity in such impressive unicellular organisms with a complex genome, a strategy that can carefully engage them in a systems view is necessary. Advances in omics technology (the collective tools of biological sciences) facilitated high-throughput studies of the genome, transcriptome, proteome, and metabolome of dinoflagellates. The omics approach was utilized to address saxitoxin-producing dinoflagellates in response to environmental stresses to improve understanding of dinoflagellates gene-environment interactions. Therefore, in this review, the progress in understanding dinoflagellate saxitoxin biosynthesis using an omics approach is emphasized. Further potential applications of metabolomics and genomics to unravel novel insights into saxitoxin biosynthesis in dinoflagellates are also reviewed.
Toxin production of a Malaysian isolate of the toxic red tide dinoflagellate Pyrodinium bahamense var. compressum was investigated at various stages of the batch culture growth cycle and under growth conditions affected by temperature, salinity, and light intensity variations. In all the experiments conducted, only 5 toxins were ever detected. Neosaxitoxin (NEO) and gonyautoxin V (GTX5) made up 80 mole percent or more of the cellular toxin content and saxitoxin (STX), GTX6 and decarbamoylsaxitoxin (dcSTX) made up the remainder. No gonyautoxins I-IV or C toxins were ever detected. In nutrient-replete batch cultures, toxin content rapidly peaked during early exponential phase and just as rapidly declined prior to the onset of plateau phase. Temperature had a marked effect on toxin content, which increased 3-fold as the temperature decreased from the optimum of 28 degrees C to 22 degrees C. Toxin content was constant at salinities of 24% or higher, but increased 3-fold at 20%. Toxin content decreased 2-fold and chlorophyll content increased 3-fold when light intensity was reduced from 90 to 15 microE m-2 s-1. This accompanied a 30% decrease in growth rate. Toxin composition (mole % individual toxin cell-1) remained constant throughout the course of the nutrient-replete culture and during growth at various salinities, but varied significantly with temperature and light intensity changes. At 22 degrees C, GTX5 was 25 mole % and NEO was 65 mole %, while at 34 degrees C, GTX5 increased to 55 mole % and NEO decreased proportionally to 40 mole %. When light intensity was reduced from 90 to 15 microE m-2 s-1, NEO decreased from 55 to 38 mole %, while GTX5 increased from 40 to 58 mole %. These data suggest that low light and high temperature both somehow enhance sulfo-transferase activity.(ABSTRACT TRUNCATED AT 250 WORDS)
Saxitoxins (STXs) constitute a family of potent sodium channel blocking toxins, causative agents of paralytic shellfish poisoning (PSP), and are produced by several species of marine dinoflagellates and cyanobacteria. Two STX-core genes, sxtA and sxtG, have been well elucidated in Alexandrium but the expression of these genes under various nutritional modes in tropical species remains unclear. This study investigates the physiological responses of a tropical Pacific strain of Alexandrium minutum growing with nitrate or ammonium, and with various nitrogen to phosphorus (N:P) supply ratios. The transcriptional responses of the sxt genes were observed. Likewise, a putative sxtI encoding O-carbamoyltransferase (herein designated as AmsxtI) was recovered from the transcriptomic data, and its expression was investigated. The results revealed that the cellular toxin quota (Qt) was higher in P-depleted, nitrate-grown cultures. With cultures at similar N:P (<16), cells grown with excess ammonium showed a higher Qt than those grown with nitrate. sxtA1 was not expressed under any culture conditions, suggesting that this gene might not be involved in STX biosynthesis by this strain. Conversely, sxtA4 and sxtG showed positive correlations with Qt, and were up-regulated in P-depleted, nitrate-grown cultures and with excess ambient ammonium. On the other hand, AmsxtI was expressed only when induced by P-depletion, suggesting that this gene may play an important role in P-recycling metabolism, while simultaneously enhancing toxin production.
The detection of tetrodotoxin (TTX) and saxitoxin (STX) in dried salted yellow puffer fish (Xenopterus naritus) eggs bought from Satok Market, Kuching, Sarawak was carried out by mouse bioassay method. The amount of TTX and STX detected in the samples ranged from 95.6-195.5 Mouse Unit (MU)/g and 1.72-3.58 MU/g respectively. The results indicate that the dried salted eggs samples were found to contain TTX 9-20 times above the regulatory limit for human consumption (10 MU/g). Although detected, the amount of STX in salted eggs extract was slightly below the accepted threshold limit (4 MU/g). The local public in Sarawak should be educated on the potential danger of consuming dried salted puffer fish eggs in addition to the current warnings on puffer fish.
The marine dinoflagellate Alexandrium minutum is known to produce saxitoxins that cause paralytic shellfish poisoning in human worldwide through consumption of the contaminated shellfish mollusks. Despite numerous studies on the growth physiology and saxitoxin production of this species, the knowledge on the molecular basis of nutrient uptakes in relation to toxin production in this species is limited. In this study, relative expressions of the high-affinity transporter genes of nitrate, ammonium, and phosphate (AmNrt2, AmAmt1 and AmPiPT1) and the assimilation genes, nitrate reductase (AmNas), glutamine synthase (AmGSIII) and carbamoyl phosphate synthase (AmCPSII) from A. minutum were studied in batch clonal culture condition with two nitrogen sources (nitrate: NO3- or ammonium: NH4+) under different N:P ratios (high-P: N:P of 14 and 16, and low-P: N:P of 155). The expression of AmAmt1 was suppressed in excess NH4+-grown condition but was not observed in AmNrt2 and AmNas. Expressions of AmAmt1, AmNrt2, AmNas, AmGSIII, AmCPSII, and AmPiPT1 were high in P-deficient condition, showing that A. minutum is likely to take up nutrients for growth under P-stress condition. Conversely, relative expression of AmCPSII was incongruent with cell growth, but was well correlated with toxin quota, suggesting that the gene might involve in arginine metabolism and related toxin production pathway. The expression of AmGSIII is found coincided with higher toxin production and is believed to involve in mechanism to detoxify the cells from excess ammonium stress. The gene regulation observed in this study has provided better insights into the ecophysiology of A. minutum in relation to its adaptive strategies in unfavorable environments.
During the months of January-February and May-June 2013 coinciding with the red tide occurrence in Kota Kinabalu, Sabah, Malaysia, six episodes involving 58 cases of paralytic shellfish poisoning (PSP) or saxitoxin (STX) poisoning and resulting in four deaths were reported. Many of them were intoxicated from consuming shellfish purchased from the markets, whereas others were intoxicated from eating shellfish collected from the beach. Levels of STX in shellfish collected from the affected areas were high (mean 2,920 ± 780 and 360 ± 140 µg STX equivalents/100 g shellfish meat respectively for the two periods). The count of toxic dinoflagellates (Pyrodinium bahamense var compressum) of the sea water sampled around the coast was also high (mean 34,200 ± 10,300 cells/L). Species of shellfish containing high levels of STX were Atrina fragilis, Perna viridis, and Crassostrea belcheri. The age of victims varied from 9 to 67 years. Symptoms presented were typical of PSP, such as dizziness, numbness, vomiting, and difficulty in breathing. Recommended steps to prevent or reduce PSP in future red tide season include better monitoring of red tide occurrence, regular sampling of shellfish for determination of STX level, wider dissemination of information on the danger of eating contaminated shellfish among the communities, fishermen, and fishmongers.