A fluorimetric method based on changes in membrane potential for screening paralytic shellfish toxins in mussels

To prevent the consumption of bivalves contaminated with paralytic shellfish poisoning (PSP), toxin levels in seafood products are estimated by using the official mouse bioassay. Because of the limitations of this bioassay other methods of monitoring toxins are clearly needed. We have developed a test to screen for PSP toxins based on its functional activity; the toxins bind to the voltage-gated Na+ channels and block their activity. The method is a fluorimetric assay that allows quantitation of the toxins by detecting changes in the membrane potential of human excitable cells. This assay gives an estimate of toxicity, since each toxin present in the sample binds to sodium channels with an affinity which is proportional to its intrinsic toxic potency. The detection limits for paralytic shellfish toxins were found to be 1 ng saxitoxin equivalents/ml compared to the regulatory limit threshold of 400 ng/ml (equivalent to 80 microg/100 g) used in most countries. Our results indicate that this fluorescent assay is a specific, very sensitive, rapid, and reliable method of monitoring PSP toxin levels in samples from seafood products and toxic algae.     

Assessment of sodium channel mutations in Makah tribal members of the U.S. Pacific Northwest as a potential mechanism of resistance to paralytic shellfish poisoning

The Makah Tribe of Neah Bay, Washington, has historically relied on the subsistence harvest of coastal seafood, including shellfish, which remains an important cultural and ceremonial resource. Tribal legend describes visitors from other tribes that died from eating shellfish collected on Makah lands. These deaths were believed to be caused by paralytic shellfish poisoning, a human illness caused by ingestion of shellfish contaminated with saxitoxins, which are produced by toxin-producing marine dinoflagellates on which the shellfish feed. These paralytic shellfish toxins include saxitoxin, a potent Na⁺ channel antagonist that binds to the pore region of voltage gated Na⁺ channels. Amino acid mutations in the Na⁺ channel pore have been demonstrated to confer resistance to saxitoxin in softshell clam populations exposed to paralytic shellfish toxins present in their environment. Because of the notion of resistance to paralytic shellfish toxins, the study aimed to determine if a resistance strategy was possible in humans with historical exposure to toxins in shellfish. We collected, extracted and purified DNA from buccal swabs of 83 volunteer Makah tribal members and sequenced the skeletal muscle Na⁺ channel (Nav1.4) at nine loci to characterize potential mutations in the relevant saxitoxin binding regions. No mutations of these specific regions were identified after comparison to a reference sequence. This study suggests that any resistance of Makah tribal members to saxitoxin, if present, is not a function of Nav1.4 modification, but may be due to mutations in neuronal or cardiac sodium channels, or some other mechanism unrelated to sodium channel function.     

Quantitation of diarrhetic shellfish poisoning toxins in Chilean mussel using pyrenyldiazomethane as fluorescent labeling reagent

Diarrhetic shellfish poisoning (DSP) is a gastrointestinal disease caused by lipid soluble polyether toxins produced by dinoflagellates and accumulated in shellfish. Diarrhetic shellfish poisoning is a worldwide threat to public health and the shellfish industry. To date, only four lipid soluble polyethers have been known as diarrhetic shellfish toxins. Among them, Okadaic acid (OA), Dinophysistoxin 1 (DTX-1, 35-methyl OA), Dinophysistoxin 2 (DTX-2, OA isomers) and Dinophysistoxin 3 (DTX-3, 7-O-acyl-35-methyl OA), all of which have free carboxilic groups. To perform quantitative analysis of DSP toxins in shellfish samples is a requirement, because DSP toxins are endemic in the Chilean mollusks of the southern regions, and although human symptoms of DSP appear relatively mild in comparison with the Paralytic Shellfish Poisoning (PSP), the necessity of monitoring the chronic effects of continued uptake of low doses of DSP toxins more closely is imperative, since DSP toxins have been described as potent tumor promoters. This paper shows the synthesis pathway of a chromophore, 1-pyrenyldiazomethane (PDAM), a fluorescent labeling reagent for determination of carboxilic acids, using High Performance Liquid Chromatography with fluorescence on-line detection. This procedure was developed in order to have a quantitative method for DSP toxins analysis that would be useful for health public services and private shellfish industries. The features of this labeling reagent are compared against ADAM and used for quantitative analysis of DSP toxins in Chilean mussels and cultured dinoflagellates samples.     

Variability and profiles of lipophilic toxins in bivalves from Great Britain during five and a half years of monitoring: azaspiracids and yessotoxins

Cefas has been responsible for the delivery of official control biotoxin testing of bivalve molluscs from Great Britain for just over a decade. Liquid chromatography tandem mass spectrometric (LC–MS/MS) methodology has been used for the quantitation of lipophilic toxins (LTs) since 2011. The temporal and spatial distribution of okadaic acid group toxins and profiles in bivalves between 2011 and 2016 have been recently reported. Here we present data on the two other groups of regulated lipophilic toxins, azaspiracids (AZAs) and yessotoxins (YTXs), over the same period. The latter group has also been investigated for a potential link with Protoceratium reticulatum and Lingulodinium polyedra, both previously recognised as YTXs producing phytoplankton.On average, AZAs were quantified in 3.2% of all tested samples but notable inter-annual variation in abundance was observed. The majority of all AZA contaminated samples were found between July 2011 and August 2013 in Scotland, while only two, three-month long, AZA events were observed in 2015 and 2016 in the south-west of England. Maximum concentrations were generally reached in late summer or early autumn. Reasons for AZAs persistence during the 2011/2012 and 2012/2013 winters are discussed. Only one toxin profile was identified, represented by both AZA1 and AZA2 toxins at an approximate ratio of 2 : 1, suggesting a single microalgal species was the source of AZAs in British bivalves. Although AZA1 was always the most dominant toxin, its proportion varied between mussels, Pacific oysters and surf clams.The YTXs were the least represented group among regulated LTs. YTXs were found almost exclusively on the south-west coast of Scotland, with the exception of 2013, when the majority of contaminated samples originated from the Shetland Islands. The highest levels were recorded in the summer months and followed a spike in Protoceratium reticulatum cell densities. YTX was the most dominant toxin in shellfish, further strengthening the link to P. reticulatum as the YTX source. Neither homo-YTX, nor 45−OH homo-YTX were detected throughout the monitored period. 45−OH YTX, thought to be a shellfish metabolite associated with YTX elimination, contributed on average 26% in mussels. Although the correlation between 45−OH YTX abundance and the speed of YTX depuration could not be confirmed, we noted the half-life of YTX was more than two-times longer in queen scallops, which contained 100% YTX, than in mussels. No other bivalve species were affected by YTXs.This is the first detailed evaluation of AZAs and YTXs occurrences and their profiles in shellfish from Great Britain over a period of multiple years.     

海洋毒素研究进展

从海洋生物中分离出大量结构新颖、生物活性独特的海洋毒素。本文对目前分离得到的海洋毒素进行了总结,包括:河豚毒素、麻痹性贝毒素、腹泻性贝毒素、肝脏毒贝毒素、神经性贝毒素、记忆丧失性贝毒素、西加毒素和其它类型的海洋毒素。海洋毒素不仅可以作为寻找新型防治心血管疾病药物和抗肿瘤药物的先导化合物,还是研究生命科学有用的工具。本文列出了67个重要的海洋毒素的结构并提供70篇参考文献。     

贝类毒素体外细胞毒性作用研究进展

贝类毒素属于非蛋白类高分子化合物,高温处理不能使之失活,严重影响着贝类食品的安全和我国贝类产品的出口贸易。目前贝类毒素最主要的检测方法是小鼠生物法(MBA),然而随着3R理念的推动,毒性评价的体外细胞毒性替代研究取得很大进展,本文综述了8组贝类毒素体外细胞毒性作用的研究成果。     

Evidence for production of paralytic shellfish toxins by bacteria associated with Alexandrium spp. (Dinophyta) in culture.

A substantial proportion of bacteria from five Alexandrium cultures originally isolated from various countries produced sodium channel blocking (SCB) toxins, as ascertained by mouse neuroblastoma assay. The quantities of SCB toxins produced by bacteria and dinoflagellates were noted, and the limitations in comparing the toxicities of these two organisms are discussed. The chemical nature of the SCB toxins in selected bacterial isolates was determined as paralytic shellfish toxins by pre- and postcolumn high-performance liquid chromatography, capillary electrophoresis-mass spectrometry, and enzyme immunoassay.     

Southern right whale (Eubalaena australis) calf mortality at Península Valdés,Argentina: Are harmful algal blooms to blame?

Península Valdés (PV) in Argentina is an important calving ground for southern right whales (SRWs, Eubalaena australis). Since 2005, right whale mortality has increased at PV, with most of the deaths (~90%) being calves <3 mo old. We investigated the potential involvement of harmful algal blooms (HABs) in these deaths by examining data that include: timing of the SRW deaths, biotoxins in samples from dead SRWs, abundances of the diatom, Pseudo‐nitzschia spp., and the dinoflagellate, Alexandrium tamarense, shellfish harvesting closure dates, seasonal availability of whale prey at PV and satellite chlorophyll data. Evidence of the whales' exposure to HAB toxins includes trace levels of paralytic shellfish toxins (PSTs) and domoic acid (DA) in tissues of some dead whales, and fragments of Pseudo‐nitzschia spp. frustules in whale feces. Additionally, whales are present at PV during both closures of the shellfish industry (due to high levels of PSTs) and periods with high levels of Pseudo‐nitzschia spp. and A. tamarense. There is a positive statistical relationship between monthly Pseudo‐nitzschia densities (but not A. tamarense) and calf deaths in both gulfs of PV.     

扇贝毒素pectenotoxins(PTXs)研究进展

扇贝毒素(pectenotoxins,PTXs)是一类聚醚大环内酯结构的脂溶性海洋生物毒素,是由海洋甲藻中的鳍藻属Dinophysisspp.的几个种产生的,1984年首次从日本的养殖扇贝Patinopecten yessoensis中发现鉴定,具有很高的小鼠腹腔注射致死毒性。近年发现的地理区域不断扩大,我国尚属空白。就这一毒素的结构、来源生物、毒性、携带生物、地理分布、降解代谢及风险评估等研究现状作一系统综述,并分析展望了今后我国藻毒素研究的重点方向。     

中国东海和南海有害赤潮高发区麻痹性贝毒素研究

用小白鼠生物检测法和高效液相色谱法对采自浙江舟山和广东深圳海域贝类的麻痹性贝毒素进行了调查和分析,结果表明,舟山海域近岸的贝类毒素检出率为14％,染毒的贝类毒素含量不高,低于小白鼠生物检测法的测定范围;深圳近岸贝类毒素检出率为30％以上,华贵栉孔扇贝是主要的染毒贝类,有1个样品毒素含量达5．1Mu·g-1,超出安全食用标准．从深圳大亚湾华贵栉孔扇贝检测出10种麻痹性贝毒素成分,消化腺的主要毒素成分为GTXl+2和GTX5,Cl+2和GTX2+3,而剔除消化腺后其余贝组织的主要成分为neoSTX和GTX5．贝毒素主要积累在扇贝的消化腺内,消化腺含有的毒素是贝肉组织的8倍．     

Purification of five azaspiracids from mussel samples contaminated with DSP toxins and azaspiracids

Human intoxications during toxic episodes in shellfish are a very important concern for public health, as well as for economic interests of producer regions. Although initially each toxin appeared in a determined geographical zone, nowadays many of them are found in multiple places worldwide. In addition, more toxic compounds (new toxins or new analogs of known toxins) are being isolated and identified, which bring about new risks for public health. An example of this situation is the group of azaspiracids (AZAs). Initially these toxins were concentrated in Irish coasts but today appear in many different geographic locations; in the first toxic episode only three analogs were isolated, but now it is known that the group is comprised of at least eleven identified compounds. A substantial problem associated with all these new toxins is the extreme difficulty associated with the study of their toxic effects and mechanisms of action due to the very small quantities of purified toxin available. Therefore, the study of procedures to isolate them from contaminated shellfish or to synthesize them is of tremendous importance. In this paper we design a complete procedure to obtain AZAs analogs from mussels contaminated with DSP toxins and azaspiracids by means of three consecutive steps: an extraction procedure to remove toxins from shellfish, a solid phase extraction (SPE) to clean the samples and separate DSP toxins and AZAs, and a preparative HPLC to isolate each analog. In all the steps LC/MS is used to detect and quantify the toxins. Large amounts of AZA1, AZA2, AZA3, AZA4 and AZA5 were obtained by use of this procedure, which can be utilized in future studies relating to the toxins such as the production of certified materials and standards.     

Biotechnological significance of toxic marine dinoflagellates

Dinoflagellates are microalgae that are associated with the production of many marine toxins. These toxins poison fish, other wildlife and humans. Dinoflagellate-associated human poisonings include paralytic shellfish poisoning, diarrhetic shellfish poisoning, neurotoxic shellfish poisoning, and ciguatera fish poisoning. Dinoflagellate toxins and bioactives are of increasing interest because of their commercial impact, influence on safety of seafood, and potential medical and other applications. This review discusses biotechnological methods of identifying toxic dinoflagellates and detecting their toxins. Potential applications of the toxins are discussed. A lack of sufficient quantities of toxins for investigational purposes remains a significant limitation. Producing quantities of dinoflagellate bioactives requires an ability to mass culture them. Considerations relating to bioreactor culture of generally fragile and slow-growing dinoflagellates are discussed. Production and processing of dinoflagellates to extract bioactives, require attention to biosafety considerations as outlined in this review.     

Recent trends in paralytic shellfish toxins in Puget Sound,relationships to climate,and capacity for prediction of toxic events

Temporal and spatial trends in paralytic shellfish toxins (PSTs) in Puget Sound shellfish and their relationships with climate are investigated using long-term monitoring data since 1957. Data are selected for trend analyses based on the sensitivity of shellfish species to PSTs and their depuration rates, and the frequency of sample collection at individual sites. These criteria limit the analyses to the shellfish species Mytilus edulis at 20 sites from 1993 to 2007. Blue mussel toxicity is highly variable, but typically exceeds the regulatory limit for human consumption from July to November annually, with most closures occurring early in fall. Using blue mussel data only, we find no robust evidence to suggest that the frequency, magnitude, duration, or geographic scope of PST events in Puget Sound increased between 1993 and 2007. However, there is a significant basin-wide trend for closures to occur earlier in the year. There are no significant correlations between annual indices of mussel toxicity and aspects of the local and large-scale climate. Case studies of daily variations in local environmental factors leading up to exceptionally toxic events identify a combination of conditions that generally precedes most closures from 1993 to 2007. These results suggest that periods of warm air and water temperatures and low streamflow on sub-seasonal timescales may facilitate toxin accumulation in mussels. No relationships were found between water residence times in the surface layer and either streamflow or mussel toxicity. Recommendations are made for future monitoring to improve forecasting of PST risks in Puget Sound, an important region for recreational, commercial, and tribal subsistence shellfish harvesting.     

Determination of paralytic shellfish poisoning (PSP) toxins in UK shellfish

A study was conducted to aid the interpretation of data generated by parallel testing of the qualitative Jellett Rapid Test (JRT) and the mouse bioassay (MBA) for detection of paralytic shellfish poisoning (PSP) toxins within the UK statutory shellfish biotoxin monitoring programme. A selection of stored sample extracts subjected to testing by MBA and/or JRT were further analysed by liquid chromatography with fluorescence detection (LC–FLD) to provide additional information on the concentrations of PSP toxins and toxin profiles.Results, from this study, demonstrate the potential of the JRT to effectively screen out PSP toxin negative shellfish samples and samples containing low concentrations of toxins from UK monitoring programmes. Additionally, data generated using LC–FLD highlights the potential of introducing alternative analytical techniques to completely replace the requirement for the MBA.     

The effect of mussel size, temperature, seston volume, food quality and volume-specific toxin concentration on the uptake rate of PSP toxins by mussels (Mytilus galloprovincialis Lmk)

The accumulation of paralytic shellfish poisoning (PSP) toxins by bivalves is a serious threat to public health all over the world. However, very little is known about the uptake kinetics of these toxins and the environmental factors that may modify this process. We have studied the effect of mussel size, temperature, seston volume, food quality, and volume-specific toxin concentration (VOSTOC), on the uptake rate of paralytic shellfish poisoning (PSP) toxins by mussels (Mytilus galloprovincialis), by means of a second order factorial experiment. Over a 3-day period, the mussels were fed artificial diets containing Alexandrium minutum AL1V (a PSP toxin producer), Tetraselmis suecica, Ensiculifera sp1 and silt, to the levels required by each treatment. Mussel size, seston volume and VOSTOC were found to be statistically significant when the total toxin accumulated per weight of wet tissue was considered. Mussel size affected the uptake negatively and latter two positively. The interactions, mussel size-VOSTOC and mussel size-food quality were also significant. The response was not linear as shown by the significance of the quadratic term of mussel size. Notwithstanding, when the PSP toxins accumulation per mussel was analysed, only one factor, the VOSTOC and the interactions, food quality-mussel size and food quality-seston volume, were found to be significant. VOSTOC was the most important factor in the accumulation of toxins, in our opinion, probably due to toxin assimilation being mainly regulated by the probability of contact between the toxins and the cellular walls of the digestive system. The size of the bivalve is also especially important because toxin concentration is usually calculated per weight of bivalve tissue and because the weight-specific ingestion increases with mussel size. The food quality, which was directly related to the assimilation of organic matter, had an inverse effect on toxin assimilation. In our opinion, this is probably due to the effect of inorganic particles in enhancing the disruption of Alexandrium cells. Temperature had no effect on the uptake rate except for the accumulation of the gonyautoxin GTX1.     

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.     

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.     

Identification of a Saxitoxin Biosynthesis Gene with a History of Frequent Horizontal Gene Transfers

The paralytic shellfish poisoning (PSP) toxins, saxitoxin, and its derivatives, are produced by a complex and unique biosynthetic pathway. It involves reactions that are rare in other metabolic pathways, however, distantly related organisms, such as dinoflagellates and cyanobacteria, produce these toxins by an identical pathway. Speculative explanations for the unusual phylogenetic distribution of this metabolic pathway have been proposed, including a polyphyletic origin, the involvement of symbiotic bacteria, and horizontal gene transfer. This study describes for the first time the identity of one gene, sxt1, that is involved in the biosynthesis of saxitoxin in cyanobacteria. It encoded an O-carbamoyltransferase (OCTASE) that was proposed to carbamoylate the hydroxymethyl side chain of saxitoxin precursor. Orthologues of sxt1 were exclusively present in PSP-toxic strains of cyanobacteria and had a high sequence similarity to each other. L. wollei had a naturally mutated sxt1 gene that encoded an inactive enzyme, and was incapable of producing carbamoylated PSP-toxin analogues, supporting the proposed function of Sxt1. Phylogenetic analysis revealed that OCATSE genes were present exclusively in prokaryotic organisms and were characterized by a high rate of horizontal gene transfer. OCTASE has most likely evolved from an ancestral O-sialoglycoprotein endopeptidase from proteobacteria, whereas the most likely phylogenetic origin of sxt1 was an ancestral alpha-proteobacterium. The phylogeny of sxt1 suggested that the entire set of genes required for saxitoxin biosynthesis may spread by horizontal gene transfer.     

An integrated digital microfluidic bioreactor for fully automatic screening of microalgal growth and stress‐induced lipid accumulation

Algae are the promising feedstock of biofuel. The screening of competent species and proper fertilizer supply is of the most important tasks. To accelerate this rather slow and laborious step, we developed an integrated high‐throughput digital microfluidic (DMF) system that uses a discrete droplet to serve as a microbioreactor, encapsulating microalgal cells. On the basis of fundamental understanding of various droplet hydrodynamics induced by the existence of different sorts of ions and biological species, incorporation of capacitance‐based position estimator, electrode‐saving‐based compensation, and deterministic splitting approach, was performed to optimize the DMF bioreactor. Thus, it enables all processes (e.g., nutrient gradient generation, algae culturing, and analyzing of growth and lipid accumulation) occurring automatically on‐chip especially in a high‐fidelity way. The ability of the system to compare different microalgal strains on‐chip was investigated. Also, the Chlorella sp. were stressed by various conditions and then growth and oil accumulation were analyzed and compared, which demonstrated its potential as a powerful tool to investigate microalgal lipid accumulation at significantly lower laborites and reduced time.