Formation of a Volunteer Harmful Algal Bloom Network in British Columbia,Canada, Following an Outbreak of Diarrhetic Shellfish Poisoning

Evidence for shellfish toxin illness in British Columbia (BC) on the west coast of Canada can be traced back to 1793. For over two hundred years, domestically acquired bivalve shellfish toxin illnesses in BC were solely ascribed to paralytic shellfish poisonings caused by algal blooms of Alexandrium. This changed in 2011, when BC experienced its first outbreak of diarrhetic shellfish poisoning (DSP). As a result of this outbreak, Canada’s first DSP symposium was held in November, 2012, in North Vancouver, BC. Three of the objectives of the symposium were to provide a forum to educate key stakeholders on this emerging issue, to identify research and surveillance priorities and to create a DSP network. The purpose of this paper is to review what is known about shellfish poisoning in BC and to describe a novel volunteer network that arose following the symposium. The newly formed network was designed for industry shellfish growers to identify harmful algae bloom events, so that they may take actions to mitigate the effects of harmful blooms on shellfish morbidity. The network will also inform public health and regulatory stakeholders of potentially emerging issues in shellfish growing areas.     

Okadaic Acid Meet and Greet: An Insight into Detection Methods,Response Strategies and Genotoxic Effects in Marine Invertebrates

Harmful Algal Blooms (HABs) constitute one of the most important sources of contamination in the oceans, producing high concentrations of potentially harmful biotoxins that are accumulated across the food chains. One such biotoxin, Okadaic Acid (OA), is produced by marine dinoflagellates and subsequently accumulated within the tissues of filtering marine organisms feeding on HABs, rapidly spreading to their predators in the food chain and eventually reaching human consumers causing Diarrhetic Shellfish Poisoning (DSP) syndrome. While numerous studies have thoroughly evaluated the effects of OA in mammals, the attention drawn to marine organisms in this regard has been scarce, even though they constitute primary targets for this biotoxin. With this in mind, the present work aimed to provide a timely and comprehensive insight into the current literature on the effect of OA in marine invertebrates, along with the strategies developed by these organisms to respond to its toxic effect together with the most important methods and techniques used for OA detection and evaluation.     

Shellfish Toxins Targeting Voltage-Gated Sodium Channels

Voltage-gated sodium channels (VGSCs) play a central role in the generation and propagation of action potentials in excitable neurons and other cells and are targeted by commonly used local anesthetics, antiarrhythmics, and anticonvulsants. They are also common targets of neurotoxins including shellfish toxins. Shellfish toxins are a variety of toxic secondary metabolites produced by prokaryotic cyanobacteria and eukaryotic dinoflagellates in both marine and fresh water systems, which can accumulate in marine animals via the food chain. Consumption of shellfish toxin-contaminated seafood may result in potentially fatal human shellfish poisoning. This article provides an overview of the structure, bioactivity, and pharmacology of shellfish toxins that act on VGSCs, along with a brief discussion on their pharmaceutical potential for pain management.     

Domoic acid toxicologic pathology: a review

Domoic acid was identified as the toxin responsible for an outbreak of human poisoning that occurred in Canada in 1987 following consumption of contaminated blue mussels [Mytilus edulis]. The poisoning was characterized by a constellation of clinical symptoms and signs. Among the most prominent features described was memory impairment which led to the name Amnesic Shellfish Poisoning [ASP]. Domoic acid is produced by certain marine organisms, such as the red alga Chondria armata and planktonic diatom of the genus Pseudo-nitzschia. Since 1987, monitoring programs have been successful in preventing other human incidents of ASP. However, there are documented cases of domoic acid intoxication in wild animals and outbreaks of coastal water contamination in many regions world-wide. Hence domoic acid continues to pose a global risk to the health and safety of humans and wildlife. Several mechanisms have been implicated as mediators for the effects of domoic acid. Of particular importance is the role played by glutamate receptors as mediators of excitatory neurotransmission and the demonstration of a wide distribution of these receptors outside the central nervous system, prompting the attention to other tissues as potential target sites. The aim of this document is to provide a comprehensive review of ASP, DOM induced pathology including ultrastructural changes associated to subchronic oral exposure, and discussion of key proposed mechanisms of cell/tissue injury involved in DOM induced brain pathology and considerations relevant to food safety and human health.     

Azaspiracid shellfish poisoning: a review on the chemistry, ecology, and toxicology with an emphasis on human health impacts

Azaspiracids (AZA) are polyether marine toxins that accumulate in various shellfish species and have been associated with severe gastrointestinal human intoxications since 1995. This toxin class has since been reported from several countries, including Morocco and much of western Europe. A regulatory limit of 160 microg AZA/kg whole shellfish flesh was established by the EU in order to protect human health; however, in some cases, AZA concentrations far exceed the action level. Herein we discuss recent advances on the chemistry of various AZA analogs, review the ecology of AZAs, including the putative progenitor algal species, collectively interpret the in vitro and in vivo data on the toxicology of AZAs relating to human health issues, and outline the European legislature associated with AZAs.     

A Feedback Mechanism to Control Apoptosis Occurs in the Digestive Gland of the Oyster Crassostrea gigas Exposed to the Paralytic Shellfish Toxins Producer Alexandrium catenella

To better understand the effect of Paralytic Shellfish Toxins (PSTs) accumulation in the digestive gland of the Pacific oyster, Crassostrea gigas, we experimentally exposed individual oysters for 48 h to a PSTs producer, the dinoflagellate Alexandrium catenella. In comparison to the effect of the non-toxic Alexandrium tamarense, on the eight apoptotic related genes tested, Bax and BI.1 were significantly upregulated in oysters exposed 48 h to A. catenella. Among the five detoxification related genes tested, the expression of cytochrome P450 (CYP1A) was shown to be correlated with toxin concentration in the digestive gland of oysters exposed to the toxic dinoflagellate. Beside this, we observed a significant increase in ROS production, a decrease in caspase-3/7 activity and normal percentage of apoptotic cells in this tissue. Taken together, these results suggest a feedback mechanism, which may occur in the digestive gland where BI.1 could play a key role in preventing the induction of apoptosis by PSTs. Moreover, the expression of CYP1A, Bax and BI.1 were found to be significantly correlated to the occurrence of natural toxic events, suggesting that the expression of these genes together could be used as biomarker to assess the biological responses of oysters to stress caused by PSTs.     

Screening Tests for the Rapid Detection of Diarrhetic Shellfish Toxins in Washington State

The illness of three people due to diarrhetic shellfish poisoning (DSP) following their ingestion of recreationally harvested mussels from Sequim Bay State Park in the summer of 2011, resulted in intensified monitoring for diarrhetic shellfish toxins (DSTs) in Washington State. Rapid testing at remote sites was proposed as a means to provide early warning of DST events in order to protect human health and allow growers to test “pre-harvest” shellfish samples, thereby preventing harvest of toxic product that would later be destroyed or recalled. Tissue homogenates from several shellfish species collected from two sites in Sequim Bay, WA in the summer 2012, as well as other sites throughout Puget Sound, were analyzed using three rapid screening methods: a lateral flow antibody-based test strip (Jellett Rapid Test), an enzyme-linked immunosorbent assay (ELISA) and a protein phosphatase 2A inhibition assay (PP2A). The results were compared to the standard regulatory method of liquid chromatography coupled with tandem mass spectroscopy (LC-MS/MS). The Jellett Rapid Test for DSP gave an unacceptable number of false negatives due to incomplete extraction of DSTs using the manufacturer’s recommended method while the ELISA antibody had low cross-reactivity with dinophysistoxin-1, the major toxin isomer in shellfish from the region. The PP2A test showed the greatest promise as a screening tool for Washington State shellfish harvesters.     

2011年黄淮海夏玉米区二点委夜蛾暴发危害的原因与防治对策

二点委夜蛾Proxenus lepigone(Moschler)是玉米上的新害虫,2005年首先在河北省发现危害夏玉米苗。2011年在黄淮海夏玉米区的河北、山东、河南、安徽、江苏、山西和北京7省(市)暴发危害,对夏玉米的生产造成严重威胁。结合二点委夜蛾近年来的发生情况及研究结果,对2011年二点委夜蛾暴发的原因进行分析,并提出防治对策。     

Biosynthetic Studies on Water-Soluble Derivative 5c (DTX5c)

The dinoflagellate Prorocentrum belizeanum is responsible for the production of several toxins involved in the red tide phenomenon known as Diarrhetic Shellfish Poisoning (DSP). In this paper we report on the biosynthetic origin of an okadaic acid water-soluble ester derivative, DTX5c, on the basis of the spectroscopical analysis of ¹³C enriched samples obtained by addition of labelled sodium [l-¹³C], [2-¹³C] acetate to artificial cultures of this dinoflagellate.     

Yessotoxins, a group of marine polyether toxins: an overview

Yessotoxin (YTX) is a marine polyether toxin that was first isolated in 1986 from the scallop Patinopecten yessoensis. Subsequently, it was reported that YTX is produced by the dinoflagellates Protoceratium reticulatum, Lingulodinium polyedrum and Gonyaulax spinifera. YTXs have been associated with diarrhetic shellfish poisoning (DSP) because they are often simultaneously extracted with DSP toxins, and give positive results when tested in the conventional mouse bioassay for DSP toxins. However, recent evidence suggests that YTXs should be excluded from the DSP toxins group, because unlike okadaic acid (OA) and dinophyisistoxin-1 (DTX-1), YTXs do not cause either diarrhea or inhibition of protein phosphatases. In spite of the increasing number of molecular studies focused on the toxicity of YTX, the precise mechanism of action is currently unknown. Since the discovery of YTX, almost forty new analogues isolated from both mussels and dinoflagellates have been characterized by NMR or LC-MS/MS techniques. These studies indicate a wide variability in the profile and the relative abundance of YTXs in both, bivalves and dinoflagellates. This review covers current knowledge on the origin, producer organisms and vectors, chemical structures, metabolism, biosynthetic origin, toxicological properties, potential risks to human health and advances in detection methods of YTXs.     

Neurotoxins from marine dinoflagellates: a brief review

Dinoflagellates are not only important marine primary producers and grazers, but also the major causative agents of harmful algal blooms. It has been reported that many dinoflagellate species can produce various natural toxins. These toxins can be extremely toxic and many of them are effective at far lower dosages than conventional chemical agents. Consumption of seafood contaminated by algal toxins results in various seafood poisoning syndromes: paralytic shellfish poisoning (PSP), neurotoxic shellfish poisoning (NSP), amnesic shellfish poisoning (ASP), diarrheic shellfish poisoning (DSP), ciguatera fish poisoning (CFP) and azaspiracid shellfish poisoning (ASP). Most of these poisonings are caused by neurotoxins which present themselves with highly specific effects on the nervous system of animals, including humans, by interfering with nerve impulse transmission. Neurotoxins are a varied group of compounds, both chemically and pharmacologically. They vary in both chemical structure and mechanism of action, and produce very distinct biological effects, which provides a potential application of these toxins in pharmacology and toxicology. This review summarizes the origin, structure and clinical symptoms of PSP, NSP, CFP, AZP, yessotoxin and palytoxin produced by marine dinoflagellates, as well as their molecular mechanisms of action on voltage-gated ion channels.     

SYBR Green实时荧光定量PCR检测利玛原甲藻的研究

建立应用SYBR Green实时荧光定量PCR技术快速定量检测利玛原甲藻的方法。结果表明,理论上,该方法对于4个利玛原甲藻细胞便可检测出,灵敏度较高;实际应用时检测出的细胞数为(505±14.72)cells/L(镜检为600 cells/L,误差为15.83%,符合海洋调查规范中浮游生物调查误差在20%以内的要求)。本检测方法具有快速、特异、准确等优点,可作为海洋环境监测中定性定量检测利玛原甲藻的方法。     

Detection of Diarrheic Shellfish Poisoning and Azaspiracid Toxins in Moroccan Mussels: Comparison of the LC-MS Method with the Commercial Immunoassay Kit

Diarrheic shellfish poisoning (DSP) is a recurrent gastrointestinal illness in Morocco, resulting from consumption of contaminated shellfish. In order to develop a rapid and reliable technique for toxins detection, we have compared the results obtained by a commercial immunoassay-“DSP-Check” kit” with those obtained by LC-MS. Both techniques are capable of detecting the toxins in the whole flesh extract which was subjected to prior alkaline hydrolysis in order to detect simultaneously the esterified and non esterified toxin forms. The LC-MS method was found to be able to detect a high level of okadaic acid (OA), low level of dinophysistoxin-2 (DTX2), and surprisingly, traces of azaspiracids 2 (AZA2) in mussels. This is the first report of a survey carried out for azaspiracid (AZP) contamination of shellfish harvested in the coastal areas of Morocco. The “DSP-Check” kit was found to detect quantitatively DSP toxins in all contaminated samples containing only OA, provided that the parent toxins were within the range of detection and was not in an ester form. A good correlation was observed between the two methods when appropriate dilutions were performed. The immunoassay kit appeared to be more sensitive, specific and faster than LC-MS for determination of DSP in total shellfish extract.     

Effect of domestic processing and cooking on selected antinutrient contents of some green leafy vegetables

Three vegetables, 5 roots and tubers, 23 fruits and 3 stored products collected and consumed by tribals of Andhra Pradesh, India were analyzed for total carotenoids (TC) and Beta carotene (BC) contents using High Performance Liquid Chromatography (HPLC). The vitamin A activity expressed as retinol equivalents (RE) was calculated based on the in vivo conversion factor given by WHO. The results of the study indicated that the vegetables, roots and tubers analyzed showed negligible vitamin A activity with TC and BC content ranging from 0.01 to 0.61 and 0.14 to 0.23 mg%, respectively. Among the fruits, Canthium parviflorum contained very high TC (9.51 mg%) and BC (6.10 mg%) contents, and the edible orange color fresh rind portion analyzed had the highest (6.8 mg%) BC content. Other fruits contained negligible content of TC (0.0015 to 1.32 mg%) and BC (0.004 to 0.49 mg%). The edible pulp portion of the fruit (Diosphros melanoxylon contained no BC but the rind portion analyzed fresh and dried had BC) 0.79 and 2.17 mg%, respectively. Tribals were found to have distinct attitudes and beliefs towards food they use or avoid in certain physiological conditions.     

A Microencapsulation Method for Delivering Tetrodotoxin to Bivalves to Investigate Uptake and Accumulation

Most marine biotoxins are produced by microalgae. The neurotoxin tetrodotoxin (TTX) has been reported in many seafood species worldwide but its source is unknown, making accumulation and depuration studies in shellfish difficult. Tetrodotoxin is a water-soluble toxin and cannot be directly ingested by shellfish. In the present study, a method was developed which involved binding TTX to solid particles of humic acid and encapsulating them in agar-gelatin capsules. A controlled quantity of TTX-containing microcapsules (size range 20–280 μm) was fed to Paphies australis, a bivalve known to accumulate TTX in the wild. The TTX-containing microcapsules were fed to P. australis every second day for 13 days. Ten P. australis (including five controls fed non-toxic microalgae) were harvested after 7 days and ten after 13 days. Paphies australis accumulated TTX, reaching concentrations of up to 103 µg kg⁻¹ by day 13, exceeding the European Food Safety Authority recommended concentration of 44 μg kg⁻¹ in shellfish. This novel method will allow future studies to explore the effects, accumulation and depuration rates of TTX in different animals and document how it is transferred through food webs.     

甘蔗细茎野生种(S.spontaneum)在杂交育种中的利用

对甘蔗栽培种与细茎野生种及其F1、BC1杂交获得的细茎野生种的F1、BC1、BC2杂交后代进行了研究.结果表明：甘蔗与细茎野生种及其F1、BC1之间的杂交容易成功,其亲和力强、结实率与杂交真实率都比较高,容易获得它们的真实杂交种,并通过抗性、糖分、产量鉴定,获得一些抗旱、抗寒、抗黑穗病、高产、高糖的优良创新种质,为选育抗逆性、抗病性强、高产、高糖的优良亲本和优良品种奠定了基础.     

Guidance Level for Brevetoxins in French Shellfish

Brevetoxins (BTXs) are marine biotoxins responsible for neurotoxic shellfish poisoning (NSP) after ingestion of contaminated shellfish. NSP is characterized by neurological, gastrointestinal and/or cardiovascular symptoms. The main known producer of BTXs is the dinoflagellate Karenia brevis, but other microalgae are also suspected to synthesize BTX-like compounds. BTXs are currently not regulated in France and in Europe. In November 2018, they have been detected for the first time in France in mussels from a lagoon in the Corsica Island (Mediterranean Sea), as part of the network for monitoring the emergence of marine biotoxins in shellfish. To prevent health risks associated with the consumption of shellfish contaminated with BTXs in France, a working group was set up by the French Agency for Food, Environmental and Occupational Health & Safety (Anses). One of the aims of this working group was to propose a guidance level for the presence of BTXs in shellfish. Toxicological data were too limited to derive an acute oral reference dose (ARfD). Based on human case reports, we identified two lowest-observed-adverse-effect levels (LOAELs). A guidance level of 180 µg BTX-3 eq./kg shellfish meat is proposed, considering a protective default portion size of 400 g shellfish meat.     

Volatile constituents, inorganic elements and primary screening of bioactivity of black coral cigarette holders

Black corals (BC) have been used for a long time in Chinese medicine, and may have some pharmaceutical functions when used as material for cigarette holders in southeast China. This study is aimed to investigate the bioactivities of volatile constituents in BC and to explore the folklore behind the use of BC cigarette holders (BCCHs). We extracted the volatile constituents of BC by supercritical fluid extraction (SFE) with carbon dioxide (CO₂-SFE), then identified and analyzed the constituents by gas chromatography-mass spectrometry (GC-MS). In total, 15 components were reliably identified in BC and found to be biologically active. These included triethyl phosphate, butylated hydroxytoluene, cedrol, n-hexadecanoic acid, squalene, and cholesterol. Meanwhile 13 inorganic elements (P, Ca, Mg, S, B, Si, Fe, Cu, Zn, Ba, etc.) were determined by inductively coupled plasma spectrometer (ICPS). In the bioactivity tests, the BC extract (BCE) showed a scavenging activity of 2,2-diphenyl-1-picrylhydrazyl free radicals and hydroxyl radicals by phenanthroline-Fe (II) oxidation and moderate inhibition of Gram-positive microorganisms. The antioxidant and antimicrobial activities of BC, which are related to the active chemical composition, may explain the perceived benefit for cigarette smokers who use BCCHs.