Comparative study on in vitro transformation of paralytic shellfish poisoning (PSP) toxins in different shellfish tissues

Dissected tissues of three shellfish species, the Chinese scallop, Chlamys farreri, Manila clam, Ruditapes philippinarurn, and Razor shell, Solen strictu were evaluated for in vitro transformation of paralytic shellfish poisoning （PSP） toxins. Tissue homogenates were incubated with extraction from toxic algae Alexandriurn rninutura to determine toxin conversion. The effects of heating and addition of a natural reductant （glutathione） on toxin conversion were also assessed. The toxin profile was investigated through high performance liquid chromatography with fluorescence detection （HPLC-FLD）. The evident variations in the toxin content were observed only in Chinese scallop viscera homogenates. The concentration of GTX4 was reduced by 45% （approximately 0.8 μmol/dm^3） and 25% （approximately 1 μmol/dm^3） for GTX1, while GTX2 and GTX3 increased by six times （approximately 1 μmol/dm^3） and 3 times （approximately 0.3μmol/dm^3） respectively. Simultaneously, the total toxicity decreased by 38% during the 48 h incubation period, the final toxicity was 20.4 nmol STXeq/g. Furthermore, heated Chinese scallop viscera homogenates samples were compared with non-heated samples. The concentration of the GTX4 and GTX1 was clearly 28% （approximately 0.53 μmol/dm^3） and 17% （approximately 0.69μmol/dm^3） higher in heated samples, GTX2 and GTX3 were four times （0.66 μmol/dm^3） and two times （0.187 μmol/dm^3） lower respectively. GSH （＋） Chinese scallop viscera homogenates samples were compared with GSH （-） samples, the concentration in the GTX4 and GTX1 was 9% （approximately 0.12 μmol/dm^3） and 11% （approximately 0.36 μmol/dm^3） lower respectively, GTX2 and GTX3 was 17% （approximately 0.14 μmol/dm^3） and 19% （approximately 0.006 μmol/dm^3） higher respectively. In contrast,there was a little change in the concentration of PSP toxins of Manila clam and Razor shell tissue ho- mogenates. These observations on three shellfish tissues confirmed that there were species-specific differences in PSP toxins transformation. PSP toxins transformation was more pronounced in viscera tissue than in muscle tissue. PSP toxins was possibly interfered by some carbamoylase enzyme, and the activity in Chinese scallop viscera tissue is more remarkable than in the other two species.

A new simple screening method for the detection of paralytic shellfish poisoning toxins

The current testing for paralytic shellfish poisoning(PSP) in shellfish is based on the mouse bioassay(MBA).To alleviate animal welfare concerns,we evaluated the utility of using sublethal indicators of toxicity as an alternative to measuring time to death.Live mice were injected with a PSP congener and the changes in neurotransmitter levels were measured 60,90,and 120 min after injection.Acetylcholine(ACh) was the most sensitive marker for PSP toxicity.The changes in neurotransmitter levels were most pronounced in the blood.Thus,measurement of Ach levels in the blood may serve as a sensitive predictor for PSP that would not require sacrifice of the mice.This method was relatively simple,sensitive(less than 1 μg/kg weight,equivalent to 20 ng/mL),low maintenance,and rapid(less than 60 min).

Evaluation of mouse bioassay results in an inter-laboratory comparison for paralytic shellfish poisoning toxins

An inter-laboratory comparison of the AOAC mouse bioassay for paralytic shellfish poisoning (PSP) toxicity in shellfish was carried out among 25 Chinese laboratories to examine the overall performance for PSP testing in China, and to analyze the main factors affecting the performance of this method. The toxic scallop Patinopecten yessoensis collected from coast of Bohai Sea, China, was used as a test sample in the comparison study. The results were reported and evaluated using robust statistical methods. The z scores showed that 80%, 8%, and 12% of laboratories reported satisfactory results, unsatisfactory results, and questionable results, respectively. This evaluation demonstrates that the PSP mouse bioassay is an appropriate method for screening and testing PSP toxicity in shellfish. However, it was found that the experience and skill of technicians, as well as the body weight and health status of mice being used significantly affected the accuracy of the method.

Transfer of Paralytic Shellfish Toxins via Marine Food Chains:A Simulated Experiment

Objective To study the transfer of paralytic shellfish toxins （PST） using four simulated marine food chains： dinoflagellate Alexandrium tamarense→Artemia Artemia salina→Mysid shrimp Neomysis awatschensis; A. tamarense→N. awatschensis; A. tamarense→A, salina→Perch Lateolabrax japonicus; and A. tamarense→L, japonicus. Methods The ingestion of A. tamarense, a producer of PST, by L. japonicus, N. awatschensis, and A. salina was first confirmed by microscopic observation of A. tamarense cells in the intestine samples of the three different organisms, and by the analysis of Chl.a levels in the samples. Toxin accumulation in L. japonicus and N. awatschensis directly from the feeding on A. tamarense or indirectly through the vector of A. salina was then studied, The toxicity of samples was measured using the AOAC mouse bioassay method, and the toxin content and profile of A. tamarense were analyzed by the HPLC method. Results Both A. salina and N. awatschensis could ingest A. tamarense cells. However, the ingestion capability of A. salina exceeded that of N. awatschensis. After the exposure to the culture of A. tamarense （2 000 cells·mL^-1） for 70 minutes, the content of Chl.a in A. salina and N. awatschensis reached 0.87 and 0.024 μg.mg^-1, respectively. Besides, A. tamarense cells existed in the intestines of L. japonicus, N. awatschensis and A. salina by microscopic observation. Therefore, the three organisms could ingest A. tamarense cells directly. A. salina could accumulate high content of PST, and the toxicity of A. salina in samples collected on days 1, 4, and 5 of the experiment was 2.18, 2.6, and 2.1 MU.g^-1, respectively. All extracts from the samples could lead to death of tested mice within 7 minutes, and the toxin content in anemia sample collected on the 1st day was estimated to be 1.65×10 ^5 μg STX equal/individual. Toxin accumulation in L japonicus and N. awatschensis directly from the feeding on A. tamarense or indirectly from the vector ofA. salina was also studied. The mice injected with extracts from L japonicus and N. awatschensis samples that accumulated PST either directly or indirectly showed PST intoxication symptoms, indicating that low levels of PST existed in these samples. Conclusion Paralytic shellfish toxins can be transferred to L. japonicus, N. awatschensis, and A. salina from A. tamarense directly or indirectly via the food chains.

Identifi cation of paralytic shellfi sh toxin-producing microalgae using machine learning and deep learning methods

Paralytic shellfi sh poisoning(PSP)microalgae,as one of the harmful algal blooms,causes great damage to the of fshore fi shery,marine culture,and marine ecological environment.At present,there is no technique for real-time accurate identifi cation of toxic microalgae,by combining three-dimensional fluorescence with machine learning(ML)and deep learning(DL),we developed methods to classify the PSP and non-PSP microalgae.The average classifi cation accuracies of these two methods for microalgae are above 90%,and the accuracies for discriminating 12 microalgae species in PSP and non-PSP microalgae are above 94%.When the emission wavelength is 650-690 nm,the fl uorescence characteristics bands(excitation wavelength)occur dif ferently at 410-480 nm and 500-560 nm for PSP and non-PSP microalgae,respectively.The identification accuracies of ML models(support vector machine(SVM),and k-nearest neighbor rule(k-NN)),and DL model(convolutional neural network(CNN))to PSP microalgae are 96.25%,96.36%,and 95.88%respectively,indicating that ML and DL are suitable for the classifi cation of toxic microalgae.

微小亚历山大藻对近江牡蛎清滤率以及麻痹性贝类毒素(PSP)蓄积的影响

为获知近江牡蛎对有毒微小亚历山大藻的滤食以及蓄积PSP毒素的规律性，本实验采用细胞毒性为（93．42±2．55）×10^3MU／个的微小亚历山大藻投喂近江牡蛎，以无毒扁藻投喂作对照，首先研究了0．5×10^3～3．0×10^3个／mL6个不同浓度的毒藻对近江牡蛎滤食的影响，然后投喂浓度为1．5×10^3个／mL的毒藻，分别研究15h短期蓄积和5d长期蓄积对近江牡蛎体内毒素的影响及规律。结果表明，在0．5×10^3～1．5×10^3个／mL毒藻浓度范围内牡蛎清滤率较高，超过1．5×10^3个／mL时牡蛎清滤率显著下降（P〈0．01），由此确定后期投喂毒藻的浓度“阀值”为1．5×10^3个／mL；短期蓄积实验牡蛎在0～6h内将有毒藻滤食尽，其体内PSP毒素在6—12h间达到最大值[（149．6±10．5）MU／100g]，随后开始下降。长期蓄积实验过程中，牡蛎的清滤率没有发生显著变化（P〉0．01），在蓄积实验的第2天牡蛎体内PSP毒素水平超过国家限量标准（400MU／100g），在5d实验结束时，牡蛎体内PSP毒素水平高达（3069．2±178．2）MU／100g，高于国家限量标准的7．7倍。实验结果作为PSP在近江牡蛎中蓄积及代谢的重要基础数据，为研究PSP的脱除及净化提供思路。

贝类中PSP毒素的脱除方法研究进展

为探讨麻痹性贝类毒素(PSP)的脱除方法,有效提高贝类食品安全性。本文从物理法、化学法以及生物法三个方面综述了PSP毒素在水产品中的脱除方法及净化效率;对温度处理,安全吸附,微生物降解,酶解转化等的研究成果进行了分析总结,以期为贝类脱除PSP毒素及贝类净化研究提供参考。

A Transcriptome Analysis of Neural Tissue of Litopenaeus vannamei After Acute Exposure to Alexandrium pacificum

Alexandrium pacificum(A.pacificum)is a typical paralytic shellfish poisonous dinoflagellate.Harmful algal blooms(HABs)caused by this species can bring serious environmental problems and economic losses to the aquaculture industry.In this study,transcriptome sequencing and analyses were performed on the neural tissue of Litopenaeus vannamei(L.vannamei)after acute exposure to A.pacificum disrupted solution for 72 h,and differentially expressed genes(DEGs)were identified.The results showed that,compared with the control samples,300 DEGs were identified in the experimental group,of which 194 were up-regulated,and 106 down-regulated.The gene ontology(GO)functional enrichment analysis showed that DEGs were significantly enriched in the cortical cytoskeleton organization,troponin complex,amylo-alpha-1,6-glucosidase and thymidine phosphorylase.Kyoto encyclopedia of genes and genomes(KEGG)enrichment analysis found that DEGs were mainly enriched in the oxidative phosphorylation process,intercellular tight junctions and mitophagy.The results showed that the proteoglycans,signaling pathways,and various metabolic processes that regulate cell proliferation,differentiation,and apoptosis all played an essential role in the response of L.vannamei to A.pacificum toxins.

致瘫贝类毒素(PSP)对鳗鱼心血管系统的影响

为了探讨PSP的降压作用是否与心脏直接有关,研究了PSP对活体和离体状态下的鳗鱼心血管系统的影响。PSP的剂量等于或大于0.4 Mu/Kg可导致体动脉血压显著下降。体动脉血压的变化与心室内压力的变化相平行,而与心率无对应的关系。PSP对鳗鱼离体心脏收缩力具有明显的影响,0.4 Mu/Kg的剂量可使心脏收缩力比对照值减少21.53％(P<0.05)。0.8 Mu/Kg的PSP阻断房室间的传导,此时心室停止跳动,而心房仍在跳动。进一步的研究表明,心脏收缩力的下降并不是由于心肌本身的收缩特性受到影响,而是由于心脏传导被逐渐阻断的结果。心脏传导阻断最容易发生在房室结部,其次在心肌细胞与细胞之间。

两种织纹螺对TTX和PSP摄食转移的初步研究

织纹螺(Nassariusspp.)是沿海地区较常见的螺种之一,对其带毒途径现还存有分歧.本文选择厦门海域常见两种织纹螺,粗肋织纹螺(Nassarius nodiferus)和红带织纹螺(Nassarius succinctus)作为研究对象,以实验室生态环境下培养的塔玛亚历山大藻和月腹刺鲀内脏投喂织纹螺.采用美国分析化学家协会推荐的小白鼠的生物检测法,对织纹螺进行毒素检测.小白鼠生物检测结果表明,红带织纹螺本身不带毒性,而粗肋织纹螺本身带有毒性,麻痹性贝毒和河豚毒素两种毒素都可以通过摄食转移累积毒性.

渤海海域唐山贝类养殖区腹泻性和麻痹性贝类毒素的监测与风险评估

为监测渤海海域唐山贝类养殖区贝类毒素的污染情况,防止食用贝类中毒事件发生,于2019年10月—2020年9月间,每月持续在渤海海域唐山贝类养殖区采集四角蛤(Mactra veneriformis)、菲律宾蛤仔(Ruditapes philippinarum)、脉红螺(Rapana venosa)、牡蛎(Crassostrea gigas)、青蛤(Cyclina sinensis)、文蛤(Meretrix meretrix)和硬壳蛤(Mercenaria mercenaria)7种经济贝类样品,采用高效液相色谱–串联质谱(HPLC-MS/MS)法测试了5种腹泻性贝类毒素(diarrhetic shellfish poisoning,DSP)和14种麻痹性贝类毒素(paralytic shellfish poisoning,PSP)。结果显示,在7种经济贝类样品中均未检出DSP。检出的PSP成分包括石房蛤毒素(Saxitoxin,STX)、膝沟藻毒素1(Gonyautoxin 1,GTX 1)、膝沟藻毒素2(Gonyautoxin 2,GTX 2)和脱氨甲酰基膝沟藻毒素3(Ddecarbamoy l gonyautoxin 3,dcGTX 3),其中,GTX 1含量最高且最高值为537.95μg/kg。不同季节贝类毒素蓄积含量有一定差异,PSP主要集中在4月检出。菲律宾蛤仔、牡蛎、文蛤和硬壳蛤中PSP的检出率分别为11.76%、47.06%、5.90%和8.82%,其他贝类均未检出。PSP总量均低于欧盟及中国的食用安全限量标准800μg STXeq/kg。应用风险熵值法和点评估法进行食用安全风险评估,显示风险熵值和暴露风险指数均在安全范围内,结果表明,渤海海域唐山贝类养殖区7种经济贝类不存在食用安全风险。

牡蛎基体麻痹性贝类毒素GTX1-4标准样品的研制

贝类食品安全是重大公共卫生问题,然而贝类毒素标准品和标准样品均很难获得。将天然产生麻痹性贝类毒素膝沟藻毒素1-4(Gonyaulax toxin 1-4,GTX1-4)的链状裸甲藻培养液与牡蛎肉均质混匀,冷冻干燥脱水后,反复过筛分处理制备成松散状细粉。采用液相色谱-串联质谱法(Liquid chromatography-tandem mass spectrometry,LC-MS/MS)测定牡蛎基体中GTX1-4含量,并比较不同制备工艺对特性值分散度的影响。样品分装成500瓶,进行均匀性、稳定性、赋值及不确定度评估。结果表明,标准样品均匀性、长期稳定性和模拟运输条件下的短期稳定性良好,特性参考值可靠,达到标准样品的技术要求,为有效开展实验室质量控制和能力评价活动,保护消费者安全具有重要意义。

大亚湾和大鹏湾麻痹性贝类毒素动态分析

于 1 997～ 1 999年在大亚湾和大鹏湾的主要贝类养殖区设立了四个采样点 ,以华贵栉孔扇贝和翡翠贻贝为对象逐月采样 ,用美国分析化学家协会推荐的生物测定法进行麻痹性贝类毒素测定。结果显示 ,1 999年 1月～ 1 999年 6月大亚湾东山海域的扇贝消化腺毒素含量为 1 2 63～ 1 0 5Mu/g ,剔除消化腺后的其他软组织毒素含量为 1 3 4至低于 1 7Mu/g ,消化腺和其他软组织毒素含量的加权平均值为 7 7～ 1 1 7Mu/g ,高于联合国粮农组织制定的贝类安全食用标准的限定值 4Mu/g ,尤其是 1月份 ,超过限定值近 30倍。同期该样点的贻贝消化腺毒素含量为 0～ 567Mu/g ,剔除消化腺后的其他软组织毒素含量为 0～ 1 4 2Mu/g ,消化腺和其他软组织毒素含量的加权平均值为 0～ 72 8Mu/g ,仅在 1月份贻贝毒素含量超标。总体上贝毒含量表现为由冬春季高峰期逐月波动下降的趋势。大亚湾澳头海域的两种贝类毒素含量都不高 ,扇贝长期含有较低的毒素 ,贻贝在大部分时间内不含毒素 ,仅在 1 998年 5月间 ,出现一低于 3 5Mu/g的高峰期。大鹏湾南澳海域的两种贝类毒素含量不高 ,贻贝毒素含量均低于生物测定法极限值 ,而扇贝消化腺毒素含量仅 1 998～ 1 999年冬季略有超标。本文还就麻痹性贝毒的分布与贝类种类、时空间的关系进行?

长江以北沿海经济贝类中的麻痹性贝毒

应用小白鼠检测方法，对我国长江以北沿岸的经济贝类进行了麻痹性贝毒检测。采样时间为１９９７ 年９ 月２３ 日～１２ 月８ 日。从鸭绿江口到长江口共调查２０ 个站位，检测经济贝类１４ 种，样品３８ 份，其中只有１ 份样品检测出麻痹性贝毒（６４ Ｍｕ／ｇ） 。

10种麻痹性贝类毒素的固相萃取及液相色谱-串联质谱测定法

建立了10种麻痹性贝类毒素（PSP）的高效液相色谱-串联质谱检测方法。样品用0.5%甲酸溶液加热提取,提取液经乙酸乙酯、三氯甲烷和HLB固相萃取柱净化,乙腈去蛋白后,在正离子模式下以电喷雾串联质谱仪进行测定,TSK改性凝胶柱为色谱分离柱,5 mmol·L-1甲酸铵（含0.5%甲酸）-乙腈（含0.5%甲酸）为流动相,采用基质匹配的标准曲线进行定量,并以虾夷扇贝（Patinopecten yessoensis）和菲律宾蛤仔（Ruditapes philippinarum）为测试对象,对方法的有效性进行了评价。评价结果为：在50～600μg·kg-1添加水平下,10种麻痹性贝类毒素平均回收率为72.3%～91.1%（批内）,相对标准偏差为3.9%～9.5%（批内）。STX、dc STX和neo STX定量限（S/N〉10）为100μg·kg-1,GTX1～GTX5、dc GTX2和dc GTX3定量限为50.0μg·kg-1。结果表明,该方法快速、准确,适合于贝类产品中麻痹性贝类毒素的测定。

广东沿海麻痹性贝类毒素的研究

于1990－1992年间在广东沿海采集24种贝类，用麻痹性贝类毒素小白鼠生物测定法对所采样品进行毒素分析研究。结果表明，所检测的大部分生物体均不同程度的积累了麻痹性毒素；大亚湾和大鹏湾2种贝体内存在着12或13种麻痹性毒素的成分；贝体中麻痹性毒素存在着一定的季节差异。还对贝体中的毒素和有毒藻类的出现进行了简单的讨论。

广州市售贝类麻痹性贝毒和腹泻性贝毒污染状况分析

目的对广州市售双壳经济贝类麻痹性贝毒(PSP)和腹泻性贝毒(DSP)污染状况进行为期一年的抽样调查,了解其食用安全性。方法采用AOAC推荐的小鼠生物检测法进行PSP和DSP的毒力测定,采用HPLC进行PSP成分分析,根据FAO、日本和欧盟水产食品卫生要求及我国渔政渔港监督管理局制订的贝类安全食用标准对贝类水产品的食用安全性进行评价。结果在所调查的7种贝类中,有2种染有PSP,毒素含量在安全食用范围内,毒力大小随季节而变化,春冬两季含量相对较高;7种贝类中有6种共计36个样品染有DSP,有10个样品毒素含量超出安全食用标准,春冬两季染毒率较高。结论广州市售贝类PSP含量和检出率整体水平较低;DSP检出率稍高,毒素含量也较高,应引起有关部门的关注,加强DSP监测工作。

几种环境因子对塔玛亚历山大藻(大鹏湾株)生长及其藻毒力影响

在室内条件下研究了温度 (θ/℃ )、盐度 (ρ/gL-1)及pH对有毒甲藻塔玛亚历山大藻 (大鹏株 )的生长及其毒力的影响 .实验表明 ,塔玛亚历山大藻θopt为 15～ 2 5℃ ,最大生长率出现在接种后 6～ 8d ;在盐度为 14～32g/L范围内 ,该藻均可生长 ,盐度 2 3～ 2 7g/L时生长最佳 ;在弱酸弱碱下 ,该藻可较好生长 ,pHopt=6～ 7;用小白鼠法测得本藻株c(HCl) =0 .1mol/L提液的麻痹性贝毒毒力为 0 .5 0× 10 -5～ 3.2× 10 -5Mu/cell,在同种藻株中属低毒藻株 .与其他作者对本藻株用不同毒素抽提方法及测定技术所得结果的比较表明 ,其藻毒力测定值最大相差达 30倍 .图 3表 2参

大亚湾麻痹性贝类毒素HPLC分析

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