Identification of Monitoring Organ in Bivalves for Early Warning of Paralytic Shellfish Toxins Accumulation

Bivalve farming plays a dominant role in mariculture in China.Paralytic shellfish toxins(PSTs)can be accumulated in bivalves and cause poisoning the consumers.A sensitive detection of PSTs can provide early warning to decrease poisoning events in bivalve consuming.PSTs are traditionally examined using the whole soft-tissues.However,PSTs accumulation varies dramatically in different tissues of bivalves.Some tough tissues/organs(such as mantle),which account for a large proportion of the total soft body,exhibit a lower accumulation of PSTs and make the toxin extraction time-and reagent-consuming,potentially decreasing the accuracy and sensitivity of PSTs monitoring in bivalves.To develop a sensitive and cost-effective approach for PSTs examination in massively farmed bivalves,we fed three commercially important bivalves,Yesso scallop Patinopecten yessoensis,Pacific oyster Crassostrea gigas,and blue mussel Mytilus edulis with PSTs-producing dinoflagellate Alexandrium catenella,and detected PSTs concentration in different tissues.For all three bivalve species,the digestive gland accumulated much more PSTs than other tissues,and the digestive gland’s toxicity was significantly correlated with the PSTs toxicity of the whole soft-tissues,with r^(2)=0.94,0.92,and 0.94 for Yesso scallop,Pacific oyster,and blue mussel,respectively.When the toxicity of the whole soft-tissues reached 80μgSTXeq(100g)^(−1),the regulatory limit for commercial shellfish,the digestive gland’s toxicity reached 571.48,498.90,and 859.20μgSTXeq(100g)^(−1) in Yesso scallop,Pacific oyster,and blue mussel,respectively.Our results indicate that digestive gland can be used for the sensitive and cost-effective monitoring of PSTs in bivalves.

Dinophysis caudata generated lipophilic shellfish toxins in bivalves from the Nanji Islands, East China Sea

A 12-month program of monitoring potentially toxic microalgae(that produce lipophilic shellfi sh toxins; LSTs) and their toxins in bivalves was conducted from April 2006 to March 2007 in the Nanji Islands, East China Sea. Two Dinophysis species, D. caudata and D. acuminata, were identifi ed, and D. caudat a was found to be the dominant species. D. caudata was detected in water samples between April and June 2006, and between February and March 2007. It reached its highest abundances in May, with a mean abundance of 1.38×10 2 cells/L in surface water and 1.25×10 2 cells/L in bottom water(<10 m deep). The temporal distribution of D. caudata was associated with the occurrence of LSTs in bivalve samples, which mostly occurred at the same time as D. caudata blooms, between April and July 2006. All of the cultured bivalves sampled between April and June were contaminated with LSTs, with an average toxicity of 85 μg okadaic acid(OA) eq./100 g meat, which was four times higher than the Chinese regulatory limit(20 μg OA eq./100 g meat). Ten out of fi fteen wild samples(66.7%) collected during the same period were positive for LSTs, and contained an average LST toxicity of 45 μg OA eq./100 g meat(more than twice the regulatory value). Cultured Patinopecten yessoensis collected on 15 May 2006 had the highest toxicity, 320 μg OA eq./100 g meat, and relatively high toxicities(80 to 160 μg OA eq./100 g meat) were found in bivalves until the end of July.

Depuration of paralytic shellfish toxins in Japanese scallop(Patinopecten yessoensis) in natural environment

To study the paralytic shellfish toxins（PSTs） depuration in Japanese scallop Patinopecten yessoensis in natural environment, Japanese scallops naturally contaminated with paralytic shellfish poisoning（PSP） toxins in the Dayao Bay in the northern Huanghai Sea are transited to Qipanmo waters in the Bohai Sea of no reported PSTs incidents. The levels and profile of PSTs during 30-day depuration are detected by the high performance liquid chromatography with fluorescence detection（HPLC-FLD）. The results show that the toxicity of the PSTs in soft tissues decreases to a relatively low level at Day 9. Moreover, the depurated ratio at the early stage of the PSTs depuration is higher than that at the later stage. The toxicity analysis of dissected organs reveals that the digestive gland is the most contaminated PSTs part, which is of important implication for the human health and scallop aquiculture. The mortality of Japanese scallops during PSTs depuration experiment is relevant to PSTs level in the soft tissue.

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.

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.

链状裸甲藻所产麻痹性贝类毒素在翡翠贻贝体内的累积、转化和排出

为研究链状裸甲藻所产麻痹性贝类毒素(paralytic shellfish toxins,PST)在翡翠贻贝体内的累积、转化和排出规律,设置试验组和对照组,采用链状裸甲藻和中肋骨条藻投喂翡翠贻贝,开展短期累积(12 h)、长期累积(10 d)和排出(28 d)试验。结果表明:翡翠贻贝具有较强的毒素累积能力,内脏团是PST累积的主要部位,PST含量与产毒藻密度呈显著正相关关系。当链状裸甲藻密度为1.0×10^(6)cells/L时,贻贝内脏团PST含量累积2 h已接近食用贝类毒素安全标准,累积8 h超标。当产毒藻密度为5.0×10^(5)cells/L时,贻贝内脏团PST含量累积2 d超标,累积8 d达到峰值(3590.4±545.7)μg/kg。贻贝对PST具有累积快排出慢的特点,内脏团PST含量在排出16 d达标,排出速率先快后慢。内脏团对PST的累积和排出速率显著高于闭壳肌和其他组织,闭壳肌和其他组织则无显著差异。PST进入贻贝体内后发生了代谢转化,贻贝可能将产毒藻中膝沟藻毒素GTX3转化为GTX2,N-磺酰氨甲酰基膝沟藻毒素C2转化为C1,部分C1转化为脱氨甲酰基膝沟藻毒素2(dcGTX2),并将高毒的石房蛤毒素(STX)转化为较低毒的脱氨甲酰基石房蛤毒素(dcSTX)。贻贝对dcSTX有较强的累积能力且排出能力较弱,肝胰腺和肾可能是PST累积的主要部位。该研究可为贝类脱毒与净化、赤潮减灾和水产品质量风险防控提供基础数据和理论依据。

Mapping the resting cysts of dinoflagellate Alexandrium catenella along the coast of Qinhuangdao,China

Dinoflagellate Alexandrium catenella is a cosmopolitan bloom-forming species with complex life cycle,the formation and germination of resting cysts are critical for its bloom dynamics.In the coastal waters of Qinhuangdao,A.catenella has been identified as the major causative agent for paralytic shellfish poisoning,but there is little knowledge concerning its resting cysts in this region.In this study,three surveys were carried out along the coast of Qinhuangdao from 2020 to 2021 to map the distribution of A.catenella resting cysts,using a quantitative PCR(qPCR)assay specific for A.catenella.The resting cysts were detected in surface sediments during all the three surveys,and their distribution patterns were similar.High abundance of resting cysts(maximum 1300 cysts/g sediment(wet weight))were found in a region(119.62°E-119.99°E,39.67°N-39.98°N)northeast to the coastal waters of Qinhuangdao,where surface sediments were mainly composed of clay and silt(percentage above 50%).Prior to the formation of the A.catenella bloom in March 2021,the abundance of A.catenella vegetative cells in seawater had extremely significant positive correlation with the abundance of resting cysts in surface sediments,reflecting the important role of resting cysts in the initiation of A.catenella blooms.As far as we know,this is the first report on the distribution of A.catenella cysts along the coast of Qinhuangdao.The results will of fer a sound basis for the future monitoring and mitigation of toxic A.catenella blooms and paralytic shellfish poisoning events in this region.

Glutathione S-transferase genes in scallops and their diverse expression patterns after exposure to PST-producing dinoflagellates

The glutathione S-transferases(GSTs)are a superfamily of enzymes that function in cellular protection against toxic substances and oxidative stress.Bivalves could accumulate high concentration of paralytic shellfish toxins(PSTs)from harmful algae.To understand the possible involvement of GSTs in protecting bivalves during PST accumulation and metabolism,the GST genes were systemically analyzed in two cultured scallops,Azumapecten farreri and Mizuhopecten yessoensis,which were reported for PST deposition during harmful algae bloom.A total of 35 and 37 GSTs were identified in A.farreri(AfGSTs)and M.yessoensis(MyGSTs)genome,respectively,and the expansion of the sigma class from the cytosolic subfamily was observed.In both scallop species,sigma class GSTs showed higher expression than other members.The high GSTs expression was detected mainly during/after larvae stages and in the two most toxic organs,hepatopancreas and kidney.After ingesting PST-producing dinoflagellates,all the regulated AfGSTs in the hepatopancrcas were from the sigma class,but with opposite regulation pattern between Alexandrium catenella and A.minutum exposure.In scallop kidneys,where PSTs transformed into higher toxicity,more AfGSTs were regulated than in the hepatopancreas,and most of them were from the sigma class,with similar regulation pattern between A.catenella and A.minutum exposure.In M.yessoensis exposed to A.catenella,MyGST-σ2 was the only up-regulated MyGST in both hepatopancreas and kidney.Our results suggested the possible diverse function of scallop GSTs and the importance of sigma class in the defense against PSTs,which would contribute to the adaptive evolution of scallops in marine environments.