Saxitoxin in octopi from Cooke Point, Port Hedland, Western Australia

Octopus (Abdopus) sp. 5 were collected from a reef flat at Cooke Point, Port Hedland in northern Western Australia. The octopi had a body size of between 5-6 cm and tentacle span of between 20 and 30 cm.Specimens were euthanased by rapid decapitation, stored at -20°C and shipped frozen to the Australian Institute of Marine Science in Townsville. Three legs from each of the four individuals were pooled for toxin extraction. Each of the four samples was extracted with 80% ethanol, homogenised, sonicated three times for 10 minutes, then clarified by centrifugation. This process was repeated two additional times with fresh extraction solvent. For each of the four extracts, the three supernatants generated were pooled, filtered through 0.2 mm nylon filters and lyophilised. Dried extracts were reconstituted in 0.05 M acetic acid and passed through a centrifugal ultra-filter and the ultra filtrates then used for radio-receptor assay and preliminary chemical analysis.Quantitation of STX concentration equivalents (STXeq) with [³H]-STX radio-receptor assays using saxiphilin and voltage gated sodium channel, was performed and octopus extracts were assayed in triplicate for inhibition of STX binding in the sodium channel and saxiphilin assay.All sample extracts which competed with [³H]-STX in the radio-receptor assays were analysed by LC-FLD for STX, decarbamoylSTX, neoSTX, 11-hydroxysulphate STXs (GTX1-6), N-sulfocarbamoyl-11-hydroxy-sulphate STXs (C1-4), hydroxybenzoate STXs and TTX.Identification of suspect toxin peaks was undertaken by mass spectrometric analysis of separated toxins using liquid chromatography-mass spectrometry (LC-MS) performed in single ion monitoring (SIM) mode.
Previous research indicates that Port Hedland on the north-west coast of Australia is a source of marine animals, encompassing a range of crustacea, gastropod and bivalve molluscs, which harbour both tetrodotoxin (TTX) and paralytic shellfish toxins (PSTs). This study was undertaken as part of a renewed investigation into paralytic shellfish toxin distribution in benthic organisms in this region.
This was the first time that saxitoxin had been reported from this family of predatory molluscs and highlights the need for greater public awareness of risks associated with consumption of previously unrecognised vectors of paralytic shellfish poisoning, such as octopus and other benthic feeders.

胴体长5-6厘米、腕足展开跨度20-30厘米的章鱼属（Abdopus）未定种5（Octopus (Abdopus) sp. 5）样本，采集自澳大利亚西澳大利亚州北部黑德兰港库克点的礁坪。

样本经快速断头处死，置于-20℃冷冻保存，随后以冷冻状态运至位于汤斯维尔的澳大利亚海洋科学研究所（Australian Institute of Marine Science）。从4只个体各取3条腕足混合以进行毒素提取：每份样品用80%乙醇提取，经均质、三次10分钟超声处理后，通过离心澄清；该步骤使用新鲜提取溶剂重复两次。将4份提取物各自得到的3份上清液合并，经0.2 mm尼龙滤膜过滤后冷冻干燥。冻干提取物用0.05 M乙酸复溶，经离心超滤处理，所得超滤液用于放射受体测定与初步化学分析。

采用沙西力蛋白（saxiphilin）与电压门控钠通道（voltage gated sodium channel），结合[³H]-标记的石房蛤毒素（saxitoxin, STX）开展放射受体测定，以定量STX浓度当量（STXeq）；针对章鱼提取物在钠通道与沙西力蛋白测定中对STX结合的抑制作用，进行三次重复实验。

所有在放射受体测定中可与[³H]-STX竞争结合的样品提取物，通过液相色谱-荧光检测器（Liquid Chromatography-Fluorescence Detector, LC-FLD）分析STX、脱氨甲酰基STX（decarbamoylSTX）、新STX（neoSTX）、11-羟硫酸盐STX类（GTX1-6）、N-磺酰氨甲酰基-11-羟硫酸盐STX类（C1-4）、羟苯甲酸酯STX类与河豚毒素（tetrodotoxin, TTX）。可疑毒素峰的鉴定通过液相色谱-质谱（Liquid Chromatography-Mass Spectrometry, LC-MS）的单离子监测（Single Ion Monitoring, SIM）模式完成。

既往研究显示，澳大利亚西北海岸的黑德兰港是多种甲壳类、腹足类与双壳类软体动物的分布区域，这些生物均携带河豚毒素（TTX）与麻痹性贝类毒素（paralytic shellfish toxins, PSTs）。本研究作为该区域底栖生物中麻痹性贝类毒素分布重新调查的一部分开展。

本研究首次在该类掠食性软体动物中报道石房蛤毒素（STX），同时凸显了提升公众对食用章鱼等此前未被认知的麻痹性贝类中毒传播媒介相关风险的认知的必要性。