The toxicity of the greater blue-ringed octopus Hapalochlaenalunulata, whose bite is fatal to humans, was examined to better understand and prevent deaths from accidental bites. Living specimens were collected from tide pools on Ishigaki Island, Okinawa Prefecture, Japan, in November and December of 2015, 2016, and 2017. The specimens were examined for the anatomical distribution of the toxicity, which was expressed in terms of mouse units (MU), by the standard bioassay method for tetrodotoxin (TTX) in Japan. Paralytic toxicity to mice was detected in all of the soft parts. The posterior salivary glands exhibited the highest toxicity score with a maximum level of 9276 MU/g, which was classified as "strongly toxic" (more than 1000 MU/g tissue) according to the classification of toxicity established by the Ministry of Health, Labor and Welfare of Japan, followed by the hepatopancreas (21.1 to 734.3 MU/g), gonads (not detectable to 167.6 MU/g), arms (5.3 to 130.2 MU/g), and other body areas (17.3 to 107.4 MU/g). Next, the toxin from the salivary glands was partially purified by a Sep-Pak C18 cartridge and an Amicon Ultra Centrifugal Filter with a 3000-Da cut-off, and analyzed by liquid chromatography-mass spectrometry (LC-MS) equipped with a φ2.0 × 150-mm (5 μm) TSKgel Amide-80 column (Tosoh, Tokyo, Japan) with a mixture of 16 mM ammonium formate buffer (pH 5.5) and acetonitrile (ratio 3:7, v/v) as a mobile phase. This study aimed to clarify the toxicity and the composition of TTX and its derivatives in this toxic octopus. The main toxin in this toxic octopus was identified as TTX, along with 4-epi TTX, 4, 9-anhydroTTX and 6-epi TTX. Further, the toxicity of this species is also significant from a food hygiene point of view.
Keywords: Hapalochlaena lunulata; Ishigaki Island; LC-MS; greater blue-ringed octopus; paralytic toxicity; posterior salivary gland; tetrodotoxin.