Toxicity and Toxin Composition of the Greater Blue-Ringed Octopus Hapalochlaena lunulata from Ishigaki Island, Okinawa Prefecture, Japan

Abstract

The toxicity of the greater blue-ringed octopus Hapalochlaena lunulata, 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.

Tetrodotoxin and Its Analogues in Cephalothrix cf. simula (Nemertea: Palaeonemertea) from the Sea of Japan (Peter the Great Gulf): Intrabody Distribution and Secretions

“…Animals using TTX in venom accumulate the toxin in the organs involved in food capture and use it to immobilize the prey. In the blue-ringed octopuses, Hapalochlaena lunulata and Hapalochlaena maculosa, the bulk of TTX was found in the posterior salivary glands, a usual tool for producing paralyzing poison [26,27]. In the polyclad flatworm Planocerid sp., a large amount of TTX and 11-norTTX-6(S)-ol is contained in the pharynx and is utilized in the process of hunting [28].…”

An Updated Review of Tetrodotoxin and Its Peculiarities

“…Records found in the literature on TTXs presence in these aquatic organisms are presented in Table 4. During the past decade, increased levels of TTXs have been reported in two blue-lined octopus species (Hapalochlaena lunulata and H. fasciata) in Taiwan and Japan [90][91][92]. The highest TTXs concentrations were detected in the posterior salivary glands, and it is believed that TTX in these species acts as a hunting and defense mechanism against predators [90].…”

“…The highest TTXs concentrations were detected in the posterior salivary glands, and it is believed that TTX in these species acts as a hunting and defense mechanism against predators [90]. These octopus species are not considered as edible; however, misidentification can result in severe morbidity and constitutes a significant risk from a food hygiene point of view [91]. In fact, the Taiwanese record is associated with a nonfatal human poisoning incident due to misidentification in the market and subsequent consumption [90].…”

Proteomic and Transcriptomic Techniques to Decipher the Molecular Evolution of Venoms

“…Most venoms are complex mixtures of proteins, making fractionation a necessary task as mass spectrometry (MS) acquisition technologies can often not handle such diversity [ 12 ]. These fractionation techniques are numerous, and some of the most common, to be reviewed below in more detail, are: Reversed-phase high-performance liquid chromatography (RP-HPLC, otherwise known as HPLC) ( Figure 3 B), capillary isoelectric focusing (CIEF) ( Figure 3 C), size-exclusion chromatography (SEC) ( Figure 3 A), capillary zone electrophoresis (CZE) ( Figure 3 D), and 2D-Gel Electrophoresis (2D-GE) [ 20 , 31 , 32 , 33 , 34 , 35 , 36 , 37 , 38 , 39 ].…”