In situ accumulation of tetrodotoxin in non-toxic Pleurobranchaea maculata (Opisthobranchia)

Tetrodotoxin (TTX) is a highly potent neurotoxin targeting voltage gated sodium channels. It is found in numerous phyla, including both marine and terrestrial taxa, however, its origin is a topic of considerable debate. The aim of this study was to investigate the origin of TTX in the Opisthobranch Pleurobranchaea maculata using in situ experimentation. Sixteen individuals sourced from non-toxic populations were transplanted to a habitat with toxic populations. These were kept in mesh net cages either; (1) anchored to the seafloor, or (2) deployed 0.5 m off the benthos. They were fed a non-toxic diet for 8 weeks before being sacrificed, and either the entire organisms or specific organs analysed for TTX via liquid chromatography–mass spectrometry. Four of the six remaining individuals from cages on the benthos contained TTX (max. 0.79 mg kg −1 ), whilst only two of eight from the suspended cages contained TTX and concentrations were lower (max. 0.43 mg kg −1 ). These were similar to the lowest concentrations (min. 0.4 mg kg −1 ) detected in free-living specimens collected during the experimental period. Among positive individuals the highest concentrations were detected in gonad tissues. These data, in concert with previous studies, suggest an environmental source of TTX for P. maculata , which may be bacterial or dietary in origin. High-Throughput Sequencing (18S ribosomal RNA gene metabarcoding) of foregut contents from toxic and non-toxic individuals was used to investigate their diet. High abundances of Cnidaria and Annelida sequences were identified and these groups should be targeted in future efforts to identify TTX-containing organisms.