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.
KeywordsTetrodotoxin High-Throughput Sequencing Cnidaria Annelida
This research was funded through a grant from the Marsden Fund of the Royal Society of New Zealand (UOW1002) to SCC, SAW and PM. We are grateful to David Taylor (Cawthron) for collection of specimens and Kati Doehring and Weimin Jiang (Cawthron) for preparation of Figs. 1 and 2. New Zealand Genomic Limited for assistance with bioinformatics and Dudley Bell and Warrick Powrie (Waikato University) for technical and field assistance.
- Do HK, Kogure K, Imada C, Noguchi T, Ohwada K, Simidu U (1991) Tetrodotoxin production of actinomycetes isolated from marine sediment. J Appl Microbiol 70:464–468Google Scholar
- Greene RR, Feldman CR (2009) Thamnophis atratus atratus diet. Herpetol Rev 40:103–104Google Scholar
- Guillou L, Bachar D, Audic S, Bass D, Berney C, Bittner L, Boutte C, Burgaud G, De Vargas C, Decelle J (2013) The protist ribosomal reference database (PR2): a catalog of unicellular eukaryote small sub-unit rRNA sequences with curated taxonomy. Nucleic Acids Res 41:D597–D604CrossRefPubMedGoogle Scholar
- McNabb P, Selwood AI, Munday R, Wood SA, Taylor DI, MacKenzie LA, van Ginkel R, Rhodes LL, Cornelisen C, Heasman K, Holland PT, King C (2010) Detection of tetrodotoxin from the grey side-gilled sea slug—Pleurobranchaea maculata, and associated dog neurotoxicosis on beaches adjacent to the Hauraki Gulf, Auckland, New Zealand. Toxicon 56:466–473CrossRefPubMedGoogle Scholar
- McNabb PS, Taylor DI, Ogilvie SC, Wilkinson L, Anderson A, Hamon D, Wood SA, Peake BM (2014) First detection of tetrodotoxin in the bivalve Paphies australis by liquid chromatography coupled to triple quadrupole mass spectrometry with and without precolumn reaction. J AOAC Int 97:325–333CrossRefPubMedGoogle Scholar
- Miyazawa K, Noguchi T (2001) Distribution and origin of tetrodotoxin. Toxin Reviews 20:11–33Google Scholar
- Noguchi T, Arakawa O, Takatani T (2006) Toxicity of pufferfish Takifugu rubripes cultured in netcages at sea or aquaria on land. Comp Biochem Phys D 1:153–157Google Scholar
- Schloss PD, Westcott SL, Ryabin T, Hall JR, Hartmann M, Hollister EB, Lesniewski RA, Oakley BB, Parks DH, Robinson CJ (2009) Introducing Mothur: open-source, platform-independent, community-supported software for describing and comparing microbial communities. Appl Environ Microbiol 75:7537–7541CrossRefPubMedPubMedCentralGoogle Scholar
- Willan RC (1983) New Zealand side-gilled sea slugs (Opisthobranchia: Notaspidea: Pluerobranchidae). Malacologia 23:221–270Google Scholar
- Wiseman KD, Pool AC (2007) Thamnophis couchii (Sierra garter snake): predator-prey interaction. Herpetol Rev 38:344Google Scholar
- Yasumoto T, Yasumura D, Yotsu M, Michishita T, Endo A, Kotaki Y (1986a) Bacterial production of tetrodotoxin and anhydrotetrodotoxin. Agr Biol Chem Tokyo 50:793–795Google Scholar
- Yotsu-Yamashita M, Nishimori K, Nitanai Y, Isemura M, Sugimoto A, Yasumoto T (2000) Binding properties of 3H-PbTx-3 and 3H-saxitoxin to brain membranes and to skeletal muscle membranes of puffer fish Fugu pardalis and the primary structure of a voltage-gated Na+ channel α-subunit (fMNa1) from skeletal muscle of F. pardalis. Biochem Biophys Res Commun 267:403–412CrossRefPubMedGoogle Scholar