Tetrodotoxin (TTX) is an extremely potent neurotoxin that acts by selectively targeting voltage gated sodium channels blocking propagation of action potentials. Long believed to be present only in pufferfish, TTX has now been detected in a wide range of phylogenetically unrelated terrestrial and aquatic taxa. Despite decades of research the exact origin of TTX remains a mystery. Current literature supports three hypotheses: endogenous, symbiotic bacteria, or bioaccumulation through a dietary source.
In 2009, the opisthobranch Pleurobranchaea maculata (grey side-gilled sea slug) was found to contain high concentrations of TTX in New Zealand. A large collaborative project, of which my research was a major part of, was initiated to explore the origin of TTX in P. maculata. During extensive benthic surveys conducted to identify possible dietary sources of TTX, high concentrations (ave. 380 mg kg-1) were detected in Stylochoplana sp. (Platyhelminthes) from Pilot Bay (Tauranga, New Zealand). Tetrodotoxin concentrations were found to vary temporally, peaking between June and August. The co-occurrence of Stylochoplana sp. and P. maculata in Pilot Bay raised the possibility that Stylochoplana sp. could be a dietary source of TTX for P. maculata. A real-time PCR assay was developed, and detected Stylochoplana sp. in seven out of nineteen P. maculata foreguts.
Symbiotic bacterial production of TTX in the tissues of P. maculata and Stylochoplana sp. was also explored. Isolated strains (102; 17 unique strains - identified using 16S rRNA gene analysis) were analyzed using a recently developed method to detect the C9 base of TTX. In addition to enhanced sensitivity, this method has the advantage that it might detect precursor and degradation products. To explore the possibility that TTX is produced by a consortium of bacteria, experiments were undertaken where homogenized tissue was spiked into marine broth and samples were collected over two weeks for toxin and molecular analysis. No C9 base or TTX production was detected in isolates or from bacterial communities, suggesting that a symbiotic microbial source of TTX is unlikely in these organisms.
The ability of non-toxic P. maculata to sequester TTX from an environment known to contain toxic populations of the same species was also assessed. Sixteen non-toxic specimens were kept in mesh cages (eight anchored to the benthos and eight suspended 0.5 m above it) for eight weeks and fed a non-toxic food source. Toxin analysis revealed that more ‘benthic’ specimens (4 verses the 2 from suspended specimens) sequestered TTX and were shown to retain higher concentrations (max. 0.79 versus 0.43 mg kg-1). These data suggest a localized microbial source of TTX that is more readily available from the benthos. Diet analysis, utilizing next generation sequencing of toxic and non-toxic P. maculata identified their diet comprised a wide array of organisms, with Thelepus sp. and Plumularia sp. being prevalent in toxic individuals, and further testing of these organisms is suggested.
Lastly, immunohistological methods, employing a monoclonal antibody targeting TTX, were conducted with tissues from P. maculata and Stylochoplana sp.. Strong TTX signals were detected in the mantle and oocytes of P. maculata and the ova and pharynx of the Stylochoplana sp.. These data suggest ecological roles for TTX including: defense in adults, protection in progeny, and prey capture in Stylochoplana sp..
A synthesis of the studies presented in this thesis, and those that were conducted as part of the larger project, are also presented and future studies to elucidate the origin of TTX in New Zealand taxa are suggested.
