Investigating Diet as the Source of Tetrodotoxin in the Grey Side-gilled Sea Slug, Pleurobranchaea maculata

Abstract

Pleurobranchaea maculata (grey-side gilled sea slug) was discovered to contain the neurotoxin tetrodotoxin (TTX) in 2009 after a spate of dog poisoning cases on the beaches of Auckland, New Zealand. One of the great mysteries of TTX is the lack of conclusive evidence about its ultimate origin. Possible sources postulated have included both endogenous and exogenous. Additionally, within a species both toxic and non-toxic strains exist. For example, in New Zealand, P. maculate from the North Island are toxic, whereas P. maculata from the South Island are not. The overarching hypothesis of this Master’s project is that TTX has a dietary origin in P. maculata and that they will preferentially feed on TTX-containing food. This Master’s thesis aimed to test this hypothesis through three distinct aims: (1) To develop a non-lethal biopsy method and determine the feasibility for future research concerning TTX in P. maculata; (2) To investigate whether nontoxic P. maculata can accumulate TTX from a dietary source, and how TTX is distributed through the organism; and (3) To investigate whether P. maculata are attracted to TTX and if this varies depending on the TTX content within P. maculata. A biopsy method was developed for taking approximately 200 mg tissue biopsies using a TemnoEvolution 18G × 11 cm Biopsy Needle inserted transversely into the foot. Six P. maculata were biopsied twice (nine days apart) and each individual was frozen immediately following the second sampling. Tetrodotoxin concentrations in biopsy samples, gonad, stomach, mantle and the remaining combined tissues and fluids were measured using liquid chromatography-mass spectrometry (LC-MS). Based on the proportional weight of the organs/tissues a total TTX concentration for each individual was calculated. There were strong correlations between biopsy TTX concentrations and the total (r² = 0.88), stomach (r² = 0.92) and gonad (r² =0.83) TTX concentrations.

To investigate the accumulation of TTX, eighteen non-toxic P. maculate were maintained in aquariums and twelve were fed a TTX-containing diet. Three P. maculata were harvested after 1 hr, 24 hrs, 17 days and 39 days and TTX concentrations in their stomach, gonad, mantle and remaining tissue/fluids determined using LC-MS. Tetrodotoxin was detected in all organs/tissue after 1 hr with an average uptake of 32%. This decreased throughout the experiment (21%, 15% and 9%, respectively). This study demonstrated that P. maculata can accumulate TTX from a dietary source. To explore whether P. maculata were attracted to TTX, three preference experiments were undertaken; (1) an aquarium zonation experiment, (2) a toxic/non-toxic agar trail experiment and, (3) a direct choice experiment using combinations of toxic/non-toxic agar blocks. A statistically significant preference for TTX was found for toxic P. maculata in the agar trails (P < 0.001) and direct choice experiments (P < 0.001). For the non-toxic P. maculata, a statistically significant preference was only found for the direct choice experiment (P = 0.002). Collectively these studies demonstrate that diet is a possible source in P. maculata. However, given the absence of identifiable TTX sources in environments where P. maculata are prevalent, in concert with their extremely high TTX concentrations and short life spans, it is unlikely to be the sole source for this species.