Further evidence in support of bacterial origins for tetrodotoxin accumulation in puffer fish
And so it seems we’ve all been inspired by toxins! Continuing the theme and complementing the post by Caroline, this paper by Auwithoothij & Noomhorm (2012) investigated the major (culturable) microbes associated with the internal organs of the puffer fish, Lagocephalus lunaris in relation to tetrodotoxin (TTX) accumulation.
The study was conducted throughout 2010 in the Gulf of Thailand, with an average of 30 samples of L. lunaris collected each month from January through to December. Aside from all the fish being the same species, all other characteristics (eg; size, sex, weight, developmental stage) appeared to be highly variable. All samples once shipboard were immediately put on ice and soaked in ethanol once in the laboratory, before aseptically removing organs to minimise any environmental contamination.
TTX accumulation within the organs of the fish was highest over two periods in the year – February to March and August to October, which reportedly coincides with cooler waters of around 25 °C. Nine dominant bacterial species were isolated from the organs of L. lunaris, however only Vibrio alginolyticus and Shewanella putrefaciens were reportedly found to produce TTX in this study. A graphical representation showed that increased loads of S. putrefaciens within the organs appeared to coincide with increased levels of TTX, although a correlation would have benefitted here.
The authors went on to culture S. putrefaciens to ascertain the effects of salinity and temperature on bacterial growth and production of TTX. Results suggest that whilst S. putrefaciens grows more slowly at lower temperatures (ie; 25 °C rather than 30 °C) it produces higher levels of TTX, which corresponds to the higher accumulation of TTX in the puffer fish organs found at lower temperatures. Salinity had no effect on growth of the bacteria but lower salinities (9 – 22 ‰ rather than 32 ‰) also increased production of TTX. Whilst not mentioned in the paper, this may correlate to the rainy season for the second period of high TTX concentration found in the organs (August to October), although data for the rainfall experienced in 2010 would need to be obtained to consider this further.
As highlighted by the authors, it must be considered that the higher abundance of S. putrefaciens is due to increased environmental abundance and may therefore be accumulated in puffer fish tissues through the food chain. Water sampling alongside the fish capture would have contributed greatly to this study, allowing comparison of background bacterial compliment against that found within the fish organs. Whilst this evidence is still circumstantial, it does provide more fuel for the bacterial origin of TTX argument which, whilst controversial, would explain how TTX is produced in such a wide range of taxa – both marine and terrestrial. What it isn’t able to answer however, is whether the bacteria are living symbiotically within the fish. The debate continues!
Auawithoothij, W., & Noomhorm, A. (2012). Shewanella putrefaciens, a major microbial species related to tetrodotoxin (TTX)‐accumulation of puffer fish Lagocephalus lunaris. Journal of applied microbiology, 113(2), 459-465.