Wednesday 29 January 2014

Further evidence in support of bacterial origins for tetrodotoxin accumulation in puffer fish



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.

8 comments:

  1. I also heart tetrodotoxon! I saw the paper counts the species of bacteria identified after and incubation period. It would be interesting to see a metagenomic analysis to determine whether any TTX producing bacteria that cannot be be cultured in the methods described here are present - and if that information could help cultured that bacteria. It would be interesting to see whether their TTX production matches Shewanella putrefaciens and Vibrio alginolyticus, or if it maintained an over all constant toxicity with optimum growth outside that of S. putrefaciens and V. alginoticus.

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    1. I agree with you Adam, metagenomic methods could help to detect other strains producing TTX. It's also likely that many bacterial species that produce TTX or related toxin in the natural environment have not been identified yet. For example, many Vibrio species go in the VBNC state and culture independent methods only are able to detect them. So it's imaginable that some TTX producing bacteria of the genus and others might do the same.

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    2. Absolutely! As we know, the vast majority of bacteria are not culturable and it is highly likely that TTX or similar structured molecules are synthesised by bacteria that we aren't even aware of yet! Genomics and proteomics of bacterial assemblages associated with TTX producing animals may go some way to answering this. Fascinating stuff!

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  2. Hi Rachel,
    have the authors only collected the puffer fish from this one site? I have recently read a paper where they have looked at TTX in a sea slug species and collected their organisms from different sites around New Zealand. The different populations showed differences in concentrations, eg organisms from one site had a much lower tissues TTX concentration than organisms from another location. They suggested that the TTX concentration of the slugs was related to their diet depending on the habitat. However they have not investigated any bacterial associations and only briefly mentioned that there might be other factors such as bacterial activity that are responsible for the toxin production.
    In most papers about TTX that I read, no conclusions could be drawn as to whether the bacteria and host live in symbiosis. I think it's very logical that they do however, it seems likely that bacteria that produce the toxin support the host to increase its survival and hence fitness, and in return the bacteria can receive nutrients and are able to accumulate and grow in almost all tissues and organs of the host (the paper mentioned before has found the toxin everywhere in the slug which could mean that the bacteria are associated everywhere in the organism possibly).

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    1. Hi Malin,
      Yes, all the fish were collected from the same area around Chang Island in the Gulf of Thailand, presumably to limit the amount of variables in this study. I agree that it does seem most likely that TTX is of bacterial origin, but it is pure speculation! I wonder whether the studies proclaiming that TTX accumulation in these animals is due to diet, have looked at whether there is bacteria within the food source?

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    2. http://www.mdpi.com/1660-3397/12/1/1

      Here is the link to the study I mentioned in case you're interested. They literally have only fed the slugs food with different concentrations of TTX to see if non-toxic animals collected could accumulate TTX through the diet. Disappointingly, the have not said anything about bacterial TTX production in the natural environment and if the food that the slug usually feeds on in nature could harbour TTX producing bacteria, and I think they have left out some important aspects that in my eyes are needed to be considered. This was more of a general study how animals accumulate TTX, and they said that diet is an important factor. As natural conditions can favour some bacteria species over others, it is thinkable that the different concentrations of the collected animals could be due to abundance and diversity of TTX producing bacteria, but clearly this needs further investigation.
      I wonder if someone has looked at algal species producing TTX which are ingested by many marine animals. Just a thought, there are so many algae that produce all sorts of toxins...

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    3. Thanks Malin. As you mention, it is possible that microalgae can produce TTX, a very similar molecule to saxitoxin that is produced by Alexandrium sp. dinoflagellates (see George's post). It is also conceivable that TTX synthesising bacteria may be associated with an algal species, conjoined as seen in Malfatti & Azam (2009) with atomic force microscopy. Certainly an area worthy of further study!

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