Bacterial production of tetrodotoxin in the pufferfish Arothron hispidus

Abstract

This study evaluates the presence, location and production source of tetrodotoxin (TTX) in two species of pufferfish, Diodon histrix and Arothron hispidus, common to Hawaiian waters. Organs from each fish were analysed for TTX and used to isolate bacteria for evaluation of possible TTX production. Comparative analyses of extracts of fish and bacterial culture media were performed with liquid chromatography–mass spectrometry (LC-MS) and a sodium channel specific bioassay. Bacterial cultivation experiments were performed in two different growth media and bacteria were identified through sequence homology of the 16S rRNA gene. Forty-two and forty-seven distinct strains were cultivated from D. histrix and A. hispidus, respectively. However, no commonality was found between the populations of bacteria isolated from the two fish. TTX was detected only in A. hispidus and was present in the flesh, pectoral fin and kidneys, as well as the skin slime. Sixteen of the forty-seven bacterial species isolated from A. hispidus were cultivated for further evaluation of TTX production. Among these sixteen bacterial species, Vibrio harveyi strains isolated from the skin slime and kidneys of A. hispidus were found to produce TTX, being the source of TTX produced in the pufferfish.

Keywords:

• tetrodotoxin
• marine toxins
• Vibrio
• fish toxins
• pufferfish

Acknowledgements

Many thanks to Paul Chong, Brian and Myrna Yamane for sample collection and to the State of Hawaii, Department of Health, Office of Hazard Evaluation Emergency Response, for providing the financial support for this work. This research was made possible in part through the Centers for Oceans and Human Health (COHH) program, National Institute of Environmental Health Sciences (P50ES012740), National Institutes of Health and the National Science Foundation (OCE04-32479). The content is solely the responsibility of the authors and does not necessarily represent the official view of the National Institute of Environmental Health Sciences, the National Institutes of Health or the National Science Foundation.