Studies On The Origin Of Tetrodotoxin In Gastropod (Nassarius Spp.)

Poisoning incidents resulted from nassariid gastropods have been reported from time to time for more than 20 years along the coast of China. Due to the limited information available on the dynamics of toxicity and the origin of toxin in nassariids, it is difficult to develop and implement effective measures to predict and prevent poisoning incidents. So far, there is no systematical research on the origin of toxin in gastropod (Nassarius spp.). Using gastropod Nassarius semiplicatus, N. variciferus, and N. semiplicatoides sp. nov., we studied the two possible sources of tetrodotoxin (TTX) in them, including symbiotic bacteria and other toxic organisms via food chains.According to pervious reports, bacterium has been considered as a potential source for TTX in many TTX-bearing marine organisms. Therefore, samples of nassariid were collected successively from Yancheng City and Lianyungang City in Jiangsu Province. The toxicity of these samples and the composition of toxin in them were tested with mouse bioassay method and liquid chromatography-mass spectrometry (LC-MS), respectively. Bacteria were isolated from the nassariid samples and from their habitat. A part of isolated bacteria were then cultured in the lab, and TTX in bacteria were screened with an ELISA method. Partial of the 16S ribosomal DNA (rDNA) of the toxic strains were then sequenced after PCR amplification, and the toxic strains of bacteria were tentatively identified based on the alignment of these sequences with published data. The toxicities of the gastropod (N. semiplicatus) samples collected from Yancheng City and Lianyungang City, were 2 mouse unit (MU) and 200 MU per gram of tissue (wet weight), respectively. High concentration of tetrodotoxin and its analogues (TTXs) were found in the digestive gland and muscle of the gastropod collected from Lianyungang City, using LC-MS. TTX was detected in 9 strains among the 14 strains of bacteria isolated from gastropod and its habitat collected from Yancheng City, and was detected in 21 strains out of the 45 strains isolated from the digestive gland of highly toxic gastropod collected from Lianyungang City. TTX content in the toxic strains was very low, which ranged from 5ng/g to 184ng/g. It was found that most of the toxic strains were closely affiliated with genus Vibrio, and the others were related to genus Shewanella, Marinomonas, Tenacibaculum, Planococcus and Aeromonas. TTX production by bacteria in genus Marinomonas and Tenacibaculum, as find in this study, is the first report to our knowledge. We had isolated several species of TTX-producing bacteria from the gastropod (Nassarius spp.) and their habitat. However, the content of TTX produced by TTX-producing bacteria isolated in this study is much lower than that in highly toxic nassaiid. Therefore, TTX-producing bacteria might play a less important role in TTX production or accumulation in nassariid.As a scavenger, nassariid may absorb and accumulate TTX after feeding TTX-bearing organisms. To confirm whether food chain play a role in TTX accumulation in gastropod, two experiments were carried on including depuration of toxicity of highly toxic gastropod and accumulation and depuration of TTX by gastropod. The content and composition of TTXs in nassariid gastropod samples collected periodically during the experiments were screened with LC-MS/MS. In the first experiment, highly toxic gastropod sample N. semiplicatus was collected from Lianyungang and maintained in the laboratory feeding with commercial marine fish feed. Dynamics of toxin compositions and contents in different anatomical compartments were analyzed. A decreasing tread of TTX content was observed during the former period, and the analogues of TTX showed similar patterns. However, a subsequent increase of TTX and its analogues contents was observed from July 9, in correspondence with the observation in the field. To further elucidate the effect of food chain in the accumulation of TTX, the experiment of accumulation and depuration of TTX by gastropod was carried on then. In gastropod (N. variciferus), after fed with toxic liver of puffer fish (Fugu vermicularis), a fast rate of TTX accumulation was observed. However, after about 16 days, TTX content stopped increasing even if the test organism were fed with toxic liver continuously. After fed with non-toxic liver of puffer fish (Takifugu rubripes), the toxin content decreased quickly. In gastropod (N. semiplicatoides sp. nov.), which had caused several poisoning incidents in China, TTXs contents increased very slowly after feeding toxic liver. However, a sharp increase of TTXs was found during the depuration stage, which coincided with that found in the control group. Moreover, a similar increase of TTXs in wild gastropod samples was found almost at the same time. These findings suggested that there should be a natural rhythm controlling the toxin production or accumulation in nassariids, which might be related to their spawning season. It suggested that high concentration of TTX in toxic nassariids could not be accumulated through food chains.All the experimental results suggested that TTX-producing bacteria might play a less important role in TTX production or accumulation in nassariid. And the high concentration of TTX in gastropod (Nassarius spp.) was not accumulated through the food chain. TTX in nassariid is most probably produced by themselves. Meanwhile, there should be a natural rhythm controlling the toxin production in gastropod Nassarius spp. These findings make it possible to predict and prevent poisoning incidents caused by nassariids. However, the true origin and biosynthetic and metabolic pathways of TTX in nassariids remains unclear. Further study along this is needed.