No evidence for a culturable bacterial tetrodotoxin producer in Pleurobranchaea maculata (Gastropoda: Pleurobranchidae) and Stylochoplana sp. (Platyhelminthes: Polycladida).

Tetrodotoxin (TTX) is a potent neurotoxin found in the tissues of many taxonomically diverse organisms. Its origin has been the topic of much debate, with suggestions including endogenous production, acquisition through diet, and symbiotic bacterial synthesis. Bacterial production of TTX has been reported in isolates from marine biota, but at lower than expected concentrations. In this study, 102 strains were isolated from Pleurobranchaea maculata (Opisthobranchia) and Stylochoplana sp. (Platyhelminthes). Tetrodotoxin production was tested utilizing a recently developed sensitive method to detect the C9 base of TTX via liquid chromatography-mass spectrometry. Bacterial strains were characterized by sequencing a region of the 16S ribosomal RNA gene. To account for the possibility that TTX is produced by a consortium of bacteria, a series of experiments using marine broth spiked with various P. maculata tissues were undertaken. Sixteen unique strains from P. maculata and one from Stylochoplana sp. were isolated, representing eight different genera; Pseudomonadales, Actinomycetales, Oceanospirillales, Thiotrichales, Rhodobacterales, Sphingomonadales, Bacillales, and Vibrionales. Molecular fingerprinting of bacterial communities from broth experiments showed little change over the first four days. No C9 base or TTX was detected in isolates or broth experiments (past day 0), suggesting a culturable microbial source of TTX in P. maculata and Stylochoplana sp. is unlikely.

Diversity and biosynthetic potential of culturable microbes associated with toxic marine animals.

Tetrodotoxin (TTX) is a neurotoxin that has been reported from taxonomically diverse organisms across 14 different phyla. The biogenic origin of tetrodotoxin is still disputed, however, TTX biosynthesis by host-associated bacteria has been reported. An investigation into the culturable microbial populations from the TTX-associated blue-ringed octopus Hapalochlaena sp. and sea slug Pleurobranchaea maculata revealed a surprisingly high microbial diversity. Although TTX was not detected among the cultured isolates, PCR screening identifiedsome natural product biosynthesis genes putatively involved in its assembly. This study is the first to report on the microbial diversity of culturable communities from H. maculosa and P. maculata and common natural product biosynthesis genes from their microbiota. We also reassess the production of TTX reported from three bacterial strains isolated from the TTX-containing gastropod Nassarius semiplicatus.

Depuration of tetrodotoxin and changes in bacterial communities in Pleurobranchea maculata adults and egg masses maintained in captivity.

Depuration of tetrodotoxin (TTX) was investigated in adult grey side-gilled sea slugs, Pleurobranchaea maculata, maintained in captivity on a TTX-free diet. Three adults were harvested every 21 days for 126 days, and TTX concentrations were measured in organs/tissues and egg masses. Automated rRNA intergenic spacer analysis (ARISA) was used to investigate bacterial community structure in selected samples. Linear modeling of adult data demonstrated a decline (P<0.001) in average total TTX concentrations over time. Temporal data obtained from a wild population showed similar depuration rates, indicating that once adults reach a certain size, or sexual maturity, TTX is no longer produced or acquired substantially. Depuration rates differed among organs, with concentrations in the heart declining the fastest. The gonads had the slowest and least significant depuration rate indicating, at most, weak depuration of this tissue. There was a strong correlation (R(2)=0.66) between TTX concentrations in the first-laid egg masses and total TTX in the corresponding adult. These data suggest that adult P. maculata transfer TTX to their offspring, and presumably that functions as a chemical defense. ARISA data showed a shift in bacterial community structure within 3 weeks of introduction to captivity. Based on the combined data, the exact origin of TTX in P. maculata is unclear, with evidence both in favor and against a dietary source, and endogenous or bacterial production.