Rough-skinned newts (Taricha granulosa) use tetrodotoxin (TTX) to block voltage-gated sodium (Nav) channels as a chemical defense against predation. Interestingly, newts exhibit extreme population-level variation in toxicity attributed to a coevolutionary arms race with TTX-resistant predatory snakes, but the source of TTX in newts is unknown. Here, we investigated whether symbiotic bacteria isolated from toxic newts could produce TTX. We characterized the skin-associated microbiota from a toxic and non-toxic population of newts and established pure cultures of isolated bacterial symbionts from toxic newts. We then screened bacterial culture media for TTX using LC-MS/MS and identified TTX-producing bacterial strains from four genera, including Aeromonas, Pseudomonas, Shewanella, and Sphingopyxis. Additionally, we sequenced the Nav channel gene family in toxic newts and found that newts expressed Nav channels with modified TTX binding sites, conferring extreme physiological resistance to TTX. This study highlights the complex interactions among adaptive physiology, animal-bacterial symbiosis, and ecological context. 

These data were generated from bacterial DNA samples collected from wild-caught rough-skinned newts (Taricha granulosa). The V4 hypervariable region of the 16S rRNA gene was amplified from each bacterial DNA sample, and resulting amplicons were sequenced on the Illumina MiSeq v3 platform with paired-end 300-bp protocol for 600 cycles. The resulting sequence data were assembled and processed using mothur (https://www.mothur.org) and the MiSeq SOP protocol (https://www.mothur.org/wiki/MiSeq_SOP). Mothur generates a "shared" file, which is an OTU abundance table (file: newts.OTU.shared). Mothur also generates a taxonomy file, with SILVA rRNA database taxonomic classifications for each OTU (file: newts.OTU.taxonomy). We manually generated a corresponding metadata file (file: newts.OTU.metadata). We also include a processed shared file that we used in our analyses, in which singletons and doubletons have been removed and each bacterial community sample has been subsampled to 5,000 seqs/sample.