Rough-skinned newts (Taricha granulosa) are poisonous salamanders that possess high concentrations of tetrodotoxin (TTX), a potent neurotoxin that blocks voltage-gated sodium channel (Nav) conductance in neurons and muscle cells. TTX is present in all species of the genus Taricha, but some populations of T. granulosa (hereafter “newts”) possess extreme quantities not seen in any other TTX-bearing species, including puffer fishes, blue-ringed octopuses, and many diverse marine invertebrates. Geographic variation in TTX toxicity across different newt populations is thought to be driven by ecological interactions with predators. Despite the central role of TTX in the physiology and evolution of newts, the mechanisms of TTX production and neurophysiological resistance are unknown. Because of the polyphyletic distribution of TTX toxicity among animals, we explored the hypothesis that TTX is produced by symbiotic skin bacteria in newts. We conducted 16S rRNA gene-based sequencing surveys to characterize skin- associated bacterial communities of newts from toxic and non-toxic populations. From here, we employed ecologically-guided cultivation strategies to target skin-associated symbionts and produce pure cultures. We screened cultures for TTX production using a customized HILIC-MS/MS method and confirmed TTX production in multiple isolated bacterial strains. Furthermore, we investigated the molecular adaptations underlying apparent TTX resistance in the Navs of newts. We cloned and sequenced the TTX binding site, the S5-S6 pore loop regions, of all six Nav genes present in this species and compared sequences from toxic and non-toxic populations, as well as from other vertebrates. As a result, we identified several mutations present in the S5-S6 pore loops of all six genes, indicating a remarkable parallel evolution of TTX resistance across the Nav gene family. To determine whether these mutations impact TTX resistance, we used site-directed mutagenesis to insert three newt mutations identified in neural subtype Nav1.6 into the TTX-sensitive mouse ortholog and examined their effects on TTX binding by heterologous expression and electrophysiological recording in Xenopus laevis oocytes. We found that each individual mutation increased TTX resistance to varying degrees, but the triple mutant was extremely resistant to TTX concentrations exceeding 100 μM. Taken together, our results indicate that TTX is derived from the skin microbiome in the extremely toxic rough-skinned newt and that multiple adaptations in newt Navs were required for the nervous system to adapt to TTX toxicity. Overall, this research contributes to a growing understanding that symbiotic microbes can affect the physiology of animal hosts and their nervous systems, and that evolution by natural selection may target genetic variation across both host and symbiont genomes, collectively termed the ‘hologenome’.
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