Vaelli PM , Theis KR , Williams JE , O'Connell LA , Foster JA , Eisthen HL .

DBI-0939454 National Science Foundation, IOS-1354089 National Science Foundation, IOS-1655392 National Science Foundation, IOS-0920505 National Science Foundation, Bauer Fellowship Harvard University, Graduate research fellowship, fellow ID: 2014165835 National Science Foundation, 2014165835 National Science Foundation

	Figure 1—figure supplement 1. The top 20 most abundant OTUs in the newt microbiome.A clearcut neighbor-joining phylogenetic tree of the top 20 most relatively abundant OTUs with a heatmap displaying the mean relative abundance of each OTU in Oregon and Idaho newts. 12 of these OTUs are unique to one population, while 8 OTUs are shared between both populations.
	Figure 1—figure supplement 2. Relative abundance of newt-associated bacteria at the phylum level.Newt skin communities are diverse, with bacterial OTUs from 12 distinct phyla present at >3% relative abundance.
	Figure 1. Characterization of the skin-associated microbiota of rough-skinned newts.(A) Rough-skinned newt (Taricha granulosa); photo by Gary Nafis (CC by ND NC 3.0). (B) Two populations of T. granulosa previously reported to possess either high concentrations of TTX (Lincoln Co, OR; red) or no TTX (Latah Co, ID; black) were compared in our study. (C) TTX measured from dorsal skin biopsies: newts from Oregon possessed 126.5 ± 42.1 ng mL−1 TTX (n = 5) while Idaho newts possessed no detectable TTX (n = 17). (D–E) Scanning electron micrographs of host-associated bacterial communities upon the dorsal skin surface and within the ducts of TTX-sequestering granular glands. Scale bars 10 µm and 1 µm for D and E, respectively. (F–G) Comparison of bacterial community richness and diversity between these two populations, along with soil samples collected contemporaneously from the ponds in which the newts were caught. Non-toxic Idaho newts possessed higher OTU richness (Chao1 index, t unequal var. = 7.90, p<0.0001) and diversity (Simpson [1-D] index, t unequal var. = 4.11, p<0.0001) than toxic Oregon newts. (H) Mean relative abundance of bacterial OTUs present in dorsal or ventral skin of each population, as well as local soil samples. Sample sizes for panels F-H were n = 12 for Oregon (TTX+) and n = 16 for Idaho (TTX-) newts.Figure 1—source data 1. Raw data for newt skin toxicity measurements by LC-MS/MS.Estimated TTX concentrations (ng mL−1) were calculated relative to a calibration curve of pure TTX standards.Figure 1—source data 2. The top 20 most abundant bacterial OTUs found among toxic (Oregon) and non-toxic (Idaho) newts.The relative abundance of each bacterial OTU within a sample was calculated and averaged across all samples for each population; these data are shown below as percentages. Taxonomy was assigned using the Ribosomal Database Project (Cole et al., 2014) and a confidence threshold of 80%. OTUs shared between the two populations are in bold.Figure 1—source data 3. Variation in alpha diversity between newt populations and sampling sites across the bodies of individual newts.Each value is shown as mean ± SEM.
	Figure 2—figure supplement 1. Principal coordinates analysis highlighting variation in bacterial community structure (Bray-Curtis index) of Oregon (red circles) and Idaho (black squares) newt skin microbiota.Dispersion (i.e. variance) of individual samples are shown as gray lines from the mean centroid, and 95% confidence ellipses are shown for each group. Permutation test of multivariate homogeneity of group dispersion (PERMDISP) indicates a significant difference between the two populations (p=0.0053).
	Figure 2—figure supplement 2. Relative abundance of all OTUs classified to the same genera as TTX-producing bacteria identified in this study (Aeromonas, Pseudomonas, Shewanella, and Sphingopyxis).Skin microbiota samples (dorsal and ventral) are clustered based on Bray-Curtis dissimilarity (Oregon n = 21; Idaho n = 27).
	Figure 2. Comparison of skin microbiota from toxic and non-toxic newt populations reveal distinct host-associated bacterial communities and enrichment of TTX-producing bacteria.(A–B) Principal coordinates analysis of bacterial community composition (OTU presence/absence; Jaccard index) and community structure (OTU relative abundance; Bray-Curtis index) of skin microbiota across four body sites from toxic Oregon and non-toxic Idaho newts reveal distinct clustering of bacterial communities within each population. Non-parametric multivariate analysis of variance (PERMANOVA) test indicates a significant effect of location on both the composition (F = 18.12, p<0.0001) and structure (F = 40.40, p<0.0001) of skin-associated bacterial communities. Newt-associated communities also cluster separately from soil communities sampled from each location. (C) Representative comparison of highly abundant OTUs from individual toxic Oregon and non-toxic Idaho newt microbiota samples (on x-axis) reveals increased relative abundance of Pseudomonas OTUs in toxic newts. (D) Linear discriminant analysis effect size (LEfSE) comparing the top 10 differentially abundant OTUs between toxic and non-toxic newt microbiota indicates that Pseudomonas OTU00042 is highly enriched in toxic newt microbiota (black arrow). Pseudomonas isolated from newt skin produced TTX (Figure 3). Sample sizes were n = 12 and n = 16 for Oregon and Idaho newts, respectively.
	Figure 3—figure supplement 1. Phylogenetic tree of bacteria cultivated from the skin of toxic rough-skinned newts.Evolutionary relationships were determined by aligning newt bacterial 16S rRNA gene sequences to the SILVA Ribosomal RNA database (Quast et al., 2013) using mothur v1.40.3 (Schloss et al., 2009). Trees were constructed using randomized axelerated maximum likelihood (RAxML) with 1000 bootstrap replicates (Stamatakis, 2014) in Geneious v11.0.5 (Kearse et al., 2012). Clades with multiple bacterial isolates are represented with triangles. Bootstrap support is shown on each node.
	Figure 3—figure supplement 2. Pairwise comparison of 16S rRNA sequences between TTX-producing strains of Pseudomonas spp. identified in this study.Gray regions represent invariable sites, while black bars show mutations. Strains TX111003, TX174011, and TX180010 and strains TX135003 and TX135004 each share >99% pairwise nucleotide identities, but the two groups appear to be distinct from each other. Sequences TX111009 and TX111008 share around 96–97% nucleotide identities with the other two groups, but each possesses unique mutations indicating that they are distinct strains of Pseudomonas. Nucleotide identities are shown as percentages (%) for each pairwise sequence comparison. Sequence groups with >99% nucleotide identities are indicated through color coding and were considered replicate isolates of the same bacterial strains.
	Figure 3. Bacterial isolates cultured from newt skin produce TTX in vitro.(A) Schematic overview of procedure for isolating and screening newt bacteria for TTX production. Bacterial samples were collected from dorsal skin of toxic newts, taxonomically identified by 16S rRNA gene sequencing, grown in liquid culture for 2 weeks, centrifuged, and the supernatant was purified by solid-phase extraction (SPE). Extracts were screened against TTX analytical standards by LC-MS/MS. (B) Representative extracted ion chromatographs showing peaks corresponding to major product ion transitions 320.1 to 162.1 m/z (black) and 320.1 to 302.1 m/z (red) for TTX in cultures from four bacterial isolates. The retention time for each peak was 2.9 min and matched that of both authentic TTX standards and culture media supplemented with 100 ng mL−1 TTX. Peaks were absent in untreated culture media. Estimated TTX concentrations in bacterial cultures are shown next to each peak. Multiple strains of Pseudomonas spp. were found to produce TTX and three additional TTX-producing genera were identified: Aeromonas, Shewanella, and Sphingopyxis.
	Figure 4—figure supplement 1. Parallel evolution of DIII and DIV P-loop substitutions in Nav1.6 of toxic newts and TTX resistant garter snakes.Structure of the Nav channel and multiple sequence alignment of Nav1.6 across representative vertebrate taxa. The consensus sequence is shown above the alignment, and orange labels indicate taxa that either possess TTX or consume TTX-laden prey. Identical amino acid substitutions were observed between rough-skinned newts (Taricha granulosa) and garter snakes (Thamnophis sirtalis) from Benton Co. OR, where T. granulosa populations are highly toxic. Furthermore, some of these mutations were also identified in the less toxic eastern newt (Notophthalmus viridescens) and the non-toxic axolotl (Ambystoma mexicanum). These observations suggest that the last common ancestor of Ambystomids and Salamandrids possessed replacements in DIII and DIV. Our physiological data indicate that these replacements provide low levels of resistance, and may have thus allowed low level exposure to TTX, possibility facilitating the evolution of TTX toxicity in some Salamandrid species. Phylogenetic relationships are based on full-length Nav1.6 assessed by RAxML with 1000 bootstrap replicates. Support values are shown on each node, and the scale bar reflects the mean number of nucleotide substitutions per site.
	Figure 4. Protein alignment of Nav channels across representative vertebrates.Sequence alignment of S5-S6 P-loops from newts and other vertebrates showing amino acid substitutions relative to the P-loop consensus sequence for each Nav channel shown here. Putative TTX resistance mutations are highlighted in orange; mutations that are not highlighted are either synapomorphic in a gene clade or are present in TTX sensitive channels. Data are missing for DI and DII of Nav1.1 in newts, which we did not recover in our sequencing efforts. The approximate locations of newt mutations are shown as orange circles, and the amino acid site of each mutation is numbered based on Nav1.6 from Mus musculus.Figure 4—source data 1. GenBank accession numbers of vertebrate Nav channel protein sequences used in multiple sequence alignments and analysis.
	Figure 5—figure supplement 1. Newt Nav1.6 mutations increase TTX resistance in the orthologous mouse Nav1.6.Current-voltage curves showing normalized peak Na+ currents measured for wild-type mouse Nav1.6 and mutant constructs DI Y371A, DIII V1407I, and DIV I1699V containing one of three mutations in the presence of the control and wash solutions (black), 1 µM TTX (blue), and 10 µM TTX (red). Each construct was coexpressed with rat β1 and β2 subunits. Data are shown as peak currents normalized to the maximum peak current recorded for each oocyte. Dotted lines indicate current after washing TTX from the oocyte. The construct containing the DI mutation was largely unaffected by TTX at 1 or 10 µM (n = 17); those containing the DIII (n = 13) and DIV (n = 15) mutations were more sensitive, but each channel shows reduced sensitivity relative to the wild-type channel (n = 21).
	Figure 5. Newts possess Nav channel mutations that confer physiological resistance to TTX.(A) Predicted topology of Nav1.6 with mutations in domains I, III, and IV. Sequence alignment of Nav1.6 pore-loop motifs revealed three amino acid differences in newts from Oregon or Idaho populations. (B) Representative currents from wild-type mouse Nav1.6 or Nav1.6 with newt substitutions Y371A, V1407I, and I1699V treated with 1 µM (blue) or 10 µM (orange) TTX. (C) Current-voltage (I–V) relationships showing normalized currents for wild-type (n = 21) and mutant Nav1.6 (n = 20) channels. Wild-type Nav1.6 was blocked by TTX (Tukey’s multiple comparisons test with Bonferroni correction: control vs. 1 µM, p<0.0001; control vs. 10 µM, p<0.0001), while mutated Nav1.6 was unaffected (repeated measures ANOVA, p=0.879). (D) Dose-response curves showing the proportion of Na+ current elicited during a step depolarization from −100 to −20 mV for wild-type, individual mutants, and triple-mutant Nav1.6 channels exposed to increasing concentrations of TTX. Sample sizes are provided in Table 2. Data were fit with a Hill equation to estimate IC50 values.

Auawithoothij, Shewanella putrefaciens, a major microbial species related to tetrodotoxin (TTX)-accumulation of puffer fish Lagocephalus lunaris. 2012, Pubmed

Auawithoothij, Shewanella putrefaciens, a major microbial species related to tetrodotoxin (TTX)-accumulation of puffer fish Lagocephalus lunaris. 2012, Pubmed
Bordenstein, Host Biology in Light of the Microbiome: Ten Principles of Holobionts and Hologenomes. 2015, Pubmed
Brodie, The evolutionary response of predators to dangerous prey: hotspots and coldspots in the geographic mosaic of coevolution between garter snakes and newts. 2002, Pubmed
Brodie, Parallel arms races between garter snakes and newts involving tetrodotoxin as the phenotypic interface of coevolution. 2005, Pubmed
Brodie, Reciprocal selection at the phenotypic interface of coevolution. 2003, Pubmed
Bucciarelli, Quantifying tetrodotoxin levels in the California newt using a non-destructive sampling method. 2014, Pubmed
Caldwell, Sodium channel Na(v)1.6 is localized at nodes of ranvier, dendrites, and synapses. 2000, Pubmed
Cardall, Secretion and regeneration of tetrodotoxin in the rough-skin newt (Taricha granulosa). 2004, Pubmed
Chau, The chemical synthesis of tetrodoxin: an ongoing quest. 2011, Pubmed
Chau, On the origins and biosynthesis of tetrodotoxin. 2011, Pubmed
Cheng, Microflora and tetrodotoxin-producing bacteria in a gastropod, Niotha clathrata. 1995, Pubmed
Cole, Ribosomal Database Project: data and tools for high throughput rRNA analysis. 2014, Pubmed
Daly, Further classification of skin alkaloids from neotropical poison frogs (Dendrobatidae), with a general survey of toxic/noxious substances in the amphibia. 1987, Pubmed
Daly, First occurrence of tetrodotoxin in a dendrobatid frog (Colostethus inguinalis), with further reports for the bufonid genus Atelopus. 1994, Pubmed
Daly, Marine toxins and nonmarine toxins: convergence or symbiotic organisms? 2004, Pubmed
Dawkins, Arms races between and within species. 1979, Pubmed
Eisthen, Animal-microbe interactions and the evolution of nervous systems. 2016, Pubmed
Ellison, The Influence of Habitat and Phylogeny on the Skin Microbiome of Amphibians in Guatemala and Mexico. 2019, Pubmed
Feldman, Constraint shapes convergence in tetrodotoxin-resistant sodium channels of snakes. 2012, Pubmed
Feldman, The Nav channel bench series: Plasmid preparation. 2014, Pubmed
Feldman, The evolutionary origins of beneficial alleles during the repeated adaptation of garter snakes to deadly prey. 2009, Pubmed
Futuyma, Macroevolution and the biological diversity of plants and herbivores. 2009, Pubmed
Gall, Tetrodotoxin levels in larval and metamorphosed newts (Taricha granulosa) and palatability to predatory dragonflies. 2011, Pubmed
Gall, Tetrodotoxin concentrations within a clutch and across embryonic development in eggs of the rough-skinned newts (Taricha granulosa). 2014, Pubmed
Gall, Female newts (Taricha granulosa) produce tetrodotoxin laden eggs after long term captivity. 2012, Pubmed
Geffeney, Mechanisms of adaptation in a predator-prey arms race: TTX-resistant sodium channels. 2002, Pubmed
Geffeney, Evolutionary diversification of TTX-resistant sodium channels in a predator-prey interaction. 2005, Pubmed
Hanifin, Tetrodotoxin levels of the rough-skin newt, Taricha granulosa, increase in long-term captivity. 2002, Pubmed
Hanifin, Evolutionary history of a complex adaptation: tetrodotoxin resistance in salamanders. 2015, Pubmed
Hanifin, The chemical and evolutionary ecology of tetrodotoxin (TTX) toxicity in terrestrial vertebrates. 2010, Pubmed
Hanifin, A predictive model to estimate total skin tetrodotoxin in the newt Taricha granulosa. 2004, Pubmed
Hanifin, Phenotypic mismatches reveal escape from arms-race coevolution. 2008, Pubmed
Hanifin, Tetrodotoxin levels in eggs of the rough-skin newt, Taricha granulosa, are correlated with female toxicity. 2003, Pubmed
Hodgson, Toxins and venoms. 2012, Pubmed
Hu, Distinct contributions of Na(v)1.6 and Na(v)1.2 in action potential initiation and backpropagation. 2009, Pubmed
Jal, An overview on the origin and production of tetrodotoxin, a potent neurotoxin. 2015, Pubmed
Jen, Tetrodotoxin poisoning evidenced by solid-phase extraction combining with liquid chromatography-tandem mass spectrometry. 2008, Pubmed
Jost, Toxin-resistant sodium channels: parallel adaptive evolution across a complete gene family. 2008, Pubmed
Kearse, Geneious Basic: an integrated and extendable desktop software platform for the organization and analysis of sequence data. 2012, Pubmed
Kozich, Development of a dual-index sequencing strategy and curation pipeline for analyzing amplicon sequence data on the MiSeq Illumina sequencing platform. 2013, Pubmed
Lehman, No evidence for an endosymbiotic bacterial origin of tetrodotoxin in the newt Taricha granulosa. 2004, Pubmed
Lorincz, Molecular identity of dendritic voltage-gated sodium channels. 2010, Pubmed
Magarlamov, Tetrodotoxin-Producing Bacteria: Detection, Distribution and Migration of the Toxin in Aquatic Systems. 2017, Pubmed
Mandel, Models and approaches to dissect host-symbiont specificity. 2010, Pubmed
McFall-Ngai, Animals in a bacterial world, a new imperative for the life sciences. 2013, Pubmed
McMurdie, phyloseq: an R package for reproducible interactive analysis and graphics of microbiome census data. 2013, Pubmed
Mebs, Further report of the occurrence of tetrodotoxin in Atelopus species (family: Bufonidae). 1995, Pubmed
Mebs, Tissue distribution of tetrodotoxin in the red-spotted newt Notophthalmus viridescens. 2010, Pubmed
Mercer, Nav1.6 sodium channels are critical to pacemaking and fast spiking in globus pallidus neurons. 2007, Pubmed
Quast, The SILVA ribosomal RNA gene database project: improved data processing and web-based tools. 2013, Pubmed
Ritz, Dose-Response Analysis Using R. 2015, Pubmed
Ross, The skin microbiome of vertebrates. 2019, Pubmed
SanMiguel, Interactions between host factors and the skin microbiome. 2015, Pubmed
Satin, A mutant of TTX-resistant cardiac sodium channels with TTX-sensitive properties. 1992, Pubmed
Schloss, Introducing mothur: open-source, platform-independent, community-supported software for describing and comparing microbial communities. 2009, Pubmed
Schoener, The newest synthesis: understanding the interplay of evolutionary and ecological dynamics. 2011, Pubmed
Shen, Structural basis for the modulation of voltage-gated sodium channels by animal toxins. 2018, Pubmed
Shropshire, Speciation by Symbiosis: the Microbiome and Behavior. 2016, Pubmed
Simidu, Taxonomy of four marine bacterial strains that produce tetrodotoxin. 1990, Pubmed
Simão, BUSCO: assessing genome assembly and annotation completeness with single-copy orthologs. 2015, Pubmed
Smith, Functional analysis of the mouse Scn8a sodium channel. 1998, Pubmed
Sommer, Advancing gut microbiome research using cultivation. 2015, Pubmed
Song, Rcorrector: efficient and accurate error correction for Illumina RNA-seq reads. 2015, Pubmed
Stamatakis, RAxML version 8: a tool for phylogenetic analysis and post-analysis of large phylogenies. 2014, Pubmed
Stevenson, New strategies for cultivation and detection of previously uncultured microbes. 2004, Pubmed
Stewart, Growing unculturable bacteria. 2012, Pubmed
Stokes, Confirmation and distribution of tetrodotoxin for the first time in terrestrial invertebrates: two terrestrial flatworm species (Bipalium adventitium and Bipalium kewense). 2014, Pubmed
Tanu, Occurrence of tetrodotoxin in the skin of a rhacophoridid frog Polypedates sp. from Bangladesh. 2001, Pubmed
Theis, Getting the Hologenome Concept Right: an Eco-Evolutionary Framework for Hosts and Their Microbiomes. 2016, Pubmed
Tsuruda, Secretory glands of tetrodotoxin in the skin of the Japanese newt Cynops pyrrhogaster. 2002, Pubmed
Wakely, The occurrence of tetrodotoxin (tarichatoxin) in amphibia and the distribution of the toxin in the organs of newts (taricha). 1966, Pubmed
Wang, Toxin-screening and identification of bacteria isolated from highly toxic marine gastropod Nassarius semiplicatus. 2008, Pubmed
Whittaker, Allelochemics: chemical interactions between species. 1971, Pubmed
Williams, Behavioral and chemical ecology of marine organisms with respect to tetrodotoxin. 2010, Pubmed
Yang, A novel TTX-producing Aeromonas isolated from the ovary of Takifugu obscurus. 2010, Pubmed
Yotsu, Distribution of tetrodotoxin, 6-epitetrodotoxin, and 11-deoxytetrodotoxin in newts. 1990, Pubmed
Yotsu, Production of tetrodotoxin and its derivatives by Pseudomonas sp. isolated from the skin of a pufferfish. 1987, Pubmed
Yotsu-Yamashita, First report on toxins in the Panamanian toads Atelopus limosus, A. glyphus and A. certus. 2010, Pubmed
Yotsu-Yamashita, The levels of tetrodotoxin and its analogue 6-epitetrodotoxin in the red-spotted newt, Notophthalmus viridescens. 2001, Pubmed
Yotsu-Yamashita, Tetrodotoxin and its analogue 6-epitetrodotoxin in newts (Triturus spp.; Urodela, Salamandridae) from southern Germany. 2007, Pubmed
Yotsu-Yamashita, Tetrodotoxin in Asian newts (Salamandridae). 2017, Pubmed
Zakon, Expansion of voltage-dependent Na+ channel gene family in early tetrapods coincided with the emergence of terrestriality and increased brain complexity. 2011, Pubmed
van Opstal, MICROBIOME. Rethinking heritability of the microbiome. 2015, Pubmed