In Silico Analysis of Tetrodotoxin Binding in Voltage-Gated Sodium Ion Channels from Toxin-Resistant Animal Lineages.

Multiple animal species have evolved resistance to the neurotoxin tetrodotoxin (TTX) through changes in voltage-gated sodium ion channels (VGSCs). Amino acid substitutions in TTX-resistant lineages appear to be positionally convergent with changes in homologous residues associated with reductions in TTX block. We used homology modeling coupled with docking simulations to test whether positionally convergent substitutions generate functional convergence at the level of TTX-channel interactions. We found little evidence that amino acids at convergent positions generated similar patterns among TTX-resistant animal lineages across several metrics, including number of polar contacts, polar contact position, and estimates of binding energy. Though binding energy values calculated for TTX docking were reduced for some TTX-resistant channels, not all TTX-resistant channels and not all of our analyses returned reduced binding energy values for TTX-resistant channels. Our results do not support a simple model of toxin resistance where a reduced number of bonds between TTX and the channel protein prevents blocking. Rather models that incorporate flexibility and movement of the protein overall may better describe how homologous substitutions in the channel cause changes in TTX block.

Chemical Ecology of the North American Newt Genera Taricha and Notophthalmus.

The North American newt genera Taricha and Notophthalmus (order Caudata) are well known for the combination of potent toxicity, aposematic coloration, and striking defense postures that protects these animals from predation. This suite of traits is centered around the neurotoxin tetrodotoxin, which causes paralysis and death in metazoans by disrupting the initiation and propagation of electrical signals in the nerves and muscles. Tetrodotoxin defends newts from predation across multiple life history stages and its role in generating arms-race coevolution between Taricha newts and garter snake (genus Thamnophis) predators is well studied. However, understanding the broader picture of chemical defenses in Taricha and Notophthalmus requires an expanded comprehension of the defensive chemical ecology of tetrodotoxin that includes possible coevolutionary interactions with insect egg predators, protection against parasites, as well as mimicry complexes associated with tetrodotoxin and aposematic coloration in both genera. Herein the authors review what is known about the structure, function, and pharmacology of tetrodotoxin to explore its evolution and chemical ecology in the North American newt. Focus is made specifically on the origin and possible biosynthesis of tetrodotoxin in these taxa as well as providing an expanded picture of the web of interactions that contribute to landscape level patterns of toxicity and defense in Taricha and Notophthalmus.

Nothing in Evolution Makes Sense Except in the Light of Biology.

A key question in biology is the predictability of the evolutionary process. If we can correctly predict the outcome of evolution, we may be better equipped to anticipate and manage species' adaptation to climate change, habitat loss, invasive species, or emerging infectious diseases, as well as improve our basic understanding of the history of life on Earth. In the present article, we ask the questions when, why, and if the outcome of future evolution is predictable. We first define predictable and then discuss two conflicting views: that evolution is inherently unpredictable and that evolution is predictable given the ability to collect the right data. We identify factors that generate unpredictability, the data that might be required to make predictions at some level of precision or at a specific timescale, and the intellectual and translational value of understanding when prediction is or is not possible.

Identification of Tricyclic Guanidino Compounds from the Tetrodotoxin-Bearing Newt Taricha granulosa.

The biosynthesis of the potent neurotoxin tetrodotoxin (TTX, 1) is still unresolved. We used MS-guided screening and nuclear magnetic resonance analyses including long-range HSQMBC to characterize two novel skeletal tricyclic guanidino compounds, Tgr-288 (2a and 2b) and Tgr-210 (3), from the TTX-bearing newt, Taricha granulosa. The presence of these compounds in toxic newts is congruent with a previously proposed pathway for TTX biosynthesis in terrestrial organisms that includes a monoterpene precursor and the production of structurally diversified guanidino compounds.

Structures of N-Hydroxy-Type Tetrodotoxin Analogues and Bicyclic Guanidinium Compounds Found in Toxic Newts.

The biosynthesis of tetrodotoxin (TTX, 1), a potent neurotoxin widely distributed in marine and terrestrial metazoans, remains unresolved. A significant issue has been identifying intermediates and shunt products associated with the biosynthetic pathway of TTX. We investigated TTX biosynthesis by screening and identifying new TTX-related compounds from Cynops ensicauda popei and Taricha granulosa. Mass spectrometry (MS)-guided screening identified two new N-hydroxy TTX analogues in newts: 1-hydroxy-8-epiTTX (2) and 1-hydroxy-8-epi-5,11-dideoxyTTX (3, previously reported as 1-hydroxy-5,11-dideoxyTTX). We prepared a new analogue, 8-epi-5,11-dideoxyTTX (4), from 3 via N-OH reduction and confirmed the presence of 4 in T. granulosa using hydrophilic interaction liquid chromatography (HILIC)-LCMS. The presence of 8-epi-type TTX analogues in both Cynops and Taricha supports a branched biosynthetic pathway of terrestrial TTX, which produces 6- and 8-epimers. In addition, new bicyclic guanidinium compounds Tgr-238 (5) and Tgr-240 (6) were identified as putative shunt products of our proposed TTX biosynthesis pathway. A structural analysis of Cep-228A (7), another bicyclic compound, was performed using NMR. Based on the structures of 5-7 and their analogues, we propose a model of the shunt and metabolic pathways of the terrestrial TTX biosynthesis.

Synthesizing and Expressing Native Ion Channels.

Synthesizing and expressing ion channels in heterologous systems enable the characterization of the functional properties of these proteins. The cDNA that encodes ion channels can be amplified directly from mRNA or synthesized de novo in its entirety before cloning into an appropriate expression vector. Gibson assembly is a powerful tool that allows rapid cloning and integration of protein-coding cDNA into a variety of expression vectors. Here we describe a method in which the cDNA encoding a native snake ion channel (NaV 1.4) is synthesized in four equal-sized pieces (or blocks), and then assembled and ligated into an expression vector. Once in an appropriate expression vector, the assembled cDNA can be used for synthesis of mRNA, and the mRNA injected and expressed in Xenopus oocytes. This method has significant advantages over traditional rtPCR and ligation-based cloning including speed, cost, ease of codon optimization, and inclusion of silent restriction sites for Gibson-based mutagenesis.

Convergent and parallel evolution in a voltage-gated sodium channel underlies TTX-resistance in the Greater Blue-ringed Octopus: Hapalochlaena lunulata.

The natural history and pharmacology of tetrodotoxin (TTX) has long intrigued biologists. This toxin has a remarkable distribution that spans two domains of life (Bacteria and Eukarya). Within Eukaryotes, TTX has only been identified in animals but is known to be present in over five-dozen species of phylogenetically distant Metazoans. Despite decades of work, the origin and biosynthetic pathways of TTX remain unresolved. Investigations in puffer fishes and salamanders have provided insights into the acquisition of auto-resistance to TTX through the evolution of voltage-gated sodium ion channels (VGSCs) that have reduced binding affinity for TTX. To date there have been no studies of these proteins in tetrodotoxic Blue-Ringed Octopuses. Here we report data demonstrating that the Greater Blue-ringed Octopus (Hapalochlaena lunulata) expresses a VGSC (HlNaV1) gene with mutations that reduce the channel's TTX-binding affinity and likely render the organism TTX resistant. We identified three amino-acid substitutions in the TTX-binding site of HlNaV1 that likely confer TTX-resistance to both the channel and the organism. These substitutions are associated with organismal TTX-resistance in other TTX-bearing taxa and are convergent with substitutions that have evolved in fish, salamanders, and some TTX-resistant invertebrates.

Large-effect mutations generate trade-off between predatory and locomotor ability during arms race coevolution with deadly prey.

Adaptive evolution in response to one selective challenge may disrupt other important aspects of performance. Such evolutionary trade-offs are predicted to arise in the process of local adaptation, but it is unclear if these phenotypic compromises result from the antagonistic effects of simple amino acid substitutions. We tested for trade-offs associated with beneficial mutations that confer tetrodotoxin (TTX) resistance in the voltage-gated sodium channel (NaV1.4) in skeletal muscle of the common garter snake (Thamnophis sirtalis). Separate lineages in California and the Pacific Northwest independently evolved TTX-resistant changes to the pore of NaV1.4 as a result of arms race coevolution with toxic prey, newts of the genus Taricha. Snakes from the California lineage that were homozygous for an allele known to confer large increases in toxin resistance (NaV1.4LVNV) had significantly reduced crawl speed compared to individuals with the ancestral TTX-sensitive channel. Heterologous expression of native snake NaV1.4 proteins demonstrated that the same NaV1.4LVNV allele confers a dramatic increase in TTX resistance and a correlated decrease in overall channel excitability. Our results suggest the same mutations that accumulate during arms race coevolution and beneficially interfere with toxin-binding also cause changes in electrophysiological function of the channel that may affect organismal performance. This trade-off was only evident in the predator lineage where coevolution has led to the most extreme resistance phenotype, determined by four critical amino acid substitutions. If these biophysical changes also translate to a fitness cost-for example, through the inability of T. sirtalis to quickly escape predators-then pleiotropy at this single locus could contribute to observed variation in levels of TTX resistance across the mosaic landscape of coevolution.

Historical Contingency in a Multigene Family Facilitates Adaptive Evolution of Toxin Resistance.

Novel adaptations must originate and function within an already established genome [1]. As a result, the ability of a species to adapt to new environmental challenges is predicted to be highly contingent on the evolutionary history of its lineage [2-6]. Despite a growing appreciation of the importance of historical contingency in the adaptive evolution of single proteins [7-11], we know surprisingly little about its role in shaping complex adaptations that require evolutionary change in multiple genes. One such adaptation, extreme resistance to tetrodotoxin (TTX), has arisen in several species of snakes through coevolutionary arms races with toxic amphibian prey, which select for TTX-resistant voltage-gated sodium channels (Nav) [12-16]. Here, we show that the relatively recent origins of extreme toxin resistance, which involve the skeletal muscle channel Nav1.4, were facilitated by ancient evolutionary changes in two other members of the same gene family. A substitution conferring TTX resistance to Nav1.7, a channel found in small peripheral neurons, arose in lizards ∼170 million years ago (mya) and was present in the common ancestor of all snakes. A second channel found in larger myelinated neurons, Nav1.6, subsequently evolved resistance in four different snake lineages beginning ∼38 mya. Extreme TTX resistance has evolved at least five times within the past 12 million years via changes in Nav1.4, but only within lineages that previously evolved resistant Nav1.6 and Nav1.7. Our results show that adaptive protein evolution may be contingent upon enabling substitutions elsewhere in the genome, in this case, in paralogs of the same gene family.

Toxicity and population structure of the Rough-Skinned Newt (Taricha granulosa) outside the range of an arms race with resistant predators.

Species interactions, and their fitness consequences, vary across the geographic range of a coevolutionary relationship. This spatial heterogeneity in reciprocal selection is predicted to generate a geographic mosaic of local adaptation, wherein coevolutionary traits are phenotypically variable from one location to the next. Under this framework, allopatric populations should lack variation in coevolutionary traits due to the absence of reciprocal selection. We examine phenotypic variation in tetrodotoxin (TTX) toxicity of the Rough-Skinned Newt (Taricha granulosa) in regions of allopatry with its TTX-resistant predator, the Common Garter Snake (Thamnophis sirtalis). In sympatry, geographic patterns of phenotypic exaggeration in toxicity and toxin-resistance are closely correlated in prey and predator, implying that reciprocal selection drives phenotypic variation in coevolutionary traits. Therefore, in allopatry with TTX-resistant predators, we expect to find uniformly low levels of newt toxicity. We characterized TTX toxicity in northwestern North America, including the Alaskan panhandle where Ta. granulosa occur in allopatry with Th. sirtalis. First, we used microsatellite markers to estimate population genetic structure and determine if any phenotypic variation in toxicity might be explained by historical divergence. We found northern populations of Ta. granulosa generally lacked population structure in a pattern consistent with northern range expansion after the Pleistocene. Next, we chose a cluster of sites in Alaska, which uniformly lacked genetic divergence, to test for phenotypic divergence in toxicity. As predicted, overall levels of newt toxicity were low; however, we also detected unexpected among- and within-population variation in toxicity. Most notably, a small number of individuals contained large doses of TTX that rival means of toxic populations in sympatry with Th. sirtalis. Phenotypic variation in toxicity, despite limited neutral genetic divergence, suggests that factors other than reciprocal selection with Th. sirtalis likely contribute to geographic patterns of toxicity in Ta. granulosa.

Evolutionary history of a complex adaptation: tetrodotoxin resistance in salamanders.

Understanding the processes that generate novel adaptive phenotypes is central to evolutionary biology. We used comparative analyses to reveal the history of tetrodotoxin (TTX) resistance in TTX-bearing salamanders. Resistance to TTX is a critical component of the ability to use TTX defensively but the origin of the TTX-bearing phenotype is unclear. Skeletal muscle of TTX-bearing salamanders (modern newts, family: Salamandridae) is unaffected by TTX at doses far in excess of those that block action potentials in muscle and nerve of other vertebrates. Skeletal muscle of non-TTX-bearing salamandrids is also resistant to TTX but at lower levels. Skeletal muscle TTX resistance in the Salamandridae results from the expression of TTX-resistant variants of the voltage-gated sodium channel NaV 1.4 (SCN4a). We identified four substitutions in the coding region of salSCN4a that are likely responsible for the TTX resistance measured in TTX-bearing salamanders and variation at one of these sites likely explains variation in TTX resistance among other lineages. Our results suggest that exaptation has played a role in the evolution of the TTX-bearing phenotype and provide empirical evidence that complex physiological adaptations can arise through the accumulation of beneficial mutations in the coding region of conserved proteins.

Confirmation and distribution of tetrodotoxin for the first time in terrestrial invertebrates: two terrestrial flatworm species (Bipalium adventitium and Bipalium kewense).

The potent neurotoxin tetrodotoxin (TTX) is known from a diverse array of taxa, but is unknown in terrestrial invertebrates. Tetrodotoxin is a low molecular weight compound that acts by blocking voltage-gated sodium channels, inducing paralysis. However, the origins and ecological functions of TTX in most taxa remain mysterious. Here, we show that TTX is present in two species of terrestrial flatworm (Bipalium adventitium and Bipalium kewense) using a competitive inhibition enzymatic immunoassay to quantify the toxin and high phase liquid chromatography to confirm the presence. We also investigated the distribution of TTX throughout the bodies of the flatworms and provide evidence suggesting that TTX is used during predation to subdue large prey items. We also show that the egg capsules of B. adventitium have TTX, indicating a further role in defense. These data suggest a potential route for TTX bioaccumulation in terrestrial systems.

Ontogeny of tetrodotoxin levels in blue-ringed octopuses: maternal investment and apparent independent production in offspring of Hapalochlaena lunulata.

Many organisms provision offspring with antipredator chemicals. Adult blue-ringed octopuses (Hapalochlaena spp.) harbor tetrodotoxin (TTX), which may be produced by symbiotic bacteria. Regardless of the ultimate source, we find that females invest TTX into offspring and offspring TTX levels are significantly correlated with female TTX levels. Because diversion of TTX to offspring begins during the earliest stages of egg formation, when females are still actively foraging and looking for mates, females may face an evolutionary tradeoff between provisioning larger stores of TTX in eggs and retaining that TTX for their own defense and offense (venom). Given that total TTX levels appear to increase during development and that female TTX levels correlate with those of offspring, investment may be an active adaptive process. Even after eggs have been laid, TTX levels continue to increase, suggesting that offspring or their symbionts begin producing TTX independently. The maternal investment of TTX in offspring of Hapalochlaena spp. represents a rare examination of chemical defenses, excepting ink, in cephalopods.

The chemical and evolutionary ecology of tetrodotoxin (TTX) toxicity in terrestrial vertebrates.

Tetrodotoxin (TTX) is widely distributed in marine taxa, however in terrestrial taxa it is limited to a single class of vertebrates (Amphibia). Tetrodotoxin present in the skin and eggs of TTX-bearing amphibians primarily serves as an antipredator defense and these taxa have provided excellent models for the study of the evolution and chemical ecology of TTX toxicity. The origin of TTX present in terrestrial vertebrates is controversial. In marine organisms the accepted hypothesis is that the TTX present in metazoans results from either dietary uptake of bacterially produced TTX or symbiosis with TTX producing bacteria, but this hypothesis may not be applicable to TTX-bearing amphibians. Here I review the taxonomic distribution and evolutionary ecology of TTX in amphibians with some attention to the origin of TTX present in these taxa.

Phenotypic mismatches reveal escape from arms-race coevolution.

Because coevolution takes place across a broad scale of time and space, it is virtually impossible to understand its dynamics and trajectories by studying a single pair of interacting populations at one time. Comparing populations across a range of an interaction, especially for long-lived species, can provide insight into these features of coevolution by sampling across a diverse set of conditions and histories. We used measures of prey traits (tetrodotoxin toxicity in newts) and predator traits (tetrodotoxin resistance of snakes) to assess the degree of phenotypic mismatch across the range of their coevolutionary interaction. Geographic patterns of phenotypic exaggeration were similar in prey and predators, with most phenotypically elevated localities occurring along the central Oregon coast and central California. Contrary to expectations, however, these areas of elevated traits did not coincide with the most intense coevolutionary selection. Measures of functional trait mismatch revealed that over one-third of sampled localities were so mismatched that reciprocal selection could not occur given current trait distributions. Estimates of current locality-specific interaction selection gradients confirmed this interpretation. In every case of mismatch, predators were "ahead" of prey in the arms race; the converse escape of prey was never observed. The emergent pattern suggests a dynamic in which interacting species experience reciprocal selection that drives arms-race escalation of both prey and predator phenotypes at a subset of localities across the interaction. This coadaptation proceeds until the evolution of extreme phenotypes by predators, through genes of large effect, allows snakes to, at least temporarily, escape the arms race.

Parallel arms races between garter snakes and newts involving tetrodotoxin as the phenotypic interface of coevolution.

Parallel "arms races" involving the same or similar phenotypic interfaces allow inference about selective forces driving coevolution, as well as the importance of phylogenetic and phenotypic constraints in coevolution. Here, we report the existence of apparent parallel arms races between species pairs of garter snakes and their toxic newt prey that indicate independent evolutionary origins of a key phenotype in the interface. In at least one area of sympatry, the aquatic garter snake, Thamnophis couchii, has evolved elevated resistance to the neurotoxin tetrodotoxin (TTX), present in the newt Taricha torosa. Previous studies have shown that a distantly related garter snake, Thamnophis sirtalis, has coevolved with another newt species that possesses TTX, Taricha granulosa. Patterns of within population variation and phenotypic tradeoffs between TTX resistance and sprint speed suggest that the mechanism of resistance is similar in both species of snake, yet phylogenetic evidence indicates the independent origins of elevated resistance to TTX.

Secretion and regeneration of tetrodotoxin in the rough-skin newt (Taricha granulosa).

Rough-skin newts (Taricha granulosa) released tetrodotoxin (TTX) in their skin secretions in response to mild electric stimulation. This release resulted in a large (21% to almost 90% of the pre-stimulation levels) reduction in the amount of TTX present in the dorsal skin of individual newts. Over the next 9 months newts significantly regenerated the levels of TTX in their skin. These data, in combination with previously published results, are consistent with the hypothesis that these newts produce their own TTX.

A predictive model to estimate total skin tetrodotoxin in the newt Taricha granulosa.

We developed a predictive model to estimate the total amount of tetrodotoxin (TTX) in the skin of individual newts (Taricha granulosa) based on measures of the amount of TTX present in dorsal skin. We found that regions of skin on a newt could be reliably differentiated using granular gland density and that patterns of variation in granular gland density matched intra-individual variation in TTX levels. Tetrodotoxin is uniformly distributed in dorsal skin and TTX levels in dorsal skin are strongly predictive of TTX levels in other regions of skin on a newt. Our model is both accurate and precise and includes the effect of body size through surface area, variation in granular gland density, as well as positional variation in toxicity apparently associated with variation in granular gland density. This technique allows us to detect patterns that would be unclear if only dorsal skin toxicity or total skin toxicity based on extracts from an entire animal were used and demonstrates that the levels of TTX present in some populations of T. granulosa are remarkably high.

Tetrodotoxin levels in eggs of the rough-skin newt, Taricha granulosa, are correlated with female toxicity.

We quantified the amount of the neurotoxin tetrodotoxin (TTX) present in females and newly deposited eggs of the rough-skin newt, Taricha granulosa, to examine the relationship between the toxicity of an individual female and the toxicity of her eggs. We found high levels of TTX in individual eggs as well as substantial variation among clutches. Variation in the amount of TTX per egg within individual clutches was extremely low. Female skin toxicity was positively correlated with the mean egg toxicity of her clutch. Neither egg volume nor female size was significantly correlated with egg TTX levels. Tetrodotoxin stereoisomer-analog profiles were identical for females and their eggs. The presence of high levels of TTX in individual eggs coupled with the relationship between levels of TTX in female skin and levels of TTX in her eggs suggests that the TTX present in eggs of T. granulosa is maternally derived. The lack of correlation between egg size and TTX levels in individual eggs, as well as the low levels of within clutch variation, may indicate that deposition of TTX in eggs of T. granulosa is not linked to the deposition of other egg resources (e.g.. lipids or other yolk components).

Tetrodotoxin levels of the rough-skin newt, Taricha granulosa, increase in long-term captivity.

We investigated the persistence of the neurotoxin tetrodotoxin (TTX) in individual captive newts (Taricha granulosa) from the Willamette Valley of Oregon using a non-lethal sampling technique. We found that the TTX levels of newts held in the laboratory for 1 yr increased. TTX stereoisomer-analog profiles were not affected by captive husbandry. Levels of TTX were high in newts from our study population and we observed substantial within population variation in quantitative levels of TTX. Females possessed more TTX than males, but the response of TTX levels to captivity did not differ between females and males. The stability of TTX toxicity in newts is consistent with other amphibian species where TTX is present and may indicate that exogenous factors play a less important role in TTX toxicity of newts than previously thought.