Genetic architecture of a feeding adaptation: garter snake (Thamnophis) resistance to tetrodotoxin bearing prey

Detailing the genetic basis of adaptive variation in natural populations is a first step towards understanding the process of adaptive evolution, yet few ecologically relevant traits have been characterized at the genetic level in wild populations. Traits that mediate coevolutionary interactions between species are ideal for studying adaptation because of the intensity of selection and the well-characterized ecological context. We have previously described the ecological context, evolutionary history and partial genetic basis of tetrodotoxin (TTX) resistance in garter snakes (Thamnophis). Derived mutations in a voltage-gated sodium channel gene (Na_v1.4) in three garter snake species are associated with resistance to TTX, the lethal neurotoxin found in their newt prey (Taricha). Here we evaluate the contribution of Na_v1.4 alleles to TTX resistance in two of those species from central coastal California. We measured the phenotypes (TTX resistance) and genotypes (Na_v1.4 and microsatellites) in a local sample of Thamnophis atratus and Thamnophis sirtalis. Allelic variation in Na_v1.4 explains 23 per cent of the variation in TTX resistance in T. atratus while variation in a haphazard sample of the genome (neutral microsatellite markers) shows no association with the phenotype. Similarly, allelic variation in Na_v1.4 correlates almost perfectly with TTX resistance in T. sirtalis, but neutral variation does not. These strong correlations suggest that Na_v1.4 is a major effect locus. The simple genetic architecture of TTX resistance in garter snakes may significantly impact the dynamics of phenotypic coevolution. Fixation of a few alleles of major effect in some garter snake populations may have led to the evolution of extreme phenotypes and an ‘escape’ from the arms race with newts.     

Adaptive evolution of tetrodotoxin resistance in animals

Tetrodotoxin (TTX), first isolated from pufferfish (tetraodontids), is a highly potent neurotoxin that selectively binds to voltage-gated sodium channels (Na(v)) in muscle and nerve tissues causing paralysis and death. Saxitoxin (STX) is a TTX-related neurotoxin produced by dinoflagellates. Recent investigations have implicated diverse substitutions in the P-loop regions of skeletal muscle and neuronal Na(v) channels in the convergent evolution of neurotoxin resistance in pufferfish, garter snakes and softshell clams, which has enabled them to feed on TTX- and STX-bearing organisms.     

Genetic basis of tetrodotoxin resistance in pufferfishes

Tetrodotoxin (TTX) is a highly potent neurotoxin that selectively binds to the outer vestibule of voltage-gated sodium channels. Pufferfishes accumulate extremely high concentrations of TTX without any adverse effect. A nonaromatic amino acid (Asn) residue present in domain I of the pufferfish, Takifugu pardalis, Na v1.4 channel has been implicated in the TTX resistance of pufferfishes . However, the effect of this residue on TTX sensitivity has not been investigated, and it is not known if this residue is conserved in all pufferfishes. We have investigated the genetic basis of TTX resistance in pufferfishes by comparing the sodium channels from two pufferfishes (Takifugu rubripes [fugu] and Tetraodon nigroviridis) and the TTX-sensitive zebrafish. Although all three fishes contain duplicate copies of Na v1.4 channels (Na v1.4a and Na v1.4b), several substitutions were found in the TTX binding outer vestibule of the two pufferfish channels. Electrophysiological studies showed that the nonaromatic residue (Asn in fugu and Cys in Tetraodon) in domain I of Na v1.4a channels confers TTX resistance. The Glu-to-Asp mutation in domain II of Tetraodon channel Na v1.4b is similar to that in the saxitoxin- and TTX-resistant Na+ channels of softshell clams . Besides helping to deter predators, TTX resistance enables pufferfishes to selectively feed on TTX-bearing organisms.     

Convergent adaptation to dangerous prey proceeds through the same first‐step mutation in the garter snake Thamnophis sirtalis

Convergent phenotypes often result from similar underlying genetics, but recent work suggests convergence may also occur in the historical order of substitutions en route to an adaptive outcome. We characterized convergence in the mutational steps to two independent outcomes of tetrodotoxin (TTX) resistance in separate geographic lineages of the common garter snake (Thamnophis sirtalis) that coevolved with toxic newts. Resistance is largely conferred by amino acid changes in the skeletal muscle sodium channel (Na_V1.4) that interfere with TTX‐binding. We sampled variation in Na_V1.4 throughout western North America and found clear evidence that TTX‐resistant changes in both lineages began with the same isoleucine‐valine mutation (I1561V) within the outer pore of Na_V1.4. Other point mutations in the pore, shown to confer much greater resistance, accumulate later in the evolutionary progression and always occur together with the initial I1561V change. A gene tree of Na_V1.4 suggests the I1561V mutations in each lineage are not identical‐by‐decent, but rather they arose independently. Convergence in the evolution of channel resistance is likely the result of shared biases in the two lineages of T. sirtalis—only a few mutational routes can confer TTX resistance while maintaining the conserved function of voltage‐gated sodium channels.     

Helminth infracommunity structure of the sympatric garter snakes Thamnophis eques and Thamnophis melanogaster from the Mesa Central of Mexico

Seventy-two Mexican garter snakes (Thamnophis eques) and 126 black-bellied garter snakes (T. melanogaster) were collected from 4 localities of the Mesa Central of Mexico between July 1996 and February 1998 and examined for helminths. Both species of garter snakes occurred sympatrically in every locality except in Lake Cuitzeo. Both species of snakes shared 9 helminth species, and in general, T. melanogaster hosted a larger number of species than T. eques. In each locality, a different helminth species showed the highest levels of prevalence and abundance (Spiroxys susanae in Ciénaga de Lerma, Telorchis corti in Lago de Pátzcuaro, Proteocephalus variabilis in Lago de Cuitzeo, and Contracaecum sp. in Lago de Chapala). Helminth communities in garter snakes of the Mesa Central are depauperate and dominated by a single parasite species. In those localities where the snakes occurred in sympatry, helminth communities were, in general, more diverse and species-rich in T. melanogaster. Differences in the ecology and physiology of these species of garter snakes may explain this pattern because black-bellied garter snakes (T. melanogaster) are more aquatic than Mexican garter snakes (T. eques) and primarily eat aquatic prey, potentially exposing themselves to a larger number of helminths transmitted by predator-prey infection. The helminth infracommunities of garter snakes in the Mesa Central of Mexico show a strong Nearctic influence because most of the species infecting these hosts have been recorded in other Nearctic colubrid snakes. However, the helminth infracommunities of these garter snakes are less species-rich and less diverse than those in colubrid snakes in more temperate latitudes. The widespread ecological perturbation of sampling sites in the Mesa Central because of human activity, and geographic differences in foraging ecology of the hosts and, thus, exposure to parasites transmitted by intermediate hosts may help to explain these patterns.     

Hematology and plasma biochemistry values for the giant garter snake (Thamnophis gigas) and valley garter snake (Thamnophis sirtalis fitchi) in the Central Valley of California

Hematology and plasma biochemistry parameters are useful in the assessment and management of threatened and endangered species. Although reference ranges are readily available for many mammalian species, reference ranges for snakes are lacking for most species. We determined hematology and plasma biochemistry reference ranges for giant garter snakes (Thamnophis gigas) and valley garter snakes (Thamnophis sirtalis fitchi) living in four management areas in the Central Valley of California. White blood cell, heterophil, lymphocyte, and azurophil counts in giant garter snakes were approximately twice the values of valley garter snakes. Statistically significant differences in aspartate aminotransferase, globulin, and potassium between the two species did not appear clinically significant. No significant differences were found in the measured parameters between male and female giant garter snakes. Some differences were found among collection sites. These reference ranges provide baseline data for comparisons over time and between collection sites.     

Biophysical costs associated with tetrodotoxin resistance in the sodium channel pore of the garter snake, <Emphasis Type="Italic">Thamnophis sirtalis</Emphasis>

Tetrodotoxin (TTX) is a potent toxin that specifically binds to voltage-gated sodium channels (NaV). TTX binding physically blocks the flow of sodium ions through NaV, thereby preventing action potential generation and propagation. TTX has different binding affinities for different NaV isoforms. These differences are imparted by amino acid substitutions in positions within, or proximal to, the TTX-binding site in the channel pore. These substitutions confer TTX-resistance to a variety of species. The garter snake Thamnophis sirtalis has evolved TTX-resistance over the course of an arms race, allowing some populations of snakes to feed on tetrodotoxic newts, including Taricha granulosa. Different populations of the garter snake have different degrees of TTX-resistance, which is closely related to the number of amino acid substitutions. We tested the biophysical properties and ion selectivity of NaV of three garter snake populations from Bear Lake, Idaho; Warrenton, Oregon; and Willow Creek, California. We observed changes in gating properties of TTX-resistant (TTXr) NaV. In addition, ion selectivity of TTXr NaV was significantly different from that of TTX-sensitive NaV. These results suggest TTX-resistance comes at a cost to performance caused by changes in the biophysical properties and ion selectivity of TTXr NaV.     

Mechanism of tetrodotoxin block and resistance in sodium channels

Tetrodotoxin (TTX) has been used for many decades to characterize the structure and function of biological ion channels. Yet, the precise mechanism by which TTX blocks voltage-gated sodium (Na_V) channels is not fully understood. Here molecular dynamics simulations are used to elucidate how TTX blocks mammalian voltage-gated sodium (Nav) channels and why it fails to be effective for the bacterial sodium channel, Na_VAb. We find that, in Na_VAb, a sodium ion competes with TTX for the binding site at the extracellular end of the filter, thus reducing the blocking efficacy of TTX. Using a model of the skeletal muscle channel, Na_V1.4, we show that the conduction properties of the channel observed experimentally are faithfully reproduced. We find that TTX occludes the entrance of Na_V1.4 by forming a network of hydrogen-bonds at the outer lumen of the selectivity filter. The guanidine group of TTX adopts a lateral orientation, rather than pointing into the filter as proposed previously. The acidic residues just above the selectivity filter are important in stabilizing the hydrogen-bond network between TTX and Na_V1.4. The effect of two single mutations of a critical tyrosine residue in the filter of Na_V1.4 on TTX binding observed experimentally is reproduced using computational mutagenesis.     

Contribution of Nav1.1 to cellular synchronization and automaticity in spontaneous beating cultured neonatal rat ventricular cells

Two factors of cell coupling influence cellular synchronization and automaticity: gap junction coupling and ion channels activity. However, the role of Na(+) channel isoforms underlying cell-to-cell interaction and cellular automaticity is not well understood. To address these questions, we studied mRNA expression of Na(+) channel isoforms and the effects of TTX on spontaneously beating cultured ventricle cells. Using RT-PCR technique we demonstrated the presence of Na(v)1.1 and Na(v)1.5 channels. The reduction of Na(v)1.1 channel activity disturbed cell-to-cell interaction and changed beating rates. Thus, Na(v)1.1 channel is involved in cellular synchronization and automaticity.     

Patterns of genetic differentiation in Thamnophis and Taricha from the Pacific Northwest

Aim The co‐evolutionary interaction between the common garter snake, Thamnophis sirtalis, and the rough‐skinned newt, Taricha granulosa, takes place throughout much of the Pacific Northwest (North America). The biogeography of the Pacific Northwest has been heavily influenced by the last Pleistocene glaciation, which reached a maximum as late as 14,000 yr bp . We researched: (1) what type of population structure is present for garter snakes and newts, (2) whether the population structure of these species is consistent with a Pleistocene glaciation hypothesis, and (3) how population structure and migration possibly affect co‐evolution between these species. Location The Pacific Northwest of North America, specifically northern California, Oregon and Washington in the USA. Methods We sampled approximately 20 populations for each species from three different transects. Using microsatellite markers and tissue samples from both species, we quantified the population structure for both species. Individual‐based assignment tests were used to estimate contemporary migration rates. Results Both Th. sirtalis and Ta. granulosa exhibited little genetic differentiation among our study sites, even among those separated by large distances. Significant population structure was detected on multiple geographic scales. Differences in population structure were observed among transects and between garter snake and newt transects. Contemporary migration rate estimates indicate high levels of genetic exchange between populations. Main conclusions Prior to this study, little was known about the fine‐scale population structure of either species in this region. Patterns of population structure for garter snakes and newts reflect a shared biogeographical history affected by the Pleistocene glaciation in the Pacific Northwest. Both species apparently migrate frequently between populations, thus potentially retarding the process of adaptive co‐evolution. We find that populations from a northern coastal transect (Washington) are most likely to be locally adapted.     

Toxin-resistant sodium channels: parallel adaptive evolution across a complete gene family

Approximately 75% of vertebrate proteins belong to protein families encoded by multiple evolutionarily related genes, a pattern that emerged as a result of gene and genome duplications over the course of vertebrate evolution. In families of genes with similar or related functions, adaptation to a strong selective agent should involve multiple adaptive changes across the entire gene family. However, we know of no evolutionary studies that have explicitly addressed this point. Here, we show how 4 taxonomically diverse species of pufferfishes (Tetraodontidae) each evolved resistance to the guanidinium toxins tetrodotoxin (TTX) and saxitoxin (STX) via parallel amino acid replacements across all 8 sodium channels present in teleost fish genomes. This resulted in diverse suites of coexisting sodium channel types that all confer varying degrees of toxin resistance, yet show remarkable convergence among genes and phylogenetically diverse species. Using site-directed mutagenesis and expression of a vertebrate sodium channel, we also demonstrate that resistance to TTX/STX is enhanced up to 15-fold by single, frequently observed replacements at 2 sites that have not previously been implicated in toxin binding but show similar or identical replacements in pufferfishes and in distantly related vertebrate and nonvertebrate animals. This study presents an example of natural selection acting upon a complete gene family, repeatedly arriving at a diverse but limited number of adaptive changes within the same genome. To be maximally informative, we suggest that future studies of molecular adaptation should consider all functionally similar paralogs of the affected gene family.     

Species specificity of methyl ketone profiles in the skin lipids of female garter snakes,genus Thamnophis

One of the relatively few vertebrate pheromones to be chemically identified, the female sex pheromone of the red-sided garter snake (Thamnophis sirtalis parietalis) is a series of saturated and monounsaturated methyl ketones contained within female skin lipids. During the breeding season, this pheromone is responsible for eliciting male courtship behaviors and males are able to utilize pheromonal variation to discriminate among females. While the pheromone system of the red-sided garter snake has been the subject of many studies, relatively little is known about the pheromone systems of other garter snakes. Through chemical analyses, we demonstrate that female skin lipids of the red-spotted garter snake (Thamnophis sirtalis concinnus), northwestern garter snake (Thamnophis ordinoides), and plains garter snake (Thamnophis radix) contain similar methyl ketones. The methyl ketone profiles of these snakes differ qualitatively from one another and from the methyl ketone profiles of red-sided garter snakes with differences particularly pronounced between sympatric species. Our results provide evidence that the use of methyl ketones in sexual signaling may be ubiquitous for Thamnophis species and suggest that these compounds could play a role in reproductive isolation between species in this genus.     

The muO-conotoxin MrVIA inhibits voltage-gated sodium channels by associating with domain-3

Several families of peptide toxins from cone snails affect voltage-gated sodium (Na(V)) channels: mu-conotoxins block the pore, delta-conotoxins inhibit channel inactivation, and muO-conotoxins inhibit Na(V) channels by an unknown mechanism. The only currently known muO-conotoxins MrVIA and MrVIB from Conus marmoreus were applied to cloned rat skeletal muscle (Na(V)1.4) and brain (Na(V)1.2) sodium channels in mammalian cells. A systematic domain-swapping strategy identified the C-terminal pore loop of domain-3 as the major determinant for Na(V)1.4 being more potently blocked than Na(V)1.2 channels. muO-conotoxins therefore show an interaction pattern with Na(V) channels that is clearly different from the related mu- and delta-conotoxins, indicative of a distinct molecular mechanism of channel inhibition.     

EVOLUTIONARY RESPONSE OF PREDATORS TO DANGEROUS PREY: PREADAPTATION AND THE EVOLUTION OF TETRODOTOXIN RESISTANCE IN GARTER SNAKES

Coevolutionary interactions typically involve only a few specialized taxa. The factors that cause some taxa and not others to respond evolutionarily to selection by another species are poorly understood. Preadaptation may render some species predisposed for evolutionary response to new pressures, whereas a lack of genetic variation may limit the evolutionary potential of other taxa. We evaluate these factors in the predator-prey interaction between toxic newts (Taricha granulosa) and their resistant garter snake predators (Thamnophis sirtalis). Using a bioassay of resistance to tetrodotoxin (TTX), the primary toxin in the prey, we examined phenotypic evolution in the genus Thamnophis. Reconstruction of ancestral character states suggests that the entire genus Thamnophis, and possibly natricine snakes in general, has slightly elevated TTX resistance compared to other lineages of snakes. While this suggests that T. sirtalis is indeed predisposed to evolving TTX resistance, it also indicates that the potential exists in sympatric congeners not expressing elevated levels of TTX resistance. We also detected significant family level variation for TTX resistance in a species of Thamnophis that does not exhibit elaborated levels of the trait. This finding suggests that evolutionary response in other taxa is not limited by genetic variability. In this predator-prey system, species and population differences in resistance appear to be largely determined by variation in the selective environment rather than preadaptation or constraint.     

Endogenous reactive oxygen species modulates voltage-gated sodium channels in dorsal root ganglia of rats

We recently reported that reactive oxygen species (ROS) plays an excitatory role in modulation of the exercise pressor reflex (EPR) in normal rats. In this study, we further tested two independent hypotheses: 1) ROS interacts with EPR-related ionotropic receptors such as the purinergic receptors (P(2)) and transient receptor potential vanilloid 1 receptors (TRPV1) to indirectly modulate the EPR function; 2) ROS directly affects excitability of muscle afferents by modulating the voltage-gated sodium (Na(v)) channels. To test the first hypothesis, we performed animal experiments to investigate the effect of the SOD mimetic 4-hydroxy-2,2,6,6-tetramethyl piperidine 1-oxyl (Tempol) on the pressor response to hindlimb intra-arterial (IA) injection of either α,β-methylene ATP (a P(2X) agonist) or capsaicin (a TRPV1 agonist) in decerebrate rats. To test the second hypothesis, we used the patch-clamp technique to determine the effect of ROS on Na(v) channels on the soma of muscle afferents. We also performed local microinjection of a sodium channel blocker, tetrodotoxin (TTX), into ipsilateral L4/L5 dorsal root ganglia (DRGs) to investigate whether the blockade of Na(v) channels by TTX affects the EPR function. We found that Tempol did not affect the pressor response to injection of either capsaicin or α,β-methylene ATP but significantly decreased the Na(v) current in small and medium-sized 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI)-labeled DRG neurons. A membrane-permeant superoxide dismutase, polyethylene glycol (PEG)-SOD, had an effect on the Na(v) current in these neurons similar to that of Tempol. Microinjection of TTX into L4/L5 DRGs dramatically attenuated the pressor response to static contraction induced by electrical stimulation of L4/L5 ventral roots. These data suggest that ROS modulates the EPR by affecting the activity of the Na(v) channels on muscle afferents.     

Interactions of the C-11 hydroxyl of tetrodotoxin with the sodium channel outer vestibule

The highly selective sodium channel blocker, tetrodotoxin (TTX) has been instrumental in characterization of voltage-gated sodium channels. TTX occludes the ion-permeation pathway at the outer vestibule of the channel. In addition to a critical guanidinium group, TTX possesses six hydroxyl groups, which appear to be important for toxin block. The nature of their interactions with the outer vestibule remains debatable, however. The C-11 hydroxyl (C-11 OH) has been proposed to interact with the channel through a hydrogen bond to a carboxyl group, possibly from domain IV. On the other hand, previous experiments suggest that TTX interacts most strongly with pore loops of domains I and II. Energetic localization of the C-11 OH was undertaken by thermodynamic mutant cycle analysis assessing the dependence of the effects of mutations of the adult rat skeletal muscle Na(+) channel (rNa(v)1.4) and the presence of C-11 OH on toxin IC(50). Xenopus oocytes were injected with the mutant or native Na(+) channel mRNA, and currents were measured by two-electrode voltage clamp. Toxin blocking efficacy was determined by recording the reduction in current upon toxin exposure. Mutant cycle analysis revealed that the maximum interaction of the C-11 OH was with domain IV residue D1532 (DeltaDeltaG: 1.0 kcal/mol). Furthermore, C-11 OH had significantly less interaction with several domain I, II, and III residues. The pattern of interactions suggested that C-11 was closest to domain IV, probably involved in a hydrogen bond with the domain IV carboxyl group. Incorporating this data, a new molecular model of TTX binding is proposed.     

Electrophysiological properties of human adipose tissue-derived stem cells

Human adipose tissue-derived stem cells (hASCs) represent a potentially valuable cell source for clinical therapeutic applications. The present study was designed to investigate properties of ionic channel currents present in undifferentiated hASCs and their impact on hASCs proliferation. The functional ion channels in hASCs were analyzed by whole-cell patch-clamp recording and their mRNA expression levels detected by RT-PCR. Four types of ion channels were found to be present in hASCs: most of the hASCs (73%) showed a delayed rectifier-like K(+) current (I(KDR)); Ca(2+)-activated K(+) current (I(KCa)) was detected in examined cells; a transient outward K(+) current (I(to)) was recorded in 19% of the cells; a small percentage of cells (8%) displayed a TTX-sensitive transient inward sodium current (I(Na.TTX)). RT-PCR results confirmed the presence of ion channels at the mRNA level: Kv1.1, Kv2.1, Kv1.5, Kv7.3, Kv11.1, and hEAG1, possibly encoding I(KDR); MaxiK, KCNN3, and KCNN4 for I(KCa); Kv1.4, Kv4.1, Kv4.2, and Kv4.3 for I(to) and hNE-Na for I(Na.TTX). The I(KDR) was inhibited by tetraethyl ammonium (TEA) and 4-aminopyridine (4-AP), which significantly reduced the proliferation of hASCs in a dose-dependent manner (P < 0.05), as suggested by bromodeoxyurindine (BrdU) incorporation. Other selective potassium channel blockers, including linopiridine, iberiotoxin, clotrimazole, and apamin also significantly inhibited I(KDR). TTX completely abolished I(Na.TTX). This study demonstrates for the first time that multiple functional ion channel currents such as I(KDR), I(KCa), I(to), and I(Na.TTX) are present in undifferentiated hASCs and their potential physiological function in these cells as a basic understanding for future in vitro experiments and in vivo clinical investigations.