Amber N. Stokes

Tetrodotoxin levels in larval and metamorphosed newts (Taricha granulosa) and palatability to predatory dragonflies.

Some populations of the newt Taricha granulosa possess extremely high concentrations of the neurotoxin tetrodotoxin (TTX). Tetrodotoxin is present in adult newts and their eggs, but has been assumed to be absent from the larval stage. We tested larval and metamorphosed juveniles for the presence of TTX and evaluated the palatability of these developmental stages to predatory dragonfly nymphs. All developmental stages retained substantial quantities of TTX and almost all individuals were unpalatable to dragonfly nymphs. Tetrodotoxin quantity varied greatly among individuals. When adjusted for mass, TTX concentrations declined steadily through metamorphosis. Several juveniles were palatable to dragonflies and these individuals had significantly lower TTX levels than unpalatable juveniles. These results suggest that despite previous assumptions, substantial quantities of TTX, originally deposited in the embryo, are retained by the developing larvae and metamorphosed juveniles and this quantity is enough to make them unpalatable to some potential predators.

An improved competitive inhibition enzymatic immunoassay method for tetrodotoxin quantification

Quantifying tetrodotoxin (TTX) has been a challenge in both ecological and medical research due to the cost, time and training required of most quantification techniques. Here we present a modified Competitive Inhibition Enzymatic Immunoassay for the quantification of TTX, and to aid researchers in the optimization of this technique for widespread use with a high degree of accuracy and repeatability.

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.

The role of corticosterone and toxicity in the antipredator behavior of the Rough-skinned Newt (Taricha granulosa).

A variety of mechanisms are responsible for enabling an organism to escape a predatory attack, including behavioral changes, alterations in hormone levels, and production and/or secretion of toxins. However, these mechanisms are rarely studied in conjunction with each other. The Rough-skinned Newt (Taricha granulosa) is an ideal organism to examine the relationships between these mechanisms because its behavioral displays and toxin secretion during a predator attack are well documented and readily characterized. While we found no direct relationship between antipredator behavior and endogenous levels of corticosterone (CORT), antipredator behavior was inhibited when exogenous CORT and adrenocorticotropic hormone (ACTH) were administered, resulting in high circulating concentrations of CORT, indicating that CORT may play a role in mediating the behavior. There was no correlation between the animals toxicity and either CORT or behavior. The results of this study provide evidence that CORT plays an important, yet complex, role in the antipredator response of these amphibians.

Female newts (Taricha granulosa) produce tetrodotoxin laden eggs after long term captivity

We investigated the presence of tetrodotoxin (TTX) in the eggs of wild-caught newts (Taricha granulosa) at capture and again after one, two, and three years in captivity. Females initially produced eggs that contained quantities of TTX similar to previous descriptions of eggs from wild-caught adults. After the first year in captivity, the egg toxicity from each female declined, ultimately remaining constant during each of the successive years in captivity. Despite declining, all females continued to produce eggs containing substantial quantities of TTX during captivity. The decline in toxicity can not be attributed to declining egg mass but may be the result of the abbreviated reproductive cycle to which the captive newts were subjected in the lab. Finally, an estimate of the amount of TTX provisioned in the entire clutch from each female is similar to the quantity of TTX regenerated in the skin after electrical stimulation. These results, coupled with other long-term studies on the maintenance and regeneration of TTX in the skin, suggests an endogenous origin of TTX in newts.

Do all portable cases constructed by caddisfly larvae function in defense

Abstract The portable cases constructed by caddisfly larvae have been assumed to act as a mechanical defense against predatory attacks. However, previous studies have compared the survival of caddisflies with different cases, thereby precluding an analysis of the survival benefits of “weaker” case materials. The level of protection offered by caddisfly cases constructed with rock, stick, or leaf material, as well as a no-case control, was investigated against predatory dragonfly nymphs (Anax junius Drury (Anisoptera: Aeshnidae)). A valid supposition is that the cases made of stronger material are more effective at deterring predators. Yet, observations revealed that there was no difference in survival between the case types. All caddisflies with a case experienced high survival in comparison to caddisflies removed from their case. In addition, larvae with stick-cases experienced fewer attacks and captures by dragonflies. These results showed that the presence of a case, regardless of the material used in its construction, offers survival benefits when faced with predatory dragonfly nymphs.

Sex-biased Predation on Newts of the Genus Taricha by a Novel Predator and its Relationship with Tetrodotoxin Toxicity

Abstract Newts of the genus Taricha have long been studied in regards to their skin toxin, tetrodotoxin (TTX). It has been shown that the TTX levels across populations of Taricha are highly variable, and this has been mostly attributed to the interaction between Taricha and their only documented predators, garter snakes of the genus Thamnophis. Here we show that predators other than Thamnophis prey extensively on some newt populations. Ledson Marsh in Annadel State Park in Santa Rosa, CA is a breeding ground for both the California newt (Taricha torosa) and the rough-skinned newt (Taricha granulosa). Predation on these newts was tracked from 1998–2009 and was most often in the form of evisceration and significantly male-biased. As TTX seems to have been developed as an antipredator defense in Taricha, we used Fluorometric High Phase Liquid Chromatography (HPLC) analysis to quantify TTX levels in the skin of ten male and ten female newts of each species to determine the influence that TTX levels may have on sex-biased predation in this population. We found Taricha females were not significantly more toxic than males. Also, we found that T. torosa were significantly more toxic than T. granulosa, which is in contrast with other newt toxicity studies.

Otter Predation on Taricha granulosa and Variation in Tetrodotoxin Levels with Elevation

Abstract Tetrodotoxin (TTX) is a low molecular weight neurotoxin that is found in a wide variety of taxa. Tetrodotoxin blocks voltage-gated sodium channels, preventing the propagation of action potentials and inducing paralysis in susceptible animals. Taricha granulosa have been documented to possess TTX in high quantities and are preyed upon by snakes of the genus Thamnophis. However, recent observations of predation events on T. granulosa by otters were documented in a high-elevation population just outside of Crater Lake National Park, Oregon. We quantified TTX levels in this population as well as 3 populations in Crater Lake National Park using a Competitive Inhibition Enzymatic Immunoassay. We further compared these high elevation populations to a known high-toxicity population from Benton County, Oregon. We found that the populations in Crater Lake have lower levels of TTX relative to populations outside of the lake, and that all high-elevation locations have relatively low levels of TTX. We then analyzed previously published whole-newt TTX levels and elevation, and found that there is a significant negative relationship. However, there is a non-significant relationship between whole-newt TTX levels and elevation when examining elevations below 500 m. This further exemplifies the potential for novel predation and previously unidentified selective pressures in high-elevation newt populations.

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

Abstract 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.

An Investigation into Tetrodotoxin (TTX) Levels Associated with the Red Dorsal Spots in Eastern Newt (Notophthalmus viridescens) Efts and Adults

We investigated the concentration of tetrodotoxin (TTX) in sections of skin containing and lacking red dorsal spots in both Eastern newt (Notophthalmus viridescens) efts and adults. Several other species, such as Pleurodeles waltl and Echinotriton andersoni, have granular glands concentrated in brightly pigmented regions on the dorsum, and thus we hypothesized that the red dorsal spots of Eastern newts may also possess higher levels of TTX than the surrounding skin. We found no difference between the concentrations of TTX in the red spots as compared to neighboring skin lacking these spots in either efts or adults. However, efts with more red dorsal spots had elevated TTX levels relative to efts with fewer spots.