Effects of predator chemical cues and behavioral biorhythms on foraging activity of terrestrial salamanders

Red-backed salamanders, Plethodon cinereus, show a variety of alarm responses to chemical cues from eastern garter snakes, Thamnophis sirtalis. We measured the foraging activity of red-backed salamanders exposed to water soiled by a garter snake (fed P. cinereus) or to unsoiled water. Salamanders exposed to snake-soiled water showed less foraging activity than salamanders exposed to unsoiled water; therefore, predators could have nonlethal effects on salamander populations. Our results also show additional factors influenced salamander foraging activity. Salamander foraging activity and responsiveness to chemical cues do not appear to have been affected by sex or food deprivation. Salamander foraging activity does appear to have been influenced by activity biorhythms. Foraging activity of animals in both treatments showed a bimodal periodicity that is consistent with natural activity patterns controlled by internal biorhythms. Exposure to snake-soiled water significantly reduced foraging activity during periods of peak foraging activity, but had a subtler effect0 on foraging activity during natural lulls in activity. We suggest that both activity biorhythms and exposure to chemical cues are important factors affecting salamander foraging behavior.     

Analysis of chemicals from earthworms and fish that elicit prey attack by ingestively naive garter snakes (Thamnophis)

Materials previously shown to elicit increased tongue-flicking and prey attack in garter snakes (Thamnophis sirtalis) were isolated from both earthworms (Lumbricus terrestris) and fish (Pimephales promelas). Both high- and low-molecular-weight components from earthworms and fish stimulated attacks and increased tongue-flicking in previously unfed neonate garter snakes relative to distilled water controls. Earthworm collagen was also effective, but even concentrated fractions were less effective than raw extract. Conflicting reports on the effectiveness of collagen suggest that the salient chemical(s) is a smaller molecule tightly bound to collagen and resisting standard purification methods.     

Prey detection by vomeronasal chemoreception in a plethodontid salamander

While chemoreception is involved in a wide variety of salamander behaviors, the chemosensory system that mediates specific behaviors is rarely known. We investigated the role of the vomeronasal system (VNS) in foraging behavior of the red-backed salamander (Plethodon cinereus) by manipulating salamanders' abilities to detect nonvolatile chemical cues emitted by potential prey. Subjects received one of three treatments: (1) impaired vomeronasal system, (2) sham manipulation, and (3) no manipulation. The role of the VNS in mediating foraging on motile prey (Drosophila melanogaster) was investigated under three light conditions (bright, dim, dark). Salamanders with impaired VNSs foraged less efficiently than either of the other experimental groups by displaying the longest latency to attack and the lowest rate of prey capture, especially in the absence of visual cues. A second experiment utilized freshly killed prey to determine whether the VNS takes on added importance in the absence of visual or tactile cues associated with moving prey. Animals with impaired VNSs showed a decreased foraging efficiency on stationary prey under both dark and light conditions. In addition, a mark–recapture study of VNS-impaired and sham salamanders in the field also indicated that salamanders with impaired VNSs consumed fewer stationary prey compared to shams. The study indicates that the VNS plays a substantial role in the foraging behavior of the plethodontid salamander, P. cinereus.     

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.     

Volatile components in scent gland secretions of garter snakes (Thamnophis spp.)

Previous analyses of the scent gland secretions of snakes have focused on the nonvolatile components. Gas chromatography-mass spectrometry of secretions from North American garter snakes (Thamnophis butleri, T. couchi, T. elegans, T. melanogaster, andT. sirtalis) indicated the following seven major volatile components: acetic, propanoic, 2-methylpropanoic, butanoic, and 3-methylbutanoic acids, trimethylamine, and 2-piperidone. Five or more of these compounds were observed in secretions of select boid, colubrid, pythonid, and viperid snakes, suggesting that they are widespread scent gland products. 3-Methylbutanal also was detected in some snake species.     

Purification and preliminary characterization of a frog-derived proteinaceous chemoattractant eliciting prey attack by checkered garter snakes (Thamnophis marcianus)

A potent proteinaceous chemoattractant, eliciting prey attack by checkered garter snakes (Thamnophis marcianus) was isolated from aqueous washes of the common frogRana temporaria and purified by preparative continuous-elution electrophoresis. The biological activity of the frog crude extract or of the purified chemoattractive protein, measured by a snake bioassay, was unaffected by freezing, lyophilization, or dialysis but was lost after proteolytic digestion. The purified chemoattractant is glycosylated, has an apparent molecular mass of 24 kDa, estimated by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS-PAGE), and a pI of 4.8. It gave one spot in two-dimensional electrophoresis. The bioassay showed that this protein is highly attractive to snakes. The lowest concentration yielding positive responses in the snake bioassay was approximately 25 µg/ml. These results suggest that a water-soluble Mr 24 kDa glycoprotein molecule produced by the common frog may be a vomeronasal stimulus used by checkered garter snakes for prey recognition.     

Population and sex differences in antipredator responses of breeding fathead minnows (Pimephales promelas) to chemical stimuli from garter snakes (Thamnophis radix andT. sirtalis)

We conducted a predator bite survey on a population of fathead minnows (Pimephales promelas) considered to be under substantial predation pressure by western plains garter snakes (Thamnophis radix). Scarring, due to failed predation attempts by garter snakes and crayfish (Orconectes virilis), was observed significantly more often in breeding males than in breeding females and nonbreeding minnows. Likely, territorial nest defense under the edges of rocks along the water's edge, a habitat occupied by crayfish and frequented by snakes, caused the breeding males to be differentially vulnerable to predation. Under controlled laboratory conditions, breeding males from this population exhibited an antipredator response to chemical stimuli from live snakes (T. sirtalis andT. radix) significantly more often than breeding female minnows from the same population and breeding minnows of both sexes from a population that was presumed to be under lower predation pressure from snakes.     

Responses of ecologically dissimilar populations of the water snakeNatrix s. sipedon to chemical cues from prey

Three populations of the water snake,Natrix s. sipedon, were tested for their responses to surface water extracts prepared from various prey species. It was clearly shown that these snakes can distinguish between different prey genera on the basis of chemical cues alone. Adult snakes caught at a fish hatchery where goldfish (Carassius auratus) were abundant responded most to goldfish extract, as did laboratory-born snakes reared on goldfish for one year. However, snakes caught in a relatively undisturbed mountain stream environment and naive young born to a mountain female responded more to the extracts of various sympatric-mountain fish and amphibians. The evidence indicates that newborn snakes have genetic predispositions for sympatric species of the classes of prey normally eaten. These preferences can be enhanced or inhibited during ontogeny.     

Naive ophiophagus lizards recognize and avoid venomous snakes using chemical cues

Monitor lizards prey on snakes. Conversely, venomous snakes prey on juvenile monitor lizards. Immediately after hatching, monitor lizards are naive to all prey items, thus correct assessment of snake prey is paramount for survival. Experiments were conducted to determine how hatchling monitor lizards (Varanus albigularis) with no previous exposure to snakes reacted to sympatric venomous and nonvenomous snakes. Hatchling lizards attacked harmless snakes, but avoided venomous species. Lizards readily accepted meat from skinned snakes, regardless of species. When invertebrate prey covered with skin segments from venomous snakes were restrained from moving, they were usually investigated by tongue-flicking and rejected. Unrestrained skin-covered prey, however, were generally attacked and eaten without prior evaluation by tongue-flicking. Attack was inhibited in trials in which unrestrained prey were tongue-flicked, suggesting that chemical cues contained in snake skins mediate avoidance of venomous snakes. Selection for the ability to perceive snake integumental chemicals may be especially strong in species that both consume and are consumed by snakes.     

Conspecific scent trailing by garter snakes (Thamnophis sirtalis) during autumn Further evidence for use of pheromones in den location

Adult garter snakes (Thamnophis sirtalis), collected in October near a traditional, communal hibernaculum in central Wisconsin, were acclimated to autumnal conditions and subjected to laboratory tests to determine whether they could follow scent trails of a conspecific. Graded responses were obtained, but 75% of the sample showed at least some inclination to follow scent trails. The results suggest that pheromone cues may be used by male and female garter snakes to locate traditional dens during autumnal migrations. Evidence from this and other studies suggests that pheromones are probably used in conjunction with other homing mechanisms and that the role of pheromones in den location may be more important in younger snakes and in populations inhabiting northern latitudes.     

Responses by corn snakes (Elaphe guttata) to chemicals from heterospecific snakes

Young corn snakes,Elaphe guttata, were tested for responses to chemicals from heterospecific snakes. Corn snakes exhibited more tongue-flicks to swabs freshly rubbed against the skin of an ophiophagous kingsnake,Lampropeltis getulus, than to blank swabs. Responses toL. getulus and a nonophiophagous western plains garter snake,Thamnophis radix haydeni, did not differ significantly. Corn snakes exhibited more tongue-flicks to swabs treated with chloroform extracts of the shed skins ofL. getulus; an ophiophagous eastern coachwhip,Masticophis flagellum; and a nonophiophagous gray ratsnake,Elaphe obsoleta, than to blank swabs, but they did not discriminate between ophiophagous and nonophiophagous species in every case. Corn snakes, when offered shelters containing bedding from the home cages of a nonophiophagous water snake,Nerodia erythrogaster, an occasionally ophiophagous water moccasin,Agkistrodon pisdvorus; orL. getulus and untreated bedding, failed to reside under snake-scented shelters at a rate significantly different from that expected by chance. The responses of corn snakes are compared with those reported for other snakes presented with heterospecific snake chemicals.     

Effect of experience with pine (Pituophis melanoleucus) and king (Lampropeltis getulus) snake odors on Y-maze behavior of pine snake hatchlings

The abilities of hatchling pine snakes (Pituophis melanoleucus) and king snakes (Lampropeltis getulus) to discriminate the chemical trails of pine and king snakes was investigated inY-maze experiments. Pine snakes were housed for 17 days either with shavings impregnated with pine snake odor, king snake odor, or no odor to test for the effect of experience on choice. Both pine and king snake hatchlings entered the arm with the pine snake odor and did not enter the arm with the king snake odor. The data support the hypothesis that hatchlings of both species can distinguish conspecific odors from other odors and that our manipulation of previous experience was without effect for pine snake hatchlings.     

Effects of Hunger and Predation Risk on Foraging Behavior of Graybelly Salamanders, Eurycea multiplicata

We examined the effects of predation and hunger levels on foraging behavior of adult neotenic graybelly salamanders, Eurycea multiplicata griseogaster. Hungry and satiated salamanders were exposed to chemical stimuli from a predatory fish (sculpin, Cottus carolinae) and from two nonpredatory species, golden redhorse fish (Moxostoma erythrurum) and tadpoles of leopard frogs (Rana sphenocephala). Latency to attack prey was lengthened in the presence of chemical stimuli from predators regardless of hunger levels, but hungry salamanders had shorter latency times than satiated salamanders. There was no interaction between hunger and threat levels. In addition, salamanders distinguished between chemical stimuli from predatory (sculpin) and nonpredatory (redhorse) fishes; responses to redhorse and tadpole stimuli were not different. Handling times were not affected by either predator treatment or hunger level. In summary, graybelly salamanders can (1) recognize sculpin predators based solely on chemical cues, (2) distinguish between chemical stimuli from predatory and nonpredatory fish, and (3) adjust their foraging behavior according to both hunger and predation risk.     

Predatory response of brown tree snakes to chemical stimuli from human skin

The brown tree snake (Boiga irregularis) is an exotic pest species on Pacific islands, most notably on Guam where it has caused considerable ecological and economic damage. On Guam, the snake commonly associates with people and can be found near or in human habitations. Bites are common, approximately 1 of 1200 emergency room visits to Guam hospitals were reported to be the result of B. irregularis bites; 80% of these victims were attacked while sleeping. Most of the attacks occurred on fingers and hands and the attacks appeared to be predatory, rather than defensive, in nature. In order to characterize the mechanism releasing this unusual behavior, we measured the predatory response of B. irregularis to chemical stimuli from humans and controls using a lab population that originated from Guam and a wild population from the species' native range in Queensland, Australia. To quantify behavior we measured the proportion of snakes displaying predatory behavior to each of the stimuli, the latency to attack, and the number of tongue-flicks displayed. We quantified predatory behavior using the tongue-flick attack score for repeated measures [TFAS(R)], a common method for quantifying predatory behavior in squamate reptiles. Captive brown tree snakes responded to human skin stimuli with feeding behavior, including predatory attacks, at the same frequency as they did to prey stimuli derived from mice, while never responding to controls with such behavior. Captive snakes also responded to human skin stimuli and prey stimuli with significantly higher TFAS(R) scores than to controls, although there were no differences between the human and mouse stimuli. Wild-caught animals in Australia also responded with predatory attacks to human skin stimuli, while not showing predatory behavior to a blank control and with higher TFAS(R) scores to human skin stimuli than to the control. As B. irregularis is a generalized predator that relies heavily on chemical signals to recognize prey, we hypothesize that the snakes recognize compounds on human skin that may be shared with other prey.     

The Identification of 2,4-diacetylphloroglucinol as an Antifungal Metabolite Produced by Cutaneous Bacteria of the Salamander <Emphasis Type="Italic">Plethodon cinereus</Emphasis>

Beneficial bacteria that live on salamander skins have the ability to inhibit pathogenic fungi. Our study aimed to identify the specific chemical agent(s) of this process and asked if any of the antifungal compounds known to operate in analogous plant–bacteria–fungi systems were present. Crude extracts of bacteria isolated from salamander skin were exposed to HPLC, UV-Vis, GC-MS, and HR-MS analyses. These investigations show that 2,4-diacetylphloroglucinol is produced by the bacteria isolate Lysobacter gummosus (AB161361), which was found on the red-backed salamander, Plethodon cinereus. Furthermore, exposure of the amphibian fungal pathogen, Batrachochytrium dendrobatidis (isolate JEL 215), to different concentrations of 2,4-diacetylphloroglucinol resulted in an IC₅₀ value of 8.73 μM, comparable to crude extract concentrations. This study is the first to show that an epibiotic bacterium on an amphibian species produces a chemical that inhibits pathogenic fungi.     

Alarm response by a plethodontid salamander (Desmognathus ochrophaeus): Conspecific and heterospecific “Schreckstoff”

The detection of chemical alarm cues plays an important role for predator avoidance in many taxonomic groups, but little is known about the presence of such chemical cues in adult or caudate amphibians. We investigated the response (i.e., aversion or nonaversion) to chemical cues from damaged salamander skin and mealworms (Tenebrio molitor) in the plethodontid salamander,Desmognathus ochrophaeus. Avoidance responses were demonstrated to skin extracts of both conspecific and heterospecific salamanders. However, salamanders (D. ochrophaeus) did not avoid heated conspecific skin, fresh conspecific viscera, fresh mealworm, or freshPlethodon richmondi skin extracts. These results indicate that chemical alarm cues are: (1) present in the skin ofDesmognathus salamanders, (2) not present in mealworm or the viscera ofDesmognathus salamanders, and (3) denatured or deactivated by heating. These results also suggest that an avoidance response to chemical cues from damaged conspecifics has adaptive value in predator avoidance in terrestrial as well as aquatic vertebrates.     

Following of conspecific and avoidance of predator chemical cues by pine snakes (Pituophis melanoleucus)

The ability of hatchling pine snakes (Pituophis melanoleucus) to follow or avoid the chemical trails of conspecifics and a king snake (Lampropeltis getulus) on paper substrates was investigated inY-maze experiments. Hatchlings entered the arm with the adult conspecific trail and avoided the arm containing the king snake trail at a frequency much greater than that due to chance. The data support the hypotheses that pine snakes follow the chemical trails of adult conspecifics and avoid the chemical trails of a predator.     

Amphibian Chemical Defense: Antifungal Metabolites of the Microsymbiont <Emphasis Type="Italic">Janthinobacterium lividum</Emphasis> on the Salamander <Emphasis Type="Italic">Plethodon cinereus</Emphasis>

Disease has spurred declines in global amphibian populations. In particular, the fungal pathogen Batrachochytrium dendrobatidis has decimated amphibian diversity in some areas unaffected by habitat loss. However, there is little evidence to explain how some amphibian species persist despite infection or even clear the pathogen beyond detection. One hypothesis is that certain bacterial symbionts on the skin of amphibians inhibit the growth of the pathogen. An antifungal strain of Janthinobacterium lividum, isolated from the skin of the red-backed salamander Plethodon cinereus, produces antifungal metabolites at concentrations lethal to B. dendrobatidis. Antifungal metabolites were identified by using reversed phase high performance liquid chromatography, high resolution mass spectrometry, nuclear magnetic resonance, and UV-Vis spectroscopy and tested for efficacy of inhibiting the pathogen. Two metabolites, indole-3-carboxaldehyde and violacein, inhibited the pathogen’s growth at relatively low concentrations (68.9 and 1.82 μM, respectively). Analysis of fresh salamander skin confirmed the presence of J. lividum and its metabolites on the skin of host salamanders in concentrations high enough to hinder or kill the pathogen (51 and 207 μM, respectively). These results support the hypothesis that cutaneous, mutualistic bacteria play a role in amphibian resistance to fungal disease. Exploitation of this biological process may provide long-term resistance to B. dendrobatidis for vulnerable amphibians and serve as a model for managing future emerging diseases in wildlife populations. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Response to prey chemical cues by hatchling pine snakes (Pituophis melanoleucus): Effects of incubation temperature and experience

The ability of hatchling pine snakes (Pituophis melanoleucus) to select and follow or avoid chemical odors of prey (mice,Mus musculus) on a shavings and paper substrate was investigated in Y-maze experiments, as a function of incubation temperature and experience. Incubation temperature affected behavior in the maze, and the maze choices of naive snakes, but not of snakes that had already eaten a mouse. The data indicate that snakes that have eaten, preferentially enter the arm bearing chemical stimuli from mice, whereas those that have not eaten show no preference.     

Comparison of earthworm- and fish-derived chemicals eliciting prey attack by garter snakes (Thamnophis)

Materials eliciting increased tongue flicking and prey attack in garter snakes were isolated from both earthworm and fish prey. New extraction methods based on chloroform-methanol mixtures are valuable adjuncts to the more typical aqueous preparations. Both high- and low-molecular weight components from earthworms and fish were active. The similarity between the active chemicals in these two classes of prey was established using several methods of analysis. These included chromatography, carbohydrate and amino acid analyses, and nuclear magnetic resonance.