Pheromonally mediated sexual isolation among denning populations of red-sided garter snakes,Thamnophis sirtalis parietalis

Utilizing behavioral experiments and chemical analyses, we examined whether pheromonally mediated sexual isolation exists between denning populations of red-sided garter snakes (Thamnophis sirtalis parietalis) in Manitoba, Canada. Simultaneous choice tests conducted during the breeding season revealed that adult males from a hibernaculum in central Manitoba displayed a strong courtship preference for females from their own population over females from a hibernaculum in western Manitoba, whereas males from the western Manitoba hibernaculum showed no such preference. In addition, trailing experiments testing the response of males from the two hibernacula to familiar and unfamiliar female trails showed similar results, demonstrating that the observed male preference is mediated through chemical cues. Subsequent chemical analysis of the female sexual attractiveness pheromone, a homologous series of long-chain saturated and -9 cis-unsaturated methyl ketones responsible for eliciting male courtship behavior and trailing behavior in garter snakes, showed significant variation in the composition of the pheromone between the two populations. Specifically, the two populations varied in the relative concentrations of individual unsaturated methyl ketones expressed by females. These results suggest that sexual isolation exists to a degree among denning populations of red-sided garter snakes due to variation in the expression of the female sexual attractiveness pheromone.     

Sex pheromone source location by garter snakes:

Male plains garter snakes,Thamnophis radix, tested in a 240-cm-long arena can detect directional information from a female pheromone trail only when the female is allowed to push against pegs while laying the trail. The female's normal locomotor activity apparently deposits pheromone on the anterolateral surfaces of vertical structures in her environment. The male sensorily assays the sides of these objects and from this information determines the female's direction of travel.     

A complex,cross-taxon,chemical releaser of antipredator behavior in amphibians

Prey species show diverse antipredator responses to chemical cues signaling predation threat. Among terrestrial vertebrates, the red-backed salamander, Plethodon cinereus, is an important species in the study of these chemical defenses. During the day and early evening, this species avoids rinses from garter snakes, Thamnophis sirtalis, independent of snake diet, but late at night, avoids only those rinses from garter snakes that have recently eaten P. cinereus. We tested whether the selective, late-night response requires the ingestion or injury of salamanders. In three experiments, we tested P. cinereus for their responses to separate or combined rinses from salamanders (undisturbed, distressed, and injured P. cinereus) and snakes (unfed, earthworm fed, and salamander-fed T. sirtalis). When paired against a water control, only rinses from salamander-fed snakes were avoided. When salamander treatments (undisturbed or distressed) were combined with the snake treatments (unfed or earthworm-fed) and tested against a water control, the combinations elicited avoidance. When selected treatments were paired against the standard rinse from salamander-fed snakes, only the combined rinses from salamanders and snakes nullified the avoidance response to the standard rinse. These data reveal a prey defense mechanism involving chemical elements from both the predator and prey that does not require injury or ingestion of the prey in the formation of the cue.     

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.     

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.     

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.     

Resistance of Neonates and Field-Collected Garter Snakes (Thamnophis spp.) to Tetrodotoxin

Prior studies of tetrodotoxin (TTX) resistance in garter snakes (Thamnophis spp.) have used laboratory-reared neonates as subjects, but the use of field-caught individuals would reduce cost and effort. We compared estimates of TTX resistance in field-caught and laboratory-born garter snakes. We found that a mass-adjusted dose of TTX administered to field-caught garter snakes produces an estimate of a population 50% dose that is comparable and unbiased with respect to those previously reported using laboratory-born neonates. Dose-response curves estimated for three field-caught populations closely matched the curves estimated from neonate data. The method was tested using populations with levels of TTX resistance ranging between approximately 5-90 mass-adjusted mouse units for their respective 50% doses. The technique of using field-caught snakes as test subjects provides larger genetically independent data sets that are more easily obtained. Our results indicate that changes in mass during development parallel ontogenetic shifts in TTX resistance.     

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.     

Variation in a female sexual attractiveness pheromone controls male mate choice in garter snakes

Male red-sided garter snakes (Thamnophis sirtalis parietalis) display a courtship preference for larger females during the breeding season. Utilizing behavioral experiments and chemical analyses, we tested the hypothesis that males can discriminate among females of varying size solely by means of the sexual attractiveness pheromone, a previously characterized sex pheromone composed of a homologous series of long-chain saturated and -9 cis-unsaturated methyl ketones contained in the skin lipids of females. When presented with skin lipid extracts from large and small females, a greater proportion of males displayed courtship behaviors to large female extracts. This demonstrates that there is an intrinsic property of the female skin lipids that allows males to differentiate among large and small females. Analysis of the sexual attractiveness pheromone revealed that the necessary variation exists for this pheromone to function as a reliable indicator to males of female body size. Specifically, we observed a strong correlation between female snout–vent length and the relative concentration of saturated and -9 cis-unsaturated methyl ketones composing the pheromone; smaller females expressed pheromone profiles higher in saturated methyl ketones, while larger females expressed pheromone profiles dominated by unsaturated methyl ketones. The results of this study suggest that male red-sided garter snakes utilize compositional variation in the female sexual attractiveness pheromone to differentiate among potential mates of varying size.     

Risk-sensitive habitat use by brook stickleback (Culaea inconstans) in areas associated with minnow alarm pheromone

Brook stickleback (Culaea inconstans) share habitat and predators with cyprinid species, and they exploit the alarm pheromone of fathead minnows (Pimephales promelas) to avoid areas of high predation risk. In this field experiment, we measured the retention and duration of area avoidance by brook stickleback from areas marked with alarm pheromone of fathead minnows. Area avoidance was greatest during the first 2 hr after the source of the alarm pheromone was removed (P<0.05), but after 4 hr, area use was not significantly different from premarking levels. This study shows that brook stickleback: (1) use the alarm pheromone of fathead minnows to avoid high risk areas, (2) continue to avoid locations associated with predation risk after the source of the pheromone has gone, and (3) avoid risky areas temporarily, and resume use of risky areas after 2–4 hr. This behavioral response by stickleback to minnow alarm pheromone could serve to minimize risk of predation.     

Chemical labeling of northern pike (Esox lucius) by the alarm pheromone of fathead minnows (Pimephales promelas)

In previous experiments, chemical stimuli from northern pike (Esox lucius) elicited fright responses from pike-naive fathead minnows (Pimephales promelas) only if the pike had recently eaten conspecific minnows. We used a behavioral assay to determine if the fright response is the result of the incorporation of the minnow alarm pheromone into the chemical signature of the pike. Because the alarm substance cells (epidermal club cells) of fathead minnows are seasonally lost by males, we used chemical stimuli from pike that had eaten breeding male minnows as a control stimulus. In independent laboratory and field experiments, pike-naive minnows exhibited fright reactions (e.g., increased shelter use, avoidance) when exposed to water from tanks containing pike that had eaten nonbreeding fatheads (with alarm substance cells), but not to water from tanks containing pike that had eaten breeding male fatheads (without alarm substance cells). These data indicate that the fathead minnow alarm pheromone chemically labels northern pike as dangerous to pike-naive receivers.     

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.     

Learned Recognition of Predation Risk by Enallagma Damselfly Larvae (Odonata, Zygoptera) on the Basis of Chemical Cues

We studied two populations of damselfly larvae (Enallagma boreale): one population cooccurred with a predatory fish (northern pike, Esox lucius); the other did not. Damselflies that cooccurred with pike adopted antipredator behavior (reduced activity) in response to chemical stimuli from injured conspecifics, and to chemical stimuli from pike, relative to a distilled water control. Damselflies from an area where pike do not occur responded only to chemical stimuli from injured conspecifics. In a second set of experiments, we conditioned pike-naive damselflies to recognize and respond to chemical stimuli from pike with antipredator behavior. Damselfly larvae that were previously unresponsive to pike stimuli learned to recognize pike stimuli after a single exposure to stimuli from pike and injured damselflies or pike and injured fathead minnows (Pimephales promelas). The response to injured fathead minnows was not a general response to injured fish because damselfly larvae did not respond to chemical stimuli from injured swordtails (Xiphophorus helleri), an allopatric fish. Taken together, these data suggest a flexible learning program that allows damselfly larvae to rapidly acquire the ability to recognize local predation risk based on chemical stimuli from predators, conspecifics, and heterospecific members of their prey guild.     

Pheromonal Mediation of Intraseasonal Declines in the Attractivity of Female Red-Sided Garter Snakes, Thamnophis sirtalis parietalis

During the breeding season, female red-sided garter snakes (Thamnophis sirtalis parietalis) produce and express a sexual attractiveness pheromone that elicits male courtship behavior. Composed of a homologous series of saturated and monounsaturated methyl ketones, this pheromone is expressed in female skin lipids. Recent studies have shown that the sexual attractivity of unmated female garter snakes declines as the breeding season progresses. Here, we investigated whether temporal changes in the quantity and/or quality of the female sexual attractiveness pheromone are responsible for the observed loss of attractivity. Female red-sided garter snakes were collected immediately following spring emergence and held under natural conditions for the duration of the breeding season. Behavioral experiments confirmed that unmated females become significantly less attractive to males within two weeks of emergence from hibernation. Additionally, these females had lower estradiol concentrations at two weeks post-emergence. Subsequent chemical analyses revealed qualitative variation between the pheromone profiles of newly emerged females and those of females at two weeks post-emergence. Together, these results support the hypothesis that changes in the female sexual attractiveness pheromone are responsible for declining post-emergence female attractivity in garter snakes.     

Conditioned Alarm Behavior in Fathead Minnows (Pimephales promelas) Resulting from Association of Chemical Alarm Pheromone with a Nonbiological Visual Stimulus

Fathead minnows (Pimephales promelas) adopt antipredator (alarm) behavior when they detect alarm pheromone released from an injured conspecific. This is an adaptive response since alarm pheromone is generally released only in the context of a predation event. Alarm reactions may also occur in response to chemical and visual stimuli that minnows learn to associate with release of alarm pheromone. Here, we tested if fathead minnows can learn to associate a nonbiological, visual stimulus with predation risk. Minnows were simultaneously exposed to red light and conspecific alarm pheromone, inducing an alarm reaction. When retested using red light alone, small shoals of minnows displayed an antipredator response: dashing movements and disorganized swimming followed by decreased height in the water column and increased shoal cohesion. This resulted from a single-trial exposure to the combined cues and demonstrates a robust ecological mechanism by which minnows learn to recognize indicators of predation risk that may vary in space and time. However, learning to associate risk with biologically irrelevant stimuli may be an ecological liability. How minnows discern between relevant and irrelevant stimuli in nature is not known.     

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.     

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.     

Field Verification of Predator Attraction to Minnow Alarm Substance

Fishes such as minnows in the superorder Ostariophysi possess specialized alarm substance cells (ASC) that contain an alarm cue. Alarm substance can only be released by damage to the epidermis; thus, the release of alarm substance is a reliable indicator of predation risk. When nearby minnows detect the cue, they adopt a range of antipredator behaviors that reduce their probability of predation. Predator–predator interactions afford prey an opportunity to escape and, thus, a fitness benefit that maintains alarm substance calls over evolutionary time. Here, we present data from a simple field experiment verifying that nearby predators are attracted to minnow alarm substance because it signals an opportunity to pirate a meal. Fishing lures were baited with sponge blocks scented with either (1) water (control for sponge odor and appearance), (2) skin extract from non-ostariophysan convict cichlids (superorder Acanthopterygii, Archocentrus Cichlasoma nigrofasciatus) to control for general injury-released cues from fish, or (3) skin extract from fathead minnows (superorder Ostariophysi, Pimephales promelas). Predator strike frequency on each sponge type was 1,1, and 7 for water, cichlid, and minnow cues, respectively. These data provide the first field test using fish predators of the predator-attraction hypothesis for the evolution of Ostariophysan alarm substance cells.     

Chemicals of predatory mosquitofish (Gambusia affinis) influence selection of oviposition site by Culex mosquitoes

Ovipositing insects may avoid aquatic sites where there is high predation risk to their offspring, but the proximate mechanisms that mediate avoidance behavior are poorly resolved. We conducted an experiment to determine whether mosquitoes would reduce oviposition rates in pools containing chemicals of the mosquitofish (Gambusia affinis), a voracious predator that is widely employed to control mosquitoes. Experimental treatments consisted of outdoor pools that contained known concentrations of fish chemicals (low, medium, or high) or no fish chemicals (control). The pools were arranged in a randomized block design, and the number of mosquito larvae in each pool served as the response variable to estimate relative oviposition rate. Members of the Culex pipiens complex were the main colonizers of the pools. The mean number of larvae per pool differed among treatments (P = 0.026) and was about three times greater in control pools compared with those receiving medium and high concentrations of fish chemicals. Pairwise comparisons indicate that only medium and high treatments differed significantly from controls, suggesting that a threshold concentration exists below which mosquitoes cannot reliably detect predators. Our data suggest that the effectiveness of Gambusia affinis in controlling mosquitoes may be compromised if adult mosquitoes respond to fish stocking by shifting to nearby breeding sites that lack fish. We discuss issues concerning the use of Gambusia in biological control programs within the context of these new findings.     

Effects of diet on localized defecation by Northern Pike,Esox lucius

Fathead minnows (Pimephales promelas) are able to detect conspecific alarm pheromone in the feces of northern pike (Esox lucius) and have been shown to avoid areas labeled with the feces of pike that were fed minnows. The minnows did not avoid areas labeled with the feces of pike that were fed swordtails (Xiphophorous helleri), which lack ostariophysan alarm pheromone. In laboratory experiments, pike fed a diet of minnows localized their defecation away from their foraging area. It has been suggested that in doing so, pike may remove chemical cues that label their foraging area as dangerous to prey species. As yet there has been no conclusive evidence to support this hypothesis. In this experiment, we test the effects of different predator diets on localized defecation by pike. Pike were fed minnows, swordtails, or mice (Mus musculus). Swordtails and mice lack ostariophysan alarm pheromones. Area use and location of feces were recorded. Pike fed minnows spent significantly more time in the home area (i.e., area of the test tank where they were fed) and defecated significantly more often in the opposite end of the tank. Pike fed swordtails also exhibited a significant preference for the home area in area use, while those fed mice showed no such preference. When fed either swordtails or mice, there was no significant difference between the proportion of time spent and proportion of feces in each area of the test tank. These data suggest that pike localize their defecation only when consuming prey items containing alarm pheromone. The current findings support the hypothesis that pike localize their defecation to remove chemical cues from the foraging area of the home range in order to avoid chemically labeling their foraging area as dangerous to prey.