Chemical defense of an Asian snake reflects local availability of toxic prey and hatchling diet.

Species that sequester toxins from prey for their own defense against predators may exhibit population-level variation in their chemical arsenal that reflects the availability of chemically defended prey in their habitat. Rhabdophis tigrinus is an Asian snake that possesses defensive glands in the skin of its neck ('nuchal glands'), which typically contain toxic bufadienolide steroids that the snakes sequester from consumed toads. In this study, we compared the chemistry of the nuchal gland fluid of R. tigrinus from toad-rich and toad-free islands in Japan and determined the effect of diet on the nuchal gland constituents. Our findings demonstrate that captive-hatched juveniles from toad-rich Ishima Island that had not been fed toads possess defensive bufadienolides in their nuchal glands, presumably due to maternal provisioning of these sequestered compounds. Wild-caught juveniles from Ishima possess large quantities of bufadienolides, which could result from a combination of maternal provisioning and sequestration of these defensive compounds from consumed toads. Interestingly, juvenile females from Ishima possess larger quantities of bufadienolides than do juvenile males, whereas a small sample of field-collected snakes suggests that adult males contain larger quantities of bufadienolides than do adult females. Captive-born hatchlings from Kinkasan Island lack bufadienolides in their nuchal glands, reflecting the absence of toads on that island, but they can sequester bufadienolides by feeding on toads (Bufo japonicus) in captivity. The presence of large quantities of bufadienolides in the nuchal glands of R. tigrinus from Ishima may reduce the risk of predation by providing an effective chemical defense, whereas snakes on Kinkasan may experience increased predation due to the lack of defensive compounds in their nuchal glands.

Does prey matter? Geographic variation in antipredator responses of hatchlings of a Japanese natricine snake (Rhabdophis tigrinus).

Rhabdophis tigrinus, which typically forages on toads, has unusual nuchal glands on its dorsal neck region containing secretions chemically comparable to cardiac steroids found in toads. R. tigrinus also has several peculiar antipredator displays involving the neck region. If the nuchal gland secretions of R. tigrinus are derived from toads obtained as prey, populations of R. tigrinus that have not eaten toads over an evolutionary time scale would be expected to lose antipredator displays related to the nuchal glands. We found that laboratory-hatched R. tigrinus from a small toad-free island exhibited displays related to nuchal glands less frequently and flight responses more frequently than hatchling snakes from areas sympatric with toads. These results are consistent with the hypothesis of the dietary origin of the nuchal gland secretions and also support the genetic origin of the behavioral differences between the populations.

The genetics of dietary experience in a restricted natural population.

The sources of individual differences in human and non-human animals remain controversial. We demonstrate that diet and genetics interact in determining the ontogenetic trajectory of chemosensory and prey preferences in the common garter snake, Thamnophis sirtalis, a dietary generalist. In litters of neonate snakes from a single small field in an earthworm-ingesting population, initial responses to chemical cues from fish and worm were similar, with zero heritabilities. After 12 meals on fish, however, the heritability of both fish and worm chemosensory responses increased markedly, the change in response to fish but not worm chemicals was heritable, the relative preference for fish versus worm was heritable, and the change in relative preference was heritable. In addition, growth rates on each diet were related to changes in chemoreceptive responses. Such genetic-environment variation that emerges only after equivalent ontogenetic experience may be a factor in responses to environmental change in many species.

Feeding behavior and an oropharyngeal component of satiety in a two-headed snake.

We studied the feeding behavior of a dicephalous Black Rat Snake (Elaphe o. obsoleta) whose heads attack and ingest prey independently. Both heads were given satiation feedings with mouse prey at either 5-day intervals (1989, n = 16 feedings) or 9-11 day intervals (1990, n = 4 feedings). In 1989, a meal pattern was manifested as a negative correlation between total (left + right) meal size (g) and total size of the last meal. Meal sizes of the heads were consistently different (L > R) despite their apparently identical genotypes. We confirmed tongue-flicking rate and swallowing time as indices of satiety/hunger in each head. In order to test whether oropharyngeal stimulation is involved in satiation, we analyzed three measures: willingness to feed, and intrameal changes in swallowing, and tongue-flicking rates. Data for these tests were taken from experimental feedings in which one head was fed to satiation (first head) before the other head (second head) was fed. The second head generally did eat (8 of 10 experimental feedings) after the first head had become satiated. In addition, while the time to swallow a mouse generally increased as the snake neared satiety, swallowing time decreased significantly (p = 0.04) from the first head's last mouse to the second head's first mouse. Finally, in two of three feedings, the first head's tongue-flicking rate decreased before the second head's. These results, which take advantage of this unique animal, indicate that oropharyngeal stimulation is involved in meal termination.

Responses to escalating predatory threat in garter and ribbon snakes (Thamnophis).

Several models suggest that animals perform a stable sequence of responses during increasing threat from a potential predator. To test the comparative utility of such models, 45 garter and ribbon snakes from four species (genus Thamnophis) were given an antipredator behavior test in which the snakes were exposed to a series of five levels of escalating threat: (a) threat is out of view, (b) threat looms over arena, (c) threat is nonmoving human hand near snake's head, (d) threat is moving human hand, and (e) threat is experimenter's tapping the snake's body. Five different antipredator behaviors were measured. It was found that both qualitative and quantitative changes in behavior occurred between threat levels. However, species differences were also found in the occurrence and amount of every behavior recorded, which indicates that even closely related species may differ in behaviors exhibited in different phases of a predatory encounter.

The comparative imperative: genetics and ontogeny of chemoreceptive prey responses in natricine snakes.

Reptiles offer a rich diversity for the study of chemoreception, and snakes are a particularly appropriate group for comparative, evolutionary, genetic, developmental, and mechanistic studies. A long-term program of research is described that attempts to integrate these approaches, focusing on the widespread North American genus Thamnophis (Natricinae). Prior to their first meal, neonatal snakes respond to aqueous surface substances from species-typical prey with increased tongue-flicking and open-mouth attacks; these responses are mediated by the vomeronasal organ. Such responses predict what prey snakes will eat and can also predict relative prey preference. Species, population, litter, and individual differences exists and are important at different levels of analysis. Chemoreceptive responses are heritable, although they may show different developmental trends. Some species respond to prey types they do not eat in nature. In the earthworm specialist, T. butleri, response to fish chemicals can be interpreted as a chemoreceptive response inertially inherited from ancestral species, decoupled from prey capture techniques, and in the process of being lost. Ontogeny and experience can modify behavior of the neonate in various ways. Feeding experience can alter response to some prey more than others, and ambient prey odor may shift prey preference. Psychophysical studies show that prey preference and threshold sensitivity to prey chemicals can be independent and differ between closely related species, indicating that neural tissue is devoted to recognition of specific types of prey. In site choice tests, garter snakes can also discriminate between feces derived from conspecific snakes fed similar or different diets.(ABSTRACT TRUNCATED AT 250 WORDS)

Development of antipredator responses in snakes: V. Species differences in ontogenetic trajectories.

The ontogeny of antipredator responses in five species of Natricine snakes was examined. In a split-litter experiment, neonates were given a standardized defensive behavior test at 1 and/or 20 days of age. Thamnophis sirtalis and Thamnophis melanogaster showed a significant increase in both striking and fleeing from a threatening stimulus. Thamnophis butleri was not reactive in terms of striking at either age but did flee frequently at both ages. Thamnophis radix showed low rates of striking and high rates of fleeing; only fleeing increased significantly between the two tests. Nerodia rhombifer exhibited moderate rates of both striking and fleeing at both ages with no developmental effects. Subsets of most of these species tested at 40 or 60 days of age found no further developmental changes.

Effect of food competition on aggregation: evidence for social recognition in the plains garter snake (Thamnophis radix).

Competition for food and aggregating behavior were examined in plains garter snakes (Thamnophis radix), in 11 groups of 1 noncompetitor and 2 competitors. Aggregation data were obtained in 48 daily scans. During food competition, success at obtaining food was asymmetrical across pairs. For aggregation, competitors were mostly associated with noncompetitors. After removal of noncompetitors, competitors were more likely to be solitary, but associations between competitors increased. After a 5-month separation, 5 of the original groups were reconstituted; aggregation patterns similar to the 1st experiment were observed. In a 3rd experiment, unfamiliar animals with and without competitive experience did not aggregate preferentially. Garter snakes discriminate on the basis of sex and species; in this study individual relationships (prior competition) affected aggregation, and the data suggest individual recognition both occurs and persists over time.

A comparative analysis of scoring methods for chemical discrimination of prey by squamate reptiles.

In studies of squamate responses to prey chemicals presented on cotton-tipped applicators, investigators typically record several responses, each of which gives only part of the overall picture. The tongue-flick/attack score (TFAS) is a widely used composite measure of response strength that accounts for attack, its latency, and number of tongue-flicks. We present data and analyses on these variables and investigate the utility of several other possible response variables. It is concluded, for both practical and theoretical reasons, that TFAS and two modifications of it are the best measures of response strength. Uses and statistical analyses of TFAS and variables derived from it are discussed. It is recommended that information on tongue-flick rate, number of individuals attacking, and latency to attack be presented.

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.

Development of antipredator responses in snakes: IV. Interspecific and intraspecific differences in habituation of defensive behavior.

Habituation of defensive attacks directed toward a threatening stimulus was investigated in neonatal garter snakes. The focus of the experiments was on differential effects of a simple experimental process in relation to species, litter, sex, and individual. In Experiment 1 newborn Thamnophis melanogaster from four liters and newborn Thamnophis butleri from three liters were given daily tests in which snakes were confronted with a nonmoving and moving human hand. Over five successive test days the T melanogaster neonates showed a decline in number of strikes directed toward the stimuli. When retested 10-13 days later the animals showed significant response recovery. Although some of the T? butleri newborns demonstrated significant habituation, there was no overall habituation of strike scores in this species. The T? melanogaster had high strike scores, more rapid habituation to moving than nonmoving stimuli, and significant liter differences in habituation rates. The T? melanogaster, but not the T? butleri neonates, showed significant habituation of flight responses over the five tests. In T? butleri, but not T? melanogaster, males were more prone to attack than were females. In both species there were large differences in both overall strike scores and habituation rates of individual newborn animals. In Experiment 2, T? melanogaster 2-months old, were tested for short-term habituation to either a moving or nonmoving stimulus for 10 successive tests on one day. Habituation of strikes was similar to both stimuli, but more animals confronted with the moving stimulus showed an initial increase in strikes, lending support to the dual-process theory of habituation. As in Experiment 1, there were large individual differences in habituation rates. The results are discussed in terms of the ecological and methodological implications for developmental studies.

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.

Diet and growth in juveniles of the garter snakes Thamnophis sirtalis infernalis and Thamnophis radix radix.

The effects of diets consisting of different food types on the growth of juvenile garter snakes was tested using a litter each of Thamnophis sirtalis infernalis and T. radix radix. The snakes were fed diets consisting of earthworms, Lumbricus terrestris, and mosquitofish, Gambusia affinis. The results reveal differences in growth per unit of mass, calories, and protein consumed on the different diets with differences in length being more pronounced than differences in mass. The results indicate that the total mass, calories, or protein consumed are insufficient to reliably predict the amount of growth to be expected in these species. All results are given plus and minus one standard error with an analysis of variance for the production-ingestion ratios.

The role of feeding regimens in the growth of neonate broad-banded water snakes, Nerodia fasciata confluens, and possible effects on reproduction.

The effect of different feeding regimens on the growth pattern of Nerodia fasciata confluens was tested using a litter of 18 captive-born neonates. The snakes were divided among three feeding groups: one group fed once per week, another fed twice per week, and the third fed on alternate days. The once per week and the twice per week groups were offered the same weight of food each week, while the alternate-day group was offered food in excess of ingestion levels during each feeding session. The results indicate that there is a shift in the allocation of energy for growth in weight, snout-vent length, and tail length with a change in the feeding regimen. Females were affected more than the males. The results are discussed in relation to their possible effect on reproduction.