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 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.     

Chemoreceptive responses of two sympatric water snakes to extracts of commonly ingested prey species

Newborn of two locally sympatric species of water snakes,Nerodia fasciata andN. erythrogaster, were exposed to skin extracts of frequently ingested prey. In southern Louisiana, fish account for 78% of the diet of adultNerodia fasciata and about 15% forNerodia erythrogaster. Anurans comprise most of the remainder of the diets of these water snakes. Responses of naive individuals were compared to the chemoreceptive response profiles of groups of snakes reared on limited diets of fish and/or frogs. All snakes were tested seven times over a nine-month period and ontogenetic changes were apparent. NewbornNerodia fasciata have a strong chemoreceptive response for fish extract that remains unchanged by early dietary restrictions for the first six months of life. After that time their responses are more variable and can be altered by recent feeding experience. In southern Louisiana, this species is the least specialized water snake, an observation supported by these laboratory results. While newbornN. erythrogaster did not respond preferentially to any of the prey extracts, a significant response rate for fish extract is established by two months of age that persists for several months regardless of dietary restrictions. A subsequent shift of the response to frog extract at eight months of age regardless of diet may reflect a maturational process. Field studies are in agreement with these results and support the notion of ontogenetic changes related to predator size and age. This is the first demonstration of an ontogenetic change in chemoreceptive response that is not related to dietary experience.     

The role of chemosensory cues in discrimination of prey odors by the amphisbaenianBlanus cinereus

Responses of amphisbaenians (Blanus cinereus) to deionized water, a control for pungency (cologne), and integumental prey odors (coleopteran larvae and adult ants) on cotton swabs were studied in experiments with a randomized blocks design to discover whether amphisbaenians use chemical cues to detect and identify prey. No individual bit the applicators. Amphisbaenians tongue-flicked at lower rates than epigean saurians, which are active foragers. Tongue-flick rate differed among treatments, but responses to prey odors were not significantly different from those to cologne. The number of directed tongue-flicks emitted during the 60-sec trials was, however, lower in response to deionized water than in response to cologne or prey odors. Response details, the low rate of tongue-flick, and absence of biting are discussed in relation to the foraging behavior and fossoriality of amphisbaenians. Evidence from this study indicates that the vomeronasal sense is used by amphisbaenians to identify odors, but our experiments failed to demonstrate that amphisbaenians discriminate between prey and nonprey odors.     

Effects of Polymorphisms in Pepsinogen (PEP), Amylase (AMY) and Trypsin (TRY) Genes on Food Habit Domestication Traits in Mandarin Fish

Mandarin fish (Siniperca chuatsi) have a peculiar feeding habit of only accepting live fish prey and refusing dead prey and artificial diets. However, previous research has shown that some individuals accept dead prey after gradual domestication. Digestive enzymes are correlated with feeding habits in fish. In the current study, SNPs in the mandarin fish genes for pepsinogen (PEP), amylase (AMY), and trypsin (TRY) were evaluated for associations with feeding habits in domesticated mandarin fish by scanning their complete genomic sequence. In total, two SNPs were found in PEP, one was found in TRY, and none were found in AMY. The D1(CTCC) and D5(TTTT) diplotypes in the PEP gene tended to show strong effects on the feeding habits of domesticated fish (p < 0.01). The results indicate that PEP may be associated with the genetic mechanism for feeding habits in mandarin fish, and the D1(CTCC) and D5(TTTT) diplotypes in the PEP gene may be useful markers for selecting mandarin fish with appropriate feeding habits for domestication.     

Acquired Predator Recognition by Fathead Minnows: Influence of Habitat Characteristics on Survival

In this study we conditioned fathead minnows (Pimephales promelas) to recognize the odor of a perch (Perca flavescens) by exposing them to perch odor coupled with minnow alarm cue. We then staged encounters between the predator and prey in order to assess whether the predator odor training had any effect on survival of the prey. We tested for a survival effect in the presence and absence of shelter. Our results indicate that fish trained with alarm signals to recognize predators gained a survival benefit during staged encounters with a predator and that habitat characteristics influenced the survival of conditioned fish.     

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.     

Discrimination of Prey, But Not Plant, Chemicals by Actively Foraging, Insectivorous Lizards, the Lacertid Takydromus sexlineatus and the Teiid Cnemidophorus gularis

Sampling environmental chemicals to reveal prey and predators and to provide information about conspecifics is highly developed in lizards. Actively foraging lizards can discriminate between prey chemicals and control stimuli, but ambush foragers do not exhibit prey chemical discrimination. Recent experiments on a few species of herbivorous lizards have also demonstrated an ability to identify plant food chemicals. We studied chemosensory responses to chemicals from prey and palatable plants in two species of actively foraging, insectivorous lizards. Both the lacertid Takydromus sexlineatus and the teiid Cnemidophorus gularis exhibited strong responses to prey chemicals, but not to plant chemicals. These findings increase confidence in the relationship between prey chemical discrimination and foraging mode, which is based on data for very few species per family. They also provide data showing that actively foraging insectivores in two families do not respond strongly to plant cues. Such information is essential for eventual comparative studies of the relationship between plant diet and responses to food chemicals. The traditional method of presenting stimuli by using hand-held cotton swabs worked well for T. sexlineatus but could not be used for C. gularis due to repeated escape attempts. When stimuli were presented to C. gularis on ceramic tiles and no experimenter was visible, the lizards responded readily. Presentation of stimuli on tiles in the absence of a visible experimenter may be a valuable approach to study of food chemical discrimination by active foragers in which antipredatory behavior interferes with responses to swabs.     

Venom variation and chemoreception of the viperid Agkistrodon contortrix: evidence for adaptation?

Previous studies of chemoreceptive behavior in vipers suggest that snakes focus on the scent of envenomated tissue to track their prey following envenomation. Other studies have indicated a correlation between qualitative differences in venom biochemistry and geographic variation in diet. The North American copperhead (Agkistrodon contortrix) varies geographically in diet and venom biochemistry; snakes were collected from three populations (Kansas, Texas, and Louisiana) that are known to have different prey preferences. Behavioral experiments were conducted to assess whether copperheads preferred envenomated prey more than nonenvenomated prey, as do other species of vipers studied thus far. Additional experiments tested the ability of copperheads to distinguish between envenomated prey from different geographic populations, and between geographic populations of copperheads and two other species of viper. Results indicated that copperheads prefer envenomated prey to nonenvenomated prey. In envenomated-prey discrimination experiments, copperheads distinguished between envenomated prey from different geographic populations, and some snakes distinguished envenomated prey of A. contortrix from those of A. piscivorus and Sistrurus catenatus. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was used to study the variation of venom biochemistry in this species and two other taxa (A. piscivorus and S. catenatus), and confirmed intraspecific and interspecific variation of venom proteins. Relative potency of the venom from different populations as indicated by time to immobilization experiments was in the order: Louisiana >Texas > Kansas. The relative potency of the venom from each population matched the order of preference in the chemoreception experiments. These results suggest that chemoreception is sensitive to subtle differences in venom biochemistry and may reflect adaptation to improve efficiency of finding envenomated prey.     

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.     

Mite predator responses to prey and predator-emitted stimuli

We found that the searching behavior of two acarine predators,Amblyseius fallacis andPhytoseiulus macropilis, for prey,Tetranychus urticae, is affected by the following stimuli: (1) prey silk and associated feces, whose combined physical and chemical properties elicit reduction in the rate of predator movements and longer halts; (2) kairomone extracted from prey silk and associated feces, which, upon contact, elicits frequent predator return to prey-inhabited locales; and (3) predator-emitted marking pheromone, which elicits shorter duration of search in presearched prey locales. We also found that treatment of filter paper with prey kairomone or silk enhanced predator location of prey eggs, leading us to speculate that application of synthetic prey kairomone could be useful in pest management programs.     

Timber Rattlesnakes (Crotalus horridus) Use Chemical Cues to Select Ambush Sites

Chemicals left by organisms moving through the environment are used by other organisms to mediate interspecific interactions. Most studies of chemical eavesdropping focus on prey responding to chemical cues from predators, despite the fact that chemical cues are frequently used by predators as a source of information about prey. Crotalus horridus uses a foraging strategy that is widespread among sedentary predators: the snake chooses a site where it is likely to encounter prey and remains immobile for many hours. I investigated this ambush hunting behavior in captive-raised timber rattlesnakes and provide evidence that sit-and-wait predators may discriminate among prey chemical cues, even when they have no prior experience with the prey. Snakes explored chemical cues with chemosensory behaviors, and more frequently adopted a stereotyped ambush foraging posture toward chemical cues from prey sympatric with their population of origin than either allopatric prey or sympatric nonprey species that are eaten by other viperids. These results support the notion that intra- and interspecific variation in diet may be mediated proximally by innate recognition of cues from particular prey items. This system also describes a bioassay that may be used in the isolation and identification of prey-derived kairomones. Studies such as this can be used to determine more realistic parameters for models of predator-prey interaction and foraging behavior that involve secretive, less active predators.     

Discriminative Response to Animal, But Not Plant, Chemicals by an Insectivorous, Actively Foraging Lizard, Scincella lateralis, and Differential Response to Surface and Internal Prey Cues

Responses by the insectivorous, actively foraging scincid lizard, Scincella lateralis, to chemical cues from a plant food favored by herbivorous lizards, its ability to discriminate prey chemicals from control substances, and its relative response to internal and surface prey chemicals were studied experimentally. We presented chemical cues to the lizards on cotton swabs and recorded their tongue-flicks and biting attacks on the swabs. The lizards exhibited significantly greater tongue-flick rates and biting frequencies to prey surface cues than to plant surface chemicals from romaine lettuce, diluted cologne (pungency control), and deionized water. Responses to the plant stimuli did not differ from those to the two control stimuli, in contrast with strong responses to the same plant cues by herbivores. This finding provides the first information suggesting that chemosensory response may be adapted to diet, with responsiveness to plant stimuli evolving de novo in herbivores. Biting and tongue-flicking responses were significantly greater to cricket chemicals than to all other stimuli, among which there were no differences. Thus, the lizards are capable of prey chemical discrimination, which may be ubiquitous among actively foraging lizards. The lizards exhibited more frequent biting and higher tongue-flick rates to internal than surface prey chemicals. Although different methods of stimulus preparation are appropriate for different purposes, we conclude that prey surface chemicals available to foraging lizards are most desirable for studies bearing on location and identification of prey.     

Nest Paper Absorbency,Toughness, and Protein Concentration of a Native vs. an Invasive Social Wasp

The amount of proteinaceous food that was allocated to nest construction by a native wasp (Polistes fuscatus) vs. an invasive wasp (Polistes dominulus) in North America was examined following a field experiment under natural and surplus prey foraging conditions. Wasps of the surplus prey foraging conditions were provided with prey ad libitum within an enclosed area, while wasps of the natural treatment foraged in an adjacent field-woodland site. At the end of the field experiment, each nest was tested for water absorbency, toughness, and protein concentration. The hypotheses were: (1) When all nests are equally sheltered, the invasive P. dominulus (PD) allocates less protein to nest paper construction (for waterproofing and strengthening) and more protein to developing larvae than the native P. fuscatus (PF). (2) Nests of P. dominulus are more absorbent (less waterproof) and less tough than nests of P. fuscatus. Results indicate that P. fuscatus nests from surplus prey foraging conditions were more absorbent (less waterproof) to artificial rain drops than P. dominulus nests. The toughness of nests was similar between wasp species. However, nests from the natural treatment were tougher than those from the surplus prey treatment. Nests from the natural foraging conditions had half as much protein as those from surplus prey foraging conditions. There was no correlation between nest protein concentration and the number of prey taken, the number of cells, the number of adult offspring produced, or the total wasp biomass produced per colony. For PF under surplus prey conditions, protein concentration and absorbency were negatively correlated, but for PD the correlation was positive. In conclusion, when prey were scarce, Polistes wasps allocated less protein to nest construction. Also, the introduced P. dominulus may increase production of offspring by allocating less to nest construction than that of the native P. fuscatus, and so more protein to offspring production.     

Chemical and behavioral ecology of foraging in prairie rattlesnakes (Crotalus viridis viridis)

Free-ranging prairie rattlesnakes (Crotalus viridis viridis) exhibit lengthy vernal migrations upon emergence from winter hibernation. A series of laboratory experiments was designed to test hypotheses regarding the function and causation of vernal movements. Rattlesnakes obtained from Wyoming and Colorado populations were used. First, we hypothesized that the function of vernal movements is to locate small mammal prey. Second, we predicted that activeC. v. viridis use prey chemicals, as well as other cues, to decide whether or not rodents are present in an area. Third, we hypothesized that vernally active males would be more responsive to rodent prey and their odors than females, given observed differences in behavior in the field. Fourth, we predicted that rattlesnakes captured in Colorado would be more sensitive to prey odors than those obtained in Wyoming, because of disparate community structure and, hence, small mammal spatial distributions. As expected, snakes exhibited reduced activity, as well as certain other dependent measures reflecting predatory investigation, in arena zones containing either live rodents or their chemicals. However, responses to the latter were reduced in Wyoming rattlesnakes tested with chemicals from deer mice (Peromyscus maniculatus), relative to Colorado animals tested with chemicals obtained from house mice (Mus musculus). In contrast to patterns observed in nature, males and females exhibited almost no differences in overall responsiveness. Results are discussed in the context of simulation modeling and ongoing studies of prairie rattlesnake behavior.     

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.     

Crayfish feeding responses to zebra mussels depend on microorganisms and learning

Three species of crayfish (Orconectes virilis, O. rusticus, andCambarus robustus) were tested for feeding responses to potential food odors from mollusks (either zebra mussels,Dreissena polymorpha, or native gastropods). In all three crayfish species, feeding responses to odor cues were shown only by individuals experienced with feeding on a prey type. Individuals exposed to just the smell of prey organisms did not show feeding responses, indicating the role of associative learning in diet breadth. Establishment of a learned association took more than one feeding experience but once established lasted more than three weeks. When microbial enzymatic degradation of food protein was eliminated, either by UV radiation or microfiltration, feeding responses were eliminated even for crayfish experienced with a prey type.     

Elevation in tongue-flick rate after biting prey in the broad-headed skink,Eumeces laticeps

A postbite elevation in tongue-flicking (PETF) rate occurs in adult male broad-headed skinks,Eumeces laticeps. Males having bitten neonatal mice showed significantly higher tongue-flicking rates in the 2 min following experimental removal of the prey than did males in several control conditions. In a second experiment designed to separate the effects of tactile and chemical stimulation of the oral cavity during biting, males tongue-flicked at significantly higher rates in response to swabs bearing surface chemicals from neonatal mice than to identical swabs lacking the surface chemicals. These findings agree with previous data showing that PETF and searching movements occur in those families of lizards that can detect prey chemicals and use the tongue to do so during active foraging. The occurrence of PETF and putative searching movements supports the interpretation that PETF represents an attempt to relocate lost prey by chemosensory means. PETF was much briefer inE. laticeps than in many snakes and in a representative species from another lizard family and was not detectable after the first minute. This brevity is consistent with the prediction that PETF should be brief in squamates that feed on prey not likely to be located by scent-trailing.     

Early warning in the predation sequence: A disturbance pheromone in Iowa darters (Etheostoma exile)

The probability of prey avoiding a predator's attack should increase if the predator's presence is detected at an early stage in the predation sequence. In this study, we tested whether threatened Iowa darters (Etheostoma exile) release disturbance pheromones that warn conspecifics of the presence of predation threat. Pairs of aquaria were visually isolated from one another, but connected chemically by water circulating between them. Darters in one aquarium were observed before and after darters in the other aquarium were chased with a model predator. In control tests, the model was moved in the same manner but there were no darters in the upstream tank. Darters receiving water from threatened fish increased vigilance behavior and decreased movement. Vigilant fish raised their head above the substratum, propping themselves up on their pectoral fins and/or arching their neck dorsally, pointing the snout upward. Exposure to water from disturbed darters suppressed exploratory behavior and resulted in movement by short rapid hops that ended abruptly in a rigid, alert posture. This study suggests that Iowa darters release a disturbance pheromone that can provide conspecifics with an early warning of predation risk.     

Identification of sex pheromone ofTetanolita mynesalis (Lepidoptera: Noctuidae), a prey species of bolas spider,Mastophora hutchinsoni

The bolas spider,Mastophora hutchinsoni, attracts adult males of four species of nocturnally active Lepidoptera through aggressive chemical mimicry of those species' sex pheromones. Here we report the identification of the sex pheromone of one prey species, the smoky tetanolita (Tetanolita mynesalis). In sex pheromone gland extracts, only two peaks stimulated an electrophysiological response as measured by a coupled gas chromatography-electroantennographic detection analysis. These two peaks had retention times identical to (3Z,6Z,9Z)-heneicosatriene (3Z,6Z,9Z-21:H) and (3Z,9Z)-cis-6,7-epoxy-heneicosadiene (3Z,9Z-cis-6,7-epoxy-21:H), respectively, and mass spectra identical to these two compounds. It was determined that 0.23±0.16 and 0.56±0.26 ng of 3Z,6Z,9Z-21:H and 3Z,9Z-cis-6,7-epoxy-21:H, respectively, were present in pheromone gland extracts from individual females. A 1:1 blend of 3Z,6Z,9Z-21:H and 3Z,9Z-6S,7R-epoxy-21:H was an effective attractant for adult males from feral populations. Blend ratios of these two components from 2:1 to 1:2 were equally effective as attractants. Greater deviation from the optimal blends resulted in diminished trap catches. The enantiomer 3Z,9Z-6R,7S-epoxy-21:H not only was not effective in attracting males, its presence in the effective blend shut down trap catches. These results indicate that the pheromone blend consists of 3Z,6Z,9Z-21:H and 3Z,9Z-6S,7R-epoxy-21:H. This is the first report of a hydrocarbon/epoxide pheromone for a prey species of this bolas spider. Sex attractants or pheromones for the other three prey species are composed of aldehydes or acetates.