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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

Concentration and preliminary characterization of a chemical attractant of the oyster drill,Urosalpinx cinerea

Predatory muricid gastropods,Urosalpinx cinerea, respond to specific chemical stimuli by creeping upcurrent. Attractant substances originate from living barnacles. Newly hatched snails have no prior predatory experience but respond strongly to attractants. We report here methods for rapidly extracting and desalting attractants from seawater. Attractants from living barnacles are relatively large, at least partially proteinaceous, heat-stable molecules (> 1000 but < 10000 dallons) that adsorb onto Amberlite XAD-7, a polyacrylate water purification resin, at neutral pH. Attractants remain adsorbed to the resin during a wash with deionized water and can be eluted in a small volume with 100% methanol. Attractant substances are effective in the bioassay in g/liter concentrations (octa- to nanomolar range). Potency is destroyed by nonspecific proteases (carboxy-peptidase and pronase) but not by trypsin. Attractant is not sequestered within barnacles.     

Evidence for Use of Chemical Cues by Male Horseshoe Crabs When Locating Nesting Females (Limulus polyphemus)

Horseshoe crabs come ashore in attached pairs during spring high tides to mate and nest on beaches of the Atlantic and Gulf coasts. Unattached males also come ashore and crowd around the nesting pairs as satellites and engage in sperm competition with the attached male. Females with no satellites and females with large numbers of satellites nest next to one another on the same tide. When females are removed and replaced by a cement model, satellite males continue to be attracted to the same location. Models over sites where females with many satellites had nested are more attractive to males than sites from which a female with no satellites had been removed or a site where no crab had been nesting recently. A second experiment demonstrated that males are responding to chemical cues. A sponge filled with seawater taken from below a female with many satellites and placed under a model female was more attractive to males than a sponge filled with seawater. This is the first demonstration that horseshoe crabs use chemical cues, in addition to visual cues, to locate mates.     

Predator-induced diel vertical migration inDaphnia: Enrichment and preliminary chemical characterization of a kairomone exuded by fish

Planktivorous fish release a cue of an unknown chemical nature into the water that induces diel vertical migration inDaphnia as predator avoidance response. We used a bioassay to obtain information about the chemical nature of the kairomone. We present a method to enrich the cue from holding water of fish by sorbent extraction. The kairomone exuded byLeucaspius delineatus can be characterized as a nonolefinic low-molecular-weight anion of intermediate lipophilicity. The presence of amino groups in the molecule can be excluded, whereas hydroxy groups are essential for activity. Separation by HPLC yielded only one active fraction. The kairomones released byCarassius carassius andRutilus rutilus showed the same chemical characteristics, suggesting that the kairomones from different species of fish, sensed byDaphia, are very similar, if not identical.     

Purification and characterization of thermophilic and alkalophilic tributyrin esterase fromBacillus strain A30-1 (ATCC 53841)

An extracellular esterase (EC 3.1.1.1) from a thermophilicBacillus A30-1 (ATCC 53841) was purified 139-fold to homogeneity by sodium chloride (6 M) treatment, ammonium sulfate fractionation (30–80%) and phenyl-Sepharose CL-6B column chromatography. The native enzyme was a single polypeptide chain with a molecular weight of about 65,000 and an isoelectric point at pH 4.8. The optimum pH for esterase activity was 9.0, and its pH stability range was 5.0–10.5. The optimum temperature for its activity was 60°C. The esterase had a half-life of 28 h at 50°C, 20 h at 60°C and 16 h at 65°C. It showed the highest activity on tributyrin, with little or no activity toward long-chain (12–20 carbon) fatty acid esters. The enzyme displayed K_m and K_cat values of 0.357 mM and 8365/min, respectively, for tributyrin hydrolysis at pH 9.0 and 60°C. Cyclodextrin (α, β, and γ), Ca²⁺, Co²⁺, Mg²⁺ and Mn²⁺ enhanced the esterase activity, and Zn²⁺ and Fe²⁺ acted as inhibitors of the enzyme activity. The enzyme activity was not affected by ethylenediaminetetraacetic acid, p-chloromercuribenzoate andN-bromosuccinimide. This paper was presented in part at the 82nd Annual Meeting and Exposition of the American Oil Chemists’ Society, held May 12–15, 1991, in Chicago, Illinois.     

Chemical protection of a fish (Abudefduf leucogaster bleeker) by a soft coral (Litophyton viridis May)

Around Laing Island (Bismarck Sea), juveniles of the fishAbudefduf leucogaster are associated with the alcyonarianLitophyton viridis which, on mechanical stimulation, can release a potent ichthyotoxin and appears to be accordingly disliked by large predators. The fish has no special immunity to high levels of toxin but is affected more slowly than others. This has been interpreted in terms of an original strategy wherebyA. leucogaster plays its long reaction time against the diffusion of the toxin, allowing the fish to remain around the soft coral and benefit from indirect chemical protection. Possible biological implications of the toxin release are discussed.Laing Island Biological Station contribution No. 39.     

Chemical defense of common antarctic shallow-water nudibranchTritoniella belli eliot (Mollusca: Tritonidae) and its prey,Clavularia frankliniana rouel (Cnidaria: Octocorallia)

Extracts of the dorid nudibranchTritoniella belli and stoloniferan coralClavularia frankliniana were chromatographed and analyzed by¹H NMR and thin-layer chromatography. Three glycerol ethers were detected inT. belli, primarily 1-O-hexadecyl glycerol (chimyl alcohol). Chimyl alcohol was also detected after gradient flash chromatography and reverse-phase HPLC purification in the tissues ofC. frankliniana. The common omnivorous predatory Antarctic sea starOdontaster validus, a likely predator of benthic invertebrates, showed feeding deterrence to small cubes ofT. belli mantle tissue placed on the tube feet along the ambulacral feeding groove, while always extruding the cardiac stomach when presented with cubes of shrimp tissue of similar size. Filter-paper disks soaked in an aqueous shrimp solution and then dried were found to elicit a broad range of feeding behaviors inO. validus, including movement of the shrimp disk to the mouth, extrusion of the cardiac stomach, and the assumption of a humped feeding posture. Chimyl alcohol-treated shrimp disks caused significant feeding deterrence in sea stars when compared with control disks (solvent plus shrimp treated disks alone).T. belli andC. frankliniana appear to employ a defensive compound that has been found in a variety of temperate and tropical mollusks, where it has been demonstrated to deter fish predators. We provide evidence for further deterrent capabilities of chimyl alcohol and of its trophic relationship in the polar ecosystem of McMurdo Sound, Antarctica.     

Defensive mechanisms of loblolly and shortleaf pine against attack by southern pine beetle,Dendroctonus frontalis Zimmermann,and its fungal associate,Ceratocystis minor (Hedgecock) Hunt

Loblolly and shortleaf pine growing on a single site in the North Carolina piedmont were examined to determine similarities and differences in their defensive mechanisms against the southern pine beetle,Dendroctonus frontalis Zimmermann, and its fungal associate,Ceratocystis minor (Hedgecock) Hunt. Both species responded to wounding and fungal inoculation by forming a hypersensitive lesion around the wound site. There were significantly less soluble sugars and more monoterpenes in the lesion tissue than in unwounded inner bark. The two species were similar in resin flow rate and inner bark soluble sugar content, but the loblolly pines had thicker bark, longer hypersensitive lesions, and a higher concentration of inner bark monoterpenes. Inner bark monoterpene composition was also significantly different between the two pine species. It is hypothesized that two different defensive strategies against southern pine beetle attack may be utilized.     

Rhodium(I) iminophosphine poly(propyleneimine) dendrimers: Synthesis, characterization and molecular structure of a mononuclear analogue

First (G1)- and second-generation (G2) poly(propyleneimine) (PPI) dendrimers were reacted with 2-(diphenylphosphino)benzaldehyde, to form iminophosphine-functionalised dendritic ligands (1, 2). The reaction of the dimeric precursor [RhCl(CO)₂]₂ with the heterobidentate dendritic ligands 1, 2 gave the corresponding rhodium(I) complexes (3, 4).     

Purification and characterization of a thermophilic chitinase produced by <Emphasis Type="Italic">Aeromonas</Emphasis> sp. DYU-Too7

An extracellular chitinase, produced by Aeromonas sp. DYU-Too7, was purified in the following procedures: ammonium sulfate precipitation, ultrafiltration, chromatographic separation of DEAE-sepharose CL-6B and sephacrylS-100HR. The resulting chitinase has a molecular mass of 36 kDa, an optimal reaction pH of 5.0, and an optimal reaction temperature of 70°C. It retains almost 100% activity in the pH range of 5.0–8.0. This chitinase has a high thermal tolerance and retained 90% of its activity at 50°C and 75% at 60°C. Enzyme activity was inhibited by Ba²⁺, Hg²⁺, Mg²⁺ and Ag⁺ cations, but was not substantially inhibited by the K⁺ cation nor the chelating agent EDTA. The K _m and V _mα , using colloidal chitin as a substrate, are 6.3 g/L and 18.69 μmol/min/mg-protein, respectively. The 36 kDa chitinase of Aeromonas sp. DYU-Too7 is an exo-type enzyme, because chitobiose was the main hydrolysate in hydrolysis of colloidal chitin.     

Iridoid glycosides ofChelone glabra (Scrophulariaceae) and their sequestration by larvae of a sawfly,Tenthredo grandis (Tenthredinidae)

Analysis ofChelone glabra (Scrophulariaceae) by gas chromatography showed that leaves of this plant contained primarily the iridoid glycoside catalpol, and in a few individuals some aucubin was also detected. There was no difference in the iridoid glycoside content of damaged compared to undamaged plants, nor was there a difference between plants collected from a population in Leverett, Massachusetts, and those from plants in an experimental garden in Cambridge, Massachusetts. Larvae and prepupae of the sawfly,Tenthredo grandis (Tenthredinidae) contained catalpol sequestered from the larval host plant. The exuvia also contained catalpol, whereas the frass contained only aucubin. These results indicate that larvae of this sawfly selectively sequester catalpol, eliminating the aucubin in the frass.