Post-bite Elevation in Tongue-flick Rate by Neonatal Garter Snakes (Thamnophis radix)

A post-biting elevation in tongue-flicking rate was demonstrated experimentally in neonatal, ingestively naive garter snakes (Thamnophis radix). That the snakes also exhibited apparent searching movements suggests that strike-induced chemosensory searching occurs in nonvenomous snakes lacking previous experience with food or prey chemicals. Two litters of neonates differed in numbers of tongue-flicks emitted, but had similar relative magnitudes of response across experimental conditions. The existence of post-bite elevation in tongue-flick rate (and presumably strike-induced chemosensory searching) argues for a genetic basis for these chemosensory behaviors in a nonvenomous species of snake, extending the recent finding that strike-induced chemosensory searching is fully developed in ingestively naive neonatal rattlesnakes. Possible patterns of evolution of post-bite elevation in tongue-flick rate, and the strike-release-trail strategy of highly venomous snakes are discussed.     

Development of Antipredator Responses in Snakes

Individual and litter differences in the number of attacks directed by Mexican garter snakes (Thamnophis melanogaster) at a threatening stimulus were studied over the first year of life. Newborn snakes born to wildcaught females from the same population were tested the day after they were born and at 7, 16, 31, and 54 weeks of age on reactivity to a potent nonmoving and moving stimulus (a human finger). There were significant individual and interlitter differences in number of strikes directed at both stimuli but no significant effect of sex. Intercorrelations among the strike scores of individuals across test days averaged .54 for the nonmoving stimulus presentations and .63 for trials with the moving stimulus. Strike scores to the moving and nonmoving stimuli were significantly correlated at each age, however, attack responses to the moving stimulus declined over the year while those to the nonmoving stimulus remained constant. Measures of concordance showed that both individual strike scores and litter means were highly consistent, whereas consistency within litters was reduced. The results indicate that litter and individual differences among newborn snakes in the tendency to engage in antipredator behavior (“defensive temperament”) remain relatively stable from the first day after birth throughout the first year of life.     

Rattlesnakes Create a Context for Localizing Their Search for Potential Prey

This paper presents systematically collected field data on what transpires between free-living rattlesnakes (Crotalus viridis oreganus) and individuals of an important prey species, California ground squirrels (Spermophilus beecheyi). In the course of two field seasons we discovered that rattlesnakes and California ground squirrels can engage in at least six different episode classes: snake watching, snake following, inspecting/probing coiled snakes, interaction, rattlesnake approaching squirrel and envenomation. If a rattlesnake is moving directly toward a squirrel and is within 3 m of its burrow an interaction may develop, but more commonly it does not. Instead of engaging the snake, the squirrels seemed to try to remain stationary in the face of the snake's advance, as if to minimize affording the snake information about the nursery burrow location. The rattlesnakes in turn behaved as though they were using the location of the squirrels that resisted moving away from their advance as the hub of a radial search pattern. We argue that if the squirrel engages the snake before it discovers the burrow, the location of the nursery burrow may be revealed and the pups' vulnerability actually increased.     

Discrimination of integumentary prey chemicals and strike-induced chemosensory searching in the ball python,Python regius

Experimental tests show that the ball python (Python regius) has the ability to discriminate prey chemicals from control substances by tongue-flicking and exhibits a poststrike elevation in tongue-flicking rate (PETF). Prey chemical discrimination was revealed by significantly higher number of tongue-flicks and tongue-flick attack score in response to integumental chemicals from mice than to cologne or distilled water and by a higher frequency of biting in response to prey than control chemicals. PETF was indicated by higher tongue-flicking rates after biting than in several control conditions. Concurrent movements of the body suggest the operation of strike-induced chemosensory searching (SICS). Ecological factors affecting responses to prey chemicals, including defensive behaviors and characteristics of foraging behavior related to reliance on different sensory modalities, are discussed. The presence of PETF and SICS in a henophidian snake and in scleroglossan lizards suggests that these behaviors are plesiomorphic in snakes.     

Past and Present Risk: Exposure to Predator Chemical Cues Before and after Metamorphosis Influences Juvenile Wood Frog Behavior

Animals are exposed to different predators over their lifespan. This raises the question of whether exposure to predation risk in an early life stage affects the response to predators in subsequent life stages. In this study, we used wood frogs (Rana sylvatica) to test whether exposure to cues indicating predation risk from dragonfly larvae during the wood frog larval stage affected post‐metamorphic activity level and avoidance of garter snake chemical cues. Dragonfly larvae prey upon wood frogs only during the larval stage, whereas garter snakes prey upon wood frogs during both the larval stage and the post‐metamorphic stage. Exposure to predation risk from dragonflies during the larval stage caused post‐metamorphic wood frog juveniles to have greater terrestrial activity than juvenile wood frogs that were not exposed to larval‐stage predation risk from dragonflies. However, exposure to predation risk as larvae did not affect juvenile wood frog responses to chemical cues from garter snakes. Wood frogs exposed as larvae to predation risk from dragonfly larvae avoided garter snake chemical cues to the same extent as wood frog larvae not exposed to predation risk from dragonfly larvae. Our results demonstrate that while some general behaviors exhibit carry‐over effects from earlier life stages, behavioral responses to predators may remain independent of conditions experienced in earlier life stages.     

Contextual Flexibility: Reassessing the Effects of Prey Size and Status on Prey Restraint Behaviour of Macrostomate Snakes

Contextual flexibility in prey restraint behaviour has been documented in advanced snakes (Colubroidea), but the degree of flexibility for earlier snake lineages has been largely unstudied. We document the prey restraint behaviour of five snake species belonging to three early macrostomate lineages: Loxocemidae, Erycinae and Boidae. Species from these lineages were chosen for this study because they utilize similar prey resources but exhibit different ecological habits that may have important consequences on prey restraint behaviour. Snakes (n = 27) were studied in a systematic experimental design assessing the effects of mouse size (small and large) and status (live and dead) across a total of 216 feeding trials. Loxocemus and Erycine snakes were highly flexible in their prey restraint behaviour patterns and these varied across prey category. Individuals of Boa constrictor exhibited very little contextual flexibility in feeding behaviour, confirming earlier reports. Flexibility in prey restraint behaviour corresponded with loop application pattern, whether the snake bent laterally or ventrally when forming a loop around prey. Our study is the first to show that early macrostomate snakes exhibit flexible prey restraint behaviours. Thus, our results suggest that flexibility in predatory behaviour may be more widespread across snake taxa than previously thought and we offer hypotheses for the observed interspecific differences in snake feeding behaviour.     

Testing an Adaptive Hypothesis Through Context-Dependence: Effects of Water Depth on Foraging Behaviour in Three Species of Garter Snakes

Adaptive hypotheses based on interspecific comparisons can be tested by evaluating the context‐dependence of the behaviour of individual organisms. Drummond (Behaviour, 86, 1983, 1) categorized garter snake species (Thamnophis) as terrestrial–aquatic generalists or aquatic specialists based on diet and aquatic foraging behaviour. He hypothesized that the characteristic foraging behaviours of aquatic specialists – including frequent crawling on the underwater substrate and a high rate of underwater predatory strikes – are adaptations for feeding on relatively widely dispersed aquatic prey. Drummond's hypothesis based on interspecific comparisons suggests that individual snakes might change their foraging in the direction of aquatic specialist behaviour with an increase in water depth (which increases prey dispersion). I tested this prediction through laboratory observations of Mexican Pacific lowlands garter snakes (T. validus) feeding on minnows in shallow (2 cm) and deep (3–7 cm) water. Members of this species are appropriate subjects because they are ecologically intermediate between the generalists and aquatic specialists studied by Drummond, and thus might be expected to show more variation in aquatic foraging behaviour than those species. T. validus showed significantly higher frequencies of crawling on the underwater substrate and of underwater strikes in the deep water than in the shallow water; i.e. increased water depth shifted the behaviour of these snakes toward that of aquatic specialists, thus supporting Drummond's hypothesis. Individuals of an aquatic specialist species, the narrow‐headed garter snake (T. rufipunctatus), showed less pronounced changes in behaviour with increased water depth. Western ribbon snakes (T. proximus), which feed primarily at the land–water interface (and are expected to act like terrestrial–aquatic generalists), typically refused to feed in deep water. Interspecific differences in underwater visual acuity may underly the behavioural differences among the three species by determining whether changes in foraging behaviour with water depth are advantageous. Information on phylogenetic relationships suggests that the facultative behaviour of T. validus may represent an intermediate stage in the evolution of aquatic specialization.     

Perceived predation risk as a function of predator dietary cues in terrestrial salamanders

Prey often avoid predator chemical cues, and in aquatic systems, prey may even appraise predation risk via cues associated with the predator's diet. However, this relationship has not been shown for terrestrial predator-prey systems, where the proximity of predators and prey, and the intensity of predator chemical cues in the environment, may be less than in aquatic systems. In the laboratory, we tested behavioural responses (avoidance, habituation and activity) of terrestrial red-backed salamanders, Plethodon cinereus, to chemical cues from garter snakes, Thamnophis sirtalis, fed either red-backed salamanders or earthworms (Lumbricus spp.). We placed salamanders in arenas lined with paper towels pretreated with snake chemicals, and monitored salamander movements during 120 min. Salamanders avoided substrates preconditioned by earthworm-fed (avoidanceX+/-SE=91.1+/-2.5%, N=25) and salamander-fed (95.2+/-2.5%, N=25) snakes, when tested against untreated substrate (control). Salamanders avoided cues from salamander-fed snakes more strongly (75.2+/-5.5%, N=25) than earthworm-fed snakes when subjected to both treatments simultaneously, implying that salamanders were sensitive to predator diet. Salamanders tended to avoid snake substrate more strongly during the last 60 min of a trial, but activity patterns were similar between salamanders exposed exclusively to control substrate versus those subject to snake cues. In another experiment, salamanders failed to avoid cues from dead conspecifics, suggesting that the stronger avoidance of salamander-fed snakes in the previous experiment was not directly due to chemical cues emitted by predator-killed salamanders. Salamanders also did not discriminate between cues from a salamander-fed snake versus a salamander-fed snake that was recently switched (i.e. <14 days) to an earthworm diet. Our results imply that terrestrial salamanders are sensitive to perceived predation risk via by-products of predator diet, and that snake predators rather than dead salamanders may be largely responsible for the release of such chemicals. Copyright 1999 The Association for the Study of Animal Behaviour.     

Discordant patterns of population structure for two co‐distributed snake species across a fragmented Ontario landscape

Aim The goal of our study was to investigate the effects of a fragmented landscape on the genetic population structure of two sympatric snake species that differ in habitat preference. The eastern garter snake (Thamnophis sirtalis sirtalis) is a common, habitat generalist, whereas the endangered eastern foxsnake (Mintonius [Elaphe] gloydi) is rarer, geographically restricted, and a marsh‐specialist. We were most interested in comparing the genetic population structure of both species and identifying any natural and human‐created features of the landscape that overlap with genetic disjunctions. Location Southwestern Ontario, Canada, surveying over half of the remaining range of the eastern foxsnake. Methods We utilized DNA microsatellite markers to examine genetic population structure of both species. The number of genetically distinct clusters for each species was determined using both Bayesian spatial assignment and spatial principal component analyses (sPCA). Genetic clusters were overlaid onto a habitat map to deduce possible physiognomic barriers to gene flow. Results Spatial assignment revealed three genetic clusters for garter snakes and five for foxsnakes. Each individual garter snake had a near equal probability of membership to two or more clusters with no cluster mapping onto a discrete geographic region, indicating that garter snakes comprise a single genetic population. The identified foxsnake clusters correspond to geographically circumscribed locations on the landscape, roughly coincident with isolated patches of suitable habitat. sPCAs revealed significant global allelic structure for foxsnakes, but not for garter snakes. No significant local structure was found for either species. Main Conclusions Our results imply that foxsnakes and garter snakes are differentially impacted by the same landscape or have dramatically different effective population sizes. Unsuitable intervening habitat such as agricultural tracts and roads between existing populations of foxsnakes appears to act as barriers to gene flow, while garter snake movement appears unrestricted by these features. Our findings have important implications for the management of eastern foxsnakes.     

Species specificity of methyl ketone profiles in the skin lipids of female garter snakes,genus Thamnophis

One of the relatively few vertebrate pheromones to be chemically identified, the female sex pheromone of the red-sided garter snake (Thamnophis sirtalis parietalis) is a series of saturated and monounsaturated methyl ketones contained within female skin lipids. During the breeding season, this pheromone is responsible for eliciting male courtship behaviors and males are able to utilize pheromonal variation to discriminate among females. While the pheromone system of the red-sided garter snake has been the subject of many studies, relatively little is known about the pheromone systems of other garter snakes. Through chemical analyses, we demonstrate that female skin lipids of the red-spotted garter snake (Thamnophis sirtalis concinnus), northwestern garter snake (Thamnophis ordinoides), and plains garter snake (Thamnophis radix) contain similar methyl ketones. The methyl ketone profiles of these snakes differ qualitatively from one another and from the methyl ketone profiles of red-sided garter snakes with differences particularly pronounced between sympatric species. Our results provide evidence that the use of methyl ketones in sexual signaling may be ubiquitous for Thamnophis species and suggest that these compounds could play a role in reproductive isolation between species in this genus.     

Foraging Behavior in the Arboreal Boid Corallus grenadensis

Corallus grenadensis is an arboreal boa endemic to the Grenada Bank. Thirty-five encounters with boas resulted in 17.65 hours of observations, including 6.3 hours of video-tape (which included two acts of predation). Boas under 100 cm are largely active foragers that move slowly through bushes and trees and tongue-flick leaf and branch surfaces apparently seeking chemosensory evidence of nocturnally quiescent lizard (Anolis) prey. Significantly more search time was directed to branches below the snake rather than to either the branches supporting the snake or to those above the snake, and tongue-flick rates were significantly higher for moving snakes than for those that were stationary. Smaller snakes prey on nocturnally quiescent lizards and they spent more time moving than did large snakes that feed on nocturnally active rodents and often employ an ambush foraging strategy. Once visual and, presumably, thermal information was received from a sleeping anole, C. grenadensis adopted a lengthy stalking process devoid of tongue-flicks. Snakes approached inactive lizards from adjacent branches with great stealth, moving at a rate of about 1 cm/min. The strike was made from close range (within 3 cm), and the prey was never released once contact was made. We conclude that, if chemosensory cues successfully lead a treeboa to a visual encounter with a sleeping lizard, subsequent behavior ensures a high rate of predation success.     

The energetic advantages of slug specialization in garter snakes (genus Thamnophis)

We tested the hypothesis that dietary specialization by foraging garter snakes is accompanied by increased assimilation efficiency on specialist prey items. Our comparison included two closely related garter snake species considered to be slug specialists (Thamnophis ordinoides and Thamnophis elegans terrestris), one fish specialist (Thamnophis couchii), and one diet generalist (Thamnophis elegans elegans). Our results suggest that slug specialists have an energetic advantage over non-slug-eating snakes when both eat slugs. Slug specialists T. ordinoides and T. e. terrestris both have higher assimilation and net assimilation efficiencies when eating slugs than do generalists T. e. elegans and T. couchii. The slug specialists did not experience decreased efficiency when eating fish. Therefore, there was no apparent digestive trade-off for the slug specialists when eating other prey.     

Ontogenetic change in blood oxygen capacity and maximum activity in garter snakes (Thamnophis sirtalis)

Summary There is an ontogenetic increase in the time that garter snakes (Thamnophis s. sirtalis) can maintain maximum activity at 25°C. Newborn snakes are exhausted by 3–5 min of activity while adults can be active for 20–25 min. The increased endurance of adult snakes results from ontogenetic increases in both aerobic and anaerobic energy generation. At rest juvenile and adult snakes have the same whole-body lactic acid concentrations, but at exhaustion adult lactic acid concentrations are 1.5 times those of juveniles. This increase in anaerobic energy production accounts for part of the endurance of adult snakes, but increased aerobic metabolism appears to be more important. Among the mechanisms increasing aerobic metabolism are more effective pulmonary ventilation and a 3-fold ontogenetic increase in blood oxygen capacity.The rapid exhaustion of small garter snakes probably limits the microhabitats they can occupy and the sorts of hunting methods they can employ. Small garter snakes feed only on small prey that are easily subdued. There is an ontogenetic increase in the relative size of prey eaten by garter snakes that parallels the ontogenetic increase in endurance. Adult feeding habits are adopted at the same body size at which adult blood oxygen capacity and endurance are attained.     

Predation Risk Avoidance by Terrestrial Amphibians: The Role of Prey Experience and Vulnerability to Native and Exotic Predators

We studied avoidance, by four amphibian prey species (Rana luteiventris, Ambystoma macrodactylum, Pseudacris regilla, Tarichia granulosa), of chemical cues associated with native garter snake (Thamnophis elegans) or exotic bullfrog (R. catesbeiana) predators. We predicted that avoidance of native predators would be most pronounced, and that prey species would differ in the intensity of their avoidance based on relative levels of vulnerability to predators in the wild. Adult R. luteiventris (presumably high vulnerability to predation) showed significant avoidance of chemical cues from both predators, A. macrodactylum (intermediate vulnerability to predation) avoided T. elegans only, while P. regilla (intermediate vulnerability to predation) and T. granulosa (low vulnerability to predation) showed no avoidance of either predator. We assessed if predator avoidance was innate and/or learned by testing responses of prey having disparate levels of prior exposure to predators. Wild‐caught (presumably predator‐exposed) post‐metamorphic juvenile R. luteiventris and P. regilla avoided T. elegans cues, while laboratory‐reared (predator‐naive) conspecifics did not; prior exposure to R. catesbeiana was not related to behavioural avoidance among adult or post‐metamorphic juvenile wild‐reared A. macrodactylum and P. regilla. These results imply that (i) some but not all species of amphibian prey avoid perceived risk from garter snake and bullfrog predators, (ii) the magnitude of this response probably differs according to prey vulnerability to predation in the wild, and (iii) avoidance tends to be largely learned rather than innate. Yet, the limited prevalence and intensity of amphibian responses to predation risk observed herein may be indicative of either a relatively weak predator–prey relationship and/or the limited importance of predator chemical cues in this particular system.     

The role of vision in the predatory behaviour of natricine snakes

Early workers concluded that ingestively naive garter snakes (Thamnophis) recognize chemical cues from their normal prey, and that such cues are sufficient to elicit prey attack, whereas visual prey cues are not sufficient. In the light of recent observations on how garter and water snakes (Nerodia) forage, new tests were made of the role of visual stimuli in the aquatic predation of several natricine species. Both experienced and ingestively naive snakes oriented to and attacked a fish model in plain water, although they made more orientations and attacks when diffuse fish odour was present in the water. Fish odour in water also elicited increased aquatic searching behaviour. Early views on the role of vision in the predation of newborn natricine snakes require modification, and there is a need for investigation of the properties of effective visual stimuli and the ontogeny of responsiveness to them.     

Microsatellite markers developed from Thamnophis elegans and Thamnophis sirtalis and their utility in three species of garter snakes

Previous studies of polyandry and population genetics in garter snakes have been hampered by the limited number of available microsatellite markers. Here we report another six dinucleotide microsatellite markers, doubling the number of previously available loci. The majority of the loci exhibit utility across a broad geographical range for the common garter snake and also cross‐amplify effectively in samples from two other Thamnophis species.     

Trade‐offs between eating and moving: what happens to the locomotion of slender arboreal snakes when they eat big prey?

The capacity to consume large prey evolved long ago in snakes. Subsequently, many specialized arboreal snake species convergently evolved slender bodies, presumably well‐suited for moving on thin branches and steep slopes, although how this accentuates changes in their shape and weight after eating and creates trade‐offs with locomotor performance is poorly understood. Hence, we tested whether the performance and modes of locomotion of a specialized arboreal snake (Boiga irregularis) changed after eating one or two mice when crawling on cylinders with and without pegs and on horizontal or 45° slopes. On surfaces with pegs: (1) only lateral undulation was used; (2) speed decreased with increased meal size; and (3) unexpectedly, more sideways toppling occurred than without pegs. On the horizontal cylinders without pegs, most unfed snakes used lateral undulation with continuous sliding contact, whereas, after eating two mice, most snakes periodically stopped and gripped the cylinder with speeds of concertina locomotion similar to those for the lateral undulation of unfed snakes. Thus, the behaviour of switching to a gripping mode of locomotion (concertina) circumvented some of the constraints of a slender limbless body plan, for which bulky meals alter shape and can impede the movement of the propulsive structures. © 2014 The Linnean Society of London, Biological Journal of the Linnean Society, 2015, 114 , 446–458.     

Prey Odor Discrimination in the Varanoid Lizards Heloderma suspectum and Varanus exanthematicus

Use of chemical senses to detect prey is believed to be an important component of foraging behavior in actively foraging lizards. Ability to detect prey odors and discriminate them from control odors by tongue-flicking was studied in representatives of two families of lizards having highly forked, elongated, retractile tongues. Responses of gila monsters (Heloderma suspectum) and savannah monitors (Varanus exanthematicus) to deionized water, a control for pungency (cologne), and mouse odor on cotton swabs were studied in experiments using repeated-measures designs and employing the tongue-flick attack score (TFAS) as the primary measure of response strength. TFAS differed among treatments for gila monsters and monitors. Both species had greater TFAS to mouse odors than to either of the control stimuli, but responses to cologne were not statistically distinguishable from those to water. Numbers of tongue-flicks elicited by prey odors were greater than those for control stimuli in V. exanthematicus, but not for H. suspectum. Gila monsters, but not savannah monitors, bit in a significantly greater proportion of tests with prey odors than with control stimuli. Details of responses, including frequency of biting, apparent search behavior in the vicinity of applicators bearing mouse odors, and responses to control stimuli are discussed in relation to the foraging behavior of these two species and their relatives. Evidence from this and other studies suggests that the vomeronasal sense (and perhaps other chemical senses) is an important means of locating and recognizing prey in widely foraging autarchoglossan lizards.     

The all-cone retina of the garter snake: spectral mechanisms and photopigment

Summary The retina of the garter snake contains 3 morphologically distinct classes of cone photoreceptor. The spectral mechanisms in the retinas of garter snakes (Thamnophis sirtalis and T. marcianus) were studied by recording a retinal gross potential, the electroretinogram, using a flicker photometric procedure. Spectral sensitivity functions recorded with stimuli presented at high temporal frequency (62.5 Hz) are broadly peaked in the region of 550–570 nm. These functions remain spectrally invariant (a) in the face of significant changes in stimulus pulse rate (8–62.5 Hz), (b) whether the eye is light or dark adapted, and (c) under conditions of intense chromatic adaptation. It is concluded that the garter snake has only a single class of cone pigment. The results from a curve fitting analysis suggests that this pigment has peak absorbance at about 556 nm.     

Purification and characterization of a chemoattractant from electric shock-induced earthworm secretion, its receptor binding, and signal transduction through the vomeronasal system of garter snakes

Following shocks with low voltage electric current, earthworms, Lumbricus terrestris, secrete a yellow mucus that has alarm properties for conspecifics and chemoattractive properties for garter snakes, Thamnophis sirtalis. A proteinaceous chemoattractant for garter snakes has been isolated and purified to homogeneity from such secretions by means of permeation chromatography and semipreparative nondenaturing polyacrylamide gel electrophoresis. The purified protein is highly attractive to garter snakes; it loses its activity after proteolytic digestion. It is a glycoprotein consisting of a single polypeptide chain with an NH2-terminal alanine. This chemoattractant has a minimum molecular mass of 15.4 kDa calculated from its amino acid and carbohydrate contents and an apparent molecular mass of about 20 kDa as estimated from sodium dodecyl sulfate-polyacrylamide gel electrophoresis. It has a pI of about 4.0, and it binds wheat germ agglutinin but not concanavalin A. This chemoattractant shows a protein to carbohydrate ratio of 2.0 +/- 0.08 (n = 5) and a ratio of total sugar to amino sugar of 1.9 +/- 0.08 (n = 3). The sequence of its NH2-terminal 15 amino acid residues has been determined. Studies were also conducted on the chemosignal transduction through the vomeronasal sensory system of the garter snake. Dot blot analysis showed that the purified chemoattractant bound to snake vomeronasal sensory epithelial membrane fractions. It did not bind to membrane extracts of the nonsensory epithelium of the vomeronasal mushroom body. The chemoattractant also bound specifically to vomeronasal sensory epithelial membrane in a reversible and saturable fashion with Kd and Bmax values of about 0.3 microM and 0.4 nmol/mg of protein, respectively. In electrophysiological studies, the chemoattractant applied to the vomeronasal epithelium caused an increase in firing rate of individual neurons in the accessory olfactory bulb of garter snakes, the projection site for vomeronasal neurons. The present results are the first clear biochemical and electrophysiological evidence for a vomeronasal epithelium response to a purified nonvolatile odorant, and this makes the garter snake vomeronasal system ideal for studying the mechanisms of chemosignal transduction.