Complexity in the timing of the first postnatal ecdysis in snakes.

Lepidosaurian reptiles, particularly snakes, periodically shed the outer epidermal layers of their skin (ecdysis) to restore or enhance vital functions such as regulating water and gaseous exchange, growth, and protection against insult, infection or physical injury. Although many studies have focused on the nature and mechanisms of skin shedding, little attention has been paid to the timing of the first ecdysis in neonates following birth or hatching. A recent study investigated patterns of the time to first postnatal ecdysis in snakes based on a large dataset taken from the literature. The analysis demonstrated patterns in the time to first postnatal ecdysis related to phylogeny as well as several life history traits. While this assessment provides important advances in our knowledge of this topic, data on known biophysical drivers of ecdysis - temperature and humidity - were largely unavailable and were not evaluated. The first postnatal ecdysis of neonatal snakes can be viewed as an adaptive adjustment to the transition from the aqueous environment of the embryo to the aerial environment of the newborn. Hence, the timing of the first postnatal ecdysis is logically influenced by the aerial environment into which a newborn snake or hatchling finds itself. Therefore, in this Commentary, we first emphasize the putative plasticity of ecdysis with respect to epidermal lipids that structure the water permeability barrier and are established or renewed during ecdysis to reduce transepidermal evaporative water loss. We then discuss the likely importance of biophysical variables as influential covariates that need future investigation as potential co-determinants of the timing of first postnatal ecdysis.

Perils of ingesting harmful prey by advanced snakes.

The advanced snakes (Alethinophidia) include the extant snakes with a highly evolved head morphology providing increased gape and jaw flexibility. Along with other physiological and morphological adaptations, this allows them to immobilize, ingest, and transport prey that may be disproportionately large or presents danger to the predator from bites, teeth, horns, or spines. Reported incidents of snakes failing to consume prey and being injured or killed during feeding mostly reflect information in the form of natural-history notes. Here we provide the first extensive review of such incidents, including 101 publications describing at least 143 cases of mortality (including six of 'multiple individuals') caused by ingestion or attempted consumption of injurious prey. We also report on 15 previously unpublished injurious feeding incidents from the USA, Austria, and Bulgaria, including mortality of five juvenile piscivorous dice snakes (Natrix tessellata) from a single location. Occurrences are spread across taxa, with mortality documented for at least 73 species from eight families and 45 genera. Incidents were generally well represented within each of three major categories: oversized prey (40.6%), potentially harmful prey (40.6%), and predator's behavioural/mechanical errors (18.9%). Reptile (33%) and fish (26%) prey caused disproportionately high mortality compared to mammals (16%). Feeding can be dangerous throughout a snake's life, with the later stages of feeding likely being more perilous. The number of reports has increased over time, and the data seem biased towards localities with a higher number of field-working herpetologists. We propose a standardized framework, comprising a set of basic information that should ideally be collected and published, and which could be useful as a template for future data collection, reporting, and analyses. We conclude that incidents of mortality during feeding are likely to be more common than previously assumed, and this hypothesis has implications for the ecology of persistence where populations are impacted by changing trophic environments.

Intraspecific investigation of dehydration-enhanced innate immune performance and endocrine stress response to sublethal dehydration in a semi-aquatic species of pit viper.

Sublethal dehydration can cause negative physiological effects, but recent studies investigating the sub-lethal effects of dehydration on innate immune performance in reptiles have found a positive correlation between innate immune response and plasma osmolality. To investigate whether this is an adaptive trait that evolved in response to dehydration in populations inhabiting water-scarce environments, we sampled free-ranging cottonmouth snakes (n=26 adult cottonmouths) from two populations inhabiting contrasting environments in terms of water availability: Snake Key (n=12), an island with no permanent sources of fresh water, and Paynes Prairie (n=14), a flooded freshwater prairie. In addition to field surveys, we manipulated the hydration state of 17 cottonmouths (Paynes Prairie n=9, Snake Key n=8) in a laboratory setting and measured the response of corticosterone and innate immune performance to dehydration with the aim of identifying any correlation or trade-offs between them. We measured corticosterone of cottonmouths at a baseline level and then again following a 60 min stress test when at three hydration states: hydrated, dehydrated and rehydrated. We found that innate immune performance improved with dehydration and then returned to baseline levels within 48 h of rehydration, which agrees with previous research in reptiles. Despite the frequent exposure of cottonmouths on Snake Key to dehydrating conditions, we did not find cottonmouths inhabiting the island to show a greater magnitude or more prolonged immune response compared with cottonmouths from Paynes Prairie. We also found a positive association between dehydration and corticosterone values.

Osmoregulatory ability predicts geographical range size in marine amniotes.

Species that are distributed over wide geographical ranges are likely to encounter a greater diversity of environmental conditions than do narrowly distributed taxa, and thus we expect a correlation between size of geographical range and breadth of physiological tolerances to abiotic challenges. That correlation could arise either because higher physiological capacity enables range expansion, or because widely distributed taxa experience more intense (but spatially variable) selection on physiological tolerances. The invasion of oceanic habitats by amniotic vertebrates provides an ideal system with which to test the predicted correlation between range size and physiological tolerances, because all three lineages that have secondarily moved into marine habitats (mammals, birds, reptiles) exhibit morphological and physiological adaptations to excrete excess salt. Our analyses of data on 62 species (19 mammals, 18 birds, 24 reptiles) confirm that more-widely distributed taxa encounter habitats with a wider range of salinities, and that they have higher osmoregulatory ability as determined by sodium concentrations in fluids expelled from salt-excreting organs. This result remains highly significant even in models that incorporate additional explanatory variables such as metabolic mode, body size and dietary habits. Physiological data thus may help to predict potential range size and perhaps a species' vulnerability to anthropogenic disturbance.

Thirst and drinking in North American watersnakes (Nerodia spp.).

We quantified drinking behavior in three species of North American watersnakes: Nerodia clarkii, which is a marine or brackish water amphibious species, and Nerodiafasciata and Nerodiataxispilota, both freshwater amphibious species. All three species have relatively small and similar thresholds of dehydration (TH, approximately -4% loss of body mass) that elicit thirst and drinking of fresh water. These species have higher thirst sensitivity than several species of hydrophiine and laticaudine sea snakes, which are characterized by much lower TH (greater dehydration, -9% to <-20%). Nerodia clarkii, which is often found in coastal oceanic water, refused to drink seawater, but drank fresh water when dehydrated. In separate trials involving dehydration of N. clarkii and N. fasciata that were concurrently fed fish at regular intervals, snakes eventually refused to eat at TH of approximately -12% of original body mass, but resumed eating after they were allowed to drink fresh water and rehydrate. The drinking behaviors of Nerodia corroborate previous data on the importance of fresh water for drinking, and they complement growing evidence that dietary water does not itself mitigate dehydration in snakes. These new data increase understanding of water relationships in the context of evolutionary transitions from land to sea, and they emphasize the importance of fresh water resources in the conservation of coastal and marine species of reptiles.

A tale of two islands: evidence for impaired stress response and altered immune functions in an insular pit viper following ecological disturbance.

The frequency and intensity of ecological perturbations affecting wild animal populations is expected to increase in the future with animals facing numerous global threats. Seahorse Key is a continental island off mainland Florida that has historically been a major rookery for several species of waterbirds. As a result of an unknown disturbance, the entire rookery abandoned Seahorse Key in April 2015 and shifted nesting activities to nearby Snake Key, resulting in an influx of food resources in the form of fish carrion to resident Florida cottonmouth snakes (Agkistrodon conanti), while snakes on Seahorse Key experienced a drastic reduction in food resources. Our objective was to assess plasma corticosterone concentrations, corticosterone negative feedback using dexamethasone, blood glucose, body condition, packed cell volume, natural antibody agglutination, white blood cell counts and ratios and erythrocyte sedimentation rate to characterize the long-term effects of differential resource availability in these two snake populations 3 years after this major ecological disturbance. We collected blood samples at three time points from cottonmouths on Seahorse Key (n = 6 individuals) and Snake Key (n = 13 individuals) in fall 2018. In due consideration of the small sample size, our study shows evidence that 3 years after the shift in waterbird nesting Seahorse Key cottonmouths exhibit a dampened acute stress response and presumptive impaired innate immune functions relative to cottonmouths on Snake Key. These results highlight the context-dependent nature of biomarkers and implicate the significant decrease in food resources on Seahorse Key in altering hormonal stress responses and innate immune functions, possibly leading to unknown long-term downstream effects. This study assessed the response of a wild population of pit viper to ecological disturbance in situ with the aim to improve our understanding of how animals cope with such perturbations and improve our capacity to make informed decisions for conservation.

Variation of organ position in snakes.

The complex and successful evolutionary history of snakes produced variation in the position and structure of internal organs. Gravity strongly influences hemodynamics, and the impact on structure and function of the cardiovascular system, including pulmonary circulation, is well established. Therefore, we hypothesized that interspecific variation in the position of the heart and vascular (faveolar) lung should exceed that of other internal organs that are less sensitive to gravity. We examined the position of selected internal organs in 72 snakes representing 5 families and 13 species including fully aquatic and scansorial/arboreal species, representing the extremes of gravitational influence. Tests for differences of variance and coefficients of variation largely confirm that interspecific variation in position of the heart and vascular lung generally exceed those of other organs that we measured, particularly posterior organs. The variance of heart position generally exceeded that of more posterior organs, was similar to that of the anterior margin of the vascular lung, and was exceeded by that of the posterior margin of the vascular lung (variance ratio = 0.23). The gravity-sensitive vascular lung exhibited the greatest variation of any organ. Importantly, these findings corroborate previous research demonstrating the influence of gravity on cardiopulmonary morphology. Snakes offer useful model systems to help understand the adaptation of organs to a spectrum of conditions related to diversity of behavior and habitat across a broad range of related taxa.

Water relations of an insular pit viper.

Colonization of novel habitats often requires plasticity or adaptation to local conditions. There is a critical need to maintain hydration in terrestrial environments having limited water. Atypical populations of Florida cottonmouth snakes, Agkistrodon conanti, inhabit continental islands with no permanent sources of fresh water. Here, we report investigations related to how these insular snakes maintain water balance considering the mainland conspecifics are semi-aquatic and typically associate with freshwater mesic habitats. We tested three hypotheses related to water relations of insular populations of cottonmouth snakes compared with those on the mainland. (1) Voluntary drinking of fresh water in free-ranging insular snakes should reflect a relationship to recency of rainfall more strongly than in mainland snakes. (2) Insular snakes will tolerate greater dehydration before drinking than will mainland snakes. (3) Insular snakes will avoid drinking seawater more strongly than will those from the mainland. Between 2001 and 2018, we quantitatively estimated the hydration status of 337 individual cottonmouth snakes from insular populations and 30 cottonmouth snakes from mainland Florida, as judged by the tendency of wild-caught snakes to drink fresh water immediately following capture. We found that insular cottonmouth snakes had a higher incidence of dehydration than did mainland cottonmouth snakes (64% versus 23%), and the hydration status of the insular snakes correlated with patterns of precipitation. We also determined experimentally the dehydration threshold for drinking fresh water in insular (mean±s.d. -5.64±4.3%, n=34) and mainland cottonmouth snakes (-5.74±4.5%, n=21), and these were not significantly different. Discrimination tests for drinking serially from a graded series of brackish water showed that mainland snakes did not discriminate against the highest brackish value (10.5 ppt or 30% seawater), whereas insular snakes showed a preference for <15% seawater. Naive neonates from insular and mainland cohorts behaved similarly. The preference of insular snakes for fresh water represents an important aspect of the maintenance of water balance that differs from the mainland conspecifics and is likely a habituated or adaptive response to dependence on rainfall.

Structure and function of skin in the pelagic sea snake, Hydrophis platurus.

We describe and interpret the functional morphology of skin of the Yellow-bellied sea snake, Hydrophis platurus. This is the only pelagic sea snake, and its integument differs from what is known for other species of snakes. In gross appearance, the scales of H. platurus consist of non-overlapping, polygonal knobs with flattened outer surfaces bearing presumptive filamentous sensillae. The deep recesses between scales ('hinge') entrap and wick water over the body surface, with mean retention of 5.1 g/cm of skin surface, similar to that determined previously for the roughened, spiny skin of marine file snakes, Acrochordus granulatus. This feature possibly serves to maintain the skin wet when the dorsal body protrudes above water while floating on calm oceanic slicks where they forage. In contrast with other snakes, including three species of amphibious, semi-marine sea kraits (Laticauda spp.), the outer corneous ß-protein layer consists of a syncytium that is thinner than seen in most other species. The subjacent α-layer is also thin, and lipid droplets and lamellar bodies are seen among the immature, cornifying α-cells. A characteristic mesos layer, comprising the water permeability barrier, is either absent or very thin. These features are possibly related to (1) permeability requirements for cutaneous gas exchange, (2) reduced gradient for water efflux compared with terrestrial environments, (3) less need for physical protection in water compared with terrestrial ground environments, and (4) increased frequency of ecdysis thought to be an anti-fouling mechanism. The lipogenic features of the α-layer possibly compensate for the reduced or absent mesos layer, or produce layers of cells that comprise what functionally might be termed a mesos layer, but where the organization of barrier lipids nonetheless appears less robust than what is characteristically seen in squamates.

Drinking by sea snakes from oceanic freshwater lenses at first rainfall ending seasonal drought.

Acquisition of fresh water (FW) is problematic for FW-dependent animals living in marine environments that are distant from sources of FW associated with land. Knowledge of how marine vertebrates respond to oceanic rainfall, and indeed the drinking responses of vertebrates generally following drought, is extremely scant. The Yellow-bellied Sea Snake (Hydrophis platurus) is the only pelagic species of squamate reptile and ranges across the Indo-Pacific oceans, having one of the largest geographic distributions of any vertebrate species. It requires FW and dehydrates at sea during periods of drought. Here we report drinking behaviors of sea snakes precisely at the transition from dry to wet season when rainfall first impacted the ocean following 6 months of seasonal drought. We show that the percentage of sea snakes that voluntarily drank FW in the laboratory when captured over eight successive days decreased from 80% to 13% before and after rainfall commenced, respectively. The percentage of snakes that drank immediately following capture exhibited a significant linear decline as the earliest rains of the wet season continued. Drinking by snakes indicates thirst related to dehydration, and thus thirsty snakes must have dehydrated during the previous six months of drought. Hence, the progressive decline in percentage of thirsty snakes indicates they were drinking from FW lenses associated with the first rainfall events of the wet season. These data reinforce the importance of accessing oceanic FW from precipitation, with implications for survival and distribution of pelagic populations that might be subjected to intensifying drought related to climate change.

Physiological and behavioral responses to salinity in coastal Dice snakes.

Secondarily marine tetrapods have evolved adaptations to maintain their osmotic balance in a hyperosmotic environment. During the transition to a marine habitat, the evolution of a euryhaline physiology likely encompassed successive changes in behavior and physiology that released organisms from regular access to fresh water. Deciphering these key steps is a complicated task. In this study, we investigated a species of freshwater natricine snake in which some populations are known to use marine environments. We experimentally subjected 30 adult Dice snakes (Natrix tessellata) from a population inhabiting the Black Sea coast to three salinities corresponding to freshwater (~0.1‰), brackish water (~15.0‰), and full-strength seawater (~34.0‰) in order to investigate their physiological (variation of body mass, osmolality) and behavioral (activity, drinking behavior) responses to salinity. Our results show that coastal Dice snakes from the study population are relatively tolerant to salinity close to that recorded in the Black Sea, but that prolonged exposure to full-strength seawater increases osmolality, stimulates thirst, decreases the activity of snakes and may ultimately jeopardize survival. Collectively with previously published data, our results strongly suggest specific physiological adaptations to withstand hyperosmolality rather than to reduce intake of salt, in coastal populations or species of semi-aquatic snakes. Future comparative investigations of Dice snakes from populations restricted to freshwater environment might reveal the functional traits and the behavioral and physiological responses of coastal N. tessellata to life in water with elevated salinity.

Feeding begets drinking: insights from intermittent feeding in snakes.

An important question related to the survival of dehydrating animals is whether feeding provides a net gain of water - contributing postprandial free water and metabolic water - or, alternatively, whether digestion and assimilation of ingested food incur a net loss of water because of requirements for digestion and the excretion of resulting metabolic wastes. Here, I address the question whether voluntary drinking increases or decreases following the ingestion of food. Increased postprandial drinking implies that food consumption increases rather than decreases the requirement for free water, whereas decreased postprandial drinking suggests there is a net profit of water from food. Snakes are ideally suited for such inquiry because they feed intermittently, and the temporal separation of meals allows relatively clear examination of the associated patterns of pre- and postprandial drinking. Voluntary drinking associated with meal consumption was quantified during consecutive feeding trials in four species representing two families of snakes. Postprandial relative to preprandial drinking increased in all four species, indicating that eating increases the physiological requirement for water. These data add to a growing literature pointing to some generality that eating can have negative rather than positive consequences for fluid homeostasis in some dehydrating animals.

Ontogenetic shifts of heart position in snakes.

Heart position relative to total body length (TL) varies among snakes, with anterior hearts in arboreal species and more centrally located hearts in aquatic or ground-dwelling species. Anterior hearts decrease the cardiac work associated with cranial blood flow and minimize drops in cranial pressure and flow during head-up climbing. Here, we investigate whether heart position shifts intraspecifically during ontogenetic increases in TL. Insular Florida cottonmouth snakes, Agkistrodon conanti, are entirely ground-dwelling and have a mean heart position that is 33.32% TL from the head. In contrast, arboreal rat snakes, Pantherophis obsoleta, of similar lengths have a mean heart position that is 17.35% TL from the head. In both species, relative heart position shifts craniad during ontogeny, with negative slopes = -.035 and -.021% TL/cm TL in Agkistrodon and Pantherophis, respectively. Using a large morphometric data set available for Agkistrodon (N = 192 individuals, 23-140 cm TL), we demonstrate there is an anterior ontogenetic shift of the heart position within the trunk (= 4.56% trunk length from base of head to cloacal vent), independent of head and tail allometry which are both negative. However, in longer snakes > 100 cm, the heart position reverses and shifts caudally in longer Agkistrodon but continues toward the head in longer individuals of Pantherophis. Examination of data sets for two independent lineages of fully marine snakes (Acrochordus granulatus and Hydrophis platurus), which do not naturally experience postural gravity stress, demonstrate both ontogenetic patterns for heart position that are seen in the terrestrial snakes. The anterior migration of the heart is greater in the terrestrial species, even if TL is standardized to that of the longer P. obsoleta, and compensates for about 5 mmHg gravitational pressure head if they are fully upright.

Oceanic circulation models help to predict global biogeography of pelagic yellow-bellied sea snake.

It is well recognized that most marine vertebrates, and especially tetrapods, precisely orient and actively move in apparently homogeneous oceanic environments. Here, we investigate the presumptive role of oceanic currents in biogeographic patterns observed in a secondarily marine tetrapod, the yellow-bellied sea snake (Hydrophis [Pelamis] platurus). State-of-the-art world ocean circulation models show how H. platurus, the only pelagic species of sea snake, can potentially exploit oceanic currents to disperse and maintain population mixing between localities that spread over two-thirds of the Earth's circumference. The very close association of these snakes with surface currents seems to provide a highly efficient dispersal mechanism that allowed this species to range extensively and relatively quickly well beyond the central Indo-Pacific area, the centre of origin, abundance and diversity of sea snakes. Our results further suggest that the pan-oceanic population of this species must be extraordinarily large.

Pelagic sea snakes dehydrate at sea.

Secondarily marine vertebrates are thought to live independently of fresh water. Here, we demonstrate a paradigm shift for the widely distributed pelagic sea snake, Hydrophis (Pelamis) platurus, which dehydrates at sea and spends a significant part of its life in a dehydrated state corresponding to seasonal drought. Snakes that are captured following prolonged periods without rainfall have lower body water content, lower body condition and increased tendencies to drink fresh water than do snakes that are captured following seasonal periods of high rainfall. These animals do not drink seawater and must rehydrate by drinking from a freshwater lens that forms on the ocean surface during heavy precipitation. The new data based on field studies indicate unequivocally that this marine vertebrate dehydrates at sea where individuals may live in a dehydrated state for possibly six to seven months at a time. This information provides new insights for understanding water requirements of sea snakes, reasons for recent declines and extinctions of sea snakes and more accurate prediction for how changing patterns of precipitation might affect these and other secondarily marine vertebrates living in tropical oceans.

Dehydration and drinking behavior of the marine file snake Acrochordus granulatus.

Dehydration and drinking behaviors were investigated in the little file snake (Acrochordus granulatus) collected from marine populations in the Philippines and in Australia. File snakes dehydrate in seawater and do not drink seawater when dehydrated in air and offered seawater to drink. Dehydrated file snakes drink freshwater, and the threshold of dehydration for first drinking response is a deficit of -7.4% ± 2.73% (mean ± SD) of original body mass. The thirst mechanism in this species is more sensitive than that recently studied in sea snakes. The volume of water ingested increases with increasing dehydration. Mean plasma osmolality was 278.89 ± 33.17 mMol/kg, mean hematocrit was 59% ± 5.45%, and both decreased in snakes that drank freshwater following acclimation in seawater. Snakes always drank freshwater at the water's surface, testing water with tongue flicks between each swallowing of water. Some snakes ingested large volumes of freshwater, approaching 50% of body mass. Visual observations and measurements of osmolality in plasma and stomach fluids suggest that water is taken up from the gut and dilutes body fluids slowly over the course of 48 h or longer. Eighty percent of snakes that were collected during the dry season (following >4 mo of drought) in Australia drank freshwater immediately following their capture, indicating that snakes were dehydrated in their marine environment even when known to have been feeding at the time. Snakes kept in seawater maintained a higher state of body condition when freshwater was periodically available. These results support a growing conclusion that diverse taxa of marine snakes require environmental sources of freshwater to maintain water balance, contrary to earlier belief. Identifying the freshwater requirements of secondarily marine vertebrates is important for better understanding how they maintain water balance in marine habitats, especially with respect to conservation in changing environments.

Non-lethal sampling of liver tissue for toxicologic evaluation of Florida cottonmouths snakes, Agkistrodon piscivorus conanti.

Due to their longevity, strong site tenure, poikilothermic metabolism, and low-energy specializations, reptiles might serve as excellent environmental sentinels. Cottonmouth snakes are generalist predators and scavengers, and as such, may have higher exposure to persistent environmental contaminants as a result of bioaccumulation. Traditionally, assessment and monitoring of contaminant exposure in reptiles have involved lethal sampling techniques. In this paper, we describe a non-destructive technique for sampling liver tissue in live anesthetized Florida cottonmouths. Wild-caught snakes (n = 21) were anesthetized with propofol, and a liver wedge biopsy was obtained by clamping the edge of the organ with two small hemostatic mosquito forceps via right-sided coeliotomy incision. A minimum required tissue sample weighing >100 mg was harvested from all except one of the animals. No mortalities occurred during the procedures or recovery from anesthesia, and all snakes were released back into the field after the animal had consumed prey and defecated, usually within 2 weeks following surgery. Hemorrhage was a minor complication in most snakes, especially those with friable discolored livers. The procedure appeared to have no short-term deleterious effects, and two biopsied individuals were captured after being released into the field and appeared to be normal and healthy. However, follow-up studies and recapture of more snakes are needed to assess long-term survivability. Our non-destructive liver sampling technique might be implemented in toxicological studies of other squamates and could help to minimize the lethal sampling of threatened species.

Sequestered defensive toxins in tetrapod vertebrates: principles, patterns, and prospects for future studies.

Chemical defenses are widespread among animals, and the compounds involved may be either synthesized from nontoxic precursors or sequestered from an environmental source. Defensive sequestration has been studied extensively among invertebrates, but relatively few examples have been documented among vertebrates. Nonetheless, the number of described cases of defensive sequestration in tetrapod vertebrates has increased recently and includes diverse lineages of amphibians and reptiles (including birds). The best-known examples involve poison frogs, but other examples include natricine snakes that sequester toxins from amphibians and two genera of insectivorous birds. Commonalities among these diverse taxa include the combination of consuming toxic prey and exhibiting some form of passive defense, such as aposematism, mimicry, or presumptive death-feigning. Some species exhibit passive sequestration, in which dietary toxins simply require an extended period of time to clear from the tissues, whereas other taxa exhibit morphological or physiological specializations that enhance the uptake, storage, and/or delivery of exogenous toxins. It remains uncertain whether any sequestered toxins of tetrapods bioaccumulate across multiple trophic levels, but multitrophic accumulation seems especially likely in cases involving consumption of phytophagous or mycophagous invertebrates and perhaps consumption of poison frogs by snakes. We predict that additional examples of defensive toxin sequestration in amphibians and reptiles will be revealed by collaborations between field biologists and natural product chemists. Candidates for future investigation include specialized predators on mites, social insects, slugs, and toxic amphibians. Comprehensive studies of the ecological, evolutionary, behavioral, and regulatory aspects of sequestration will require teams of ecologists, systematists, ethologists, physiologists, molecular biologists, and chemists. The widespread occurrence of sequestered defenses has important implications for the ecology, evolution, and conservation of amphibians and reptiles.