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.

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.

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.

Stressed snakes strike first: Hormone levels and defensive behavior in free ranging cottonmouths (Agkistrodon piscivorus).

Stress is believed to be an important factor mediating animal behavior. Here we explore the relationship between concentrations of a stress hormone and defensive behavior of a snake. The cottonmouth (Agkistrodon piscivorus) is an abundant, large-bodied pitviper that is well known for its intense defensive behaviors. The defensive behaviors and hormonal ecology of cottonmouths have been studied extensively, but the interaction between these is not well understood. We conducted field trials, recording the snake's behavior and obtaining blood samples to quantify plasma CORT concentrations, both upon first encountering a snake and after a 30min standardized confinement stressor. We found that snakes with elevated levels of baseline CORT at first encounter were more likely to strike than exhibit a threat display when approached in the field. However, this behavior was not related to the magnitude of the snake's CORT increase following confinement, suggesting that more stress-prone snakes are not more defensive. Post-stressor antipredator behavior was also not related to any of our CORT measures. This study suggests that baseline CORT levels can be important correlates of defensive behavior. If this is a causative relationship, environmental challenges that increase baseline stress levels of populations may elevate cottonmouth defensive behavior. This would increase costs associated with defensive behavior (energetic, lost opportunity, etc.) and have important consequences for animal-human interactions.

Debunking the viper's strike: harmless snakes kill a common assumption.

To survive, organisms must avoid predation and acquire nutrients and energy. Sensory systems must correctly differentiate between potential predators and prey, and elicit behaviours that adjust distances accordingly. For snakes, strikes can serve both purposes. Vipers are thought to have the fastest strikes among snakes. However, strike performance has been measured in very few species, especially non-vipers. We measured defensive strike performance in harmless Texas ratsnakes and two species of vipers, western cottonmouths and western diamond-backed rattlesnakes, using high-speed video recordings. We show that ratsnake strike performance matches or exceeds that of vipers. In contrast with the literature over the past century, vipers do not represent the pinnacle of strike performance in snakes. Both harmless and venomous snakes can strike with very high accelerations that have two key consequences: the accelerations exceed values that can cause loss of consciousness in other animals, such as the accelerations experienced by jet pilots during extreme manoeuvres, and they make the strikes faster than the sensory and motor responses of mammalian prey and predators. Both harmless and venomous snakes can strike faster than the blink of an eye and often reach a target before it can move.

Roads are associated with a blunted stress response in a North American pit viper.

Whereas numerous studies have examined roads as anthropogenic stressors in birds and mammals, comparatively few studies have been undertaken on reptiles. We investigated plasma corticosterone (CORT) levels at baseline and following 30min of restraint stress in free-ranging copperhead snakes (Agkistrodon contortrix) captured within the forest interior or while in contact with public roads. There was no difference in baseline CORT levels between snakes in the forest and on roads. Copperheads responded to restraint stress by increasing plasma levels of CORT; however snakes on roads exhibited a lower CORT stress response compared to forest snakes. Additionally, among snakes captured on roads there was a negative association between road traffic and baseline CORT, stressed CORT, and the magnitude of the CORT response. Our results suggest that roads are associated with a blunted stress response in copperheads. Reduced stress responses may be indicative of acclimation, the inhibited ability to mount a stress response in the face of prolonged chronic stress, or that road environments select for individuals with lower CORT responsiveness. Either scenario could result in increased road mortality if snakes do not perceive roads as a potential threat.

Facultative parthenogenesis discovered in wild vertebrates.

Facultative parthenogenesis (FP)-asexual reproduction by bisexual species-has been documented in a variety of multi-cellular organisms but only recently in snakes, varanid lizards, birds and sharks. Unlike the approximately 80 taxa of unisexual reptiles, amphibians and fishes that exist in nature, FP has yet to be documented in the wild. Based on captive documentation, it appears that FP is widespread in squamate reptiles (snakes, lizards and amphisbaenians), and its occurrence in nature seems inevitable, yet the task of detecting FP in wild individuals has been deemed formidable. Here we show, using microsatellite DNA genotyping and litter characteristics, the first cases of FP in wild-collected pregnant females and their offspring of two closely related species of North American pitviper snakes-the copperhead (Agkistrodon contortrix) and cottonmouth (Agkistrodon piscivorus). Our findings support the view that non-hybrid origins of parthenogenesis, such as FP, are more common in squamates than previously thought. With this confirmation, FP can no longer be viewed as a rare curiosity outside the mainstream of vertebrate evolution. Future research on FP in squamate reptiles related to proximate control of induction, reproductive competence of parthenogens and population genetics modelling is warranted.

Reproduction in female copperhead snakes (Agkistrodon contortrix): plasma steroid profiles during gestation and post-birth periods.

We investigated levels of plasma progesterone (P4), 17ß-estradiol (E2), testosterone (T), and corticosterone (CORT) during gestation and post-birth periods in wild-collected female copperhead snakes (Viperidae; Agkistrodon contortrix). We also sought to determine whether CORT levels at (or near) birth dramatically increase and were correlated with duration of labor and litter size. Specifically, pregnant subjects (N = 14) were collected during early- to mid-gestation, held in the laboratory, and repeatedly bled to obtain plasma for steroid analyses. Progesterone showed significant changes during gestation, with the highest levels at the onset of sampling (circa 50 days prior to birth); P4 progressively declined up to parturition, and basal levels were observed thereafter. At the onset of sampling, E2 was at peak levels and fell sharply at circa 30 days prior to birth, a trend observed throughout the post-birth sampling period. Throughout the entire sampling period, T was undetectable. Although CORT showed no significant changes during gestation and several days following parturition, there was a highly significant peak at the time of birth. Our findings mirror the results of previous studies on pregnancy and steroid hormones of other live-bearing snakes, lizards, and mammals. As expected, there was a significant relationship between duration of labor and litter size; however, although levels of CORT did not achieve significance, there was a positive trend with litter size. We suggest that elevation of CORT at birth is involved in the mobilization and regulation of energy stores necessary for the physiological process of parturition and as a possible mechanism to trigger birth.

Innate immune performance and steroid hormone profiles of pregnant versus nonpregnant cottonmouth snakes (Agkistrodon piscivorus).

Squamates (lizards and snakes) have independently evolved viviparity over 100 times, and exhibit a wide range of maternal investment in developing embryos from the extremes of lecithotrophic oviparity to matrotrophic viviparity. This group therefore provides excellent comparative opportunities for studying endocrine and immune involvement during pregnancy, and their possible interactions. We studied the cottonmouth (Agkistrodon piscivorus), since they exhibit limited placentation (e.g., ovoviviparity), allowing comparison with squamate species hypothesized to require considerable maternal immune modulation due to the presence of a more extensive placental connection. Furthermore, the cottonmouth's biennial reproductive cycle provides an opportunity for simultaneously comparing pregnant and non-pregnant females in the wild. We document significantly elevated concentrations of progesterone (P4) and significantly lower concentrations of estradiol (E2) in pregnant females relative to non-pregnant females. Pregnant females had lower plasma bacteria lysis capacity relative to non-pregnant females. This functional measure of innate immunity is a proxy for complement performance, and we also determined significant correlations between P4 and decreased complement performance in pregnant females. These findings are consistent with studies that have determined P4's role in complement modulation during pregnancy in mammals, and thus this study joins a growing number of studies that have demonstrated convergent and/or conserved physiological mechanisms regulating viviparous reproduction in vertebrates.

Structural, enzymatic and pharmacological profiles of AplTX-II - A basic sPLA2 (D49) isolated from the Agkistrodon piscivorus leucostoma snake venom.

A basic sPLA2 (D49) from the venom of snake Agkistrodon piscivorus leucostoma (AplTX-II) was isolated, purified and characterized. We determined the enzymatic and pharmacological profiles of this toxin. AplTX-II was isolated with a high level of purity through reverse phase chromatography and molecular exclusion. The enzyme showed pI 9.48 and molecular weight of 14,003 Da. The enzymatic activity of the AplTX-II depended on Ca2+ pH and temperature. The comparison of the primary structure with other sPLA2s revealed that AplTX-II presented all the structural reasons expected for a basic sPLA2s. Additionally, we have resolved its structure with the docked synthetic substrate NOBA (4-nitro-3-octanoyloxy benzoic acid) by homology modeling, and performed MD simulations with explicit solvent. Structural similarities were found between the enzyme's modeled structure and other snake sPLA2 X-Ray structures, available in the PDB database. NOBA and active-site water molecules spontaneously adopted stable positions and established interactions in full agreement with the reaction mechanism, proposed for the physiological substrate, suggesting that NOBA hydrolysis is an excellent model to study phospholipid hydrolysis.

Comparative energetics and thermal responses to feeding in allied Agkistrodon snakes with contrasting diet and habitat use.

Variation in animal responses to feeding can be attributed to a variety of ecological factors, including foraging mode and dietary specialization. Specialization often favors species that have traits for exploiting food resources that are rare and that are not commonly shared by dietary generalists. We investigated physiological and behavioral responses to feeding between two snake species with different degrees of mammal feeding specialization: Agkistrodon contortrix (copperheads; a terrestrial species in which adults feed almost exclusively on mammals) and Agkistrodon piscivorus (cottonmouths; a semi-aquatic species feeding less on mammals and primarily on ectothermic prey). We measured metabolic rates (at 20, 25, and 30 °C) and body temperature (Tb) selection of snakes both pre- and post-feeding. Following the consumption of rodent meals, post-feeding energy use was higher in A. piscivorus than A. contortrix at both 25 and 30 °C. After feeding, A. piscivorus maintained body temperatures that were 3-4 °C higher, whereas A. contortrix remained within 1 °C of their pre-feeding Tb. Our results support the contention that dietary specialization leads to potential energetic advantages and that generalist species may change their behavior to offset energy used to digest prey.

Venous hematology, biochemistry, and blood gas analysis of free-ranging Eastern Copperheads (Agkistrodon contortrix) and Eastern Ratsnakes (Pantherophis alleghaniensis).

Hematology, plasma biochemistry, and blood gas analysis were performed on venous samples obtained from free-ranging Eastern Copperheads (Agkistrodon contortrix) and Eastern Ratsnakes (Pantherophis alleghaniensis) in central North Carolina during a mark-recapture study conducted from April to October 2015 at the North Carolina Zoo. Blood samples were collected from 31 (15 male and 16 female) free-ranging copperheads and 34 (20 male and 14 female) free-ranging ratsnakes at the beginning and end of restraint. Restraint was performed for morphometric measurements, sex determination, and identification via placement of intracelomic passive integrated transponder (PIT) tags and marking of ventral scutes with a handheld electrocautery unit. Blood gas analytes were measured at the beginning of restraint and compared to analytes measured at the end to evaluate for changes secondary to handling. Total restraint time prior to the first blood sampling was 1.4 ± 0.4 mins (mean ± SD) and 1.0 ± 0.2 mins (mean ± SD) and restraint time prior to second blood sampling was 12.5 ± 2.4 mins (mean ± SD) and 13.5 ± 3.4 mins (mean ± SD) for copperheads and ratsnakes, respectively. Blood lactate concentrations at the beginning of restraint were similar for both species. Lactate concentrations increased significantly and pH decreased significantly for both species at the end of restraint when compared to the beginning of restraint. Furthermore, lactate concentrations at the end of restraint were significantly elevated in ratsnakes compared to copperheads. This study provides guidelines for interpretation of venous hematology, plasma biochemistry, and blood gas values for free-ranging copperheads and ratsnakes in central North Carolina and demonstrates the physiological response to venous blood gas analytes secondary to capture and restraint.

Drought-induced Suppression of Female Fecundity in a Capital Breeder.

Human-induced global climate change is exerting increasingly strong selective pressures on a myriad of fitness traits that affect organisms. These traits, in turn, are influenced by a variety of environmental parameters such as temperature and precipitation, particularly in ectothermic taxa such as amphibians and reptiles. Over the past several decades, severe and prolonged episodes of drought are becoming commonplace throughout North America. Documentation of responses to this environmental crisis, however, is often incomplete, particularly in cryptic species. Here, we investigated reproduction in a population of pitviper snakes (copperhead, Agkistrodon contortrix), a live-bearing capital breeder. This population experienced a severe drought from 2012 through 2016. We tested whether declines in number of progeny were linked to this drought. Decline in total number offspring was significant, but offspring length and mass were unaffected. Reproductive output was positively impacted by precipitation and negatively impacted by high temperatures. We hypothesized that severe declines of prey species (e.g., cicada, amphibians, and small mammals) reduced energy acquisition during drought, negatively impacting reproductive output of the snakes. Support for this view was found using the periodical cicada (Magicicada spp.) as a proxy for prey availability. Various climate simulations, including our own qualitative analysis, predict that drought events will continue unabated throughout the geographic distribution of copperheads which suggests that long-term monitoring of populations are needed to better understand geographic variation in drought resilience and cascading impacts of drought phenomena on ecosystem function.

Phylogenetic origins for severe acetaminophen toxicity in snake species compared to other vertebrate taxa.

While it has been known for a while that some snake species are extremely sensitive to acetaminophen, the underlying mechanism for this toxicity has not been reported. To investigate if essential detoxification enzymes are missing in snake species that are responsible for biotransformation of acetaminophen in other vertebrate species, livers were collected from a variety of snake species, together with samples from alligator, snapping turtle, cat, rat, and cattle. Subcellular fractions were analyzed for enzymatic activities of phenol-type sulfotransferase and UDP­glucuronosyltransferase, total glutathione S­transferase, and N­acetyltransferase. The results showed that none of the snake species, together with the cat samples, had any phenol-type glucuronidation activity, and that this activity was much lower in alligator and turtle samples than in the mammalian species. Combined with the lack of N­acetyltransferase activity in snakes and cats, this would explain the accumulation of the aminophenol metabolite, which induces methemoglobinemia and subsequent suffocation of snakes and cats after acetaminophen exposure. While previous investigations have concluded that in cats the gene for the phenol-type glucuronosyltransferase isoform has turned into a pseudogene because of several point mutations, evaluation of genomic information for snake species revealed that they have only 2 genes that may code for glucuronosyltransferase isoforms. Similarity of these genes with mammalian genes is <50%, and suggests that the expressed enzymes may act on other types of substrates than aromatic amines. This indicates that the extreme sensitivity for acetaminophen in snakes is based on a different phylogenetic origin than the sensitivity observed in cats.

Sperm aggregations in female Agkistrodon piscivorus (Reptilia:Squamata): a histological and ultrastructural investigation.

Upon copulation in female Agkistrodon piscivorus, sperm migrate up the oviduct to sperm storage tubules (SSTs) in the posterior infundibulum. The epithelium of the SSTs is composed of ciliated and secretory cells and differs ultrastructurally from that of the epithelium lining the lumen of the posterior infundibulum. Sperm pass through an area composed primarily of ciliated cells at the opening of each gland before aligning themselves in parallel arrays with their nuclei facing an area composed primarily of secretory cells at the base of the tubules. Sperm are also found embedded inter- and intracellularly in the SSTs. The secretory vacuoles in the SSTs become highly electron dense after the start of the fall mating season along with the synthesis of lipid droplets. Histochemical analysis reveals that the alteration in secretory material density is caused by the production of neutral carbohydrates. Some sperm remain in aggregates in the nonglandular section of the posterior uterus until the time of ovulation. However, ultrastructural evidence indicates these sperm degrade before ovulation. Therefore, sperm in posterior aggregates have no role in fertilization of ovulated ova. The data presented here support the hypothesis that infundibular sperm storage is the mode that snakes utilize to sequester viable sperm until ovulation.

The reproductive biology of male cottonmouths (Agkistrodon piscivorus): do plasma steroid hormones predict the mating season?

To better understand the proximate causation of the two major types of mating seasons described for North American pitvipers, we conducted a field study of the cottonmouth (Agkistrodon piscivorus) in Georgia from September 2003 to May 2005 that included an extensive observational regime and collection of tissues for behavioral, anatomical, histological, and hormone analysis. Enzyme immunoassays (EIA) of plasma samples and standard histological procedures were conducted on reproductive tissues. Evidence from the annual testosterone (T) and sexual segment of the kidney (SSK) cycle and their relationship to the spermatogenic cycle provide correlative evidence of a unimodal mating pattern in this species of pitviper, as these variables consistently predict the mating season in all snake species previously examined under natural conditions. In most reptiles studied to date, high plasma levels of T and corticosterone (CORT) coincide during the mating period, making the cottonmouth an exception to this trend; we suggest two possible explanations for increased CORT during spring (regulation of a spring basking period), and decreased CORT during summer (avoiding reproductive behavioral inhibition), in this species.

Phospholipases A2 purified from cottonmouth snake venoms display no antibacterial effect against four representative bacterial species.

Snake venom phospholipase A2 (PLA2) has widely been reported to possess antibacterial effects, and PLA2 is the major component of cottonmouth snake venoms. We assessed the antibacterial activities of crude venoms from Western cottonmouth (Agkistrodon piscivorus leucostoma), Eastern cottonmouth (Agkistrodon piscivorus piscivorus), and Florida cottonmouth (Agkistrodon piscivorus conanti) snakes against two gram-positive (Bacillus subtilis, Staphylococcus aureus), and two gram-negative (Escherichia coli, Vibrio cholerae) bacteria. Antibacterial activity of PLA2 proteins, AplAsp49 and AplLys49 purified from A. p. leucostoma venom, was also examined. Disk-diffusion assays revealed that A. p. leucostoma crude venom is most effective in inhibiting the growth of the bacteria tested, compared to the other two. Surprisingly, AplAsp49 and AplLys49 PLA2s purified from A. p. leucostoma venom did not display detectable antibacterial activity against any bacteria tested neither by disk-diffusion nor by minimum inhibitory concentrations (MIC) and minimum bactericidal concentrations (MBC). The lack of antibacterial activity of cottonmouth venom PLA2s is discussed.

The effect of the myotoxic Lys49 phospholipase A(2) from Agkistrodon contortrix laticinctus snake venom on Na+/K+ -ATPase activity of toad bladders.

ACLMT is a myotoxic Lys49 phospholipase A(2) isolated from the venom of the snake Agkistrodon contortrix laticinctus. We have previously shown that ACLMT increases baseline water transport and partially inhibits vasopressin-stimulated water transport across toad bladders due to an increase in cytosolic calcium. However, these evidences provide insufficient insight into the mechanisms involved in the effects of ACLMT on membrane permeability. In an attempt to better understand such mechanisms, the current study aimed to investigate whether the Na(+)/K(+)-ATPase activity of isolated toad bladders can be affected by the ACLMT and the synthetic peptide from its C-terminal region. The toxin significantly decreased the Na(+)/K(+)-ATPase, while the peptide did not alter it. These findings suggest that the effects of ACLMT on membrane permeability may be due to the inhibition of the Na(+)/K(+)-ATPase activity, and that the C-terminal region may not play a relevant role in this effect. This study contributes toward a better understanding of the mechanisms involved in the toxicity of the snake venom Lys49 PLA(2) myotoxins on biological tissues.

Distribution of myelinated and unmyelinated nerve fibers and their possible role in blood flow control in crotaline snake infrared receptor organs.

We used transmission electron microscopic montages to examine the composition of nerve bundles serving the infrared pit organs of two species of crotaline snakes, Agkistrodon blomhoffii and A. brevicaudus. In the three main bundles, the myelinated fibers totaled 2,200-3,700, and unmyelinated fibers 2,400. We also discovered for the first time two accessory bundles composed almost entirely of unmyelinated fibers running alongside the main bundles, containing an average total of 3,300 unmyelinated fibers vs. an average of 10 myelinated fibers. Thus, the average total of unmyelinated fibers was nearly twice that of myelinated fibers. To study the nature of the unmyelinated fibers, we did double staining immunohistochemistry with antibodies for substance P (SP) and vasoactive intestinal polypeptide (VIP) in combination with and without capsaicin pretreatment. SP and VIP immunoreactive varicose fibers ran straight toward the center of the pit membrane in parallel with arterioles and venules, and also formed a dense network around the periphery of the membrane. There were three types of fibers: fibers containing only SP, fibers containing only VIP, and fibers containing both peptides. SP-only fibers were distributed singly throughout the pit membrane and in small bundles around the periphery. SP+VIP fibers were distributed sparsely in the pit membrane and around its periphery. VIP-only fibers were distributed throughout the pit membrane and were of smaller diameter than SP and SP+VIP fibers. After treatment with capsaicin, most of the three types of varicose fibers disappeared from the central part of the pit membrane, but those around the periphery remained unaffected. The capsaicin-sensitive fibers may be unmyelinated sensory types, and the unaffected ones may be autonomic nerve fibers.