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.

Long-term monitoring of two snake species reveals immune-endocrine interactions and the importance of ecological context.

While there is huge promise in monitoring physiological parameters in free-living organisms, we also find high amounts of variability over time and space. This variation requires us to capitalize on long-term physiological monitoring to adequately address questions of population health, conservation status, or evolutionary trends as long-term sampling can examine ecoimmunological and endocrine interactions in wild populations while accounting for the variation that often makes ecophysiological field studies difficult to compare. In this study, we tested how immune efficacy and endocrinology interact while accounting for ecological context and environmental conditions in two snake species. Specifically, we measured bacterial killing ability, steroid hormones, and morphological characteristics in multiple populations of the Western Terrestrial Gartersnake (Thamnophis elegans) and Common Gartersnake (T. sirtalis) for multiple seasons over 6 years. Leveraging this long-term dataset, we tested how a broad immune measure and endocrine endpoints interact while accounting for individual traits, sampling date, and environmental conditions. Across both species, we found bacterial killing ability to be directly related to corticosterone (CORT) and temperature and greater overall in the spring compared to the fall. We found CORT and testosterone yielded relationships with individual sex, sampling temperature, and time of year. Wild populations can exhibit high amounts of variation in commonly collected physiological endpoints, highlighting the complexity and difficulty inherent in interpreting single endpoints without taking ecological and environmental conditions into account. Our study emphasizes the importance of reporting the environmental conditions under which the sampling occurred to allow for better contextualization and comparison between studies.

Investigating the relationship between corticosterone and glucose in a reptile.

The glucocorticoid hormone corticosterone (CORT) has classically been used in ecophysiological studies as a proxy for stress and energy mobilization, but rarely are CORT and the energy metabolites themselves concurrently measured. To examine CORT's role in mobilizing glucose in a wild reptile, we conducted two studies. The first study measured natural baseline and stress-induced blood-borne CORT and glucose levels in snakes during spring emergence and again when snakes return to the denning sites in autumn. The second study manipulated the hypothalamic-pituitary-adrenal (HPA) axis in male snakes in the autumn by taking a baseline blood sample, then subjecting individuals to one of five treatments (no injection, saline, CORT, adrenocorticotropin hormone and metyrapone). Subsequent samples were taken at 30 and 60 min. In both studies, we found that glucose levels do increase with acute stress, but that the relationship was not directly related to CORT elevation. In the second study, we found that none of the HPA axis manipulations directly affected blood glucose levels, further indicating that CORT may play a complex but not direct role in glucose mobilization in snakes. This study highlights the need for testing mechanisms in wild organisms by combining in situ observations with manipulative studies.

Sirtilins - the new old members of the vitamin K-dependent coagulation factor family.

Essentials Blood coagulation is driven by vitamin K (VK)-dependent proteases. We have identified and characterized 'sirtilin' as an additional VK-dependent protease. Sirtilins emerged early in the evolution of the coagulation system of vertebrates. Ubiquitous occurrence might indicate an important functional role of sirtilins. SUMMARY: Background Vitamin K (VK)-dependent proteases are major players in blood coagulation, including both the initiation and the regulation of the cascade. Five different members of this protease family have been described, comprising the following coagulation factors: factor VII, FIX, FX, protein C (PC), and prothrombin (FII). FVII, FIX, FX and PC share a typical domain architecture, with an N-terminal γ-carboxyglutamate (Gla) domain, two epidermal growth factor-like (EGF) domains, and a C-terminal trypsin-like serine protease (SP) domain. Objectives We have identified uncharacterized proteins in snake genomes showing the typical Gla-EGF1-EGF2-SP domain architecture but relatively low sequence conservation compared to known VK-dependent proteases. On the basis of sequence analysis, we hypothesized that these proteins are functional members of the VK-dependent protease family. Methods/results Using phylogenetic analyses, we confirmed the so-called 'sirtilins' as an additional VK-dependent protease class. These proteases were found in several vertebrates, including jawless fish, cartilaginous fish, bony fish, reptiles, birds, and marsupials, but not in other mammals. The recombinant zymogen form of Thamnophis sirtalis sirtilin was produced by in vitro renaturation, and was activated with human activated FXI. The activated form of sirtilin proteolytically cleaved peptide and protein substrates, including prothrombin. Mass spectrometry-based substrate profiling of sirtilin revealed a narrower sequence specificity than those of FIX and FX. Conclusions The ubiquitous occurrence of sirtilins in many vertebrate classes might indicate an important functional role. Understanding the detailed functions of sirtilins might contribute to a deeper understanding of the evolution and function of the vertebrate coagulation system.

Immune configuration in hatchling snakes is affected by incubation moisture, and is linked to subsequent growth and survival in the field.

We incubated the eggs of field-caught keelback snakes (Tropidonophis mairii) on wet versus dry substrates to explore impacts of incubation conditions on white blood cell (WBC) concentrations and differential WBC counts of hatchlings. In a second, independent study young snakes were released into the field, allowing us to explore fitness correlates of WBC profiles. Dry incubation reduced embryonic survival and hatchling body size, thus decoupling egg size from hatchling size. Incubation conditions also altered WBC profiles. Lymphocyte and azurophil counts were related to hatchling body size but not to initial egg mass, whereas heterophil counts were related to both of these traits. The egg-size effect on heterophil counts may reflect a maternal effect on offspring immune configuration. The ratio of heterophils to lymphocytes (an index of stress) was higher in hatchlings from eggs incubated on dry substrates. Snakes with higher counts of lymphocytes at hatching were more likely to be recaptured as adults (H:L; an index of survival), whereas snakes with higher basophil counts exhibited more rapid growth. In summary, our experiments show that incubation moisture levels influence the immune configuration of hatchling snakes, and that variation among individuals in WBC counts at hatching is a significant predictor of an individual's fitness after it is released into the wild. The demonstrated link between incubation conditions and offspring fitness is likely to impose strong selection on maternal nest-site choice.

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.

Investigation of the inhibitory potential of phospholipase A2 inhibitor gamma from Sinonatrix annularis to snake envenomation.

SaPLIγ is a novel gamma phospholipase A2 inhibitor (PLI) recently isolated from Sinonatrix annularis, a Chinese endemic non-venomous snake. To explore the neutralization effects of saPLIγ in snakebite envenomation, a dose equivalent to LD50 of Deinagkistrodon acutus, Agkistrodon halys and Naja atra venom with/without saPLIγ was inoculated into the gastrocnemius muscle of female Kunming mice. The ability of saPLIγ to inhibit myonecrosis and systemic toxicity were evaluated through investigations of muscle histopathology, and determination of the serum levels of creatine kinase (CK), lactate dehydrogenase isoenzyme1 (LDH1) and aspartate transferase (AST). Edema of the gastrocnemius muscle was evaluated by calculating the width difference between the inoculated limb and the contralateral leg. Desmin loss in the gastrocnemius muscle was determined by Western blot analysis. Co-immunoprecipitation and shotgun LC-MS/MS analyses were performed to identify venom proteins that interact with saPLIγ. All the envenomed mice had significantly elevated serum CK, LDH1 and AST levels, whereas the levels were decreased significantly in the presence of saPLIγ. Histopathological evaluation of gastrocnemius muscle sections showed severe snake venom-induced damage, characterized by leukocyte infiltration and erythrocyte leakage, leading to local edema. Myonecrosis, hemorrhage and desmin loss were significantly attenuated by saPLIγ. SaPLIγ interacted with a wide range of venom proteins, including PLA2s, metalloproteinases and C type lectins, which may contribute to broad anti-venom effects.

Corticosteroid responses of snakes to toxins from toads (bufadienolides) and plants (cardenolides) reflect differences in dietary specializations.

Toads are chemically defended by cardiotonic steroids known as bufadienolides. Resistance to the acute effects of bufadienolides in snakes that prey on toads is conferred by target-site insensitivity of the toxin's target enzyme, the Na+/K+-ATPase. Previous studies have focused largely on the molecular mechanisms of resistance but have not investigated the physiological mechanisms or consequences of exposure to the toxins. Adrenal enlargement in snakes often is associated with specialization on a diet of toads. These endocrine glands are partly composed of interrenal tissue, which produces the corticosteroids corticosterone and aldosterone. Corticosterone is the main hormone released in response to stress in reptiles, and aldosterone plays an important role in maintaining ion balance through upregulation of Na+/K+-ATPase. We tested the endocrine response of select species of snakes to acute cardiotonic steroid exposure by measuring circulating aldosterone and corticosterone concentrations. We found that Rhabdophis tigrinus, which specializes on a diet of toads, responds with lower corticosterone and higher aldosterone compared to other species that exhibit target-site resistance to the toxins but do not specialize on toads. We also found differences between sexes in R. tigrinus, with males generally responding with higher corticosterone and aldosterone than females. This study provides evidence of physiological adaptations, beyond target-site resistance, associated with tolerance of bufadienolides in a specialized toad-eating snake.

Geographic variation and within-individual correlations of physiological stress markers in a widespread reptile, the common garter snake (Thamnophis sirtalis).

Characterizing the baseline and stress-induced hormonal, metabolite, and immune profiles of wild animals is important to assess the impacts of variable environments, including human-induced landscape changes, on organismal health. Additionally, the extent to which these profiles are coordinated across physiological systems within individuals remains an important question in understanding how stressors can differentially affect aspects of an individual's physiology. Here, we present data from wild populations of the common garter snake (Thamnophis sirtalis) on both baseline and stress-induced biomarkers: plasma corticosterone (CORT) concentration, plasma glucose concentration, and whole blood heterophil:lymphocyte ratio. Using a standardized restraint protocol with individuals from populations in disparate portions of this species' range - the Sierra Nevada Mountains of California and the plains of Iowa - we collected blood plasma samples at nine time points over three days. Both CORT and glucose response curves differed between georegions, with Iowa snakes attaining higher glucose concentration and maintaining elevated CORT and glucose levels for a longer duration. Additionally, both the total amount and proportional increases of CORT and glucose were lower in larger and therefore older snakes, suggesting ontogenetic shifts in stress perception or response. Within-individual correlation among the three physiological indicators was significant at the time of capture, absent after 3h in captivity, and partially restored after 3days in captivity, demonstrating the effect of stress on the relationships among these physiological systems. Together, these results provide further evidence for the great physiological flexibility of ectothermic tetrapods in maintaining homeostasis across a range of factors.

Isolation and biochemical characterization of a gamma-type phospholipase A2 inhibitor from Macropisthodon rudis snake serum.

A novel phospholipaseA2 (PLA2) inhibitory protein (PLI) was purified from the serum of Macropisthodon rudis, a non-venomous snake mainly found in southern China. The molecular mass of the purified PLI was 160 kDa as determined by Superdex 200HR; however, the PLI protein had only one subunit of 25.4 kDa as determined by 12% SDS-PAGE, indicating an oligomeric protein. PLI cDNA obtained by PCR from the liver of Macropisthodon rudis, revealed 549 bps coding for a mature protein of 183 amino acid residues. Based on an amino acid sequence alignment with venomous and non-venomous snakes, this inhibitor was determined to be in the γ type family of PLI. In vitro experiments showed that PLIγ inhibited enzymatic, inflammatory, and antibacterial activities of snake venom PLA2 isolated from Agkistrodon acutus.

Orthogonal optimization of prokaryotic expression of a natural snake venom phospholipase A2 inhibitor from Sinonatrix annularis.

Phospholipase A2 (PLA2) is a calcium-dependent enzyme that is involved in inflammatory processes such as the liberation of free arachidonic acid from the membrane pool for the biosynthesis of eicosanoids. Snake venom are known containing PLA2s (svPLA2s) which exhibit a wide variety of pharmacological effects including neurotoxicity, cardiotoxicity, myotoxicity and hemorrhage. Therefore, inhibition of svPLA2 would be advantageous to successful envenomation treatment. A gamma type PLI (PLA2 inhibitor) has been extracted from the serum of Sinonatrix annularis, a non-venomous snake indigenous to China. This showed strong inhibition of Deinagkistrrodon acutus PLA2, however, the PLIγ level in the serum and snake resource are not sufficiently sustainable for further research. To overcome these limitations, we constructed a His6-PLIγ pET28 fusion expression vector and transformed Escherichia coli BL21. To improve the expression of PLIγ, an orthogonal experiment [L16(4)(5)] was performed to optimize induction parameters. The optimized condition was determined to be: induction by 0.4 mM isopropyl-ß-D-thiogalactoside (IPTG) for 6 h to the recombinant BL21 after its OD600 was 0.8, with continuous shaking cultivation at 190 rpm and 35 °C. Under these conditions, the amount of expressed protein could reach 57 mg/L. The His6-PLIγ was purified by nickel affinity chromatography and renatured by On-column refolding. The resulting PLIγ showed a good inhibitory effect of enzymatic activities to venom PLA2 isolated from D. acutus. Moreover, the PLIγ had a wide anti-hemorrhage activities to D. acutus, Naja atra and Agkistrodon halys venom.

The role of gamont entry into erythrocytes in the specificity of Hepatozoon species (Apicomplexa: Adeleida) for their frog hosts.

Hepatozoon species are apicomplexan parasites that infect blood cells and viscera of terrestrial vertebrates. One species, Hepatozoon clamatae, primarily infects green frogs, Rana clamitans , whereas another, Hepatozoon catesbianae, primarily infects bullfrogs, Rana catesbeiana , although both species of parasite are capable of infecting either species of frog. The aim of this study was to determine whether the basis for this partial host specificity is manifested at the gamont, or intraerythrocytic, stage of the parasite's life cycle. Blood was drawn from infected frogs and treated in vitro with a saline solution to induce intracellular gamonts to emerge from host erythrocytes. This treated blood was added to in vitro samples of uninfected blood of green frogs and bullfrogs. After 1 hr, samples were analyzed to determine the level of re-entry of the parasites into uninfected erythrocytes. Results obtained using multiple combinations of donor and recipient frogs indicate that extracellular gamonts of both parasite species do not exhibit preference for erythrocytes of 1 frog species over those of another. These results suggest that the basis for the observed host specificity is not determined at the gamont stage and is more likely dependent on another stage in the parasite life cycle.

Hematology and plasma biochemistry values for the giant garter snake (Thamnophis gigas) and valley garter snake (Thamnophis sirtalis fitchi) in the Central Valley of California.

Hematology and plasma biochemistry parameters are useful in the assessment and management of threatened and endangered species. Although reference ranges are readily available for many mammalian species, reference ranges for snakes are lacking for most species. We determined hematology and plasma biochemistry reference ranges for giant garter snakes (Thamnophis gigas) and valley garter snakes (Thamnophis sirtalis fitchi) living in four management areas in the Central Valley of California. White blood cell, heterophil, lymphocyte, and azurophil counts in giant garter snakes were approximately twice the values of valley garter snakes. Statistically significant differences in aspartate aminotransferase, globulin, and potassium between the two species did not appear clinically significant. No significant differences were found in the measured parameters between male and female giant garter snakes. Some differences were found among collection sites. These reference ranges provide baseline data for comparisons over time and between collection sites.

Temperature-dependent regulation of blood distribution in snakes.

Regional control of blood flow is often suggested as a mechanism for fine thermoregulatory adjustments in snakes. However, the flow of blood to different body regions at various temperatures has never been visualized to confirm this mechanism. We used (99m)technetium-labelled macroaggregated albumin ((99m)Tc-MAA), a radioactive tracer, to follow the flow of blood through the bodies of garter snakes (Thamnophis sirtalis) near their thermal maxima and minima. We injected snakes with(99m)Tc-MAA at cold (6-8°C) and hot (27-32°C) temperatures and imaged them using a gamma scanner. At cold ambient temperatures, snakes significantly reduced the blood flow to their tails and significantly increased the blood flow to their heads. Conversely, at hot ambient temperatures, snakes significantly increased the blood flow to their tails and significantly reduced the blood flow to their heads. This confirms that snakes are able to use differential blood distribution to regulate temperature. Our images confirm that snakes use regional control of blood flow as a means of thermoregulation and that vasomotor control of vascular beds is likely to be the mechanism of control.

Identification and characterization of phospholipase A2 inhibitors from the serum of the Japanese rat snake, Elaphe climacophora.

Two distinct phospholipase A2 (PLA2) inhibitory proteins (PLIs) were purified from the serum of the Japanese rat snake, Elaphe climacophora. The 150-kDa inhibitor, a trimer of a 50-kDa subunit, specifically inhibited the basic PLA2 purified from the venom of Gloydius brevicaudus, whereas the 120-kDa one composed of two distinct 25-kDa subunits. A and B, inhibited both the acidic and basic PLA2s of G. brevicaudus. On the basis of their amino acid sequences, these inhibitors were assigned as PLI beta and PLI gamma, respectively. A PLI alpha homolog (PLI alpha-like protein; PLI alpha-LP) having an apparent molecular weight of 50-kDa and composed of 15-kDa subunits was also purified from the E. climacophora serum. This homolog was immunoreactive with antibody raised against the G. brevicaudus PLI alpha, but lacked in the inhibitory activity toward the acidic and basic PLA2s. The cDNAs encoding PLI alpha-LP, PLI beta, PLI gamma-A, and PLI gamma-B were cloned from liver RNA, and their nucleotide sequences were compared with those of other venomous and non-venomous snakes.

A test of life-history theories of immune defence in two ecotypes of the garter snake, Thamnophis elegans.

1. Life-history theorists have long observed that fast growth and high reproduction tend to be associated with short life span, suggesting that greater investment in such traits may trade off with self-maintenance. The immune system plays an integral role in self-maintenance and has been proposed as a mediator of life-history trade-offs. 2. Ecoimmunologists have predicted that fast-living organisms should rely more heavily on constitutive innate immunity than slow-living organisms, as constitutive innate defences are thought to be relatively inexpensive to develop and can provide a rapid, general response to pathogens. 3. We present the first study to examine this hypothesis in an ectothermic vertebrate, by testing for differences in three aspects of constitutive innate immunity in replicate populations of two life-history ecotypes of the garter snake Thamnophis elegans, one fast-living and one slow-living. 4. As predicted, free-ranging snakes from the fast-living ecotype had higher levels of all three measures of constitutive innate immunity than the slow-living ecotype. These differences in immunity were not explained by parasite loads measured. Furthermore, both ecotypes exhibited a positive relationship between innate immunity and body size/age, which we discuss in the context of ectotherm physiology and ecotype differences in developmental rates.

Evolutionary ecology of endocrine-mediated life-history variation in the garter snake Thamnophis elegans.

The endocrine system plays an integral role in the regulation of key life-history traits. Insulin-like growth factor-1 (IGF-1) is a hormone that promotes growth and reproduction, and it has been implicated in the reduction of lifespan. IGF-1 is also capable of responding plastically to environmental stimuli such as resource availability and temperature. Thus pleiotropic control of life-history traits by IGF-1 could provide a mechanism for the evolution of correlated life-history traits in a new or changing environment. An ideal system in which to investigate the role of IGF-1 in life-history evolution exists in two ecotypes of the garter snake Thamnophis elegans, which derive from a single recent ancestral source but have evolved genetically divergent life-history characteristics. Snakes from meadow populations near Eagle Lake, California (USA) exhibit slower growth rates, lower annual reproductive output, and longer median adult lifespans relative to populations along the lakeshore. We hypothesized that the IGF-1 system has differentiated between these ecotypes and can account for increased growth and reproduction and reduced survival in lakeshore vs. meadow snakes. We tested for a difference in plasma IGF-1 levels in free-ranging snakes from replicate populations of each ecotype over three years. IGF-1 levels were significantly associated with adult body size, reproductive output, and season in a manner that reflects established differences in prey ecology and age/size-specific reproduction between the ecotypes. These findings are discussed in the context of theoretical expectations for a tradeoff between reproduction and lifespan that is mediated by pleiotropic endocrine mechanisms.

Effects of acute temperature change, confinement and housing on plasma corticosterone in water snakes, Nerodia sipedon (Colubridae: Natricinae).

Body temperature affects many aspects of reptilian behavior and physiology, but its effect on hormonal secretion has been little studied, especially in snakes. Major objectives of this study were to determine if acute changes in body temperature during confinement influenced plasma corticosterone levels and if initial body temperatures upon capture in the field were related to baseline corticosterone levels in water snakes (Nerodia sipedon). Water snakes were bled upon capture in the field and after one hour of confinement in a cooled, control, or heated incubator. Since little is known about the potential metabolic changes in response to stress in reptiles, plasma triglyceride levels were also measured. Upon completion of the field study, snakes were housed for 5-8 days without food to determine the effect of chronic stress on both corticosterone and triglyceride levels. Plasma corticosterone concentrations were measured using enzyme-linked immunosorbant assay (ELISA) and plasma triglycerides were determined enzymatically. In the field, experimental alterations of body temperature during confinement had no effect on corticosterone levels. Similarly, there was no correlation between initial body temperature and baseline plasma corticosterone concentrations. However, post-confinement corticosterone levels were approximately three-times greater in females than males. Plasma triglyceride levels were not affected by temperature treatment, confinement, or sex. Compared to field values, both baseline and post-confinement corticosterone levels were elevated after the chronic stress of short-term laboratory housing but triglyceride levels decreased. Overall, these results indicate that sex but not body temperature has a major influence on the adrenocortical stress response in Nerodia sipedon.

Geographic variation in timekeeping systems among three populations of garter snakes (Thamnophis sirtalis) in a common garden.

Transduction of environmental cues into endocrine signals that synchronize physiology and behavior with optimal environmental conditions is central to an animal's timekeeping system. Using a common garden approach, we investigated possible geographic variation in timekeeping systems by comparing 24-h melatonin and corticosterone rhythms and reproductive behavior among three populations of garter snakes with very different life histories: red-sided garter snakes (Thamnophis sirtalis parietalis) from Manitoba, Canada; red-spotted garter snakes (Thamnophis sirtalis concinnus) from western Oregon; and eastern garter snakes (Thamnophis sirtalis sirtalis) from southern Florida. Melatonin and corticosterone cycles differed significantly among the three snake populations in a majority of the sampling periods. Population differences were observed across a wide range of acclimatization conditions and were themselves plastic (i.e., one snake population was not consistently different from the others). Changes in courtship behavior during emergence also varied significantly among populations. Our data support the hypothesis that endogenous timekeeping systems have evolved in the presence of unique environmental conditions. Further research is necessary to determine whether this geographic variation results from inherent genetic differences or whether it is a product of development. These studies provide insight into the evolution of timekeeping systems and may aid in understanding the potential effects of environmental perturbations on seasonal physiology and behavior.

Temporal and spatial patterns of contaminants in Lake Erie watersnakes (Nerodia sipedon insularum) before and after the round goby (Apollonia melanostomus) invasion.

Temporal and spatial trends in contaminant concentrations were assessed in Lake Erie watersnakes, a threatened (USA)/endangered (Canada) species restricted to western Lake Erie. Temporal changes in plasma contaminant levels were determined in 1990 and 2003, and spatial patterns in 2003 at 12 sites, throughout the species' range. During this period, the watersnakes' diet changed from fish (75%) and amphibians (25%) that avoid zebra mussels, to round gobies (95%) that feed extensively on zebra mussels. Temporal trends indicate that watersnakes on Pelee and North Bass Islands showed a marginal increase in hexachlorobenzene levels, and a significant decline in dieldrin, oxychlordane, and heptachlor epoxide, likely reflecting declines in aerial deposition and clearing of local vineyards. The contaminants with the greatest burdens, sum PCBs and p,p'-DDE, remained stable in the snakes, consistent with trends in other local biota, suggesting that although the dietary switch to round gobies meant consumption of a more contaminated diet, their diet remained at the same trophic position. We suggest that the watersnakes' PCB and p,p'-DDE temporal patterns reflect the lack of change in sediment concentrations with minimal influence from their dietary switch. Similar to top avian predators, PCBs, p,p'-DDE, and technical chlordane, are most prevalent in watersnakes; this ranking remains unchanged. In 2003, the watersnakes demonstrated significant spatial differences in concentrations of p,p'-DDE, dieldrin, technical chlordane and its metabolites. Their 2003 concentrations of p,p'-DDE, and to a lesser extent PCBs, exceed the recommended interim no-observable effects levels on watersnake embryonic survival. Further investigations are required to determine if these higher levels of PCBs, p,p'-DDE, and technical chlordane, affect reproductive and physiological parameters of the Lake Erie watersnake. Until concentrations of sediment contaminants decline in western Lake Erie, these endangered/threatened watersnakes will continue to be exposed to higher concentrations of persistent organic pollutants.