BODY CONDITION OF EASTERN BOX TURTLES (TERRAPENE CAROLINA CAROLINA) EVALUATED BY COMPUTED TOMOGRAPHY.

Conservation efforts determining the health status of individuals can aid in assessing population health and sustainability. Body condition was determined in 65 free-ranging Eastern box turtles (Terrapene carolina carolina) from four locations in Vermilion County, Illinois, in the months of May, July, and September 2014, 2015, and 2016 using computed tomography (CT). Physical examinations were performed to determine morphometric measurements, and CT scans measured body fat. Twenty-three linear models were constructed to determine which morphometric measurements best correlated to fat content. The top two models of body fat included a relationship between mass and carapace width. Fat increases as both mass and carapace width increase. CT, while impractical for daily use, improved on previous methods of measuring body condition and created a calculation that can be applied broadly to Eastern box turtles. By understanding how measurements of mass and carapace width correlate to body condition, practitioners, researchers, and conservationists can evaluate chelonians with increased confidence in the accuracy of their assessment.

Predicting the range of a regionally threatened, benthic fish using species distribution models and field surveys.

Understanding a species' historical and current distribution is critical when making conservation and management decisions. Recent observations in headwater streams of northern Illinois, USA, where no previous records of Iowa Darters Etheostoma exile occurred, revealed the need to re-evaluate its state-wide distribution. We conducted a series of species distribution modelling procedures coupled with targeted field surveys to generate historical and contemporary distribution models. The historical distribution model indicated E. exile distributions were concentrated across extreme northern Illinois. Both contemporary models included results of the historical model as a model variable. Based on the initial contemporary model, 30 potential Iowa Darter sites, 10 each of three groups representing low, medium and high model scores, were sampled during the summer of 2016. Field surveys provided nine new E. exile localities and presence-absence data from field surveys were then applied to revise the contemporary model. The revised contemporary model suggests a decrease in certain areas of its historic Illinois range, as well as areas where it has potentially expanded its range.

Select metal and metalloid surveillance of free-ranging Eastern box turtles from Illinois and Tennessee (Terrapene carolina carolina).

The Eastern box turtle (Terrapene carolina carolina) is a primarily terrestrial chelonian distributed across the eastern US. It has been proposed as a biomonitor due to its longevity, small home range, and reliance on the environment to meet its metabolic needs. Plasma samples from 273 free-ranging box turtles from populations in Tennessee and Illinois in 2011 and 2012 were evaluated for presence of heavy metals and to characterize hematologic variables. Lead (Pb), arsenic (As), zinc (Zn), chromium (Cr), selenium (Se), and copper (Cu) were detected, while cadmium (Cd) and silver (Ag) were not. There were no differences in any metal detected among age class or sex. However, Cr and Pb were higher in turtles from Tennessee, while As, Zn, Se, and Cu were higher in turtles from Illinois. Seasonal differences in metal concentrations were observed for Cr, Zn, and As. Health of turtles was assessed using hematologic variables. Packed cell volume was positively correlated with Cu, Se, and Pb in Tennessee. Total solids, a measure of plasma proteins, in Tennessee turtles were positively correlated with Cu and Zn. White blood cell count, a measure of inflammation, in Tennessee turtles was negatively correlated with Cu and As, and positively correlated with Pb. Metals are a threat to human health and the health of an ecosystem, and the Eastern Box Turtle can serve as a monitor of these contaminants. Differences established in this study can serve as baseline for future studies of these or related populations.

Phenology and predictors of spring emergence for the Timber Rattlesnake (Crotalus horridus).

Many temperate reptiles survive winter by using subterranean refugia until external conditions become suitable for activity. Determining when to emerge from refugia relies on the ability to interpret when above-ground environmental conditions are survivable. If temperate reptiles rely on specific environmental cues such as temperature to initiate emergence, we should expect emergence phenologies to be predictable using local climatic data. However, specific predictors of emergence for many temperate reptiles, including the Timber Rattlesnake (Crotalus horridus), remain unclear, limiting our understanding of their overwintering phenology and restricting effective conservation and management. Our objectives were to identify environmental cues of spring emergence for C. horridus in Illinois to determine the species' emergence phenology, and to examine the applicability of identified cues in predicting emergence phenology across the species' range. We used wildlife cameras and weather station-derived environmental data to observe and predict the daily surface presence of C. horridus throughout the late winter and early spring at communal refugia in west-central and northern Illinois. The most parsimonious model for predicting surface presence included the additive effects of maximum daily temperature, accumulated degree days, and latitude. With a notable exception in the southeastern U.S., the model accurately predicted the average emergence day for eight other populations range wide, emphasizing the importance of temperature in influencing the phenological plasticity observed across the species' range. The apparent broad applicability of the model to other populations suggests it can be a valuable tool in predicting spring emergence phenology. Our results provide a foundation for further ecological enquiries and improved management and conservation strategies.

Limited gene flow and pronounced population genetic structure of Eastern Massasauga (Sistrurus catenatus) in a Midwestern prairie remnant.

As anthropogenic changes continue to ecologically stress wildlife, obtaining measures of gene flow and genetic diversity are crucial for evaluating population trends and considering management and conservation strategies for small, imperiled populations. In our study, we conducted a molecular assessment to expand on previous work to elucidate patterns of diversity and connectivity in the remaining disjunct Eastern Massasauga Rattlesnake (Sistrurus catenatus) hibernacula in Illinois. We assayed genetic data for 327 samples collected during 1999-2015 from the Carlyle Lake study area across 21 microsatellite loci. We found hibernacula formed distinct genetic clusters corresponding to the three main study areas (Dam Recreation Areas, Eldon Hazlet State Park, and South Shore State Park). Genetic structuring and low estimates of dispersal indicated that connectivity among these study areas is limited and each is demographically independent. Hibernacula exhibited moderate levels of heterozygosity (0.60-0.73), but estimates of effective population size (5.2-41.0) were low and track census sizes generated via long-term mark-recapture data. Hibernacula at Carlyle Lake, which represent the only Eastern Massasauga remaining in Illinois, are vulnerable to future loss of genetic diversity through lack of gene flow as well as demographic and environmental stochastic processes. Our work highlights the need to include population-level genetic data in recovery planning and suggests that recovery efforts should focus on managing the three major study areas as separate conservation units in order to preserve and maintain long-term adaptive potential of these populations. Specific management goals should include improving connectivity among hibernacula, maintaining existing wet grassland habitat, and minimizing anthropogenic sources of mortality caused by habitat management (e.g., mowing, prescribed fire) and recreational activities. Our molecular study provides additional details about demographic parameters and connectivity at Carlyle Lake that can be used to guide recovery of Eastern Massasauga in Illinois and throughout its range.

Growing as slow as a turtle: Unexpected maturational differences in a small, long-lived species.

Turtle body size is associated with demographic and other traits like mating success, reproductive output, maturity, and survival. As such, growth analyses are valuable for testing life history theory, demographic modeling, and conservation planning. Two important but unsettled research areas relate to growth after maturity and growth rate variation. If individuals exhibit indeterminate growth after maturity, older adults may have an advantage in fecundity, survival, or both over younger/smaller adults. Similarly, depending on how growth varies, a portion of the population may mature earlier, grow larger, or both. We used 23-years of capture-mark-recapture data to study growth and maturity in the Spotted Turtle (Clemmys guttata), a species suffering severe population declines and for which demographic data are needed for development of effective conservation and management strategies. There was strong support for models incorporating sex as a factor, with the interval growth model reparametrized for capture-mark-recapture data producing later mean maturation estimates than the age-based growth model. We found most individuals (94%) continued growing after maturity, but the instantaneous relative annual plastral growth rate was low. We recommend future studies examine the possible contribution of such slow, continued adult growth to fecundity and survival. Even seemingly negligible amounts of annual adult growth can have demographic consequences affecting the population vital rates for long-lived species.

Measuring agreement and discord among hemagglutination inhibition assays against different ophidian paramyxovirus strains in the Eastern massasauga (Sistrurus catenatus catenatus).

At present, the hemagglutination inhibition (HI) assay is the sole commercially available serologic method available to detect exposure to ophidian paramyxovirus (OPMV) in snakes. During 2006, 26 eastern massasaugas (Sistrurus catenatus catenatus) were collected, and blood was sampled to determine their OPMV status. Samples from each snake were divided into 3 aliquots and tested by using commercially available HI assays against the 4 OPMV isolates used in the 3 laboratories that offer the service. All snakes were tested for antibodies by using HI assays against the green tree python (GTP), San Lucan rattlesnake (SLR), and Aruba Island rattlesnake (AIR) isolates. Twenty-five snakes were tested for antibodies against the western diamondback rattlesnake (WDR) isolate. All samples tested against the GTP and SLR were positive (26/26), whereas 56% (14/25) of the WDR assays were positive, and none (0/26) of the AIR assays yielded a positive result. There was 100% agreement between the GTP and SLR assays, and complete disagreement between the SLR and AIR, as well as the GTP and AIR assays. Kappa statistics for the GTP-WDR, SLR-WDR, GTP-AIR, SLR-AIR, and WDR-AIR indicated that the assays had less than chance agreement. The results demonstrate that current HI assays are not reliable as a sole diagnostic assay in the eastern massasauga. Furthermore, HI assays need to be evaluated by using other parameters to determine OPMV exposure in eastern massasaugas.

Climatic and geographic predictors of life history variation in Eastern Massasauga (Sistrurus catenatus): A range-wide synthesis.

Elucidating how life history traits vary geographically is important to understanding variation in population dynamics. Because many aspects of ectotherm life history are climate-dependent, geographic variation in climate is expected to have a large impact on population dynamics through effects on annual survival, body size, growth rate, age at first reproduction, size-fecundity relationship, and reproductive frequency. The Eastern Massasauga (Sistrurus catenatus) is a small, imperiled North American rattlesnake with a distribution centered on the Great Lakes region, where lake effects strongly influence local conditions. To address Eastern Massasauga life history data gaps, we compiled data from 47 study sites representing 38 counties across the range. We used multimodel inference and general linear models with geographic coordinates and annual climate normals as explanatory variables to clarify patterns of variation in life history traits. We found strong evidence for geographic variation in six of nine life history variables. Adult female snout-vent length and neonate mass increased with increasing mean annual precipitation. Litter size decreased with increasing mean temperature, and the size-fecundity relationship and growth prior to first hibernation both increased with increasing latitude. The proportion of gravid females also increased with increasing latitude, but this relationship may be the result of geographically varying detection bias. Our results provide insights into ectotherm life history variation and fill critical data gaps, which will inform Eastern Massasauga conservation efforts by improving biological realism for models of population viability and climate change.

HEMATOLOGY IN AN EASTERN MASSASAUGA (SISTRURUS CATENATUS) POPULATION AND THE EMERGENCE OF OPHIDIOMYCES IN ILLINOIS, USA.

Disease events are threatening wildlife populations across North America. Specifically, mortality events due to Ophidiomyces (snake fungal disease; SFD) have been observed recently in snakes in Illinois, US. We investigated the health of a population of eastern massasaugas ( Sistrurus catenatus ) in south-central Illinois using 1) a meta-analysis of hematologic findings from 2004, 2011, 2013, and 2014; 2) a determination of the prevalence of SFD in snakes examined in 2013 and 2014; and 3) the examination of 184 museum specimens collected from 1999-2013 for signs and presence of SFD. For the meta-analysis and prevalence of SFD, hematologic analytes were reduced to three principle components that explained 67.5% of the cumulative variance. There were significant differences among one principle component (total white blood cell counts, monocytes, lymphocytes, and basophils) across years when it was highest in 2004 and 2014. The top general linear model explaining the difference in principle components included the main effects of year and stage, body condition index (BCI), and the interaction between stage and BCI. The prevalence of SFD was 18% (n=7) in 2013 and 24% (n=11) in 2014, and no hematologic analytes were associated with SFD. In museum specimens, Ophidiomyces DNA was first detected from an individual collected in 2000. Studies such as these, integrating multiple modalities of health, can elucidate the epidemiology of diseases that may pose conservation threats.

Prevalence and Seasonality of the Amphibian Chytrid Fungus Batrachochytrium dendrobatidis Along Widely Separated Longitudes Across the United States.

The chytrid fungus Batrachochytrium dendrobatidis (Bd) has been implicated in amphibian declines on almost all continents. We report on prevalence and intensity of Bd in the United States amphibian populations across three longitudinally separated north-to-south transects conducted at 15 Department of Defense installations during two sampling periods (late-spring/early summer and mid to late summer). Such a standardized approach minimizes the effects of sampling and analytical bias, as well as human disturbance (by sampling restricted military bases), and therefore permits a cleaner interpretation of environmental variables known to affect chytrid dynamics such as season, temperature, rainfall, latitude, and longitude. Our prevalence of positive samples was 20.4% (137/670), and our mean intensity was 3.21 zoospore equivalents (SE = 1.03; range 0.001-103.59). Of the 28 amphibian species sampled, 15 tested positive. Three sites had no evidence of Bd infection; across the remaining 12 Bd-positive sites, neither infection prevalence nor intensity varied systematically. We found a more complicated pattern of Bd prevalence than anticipated. Early season samples showed no trend associated with increasing temperature and precipitation and decreasing (more southerly) latitudes; while in late season samples, the proportion of infected individuals decreased with increasing temperature and precipitation and decreasing latitudes. A similar pattern held for the east-west gradient, with the highest prevalence associated with more easterly/recently warmer sites in the early season then shifting to more westerly/recently cooler sites in the later season. Bd intensity across bases and sampling periods was comparatively low. Some of the trends in our data have been seen in previous studies, and our results offer further continental-level Bd sampling over which more concentrated local sampling efforts can be overlaid.

Microsatellite and major histocompatibility complex variation in an endangered rattlesnake, the Eastern Massasauga (Sistrurus catenatus).

Genetic diversity is fundamental to maintaining the long-term viability of populations, yet reduced genetic variation is often associated with small, isolated populations. To examine the relationship between demography and genetic variation, variation at hypervariable loci (e.g., microsatellite DNA loci) is often measured. However, these loci are selectively neutral (or near neutral) and may not accurately reflect genomewide variation. Variation at functional trait loci, such as the major histocompatibility complex (MHC), can provide a better assessment of adaptive genetic variation in fragmented populations. We compared patterns of microsatellite and MHC variation across three Eastern Massasauga (Sistrurus catenatus) populations representing a gradient of demographic histories to assess the relative roles of natural selection and genetic drift. Using 454 deep amplicon sequencing, we identified 24 putatively functional MHC IIB exon 2 alleles belonging to a minimum of six loci. Analysis of synonymous and nonsynonymous substitution rates provided evidence of historical positive selection at the nucleotide level, and Tajima's D provided support for balancing selection in each population. As predicted, estimates of microsatellite allelic richness, observed, heterozygosity, and expected heterozygosity varied among populations in a pattern qualitatively consistent with demographic history and abundance. While MHC allelic richness at the population and individual levels revealed similar trends, MHC nucleotide diversity was unexpectedly high in the smallest population. Overall, these results suggest that genetic variation in the Eastern Massasauga populations in Illinois has been shaped by multiple evolutionary mechanisms. Thus, conservation efforts should consider both neutral and functional genetic variation when managing captive and wild Eastern Massasauga populations.

Development of Snake Fungal Disease after Experimental Challenge with Ophidiomyces ophiodiicola in Cottonmouths (Agkistrodon piscivorous).

Snake fungal disease (SFD) is a clinical syndrome associated with dermatitis, myositis, osteomyelitis, and pneumonia in several species of free-ranging snakes in the US. The causative agent has been suggested as Ophidiomyces ophiodiicola, but other agents may contribute to the syndrome and the pathogenesis is not understood. To understand the role of O. ophiodiicola in SFD, a cottonmouth snake model of SFD was designed. Five cottonmouths (Agkistrodon piscivorous) were experimentally challenged by nasolabial pit inoculation with a pure culture of O. ophiodiicola. Development of skin lesions or facial swelling at the site of inoculation was observed in all snakes. Twice weekly swabs of the inoculation site revealed variable presence of O. ophiodiicola DNA by qPCR in all five inoculated snakes for 3 to 58 days post-inoculation; nasolabial flushes were not a useful sampling method for detection. Inoculated snakes had a 40% mortality rate. All inoculated snakes had microscopic lesions unilaterally on the side of the swabbed nasolabial pit, including erosions to ulcerations and heterophilic dermatitis. All signs were consistent with SFD; however, the severity of lesions varied in individual snakes, and fungal hyphae were only observed in 3 of 5 inoculated snakes. These three snakes correlated with post-mortem tissue qPCR evidence of O. ophiodiicola. The findings of this study conclude that O. ophiodiicola inoculation in a cottonmouth snake model leads to disease similar to SFD, although lesion severity and the fungal load are quite variable within the model. Future studies may utilize this model to further understand the pathogenesis of this disease and develop management strategies that mitigate disease effects, but investigation of other models with less variability may be warranted.