Emydomyces testavorans Surveillance in Multiple Free-Ranging Terrestrial and Aquatic Chelonian Species in Illinois, USA.

The order Onygenales contains multiple fungal pathogens that affect free-ranging and zoo-housed reptilian species. Emydomyces testavorans, an onygenalean fungus associated with skin and shell disease, has been sporadically detected in aquatic chelonians. Because of the recent discovery of this organism, little is known about its prevalence in free-ranging chelonians. The objective of this study was to perform surveillance for E. testavorans in six free-ranging aquatic and terrestrial chelonian species in Illinois, USA: Blanding's turtles (n=437; Emydoidea blandingii), painted turtles (n=199; Chrysemys picta), common snapping turtles (n=35; Chelydra serpentina), red-eared sliders (n=62; RES; Trachemys scripta elegans), eastern box turtles (n=73; Terrapene carolina carolina) and ornate box turtles (n=29; Terrapene carolina ornata). Combined cloacal-oral swabs (COSs) or shell (carapace and plastron surfaces) swabs were collected from 2019 to 2021 and tested for E. testavorans using quantitative PCR. The PCR detected E. testavorans in COSs of an adult male, subadult female, and juvenile male Blanding's turtle (0.6%; 95% confidence interval [CI], 0.2-1.9%) and a shell swab from an adult female RES (1.6%; 95% CI, 0-8.7%). Shell lesions consistent with E. testavorans infection were present in two of the positive Blanding's turtles. These results document the rarity of this pathogen on the landscape in Illinois. Additional studies should determine this pathogen's impact on individuals and clarify its significance for conservation efforts of Blanding's turtle, in which E. testavorans has not been reported previously.

Evaluating Different Methods For Determining Erythrocyte Sedimentation Rate In Free-Living Blanding's Turtles (Emydoidea Blandingii).

Characterizing host factors affecting individual health can be important in the conservation of many chelonians, including the Blanding's turtle (Emydoidea blandingii). However, many routine clinical pathology tests do not reliably detect differences in health status in reptiles. The erythrocyte sedimentation rate (ESR) has been shown to characterize inflammation in many species, including tortoises, and may be useful in assessing Blanding's turtles. The purpose of our study was to evaluate two methods of ESR measurement in free-ranging Blanding's turtles. Sixty Blanding's turtles captured at two sites in Illinois received a complete physical examination, and blood was collected for determination of packed cell volume, total solids, and ESR with both microhematocrit tubes and commercial kits (Winpette). Method agreement was assessed with Passing-Bablok regression. Associations between ESR and demographic, environmental, and health factors were determined by linear regression. The microhematocrit tube method proportionately overestimated ESR compared with the Winpette. With the use of both measurement methods, ESR was significantly different between study sites, higher in adult Blanding's turtles, and higher in females that were gravid compared with those that were not. ESR was also negatively associated with packed cell volume. ESR values in Blanding's turtles are comparable to other reptiles, and although microhematocrit tube and Winpette results are not interchangeable, their associations with biological predictors are similar, and either method has the potential to be used with separate reference intervals to assess turtles for inflammatory conditions.

Plasma electrophoresis profiles of Blanding's turtles (Emydoidea blandingii) and influences of month, age, sex, health status, and location.

Baseline plasma electrophoresis profiles (EPH) are important components of overall health and may aid in the conservation and captive management of species. The aim of this study was to establish plasma protein fractions for free-ranging Blanding's turtles (Emydoidea blandingii) and evaluate differences due to age class (adult vs. sub-adult vs. juvenile), sex (male, female, or unknown), year (2018 vs. 2019), month (May vs. June vs. July), health status, and geographical location (managed vs. unmanaged sites). Blood samples were obtained from 156 Blanding's turtles in the summer of 2018 and 129 in 2019 at two adjacent sites in Illinois. Results of the multivariate analysis demonstrated that age class, sex, year, month, health status, and geographical location all contributed to the variation observed in free-ranging populations. Adult females had the highest concentration of many protein fractions, likely associated with reproductive activity. Juveniles had lower protein concentrations. Temperature and rainfall differences between years impacted concentrations between 2018 and 2019, while May and June of both years saw higher levels in some protein fractions likely due to peak breeding and nesting season. Individuals with evidence of trauma or disease also showed increased plasma protein fractions when compared to those that were considered healthy. The two sites showed a wide/large variation over the two years. All of these factors emphasize the importance of considering multiple demographic or environmental factors when interpreting the EPH fractions. Establishing ranges for these analytes will allow investigation into disease prevalence and other environmental factors impacting this endangered species.

Detection of a novel herpesvirus associated with squamous cell carcinoma in a free-ranging Blanding's turtle.

The spread of both infectious and noninfectious diseases through wildlife populations is of increasing concern. Neoplastic diseases are rarely associated with population-level impacts in wildlife; however, impacts on individual health can be severe and might reflect deteriorating environmental conditions. An adult male free-ranging Blanding's turtle (Emydoidea blandingii) originally captured in 2005 and deemed healthy, was recaptured in 2018 with a 1 × 1.5 cm intra-oral broad-based right mandibular mass. An excisional biopsy was performed, and histopathology revealed squamous cell carcinoma (SCC). Consensus herpesvirus PCR identified a novel herpesvirus (proposed name Emydoidea herpesvirus 2 [EBHV-2]) within the tumor. EBHV-2 shares 85% sequence homology with Terrapene herpesvirus 2 (TerHV-2), a herpesvirus linked to fibropapillomas in eastern box turtles (Terrapene carolina carolina). Virus-associated fibropapillomas have been identified in multiple marine turtle species and have had debilitating effects on their populations, but to date, virus-associated SCCs are rarely reported.

CHARACTERIZING THE EPIDEMIOLOGY OF HISTORIC AND NOVEL PATHOGENS IN BLANDING'S TURTLES (EMYDOIDEA BLANDINGII).

Pathogens such as herpesviruses, Mycoplasma spp., and frog virus 3-like ranavirus have contributed to morbidity and mortality in many species of free-living and zoo-maintained chelonians. However, their prevalence is understudied in Blanding's turtles (Emydoidea blandingii) across North America. To assess the presence of these pathogens, Blanding's turtles were sampled in Lake County, Illinois, in 2017 (N = 213) and 2018 (N = 160). DNA from cloacal-oral swabs was assayed for four ranaviruses, three Mycoplasma spp., two Salmonella spp., Emydoidea herpesvirus 1 (EBHV1), and tortoise intranuclear coccidiosis (TINC) using a multiplex quantitative polymerase chain reaction (qPCR). Pathogens were most frequently detected in adult turtles (n = 25) and rarely in subadults (n = 2) or juveniles (n = 1). EBHV1 was detected in 22 individuals with no clinical signs of illness, most (n = 20) occurring in the month of May (P < 0.0001). EBHV1 cases at one study site significantly clustered within the same 0.64-km area from 17 to 22 May 2017 (P < 0.0001) and 14 to 15 May 2018 (P = 0.0006). Individuals were rarely positive for Salmonella typhimurium (n = 6). A novel Mycoplasma sp. sharing high homology with other emydid Mycoplasma spp. was detected in one turtle with nasal discharge. Neither TINC nor any ranaviruses were detected. Continued monitoring of this population and habitat may facilitate identification of risk factors for pathogen occurrence and clarify the impact of infectious diseases on Blanding's turtle conservation outcomes.

Hematology and plasma biochemistries in the Blanding's turtle (Emydoidea blandingii) in Lake County, Illinois.

Chelonians are one of the most imperiled vertebrate taxa on the planet due to changes in the environment, anthropogenic influences, and disease. Over the last two decades, conservation strategies including nest protection, head-starting and meso-predator control have been successfully adopted by the Lake County Forest Preserve District for a population of state-endangered Blanding's turtles (Emydoidea blandingii) in Illinois. Only recently have efforts expanded to assess the effects of management action on turtle health. The objectives of this study were to 1) establish reference intervals for 16 hematologic and plasma biochemical analytes in free-ranging Blanding's turtles, 2) characterize demographic and temporal drivers of clinical pathology values including age class, sex, month, and year, and 3) describe bloodwork differences between a managed (SBCP) and unmanaged (IBSP) study site. Hematology and plasma biochemistries were performed for 393 turtles from 2017-18 at two sites in the Lake Plain region. Subject or population-based reference intervals were established based on the index of individuality per American Society for Veterinary Clinical Pathology guidelines. Analytes differed by age class [packed cell volume (PCV), total solids (TS), total white blood cell counts (WBC), heterophils, lymphocytes, heterophil:lymphocyte ratio (H:L), total calcium (Ca), calcium:phosphorous (Ca:P), bile acids (BA), aspartate aminotransferase (AST)], sex [H:L, Ca, phosphorus (P), Ca:P, creatine kinase (CK)], month [eosinophils, H:L, Ca, P, uric acid (UA), AST], and year [PCV, WBC, lymphocytes, basophils, H:L, Ca, P, UA]. Several analytes also varied by site [PCV, TS, monocytes, eosinophils, P, UA, AST], suggesting that health status may be affected by habitat management or lack thereof. The results of this study provide a baseline for ongoing health assessments in this region as well as across the Blanding's turtle range.

EPIDEMIOLOGY OF EMYDOIDEA HERPESVIRUS 1 IN FREE-RANGING BLANDING'S TURTLES (EMYDOIDEA BLANDINGII) FROM ILLINOIS.

Herpesvirus infections have been associated with high morbidity and mortality in populations of captive emydid chelonians worldwide, but novel herpesviruses have also recently been identified in apparently healthy free-ranging emydid populations. Blanding's turtle (Emydoidea blandingii), an endangered species in Illinois, has experienced range-wide declines because of habitat loss, degradation, and fragmentation. A novel herpesvirus, Emydoidea herpesvirus 1 (EBHV1), was identified in Blanding's turtles in DuPage County, IL, in 2015. Combined oral-cloacal swabs were collected from radio transmitter-fitted and trapped (n = 54) turtles multiple times over the 2016 activity season. In addition, swabs were collected at a single time point from trapped and incidentally captured (n = 84) Blanding's turtles in DuPage (n = 33) and Lake (n = 51) counties over the same field season. Each sample was tested for EBHV1 using quantitative polymerase chain reaction (qPCR). EBHV1 was detected in 15 adult females for an overall prevalence of 10.8% (n = 15/138; 95% confidence interval [CI]: 6.2-17.3%). In radio transmitter-fitted females, there was a significantly higher prevalence of EBHV1 DNA in May (23.8%, n = 10/42) than June (3.6%, n = 1/28), July (0%, n = 0/42), August (0%, n = 0/47), or September (7.7%, n = 3/39) (odds ratio: 12.19; 95% CI: 3.60-41.30). The peak in May corresponds to the onset of nesting and may be associated with increased physiologic demands. Furthermore, all positive turtles were qPCR negative in subsequent months. There were no clinical signs associated with EBHV1 detection. This investigation is the critical first step to characterizing the implications of EBHV1 for Blanding's turtle population health and identifying management changes that may improve sustainability.

MEASURING FAT CONTENT USING COMPUTED TOMOGRAPHY TO ESTABLISH A BODY CONDITION INDEX IN FREE-RANGING BLANDING'S TURTLES (EMYDOIDEA BLANDINGII) IN ILLINOIS.

Health assessment of free-ranging populations requires an integrated approach, often incorporating a method to measure mass as a representation of the animals' ability to utilize environmental resources. In chelonians, direct measurements of mass have historically served as a corollary for body condition. However, this method may not accurately represent the true fat volume (FV) and may be skewed by the presence of eggs, shell size, or muscle mass. The objective of this study was to use computed tomography (CT) to develop a model for determining body condition index (BCI) in free-ranging Blanding's turtles (Emydoidea blandingii). Mass, shell measurements, and FV were measured by CT in 65 free-ranging Blanding's turtles from Lake and DuPage counties in Illinois. Twenty-one different models were built for BCI using both FV and fat percentage (FP) as dependent variables. The best fit model for FP included the relationship between mass and carapace length with nearly 60% model support. The model for FV demonstrated a similar relationship but had only 18% support. Linear models with BCI as the dependent variable showed that juveniles had a higher FP than adults and females with more eggs had a lower FP. FP can be calculated in the field with nearly 60% accuracy compared to CT-assessed FP as a component of a physical exam and population health survey to assess the effectiveness of conservation efforts for the endangered Blanding's turtle.

Geographic coincidence of richness, mass, conservation value, and response to climate of U.S. land birds.

Distributional patterns across the United States of five avian community breeding-season characteristics--community biomass, richness, constituent species' vulnerability to extirpation, percentage of constituent species' global abundance present in the community (conservation index, CI), and the community's position along the ecological gradient underlying species composition (principal curve ordination score, PC--were described, their covariation was analyzed, and projected effects of climate change on the characteristics and their covariation were modeled. Higher values of biomass, richness, and CI were generally preferred from a conservation perspective. However, higher values of these characteristics often did not coincide geographically; thus regions of the United States would differ in their value for conservation depending on which characteristic was chosen for setting conservation priorities. For instance, correlation patterns between characteristics differed among Landscape Conservation Cooperatives. Among the five characteristics, community richness and the ecological gradient underlying community composition (PC) had the highest correlations with longitude, with richness declining from east to west across the contiguous United States. The ecological gradient underlying composition exhibited a demarcation near the 100th meridian, separating the contiguous United States grossly into two similar-sized avian ecological provinces. The combined score (CS), a measure of species' threat of decline or extirpation, exhibited the strongest latitudinal pattern, declining from south to north. Over -75% of the lower United States, projected changes in June temperature and precipitation to year 2080 were associated with decreased averaged values of richness, biomass, and CI, implying decreased conservation value for birds. The two ecological provinces demarcated near the 100th meridian diverged from each other, with projected changes in June temperatures and precipitation from the year 2000 to 2080 suggesting increased ecological dissimilarity between the eastern and western halves of the lower United States with changing climate. Anticipated climate-related changes in the five characteristics by 2080 were more weakly correlated with latitude or longitude then the responses themselves, indicating less distinct geographic patterns of characteristic change than in the characteristics themselves. Climate changes projected for 2080 included geographic shifts in avian biomass, CS, and PC values, a moderate overall decline in CI, and general decline in species richness per site.

Floral and nesting resources, habitat structure, and fire influence bee distribution across an open-forest gradient.

Given bees' central effect on vegetation communities, it is important to understand how and why bee distributions vary across ecological gradients. We examined how plant community composition, plant diversity, nesting suitability, canopy cover, land use, and fire history affected bee distribution across an open-forest gradient in northwest Indiana, USA, a gradient similar to the historic Midwest United States landscape mosaic. When considered with the other predictors, plant community composition was not a significant predictor of bee community composition. Bee abundance was negatively related to canopy cover and positively to recent fire frequency, bee richness was positively related to plant richness and abundance of potential nesting resources, and bee community composition was significantly related to plant richness, soil characteristics potentially related to nesting suitability, and canopy cover. Thus, bee abundance was predicted by a different set of environmental characteristics than was bee species richness, and bee community composition was predicted, in large part, by a combination of the significant predictors of bee abundance and richness. Differences in bee community composition along the woody vegetation gradient were correlated with relative abundance of oligolectic, or diet specialist, bees. Because oligoleges were rarer than diet generalists and were associated with open habitats, their populations may be especially affected by degradation of open habitats. More habitat-specialist bees were documented for open and forest/scrub habitats than for savanna/woodland habitats, consistent with bees responding to habitats of intermediate woody vegetation density, such as savannas, as ecotones rather than as distinct habitat types. Similarity of bee community composition, similarity of bee abundance, and similarity of bee richness between sites were not significantly related to proximity of sites to each other. Nestedness analysis indicated that species composition in species-poor sites was not merely a subset of species composition at richer sites. The lack of significant proximity or nestedness effects suggests that factors at a small spatial scale strongly influence bees' use of sites. The findings indicate that patterns of plant diversity, nesting resource availability, recent fire, and habitat shading, present at the scale of a few hundred meters, are key determinants of bee community patterns in the mosaic open-savanna-forest landscape.