The innate immune system serves as a critical first line of defense in reptiles, particularly under the energetic constraints of ectothermy. This study characterizes complement-mediated innate immune responses in the diamondback terrapin (Malaclemys terrapin), an estuarine turtle species facing increasing environmental and disease pressures. We evaluated the antibacterial capacity of terrapin plasma against Aeromonas hydrophila, Vibrio vulnificus, Staphylococcus aureus, and Streptococcus pyogenes and assessed complement activity through sheep red blood cell (SRBC) hemolysis assays. Plasma showed strong, concentration-dependent antibacterial activity, with complete inhibition of S. aureus at 100% plasma and substantial inhibition of the other species at ≥25%. Hemolytic activity was rapid, occurring primarily within 10 min, and peaked at 25°C–30°C. Complement activity was abolished by heat, protease, and EDTA treatments and restored with divalent cations, indicating a heat-labile, protease-sensitive mechanism consistent with the alternative complement pathway. Mannan affinity chromatography and SDS-PAGE revealed two major lectins (~25.8 and ~33.4 kDa), consistent with known complement-associated lectins in other emydid turtles. These findings demonstrate that diamondback terrapins possess robust, complement-mediated innate immune responses, potentially shaped by evolutionary exposure to estuarine pathogens like Vibrio spp., and support their use in comparative eco-immunological research and conservation health assessments.

Hematologic assessment is a common clinical tool used to characterize both individual and population health. In ectothermic organisms, the hematologic response is influenced by external factors such as temperature and season, especially in temperate species. Yet, specific environmental effects have received little direct attention. Therefore, to improve the diagnostic utility of hematologic assessments in reptiles, it is imperative to characterize physiological variation in diagnostic parameters. Our objective was to determine the impacts of temperature and season on commonly assessed components of the complete blood count, including packed cell volume (PCV), total solids (TS) and white blood cell count (WBC). We used a group of 17 long-term captive Prairie Rattlesnakes (Crotalus viridis). Animals were randomly assigned to either a control group kept in an environmental chamber at a constant temperature of 25°C or an experimental group housed in an environmental chamber which reflected average ambient temperature throughout the year (5°C-32°C). We collected blood samples twice monthly for 13 months. We found WBC decreased as the mean previous 14-day temperature increased, with no effect of season. Changes in total WBC were driven by increased numbers of lymphocytes and decreased number of heterophils and azurophils at higher temperatures. We found a difference in the effect of time since the start of the experiment between control and experimental groups in PCV, with no change in our control group, but a decrease in our experimental group through time. Total solids were higher in the control group, but there was no direct effect of temperature or season. Therefore, we conclude environmental conditions, rather than season, drive variation in hematologic parameters of otherwise healthy snakes and should be included in the interpretation of hematologic assessments in wild reptiles.

Ectotherms are particularly vulnerable to climate change because they rely on the surrounding environment for thermoregulation. In snakes, prolonged warmer temperatures and precipitation changes may lead to faster growth and smaller body size. Our goal was to assess how climate change may impact future sizes and growth rates on an endangered pitviper species as a model organism. We hypothesized that climate change-induced shifts in growing season temperature, early growing season precipitation, and snow residence time (SRT) would impact growth rate and asymptotic size in the threatened Eastern Massasauga (Sistrurus catenatus) rattlesnake. We used capture-mark-recapture data from eleven sites across the species’ range to fit a set of non-linear models evaluating the effects of these three climate variables on asymptotic size and growth coefficient in a modified von Bertalanffy growth curve. We found that longer SRT resulted in larger asymptotic size. We also found support for both a negative relationship between SRT and growth rate and a positive relationship between precipitation and growth rate. We averaged all supported models and predicted growth and size in the 2080s under a stabilization (RCP 4.5) and a high (RCP 8.5) emission scenario. Under RCP 8.5, predicted increases in female growth rate ranged from 10% to 33%, whereas decreases in female size across sites ranged from 9% to 17%. Under this scenario female lifetime reproductive success will decrease, as faster growth is associated with increased mortality, early senescence, and poor offspring quality, and smaller body sizes will result in smaller and fewer young.

Ophidiomycosis, caused by the fungus Ophidiomyces ophidiicola, poses a threat to the health of wild and managed snakes worldwide. Variation in snake innate immunity, the primary defense against infection in reptiles, may explain the observed variation in ophidiomycosis clinical disease severity among snakes. In this study, two components of the innate immune response were examined in snake plasma. We investigated whether complement activity, as measured by sheep red blood cell hemolysis, and chitotriosidase activity were associated with ophidiomycosis disease severity and time in captivity in Lake Erie watersnakes (Nerodia sipedon insularum). There was no difference in complement-mediated hemolysis or chitotriosidase activities between snakes with varying levels of ophidiomycosis clinical severity sampled in the field. However, among snakes with skin lesions kept in captivity, chitotriosidase activity was significantly higher in snakes with mild disease, compared with snakes with severe disease, and hemolysis activity increased with time in captivity. Overall, Lake Erie watersnakes had higher complement activity, but lower chitotriosidase activity, compared with other reptile species. To our knowledge, this study is the first to describe chitotriosidase activity in a snake species. These results provide mixed evidence of associations between innate immune function and ophidiomycosis severity, and more work is needed to investigate differences among snake species.

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.

Abstract. Treatment of bacterial cultures with hemolymph collected from desert hairy scorpions (Hadrurus arizonensis Ewing, 1928, Hadruridae) resulted in a time- and concentration-dependent inhibition of bacterial proliferation. The hemolymph proved effective in inhibiting growth of both Gram-negative and Gram-positive bacterial species. Incubation of E. coli bacteria with hemolymph at different temperatures (20–40°C) showed that the antibacterial effects increased from 20–30°C, but the hemolymph was largely ineffective in killing the bacteria at 35 and 40°C. Incubation of E. coli with hemolymph at 25°C for different time periods revealed that the antibacterial activities were extremely rapid and resulted in killing of bacteria within 1–2 minutes of contact. Interestingly, the hemolymph exhibited no phenoloxidase enzyme activity, hemolytic activity against sheep red blood cells, or melanization activity, which is a common mechanism of immunity among many diverse arthropods. This study is the first characterization of immune function of hemolymph from any scorpion species.

Environmental DNA represents a potentially cost-effective method for detection of rare and cryptic species, for which traditional sampling can be logistically costly. We tested species-specific probe-based assays to detect eDNA for the federally threatened eastern massasauga rattlesnake and the causative agent of snake fungal disease, Ophidiomyces ophiodiicola . We collected water samples from 100 crayfish burrows (over-wintering refugia) during spring emergence and screened them for snake and fungal DNA. We detected snake DNA in two samples and failed to detect O. ophiodiicola DNA. Given the high density of both target species at the sites, we suggest these methods may not offer a logistical and financial improvement over traditional sampling protocols.

Background Plasma phospholipase A 2 (PLA 2 ) enzyme activity is a key component of innate immunity in most vertebrates. We evaluated circulating secreted PLA 2 activity of prairie rattlesnakes ( Crotalus viridis ) by incubation of plasma with bacteria labeled with fluorescent membrane lipids. Results Incubation of bacteria with increasing volumes of plasma resulted in volume-dependent lysis of fatty acids from bacterial membranes. The activity was rapid, with substantial activity recorded after only 5 min of incubation with labeled bacteria, and a linear response for 20 min. In addition, the lysis activity was temperature-dependent, increasing activities from 5 to 20 °C, peak activities at 25–30 °C, and then decreasing activities from 35 to 40 °C. Furthermore, the activity was inhibited in a concentration-dependent manner by p -bromophenacyl bromide, a specific inhibitor of PLA 2 activity, which indicated that the observed activities were due to the presence of PLA 2 in the plasma of C. viridis . Conclusions This study represents the first description of secretory PLA 2 activity in the plasma of a snake. Our study shows that in addition to being an important component of snake venom, PLA 2 enzymes play an important role in the snake’s immune response.

Eastern (Terrapene carolina carolina) and ornate (Terrapene ornata ornata) box turtles have robust plasma antibacterial activity, however, the mechanism behind this activity is unknown. We used sheep red blood cell (SRBC) hemolysis assays, mannan-affinity chromatography, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) to explore the mechanisms of complement activity in box turtles. Plasma from both species demonstrated volume, time, and temperature-dependent SRBC hemolysis, with significantly greater hemolytic activity in ornate box turtle plasma. Hemolytic activity was highly attenuated following treatment with heat, EDTA, and salicylaldoxime in both species, but was unchanged after treatment with methylamine and ammonium hydroxide. Two abundant mannan-binding proteins (presumed C-type lectins) were identified in eastern box turtle plasma using SDS-PAGE and MALDI-TOF, but ornate box turtles did not express either protein. Eastern box turtles appear to rely on the lectin pathway of complement activation while ornate box turtles utilize the alternative pathway. This study provides further evidence that mechanisms underlying immune function are not always conserved between closely related species. This finding may have important implications for explaining species differences in susceptibility to emerging threats such as disease, toxicants, and climate change.

Chelonians are one of the most imperiled vertebrate taxa and many species are increasingly threatened by disease, however, the immune response in this group is understudied. We quantified the innate immune response of eastern (Terrapene carolina; EBT) and ornate (Terrapene ornate; OBT) box turtles using plasma antibacterial activity assays. Plasma from both species abolished or significantly reduced the growth of all eight bacterial species evaluated, including Salmonella typhimurium, Escherichia coli, Enterobacter cloacae, Citrobacter freundi, Bacillus subtilis, Staphylococcus epidermidis, and Staphylococcus aureus. Bactericidal capacity was greater in OBT compared to EBT, and OBT plasma retained high antibacterial activities at a broader temperature range (20-40°C) compared to EBT (30-40°C). Plasma antibacterial activity was abolished following treatment with heat, protease, and ethylenediaminetetraacetic acid, indicating that complement is likely responsible for the observed effects. Further characterization of the box turtle immune response may provide insight into the importance of infectious diseases for species conservation, enabling the development of more efficient and effective population management strategies.

Population surveying and monitoring are important for identifying conservation needs and tracking trends in populations, communities, and ecosystems over time and laying the groundwork for conservation management and policy decisions. If species or populations go undetected because of inadequate effort or sampling design, protection and management cannot be properly provided. Due to the widespread loss of populations, the Eastern Massasauga (a rattlesnake) was recently listed as a federally threatened species in the United States; it is also listed as threatened in Canada. Given its current conservation status, there is considerable interest at state and federal levels in determining how to best survey for Eastern Massasaugas to aid in management decisions. Using a 16-year dataset, we examined the relationships among environmental, temporal, area, management, and search effort factors on the detection probability of Eastern Massasaugas. We found that four abiotic parameters (solar irradiance, shaded air temperature, three-day maximum air temperature, and humidity) and three search parameters (effort per researcher, search area, and search time of day) influenced detection of Eastern Massasaugas. As the current biodiversity crisis continues, the cost-effective use of resources and scientific expertise will continue to increase in importance. We hope our results stimulate similar analyses in other taxa, which will be critical for designing and implementing regional survey and monitoring programs.

The acute phase response is a highly conserved reaction to infection, inflammation, trauma, stress, and neoplasia. Acute phase assays are useful for wildlife health assessment, however, they are infrequently utilized in reptiles. This study evaluated erythrocyte sedimentation rate (ESR) in eastern (Terrapene carolina carolina) and ornate box turtles (Terrapene ornata ornata) and hemoglobin-binding protein (HBP) in T. ornata. Erythrocyte sedimentation rate in 90 T. carolina and 105 T. ornata was negatively associated with packed cell volume and was greater in unhealthy turtles (p < 0.05). Female T. ornata had higher ESR values than males (p < 0.05). Measurement of ESR with a microhematocrit tube proportionally overestimated values from a commercial kit (Winpette), though both methods may retain utility with separate reference intervals. Hemoglobin-binding protein concentration in 184 T. ornata was significantly increased in adults and unhealthy turtles (p < 0.05). Erythrocyte sedimentation rate values were similar between seasons and populations, and HBP values were consistent between years, indicating that these analytes may have more stable baseline values than traditional health metrics in reptiles. This study demonstrates that ESR and HBP are promising diagnostics for health assessment in wild box turtles. Incorporating these tests into wild herptile health assessment protocols may support conservation efforts and improve ecosystem health monitoring.

Reptiles are declining worldwide yet our understanding of their immune function lags far behind other taxa. The innate immune system is the primary mode of defense in reptiles, and the serum complement cascade is its major component. We assessed serum complement activity of plasma in two closely related aquatic turtle species, the common snapping turtle (CST; Chelydra serpentina) and alligator snapping turtle (AST; Macrochelys temminckii). We used a sheep red blood cell (SRBC) hemolysis assay to assess serum complement activity. Although the antibacterial activities of the plasma of these turtle species are similar, the hemolytic activity was much stronger in CST than AST. Treatment with inhibitors of the serum complement cascade indicated differences in the mechanisms of complement activation between the turtle species. We subjected plasma from both turtle species to mannan affinity chromatography and analyzed the eluate with SDS-PAGE, which revealed that plasma from the CSTs contained only small amounts of one C-type lectin protein while the AST plasma contained high concentrations of two C-type lectins (31.0 and 35.9 kDa). Edman degradation analyses confirmed that the two AST proteins contained identical N-terminal sequences. Thus, the CST appears to rely more heavily on the alternative mechanism of serum complement activation, while the AST appears to rely more on the lectin-mediated pathway, which is a pattern recognition response to prokaryotes not activated by the SRBCs. These results are unique in that the use of serum complement pathways are generally assumed to be conserved within clades.

Ophidiomycosis, historically referred to as snake fungal disease (SFD), caused by Ophidiomyces ophiodiicola, is a significant disease of snakes characterized by crusty scales, pustules, subcutaneous nodules, and death. Ophidiomycosis is a proposed threat to sustainability of free-ranging snake populations throughout the United States and Europe, but the clinical progression during periods of reproductive activity (gravid females, neonates) is unknown. In spring 2012, five apparently healthy gravid eastern massasauga (Sistrurus catenatus) rattlesnakes from Clinton County, Illinois, were brought into captivity to give birth and be returned into the population. While in captivity, one adult female and 21 neonates died. Five individuals were subsequently confirmed positive for O. ophiodiicola by using quantitative polymerase chain reaction (qPCR). In 2016, a gravid timber rattlesnake (Crotalus horridus) with ophidiomycosis from Jackson County, Illinois, gave birth in captivity to 13 neonates. Skin swabs were taken from all neonates immediately after birth and confirmed negative for O. ophiodiicola by using qPCR. The neonates remained housed with the positive female for 10 days before all animals were reswabbed and released back into the wild. One neonate was O. ophiodiicola positive at time of release. The initial negative result followed by a positive result several days postpartum suggests that the neonate was infected by the female after direct contact. Both case series represent natural infection of neonates after parturition and highlight the importance of this disease in a demographically important age class.

Snake Fungal Disease (SFD), caused by Ophidiomyces ophiodiicola, is the most recently described fungal disease afflicting wildlife populations across North America and Europe. It has been proposed as a significant conservation threat yielding high mortality and yet much its ecology is unknown. We collected 144 skin swabs from Eastern Massasaugas (Sistrurus catenatus) in 2015 and 2016 to determine document ongoing prevalence and assess differences in microbial assemblages between positive and negative individuals. Alpha diversity of fungi was reduced in SFD positive animals, while beta diversity identified distinct assemblages of microbes between SFD-positive and -negative samples. Ophidiomyces was present on the skin of affected animals, even on body sites distant to lesions indicating that the microbiome on entire surface of the skin is altered. Ophidiomyces was not detected in any non-SFD snake. There were smaller, but significant, influences of year sampled. Bacterial genera Janthinobacterium and Serratia were significantly increased in SFD snakes, while Xylanimicrobium, Cellulosimicrobium, and Rhodococcus were the only bacterial taxa significantly reduced. The relative abundance of fungi within the orders Pleosporales and Canopdiales was reduced in SFD-positive samples, though Pyrenochaetopsis pratorum was the only species found to differ significantly. This is the first study to determine the impact that this fungal pathogen has on the skin microbiome.

Innate immunity provides a fast-acting and nonspecific defense against microbial infection, and appears to have particular importance in the immune response of ectothermic vertebrates. Chelonians are a globally distributed and diverse group, yet little is known about their basic immune function. The chelonian family Chelydridae is made up of two genera (Chelydra and Macrochelys), represented in our study by the widespread common snapping turtle ( Chelydra serpentina; CST) and the southeast USA endemic alligator snapping turtle ( Macrochelys temminckii; AST). Our goal was to quantify the innate immune response of the family Chelydridae, using the antibacterial activity of plasma as a measure of immune function. Our results show that the plasma of both species has strong antibacterial properties, but CST plasma kills a higher percentage of bacteria than AST plasma. In addition, while both species showed the highest antibacterial activity at 25 to 30°C, CST plasma retained its antibacterial properties at lower and higher temperatures than AST plasma. Our results indicate that, like many ectotherms, Chelydridae have a relatively strong innate immune response. The stronger, more robust immune response of CSTs compared with ASTs is likely correlated to the differences in geographic ranges but may also have implications for each species' tolerance to anthropogenic habitat degradation and global climate change.