Notes on the Courtship and Captive Reproduction of the Southern Vietnam Box Turtle, Cuora picturata, at the Turtle Conservation Center in Cuc Phuong National Park, Vietnam.

Endemic to only three known provinces in South Central Vietnam, Cuora picturata have faced extensive collection pressures for the food, medicinal, and pet trades. Further exacerbating their decline is the lack of protected areas where wild populations exist, with only one known population occurring within a protected area. With threats to wild populations persisting, the development of an assurance colony has been prioritized for C. picturata. The Asian Turtle Program of Indo-Myanmar Conservation has been operating the Turtle Conservation Center (TCC) in Cuc Phuong National Park since 1998. From 2020 to 2022, 24 C. picturata were acquired by the TCC from the illegal trade. With no ideal repatriation plan, these animals provided an opportunity for developing a captive assurance colony at the TCC to safeguard the species from extinction. As semi-aquatic species, box turtles in the genus Cuora are a unique group that shows variations in habitat use and behavior between species. Herein, we documented some specific courtship behaviors not yet described for C. picturata, providing insight into the unique reproductive behaviors of this species.

Ranavirus (Frog Virus 3) Infection in Free-Living Three-Toed Box Turtles (Terrapene mexicana triunguis) in Missouri, USA.

Frog virus 3 (FV3) and related ranaviruses are emerging infectious disease threats to ectothermic vertebrate species globally. Although the impact of these viruses on amphibian health is relatively well studied, less is understood about their effects on reptile health. We report two cases of FV3 infection, 11 mo apart, in three-toed box turtles (Terrapene mexicana triunguis) from a wildlife rehabilitation center. Case 1 had upper respiratory signs upon intake but had no clinical signs at the time of euthanasia 1 mo later. Case 2 presented for vehicular trauma, had ulcerative pharyngitis and glossitis, and died overnight. In case 1, we detected FV3 nucleic acid with qPCR in oral swabs, kidney, liver, spleen, and tongue. In case 2, we detected FV3 in an oral swab, an oral plaque, heart, kidney, lung, liver, spleen, and tongue. We also detected FV3 nucleic acid with in situ hybridization for case 2. For both cases, FV3 was isolated in cell culture and identified with DNA sequencing. Histopathologic examination of postmortem tissue from case 1 was unremarkable, whereas acute hemorrhagic pneumonia and splenic necrosis were noted in case 2. The difference in clinical signs between the two cases may have been due to differences in the temporal course of FV3 disease at the time of necropsy. Failure to detect this infection previously in Missouri reptiles may be due to lack of surveillance, although cases may also represent a novel spillover to box turtles in Missouri. Our findings reiterate previous suggestions that the range of FV3 infection may be greater than previously documented and that infection may occur in host species yet to be tested.

Anesthetic efficacy of dexmedetomidine-ketamine in eastern box turtles (Terrapene carolina carolina) is enhanced with the addition of midazolam and when administered in the forelimb versus the hindlimb.

OBJECTIVE: To compare dexmedetomidine-ketamine (DK; 0.1 and 10 mg/kg, respectively) with midazolam (M; 1.0 mg/kg) or 0.9% sodium chloride (S; 0.2 mL/kg) administered IM in the forelimb (F) or hindlimb (H) in eastern box turtles (Terrapene carolina carolina). ANIMALS: 20 clinically healthy, captive adult eastern box turtles. METHODS: In a randomized, blinded, complete crossover study with 1-week washout periods, turtles were administered each of 3 treatments: F-DKS, F-DKM, or H-DKM. Palpebral reflex, muscle tone, and withdrawal responses were serially assessed and used to calculate cumulative sedation scores at each 5-minute time point. The ability to intubate was evaluated. At 60 minutes, atipamezole (1.0 mg/kg) and either flumazenil (F-DKM, H-DKM; 0.05 mg/kg) or 0.9% sodium chloride (F-DKS; 0.5 mL/kg) were administered IM. RESULTS: All treatments resulted in clinically relevant anesthetic effects. F-DKM produced significantly higher sedation scores than H-DKM or F-DKS at all time points between 10 and 60 minutes (P < .05). Sedation score variability was observed with all treatments with significantly higher variability for H-DKM (P < .05). Intubation was successful in 32, 89, and 11% of turtles in F-DKS, F-DKM, and H-DKM, respectively. Median (range) recovery time was 10 (5-22), 16 (7-45), and 12 (4-28) minutes for F-DKS, F-DKM, and H-DKM, respectively. CLINICAL RELEVANCE: In eastern box turtles, forelimb dexmedetomidine-ketamine resulted in clinically relevant anesthetic effects that were heightened with the addition of midazolam. Hindlimb administration of midazolam-dexmedetomidine-ketamine resulted in reduced and more variable anesthetic effects compared to forelimb administration, supporting a hepatic first-pass effect.

Ecological and Fitness Correlates of Personality in a Long-lived Terrestrial Turtle.

An individual's behavioral tendencies (i.e., personality or temperament) can influence its interactions with the environment and thus have important ecological and evolutionary consequences for animal populations. Boldness, defined as an individual's tendency to engage in risk-taking activities, is a phenotypically variable trait linked with numerous behavioral and fitness outcomes in free-ranging animals. We examined variation and repeatability of boldness and other behavioral characteristics in two wild Eastern Box Turtle (Terrapene carolina carolina) populations using radiotelemetry, and assessed fitness correlates of boldness over multiple years. We observed large amounts of among-individual variation and within-individual consistency (i.e., repeatability) of boldness as measured by their head emergence latency following a standardized confinement assay. Individuals were also consistent in several in-field behaviors including movement rate, home range size, and date of emergence from overwintering refuges. Individuals with shorter head emergence latencies (i.e., bolder turtles) had larger home ranges, emerged earlier from overwintering dormancy, and experienced moderately lower survival compared with shy individuals. Boldness did not affect time spent within the thermal preference range, somatic growth rates, or the frequency of mating or same-sex aggressive encounters. Boldness and its effects on in-field behaviors differed between sexes and populations, and the relationship between boldness and survival was temporally variable. Our results suggest possible intrinsic behavioral types in T. c. carolina and highlight the importance of long-term and multipopulation studies when examining ecological and evolutionary processes that shape personality phenotypes in turtles.

Systemic Exophiala equina infection in an Eastern box turtle (Terrapene carolina carolina): a case report and literature review.

Phaeohyphomycosis is an infection caused by melanized fungi. This disease has been reported in several animal species including invertebrates, cold-blooded vertebrates, mammals, and humans. Melanized fungi have similar phenotypical features and confirmation requires culture and molecular diagnostics. To exemplify this we present a case of a 333 g adult of unknown age, free-ranging, male Eastern box turtle (Terrapene carolina carolina) that was referred to the Turtle Rescue Team at North Carolina State University for evaluation of multilobulated masses occupying the entire left orbit and at the right forelimb on the plantarolateral aspect of the foot. A fine needle aspirate cytologic examination of the mass on the right forelimb revealed large numbers of inflammatory cells and fungal organisms. Histopathology of the skin biopsies from the right forefoot was consistent with phaeohyphomycosis. A course of antifungal medication was started (Fluconazole 21 mg/kg loading dose IV then 5 mg/kg PO SID q 30 days). Due to concern for the patient's quality of life and the lack of a curative treatment plan, humane euthanasia was elected. Gross and histological postmortem examination confirmed the presence of multiple coelomic masses similar in appearance to those observed in the left orbit and right forefoot indicating disseminated phaeohyphomycosis. A swab of the periocular mass was submitted for fungal culture and phenotypic identification. The isolate was later identified as Exophiala equina through a combination of phenotypic characterization and sequencing of the ITS region of the nuclear rDNA. Exophiala is a genus in the family Herpotrichiellaceae, order Chaetothyriales and is considered an opportunistic "black yeast" causing infection in aquatic invertebrates, fish, amphibians, reptiles, and mammals including humans. Exophiala equina is infrequently reported in animals, with only three cases in the literature including the herein report.

Evaluation of lidocaine for brachial plexus blockade in eastern box turtles (Terrapene carolina carolina).

OBJECTIVE: To evaluate latency and duration of a brachial plexus block technique in eastern box turtles performed with 2% lidocaine at three dose rates. STUDY DESIGN: Prospective, randomized, blinded crossover study. ANIMALS: Adult eastern box turtles, two for drug dose evaluation and a group of six (three male, three female) weighing 432 ± 40 g (mean ± standard deviation) for the main study. METHODS: Animals were randomly assigned to four brachial plexus blocks with lidocaine at 5, 10 and 20 mg kg-1 or 0.9% saline (treatments LID5, LID10, LID20 and CON, respectively), separated by 1 week. Treatment side was randomized and blocks were performed unilaterally. Baseline observations of mentation, heart rate (HR), respiratory rate (fR), skin temperature and limb response to manipulation or toe pinch were evaluated. Assessments were made every 10 minutes until 1 hour of normal sensory and motor function to the treated thoracic limb, or for a total of 2 hours if no block was evident. RESULTS: Motor and sensory blockade was achieved in treatments LID10 and LID20 in one turtle, with a latency of 10 minutes and duration of 50 minutes for both doses. Raising of the ipsilateral lower palpebra occurred with both blocks. Turtles administered lidocaine experienced higher HR compared with CON, and HR decreased over time for all individuals. Mentation and fR were not changed with any lidocaine dose. CONCLUSIONS: The technique was unreliable in producing brachial plexus motor and sensory blockade at the lidocaine doses evaluated in this study. HR was higher in lidocaine-administered turtles but remained within normal limits for the species. No change in mentation or fR was observed among treatments. CLINICAL RELEVANCE: General anesthesia with systemic analgesia is recommended for surgical procedures involving the chelonian thoracic limb. Further studies are needed to optimize a brachial plexus block in this species.

Rapid and sensitive detection of box turtles using an electrochemical DNA biosensor based on a gold/graphene nanocomposite.

The Southeast Asian box turtle, Cuora amboinensis, is an ecologically important endangered species which needs an onsite monitoring device to protect it from extinction. An electrochemical DNA biosensor was developed to detect the C. amboinensis mitochondrial cytochrome b gene based on an in silico designed probe using bioinformatics tools, and it was also validated in wet-lab experiments. As a detection platform, a screen-printed carbon electrode (SPCE) enhanced with a nanocomposite containing gold nanoparticles and graphene was used. The morphology of the nanoparticles was analysed by field-emission scanning electron microscopy and structural characteristics were analysed by using energy-dispersive X-ray, UV-vis, and Fourier-transform infrared spectroscopy. The electrochemical characteristics of the modified electrodes were studied by cyclic voltammetry, differential pulse voltammetry (DPV), and electrochemical impedance spectroscopy. The thiol-modified synthetic DNA probe was immobilised on modified SPCEs to facilitate hybridisation with the reverse complementary DNA. The turtle DNA was distinguished based on hybridisation-induced electrochemical change in the presence of methylene blue compared to their mismatches, noncomplementary, and nontarget species DNA measured by DPV. The developed biosensor exhibited a selective response towards reverse complementary DNAs and was able to discriminate turtles from other species. The modified electrode displayed good linearity for reverse complementary DNAs in the range of 1 × 10-11-5 × 10-6 M with a limit of detection of 0.85 × 10-12 M. This indicates that the proposed biosensor has the potential to be applied for the detection of real turtle species.

Enhancing Chelonian Health Assessments by Determining Agreement in Pathogen Detection between Sampling Sites Using Quantitative PCR.

Pathogen surveillance is common in chelonians and multiple anatomical sampling sites are used for pathogen detection, but agreement between these sites has not been assessed. The objective of this study was to assess agreement between three sampling sites, oral swab (OS), cloacal swab (CS), and combined oral/cloacal swab (OCS), for detecting three pathogens in eastern box turtles (Terrapene carolina carolina). Box turtles (n=88) were assayed for Terrapene adenovirus 1 (TerADV1), Terrapene herpesvirus 1 (TerHV1), and Mycoplasmopsis sp. using quantitative PCR. Agreement between pathogen status based on sampling site was assessed using the Cohen kappa. Agreement was highest for Mycoplasmopsis sp. between OCS and OS (k=0.941), whereas moderate and minimal agreement were noted between OCS and CS (k=0.64) and OS and CS (k=0.538). For TerADV1, agreement was weak between OCS and OS (k=0.559), minimal between OS and CS (k=0.283), and absent between OCS and CS (k=0.204). TerHV1 agreement was moderate between OCS and OS (k=0.783) and absent between OCS and CS (k=0.106) and OS and CS (k=0.052). All pathogens were most frequently detected in OCS samples and DNA concentrations differed between sampling sites (P<0.0001). If testing multiple samples is not possible, OCS sampling improves the detection of these three pathogens over OS and CS alone.

Feasibility of a blind perineural injection technique for brachial plexus blockade in eastern box turtles (Terrapene carolina carolina): a cadaver study.

OBJECTIVE: To describe the anatomy of the brachial plexus in eastern box turtles (Terrapene carolina carolina), develop a blind perineural injection technique for brachial plexus blockade and evaluate the distribution of three volumes of new methylene blue dye for injection in cadavers. STUDY DESIGN: Prospective, randomized, blinded cadaveric study. ANIMALS: A total of 24 frozen-thawed box turtle cadavers; two turtles identified with shoulder injuries were subsequently excluded from the study. The remaining 22 turtles weighed 397 (190-581) g, median (range). METHODS: The brachial plexus and regional anatomy were identified by dissection of seven cadavers to determine anatomic landmarks for a perineural injection technique. This technique was tested by randomizing 15 cadavers into one of three groups to be injected bilaterally with one of three volumes (0.1, 0.2 or 0.3 mL) of methylene blue dye 1% aqueous solution. Investigators blinded to the assigned group dissected cadavers 15 minutes after injection and used staining of the four cervical spinal nerves (C5-C8; 25% for each nerve) to record a staining score of the brachial plexus (0-100%). RESULTS: Based on descriptions of the anatomy of the brachial plexus, an injection technique was designed. Injections of 0.1 mL methylene blue dye resulted in nine/10 injections with 100% nerve stained, and one/10 injection with 50% (two) nerves stained. All injections of 0.2 or 0.3 mL of methylene blue dye resulted in 100% nerves stained. CONCLUSIONS AND CLINICAL RELEVANCE: Perineural injection of the brachial plexus with 0.1, 0.2 or 0.3 mL methylene blue dye was successful in 29/30 injections in box turtle cadavers weighing 190-581 g. Further studies are needed to determine the minimum volume of injectate that can be successfully used for this technique, and to evaluate its application and efficacy in live turtles.

Overwintering behavior reduces mortality for a terrestrial turtle in forests managed with prescribed fire.

Prescribed fire is an essential management practice in pyrogenic ecosystems, but fire can also be a significant disturbance and source of mortality for both target and non-target species. Seasonal periods of animal inactivity may provide opportunities to design burn plans that minimize negative impacts to species of conservation concern, but few studies have rigorously examined this possibility. Using radiotelemetry, we studied overwintering behavior and interactions with fire in a forest-dwelling terrestrial turtle, the Eastern Box Turtle (Terrapene carolina carolina), over an eight-year period at two sites that use prescribed fire in forest management. Turtles at both sites selected predominantly hardwood forests and mesic habitats and avoided upland pine forests. Turtles buried deepest (2.9 - 3.2 cm) below the soil-litter interface in late February and then moved gradually shallower until emergence in early April. Emergence timing varied over a 58-day period, but was consistent within individuals from year to year. Turtles also maintained fidelity to refuge locations, but those overwintering in burned areas selected sites over twice as far from refuges used in previous years compared to those in unburned areas. The areas and habitats selected by turtles during winter served as refugia from fire, and those whose refuges did burn remained buffered from lethal temperatures even at shallow burial depths. The only fire-related injury or mortality occurred during seasons of surface activity. Timing burning and other forest management practices during periods of winter dormancy may thus minimize threats to turtle populations, but modifications to prescribed fire regimes must also be balanced with other management objectives.

Plasma complement activation mechanisms differ in ornate (Terrapene ornata ornata) and eastern box turtles (Terrapene carolina carolina).

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.

Development and validation of a quantitative PCR assay for detection of Terrapene herpesvirus 2 in eastern box turtles (Terrapene carolina carolina).

Herpesviruses are associated with disease in many chelonian species, resulting in pathology such as respiratory tract infection, stomatitis, conjunctivitis, hepatitis, and papillomatosis. Herpesvirus-associated fibropapillomas cause significant morbidity and mortality in marine turtles, and have been identified in an eastern box turtle (Terrapene carolina carolina) infected with Terrapene herpesvirus 2 (TerHV2). Further investigation is necessary to understand the impact of carcinogenic herpesviruses on chelonian health; however, reliable and specific methods for detection and quantitation of herpesviral load are lacking. The purpose of this study was to develop and validate a quantitative PCR assay for detection of TerHV2 in box turtles. TaqMan primer-probes were developed targeting the DNA polymerase gene. Inter- and intra-assay variability, linear range of detection, limit of detection, and specificity were assessed. The assay was highly specific for TerHV2, failing to amplify seven closely-related chelonian herpesviruses. It performed with high efficiency (slope = -3.52, R2 = 1, efficiency = 92.29 %), low intra-assay variability and low inter-assay variability (coefficient of variation ≤ 1.25 % at all standard dilutions). Reaction efficiency was not impacted in the presence of box turtle DNA from combined oral/cloacal swabs or whole blood. This qPCR assay has a linear range of detection from 107 to 101 viral copies per reaction and provides a valuable tool in the surveillance and characterization of TerHV2 in box turtles.

TEMPORAL PATTERNS IN ADMISSION OF EASTERN BOX TURTLES (TERRAPENE CAROLINA CAROLINA) AT A NORTH CAROLINA WILDLIFE CLINIC AS A REFLECTION OF CLIMATE.

Significant impacts of global climate change on wildlife have been documented and are projected to continue. Reptiles have been suggested as being especially susceptible to these effects along with other anthropogenic impacts on their environment. A retrospective review of medical records for 2,356 wild eastern box turtles (Terrapene carolina carolina) admitted to the Turtle Rescue Team (TRT) at the North Carolina State University College of Veterinary Medicine between 1996 and 2017 was performed in order to: 1) report common presenting complaints, 2) describe the timeline of when box turtles were admitted to the TRT for each year of the study, and 3) investigate temporal shifts in turtle admissions and associations with changes in environmental temperature over a 22-yr period. The most common presenting complaint was vehicular trauma (n = 1,195) with over 70% of the caseload associated with anthropogenic impacts. Average monthly temperatures from 1996 to 2017 for Raleigh, North Carolina, USA, were extracted from the National Oceanic and Atmospheric Administration climate database. By comparing the pre- and post-2006 years using the Mann-Whitney test, we found that both the annual peak temperature and the annual timing of admission to the TRT have shifted significantly or with a strong tendency towards significance (P = 0.0008 and 0.052, respectively). Annual peak temperature has increased by 1.3°C, and timing of admission has shifted 18 days earlier between pre- and post-2006 years. This supports the hypothesis that box turtle activity patterns are shifting and that these changes are potentially related to climate.

Hematology of the keeled box turtle (Cuora mouhotii).

A healthy adult, intact female keeled box turtle (Cuora mouhotii) was found to have a marked heterophilic leukocytosis using normal hematologic parameters established for the eastern box turtle (Terrapene carolina carolina), a related chelonian species. This animal was monitored with serial complete blood counts (CBCs) over the next 15 years despite remaining asymptomatic for an infectious condition. Retrospective CBC data were compiled from 38 presumably healthy keeled box turtles to establish hematologic values for comparison in this species. Using this species-specific data, over the 15-year period, the female keeled box turtle had two times where the white blood cell (WBC) count was greater than 2 standard deviations (SD) above the mean, six times where the WBC count was greater than 1 SD above the mean, six times where the PCV was greater than 2 SD above the mean, and eight times where the PCV was greater than 1 SD above the mean. Infection and inflammation are the most common causes of leukocytosis in reptiles; however, given the clinical presentation of this patient, it was postulated that these clinicopathologic changes could be secondary to a stress response. Establishing reference intervals and understanding how stress impacts CBC parameters are important for evaluating the health status of keeled box turtles kept in captivity and for assessing the effects of environmental changes on the health status of wild populations of this endangered chelonian species.

Plasma antibacterial activities in ornate (Terrapene ornata) and eastern box turtles (Terrapene carolina).

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.

PREVALENCE OF BOX TURTLE ADENOVIRUS IN EASTERN BOX TURTLES (TERRAPENE CAROLINA CAROLINA) PRESENTED TO A WILDLIFE REHABILITATION CENTER IN VIRGINIA, USA.

Eastern box turtles (Terrapene carolina carolina) are a native North American species with a declining population trend that may be attributable to habitat fragmentation, vehicle collisions, and disease. Adenoviral infections can cause significant morbidity and mortality in captive reptile populations. Adenoviruses have been documented in box turtles, but their occurrence and impact in wild populations are unknown. A disease survey was performed at The Wildlife Center of Virginia, USA, to assess the prevalence of box turtle adenovirus (BTAdV) in wild eastern box turtles and evaluate potential associations with clinical disease. Swabs from the oral cavity, including the choanal slit, and the cloaca were collected from 106 eastern box turtles from July 2015 through June 2016. The quantitative polymerase chain reaction (qPCR) primer detected both ornate box turtle adenovirus 1 and eastern box turtle adenovirus. The resulting qPCR adenovirus prevalence was 55.7% (n = 59). Most animals (99.3%) that tested positive for BTAdV had fewer than 100 viral copies/ng DNA. This study did not find a statistically significant association between cause of admission, age, sex, outcome, and BTAdV qPCR status. However, the probability of BTAdV detection was 1.5 times higher in rehabilitation turtles compared with wild turtles (P = 0.01). Albumin was significantly lower in qPCR BTAdV-positive turtles (P = 0.007). Hypoalbuminemia is not generally associated with adenovirus infections in other species, and no obvious clinical cause for this abnormality was identified. The results of this study suggest that eastern box turtles may harbor BTAdV infections at low levels and that infection is rarely associated with clinical disease, potentially identifying BTAdV as a host-adapted pathogen. Future studies should focus on this pathogen's ability to induce clinical disease and its potential impact on recovery efforts for this species.

Variation and repeatability of home range in a forest-dwelling terrestrial turtle: implications for prescribed fire in forest management.

Animal movements and use of space are in part determined by interactions between individual attributes such as sex and body size and extrinsic environmental factors such as the seasonal availability, quality and spatial configuration of resource patches in the landscape. Fire is a common and widespread disturbance process that has the potential to affect animal movements through modifications to the environment. Using radiotelemetry, we examined the contribution of these factors to variation in movements and home range over a 5-year period in a forest-dwelling terrestrial turtle, Terrapene carolina, at fire-maintained and unburned habitats in the southeastern United States. Female turtles had annual home-range sizes twice as large as males and moved longer distances per day during the nesting season (June and July), but males exhibited greater spatial fidelity from year to year. Turtles at the unburned site had home-range sizes twice as large as those at the fire-maintained site, and home-range size also decreased with increasing frequency and extent of fire, but this latter effect was strongest in females. Home-range behavior was highly repeatable within individuals of both sexes over time. This is the first evidence that fire influences the spatial ecology and movements of turtles, most likely through fire's impact on the spatial configuration, availability and quality of critical resources. That individuals behaved consistently through time, but differently from one another according to both intrinsic individual attributes and extrinsic environmental factors provides strong evidence of consistent inter- and intra-population variation in space use and movement behaviors in T. carolina. Such intra-specific behavioral variation suggests applying caution when extrapolating results to other sites across the geographic range of a species for use in conservation and management.

TISSUE ENZYME ACTIVITIES IN FREE-LIVING EASTERN BOX TURTLES (TERRAPENE CAROLINA CAROLINA).

Plasma biochemical enzymes are commonly assayed as part of a diagnostic evaluation for zoological species, but their interpretation is complicated by a lack of knowledge about tissue of origin in many reptiles. This study evaluated tissue specificity of six biochemical enzymes (alanine aminotransferase [ALT], aspartate aminotransferase [AST], alkaline phosphatase [ALP], creatine kinase [CK], gamma-glutamyl transferase [GGT], and glutamate dehydrogenase [GLDH]) in 10 tissues (skeletal muscle, cardiac muscle, lung, liver, gallbladder, pancreas, gastrointestinal tract, kidney, spleen, and reproductive tract) from 10 free-living eastern box turtles (Terrapene carolina carolina). CK activity was highest in skeletal muscle, cardiac muscle, and gastrointestinal tract; GLDH and ALT activities were highest in liver, kidney, and gallbladder; ALP and GGT activities were elevated in kidney and gastrointestinal tract; and AST was relatively nonspecific, with significantly higher activity in the cardiac muscle, liver, kidney, skeletal muscle, and gallbladder compared to other tissues (P < 0.05). These results serve as a first step toward improving clinical interpretation of plasma biochemistry panels in box turtles.

DETECTION OF MYCOPLASMA SP. IN INDOCHINESE BOX TURTLES (CUORA BOURRETI).

Mycoplasma species are important pathogens of captive and free-ranging chelonians. Bourret's box turtle (Cuora bourreti) is a critically endangered species of Indochinese box turtle in the family Geoemydidae. Four privately owned wild-caught Bourret's box turtles were presented for clinical evaluation for anorexia and lethargy following shipment from a reptile wholesaler 3 wk prior. Choanal-cloacal swabs of two of the turtles were positive for Mycoplasma sp. by polymerase chain reaction. Sequencing of the 16S rRNA gene and 16S-23S rRNA intergenic spacer was 99% homologous to an unclassified Mycoplasma sp. previously documented in free-ranging and captive North American species of the family Emydidae. The potential of Mycoplasma sp. to induce disease in Bourret's box turtles is unknown. Global trade in live reptiles is believed to have facilitated this potential expansion of host range.

Graphene oxide and gold nanoparticle based dual platform with short DNA probe for the PCR free DNA biosensing using surface-enhanced Raman scattering.

Surface-enhanced Raman scattering (SERS) based DNA biosensors have considered as excellent, fast and ultrasensitive sensing technique which relies on the fingerprinting ability to produce molecule specific distinct spectra. Unlike conventional fluorescence based strategies SERS provides narrow spectral bandwidths, fluorescence quenching and multiplexing ability, and fitting attribute with short length probe DNA sequences. Herein, we report a novel and PCR free SERS based DNA detection strategy involving dual platforms and short DNA probes for the detection of endangered species, Malayan box turtle (MBT) (Cuora amboinensis). In this biosensing feature, the detection is based on the covalent linking of the two platforms involving graphene oxide-gold nanoparticles (GO-AuNPs) functionalized with capture probe 1 and gold nanoparticles (AuNPs) modified with capture probe 2 and Raman dye (Cy3) via hybridization with the corresponding target sequences. Coupling of the two platforms generates locally enhanced electromagnetic field 'hot spot', formed at the junctions and interstitial crevices of the nanostructures and consequently provide significant amplification of the SERS signal. Therefore, employing the two SERS active substrates and short-length probe DNA sequences, we have managed to improve the sensitivity of the biosensors to achieve a lowest limit of detection (LOD) as low as 10 fM. Furthermore, the fabricated biosensor exhibited sensitivity even for single nucleotide base-mismatch in the target DNA as well as showed excellent performance to discriminate closely related six non-target DNA sequences. Although the developed SERS biosensor would be an attractive platform for the authentication of MBT from diverse samples including forensic and/or archaeological specimens, it could have universal application for detecting gene specific biomarkers for many diseases including cancer.