A novel bioaugmentation technique effectively increases the skin-associated microbial diversity of captive eastern hellbenders.

Captive environments are maintained in hygienic ways that lack free-flowing microbes found in animals' natural environments. As a result, captive animals often have depauperate host-associated microbial communities compared to conspecifics in the wild and may have increased disease susceptibility and reduced immune function. Eastern hellbenders (Cryptobranchus alleganiensis alleganiensis) have suffered precipitous population declines over the past few decades. To bolster populations, eastern hellbenders are reared in captivity before being translocated to the wild. However, the absence of natural microbial reservoirs within the captive environment diminishes the diversity of skin-associated bacteria on hellbender skin and may negatively influence their ability to defend against pathogenic species once they are released into the wild. To prepare hellbenders for natural bacteria found in riverine environments, we devised a novel bioaugmentation method to increase the diversity of skin microbial communities within a captive setting. We exposed juvenile hellbenders to increasing amounts of river water over 5 weeks before translocating them to the river. We genetically identified and phylogenetically compared bacteria collected from skin swabs and river water for alpha (community richness) and beta (community composition) diversity estimates. We found that hellbenders exposed to undiluted river water in captivity had higher alpha diversity and distinct differentiation in the community composition on their skin, compared to hellbenders only exposed to well water. We also found strong evidence that hellbender skin microbiota is host-specific rather than environmentally driven and is colonized by rare environmental operational taxonomic units in river water. This technique may increase hellbender translocation success as increasing microbial diversity is often correlated with elevated disease resistance. Future work is necessary to refine our methods, investigate the relationship between microbial diversity and hellbender health and understand how this bioaugmentation technique influences hellbenders' survival following translocation from captivity into the wild.

Captivity-Induced Changes in the Skin Microbial Communities of Hellbenders (Cryptobranchus alleganiensis).

Variation in environmental conditions can result in disparate associations between hosts and microbial symbionts. As such, it is imperative to evaluate how environmental variables (e.g., habitat quality) can influence host-associated microbiome composition. Within wildlife conservation programs, captive conditions can negatively influence the establishment and maintenance of "wild-type" microbiotas within a host. Alternative microbial communities can result in the proliferation of disease among captive stock or upon reintroduction. Hellbenders (Cryptobranchus alleganiensis) are a threatened salamander for which extensive captive management is currently employed. Using metabarcoding, we characterized the skin microbiota of wild and captive hellbenders from two subspecies in the state of Missouri, the eastern (C. a. alleganiensis) and the Ozark hellbender (C. a. bishopi). Both subspecies in our study included wild adults and captive juveniles that were collected from the wild as eggs. Our objectives were to investigate differences in the skin microbial communities' richness/diversity, composition, and functional profiles of microbes between wild and captive individuals. Captive eastern hellbenders possessed richer communities than wild cohorts, whereas the opposite pattern was observed within the Ozark subspecies. We found significant microbial community structure between wild and captive populations of both subspecies. Microbiota structure translated into differences in the predicted metagenome of wild and captive individuals as well. As such, we can expect captive hellbenders to experience alternative microbial structure and function upon reintroduction into the wild. Our study provides a baseline for the effect of captivity on the skin microbial communities of hellbenders, and highlights the need to incorporate microbiota management in current captive-rearing programs.

Influence of immunogenetics, sex and body condition on the cutaneous microbial communities of two giant salamanders.

The complex association between hosts and microbial symbionts requires the implementation of multiple approaches to evaluate variation in host physiology. Within amphibians, heterogeneity in immunogenetic traits and cutaneous microbiota is associated with variation in disease resistance. Ozark (Cryptobranchus alleganiensis bishopi) and eastern hellbenders (C. a. alleganiensis) provide a model system to assess variation in host traits and microbial communities. Ozark hellbenders have experienced declines throughout their range, are federally endangered and experience wound retardation that is absent in the eastern subspecies. Previous microbial investigations indicate differentiation in the composition of the skin microbiota of both hellbender subspecies, but it is not clear whether these patterns are concurrent with diversity in the major histocompatibility complex (MHC) genes. We characterized the MHC IIB and the skin microbiota of hellbenders in Missouri, where both subspecies co-occur though not sympatric. We compared the microbiota composition and MHC diversity between both subspecies and investigated whether individual-level MHC diversity, sex and body condition were associated with microbiota composition. Overall, MHC IIB diversity was lower in Ozark hellbenders compared to the eastern subspecies. Multivariate statistical comparisons identified microbiota differentiation between Ozark and eastern hellbenders. MHC IIB allele presence/absence, allele divergence, body composition and sex defined grouping of hellbender microbiotas within populations. Differentiation of the cutaneous microbiotas and MHC IIB genes between eastern and Ozark hellbenders suggests that differences exist in immunity between the two subspecies. This study demonstrates how simultaneous assessments of host genetic traits and microbiotas can inform patterns of microbial community structure in natural systems.

Cutaneous Microbial Community Variation across Populations of Eastern Hellbenders (Cryptobranchus alleganiensis alleganiensis).

Multicellular hosts maintain complex associations with microbial communities. While microbial communities often serve important functional roles for their hosts, our understanding of the local and regional processes that structure these communities remains limited. Metacommunity analyses provide a promising tool for investigating mechanisms shaping microbiome heterogeneity, which is essential for predicting functional variation between hosts. Using a metacommunity framework, we examined heterogeneity in the skin microbiome of the eastern hellbender (Cryptobranchus alleganiensis alleganiensis). Hellbenders are broadly distributed throughout river systems in the eastern United States, but are present in specific environmental locations throughout their range. The large range of the species and history of population fragmentation suggest that local and regional processes contribute to the distribution of cutaneous symbiont diversity. Therefore, we characterized the skin and environmental bacterial communities at eight rivers throughout the range of the species. We observed variation among hellbender populations in skin microbial community diversity and proportion of shared operational taxonomic units (OTUs) between animal and river water communities. Among populations sampled, we noted significant clumped OTU turnover (i.e., Clementsian structure) resulting in unique cutaneous communities. In addition, we observed a significant positive correlation between skin community divergence and hellbender population genetic divergence. Host-population skin community dissimilarity did not correlate strongly with distance between sampling locations, indicating a weak spatial effect on the distribution of symbionts. These results suggest that species sorting mechanisms (i.e., local processes) structure local skin microbial communities in hellbenders. The variation in skin community composition observed among host populations foreshadows a similar pattern in important functional characteristics (e.g., resistance to dysbiosis). Future work should focus on investigating forces shaping microbiome structure in eastern hellbenders, examining functional variation among populations, and evaluating effectiveness of microbiome management recommendations.

A Severe Ranavirus Outbreak in Captive, Wild-Caught Box Turtles.

A Ranavirus outbreak in a captive population of wild-caught individuals was monitored using clinical evaluations and real-time PCR in 317 wild box turtles held in captivity during translocation. During the 2-year study period, the population experienced 71.6% mortality, suggesting that ranaviruses can rapidly attenuate populations. Wide variation in infection rate (7-94% per sampling period) was observed, which may have been driven by clearing and reinfection, adaptive immunity, or imperfect detection using noninvasive samples. Only nasal clinical signs were significantly related to infection status, and agreement among sample types was low. Subsequent to the initial outbreak, low mortality but high real-time PCR prevalence of Ranavirus was observed, suggesting that surviving individuals might be tolerant.

Characterization of the Cutaneous Bacterial Communities of Two Giant Salamander Subspecies.

Pathogens currently threaten the existence of many amphibian species. In efforts to combat global declines, researchers have characterized the amphibian cutaneous microbiome as a resource for disease management. Characterization of microbial communities has become useful in studying the links between organismal health and the host microbiome. Hellbender salamanders (Cryptobranchus alleganiensis) provide an ideal system to explore the cutaneous microbiome as this species requires extensive conservation management across its range. In addition, the Ozark hellbender subspecies (Cryptobranchus alleganiensis bishopi) exhibits chronic wounds hypothesized to be caused by bacterial infections, whereas the eastern hellbender (Cryptobranchus alleganiensis alleganiensis) does not. We assessed the cutaneous bacterial microbiome of both subspecies at two locations in the state of Missouri, USA. Through 16S rRNA gene-based amplicon sequencing, we detected more than 1000 distinct operational taxonomic units (OTUs) in the cutaneous and environmental bacterial microbiome. Phylogenetic and abundance-based dissimilarity matrices identified differences in the bacterial communities between the two subspecies, but only the abundance-based dissimilarity matrix identified differences between wounds and healthy skin on Ozark hellbenders. The higher abundance of OTUs on Ozark wounds suggests that commensal bacteria present on the skin and environment may be opportunistically colonizing the wounds. This brief exploration of the hellbender cutaneous bacterial microbiome provides foundational support for future studies seeking to understand the hellbender cutaneous bacterial microbiome and the role of the bacterial microbiota on chronic wounds of Ozark hellbenders.

Genetic analysis reveals multiple parentage in captive reared eastern hellbender salamanders (Cryptobranchus alleganiensis).

Information on the parentage of captive reared clutches is vital for conservation head-starting programs. Molecular methods, such as genotyping individuals with hyper-variable markers, can elucidate the genealogical contribution of captive-reared, reintroduced individuals to native populations. In this study, we used 12 polymorphic microsatellite loci to infer parentage of a clutch of 18 eastern hellbenders collected from a single nest from Buffalo Creek, West Virginia, subsequently reared in captivity, and used for translocations in Indiana. Collectively, these markers successfully detected the presence of multiple parentage for this species of conservation concern presently used in captive management programs in zoos across many states. This study highlights the need for genetic analysis of captive reared clutches used in translocations to minimize the loss of genetic diversity and potential for genetic swamping at release sites.

Mosquitoes as a Potential Vector of Ranavirus Transmission in Terrestrial Turtles.

Ranaviruses are significant pathogens of amphibians, reptiles, and fishes, contributing to mass mortality events worldwide. Despite an increasing focus on ranavirus ecology, our understanding of ranavirus transmission, especially among reptilian hosts, remains limited. For example, experimental evidence for oral transmission of the virus in chelonians is mixed. Consequently, vector-borne transmission has been hypothesized in terrestrial turtle species. To test this hypothesis, mosquitoes captured during a 2012/2013 ranavirus outbreak in box turtles from southwestern Indiana were pooled by genus and tested for ranavirus DNA using qPCR. Two of 30 pools tested positive for ranavirus. Additionally, an individual Aedes sp. mosquito observed engorging on a box turtle also tested positive for ranavirus. Although our approach does not rule out the possibility that the sequenced ranavirus was simply from virus in bloodmeal, it does suggests that mosquitoes may be involved in virus transmission as a mechanical or biological vector among ectothermic vertebrates. While additional studies are needed to elucidate the exact role of mosquitoes in ranavirus ecology, our study suggests that a greater focus on vector-borne transmission may be necessary to fully understand ranaviral disease dynamics in herpetofauna.

Effects of timber harvests and silvicultural edges on terrestrial salamanders.

Balancing timber production and conservation in forest management requires an understanding of how timber harvests affect wildlife species. Terrestrial salamanders are useful indicators of mature forest ecosystem health due to their importance to ecosystem processes and sensitivity to environmental change. However, the effects of timber harvests on salamanders, though often researched, are still not well understood. To further this understanding, we used artificial cover objects to monitor the relative abundance of terrestrial salamanders for two seasons (fall and spring) pre-harvest and five seasons post-harvest in six forest management treatments, and for three seasons post-harvest across the edge gradients of six recent clearcuts. In total, we recorded 19,048 encounters representing nine species of salamanders. We observed declines in mean encounters of eastern red-backed salamanders (Plethodon cinereus) and northern slimy salamanders (P. glutinosus) from pre- to post-harvest in group selection cuts and in clearcuts. However, we found no evidence of salamander declines at shelterwoods and forested sites adjacent to harvests. Edge effects induced by recent clearcuts influenced salamanders for approximately 20 m into the forest, but edge influence varied by slope orientation. Temperature, soil moisture, and canopy cover were all correlated with salamander counts. Our results suggest silvicultural techniques that remove the forest canopy negatively affect salamander relative abundance on the local scale during the years immediately following harvest, and that the depth of edge influence of clearcuts on terrestrial salamanders is relatively shallow (<20 m). Small harvests (<4 ha) and techniques that leave the forest canopy intact may be compatible with maintaining terrestrial salamander populations across a forested landscape. Our results demonstrate the importance of examining species-specific responses and monitoring salamanders across multiple seasons and years. Long-term monitoring will be necessary to understand the full impacts of forest management on terrestrial salamanders.

Seasonal variation in hematology and blood plasma chemistry values of the timber rattlesnake (Crotalus horridus).

Hematology, biochemical analyses, and body condition indices are useful tools for describing animal health, especially when making management decisions for species of conservation concern. We report hematologic, biochemical, and body condition index data for 13 free-ranging timber rattlesnakes (Crotalus horridus) sampled repeatedly over an active season in Indiana, USA.

Unexpectedly low rangewide population genetic structure of the imperiled eastern box turtle Terrapene c. carolina.

Rangewide studies of genetic parameters can elucidate patterns and processes that operate only over large geographic scales. Herein, we present a rangewide population genetic assessment of the eastern box turtle Terrapene c. carolina, a species that is in steep decline across its range. To inform conservation planning for this species, we address the hypothesis that disruptions to demographic and movement parameters associated with the decline of the eastern box turtle has resulted in distinctive genetic signatures in the form of low genetic diversity, high population structuring, and decreased gene flow. We used microsatellite genotype data from (n = 799) individuals from across the species range to perform two Bayesian population assignment approaches, two methods for comparing historical and contemporary migration among populations, an evaluation of isolation by distance, and a method for detecting barriers to gene flow. Both Bayesian methods of population assignment indicated that there are two populations rangewide, both of which have maintained high levels of genetic diversity (HO = 0.756). Evidence of isolation by distance was detected in this species at a spatial scale of 300-500 km, and the Appalachian Mountains were identified as the primary barrier to gene flow across the species range. We also found evidence for historical but not contemporary migration between populations. Our prediction of many, highly structured populations across the range was not supported. This may point to cryptic contemporary gene flow, which might in turn be explained by the presence of rare transients in populations. However these data may be influenced by historical signatures of genetic connectivity because individuals of this species can be long-lived.

Population genetics of the Eastern Hellbender (Cryptobranchus alleganiensis alleganiensis) across multiple spatial scales.

Conservation genetics is a powerful tool to assess the population structure of species and provides a framework for informing management of freshwater ecosystems. As lotic habitats become fragmented, the need to assess gene flow for species of conservation management becomes a priority. The eastern hellbender (Cryptobranchus alleganiensis alleganiensis) is a large, fully aquatic paedamorphic salamander. Many populations are experiencing declines throughout their geographic range, yet the genetic ramifications of these declines are currently unknown. To this end, we examined levels of genetic variation and genetic structure at both range-wide and drainage (hierarchical) scales. We collected 1,203 individuals from 77 rivers throughout nine states from June 2007 to August 2011. Levels of genetic diversity were relatively high among all sampling locations. We detected significant genetic structure across populations (Fst values ranged from 0.001 between rivers within a single watershed to 0.218 between states). We identified two genetically differentiated groups at the range-wide scale: 1) the Ohio River drainage and 2) the Tennessee River drainage. An analysis of molecular variance (AMOVA) based on landscape-scale sampling of basins within the Tennessee River drainage revealed the majority of genetic variation (∼94-98%) occurs within rivers. Eastern hellbenders show a strong pattern of isolation by stream distance (IBSD) at the drainage level. Understanding levels of genetic variation and differentiation at multiple spatial and biological scales will enable natural resource managers to make more informed decisions and plan effective conservation strategies for cryptic, lotic species.

Seasonal variations in plasma vitellogenin and sex steroids in male and female Eastern Box Turtles, Terrapene carolina carolina.

The Eastern Box Turtle (Terrapene carolina carolina) is a widespread species that has recently experienced precipitous declines throughout its range. Although many studies have documented aspects of reproduction in box turtles, reproductive physiology of free-ranging animals is unknown and can be crucial in this species' recovery. Over a two-year period, we measured reproductive parameters, (vitellogenin [Vtg], estradiol-17ß [E2], and total testosterone [TT]), in plasma of 116 free-ranging Eastern Box Turtles across their active season. We found similar seasonal variations of Vtg and E2 within females. Mid-season, females showed a sharp peak in E2 that correlates with the putative beginning of the ovarian cycle. Individual females lacking these expected peaks of both Vtg and E2 suggest that some female T. c. carolina may not reproduce annually. Females typically expressed undetectable levels of TT, yet there was a small peak in TT early in the active season. Male Eastern Box Turtles exhibited a dual peak in TT. Elevated TT in males was significantly associated with observed sexual behaviors and smaller home ranges. Body condition had no effect on the concentration of TT or E2 in either sex. This is the first study to (1) document Vtg and sex steroid hormones in free-ranging animals of this genus, and (2) relate those metrics to individuals, the population, the purported annual cycle, and to other chelonian species.

Short-term forest management effects on a long-lived ectotherm.

Timber harvesting has been shown to have both positive and negative effects on forest dwelling species. We examined the immediate effects of timber harvests (clearcuts and group selection openings) on ectotherm behavior, using the eastern box turtle as a model. We monitored the movement and thermal ecology of 50 adult box turtles using radiotelemetry from May-October for two years prior to, and two years following scheduled timber harvests in the Central Hardwoods Region of the U.S. Annual home ranges (7.45 ha, 100% MCP) did not differ in any year or in response to timber harvests, but were 33% larger than previous estimates (range 0.47-187.67 ha). Distance of daily movements decreased post-harvest (from 22 m±1.2 m to 15 m±0.9 m) whereas thermal optima increased (from 23±1°C to 25±1°C). Microclimatic conditions varied by habitat type, but monthly average temperatures were warmer in harvested areas by as much as 13°C. Animals that used harvest openings were exposed to extreme monthly average temperatures (∼40°C). As a result, the animals made shorter and more frequent movements in and out of the harvest areas while maintaining 9% higher body temperatures. This experimental design coupled with radiotelemetry and behavioral observation of a wild ectotherm population prior to and in response to anthropogenic habitat alteration is the first of its kind. Our results indicate that even in a relatively contiguous forested landscape with small-scale timber harvests, there are local effects on the thermal ecology of ectotherms. Ultimately, the results of this research can benefit the conservation and management of temperature-dependent species by informing effects of timber management across landscapes amid changing climates.

Temporal variance in hematologic and plasma biochemical reference intervals for free-ranging eastern box turtles (Terrapene carolina carolina).

Eastern box turtle (Terrapene carolina carolina) populations are in decline, likely due to anthropogenic forces and disease, necessitating hematologic and biochemical data from healthy individuals for evaluation of wild populations. We repeatedly sampled 21 free-ranging eastern box turtles from May to September 2009 in the spring, summer, and fall to establish temporal hematologic and biochemical reference intervals. Packed cell volume, aspartate aminotransferase, and potassium levels declined significantly as the active season progressed. High levels of albumin, globulin, and calcium coincided with the presence of eggs in females. These reference intervals should provide baseline data for the clinical evaluation of wild box turtles presented for veterinary care or for studies of wild populations.

Health and habitat quality assessment for the eastern hellbender (Cryptobranchus alleganiensis alleganiensis) in Indiana, USA.

The eastern hellbender (Cryptobranchus alleganiensis alleganiensis) has experienced precipitous population declines throughout its range. Numerous factors are speculated to be involved, but no empirical evidence has been presented for any. We implemented a population-wide health assessment in Indiana, USA, examining both the physical well-being of individuals and the quality of their habitat. Physicochemical parameters were analyzed directly in the field and later in the laboratory, when appropriate. Samples were collected June 2008-October 2008 and June 2009-September 2009 for reproductive analysis, blood screening, and disease prevalence. Of 27 chemicals screened in water samples, three were found in the study site, including atrazine. Atrazine was found at levels reported to cause reproductive problems in other amphibians. Vitellogenin was detected only in females and proved a reliable indicator of sex. Sperm parameters were generally of high quality and similar to other populations. Most plasma parameters were similar between sexes, although there were significant differences in calcium and potassium concentrations. Abnormalities were common, occurring in 68% of individuals. No hemoparasites were found, but amphibian chytrid fungus (Batrachochytrium dendrobatidis) was detected on one individual. Our findings establish a baseline for hematology and water-quality parameters that can be used as a model for evaluating population health throughout the hellbender range.

Condition-dependent mate choice and a reproductive disadvantage for MHC-divergent male tiger salamanders.

Major histocompatibility complex (MHC) alleles likely have adaptive value because of overdominance, in which case MHC heterozygous individuals have increased fitness relative to homozygotes. Because of this potential benefit, the evolution of sexual reproduction between MHC-divergent individuals (i.e. negative assortative mating, NAM) may be favoured. However, the strongest evidence for MHC-based NAM comes from inbred animals, and context-dependent mating preferences have rarely been evaluated although they often occur in nature. We assessed the extent MHC-based mating preferences among wild tiger salamanders (Ambystoma tigrinum) using multiple molecular approaches. We genotyped 102 adults and 864 larvae from 36 breeding trials at both microsatellite and MHC loci. Parentage analysis revealed that reproductive success among males was positively associated with increased tail length and that with respect to the focal female, MHC-similar males sired a significantly higher number of offspring than more dissimilar males. This trend was consistent, even under context-dependent scenarios that favour traditional MHC-based NAM. These results suggest that the most MHC-divergent males may be at a reproductive disadvantage in pairwise breeding trials. Our data add to a growing body of evidence that suggests where it exists, MHC-based choice is probably dynamic and mediated by many factors that vary in the wild, notably signals from other indicator traits and by the quality and quantity of potential mates.

Microsatellite mutation rates in the eastern tiger salamander (Ambystoma tigrinum tigrinum) differ 10-fold across loci.

Microsatellites are commonly used for mapping and population genetics because of their high heterozygosities and allelic variability (i.e., polymorphism). Microsatellite markers are generally more polymorphic than other types of molecular markers such as allozymes or SNPs because the insertions/deletions that give rise to microsatellite variability are relatively common compared to nucleotide substitutions. Nevertheless, direct evidence of microsatellite mutation rates (MMRs) is lacking in most vertebrate groups despite the importance of such estimates to key population parameters (e.g., genetic differentiation or theta = 4N (e)micro). Herein, we present empirical data on MMRs in eastern tiger salamanders (Ambystoma tigrinum tigrinum). We conducted captive breeding trials and genotyped over 1,000 offspring at a suite of microsatellite loci. These data on 7,906 allele transfers provide the first direct estimates of MMRs in amphibians, and they illustrate that MMRs can vary by more than an order of magnitude across loci within a given species (one locus had ten mutations whereas the others had none).

Amphibian malformations and inbreeding.

Inbreeding may lead to morphological malformations in a wide variety of taxa. We used genetic markers to evaluate whether malformed urodeles were more inbred and/or had less genetic diversity than normal salamanders. We captured 687 adult and 1,259 larval tiger salamanders (Ambystoma tigrinum tigrinum), assessed each individual for gross malformations, and surveyed genetic variation among malformed and normal individuals using both cytoplasmic and nuclear markers. The most common malformations in both adults and larvae were brachydactyly, ectrodactyly and polyphalangy. The overall frequency of adults with malformations was 0.078 compared to 0.081 in larval samples. Genetic diversity was high in both normal and malformed salamanders, and there were no significant difference in measures of inbreeding (f and F), allele frequencies, mean individual heterozygosity or mean internal relatedness. Environmental contaminants or other extrinsic factors may lead to genome alternations that ultimately cause malformations, but our data indicate that inbreeding is not a causal mechanism.

Inferring population history and demography using microsatellites, mitochondrial DNA, and major histocompatibility complex (MHC) genes.

Microsatellites and mitochondrial DNA (mtDNA) have traditionally been used in population genetics because of their variability and presumed neutrality, whereas genes of the major histocompatibility complex (MHC) are increasingly of interest because strong selective pressures shape their standing variation. Despite the potential for MHC genes, microsatellites, and mtDNA sequences to complement one another in deciphering population history and demography, the three are rarely used in tandem. Here we report on MHC, microsatellite, and mtDNA variability in a single large population of the eastern tiger salamander (Ambystoma tigrinum tigrinum). We use the mtDNA mismatch distribution and, on microsatellite data, the imbalance index and bottleneck tests to infer aspects of population history and demography. Haplotype and allelic variation was high at all loci surveyed, and heterozygosity was high at the nuclear loci. We find concordance among neutral molecular markers that suggests our study population originated from post-Pleistocene expansions of multiple, fragmented sources that shared few migrants. Differences in N(e) estimates derived from haploid and diploid genetic markers are potentially attributable to secondary contact among source populations that experienced rapid mtDNA divergence and comparatively low levels of nuclear DNA divergence. We find strong evidence of natural selection acting on MHC genes and estimate long-term effective population sizes (N(e)) that are very large, making small selection intensities significant evolutionary forces in this population.