Steven J. A. Kimble

Of contigs and quagmires: next-generation sequencing pitfalls associated with transcriptomic studies.

Molecular ecologists have good reasons to be excited about the newest DNA/RNA sequencing technologies. However, this exuberance should be tempered with a hefty dose of reality: new sequencing technologies come with significant new challenges. Herein, we offer a brief overview of some practical problems encountered during transcriptomics studies conducted in our laboratory, and of nontrivial issues that prospective practitioners should consider. These include template contamination (e.g. from xenobiotics) and the cutting‐room floor problem, whereby most of the data are often unassembled, unannotated and unused. We also highlight computational requirements, including hardware, personnel time and associated skill sets. We are very optimistic about the future of molecular ecology, but we hope this cautionary overview will help neophytes better recognize some key challenges associated with new technologies.

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.

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.

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.

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.

Identification of 12 polymorphic microsatellite loci for the eastern box turtle (Terrapene carolina carolina)

We developed 12 highly polymorphic microsatellite markers for the eastern box turtle (Terrapene carolina carolina). The number of alleles per locus ranged from 5 to 27 (mean: 15.2 per locus) and the observed heterozygosities ranged from 0.371 to 0.943 among 35 individuals. Two loci exhibited significant deviations from Hardy–Weinberg equilibrium. Five loci exhibited putative null alleles but this is probably due to the high number of alleles relative to the sample size. There was no evidence of linkage disequilibrium between pairs of loci. This library should facilitate research on the population structure and ecology of this threatened species.

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.

Seasonal Variation in Hematology and Blood Plasma Chemistry Values of the Timber Rattlesnake (Crotalus horridus)

Abstract 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.