Twenty-first Annual Meeting

of the

Tennessee Herpetological Society



Twenty-First Annual Meeting

of the

Tennessee Herpetological Society


Horse Creek Wildlife Sanctuary and Animal Refuge

Savannah, TN

Hosted By

Freed-Hardeman University

Thursday PM

Location: Rockhouse Lodge (use the Rockhouse Lodge entrance)

35°06’41.11”N; 88°09’38.59”W

1:00 Welcome

1:15 Joshua R. Ennen Keynote: The where and why of North American turtles: an

explanation of species richness and composition patterns. Ennen, J.R., W.

Matamoros, S.C. Hazzard, M. Agha, and J.E. Lovich

2:15 Break

2:30 Metler, A. If you build it, will they come? Temporary backyard ponds as an

alternative to traditional frog metamorphosis projects

2:50 Lillard, C.E., D.L. Miller, J.L. Howard, B.S. Wilson, R.T. Jackson, J.R. Asper, R.P.

Wilkes, L. Rollins-Smith, R.D. Hill, Y. Geng, W.B. Sutton, B. Reeves, S. Reinsch,

D. McGinnity, and M.J. Gray. Pathogens vs. pesticides: the threat to eastern


3:10 Carter, E.D., M.J. Gray, J.A. Spatz, and D.L. Miller. Rapid transmission of

ranavirus: role of direct contact

3:30 Spatz, J.A., M.J. Gray, E.D. Carter, and D.L. Miller. Poor biosecurity could lead to

disease outbreaks in amphibian populations

3:50 Kirkpatrick, S.J. and V.A. Cobb. Does digestion affect thermoregulation in

freeranging timber rattlesnakes (Crotalus horridus)?

4:10 Flaherty, J.P. and C.M. Gienger. Habitat selection and movement patterns of

copperheads (Agkistrodon contortrix) in fire-altered landscapes

4:30 Hamed, K. and K. Cecala. Proposal for a journal published by THS

5:00 Business Meeting

6:00 Dinner Catered

7:00-8:30 Lewis Memorial Scholarship Auction

8:30 Field Trip

Friday AM

8:30 Colvin, R. An update of Alligator Snapping Turtle, Macrochelys temminckii,

surveys in West Tennessee and potential future research

8:50 Nelson, S., B. Fitzpatrick, M. Niemiller, and M. Ogle. Evidence of a new species of

mudpuppy in Tennessee

9:10 Vannatta, J. Demographic characteristics, incidence of ranavirus infection, and

corticosterone levels in the eastern box turtle, Terrapene carolina carolina, in a

suburban wetlands habitat of Middle Tennessee

9:30 Break

9:50 Ennen, J.R., J.M. Davenport, and K.F. Alford. Interspecific competition among

headwater stream vertebrates

10:10 Walker, D.M., S.P. Graham, C. Kelehear, C. Camp, A.J. Hill, M. Edelbrock, J.

Rheubert, and J. Wooten. A next-generation sequencing approach to assess the

diversity of the skin and gut microbiota in the slimy salamander (Plethodon

glutinosus) species complex, with an emphasis on gastrointestinal fungal pathogens

10:30 Ogle, C. An update of amphibian and reptile surveys on wildlife management areas

within TWRA’s administrative region 4.

10:50 Barton, L.J., B.P. Butterfield, and J.B. Hauge. A preliminary survey of anurans

from Laguna Del Lagarto Lodge, Boca Tapada, Costa Rica

11:10 Butterfield, B.P. and L.J. Barton. Amphibians and reptiles of Horse Creek with an

emphasis on the turtle community

Poster Burton, E., K. Hudson, and T. Blanchard. Differences in habitat use among

species of aquatic snakes in the Reelfoot Lake area of Northwestern Tennessee

Poster Walker, D.M., S.P. Graham, C. Kelehear, C. Camp, A.J. Hill, M. Edelbrock, J.

Rheubert, and J. Wooten. A next-generation sequencing approach to assess the

diversity of the skin and gut microbiota in the slimy salamander (Plethodon

glutinosus) species complex, with an emphasis on gastrointestinal fungal pathogens

11:30 Lunch

12:30 Field Trip




A Preliminary Survey of Anurans from Laguna Del Lagarto Lodge, Boca Tapada,

Costa Rica

Lee J. Barton1, Brian P. Butterfield1, and J. Brian Hauge2

1Freed-Hardeman University, 2Peninsula College

The number of anurans species found in Mesoamerica is astounding. However, our

understanding of the basic biology of many of these species is incomplete, even in many

well-studied regions. Here we report preliminary results of an ongoing survey of the

anurans of La Laguna Del Lagarto Lodge, Boca Tapada, Costa Rica. To date, we have

documented 20 species at this site. Determining the geographic distribution of species is a

necessary step for the successful development and implementation of conservation plans

designed to protect biodiversity.


Differences in Habitat Use Among Species of Aquatic Snakes in the Reelfoot Lake

Area of Northwestern Tennessee

Erica Burton, Kelsey Hudson, and Tom Blanchard

University of Tennessee at Martin

Five species of water snakes occur in Tennessee and all of them are commonly observed

in the Reelfoot Lake area. Although several studies of habitat use in these animals have

been published, few address large-scale differences in habitat use among them in regions

where they occur together. Published information on water snakes in Tennessee suggests

that all five species are habitat generalists and regularly occur in cypress swamps,

bayous, lakes, streams, and ditches. Personal observations of water snakes at Reelfoot

Lake however, suggest a more restrictive habitat use for some. The purpose of this study

was to determine if meso-scale habitat use was different among the five species of water

snakes that occur at Reelfoot Lake. We collected data from May - October of 2014 and

from March - April 2015. A variety of habitat types were searched by boat or on foot and

snakes were captured if possible, identified, marked, and released. A location for each

animal was recorded with a hand-held GPS unit and a variety of habitat variables were

obtained. Our preliminary results suggest that some species of water snakes in the

Reelfoot Lake area are generalist, whereas others are more restrictive in their habitat use.


Amphibians and Reptiles of Horse Creek with an Emphasis on the Turtle


Brian P. Butterfield and Lee J. Barton

Freed-Hardeman University

We began surveying the herpetofauna of Horse Creek Wildlife Sanctuary and Animal

Rescue in 2012. Our purposes for this ongoing study are to: 1) construct a species list, 2)

establish a long-term turtle community study modeled after North American Freshwater

Turtle Research Group (NAFTRG) studies, and 3) provide undergraduates with

opportunities to conduct field research and enhance their educational experiences. We

have documented 38 species that include 10 records for Hardin County. We have

captured and marked 152 individual turtles representing eight species. The most abundant

turtle species is T. scripta. Our data provide insight into amphibian and reptile diversity

within a protected area and should serve as a baseline from which to assess assemblages

from more disturbed systems.


Rapid Transmission of Ranavirus: Role of Direct Contact

E. Davis Carter1, Matthew J. Gray1, Jennifer A. Spatz1, and Debra L. Miller1,2

1Center for Wildlife Health, University of Tennessee, Knoxville, TN, 2College of

Veterinary Medicine, University of Tennessee, Knoxville, TN

Amphibian populations are declining globally in part due to viral pathogens from the

genus Ranavirus. Ranaviruses can be transmitted through various routes including

contact with contaminated water and soil, ingestion of infected tissue, and direct contact

with infected individuals. Our objective was to determine how quickly an outbreak of

ranavirus could occur through direct contact of infected individuals. We exposed five

wood frog (Lithobates sylvaticus) tadpoles to a lethal dose of ranavirus (103 PFU/mL) in

water for 24 hours. We touched each exposed individual for 1 sec to five unexposed L.

sylvaticus and five unexposed Cope’s gray tree frog (Hyla chrysoscelis) tadpoles at 24,

48, 72, and 96 hours. Significant transmission of ranavirus between wood frog tadpoles

started at 48 hours, with the resulting mortality increasing at each consecutive time step

(48 h: 36%, 72 h: 76%, 96 h: 92%). Transmission of ranavirus between wood frog and

Cope’s gray tree frog tadpoles was less, with resulting mortality <36% among the postexposure

contact durations. Our results indicate that outbreaks of ranavirus in wood frog

populations can occur quickly through direct contact of conspecifics; however, spillover

effects to other species may be dependent on other factors, such as virion concentrations

increasing beyond lethal dose levels.


An update of Alligator Snapping Turtle, Macrochelys temminckii, Surveys in West

Tennessee and Potential Future Research

Rob Colvin

Tennessee Wildlife Resources Agency

From 2000 through 2006, the Tennessee Wildlife Resources Agency (TWRA) conducted

an alligator snapping turtle (Macrochelys temminckii) restoration project involving the

release of adult and juvenile turtles in various watersheds. Since then, there has been

limited work done with this species regarding their distribution and population status in

Tennessee. Between May and June 2015 we sampled two different locations in Tipton

and Dyer Counties using baited hoop nets and we were able to locate a new population of

alligator snapping turtles in Tipton County. Two additional records were also

documented by other individuals in the Beech River and at Radnor Lake State Park. We

plan to start a multi-year survey beginning in 2016 to gather distribution and population

data to better understand the status of this species in Tennessee.


Interspecific Competition Among Headwater Stream Vertebrates

Joshua R. Ennen*, Jon M. Davenport, and Kathlina F. Alford*

1Tennessee Aquarium Conservation Institute, Chattanooga, Tennessee, United States of

America. Department of Biology, Southeast Missouri State University, Cape Girardeau,

Missouri, United States of America.

Species interactions, such as interspecific competition, often influence species

distribution and abundance over space and time. Some stream vertebrates, such as

salamanders, can reach high abundances in headwater systems and account for a

disproportionate amount of total vertebrate biomass. However, the importance of stream

salamanders in structuring these headwater systems is still understudied. We conducted

an artificial stream experiment to evaluate interactions among three common vertebrates.

We investigated the scaled mass growth of salamanders in the black-bellied dusky

complex (Desmognathus quadramaculatus/folkertsi) in the presence of two different fish

species, common creek chub (Semotilus atromaculatus) and Coosa darter (Etheostoma

coosae). There was no statistical significance (t = 0.71, df = 5, P = 0.51) between the

control and darter present treatments (mean with darters: 0.09±0.64 SE; mean with

conspecifics: 0.47±0.10 SE). However, the body condition of salamanders (-0.25±0.26

SE) were significantly reduced (t = -2.65, df = 6, P = 0.04) in the presence of the creek

chubs suggesting an asymmetric competitive interaction between those two species. Our

results suggest that asymmetric competition, and not solely predation, may play an

important role in limiting the distribution and abundance of stream vertebrates in

headwater streams.


The Where and Why of North American Turtles: An Explanation of Species

Richness and Composition Patterns

Joshua R. Ennen, Wilfredo Matamoros, Sarah C. Hazzard, Mickey Agha, and Jeffrey E.


Tennessee Aquarium Conservation Institute, Tennessee Aquarium, 201 Chestnut Street,

Chattanooga, TN, 37402 USA

Species richness and composition is not uniformly distributed across the landscape, and

these patterns are often associated with environmental gradients. In this study, we

examine species richness and composition patterns within North America turtles and use

14 abiotic variables (8 climatic, 2 topographical, 2 stream variables, as well as latitude

and longitude) to construct regression models fitted to spatial data that predict species

richness for all turtles (n = 84), freshwater aquatic and semiaquatic turtles, turtles in the

family Emydidae, and those in the Kinosternidae. Overall, species richness was positively

related to maximum temperature of the warmest month (K), precipitation of the wettest

month (mm), total stream length (km), and temperature seasonality. Species richness of

aquatic and semiaquatic turtles was positively related with precipitation of the wettest

month, total stream length, and temperature seasonality. Emydid species richness was

only related (positively) to precipitation of the wettest month. Interestingly, only

kinosternid richness was related (negatively) with latitude. Also, species richness within

kinosternid was positively related to precipitation of the wettest month and total stream

length. Given current and projected climate trends, especially alterations of temperature

and precipitation patterns, species richness of North American turtles could be impacted.


Habitat Selection and Movement Patterns of Copperheads (Agkistrodon contortrix)

in Fire-altered Landscapes

James P. Flaherty and C.M. Gienger

Center of Excellence for Field Biology & Department of Biology

Austin Peay State University, Clarksville, TN 37044

Fire can functionally alter habitat available to wildlife through alteration of structural and

micro-climatic characteristics. Reptiles, due to their reliance on behavioral

thermoregulation and crypsis, can be especially susceptible to these post-fire changes in

habitat characteristics. The copperhead (Agkistrodon contortrix) is a widely distributed

snake throughout the southeastern US, and is found in a variety of habitats. Previous

studies suggest that copperheads decrease in abundance immediately after fire events, but

to our knowledge there has been no work investigating this species’ habitat selection and

movements in a fire-altered landscape. We tracked 11 adult male copperheads captured

in burned and unburned sites at Land Between the Lakes National Recreation Area, KY

using radio-telemetry. Copperheads were tracked up to three times per week during the

active season (May-October) of 2014. Home ranges were calculated by minimum

convex polygons (MCP) and fixed kernel density estimation (KDE). Movement patterns

were assessed by measurement of straight-line distances between successive copperhead

relocations. At each copperhead location, a suite of structural and environmental habitat

variables were recorded. In order to measure habitat availability, each snake relocation

point was paired with a randomly selected point. There was no significant difference

between MCP home ranges and movement patterns of copperheads captured in burned

and unburned sites. KDE home ranges were significantly larger in copperheads captured

in unburned sites than those captured in burned sites. Among the available habitats,

burned sites exhibited significantly greater habitat heterogeneity than unburned sites, but

overall, copperheads selected habitats more similar to those available in burned sites than

in unburned sites.


Proposal for a Journal Published by THS

Kevin Hamed1, Kristen Cecala2

1Virginia Highlands Community College, 2Sewanee; University of the South

We propose the development of a peer-reviewed journal published by the Tennessee

Herpetological Society. Many individuals are currently contributing to improving

knowledge about Tennessee’s amphibians and reptiles. We recommend building an

avenue to increase awareness of new discoveries and findings by and for any individual

interested in Tennessee’s herpetofauna.

An option successfully implemented by Virginia’s Herpetological Society is the journal

Catesbeiana. They share short papers, the results of bioblitzes and other surveys, field

notes, range extensions and new species localities, as well as society business.

We propose that a journal published by the Tennessee Herpetological Society include the

following components:

1) Survey Results – We encourage the publication of habitat types and species

found during herpetological surveys of private and public lands in Tennessee.

2) Field Notes – Publication of field observations of amphibians and reptiles. This

may include observations of aspects of the natural history of a species as well as

identification of new localities. We suggest conversations with Floyd Scott and

William Redmond and TWRA to facilitate incorporation of these localities into

state-wide databases.

3) Original Research Papers – Publication of results not published in other venues.

4) Recent Study Reviews– Short descriptions of research published about the

natural history, distribution, and ecology of Tennessee’s herpetofauna in other

venues. We encourage these pieces to be less than 250 words and be written for

public audiences.

5) Biographies – To facilitate getting to know the diversity of people involved in the

Tennessee Herpetological Society, we encourage less than 500 word profiles

highlighting the background and interests of our various members.

6) Minutes or summary of annual Tennessee Herpetological Society meetings.

Survey results, field notes, and original research papers would all be peer reviewed and

submitted electronically to an editor. We would encourage an online publication once or

twice per year depending on popularity. Content could be grouped and published into a

pdf file for posting on the THS website.


Does Digestion Affect Thermoregulation in Free-ranging Timber Rattlesnakes

(Crotalus horridus)?

Sarah J. Kirkpatrick and Vincent A. Cobb

Middle Tennessee State University

For terrestrial ectotherms, increasing body temperature (Tb) during digestion can facilitate

localized biochemical reactions and consequently increase passage rate of food through

the gut. Snakes benefit particularly from an increase in digestion rate; they typically

undergo a relatively large increase in body mass after feeding, which can have a negative

effect on mobility and defensive behavior. Therefore, a common assumption is that

digestion can alter thermoregulatory behavior, i.e., digesting snakes choose warmer

environmental temperatures than non-digesting snakes, and snakes actively

thermoregulate to optimize digestive efficiency. However, a predominantly inactive,

infrequently-feeding ambush predator may sacrifice optimal body temperature to

conserve energy and/or to avoid detection. This study investigated thermoregulation and

microhabitat selection before and after feeding in telemetered timber rattlesnakes

(Crotalus horridus) in central Tennessee. Snakes were offered large food items

(laboratory rats weighing 30-50% of snake body mass) or were observed feeding

naturally. Data from a total of eleven individuals monitored over four active seasons

indicated that neither Tb nor environmental temperatures differed significantly between

pre-fed and post-fed treatments. This lack of a difference in temperature selection is

counter to what we hypothesized and what is generally assumed for snakes. A possible

explanation for this result is the tendency of C. horridus to be a thermoconformer.


Pathogens Vs. Pesticides: The Threat to Eastern Hellbenders

Carson E. Lillard1, Debra L. Miller1,2, Jennifer L. Howard2, Benjamin S. Wilson1, Reilly

T. Jackson1, Jennifer R. Asper1, Rebecca P. Wilkes3, Louise Rollins-Smith4, Rachel D.

Hill1, Yi Geng1,5, William B. Sutton1,6, Bill Reeves7, Sherri Reinsch8, Dale McGinnity8,

and Matthew J. Gray1

1Center for Wildlife Health, University of Tennessee, Knoxville, TN, USA

2College of Veterinary Medicine, University of Tennessee, Knoxville, TN, USA

3Veterinary Diagnostic and Investigational Laboratory, University of Georgia, Tifton


4Department of Pathology, Microbiology, and Immunology, Vanderbilt University

Medical Center, Nashville, TN, USA

5College of Veterinary Medicine, Sichuan Agricultural University, Ya’an, China

6Department of Agricultural and Environmental Sciences, Tennessee State University,

Nashville, TN, USA

7Tennessee Wildlife Resources Agency, Nashville, TN, USA

8Nashville Zoo at Grassmere, Nashville, TN, USA

The eastern hellbender (Cryptobranchus alleganiensis alleganiensis) of North America is

declining in several watersheds across its geographic range. Frequently, these watersheds

are degraded due to surrounding agricultural land use. The increase in application of

glyphosate in the 1990s coincided with the decline of hellbenders in some watersheds.

To date, no studies have tested the toxicity of glyphosate herbicides to hellbenders. The

1990s also were a period when several pathogens emerged in amphibian populations, but

their effects on hellbenders remain unclear. We tested the toxicity Cornerstone® and

Roundup® formulations of glyphosate on larval hellbenders, the susceptibility of

hellbenders to ranavirus and Batrachochytrium dendrobatidis (Bd), and if there was an

interaction between these pathogens and a sub-lethal exposure to Roundup®. We found

that larval hellbenders could withstand high concentrations of Cornerstone® (24 ppm)

and Roundup® (12 ppm), larval and juvenile hellbenders experienced 67 – 80% mortality

when exposed to ranavirus, and juvenile hellbenders did not die when exposed to Bd.

There was evidence of interactive effects, with 20% greater mortality when exposed to

Roundup® and either of these pathogens. Our results indicate that, of the factors we

tested, ranaviruses are the greatest threat to larval and juvenile hellbenders, and sub-lethal

exposure to Roundup® could contribute to mortality when pathogens are present.


If You Build it, Will They Come? Temporary Backyard Ponds as an Alternative to

Traditional Frog Metamorphosis Projects

Alli Metler

Homeschooled, 6th grade, 7007 Scepter Dr., Bartlett, TN 38135

For years, homeschool curricula and schools everywhere have done the traditional project

of watching a tadpole metamorphose into a frog. We sought to redefine that project by

setting up a wading pool in our suburban Memphis area property with assorted cover

objects, both commercial and found objects. In Year 2, plants were put near the pool. Our

question was whether or not frogs would do amplexus in this pool. In Year 1, there was

definite use by American Toads, Bronze Frogs, and Southern Leopard Frogs, as well as

Gray Treefrogs, Copes’ Gray Treefrogs, and Green Treefrogs. In Year 2, tadpoles were

found from Gray Treefrogs and there was definite colonization from Bronze Frogs,

Southern Leopard Frogs, American Toads, Gray Treefrogs, and Copes’ Gray Treefrogs.

Other animal use includes birds as well as mammalian species such as squirrels, possums,

and cats. Others around the United States have agreed to take part in this project and

record results.


Evidence of a New Species of Mudpuppy in Tennessee

Stephen Nelson1, Ben Fitzpatrick2, Matthew Niemiller3, and Michael Ogle4

1Knoxville Zoo, 2University of Tennessee (Knoxville), 3Illinois Natural History Survey,

and 4Knoxville Zoo

There is currently only one necturid species (family Necturidae) currently known to occur

in Tennessee, the Common Mudpuppy (Necturus maculosus), (Pasachnik and Niemiller

2011). However, there is evidence from color patterns, mitochondrial DNA (mtDNA) and

nuclear DNA (nDNA) that an additional species also occurring in Tennessee. The

taxonomy of mudpuppies in the southeastern United States has been in dispute for several

years. Current taxonomy recognizes five species and one subspecies. Our evidence

suggests that there is a new species of mudpuppy that coexists with the most well-known

and widely distributed of the water dogs and mudpuppies, the Common Mudpuppy

(Necturus maculosus). The putative new species co-occurs with the Common Mudpuppy

in one watershed in Southeastern Tennessee, but the extent of its geographic range is



An Update of Amphibian and Reptile Surveys on Wildlife Management Areas

Within TWRA’s Administrative Region 4.

Chris Ogle

Tennessee Wildlife Resources Agency

Since 2004, the Tennessee Wildlife Resources Agency (TWRA) has been inventorying

nongame species on numerous Wildlife Management Areas (WMA’s). More recently,

this inventory has begun to focus on species of Greatest Conservation Need (GCN) in

each of the following classes: Amphibia, Reptilia, Mammalia, and Aves. Within each of

TWRA’s four administrative Regions, one Wildlife Manager 1 has been tasked with this

responsibility. A variety of techniques have been implemented to accomplish this goal,

and presented here is an update of the work and species captured within TWRA’s

administrative Region IV since the 2014 meeting.


Poor Biosecurity Could Lead to Disease Outbreaks in Amphibian Populations

Jennifer A. Spatz1, Matthew J. Gray1, E. Davis Carter1, and Debra L. Miller1,2

1Center for Wildlife Health, University of Tennessee, Knoxville, TN 37996

2College of Veterinary Medicine, University of Tennessee, Knoxville, TN 37996

Outbreaks of ranavirus and chytrid fungus have contributed to amphibian population

declines. It has been suspected that biologists could contribute to pathogen outbreaks

through poor biosecurity practices during sampling. Biologists frequently co-house

captured amphibians and do not change gloves between handling different individuals.

We tested whether these poor biosecurity practices could facilitate transmission of

ranavirus from infected to uninfected wood frog (Lithobates sylvaticus) tadpoles, and

increase the likelihood of mortality. Co-housing tadpoles for only 15 minutes with 10%

of individuals initially infected resulted in transmission and mortality of 50% of

uninfected tadpoles. Not changing gloves between individuals when 10% were initially

infected resulted in transmission of ranavirus and mortality of 70% of uninfected

tadpoles. More extreme mortality was observed when tadpoles were co-housed for longer

durations, or when the initial infection prevalence was >10%. Our results indicate that

poor biosecurity practices can cause pathogen transmission between individuals, which

could lead to disease outbreaks and decrease survival in populations. Biologists should

change gloves or decontaminate them between handling individuals, and not co-house



Demographic Characteristics, Incidence of Ranavirus Infection, and Corticosterone

Levels in the Eastern Box Turtle, Terrapene carolina carolina, in a Suburban

Wetlands Habitat of Middle Tennessee.

Jessica Vannatta

Tennessee Technological University

The Eastern Box Turtle, Terrapene carolina carolina, is a species of concern in

Tennessee because of population decline. Decline is due mainly to anthropogenic causes

including, but not limited to, road mortality, disease, and habitat fragmentation. The

purpose of this study was to assess the general health of the T. c. carolina population in a

suburban wetlands habitat in Murfreesboro, TN, USA (Nickajack) by measuring

demographic characteristics, infection status, and physiological characteristics.

Demographic characteristics recorded include population density, age class, sex ratio, and

several body size measurements (e.g., carapace length and carapace height). A small

blood sample (0.2 ml) was drawn to measure corticosterone levels, triglycerides, uric

acid, innate immunity, and to determine Ranavirus infection status. The population

density was estimated to be approximately 14-15.5 turtles per hectare. The age class

structure had a normal distribution with most turtles falling within the middle age class

(10-14 years). The sex ratio was 1.26 male:1 female. For body size, the only variable

with a significant difference between males and females was straight-line carapace length

in which males were longer than females. A single turtle (LPW) was positive for

Ranavirus infection out of the 102 turtles sampled and tested, accounting for 1%

prevalence in the sampled population. Females had higher body condition indices than

males. In 2013, corticosterone levels were significantly higher in summer than in spring.

In 2014, corticosterone levels were significantly higher in fall than in both spring and

summer. The difference in the seasonal timing of peak corticosterone levels between the

years may be related to weather conditions or associated with opportunistic mating.

Corticosterone levels were positively correlated with hemolysis titer, possibly because of

an immunoenhancing effect of stress hormones. In both 2013 and 2014, triglyceride

levels were significantly higher in females than in males. The higher triglyceride levels of

females were most likely associated with the energetic demands of egg production,

carrying eggs, and nesting. Triglycerides were positively correlated with body condition,

indicating greater lipid reserves in individuals with a better body condition. All other

physiological measures, including total leukocytes and heterophil counts, failed to exhibit

significant seasonal or sex related differences. To our knowledge, this is the first report of

baseline corticosterone values and innate immunity in free-ranging T. c. carolina. The

results obtained will aid in the conservation and protection of this species of concern.


A Next-generation Sequencing Approach to Assess the Diversity of the Skin and Gut

Microbiota in the Slimy Salamander (Plethodon glutinosus) Species Complex, with

an Emphasis on Gastrointestinal Fungal Pathogens

1Donald M. Walker, 2Sean P. Graham, 3Crystal Kelehear, 4Carlos Camp, 1Aubree J. Hill,

5Michael Edelbrock, 5Justin Rheubert, 6Jessica Wooten

1Department of Biology, Tennessee Technological University, Cookeville, TN 38505

2Department of Biology, Geology, and Physical Sciences, Sul Ross State University,

Alpine, TX 79832

3 Smithsonian Tropical Research Institute, Apartado 0843-03092, Balboa, Ancon,

Panama, Republic of Panama

4Department of Biology, Piedmont College, Demorest, GA 30535

5Department of Natural Sciences, The University of Findlay, Findlay, OH 45840

6Department of Biology, Centre College, Danville, KY 40422

Amphibians have become an increasing conservation concern due to documentation of

widespread declines in populations throughout the world. Many of these declines are

attributable to outright habitat loss and degradation; however, many are linked to an

emerging infectious fungal pathogen: Batrachochytrium dendrobatidis (Bd). Some

amphibians have developed an active and potent battery of cutaneous antimicrobial

peptides to control potential pathogen growth. In addition, a few studies have shown that

amphibians have a symbiotic gut microbial community that either assists with digestion

or is entirely benign to the host organism. Therefore, studies simultaneously considering

the gut and skin microbial community of amphibians may contribute to our understanding

of their peculiar susceptibility to emerging infectious diseases. Skin swabs (n=57) and

fecal samples (n=57) were collected from eight species of Plethodon (n=57 individuals)

in the southeastern US. DNA was extracted, the 16S rRNA gene (bacteria) and ITS

rDNA gene (fungi) were PCR amplified, and total microbial communities sequenced on

the Illumina MiSeq platform. The most common bacterial phyla on the skin and gut were

Bacteroidetes, Firmicutes, Proteobacteria, and Verrucomicrobia. A non-metric

multidimensional scaling ordination indicated strong differences (stress 0.13) in skin and

gut microbial communities across all Plethodon species. Two-way ANOSIM analyses

were performed to assess whether host species and/or skin/fecal communities were

significant predictors of variability across Plethodon species. The ANOSIM confirmed

skin and fecal communities were a significant predictor of variability (R=0.976,

p<0.001), whereas, host species was not (R=0.149, p<0.001). Gastrointestinal fungal

pathogens in the genera Basidiobolus was detected in all eight and Mariannaea in seven

of eight salamander species. This comparative analysis documents the bacterial and

fungal diversity of both the skin and gut microbiome for species of slimy salamanders.



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