Biological sex identification in the endangered dusky gopher frog (Lithobates sevosa): a comparison of body size measurements, secondary sex characteristics, ultrasound imaging, and urinary hormone analysis methods.

Katherine M Graham,Scott T Willard,Cecilia J Langhorne,Andrew J Kouba,Ruth M Marcec

doi:10.1186/s12958-016-0174-9

Katherine M Graham, Scott T Willard + Show 3 more

Open Access

https://doi.org/10.1186/s12958-016-0174-9

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Abstract

BackgroundAccurate sex identification techniques are important for wildlife demographic studies and for genetic management of captive breeding colonies. Various non-invasive methods for identification of biological sex in the weakly dimorphic endangered dusky gopher frog (DGF; Lithobates sevosa) were explored to support planned recovery efforts for this species including breeding and augmentation of wild populations.MethodsBody size (snout-vent length and body weight) measurements, observation of nuptial pads, ultrasound imaging, and urinary hormone analysis for testosterone and estrone were performed on 27 male and 19 female DGFs. For each method, the mean and range of measurement values were determined for male and female DGFs housed in a captive breeding population. The ability of these methods to accurately predict the true biological sex of the individuals was assessed retrospectively.ResultsBody size measurements were of limited use for sex identification purposes, as males and females demonstrated overlapping body lengths and weights. Observation of the presence/absence of nuptial pads in males and females, respectively, proved to be accurate and easy to perform in most cases. Ultrasound imaging was useful for predicting the sex of female frogs, particularly when females were gravid. Commercial enzyme immunoassay kits were validated to measure urinary hormones in the DGF. Mean urinary testosterone (males: 2.22 ± 0.38 ng/ml; females: 0.92 ± 0.11 ng/ml) and estrone (males: 0.08 ± 0.01 ng/ml; females: 1.50 ± 0.39 ng/ml) concentrations were significantly (p < 0.05) different between the sexes. However, there was some overlap in hormone concentrations between the sexes. When a ratio of testosterone (T) to estrone (E) concentrations was calculated for each individual, males demonstrated significantly greater T/E ratios compared to females (p < 0.05). Use of this ratio showed greater accuracy in predicting the sex of the animal compared to using testosterone or estrone concentrations alone.ConclusionsMonitoring for presence/absence of nuptial pads and using urinary testosterone to estrone hormone ratios were the most accurate methods for identifying the biological sex of adult DGFs. Urinary hormone measurements for sex identification may be useful in other weakly dimorphic and monomorphic amphibian species in both ex situ and in situ settings.

Highlights

Accurate sex identification techniques are important for wildlife demographic studies and for genetic management of captive breeding colonies
Research groups must find a balance between the need for an accurate answer and the amount of time and cost they are willing to invest into sex identification, especially for monomorphic species
Ultrasound may be useful in identifying females with well-developed follicles when the equipment is available; the technique can be somewhat subjective and follicles can be difficult to discern in some cases

Summary

Introduction

Accurate sex identification techniques are important for wildlife demographic studies and for genetic management of captive breeding colonies. An obvious but necessary initial management step for meeting reproduction and genetic management goals is to identify the biological sex of the individuals present within an ex situ population. This is not always accomplished, when working with species that have monomorphic or weakly dimorphic secondary sexual characteristics. Dark nuptial pads can frequently identify adult males when they are captured migrating to breeding ponds [6, 7] It is unclear why captive males may fail to display secondary sex characteristics; it may be that artificial habitats are missing important environmental cues that could result in lower steroid concentrations leading to poorer representation of these secondary sexual characteristics [5, 7]

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