Identifying Sex of Neonate Turtles with Temperature-dependent Sex Determination via Small Blood Samples

Itzel Sifuentes-Romero,Sarah Milton,Jeanette Wyneken,Boris Tezak

doi:10.1038/s41598-020-61984-2

Abstract

Temperature-dependent sex determination, present in most turtle species, is a mechanism that uses temperature to direct the sex of the embryo. The rapid increase of global temperatures highlights the need for a clear assessment of how sex ratios of organisms with TSD are affected. In turtles with TSD, quantifying primary sex ratios is challenging because they lack external dimorphism and heteromorphic sex chromosomes. Here we describe a new technique used to identify sex in neonate turtles of two TSD species, a freshwater turtle (Trachemys scripta) and a marine turtle (Caretta caretta) via analysis of small blood samples. We used an immunoassay approach to test samples for the presence of several proteins known to play an important role in sex differentiation. Our results show that Anti-Mullerian Hormone (AMH) can be reliably detected in blood samples from neonate male turtles but not females and can be used as a sex-specific marker. Verification of sex via histology or laparoscopy revealed that this method was 100% reliable for identifying sex in both T. scripta and C. caretta 1–2 day-old hatchlings and 90% reliable for identifying sex in 83–177 day-old (120–160 g) loggerhead juveniles. The method described here is minimally invasive, and for the first time, greatly enhances our ability to measure neonate turtle sex ratios at population levels across nesting sites worldwide, a crucial step in assessing the impact of climate change on imperiled turtle species.

Highlights

Environmentally-dependent sex determining systems (ESD) are common in vertebrate animals[1], but the developmental mechanisms involved remain poorly understood
Valenzuela et al, (2004) describes a landmark-based geometric morphometric method to detect subtle sexual dimorphism in hatchlings of two freshwater turtle species which were incubated under constant temperatures[26]; this approach has since been used in other freshwater turtle species[27,28,29,30]
We examined 1–2 day-old hatchling red-eared slider (Trachemys scripta elegans) and loggerhead (Caretta caretta) turtles to determine if sex specific proteins (Dmrt[1], Sox[9], Amh, Aromatase and Cold-inducible RNA-binding protein (CIRBP)) could be detected in blood samples via Western Blot analysis

Summary

Introduction

Environmentally-dependent sex determining systems (ESD) are common in vertebrate animals[1], but the developmental mechanisms involved remain poorly understood. We used previously published data from studies on the molecular mechanisms of sex determination and differentiation in vertebrates[48,49] to identify a list of candidate proteins that could potentially be detected in blood samples to serve as sex specific markers of hatchling sex. To our knowledge, differences in sex specific protein expression patterns have never been identified in blood samples of hatchlings with TSD.

Results

Conclusion