An Immunohistochemical Approach to Identify the Sex of Young Marine Turtles

ABSTRACT Marine turtles exhibit temperature‐dependent sex determination (TSD). During critical periods of embryonic development, the nest's thermal environment directs whether an embryo will develop as a male or female. At warmer sand temperatures, nests tend to produce female‐biased sex ratios. The rapid increase of global temperature highlights the need for a clear assessment of its effects on sea turtle sex ratios. However, estimating hatchling sex ratios at rookeries remains imprecise due to the lack of sexual dimorphism in young marine turtles. We rely mainly upon laparoscopic procedures to verify hatchling sex; however, in some species, morphological sex can be ambiguous even at the histological level. Recent studies using immunohistochemical (IHC) techniques identified that embryonic snapping turtle ( Chelydra serpentina ) ovaries overexpressed a particular cold‐induced RNA‐binding protein in comparison to testes. This feature allows the identification of females vs. males. We modified this technique to successfully identify the sexes of loggerhead sea turtle ( Caretta caretta ) hatchlings, and independently confirmed the results by standard histological and laparoscopic methods that reliably identify sex in this species. We next tested the CIRBP IHC method on gonad samples from leatherback turtles ( Dermochelys coriacea ). Leatherbacks display delayed gonad differentiation, when compared to other sea turtles, making hatchling gonads difficult to sex using standard H&E stain histology. The IHC approach was successful in both C. caretta and D. coriacea samples, offering a much‐needed tool to establish baseline hatchling sex ratios, particularly for assessing impacts of climate change effects on leatherback turtle hatchlings and sea turtle demographics. Anat Rec, 300:1512–1518, 2017. © 2017 Wiley Periodicals, Inc.