Using an immunohistochemical approach to identify the sex of marine turtles

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 a female. At warmer sand temperatures the nest tends to produce female-biased sex ratios.
The rapid increase of global temperature highlights the need for a clear assessment of effects on sea
turtle sex ratios. However, identifying hatchling sex ratios at rookeries remain coarse estimates due to
the lack of any external gender markers. We rely mainly upon laparoscopic procedures to verify a
hatchling sex; however, in some species, morphological sex can be ambiguous even at the histological
level. Recent studies using immunohistochemical techniques identified that red-eared slider
(Trachemys scripta) embryos over-expressed a particular cold-induced RNA binding protein in the
ovaries in comparison to the testes. This principle allows the distinction between females and males.
We developed a variation of this technique and successfully identified the sexes of loggerhead sea
turtle (Caretta caretta) hatchlings, as confirmed by standard histological and laparoscopic methods that
reliably identifies the sex in this species. Next, we tested a more challenging species, the leatherback
turtle (Dermochelys coriacea), which retains many neotenic features. The morphology of leatherback
hatchling gonads remains difficult to interpret, particularly when dead-in-nest hatchlings and embryos
are the source tissues. In summary, this new and more efficient technique enhances our ability to investigate and identify baseline hatchling sex ratios, a critical progression in assessing global climate
change on sea turtle populations.