Incubation history prior to the canonical thermosensitive period determines sex in the American alligator.

Despite the widespread occurrence of environmental sex determination (ESD) among vertebrates, our knowledge of the temporal dynamics by which environmental factors act on this process remains limited. In many reptiles, incubation temperature determines sex during a discrete developmental window just prior to and coincident with the differentiation of the gonads. Yet, there is substantial variation in sex ratios among different clutches of eggs incubated at identical temperatures during this period. Here, we test the hypothesis that temperatures experienced prior to the reported thermosensitive period for alligators (Alligator mississippiensis) can impact how the sex determination system responds to thermal cues later in development. Temperature shift experiments on eggs collected from the field within 24  h of oviposition were employed to decouple various maternal influences from thermal effects, and results demonstrate a previously undefined window of thermosensitivity occurring by stage 15 of embryonic development, six stages earlier than previously reported. We also examine the intrasexual expression of several male- and female-biased genes and show that while male-biased genes display no intrasexual differences, ovarian CYP19A1 (aromatase) transcript abundance differs by approximately twofold depending on thermal exposures experienced at early stages of embryonic development. These findings expand our understanding of the ESD in the alligator and provide the rationale for reevaluation of the temporal dynamics of sex determination in other crocodilians.