Heat Waves Produce Ecologically Relevant Expression Profiles for Temperature‐responsive Genes

The red‐eared slider turtle ( Trachemys scripta elegans ), exhibits temperature‐dependent sex determination (TSD), where sex‐specific genetic profiles are produced in response to incubation temperature. Despite over 50 years of study on this topic, we still do not fully understand how temperature affects the molecular mechanisms underlying TSD, and prior studies aimed at characterizing genes in the sex‐determining pathway have primarily used constant incubation temperatures. Such studies fail to capture the thermal variability that developing embryos naturally experience, but have been used to identify potential candidate genes in the sex‐determining pathway. One candidate, cold‐inducible RNA‐binding protein ( Cirp ), has sex‐specific expression in several species of turtles and other reptiles under constant incubation conditions. We characterized Cirp expression in the gonads of T. s. elegans embryos exposed to temperatures that are a better mimic of natural nest conditions. All eggs were incubated for the first 24 days at male‐producing temperatures (MPT; 25.0 ± 3°C) before a subset were shifted to female‐producing temperatures (FPT; 29.5 ± 3°C) to induce ovarian development via a simulated heatwave. Embryonic gonads were sampled on day 24, then sampled from both FPT and MPT for five consecutive days. In contrast to results from constant temperature studies, we found that Cirp expression did not differ in embryos at MPT and FPT. We then submitted a subset of our samples for RNAseq to identify genes exhibiting differential expression (DE) in the gonads of embryos at FPT and MPT. A preliminary analysis found 74 genes to be upregulated and 102 genes to be downregulated by day 5 of the heatwave. Similar work using constant temperature incubation found 1887 DE genes in the gonads of embryos at the same stage of development. Only 25 DE genes were shared between the constant and fluctuating temperature datasets. Our initial comparison of gene expression under constant and fluctuating conditions suggests that while constant incubation conditions result in many more DE genes, many of these genes may not be differentially expressed under more natural incubation conditions. Further, these findings highlight the importance of using ecologically relevant conditions to identify genes expressed during early gonadogenesis in TSD species. Support or Funding Information American Physiological Society William Townsend Porter Pre‐Doctoral Fellowship, Society for Integrative and Comparative Biology Grants‐in‐Aid‐of‐Research, Beta Lambda Phi Sigma Biological Honors Society Weigel Grant