Experiencing short heat waves early in development changes thermal responsiveness of turtle embryos to later heat waves.

Although physiological responses to the thermal environment are most frequently investigated using constant temperatures, the incorporation of thermal variability can allow for a more accurate prediction of how thermally sensitive species respond to a rapidly changing climate. In species with temperature-dependent sex determination (TSD), developmental responses to incubation temperature are mediated by several genes involved in gonadal differentiation. Kdm6b and Dmrt1 respond to cool incubation temperatures and are associated with testis development, while FoxL2 and Cyp19A1 respond to warm incubation temperatures and are associated with ovary development. Using fluctuating incubation temperatures, we designed two studies, one investigating how conflicting thermal cues affect the timing of commitment to gonadal development, and another investigating the rapid molecular responses to conflicting thermal cues in the red-eared slider turtle (Trachemys scripta). Using gene expression as a proxy of timing of commitment to gonadal fate, results from the first study show that exposure to high amounts of conflicting thermal cues during development delays commitment to gonadal fate. Results from the second study show that Kdm6b splice variants exhibit differential responses to early heat wave exposure, but rapidly (within 2 days) recover to pre-exposure levels after the heat wave. Despite changes in the expression of Kdm6b splice variants, there was no effect on Dmrt1 expression. Collectively, these findings demonstrate how short exposures to heat early in development can change how embryos respond to heat later in development.

Understanding how variable thermal environments affect the molecular mechanisms underlying temperature-sensitive phenotypes: lessons from sex determination.

The thermal environment that organisms experience can affect many aspects of their phenotype. As global temperatures become more unpredictable, it is imperative that we understand the molecular mechanisms by which organisms respond to variable, and often transient, thermal environments. Beyond deciphering the mechanisms through which organisms respond to temperature, we must also appreciate the underlying variation in temperature-dependent processes, as this variation is essential for understanding the potential to adapt to changing climates. In this Commentary, we use temperature-dependent sex determination as an example to explore the mechanistic processes underlying the development of temperature-sensitive phenotypes. We synthesize the current literature on how variable thermal conditions affect these processes and address factors that may limit or allow organisms to respond to variable environments. From these examples, we posit a framework for how the field might move forward in a more systematic way to address three key questions: (1) which genes directly respond to temperature-sensitive changes in protein function and which genes are downstream, indirect responders?; (2) how long does it take different proteins and genes to respond to temperature?; and (3) are the experimental temperature manipulations relevant to the climate the organism experiences or to predicted climate change scenarios? This approach combines mechanistic questions (questions 1 and 2) with ecologically relevant conditions (question 3), allowing us to explore how organisms respond to transient thermal environments and, thus, cope with climate change.

Effects of Constant and Fluctuating Temperatures on Gene Expression During Gonadal Development.

There is ample research demonstrating that temperature can have complex effects on biological processes, including the timing of when organisms respond to temperature; some responses occur rapidly while others require an extended exposure time. However, most of what we know about temperature effects comes from studies using constant temperature conditions, which are not reflective of natural, fluctuating temperatures. Species with temperature-dependent sex determination (TSD) present an ideal system to study the temporal aspects of the temperature response because prior research has established a number of temperature-responsive genes involved in TSD, albeit under constant temperatures. To investigate potential differences in timing of sexual development between constant and fluctuating incubation temperatures, we exposed Trachemys scripta embryos to two conditions that produce males (constant 26°C and 26 ± 3°C) and two that produce females (constant 31°C and 31 ± 3°C), and sampled embryonic gonads for gene expression analysisvia qPCR. We analyzed three genes involved in testis differentiation (Kdm6b,Dmrt1, andSox9) and two genes involved in ovary differentiation (Foxl2andCyp19A1). Results show that Kdm6b expression was significantly lower under fluctuating temperatures compared to constant temperatures. Foxl2 and Cyp19A1 expression were also lower under fluctuating temperatures, but not at all stages of development. These results suggest that constant temperatures caused increases in both Foxl2 and Cyp19A1 expression earlier (developmental stage 20) than fluctuating temperatures (stages 22 and 23). Dmrt1 and Sox9 expression did not differ between constant and fluctuating temperatures. These results highlight that not all genes in a temperature-dependent process respond to temperature in the same manner. Whether there are functional consequences of this variation remains to be determined.

Using naturalistic incubation temperatures to demonstrate how variation in the timing and continuity of heat wave exposure influences phenotype.

Most organisms are exposed to bouts of warm temperatures during development, yet we know little about how variation in the timing and continuity of heat exposure influences biological processes. If heat waves increase in frequency and duration as predicted, it is necessary to understand how these bouts could affect thermally sensitive species, including reptiles with temperature-dependent sex determination (TSD). In a multi-year study using fluctuating temperatures, we exposed Trachemys scripta embryos to cooler, male-producing temperatures interspersed with warmer, female-producing temperatures (heat waves) that varied in either timing during development or continuity and then analysed resulting sex ratios. We also quantified the expression of genes involved in testis differentiation (Dmrt1) and ovary differentiation (Cyp19A1) to determine how heat wave continuity affects the expression of genes involved in sexual differentiation. Heat waves applied during the middle of development produced significantly more females compared to heat waves that occurred just 7 days before or after this window, and even short gaps in the continuity of a heat wave decreased the production of females. Continuous heat exposure resulted in increased Cyp19A1 expression while discontinuous heat exposure failed to increase expression in either gene over a similar time course. We report that even small differences in the timing and continuity of heat waves can result in drastically different phenotypic outcomes. This strong effect of temperature occurred despite the fact that embryos were exposed to the same number of warm days during a short period of time, which highlights the need to study temperature effects under more ecologically relevant conditions where temperatures may be elevated for only a few days at a time. In the face of a changing climate, the finding that subtle shifts in temperature exposure result in substantial effects on embryonic development becomes even more critical.