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

Abstract

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.

1. Introduction

The thermal environment can have complex effects on an organism, but much of what we know about how temperature influences organisms comes from studies that used constant temperature conditions [1–4]. Because almost all organisms live in environments where temperatures fluctuate, using constant conditions, or even the mean of fluctuating conditions, can produce spurious results [5–8]. With the rapidly changing climate, there is an increased urgency to understand how organisms respond to temperature extremes. For example, dramatically different phenotypic outcomes may result depending upon when (during its lifespan) an organism experiences a heat wave. As defined by the United States National Oceanic and Atmospheric Administration, a heat wave is simply abnormally hot weather lasting at least 2 days [9], and heat waves are predicted to increase in both frequency and duration as a result of climate change [10–14]. Prior research has found that the timing or continuity of heat wave exposure can affect biological processes ranging from population dynamics [15,16] to phenology [17], making it necessary to develop a more detailed understanding of how these effects arise.

Early life stages tend to be understudied compared to later life stages, but recent evidence suggests that embryos are far more thermally sensitive than are adults [18–23], with effects on physiology and phenotype lasting into adulthood [24,25]. An interesting example of long-lasting temperature effects on phenotype is temperature-dependent sex determination (TSD). In species with TSD, including many reptiles [26–28] and some teleost fishes [29,30], sex is principally determined by the incubation temperatures experienced during embryonic development [26,31]. For decades, studies have been conducted using constant temperature incubation conditions to characterize the effect of temperature on sex determination in turtles. In many turtle species with TSD, including the red-eared slider turtle (Trachemys scripta), cooler incubation temperatures (26°C) result in male hatchlings and warmer incubation temperatures (31°C) result in female hatchlings [26,32,33], and the temperature that produces a population-wide 50 : 50 sex ratio is known as the pivotal temperature [31], which is approximately 29°C in multiple turtle species [34]. This foundational work has also shown that incubation temperature affects sex determination most acutely during the thermosensitive period (TSP), which, under constant conditions, comprises approximately the middle third of development [34–36]. In T. scripta, the TSP is approximately stages 15 through 21 at cooler incubation temperatures (26°C) and stages 15 through 19 at warmer incubation temperatures (31°C) [34,37]. Adding to this, further research has investigated the underlying genetic mechanisms of TSD [38–43].

In T. scripta and other species with TSD, the development of the bipotential gonads into testes or ovaries is mediated by expression of a suite of genes involved in sexual differentiation [44,45]. Interestingly, even in some species of reptiles with sex chromosomes (e.g. male heterogamety (XX/XY), female heterogamety (ZZ/ZW)), the sex of an individual can be affected by temperature. For example, warmer incubation temperatures can override the gene(s) involved in male differentiation and subsequently cause sex reversal in the Australian central bearded dragon (Pogona vitticeps), suggesting a dosage effect of a gene on the Z chromosome rather than a female-determining gene on the W as the mechanism underlying sex determination [46]. More recent work has proposed that, rather than sex being determined by a single master gene, the genes underlying TSD are thought to function in a parliamentary system of sex determination, in which networks of genes have simultaneous input to components downstream in the cascade [47], and the communication among these gene networks is displaced by temperature [48]. For example, in P. vitticeps, sex reversal of ZZ males caused by warmer incubation temperatures leads to a decrease in the frequency of the W chromosome via negative frequency-dependent selection [49]. In sex-reversed genotypic males without a W chromosome, sexual development is canalized by the networks of genes comprising this parliamentary system of sex determination [47,48]. One gene that is involved in the gene networks of the male sexual development cascade, and therefore is upregulated during testis development, is Dmrt1. The gene product of Dmrt1 is a transcription factor that upregulates genes associated with testicular development [42,50–52]. Constant temperature studies in species with TSD have demonstrated that the dimorphic expression of Dmrt1 in response to temperature begins early in the TSP [42,53]. Similarly, Cyp19A1 (which codes for aromatase, the enzyme responsible for converting androgens into oestrogens) is a gene involved in the gene networks of the female sexual development cascade and is upregulated during ovarian development [54,55]. While research has identified numerous genes involved in the parliamentary system of sex determination in the developing gonads that exhibit differential expression across temperatures [53–55], these genes have only been examined under constant incubation temperatures.

Recent work has focused on leveraging the insights from constant temperature studies while emphasizing the need to increase biological realism [56–59]. As embryos naturally experience a range of temperatures, including both male- and female-producing temperatures, and often within a single day (figure 1), it is necessary to understand how sex ratios and the expression of genes involved in sex determination respond to thermal fluctuations. Studies using incubation temperatures that fluctuate daily demonstrate that more naturalistic conditions have very different effects on sex ratios than constant temperature conditions [59–63]. For species in which females are produced at warmer temperatures, fluctuating conditions result in the production of more females compared to constant conditions. For example, incubating T. scripta eggs at 28.5 ± 3°C produced significantly more female hatchlings than incubating at a constant 28.5°C, despite both conditions having the same mean incubation temperature across development [61]. Fluctuating temperatures probably produce more females because more development is occurring when thermal conditions surpass the pivotal temperature [60,64]. Mechanistically, a biochemical signal (possibly a transcript or a gene product) is thought to accumulate during development at warm temperatures until it reaches a threshold to trigger ovarian development [34,60,65,66]. But little work has been done to characterize how ovarian differentiation may be triggered when incubation temperatures periodically shift between male- and female-producing conditions despite such fluctuations occurring frequently in natural nests (figure 1). Recent studies using a heat wave context, in which embryos were exposed to cooler, male-producing fluctuations with episodes of warmer, female-producing fluctuations to simulate heat waves, demonstrated that short exposures to warm temperatures are sufficient to produce females in T. scripta [67,68]. Specifically, eggs incubated at 25.0 ± 3°C for all of development produced 100% male hatchlings, but females were produced after only a 5-day heat wave (at 29.5° ± 3°C), and approximately 8 days of heat exposure produced a 50 : 50 sex ratio [67,68]. These findings demonstrate that relatively short exposures to warm temperatures are sufficient to trigger ovarian development, further emphasizing the importance of studying how temperature affects biological processes under more natural conditions. However, we lack an understanding of how the timing of when a heat wave occurs during development or the continuity of a heat wave (e.g. two short heat waves versus one long one) might influence the resulting phenotypic outcome to heat exposure. To address this, we conducted a multi-year study to test the hypothesis that the timing and continuity of heat exposure would affect both gonadal development and the expression of genes involved in sex differentiation.

Figure 1.

Thermal trace from a T. scripta nest. Shown are temperature readings taken every 90 min. A reference line for the pivotal temperature is drawn at 29°C.

2. Material and methods

(a). Heat wave timing

Heat wave treatments were designed to cover the middle third of development to allow us to delineate the timing of the TSP under fluctuating conditions. We collected 15 clutches of T. scripta eggs between 31 May and 1 June 2017 at Banner Marsh State Fish and Wildlife Area (Canton, IL) by either digging up nests soon after oviposition or capturing gravid females in baited hoop traps and inducing oviposition in the laboratory via intraperitoneal oxytocin injection [69,70]. The mean clutch size for all collected clutches was (mean ± s.d.) 11.55 ± 4.11 eggs. Eggs were randomly assigned to treatments using a split-clutch design and put into plastic containers containing moist vermiculite and maintained at approximately −150 kPa. All eggs were initially placed into one of two incubators programmed to run a daily sinusoidal cycle of 25.0 ± 3°C (IPP 110 Plus, Memmert GmbH + Co.KG, Schwabach, Germany) within 3 days of being laid; 25.0 ± 3°C will produce 100% males when applied for the entirety of incubation [67]. This fluctuating regime is also within the range of naturally occurring nest temperatures, as verified by sub-surface soil temperatures (figure 1) [62,67,71]. All eggs (except those in the continuous 25.0 ± 3°C group) were moved into one of three incubators programmed to run a daily sinusoidal cycle of 29.5 ± 3°C (IPP 400, Memmert GmbH + Co.KG, Schwabach, Germany) to expose them to a 15-day heat wave (29.5 ± 3°C) starting after 10, 17, 24, 31, 38 or 45 days of development, before being returned to 25.0 ± 3°C for the remainder of incubation (figure 2a). Incubation at 29.5 ± 3°C has been shown to produce 100% females when applied for the entirety of incubation [67,70]. The mean incubation length under these thermal conditions has been shown to be approximately 72 days [68], so eggs were checked daily, starting at approximately incubation day 65 for any breaches in the eggshell by the hatchling. Once the eggshell was breached, the hatchling was placed into an individual cup to maintain identity. Approximately six weeks post-hatch, hatchlings were euthanized, and sex was diagnosed via macroscopic gonad examination [70]. On a morphological level, testes are short in length and opaque while ovaries are longer, transparent and accompanied by ovarian ducts. All hatchling and adult work was carried out in accordance with the methodology approved by Illinois State University's Institutional Animal Care and Use Committee (IACUC) and the Illinois Department of Natural Resources.

Figure 2.

(a) Incubation treatments for the heat wave timing study. (b) Incubation treatments for the heat wave continuity study. (c) Incubation treatments for the heat wave gap study. (d) Incubation treatments for the gene expression study.

(b). Heat wave continuity

Heat wave treatments were designed to investigate the continuity effects of heat waves applied within the middle third of development. For this study, 18 clutches of eggs were collected from 29 to 31 May 2018 and eggs were prepared for incubation as described above. All eggs were incubated at 25.0 ± 3°C until day 24 (with the exception of one treatment, which remained at 25.0 ± 3°C until day 30). Then, each treatment was exposed to 12 days at 29.5 ± 3°C with varying continuity: (i) an early 12-day heat wave that started on day 24, (ii) a late 12-day heat wave that started on day 30, (iii) two 6-day heat waves broken up by 6 days of 25.0 ± 3°C and (iv) four 3-day heat waves, each separated by 2 days at 25.0 ± 3°C (figure 2b). The early and late 12-day heat waves were designed to control for differential sensitivity across the middle third of development. All eggs were returned to 25.0 ± 3°C after experiencing 12 total days at 29.5 ± 3°C. We selected a heat wave of 12 days because recent work shows that under these temperature conditions, a 12-day heat wave applied during the TSP should produce mostly females [68]. Hatchlings were maintained and sex ratios determined as described above.

(c). Heat wave gap

Treatments were designed to investigate the effects of heat wave continuity during the window of sensitivity determined from the heat wave timing study. For this study, 16 clutches of eggs were collected from 7 to 8 June 2019 and eggs were prepared for incubation as described above. All eggs were initially incubated at 25.0 ± 3°C before switching to heat wave condition (29.5 ± 3°C). As in the heat wave continuity study, we designed continuous treatment groups to control for differential sensitivity across the middle third of development. The remaining non-control treatments each consisted of two 7-day heat waves separated by a period of cooler temperatures ranging from 1 to 12 days (figure 2c). The first 7-day heat wave for all non-control treatments started on incubation day 17, as results from the heat wave timing study demonstrated that a heat wave starting on incubation day 17 should result in female production similar to that of a heat wave of equal length starting on day 24. All eggs were returned to 25.0 ± 3°C after experiencing 14 days at 29.5 ± 3°C. Hatchlings were maintained and sex ratios determined as described above.

(d). Gene expression

For the gene expression studies, we used eggs from clutches collected for the heat wave continuity study (continuous exposure treatment), and the heat wave gap study (discontinuous exposure treatment). In the continuous exposure treatment, eggs were initially exposed to 25.0 ± 3°C, then starting on day 24 (early TSP [68]), all eggs were switched to 29.5 ± 3°C. Embryonic gonads were sampled in this treatment on days 6-, 9-, 12- and 16 of the heat wave (or days 30, 33, 36 and 40 of development; figure 2d). In the discontinuous exposure treatment, eggs were initially exposed to 25.0 ± 3°C before switching to 29.5 ± 3°C on day 24 to simulate a 9-day heat wave, after which all eggs (except those in the earliest sampling group) were returned to 25.0 ± 3°C. Embryonic gonads were collected at the end of the 9-day heat wave, and 10, 12, 14 and 16 days after the start of the heat wave (or days 33, 34, 36, 38 and 40 of development). The timing of sampling points in both treatments was designed to analyse gene expression patterns within the TSP [67,68], when the trajectory of the bipotential gonads is sensitive to temperature [55]. At each sampling point, both gonads were removed from each embryo and stored in TRIzol Reagent (Ambion) at −80°C prior to RNA extraction using 2-propanol (Fisher Chemical) and chloroform. cDNA was synthesized from 1 µg RNA using the Maxima First Strand cDNA Synthesis Kit for RT-qPCR with dsDNase (Thermo Scientific) based upon the manufacturer's protocol. RT-qPCR was done using triplicates of each sample and PowerUp SYBR Green Master Mix (Applied Biosystems). Expression of Cyp19A1 and Dmrt1 was normalized with Gapdh as a housekeeping gene, and relative expression was calculated using the ΔΔCt method [72]. Efficiencies for each primer pair (Cyp19A1, Dmrt1, Gapdh) were calculated using standard curves in QuantStudio 3 Real-Time PCR System Software (Applied Biosystems), and these efficiency values were used when calculating relative expression levels. Published primers were used for Cyp19A1 [55] and Dmrt1 [39]. Gapdh primers were constructed using the T. scripta transcriptome [41]. PCR product was sent to the Core/Sanger Facility (Roy J. Carver Biotechnology Center, University of Illinois at Urbana-Champaign) for sequencing to confirm that the primer pair was amplifying T. scripta Gapdh.

(e). Statistical analysis

All statistical analyses were conducted using R (The R Project for Statistical Computing, Vienna, Austria). Sex ratios from the sex ratio experiments were analysed using a generalized linear model, which specified a binomial distribution and a logit link function. Temperature treatment was used as a fixed effect, and initially, the clutch was used as a random effect. Dropping clutch as a random effect did not affect the statistical conclusions for any of the sex ratio studies, so the clutch was ultimately dropped from the models. Data from the gene expression experiment were analysed using an ANOVA with sampling day as a fixed effect for each gene. Expression data for both genes were transformed to log scale to meet model assumptions, and Tukey's HSD was used for the post hoc comparison between sampling days for each gene.

3. Results

(a). Heat wave timing

The timing of heat wave exposure had a strong effect on resulting sex ratios (χ² = 28.94, d.f. = 4, p < 0.001; figure 3). Embryos exposed to a heat wave in the middle of development, specifically heat waves starting on incubation day 17 and ending before incubation day 45, were significantly more likely to develop as females compared to embryos that were exposed to a heat wave just seven days earlier or later in development. The earliest heat wave treatment (days 10–24) only produced 12% females while the next three heat wave treatments (days 17–31, 24–38 and 31–45), all produced female-biased sex ratios over 70% female. The latest heat wave treatment (days 38–52) produced only 6% females. The control group produced 100% males as expected [67,70] (data not included in statistical analysis).

Figure 3.

Effect of heat wave timing on sex ratios. Error bars represent a 95% confidence interval. Treatments that do not share a letter significantly differ in their potency to produce females. Sample sizes are listed as follows (treatment:n) day 10–24: 25, day 17–31: 25, day 24–38: 19, day 31–45: 19, day 38–52: 17.

(b). Heat wave continuity

The continuity of the heat wave significantly affected resulting sex ratios (χ² = 20.75, d.f. = 3, p < 0.001; figure 4a), with the early continuous heat wave producing more females than any of the other heat wave treatments. Embryos exposed to a 12-day heat wave starting on day 24 of incubation were significantly more likely to develop as females compared to those that were exposed to a 12-day heat wave later in development, multiple 6-day heat waves spread across this period or multiple 3-day heat waves spread across this period. The 12-day heat wave applied on day 24 of development produced 64% female hatchlings while the 12-day heat wave applied just 6 days later only produced 25% females. Despite having conditions identical to the early 12-day group for the first 6 days starting at day 24, the multiple 6-day heat waves only produced 12.5% females and the multiple 3-day heat waves also produced 12.5% females.

Figure 4.

(a) Effect of heat wave continuity on sex ratios. Sample sizes are listed as follows (treatment: n) early 12-day: 25, late 12-day: 24, two 6-day: 24, four 3-day: 24. (b) Effects of heat wave gaps on sex ratios. Sample sizes are listed as follows (treatment: n) early control: 15, late control: 17, 1-day gap: 16, 3-day gap: 17, 6-day gap: 16, 9-day gap: 13, 12-day gap: 16. Error bars represent a 95% confidence interval. Treatments that do not share a letter significantly differ in their potency to produce females.

(c). Heat wave gap

The results of the heat wave gap study clarify how the continuity of exposure to warm temperatures affects resulting sex ratios (χ² = 15.05, d.f. = 6, p = 0.02; figure 4b). Embryos exposed to a 3-day gap or longer between heat waves were significantly less likely to develop as females compared to embryos exposed to no gap or a 1-day gap between heat waves. Percentages of females produced by each treatment are listed as follows (treatment: percentage female): early continuous control: 40%, late continuous control: 29%, 1-day gap: 50%, 3-day gap: 12%, 6-day gap: 19%, 9-day gap: 8%, 12-day gap: 6%.

(d). Gene expression

The data show that continuous and discontinuous heat exposure affect gene expression differently. In the continuous exposure treatment, Cyp19A1 expression was significantly affected by heat wave duration (F_3,25 = 16.77, p < 0.0001), but Dmrt1 expression was not (F_3,25 = 0.68, p = 0.57; figure 5a). Cyp19A1 was upregulated on days 12 and 16 of the heat wave. However, neither Cyp19A1 nor Dmrt1 were upregulated in the discontinuous exposure treatment when temperatures returned to baseline after 9 days (figure 5b). Both Cyp19A1 (F_4,35 = 2.98, p = 0.03) and Dmrt1 (F_4,35 = 4.233, p = 0.0067) were significantly affected by exposure to a 9-day heat wave and subsequent return to cooler conditions, but the effect was actually a drop in expression values after the heat wave. Post hoc analyses demonstrate that both Cyp19A1 (p = 0.03) and Dmrt1 (p = 0.0035) expression was significantly lower the day after the end of the heat wave compared to 7 days after the heat wave.

Figure 5.

(a) Changes in Cyp19A1 and Dmrt1 expression over the course of a heat wave. The analysis was performed using both gonads from each sampled embryo. Sample sizes are listed as follows (days since start of heat wave: embryos sampled): 6: 6, 9: 6, 12: 10, 16: 7. (b) Changes in Cyp19A1 and Dmrt1 expression following a heat wave. The analysis was performed using both gonads from each sampled embryo. Sample sizes are listed as follows (days since start of heat wave:embryos sampled): 9: 7, 10: 8, 12: 8, 14: 8, 16: 9. The heat waves used in both conditions started on day 24 of incubation. Data shown with the median (thick black line), 25th and 75th percentiles (lower and upper boundaries of box plots, respectively), maximum and minimum values (whiskers) and outliers (closed circles). Sampling points that do not share an uppercase letter had significantly different levels of Cyp19A1 expression, while sampling points that do not share a lowercase letter had significantly different levels of Dmrt1 expression.

4. Discussion

Although organisms will probably be exposed to heat waves more frequently as a result of climate change [10–14], we have a limited understanding of how heat waves affect biological processes. To address this, we studied how heat wave timing and continuity affect sex ratios and the underlying expression of two genes involved in sexual differentiation, Cyp19A1 and Dmrt1 (involved in female and male sexual development, respectively). We found that varying either heat wave timing or continuity significantly affected sex ratios, and that gene expression was affected differently depending on the continuity of heat exposure. These results have important implications for understanding the potential consequences of heat wave exposure in species with TSD as well as for studying how temperature affects biological processes in general. Here, we show that even slight differences in the timing and/or continuity of heat waves can greatly affect phenotypic outcomes.

While the finding that sensitivity to an environment can change over time is not novel, our findings demonstrate just how quickly this sensitivity can shift. In the timing study, embryos were relatively insensitive to heat waves that began on day 10 but were sensitive to heat waves just 7 days later, and the developmental window in which heat exposure is likely to have the most acute impact on sex ratios is between incubation days 17 and 45 under these temperature conditions (approximately the middle third of development). Further, the embryos remained sensitive to heat waves for approximately three weeks, after which they were again insensitive to heat. The finding that sensitivity to temperature can change quickly has implications for interpreting and predicting how transient increases in temperatures such as heat waves influence the phenotypic response of individuals and populations. For any given individual, whether or not a heat wave hits during their period of sensitivity will dictate their phenotypic response, and at the population level, the proportion of individuals that are sensitive when a heat wave hits will dictate how the population responds. In the example of TSD, it is plausible that one heat wave could induce a sharp increase in the production of female hatchlings while a similar heat wave occurring two weeks later might minimally affect sex ratios. It is worth noting that the treatment with heat exposure on days 38–52 produced only a single female despite the heat exposure occurring almost entirely within the middle third of development (average incubation duration = 75.35 ± 0.42 days for this treatment), suggesting that the exact timing of the TSP is dependent upon the thermal conditions experienced during incubation, as previous research has demonstrated [73]. Aside from this difference, the results generally corroborate with those of constant temperature studies [34–36] demonstrating that temperature affects sex most acutely during the middle third of development (the TSP) under fluctuating conditions. Our results support the previous hypotheses that sex is determined once an embryo has been exposed to a ‘specific dose' of a sex-determining factor [74], and that there is variation between individuals in the threshold of the signal needed before an embryo is committed to one sex or the other. This is evidenced by the fact that none of the temperature treatments produced 100% sex ratios and suggests that variable sex ratios in populations are the result of individual variation in this exposure threshold. These findings highlight the importance of working with a fine temporal resolution (e.g. days to weeks) when examining the effects of temperature on phenotype across an entire population.

In addition to finding that sensitivity to temperature changes over time, we found that the continuity of exposure to warm temperatures also affects phenotypic responses. In both continuity studies, we found that continuous exposure to warm temperatures had a stronger effect on sex ratios, with a continuous 12-day heat wave producing more females compared to treatments with lower continuity. Knowing that embryonic sensitivity to temperature drops later in development, one possible explanation for the effects of continuity on sex ratios is that the discontinuous exposure simply pushed some heat exposure into periods of development when embryos are less sensitive. Another potential explanation is that breaks in the exposure to warm temperatures alter the underlying physiology in a manner that reverses the effects of earlier heat exposure. These two explanations are not mutually exclusive and are likely both operating in nature. Spreading out heat exposure (i.e. introducing discontinuity), automatically results in embryos receiving heat exposure on different days than those receiving a continuous exposure, and if those days have altered sensitivity, then the phenotypic response will be likely to differ. However, our second continuity study demonstrated that just a 3-day break in heat exposure had a significant effect on sex ratios, further emphasizing the importance of analysing how temperature affects biological processes on a finer time scale. The results from the timing study would suggest that the extension of heat exposure by 3 days still remained within periods of similar embryonic sensitivity. Thus, while recent research has demonstrated that even short bouts of heat exposure are sufficient to produce females [67,68], our results show that these short bouts of heat are more likely to induce ovarian development if they are continuous, and that sensitivity to temperature changes quickly. While exposure to warmer temperatures can have a cumulative effect on sex determination [34,60,65,66], our results demonstrate that accumulation can be reversed if exposure to warmer temperatures is discontinuous. Regardless of the underlying mechanisms, both of our continuity studies demonstrate that experiencing a similar number of warm days, but in a discontinuous manner, affects phenotype differently than experiencing those warm days in a continuous manner, and this was supported at the genetic level as well.

The results from our gene expression studies provide insight into how part of the parliamentary system of sex determination responds to heat waves. In the first study that exposed embryos to continuous warm temperatures, Cyp19A1 expression increased 12 days after the start of the heat wave. In the second study, when exposure to warm temperatures only lasted 9 days before returning to cooler temperatures, neither gene exhibited an increase in expression as would be expected if gonadal differentiation was occurring [45]. Therefore, results from the gene expression studies, as well as the heat wave timing study, suggest that gonadal differentiation is probably delayed under the more naturalistic fluctuating conditions that we employed compared to the more traditionally used constant temperature incubations. When eggs are incubated at a constant 26.0°C, Dmrt1 expression starts to rise at stage 16 [45], which would be approximately day 24 at 25.0 ± 3°C in our studies reported here. When eggs are incubated at a constant 31.0°C, Cyp19A1 expression starts to rise at stage 18 [40,55], which would be approximately day 32 at 25.0 ± 3°C in our studies reported here [39,75]. Using an increase in Dmrt1 and Cyp19A1 expression as an indicator that testis or ovary differentiation is taking place, respectively, both studies suggest that this process can take place relatively late in development depending on the timing and continuity of heat exposure. While much more work needs to be done on how gonadal differentiation takes place under fluctuating incubation temperatures, our results demonstrate that the timing of when this takes place is variable, is influenced by the continuity of heat exposure, and is probably delayed compared to constant temperature conditions.

Overall, these findings have important implications for understanding how environmental temperatures affect biological processes. If the continuity of heat exposure can produce different phenotypic outcomes, and if heat waves increase in intensity, duration and frequency as climate change models predict [10–14], there is an increased urgency to investigate how the timing and continuity of heat exposure affects phenotype across taxa. The heat wave conditions that we used in some experiments inserted periods of cooler temperatures between heat waves, providing breaks from heat exposure as is often seen in nature (figure 1). These periods of cooler temperatures resulted in the production of more males and the failure of Cyp19A1 expression to upregulate. However, as climate change progresses, it is likely that these periods of cooler temperatures will decrease in duration and frequency. Therefore, it is imperative to investigate how phenotypic responses to heat exposure change temporally as well as across different degrees of continuity, because continuity of heat exposure will likely increase in the future. We recommend that future studies investigating how more variable temperatures affect biological processes incorporate timing (during development) and continuity of exposure to warm temperatures, as our results show that even small changes in timing or continuity can lead to large differences in phenotype.

5. Conclusion

Here, we demonstrate that heat wave timing and continuity can affect biological processes, producing drastically different phenotypic outcomes, despite no changes in mean conditions across incubation. Thus, accounting for heat wave dynamics other than mean conditions, such as timing and continuity, in ecophysiological studies will allow us to more accurately define how systems operate under natural conditions and inform conservation efforts of thermally sensitive species. More continuous heat conditions produced more females and the upregulation of Cyp19A1, while discontinuous heat conditions produced fewer females and a delayed Cyp19A1 expression response. Current climatic conditions seem to provide periods of cooler temperatures as relief from heat wave conditions, but this may change in the face of a changing climate. In the future, these periods of cooler temperatures may become shorter or less frequent, which could drastically affect sex ratios in species with TSD and other phenotypes in other systems. Collectively, the results show that even short breaks between bouts of warmer temperatures can affect sex ratios and underlying gene expression. Designing and using temperature treatments with high ecological relevancy in studies in species with TSD will further our understanding of how TSD operates under natural conditions, as it will also further our understanding of how temperature affects biological processes across taxa.

Ethics

All work was carried out in accordance with the methodology approved by Illinois State University's Institutional Animal Care and Use Committee as well as the Illinois Department of Natural Resources.

Data accessibility

The datasets generated and analysed during the current studies are available from the Dryad Digital Repository: https://doi.org/10.5061/dryad.hhmgqnkdg [76].

Authors' contributions

All authors contributed to the design of the studies; A.T.B. and A.W.C. conducted field collections; A.T.B. conducted qPCR sampling and gene expression analysis; all authors contributed to the interpretation of results and the writing of the manuscript.

Competing interests

We declare we have no competing interests.

Funding

This multi-year study was carried out under the support of three Weigel grants from the Beta Lambda Chapter of the Phi Sigma Biological Sciences Honors Society, two Grants-in-aid of Research from Sigma Xi, a Steve Kolsto Graduate Scholarship from Illinois Lakes Management Association, and a Helen T. and Frederick M. Gaige award from the American Society of Ichthyologists and Herpetologists to A.T.B.