Dung beetles show metabolic plasticity as pupae and smaller adult body size in response to increased temperature mean and variance.

Though organisms may use thermal plasticity to cope with novel temperature regimes, our understanding of plastic responses is limited. Research on thermal plasticity has traditionally focused on the response of organisms to shifts in mean temperatures. However, increased temperature variation can have a greater impact on organismal performance than mean temperature alone. In addition, thermal plasticity studies are often designed to investigate plasticity in response to more extreme temperatures despite the fact that organisms make physiological adjustments to diurnal temperature fluctuations that they experience. Using pupae of the dung beetle Onthophagus taurus, we investigated the potential for plasticity in response to increasing temperature mean and variance using thermal regimes that were well within the species critical thermal limits. We reared 40 beetles from egg to pupae (n = 20) or adults (n = 20) at one of nine incubation treatments, including all combinations of three mean temperatures (22, 24, 26 °C) and three amplitudes of fluctuation (±2, ±4, ±8 °C). To measure thermal plasticity of pupae, we quantified CO2 production across a range of temperatures (i.e., 15, 20, 25, and 30 °C) for 20 beetles per treatment. The relationship between CO2 production and temperature provides an estimate of energetic costs at a given temperature (i.e., using the intercept) and thermal sensitivity (i.e., using the slope). We reared the remaining O. taurus in each treatment (n = 20) to adulthood and then recorded mass (g) to determine body size, a proxy for fitness. Pupae exhibited thermal plasticity in response to the additive and interactive effects of temperature mean and variance. Pupae reared in the warmest and most variable treatment (26 ± 8 °C) showed the greatest decrease in overall metabolism compared to all other treatments, and adult beetles from this treatment (26 ± 8 °C) were also significantly smaller than adult beetles from any other treatment. We found that both temperature mean and variance contributed to thermal plasticity of pupae and had consequences for adult body size, a trait related to dung beetle fitness. Importantly, the temperatures we used in our treatments are not extreme and are likely well below the critical thermal maxima of the species, demonstrating that organisms can make adjustments to temperatures they experience across diurnal or seasonal timescales.

Different amplitudes of temperature fluctuation induce distinct transcriptomic and metabolomic responses in the dung beetle Phanaeus vindex.

Most studies exploring molecular and physiological responses to temperature have focused on constant temperature treatments. To gain a better understanding of the impact of fluctuating temperatures, we investigated the effects of increased temperature variation on Phanaeus vindex dung beetles across levels of biological organization. Specifically, we hypothesized that increased temperature variation is energetically demanding. We predicted that thermal sensitivity of metabolic rate and energetic reserves would be reduced with increasing fluctuation. To test this, we examined the responses of dung beetles to constant (20°C), low fluctuation (20±5°C), or high fluctuation (20±12°C) temperature treatments using respirometry, assessment of energetic reserves and HPLC-MS-based metabolomics. We found no significant differences in metabolic rate or energetic reserves, suggesting increased fluctuations were not energetically demanding. To understand why there was no effect of increased amplitude of temperature fluctuation on energetics, we assembled and annotated a de novo transcriptome, finding non-overlapping transcriptomic and metabolomic responses of beetles exposed to different fluctuations. We found that 58 metabolites increased in abundance in both fluctuation treatments, but 15 only did so in response to high-amplitude fluctuations. We found that 120 transcripts were significantly upregulated following acclimation to any fluctuation, but 174 were upregulated only in beetles from the high-amplitude fluctuation treatment. Several differentially expressed transcripts were associated with post-translational modifications to histones that support a more open chromatin structure. Our results demonstrate that acclimation to different temperature fluctuations is distinct and may be supported by increasing transcriptional plasticity. Our results indicate for the first time that histone modifications may underlie rapid acclimation to temperature variation.

Life stages differ in plasticity to temperature fluctuations and uniquely contribute to adult phenotype in Onthophagus taurus dung beetles.

Adaptive thermal plasticity allows organisms to adjust their physiology to cope with fluctuating environments. However, thermal plasticity is rarely studied in response to thermal variability and is often measured in a single life stage. Plasticity in response to thermal variability likely differs from responses to constant temperature or acute stress. In addition, life stages likely differ in their plasticity, and responses in one stage may be affected by the experiences in a previous stage. Increasing the resolution with which we understand thermal plasticity in response to thermal variation across ontogeny is crucial to understanding how organisms cope with the thermal variation in their environment and to estimating the capacity of plasticity to mitigate costs of rapid environmental change. We wanted to know whether life stages differ in their capacity for thermal plasticity under temperature fluctuations. We reared Onthophagus taurus dung beetles in either low or high temperature fluctuation treatments and quantified thermal plasticity of metabolism of pupae and adults. We found that adults were thermally plastic and pupae were not. Next, we tested whether the plasticity observed in the adult life stage was affected by the thermal conditions during development. We again used low and high temperature fluctuation treatments and reared individuals in one condition through all egg to pupal stages. At eclosion, we switched half of the individuals in each treatment to the opposite fluctuation condition and, later, measured thermal plasticity of metabolism in adults. We found that temperature conditions experienced during the adult stage, but not egg to pupal stages, affect adult thermal plasticity. However, temperature fluctuations during development affect adult body size, suggesting that some aspects of the adult phenotype are decoupled from previous life stages and others are not. Our data demonstrate that life stages mount different responses to temperature variability and uniquely contribute to the adult phenotype. These findings emphasize the need to broadly integrate the life cycle into studies of phenotypic plasticity and physiology; doing so should enhance our ability to predict organismal responses to rapid global change and inform conservation efforts.

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.

Red-eared slider hatchlings (Trachemys scripta) show a seasonal shift in behavioral types.

Correlated and repeatable patterns of behavior, termed behavioral types, can affect individual fitness. The most advantageous behavioral type may differ across predictable environments (e.g., seasonally), and maternally mediated effects may match hatchling behavior to the environment. We measured righting response, an indicator of behavioral type, of juvenile red-eared slider turtles (Trachemys scripta) emerging from early and late season clutches to understand if the production of behavioral types differs across the nesting season. There was a significant effect of season, with early season hatchlings righting more quickly than late season hatchlings, and we explored two potential underlying mechanisms, maternal estrogens and maternal investment (e.g., yolk allocation). We dosed early season eggs with an estrogen mixture to mimic late season eggs and assayed hatchling righting response, but found no significant effect of this maternal effect. We assessed maternal investment by measuring egg, hatchling, and residual yolk masses. We found a seasonal pattern in yolk allocation, where early season eggs have more yolk than late season eggs. Early season hatchlings used more yolk for growth rather than maintenance of existing tissues, resulting in larger hatchlings. Interestingly, across both seasons, hatchlings that received less maternal yolk appeared to be more efficient at converting yolk to tissue, but we found no direct correlation with righting behavior. We demonstrate that the prevalence of behavioral types varies across the nesting season, creating correlated suites of seasonal phenotypes in turtle hatchlings, but it appears that neither maternal estrogens or investment in yolk directly underlie this shift in behavior.

Evidence of embryonic regulation of maternally derived yolk corticosterone.

In recent years, the potential for maternal stress effects to adaptively alter offspring phenotype has received considerable attention. This research has identified offspring traits that are labile in response to maternal stress; however, an understanding of the mechanisms underlying these effects is lagging and is crucial to appreciating the significance of this maternal effect. In the present study, we sought to better understand maternal stress effects by examining the potential for embryonic regulation of corticosterone exposure, determining the phenotypic consequences of elevated corticosterone during development, and characterizing the levels of maternally transferred corticosterone in unmanipulated eggs using Trachemys scripta By dosing eggs with tritiated corticosterone and tracking the steroid throughout development, we found that most corticosterone is metabolized, and less than 1% of the corticosterone dose reaches the embryo as free corticosterone. We also found that exogenous dosing of corticosterone, in concentrations sufficient to overwhelm embryonic metabolism, reduces embryonic survival and negatively impacts hatchling traits important to fitness. Our results demonstrate that concentrations of maternal corticosterone in the yolks of unmanipulated eggs are low and are significantly lower than the doses of corticosterone required to elicit phenotypic effects in hatchlings. Taken together, these results provide evidence that both the embryo and the female may minimize corticosterone accumulation in the embryo to avoid reductions in embryonic survival and negative impacts on offspring phenotype and fitness.

Seasonal shifts in sex ratios are mediated by maternal effects and fluctuating incubation temperatures.

Sex-specific maternal effects can be adaptive sources of phenotypic plasticity. Reptiles with temperature-dependent sex determination (TSD) are a powerful system to investigate such maternal effects because offspring phenotype, including sex, can be sensitive to maternal influences such as oestrogens and incubation temperatures.In red-eared slider turtles (Trachemys scripta), concentrations of maternally derived oestrogens and incubation temperatures increase across the nesting season; we wanted to determine if sex ratios shift in a seasonally concordant manner, creating the potential for sex-specific maternal effects, and to define the sex ratio reaction norms under fluctuating temperatures across the nesting season.Eggs from early and late season clutches were incubated under a range of thermally fluctuating temperatures, maternally derived oestradiol concentrations were quantified via radioimmunoassay, and hatchling sex was identified. We found that late season eggs had higher maternal oestrogen concentrations and were more likely to produce female hatchlings. The sex ratio reaction norm curves systematically varied with season, such that with even a slight increase in temperature (0.5°C), late season eggs produced up to 49% more females than early season eggs.We found a seasonal shift in sex ratios which creates the potential for sex-specific phenotypic matches across the nesting season driven by maternal effects. We also describe, for the first time, systematic variation in the sex ratio reaction norm curve within a single population in a species with TSD.

Incubation temperature causes skewed sex ratios in a precocial bird.

Many animals with genetic sex determination are nonetheless capable of manipulating sex ratios via behavioral and physiological means, which can sometimes result in fitness benefits to the parent. Sex ratio manipulation in birds is not widely documented, and revealing the mechanisms for altered sex ratios in vertebrates remains a compelling area of research. Incubation temperature is a key component of the developmental environment for birds, but despite its well-documented effects on offspring phenotype it has rarely been considered as a factor in avian sex ratios. Using ecologically relevant manipulations of incubation temperature within the range 35.0-37.0°C, we found greater mortality of female embryos during incubation than males regardless of incubation temperature, and evidence that more female than male embryos die at the lowest incubation temperature (35.0°C). Our findings in conjunction with previous work in brush turkeys suggest incubation temperature is an important determinant of avian secondary sex ratios that requires additional study, and should be considered when estimating the impact of climate change on avian populations.

Constancy in an inconstant world: moving beyond constant temperatures in the study of reptilian incubation.

Variable environmental conditions can alter the phenotype of offspring, particularly in ectothermic species such as reptiles. Despite this, the majority of studies on development in reptiles have been carried out under constant conditions in the laboratory, raising the question of just how applicable those investigations are to natural conditions? Here, we first review what we have learned from these constant-temperature studies. Second, we examine the importance of temperature fluctuations for development in reptiles and highlight the outcomes of studies conducted under fluctuating conditions. Next, we report our findings from a new study that examines how the frequency of fluctuations in temperature experienced during development affects phenotype. Finally, we suggest some areas in need of additional research so that we can better understand the complex interactions of temperature and physiology, particularly in species with temperature-dependent sex determination. For questions aimed at understanding the complex effects of the environment on phenotype, we must move toward studies that better capture environmental variation. By taking such an approach, it may be possible to predict more accurately how these thermally sensitive organisms will respond to environmental perturbations, including climatic change.

Deposition of pathogenic Mycoplasma gallisepticum onto bird feeders: host pathology is more important than temperature-driven increases in food intake.

Although ambient temperature has diverse effects on disease dynamics, few studies have examined how temperature alters pathogen transmission by changing host physiology or behaviour. Here, we test whether reducing ambient temperature alters host foraging, pathology and the potential for fomite transmission of the bacterial pathogen Mycoplasma gallisepticum (MG), which causes seasonal outbreaks of severe conjunctivitis in house finches (Haemorhous mexicanus). We housed finches at temperatures within or below the thermoneutral zone to manipulate food intake by altering energetic requirements of thermoregulation. We predicted that pathogen deposition on bird feeders would increase with temperature-driven increases in food intake and with conjunctival pathology. As expected, housing birds below the thermoneutral zone increased food consumption. Despite this difference, pathogen deposition on feeders did not vary across temperature treatments. However, pathogen deposition increased with conjunctival pathology, independently of temperature and pathogen load, suggesting that MG could enhance its transmission by increasing virulence. Our results suggest that in this system, host physiological responses are more important for transmission potential than temperature-dependent alterations in feeding. Understanding such behavioural and physiological contributions to disease transmission is critical to linking individual responses to climate with population-level disease dynamics.