In nature, many organisms experience a daily range of body temperatures. Thermal performance at stable temperatures is often extrapolated to predict function in cyclical environments. However, temperature order and cyclicity may influence physiological processes. The current study compared energy intake, digestive passage time, and energy budgets across stable temperature (30˚C, 33˚C, 36˚C) and two temperature cycles in lizards (Sceloporus conosbrinus), determining 1) if stable treatments adequately project performance in a cycling environment and 2) if temperature order influences performance. Cycles rotated through 30˚C, 33˚C, 36˚C daily, with equal durations of time at each temperature but differing temperature order, with warm days and cool nights in cycle 1 and cool days and warm nights in cycle 2. For analyses, stable treatments were compiled into a single dataset and compared to cycles. If temperature is the primary factor regulating performance, then performance averages from stable treatments and cycles should compare favorably. However, physiological performance varied based on temperature treatment. Intake and energy budgets were similar between stable trials and cycle 1 but not cycle two. Passage time was quicker in cycle 1 than cycle 2 and stable treatment predictions. Notably, the two cycling regimes consistently varied in performance, indicating that temperature order plays a primary role in regulating performance. Physiological data collection requires careful consideration of effects of cycling versus stable temperature treatments. Stable temperatures do not consistently represent performance in cycling regimes and consideration should be paid not only to which temperatures animals experience, but how temperature is experienced in nature.

Field Collection

            Male and female adult S. consobrinus were collected in northwest Arkansas from 2020 – 2022, ranging in size from 48 – 70mm snout-vent length (n = 39).  All necessary permits were acquired for the research conducted (IACUC #19080, Arkansas Game and Fish Commission Permit #050120211).

Temperature Treatments

Lizards were randomly assigned to one of three temperature treatments, stable 33°C (n = 12), cycle 1 (n = 12), or cycle 2 (n = 15), with males and females as evenly divided as possible. The first cycling temperature treatment (referred to as cycle 1) consisted of a nighttime temperature of 30°C and a daytime temperature of 36°C, with a sunrise and sunset temperature of 33°C during the transition between the low and high temperatures. To compare performance under different orders of temperature cycles the second cycling temperature treatment (referred to as cycle 2) consisted of a nighttime temperature of 36°C and a daytime temperature of 30°C, with a sunrise and sunset temperature of 33°C during the transition between the low and high temperatures.

The amount of time spent at each temperature during cycling trials was identical for a 24-hour period. One full daily cycle totaled 6 hours and 40 minutes at each temperature, with a one-hour transition period for the environmental chamber to reach the next set point. The average body temperature experienced by lizards in both cycles was 33˚C, which is why it was selected for comparison as the stable temperature treatment. The temperature treatments were selected based on body temperature profiles in the field, quantified by inserting an Omega thermocouple (K) into the cloaca immediately upon capture. Body temperatures were maintained during trials using an environmental chamber.

Physiological Data Collection

For lab trials, lizards were housed in plastic containers (41.9 cm x 33 cm x 16.8 cm) lined with butcher paper, a hide box, and water provided ad libitum. Prior to beginning trials lizards were acclimated to their respective temperature treatment for 5 days. At the beginning of the acclimation period lizards were fed one meal to allow for digestion at the treatment temperature, and then fasted to ensure the gut was clear. Temperature was maintained using two environmental chambers ( 0.5ºC). During trials, live Fluker’s 2- and 3-week-old crickets were weighed and then fed to lizards ad-libitum every morning. The uneaten crickets were removed after ~3 hours, and re-weighed and subtracted from the mass offered to determine grams of crickets consumed (weighed to the nearest 0.1 mg). Digestive passage time (hours) represents the time it takes to pass food from consumption to excretion. Metabolizable energy intake is a measure of the maximum potential energy to be allocated to growth, maintenance, storage, and reproduction, and is calculated using the formula:

MEI = C – F – U

where C is energy consumed, F is energy lost as feces, and U is energy lost as uric acid, measured in kilojoules. Assimilated energy represents digestible energy, and is calculated using the formula:

AE = C - F

To begin trials, a single cricket was injected with a marker, which was a slurry made by mixing inert UV-fluorescent powder (Scientific Marking Materials Inc, Seattle, WA) with water (Beaupre et al. 1993; Beaupre and Zaidan 2012). The fluorescent powder associates with feces and does not influence the edibility of crickets, so lizards consumed the mark voluntarily. Trials began when lizards ate the first mark, and time of feeding was noted. Lizard tanks were then monitored every 2 – 4 hours during the day for feces until the fluorescent powder was identified using a UV blacklight, indicating passage time (amount of time from mark consumption to first appearance in feces). After the first mark was excreted, typically 10 or more days were allotted to feed lizards and collect feces and urates for adequate measurement of consumption and bomb calorimetry. Afterwards, a second marker was fed, and tanks were monitored again every 2 – 4 hours. Once the second marker appeared in feces, the trial was considered complete.

            During trials all feces and urates were collected, separated, frozen, and freeze dried. To quantify energy ingested (consumption rate), a wet mass was taken for 10 crickets, which were then freeze dried, and re-weighed. The relationship between wet and dry cricket mass allowed for conversion of wet cricket mass consumed to dry cricket mass consumed. Using a Parr Semimicro Calorimeter, the energy density of crickets was determined, which was used to convert dry cricket mass consumed to kilojoules consumed for each lizard. To determine fecal and urate production (kJ), excrement samples collected during the trials were pooled for individual lizards, weighed, homogenized, and analyzed using a Parr Semimicro Calorimeter. Metabolizable energy intake and AE could then be calculated.

Statistical Analyses

Analyses of Covariance (ANCOVAs) were performed to determine the effect of temperature treatment on energy consumption (kJ), digestive passage time, MEI (kJ) and AE (kJ). Comparisons were made among performance at stable 33˚C, cycle 1, and cycle 2, as the 33 ˚C represents the meant temperature experienced by lizards in each cycle. When assessing energy consumption, snout-vent length (SVL) was also included as a covariate and an interaction term of SVL*treatment to test for heterogenous slopes. For MEI and AE, energy consumption was included as a covariate and an interaction term of energy consumption*treatment was included to test for heterogeneity of slopes. Post-hoc analyses were made by comparing adjusted means and 95% confidence intervals generated from adjusted means (Day and Quinn 1989). Significance among treatments was determined based on non-overlapping confidence intervals with adjusted treatment means, assuming the probability of a type 1 error is 0.05. The residuals of analyses were examined to determine if the assumptions of parametric statistics were met.