DOI: 10.5061/dryad.2rbnzs7w2
title: Cycling temperature treatments and digestion in prairie lizards (Sceloporus consobrinus)

description: 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.
description: <em>Field Collection</em>
             Male and female adult <em>S. consobrinus
 </em>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).
 <em>Temperature Treatments</em>
 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.
 <em>Physiological Data Collection </em>
 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.
 <em>Statistical Analyses</em>
 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 (<em>Day and Quinn 1989)</em>. 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.
description: # Cycling Temperature Treatments and Digestion in Prairie Lizards
 (Sceloporus consobrinus) 
 The dataset contains all information used in the analysis. Physiological
 data were collected at 33C stable temperature, and under two cycling
 temperature treatments. We looked at food consumption rate using voluntary
 consumption with crickets, in first grams which was converted to
 kilojoules. We also quantified digestive passage time in hours, the time
 from consuming food to excreting it. We quantified energy excreted in
 grams which was converted to kilojoules, for both urates and feces.
 Lastly, we calculated metabolizable energy intake and assimilated energy
 considering food consumed (kJ) and excreted (kJ). ## Description of the
 data and file structure The columns are titled according to the data
 contained: * Lizard - This column is a unique ID indicating which lizard
 was used. These are specific to our lab, but indicate each lizard was
 unique. * Temperature - Temperature refers to the temperature treatment
 the data were collected at. "33" is a stable, constant, 33C
 temperature at which data were collected for the entire row. "cycle
 1" refers to lizards who were at 36C during the day, 33C at sunrise
 and sunset, and 30C at night, for the entire row of data collected.
 "cycle 2" refers to lizards who were at 30C during the day, 33C
 at sunrise and sunset, and 36C at night, for the entire row of data
 collected. * total excrement mass (g) urate - This refers to grams
 excreted per lizard for the whole trial * calories/gram urate - This
 refers to the output from the calorimeter of calories per gram in the
 urate sample for that lizard. * kJ/gram fecal - This refers to the
 kilojoules per gram in the fecal sample for that lizard. * kJ excreted
 urate - This refers to the total kilojoules excreted by that lizard in
 urates. * wet consumed - This refers to wet grams of crickets consumed by
 that lizard. * dry consumed - This refers to the dry mass of crickets
 consumed by that lizard. * calories consumed - This refers to total
 calories the lizard consumed. * kJ consumed - This refers to the
 kilojoules of energy the lizard consumed. * MEI - This is metabolizable
 energy intake calculated as kJ consumed - fecal kJ - urate kJ. * AE - This
 is assimilated energy calculated as kJ consumed - fecal kJ. * total
 excreted mass (g) fecal - This refers to grams of feces for that lizard. *
 calories per gram fecal - This refers to the calorimeter output of
 calories/gram in feces for that lizard. * kJ/gram urate - This refers to
 kilojoules/gram in urates for that lizard. * kJ excreted fecal - This is
 the total kJ excreted for that lizard. * passagetime - This is hours on
 average to excrete the marked food item, based on mark 1 and 2. * pt1 -
 This is the hours to pass mark 1. * pt2 - This is the hours to pass mark
 2. * svl - This is the lizard's snout-vent length in mm. * days -
 This refers to days in the trial. * meiday - This refers to mei per day,
 by dividing mei/days. * conday - This refers to kJ consumed per day, by
 dividing kJ/days. * aeday - This refers to ae per day, by dividing
 ae/days.