The effect of thermopreference on circadian thermoregulation in sprague-dawley and fisher 344 rats

Interstrain differences in thermoregulation of rats are important in biomedical research because subtleties in thermoregulatory sensitivities may greatly affect data collected. Little is known regarding how individual rodent strains differentially utilize behavioral thermal preference to regulate core temperature (Tc). Sprague-Dawley (SD) and Fischer 344 (F344) rats are known to have differences in thermoregulation including heat tolerance and are useful models to study interstrain differences in thermoregulation. Adult male SD and F344 rats of similar body size were implanted with radiotelemetry thermoprobes (DSI) to measure Tc and MA and housed in either a longitudinal temperature gradient with an ambient temperature (Ta) range of ∼15–40 °C to measure selected Ta (STa) or control environment maintained at a Ta of 23 °C. When continuously monitored for 48 h, Tc and MA increased at night, while STa decreased, according to their normal circadian cycle in both strains. SD rats were more active than F344 rats throughout the circadian cycle (SD gradient: day=12.9±1.2 m/h, night=32.1±2.4 m/h; F344 gradient: day=4.1±0.6 m/h, night=16.8±1.8 m/h; p<0.05 interstrain and circadian effects). The STa of each strain was greater during the daytime (SD: 26.4±0.2 °C; F344: 27.8±0.3 °C) than at night (SD: 24.7±0.3 °C; F344: 25.7±0.3 °C) confirming past studies that thermopreference during the day and night is greater than standard room temperature (∼23 °C). Correlations between MA and Tc suggest that MA has a greater effect on Tc in the F344 but not the SD strain when housed in a temperature gradient. There were significant strain differences in Tc depending on whether rats were housed in a temperature gradient. That is, the control F344 rats had a lower Tc during the transition from dark to light compared to rats housed in a gradient. Tc of the SD strain was unaffected by housing in the gradient. Rats are typically housed at a standard room temperature of 23 °C. However, the results demonstrate that when given the opportunity to behaviorally thermoregulate in a temperature gradient, the F344 strain selects a warmer environment that affects the regulation of Tc. This may be important in the experimenters' choice of ambient temperatures to house and study rats and other rodents.     

Ambient temperature-dependent thermoregulatory role of REM sleep

Changes in ambient temperature produce complex effects on sleep–wakefulness. In order to find out the mechanisms involved in temperature-sensitive changes in sleep in rats, their thermal preference, body temperature and sleep were studied before and after the destruction of both peripheral and central warm receptors, by systemic administration of 375 mg/kg capsaicin. Though the pre-treated rats preferred to stay mostly at the ambient temperature of 27 °C, post-treated rats strayed freely into chambers having ambient temperature of 30 °C and 33 °C. Sleep and body temperature of these rats were studied for six hours each, when they were kept at an ambient temperature of 18–36 °C. Total sleep time, especially REM sleep, was maximum at 30 °C in pre-treated rats, but this REM sleep peak at 30 °C disappeared after capsaicin administration. Body temperature increased sharply in post-treated rats, at ambient temperatures above 30 °C. Apart from the ability to defend body temperature at high ambient temperature, avoidance of warm ambient temperature and increase in REM sleep are the behavioral measures which are lost in post-treated rats. Results of this study suggest that the ambient temperature-related increase in REM sleep at 30 °C could be part of the thermoregulatory measures.     

Metabolic depression is delayed and mitochondrial impairment averted during prolonged anoxia in the ghost shrimp, Lepidophthalmus louisianensis (Schmitt, 1935)

Lepidophthalmus louisianensis burrows deeply into oxygen-limited estuarine sediments and is subjected to extended anoxia at low tides. Large specimens (> 2 g) have a lethal time for 50% mortality (LT₅₀) of 64 h under anoxia at 25 °C. Small specimens (< 1 g) have a significantly higher LT₅₀ of 113 h, which is the longest ever reported for a crustacean. Whole body lactate levels rise dramatically under anoxia and exceed 120 μmol g.f.w.^− 1 by 72 h. ATP, ADP, and AMP do not change during 48 h of anoxia, but arginine phosphate declines by over 50%. Thus arginine phosphate may help stabilize the ATP pool. Surprisingly, when compared to the aerobic resting rate, ATP production under anoxia declines only moderately during the first 12 h, and drops to only about 30% between 12 and 48 h. Finally, after 48 h of anoxia, a major metabolic depression to less than 5% occurs. Downregulation of metabolism is delayed in L. louisianensis compared to many invertebrates that exhibit facultative anaerobiosis. Bioenergetic constraints as a result of eventual metabolic depression lead to ionic disturbances like calcium overload and compromised membrane potential of mitochondria. Because these phenomena trigger apoptosis in mammalian species, we evaluated the susceptibility of ghost shrimp mitochondria to opening of the mitochondrial permeability transition pore (MPTP) and associated damage. Energized mitochondria isolated from hepatopancreas possess a pronounced capacity for calcium uptake. Exogenous calcium does not stimulate opening of the MPTP, which potentially could reduce cell death during prolonged anoxia.     

Effect of temperature on isoproterenol-induced increases in left ventricular developed pressure

The goal of this project was to determine the effects of elevated cardiac temperature on preload-dependent and preload-independent regulation of left ventricular developed pressure (LVDP) in Langendorff-perfused, electrically paced (420 bpm), Sprague-Dawley rat hearts. LVDP responses to steady-state isoproterenol infusions (10⁻⁸ M) were determined at 37, 38, 39, and 40 °C. Preload-dependent LVDP was determined at 37 and 40 °C. Isoproterenol-induced LVDP and preload-dependent LVDP time controls were conducted in a separate group maintained at 37 °C. The percent increase in LVDP during isoproterenol infusion significantly decreased at 40 °C to 42±6 (SE), compared to 55±9, 55±6, and 53±7% at 37, 38, and 39 °C, respectively. No significant differences were observed in the percent increase in LVDP to isoproterenol among the corresponding time controls (50±6, 47±3, 56±4, and 56±5%). Preload-dependent LVDP decreased across the experimental protocol, but there were no cardiac temperature effects. These data indicate that β-adrenergic mediated contractility is not altered by moderate heating from normothermia but is compromised at very high temperatures (40 °C). Cardiac temperatures from 37 to 40 °C do not alter the inherent preload-dependent LVDP, indicating that the Frank–Starling relation is not directly affected within this temperature range.     

Endothelial cell preservation at hypothermic to normothermic conditions using clinical and experimental organ preservation solutions

Introduction

Endothelial barrier function is pivotal for the outcome of organ transplantation. Since hypothermic preservation (gold standard) is associated with cold-induced endothelial damage, endothelial barrier function may benefit from organ preservation at warmer temperatures. We therefore assessed endothelial barrier integrity and viability as function of preservation temperature and perfusion solution, and hypothesized that endothelial cell preservation at subnormothermic conditions using metabolism-supporting solutions constitute optimal preservation conditions.Methods: Human umbilical vein endothelial cells (HUVEC) were preserved at 4–37 °C for up to 20 h using Ringer's lactate, histidine–tryptophan–ketoglutarate solution, University of Wisconsin (UW) solution, Polysol, or endothelial cell growth medium (ECGM). Following preservation, the monolayer integrity, metabolic capacity, and ATP content were determined as positive parameters of endothelial cell viability. As negative parameters, apoptosis, necrosis, and cell activation were assayed. A viability index was devised on the basis of these parameters.Results: HUVEC viability and barrier integrity was compromised at 4 °C regardless of the preservation solution. At temperatures above 20 °C, the cells' metabolic demands outweighed the preservation solutions' supporting capacity. Only UW maintained HUVEC viability up to 20 °C. Despite high intracellular ATP content, none of the solutions were capable of sufficiently preserving HUVEC above 20 °C except for ECGM.Conclusion: Optimal HUVEC preservation is achieved with UW up to 20 °C. Only ECGM maintains HUVEC viability at temperatures above 20 °C.     

Diurnally cycling temperature and ventilation affect young turkeys' performance and sensible heat loss

The goals were to elucidate the effects of ventilation rate (VR) coupled with exposure to constant 20 °C or to diurnal temperature cycling on young turkeys' performance and sensible heat loss (SHL). In three experiments male British United Turkeys (BUT), from 3 to 6 weeks of age, were exposed to constant 20 °C, or to 35/25 °C or 30/20 °C diurnal temperature cycling, all at 50% RH and with VR (expressed as air velocity (AV)) ranging from 0.8 to 3.0 m s⁻¹. The 2nd and 3rd of these experiments included a positive control at constant 30 °C and VR of 1.5 m s⁻¹. Weight gain, feed intake, and feed efficiency were measured or calculated, as appropriate; SHL was calculated from measured surface temperature, and plasma concentrations of triiodothyronine (T₃) was determined in the 1st experiment. Changes of VR at constant 20 °C did not affect performance, but total SHL increased significantly with increasing VR. Under the 35/25 °C regime a significantly higher BW was recorded, with a similar pattern of feed efficiency, when VR during the hot part of the cycle was 1.5 or 2.0 m s⁻¹ than when it was 0.8 m s⁻¹. In the 3rd experiment, BW in the 35/20 °C treatment was significantly lower than that of the controls. In all experiments, turkeys maintained body temperature (T_b) within the normothermic range, and SHL varied with VR. It can be concluded that although diurnal temperature cycling reflects the natural situation, exposing young turkeys to constant 30 °C combined with optimal ventilation might yield the best performance results.     

Effect of cold on lipid peroxidation in the brown adipose tissue and liver of rats

1. Lipid peroxidation in the interscapular brown adipose tissue (iBAT) and liver was studied in rats acclimated to room (23±1 °C) and low temperature (5±1 °C, 42 days), as well as in animals exposed to 5±1 °C for 24 h; in addition, the tissue metallothionein (MT) and iron were determined.     

Thermal response and reversibility of prolonged larval diapause in the chestnut weevil, Curculio sikkimensis

Curculio sikkimensis undergoes prolonged larval diapause that is terminated by chilling and warming cycles. To examine the effects of warming temperatures and their duration on diapause termination, we exposed diapause larvae that had not been reactivated after chilling at 5 °C to 20 or 25 °C and chilled them again before incubation at 20 °C. With increasing warming duration at 20 °C, diapause termination after chilling increased and shorter chilling durations became effective. In contrast, few or no larvae warmed at 25 °C terminated diapause after chilling, irrespective of the warming duration. To investigate the effect of warming temperature on diapause intensity, larvae with diapause weakened by initial incubation at 20 °C after the first chilling were subsequently incubated at 15, 20, or 25 °C, then chilled at 5 °C before incubation at 20 °C. Diapause termination increased significantly after the larvae were treated at 15 or 20 °C but decreased significantly after they were treated at 25 °C. The intensification of prolonged diapause at 25 °C was reversed when the larvae were transferred to 20 °C. Diapause intensity in C. sikkimensis therefore decreases at 20 °C, increases at 25 °C, and can be reversed by alternately exposing diapause larvae to 20 and 25 °C. In C. sikkimensis, prolonged diapause does not always proceed in one direction, and its intensity fluctuates in response to ambient temperature conditions.     

Thermal responses of juvenile squaretail mullet (Liza vaigiensis) and juvenile crescent terapon (Terapon jarbua) acclimated at near-lethal temperatures, and the implications for climate change

The negative effects of climate alteration on coral reef fishes receive ever increasing attention; however, implications of rising sea temperatures on fishes inhabiting marine nursery environments are poorly understood. We used critical thermal methodology to quantify critical thermal maxima (CTmaxima) of juvenile squaretail mullet (Liza vaigiensis) and juvenile crescent terapon (Terapon jarbua) captured from shallow seagrass nursery areas around Hoga Island, southeast Sulawesi, Indonesia. We tested the hypothesis that these distantly related fishes, when acclimated to cycling temperatures, would display higher CTmaxima than groups acclimated at constant temperatures. Groups of mullet acclimated to a constant temperature of 37 °C and temperature cycles of 35 to 39 °C or 37 to 41 °C displayed statistically similar mean CTmaxima of 44.7, 44.4 and 44.8 °C, respectively. Likewise, terapon acclimated at temperature cycles of 37 to 40 °C did not display a higher CTmaxima than fish acclimated at a constant temperature of 37 °C, with both acclimation groups' mean CTmaxima equal to 43.8 °C. Acclimation to higher cycling temperatures did not result in significant upper temperature tolerance acquisition for either species; however, mullet values were significantly higher than those seen in terapon (P < 0.0001). These data suggest that mullet and terapon will not suffer direct thermal effects should shallow nursery temperature increases be marginally higher than 1-2 °C above ~ 27 °C, and they provide evidence that the upper thermal tolerance of fishes inhabiting shallow seagrass and mangrove areas can approach the biokinetic limits for vertebrate life. Tropical marine fishes inhabiting fringing nursery environments may have the upper thermal tolerance necessary to endure substantial increases in sea temperatures.     

Effect of temperature on sporulation of Neozygites floridana isolates from different climates and their virulence against the tomato red spider mite, Tetranychus evansi

The fungal pathogen Neozygites floridana Weiser and Muma has been evaluated as a classical biological candidate for introduction into Africa against the invasive tomato red spider mite Tetranychus evansi Baker and Pritchard. In this study, the effect of temperature on sporulation, germination and virulence of three isolates of N. floridana collected from T. evansi in three climatically distinct regions of Brazil and Argentina was determined. Six constant temperatures of 13 °C, 17 °C, 21 °C, 25 °C, 29 °C and 33 °C were tested for their effect on the ability of the three fungal isolates to sporulate, germinate and kill the mites. Six alternating-temperature regimes of 17-13 °C, 21-13 °C, 29-13 °C, 33-13 °C, 33-23 °C, 33-28 °C under a 12 h photophase were also tested to estimate virulence of the three isolates against T. evansi. The Vipos isolate discharged more conidia than isolates from Recife or Piracicaba at all temperatures and sporulation was strongly temperature dependent. Optimal sporulation rates were observed at 25 °C while optimal germination rates were observed at 25 °C and 29 °C. At 29 °C, the shortest mean survival time of T. evansi (3.16 days, 95% CI of 3.05-3.27) was observed for the isolate from Vipos, while the longest LT₅₀ (3.47 days, 95% CI 3.34-3.59) was observed for the isolate from Piracicaba. Mortality of mites increased as the differences between alternating day and night temperatures increased from 8 °C (21-13 °C), to 10 °C (33-23 °C), to 16 °C (29-13 °C), with smallest and highest temperature differences of 4 °C (17-13 °C) and 20 °C (33-13 °C), both producing low mortalities. The overall results suggest that the Vipos isolate is better adapted to a wider range of temperatures than the other isolates tested.     

Hypothermia induction and recovery in free-ranging rats

1.

To avoid anesthesia confounders, free-ranging rats were exposed (4 h) to cool water (CW; 10 °C; 5 cm), warm water (WW; 35 °C; 5 cm) or temperate air (TA; 25 °C) to induce hypothermia, or control for water or novel environment stress, respectively.     

Rapid thermal responses and thermal tolerance in adult codling moth Cydia pomonella (Lepidoptera: Tortricidae)

In order to preserve key activities or improve survival, insects facing variable and unfavourable thermal environments may employ physiological adjustments on a daily basis. Here, we investigate the survival of laboratory-reared adult Cydia pomonella at high or low temperatures and their responses to pre-treatments at sub-lethal temperatures over short time-scales. We also determined critical thermal limits (CTLs) of activity of C. pomonella and the effect of different rates of cooling or heating on CTLs to complement the survival assays. Temperature and duration of exposure significantly affected adult C. pomonella survival with more extreme temperatures and/or longer durations proving to be more lethal. Lethal temperatures, explored between −20 °C to −5 °C and 32 °C to 47 °C over 0.5, 1, 2, 3 and 4 h exposures, for 50% of the population of adult C. pomonella were −12 °C for 2 h and 44 °C for 2 h. Investigation of rapid thermal responses (i.e. hardening) found limited low temperature responses but more pronounced high temperature responses. For example, C. pomonella pre-treated for 2 h at 5 °C improved survival at −9 °C for 2 h from 50% to 90% (p < 0.001). At high temperatures, pre-treatment at 37 °C for 1 h markedly improved survival at 43 °C for 2 h from 20% to 90% (p < 0.0001). We also examined cross-tolerance of thermal stressors. Here, low temperature pre-treatments did not improve high temperature survival, while high temperature pre-treatment (37 °C for 1 h) significantly improved low temperature survival (−9 °C for 2 h). Inducible cross-tolerance implicates a heat shock protein response. Critical thermal minima (CTmin) were not significantly affected by cooling at rates of 0.06, 0.12 and 0.25 °C min⁻¹ (CTmin range: 0.3-1.3 °C). By contrast, critical thermal maxima (CTmax) were significantly affected by heating at these rates and ranged from 42.5 to 44.9 °C. In sum, these results suggest pronounced plasticity of acute high temperature tolerance in adult C. pomonella, but limited acute low temperature responses. We discuss these results in the context of local agroecosystem microclimate recordings. These responses are significant to pest control programmes presently underway and have implications for understanding the evolution of thermal tolerance in these and other insects.     

Thermal adaptability and disease association in common carp (Cyprinus carpio communis) acclimated to different (four) temperatures

The present study was designed to investigate the effect of temperature (20 °C, 24 °C, 28 °C and 32 °C) on the heamato-biochemical and histological alterations of Cyprinus carpio communis. Increase in the temperature showed significant decrease in the serum protein, while a reduced level of blood glucose at high temperature of 32 °C was observed leading to hypoglycemic conditions in the experimental fishes. A significant correlation (P<0.01) was observed between cholesterol (Cho) and triglycerides (TG) for different temperature treatments. Elevated blood urea nitrogen (BUN) at high temperatures was a good indicator of gill osmoregulatory failure. A variation of 86.40% and 38.33%, respectively, was noticed in alanine aminotransferase (ALT) and aspartate aminotransferase (AST) at 32 °C over minimum experimental temperature of 20 °C. The increase in red blood cell (RBC) and Heamoglobin (Hb) concentration is associated with the decrease of mean corpuscular hemoglobin (MCH) and mean corpuscular hemoglobin concentration (MCHC), could be the reason for observed poikilo-anisocytosis. Histological studies of different organs of experimental fishes showed accumulation of MMC's (melanomacrophagic centers) and atrophy of the interrenal tissue on exposure to various levels of temperature. These changes were related to severity of thermal stress, being most marked when high temperature was prolonged during acclimatization. Some fishes were found infested by protozoan parasite at elevated temperature of 32 °C. Increased levels of certain biochemical and haemotological parameters studied were strongly correlated with disease in the Cyprinus carpio communis species.     

Duration tenacity: A method for assessing acclimatory capacity of the Antarctic limpet, Nacella concinna

The limpet, Nacella concinna, collected from the Antarctic Peninsula (67°S), was incubated at − 0.3 °C and 2.9 °C for 9 months to test if the previously reported absence of acclimation capacity in Antarctic marine ectotherms could be due to the extended time it takes for them to adjust their physiology to a new stable state. Acclimation was tested through acute measurements of upper lethal limit and a modified measure of tenacity, that tested muscle capacity by measuring the length of time that N. concinna were able to remain attached to the substratum at different temperatures. Both measures acclimated in response to incubation to the higher temperature. Lethal limits were elevated in N. concinna incubated at 2.9 °C (8.1 ± 0.3 °C) compared to those incubated at − 0.3 °C (6.9 ± 0.4 °C). 2.9 °C incubated N. concinna also had a maximum tenacity at 2.1 °C, a higher temperature than the maximum tenacity of those incubated at − 0.3 °C, which occurred at − 1.0 °C. This study is the first to show that the Antarctic limpet can acclimate its physiology, but that it requires a greater period of time for acclimation to occur than previous studies have allowed for.     

Effects of acclimation and egg-incubation temperature on selected temperature by hatchling western painted turtles (Chrysemys picta bellii)

The ability of hatchling turtles to detect environmental temperature differences and to effectively select preferred temperature is a function that critically impacts survival. In some turtle species, temperature preference may be influenced by embryonic and post-hatching conditions, such as egg-incubation and acclimation temperature. We tested for effects of embryonic incubation temperature (27.5 °C, 30 °C) and acclimation temperature (20 °C, 25 °C) on the selected temperature and movement patterns of 32 Chrysemys picta bellii (Reptilia: Emydidae) hatchlings in an aquatic thermal gradient of 14-34 °C and in single-temperature (20 °C, 25 °C) control tests. Among 10-11 month old hatchlings, acclimation temperature and egg-incubation temperature influenced temperature selection and movement patterns. Acclimation temperature affected activity and movement: in thermal gradient and single-temperature control tests, 25 °C-acclimated turtles relocated between chambers significantly more frequently than individuals acclimated to 20 °C. Acclimation temperature also affected temperature selection: 20 °C-acclimated turtles selected a specific temperature during gradient tests, but 25 °C-acclimated turtles did not. Among 20 °C-acclimated turtles, egg-incubation temperature was inversely related to selected temperature: hatchling turtles incubated at 27.5 °C selected the warmest temperature available (34 °C); individuals incubated at 30 °C selected the coldest temperature (14 °C). These results suggest that interactions of environmental conditions may influence post-hatching thermoregulatory behavior in C. picta bellii, a factor that ultimately affects fitness.     

Effects of gestation temperature on offspring sex and maternal reproduction in a viviparous lizard (Eremias multiocellata) living at high altitude

Temperature-dependent sex determination (TSD) is well studied in many species of reptiles, but little is known on how geographic distribution and altitude affect the sex ratio. In the present study, we focused on a population of a viviparous lizard with TSD (Eremias multiocellata) that lives at high altitudes (≈2900 m) in Tianzhu, Gansu province, China. Gestation temperature had a notable effect on the offspring sex ratio, gestation period, and the mother's body mass. The mothers produced female biased offspring at 25 °C but male biased offspring at 35 °C. All female lizards lost weight during pregnancy, and the least loss of the body mass was observed at 31 °C. The gestation period increased in a non-linear fashion as ambient temperature was reduced. Average litter size was elevated with an increase of gestation temperatures, reached a maximum at 31 °C, and then declined at 35 °C. Compared with a previous study on a Minqin population which lives at a lower altitude (≈1400 m) and warmer climate, the present study obtained a less skewed sex ratio of offspring in the Tianzhu population. Geographic variations also affected offspring morphology between the two populations; females collected from Tianzhu produced larger litters but with a smaller body weight of offspring. These differences may be caused by the adaptive response to the cool climatic and high-altitude environmental conditions.     

Diapause pupal color diphenism induced by temperature and humidity conditions in Byasa alcinous (Lepidoptera: Papilionidae)

We investigated whether diapause pupae of Byasa alcinous exhibit pupal color diphenism (or polyphenism) similar to the diapause pupal color polyphenism shown by Papilio xuthus. All diapause pupae of B. alcinous observed in the field during winter showed pupal coloration of a dark-brown type. When larvae were reared and allowed to reach pupation under short-day conditions at 18 °C under a 60 ± 5% relative humidity, diapause pupae exhibited pupal color types of brown (33%), light-brown (25%), yellowish-brown (21%), diapause light-yellow (14%) and diapause yellow (7%). When mature larvae reared at 18 °C were transferred and allowed to reach pupation at 10 °C and 25 °C under a 60 ± 5% relative humidity after a gut purge, the developmental ratio of brown and light-brown, yellowish-brown, and diapause light-yellow and diapause yellow types was 91.2, 8.8 and 0.0% at 10 °C, and 12.2, 48.8 and 39.0% at 25 °C, respectively. On the other hand, when mature larvae reared at 18 °C were transferred and allowed to reach pupation at 10 °C, 18 °C and 25 °C under an over 90% relative humidity after a gut purge, the developmental ratio of brown and light-brown, yellowish-brown, and diapause light-yellow and diapause yellow types was 79.8, 16.9 and 3.3% at 10 °C, 14.5, 26.9 and 58.6% at 18 °C, and 8.3, 21.2 and 70.5% at 25 °C, respectively. These results indicate that diapause pupae of brown types are induced by lower temperature and humidity conditions, whereas yellow types are induced by higher temperature and humidity conditions. The findings of this study show that diapause pupae of B. alcinous exhibit pupal color diphenism comprising brown and diapause yellow types, and suggest that temperature and humidity experienced after a gut purge are the main factors that affect the diapause pupal coloration of B. alcinous as environmental cues.     

Phenotypic variation in hatchling Chinese ratsnakes (Zaocys dhumnades) from eggs incubated at constant temperatures

We incubated eggs of the Chinese ratsnake Zaocys dhumnades at four constant temperatures (24, 27, 30 and 30 °C) to examine the effects of incubation temperature on hatching success and hatchling phenotypes. Incubation length increased nonlinearly as temperature decreased, with the mean incubation length being 76.7 d at 24 °C, 57.4 d at 27 °C, 47.3 d at 30 °C, and 44.1 d at 33 °C. Hatching successes were lower at the two extreme temperatures (69% at 24 °C, and 44% at 33 °C) than at the other two moderate temperatures (96% at 27 °C, and 93% at 30 °C). Incubation temperature affected nearly all hatchling traits examined in this study. Incubation of Z. dhumnades eggs at 33 °C resulted in production of smaller hatchlings that characteristically had less-developed carcasses but contained more unutilized yolks. Hatchlings from eggs incubated at 27 and 30 °C did not differ in any examined traits. Taking the rate of embryonic development, hatching success and hatchling phenotypes into account, we conclude that the temperature range optimal for incubation of Z. dhumnades eggs is narrower than the range of 24−33 °C but should be wider than the range of 27−30 °C.