Classification of sex-dependent specific behaviours by tri-axial acceleration in the tegu lizard Salvator merianae.

Validated patterns of behaviour detected by tri-axial acceleration in the laboratory can be used for remote measurements of free-living animals. The tegu lizard naturally occupies diverse biomes in South America and presents ecological threats in regions where it was artificially introduced. We aimed to validate the use of tri-axial acceleration to distinguish among behaviours of male and female tegus in captivity by comparing observed behaviours to recorded acceleration data. Adult animals were externally fitted with an accelerometer fixed between their scapulae to quantify anteroposterior, lateral, and dorsoventral acceleration. Video recordings of cameras positioned on the walls of the animal-holding arena documented behaviours. Behaviour patterns, such as resting, walking, and eating, were identified for both sexes, and nest building in females and courtship and copulation in males. Random Forest algorithm was used to validate the behaviour patterns from accelerometry data based on two models, random split (70 % training-30 % validation; RS) and leave-one-out (divided by individual; LOO). Although LOO showed lower accuracies than RS for all the acceleration data, nest building in females and copulation in males had high accuracies in both models. In contrast, the lowest accuracies for walking and eating indicates they may involve more inconsistent movement patterns. Comparing the results from RS and LOO, female behaviours may be more identifiable in the field using triaxial accelerometry than males. The identification of behaviours by accelerometry, especially related to reproduction, without the necessity for direct observation of the tegus would be helpful for conservation purposes, for both natural and invasive populations.

Long-term effects on cardiorespiratory and behavioral responses in male and female rats prenatally exposed to cannabinoid.

Development of the respiratory system can be affected by the use of drugs during pregnancy, as the prenatal phase is highly sensitive to pharmacological interventions, resulting in long-term consequences. The deleterious effects of external cannabinoids during gestation may be related to negative interference in central nervous system formation, cardiorespiratory system function, and behavioral disorders. Nevertheless, the impact of external cannabinoids on cardiorespiratory network development, chemosensitivity, and its future consequences in adulthood is still unclear. We evaluated the effects of prenatal exposure to a synthetic cannabinoid (WIN 55,212-2, 0.5 mg·kg-1·day-1) on the cardiorespiratory control and panic-like behavior of male and female rats in adulthood. Exogenous cannabinoid exposure during pregnancy resulted in a sex-dependent difference in breathing control. Specifically, males showed increased chemosensitivity to CO2 and O2, whereas females exhibited decreased sensitivity. Altered cardiovascular control was evident, with prenatally treated males and females being more susceptible to hypertension and tachycardia under adverse environmental conditions. Moreover, WIN-treated males exhibited higher fragmentation of sleep episodes, whereas females displayed anxiolytic and panicolytic behavioral responses to CO2. However, no changes were observed in the mechanical component of the respiratory system, and there were no neuroanatomical alterations, such as changes in the expression of CB1 receptors in the brainstem or in the quantification of catecholaminergic and serotonergic neurons. These findings highlight that external interference in cannabinoid signaling during fetal development causes sex-specific, long-lasting effects for the cardiorespiratory system and behavioral responses in adulthood.NEW & NOTEWORTHY The surge in recreational cannabis use and cannabinoid-based medication prescription among pregnant women has been notable in recent years, fueled by the misconception that natural products are inherently safe. Significant gaps persist regarding the potential risks of maternal consumption of cannabinoids and the long-term effects on the cardiorespiratory system of their offspring, which may be determined by sex. Accordingly, this research aims to diminish this lack of information and raise a note of caution.

Locus coeruleus noradrenaline depletion and its differential impact on CO2-induced panic and hyperventilation in male and female mice.

CO2 exposure has been used to investigate the panicogenic response in patients with panic disorder. These patients are more sensitive to CO2, and more likely to experience the "false suffocation alarm" which triggers panic attacks. Imbalances in locus coeruleus noradrenergic (LC-NA) neurotransmission are responsible for psychiatric disorders, including panic disorder. These neurons are sensitive to changes in CO2/pH. Therefore, we investigated if LC-NA neurons are differentially activated after severe hypercapnia in mice. Further, we evaluated the participation of LC-NA neurons in ventilatory and panic-like escape responses induced by 20% CO2 in male and female wild type mice and two mouse models of altered LC-NA synthesis. Hypercapnia activates the LC-NA neurons, with males presenting a heightened level of activation. Mutant males lacking or with reduced LC-NA synthesis showed hypoventilation, while animals lacking LC noradrenaline present an increased metabolic rate compared to wild type in normocapnia. When exposed to CO2, males lacking LC noradrenaline showed a lower respiratory frequency compared to control animals. On the other hand, females lacking LC noradrenaline presented a higher tidal volume. Nevertheless, no change in ventilation was observed in either sex. CO2 evoked an active escape response. Mice lacking LC noradrenaline had a blunted jumping response and an increased freezing duration compared to the other groups. They also presented fewer racing episodes compared to wild type animals, but not different from mice with reduced LC noradrenaline. These findings suggest that LC-NA has an important role in ventilatory and panic-like escape responses elicited by CO2 exposure in mice.

Exercise derived myokine irisin as mediator of cardiorespiratory, metabolic and thermal adjustments during central and peripheral chemoreflex activation.

Exercise elicits physiological adaptations, including hyperpnea. However, the mechanisms underlying exercise-induced hyperpnea remain unresolved. Skeletal muscle acts as a secretory organ, releasing irisin (IR) during exercise. Irisin can cross the blood-brain barrier, influencing muscle and tissue metabolism, as well as signaling in the central nervous system (CNS). We evaluated the effect of intracerebroventricular or intraperitoneal injection of IR in adult male rats on the cardiorespiratory and metabolic function during sleep-wake cycle under room air, hypercapnia and hypoxia. Central IR injection caused an inhibition on ventilation (VE) during wakefulness under normoxia, while peripheral IR reduced VE during sleep. Additionally, central IR exacerbates hypercapnic hyperventilation by increasing VE and reducing oxygen consumption. As to cardiovascular regulation, central IR caused an increase in heart rate (HR) across all conditions, while no change was observed following peripheral administration. Finally, central IR attenuated the hypoxia-induced regulated hypothermia and increase sleep episodes, while peripheral IR augmented CO2-induced hypothermia, during wakefulness. Overall, our results suggest that IR act mostly on CNS exerting an inhibitory effect on breathing under resting conditions, while stimulating the hypercapnic ventilatory response and increasing HR. Therefore, IR seems not to be responsible for the exercise-induced hyperpnea, but contributes to the increase in HR.

Temperature effects on metabolism and energy requirement during the fast growth phase in the red-footed tortoise, Chelonoidis carbonaria.

Early life is a challenging phase because of the high rates of morphophysiological development and growth. Changes in ambient temperature, which directly affect energy metabolism and digestive functions in ectotherms, may be of great impact during this phase. We addressed this issue in red-footed tortoise (Chelonoidis carbonaria) hatchlings kept in captivity. To this end, we investigated the effect of temperature (28 °C and 18 °C) on mass-specific gross energy intake (GEIm), daily body mass gain (MG), daily intake of gross energy (GEI), digestible energy (DEI), resting metabolic rate (RMR), and specific dynamic action (SDA) components during different seasons in the first 13 months after hatching. Greater GEIm and MG were observed in spring (381.7 ± 84.9 J.g-0.86.day-1 and 0.9 ± 0.4 g.day-1) and summer (356.9 ± 58.9 J.g-0.86.day-1 and 1.0 ± 0.4 g.day-1). The highest and lowest RMRs at 28 °C were observed in spring (36.4 ± 5.1 kJ.kg-1.day-1) and winter (22.4 ± 6.2 kJ.kg-1.day-1), respectively. Regardless season, hatchlings showed greater GEI and DEI, O2 consumption, CO2 production, RMR, maximum metabolic rate after feeding (FMRMAX), and heat increment (FMRMAX- RMR) at 28 °C compared to 18 °C. In addition, the significant body mass influence showed allometric exponents of 0.62 at 28 °C and 0.92 at 18 °C for RMR. Our results indicate an important effect of environmental temperature on energy requirements and utilization in C. carbonaria hatchlings, which is seasonally influenced even in this early phase of life.

Inhibition of Pro-Inflammatory Microglia with Minocycline Improves Cognitive and Sleep-Wake Dysfunction Under Respiratory Stress in a Sporadic Model for Alzheimer's Disease.

BACKGROUND: Neuroinflammation in Alzheimer's disease (AD) can occur due to excessive activation of microglia in response to the accumulation of amyloid-ß peptide (Aß). Previously, we demonstrated an increased expression of this peptide in the locus coeruleus (LC) in a sporadic model for AD (streptozotocin, STZ; 2 mg/kg, ICV). We hypothesized that the STZ-AD model exhibits neuroinflammation, and treatment with an inhibitor of microglia (minocycline) can reverse the cognitive, respiratory, sleep, and molecular disorders of this model. OBJECTIVE: To evaluate the effect of minocycline treatment in STZ model disorders. METHODS: We treated control and STZ-treated rats for five days with minocycline (30 mg/kg, IP) and evaluated cognitive performance, chemoreflex response to hypercapnia and hypoxia, and total sleep time. Additionally, quantification of Aß, microglia analyses, and relative expression of cytokines in the LC were performed. RESULTS: Minocycline treatment improved learning and memory, which was concomitant with a decrease in microglial cell density and re-establishment of morphological changes induced by STZ in the LC region. Minocycline did not reverse the STZ-induced increase in CO2 sensitivity during wakefulness. However, it restored the daytime sleep-wake cycle in STZ-treated animals to the same levels as those observed in control animals. In the LC, levels of A and expression of Il10, Il1b, and Mcp1 mRNA remained unaffected by minocycline, but we found a strong trend of minocycline effect on Tnf- α. CONCLUSION: Our findings suggest that minocycline effectively reduces microglial recruitment and the inflammatory morphological profile in the LC, while it recovers cognitive performance and restores the sleep-wake pattern impaired by STZ.

Heat stress affects breathing and metabolism of chicks incubated at high temperature.

Global warming poses serious implications to animal physiology and a gradual increase in ambient temperature affects all living organisms, particularly fast-growing selected species. We recorded ventilation (V̇E), body temperature (TB), oxygen consumption (V̇O2) and respiratory equivalent (V̇E/V̇O2) of 14-day-old (14d) male and female chicks at room air conditions, hypercapnia and hypoxia at heat stress (HS, 32 °C). These chicks had previously been exposed to control (CI, 37.5 °C) and high (HI, 39 °C) temperatures during the first 5 days of incubation. Under resting conditions, acute HS increased V̇E in HI females but not in HI males. Hypercapnia combined with heat promoted a potentiation of CO2-hyperventilatory response in HI females when compared with thermoneutral condition, whereas in HI incubated males a hypoventilation under hypercapnia and heat stress was observed compared to the CI group. Hypoxia associated with heat stress increased V̇E only in HI females. Our data indicates that females are more sensitive to thermal manipulation during incubation and it seems that the thermal embryonic manipulation, at least during the first days of development, does not improve the adaptive response of chicks to heat stress.

Sex- and age-specific respiratory alterations induced by prenatal exposure to the cannabinoid receptor agonist WIN 55,212-2 in rats.

BACKGROUND AND PURPOSE: Cannabis legalization has risen in many countries, and its use during pregnancy has increased. The endocannabinoid system is present in the CNS at early stages of embryonic development, and regulates functional brain maturation including areas responsible for respiratory control, data on the influence of external cannabinoids on the development of the respiratory system and possible consequences during postnatal life are limited. EXPERIMENTAL APPROACH: We evaluated the effects of prenatal exposure to synthetic cannabinoid (WIN 55,212-2 [WIN], 0.5 mg·kg-1 ·day-1 ) on the respiratory control system in neonatal (P0, P6-7 and P12-13) and juvenile (P27-28) male and female rats. KEY RESULTS: WIN administration to pregnant rats interfered sex-specifically with breathing regulation of offspring, promoting a greater sensitivity to CO2 at all ages in males (except P6-7) and in juvenile females. An altered hypoxic chemoreflex was observed in P0 (hyperventilation) and P6-7 (hypoventilation) males, which was absent in females. Along with breathing alterations, brainstem analysis showed an increase in the number of catecholaminergic neurons and cannabinoid receptor type 1 (CB1 ) and changes in tissue respiration in the early males. A reduction in pulmonary compliance was observed in juvenile male rats. Preexposure to WIN enhanced spontaneous apnoea and reduced the number of serotoninergic (5-HT) neurons in the raphe magnus nucleus of P0 females. CONCLUSIONS AND IMPLICATIONS: These data demonstrate that excess stimulation of the endocannabinoid system during gestation has prolonged and sex-specific consequences for the respiratory control system.

Intra-uterine diazepam exposure decreases the number of catecholaminergic and serotoninergic neurons of neonate rats.

Benzodiazepines, such as diazepam (DZP), are used to treat anxiety disorders, and are prescribed to pregnant woman for therapeutic purposes. Concerns regarding their consequences on postnatal development rise as they cross the placenta and interact with the embryo. Occurrence of malformation and behavioral syndromes have been reported for different ages, but little is known about their effects on the brain after exposure during intrauterine life. Thus, we sought to evaluate the effects of intrauterine exposure to DZP on the number of brainstem's catecholaminergic and serotonergic neurons, implicated in respiratory control, in male and female rats on postnatal (P) day 12-13, using immunofluorescence labeling for tyrosine-hydroxylase (TH) and serotonin (5-HT). We observed a reduction in the number of catecholaminergic neurons for males and females. Special attention is given to the reduction in the density of neurons in the A6 region, involved in ventilatory responses to CO2. Interestingly, only males showed a reduction in the number of serotonergic neurons, while females were not affected. These findings suggest that in utero exposure to DZP results in deleterious neuroanatomical effects on P12-13 rats and raises a note of concern for women clinicians to make more informed choices about the use of anxiolytic treatments during gestation.

Metabolic and respiratory chemosensitivity and brain monoaminergic responses to cold exposure in chicks subjected to thermal manipulation during incubation.

We evaluated ventilation (V˙E), body temperature (TB), oxygen consumption (V˙ O2), respiratory equivalent (V˙E/ V˙ O2), and monoamine concentrations of 14-day-old (14d) male and female chicks from eggs incubated at low (LT, 36 °C), control (CT, 37.5 °C) and high (HT, 39 °C) temperature during the early embryonic phase, to normoxia, hypercapnia and hypoxia under exposure to cold environment (20 °C). At normoxia, acute cold exposure did not affect the ventilatory variables, with the exception of HT males, in which cold prevented the reduced V˙E observed under thermoneutral conditions. Exposure to 20 °C caused a decrease in TB in both sexes, and LT and HT females presented a greater hypothermic response. Hypercapnia combined with cold did not alter the ventilatory variables, but LT females and CT males and females showed a blunted CO2-induced hyperventilation due to a higher V˙ O2, compared to the same groups in thermoneutral conditions. Unlike with thermoneutral conditions, the blunted hypercapnic hyperventilation observed in the HT groups was not observed during cold challenge. CO2 exposure promoted a similar decrease in TB in the thermoneutral and acutely cold exposed groups, while LT females under cold condition presented a blunted hypothermic response. During hypoxia, cold challenge attenuated the increase in V˙E in LT females and HT males, due to changes in VT. Hypoxic metabolic depression was greater in LT females and males and HT males during cold exposure, while no change in V˙E/ V˙ O2 was observed. The only alteration in monoaminergic concentration under cold challenge was an increase in brainstem 5-HIAA and 5-HIAA/5-HT ratio in HT females, and an enhanced 5-HT concentration in HT males. In summary, thermal manipulation during embryogenesis induces 14d old chicks to respond differently to cold stress with LT females and HT males being more sensitive.

Cutaneous TRPV4 Channels Activate Warmth-Defense Responses in Young and Adult Birds.

Transient receptor potential vanilloid 4 (TRPV4) channels are sensitive to warm ambient temperatures (Tas), triggering heat loss responses in adult rats in a Tas range of ∼26-30°C. In birds, however, the thermoregulatory role of TRPV4 has never been shown. Here, we hypothesized that stimulation of TRPV4 induces thermolytic responses for body temperature (Tb) maintenance in birds, and that this function is already present in early life, when the Ta range for TRPV4 activation does not represent a warm condition for these animals. We first demonstrated the presence of TRPV4 in the dorsal and ventral skin of chickens (Gallus gallus domesticus) by immunohistochemistry. Then, we evaluated the effects of the TRPV4 agonist, RN1747, and the TRPV4 antagonists, HC067047 and GSK2193874, on Tb and thermoeffectors at different Tas in 5-day-old chicks and 60-day-old adult chickens. For the chicks, RN1747 transiently reduced Tb both in thermoneutrality (31°C) and in a cold Ta for this phase (26°C), which relied on huddling behavior inhibition. The TRPV4 antagonists alone did not affect Tb or thermoeffectors but blocked the Tb decrease and huddling inhibition promoted by RN1747. For the adults, TRPV4 antagonism increased Tb when animals were exposed to 28°C (suprathermoneutral condition for adults), but not to 19°C. In contrast, RN1747 decreased Tb by reducing metabolic rate and activating thermal tachypnea at 19°C, a Ta below the activation range of TRPV4. Our results indicate that peripheral TRPV4 receptors are functional in early life, but may be inhibited at that time when the range of activation (∼26-30°C) represents cold Ta for chicks, and become physiologically relevant for Tb maintenance when the activation Ta range for TRPV4 becomes suprathermoneutral for adult chickens.

Sexually dimorphic effects of prenatal diazepam exposure on respiratory control and the monoaminergic system of neonate and young rats.

Pregnancy is highly affected by anxiety disorders, which may be treated with benzodiazepines, especially diazepam (DZP), that can cross the placental barrier and interact with the fetal GABAergic system. We tested whether prenatal exposure to DZP promotes sex-specific postnatal changes in the respiratory control of rats. We evaluated ventilation ([Formula: see text]) and oxygen consumption ([Formula: see text] O2) in resting conditions and under hypercapnia (7% CO2) and hypoxia (10% O2) in newborn [postnatal day (P) 0-1 and P12-13)] and young (P21-22) rats from mothers treated with DZP during pregnancy. We also analyzed brainstem monoamines at the same ages. DZP exposure had minimal effects on room air-breathing variables in females, but caused hypoventilation (drop in [Formula: see text]/[Formula: see text] O2) in P12-13 males, lasting until P21-22. The hypercapnic ventilatory response was attenuated in P0-1 and P12-13 DZP-treated females mainly by a decrease in tidal volume (VT), whereas males had a reduction in respiratory frequency (fR) at P12-13. Minor changes were observed in hypoxia, but an attenuation in [Formula: see text] was seen in P12-13 males. In the female brainstem, DZP increased dopamine concentration and decreased 5-hydroxyindole-3-acetic acid (5-HIAA) and the 3,4-dihydroxyphenylacetic acid (DOPAC)/dopamine ratio at P0-1, and reduced DOPAC concentration at P12-13. In males, DZP decreased brainstem noradrenaline at P0-1. Our results demonstrate that prenatal DZP exposure reduces CO2 chemoreflex only in postnatal females and does not affect hypoxia-induced hyperventilation in both sexes. In addition, prenatal DZP alters brainstem monoamine concentrations throughout development differently in male and female rats.

Prenatal fluoxetine has long-lasting, differential effects on respiratory control in male and female rats.

Serotonin (5-HT) is an important modulator of brain networks that control breathing. The selective serotonin reuptake inhibitor fluoxetine (FLX) is the first-line antidepressant drug prescribed during pregnancy. We investigated the effects of prenatal FLX exposure on baseline breathing, ventilatory and metabolic responses to hypercapnia and hypoxia as well as number of brainstem 5-HT and tyrosine hydroxylase (TH) neurons of rats during postnatal development (P0-82). Prenatal FLX exposure of males showed a lower baseline V̇e that appeared in juveniles and remained in adulthood, with no sleep-wake state dependency. Prenatal FLX exposure of females did not affect baseline breathing. Juvenile male FLX showed increased CO2 and hypoxic ventilatory responses, normalizing by adulthood. Alterations in juvenile FLX-treated males were associated with a greater number of 5-HT neurons in the raphe obscurus (ROB) and raphe magnus (RMAG). Adult FLX-exposed males showed greater number of 5-HT neurons in the raphe pallidus (RPA) and TH neurons in the A5, whereas reduced number of TH neurons in A7. Prenatal FLX exposure of female rats was associated with greater hyperventilation induced by hypercapnia at P0-2 and juveniles, whereas P12-14 and adult FLX (non-rapid eye movement, NREM sleep) rats showed an attenuation of the hyperventilation induced by CO2. FLX-exposed females had fewer 5-HT neurons in the RPA and reduced TH A6 density at P0-2; and greater number of TH neurons in the A7 at P12-14. These data indicate that prenatal FLX exposure affects the number of some monoaminergic regions in the brain and results in long-lasting, sex-specific changes in baseline breathing pattern and ventilatory responses to respiratory challenges.NEW & NOTEWORTHY Selective serotonin reuptake inhibitors (SSRIs) readily cross the placental and the fetal blood-brain barrier where it will affect 5-HT levels in the developing brain. Although SSRI is used during pregnancy, there are no studies showing SSRI exposure during late pregnancy and postnatal effects on breathing control in males and females. We demonstrated that fluoxetine exposure during late pregnancy in rats was associated with long-lasting, sex-specific effects on breathing and brainstem monoaminergic groups.

Starch and fiber intake effects on energy metabolism, growth, and carapacial scute pyramiding of red-footed tortoise hatchlings (Chelonoidis carbonaria).

Tortoise husbandry includes reports of excessive growth and carapace pyramiding, although triggers still remain to be fully elucidated. Juvenile red-footed tortoises (Chelonoidis carbonaria) were fed with two different diets, one high in fiber (HF; 14.2% crude fiber; 39.2% neutral detergent fiber, NDF; dry matter basis, DMB) and one high in starch (HS; 27.7% DMB), to assess effects on energy metabolism, nutrient digestibility, and growth. A total of 20 hatchlings (10 per diet) were used to evaluate: apparent digestibility coefficients (Da) of nutrients and gross energy (GE), passage times at 5 and 11 months of age; resting and post-prandial metabolic rates at 6 and 12 months of age; growth rates; pyramiding; and estimated body composition. Animals fed HS showed higher mass-specific intake of digestible energy (113.9 ± 32.1 kJ kg-1 day-1 vs. 99.6 ± 35.3 kJ kg-1 day-1; P < 0.05), digestible DM (6.1 ± 1.8 g kg-1 day-1 vs. 5.0 ± 1.8 g kg-1 day-1; P < 0.01), shorter transit (3 ± 1 days vs. 4 ± 1 days; P < 0.01) and retention times (8 ± 2 days vs. 10 ± 2 days; P < 0.01), and higher Da of DM, starch, NDF, and GE. Crude protein Da was higher for HF. Rest and post-prandial metabolic rates, and pyramiding degree were not affected by diets. At 13 months, the animals from HS presented wider plastrons and carapaces, and higher carapace width growth rates. In addition, these animals had lower body mineral content (1.88 ± 0.15% vs. 2.15 ± 0.19%; P < 0.01) and bone density (0.13 ± 0.01 g mm-2 vs. 0.15 ± 0.02 g mm-2; P < 0.02). Results provide evidence that highly digestible foods can accelerate shell growth and lower mineralization in this species.

Seasonal changes in steroid and thyroid hormone content in shed skins of the tegu lizard Salvator merianae.

Sampling blood for endocrine analysis from some species may not be practical or ethical. Quantification of hormones extracted from nontypical sample types, such as keratinized tissues, offers a less invasive alternative to the traditional collection and analysis of blood. Here, we aimed to validate assays by using parallelism and accuracy tests for quantification of testosterone, corticosterone, progesterone, and triiodothyronine (T3) in shed skins of tegu lizards. We assessed whether hormone content of sheds varied across one year similar to what was previously detected in plasma samples. In addition, we aimed to identify the phase relationship between hormone levels of shed skin and plasma levels obtained from the same animals. High frequency of shedding occurred during the active season for tegus (spring/summer), while shedding ceased during hibernation (winter). All hormones measured in shed skins exhibited seasonal changes in concentration. Levels of testosterone in shed skins of male tegus correlated positively with plasma testosterone levels, while corticosterone in both males and females exhibited an inverse relationship between sample types for the same month of collection. An inverse relationship was found when accounting for a lag time of 3 and 4 months between sheds and plasma testosterone. These results indicate that endocrine content of sheds may be confounded by factors (i.e., seasons, environmental temperature, thermoregulatory behavior, among others) that affect frequency of molting, skin blood perfusion, and therefore hormone transfer from the bloodstream and deposition in sheds of squamates.

Thermal Acclimation to the Highest Natural Ambient Temperature Compromises Physiological Performance in Tadpoles of a Stream-Breeding Savanna Tree Frog.

Amphibians may be more vulnerable to climate-driven habitat modification because of their complex life cycle dependence on land and water. Considering the current rate of global warming, it is critical to identify the vulnerability of a species by assessing its potential to acclimate to warming temperatures. In many species, thermal acclimation provides a reversible physiological adjustment in response to temperature changes, conferring resilience in a changing climate. Here, we investigate the effects of temperature acclimation on the physiological performance of tadpoles of a stream-breeding savanna tree frog (Bokermannohyla ibitiguara) in relation to the thermal conditions naturally experienced in their microhabitat (range: 18.8-24.6°C). We quantified performance measures such as routine and maximum metabolic rate at different test (15, 20, 25, 30, and 34°C) and acclimation temperatures (18 and 25°C). We also measured heart rate before and after autonomic blockade with atropine and sotalol at the respective acclimation temperatures. Further, we determined the critical thermal maximum and warming tolerance (critical thermal maximum minus maximum microhabitat temperature), which were not affected by acclimation. Mass-specific routine and mass-specific maximum metabolic rate, as well as heart rate, increased with increasing test temperatures; however, acclimation elevated mass-specific routine metabolic rate while not affecting mass-specific maximum metabolic rate. Heart rate before and after the pharmacological blockade was also unaffected by acclimation. Aerobic scope in animals acclimated to 25°C was substantially reduced, suggesting that physiological performance at the highest temperatures experienced in their natural habitat is compromised. In conclusion, the data suggest that the tadpoles of B. ibitiguara, living in a thermally stable environment, have a limited capacity to physiologically adjust to the highest temperatures found in their micro-habitat, making the species more vulnerable to future climate change.

Embryonic Thermal Manipulation Affects Ventilation, Metabolism, Thermal Control and Central Dopamine in Newly Hatched and Juvenile Chicks.

The first third of incubation is critical for embryonic development, and environmental changes during this phase can affect the physiology and survival of the embryos. We evaluated the effects of low (LT), control (CT), and high (HT) temperatures during the first 5 days of incubation on ventilation ( V . E ), body temperature (Tb), oxygen consumption ( V . O2), respiratory equivalent ( V . E / V . O2), and brain monoamines on 3-days-old (3d) and 14-days-old (14d) male and female chickens. The body mass of LT animals of both ages and sexes was higher compared to HT and CT animals (except for 3d males). The heart mass of 14d HT animals was higher than that of CT animals. Thermal manipulation did not affect V . E , V . O2 or V . E / V . O2 of 3d animals in normoxia, except for 3d LT males V . E , which was lower than CT. Regarding 14d animals, the HT females showed a decrease in V . E and V . O2 compared to CT and LT groups, while the HT males displayed a lower V . O2 compared to CT males, but no changes in V . E / V . O2. Both sexes of 14d HT chickens presented a greater Tb compared to CT animals. Thermal manipulations increased the dopamine turnover in the brainstem of 3d females. No differences were observed in ventilatory and metabolic parameters in the 3d animals of either sexes, and 14d males under 7% CO2. The hypercapnic hyperventilation was attenuated in the 14d HT females due to changes in V . O2, without alterations in V . E . The 14d LT males showed a lower V . E , during hypercapnia, compared to CT, without changes in V . O2, resulting in an attenuation in V . E / V . O2. During hypoxia, 3d LT females showed an attenuated hyperventilation, modulated by a higher V . O2. In 14d LT and HT females, the increase in V . E was greater and the hypometabolic response was attenuated, compared to CT females, which resulted in no change in the V . E / V . O2. In conclusion, thermal manipulations affect hypercapnia-induced hyperventilation more so than hypoxic challenge, and at both ages, females are more affected by thermal manipulation than males.

5-HT neurons of the medullary raphe contribute to respiratory control in toads.

Air-breathing vertebrates undergo respiratory adjustments when faced with disturbances in the gas composition of the environment. In mammals, the medullary raphe nuclei are involved in the neuronal pathway that mediates the ventilatory responses to hypoxia and hypercarbia. We investigate whether the serotoninergic neurons of the medullary raphe nuclei of toads (Rhinella diptycha) play a functional role in respiratory control during resting conditions (room air), hypercarbia (5% CO2), and hypoxia (5% O2). The raphe nuclei were located and identified based on the location of the serotoninergic neurons in the brainstem. We then lesioned the medullary raphe (raphe pallidus, obscurus and magnus) with anti-SERT-SAP and measured ventilation in both control and lesioned groups and we observed that serotonin (5-HT) specific chemical lesions of the medullary raphe caused reduced respiratory responses to both hypercarbia and hypoxia. In summary, we report that the serotoninergic neurons of the medullary raphe of the cururu toad Rhinella diptycha participate in the chemoreflex responses during hypercarbia and hypoxia, but not during resting conditions. This current evidence in anurans, together with the available data in mammals, brings insights to the evolution of brain sites, such as the medullary raphe, involved in the ventilatory chemoreflex in vertebrates.

Influence of incubation temperature on embryo development, hatchling morphology and early growth rate in red-footed tortoise (Chelonoidis carbonaria).

Reptile embryos respond to temperature changes with metabolic and physiological adjustments that influence hatchling success, phenotype, behaviour, and growth rate. Climate change and global warming can affect the reptile population by altering the frequencies of hatchling survival and phenotypes. Therefore, previous studies proposed artificial incubation as a potential strategy for mitigating these effects. Red-footed tortoise (Chelonoidis carbonaria) eggs were collected and incubated at constant temperatures of 27.5 °C and 29.5 °C to investigate the physiological effects of temperature on embryo development, hatchling morphology, and early post-hatch growth rate. The direct effect of temperature on the incubation period, egg mass loss, hatching success, hatchling size, and mass was evaluated at hatching and three months of age. Hatchlings from 29.5 °C presented a shorter incubation period (141 days) than those from 27.5 °C (201 days; p < 0.05). Egg mass loss, hatchling mass, and size at hatching were not different between the incubation temperatures (p > 0.05). However, the hatching success (survival rate) was lower (64.5% versus 100%) in eggs incubated at 29.5 °C, but the hatchling mass and straight plastron width were higher at three months of age than those from eggs incubated at 27.5 °C (p < 0.05). These results indicate that incubation temperature influences hatching success and hatchling size and mass in the first months by influencing the early growth rate.

Who Rules Over Immunology? Seasonal Variation in Body Temperature, Steroid Hormones, and Immune Variables in a Tegu Lizard.

Multiple factors can influence the immune response of ectothermic vertebrates, including body temperature (Tb), gonadal steroids, and seasonality, in ways that are thought to reflect trade-offs between energetic investment in immunity versus reproduction. Hibernating tegu lizards (Salvator merianae) are a unique model to investigate how immunocompetence might be influenced by different factors during their annual cycle. We assessed immunological measures (plasma bacterial killing ability, total and differential leukocyte count), plasma hormone levels (testosterone in males, estradiol and progesterone in females, and corticosterone [CORT] in both sexes), Tb, and body condition from adult tegus during each stage of their annual cycle: reproduction, post-reproduction/preparation for hibernation, and hibernation. Our hypothesis that immune traits present higher values during the reproductive phase, and a sharp decrease during hibernation, was partially supported. Immune variables did not change between life history stages, except for total number of leukocytes, which was higher at the beginning of the reproductive season (September) in both males and females. Average Tb of the week prior to sampling was positively correlated with number of eosinophils, basophils, monocytes, and azurophils, corroborating other studies showing that when animals maintain a high Tb, there is an increase in immune activity. Surprisingly, no clear relationship between immune traits and gonadal steroids or CORT levels was observed, even when including life history stage in the model. When gonadal hormones peaked in males and females, heterophil: lymphocyte ratio (which often elevates during physiological stress) also increased. Additionally, we did not observe any trade-off between reproduction and immunity traits, sex differences in immune traits, or a correlation between body condition and immune response. Our results suggest that variation in patterns of immune response and correlations with body condition and hormone secretion across the year can depend upon the specific hormone and immune trait, and that experienced Tb is an important variable determining immune response in ectotherms.