Role of Akt signaling pathway regulation in the speckled mousebird (Colius striatus) during torpor displays tissue specific responses

Pronounced heterothermic responses are relatively rare among birds. Along with taxa such as hummingbirds and caprimulgids, the order Coliiformes (mousebirds) is known to possess the physiological capacity for torpor. During torpor, body temperature is greatly reduced and a bird becomes unresponsive to external stimuli until ambient temperatures return to more favorable conditions. Under such conditions, these birds are forced to rely only on their internal fuel storage for energy and show great reduction in metabolic rates by decreasing energy-expensive processes. This study investigated the role of the key insulin-Akt signaling kinase pathway involved in regulating energy metabolism and protein translation in the liver, kidney, heart, skeletal muscle, and brain of the speckled mousebird (Colius striatus). The degree of phosphorylation of well-conserved target residues with important regulatory function was examined in both the euthermic control and torpid birds. The results demonstrated marked differences in responses between the tissues with decreases in RPS6 S235/236 phosphorylation in the kidney (0.52 fold of euthermic) and muscle (0.29 fold of euthermic) as well as decreases in GS3K3β S9 in muscle (0.60 fold of euthermic) and GSK3α S21 (0.71 fold of euthermic) phosphorylation in kidney during torpor, suggesting a downregulation of this pathway. Interestingly, the liver demonstrated an increase in RPS6 S235/236 (2.89 fold increase) and P70S6K T412 (1.44 fold increase) phosphorylation in the torpor group suggesting that protein translation is maintained in this tissue. This study demonstrates that avian torpor is a complex phenomenon and alterations in this signaling pathway follow a tissue specific pattern.Supplementary material 1: Western blots for quantifying the relative levels of phosphorylated S209 eIF-4E and total mTOR levels in euthermic and torpid C. striatus tissuesSupplementary material 2: Sequence alignment of mousebird protein targets involved in the Akt signaling kinase pathway and protein translation in comparison to human sequencesThe Natural Sciences and Engineering Research Council of Canada (NSERC), the Canada Research Chair in Molecular Physiology, an NSERC funded Alexander Graham Bell Canada Graduate Scholarship, an Ontario Graduate Scholarship and the National Research Foundation of South Africa.https://www.elsevier.com/locate/cellsig2021-11-01hj2020Zoology and Entomolog

Regulation of the PI3K/AKT Pathway and Fuel Utilization During Primate Torpor in the Gray Mouse Lemur, Microcebus murinus

Gray mouse lemurs (Microcebus murinus) from Madagascar present an excellent model for studies of torpor regulation in a primate species. In the present study, we analyzed the response of the insulin signaling pathway as well as controls on carbohydrate sparing in six different tissues of torpid versus aroused gray mouse lemurs. We found that the relative level of phospho-insulin receptor substrate (IRS-1) was significantly increased in muscle, whereas the level of phospho-insulin receptor (IR) was decreased in white adipose tissue (WAT) of torpid animals, both suggesting an inhibition of insulin/insulin-like growth factor-1 (IGF-1) signaling during torpor in these tissues. By contrast, the level of phospho-IR was increased in the liver. Interestingly, muscle, WAT, and liver occupy central roles in whole body homeostasis and each displays regulatory controls operating at the plasma membrane. Changes in other tissues included an increase in phospho-glycogen synthase kinase 3α (GSK3α) and decrease in phospho-ribosomal protein S6 (RPS6) in the heart, and a decrease in phospho-mammalian target of rapamycin (mTOR) in the kidney. Pyruvate dehydrogenase (PDH) that gates carbohydrate entry into mitochondria is inhibited via phosphorylation by pyruvate dehydrogenase kinase (e.g., PDK4). In the skeletal muscle, the protein expression of PDK4 and phosphorylated PDH at Ser 300 was increased, suggesting inhibition during torpor. In contrast, there were no changes in levels of PDH expression and phosphorylation in other tissues comparing torpid and aroused animals. Information gained from these studies highlight the molecular controls that help to regulate metabolic rate depression and balance energetics during primate torpor

Thermoregulatory capacity of arboreal small mammals in the tropics : insights from the past and implications for the future.

Doctoral Degree. University of KwaZulu-Natal, Pietermaritzburg.Species are expected to respond to global warming through range shifts that are either poleward or towards higher altitude such that they track the movement of their current thermal niches. This generalized view is fundamentally flawed because it marginalizes the role of key biological aspects in shaping ecosystems. For example, it disregards any potential influence that phenotypic flexibility may convey, the influence of habitat heterogeneity and the availability of microclimates as thermal refugia, differences between species dispersal ability, the importance of species interactions, and the influence of phenological mismatches. Given the limitations associated with the view that species will deal with global warming by continually migrating, as well as the rapid rate of warming, there is an urgent need to improve the understanding of how species potentially may respond. Thus, it is crucial that mechanistic approaches are adopted to generate a holistic perspective of the factors that govern species distributions and use this information to forecast responses to global warming. Physiological studies are vital because knowing the physiological tolerances of a species provides insight into their fundamental niche, and also provides a means of identifying species that face higher risks of experiencing more immediate effects due to global warming. Autecological knowledge would also serve to refine the species fundamental niche to more closely resemble their realized niche. In this regard, this thesis identifies arboreal mammals in the tropics as being vulnerable to hyperthermia due to global warming. The basis of this claim is related to the exposed lifestyle of many arboreal species, the biphasic effect of temperature on biological processes and the hypothesis that natural selection would have favoured the optimisation of bodily functions at or close to the species-specific body temperatures (Tb). Initially, there is a positive relationship between biological process and temperature as the rate of processes increase with temperature, up to a maximum point. Thereafter, hyperthermia ensues as further increases in temperature results in a rapid decline in the rate of said processes. Thus, it is plausible to expect that species with lower Tb risk deleterious effects at lower absolute Tbs relative to their higher Tb counterparts. Therefore, it is concerning that many small, tropical endotherms have low and thermolabile Tbs that, because of the small temperature differential between themselves and the ambient (Ta), compromises their capacity to passively off-load excess stored body heat. In addition, the high humidity of tropical environments would theoretically reduce their capacity to retard heat storage by off-loading body heat via evaporation. This reduced capacity to dissipate excess stored body heat, in combination with the exposed life-style of an arboreal species, suggests that small, tropical arboreal mammals are vulnerable to hyperthermia should even minor increases in Ta occur. The aim of this thesis was to assess the vulnerability of tropical, arboreal small mammals to hyperthermia due to global warming. This was achieved by determining and integrating the physiological susceptibility for heat stress in two species and relate that to the microclimate experienced within their habitats. Given the growing argument that adaptive heterothermy - the capacity for species to facultatively down-regulate metabolism and enter torpor or hibernation - may be employed at high Ta to cope better with hyperthermia, this thesis in addition investigated whether heterotherms use torpor at high Ta and identified the putative benefits of hyperthermic torpor. Furthermore, by considering the phylogenetic placement of the study species, this thesis also sought to provide insights into the evolution of endothermy in placental mammals. Flow-through respirometry was used to measure resting metabolic rate (RMR) and evaporative water loss (EWL) at a range of Ta in a heterothermic bat, the lesser dog-faced fruit bat (Cynopterus brachyotis), and a suspected heterothermic primate, the western tarsier (Cephalopachus bancanus). The animals were injected with temperature-sensitive passive integrated transponder tags to obtain concurrent Tb readings during respirometry measurements in freshly-caught individuals. In addition, Tb was measured in free-ranging tarsiers using custom designed temperature data-loggers. The Ta at capture sites were measured using commercially available data-loggers. The laboratory data show that, whereas tarsiers endeavoured to remain normothermic, lesser dog-faced fruit bats readily entered torpor at low temperatures. The free-ranging Tb data support the assertion that tarsiers may be incapable of adaptive heterothermy. The onset of heat storage in tarsiers occurred at approximately 30°C, once the thermal gradient (ΔT = Tb-Ta) approached 4°C, whereas the onset of heat storage in lesser dog-faced fruit bats occurred at approximately 31°C, which was only when ΔT approached 1°C. Given that both species have low normothermic resting Tbs (tarsiers: Tb ≈ 34.5°C; lesser dog-faced fruit bats: Tb ≈ 32.5°C), they seem physiologically susceptible to heat stress at moderately low Ta. Notably, though, lesser dog-faced fruit bats appeared to thermoconform at Ta above their thermoneutral zone suggesting that they may have entered torpor. Torpor seems to have allowed them to reduce heat storage. Field data suggest that lesser dog-faced fruit bats may have the option to exploit cool microclimates at their capture sites, but the data at the capture site of tarsiers suggest that they may not. However, even though the population of tarsiers studied may not have access to cool microclimates, the same may not be true for other populations of tarsiers. Thus, the empirical results support the argument that tropical, arboreal small mammals are physiologically susceptible to heat stress due to global warming, but they also suggest that thermal refugia are an important consideration as they may allow species to escape the predicted future high Ta and its related deleterious effects. This thesis also presents a meta-analysis on the thermoregulatory pattern of bats in general. The aim of this meta-analysis was to determine whether corroborating physiological support for the use of torpor at high Ta exists. A comprehensive literature search was conducted based on the availability of concurrent measures of Tb and RMR; a new dataset of thermoregulatory variables was generated for 29 species of bats (18 heterothermic spp. and 11 homeothermic spp.). The dataset was standardized, and phylogenetic relatedness was considered before any comparative analyses were performed. The results show that heterothermic bats maintain lower Tbs than homeothermic bats, yet they have similar upper limits of thermoneutrality (Tuc). In contrast to expectations, heterothermic bats had a lower rate of evaporative water loss at similar Ta, especially at Tuc. Crucially, in the case of heterothermic bats, Tuc exceeds Tb. The only manner in which heterothermic bats could achieve this, would be through a reduction in metabolic rate with the onset of heat storage at high Tas. Moreover, heterothermic species thermoconform even at comparatively moderate Ta, which presumably also minimizes heat storage and lowers evaporative water loss. Thus, these results support the hypothesis of torpor use at high Ta and suggest that heterotherms, in particular small, tropical, arboreal heterotherms, could benefit from a reduction in water use associated with evaporative cooling and tolerate higher Ta. Overall, the results presented in this thesis illustrate that even though small mammal species living in tropical regions may be physiologically susceptible to heat stress due to global warming, they could minimize their risk of lethal hyperthermia through behavioural mechanisms such as exploiting cooler microclimates; provided that suitable habitats are available. In addition, adaptive heterothermy may convey a physiological advantage that allows heterotherms to better cope with heat. As such, heterotherms may be resilient to the negative effects associated with global warming because they are able to employ torpor to conserve energy during periods of low resource availability, as well as to minimize heat storage and endure moderate hyperthermia to conserve water to use at more extreme Tas. Given the phylogenetic placement of tarsiers at the base of the Haplorrhini clade (Anthropoidea and Tarsiidae), the current lack of evidence for adaptive heterothermy in tarsiers, in combination with the lack of evidence in anthropoids, suggest that adaptive heterothermy in haplorrhine primates may have been lost at the Strepsirrhini-Haplorrhini split. The implication of the aforementioned idea is that many primates may not have the benefits associated with adaptive heterothermy to improve their future survivability as global warming continues. By adopting a mechanistic approach, this thesis highlighted the potential for species to respond to global warming using behavioural and physiological mechanisms that could allow them to persist in their current habitats until the end of the century at least. However, to improve the likelihood of arboreal tropical small mammals to persist in their current habitats for the foreseeable future, especially those mammals that are strictly homeothermic, conservation efforts must prioritise the preservation of areas that could serve as thermal refugia