Cyclorana Alboguttata Research Articles

Frogs seek hypoxic microhabitats that accentuate metabolic depression during dormancy.

Many animals occupy microhabitats during dormancy where they may encounter hypoxic conditions (e.g. subterranean burrows). We used the green-striped burrowing frog (Cyclorana alboguttata) to test the hypothesis that animals seek hypoxic microhabitats that accentuate metabolic depression during dormancy. We first measured the partial pressure of oxygen (PO2 ) within artificial cavities excavated in wet clay soil, which simulated C. alboguttata underground aestivation chambers, and recorded hypoxic conditions (PO2 as low as 8.9 kPa). Using custom-built tunnels that maintained a longitudinal PO2 gradient (hypoxic to normoxic), we then examined the PO2 preference of C. alboguttata in response to drying habitat conditions. In support of our hypothesis, we found that C. alboguttata chose to spend a greater proportion of time at the hypoxic end of the PO2 gradient compared with the normoxic end. To determine whether hypoxia accentuates metabolic depression in C. alboguttata, we exposed frogs to normoxia (21.0 kPa) or hypoxia (10.5 kPa) for 7 weeks during the transition from an active to an aestivating state. We found that hypoxia exposure accelerated the onset of metabolic depression in C. alboguttata by 2 weeks. Furthermore, we found that frogs exposed to hypoxia exhibited a 66% reduction in O2 consumption after 7 weeks compared with active frogs in normoxia, whereas frogs exposed to normoxia reduced O2 consumption by only 51%. Overall, our findings indicate that some animals may seek microhabitats to maximally depress metabolic rate during dormancy, and that microhabitat O2 availability can have significant implications for energy metabolism.

Survey of the reptiles and amphibians of Merriwindi State Conservation Area in the Pilliga forest of northern inland New South Wales

A survey of the herpetofauna of Merriwindi State Conservation Area, a small conservation reserve in the western Pilliga forest in northern inland New South Wales, was done over the period 2009 to 2011. In total, 14 frog species (three families) and 25 reptile species (eight families) were recorded by a combination of diurnal active searches, nocturnal road transects and nocturnal searches of water bodies. The herpetofauna community includes a mix of western (Eyrean), northern (Torresian) and eastern (Bassian) species and demonstrates the significant species diversity and conservation value of the Pilliga forest's herpetofauna. Noteworthy findings from this study included the threatened Pale-headed Snake Hoplocephalus bitorquatus, a number of declining woodland reptile species such as the Nobbi Diporiphora nobbi , Brown-blazed Ctenotus Ctenotus allotropis and Australian Coral Snake Brachyurophis australis, and the first documented record of the Striped Burrowing Frog Cyclorana alboguttata from the western ...

Activity, abundance and expression of Ca²⁺-activated proteases in skeletal muscle of the aestivating frog, Cyclorana alboguttata.

In most mammals, prolonged muscle disuse (e.g. bed-rest, limb casting or spaceflight) results in atrophy of muscle fibres which is largely due to unregulated proteolysis. Although numerous proteolytic pathways are known to participate in muscle disuse atrophy, recent evidence suggests that activation of Ca²⁺-dependent cysteine proteases (calpains) is required for disuse atrophy in limb skeletal muscles. In contrast to typical models of muscle disuse (humans and rodents), animals that experience natural bouts of chronic muscle inactivity, such as hibernating mammals and aestivating frogs, consistently exhibit limited or no change in skeletal muscle size. In the current study, we examined enzyme activity, protein abundance and gene expression levels of calpain isoforms in gastrocnemius muscle of the aestivating frog, Cyclorana alboguttata. We predicted that in aestivating C. alboguttata there would be a downregulation of the abundance, activity and gene expression of calpain 1 and calpain 2. In contrast to our hypothesis, there was no significant decrease in the enzyme activity levels or the relative protein abundances of calpain 1 and calpain 2. Similarly, gene expression assays (both qRT-PCR and RNA Seq data) indicated that calpains were unaffected by aestivation. Western blotting of 'muscle-specific' calpain 3, which is consistently downregulated during atrophic conditions, indicated that this isoform is present in C. alboguttata muscle where it appears to be in its autolysed state. The absence of any increase in enzyme activity, protein and mRNA abundance of calpains in aestivators is consistent with the protection of gastrocnemius muscle against uncontrolled proteolysis throughout aestivation.

A preliminary evaluation of frog assemblages in the Pilliga forests

This paper provides a preliminary evaluation of frog assemblages in the Pilliga forests, a geographically significant region in northern New South Wales. Spotlighting and call recognition surveys were conducted at 19 sites: 14 fire dams, three woodland sites and two built environs. A total of 11 species were detected, the most frequently recorded being Litoria latopalmata (broad-palmed rocket frog). Five species were also found at sites on built environs. Of particular interest, Cyclorana alboguttata (striped burrowing frog), a rarely seen species in this region, was recorded at one dam. A desktop assessment of species records was conducted to support the field data, accounting for 10 species that were not located in the surveys.

LTR retroelements are intrinsic components of transcriptional networks in frogs.

BackgroundLTR retroelements (LTR REs) constitute a major group of transposable elements widely distributed in eukaryotic genomes. Through their own mechanism of retrotranscription LTR REs enrich the genomic landscape by providing genetic variability, thus contributing to genome structure and organization. Nonetheless, transcriptomic activity of LTR REs still remains an obscure domain within cell, developmental, and organism biology.ResultsHere we present a first comparative analysis of LTR REs for anuran amphibians based on a full depth coverage transcriptome of the European pool frog, Pelophylax lessonae, the genome of the African clawed frog, Silurana tropicalis (release v7.1), and additional transcriptomes of S. tropicalis and Cyclorana alboguttata. We identified over 1000 copies of LTR REs from all four families (Bel/Pao, Ty1/Copia, Ty3/Gypsy, Retroviridae) in the genome of S. tropicalis and discovered transcripts of several of these elements in all RNA-seq datasets analyzed. Elements of the Ty3/Gypsy family were most active, especially Amn-san elements, which accounted for approximately 0.27% of the genome in Silurana. Some elements exhibited tissue specific expression patterns, for example Hydra1.1 and MuERV-like elements in Pelophylax. In S. tropicalis considerable transcription of LTR REs was observed during embryogenesis as soon as the embryonic genome became activated, i.e. at midblastula transition. In the course of embryonic development the spectrum of transcribed LTR REs changed; during gastrulation and neurulation MuERV-like and SnRV like retroviruses were abundantly transcribed while during organogenesis transcripts of the XEN1 retroviruses became much more active.ConclusionsThe differential expression of LTR REs during embryogenesis in concert with their tissue-specificity and the protein domains they encode are evidence for the functional roles these elements play as integrative parts of complex regulatory networks. Our results support the meanwhile widely accepted concept that retroelements are not simple “junk DNA” or “harmful genomic parasites” but essential components of the transcriptomic machinery in vertebrates.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2164-15-626) contains supplementary material, which is available to authorized users.

Effect of Opioids on Tissue Metabolism in Aestivating and Active Green-Striped Burrowing Frogs,Cyclorana alboguttata

Metabolic depression is a common strategy used during stressful environmental conditions, allowing animals to survive for prolonged periods on limited endogenous fuel stores; yet, the mechanisms involved in regulating metabolic depression are poorly understood. The endogenous opioid system (EOS) has been implicated in the regulation of metabolism during hibernation in vertebrates, and we hypothesized that opioids also may regulate metabolic rate during aestivation in the Green-Striped Burrowing Frog, Cyclorana (Litoria) alboguttata (Gunther, 1867). When incubated in the presence of opioid agonists, liver and muscle tissue slices from C. alboguttata underwent tissue- and agonist-specific metabolic depression, and the effect of opioid agonists on metabolism was more pronounced in tissue slices from aestivating animals. Gene transcript levels for the δ-opioid receptor remained constant in the brain and muscle of aestivating C. alboguttata compared with that of active frogs, as did brain transcript levels of the opioid receptor XOR1. The results of this study demonstrate that opioids can influence metabolic rate in isolated tissues from C. alboguttata. The maintenance of key opioid receptor gene transcript levels in tissues of aestivating frogs suggests that despite the energetic costs, maintenance of receptor abundance is important. We propose that the maintenance of opioid receptor abundance during aestivation in C. alboguttata is indicative of a role for the EOS in the regulation of metabolic rate during aestivation.

Frogs and estivation: transcriptional insights into metabolism and cell survival in a natural model of extended muscle disuse

Green-striped burrowing frogs (Cyclorana alboguttata) survive in arid environments by burrowing underground and entering into a deep, prolonged metabolic depression known as estivation. Throughout estivation, C. alboguttata is immobilized within a cast-like cocoon of shed skin and ceases feeding and moving. Remarkably, these frogs exhibit very little muscle atrophy despite extended disuse and fasting. Little is known about the transcriptional regulation of estivation or associated mechanisms that may minimize degradative pathways of atrophy. To investigate transcriptional pathways associated with metabolic depression and maintenance of muscle function in estivating burrowing frogs, we assembled a skeletal muscle transcriptome using next-generation short read sequencing and compared gene expression patterns between active and 4 mo estivating C. alboguttata. This identified a complex suite of gene expression changes that occur in muscle during estivation and provides evidence that estivation in burrowing frogs involves transcriptional regulation of genes associated with cytoskeletal remodeling, avoidance of oxidative stress, energy metabolism, the cell stress response, and apoptotic signaling. In particular, the expression levels of genes encoding cell cycle and prosurvival proteins, such as serine/threonine-protein kinase Chk1, cell division protein kinase 2, survivin, and vesicular overexpressed in cancer prosurvival protein 1, were upregulated during estivation. These data suggest that estivating C. alboguttata are able to regulate the expression of genes in several major cellular pathways critical to the survival and viability of cells, thus preserving muscle function while avoiding the deleterious consequences often seen in laboratory models of muscle disuse.

Hot and steady: Elevated temperatures do not enhance muscle disuse atrophy during prolonged aestivation in the ectothermCyclorana alboguttata

Cover illustration. The cover image shows a Australian Green-striped burrowing frog, Cyclorana albogutatta, emerging from its burrow after aestivation. In this issue of the Journal of Morphology, Young et al. study the effect of temperature on the extent of muscle disuse atrophy in the gastrocnemius and iliofibularis muscles during aestivation in the burrowing frog C. alboguttata. (pp. 165-174). They show that despite being inactive and immobile for many months during aestivation this frogs protects it skeletal muscles from extensive disuse atrophy even at elevated temperatures. Photo: Karen Young.

Decreased hydrogen peroxide production and mitochondrial respiration in skeletal muscle but not cardiac muscle of the green-striped burrowing frog, a natural model of muscle disuse

Suppression of disuse-induced muscle atrophy has been associated with altered mitochondrial reactive oxygen species (ROS) production in mammals. However, despite extended hindlimb immobility, aestivating animals exhibit little skeletal muscle atrophy compared with artificially immobilised mammalian models. Therefore, we studied mitochondrial respiration and ROS (H2O2) production in permeabilised muscle fibres of the green-striped burrowing frog, Cyclorana alboguttata. Mitochondrial respiration within saponin-permeabilised skeletal and cardiac muscle fibres was measured concurrently with ROS production using high-resolution respirometry coupled to custom-made fluorometers. After 4 months of aestivation, C. alboguttata had significantly depressed whole-body metabolism by ~70% relative to control (active) frogs, and mitochondrial respiration in saponin-permeabilised skeletal muscle fibres decreased by almost 50% both in the absence of ADP and during oxidative phosphorylation. Mitochondrial ROS production showed up to an 88% depression in aestivating skeletal muscle when malate, succinate and pyruvate were present at concentrations likely to reflect those in vivo. The percentage ROS released per O2 molecule consumed was also ~94% less at these concentrations, indicating an intrinsic difference in ROS production capacities during aestivation. We also examined mitochondrial respiration and ROS production in permeabilised cardiac muscle fibres and found that aestivating frogs maintained respiratory flux and ROS production at control levels. These results show that aestivating C. alboguttata has the capacity to independently regulate mitochondrial function in skeletal and cardiac muscles. Furthermore, this work indicates that ROS production can be suppressed in the disused skeletal muscle of aestivating frogs, which may in turn protect against potential oxidative damage and preserve skeletal muscle structure during aestivation and following arousal.

Hot and steady: Elevated temperatures do not enhance muscle disuse atrophy during prolonged aestivation in the ectothermCyclorana alboguttata

Animals that undergo prolonged dormancy experience minimal muscle disuse atrophy (MDA) compared to animals subjected to artificial immobilisation over shorter timeframes. An association between oxidative stress and MDA suggests that metabolic depression presumably affords dormant animals some protection against muscle disuse. Because aerobic metabolism is temperature sensitive, we proposed that MDA in dormant (aestivating) ectotherms would be enhanced at elevated temperatures. In the green-striped burrowing frog, Cyclorana alboguttata, the thermal sensitivity of skeletal muscle metabolic rate is muscle-specific. We proposed that the degree of atrophy experienced during aestivation would correlate with the thermal sensitivity of muscle metabolic rate such that muscles with a relatively high metabolic rate at high temperatures would experience more disuse atrophy. To test this hypothesis, we examined the effect of temperature and aestivation on the extent of MDA in two functionally different muscles: the M. gastrocnemius (jumping muscle) and M. iliofibularis (non-jumping muscle), in C. alboguttata aestivating at 24 or 30 °C for 6 months. We compared a range of morphological parameters from muscle cross-sections stained with succinic dehydrogenase to show that muscle-specific patterns of disuse atrophy were consistent with the relative rates of oxygen consumption of those muscle types. However, despite muscle-specific differences in thermal sensitivity of metabolic rate, aestivation temperature did not influence the extent of atrophy in either muscle. Our results suggest that the muscles of frogs aestivating at high temperatures are defended against additional atrophy ensuring protection of muscle function during long periods of immobilisation.

Each to their own: skeletal muscles of different function use different biochemical strategies during aestivation at high temperature

Preservation of muscle morphology depends on a continuing regulatory balance between molecules that protect and molecules that damage muscle structural integrity. Excessive disruption of the biochemical balance that favours reactive oxygen species (ROS) in disused muscles may lead to oxidative stress, which in turn is associated with increased atrophic or apoptotic signalling and/or oxidative damage to the muscle and thus muscle disuse atrophy. Increases in the rate of oxygen consumption likely increase the overall generation of ROS in vivo. Temperature-induced increases in oxygen consumption rate occur in some muscles of ectotherms undergoing prolonged muscular disuse during aestivation. In the green-striped burrowing frog, Cyclorana alboguttata, both large jumping and small non-jumping muscles undergo atrophy seemingly commensurate with their rate of oxygen consumption during aestivation. However, because the extent of atrophy in these muscles is not enhanced at higher temperatures, despite a temperature-sensitive rate of oxygen consumption in the jumping muscle, we proposed that muscles are protected by biochemical means that, when mobilised at higher temperatures, inhibit atrophy. We proposed that the biochemical response to temperature would be muscle-specific. We examined the effect of temperature on the antioxidant and heat shock protein systems and determined the extent of oxidative damage to lipids and proteins in two functionally different skeletal muscles, the gastrocnemius (jumping muscle) and the iliofibularis (non-jumping muscle), by aestivating frogs at 24 and 30°C for 6 months. We assayed small molecule antioxidant capacity, mitochondrial and cytosolic superoxide dismutase activities and Hsp70 concentrations to show that protective mechanisms in disused muscles are differentially regulated with respect to both temperature and aestivation. High aestivation temperature results in an antioxidant response in the metabolically temperature-sensitive jumping muscle. We assayed lipid peroxidation and protein oxidation to show that oxidative damage is apparent during aestivation and its pattern is muscle-specific, but unaffected by temperature. Consideration is given to how the complex responses of muscle biochemistry inform the different strategies muscles may use in regulating their oxidative environment during extended disuse and disuse at high temperature.

Influence of elevated temperature on metabolism during aestivation: implications for muscle disuse atrophy

Reactive oxygen species (ROS), produced commensurate with aerobic metabolic rate, contribute to muscle disuse atrophy (MDA) in immobilised animals by damaging myoskeletal protein and lipids. Aestivating frogs appear to avoid MDA in part by substantially suppressing metabolic rate. However, as ectotherms, metabolic rate is sensitive to environmental temperature, and the high ambient temperatures that may be experienced by frogs during aestivation could in fact promote MDA. In this study, we investigated the effect of temperature on the metabolic rate of the aestivating frog Cyclorana alboguttata and its skeletal muscles in order to determine their likely susceptibility to MDA. Compared with non-aestivating frogs, a significant decrease in metabolic rate was recorded for aestivating frogs at 20, 24 and 30°C. At 30°C, however, the metabolic rate of aestivating frogs was significantly higher, approximately double that of frogs aestivating at 20 or 24°C, and the magnitude of the metabolic depression was significantly reduced at 30°C compared with that at 20°C. Temperature effects were also observed at the tissue level. At 24 and 30°C the metabolic rate of all muscles from aestivating frogs was significantly depressed compared with that of muscles from non-aestivating frogs. However, during aestivation at 30°C the metabolic rates of gastrocnemius, sartorius and cruralis were significantly elevated compared with those from frogs aestivating at 24°C. Our data show that the metabolism of C. alboguttata and its skeletal muscles is elevated at higher temperatures during aestivation and that the capacity of the whole animal to actively depress metabolism is impaired at 30°C.

Enzyme activity in the aestivating Green-striped burrowing frog (Cyclorana alboguttata)

Green-striped burrowing frogs (Cyclorana alboguttata) can depress their resting metabolism by more than 80% during aestivation. Previous studies have shown that this species is able to withstand long periods of immobilisation during aestivation while apparently maintaining whole muscle mass and contractile performance. The aim of this study was to determine the effect of prolonged aestivation on the levels of metabolic enzymes (CCO, LDH and CS) in functionally distinct skeletal muscles (cruralis, gastrocnemius, sartorius, iliofibularis and rectus abdominus) and liver of C. alboguttata. CS activity was significantly reduced in all tissues except for the cruralis, gastrocnemius and the liver. LDH activity was significantly reduced in the sartorius and rectus abdominus, but remained at control (active) levels in the other tissues. CCO activity was significantly reduced in the gastrocnemius and rectus abdominus, and unchanged in the remaining tissues. Muscle protein was significantly reduced in the sartorius and iliofibularis during aestivation, and unchanged in the remaining muscles. The results suggest that the energy pathways involved in the production and consumption of ATP are remodelled during prolonged aestivation but selective. Remodelling and subsequent down-regulation of metabolic activity seem to target the smaller non-jumping muscles, while the jumping muscles retain enzyme activities at control levels during aestivation. These results suggest a mechanism by which aestivating C. alboguttata are able to maintain metabolic depression while ensuring that the functional capacity of critical muscles is not compromised upon emergence from aestivation.

Ups and downs of intestinal function with prolonged fasting during aestivation in the burrowing frog, Cyclorana alboguttata

Although green striped burrowing frogs (Cyclorana alboguttata) experience large reductions in the mass and absorptive surface area of the small intestine (SI) during aestivation, little is known about how this may affect the functional capacity of the SI. We examined changes in the function (l-proline uptake rate and capacity) and metabolism of the SI (in vitro oxygen consumption, Na(+)/K(+)-ATPase activity and abundance) of C. alboguttata following 6 months of aestivation. l-Proline uptake rate was significantly higher in aestivating frogs, but overall uptake capacity was lower than in active frogs. Total SI oxygen consumption rate (V(O(2))) was also lower in aestivating frogs, despite no difference in mass-specific V(O(2)). The proportion of intestinal V(O(2)) associated with Na(+)/K(+)-ATPase activity and protein synthesis was equivalent between active and aestivating frogs, suggesting these processes were unaffected by aestivation. Indeed, the activity of Na(+)/K(+)-ATPase transporters in the SI of aestivating frogs was not different from that of active animals. Aestivating frogs maintained Na(+)/K(+)-ATPase activity, despite experiencing a reduction in the density of Na(+)/K(+)-ATPase transporters, by increasing the molecular activity of the remaining pumps to 2-3 times that of active frogs. These results show that functionality of the SI is maintained at the cellular level, potentially facilitating the reclamation of nutrients from the intestinal lumen while in aestivation. Despite this, the functional capacity of the SI in aestivating C. alboguttata is significantly reduced due to a reduction in tissue mass, helping frogs to conserve energy while in aestivation.

Skeletal muscle atrophy occurs slowly and selectively during prolonged aestivation inCyclorana alboguttata(Günther 1867)

We investigated the effect of prolonged immobilisation of six and nine months duration on the morphology and antioxidant biochemistry of skeletal muscles in the amphibian aestivator Cyclorana alboguttata. We hypothesised that, in the event of atrophy occurring during aestivation, larger jumping muscles were more likely to be preserved over smaller non-jumping muscles. Whole muscle mass (g), muscle cross-sectional area (CSA) (microm(2)), water content (%) and myofibre number (per mm(2)) remained unchanged in the cruralis muscle after six to nine months of aestivation; however, myofibre area (microm(2)) was significantly reduced. Whole muscle mass, water content, myofibre number and myofibre CSA remained unchanged in the gastrocnemius muscle after six to nine months of aestivation. However, iliofibularis dry muscle mass, whole muscle CSA and myofibre CSA was significantly reduced during aestivation. Similarly, sartorius dry muscle mass, water content and whole muscle CSA was significantly reduced during aestivation. Endogenous antioxidants were maintained at control levels throughout aestivation in all four muscles. The results suggest changes to muscle morphology during aestivation may occur when lipid reserves have been depleted and protein becomes the primary fuel substrate for preserving basal metabolic processes. Muscle atrophy as a result of this protein catabolism may be correlated with locomotor function, with smaller non-jumping muscles preferentially used as a protein source during fasting over larger jumping muscles. Higher levels of endogenous antioxidants in the jumping muscles may confer a protective advantage against oxidative damage during aestivation; however, it is not clear whether they play a role during aestivation or upon resumption of normal metabolic activity.

Metabolic depression during aestivation in Cyclorana alboguttata

The green striped burrowing frog, Cyclorana alboguttata, spends, on average, nine to ten months of every year in aestivation. Recently, C. alboguttata has been the focus of much investigation regarding the physiological processes involved in aestivation, yet our understanding of this frog's capacity to metabolically depress remains limited. This study aimed to extend our current knowledge of metabolic depression during aestivation in C. alboguttata. C. alboguttata reduced whole animal metabolism by 82% within 5 weeks of aestivation. The effects of aestivation on mass specific in vitro tissue metabolic rate (VO 2) varied among individual organs, with muscle and liver slices showing significant reductions in metabolism; kidney VO 2 was elevated and there was no change in the VO 2 of small intestine tissue slices. Organ size was also affected by aestivation, with significant reductions in the mass of all tissues, except the gastrocnemius. These reductions in organ size, combined with changes in mass specific VO 2 of tissue slices, resulted in further energy savings to aestivating animals. This study shows that C. alboguttata is capable of selectively down- or up-regulating individual tissues, using both changes in metabolic rate and morphology. This strategy allows maximal energy savings during aestivation without compromising organ functionality and survival at arousal.

Surviving the drought: burrowing frogs save energy by increasing mitochondrial coupling

During dormancy energy conservation is a key priority and as such dormant animals undergo a major metabolic depression to conserve their limited endogenous fuel supplies. Mitochondrial coupling efficiency, the efficiency with which mitochondria convert oxygen into ATP, significantly affects aerobic metabolism and thus to maximise energy savings during dormancy it has been hypothesised that coupling efficiency should increase. However, previous studies have shown coupling efficiency to be maintained or even to decrease. In this study we measured state 3 and state 4 mitochondrial respiration in the muscle of the burrowing frog, Cyclorana alboguttata and calculated the respiratory control ratio as a measure of coupling efficiency. After 7 months in aestivation, C. alboguttata significantly reduced oxygen consumption of isolated mitochondria by 83% and, unlike other dormant animals, the frogs appeared to decrease rates of proton leak to a greater extent than ATP synthesis, consistent with an increase in mitochondrial coupling efficiency. The significant energy savings observed at the mitochondrial level were reflected at higher levels of biological organisation, with tissue oxygen consumption depressed by as much as 81% and whole animal metabolic rate by 82%. Cyclorana alboguttata can survive in a dormant state for several years and we propose the hypothesis that energy efficiency is increased during aestivation.

Opioids as triggers for metabolic depression in the frog, Cyclorana alboguttata

Many species of Australian arid zone frogs survive the dry winter months by burrowing underground and entering a state of metabolic depression known as aestivation. While in aestivation these frogs can survive months to years in the absence of food and water, yet the endogenous mechanisms involved in initiation and maintenance of metabolic depression remain poorly understood. The aim of this study was to investigate the potential role of opioids in initiating a metabolic depression in the aestivating frog, Cyclorana alboguttata. To do this we took a two fold approach. First, the presence of opioid receptors was confirmed using PCR and degenerate primers designed from known opioid receptor sequences. Secondly, we used closed box respirometry to measure the direct effects of opioid agonists on tissue metabolic rate. The delta opioid DADLE was found to decrease oxygen consumption of liver and muscle slices by 20% and 25%, respectively, compared to control slices, whereas the kappa opioid Dynorphin A and the mu opioid Morphiceptin had no effect on tissue metabolic rate. This is the first time that opioids have been shown to directly affect metabolic rate in an aestivating species, indicating that endogenous opioids may play an important role in initiating a metabolic depression. Copyright © 2008 Published by Elsevier Inc.

Morphological and functional responses of the small intestine during aestivation in the green-striped burrowing frog, Cyclorana alboguttata

Morphological and functional responses of the small intestine during aestivation in the green-striped burrowing frog, Cyclorana alboguttata

Epigenetic silencers are enriched in dormant desert frog muscle

Green-striped burrowing frogs, Cyclorana alboguttata, survive droughts by entering a metabolic depression called aestivation, characterised by a reduction in resting oxygen consumption by 80%. Aestivation in C. alboguttata is manifest by transcriptional silencing of skeletal muscle bioenergetic genes, such as NADH ubiquinone oxidoreductase 1, ATP synthase and superoxide dismutase 2. In this study, we hypothesised that aestivation is associated with epigenetic change in frog muscle. We assessed mRNA transcript abundance of seven genes that code for proteins with established roles in epigenetically-mediated gene silencing [transcriptional co-repressor SIN3A, DNA (cytosine-5-) methyltransferase 1, methyl CpG binding protein 2, chromodomain helicase DNA binding protein 4, histone binding protein rbbp4, histone deacetylase 1 and nuclear receptor co-repressor 2] using qRT-PCR. These seven genes showed a modest (1.1-3.5-fold) but coordinated upregulation in 6-month aestivating muscle. This reached significance for SIN3A and DNA cytosine-5-methyltransferase 1 in standard pair-wise comparisons (p < 0.05), and the candidates as a whole when analysed by Fisher's combined probability test (p < 0.01). These data are consistent with the hypothesis that the transcriptional silencing and metabolic depression that occurs during seasonal dormancy are associated with chromatin remodelling, and present a novel example of an environmentally induced epigenetic modification in an adult vertebrate.