Months Of Aestivation Research Articles

Physiology of the Apple Snail Pomacea maculata: Aestivation and Overland Dispersal.

Apple snails, in the genus Pomacea, have gained considerable notoriety for their impact on invaded habitats. Louisiana is currently under invasion by Pomacea maculata, which represents a potential threat to the state's valuable plants and cash crops. Insight into the physiology of the invasive snail may assist in developing control measures and enhance our understanding of the processes of adaptation and coevolution that accompany introductions. This paper addresses the capacity, extent, and means by which aquatic apple snails in Louisiana tolerate aerial exposure, as well as the factors that contribute to desiccation tolerance in P. maculata. Invasive P. maculata in Louisiana survived about 10 months of aestivation before 50% mortality was incurred, during which body mass was reduced by only about 30%; mortality and loss of body mass were positively correlated during aestivation. Size affects the loss of body mass in snails under 20 grams. Relative humidity interfered with the induction of aestivation, but it did not significantly affect the loss of body mass. Invasive apple snails in Louisiana also demonstrated the inclination and ability to sustain travel for at least three hours over dry land, at a rate of two meters per hour. The results of this study show that P. maculata is well adapted for survival in the absence of water. The ability to sustain travel over land and to endure long periods of aerial exposure suggests that the dry-down of infested bodies of water would not significantly impact populations of P. maculata in Louisiana.

Molecular characterization of three Rhesus glycoproteins from the gills of the African lungfish, Protopterus annectens, and effects of aestivation on their mRNA expression levels and protein abundance.

African lungfishes are ammonotelic in water. They can aestivate for long periods on land during drought. During aestivation, the gills are covered with dried mucus and ammonia excretion ceases. In fishes, ammonia excretion through the gills involves Rhesus glycoproteins (RhGP/Rhgp). This study aimed to obtain the complete cDNA coding sequences of rhgp from the gills of Protopterus annectens, and to determine their branchial mRNA and protein expression levels during the induction, maintenance and arousal phases of aestivation. Three isoforms of rhgp (rhag, rhbg and rhcg) were obtained in the gills of P. annectens. Their complete cDNA coding sequences ranged between 1311 and 1398 bp, coding for 436 to 465 amino acids with estimated molecular masses between 46.8 and 50.9 kDa. Dendrogramic analyses indicated that Rhag was grouped closer to fishes, while Rhbg and Rhcg were grouped closer to tetrapods. During the induction phase, the protein abundance of Rhag, but not its transcript level, was down-regulated in the gills, suggesting that there could be a decrease in the release of ammonia from the erythrocytes to the plasma. Furthermore, the branchial transcript levels of rhbg and rhcg decreased significantly, in preparation for the subsequent shutdown of gill functions. During the maintenance phase, the branchial expression levels of rhag/Rhag, rhbg/Rhbg and rhcg/Rhcg decreased significantly, indicating that their transcription and translation were down-regulated. This could be part of an overall mechanism to shut down branchial functions and save metabolic energy used for transcription and translation. It could also be regarded as an adaptive response to stop ammonia excretion. During the arousal phase, it is essential for the lungfish to regain the ability to excrete ammonia. Indeed, the protein abundance of Rhag, Rhbg and Rhcg recovered to the corresponding control levels after 1 day or 3 days of recovery from 6 months of aestivation.

Molecular characterization of myostatin from the skeletal muscle of the African lungfish, Protopterus annectens, and changes in its mRNA and protein expression levels during three phases of aestivation.

African lungfishes can aestivate and remain torpid without food and water for years, but disuse muscle atrophy is not prominent during aestivation. This study aimed to clone myostatin (mstn/Mstn), a factor associated with disuse muscle atrophy in mammals, from the skeletal muscle of the African lungfish Protopterus annectens, and to determine its mRNA expression level and protein abundance therein during the induction, maintenance, and arousal phases of aestivation. The complete coding cDNA sequence of mstn comprised 1128bp, encoding for 376 amino acids with an estimated molecular mass of 42.9kDa. It was grouped together with Mstn/MSTN of coelacanth and tetrapods in a clade separated from teleost Mstn. After 6 days (the induction phase) of aestivation, the mstn transcript level in the muscle increased significantly, while the protein abundance of Mstn remained comparable to the control. Following that, a significant increase in the expression levels of mstn/Mstn occurred on day 12 (the early maintenance phase) of aestivation. After 6 months of aestivation (the prolonged maintenance phase), the expression levels of mstn/Mstn returned to control levels, indicating the possible impediment of a drastic increase in muscle degradation to prevent muscle atrophy. During 1-3 days of arousal from aestivation, the expression levels of mstn/Mstn in the muscle remained comparable to the control. Hence, tissue reconstruction/regeneration of certain organs might not involve the mobilization of amino acids from the muscle during the early arousal. These results provide insights into how aestivating P. annectens regulates the expression of mstn/Mstn possibly to ameliorate disuse muscle atrophy.

Differential gene expression in the liver of the African lungfish, Protopterus annectens, after 6 months of aestivation in air or 1 day of arousal from 6 months of aestivation.

The African lungfish, Protopterus annectens, can undergo aestivation during drought. Aestivation has three phases: induction, maintenance and arousal. The objective of this study was to examine the differential gene expression in the liver of P. annectens after 6 months (the maintenance phase) of aestivation as compared with the freshwater control, or after 1 day of arousal from 6 months aestivation as compared with 6 months of aestivation using suppression subtractive hybridization. During the maintenance phase of aestivation, the mRNA expression of argininosuccinate synthetase 1 and carbamoyl phosphate synthetase III were up-regulated, indicating an increase in the ornithine-urea cycle capacity to detoxify ammonia to urea. There was also an increase in the expression of betaine homocysteine-S-transferase 1 which could reduce and prevent the accumulation of hepatic homocysteine. On the other hand, the down-regulation of superoxide dismutase 1 expression could signify a decrease in ROS production during the maintenance phase of aestivation. In addition, the maintenance phase was marked by decreases in expressions of genes related to blood coagulation, complement fixation and iron and copper metabolism, which could be strategies used to prevent thrombosis and to conserve energy. Unlike the maintenance phase of aestivation, there were increases in expressions of genes related to nitrogen, carbohydrate and lipid metabolism and fatty acid transport after 1 day of arousal from 6 months aestivation. There were also up-regulation in expressions of genes that were involved in the electron transport system and ATP synthesis, indicating a greater demand for metabolic energy during arousal. Overall, our results signify the importance of sustaining a low rate of waste production and conservation of energy store during the maintenance phase, and the dependence on internal energy store for repair and structural modification during the arousal phase, of aestivation in the liver of P. annectens.

Molecular characterization of betaine-homocysteine methyltransferase 1 from the liver, and effects of aestivation on its expressions and homocysteine concentrations in the liver, kidney and muscle, of the African lungfish, Protopterus annectens.

Homocysteine accumulation has numerous deleterious effects, and betaine-homocysteine S-methyltransferase (BHMT) catalyses the synthesis of methionine from homocysteine and betaine. This study aimed to determine homocysteine concentrations, and mRNA expression levels and protein abundances of bhmt1/Bhmt1 in the liver, kidney and muscle of the African lungfish, Protopterus annectens, during the induction (6 days), maintenance (6 months) or arousal (3 days after arousal) phase of aestivation. The homocysteine concentration decreased significantly in the liver of P. annectens after 6 days or 6 months of aestivation, but it returned to the control level upon arousal. By contrast, homocysteine concentrations in the kidney and muscle remained unchanged during the three phases of aestivation. The complete coding cDNA sequence of bhmt1 from P. annectens consisted of 1236 bp, coding for 412 amino acids. The Bhmt1 from P. annectens had a close phylogenetic relationship with those from tetrapods and Callorhinchus milii. The expression of bhmt1 was detected in multiple organs/tissues of P. annectens, and this is the first report on the expression of bhmt1/Bhmt1 in animal skeletal muscle. The mRNA and protein expression levels of bhmt1/Bhmt1 were up-regulated in the liver of P. annectens during the induction and maintenance phases of aestivation, possibly to regulate the hepatic homocysteine concentration. The significant increase in hepatic Bhmt1 protein abundance during the arousal phase could be a response to increased cellular methylation for the purpose of tissue reconstruction. Unlike the liver, Bhmt1 expression in the kidney and muscle of P. annectens was regulated translationally, and its up-regulation could be crucial to prevent homocysteine accumulation.

Aestivation induces changes in transcription and translation of coagulation factor II and fibrinogen gamma chain in the liver of the African lungfish Protopterus annectens.

This study aimed to sequence and characterize two pro-coagulant genes, coagulation factor II (f2) and fibrinogen gamma chain (fgg), from the liver of the African lungfish Protopterus annectens, and to determine their hepatic mRNA expression levels during three phases of aestivation. The protein abundance of F2 and Fgg in the liver and plasma was determined by immunoblotting. The results indicated that F2 and Fgg of P. annectens were phylogenetically closer to those of amphibians than those of teleosts. Three days of aestivation resulted in an up-regulation in the hepatic fgg mRNA expression level, while 6 days of aestivation led to a significant increase (3-fold) in the protein abundance of Fgg in the plasma. Hence, there could be an increase in the blood-clotting ability in P. annectens during the induction phase of aestivation. By contrast, the blood-clotting ability in P. annectens might be reduced in response to decreased blood flow and increased possibility of thrombosis during the maintenance phase of aestivation, as 6 months of aestivation led to significant decreases in mRNA expression levels of f2 and fgg in the liver. There could also be a decrease in the export of F2 and Fgg from the liver to the plasma so as to avert thrombosis. Three to 6 days after arousal from 6 months of aestivation, the protein abundance of F2 and Fgg recovered partially in the plasma of P. annectens; a complete recovery of the transcription and translation of f2/F2 in the liver might occur only after refeeding.

Signal molecule changes in the gills and lungs of the African lungfish Protopterus annectens, during the maintenance and arousal phases of aestivation

African lungfishes are obligate air breathers, with reduced gills and pulmonary breathing throughout their life. During the dry season they aestivate on land, with the collapse of secondary lamellae of their gills and the establishment of an exclusive aerial ventilation through the vascularization and expansion of their lungs. To date, the mechanisms underlining the respiratory organ remodeling in aestivating lungfishes are unknown. This study aimed to identify key switch components of the stress-induced signal transduction networks implicated in both rapid and medium–long term remodeling of the gills and lungs of the African lungfish Protopterus annectens during aestivation. Through immunofluorescence microscopy and Western blotting, the localization and the expression of nitric oxide synthase (NOS), Akt, Hsp-90 and HIF-1α were evaluated in both gills and lungs exposed to three experimental conditions: freshwater (FW), 6 months of experimentally induced aestivation (6mAe), and 6 days after arousal from 6 months of aestivation (6mAe6d). After 6mAe, the expression of NOS (p-eNOS antibody), Akt (p-Akt antibody), and Hsp-90 decreased in the gills, while NOS and Hsp-90 expression increased with Akt remained unchanged in the lungs. Upon 6mAe6d, NOS, Akt and Hsp-90 expression in the gills returned to the respective FW values. In the lungs of the aroused fish, NOS and Akt decreased to their respective FW levels, while Hsp-90 expression was enhanced with respect to aestivation. In both respiratory organs, the qualitative and quantitative patterns of HIF-1α expression correlated inversely to those of NOS. Overall, our findings suggest that the molecular components of the NOS/NO system changed in a tissue-specific manner in parallel with organ readjustment in the gills and lungs of P. annectens during aestivation and arousal.

Molecular characterization of argininosuccinate synthase and argininosuccinate lyase from the liver of the African lungfish Protopterus annectens, and their mRNA expression levels in the liver, kidney, brain and skeletal muscle during aestivation.

Argininosuccinate synthase (Ass) and argininosuccinate lyase (Asl) are involved in arginine synthesis for various purposes. The complete cDNA coding sequences of ass and asl from the liver of Protopterus annectens consisted of 1,296 and 1,398bp, respectively. Phylogenetic analyses revealed that the deduced Ass and Asl of P. annectens had close relationship with that of the cartilaginous fish Callorhinchus milii. Besides being strongly expressed in the liver, ass and asl expression were detectable in many tissues/organs. In the liver, mRNA expression levels of ass and asl increased significantly during the induction phase of aestivation, probably to increase arginine production to support increased urea synthesis. The increases in ass and asl mRNA expression levels during the prolonged maintenance phase and early arousal phase of aestivation could reflect increased demand on arginine for nitric oxide (NO) production in the liver. In the kidney, there was a significant decrease in ass mRNA expression level after 6months of aestivation, indicating possible decreases in the synthesis and supply of arginine to other tissues/organs. In the brain, changes in ass and asl mRNA expression levels during the three phases of aestivation could be related to the supply of arginine for NO synthesis in response to conditions that resemble ischaemia and ischaemia-reperfusion during the maintenance and arousal phase of aestivation, respectively. The decrease in ass mRNA expression level, accompanied with decreases in the concentrations of arginine and NO, in the skeletal muscle of aestivating P. annectens might ameliorate the potential of disuse muscle atrophy.

Brain Na+/K+-ATPase α-subunit isoforms and aestivation in the African lungfish, Protopterus annectens

This study aimed to clone and sequence Na (+) / K (+)-ATPase (nka) α-subunit isoforms from, and to determine their mRNA expression levels and protein abundance in the brain of the African lungfish, Protopterus annectens during the induction, maintenance and arousal phases of aestivation in air. We obtained the full cDNA sequences of nkaα1, nkaα2 and nkaα3 from the brain of P. annectens. Phylogenetic analysis of their deduced amino acid sequences revealed that they are closer to the corresponding NKA α-subunits of tetrapods than to those of fishes. The mRNA expression of these three nkaα isoforms showed differential changes in the brain of P. annectens during the three phases of aestivation. After 12 days of aestivation, there was a significant increase in the protein abundance of Nkaα1 in the brain of P. annectens. This could be an important response to maintain cellular Na(+) and K(+) concentrations and regulate cell volume during the early maintenance phase of aestivation. On the other hand, the mRNA expression of nkaα2 decreased significantly in the brain of P. annectens after 6 months of aestivation, which could be a result of a suppression of transcriptional activities to reduce energy expenditure. The down-regulation of mRNA expression of nkaα1, nkaα2 and nkaα3 and the overall protein abundance of Nka α-subunit isoforms in the brain of P. annectens after 1 day of arousal from 6 months of aestivation were novel observations, and it could be an adaptive response to restore blood pressure and/or to prevent brain oedema.

Lympho-granulocytic tissue associated with the wall of the spiral valve in the African lungfish Protopterus annectens

We describe the structure of the lympho-granulocytic tissue associated with the wall of the spiral valve of the African lungfish Protopterus annectens. The study was performed under freshwater conditions and after 6 months of aestivation. The lympho-granulocytic tissue consists of nodes surrounded by reticular tissue. The nodes are formed by an outer and an inner component separated by a thin collagenous layer. The outer component is a reticular-like tissue that contains two types of granulocytes, developing and mature plasma cells and melanomacrophage centres (MMCs). The inner component, the parenchyma, contains a meshwork of trabeculae and vascular sinusoids and shows dark and pale areas. The dark areas contain diffuse lymphoid tissue, with a large number of mitoses and plasma cell clusters. The pale areas contain a small number of macrophages and lymphocytes. Macrophages and sinus endothelial cells are filled with haemosiderin granules and appear to form part of the reticuloendothelial system of the lungfish. The reticular tissue houses granulocytes, plasma cells and MMCs and might serve for the housing and maturation of cells of the white series. After aestivation, the nodes undergo lymphocyte depletion, the suppression of mitosis, granulocyte invasion and the occurrence of cell death. By contrast, few histological changes occur in the reticular tissue. Whereas the nodes appear to be involved in lymphocyte proliferation and plasma cell maturation, the function of the reticular tissue remains obscure.

Differential gene expression in the brain of the African lungfish, Protopterus annectens, after six days or six months of aestivation in air.

The African lungfish, Protopterus annectens, can undergo aestivation during drought. Aestivation has three phases: induction, maintenance and arousal. The objective of this study was to examine the differential gene expression in the brain of P. annectens during the induction (6 days) and maintenance (6 months) phases of aestivation as compared with the freshwater control using suppression subtractive hybridization. During the induction phase of aestivation, the mRNA expression of prolactin (prl) and growth hormone were up-regulated in the brain of P. annectens, which indicate for the first time the possible induction role of these two hormones in aestivation. Also, the up-regulation of mRNA expression of tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein γ polypeptide and the down-regulation of phosphatidylethanolamine binding protein, suggest that there could be a reduction in biological and neuronal activities in the brain. The mRNA expression of cold inducible RNA-binding protein and glucose regulated protein 58 were also up-regulated in the brain, probably to enhance their cytoprotective effects. Furthermore, the down-regulation of prothymosin α expression suggests that there could be a suppression of transcription and cell proliferation in preparation for the maintenance phase. In general, the induction phase appeared to be characterized by reduction in glycolytic capacity and metabolic activity, suppression of protein synthesis and degradation, and an increase in defense against ammonia toxicity. In contrast, there was a down-regulation in the mRNA expression of prl in the brain of P. annectens during the maintenance phase of aestivation. In addition, there could be an increase in oxidative defense capacity, and up-regulation of transcription, translation, and glycolytic capacities in preparation for arousal. Overall, our results signify the importance of reconstruction of protein structures and regulation of energy expenditure during the induction phase, and the needs to suppress protein degradation and conserve metabolic fuel stores during the maintenance phase of aestivation.

Nitric oxide synthase-dependent “On/Off” switch and apoptosis in freshwater and aestivating lungfish, Protopterus annectens: Skeletal muscle versus cardiac muscle

African lungfishes (Protopterus spp.) are obligate air breathers which enter in a prolonged torpor (aestivation) in association with metabolic depression, and biochemical and morpho-functional readjustments during the dry season. During aestivation, the lungfish heart continues to pump, while the skeletal muscle stops to function but can immediately contract during arousal. Currently, nothing is known regarding the orchestration of the multilevel rearrangements occurring in myotomal and myocardial muscles during aestivation and arousal. Because of its universal role in cardio-circulatory and muscle homeostasis, nitric oxide (NO) could be involved in coordinating these stress-induced adaptations.Western blotting and immunofluorescence microscopy on cardiac and skeletal muscles of Protopterus annectens (freshwater, 6months of aestivation and 6days after arousal) showed that expression, localization and activity of the endothelial-like nitric oxide synthase (eNOS) isoform and its partners Akt and Hsp-90 are tissue-specifically modulated. During aestivation, phospho-eNOS/eNOS and phospho-Akt/Akt ratios increased in the heart but decreased in the skeletal muscle. By contrast, Hsp-90 increased in both muscle types during aestivation. TUNEL assay revealed that increased apoptosis occurred in the skeletal muscle of aestivating lungfish, but the myocardial apoptotic rate of the aestivating lungfish remained unchanged as compared with the freshwater control. Consistent with the preserved cardiac activity during aestivation, the expression of apoptosis repressor (ARC) also remained unchanged in the heart of aestivating and aroused fish as compared with the freshwater control. Contrarily, ARC expression was strongly reduced in the skeletal muscle of aestivating lungfish.On the whole, our data indicate that changes in the eNOS/NO system and cell turnover are implicated in the morpho-functional readjustments occurring in lungfish cardiac and skeletal muscle during the switch from freshwater to aestivation, and between the maintenance and arousal phases of aestivation.

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.

The spleen of the African lungfish Protopterus annectens: freshwater and aestivation

We describe the structure of the spleen of the African lungfish Protopterus annectens in freshwater conditions, and after 6months of aestivation. The spleen is formed by cortical tissue that surrounds the splenic parenchyma. The cortex is a reticulum that contains two types of granulocytes, developing and mature plasma cells, and melanomacrophage centres (MMCs). The parenchyma is divided into lobules that show a subcapsular sinus and areas of red pulp and white pulp. Red pulp contains vascular sinuses and atypical cords formed by delicate trabeculae. White pulp also contains vascular sinuses and cords. Structural data indicate that red pulp is involved in erythropoiesis, destruction of effete erythrocytes, and plasma cell differentiation. White pulp appears to be involved in the production of immune responses. Macrophages and sinus endothelial cells constitute the reticulo-endothelial system of the spleen. After aestivation, the number of MMCs increases, and spleen tissue is infiltrated by lymphocytes, granulocytes, and monocytes. Also, white pulp is reduced, and sinus endothelial cells undergo vacuolar degeneration. Lungfish spleen shares structural characteristics with secondary lymphoid organs of both ectothermic and endothermic vertebrates, but appears to have evolved in unique ways.

The Alimentary Canal of the African Lungfish Protopterus annectens During Aestivation and After Arousal

We describe the structural modifications that occur in the alimentary canal of the African lungfish Protopterus annectens during aestivation and after arousal. With fasting, all gut segments undergo structural modifications. The epithelium covering the intestinal vestibule undergoes bursts of activation at 4 months of aestivation, adopting a more quiescent appearance at 6 months. The ridge area of the spiral intestine shows, at 4 months of aestivation, epithelial disintegration, cell desquamation, cell death, and loss of the freshwater phenotype. Surprisingly, the epithelium adopts a stratified appearance at 6 months of aestivation. Except for epithelial disintegration, the smooth portion of the spiral intestine follows a similar pattern of modifications than the ridge area. The entire epithelium of spiral intestine appears to be renewed during aestivation. The presence of intraepithelial mast cells suggests that inflammation is part of the cellular response to aestivation. After arousal, cell phenotypes are restored in about 6 days, but full structural recovery is not attained during the experimental period (15 days post-aestivation). Several aspects of the cellular response to fasting are shared by a wide range of animal groups. This commonality agrees with the presence of a character that allows to adjust the structural and functional properties of the gut to food availability and food quality, and to the characteristics of the fasting episodes.

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.

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.

Getting the jump on skeletal muscle disuse atrophy: preservation of contractile performance in aestivatingCyclorana alboguttata(Günther 1867)

Prolonged immobilisation or unloading of skeletal muscle causes muscle disuse atrophy, which is characterised by a reduction in muscle cross-sectional area and compromised locomotory function. Animals that enter seasonal dormancy, such as hibernators and aestivators, provide an interesting model for investigating atrophy associated with disuse. Previous research on the amphibian aestivator Cyclorana alboguttata (Günther 1867) demonstrated an absence of muscle disuse atrophy after 3 months of aestivation, as measured by gastrocnemius muscle contractile properties and locomotor performance. In this study, we aimed to investigate the effect of aestivation on iliofibularis and sartorius muscle morphology and contractile function of C. alboguttata over a longer, more ecologically relevant time-frame of 9 months. We found that whole muscle mass, muscle cross-sectional area, fibre number and proportions of fibre types remained unchanged after prolonged disuse. There was a significant reduction in iliofibularis fibre cross-sectional area (declined by 36% for oxidative fibre area and 39% for glycolytic fibre area) and sartorius fibre density (declined by 44%). Prolonged aestivation had little effect on the isometric properties of the skeletal muscle of C. alboguttata. There was a significant reduction in the isometric contraction times of the relatively slow-twitch iliofibularis muscle, suggesting that the muscle was becoming slower after 9 months of aestivation (time to peak twitch increased by 25%, time from peak twitch to half relaxation increased by 34% and time from last stimulus to half tetanus relation increased by 20%). However, the results of the work-loop analysis clearly demonstrate that, despite changes to muscle morphology and isometric kinetics, the overall contractile performance and power output levels of muscles from 9-month aestivating C. alboguttata are maintained at control levels.

Effect of soil type on burrowing behavior and cocoon formation in the green-striped burrowing frog,Cyclorana alboguttata

This study examined the effect of soil type on burrowing behaviour and cocoon formation during aestivation in the green-striped burrowing frog, Cyclorana alboguttata (Günther, 1867). Given a choice, frogs always chose to burrow in wet sand in preference to wet clay. Frogs buried themselves faster and dug deeper burrows in sandy soil. However, under my laboratory conditions, there was little difference in the pattern of soil drying between the two soil types. Frogs in both sand and clay soil experienced hydrating conditions for the first 3 months and dehydrating conditions for the last 3 months of the 6-month aestivation period, and cocoons were not formed until after 3 months of aestivation. After 6 months, there were more layers in the cocoons of frogs aestivating in sand than those aestivating in clay. Frogs were able to absorb water from sandy soil with water potentials greater than –400 kPa, but lost water when placed on sand with a water potential of –1000 kPa.

Arousal and re-feeding rapidly restores digestive tract morphology following aestivation in green-striped burrowing frogs

During aestivation, the gut of the green-striped burrowing frog, Cyclorana alboguttata undergoes significant morphological down-regulation. Despite the potential impact such changes might have on the re-feeding efficiency of these animals following aestivation, they appear to be as efficient at digesting their first meals as active, non-aestivating animals. Such efficiency might come about by the rapid restoration of intestinal morphology with both arousal from aestivation and the initial stages of re-feeding. Consequently, this study sought to determine what morphological changes to the intestine accompany arousal and re-feeding following 3 months of aestivation. Arousal from aestivation alone had a marked impact on many morphological parameters, including small and large intestine masses, small intestinal length, LF heights, enterocyte cross-sectional area and microvilli height and density. In addition, the onset of re-feeding was correlated with an immediate reversal of many morphological parameters affected by 3 months of aestivation. Those parameters that had not returned to control levels within 36 h of feeding generally had returned to control values by the completion of digestion (i.e. defecation of the meal). Re-feeding was also associated with several changes in enterocyte morphology including the incorporation in intracytoplasmic lipid droplets and the return of enterocyte nuclear material to the ‘active’ euchromatin state. In conclusion, morphological changes to the gut of aestivating frogs which occur during aestivation are transitory and rapidly reversible with both arousal from aestivation and re-feeding. The proximate causes behind these transitions and their functional significance are discussed.