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Aestivation and hibernation represent distinct forms of animal quiescence, characterized by physiological changes, including ion composition. Intracellular ion flows play a pivotal role in eliciting alterations in membrane potential and facilitating cellular communication, while outward K+ currents aid in the restitution and upkeep of the resting membrane potential. This study explores the relationship between inward and outward currents during aestivation in Achatina fulica snails. Specimens were collected near MSUBIT University in Shenzhen and divided into two groups. The first group was kept on a lattice diet, while the second one consisted of aestivating individuals, that were deprived of food and water until a cork-like structure sealed their shells. Recording of current from isolated neurons were conducted using the single-electrode voltage clamp mode with an AxoPatch 200B amplifier. Electrophysiological recordings on pedal ganglia neurons revealed significant differences in the inactivation processes of the Ia and Ikdr components. Alterations in the Ikdr component may inhibit pacemaker activity in pedal ganglion neurons, potentially contributing to locomotion cessation in aestivated animals. The KS current remains unaffected during aestivation. Changes in slow K+ current components could disrupt the resting membrane potential, possibly leading to cell depolarization and influx of Ca2+ and Na+ ions, impacting cell homeostasis. Thus, maintaining the constancy of outward K+ current is essential for cell stability.

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The invasive Pomacea canaliculata estivates during periods of drought and should cope with harmful effects of reoxygenation during arousal. We studied thiobarbituric acid reactive substances (TBARS), enzymatic (superoxide dismutase, SOD and catalase, CAT) and non-enzymatic antioxidants (uric acid and reduced glutathione), and heat shock protein expression (Hsc70, Hsp70 and Hsp90) in (1) active control snails, (2) snails after 45 days of estivation, and (3) aroused snails 20 min and (4) 24 h after water exposure, in midgut gland, kidney and foot. Both kidney and foot (but not the midgut gland) showed a TBARS increase during estivation and a decrease after arousal. Tissue SOD and CAT did not change in any experimental groups. Uric acid increased during estivation in all tissues, and it decreased after arousal in the kidney. Allantoin, the oxidation product of uric acid, remained constant in the midgut gland but it decreased in the kidney until 20 min after arousal; however, allantoin levels rose in both kidney and foot 24 h after arousal. Reduced glutathione decreased during estivation and arousal, in both midgut gland and kidney, and it remained constant in the foot. Hsc70 and Hsp70 kidney levels were stable during the activity-estivation cycle and Hsp90 expression decreases during estivation and recovers in the early arousal. In foot, the expression of Hsp70 and Hsp90 was high during activity and estivation periods and disminished after arousal. Results indicate that a panoply of antioxidant and molecular chaperone defences may be involved during the activity-estivation cycle in this freshwater gastropod.

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The physiological ability to estivate is relevant for the maintenance of population size in the invasive Pomacea canaliculata. However, tissue reoxygenation during arousal from estivation poses the problem of acute oxidative stress. Uric acid is a potent antioxidant in several systems and it is stored in specialized tissues of P. canaliculata. Changes in tissue concentration of thiobarbituric acid reactive substances (TBARS), uric acid and allantoin were measured during estivation and arousal in P. canaliculata. Both TBARS and uric acid increased two-fold during 45 days estivation, probably as a consequence of concomitant oxyradical production during uric acid synthesis by xanthine oxidase. However, after arousal was induced, uric acid and TBARS dropped to or near baseline levels within 20 min and remained low up to 24h after arousal induction, while the urate oxidation product allantoin continuously rose to a maximum at 24h after induction, indicating the participation of uric acid as an antioxidant during reoxygenation. Neither uric acid nor allantoin was detected in the excreta during this 24h period. Urate oxidase activity was also found in organs of active snails, but activity shut down during estivation and only a partial and sustained recovery was observed in the midgut gland.

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In situations of food and water deprivation associated with unfavorable environmental conditions, a number of animal species undergo estivation. This state of locomotor inactivity involves a drastic reduction in the metabolic rate, allowing the estivator to survive long periods of adverse situations. However, the arousal from dormancy causes a rapid increase in oxygen consumption, which may elevate the production of oxygen radicals. Thus, it is expected that animals that arouse from estivation suffer a physiological oxidative stress. The reported mechanisms that protect estivators (anurans and gastropods) from the potential dangers of increased oxyradical formation are discussed. This includes the modulation of endogenous antioxidant defenses (enzymes and glutathione) of gastropods during dormancy, preparing them for arousal. A different strategy used for estivating anurans is also discussed.

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The physiological mechanisms, behavioral adjustments, and ecological associations that allow animal species to live in extreme environments have evoked the attention of many zoologists. Often, extreme environments are defined as those believed to be limiting to life in terms of water, energetic availability, and temperature. These three elements seem extreme in a number of arid and semi-arid settings that even so have been colonized by amphibians. Because this taxon is usually seen as the quintessential water-dependent ectotherm tetrapods, their presence in a number of semi-arid environments poses a number of intriguing questions regarding microhabitat choice and physiological plasticity, particularly regarding the ecological and physiological correlates of behaviors granting avoidance of the harshest conditions of semi-arid environments. Such avoidance states, generally associated to the concept of aestivation, are currently seen as a diverse and complex phenomena varying from species to species and involving numerous behavioral and metabolic adjustments that enhance survival during the drought. This chapter reviews the physiological ecology of anuran aestivation, mainly from the perspective of water and energy balance.

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The African (Protopterus sp.) and South American lungfish (Lepidosiren paradoxa) inhabit shallow waters, that seasonally dry out, which induces aestivation and cocoon formation in Protopterus. Differently, L. paradoxa has no cocoon, and it aestivates in a simple burrow. In water PaCO(2) is 21.8+/-0.4mmHg (mean values+/-S.E.M.; n=5), whereas aestivation for 20 days increased PaCO(2) to as much as 37.6+/-2.1mmHg, which remained the same after 40 days (35.8+/-3.3mmHg). Concomitantly, the plasma [HCO(3)(-)]-values for animals in water were 22.5+/-0.5mM, which after 20 days increased to 40.2+/-2.3mM and after 40 days to 35.8+/-3.3mM. Initially in water, PaO(2) was 87.7+/-2.0mmHg, but 20 days in aestivation reduced the value to 80.5+/-2.2 and later (40 days) to 77.1+/-3.0mmHg. Meanwhile, aestivation had no effect on pHa and hematocrit. The blood pressures were equal for animals in the water or in the burrow (P(mean) approximately 30mmHg), and cardiac frequency (f(H)) fell from 31beats min(-1) to 22beats min(-1) during 40 days of aestivation. The osmolality (mOsmkgH(2)O(-1)) was elevated after 20 and 40 days of aestivation but declined upon return to water. The transition from activity to aestivation involves new set-points for the variables that determine the acid-base status and PaO(2) of the animals, along with a reduction of cardiac frequency.

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Metabolic changes, principally in intermediary metabolism and nitrogen excretion, were investigated in the marble swamp eel (Synbranchus marmoratus) after 15 and 45 days of artificially induced semi-aestivation. Glucose, glycogen, lactate, pyruvate, free amino acids, triglycerides, ammonia, urea, and urate contents were determined in liver, kidney, white muscle, heart, brain, and plasma. Lactate dehydrogenase, glutamate dehydrogenase, malate dehydrogenase, aspartate amino transferase, alanine amino transferase, glutamine synthase, ornithine carbamoyl transferase, and arginase enzymes were assayed. The teleost S. marmoratus maintained initial energetic demands by lipid oxidation. The course of normal oxidative processes was observed through tissue enzyme profiles. After the lipid stores were exhausted, the fish consumed body proteins. Constant values of hematocrit during induced semi-aestivation suggested that the water balance remained normal. Therefore, the surrounding water was probably did not trigger the semi-aestivation in this teleost. Decrease of ammonia and increase of renal urea synthesis after 45 days of semi-aestivation led to the assumption that an alternative form of eliminating ammonia exists. Metabolic changes entailed by starvation were proposed to explain the biosynthesis of small molecules involved in the semi-aestivation of S. marmoratus.

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We investigated the regulation of free radical metabolism in Helix aspersa snails during a cycle of 20-day estivation and 24-h arousal in summer in comparison with estivation/arousal in winter-snails. In winter-snails (J. Exp. Biol. 206, 675-685, 2003), we had already observed an increase in the selenium-dependent glutathione-peroxidase (Se-GPX) activity in foot muscle and hepatopancreas and in the contents of hepatopancreas GSH-equivalents (GSH-eq=GSH+2 GSSG) during estivation compared with 24-h aroused snails. Summer-estivation prompted a 3.6-fold increase in Se-GPX activity in hepatopancreas, though not in foot muscle. Total-superoxide dismutase and catalase activities in hepatopancreas decreased (by 30-40%) during summer-estivation; however, no changes occurred in the activities of glutathione reductase, glutathione S-transferase and glucose-6-phosphate dehydrogenase in the two organs. GSH-eq levels were increased (by 54%) in foot muscle during estivation, but were unchanged in hepatopancreas. In contrast with winter-snails, oxidative stress markers (lipid peroxidation, carbonyl protein, and the GSSG/GSH-eq ratio) were unaltered during estivation/arousal in summer. These results demonstrate that seasonality modulates not only the absolute activities/levels of antioxidants (enzymes and GSH-eq) in H. aspersa, but also the regulatory process that controls the snail's antioxidant capacity during estivation/arousal. These results suggest that H. aspersa has an "internal clock" controlling the regulation of free radical metabolism in the different seasons.

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Foram investigadas variações metabólicas em muçum (Synbranchus marmoratus) após 15 e 45 dias de semi-estivação artificial induzida, com ênfase no metabolismo intermediário e excreção de nitrogênio. Glicose, glicogênio, lactato, piruvato, aminoácidos livres, triglicerídios, amônia, uréia e uratos foram determinados em fígado, rim, músculo branco, coração, cérebro, e plasma. As enzimas lactato desidrogenase, glutamato desidrogenase, malato desidrogenase, aspartato amino transferase, alanina amino transferase, glutamina sintetase, ornitina carbamil transferase e arginase foram ensaiadas. O teleósteo S. marmoratus manteve sua demanda energética inicial pela oxidação de lipídios. A seqüência normal dos processos oxidativos foi observada por meio do perfil enzimático. Após o consumo das reservas lipídicas, os peixes passaram a consumir proteína. Valores constantes de hematócrito durante a semi-estivação induzida sugeriu que o balanço de água foi mantido normal. Todavia, a água ambiental provavelmente não foi o sinal inicial da semi-estivação em S. marmoratus. A diminuição de amônia e o aumento da síntese de uréia renal após 45 dias de semi-estivação levaram a assumir que existe uma forma alternativa de eliminar amônia. As variações metabólicas conseqüentes do jejum prolongado foram propostas como responsáveis pela síntese de pequenas moléculas envolvidas na semi-estivação de S. marmoratus.

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The aim of this work was to evaluate the effect of a cycle of estivation and awakening on free radical metabolism in selected organs of the land snail Helix aspersa. Estivation for 20 days induced a 4.9- and 1.8-fold increase in selenium-dependent glutathione peroxidase activity (Se-GPX) and in total glutathione levels (GSH-eq), respectively, in hepatopancreas when compared to activity in active animals 24 h after awakening. Foot muscle Se-GPX activity was also increased 3.9-fold during estivation, whereas GSH-eq did not vary. The activities of other antioxidant enzymes (catalase, superoxide dismutase, glutathione reductase and glutathione S-transferase) and glucose 6-phosphate dehydrogenase were unchanged in both organs. After 15 min of awakening, the glutathione disulphide (GSSG)/GSH-eq ratio increased significantly by 55% in hepatopancreas, slowly returning to the levels observed during estivation. The higher GSSG/GSH-eq ratio may be caused by increased formation of reactive oxygen species (ROS) during awakening. The levels of thiobarbituric acid reactive substances (TBARS) decreased from 49 to 30.7 nmol g(-1) wet mass in hepatopancreas after 5 min arousal and, after 30 min, TBARS rose significantly to 39.6 nmol g(-1) wet mass, gradually declining thereafter. The levels of lipid hydroperoxides in hepatopancreas and of carbonyl protein in foot muscle both decreased during awakening. The higher levels of products of free radical damage during estivation may have resulted from low levels of ROS formation associated with decreased rates of lipid hydroperoxide detoxification and oxidized protein turnover caused by metabolic depression. The regulation of the antioxidant system during hypometabolism may constitute a mechanism to minimize oxidative stress during cycles of estivation and awakening.

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Lepidosiren paradoxa (pirambóia) is the single representative of Dipnoan (lungfish) in South America. This species is considered a living fossil, in spite of some reports describing this fish as having a very specialized life style. It aestivates during the dry season, and has developed metabolic adaptations to cope with both flooding and drought. The literature describing its tissue ultra-structure shows high glycogen stored in the muscle, suggesting a strong dependence on anaerobic glycolysis. The present paper reports tissue enzyme levels of LDH, MDH, and CS, and isozymic tissue distribution of LDH, MDH, ADH, PGI, SOD, and PGM of 7 aestivating specimens from Lago do Canteiro in the Amazonas River. Animals were caught while burrowed in mud during the aestivation period. Our findings reveal high anaerobic capacity of both skeletal and heart muscles, even during the aestivation period, when enzymes showed suppressed levels compared to those of non-aestivating animals (data from the literature). Isozymic patterns suggest loss of duplicate condition in most analyzed loci, a characteristic that occurs mainly in higher vertebrate categories. These data indicate that, compared to the fish group, lungfish may be considered advanced, despite retaining primitive morphological characters.

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Using high performance liquid chromatography (HPLC) analysis it was possible to determine simultaneously the concentration of organic acids (pyruvate, lactate, succinate, fumarate, malate, acetate, propionate, acetoacetate, and ss-hydroxybutyrate) in the digestive gland and the extracellular concentration of these same acids in the hemolymph of estivating Biomphalaria glabrata, the intermediate host of Schistosoma mansoni. After a 7 day period of estivation, there was a significant increase in the tissue levels of lactate, succinate, malate and acetate compared to non-estivating snails. After 14 days of estivation, the levels of lactate and acetate were also significantly elevated. The hemolymph concentrations of pyruvate and acetate increased significantly after 7 days and acetate concentrations continued to be significantly increased up to 14 days of estivation. The other organic acids studied, such as ketone body acetoacetate and ss-hydroxybutyrate or the volatile acid propionate, did not accumulate. Their tissue concentrations, however, increased on the 7th day of estivation and reached normal levels within two weeks of estivation for some of them. One should take into consideration how the reduction in metabolism can be handled under aerobic conditions, and what role anaerobic pathways may play in both energy formation and redox balance processes.

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Using high performance liquid chromatography (HPLC) analysis it was possible to determine simultaneously the concentration of organic acids (pyruvate, lactate, succinate, fumarate, malate, acetate, propionate, acetoacetate, and ß-hydroxybutyrate) in the digestive gland and the extracellular concentration of these same acids in the hemolymph of estivating Biomphalaria glabrata, the intermediate host of Schistosoma mansoni. After a 7 day period of estivation, there was a significant increase in the tissue levels of lactate, succinate, malate and acetate compared to non-estivating snails. After 14 days of estivation, the levels of lactate and acetate were also significantly elevated. The hemolymph concentrations of pyruvate and acetate increased significantly after 7 days and acetate concentrations continued to be significantly increased up to 14 days of estivation. The other organic acids studied, such as ketone body acetoacetate and ß-hydroxybutyrate or the volatile acid propionate, did not accumulate. Their tissue concentrations, however, increased on the 7th day of estivation and reached normal levels within two weeks of estivation for some of them. One should take into consideration how the reduction in metabolism can be handled under aerobic conditions, and what role anaerobic pathways may play in both energy formation and redox balance processes

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The roles of enzymatic antioxidant defenses in the natural tolerance of environmental stresses that impose changes in oxygen availability and oxygen consumption on animals is discussed with a particular focus on the biochemistry of estivation and metabolic depression in pulmonate land snails. Despite reduced oxygen consumption and PO2 during estivation, which should also mean reduced production of oxyradicals, the activities of antioxidant enzymes, such as superoxide dismutase and catalase, increased in 30 day-estivating snails. This appears to be an adaptation that allows the snails to deal with oxidative stress that takes place during arousal when PO2 and oxygen consumption rise rapidly. Indeed, oxidative stress was indicated by increased levels of lipid peroxidation damage products accumulating in hepatopancreas within minutes after arousal was initiated. The various metabolic sites responsible for free radical generation during arousal are still unknown but it seems unlikely that the enzyme xanthine oxidase plays any substantial role in this despite being implicated in oxidative stress in mammalian models of ischemia/reperfusion. We propose that the activation of antioxidant defenses in the organs of Otala lactea during estivation is a preparative mechanism against oxidative stress during arousal. Increased activities of antioxidant enzymes have also observed under other stress situations in which the actual production of oxyradicals should decrease. For example, antioxidant defenses are enhanced during anoxia exposure in garter snakes Thamnophis sirtalis parietalis (10 h at 5 degrees C) and leopard frogs Rana pipiens (30 h at 5 degrees C) and during freezing exposure (an ischemic condition due to plasma freezing) in T. sirtalis parietalis and wood frogs Rana sylvatica. It seems that enhancement of antioxidant enzymes during either anoxia or freezing is used as a preparatory mechanism to deal with a physiological oxidative stress that occurs rapidly within the early minutes of recovery during reoxygenation or thawing. Thus, a wide range of stress tolerant animals display coordinated changes in antioxidant defenses that allow them to deal with oxidative stress that occurs as part of natural cycles of stress/recovery that alter oxygen levels in tissues. The molecular mechanisms that trigger and regulate changes in antioxidant enzyme activities in these species are still unknown but could prove to have key relevance for the development of new intervention strategies in the treatment of cardiovascular ischemia/reperfusion injuries in humans.

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The cardiodynamic consequences of dry season dormancy in ectothermic vertebrates is not well known. Our hypothesis was that dormancy would reduce cardiac activity. We therefore determined oxygen uptake and cardiovascular function in aestivating toads, Bufo paracnemis, native to São Paulo State, Brazil. Specimens were collected and kept in the laboratory under controlled temperature and light regimes. We compared oxygen uptake, heart rate, blood pressure, rate-pressure product (RPP), and blood gases in toads during aestivation (dry winter season) and their early active season (spring). Oxygen uptake of winter toads at 25 degrees C was considerably lower than that of spring toads (winter: 24.0 +/- 1.8 ml/(kgh); early spring: 44.4 +/- 5.1 ml/(kgh); mean +/- SE; same in the following). A seasonal dichotomy was also observed at 15 degrees C although the differences was less pronounced (15.8 +/- 1.8 ml/(kgh) winter; 23 +/- 2.1 ml/(kgh) early spring). Chronic arterial cannulation permitted measurements of cardiodynamic variables without any undesired change in VO2. Heart rates of winter toads were significantly lower than those of early spring animals at both experimental temperatures (25 degrees C: winter 25 +/- 1.4 beats/min.; early spring: 35.2 +/- 5.1 beats/min. 15 degrees C: winter 15, 4 +/- 1.8 beats/min.; early spring: 23.9 +/- 2.1 beats/min). Systemic, diastolic and mean arterial pressures decreased slightly but not significantly during aestivation. We conclude that: (1) Bufo paracnemis downregulates metabolic rate during the dry season and (2) heart rate is also downregulated with little change of blood pressure. While the energetics of these responses are probably beneficial for survival during aestivation, the underlying biochemical mechanisms remain obscure.

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As a consequence of aerobic life, an organism must deal with the continuous generation of reactive oxygen species (O2-, H202, .OH) as byproducts of metabolism and defend itself against the harm that these can do to cellular macromolecules. Organisms protect themselves from such damage with both enzymatic and nonenzymatic antioxidant defenses. However, the reperfusion injuries noted after ischemic insult in mammalian organs and ascribed to a burst of reactive oxygen species produced when oxygenated blood is reintroduced demonstrate that the antioxidant defenses of many organisms can be overwhelmed, Although unusual among most mammals, many organisms routinely experience wide variation in oxygen availability to their tissues due to factors such as environmental oxygen lack, breath-hold diving, extracellular freezing, or apnoeic breathing patterns in arrested metabolic states. In recent studies using various animal models (anoxia-tolerant turtles, freeze-tolerant snakes and frogs, estivating snails) our laboratory has explored the adaptations of antioxidant defenses that allow such organisms to deal with rapid changes in tissue oxygenation with little or no accumulation of damage products. The key to successful transitions in several systems is the induction, during the oxygen-limited state, of elevated activities of antioxidant and associated enzymes, such as catalase, superoxide dismutase, glutathione-S-transferase, and glutathione peroxidase, so that damage during the reintroduction of oxygen (such as lipid peroxidation) is minimized. However, animals that are excellent facultative anaerobes, such as freshwater turtles, appear to deal with potential of oxidative stress during the anoxic-aerobic transition by maintaining constitutively high antioxidant defenses (e.g. enzyme activities similar to those of mammals and much higher than those of anoxia-intolerant lower vertebrates) that can readily accommodate the burst of reactive oxygen species generation when breathing is renewed.

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A number of amphibians and reptiles have cyclic behavior, becoming inactive with the coming of the dry season. In South America this pattern of activity is common, particularly in savannah-like vegetation. During the dry season amphibians burrow into the mud or soil, and either form a cocoon or increase the osmotic concentration of body fluids to reduce evaporative water loss. Some phyllomedusid tree frogs coat their body surface with skin secretion and excrete uric acid to minimize water loss. Reptiles also retreat into shelter deep enough to avoid temperature fluctuation during estivation or reduce metabolic response to temperature. Reduction of temperature sensitivity of the metabolism seems to be a strategy common to estivating amphibians and reptiles. Despite seasonal change of the environment, some species of reptiles are active all year round.

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A number of amphibians and reptiles have cyclic behavior, becoming inactive with the coming of the dry season. In South America this pattern of activity is common, particularly in savannah-like vegetation. During the dry season amphibians burrow into the mud or soil, and either form a cocoon or increase the osmotic concentration of body fluids to reduce evaporative water loss. Some phyllomedusid tree frogs coat their body surface with skin secretion and excrete uric acid to minimize water loss. Reptiles also retreat into shelter deep enough to avoid temperature fluctuation during estivation or reduce metabolic response to temperature. Reduction of temperature sensitivity of the metabolism seems to be a strategy common to estivating amphibians and reptiles. Despite seasonal change of the environment, some species of reptiles are active all year round.

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The frog, L. fuscus, becomes dormant during the dry season in southeastern Brazil. Plasma and urine were obtained and analyzed for K+, Na+, and osmotic concentrations in active and estivating frogs. Soil water potential from the estivation sites was compared with the osmotic concentrations of the frog. Plasma and urine osmotic concentrations (286.2 +/- 13.8 and 242.3 +/- 17.2 mOsm1(-1), respectively) were higher in the estivating than in active frogs (240.3 +/- 12.8 and 112.7 +/- 15.6 mOsm1(-1); plasma and urine), and the same holds true for plasma K+ content. The Na+ concentration was the same for active and estivating frogs. Soil water potential corresponded to osmotic pressure of 110 mOsm1(-1), showing that L. fuscus may uptake water from the soil during the estivation.

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