Effect of prolonged inactivity on skeletal motor nerve terminals during aestivation in the burrowing frog, <Emphasis Type="Italic">Cyclorana alboguttata</Emphasis>

This study examined the effect of prolonged inactivity, associated with aestivation, on neuromuscular transmission in the green-striped burrowing frog, Cyclorana alboguttata. We compared the structure and function of the neuromuscular junctions on the iliofibularis muscle from active C. alboguttata and from C. alboguttata that had been aestivating for 6 months. Despite the prolonged period of immobility, there was no significant difference in the shape of the terminals (primary, secondary or tertiary branches) or the length of primary terminal branches between aestivators and non-aestivators. Furthermore, there was no significant difference in the membrane potentials of muscle fibres or in miniature end plate potential (EPP) frequency and amplitude. However, there was a significant decrease in evoked transmitter release characterised by a 56% decrease in mean EPP amplitude, and a 29% increase in the failure rate of nerve terminal action potentials to evoke transmitter release. The impact of this suite of neuromuscular characteristics on the locomotor performance of emergent frogs is discussed.     

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.     

Hot and steady: Elevated temperatures do not enhance muscle disuse atrophy during prolonged aestivation in the ectotherm Cyclorana 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. J. Morphol., 2013. © 2012 Wiley Periodicals, Inc.     

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 (VO₂) varied among individual organs, with muscle and liver slices showing significant reductions in metabolism; kidney VO₂ was elevated and there was no change in the VO₂ 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 VO₂ 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.     

The impact of prolonged fasting during aestivation on the structure of the small intestine in the green-striped burrowing frog, Cyclorana alboguttata

The effects of short‐term fasting and prolonged fasting during aestivation on the morphology of the proximal small intestine and associated organs were investigated in the green‐striped burrowing frog, Cyclorana alboguttata (Anura: Hylidae). Animals were fasted for 1 week while active or for 3–9 months during aestivation. Short‐duration fasting (1 week) had little effect on the morphology of the small intestine, whilst prolonged fasting during aestivation induced marked enteropathy including reductions in intestinal mass, length and diameter, longitudinal fold height and tunica muscularis thickness. Enterocyte morphology was also affected markedly by prolonged fasting: enterocyte cross‐sectional area and microvillous height were reduced during aestivation, intercellular spaces were visibly reduced and the prevalence of lymphocytes amongst enterocytes was increased. Mitochondria and nuclei were also affected by 9 months of aestivation with major disruptions to mitochondrial cristae and increased clumping of nuclear material and increased infolding of the nuclear envelope. The present study demonstrates that the intestine of an aestivating frog responds to prolonged food deprivation during aestivation by reducing in size, presumably to reduce the energy expenditure of the organ.     

Metabolic depression and Na+/K+ gradients in the aestivating Australian goldfields frog,Neobatrachus wilsmorei

During aestivation the metabolic rate of the Australian goldfields frog Neobatrachus wilsmorei was reduced by 80% from its standard metabolic rate. The in vitro rate of oxygen consumption of isolated muscle and skin from aestivating frogs was up to 50% lower than that of the non-aestivating frogs. This in vitro rate of oxygen consumption was maintained for 6–12 h, indicating an intrinsic metabolic depression of tissues during aestivation. Frogs became dehydrated during aestivation. Muscle, skin and liver also became dehydrated during aestivation, but brain and kidney did not. Na⁺ and K⁺ contents and extracellular space measurement for muscle indicated that ion gradients were maintained across the muscle cell membrane during aestivation. Increases in plasma concentrations of Na⁺ and K⁺ were matched with similar increases in muscle intracellular ion concentrations. Extracellular space measurements were unsuccessful in the other tissues, but K⁺ content in all tissues (per dry weight) was maintained during aestivation, and the concentration of plasma K⁺ did not increase above that which can be accounted for by dehydration, indicating that K⁺ gradients were maintained.Abbreviations bm body mass - DPM disintegrations per minute - dw dry weight - MR metabolic rate - vO₂ rate of oxygen consumption - ww wet weight     

Effect of aestivation on muscle characteristics and locomotor performance in the Green-striped burrowing frog, Cyclorana alboguttata

The Green-striped burrowing frog, Cyclorana alboguttata survives extended drought periods by burrowing underground and aestivating. These frogs remain immobile within cocoons of shed skin and mucus during aestivation and emerge from their burrows upon heavy rains to feed and reproduce. Extended periods of immobilisation in mammals typically result in muscle atrophy and a decrease in muscle performance. We examined the effect of aestivation and hence prolonged immobilisation, on skeletal muscle mass, in vitro muscle performance, and locomotor performance in C. alboguttata. Frogs were aestivated in soil for 3 months and were compared with control animals that remained active, were fed, and had a continual supply of water. Compared to the controls, the wet mass of the gastrocnemius, sartorius, gracilus major, semimembranosus, peroneus, extensor cruris, tibialis posticus and tibialis anticus longus of aestivators remained unchanged indicating no muscle atrophy. The in-vitro performance characteristics of the gastrocnemius muscle were maintained and burst swimming speed was unaffected, requiring no recovery from the extended period of immobilisation associated with aestivation. This preservation of muscle size, contractile condition and locomotor performance through aestivation enables C. alboguttata to compress their life history into unpredictable windows of opportunity, whenever heavy rains occur.     

Metabolic depression and sodium-potassium ATPase in the aestivating frog, Neobatrachus kunapalari

In aestivation the metabolic rate of the Australian desert frog Neobatrachus kunapalari was 50–67% lower than in the non-aestivating state. The rate of O₂ consumption of isolated muscle, skin and brain was measured in both metabolic states. The average rate of O₂ consumption of muscle was 30% lower and brain 50% lower in aestivating frogs, while the rate of O₂ consumption of skin was the same. The reduction in muscle could account for a large proportion of whole animal metabolic depression. To look for evidence of a reduction in energy demand in the tissues we measured the ouabain-sensitive fraction of tissue rate of O₂ consumption, which is considered to be the proportion of metabolism used for transmembrane Na⁺/K⁺ pumping. Ouabain inhibited the in vitro rate of O₂ consumption of skin by a average of 20% and of brain by an average of 30%. However, in muscle, ouabain stimulated in vitro O₂ consumption. Despite the 50% reduction in the in vitro rate of O₂ consumption of brain during aestivation, neither the ouabain-sensitive nor ouabain-insensitive fractions were found be statistically different, possibly because of the large individual variation in the degree of ouabain inhibition. A reduction in the level of ion pumping during aestivation was therefore not demonstrated in any tissue. Measurement of the level of the enzyme Na⁺K⁺-ATPase in skeletal muscle, ventricle, kidney and brain showed that there was no change in the amount of this enzyme in the aestivating frogs. Measurement of the levels of adenylates in muscle and liver showed that the adenylate energy charge was maintained in aestivation, but that there was a reduction in ATP in liver and a reduction in the level of total adenylates in both tissues, which could be an adaptation of the tissues to a lower energy turnover. Accepted: 22 July 1996     

Energy loss in an aestivating population of the tropical snail pila globosa

The dried peripheral area of pond Idumban (62 ha) increased from 3.2 ha in January to 3 1.9 ha in April. Pila globosa, which were abundant in the littoral area, did not commence aestivation during this period, perhaps due to low temperature and/or high dissolved oxygen content. The number of aestivating snails averaged 0.5/m² in May, 1973 (3.6% of the total population) and it increased to 1.1/M² in September (26.2%). Biomass of the snail increased from 3.5 to 19.9 g dry weight (including shell)/M². Number of aestivating snails increased from 0.4/m² (5.2% of the total population) in May 1974 to 0.8/m² (11.1%) in July and the biomass from 4.1 g/m² to 10.7 g/m². Availability of dried area for aestivation increased from 5.3 to 23.7 ha in 1973 and from 13.5 to 30.2 ha in 1974.Monthly observations made on the marked snails forced to aestivate at 7.5, 15.0, 22.5 and 30.0 cm depth in the pond during May, revealed that temperature above 35°C and moisture below 5% were critical. Mortality and weight loss decreased in the snails forced to aestivate at increasing depth. Random observations indicated that 83% of the aestivating snails buried themselves at 15 cm depth in the pond. On the whole, 98,480 snails (592 Kg) and 115,270 (758 Kg) died during aestivation in 1973 and 1974 respectively. Of the total weight loss, the energy lost via metabolism contributed only a small fraction of 2.2% (12 Kg) and 2.1% (15 Kg) during these years. Considering the total aestivation area, the snails which succumbed averaged only 0.4/m²/year (2.5 g/m²/year). On an average, dry substance equivalent to about 2.6 mg dry weight/ g dry weight of snail/ day (3.7 gcal/ g live snail/ day) was lost on metabolism by the aestivating snails, i.e. the metabolic level of the aestivating snail was about 1 / 18th of that of the actively feeding snail.     

Structure and function of mitochondria in hepatopancreas cells from metabolically depressed snails

Mitochondria in cells isolated from the hepatopancreas of aestivating land snails (Helix aspersa) consume oxygen at 30% of the active control rate. The aim of this study was to investigate whether the lower respiration rate is caused by a decrease in the density of mitochondria or by intrinsic changes in the mitochondria. Mitochondria occupied 2% of cellular volume, and the mitochondrial inner membrane surface density was 17 microm(-1), in cells from active snails. These values were not different in cells from aestivating snails. The mitochondrial protein and mitochondrial phospholipid contents of cells were also similar. There was little difference in the phospholipid fatty acyl composition of mitochondria isolated from metabolically depressed or active snails, except for arachidonic acid, which was 18% higher in mitochondria from aestivating snails. However, the activities of citrate synthase and cytochrome c oxidase in mitochondria isolated from aestivating snails were 68% and 63% of control, respectively. Thus the lower mitochondrial respiration rate in hepatopancreas cells from aestivating snails was not caused by differences in mitochondrial volume or surface density but was associated with intrinsic changes in the mitochondria.     

Morpho-functional modifications of human syncytiotrophoblast plasma membrane during Pregnancy Induced Hypertension

Changes in [³⁵S]methionine protein labeling patterns were examined by following incorporation into the acid precipitate protein fraction of land snails,Otala lactea (Müller) (Pulmonata, Helicidae). Labeled proteins were analyzed by SDS polyacrylamide gel electrophoresis and isoelectric focusing columns. Snails in four different physiological states were compared: active controls, short term aestivating snails (injected and allowed to enter aestivation), long term aestivating snails (aestivated for 14 days, injected, and maintained in the aestivating state), and snails aroused after aestivation (aestivated, injected, and aroused). Protein associated radioactivity was measured over a 7 day time course post injection. Autoradiographic analysis of SDS-polyacrylamide gels showed increases in the radioactivity of four proteins: 91 kDa (hepatopancreas, day 1 in long term aestivating animals), 50 kDa (hepatopancreas, day 2 in short term aestivating snails), 70 kDa and 30 kDa (foot, day 2 in short term aestivating animals). Hepatopancreas and foot from day 1 long term aestivating and day 2 short term aestivating animals were also analyzed by isoelectric focusing columns. Several pH-specific differences were apparent when controls and aestivating animals were analyzed. In particular a peak of radioactivity was observed at pH 5.05 in 1 d long term aestivating hepatopancreas and at pH 4.30 in 2d short term aestivating animals. Several differences were noted in foot with no specific pattern emerging. SDS-polyacrylamide gel electrophoresis analysis of the hepatopancreas peaks showed the appearance of several bands with increased radioactivity, including the 91 kDa and 50 kDa proteins described above. These results suggest thatO. lactea aestivation specific proteins may be involved in the transition to a depressed metabolic state.Abbreviations dpm radioactive disintegrations per minute - PAGE polyacrylamide gel electrophoresis - SDS sodium dodecyl sulphate - SRP stress related protein     

Protein phosphorylation patterns during aestivation in the land snailOtala lactea

Protein phosphorylation patterns were investigated in whole tissues and subcellular fractions of active and aestivatingOtala lactea (Müller) (Pulmonata, Helicidae). Measurement of overall protein phosphorylation showed that incorporation of³²P increased until the second day after injection and remained constant for the remaining 4 days of the time course. Comparison of tissues from aestivating and active snails on day 3 showed a decreased protein phosphorylation in aestivating snails (44% of active). No differences in total and protein-associated radioactivity for foot, mantle or haemolymph were observed. Subcellular fractionation of the hepatopancreas localized the changes to plasma membrane, microsomal, and cytosolic fractions: values for aestivating animals were reduced to 71, 37 and 58% of the corresponding active values. Separation of the individual subcellular fractions on isoelectric focusing columns revealed differences in the phosphate incorporation patterns. Plasma membrane from aestivating animal hepatopancreas had a lower overall level of incorporation and fewer radioactive peaks in the pH 7–10 region than did the plasma membrane fraction from active animals. SDS-PAGE analysis of plasma membrane fractions from active and aestivating snails showed a relative decrease in phosphorylation between 60–80 kDa and 30–40 kDa. IEF analysis of cytosolic proteins from aestivating snail hepatopancreas also showed peaks of radioactivity that were apparently shifted by 0.3 pH units toward higher pI values. Increased phosphate incorporation was observed at a peak that corresponded to the pI value for pyruvate kinase in aestivating snails but definite assignment of peaks was not possible. SDS-PAGE analysis of cytosolic proteins showed an aestivation-related decrease in relative protein phosphorylation between 30–35 kDa and 40–45 kDa. A relative increase in phosphorylation during aestivation was observed for proteins between 16–22 kDa. Overall, the data indicate that snails dramatically alter their protein phosphorylation pattern in hepatopancreas during aestivation. (Mol Cell Biochem143: 7–13, 1995)Abbreviations CY cytosol - dpm radioactive disintegrations per minute - IEF isoelectrofocusing - GP glycogen phosphorylase - MC microsomes - MT mitochondria - PAGE polyacrylamide gel electrophoresis - PKF phosphofructokinase - PK pyruvate kinase - PM plasma membrane - SDS sodium dodecyl sulphate     

Do invasive cane toads (Chaunus marinus) compete with Australian frogs (Cyclorana australis)?

Abstract Despite widespread concern about the ecological impacts of invasive species, mechanisms of impact remain poorly understood. Cane toads (Chaunus [Bufo] marinus) were introduced to Queensland in 1935, and have now spread across much of tropical Australia. One plausible impact of toad invasion concerns competition between toads and native frogs, but there has been no previous experimental evaluation of this possibility. We examined interactions between toads and a morphologically similar species of native frog (Cyclorana australis) by manipulating toad and frog densities within large outdoor enclosures beside a floodplain in the wet‐dry tropics of the Northern Territory. Toads differed from frogs significantly in dietary composition and feeding rates, even in comparisons controlling for body‐size differences between these two taxa. Perhaps reflecting the abundant insect biomass, manipulating anuran densities or the presence of the putatively competing species did not influence food intake or dietary composition. However, the presence of toads suppressed activity levels of native frogs. The degree to which the invasion of cane toads influences attributes such as the activity levels, food intake and dietary composition of native frogs warrants further study, but our study suggests that competitive effects are likely to be minor compared with other pathways (such as direct poisoning during ingestion attempts) by which toads can affect frog populations.     

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.     

Lipid, ketone body and oxidative metabolism in the African lungfish, Protopterus dolloi following 60 days of fasting and aestivation

The potential importance of lipids and ketone bodies as fuels in the African lungfish, Protopterus dolloi, and the role of oxidative metabolism, were examined under control, fasted and aestivated conditions. In aestivating but not fasting lungfish, the activities of citrate synthase (CS) and cytochrome c oxidase (CCO) (enzymes of oxidative metabolism) showed tissue-specific changes. Significant reductions in CS activity occurred in the kidney, heart, gill and muscle, and in CCO in the liver and kidney tissues. Aestivation, but not fasting, also had a tissue-specific effect on mitochondrial state 3 respiration rates (using succinate as a substrate), with a >50% reduction in the liver, yet no change within muscle mitochondria. There is no indication that enzymes involved in lipid catabolism are up-regulated during periods of fasting or aestivation; however, both 3-hydroxyacyl CoA dehydrogenase (HOAD) and carnitine palmitoyl CoA transferase (CPT) activities were sustained in the liver despite the approximately 42% reduction in CCO activity, potentially indicating lipid metabolism is of importance during aestivation. Lungfish are able to utilize both the d- and l-stereoisomers of the ketone body beta-hydroxybutyrate (beta-HB); however, beta-HB does not appear to be an important fuel source during aestivation or fasting as no changes were observed in beta-HB tissue levels. This study demonstrates that an important aspect of metabolic depression during aestivation in lungfish is the tissue-specific down regulation of enzymes of aerobic metabolism while maintaining the activities of enzymes in pathways that supply substrates for aerobic metabolism.     

Water content, body weight and acid mucopolysaccharides, hyaluronidase and beta-glucuronidase in response to aestivation in Australian desert frogs

This study investigates the effects of aestivation on body water content, body mass, acid mucopolysaccharide (AMPS) and some of its degrading enzymes in different tissues for some Australian desert frogs. The AMPS component of the liver, kidney, skin and cocoon alter during aestivation to help retain water, which is unchanged in most tissues of all frog species, and to protect the frogs from desiccation during extended periods of aestivation. Hepatic AMPS was unaltered in Cyclorana maini, C. platycephala and Neobatrachus sutor but increased significantly after 2 months of aestivation in C. australis. The level of AMPS in the kidney was elevated in all four frog species after 5 months of aestivation. Skin AMPS content in the skin of awake frogs decreases with aestivation period and increases in the cocoon. AMPS in the cocoon probably works as a cement between the cocoons' layers and its physical presence presumably contributes to preventing water flux. Changes in AMPS content in different tissues were accompanied by significant changes in both hyaluronidase and beta-glucuronidase activities, which play an important role in AMPS metabolism. Alcian blue staining of control and digested skin of C. australis and C. platycephala with testicular hyaluronidase indicated the presence of AMPS, concentrated in a thin layer (called ground substance, GS) located between stratum compactum and stratum spongiosum, and acid mucin concentrated in the mucous glands and in a 'tubular' structure which could be observed in the epidermal layer. Hyaluronidase digestion of the cocoon slightly changed the Alcian Blue colour, suggesting the presence of a large amount of acid mucin similar to that found in the skin mucous gland. The results of this study present data for the redistribution of AMPS, which may help in reducing water loss across the cocoon and reabsorption of water in the kidney during aestivation.     

Relictual physiological ecology in the threatened land snail Codringtonia helenae: A cause for decline in a changing environment?

Land snails often exhibit intra-annual cycles of activity interspersed by periods of dormancy (hibernation/aestivation), accompanied by a range of behavioural and physiological adaptations to ensure their survival under adverse environmental conditions. These adaptations are useful to understand species-specific habitat requirements and to predict their response to environmental changes. We examined the seasonal physiological and biochemical composition patterns of the threatened land snail Codringtonia helenae, endemic to Greece, in relation to its behavioural ecology and climatic conditions. Fuel reserves (glycogen, lipids, proteins) and water were accumulated prior to aestivation, but subsequently were rapidly depleted. LDH exhibited substantial rise during aestivation, suggesting that anaerobic pathways may provide additional energy. The major outcome of our study was the unambiguous discrimination of the four life-cycle periods. Most remarkable was the clear distinction of the aestivation period, with hibernation, the other dormancy period, showing similarity with the two active periods but not with aestivation. We observed disassociation between behavioural and physiological responses and climatic conditions. The physiological responses of C. helenae were effective during hibernation, but only partly compensate the effect of adverse conditions during aestivation, since its aestivating behaviour is occasional and time limited. Perhaps, the behavioural ecology of Codringtonia is relictual and shaped during past environmental conditions. This constitutes an important extinction threat considering the current climatic trends and the deterioration of the habitat of that species due to human activities.