Desert Toad Research Articles

Plasma and brain angiotensin concentrations associated with water response behavior in the desert anuran, Scaphiopus couchii under natural conditions in the field

Terrestrial amphibians obtain water by absorption across a specialized region of the ventral skin and exhibit a behavior, the water absorption response (WR) to place that region in contact with moist surfaces. Spadefoot toads ( Scaphiopus couchii) spend dry months of the year in burrows, then emerge during brief periods of summer rainfall and seek water sources for rehydration and reproduction. We tested the hypothesis that these toads have changes in plasma and/or central angiotensin concentrations that are associated with seasonal emergence and WR behavior. Immunoreactive concentrations of combined angiotensin II and III (ir-ANG) were measured in plasma samples and microdissected regions of brain tissue taken from toads moving across the road or toads showing WR behavior in shallow puddles on the road. Plasma ir-ANG concentrations were not significantly different between these groups, but were significantly higher in the periventricular region of the hypothalamus in toads showing WR behavior. Concentrations in other brain regions, while highly variable among individuals, were not different between groups. Within the context of the natural history of a specialized desert toad, these results support the hypothesis that ir-ANG is associated with WR behavior in spadefoot toads in a manner analogous to oral drinking exhibited by other vertebrate clades.

Evaluation of MS-222 (Tricaine Methanesulfonate) and Propofol as Anesthetic Agents in Sonoran Desert Toads (Bufo alvarius)

Toads in the genus Bufo are commonly kept in pet, research, and zoological settings and may require anesthesia during veterinary care. Limited information is available comparing anesthetic protocols in most amphibian species. The purpose of this study was to evaluate the clinical and cardiopulmonary effects of tricaine methanesulfonate (MS-222) and propofol in Sonoran desert toads (Bufo alvarius). Nine juvenile Sonoran desert toads were anesthetized with an immersion bath of 1 g/L MS-222 and 35 mg/kg intracoelomic propofol with a minimum 2-wk wash-out period between trials. Heart rate, respiratory rate, and reflexes (righting, escape, corneal, superficial pain, and deep pain) were monitored every 5 min for the first 90 min and then every 10 min for the next 90 min during both anesthetic trials. Surgical anesthesia was defined as complete loss of all measured reflexes. MS-222 produced surgical anesthesia in 100% (9/9) of toads, whereas propofol produced surgical anesthesia in 11.1% (1/9). Mean induction time for the MS-222 trial was 19.9 min (SD: 5.4, Min–Max: 13–30), with mean duration of surgical anesthesia 23.9 min (SD: 10.8, Min–Max: 10–42). Mean recovery time after removal from the MS-222 bath was 85.3 min (SD: 18.5, Min–Max: 60–110). Righting reflex was lost in all animals in the propofol trial at a mean of 23.9 min (SD: 5.5, Min–Max: 20–35) following administration. A single animal in the propofol trial reached a surgical plane of anesthesia at 25 min post-administration, with surgical anesthesia lasting for 50 min. Mean time to recovery following administration of propofol was 145 min (SD: 47.2, Min–Max: 60–180); one toad was not fully recovered at the end of the monitoring period. Heart rate was not found to significantly (P > 0.05) change from baseline at any monitoring point for either anesthetic protocol. Respiratory rate was found to decrease significantly (P < 0.05) at all time points between 5 and 65 min in the MS-222 trial and between 10 and 130 min in the propofol trial. MS-222 at 1 g/L immersion was found to reliably produce surgical anesthesia in Sonoran desert toads with a faster onset of action and recovery when compared to propofol administered intracoelomically at 35 mg/kg.

Epigenetic silencers are enriched in dormant desert frog muscle

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

One year in the life of Bufo punctatus: annual patterns of body temperature in a free-ranging desert anuran

The Mojave Desert is characterized by hot dry summers and cold winters. The red-spotted toad (Bufo (Anaxyrus) punctatus) is the predominant anuran species; yet little is known of their thermal histories and strategies to avoid temperature extremes. We measured body temperature (T(b)) in free-ranging adult toads across all four seasons of a year using implanted data loggers. There is marked individual variation in the temperatures experienced by these toads. As expected, toads generally escape extreme seasonal and diel temperature fluctuations. However, our data demonstrate a much wider estimated T(b) range than was previously assumed. Though often for short periods, red-spotted toads do experience T(b) as low as 3.1 degrees C and as high as 39.1 degrees C. All animals showed periods of prolonged thermal stability in cooler months and wider diel oscillations in warmer months. Red-spotted toad thermal history is likely a function of site choice; the exploitation of different refuges results in diverse thermal experiences. These data represent the most complete record of thermal experiences for a desert anuran and reveal greater extremes in body temperature than previously suggested.

Role of calcium and vesicle-docking proteins in remobilising dormant neuromuscular junctions in desert frogs

Despite prolonged immobility the desert frog, Cyclorana alboguttata, suffers little impairment in muscle function. To determine compensatory mechanisms at neuromuscular junctions, transmitter release was examined along primary terminals in C. alboguttata iliofibularis muscle. Using extracellular recording we found the amplitudes of evoked endplate currents were significantly smaller in dormant frogs. In active frogs we identified two negatively sloping proximal-distal gradients of transmitter frequency and quantal content; a shallow proximal-distal gradient with low probability of transmitter release (<0.2) and a second much steeper proximal-distal gradient for quantal content with high probability release sites (>0.6). During aestivation, only a shallow gradient was identified. The high probability release sites in control frogs were inhibited during aestivation by a mechanism that could be reversed by (1) increasing the extracellular calcium concentration, and (2) increasing the frequency of stimulation. This suggests that transmitter vesicles are available during aestivation but not released. We quantified expression of messenger RNA transcripts coding for the transmitter vesicle-docking proteins synaptotagmin 1, syntaxin 1B and UNC-13. All three were rare transcripts maintained at control values during aestivation. Neuromuscular remobilisation after dormancy in C. alboguttata is more likely a product of rapidly reversible physiologic mechanisms than reorganisations of the neuromuscular transcriptome.

Variation in thyroid hormone action and tissue content underlies species differences in the timing of metamorphosis in desert frogs

Hormonal control of post-embryonic morphogenesis is well established, but it is not clear how differences in developmental endocrinology between species may underlie animal diversity. We studied this issue by comparing metamorphic thyroid hormone (TH) physiology and gonad development across spadefoot toad species divergent in metamorphic rate. Tissue TH content, in vitro tail tip sensitivity to TH, and rates of TH-induced tail tip shrinkage correlated with species differences in larval period duration. Gonad differentiation occurred before metamorphosis in species with long larval periods and after metamorphosis in the species with short larval periods. These differences in TH physiology and gonad development, informed by phylogeny and ecology of spadefoot metamorphosis, provide evidence that selection for the short larval periods in spadefoot toads acted via TH physiology and led to dramatic heterochronic shifts in metamorphic climax relative to gonad development.

Priority effects and desert anuran communities

The roles of priority effects and predation in structuring anuran communities have not been considered important in desert environments characterized by highly ephemeral pools and anuran species that breed synchronously. Predation is one mechanism that can set the stage for priority effects to be important, especially in ephemeral pools where resources are limiting and densities are high. We observed oophagy by Scaphiopus couchii Baird, 1854 on eggs of Bufo speciosus Girard, 1854 in the field and conducted laboratory experiments to test the hypothesis that S. couchii tadpoles would reduce B. speciosus survivorship via predation. Three-day-old S. couchii tadpoles reduced hatching success of B. speciosus eggs by nearly 90%. When S. couchii and B. speciosus eggs were laid the same day, S. couchii tadpoles reduced B. speciosus egg survivorship by 56%. Our results indicate that priority effects and predation may play an important role in influencing species composition of anuran assemblages in desert regions. Our study provides more support for the need to reevaluate the current paradigm in aquatic ecology that suggests predation does not play a role in structuring community assemblages in highly ephemeral pools.

Large-scale Habitat Associations of Four Desert Anurans in Big Bend National Park, Texas

We used night driving to examine large scale habitat associations of four common desert anurans in Big Bend National Park, Texas. We examined association of soil types and vegetation communities with abundance of Couch's Spadefoots (Scaphiopus couchii), Red-spotted Toads (Bufo punctatus), Texas Toads (Bufo speciosus), and Western Green Toads (Bufo debilis). All four species were disproportionately associated with frequently inundated soils that are relatively high in clay content. Bufo punctatus was associated with rocky soil types more frequently than the other three species. Association between all four species and vegetation types was disproportionate in relation to availability. Bufo debilis and Bufo punctatus were associated with creosote and mixed scrub vegetation. Bufo speciosus and Scaphiopus couchii were associated with mesquite scrub vegetation. Bufo debilis, Scaphiopus couchii, and B. speciosus were more tightly associated with specific habitat types, whereas B. punctatus exhibited a broader distribution across the habitat categories. Examining associations between large-scale habitat categories and species abundance is an important first step in understanding factors that influence species distributions and presence-absence across the landscape.

Spermatogenesis and plasma testosterone levels in Western Australian burrowing desert frogs, Cyclorana platycephala, Cyclorana maini, and Neobatrachus sutor, during aestivation

Changes in testis size, histological status, and plasma levels of testosterone were monitored for males of three species of Western Australian desert frogs, Cyclorana maini, Cyclorana platycephala, and Neobatrachus sutor during aestivation. The frogs were induced to burrow and form cocoons soon after their capture and then disinterred at intervals in order to monitor changes in reproductive activity of the testes. All stages of spermatogenesis were evident in active frogs, which were collected a few days following rain from breeding choruses. Relative testis mass declined gradually in all species during the first 7 months of aestivation and then increased significantly at 16–19 months in the two species for which extended data were available ( C. maini and N. sutor). A decrease in the number of sperm bundles 2–4 months after cocooning was associated with an initial increase in the number of free spermatazoa in all three species, which then returned to the levels seen in active animals after 7 months. Increases in the number of primary and secondary spermatogonia were most evident in C. platycephala after 4–7 months of aestivation, but early stages of spermatocytogenesis were evident in all species after 7 months of aestivation, especially in individuals that contained neither sperm bundles nor mature spermatazoa. Changes in plasma testosterone levels correlated significantly with variations in the diameter of the seminiferous tubules and the GSI, suggesting that this hormone plays a major role in controlling testicular recrudescence in aestivating, cocooned, desert frogs. Data from this study show that, in the absence of any external cues, testicular recrudescence is evident after approximately one year of aestivation in desert frogs which prepares them to breed again, once rain falls.

Pathological effects of Pseudodiplorchis americanus (Monogenea: Polystomatidae) on the lung epithelium of its host, Scaphiopus couchii.

Infection of the desert toad, Scaphiopus couchii, by the monogenean Pseudodiplorchis americanus involves 2 principal sites: post-invasion juveniles reside in the respiratory tract for 1 month before migrating to the urinary bladder where they reach sexual maturity and may live up to 4 years. While previous work has demonstrated the long-term impact on host condition of the blood-feeding adults, this study assesses pathological effects of the short-term pulmonary infection. Lung ultrastructure was compared in toads (i) maintained in captivity for 1 year without invasion, and (ii) experimentally infected with 50-300 juveniles/host, equivalent to burdens in the wild, and examined 23-44 days p.i. Typically, the alveolar lining of S. couchii is composed of a single cell type with characteristics of both Type I and Type II pneumocytes. However, infected lung tissue exhibited an inflammatory reaction with epithelial cell vacuolation, interstitial oedema, and an increase of alveolar exudate, leucocytes and fibrous tissue. Accompanying a post-infection increase in host immune cells in the lungs, there was evidence of reciprocal tegumental damage to the parasites. Lung epithelium of toads free of infection for 1 year exhibited scar tissue representing a residual effect of past infection. The pathological consequences of P. americanus infection therefore have 2 components. Acute lung infection coincides with the host's brief activity season: impaired respiratory function could compromise feeding and accumulation of reserves and hence ability to survive following a 10 month period of hibernation. Additionally, adult toads are normally exposed annually to re-infection and may accumulate chronic lung damage with extended effects on host survival.

Water content, body weight and acid mucopolysaccharides, hyaluronidase and β-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 β-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.

Competition, predation, and the distributions of four desert anurans.

Several studies have shown that larval competition and susceptibility to predation affect distributions of amphibian assemblages across ephemeral and perennial habitats. However, few studies have examined mechanisms affecting distribution patterns and site use of anurans adapted to highly ephemeral habitats. This study examines hypotheses about competition and predation as mechanisms creating non-overlapping patterns of site use in four anurans that breed in highly ephemeral habitats: Scaphiopus couchii (Couch's spade-foot toad), Gastrophryne olivacea (narrow-mouthed toad), Bufo speciosus (Texas toad), and Bufo punctatus (red-spotted toad). These four anurans showed a significantly nonrandom pattern of co-occurrence. Only 12% of 95 ephemeral breeding sites surveyed were occupied by more than one species. We tested the hypotheses that non-overlapping use of breeding sites was due to activity rates of their tadpoles that in turn reflect their competitive ability and susceptibility to predation. Tadpoles of S. couchii were significantly more active and more susceptible to predation than were tadpoles of G. olivacea, B. speciosus, and B. punctatus. The masses of G. olivacea, B. speciosus, and B. punctatus were less when they were reared with S. couchii, demonstrating the possible competitive dominance of S. couchii. These results suggest that the competitive ability of S. couchii may play a role in excluding G. olivacea, B. speciosus, and B. punctatus from very ephemeral breeding sites, and that susceptibility to predation could play a role in excluding S. couchii from breeding sites of longer duration that are more likely to be colonized by aquatic predators.

The spinal nerves innervate putative chemosensory cells in the ventral skin of desert toads, Bufo alvarius.

Toads normally obtain water by absorption across their skin from osmotically dilute sources. When hyperosmotic salt solutions are presented as a hydration source to dehydrated desert toads, they place the ventral skin onto the source but soon afterwards escape to avoid dehydration. The escape behavior coincides with neural excitation of the spinal nerves that innervate putative chemosensory cells in the ventral skin. In the present study, fluorescent dye translocated through the spinal nerves to those receptor cells in the epidermis was photoconverted in the presence of 3, 3'-diaminobenzidine tetrahydrochloride for electron-microscopic observation of the cells and associated nerve terminals. Most of the photoconverted cells were located in the deepest layer of the epidermis, with some being in more intermediate layers. No labeled cell was seen in the outermost layer of living cells. In desert toads, flask cells and Merkel cells are occasionally seen in the epidermis. An association of nerve fibers with these epidermal cells has been reported in some species of the anurans. In the present study, however, the cytological features of the photoconverted cells are neither reminiscent of flask cells nor Merkel cells, but are similar to those of surrounding epithelial cells in each layer of the epidermis. We hypothesize a sensory function for these cells, because they have a close association with nerve fibers and participate in the transepithelial transport of salts that must pass through all cell layers of the skin.

Parasite adaptation to extreme conditions in a desert environment

Deserts represent universally recognized extreme environments for animal life. This paper documents the highly specialized adaptations of Pseudodiplorchis americanus, a monogenean parasite of the desert toad, Scaphiopus couchii. Building on a long-term record of parasite population ecology (continuing since the early 1980s), field studies focus on the effects of severe drought in the Sonoran Desert, Arizona, in the mid 1990s. This provides a test of the ability of the host-parasite system to tolerate exceptional perturbation. The analysis provides new insight into parasite infection dynamics in a natural wildlife system through integration of host and parasite population age structure. The environmental check interrupted host recruitment in 1993-95 and parasite recruitment in 1995-97. This produced an imprint in age structure and infection levels recognizable over several years: parasite recruitment failure reduced transmission 2-3 years later. The host (maximum life span 17 years) tolerated the disruption but the impact was more serious for the parasite (life span 3 years) leading to extinction of some previously stable populations. Despite this demonstration of a rare event exacerbating external environmental constraints, experimental studies suggest that the internal (host) environment normally creates the most severe conditions affecting P. americanus. Only about 3% of parasites survive from invasion until first reproduction. Post-invasion factors including host immunity, characteristic of most parasite life cycles, constitute a greater constraint upon survival than external conditions, even in a desert environment.

Desert toads discriminate salt taste with chemosensory function of the ventral skin.

Toads obtain water by absorption across their skin. When dehydrated, desert toads exhibit stereotyped hydration behavior in which they press their ventral skin onto a moist surface. However, dehydrated toads avoid surfaces moistened with hyperosmotic NaCl and KCl solutions (Hoff KvS, Hillyard SD. 1993. J. Exp. Biol. 183:347-351). We have studied neural mechanisms for this avoidance with physiologic, behavioral, and morphologic approaches. Spinal nerves innervating the ventral skin could be stimulated by exposure to a hyperosmotic NaCl solution applied to the outer surface of the skin. This neural response occurred with much longer latency than to mechanical stimulation and could be reduced by amiloride, a blocker for Na+ channels known to be responsible for epithelial ion transport and salt taste transduction. In behavioral experiments, avoidance of a NaCl solution was also reduced by adding amiloride to the solution, suggesting involvement of amiloride-sensitive Na+ channels for detecting the hyperosmotic salt solution. Neural tracing with fluorescent dye revealed spinal nerve endings and connections to putative receptor cells, both located in the deeper layer of the epidermis. Either of these or both may be associated with the transduction of Na+ flowing into the skin. The ability of toads to detect hyperosmotic salt solutions in their environment reveals a previously unknown chemosensory function for spinal nerves in anuran amphibians.

The Role of Protein Synthesis During Metabolic Depression in the Australian Desert Frog Neobatrachus centralis

Little is known about the role of energy consuming processes during metabolic depression. We have shown that aestivation in the Australian desert frog Neobatrachus centralis is accompanied by an in vivo metabolic depression of 77%. Using an in vitro liver slice preparation, we have measured an in vitro metabolic depression in liver of 55% with a concomitant 67% decrease in the rate of protein synthesis. The decrease in protein synthesis accounts for 52% of the metabolic depression of the tissue, but only 4.9% of the metabolic depression of the whole animal. No in vitro metabolic depression or decrease in protein synthesis during aestivation was measured in muscle, but a decrease in the low rate of protein synthesis in muscle in vivo could not, in any case, account for more than 3% of the metabolic depression of the whole animal. The liver, although not a quantitatively important tissue in terms of metabolic depression in vivo, offers the opportunity to characterise the regulation of protein synthesis in a system in which metabolic depression is not confounded by changes in ambient temperature and P O 2 .

Histological analysis of the egg capsule of the ovoviviparous polystomatid monogenean, Pseudodiplorchis americanus

Transmission of Pseudodiplorchis americanus is restricted to the brief period when its host, a desert toad, enters water to spawn. The parasite accumulates its entire annual reproductive output within an elongated uterus during the 10–11-month period of host hibernation. Embryos of P. americanus, at all stages of development, are retained within the uterus which eventually becomes packed with around 150 encapsulated infective larvae. Recently formed eggs, which comprise a fertilized ovum and 2–3 vitelline cells, are closely surrounded by a primary eggshell which stains positively for acidic proteins and keratin. Initially, during passage along the proximal uterus, the egg capsule is only 60 μm in diameter, but as it passes to the distal uterus it expands to 800 μm in diameter to accommodate the growing larva. Due to chemical alterations or complete replacement of the shell, the final (secondary) egg capsule is a large sac-like structure composed of elastin. The flexible nature of this shell maximizes the numbers of infective larvae which can be stored in utero. TEM studies have revealed this capsule to be composed of multi-laminate membranes with a specialized cytoplasmic lining involved in a unique mechanism for embryo nutrition. This is the first report of an elastintype eggshell within the Monogenea.

Effects of Urea on M4-Lactate Dehydrogenase from Elasmobranchs and Urea-Accumulating Australian Desert Frogs

We measured the effect of urea on M4-lactate dehydrogenase (M4-LDH) from elasmobranchs and Australian desert frogs (urea accumulators) and from two animals that do not accumulate urea, the axolotl and the rabbit. An analysis of the effect of urea on the Kd(NADH), V, V/Km(pyr) and V/Km(NADH) shows that in all cases the major effect of urea was on the binding of pyruvate, which fits with data in the literature that show that urea acts as a competitive inhibitor of LDH. The characteristics of the elasmobranch enzymes are consistent with a proposed adaptation model, but the situation for the enzymes from the aestivating frogs is equivocal. Urea (400 mM) had less effect on the Km(pyr) of M4-LDH from the urea accumulators than it did on the non-accumulators, suggesting a general adaptation and that the enzyme produced by the aestivating frogs (urea accumulators) is kinetically different from that of non-aestivating frogs (non-accumulators). A new approach is used to characterize the overall pattern of adaptation to urea. The pattern is similar in an enzyme from an elasmobranch and an aestivating frog despite the temporary presence of urea in the latter and the phylogenetic difference between these animals.

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.

Do Australian desert frogs co-accumulate counteracting solutes with urea during aestivation?

Australian desert frogs of the genera Neobatrachus, Cyclorana and Heleioporus experience significant dehydration, and iono- and osmoconcentration, during aestivation in the laboratory and accumulate substantial amounts of urea (100-200 mmol)(l-1). We expected a priori that aestivating frogs probably would not need to accumulate balancing osmolytes but would accumulate trimethylamine oxide (TMAO) or betaine as counteracting solutes to urea. These aestivating frogs did not co-accumulate a substantial quantity of any particular balancing osmolyte or counteracting solute, such as a methylamine [TMAO, trimethylamine amine (TMA), betaine, sarcosine, glycerophosphorylcholine (GPC)] or polyol (inositol, mannitol, sorbitol) in plasma or muscle relative to urea accumulation. However, for aestivating frogs, the total concentration of all measured methylamines and polyols (TMAO + TMA + betaine + sarcosine + GPC + inositol) in muscle was approximately 35-45 mmol kg-1, and so it is possible that all of these solutes have a combined counteracting osmolyte role in aestivating frogs at a ratio to urea of approximately 1:2.5, as has been described for elasmobranch fishes. Alternatively, the absence of substantial co-accumulation with urea of any particular solute suggests that aestivating frogs might not require any major extracellular or intracellular counteracting solutes (TMAO, betaine, GPC). The enzyme systems of these aestivating frogs may be insensitive to the perturbing effects of urea, or the perturbing effects of accumulated urea may be a mechanism for metabolic depression, during aestivation.