Rebecca L. Cramp

Branchial osmoregulation in the euryhaline bull shark, Carcharhinus leucas: a molecular analysis of ion transporters.

SUMMARY Bull sharks, Carcharhinus leucas, are one of only a few species of elasmobranchs that live in both marine and freshwater environments. Osmoregulation in euryhaline elasmobranchs is achieved through the control and integration of various organs (kidney, rectal gland and liver) in response to changes in environmental salinity. However, little is known regarding the mechanisms of ion transport in the gills of euryhaline elasmobranchs and how they are affected by osmoregulatory challenges. This study was conducted to gain insight into the branchial ion and acid-base regulatory mechanisms of C. leucas by identifying putative ion transporters and determining whether their expression is influenced by environmental salinity. We hypothesised that expression levels of the Na+/K+-ATPase (NKA) pump, Na+/H+ exchanger 3 (NHE3), vacuolar-type H+-ATPase (VHA) and anion exchanger pendrin (PDN) would be upregulated in freshwater (FW) C. leucas. Immunohistochemistry was used to localise all four ion transporters in gills of bull sharks captured in both FW and estuarine/seawater (EST/SW) environments. NHE3 immunoreactivity occurred in the apical region of cells with basolateral NKA expression whereas PDN was apically expressed in cells that also exhibited basolateral VHA immunoreactivity. In accordance with our hypotheses, quantitative real-time PCR showed that the mRNA expression of NHE3 and NKA was significantly upregulated in gills of FW-captured C. leucas relative to EST/SW-captured animals. These data suggest that NHE3 and NKA together may be important in mediating branchial Na+ uptake in freshwater environments, whereas PDN and VHA might contribute to Cl-/HCO3- transport in marine and freshwater bull shark gills.

Changes in cutaneous microbial abundance with sloughing: possible implications for infection and disease in amphibians

The emergence of disease as a significant global threat to amphibian diversity has generated considerable interest in amphibian defenses against cutaneous microbial infection and disease. To date, however, the influence of sloughing on the susceptibility of amphibians to infection and disease has been largely overlooked. To investigate the potential for sloughing to regulate topical microbial loads, the abundance of cultivable cutaneous bacteria and fungi in the cane toad Rhinella marina were compared before and after sloughing. Toads were also exposed to fluctuating thermal regimes (10-20 and 20-30°C) and variable photoperiods to investigate possible effects of season and climate on sloughing periodicity. Sloughing substantially reduced the abundance of cultivable cutaneous bacteria and fungi by up to 100%. The intermoult interval of toads maintained at 10-20°C was twice that of animals at 20-30°C and did not appear to thermally acclimate. Photoperiod had no discernable influence on sloughing periodicity. Results of this study suggest that normal sloughing cycles could play a significant role in controlling the persistence and build-up of cutaneous microbes, including pathogens. The loss of non-pathogenic commensal and protective skin microbiota after sloughing may also influence host susceptibility to cutaneous pathogens. We suggest that the spatio-temporal dynamics of chytridiomycosis, the widespread and often fatal disease caused by the fungal pathogen Batrachochytrium dendrobatidis, are related to temperature not only because of its effect on the growth of the fungus, but also because of its effect on the frequency of host sloughing.

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 frogs 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 5weeks of aestivation. The effects of aestivation on mass specific in vitro tissue metabolic rate (VO(2)) varied among individual organs, with muscle and liver slices showing significant reductions in metabolism; kidney VO(2) was elevated and there was no change in the VO(2) of small intestine tissue slices. Organ size was also affected by aestivation, with significant reductions in the mass of all tissues, except the gastrocnemius. These reductions in organ size, combined with changes in mass specific VO(2) of tissue slices, resulted in further energy savings to aestivating animals. This study shows that C. alboguttata is capable of selectively down- or up-regulating individual tissues, using both changes in metabolic rate and morphology. This strategy allows maximal energy savings during aestivation without compromising organ functionality and survival at arousal.

Skeletal muscle atrophy occurs slowly and selectively during prolonged aestivation in Cyclorana alboguttata (Gunther 1867).

SUMMARY 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) (μm2), water content (%) and myofibre number (per mm2) remained unchanged in the cruralis muscle after six to nine months of aestivation; however, myofibre area (μm2) 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.

Physiological responses of ectotherms to daily temperature variation

ABSTRACT Daily thermal fluctuations (DTFs) impact the capacity of ectotherms to maintain performance and energetic demands because of thermodynamic effects on physiological processes. Mechanisms that reduce the thermal sensitivity of physiological traits may buffer ectotherms from the consequences of DTFs. Species that experience varying degrees of DTFs in their environments may differ in their responses to thermally variable conditions, if thermal performance curves reflect environmental conditions. We tested the hypothesis that in response to DTFs, tadpoles from habitats characterised by small DTFs would show greater plasticity in the thermal sensitivity of physiological processes than tadpoles from environments characterised by large DTFs. We tested the thermal sensitivity of physiological traits in tadpoles of three species that differ naturally in their exposure to DTFs, raised in control (24°C) and DTF treatments (20–30°C and 18–38°C). DTFs reduced growth in all species. Development of tadpoles experiencing DTFs was increased for tadpoles from highly thermally variable habitats (∼15%), and slower in tadpoles from less thermally variable habitats (∼30%). In general, tadpoles were unable to alter the thermal sensitivity of physiological processes, although DTFs induced plasticity in metabolic enzyme activity in all species, although to a greater extent in species from less thermally variable environments. DTFs increased upper thermal limits in all species (between 0.89 and 1.6°C). Our results suggest that the impact of increased thermal variability may favour some species while others are negatively impacted. Species that cannot compensate for increased variability by buffering growth and development will probably be most affected. Summary: When developing ectotherms cannot reduce the costs of daily temperature fluctuations through phenotypic plasticity, effects on the rate of development may determine fitness consequences.

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

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

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

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

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

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

First line of defence: the role of sloughing in the regulation of cutaneous microbes in frogs

The skin is the first line of defence in preventing the establishment of pathogens and associated infections in frogs. Regular sloughing of the outer layer can reduce the abundance of cultivable cutaneous microbes in green tree frogs which has ramifications for our understanding of cutaneous pathogens like the amphibian chytrid fungus.

Functional and morphological plasticity of crocodile (Crocodylus porosus) salt glands.

SUMMARY The estuarine crocodile, Crocodylus porosus, inhabits both freshwater and hypersaline waterways and maintains ionic homeostasis by excreting excess sodium and chloride ions via lingual salt glands. In the present study, we sought to investigate the phenotypic plasticity, both morphological and functional, in the lingual salt glands of the estuarine crocodile associated with chronic exposure to freshwater (FW) and saltwater (SW) environments. Examination of haematological parameters indicated that there were no long-term disruptions to ionic homeostasis with prolonged exposure to SW. Maximal secretory rates from the salt glands of SW-acclimated animals (100.8±14.7 μmol 100 g–0.7 body mass h–1) were almost three times greater than those of FW-acclimated animals (31.6±6.2 μmol 100 g–0.7 body mass h–1). There were no differences in the mass-specific metabolic rate of salt gland tissue slices from FW- and SW-acclimated animals (558.9±49.6 and 527.3±142.8 μl O2 g–1 h–1, respectively). Stimulation of the tissue slices from SW-acclimated animals by methacholine resulted in a 33% increase in oxygen consumption rate. There was no significant increase in the metabolic rate of tissues from FW-acclimated animals in response to methacholine. Morphologically, the secretory cells from the salt glands of SW-acclimated animals were larger than those of FW-acclimated animals. In addition, there were significantly more mitochondria per unit volume in secretory tissue from SW-acclimated animals. The results from this study demonstrate that the salt glands of C. porosus are phenotypically plastic, both morphologically and functionally and acclimate to changes in environmental salinity.