Physiological and morphological correlates of extreme acid tolerance in larvae of the acidophilic amphibian Litoria cooloolensis.

The Cooloola sedgefrog (Litoria cooloolensis) is one of a number of frog species endemic to the coastal sandy lowlands of east Australia exhibiting remarkable tolerance to dilute waters of low pH (< pH 3.5). To investigate the physiological and morphological underpinnings of acid tolerance in L. cooloolensis larvae, we compared Na+ balance, uptake and efflux rates, and gill and skin morphology in larvae reared in circum-neutral (pH 6.5) and pH 3.5 water. We hypothesised that L. cooloolensis larvae would be more resistant to ionregulatory disturbance and epithelial damage at low pH relative to acid-sensitive species. Net Na+ flux rates were not significantly different from zero in L. cooloolensis larvae reared at pH 3.5 and in acid-naïve animals maintained in pH 6.5 water. Animals reared at pH 6.5 and acutely exposed to pH 3.5, however, exhibited a net loss of Na+ due to inhibition of Na+ uptake. In contrast, L. cooloolensis larvae reared at pH 3.5 maintained Na+ balance at pH 3.5 and did not exhibit inhibition of Na+ uptake at this pH. Investigation of Na+ transport kinetics and the morphology of the gills and integument suggests tolerance of L. cooloolensis larvae to low pH may be attributed to a high capacity for branchial Na+ uptake, increased tight junction length and elevated mucus production at the gills and integument. These factors confer resistance to acid damage and disruption of ionic homeostasis which would otherwise result in the death of amphibian larvae exposed to waters of pH 4.0 and less.

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

Amphibian populations worldwide are currently experiencing unprecedented declines due to the combined effects of emerging infectious disease and climate change. The skin is the first line of defence in preventing establishment of pathogens and associated infections. Although amphibians undergo regular sloughing of the outer layer of the skin, the potential for regular sloughing to play a role in influencing cutaneous microbial populations and pathogens has been largely overlooked. In the present study, we assessed the effect of skin sloughing on cultivable cutaneous bacterial abundance in the green tree frog (Litoria caerulea). We also examined the effects of temperature and hydric environment on sloughing frequency and microbial re-establishment rates. Our data showed that cultivable cutaneous bacterial abundance was significantly reduced by sloughing events, and frogs kept at 'summer' temperatures (23-33°C) sloughed almost twice as frequently as those maintained at 'winter' temperatures (13-23°C). No effect of hydric environment on sloughing frequency was observed, but we did find that sloughing in L. caerulea appeared to be linked to ambient light cycles. Examination of the effect of sloughing on microbial recolonization indicated that at cool temperatures, an extended intermoult interval allowed microbial abundance to reach higher levels than at warmer 'summer' temperatures (when the intermoult interval was significantly reduced). Our data suggest that sloughing may significantly influence the establishment and/or maintenance of cutaneous bacterial populations (pathogenic, mutualistic and/or commensal) and this, in turn, may be affected by environmental factors, such as ambient light and temperature. These findings are likely to be important for our understanding of the ecology of skin-based pathogens, such as the amphibian chytrid fungus, Batrachochytrium dendrobatidis.

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.

Damage to the gills and integument of Litoria fallax larvae (Amphibia: Anura) associated with ionoregulatory disturbance at low pH.

In fish, exposure to waters of low pH causes significant damage to the gill resulting in fatal iono- and osmoregulatory disturbance. In amphibians, exposure to acid waters also disrupts ionic homeostasis, however the extent and nature of injuries to amphibian larvae from acid exposure are poorly understood. Changes in gross morphology and ultrastructure of the gills and integument were examined, together with measures of Na(+) efflux/uptake, in larval Litoria fallax (Amphibia: Anura) following acute acid exposure. Examination of tissues revealed significant changes in morphology and ultrastructure of both gills and the integument following acutely lethal exposure to low pH water. Changes to the gills of acid-exposed L. fallax larvae included lifting of the branchial epithelium and opening of tight junctions between pavement cells (with a consequent reduction in tight junction length). Damage to epithelial cell-cell tight junctions was also apparent at the integument along with widespread oncosis and localised epithelial necrosis. Mucous secretory activity at the gills and body surface was largely unaffected by acid exposure, with little or no difference in density, cross-sectional area and number of epithelial mucous secretory vesicles in acid-exposed and control larvae. Changes in morphology and ultrastructure at low pH were accompanied by significant Na(+) loss (up to 50% of the total body Na(+) content) attributable in large part to increased paracellular ionic efflux across the gills as well as increased transcellular and paracellular efflux of ions across the integument.

Ups and downs of intestinal function with prolonged fasting during aestivation in the burrowing frog, Cyclorana alboguttata.

Although green striped burrowing frogs (Cyclorana alboguttata) experience large reductions in the mass and absorptive surface area of the small intestine (SI) during aestivation, little is known about how this may affect the functional capacity of the SI. We examined changes in the function (l-proline uptake rate and capacity) and metabolism of the SI (in vitro oxygen consumption, Na(+)/K(+)-ATPase activity and abundance) of C. alboguttata following 6 months of aestivation. l-Proline uptake rate was significantly higher in aestivating frogs, but overall uptake capacity was lower than in active frogs. Total SI oxygen consumption rate (V(O(2))) was also lower in aestivating frogs, despite no difference in mass-specific V(O(2)). The proportion of intestinal V(O(2)) associated with Na(+)/K(+)-ATPase activity and protein synthesis was equivalent between active and aestivating frogs, suggesting these processes were unaffected by aestivation. Indeed, the activity of Na(+)/K(+)-ATPase transporters in the SI of aestivating frogs was not different from that of active animals. Aestivating frogs maintained Na(+)/K(+)-ATPase activity, despite experiencing a reduction in the density of Na(+)/K(+)-ATPase transporters, by increasing the molecular activity of the remaining pumps to 2-3 times that of active frogs. These results show that functionality of the SI is maintained at the cellular level, potentially facilitating the reclamation of nutrients from the intestinal lumen while in aestivation. Despite this, the functional capacity of the SI in aestivating C. alboguttata is significantly reduced due to a reduction in tissue mass, helping frogs to conserve energy while in aestivation.

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

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 micromol 100 g(-0.7) body mass h(-1)) were almost three times greater than those of FW-acclimated animals (31.6+/-6.2 micromol 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 microl O(2) 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.