Observations on spindly leg syndrome in a captive population of Andinobates geminisae.

Amphibian health problems of unknown cause limit the success of the growing number of captive breeding programs. Spindly leg syndrome (SLS) is one such disease, where affected individuals with underdeveloped limbs often require euthanization. We experimentally evaluated husbandry-related factors of SLS in a captive population of the critically endangered frog, Andinobates geminisae. SLS has been linked to tadpole nutrition, vitamin B deficiency, water filtration methods, and water quality, but few of these have been experimentally tested. We tested the effects of water filtration method and vitamin supplementation (2017) and the effects of tadpole husbandry protocol intensity (2018) on time to metamorphosis and the occurrence of SLS. We found that vitamin supplementation and reconstituted reverse osmosis filtration of tadpole rearing water significantly reduced SLS prevalence and that reduced tadpole husbandry delayed time to metamorphosis. A fortuitous accident in 2018 resulted in a decrease in the phosphate content of rearing water, which afforded us an additional opportunity to assess the influence of phosphate on calcium sequestration. We found that tadpoles that had more time to sequester calcium for ossification during development had decreased the prevalence of SLS. Taken together, our results suggest that the qualities of the water used to rear tadpoles plays an important role in the development of SLS. Specifically, filtration method, vitamin supplementation, and calcium availability of tadpole rearing water may play important roles. Focused experiments are still needed, but our findings provide important information for amphibian captive rearing programs affected by high SLS prevalence.

Monoaminergic integration of diet and social signals in the brains of juvenile spadefoot toads.

Social behavior often includes the production of species-specific signals (e.g. mating calls or visual displays) that evoke context-dependent behavioral responses from conspecifics. Monoamines are important neuromodulators that have been implicated in context-dependent social behavior, yet we know little about the development of monoaminergic systems and whether they mediate the effects of early life experiences on adult behavior. We examined the effects of diet and social signals on monoamines early in development in the plains spadefoot toad (Spea bombifrons), a species in which diet affects the developmental emergence of species recognition and body condition affects the expression of adult mating preferences. To do so, we manipulated the diet of juveniles for 6 weeks following metamorphosis and collected their brains 40 min following the presentation of either a conspecific or a heterospecific call. We measured levels of monoamines and their metabolites using high pressure liquid chromatography from tissue punches of the auditory midbrain (i.e. torus semicircularis), hypothalamus and preoptic area. We found that call type affected dopamine and noradrenaline signaling in the auditory midbrain and that diet affected dopamine and serotonin in the hypothalamus. In the preoptic area, we detected an interaction between diet and call type, indicating that diet modulates how the preoptic area integrates social information. Our results suggest that the responsiveness of monoamine systems varies across the brain and highlight preoptic dopamine and noradrenaline as candidates for mediating effects of early diet experience on later expression of social preferences.

Testing the effect of dietary carotenoids on larval survival, growth and development in the critically endangered southern corroboree frog.

The success of captive breeding programs (CBPs) for threatened species is often limited due to a lack of knowledge of the nutritional conditions required for optimal growth and survival. Carotenoids are powerful antioxidants known to accelerate vertebrate growth and reduce mortality. However, the effect of carotenoids on amphibian life-history traits remains poorly understood. The aim of our study was to use a manipulative laboratory experiment to test the effect of dietary-carotenoid supplementation during the larval life stage on the survival, growth and development of the critically endangered southern corroboree frog (Pseudophryne corroboree). Larvae were fed either a carotenoid supplemented diet or an unsupplemented diet and the survival, growth and development of individuals was monitored and compared. There was no significant effect of dietary treatment on larval survival, growth rate, time taken to reach metamorphosis, or body size at metamorphosis. Our findings provide no evidence that carotenoid supplementation during the larval life stage improves the growth and development of southern corroboree frogs. However, because the carotenoid dose used in our study did not have any detrimental effects on P. corroboree larvae, but has previously been shown to improve adult coloration, immunity, and exercise performance, carotenoid supplementation should be considered when evaluating the nutritional requirements of P. corroboree in captivity. Carotenoid supplementation studies are now required for a diversity of anuran species to determine the effects of carotenoids on amphibian survival, growth and development. Understanding the effects of dietary carotenoids on different life-history traits may assist with amphibian captive breeding and conservation.

Dietary Carotenoid Supplementation Enhances the Cutaneous Bacterial Communities of the Critically Endangered Southern Corroboree Frog (Pseudophryne corroboree).

The rapid spread of infectious disease has resulted in the decline of animal populations globally. Amphibians support a diversity of microbial symbionts on their skin surface that help to inhibit pathogen colonisation and reduce disease susceptibility and virulence. These cutaneous microbial communities represent an important component of amphibian immune defence, however, very little is known about the environmental factors that influence the cutaneous microbiome. Here, we characterise the cutaneous bacterial communities of a captive colony of the critically endangered Australian southern corroboree frog, Pseudophyrne corroboree, and examine the effect of dietary carotenoid supplementation on bacterial abundance, species richness and community composition. Individuals receiving a carotenoid-supplemented diet exhibited significantly higher bacterial abundance and species richness as well as an altered bacterial community composition compared to individuals that did not receive dietary carotenoids. Our findings suggest that dietary carotenoid supplementation enhances the cutaneous bacteria community of the southern corroboree frog and regulates the presence of bacteria species within the cutaneous microbiome. Our study is the second to demonstrate that carotenoid supplementation can improve amphibian cutaneous bacterial community dynamics, drawing attention to the possibility that dietary manipulation may assist with the ex situ management of endangered species and improve resilience to lethal pathogens such as Batrachochytrium dendrobatidis (Bd).

The influence of carotenoid supplementation at different life-stages on the foraging performance of the Southern Corroboree frog (Pseudophryne corroboree): A test of the Silver Spoon and Environmental Matching Hypotheses.

Organismal performance can be significantly affected by the nutritional conditions experienced during different life-stages. The Silver Spoon Hypothesis predicts that individuals will always perform better as adults when they experience advantageous conditions during development. In contrast, the Environmental Matching Hypothesis predicts that individuals will perform better if they experience similar conditions during development and adulthood. Past tests of these hypotheses have focussed on the effect of food quantity on growth and development, with little attempt to investigate the effect of individual nutrients on behavioural traits. This study aimed to test the predictions of the Environmental Matching and Silver Spoon Hypotheses by investigating the influence of carotenoid supplementation at different life-stages on the foraging performance of Pseudophryne corroboree. To assess foraging performance, adults were presented with prey in either a cryptic or conspicuous foraging matrix. There was no effect of diet treatment on time to first movement towards prey, number of stalking events, time spent actively foraging, proportion of successful strikes, proportion of prey consumed or number of pedal luring events. These findings indicate that carotenoid supplementation at different life-stages does not influence the foraging performance of P. corroboree, providing no support for either the Silver Spoon or Environmental Matching Hypotheses.

Strong expression of the calreticulin gene in the liver of Rana rugosa tadpoles, but not adult frogs.

In the present paper we report the purification of calreticulin (CLT) from livers of the frog, Rana rugosa, the cloning and sequencing of its cDNA, and the CLT gene expression. CLT with M(r) = 52 kDa, estimated by SDS-PAGE, was purified from frog livers. Using rat CLT cDNA as a probe, a 2.4-kilobase frog cDNA clone was isolated from a frog liver cDNA library. The cDNA encoded 419 amino acids including an 18-residue NH2-terminal signal sequence that was 76% homologous to the rat CLT sequence and was 84% homologous to the partial sequence of Xenopus laevis CLT (Treves et al. [1992] Biochem. J. 287:579-581). Phylogenetic relationships estimated from the amino acid sequence of CLTs showed no pronounced variation between the two frog species, R. rugosa and X. laevis. Northern blot analysis indicated that the CLT mRNA level was very high in the liver of tadpoles, but extremely low in adult frogs. Expression levels were also very high in the premature ovary, while moderate expression was observed in the testis and brain of adult frogs. However, there was little histological change in the liver of tadpoles during development. Furthermore, CLT was recognized by Western blot analysis of total proteins in the liver of adult frogs. Immunostaining showed that CLT was distributed in the cytoplasm of liver cells. These results suggest that the expression of the CLT gene is tissue-dependent in the frog, R. rugosa, and that CLT probably functions biochemically in liver cells even when its gene expression is low.

Developmental regulation of the urea-cycle enzyme arginase in the direct developing frog Eleutherodactylus coqui.

Direct developing organisms obviate the larval intermediary from their ontogeny, hatching as miniature adults. To investigate this phenomenon, we have examined the developmental expression of arginase in the direct developing frog Eleutherodactylus coqui. An enzyme in the ornithine-urea cycle, the activation of liver arginase is necessary for the switch from ammonotelism to ureotelism which occurs when many frogs metamorphose and assume a terrestrial existence. Arginase enzyme activity is detectable at low levels in late prehatching stages of E. coqui, and increases at hatching, at which point the protein becomes detectable on Western blots. The activity increases gradually during posthatching development, reaching maximal levels at approximately the same time as yolk resorption is completed. Thyroid hormone is responsible for upregulating arginase activity during metamorphosis in Rana, but the role of thyroid hormone in direct developing frogs is unknown. A high dose (250 nM) of the thyroid hormone analogue 3,3'5-triiodo-L-thyronine (T3) caused precocious induction of arginase protein and activity, showing that even in a direct developing frog, some level of responsiveness to the metamorphic trigger, thyroid hormone, has been retained.