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Variable harlequin frogs Atelopus varius have declined significantly throughout their range as a result of infection with the fungal pathogen Batrachochytrium dendrobatidis (Bd). The Panama Amphibian Rescue and Conservation Project maintains an ex situ population of this Critically Endangered species. We conducted a release trial with surplus captive-bred A. varius individuals to improve our ability to monitor frog populations post-release, observe dispersal patterns after freeing them into the wild and learn about threats to released frogs, as well as to determine whether natural skin toxin defences of frogs could be restored inside mesocosms in the wild and to compare Bd dynamics in natural amphibian communities at the release site vs a non-release site. The 458 released frogs dispersed rapidly and were difficult to re-encounter unless they carried a radio transmitter. No frog was seen after 36 days following release. Thirty frogs were fitted with radio transmitters and only half were trackable by day 10. Tetrodotoxin was not detected in the skins of the frogs inside mesocosms for up to 79 days. Bd loads in other species present at sites were high prior to release and decreased over time in a pattern probably driven by weather. No differences were observed in Bd prevalence between the release and non-release sites. This trial showed that refinements of our methods and approaches are required to study captive Atelopus frogs released into wild conditions. We recommend continuing release trials of captive-bred frogs with post-release monitoring methods, using an adaptive management framework to advance the field of amphibian reintroduction ecology.

Abstract Biodiversity loss is extreme in amphibians. Despite ongoing conservation action, it is difficult to determine where we stand in overcoming their extinction crisis. Among the most threatened amphibians are the 131 Neotropical harlequin toads. Many of them declined since the 1980s with several considered possibly extinct. Recently, more than 30 species have been rediscovered, raising hope for a reversing trend in the amphibian extinction crisis. We use past and present data available for harlequin toads (Atelopus), to examine whether the amphibian extinction crisis is still in an emergency state. Since 2004 no species has improved its population status, suggesting that recovery efforts have not been successful. Threats include habitat change, pathogen spread and climate change. More mitigation strategies need implementation, especially habitat protection and disease management, combined with captive conservation breeding. With harlequin toads serving as a model, it is clear that the amphibian extinction crisis is still underway.

The Vanishing Rainfrog (Craugastor evanesco) is an endemic and critically endangered frog species of Panama. It is suspected that 90% of the population has disappeared from the wild. Frogs were collected from the wild and brought to a Captive Breeding Program; however, accomplishing regular reproductive events for this species has been difficult. The objective of this study was to determine the effect of hormonal stimulation on the production and quality of C. evanesco spermatozoa, aiming to develop an efficient and safe sperm collection protocol as a tool to help reproduce this endangered species. Mature males received intra-peritoneal injections with one of six hormone treatments, including des-Gly10, D-Ala6, Pro-NHEt9—GnRH-A, Amphiplex or hCG. Urine samples were collected at 10 different time points post-injection. Quality assessments included sperm concentration, percentage motility, percentage forward progressive motility (FPM), osmolality, pH and morphology analysis. Our results indicate that the optimal treatment for the collection of highly concentrated sperm samples of C. evanesco is 4 µg/gbw GnRH, followed by Amphiplex and 2 µg/gbw GnRH as sub-optimal treatments and finally, 6 µg/gbw GnRH and 5 and 10 IU/gbw hCG as non-optimal treatments. GnRH-A at 4 μg/gbw and Amphiplex stimulated the production of samples with the highest sperm concentrations and quality, despite Amphiplex producing lower percentages of intact acrosome and tail. In contrast, hCG concentrations were not reliable inducers of sperm production, consistently showing lower concentrations, higher percentages of sperm abnormalities and more acidic spermic urine than that induced by Amphiplex and GnRH-A. Morphological assessments revealed that C. evanesco spermatozoa have a filiform shape with a large acrosome on the anterior part of an elongated head, a small midpiece and a long tail with two filaments joined together by an undulating membrane.

Spindly leg syndrome (SLS) is a relatively common musculoskeletal abnormality associated with captive-rearing of amphibians with aquatic larvae. We conducted an experiment to investigate the role of environmental calcium and phosphate in causing SLS in tadpoles. Our 600-tadpole experiment used a fully-factorial design, rearing Atelopus varius tadpoles in water with either high (80mg/l CaCO3), medium (50mg/l CaCO3), or low calcium hardness (20mg/l CaCO3), each was combined with high (1.74 mg/l PO4) or low (0.36 mg/l PO4) phosphate levels. We found that calcium supplementation significantly improved tadpole survival from 19% to 49% and that low calcium treatments had 60% SLS that was reduced to about 15% at the medium and high calcium treatments. Phosphate supplementation significantly reduced SLS prevalence in low calcium treatments. This experimental research clearly links SLS to the calcium: phosphate homeostatic system, but we were unable to completely eliminate the issue, suggesting an interactive role of other unidentified factors.

Spindly Leg Syndrome (SLS) is a persistent animal welfare issue associated with the rearing of amphibians in captivity. We conducted two experiments to investigate the effects of diet, water composition and overfeeding on prevalence of SLS in newly metamorphosed harlequin frogs (Atelopus spp.). In our first experiment, we offered 400 full-sibling tadpoles of Atelopus certus isocaloric diets in treatments of 31%, 37%, 42% and 48% crude protein respectively. Tadpoles fed higher protein diets metamorphosed faster, but the incidence of SLS exceeded 80% in all treatments leading to the conclusion that variation in dietary protein was not responsible for causing SLS. We used 720 full-sibling Atelopus glyphus tadpoles in a second experiment to examine the effects of diet type, water composition and diet ration on SLS. We found that an overall incidence of 58% spindly leg in tadpoles reared in tap water, but reduced to about 10% in water treated by reverse osmosis and then reconstituted. It is possible that the reverse osmosis treatment removed some factor that caused the SLS, or that the reconstitution may have added a mineral lacking in the original tap water. Within tap water treatments, overfeeding tadpoles in tanks increased the incidence of SLS. We recommend further experimental research into this condition to identify the causative factors in the water. Additional research into the nutritional composition of food available to wild tadpoles would be useful in formulating captive diets, that have to date been solely based on surrogate species.

We have established ex situ assurance colonies of two endangered Panamanian harlequin frogs, Atelopus certus and Atelopus glyphus, but observed that males fought with each other when housed as a group. Housing frogs individually eliminated this problem, but created space constraints. To evaluate the potential stress effects from aggressive interactions when grouping frogs, we housed male frogs in replicated groups of one, two, and eight. We measured aggressive behavioral interactions and fecal glucocorticoid metabolite (GC) concentrations as indicators of stress in each tank. In both small and large groups, frogs initially interacted aggressively, but aggressive interactions and fecal GCs declined significantly after the first 2 weeks of being housed together, reaching the lowest levels by week 4. We conclude that aggressive interactions in same-sex groups of captive Atelopus may initially cause stress, but the frogs become habituated within a few weeks and they can safely be housed in same-sex groups for longer periods of time.

El objetivo de este estudio fue determinar el estado de conservación del bosque nuboso de Cerro Azul, considerando la riqueza de especies de anfibios existentes. El muestreo se realizó por un periodo de seis meses, desde febrero de 2009 hasta julio del mismo año. Los muestreos fueron llevados a cabo mediante la técnica de búsqueda por transectos, realizando incursiones diurnas y nocturnas a lo largo de fuentes naturales de agua (ríos y quebradas), ubicadas en los tres sitios que fueron seleccionados para monitorear. La abundancia registrada de anfibios fue de 661 individuos, agrupados en dos órdenes (Anura y Caudata), 9 familias, 15 géneros y 26 especies. Se obtuvo una riqueza de 26 especies de anfibios, la cual presentó un patrón estacional, siendo los meses con menor precipitación los que aportaron el mayor número de especies. El área de Cerro Azul presentó el mayor índice de diversidad durante el período de transición, entre la estación seca y la estación lluviosa. Al aplicar el análisis de viabilidad ecológica (AVE) se determinó que el estado de conservación del bosque nuboso para Cerro Azul, basado en la riqueza de especies de anfibios, se encuentra en una condición de Pobre. Los rangos de abundancia indicaron que más de la mitad de las especies de anfibios registradas (62%) disminuyeron su abundancia desde el último inventario realizado hace 15 años.