Anti-Trypanosomal Bufadienolides from the Oocytes of the Toad Rhinella alata (Anura, Bufonidae).

Amphibians are widely known as a prolific source of bioactive metabolites. In this work, we isolated and characterized compounds with antiparasitic activity from the oocytes of the toad Rhinella alata collected in Panama. Bio-guided isolation and structural elucidation were carried out using chromatographic and spectroscopic techniques, respectively. The organic extract was subjected to solid phase extraction followed by HPLC purification of the fraction with in vitro activity against Trypanosoma cruzi trypomastigotes. Seven steroids (1-7) of the bufadienolide family were isolated, and their structures were determined using NMR and MS analyses; of these 19-formyl-dyscinobufotalin, (3) is reported as a new natural product. Compounds 1 and 3-7 resulted in a good anti-trypanosomal activity profile. Among these, 16ß-hydroxyl-hellebrigenin (1) and bufalin (7) showed significant selectivity values of >5 and 2.69, respectively, while the positive control benznidazole showed a selectivity of 18.81. Furthermore, molecular docking analysis showed compounds 1, 3 and 7 interact through H-bonds with the amino acid residues GLN-19, ASP-158, HIS-159 and TRP-177 from cruzipain at the catalytic site. Given the lack of therapeutic options to treat American trypanosomiasis, this work can serve as the basis for further studies that aim for the development of bufadienolides or their derivatives as drugs against Chagas disease.

Body condition, skin bacterial communities and disease status: insights from the first release trial of the limosa harlequin frog, Atelopus limosus.

Many endangered amphibian species survive in captive breeding facilities, but there have been few attempts to reintroduce captive-born individuals to rebuild wild populations. We conducted a soft-release trial of limosa harlequin frogs, Atelopus limosus, which are highly susceptible to the amphibian chytrid fungus Batrachochytrium dendrobatidis (Bd), to understand changes associated with the transition from captivity to the wild. Specifically, we assessed changes in body condition, skin-associated bacterial communities and disease status after release. Frogs were housed individually in field mesocosms and monitored for 27 days. Body condition did not significantly change in the mesocosms, and was similar to, or higher than, that of wild conspecifics at day 27. The skin bacteria of captive-born frogs, based on 16S rRNA gene amplicons, became similar to that of wild conspecifics after 27 days in mesocosms. Prevalence of Bd in wild conspecifics was 13-27%, and 15% of the A. limosus in mesocosms became infected with Bd, but no mortality of infected frogs was observed. We conclude that mesocosms are suitable for systematically and repeatedly monitoring amphibians during release trials, and that body condition, the skin microbiome, and Bd status can all change within one month of placement of captive-born individuals back into the wild.

Whole exome sequencing identifies the potential for genetic rescue in iconic and critically endangered Panamanian harlequin frogs.

Avoiding extinction in a rapidly changing environment often relies on a species' ability to quickly adapt in the face of extreme selective pressures. In Panamá, two closely related harlequin frog species (Atelopus varius and Atelopus zeteki) are threatened with extinction due to the fungal pathogen Batrachochytrium dendrobatidis (Bd). Once thought to be nearly extirpated from Panamá, A. varius have recently been rediscovered in multiple localities across their historical range; however, A. zeteki are possibly extinct in the wild. By leveraging a unique collection of 186 Atelopus tissue samples collected before and after the Bd outbreak in Panama, we describe the genetics of persistence for these species on the brink of extinction. We sequenced the transcriptome and developed an exome-capture assay to sequence the coding regions of the Atelopus genome. Using these genetic data, we evaluate the population genetic structure of historical A. varius and A. zeteki populations, describe changes in genetic diversity over time, assess the relationship between contemporary and historical individuals, and test the hypothesis that some A. varius populations have rapidly evolved to resist or tolerate Bd infection. We found a significant decrease in genetic diversity in contemporary (compared to historical) A. varius populations. We did not find strong evidence of directional allele frequency change or selection for Bd resistance genes, but we uncovered a set of candidate genes that warrant further study. Additionally, we found preliminary evidence of recent migration and gene flow in one of the largest persisting A. varius populations in Panamá, suggesting the potential for genetic rescue in this system. Finally, we propose that previous conservation units should be modified, as clear genetic breaks do not exist beyond the local population level. Our data lay the groundwork for genetically informed conservation and advance our understanding of how imperiled species might be rescued from extinction.

Expanded host and geographic range of tadpole associations with the Severe Perkinsea Infection group.

Severe Perkinsea infection is an emerging disease of amphibians, specifically tadpoles. Disease presentation correlates with liver infections of a subclade of Perkinsea (Alveolata) protists, named Pathogenic Perkinsea Clade (PPC). Tadpole mortality events associated with PPC infections have been reported across North America, from Alaska to Florida. Here, we investigate the geographic and host range of PPC associations in seemingly healthy tadpoles sampled from Panama, a biogeographic provenance critically affected by amphibian decline. To complement this work, we also investigate a mortality event among Hyla arborea tadpoles in captive-bred UK specimens. PPC SSU rDNA was detected in 10 of 81 Panama tadpoles tested, and H. arborea tadpoles from the UK. Phylogenies of the Perkinsea SSU rDNA sequences demonstrate they are highly similar to PPC sequences sampled from mortality events in the USA, and phylogenetic analysis of tadpole mitochondrial SSU rDNA demonstrates, for the first time, PPC associations in diverse hylids. These data provide further understanding of the biogeography and host range of this putative pathogenic group, factors likely to be important for conservation planning.

Bufadienolides from the Skin Secretions of the Neotropical Toad Rhinella alata (Anura: Bufonidae): Antiprotozoal Activity against Trypanosoma cruzi.

Toads in the family Bufonidae contain bufadienolides in their venom, which are characterized by their chemical diversity and high pharmacological potential. American trypanosomiasis is a neglected disease that affects an estimated 8 million people in tropical and subtropical countries. In this research, we investigated the chemical composition and antitrypanosomal activity of toad venom from Rhinella alata collected in Panama. Structural determination using mass spectrometry (MS) and nuclear magnetic resonance (NMR) spectroscopy led to the identification of 10 bufadienolides. Compounds identified include the following: 16ß-hydroxy-desacetyl-bufotalin-3-adipoyl-arginine ester (1), bufotalin (2), 16ß-hydroxy-desacetyl-bufotalin-3-pimeloyl-arginine ester (3), bufotalin-3-pimeloyl-arginine ester (4), 16ß-hydroxy-desacetyl-bufotalin-3-suberoyl-arginine ester (5), bufotalin-3-suberoyl-arginine ester (6), cinobufagin-3-adipoyl-arginine ester (7), cinobufagin-3-pimeloyl-arginine ester (8), cinobufagin-3-suberoyl-arginine ester (9), and cinobufagin (10). Among these, three new natural products, 1, 3, and 5, are described, and compounds 1-10 are reported for the first time in R. alata. The antitrypanosomal activity assessed in this study revealed that the presence of an arginyl-diacid attached to C-3, and a hydroxyl group at C-14 in the structure of bufadienolides that is important for their biological activity. Bufadienolides showed cytotoxic activity against epithelial kidney Vero cells; however, bufagins (2 and 10) displayed low mammalian cytotoxicity. Compounds 2 and 10 showed activity against the cancer cell lines MCF-7, NCI-H460, and SF-268.

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.

Being red, blue and green: the genetic basis of coloration differences in the strawberry poison frog (Oophaga pumilio).

BACKGROUND: Animal coloration is usually an adaptive attribute, under strong local selection pressures and often diversified among species or populations. The strawberry poison frog (Oophaga pumilio) shows an impressive array of color morphs across its distribution in Central America. Here we quantify gene expression and genetic variation to identify candidate genes involved in generating divergence in coloration between populations of red, green and blue O. pumilio from the Bocas del Toro archipelago in Panama. RESULTS: We generated a high quality non-redundant reference transcriptome by mapping the products of genome-guided and de novo transcriptome assemblies onto a re-scaffolded draft genome of O. pumilio. We then measured gene expression in individuals of the three color phenotypes and identified color-associated candidate genes by comparing differential expression results against a list of a priori gene sets for five different functional categories of coloration - pteridine synthesis, carotenoid synthesis, melanin synthesis, iridophore pathways (structural coloration), and chromatophore development. We found 68 candidate coloration loci with significant expression differences among the color phenotypes. Notable upregulated examples include pteridine synthesis genes spr, xdh and pts (in red and green frogs); carotenoid metabolism genes bco2 (in blue frogs), scarb1 (in red frogs), and guanine metabolism gene psat1 (in blue frogs). We detected significantly higher expression of the pteridine synthesis gene set in red and green frogs versus blue frogs. In addition to gene expression differences, we identified 370 outlier SNPs on 162 annotated genes showing signatures of diversifying selection, including eight pigmentation-associated genes. CONCLUSIONS: Gene expression in the skin of the three populations of frogs with differing coloration is highly divergent. The strong signal of differential expression in pteridine genes is consistent with a major role of these genes in generating the coloration differences among the three morphs. However, the finding of differentially expressed genes across pathways and functional categories suggests that multiple mechanisms are responsible for the coloration differences, likely involving both pigmentary and structural coloration. In addition to regulatory differences, we found potential evidence of differential selection acting at the protein sequence level in several color-associated loci, which could contribute to the color polymorphism.

Lens transmittance shapes ultraviolet sensitivity in the eyes of frogs from diverse ecological and phylogenetic backgrounds.

The amount of short wavelength (ultraviolet (UV), violet and blue) light that reaches the retina depends on the transmittance properties of the ocular media, especially the lens, and varies greatly across species in all vertebrate groups studied previously. We measured the lens transmittance in 32 anuran amphibians with different habits, geographical distributions and phylogenetic positions and used them together with eye size and pupil shape to evaluate the relationship with diel activity pattern, elevation and latitude. We found an unusually high lens UV transmittance in the most basal species, and a cut-off range that extends into the visible spectrum for the rest of the sample, with lenses even absorbing violet light in some diurnal species. However, other diurnal frogs had lenses that transmit UV light like the nocturnal species. This unclear pattern in the segregation of ocular media transmittance and diel activity is shared with other vertebrates and is consistent with the absence of significant correlations in our statistical analyses. Although we did not detect a significant phylogenetic effect, closely related species tend to have similar transmittances, irrespective of whether they share the same diel pattern or not, suggesting that anuran ocular media transmittance properties might be related to phylogeny.

Phylogenetic relationships of toads of the Rhinella granulosa group (Anura: Bufonidae): a molecular perspective with comments on hybridization and introgression.

The Rhinella granulosa group consists of 13 species of toads distributed throughout open areas of South America and Panama. In this paper we perform a phylogenetic analysis considering all but one species of the group, employing five nuclear and four mitochondrial genes, for up to 7910 bp per specimen. Separate phylogenetic analyses under direct optimization (DO) of nuclear and mitochondrial sequences recovered the R. granulosa group as monophyletic and revealed topological incongruence that can be explained mainly by multiple events of hybridization and introgression, both mitochondrial and nuclear. The DO combined analysis, after the exclusion of putatively introgressed or heterozygous genomes, resulted in a phylogenetic hypothesis for the R. granulosa group in which most of the species are recovered as monophyletic, but with interspecific relationships poorly supported. The optimization of morphological (adult and larval), chromosomal, and behavioural characters resulted in 12 putative phenotypic synapomorphies for this species group and some other synapomorphies for internal clades. Our results indicate the need for additional population genetic studies on R. dorbignyi and R. fernandezae to corroborate the taxonomic status of both taxa. Finally, we discuss biological and genetic characteristics of Bufonidae, as possible explanations for the common occurrence of hybridization and introgression observed in some lineages of this family.

Testing the role of ecology and life history in structuring genetic variation across a landscape: a trait-based phylogeographic approach.

Hypotheses to explain phylogeographic structure traditionally invoke geographic features, but often fail to provide a general explanation for spatial patterns of genetic variation. Organisms' intrinsic characteristics might play more important roles than landscape features in determining phylogeographic structure. We developed a novel comparative approach to explore the role of ecological and life-history variables in determining spatial genetic variation and tested it on frog communities in Panama. We quantified spatial genetic variation within 31 anuran species based on mitochondrial DNA sequences, for which hierarchical approximate Bayesian computation analyses rejected simultaneous divergence over a common landscape. Regressing ecological variables, on genetic divergence allowed us to test the importance of individual variables revealing that body size, current landscape resistance, geographic range, biogeographic origin and reproductive mode were significant predictors of spatial genetic variation. Our results support the idea that phylogeographic structure represents the outcome of an interaction between organisms and their environment, and suggest a conceptual integration we refer to as trait-based phylogeography.

Recovery and Characterization of Spermatozoa in a Neotropical, Terrestrial, Direct-Developing Riparian Frog (Craugastor evanesco) through Hormonal Stimulation.

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.

The Great American Biotic Interchange in frogs: multiple and early colonization of Central America by the South American genus Pristimantis (Anura: Craugastoridae).

The completion of the land bridge between North and South America approximately 3.5-3.1 million years ago (Ma) initiated a tremendous biogeographic event called the Great American Biotic Interchange (GABI), described principally from the mammalian fossil record. The history of biotic interchange between continents for taxonomic groups with poor fossil records, however, is not well understood. Molecular and fossil data suggest that a number of plant and animal lineages crossed the Isthmus of Panama well before 3.5 Ma, leading biologists to speculate about trans-oceanic dispersal mechanisms. Here we present a molecular phylogenetic analysis of the frog genus Pristimantis based on 189 individuals of 137 species, including 71 individuals of 31 species from Panama and Colombia. DNA sequence data were obtained from three mitochondrial (COI, 12S, 16S) and two nuclear (RAG-1 and Tyr) genes, for a total of 4074 base pairs. The resulting phylogenetic hypothesis showed statistically significant conflict with most recognized taxonomic groups within Pristimantis, supporting only the rubicundus Species Series, and the Pristimantis myersi and Pristimantis pardalis Species Groups as monophyletic. Inference of ancestral areas based on a likelihood model of geographic range evolution via dispersal, local extinction, and cladogenesis (DEC) suggested that the colonization of Central America by South American Pristimantis involved at least 11 independent events. Relaxed-clock analyses of divergence times suggested that at least eight of these invasions into Central America took place prior to 4 Ma, mainly in the Miocene. These findings contribute to a growing list of molecular-based biogeographic studies presenting apparent temporal conflicts with the traditional GABI model.

An experimental test of disease resistance function in the skin-associated bacterial communities of three tropical amphibian species.

Variation in the structure of host-associated microbial communities has been correlated with the occurrence and severity of disease in diverse host taxa, suggesting a key role of the microbiome in pathogen defense. However, whether these correlations are typically a cause or consequence of pathogen exposure remains an open question, and requires experimental approaches to disentangle. In amphibians, infection by the fungal pathogen Batrachochytrium dendrobatidis (Bd) alters the skin microbial community in some host species, whereas in other species, the skin microbial community appears to mediate infection dynamics. In this study, we completed experimental Bd exposures in three species of tropical frogs (Agalychnis callidryas, Dendropsophus ebraccatus,andCraugastor fitzingeri) that were sympatric with Bd at the time of the study. For all three species, we identified key taxa within the skin bacterial communities that were linked to Bd infection dynamics. We also measured higher Bd infection intensities in D. ebraccatus and C. fitzingeri that were associated with higher mortality in C. fitzingeri. Our findings indicate that microbially mediated pathogen resistance is a complex trait that can vary within and across host species, and suggest that symbiont communities that have experienced prior selection for defensive microbes may be less likely to be disturbed by pathogen exposure.

DNA barcoding of the National Museum of Natural History reptile tissue holdings raises concerns about the use of natural history collections and the responsibilities of scientists in the molecular age.

Natural history collections are essential to a wide variety of studies in biology because they maintain large collections of specimens and associated data, including genetic material (e.g., tissues) for DNA sequence data, yet they are currently under-funded and collection staff have high workloads. With the advent of aggregate databases and advances in sequencing technologies, there is an increased demand on collection staff for access to tissue samples and associated data. Scientists are rapidly developing large DNA barcode libraries, DNA sequences of specific genes for species across the tree of life, in order to document and conserve biodiversity. In doing so, mistakes are made. For instance, inconsistent taxonomic information is commonly taken from different lending institutions and deposited in data repositories, such as the Barcode of Life Database (BOLD) and GenBank, despite explicit disclaimers regarding the need for taxonomic verification by the lending institutions. Such errors can have profound effects on subsequent research based on these mis-labelled sequences in data repositories. Here, we present the production of a large DNA barcode library of reptiles from the National Museum of Natural History tissue holdings. The library contains 2,758 sequences (2,205 COI and 553 16S) from 2260 specimens (four crocodilians, 37 turtles, and 2,219 lizards, including snakes), representing 583 named species, from 52 countries. In generating this library, we noticed several common mistakes made by scientists depositing DNA barcode data in public repositories (e.g., BOLD and GenBank). Our goal is to raise awareness of these concerns and offer advice to avoid such mistakes in the future to maintain accurate DNA barcode libraries to properly document Earth's biodiversity.

Reptiles as potential vectors and hosts of the amphibian pathogen Batrachochytrium dendrobatidis in Panama.

Chytridiomycosis, the disease caused by Batrachochytrium dendrobatidis, is considered to be a disease exclusively of amphibians. However, B. dendrobatidis may also be capable of persisting in the environment, and non-amphibian vectors or hosts may contribute to disease transmission. Reptiles living in close proximity to amphibians and sharing similar ecological traits could serve as vectors or reservoir hosts for B. dendrobatidis, harbouring the organism on their skin without succumbing to disease. We surveyed for the presence of B. dendrobatidis DNA among 211 lizards and 8 snakes at 8 sites at varying elevations in Panama where the syntopic amphibians were at pre-epizootic, epizootic or post-epizootic stages of chytridiomycosis. Detection of B. dendrobatidis DNA was done using qPCR analysis. Evidence of the amphibian pathogen was present at varying intensities in 29 of 79 examined Anolis humilis lizards (32%) and 9 of 101 A. lionotus lizards (9%), and in one individual each of the snakes Pliocercus euryzonus, Imantodes cenchoa, and Nothopsis rugosus. In general, B. dendrobatidis DNA prevalence among reptiles was positively correlated with the infection prevalence among co-occurring anuran amphibians at any particular site (r = 0.88, p = 0.004). These reptiles, therefore, may likely be vectors or reservoir hosts for B. dendrobatidis and could serve as disease transmission agents. Although there is no evidence of B. dendrobatidis disease-induced declines in reptiles, cases of coincidence of reptile and amphibian declines suggest this potentiality. Our study is the first to provide evidence of non-amphibian carriers for B. dendrobatidis in a natural Neotropical environment.

Antimicrobial Secretions of Toads (Anura, Bufonidae): Bioactive Extracts and Isolated Compounds against Human Pathogens.

Species of the family Bufonidae, better known as true toads, are widespread and produce bioactive substances in the secretions obtained from specialized skin macroglands. Some true toads have been employed as a folk remedy to treat infectious diseases caused by microbial pathogens. Recent publications based on in silico analysis highlighted the Bufonidae as promising sources of antimicrobial peptides. A review of the literature reveals that Bufonidae skin secretion extracts show inhibitory activity in vitro against clinical isolates of bacteria, resistant and standard strains of bacterial, and fungal and parasitic human pathogens. Secondary metabolites belonging to the classes of alkaloids, bufadienolides, and peptides with antimicrobial activity have been isolated from species of the genera Bufo, Bufotes, Duttaphrynus, and Rhinella. Additionally, some antimicrobial extracts and purified compounds display low cytotoxicity against mammal cells.

19-Hydroxy-bufalin, a major bufadienolide isolated from the parotoid gland secretions of the Panamanian endemic toad Rhinella centralis (Bufonidae), inhibits the growth of Trypanosoma cruzi.

American trypanosomiasis is a parasitic neglected disease, responsible for the death of approximately 10,000 people every year. Amphibians are recognized for producing in their cutaneous glands substances with pharmacological potential against a variety of pathologies. Here we investigated the antiprotozoal activity against Trypanosoma cruzi of bufadienolides isolated from the parotoid glands secretions of the toad Rhinella centralis from Panama. NMR and mass spectrometry analysis led to the identification of the active compound 19-hydroxy-bufalin, for which its antitrypanosomal activity and occurrence in the genus Rhinella are reported for the first time. This compound showed low cytotoxicity and significant selectivity which confers to it a potential role for the treatment of Chagas disease.

Sarcophagid Myiasis in the Bufonid Rhinella alata in Panama.

Rhinella alata is a small terrestrial bufonid that occurs in Ecuador, Colombia, and Panama. Between January 2014 and October 2015, we inspected 339 R. alata from Panama and report myiasis in eight of these toads. All infested toads were male and presented with mobile dark fly larvae visible beneath the ventral skin. At necropsy, we identified the larvae as belonging to the family Sarcophagidae (flesh flies). Flesh flies have been variously considered as predators, parasitoids, and parasites of anurans. There are at least four species of flesh flies that infect adult amphibians in the Neotropics, with the most common and widespread being Lepidodexia bufonivora. Myiasis has only rarely been reported in Panamanian anurans. Anuran cases of sarcophagid myiasis are usually fatal and we suspect myiasis as the cause of death for the R. alata that died in the current study.

Metabolites from Microbes Isolated from the Skin of the Panamanian Rocket Frog Colostethus panamansis (Anura: Dendrobatidae).

The Panamanian rocket frog Colostethus panamansis (family Dendrobatidae) has been affected by chytridiomycosis, a deadly disease caused by the fungus Batrachochytrium dendrobatidis (Bd). While there are still uninfected frogs, we set out to isolate microbes from anatomically distinct regions in an effort to create a cultivable resource within Panama for potential drug/agricultural/ecological applications that perhaps could also be used as part of a strategy to protect frogs from infections. To understand if there are specific anatomies that should be explored in future applications of this resource, we mapped skin-associated bacteria of C. panamansis and their metabolite production potential by mass spectrometry on a 3D model. Our results indicate that five bacterial families (Enterobacteriaceae, Comamonadaceae, Aeromonadaceae, Staphylococcaceae and Pseudomonadaceae) dominate the cultivable microbes from the skin of C. panamansis. The combination of microbial classification and molecular analysis in relation to the anti-Bd inhibitory databases reveals the resource has future potential for amphibian conservation.

Spindly leg syndrome in Atelopus varius is linked to environmental calcium and phosphate availability.

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.