Nutrition and health in amphibian husbandry.

Amphibian biology is intricate, and there are many inter-related factors that need to be understood before establishing successful Conservation Breeding Programs (CBPs). Nutritional needs of amphibians are highly integrated with disease and their husbandry needs, and the diversity of developmental stages, natural habitats, and feeding strategies result in many different recommendations for proper care and feeding. This review identifies several areas where there is substantial room for improvement in maintaining healthy ex situ amphibian populations specifically in the areas of obtaining and utilizing natural history data for both amphibians and their dietary items, achieving more appropriate environmental parameters, understanding stress and hormone production, and promoting better physical and population health. Using a scientific or research framework to answer questions about disease, nutrition, husbandry, genetics, and endocrinology of ex situ amphibians will improve specialists' understanding of the needs of these species. In general, there is a lack of baseline data and comparative information for most basic aspects of amphibian biology as well as standardized laboratory approaches. Instituting a formalized research approach in multiple scientific disciplines will be beneficial not only to the management of current ex situ populations, but also in moving forward with future conservation and reintroduction projects. This overview of gaps in knowledge concerning ex situ amphibian care should serve as a foundation for much needed future research in these areas.

Leaping forward in amphibian health and nutrition.

The Epidemiology Working Group, a subgroup of the participants of the Disney's Animal Kingdom Workshop on "Ex situ Amphibian Medicine and Nutrition," identified a critical need to design and implement approaches that will facilitate the assessment and evaluation of factors impacting amphibian health. In this manuscript, we describe and summarize the outcomes of this workshop with regards (a) the identified gaps in knowledge, (b) identified priorities for closing these gaps, and (c) compile a list of actions to address these priorities. Four general areas of improvement were identified in relation to how measurements are currently being taken to evaluate ex situ amphibian health: nutrition, infectious diseases, husbandry, and integrated biology including genetics and endocrinology. The proposed actions that will be taken in order to address the identified gaps include: (1) identify and quantify major health issues affecting ex situ amphibian populations, (2) identify and coordinate laboratories to conduct analyses using standardized and validated protocols to measure nutritional, infectious diseases, genetic, and hormonal parameters, (3) determine in situ baseline distribution of parameters related to amphibian health, and (4) establish an inter-disciplinary research approach to target specific hypotheses related to amphibian health such as the effects of population genetics (e.g., relatedness, inbreeding) on disease susceptibility, or how environmental parameters are related to chronic stress and hormone production. We think is important to address current gaps in knowledge regarding amphibian health in order to increase the probability to succeed in addressing the issues faced by in situ and ex situ amphibians populations. We are confident that the recommendations provided in this manuscript will facilitate to address these challenges and could have a positive impact in both the health of in situ and ex situ amphibian populations, worldwide.

Novel dermaseptin, adenoregulin and caerin homologs from the Central American red-eyed leaf frog, Agalychnis callidryas, revealed by functional peptidomics of defensive skin secretion.

By integrating systematic peptidome and transcriptome studies of the defensive skin secretion of the Central American red-eyed leaf frog, Agalychnis callidryas, we have identified novel members of three previously described antimicrobial peptide families, a 27-mer dermaseptin-related peptide (designated DRP-AC4), a 33-mer adenoregulin-related peptide (designated ARP-AC1) and most unusually, a 27-mer caerin-related peptide (designated CRP-AC1). While dermaseptin and adenoregulin were originally isolated from phyllomedusine leaf frogs, the caerins, until now, had only been described in Australian frogs of the genus, Litoria. Both the dermaseptin and adenoregulin were C-terminally amidated and lacked the C-terminal tripeptide of the biosynthetic precursor sequence. In contrast, the caerin-related peptide, unlike the majority of Litoria analogs, was not C-terminally amidated. The present data emphasize the need for structural characterization of mature peptides to ensure that unexpected precursor cleavages and/or post-translational modifications do not produce mature peptides that differ in structure to those predicted from cloned biosynthetic precursor cDNA. Additionally, systematic study of the secretory peptidome can produce unexpected results such as the CRP described here that may have phylogenetic implications. It is thus of the utmost importance in the functional evaluation of novel peptides that the primary structure of the mature peptide is unequivocally established -- something that is often facilitated by cloning biosynthetic precursor cDNAs but obviously not reliable using such data alone.

Elements of the granular gland peptidome and transcriptome persist in air-dried skin of the South American orange-legged leaf frog, Phyllomedusa hypocondrialis.

The defensive strategy of amphibians against predator attack relies heavily on the secretion of noxious/toxic chemical cocktails from specialized skin granular glands. Bioactive peptides constitute a major component of secretions in many species and the most complex are produced by neotropical leaf frogs of the sub-family Phyllomedusinae. We recently reported that these skin secretions contain elements of both the granular gland peptidome and transcriptome and that polyadenylated mRNAs constituting the latter are protected from degradation by interactions with endogenous amphipathic peptides. This thus permits parallel amino acid sequencing of peptides and nucleic acid sequencing of cloned precursor transcripts from single lyophilized samples of secretion. Here we report that the protection afforded is sufficiently robust to permit transcriptome studies by cloning of full-length polyadenylated peptide precursor encoding mRNAs from libraries constructed using ambient temperature air-dried skin from recently deceased specimens as source material. The technique was sufficiently sensitive to permit the identification of cDNAs encoding antimicrobial peptides constituted by six different isoforms of phylloseptin and two dermaseptins. Also, for the first time, establishment of the nucleic acid and amino acid sequence of the precursor encoding the phyllomedusine frog skin bradykinin-related peptide, phyllokinin, from cloned cDNA, was achieved. These data unequivocally demonstrate that the granular gland transcriptome persists in air-dried amphibian skin--a finding that may have fundamental implications in the study of archived materials but also in the wider field of molecular biology.

Partial structure of the phylloxin gene from the giant monkey frog, Phyllomedusa bicolor: parallel cloning of precursor cDNA and genomic DNA from lyophilized skin secretion.

Phylloxin is a novel prototype antimicrobial peptide from the skin of Phyllomedusa bicolor. Here, we describe parallel identification and sequencing of phylloxin precursor transcript (mRNA) and partial gene structure (genomic DNA) from the same sample of lyophilized skin secretion using our recently-described cloning technique. The open-reading frame of the phylloxin precursor was identical in nucleotide sequence to that previously reported and alignment with the nucleotide sequence derived from genomic DNA indicated the presence of a 175 bp intron located in a near identical position to that found in the dermaseptins. The highly-conserved structural organization of skin secretion peptide genes in P. bicolor can thus be extended to include that encoding phylloxin (plx). These data further reinforce our assertion that application of the described methodology can provide robust genomic/transcriptomic/peptidomic data without the need for specimen sacrifice.