Using “omics” and integrated multi-omits approaches to guide probiotic selection to mitigate chytridiomycosis and other emerging infectious diseases

Emerging infectious diseases in wildlife are responsible for massive population declines. In amphibians, chytridiomycosis caused by Batrachochytrium dendrobatidis, Bd, has severely affected many amphibian populations and species around the world. One promising management strategy is probiotic bioaugmentation of antifungal bacteria on amphibian skin. In vivo experimental trials using bioaugmentation strategies have had mixed results, and therefore a more informed strategy is needed to select successful probiotic candidates. Metagenomic, transcriptomic, and metabolomic methods, colloquially called “omics,” are approaches that can better inform probiotic selection and optimize selection protocols. The integration of multiple omic data using bioinformatic and statistical tools and in silico models that link bacterial community structure with bacterial defensive function can allow the identification of species involved in pathogen inhibition. We recommend using 16S rRNA gene amplicon sequencing and methods such as indicator species analysis, the Kolmogorov–Smirnov Measure, and co-occurrence networks to identify bacteria that are associated with pathogen resistance in field surveys and experimental trials. In addition to 16S amplicon sequencing, we recommend approaches that give insight into symbiont function such as shotgun metagenomics, metatranscriptomics, or metabolomics to maximize the probability of finding effective probiotic candidates, which can then be isolated in culture and tested in persistence and clinical trials. An effective mitigation strategy to ameliorate chytridiomycosis and other emerging infectious diseases is necessary; the advancement of omic methods and the integration of multiple omic data provide a promising avenue toward conservation of imperiled species

Antifungal isolates database of amphibian skin-associated bacteria and function against emerging fungal pathogens

Microbial symbionts of vertebrate skin have an important function in defense of the host against pathogens. In particular, the emerging chytrid fungus Batrachochytrium dendrobatidis, causes widespread disease in amphibians but can be inhibited via secondary metabolites produced by many different skin-associated bacteria. Similarly, the fungal pathogens of terrestrial salamander eggs Mariannaea elegans and Rhizomucor variabilis are also inhibited by a variety of skin-associated bacteria. Indeed, probiotic therapy against fungal diseases is a recent approach in conservation medicine with growing experimental support. We present a comprehensive Antifungal Isolates Database of amphibian skin-associated bacteria that have been cultured, isolated, and tested for antifungal properties. At the start, this database includes nearly 2000 cultured bacterial isolates from 37 amphibian host species across 18 studies on five continents: Africa, Oceania, Europe, and North and South America. As the research community gathers information on additional isolates, the database will be updated periodically. The resulting database can serve as a conservation tool for amphibians and other organisms, and provides empirical data for comparative and bioinformatic studies. The database consists of a FASTA file containing 16S rRNA gene sequences of the bacterial isolates, and a metadata file containing information on the host species, life-stage, geographic region, and antifungal capacity and taxonomic identity of the isolate

Cross-ecosystem effects of terrestrial predators link treefrogs, zooplankton, and aquatic primary production

Predators can directly or indirectly shape food webs through a combination of consumptive and non-consumptive effects. Yet, how these effects vary across natural populations and their consequences for adjacent ecosystems remains poorly resolved. We examined links between terrestrial predators and aquatic ecosystems through their effects on a locally abundant amphibian, the red-eyed treefrog (Agalychnis callidryas), which has arboreal eggs (heavily predated by snakes and wasps) and aquatic larvae; embryos can escape terrestrial threats by hatching at an earlier age and smaller size. Our multi-site field survey indicates that in natural populations, the relative contributions of these consumptive and non-consumptive effects of predators can be substantial and remarkably similar. However, in mesocosms where we experimentally mimicked these predator effects, changes in the density and initial hatching age of tadpoles carried distinct consequences for aquatic food webs. Density-dependent growth resulted in peak tadpole biomass at intermediate densities (reflecting intermediate predation), and early-hatched tadpoles grew 16% faster and produced 26% more biomass than their late-hatched counterparts. These changes in tadpole growth and size differentially affected zooplankton communities, and the production and stability of phytoplankton. Together, these results illustrate multiple pathways through which predators in one ecosystem can modulate the structure of adjacent food webs

Sequences

Raw, demultiplexed sequence reads collected from swabs of eggs and adults during male removal experiment

Sequence metadata

Metadata for all sequences contained in adjoining sequence (.fastq) file

Clutch monitoring data

Summary of fates of embryos from male removal experiment

Skin bacterial microbiome of a generalist Puerto Rican frog varies along elevation and land use gradients

Host-associated microbial communities are ubiquitous among animals, and serve important functions. For example, the bacterial skin microbiome of amphibians can play a role in preventing or reducing infection by the amphibian chytrid fungus, Batrachochytrium dendrobatidis. Evidence suggests that environmental bacteria likely serve as a source pool for at least some of the members of the amphibian skin bacterial community, underscoring the potential for local environmental changes to disrupt microbial community source pools that could be critical to the health of host organisms. However, few studies have assessed variation in the amphibian skin microbiome along clear environmental gradients, and so we know relatively little about how local environmental conditions influence microbiome diversity. We sampled the skin bacterial communities of Coqui frogs, Eleutherodactylus coqui (N = 77), along an elevational gradient in eastern Puerto Rico (0–875 m), with transects in two land use types: intact forest (N = 4 sites) and disturbed (N = 3 sites) forest. We found that alpha diversity (as assessed by Shannon, Simpson, and Phylogenetic Diversity indices) varied across sites, but this variation was not correlated with elevation or land use. Beta diversity (community structure), on the other hand, varied with site, elevation and land use, primarily due to changes in the relative abundance of certain bacterial OTUs (∼species) within these communities. Importantly, although microbiome diversity varied, E. coqui maintained a common core microbiota across all sites. Thus, our findings suggest that environmental conditions can influence the composition of the skin microbiome of terrestrial amphibians, but that some aspects of the microbiome remain consistent despite environmental variation

Escape-hatching responses of individual treefrog embryos vary with threat level in wasp attacks: a mechanistic analysis

<p>Theory predicts that prey behavioural responses should reflect the level of risk posed by predators. We investigated how red-eyed treefrog embryos perceive and respond to spatially variable risk during wasp attacks on their clutches. First, we spatially restricted wasp activity on clutches and compared hatching of wasp-exposed, adjacent, and protected embryos. Hatching occurred in all zones but increased with exposure, being highest in directly exposed embryos. Second, we videotaped wasps attacking clutches and compared the experiences of embryos that hatched first and those that did not hatch until later. Embryos that hatched first experienced more predatory wasp activity directed at themselves or at siblings within a 2-egg radius. Models predicting hatching indicate that cues used to assess risk originate from the behaviour of wasps, rather than other embryos. This research demonstrates that embryos can integrate information about predator behaviour and proximity to respond appropriately based on their level of risk.</p