Pathogenesis is strongly dependent on microbial context, but development of probiotic therapies has neglected the impact of ecological interactions. Dynamics among microbial communities, host immune responses, and environmental conditions may alter the effect of probiotics in human and veterinary medicine, agriculture and aquaculture, and the proposed treatment of emerging wildlife and zoonotic diseases such as those occurring on amphibians or vectored by mosquitoes. Here we use a holistic measure of amphibian mucosal defenses to test the effects of probiotic treatments and to assess disease risk under different ecological contexts. We developed a non-invasive assay for antifungal function of the skin mucosal ecosystem (mucosome function) integrating host immune factors and the microbial community as an alternative to pathogen exposure experiments. From approximately 8500 amphibians sampled across Europe, we compared field infection prevalence with mucosome function against the emerging fungal pathogen Batrachochytrium dendrobatidis. Four species were tested with laboratory exposure experiments, and a highly susceptible species, Alytes obstetricans, was treated with a variety of temperature and microbial conditions to test the effects of probiotic therapies and environmental conditions on mucosome function. We found that antifungal function of the amphibian skin mucosome predicts the prevalence of infection with the fungal pathogen in natural populations, and is linked to survival in laboratory exposure experiments. When altered by probiotic therapy, the mucosome increased antifungal capacity, while previous exposure to the pathogen was suppressive. In culture, antifungal properties of probiotics depended strongly on immunological and environmental context including temperature, competition, and pathogen presence. Functional changes in microbiota with shifts in temperature provide an alternative mechanistic explanation for patterns of disease susceptibility related to climate beyond direct impact on host or pathogen. This nonlethal management tool can be used to optimize and quickly assess the relative benefits of probiotic therapies under different climatic, microbial, or host conditions.

The utilization of similar habitats by different species provides an ideal opportunity to identify genes underlying adaptation and acclimatization. Here, we analysed the gene expression of two closely related salamander species: Salamandra salamandra in Central Europe and Salamandra infraimmaculata in the Near East. These species inhabit similar habitat types: 'temporary ponds' and 'permanent streams' during larval development. We developed two species-specific gene expression microarrays, each targeting over 12 000 transcripts, including an overlapping subset of 8331 orthologues. Gene expression was examined for systematic differences between temporary ponds and permanent streams in larvae from both salamander species to establish gene sets and functions associated with these two habitat types. Only 20 orthologues were associated with a habitat in both species, but these orthologues did not show parallel expression patterns across species more than expected by chance. Functional annotation of a set of 106 genes with the highest effect size for a habitat suggested four putative gene function categories associated with a habitat in both species: cell proliferation, neural development, oxygen responses and muscle capacity. Among these high effect size genes was a single orthologue (14-3-3 protein zeta/YWHAZ) that was downregulated in temporary ponds in both species. The emergence of four gene function categories combined with a lack of parallel expression of orthologues (except 14-3-3 protein zeta) suggests that parallel habitat adaptation or acclimatization by larvae from S. salamandra and S. infraimmaculata to temporary ponds and permanent streams is mainly realized by different genes with a converging functionality.

The postembryonic development of amphibians has been characterized as divided into three predominant periods, hereafter named primary developmental stages: premetamorphosis (PreM), prometamorphosis (ProM), metamorphic climax (Meta), and completion of metamorphosis (PostM), largely based on examination of anuran development. Here, we categorized the postembryonic development of larvae of a poisonous fire salamander (Salamandra salamandra) by integrating morphology and gene expression (transcriptomic) data. Morphological analysis revealed three distinct clusters suggestive of PreM, ProM, and Meta, which were confirmed in parallel by microarray-derived gene expression analysis. In total, 3,510 probes targeted transcripts differentially expressed between the clusters we identified. Genes upregulated in PreM related to organogenesis, and those upregulated in Meta underlie structural proteins and related to development of anatomical structures and pigmentation. Biosynthesis pathways of pigments (pteridines and melanin) were upregulated during late ProM and Meta. Gas chromatographic analysis of alkaloids indicated the onset of steroidal alkaloid biosynthesis at ProM. When comparing gene expression in the fire salamander to that in other amphibians-three anurans, Xenopus laevis, X. tropicalis, and Michrohyla fissipes, and one caudate, Ambystoma mexicanum- we identified genes with conserved expression patterns involved in basic metamorphic processes such as skin restructuring and tail fin resorption. Our results support that primary stages of postembryonic development in caudates are homologous to those of anurans, and offer a baseline for the study of the evolution of developmental modes.

Tail-tip clipping is a common technique for collecting tissue samples from amphibian larvae and adults. Surprisingly, studies of this invasive sampling procedure or of natural tail clipping – i.e., bites inflicted by predators including conspecifics - on the performance and fitness of aquatic larval stages of urodeles are scarce. We conducted two studies in which we assessed the effects of posterior tail clipping (~30 percent of tail) on Near Eastern fire salamander (Salamandra infraimmaculata) larvae. In a laboratory study, we checked regeneration rates of posterior tail-tip clipping at different ages. Regeneration rates were hump-shaped, peaking at the age of ~30 days and then decreasing. This variation in tail regeneration rates suggests tradeoffs in resource allocation between regeneration and somatic growth during early and advanced development. In an outdoor artificial pond experiment, under constant larval densities, we assessed how tail clipping of newborn larvae affects survival to, time to, and size at metamorphosis. Repeated measures ANOVA on mean larval survival per pond revealed no effect of tail clipping. Tail clipping had correspondingly no effect on larval growth and development expressed in size (mass and snout-vent length) at, and time to, metamorphosis. We conclude that despite the given variation in tail regeneration rates throughout larval ontogeny, clipping of 30% percent of the posterior tail area seems to have no adverse effects on larval fitness and survival. We suggest that future use of this imperative tool for the study of amphibian should take into account larval developmental stage during the time of application and not just the relative size of the clipped tail sample.