Interacting symbionts and immunity in the amphibian skin mucosome predict disease risk and probiotic effectiveness

Abstract

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.

Figure 1. Infection prevalence (mean, 95% binomial CI) of amphibians sampled across Europe and within Switzerland predicted by mucosome function and skin defense peptide activity against Batrachochytrium dendrobatidis (Bd) zoospores.

Mucosome function (mean, SE) indicates Bd viability after a 1 hr exposure to amphibian mucus (a,b) and units represent green:red fluorescence. Peptide efficiency (mean, SE) indicates quantity of natural mixtures of skin peptides induced from granular glands multiplied by activity of a standard concentration of peptides against Bd zoospore growth. Only post-metamorphic amphibians sampled upon subcutaneous injection with norepinephrine are plotted in (c) and (d). Amphibian skin mucosome function is a better predictor of infection prevalence than induced skin peptide efficiency (logistic regression, see text). Summary data for all species and life-history stages are presented in Table 1.

Figure 2. Relative survival (95% binomial CI; a) and Proportion of infected frogs (95% binomial CI; b) predicted by Mucosome function.

Post-metamorphosis survival was measured from four Swiss amphibian species after exposure to zoospores of a Swiss Bd isolate, TG 739. Survival curves for each species are presented in Supporting Information (Figs. S2, S3 in File S1) and relative survival was calculated as the proportion of infected frogs surviving/proportion of unexposed control frogs surviving. Alytes obstetricans showed the highest infection and mortality, and Rana temporaria the lowest, with Bombina variegata and Pelophylax esculentus intermediate. All frogs were raised in captivity from egg clutches and had no history of natural exposure to Bd. Mucosome function (mean, SE) indicates Bd viability after exposure to amphibian mucus and is a significant predictor of both survival (binomial logistic regressions, P<0.0001) and infection prevalence (P = 0.0106).

Figure 3. Temperature and probiotic treatments of recently metamorphosed midwife toads, A. obstetricans, influence skin mucosome function (a) but not induced skin peptide defenses (b).

(a) Mucosome function indicates B. dendrobatidis (Bd) viability after exposure to amphibian mucus quantified by green: red fluorescence. Significantly different subsets are indicated by letters above bars (Tukey post-hoc test). Bd zoospore viability was reduced after exposure to mucus from frogs treated with the bacterium F. johnsoniae and the fungus P. expansum, and zoospore viability was highest after exposure to mucus from toads previously exposed to Bd. (b) Skin peptide effectiveness against Bd did not differ significantly among treatments (ANOVA, F⁶ = 0.952, P = 0.466).

Figure 4. Environmental context determines antifungal capacity of probiotics.

Tested temperatures (14, 19, 22°C) significantly affected the production of bacterial metabolites in liquid media that could inhibit B. dendrobatidis (Bd; GPL isolate VMV 813) zoospore growth in a dose-dependent fashion (a  =  full strength metabolites, b = 1∶10 dilution). * indicates that Bd growth differed among metabolite temperature treatments (ANOVA, Bonferroni-corrected P's<0.05). (c) Representative replicates are shown of two isolates of Serratia plymuthica isolated from egg clutches of common midwife toads, Alytes obstetricans, grown on solid media under different temperature conditions. Filtrate from isolate 27 always inhibited growth of Bd, but filtrate from isolate 28 inhibited Bd growth at 18°C, and enhanced Bd growth at 25°C. Filtrate from sterile media (R2A agar supplemented with 1% tryptone) caused enhanced growth of Bd. Note that colony color can be an indication of antifungal metabolites such as prodiginines from red Serratia spp. , , but are produced only under certain growth conditions.

Figure 5. Choosing probiotics with the greatest potential against amphibian chytridiomycosis.

Candidate probiotic bacteria (or fungi) are isolated from populations of amphibians that are able to persist in the presence of B. dendrobatidis (Bd) . To increase the chances of successful prophylactic biotherapy, candidate probiotics should be tested for at least three characteristics: (a) capacity to inhibit Bd growth as a pure isolate without specific competitive interactions to induce antifungal metabolites, (b) capacity to inhibit Bd at a temperature range consistent with host habitat, and (c) resistance to host skin immune defenses that would complicate probiotic establishment. Remedial biotherapy of already infected individuals should maintain antifungal capacity when grown in competition with Bd and withstand the sometimes lethal effects of Bd metabolites (Fig. S6 in File S1). Testing probiotic effect in vivo can be accomplished without resorting to pathogen exposure experiments by using the mucosome function assay described here.