Increased numbers of culturable inhibitory bacterial taxa may mitigate the effects of Batrachochytrium dendrobatidis in Australian wet tropics frogs

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Abstract

Symbiotic bacterial communities resident on amphibian skin can benefit their hosts. For example, antibiotic production by community members can control the pathogen Batrachochytrium dendrobatidis (Bd) and it is possible for these community members to be used as probiotics to reduce infection levels. In the early 1990s, the emergence of Bd caused declines and disappearances of frogs in the Australian Wet Tropics; the severity of its effects varied among species and sites. Some species have since recolonized despite enzootic Bd within their populations. This variation in history among species and sites provided an opportunity to investigate the role of anti-fungal cutaneous bacteria in protecting frogs against Bd infection. We collected cutaneous swab samples from three species of frogs at two upland and two lowland sites in the Wet Tropics, and used in vitro challenge assays to identify culturable Bd-inhibitory bacterial isolates for further analysis. We sequenced DNA from cultured inhibitory isolates to identify taxa, resulting in the classification of 16 Bd-inhibitory OTUs, and determined whether inhibitory taxa were associated with frog species, site, or intensity of infection. We present preliminary results showing that the upper limit of Bd infection intensity was negatively correlated with number of inhibitory OTUs present per frog indicating that increased numbers of Bd-inhibiting taxa may play a role in reducing the intensity of Bd infections, facilitating frog coexistence with enzootic Bd. One upland site had a significantly lower prevalence of Bd infection, a significantly higher proportion of frogs with one or more culturable Bd-inhibitory OTUs, a greater number of inhibitory bacterial genera present per frog, and statistically significant clustering of individual frogs with similar Bd-inhibitory signatures when compared to all other sites. This suggests that Bd-inhibitory taxa are likely to be particularly important to frogs at this site and may have played a role in their ability to recolonize following population declines. Our findings suggest that the use of multi-taxon Bd-inhibitory probiotics to support at-risk amphibian populations may be more effective than single-taxon alternatives.

Figures

  • TABLE 1 | Locations, species and Batrachochytrium dendrobatidis infection status of frogs sampled for each survey site, and overall Bd infection prevalence by site.
  • FIGURE 1 | The proportion of culturable isolates as categorized by challenge assay results for each frog species at each site. Category definitions follow Bell et al. (2013). Species are Litoria serrata (LS), L. nannotis (LN), and L. rheocola (LR). Sites are Kirrama upland (KU), Kirrama lowland (KL), Wooroonooran upland (WU), and Wooroonooran lowland (WL).
  • FIGURE 2 | Neighbor-joining tree showing OTUs with representative type species for comparison. Nodes are labeled with percentage bootstrap support values. Labels with 100% bootstrap support were removed from some nodes to improve visualization.
  • TABLE 2 | Taxonomic classification of 16S rRNA OTUs by frog species and site.
  • FIGURE 4 | Intensity of Bd infection decreased as the number of inhibitory OTUs increased. The ninetieth percentile regression line shows the negative relationship between the number of OTUs per infected frog and the upper limits of Bd infection intensity (n = 14, t = -2.818, p = 0.0155).
  • FIGURE 3 | The relationships between (A) the number of totally inhibitory bacterial isolates identified and sequenced and the expected number of totally inhibitory OTUs (EstimateS; Colwell, 2013), and (B) the total number of isolates obtained from each individual frog and the number of totally inhibitory OTUs detected. The size of circles corresponds to the number of frogs at each combination of number of isolates and number of OTUs, with the smallest indicating one individual and the largest indicating eight. The best fit accumulation curve is a power curve fitted to the number of OTUs plus 1 (y = 0.717x0.449 with R2 = 0.505, p = 0.001).
  • FIGURE 5 | Results of an nMDS illustrating how the OTU signatures of frogs cluster by site. Stress = 0.124. Sites are KL, Kirrama lowland; KU, Kirrama upland; WL, Wooroonooran lowland; WU, Wooroonooran upland.
  • FIGURE 6 | Proportion of frogs with isolates in each OTU by site. For ease of visualization, OTUs that occurred on fewer than two frogs at each site have been excluded. KU, Kirrama upland; KL, Kirrama lowland; WU, Wooroonooran upland; WL, Wooroonooran lowland.

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Bell, S. C., Garland, S., & Alford, R. A. (2018). Increased numbers of culturable inhibitory bacterial taxa may mitigate the effects of Batrachochytrium dendrobatidis in Australian wet tropics frogs. Frontiers in Microbiology, 9(JUL). https://doi.org/10.3389/fmicb.2018.01604

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