The role of cutaneous bacteria in resistance of Australian tropical rainforest frogs to the amphibian chytrid fungus Batrachochytrium dendrobatidis
Bell, Sara Christiane (2012) The role of cutaneous bacteria in resistance of Australian tropical rainforest frogs to the amphibian chytrid fungus Batrachochytrium dendrobatidis. PhD thesis, James Cook University.
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Abstract
Fungal diseases pose a serious threat to animal and plant health. The emergence of the disease chytridiomycosis, caused by the fungus Batrachochytrium dendrobatidis (Bd), has resulted in the declines or extinctions of many amphibian species worldwide, although the severity of its effects varies widely among host species. Antibiotic-producing bacterial symbionts present on amphibian skin can protect their hosts from disease-related mortality. Bioaugmentation of naturally occurring bacteria that are antagonistic to Bd offers great potential for disease prevention in field settings. However, there is a paucity of knowledge on the ecological interactions of cutaneous bacteria with their hosts and Bd; this needs to be addressed prior to development and application of probiotics. This thesis addresses technical issues associated with culture and testing of bacterial isolates in the laboratory, investigates variation among rainforest-dwelling frogs in the activity of their bacterial symbionts against Bd, and assesses the temporal stability of frog bacterial assemblages.
One factor that might limit the development of effective probiotics is the potentially low proportion of bacteria that can be cultured from environmental samples; commonly believed to be approximately 1%. If the great majority of bacteria on amphibian skin are not culturable, many species with desirable properties are likely to be unavailable for bioaugmentation research. I used both culture and culture-independent techniques to estimate the proportion of isolates that are not culturable and determine the proportion of cultured bacteria that are missed during the process of colony selection for isolation. On average, 75% of the bacteria on individual green-eyed treefrogs, Litoria serrata, were culturable. My results thus indicate that the proportion of culturable bacteria present on frog skin is much higher than the traditional 1%. However, of those culturable bacteria, only 53% were selected for isolation. This discrepancy is likely due to morphologically identical colonies present on agar plates but not selected for isolation. Therefore, apparently morphologically identical colonies should be over-selected during initial isolation to reduce the proportion that are missed, and ensure that most bacteria with potential for bioaugmentation research are likely to be cultured. Another factor that might affect the detection of bacterial symbionts with activity against Bd is the method used to screen bacterial isolates for inhibition. Previous studies have used agar-based in vitro challenge assays to identify candidates for bacterial supplementation trials. However, agar-based assays can be difficult to set up and to replicate reliably. To overcome these difficulties, I developed a semi-quantitative spectrophotometric challenge assay technique. Cell-free supernatants were prepared from filtered bacterial cultures and added to 96-well plates in replicated wells containing Bd zoospores suspended in TGhL broth medium. Plates were then read daily on a spectrophotometer until positive controls reached maximum growth in order to determine growth curves for Bd. I tested the technique by screening skin bacteria from L. serrata. Thirty-one percent of bacteria tested showed strong Bd inhibition, while some may have promoted Bd growth, a previously unknown effect. My technique avoids a number of issues associated with agar-plate assays and thus provides a useful contribution to the expanding field of bioaugmentation research.
One aspect of community ecology theory suggests that complex communities, with higher numbers of taxa and hence more potential interactions, are more resistant to invasion than simple communities with fewer taxa. In the Australian Wet Tropics, the severity of Bd effects varied among sites and frog species, and some species have since reappeared or recolonised, despite Bd now being enzootic. The available range of variation in history among species and sites provided an opportunity to investigate the role of anti-fungal cutaneous bacteria in protection of frogs against Bd infection. I conducted in vitro challenge assays to determine the capacity of bacteria, isolated from five species of rainforest frogs at five sites in northern Queensland, Australia, to inhibit Bd. I then used DNA sequencing to identify Bd-inhibitory bacteria and determine whether cutaneous bacterial taxa were associated with particular frog species, sites, infection status of frogs or intensity of Bd infection. Ninety-four percent of bacterial isolates came from just three families; Pseudomonadaceae, Enterobacteriaceae and Xanthomonadaceae, which were present across all frog species and sites. Bd infection intensity was negatively correlated with number of inhibitory genera present on frogs, suggesting that increased diversity of Bd-inhibitory taxa may play a role in reducing the intensity of Bd infections, hence facilitating frog coexistence with enzootic Bd. There was evidence suggesting that Bd-inhibitory bacteria may have facilitated the reappearance of frogs at one of two upland Wet Tropics sites I surveyed. Frogs at this site had greater cultured bacterial isolate richness, a greater number of inhibitory bacterial genera, and a higher proportion of inhibitory isolates than their lowland conspecifics. They also had a significantly higher proportion of individuals with one or more Bd-inhibitory bacterial species. My results also suggest that alternative mechanisms for decreasing susceptibility to Bd infection are likely to have evolved at the other Wet Tropics upland site I monitored where frogs have reappeared.
While the possibility of bioaugmentation offers hope for frogs threatened with extirpation, the stability of the symbiotic microbial assemblage is likely to affect its susceptibility to manipulation, and thus the success of bacterial supplementation. A microbial assemblage that changes little with time may have highly stable species interactions and therefore be difficult to supplement, while a dynamic microbial assemblage may be more amenable to manipulation. I collected swab samples from 14 individual rainforest frogs captured multiple times over the course of a year, and compared the bacterial assemblage profile generated for each swab sample among frogs grouped by date sampled and by patterns of Bd infection. Twenty-five of 114 unique bacterial strains found eight times or more constituted the dominant assemblage members, with five core strains each occurring in greater than 65% of samples. The proportion of core bacterial strains was significantly higher in frogs that were not infected with Bd prior to sampling than in frogs that were infected or had recently lost infection. The taxonomic composition of the dominant microbial assemblage also differed significantly between these two groups of frogs. Bacterial species richness decreased over the year, with a corresponding statistically significant change in assemblage composition. The temporal changes observed suggest that the microbiota would be amenable to manipulation through bioaugmentation of Bd-inhibitory bacteria. However, turnover of core strains was low, suggesting that bioaugmentation success might be enhanced by application of dominant assemblage members.
I have presented evidence for the importance of amphibian cutaneous microbiota in protection of wild amphibians against Bd; greater taxonomic richness of cutaneous bacteria is associated with lower Bd infection intensity. With this knowledge, an improved technique to screen bacteria for inhibitory effects against Bd, and an understanding of the natural changes that occur in bacterial assemblages over time, there is now further support for the use of bioaugmentation as a tool to protect amphibians against the devastating effects of chytridiomycosis.
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