Genetic variability and ontogeny predict microbiome structure in a disease-challenged montane amphibian.
ABSTRACT: Amphibian populations worldwide are at risk of extinction from infectious diseases, including chytridiomycosis caused by the fungal pathogen Batrachochytrium dendrobatidis (Bd). Amphibian cutaneous microbiomes interact with Bd and can confer protective benefits to the host. The composition of the microbiome itself is influenced by many environment- and host-related factors. However, little is known about the interacting effects of host population structure, genetic variation and developmental stage on microbiome composition and Bd prevalence across multiple sites. Here we explore these questions in Amietia hymenopus, a disease-affected frog in southern Africa. We use microsatellite genotyping and 16S amplicon sequencing to show that the microbiome associated with tadpole mouthparts is structured spatially, and is influenced by host genotype and developmental stage. We observed strong genetic structure in host populations based on rivers and geographic distances, but this did not correspond to spatial patterns in microbiome composition. These results indicate that demographic and host genetic factors affect microbiome composition within sites, but different factors are responsible for host population structure and microbiome structure at the between-site level. Our results help to elucidate complex within- and among- population drivers of microbiome structure in amphibian populations. That there is a genetic basis to microbiome composition in amphibians could help to inform amphibian conservation efforts against infectious diseases.
Project description:Symbiotic microbial communities may interact with infectious pathogens sharing a common host. The microbiome may limit pathogen infection or, conversely, an invading pathogen can disturb the microbiome. Documentation of such relationships during naturally occurring disease outbreaks is rare, and identifying causal links from field observations is difficult. This study documented the effects of an amphibian skin pathogen of global conservation concern [the chytrid fungus Batrachochytrium dendrobatidis (Bd)] on the skin-associated bacterial microbiome of the endangered frog, Rana sierrae, using a combination of population surveys and laboratory experiments. We examined covariation of pathogen infection and bacterial microbiome composition in wild frogs, demonstrating a strong and consistent correlation between Bd infection load and bacterial community composition in multiple R. sierrae populations. Despite the correlation between Bd infection load and bacterial community composition, we observed 100% mortality of postmetamorphic frogs during a Bd epizootic, suggesting that the relationship between Bd and bacterial communities was not linked to variation in resistance to mortal disease and that Bd infection altered bacterial communities. In a controlled experiment, Bd infection significantly altered the R. sierrae microbiome, demonstrating a causal relationship. The response of microbial communities to Bd infection was remarkably consistent: Several bacterial taxa showed the same response to Bd infection across multiple field populations and the laboratory experiment, indicating a somewhat predictable interaction between Bd and the microbiome. The laboratory experiment demonstrates that Bd infection causes changes to amphibian skin bacterial communities, whereas the laboratory and field results together strongly support Bd disturbance as a driver of bacterial community change during natural disease dynamics.
Project description:Symbiotic microbial communities play key roles in the health and development of their multicellular hosts. Understanding why microbial communities vary among different host species or individuals is an important step toward understanding the diversity and function of the microbiome. The amphibian skin microbiome may affect resistance to the fungal pathogen Batrachochytrium dendrobatidis (Bd). Still, the factors that determine the diversity and composition of the amphibian skin microbiome, and therefore may ultimately contribute to disease resistance, are not well understood. We conducted a two-phase experiment to first test how host and environment shape the amphibian skin microbiome, and then test if the microbiome affects or is affected by Bd infection. Most lab experiments testing assembly of the amphibian skin microbiome so far have compared sterile to non-sterile environments or heavily augmented to non-augmented frogs. A goal of this study was to evaluate, in an experimental setting, realistic potential drivers of microbiome assembly that would be relevant to patterns observed in nature. We tested effects of frog genetic background (2 source populations) and 6 natural lake water sources in shaping the microbiome of the frog Rana sierrae. Water in which frogs were housed affected the microbiome in a manner that partially mimicked patterns observed in natural populations. In particular, frogs housed in water from disease-resistant populations had greater bacterial richness than frogs housed in water from populations that died out due to Bd. However, in the experiment this difference in microbiomes did not lead to differences in host mortality or rates of pathogen load increase. Frog source population also affected the microbiome and, although none of the frogs in this study showed true resistance to infection, host source population had a small effect on the rate of pathogen load increase. This difference in infection trajectories could be due to the observed differences in the microbiome, but could also be due to other traits that differ between frogs from the two populations. In addition to examining effects of the microbiome on Bd, we tested the effect of Bd infection severity on the microbiome. Specifically, we studied a time series of the microbiome over the course of infection to test if the effects of Bd on the microbiome are dependent on Bd infection severity. Although limited to a small subset of frogs, time series analysis suggested that relative abundances of several bacterial phylotypes changed as Bd loads increased through time, indicating that Bd-induced disturbance of the R. sierrae microbiome is not a binary effect but instead is dependent on infection severity. We conclude that both host and aquatic environment help shape the R. sierrae skin microbiome, with links to small changes in disease resistance in some cases, but in this study the effect of Bd on the microbiome was greater than the effect of the microbiome on Bd. Assessment of the microbiome differences between more distantly related populations than those studied here is needed to fully understand the role of the microbiome in resistance to Bd.
Project description:Infectious diseases have serious impacts on human and wildlife populations, but the effects of a disease can vary, even among individuals or populations of the same host species. Identifying the reasons for this variation is key to understanding disease dynamics and mitigating infectious disease impacts, but disentangling cause and correlation during natural outbreaks is extremely challenging. This study aims to understand associations between symbiotic bacterial communities and an infectious disease, and examines multiple host populations before or after pathogen invasion to infer likely causal links. The results show that symbiotic bacteria are linked to fundamentally different outcomes of pathogen infection: host-pathogen coexistence (endemic infection) or host population extirpation (epidemic infection). Diversity and composition of skin-associated bacteria differed between populations of the frog, Rana sierrae, that coexist with or were extirpated by the fungal pathogen, Batrachochytrium dendrobatidis (Bd). Data from multiple populations sampled before or after pathogen invasion were used to infer cause and effect in the relationship between the fungal pathogen and symbiotic bacteria. Among host populations, variation in the composition of the skin microbiome was most strongly predicted by pathogen infection severity, even in analyses where the outcome of infection did not vary. This result suggests that pathogen infection shapes variation in the skin microbiome across host populations that coexist with or are driven to extirpation by the pathogen. By contrast, microbiome richness was largely unaffected by pathogen infection intensity, but was strongly predicted by geographical region of the host population, indicating the importance of environmental or host genetic factors in shaping microbiome richness. Thus, while both richness and composition of the microbiome differed between endemic and epidemic host populations, the underlying causes are most likely different: pathogen infection appears to shape microbiome composition, while microbiome richness was less sensitive to pathogen-induced disturbance. Because higher richness was correlated with host persistence in the presence of Bd, and richness appeared relatively stable to Bd infection, microbiome richness may contribute to disease resistance, although the latter remains to be directly tested.
Project description:Describing the structure and function of the amphibian cutaneous microbiome has gained importance with the spread of Batrachochytrium dendrobatidis (Bd), the fungal pathogen that can cause the skin disease chytridiomycosis. Sampling amphibian skin microbiota is needed to characterize current infection status and to help predict future susceptibility to Bd based on microbial composition since some skin microbes have antifungal capabilities that may confer disease resistance. Here, I use 16S rRNA sequencing to describe the composition and structure of the cutaneous microbiota of six species of amphibians. Frog skin samples were also tested for Bd, and I found 11.8% Bd prevalence among all individuals sampled (n = 76). Frog skin microbiota varied by host species and sampling site, but did not differ among Bd-positive and Bd-negative individuals. These results suggest that bacterial composition reflects host species and the environment, but does not reflect Bd infection among the species sampled here. Of the bacterial OTUs identified using an indicator species analysis as strongly associated with amphibians, significantly more indicator OTUs were putative anti-Bd taxa than would be expected based on the proportion of anti-Bd OTUs among all frog OTUs, suggesting strong associations between host species and anti-Bd OTUs. This relationship may partially explain why some of these frogs are asymptomatic carriers of Bd, but more work is needed to determine the other factors that contribute to interspecific variation in Bd susceptibility. This work provides important insights on inter- and intra-specific variation in microbial community composition, putative function, and disease dynamics in populations of amphibians that appear to be coexisting with Bd.
Project description:Many pathogens infect more than one host species, and clarifying how these different hosts contribute to pathogen dynamics can facilitate the management of pathogens and can lend insight into the functioning of pathogens in ecosystems. In this study, we investigated a suite of native and non-native amphibian hosts of the pathogen Batrachochytrium dendrobatidis (Bd) across multiple scales to identify potential mechanisms that may drive infection patterns in the Colorado study system. Specifically, we aimed to determine if: 1) amphibian populations vary in Bd infection across the landscape, 2) amphibian community composition predicts infection (e.g., does the presence or abundance of any particular species influence infection in others?), 3) amphibian species vary in their ability to produce infectious zoospores in a laboratory infection, 4) heterogeneity in host ability observed in the laboratory scales to predict patterns of Bd prevalence in the landscape. We found that non-native North American bullfrogs (Lithobates catesbeianus) are widespread and have the highest prevalence of Bd infection relative to the other native species in the landscape. Additionally, infection in some native species appears to be related to the density of sympatric L. catesbeianus populations. At the smaller host scale, we found that L. catesbeianus produces more of the infective zoospore stage relative to some native species, but that this zoospore output does not scale to predict infection in sympatric wild populations of native species. Rather, landscape level infection relates most strongly to density of hosts at a wetland as well as abiotic factors. While non-native L. catesbeianus have high levels of Bd infection in the Colorado Front Range system, we also identified Bd infection in a number of native amphibian populations allopatric with L. catesbeianus, suggesting that multiple host species are important contributors to the dynamics of the Bd pathogen in this landscape.
Project description:In Brazil’s Atlantic Forest (AF) biodiversity conservation is of key importance since the fungal pathogen Batrachochytrium dendrobatidis (Bd) has led to the rapid loss of amphibian populations here and worldwide. The impact of Bd on amphibians is determined by the host's immune system, of which the skin microbiome is a critical component. The richness and diversity of such cutaneous bacterial communities are known to be shaped by abiotic factors which thus may indirectly modulate host susceptibility to Bd. This study aimed to contribute to understanding the environment-host–pathogen interaction determining skin bacterial communities in 819 treefrogs (Anura: Hylidae and Phyllomedusidae) from 71 species sampled across the AF. We investigated whether abiotic factors influence the bacterial community richness and structure on the amphibian skin. We further tested for an association between skin bacterial community structure and Bd co-occurrence. Our data revealed that temperature, precipitation, and elevation consistently correlate with richness and diversity of the skin microbiome and also predict Bd infection status. Surprisingly, our data suggest a weak but significant positive correlation of Bd infection intensity and bacterial richness. We highlight the prospect of future experimental studies on the impact of changing environmental conditions associated with global change on environment-host–pathogen interactions in the AF.
Project description:<h4>Background</h4>Microorganisms have intimate functional relationships with invertebrate and vertebrate taxa, with the potential to drastically impact health outcomes. Perturbations that affect microbial communities residing on animals can lead to dysbiosis, a change in the functional relationship, often associated with disease. Batrachochytrium dendrobatidis (Bd), a fungal pathogen of amphibians, has been responsible for catastrophic amphibian population declines around the globe. Amphibians harbor a diverse cutaneous microbiome, including some members which are known to be antagonistic to Bd (anti-Bd). Anti-Bd microorganisms facilitate the ability of some frog populations to persist in the presence of Bd, where other populations that lack anti-Bd microorganisms have declined. Research suggests disease-antagonistic properties of the microbiome may be a function of microbial community interactions, rather than individual bacterial species. Conservation efforts have identified amphibian-associated bacteria that exhibit anti-fungal properties for use as 'probiotics' on susceptible amphibian populations. Probiotic application, usually with a single bacterial species, may benefit from a greater understanding of amphibian species-specific microbiome responses to disturbances (e.g. dysbiosis vs. recovery). We assessed microbiome responses to two microbial disturbance events over multiple time points.<h4>Results</h4>Exposing Lithobates sphenocephalus (southern leopard frog) adults to the biopesticidal bacteria Bacillus thuringiensis, followed by exposure to the fungal pathogen Bd, did not have long term impacts on the microbiome. After initial shifts, microbial communities recovered and returned to a state that resembled pre-disturbance.<h4>Conclusions</h4>Our results indicate microbial communities on L. sphenocephalus are robust and resistant to permanent shifts from some disturbances. This resiliency of microbial communities may explain why L. sphenocephalus is not experiencing the population declines from Bd that impacts many other species. Conservation efforts may benefit from studies outlining amphibian species-specific microbiome responses to disturbances (e.g. dysbiosis vs. recovery). If microbial communities on a threatened amphibian species are unlikely to recover following a disturbance, additional measures may be implemented to ameliorate the impacts of physical and chemical stressors on host-associated microbial communities.
Project description:North American amphibians have recently been impacted by two major emerging pathogens, the fungus Batrachochytrium dendrobatidis (Bd) and iridoviruses in the genus Ranavirus (Rv). Environmental factors and host genetics may play important roles in disease dynamics, but few studies incorporate both of these components into their analyses. Here, we investigated the role of environmental and genetic factors in driving Bd and Rv infection prevalence and severity in a biodiversity hot spot, the southeastern United States. We used quantitative PCR to characterize Bd and Rv dynamics in natural populations of three amphibian species: Notophthalmus perstriatus, Hyla squirella and Pseudacris ornata. We combined pathogen data, genetic diversity metrics generated from neutral markers, and environmental variables into general linear models to evaluate how these factors impact infectious disease dynamics. Occurrence, prevalence and intensity of Bd and Rv varied across species and populations, but only one species, Pseudacris ornata, harbored high Bd intensities in the majority of sampled populations. Genetic diversity and climate variables both predicted Bd prevalence, whereas climatic variables alone predicted infection intensity. We conclude that Bd is more abundant in the southeastern United States than previously thought and that genetic and environmental factors are both important for predicting amphibian pathogen dynamics. Incorporating both genetic and environmental information into conservation plans for amphibians is necessary for the development of more effective management strategies to mitigate the impact of emerging infectious diseases.
Project description:BACKGROUND:The skin microbiome serves as a first line defense against pathogens in vertebrates. In amphibians, it has the potential to protect against the chytrid fungus Batrachochytrium dendrobatis (Bd), a likely agent of amphibian declines. Alteration of the microbiome associated with unfavorable environmental changes produced by anthropogenic activities may make the host more susceptible to pathogens. Some amphibian species that were thought to be "extinct" have been rediscovered years after population declines in the late 1980s probably due to evolved Bd-resistance and are now threatened by anthropogenic land-use changes. Understanding the effects of habitat disturbance on the host skin microbiome is relevant for understanding the health of these species, along with its susceptibility to pathogens such as Bd. Here, we investigate the influence of habitat alteration on the skin bacterial communities as well as specifically the putative Bd-inhibitory bacterial communities of the montane frog Lithobates vibicarius. This species, after years of not being observed, was rediscovered in small populations inhabiting undisturbed and disturbed landscapes, and with continuous presence of Bd. RESULTS:We found that cutaneous bacterial communities of tadpoles and adults differed between undisturbed and disturbed habitats. The adults from disturbed habitats exhibited greater community dispersion than those from undisturbed habitats. We observed a higher richness of putative Bd-inhibitory bacterial strains in adults from disturbed habitats than in those from undisturbed habitats, as well as a greater number of these potential protective bacteria with a high relative abundance. CONCLUSIONS:Our findings support the microbial "Anna Karenina principle", in which disturbance is hypothesized to cause greater microbial dispersion in communities, a so-called dysbiosis, which is a response of animal microbiomes to stress factors that decrease the ability of the host or its microbiome to regulate community composition. On the positive side, the high richness and relative abundance of putative Bd-inhibitory bacteria may indicate the development of a defense mechanism that enhances Bd-protection, attributed to a co-occurrence of more than 30-years of host and pathogen in these disturbed habitats. Our results provide important insight into the influence of human-modified landscapes on the skin microbiome and health implications of Bd-survivor species.
Project description:Deforestation has detrimental consequences on biodiversity, affecting species interactions at multiple scales. The associations among vertebrates, pathogens and their commensal/symbiotic microbial communities (i.e. microbiomes) have important downstream effects for biodiversity conservation, yet we know little about how deforestation contributes to changes in host microbial diversity and pathogen abundance. Here, we tested the effects of landcover, forest connectivity and infection by the chytrid fungus Batrachochytrium dendrobatidis (Bd) on amphibian skin bacterial diversity along deforestation gradients in Brazilian landscapes. If disturbance to natural habitat alters skin microbiomes as it does in vertebrate host communities, then we would expect higher host bacterial diversity in natural forest habitats. Bd infection loads are also often higher in these closed-canopy forests, which may in turn impact skin-associated bacterial communities. We found that forest corridors shaped composition of host skin microbiomes; high forest connectivity predicted greater similarity of skin bacterial communities among host populations. In addition, we found that host skin bacterial diversity and Bd loads increased towards natural vegetation. Because symbiotic bacteria can potentially buffer hosts from Bd infection, we also evaluated the bi-directional microbiome-Bd link but failed to find a significant effect of skin bacterial diversity reducing Bd infections. Although weak, we found support for Bd increasing bacterial diversity and/or for core bacteria dominance reducing Bd loads. Our research incorporates a critical element in the study of host microbiomes by linking environmental heterogeneity of landscapes to the host-pathogen-microbiome triangle.