RESUMO
Host-associated microbial communities, like other ecological communities, may be impacted by the colonization order of taxa through priority effects. Developing embryos and their associated microbiomes are subject to stochasticity during colonization by bacteria. For amphibian embryos, often developing externally in bacteria-rich environments, this stochasticity may be particularly impactful. For example, the amphibian microbiome can mitigate lethal outcomes from disease for their hosts; however, this may depend on microbiome composition. Here, we examined the assembly of the bacterial community in spring peeper (Pseudacris crucifer) embryos and tadpoles. First, we reared embryos from identified mating pairs in either lab or field environments to examine the relative impact of environment and parentage on embryo and tadpole bacterial communities. Second, we experimentally inoculated embryos to determine if priority effects (i) could be used to increase the relative abundance of Janthinobacterium lividum, an amphibian-associated bacteria capable of preventing fungal infection, and (ii) would lead to observed differences in the relative abundances of two closely related bacteria from the genus Pseudomonas. Using 16S rRNA gene amplicon sequencing, we observed differences in community composition based on rearing location and parentage in embryos and tadpoles. In the inoculation experiment, we found that priority inoculation could increase the relative abundance of J. lividum, but did not find that either Pseudomonas isolate was able to prevent colonization by the other when given priority. These results highlight the importance of environmental source pools and parentage in determining microbiome composition, while also providing novel methods for the administration of a known amphibian probiotic. IMPORTANCE: Harnessing the functions of host-associated bacteria is a promising mechanism for managing disease outcomes across different host species. In the case of amphibians, certain frog-associated bacteria can mitigate lethal outcomes of infection by the fungal pathogen Batrachochytrium dendrobatidis. Successful probiotic applications require knowledge of community assembly and an understanding of the ecological mechanisms that structure these symbiotic bacterial communities. In our study, we show the importance of environment and parentage in determining bacterial community composition and that community composition can be influenced by priority effects. Further, we provide support for the use of bacterial priority effects as a mechanism to increase the relative abundance of target probiotic taxa in a developing host. While our results show that priority effects are not universally effective across all host-associated bacteria, our ability to increase the relative abundance of specific probiotic taxa may enhance conservation strategies that rely on captive rearing of endangered vertebrates.
Assuntos
Anuros , Larva , Microbiota , Probióticos , RNA Ribossômico 16S , Animais , Larva/microbiologia , Larva/crescimento & desenvolvimento , Anuros/microbiologia , Probióticos/administração & dosagem , Probióticos/farmacologia , RNA Ribossômico 16S/genética , Bactérias/classificação , Bactérias/isolamento & purificação , Bactérias/genética , Oxalobacteraceae/fisiologia , Pseudomonas/fisiologia , Embrião não Mamífero/microbiologiaRESUMO
Priority effects, or impacts of colonization order, may have lasting influence on ecological community composition. The embryonic microbiome is subject to stochasticity in colonization order of bacteria. Stochasticity may be especially impactful for embryos developing in bacteria-rich environments, such as the embryos of many amphibians. To determine if priority effects experienced as embryos impacted bacterial community composition in newly hatched tadpoles, we selectively inoculated the embryos of laboratory-raised hourglass treefrogs, Dendropsophus ebraccatus, with bacteria initially isolated from the skin of wild D. ebraccatus adults over 2 days. First, embryos were inoculated with two bacteria in alternating sequences. Next, we evaluated the outcomes of priority effects in an in vitro co-culture assay absent of host factors. We then performed a second embryo experiment, inoculating embryos with one of three bacteria on the first day and a community of five target bacteria on the second. Through 16S rRNA gene amplicon sequencing, we observed relative abundance shifts in tadpole bacteria communities due to priority effects. Our results suggest that the initial bacterial source pools of embryos shape bacterial communities at later life stages; however, the magnitude of those changes is dependent on the host environment and the identity of bacterial colonists.
Assuntos
Microbiota , Animais , Larva/microbiologia , RNA Ribossômico 16S/genética , Microbiota/genética , Anuros/genética , Biota , BactériasRESUMO
Amphibian population declines caused by the fungus Batrachochytrium dendrobatidis (Bd) have prompted studies on the bacterial community that resides on amphibian skin. However, studies addressing the fungal portion of these symbiont communities have lagged behind. Using ITS1 amplicon sequencing, we examined the fungal portion of the skin microbiome of temperate and tropical amphibian species currently coexisting with Bd in nature. We assessed cooccurrence patterns between bacterial and fungal OTUs using a subset of samples for which bacterial 16S rRNA gene amplicon data were also available. We determined that fungal communities were dominated by members of the phyla Ascomycota and Basidiomycota, and also by Chytridiomycota in the most aquatic amphibian species. Alpha diversity of the fungal communities differed across host species, and fungal community structure differed across species and regions. However, we did not find a correlation between fungal diversity/community structure and Bd infection, though we did identify significant correlations between Bd and specific OTUs. Moreover, positive bacterial-fungal cooccurrences suggest that positive interactions between these organisms occur in the skin microbiome. Understanding the ecology of amphibian skin fungi, and their interactions with bacteria will complement our knowledge of the factors influencing community assembly and the overall function of these symbiont communities.
Assuntos
Anuros/microbiologia , Quitridiomicetos , Micobioma , Micoses/veterinária , Animais , Quitridiomicetos/genética , Fungos/classificação , Fungos/isolamento & purificação , Especificidade de Hospedeiro , Microbiota , Tipagem Molecular , Micoses/microbiologia , Pele/microbiologia , SimbioseRESUMO
Animals host a wide diversity of symbiotic microorganisms that contribute important functions to host health, and our knowledge of what drives variation in the composition of these complex communities continues to grow. Microbiome studies at larger spatial scales present opportunities to evaluate the contribution of large-scale factors to variation in the microbiome. We conducted a large-scale field study to assess variation in the bacterial symbiont communities on adult frog skin (Pseudacris crucifer), characterized using 16S rRNA gene amplicon sequencing. We found that skin bacterial communities on frogs were less diverse than, and structurally distinct from, the surrounding habitat. Frog skin was typically dominated by one of two bacterial OTUs: at western sites, a Proteobacteria dominated the community, whereas eastern sites were dominated by an Actinobacteria. Using a metacommunity framework, we then sought to identify factors explaining small- and large-scale variation in community structure-that is, among hosts within a pond, and among ponds spanning the study transect. We focused on the presence of a fungal skin pathogen, Batrachochytrium dendrobatidis (Bd) as one potential driver of variation. We found no direct link between skin bacterial community structure and Bd infection status of individual frog hosts. Differences in pond-level community structure, however, were explained by Bd infection prevalence. Importantly, Bd infection prevalence itself was correlated with numerous other environmental factors; thus, skin bacterial diversity may be influenced by a complex suite of extrinsic factors. Our findings indicate that large-scale factors and processes merit consideration when seeking to understand microbiome diversity.
Assuntos
Anuros/microbiologia , Bactérias/isolamento & purificação , Microbiota , Pele/microbiologia , Animais , Bactérias/classificação , RNA Bacteriano/análise , RNA Ribossômico 16S/análise , VirginiaRESUMO
Some amphibian skin bacteria inhibit growth of a fungal amphibian pathogen, Batrachochytrium dendrobatidis (Bd), but it is unclear how dominant these anti-Bd bacteria are in skin communities. Using in vitro co-culture challenge assays, we quantified Bd inhibition by bacterial isolates collected from the skin of four amphibian species: bullfrogs, Eastern newts, spring peepers and American toads. The 16S rRNA sequences for each isolate were matched to culture-independent amplicon sequences from the same individuals to assess inhibitory function versus relative abundance. Dominant bacteria had higher Bd inhibition than rare bacteria in bullfrog and newt populations, in which Bd was prevalent (> 25%). Dominant and rare bacteria did not differ in Bd inhibition in spring peeper and toad populations, in which Bd was absent or at low prevalence (< 7%). In addition, over half of the relative abundance of cultured bacteria on bullfrogs and newts was comprised of inhibitory bacteria, while only 25% and 37% of the relative abundance was inhibitory on spring peepers and toads, respectively. These results suggest that the dominant members of the amphibian skin bacterial community may be functionally important in terms of disease-resistance, and that Bd prevalence and/or host species identity may impact the relative abundance and inhibitory properties of skin bacteria.
Assuntos
Antibiose/fisiologia , Anuros/microbiologia , Bactérias/classificação , Quitridiomicetos/crescimento & desenvolvimento , Pele/microbiologia , Animais , Bactérias/genética , Bactérias/isolamento & purificação , Microbiota , RNA Ribossômico 16S/genéticaRESUMO
Both the structure and function of host-associated microbial communities are potentially impacted by environmental conditions, just as the outcomes of many free-living species interactions are context-dependent. Many amphibian populations have declined around the globe due to the fungal skin pathogen, Batrachochytrium dendrobatidis (Bd), but enivronmental conditions may influence disease dynamics. For instance, in Panamá, the most severe Bd outbreaks have occurred at high elevation sites. Some amphibian species harbor bacterial skin communities that can inhibit the growth of Bd, and therefore, there is interest in understanding whether environmental context could also alter these host-associated microbial communities in a way that might ultimately impact Bd dynamics. In a field survey in Panamá, we assessed skin bacterial communities (16S rRNA amplicon sequencing) and metabolite profiles (HPLC-UV/Vis) of Silverstoneia flotator from three high- and three low-elevation populations representing a range of environmental conditions. Across elevations, frogs had similar skin bacterial communities, although one lowland site appeared to differ. Interestingly, we found that bacterial richness decreased from west to east, coinciding with the direction of Bd spread through Panamá. Moreover, metabolite profiles suggested potential functional variation among frog populations and between elevations. While the frogs have similar bacterial community structure, the local environment might shape the metabolite profiles. Ultimately, host-associated community structure and function could be dependent on environmental conditions, which could ultimately influence host disease susceptibility across sites.
Assuntos
Anuros/microbiologia , Bactérias/metabolismo , Metaboloma , Pele/microbiologia , Animais , Quitridiomicetos/patogenicidade , Panamá , RNA Ribossômico 16S/genéticaRESUMO
UNLABELLED: Disruptions to the microbiome can impact host health as can exposure to environmental contaminants. However, few studies have addressed how environmental contaminants impact the microbiome. We explored this question for frogs that breed in wetlands contaminated with fly ash, a by-product of coal combustion that is enriched in trace elements. We found differences in the bacterial communities among a fly ash-contaminated site and several reference wetlands. We then experimentally assessed the impacts of fly ash on the skin microbiome of adult spring peepers (Pseudacris crucifer). Frogs were exposed to fly ash in the laboratory for 12 h, the duration of a typical breeding event, and the skin microbiome was assessed after 5 days (experiment 1) or after 5 and 15 days (experiment 2). We examined bacterial community structure using 16S rRNA gene amplicon sequencing and metabolite profiles using high-pressure liquid chromatography-mass spectrometry (HPLC-MS). We found little impact as the result of acute exposure to fly ash on the bacterial communities or metabolite profiles in either experiment, suggesting that the bacterial symbiont communities of adults may be relatively resistant to brief contaminant exposure. However, housing frogs in the laboratory altered bacterial community structure in the two experiments, which supports prior research suggesting that environmental source pools are important for maintaining the amphibian skin microbiome. Therefore, for contaminants like fly ash that may alter the potential source pool of symbionts, we think it may be important to explore how contaminants affect the initial assembly of the amphibian skin microbiome in larval amphibians that develop within contaminated sites. IMPORTANCE: Animals are hosts to many symbiotic microorganisms, collectively called the microbiome, that play critical roles in host health. Therefore, environmental contaminants that alter the microbiome may impact hosts. Some of the most widespread contaminants, produced worldwide, are derived from the mining, storage, and combustion of coal for energy. Fly ash, for example, is a by-product of coal combustion. It contains compounds such as arsenic, selenium, cadmium, and strontium and is a recognized source of ground and surface water contamination. Here, we experimentally investigated the impacts of short-term fly ash exposure on the skin microbiome of spring peepers, one of many species of amphibian that sometimes breed in open fly ash disposal ponds. This research provides a look into the potential impacts of fly ash on an animal's microbiome and suggests important future directions for research on the effects of environmental contaminants on the microbiome.
Assuntos
Anuros , Bactérias/classificação , Bactérias/genética , Biota/efeitos dos fármacos , Poluentes Ambientais/metabolismo , Pele/efeitos dos fármacos , Animais , Análise por Conglomerados , Carvão Mineral , DNA Ribossômico/química , DNA Ribossômico/genética , Filogenia , RNA Ribossômico 16S/genética , Análise de Sequência de DNARESUMO
Currently, it is estimated that only 0.001% to 15% of bacteria in any given system can be cultured by use of commonly used techniques and media, yet culturing is critically important for investigations of bacterial function. Despite this situation, few studies have attempted to link culture-dependent and culture-independent data for a single system to better understand which members of the microbial community are readily cultured. In amphibians, some cutaneous bacterial symbionts can inhibit establishment and growth of the fungal pathogen Batrachochytrium dendrobatidis, and thus there is great interest in using these symbionts as probiotics for the conservation of amphibians threatened by B. dendrobatidis. The present study examined the portion of the culture-independent bacterial community (based on Illumina amplicon sequencing of the 16S rRNA gene) that was cultured with R2A low-nutrient agar and whether the cultured bacteria represented rare or dominant members of the community in the following four amphibian species: bullfrogs (Lithobates catesbeianus), eastern newts (Notophthalmus viridescens), spring peepers (Pseudacris crucifer), and American toads (Anaxyrus americanus). To determine which percentage of the community was cultured, we clustered Illumina sequences at 97% similarity, using the culture sequences as a reference database. For each amphibian species, we cultured, on average, 0.59% to 1.12% of each individual's bacterial community. However, the average percentage of bacteria that were culturable for each amphibian species was higher, with averages ranging from 2.81% to 7.47%. Furthermore, most of the dominant operational taxonomic units (OTUs), families, and phyla were represented in our cultures. These results open up new research avenues for understanding the functional roles of these dominant bacteria in host health.
Assuntos
Anuros/microbiologia , Bactérias/crescimento & desenvolvimento , Bactérias/isolamento & purificação , Pele/microbiologia , Animais , Anuros/classificação , Bactérias/classificação , Bactérias/genética , Biodiversidade , Dados de Sequência Molecular , FilogeniaRESUMO
Greater knowledge of how host-microbiome interactions vary with anthropogenic environmental change and influence pathogenic infections is needed to better understand stress-mediated disease outcomes. We investigated how increasing salinization in freshwaters (e.g. due to road de-icing salt runoff) and associated increases in growth of nutritional algae influenced gut bacterial assembly, host physiology and responses to ranavirus exposure in larval wood frogs (Rana sylvatica). Elevating salinity and supplementing a basic larval diet with algae increased larval growth and also increased ranavirus loads. However, larvae given algae did not exhibit elevated kidney corticosterone levels, accelerated development or weight loss post-infection, whereas larvae fed a basic diet did. Thus, algal supplementation reversed a potentially maladaptive stress response to infection observed in prior studies in this system. Algae supplementation also reduced gut bacterial diversity. Notably, we observed higher relative abundances of Firmicutes in treatments with algae-a pattern consistent with increased growth and fat deposition in mammals-that may contribute to the diminished stress responses to infection via regulation of host metabolism and endocrine function. Our study informs mechanistic hypotheses about the role of microbiome mediation of host responses to infection that can be tested in future experiments in this host-pathogen system. This article is part of the theme issue 'Amphibian immunity: stress, disease and ecoimmunology'.
Assuntos
Microbioma Gastrointestinal , Microbiota , Ranavirus , Animais , Salinidade , Dieta , Larva , MamíferosRESUMO
Spatial contagion occurs when the perceived suitability of neighbouring habitat patches is not independent. As a result, organisms may colonize less-preferred patches near preferred patches and avoid preferred patches near non-preferred patches. Spatial contagion may thus alter colonization dynamics as well as the type and frequency of post-colonization interactions. Studies have only recently documented the phenomenon of spatial contagion and begun to examine its consequences for local recruitment. Here, we test for spatial contagion in the colonization of arboreal egg clutches of red-eyed treefrogs by a frogfly and examine the consequences of contagion for fly recruitment. In laboratory choice experiments, flies oviposit almost exclusively on clutches containing dead frog eggs. In nature, however, flies often colonize intact clutches without dead eggs. Consistent with predictions of contagion-induced oviposition, we found that flies more frequently colonize intact clutches near damaged clutches and rarely colonize intact clutches near other intact clutches. Moreover, contagion appears to benefit flies. Flies survived equally well and suffered less parasitism on clutches lacking dead eggs. This study demonstrates how reward contagion can influence colonization dynamics and suggests that colonization patterns caused by contagion may have important population- and community-level consequences.
Assuntos
Anuros/fisiologia , Ecossistema , Animais , Comportamento Animal , Dípteros/fisiologia , Modelos Lineares , Modelos Estatísticos , Oviposição , Dinâmica Populacional , Comportamento Predatório , Territorialidade , ÁrvoresRESUMO
Community ecology aims to understand how species interactions shape species diversity and abundance. Although less studied than predatory or competitive interactions, facilitative interactions can be important in communities associated with ephemeral microhabitats. Successful recruitment from these habitats requires species to rapidly colonize, develop, and disperse during brief periods of habitat suitability. Interactions between organisms, including processing chain interactions whereby initial consumers alter resources in ways that improve their quality for subsequent consumers, could aid these processes. The terrestrial egg masses of red-eyed treefrogs (Agalychnis callidryas) are a resource for predatory wasps (Agelaia spp., Polybia rejecta) and a microhabitat and resource for saprovoric and pathogenic fungi and saprovoric flies (Megaselia spp., Psychoda savaiiensis). We investigate how interactions with wasps might facilitate fly and fungal colonization of and survival on frog egg masses. Our results indicate that wasps facilitate fungal colonization, whereas flies appear not to, and that both wasps and fungi generate frog egg carrion that attracts saprovoric flies to oviposit and increases the survival of fly larvae. While studies of colonization order often focus on inhibition by early colonizers of subsequent arrivals, this study demonstrates how early colonizers can facilitate the establishment of later ones, by modifying resources in ways that promote the location of and survival in habitat patches. This research draws attention to the diversity of interactions that can occur within ephemeral communities and emphasizes the role that positive interactions may play. Processing chain interactions may be a generally important mechanism increasing the diversity of local communities, including very ephemeral ones.
Assuntos
Anuros , Biota , Dípteros/fisiologia , Ecossistema , Fungos , Modelos Biológicos , Vespas/fisiologia , Animais , Óvulo/microbiologia , Óvulo/parasitologia , Panamá , Densidade Demográfica , Comportamento Predatório/fisiologia , Análise de SobrevidaRESUMO
Variation in the structure of host-associated microbial communities has been correlated with the occurrence and severity of disease in diverse host taxa, suggesting a key role of the microbiome in pathogen defense. However, whether these correlations are typically a cause or consequence of pathogen exposure remains an open question, and requires experimental approaches to disentangle. In amphibians, infection by the fungal pathogen Batrachochytrium dendrobatidis (Bd) alters the skin microbial community in some host species, whereas in other species, the skin microbial community appears to mediate infection dynamics. In this study, we completed experimental Bd exposures in three species of tropical frogs (Agalychnis callidryas, Dendropsophus ebraccatus,andCraugastor fitzingeri) that were sympatric with Bd at the time of the study. For all three species, we identified key taxa within the skin bacterial communities that were linked to Bd infection dynamics. We also measured higher Bd infection intensities in D. ebraccatus and C. fitzingeri that were associated with higher mortality in C. fitzingeri. Our findings indicate that microbially mediated pathogen resistance is a complex trait that can vary within and across host species, and suggest that symbiont communities that have experienced prior selection for defensive microbes may be less likely to be disturbed by pathogen exposure.
Assuntos
Quitridiomicetos , Microbiota , Anfíbios/microbiologia , Animais , Bactérias/genética , Resistência à Doença , Pele/microbiologiaRESUMO
Chytridiomycosis, caused by the pathogen Batrachochytrium dendrobatidis (Bd), has led to population declines and extinctions of frog species around the world. While it is known that symbiotic skin bacteria can play a protective role against pathogens, it is not known how these defensive bacteria are integrated into the bacterial community on amphibian skin. In this study, we used 16S rRNA gene amplicon sequencing, culturing and Bd inhibition bioassays to characterize the communities of skin bacteria on three Neotropical frog species that persist in a Bd-infected area in Panama and determined the abundance and integration of anti-Bd bacteria into the community. We found that the two treefrog species had a similar bacterial community structure, which differed from the more diverse community found on the terrestrial frog. Co-occurrence networks also revealed differences between frog species such that the treefrogs had a significantly higher number of culturable Bd-inhibitory OTUs with high centrality scores compared with the terrestrial frog. We found that culture-dependent OTUs captured between 21 and 39% of the total relative abundance revealed in culture-independent communities. Our results suggest different ecological strategies occurring within skin antifungal communities on host species that have not succumbed to Bd infections in the wild.
Assuntos
Antifúngicos/farmacologia , Anuros/microbiologia , Bactérias/isolamento & purificação , Quitridiomicetos/fisiologia , Especificidade de Hospedeiro , Microbiota , Micoses/veterinária , Animais , Bactérias/química , Bactérias/genética , Quitridiomicetos/efeitos dos fármacos , Micoses/microbiologia , Micoses/prevenção & controle , RNA Ribossômico 16S/genética , Pele/microbiologia , SimbioseRESUMO
The amphibian skin microbiome has been the focus of recent studies aiming to better understand the role of these microbial symbionts in host defense against disease. However, host-associated microbial communities are complex and dynamic, and changes in their composition and structure can influence their function. Understanding temporal variation of bacterial communities on amphibian skin is critical for establishing baselines from which to improve the development of mitigation techniques based on probiotic therapy and provides long-term host protection in a changing environment. Here, we investigated whether microbial communities on amphibian skin change over time at a single site. To examine this, we collected skin swabs from two pond-breeding species of treefrogs, Agalychnis callidryas and Dendropsophus ebraccatus, over 4 years at a single lowland tropical pond in Panamá. Relative abundance of operational taxonomic units (OTUs) based on 16S rRNA gene amplicon sequencing was used to determine bacterial community diversity on the skin of both treefrog species. We found significant variation in bacterial community structure across long and short-term time scales. Skin bacterial communities differed across years on both species and between seasons and sampling days only in D. ebraccatus. Importantly, bacterial community structures across days were as variable as year level comparisons. The differences in bacterial community were driven primarily by differences in relative abundance of key OTUs and explained by rainfall at the time of sampling. These findings suggest that skin-associated microbiomes are highly variable across time, and that for tropical lowland sites, rainfall is a good predictor of variability. However, more research is necessary to elucidate the significance of temporal variation in bacterial skin communities and their maintenance for amphibian conservation efforts.
RESUMO
Animal-associated microbiomes are integral to host health, yet key biotic and abiotic factors that shape host-associated microbial communities at the global scale remain poorly understood. We investigated global patterns in amphibian skin bacterial communities, incorporating samples from 2,349 individuals representing 205 amphibian species across a broad biogeographic range. We analysed how biotic and abiotic factors correlate with skin microbial communities using multiple statistical approaches. Global amphibian skin bacterial richness was consistently correlated with temperature-associated factors. We found more diverse skin microbiomes in environments with colder winters and less stable thermal conditions compared with environments with warm winters and less annual temperature variation. We used bioinformatically predicted bacterial growth rates, dormancy genes and antibiotic synthesis genes, as well as inferred bacterial thermal growth optima to propose mechanistic hypotheses that may explain the observed patterns. We conclude that temporal and spatial characteristics of the host's macro-environment mediate microbial diversity.
Assuntos
Anuros/microbiologia , Clima , Microbiota , Urodelos/microbiologia , Animais , Bactérias/classificação , Fenômenos Fisiológicos Bacterianos , Pele/microbiologiaRESUMO
Skin symbiotic bacteria on amphibians can play a role in protecting their host against pathogens. Chytridiomycosis, the disease caused by Batrachochytrium dendrobatidis, Bd, has caused dramatic population declines and extinctions of amphibians worldwide. Anti-Bd bacteria from amphibian skin have been cultured, and skin bacterial communities have been described through 16S rRNA gene amplicon sequencing. Here, we present a shotgun metagenomic analysis of skin bacterial communities from a Neotropical frog, Craugastor fitzingeri. We sequenced the metagenome of six frogs from two different sites in Panamá: three frogs from Soberanía (Sob), a Bd-endemic site, and three frogs from Serranía del Sapo (Sapo), a Bd-naïve site. We described the taxonomic composition of skin microbiomes and found that Pseudomonas was a major component of these communities. We also identified that Sob communities were enriched in Actinobacteria while Sapo communities were enriched in Gammaproteobacteria. We described gene abundances within the main functional classes and found genes enriched either in Sapo or Sob. We then focused our study on five functional classes of genes: biosynthesis of secondary metabolites, metabolism of terpenoids and polyketides, membrane transport, cellular communication and antimicrobial drug resistance. These gene classes are potentially involved in bacterial communication, bacterial-host and bacterial-pathogen interactions among other functions. We found that C. fitzingeri metagenomes have a wide array of genes that code for secondary metabolites, including antibiotics and bacterial toxins, which may be involved in bacterial communication, but could also have a defensive role against pathogens. Several genes involved in bacterial communication and bacterial-host interactions, such as biofilm formation and bacterial secretion systems were found. We identified specific genes and pathways enriched at the different sites and determined that gene co-occurrence networks differed between sites. Our results suggest that skin microbiomes are composed of distinct bacterial taxa with a wide range of metabolic capabilities involved in bacterial defense and communication. Differences in taxonomic composition and pathway enrichments suggest that skin microbiomes from different sites have unique functional properties. This study strongly supports the need for shotgun metagenomic analyses to describe the functional capacities of skin microbiomes and to tease apart their role in host defense against pathogens.
RESUMO
Host-associated microbial communities are ubiquitous among animals, and serve important functions. For example, the bacterial skin microbiome of amphibians can play a role in preventing or reducing infection by the amphibian chytrid fungus, Batrachochytrium dendrobatidis. Evidence suggests that environmental bacteria likely serve as a source pool for at least some of the members of the amphibian skin bacterial community, underscoring the potential for local environmental changes to disrupt microbial community source pools that could be critical to the health of host organisms. However, few studies have assessed variation in the amphibian skin microbiome along clear environmental gradients, and so we know relatively little about how local environmental conditions influence microbiome diversity. We sampled the skin bacterial communities of Coqui frogs, Eleutherodactylus coqui (N = 77), along an elevational gradient in eastern Puerto Rico (0-875 m), with transects in two land use types: intact forest (N = 4 sites) and disturbed (N = 3 sites) forest. We found that alpha diversity (as assessed by Shannon, Simpson, and Phylogenetic Diversity indices) varied across sites, but this variation was not correlated with elevation or land use. Beta diversity (community structure), on the other hand, varied with site, elevation and land use, primarily due to changes in the relative abundance of certain bacterial OTUs (â¼species) within these communities. Importantly, although microbiome diversity varied, E. coqui maintained a common core microbiota across all sites. Thus, our findings suggest that environmental conditions can influence the composition of the skin microbiome of terrestrial amphibians, but that some aspects of the microbiome remain consistent despite environmental variation.
RESUMO
Symbiotic bacteria on amphibian skin can inhibit growth of the fungus Batrachochytrium dendrobatidis (Bd) that has caused dramatic population declines and extinctions of amphibians in the Neotropics. It remains unclear how the amphibians' skin microbiota is influenced by environmental bacterial reservoirs, host-associated factors such as susceptibility to pathogens, and pathogen presence in tropical amphibians. We sampled skin bacteria from five co-occurring frog species that differ in Bd susceptibility at one Bd-naive site, and sampled one of the non-susceptible species from Bd-endemic and Bd-naive sites in Panama. We hypothesized that skin bacterial communities (1) would be distinct from the surrounding environment regardless of the host habitat, (2) would differ between Bd susceptible and non-susceptible species and (3) would differ on hosts in Bd-naive and Bd-endemic sites. We found that skin bacterial communities were enriched in bacterial taxa that had low relative abundances in the environment. Non-susceptible species had very similar skin bacterial communities that were enriched in particular taxa such as the genera Pseudomonas and Acinetobacter. Bacterial communities of Craugastor fitzingeri in Bd-endemic sites were less diverse than in the naive site, and differences in community structure across sites were explained by changes in relative abundance of specific bacterial taxa. Our results indicate that skin microbial structure was associated with host susceptibility to Bd and might be associated to the history of Bd presence at different sites.
Assuntos
Anuros/microbiologia , Bactérias/genética , Quitridiomicetos/fisiologia , Microbiota/imunologia , Animais , Anuros/imunologia , Bactérias/classificação , Suscetibilidade a Doenças , Pele/microbiologiaRESUMO
Emerging infectious diseases in wildlife are responsible for massive population declines. In amphibians, chytridiomycosis caused by Batrachochytrium dendrobatidis, Bd, has severely affected many amphibian populations and species around the world. One promising management strategy is probiotic bioaugmentation of antifungal bacteria on amphibian skin. In vivo experimental trials using bioaugmentation strategies have had mixed results, and therefore a more informed strategy is needed to select successful probiotic candidates. Metagenomic, transcriptomic, and metabolomic methods, colloquially called "omics," are approaches that can better inform probiotic selection and optimize selection protocols. The integration of multiple omic data using bioinformatic and statistical tools and in silico models that link bacterial community structure with bacterial defensive function can allow the identification of species involved in pathogen inhibition. We recommend using 16S rRNA gene amplicon sequencing and methods such as indicator species analysis, the Kolmogorov-Smirnov Measure, and co-occurrence networks to identify bacteria that are associated with pathogen resistance in field surveys and experimental trials. In addition to 16S amplicon sequencing, we recommend approaches that give insight into symbiont function such as shotgun metagenomics, metatranscriptomics, or metabolomics to maximize the probability of finding effective probiotic candidates, which can then be isolated in culture and tested in persistence and clinical trials. An effective mitigation strategy to ameliorate chytridiomycosis and other emerging infectious diseases is necessary; the advancement of omic methods and the integration of multiple omic data provide a promising avenue toward conservation of imperiled species.
RESUMO
Vertebrates, including amphibians, host diverse symbiotic microbes that contribute to host disease resistance. Globally, and especially in montane tropical systems, many amphibian species are threatened by a chytrid fungus, Batrachochytrium dendrobatidis (Bd), that causes a lethal skin disease. Bd therefore may be a strong selective agent on the diversity and function of the microbial communities inhabiting amphibian skin. In Panamá, amphibian population declines and the spread of Bd have been tracked. In 2012, we completed a field survey in Panamá to examine frog skin microbiota in the context of Bd infection. We focused on three frog species and collected two skin swabs per frog from a total of 136 frogs across four sites that varied from west to east in the time since Bd arrival. One swab was used to assess bacterial community structure using 16S rRNA amplicon sequencing and to determine Bd infection status, and one was used to assess metabolite diversity, as the bacterial production of anti-fungal metabolites is an important disease resistance function. The skin microbiota of the three Panamanian frog species differed in OTU (operational taxonomic unit, ~bacterial species) community composition and metabolite profiles, although the pattern was less strong for the metabolites. Comparisons between frog skin bacterial communities from Panamá and the US suggest broad similarities at the phylum level, but key differences at lower taxonomic levels. In our field survey in Panamá, across all four sites, only 35 individuals (~26%) were Bd infected. There was no clustering of OTUs or metabolite profiles based on Bd infection status and no clear pattern of west-east changes in OTUs or metabolite profiles across the four sites. Overall, our field survey data suggest that different bacterial communities might be producing broadly similar sets of metabolites across frog hosts and sites. Community structure and function may not be as tightly coupled in these skin symbiont microbial systems as it is in many macro-systems.