From Skin to Gut: Understanding Microbial Diversity in Rana amurensis and R. dybowskii.

Amphibians face the threat of decline and extinction, and their health is crucially affected by the microbiota. Their health and ecological adaptability essentially depend on the diverse microbial communities that are shaped by unique host traits and environmental factors. However, there is still limited research on this topic. In this study, cutaneous (C) and gut (G) microbiota in Rana amurensis (A) and R. dybowskii (D) was analyzed through 16S amplicon sequencing. Groups AC and DC significantly differed in alpha diversity, while the gut groups (AG and DG) showed no such differences. Analyses of Bray-Curtis dissimilarity matrix and unweighted UniFrac distances showed significant differences in cutaneous microbiota between groups AC and DC, but not between groups AG and DG. Stochastic processes significantly influenced the assembly of cutaneous and gut microbiota in amphibians, with a notably higher species dispersal rate in the gut. The predominant phyla in the skin of R. amurensis and R. dybowskii were Bacteroidetes and Proteobacteria, respectively, with significant variations in Bacteroidota. Contrarily, the gut microbiota of both species was dominated by Firmicutes, Proteobacteria, and Bacteroidetes, without significant phylum-level differences. Linear discriminant analysis effect size (LEfSe) analysis identified distinct microbial enrichment in each group. Predictive analysis using phylogenetic investigation of communities by reconstruction of unobserved states 2 (PICRUSt2) revealed the significant functional pathways associated with the microbiota, which indicates their potential roles in immune system function, development, regeneration, and response to infectious diseases. This research underscores the critical impact of both host and environmental factors in shaping amphibian microbial ecosystems and emphasizes the need for further studies to explore these complex interactions for conservation efforts.

Gut dysbiosis of Rana zhenhaiensis tadpoles after lead (Pb) exposure based on integrated analysis of microbiota and gut transcriptome.

Lead (Pb) is a ubiquitously detected heavy metal pollutant in aquatic ecosystems. Previous studies focused mainly on the response of gut microbiota to Pb stress, with less emphasis on gene expression in intestine, thereby limiting the information about impacts of Pb on intestinal homeostasis in amphibians. Here, microbial community and transcriptional response of intestines in Rana zhenhaiensis tadpoles to Pb exposure were evaluated. Our results showed that 10 µg/L Pb significantly decreased bacterial diversity compared to the controls by the Simpson index. Additionally, 1000 µg/L Pb exposure resulted in a significant reduction in the abundance of Fusobacteriota phylum and Cetobacterium genus but a significant expansion in Hafnia-Obesumbacterium genus. Moreover, transcriptome analysis revealed that about 90 % of the DEGs (8458 out of 9450 DEGs) were down-regulated in 1000 µg/L Pb group, mainly including genes annotated with biological functions in fatty acid degradation, and oxidative phosphorylation, while up-regulated DEGs involved in metabolism of xenobiotics by cytochrome P450. The expression of Gsto1, Gsta5, Gstt4, and Nadph showed strong correlation with the abundance of genera Serratia, Lactococcus, and Hafnia-Obesumbacterium. The findings of this study provide important insights into understanding the influence of Pb on intestinal homeostasis in amphibians.

Co-infections of Klebsiella pneumoniae and Elizabethkingia miricola in black-spotted frogs (Pelophylax nigromaculatus).

Pelophylax nigromaculatus is a common commercial specie of frogs that generally cultured throughout China. With the application of high-density culture, P. nigromaculatus can be co-infected by two or more pathogens, which thereby induce synergistic influence on the virulence of the infection. In this study, two bacterial strains were simultaneously isolated from diseased frogs by incubating on Luria-Bertani (LB) agar. Isolates were identified as Klebsiella pneumoniae and Elizabethkingia miricola by morphological, physiological and biochemical features, as well as 16S rRNA sequencing and phylogenetic analysis. The whole genome of K. pneumoniae and E. miricola isolates consist single circular chromosome of 5,419,557 bp and 4,215,349 bp, respectively. The genomic sequence analysis further indicated that K. pneumoniae isolate conserved 172 virulent and 349 antibiotic-resistance genes, whereas E. miricola contained 24 virulent and 168 antibiotic resistance genes. In LB broth, both isolates could grow well at 0%-1% NaCl concentration and pH 5-7. Antibiotic susceptibility testing revealed that both K. pneumoniae and E. miricola were resistant to kanamycin, neomycin, ampicillin, piperacillin, carbenicillin, enrofloxacin, norfloxacin and sulfisoxazole. Histopathological studies showed that co-infection caused considerable lesions in the tissues of brain, eye, muscle, spleen, kidney and liver, including cell degeneration, necrosis, hemorrhage and inflammatory cell infiltration. The LD50 of K. pneumoniae and E. miricola isolates were 6.31 × 105 CFU/g and 3.98 × 105 CFU/g frog weight, respectively. Moreover, experimentally infected frogs exhibited quick and higher mortality under coinfection with K. pneumoniae and E. miricola than those single challenge of each bacterium. To date, no natural co-infection by these two bacteria has been reported from frogs and even amphibians. The results will not only shed light on the feature and pathogenesis of K. pneumoniae and E. miricola, but also highlight that co-infection of these two pathogen is a potential threat to black-spotted frog farming.

A Variety of Fungal Species on the Green Frogs' Skin (Pelophylax esculentus complex) in South Banat.

In the last several decades, amphibian populations have been declining worldwide. Many factors have been linked to global amphibian decline, including habitat destruction, pollution, introduced species, global environmental changes, and emerging infectious diseases. Recent studies of amphibian skin infections were mainly focused on the presence of chytridiomycosis, neglecting other members of the frogs' skin communities. The diversity pattern of fungal dwellers on the skin of green frogs (Pelophylax esculentus complex) was investigated. A total of 100 adults were sampled from three localities in South Banat (northern Serbia) over three consecutive years and detected fungal dwellers were identified using light microscopy and ITS and BenA gene sequencing. Structures belonging to fungi and fungus-like organisms including a variety of spores and different mycelia types were documented in the biofilm formed on amphibian skin, and are classified into 10 groups. In total, 42 fungal isolates were identified to species, section, or genus level. The difference in mycobiota composition between sampling points (localities and green frog taxa) was documented. The highest number of fungal structures and isolates was recorded on the hybrid taxon P. esculentus and locality Stevanove ravnice. Parental species showed a markedly lower diversity than the hybrid taxon and were more similar in diversity patterns and were placed in the same homogenous group. The locality Stevanove ravnice exhibited more pronounced differences in diversity pattern than the other two localities and was placed in a distinct and separate homogenous group. Among the fungal isolates, the highest isolation frequency was documented for Alternaria alternata, Aspergillus sp. sect. Nigri, Epicoccum nigrum, Fusarium proliferatum, and Trichoderma atroviride. Among the documented species, dematiaceous fungi, causative agents of chromomycosis in amphibians, were also recorded in this research with high isolation frequency. Also, some rare fungal species such as Quambalaria cyanescens and Pseudoteniolina globosa are documented for the first time in this research as microbial inhabitants of amphibian skin.

Habitat Disturbance Linked with Host Microbiome Dispersion and Bd Dynamics in Temperate Amphibians.

Anthropogenic habitat disturbances can dramatically alter ecological community interactions, including host-pathogen dynamics. Recent work has highlighted the potential for habitat disturbances to alter host-associated microbial communities, but the associations between anthropogenic disturbance, host microbiomes, and pathogens are unresolved. Amphibian skin microbial communities are particularly responsive to factors like temperature, physiochemistry, pathogen infection, and environmental microbial reservoirs. Through a field survey on wild populations of Acris crepitans (Hylidae) and Lithobates catesbeianus (Ranidae), we assessed the effects of habitat disturbance and connectivity on environmental bacterial reservoirs, Batrachochytrium dendrobatidis (Bd) infection, and skin microbiome composition. We found higher measures of microbiome dispersion (a measure of community variability) in A. crepitans from more disturbed ponds, supporting the hypothesis that disturbance increases stochasticity in biological communities. We also found that habitat disturbance limited microbiome similarity between locations for both species, suggesting greater isolation of bacterial assemblages in more disturbed areas. Higher disturbance was associated with lower Bd prevalence for A. crepitans, which could signify suboptimal microclimates for Bd in disturbed habitats. Combined, our findings show that reduced microbiome stability stemming from habitat disturbance could compromise population health, even in the absence of pathogenic infection.

Composition of the North American Wood Frog (Rana sylvatica) Bacterial Skin Microbiome and Seasonal Variation in Community Structure.

While a number of amphibian skin microbiomes have been characterized, it is unclear how these communities might vary in response to seasonal changes in the environment and the corresponding behaviors that many amphibians exhibit. Given recent studies demonstrating the importance of the skin microbiome in frog innate immune defense against pathogens, investigating how changes in the environment impact the microbial species present will provide a better understanding of conditions that may alter host susceptibility to pathogens in their environment. We sampled the bacterial skin microbiome of North American wood frogs (Rana sylvatica) from two breeding ponds in the spring, along with the bacterial community present in their vernal breeding pools, and frogs from the nearby forest floor in the summer and fall to determine whether community composition differs by sex, vernal pond site, or temporally across season (spring, summer, fall). Taxon relative abundance data reveals a profile of bacterial phyla similar to those previously described on anuran skin, with Proteobacteria, Bacteroidetes, and Actinobacteria dominating the wood frog skin microbiome. Our results indicate that sex had no significant effect on skin microbiota diversity; however, this may be due to our limited female frog sample size. Vernal pool site had a small but significant effect on skin microbiota, but skin-associated communities were more similar to each other than to the communities observed in the frogs' respective pond water. Across seasons, diversity analyses suggest that there are significant differences between the bacterial skin microbiome of frogs from spring and summer/fall groups while the average α-diversity per frog remained consistent. These results illustrate seasonal variation in wood frog skin microbiome structure and highlight the importance of considering temporal trends in an amphibian microbiome, particularly for species whose life history requires recurrent shifts in habitat and behavior.

Changes in the gut microbiota diversity of brown frogs (Rana dybowskii) after an antibiotic bath.

BACKGROUND: Captive amphibians frequently receive antibiotic baths to control bacterial diseases. The potential collateral effect of these antibiotics on the microbiota of frogs is largely unknown. To date, studies have mainly relied on oral administration to examine the effects of antibiotics on the gut microbiota; in contrast, little is known regarding the effects of bath-applied antibiotics on the gut microbiota. The gut microbiota compositions of the gentamicin, recovery, and control groups were compared by Illumina high-throughput sequencing, and the functional profiles were analysed using Phylogenetic Investigation of Communities by Reconstruction of Unobserved States (PICRUSt). Furthermore, the relationship between the structure and predicted functional composition of the gut microbiota was determined. RESULTS: The alpha diversity indices were significantly reduced by the gentamicin bath, illustrating that this treatment significantly changed the composition of the gut microbiota. After 7 days, the gut microbiota of the recovery group was not significantly different from that of the gentamicin group. Forty-four indicator taxa were selected at the genus level, comprising 42 indicators representing the control group and 2 indicators representing the gentamicin and recovery groups. Potential pathogenic bacteria of the genera Aeromonas, Citrobacter, and Chryseobacterium were significantly depleted after the gentamicin bath. There was no significant positive association between the community composition and functional composition of the gut microbiota in the gentamicin or control frogs, indicating that the functional redundancy of the gut bacterial community was high. CONCLUSIONS: Gentamicin significantly changed the structure of the gut microbiota of R. dybowskii, and the gut microbiota exhibited weak resilience. However, the gentamicin bath did not change the functional composition of the gut microbiota of R. dybowskii, and there was no significant correlation between the structural composition and the functional composition of the gut microbiota.

Habitat disturbance influences the skin microbiome of a rediscovered neotropical-montane frog.

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.

Effects of Seasonal Hibernation on the Similarities Between the Skin Microbiota and Gut Microbiota of an Amphibian (Rana dybowskii).

Both the gut and skin microbiotas have important functions for amphibians. The gut microbiota plays an important role in both the health and evolution of the host species, whereas the role of skin microbiota in disease resistance is particularly important for amphibians. Many studies have examined the effects of environmental factors on the skin and gut microbiotas, but no study has yet explored the similarities between the skin and gut microbiotas. In this study, the gut and skin microbiotas of Rana dybowskii in summer and winter were investigated via high-throughput Illumina sequencing. The results showed that the alpha diversity of gut and skin microbiotas decreased significantly from summer to winter. In both seasons, the microbial composition and structure differed significantly between the gut and skin, and the similarities between these microbiotas differed between seasons. The pairwise distances between the gut and skin microbiotas were greater in winter than in summer. The ratio of core OTUs and shared OTUs to the sum of the OTUs in the gut and skin microbiotas in summer was significantly higher than that in winter. The similarities between the gut and skin microbiotas are important for understanding amphibian ecology and life history.

Probiotics Modulate a Novel Amphibian Skin Defense Peptide That Is Antifungal and Facilitates Growth of Antifungal Bacteria.

Probiotics can ameliorate diseases of humans and wildlife, but the mechanisms remain unclear. Host responses to interventions that change their microbiota are largely uncharacterized. We applied a consortium of four natural antifungal bacteria to the skin of endangered Sierra Nevada yellow-legged frogs, Rana sierrae, before experimental exposure to the pathogenic fungus Batrachochytrium dendrobatidis (Bd). The probiotic microbes did not persist, nor did they protect hosts, and skin peptide sampling indicated immune modulation. We characterized a novel skin defense peptide brevinin-1Ma (FLPILAGLAANLVPKLICSITKKC) that was downregulated by the probiotic treatment. Brevinin-1Ma was tested against a range of amphibian skin cultures and found to inhibit growth of fungal pathogens Bd and B. salamandrivorans, but enhanced the growth of probiotic bacteria including Janthinobacterium lividum, Chryseobacterium ureilyticum, Serratia grimesii, and Pseudomonas sp. While commonly thought of as antimicrobial peptides, here brevinin-1Ma showed promicrobial function, facilitating microbial growth. Thus, skin exposure to probiotic bacterial cultures induced a shift in skin defense peptide profiles that appeared to act as an immune response functioning to regulate the microbiome. In addition to direct microbial antagonism, probiotic-host interactions may be a critical mechanism affecting disease resistance.

Out in the cold and sick: low temperatures and fungal infections impair a frog's skin defenses.

Amphibians worldwide continue to battle an emerging infectious disease, chytridiomycosis, caused by Batrachochytrium dendrobatidis (Bd). Southern leopard frogs, Rana sphenocephala, are known to become infected with this pathogen, yet they are considered 'of least concern' for declines due to chytridiomycosis. Previous studies have shown that R. sphenocephala secretes four antimicrobial peptides (AMPs) onto their skin which may play an important role in limiting susceptibility to chytridiomycosis. Here, we examined (1) the effects of temperature and AMP depletion on infections with Bd and (2) the effects of temperature and Bd infection on the capacity to secrete AMPs in juvenile leopard frogs. Pathogen burden and mortality were greater in frogs exposed to Bd at low temperature but did not increase following monthly AMP depletion. Both low temperature and Bd exposure reduced the capacity of juvenile frogs to restore peptides after monthly depletions. Frogs held at 14°C were poorly able to restore peptides in comparison with those at 26°C. Frogs held at 26°C were better able to restore their peptides, but when exposed to Bd, this capacity was significantly reduced. These results strongly support the hypothesis that both colder temperatures and Bd infection impair the capacity of juvenile frogs to produce and secrete AMPs, an important component of their innate defense against chytrid fungi and other pathogens. Thus, in the face of unpredictable climate changes and enzootic pathogens, assessments of disease risk should consider the potential for effects of environmental variation and pathogen exposure on the quality of host defenses.

Bacterial Community Analysis on the Skin of Odorrana grahami and Proposal of Comamonas aquatica subsp. aquatica subsp. nov. and Comamonas aquatica subsp. rana subsp. nov.

Several studies indicated that Odorrana grahami (O. grahami) skin contains abundant antimicrobial peptides, and the skin was recognized as hostile habitat for microorganisms. In this study, the microbial community was evaluated by 16S rRNA gene sequencing, and two associated bacterial isolates were obtained and characterized from the skin of O. grahami. Sixteen bacterial genera were identified from the O. grahami skin by uncultured clone sequences. The dominant groups were Comamonas, Bacillus and Morganella, and the genus Comamonas was the most abundant group (41.7% of the total) of the community. Fortunately, strains CW-25T and CW-518 belonging to genus Comamonas were isolated by plating dilutions. The polyphasic taxonomy results indicated that strain CW-25T was a member of Comamonas aquatica, it showed much higher antimicrobials resistance than the closest C. aquatica strains of LMG 2370T, LMG5937 and LMG 6112 isolated from freshwater. Based on the polyphasic taxonomic studies and antimicrobials resistance characteristics, two subspecies of Comamonas aquatica subsp. aquatica nov. and Comamonas aquatica subsp. rana nov. were proposed. The super-antimicrobial resistance endows the strains of Comamonas aquatica subsp. rana inhabit the O. grahami skin, and the primary defense of O. grahami might be composed by the antimicrobial peptides and the native bacteria.

Urea hydrolysis by gut bacteria in a hibernating frog: evidence for urea-nitrogen recycling in Amphibia.

Gut bacteria that produce urease, the enzyme hydrolysing urea, contribute to nitrogen balance in diverse vertebrates, although the presence of this system of urea-nitrogen recycling in Amphibia is as yet unknown. Our studies of the wood frog (Rana sylvatica), a terrestrial species that accrues urea in winter, documented robust urease activity by enteric symbionts and hence potential to recoup nitrogen from the urea it produces. Ureolytic capacity in hibernating (non-feeding) frogs, whose guts hosted an approximately 33% smaller bacterial population, exceeded that of active (feeding) frogs, possibly due to an inductive effect of high urea on urease expression and/or remodelling of the microbial community. Furthermore, experimentally augmenting the host's plasma urea increased bacterial urease activity. Bacterial inventories constructed using 16S rRNA sequencing revealed that the assemblages hosted by hibernating and active frogs were equally diverse but markedly differed in community membership and structure. Hibernating frogs hosted a greater relative abundance and richer diversity of genera that possess urease-encoding genes and/or have member taxa that reportedly hydrolyse urea. Bacterial hydrolysis of host-synthesized urea probably permits conservation and repurposing of valuable nitrogen not only in hibernating R. sylvatica but, given urea's universal role in amphibian osmoregulation, also in virtually all Amphibia.

First isolation and characterization of the pathogenic Aeromonas veronii bv. veronii associated with ulcerative syndrome in the indigenous Pelophylax ridibundus of Al-Ahsaa, Saudi Arabia.

Virulent microbial pathogen infections are the main cause for amphibian decline worldwide. In the present study, a bacterial strain named RDL-2, which was isolated from the skin of infected Pelophylax ridibundus larvae, was cultured and then phenotypically and biochemically characterized using scanning electron microscopic observations and the API20E strip and Biolog Gen III MicroPlate system. The 16S rRNA gene sequence of this strain was also obtained and used in strain identification and phylogenetic analysis. Healthy P. ridibundus larvae were challenged with RDL-2 and monitored to determine clinical signs consistent with the disease. Strain RDL-2 was identified as Aeromonas veronii based on its phenotypic and biochemical characteristics and on 16S rRNA gene sequence and phylogenetic analysis: RDL-2 was gram-negative, rod-shaped, and reacted positively for 69 (73%) of the 94 traits analysed; its 16S rRNA gene sequence displayed the highest homology to that of A. veronii bv. veronii (99.9%). Koch's postulates were fulfilled confirming that A. veronii is the causal agent of ulcerative syndrome. Thus, this study is the first to report A. veronii as a marsh frog pathogen in the Al-Ahsaa region. The aetiology of A. veronii as a potential poikilothermic pathogen shown here will expedite the development of diagnostic tests and methods for eradicating ulcerative syndrome.

High juvenile mortality in amphibians during overwintering related to fungal pathogen exposure.

The result of pathogen exposures may depend upon trade-offs in energetic demands for immune responses against host growth and survival. Environmental conditions may influence these trade-offs by affecting host size, or trade-offs may change across seasons, altering impacts of pathogens. We exposed northern leopard frog Lithobates pipiens tadpoles to different larval environments (low leaf litter, high density of conspecifics, atrazine, caged fish, or controls) that influenced size at metamorphosis. Subsequently, we exposed metamorphs to Batrachochytrium dendrobatidis (Bd), a fungal pathogen, just after metamorphosis and/or prior to overwintering 12 wk later. Bd exposure dramatically reduced survival during overwintering, with the strongest effects when hosts were exposed at both time points. Larval environments resulted in differences in host size. Those exposed to caged fish were 2.5 times larger than the smallest (those exposed to high density of conspecifics), but larval environment did not influence Bd effects on growth and survival. The largest frogs exposed to caged fish had greater survival through overwintering, but in the absence of Bd. We built stage-structured models to evaluate if overwinter mortality from Bd is capable of having effects on host populations. Our models suggest that Bd exposure after metamorphosis or before overwintering can reduce population growth rates. Our study demonstrates that hosts suffer little effects of Bd exposures following metamorphosis and that small body size did not hamper growth and survival. Instead, we provide evidence that winter mortality from Bd exposure is capable of reducing population sizes, providing a plausible mechanism for amphibian declines in temperate regions.

Amphibian chytrid infection is influenced by rainfall seasonality and water availability.

Amphibians suffer from a number of factors that make them the most threatened group of vertebrates. One threat is the fungal disease chytridiomycosis caused by the emerging pathogen Batrachochytrium dendrobatidis (Bd), which has rapidly spread and caused the loss of massive amphibian biodiversity worldwide. Recently, Bd was associated with a few amphibian population declines and extinctions in some areas of the Brazilian Atlantic Forest. However, the mechanisms underlying such declines are not fully understood. Therefore, it is essential to improve our knowledge of abiotic factors that can possibly influence Bd prevalence and chytridiomycosis disease severity. Herein we tested the hypothesis that water availability (such as in perennial streams, where Bd is frequently present in larvae) and rainfall would increase the prevalence of Bd. To test this, we sampled frogs from 6 transects with different numbers of perennial waterbodies, and we report that the more water available in the area, the higher the probability of Bd infection on anurans. Seasonality also influenced both the Bd prevalence in the area and the intensity of infection in infected frogs. However, Bd prevalence was higher during the rainy months whereas the infection burden was lower. We suggest that Bd is likely spread during the summer, when most anuran species gather near the water for spawning and when rainfall overfills ephemeral wetlands. On the other hand, during the drier months, a higher infection burden may be explained by increased disease susceptibility.

Pathogenic Elizabethkingia miricola Infection in Cultured Black-Spotted Frogs, China, 2016.

Multiregional outbreaks of meningitis-like disease caused by Elizabethkingia miricola were confirmed in black-spotted frog farms in China in 2016. Whole-genome sequencing revealed that this amphibian E. miricola strain is closely related to human clinical isolates. Our findings indicate that E. miricola can be epizootic and may pose a threat to humans.

Epidemic and endemic pathogen dynamics correspond to distinct host population microbiomes at a landscape scale.

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.

Greater Species Richness of Bacterial Skin Symbionts Better Suppresses the Amphibian Fungal Pathogen Batrachochytrium Dendrobatidis.

The symbiotic microbes that grow in and on many organisms can play important roles in protecting their hosts from pathogen infection. While species diversity has been shown to influence community function in many other natural systems, the question of how species diversity of host-associated symbiotic microbes contributes to pathogen resistance is just beginning to be explored. Understanding diversity effects on pathogen resistance could be particularly helpful in combating the fungal pathogen Batrachochytrium dendrobatidis (Bd) which has caused dramatic population declines in many amphibian species and is a major concern for amphibian conservation. Our study investigates the ability of host-associated bacteria to inhibit the proliferation of Bd when grown in experimentally assembled biofilm communities that differ in species number and composition. Six bacterial species isolated from the skin of Cascades frogs (Rana cascadae) were used to assemble bacterial biofilm communities containing 1, 2, 3, or all 6 bacterial species. Biofilm communities were grown with Bd for 7 days following inoculation. More speciose bacterial communities reduced Bd abundance more effectively. This relationship between bacterial species richness and Bd suppression appeared to be driven by dominance effects-the bacterial species that were most effective at inhibiting Bd dominated multi-species communities-and complementarity: multi-species communities inhibited Bd growth more than monocultures of constituent species. These results underscore the notion that pathogen resistance is an emergent property of microbial communities, a consideration that should be taken into account when designing probiotic treatments to reduce the impacts of infectious disease.

Transcriptome profiling and digital gene expression analysis of the skin of Dybowski's frog (Rana dybowskii) exposed to Aeromonas hydrophila.

Recently, populations of Rana dybowskii, an important amphibian species in Northeast China, have decreased, mainly owing to the disease caused by Aeromonas hydrophila. However, effective control methods have not yet been developed. In order to explore the immune responses of R. dybowskii upon exposure to A. hydrophila infection, Illumina high-throughput transcriptome sequencing and digital gene expression (DGE) technology were employed to investigate transcriptomic changes in the skin of R. dybowskii exposed to A. hydrophila. In this work, a total of 26,244,446 transcriptome sequencing reads were obtained and assembled into 109,089 unique unigenes using de novo assembly, and a total of 37,105 unigenes (34.0%) were functionally annotated against the non-redundant (Nr), Swiss-Prot, Cluster of Orthologous Groups of Proteins (COG), Kyoto Encyclopedia of Genes and Genomes (KEGG), and Gene Ontology (GO) databases. Gene expression changes in the skin tissue of R. dybowskii exposed to A. hydrophila were investigated by a tag-based DGE system, and a total of 1435 significantly differentially expressed genes were identified, including 460 that were up-regulated and 975 that were down-regulated, indicating a large change in the host transcriptome profile exposed to A. hydrophila. Among these, 478 genes were associated with immune-relevant pathways, metabolic pathways, cellular components, growth, migration, and muscle and hormone signaling pathways. We confirmed the differential expression of 106 immune-relevant genes associated with innate and adaptive immune responses. Our data provide a fairly comprehensive molecular biology background for the deeper understanding of the amphibian immune system following A. hydrophila infection.