Antifungal Bacteria on Woodland Salamander Skin Exhibit High Taxonomic Diversity and Geographic Variability

ABSTRACT Diverse bacteria inhabit amphibian skin; some of those bacteria inhibit growth of the fungal pathogen Batrachochytrium dendrobatidis. Yet there has been no systematic survey of anti-B. dendrobatidis bacteria across localities, species, and elevations. This is important given geographic and taxonomic variations in amphibian susceptibility to B. dendrobatidis. Our collection sites were at locations within the Appalachian Mountains where previous sampling had indicated low B. dendrobatidis prevalence. We determined the numbers and identities of anti-B. dendrobatidis bacteria on 61 Plethodon salamanders (37 P. cinereus, 15 P. glutinosus, 9 P. cylindraceus) via culturing methods and 16S rRNA gene sequencing. We sampled co-occurring species at three localities and sampled P. cinereus along an elevational gradient (700 to 1,000 meters above sea level [masl]) at one locality. We identified 50 anti-B. dendrobatidis bacterial operational taxonomic units (OTUs) and found that the degree of B. dendrobatidis inhibition was not correlated with relatedness. Five anti-B. dendrobatidis bacterial strains occurred on multiple amphibian species at multiple localities, but none were shared among all species and localities. The prevalence of anti-B. dendrobatidis bacteria was higher at Shenandoah National Park (NP), VA, with 96% (25/26) of salamanders hosting at least one anti-B. dendrobatidis bacterial species compared to 50% (7/14) at Catoctin Mountain Park (MP), MD, and 38% (8/21) at Mt. Rogers National Recreation Area (NRA), VA. At the individual level, salamanders at Shenandoah NP had more anti-B. dendrobatidis bacteria per individual (μ = 3.3) than those at Catoctin MP (μ = 0.8) and at Mt. Rogers NRA (μ = 0.4). All salamanders tested negative for B. dendrobatidis. Anti-B. dendrobatidis bacterial species are diverse in central Appalachian Plethodon salamanders, and their distribution varied geographically. The antifungal bacterial species that we identified may play a protective role for these salamanders. IMPORTANCE Amphibians harbor skin bacteria that can kill an amphibian fungal pathogen, Batrachochytrium dendrobatidis. Some amphibians die from B. dendrobatidis infection, whereas others do not. The bacteria that can kill B. dendrobatidis, called anti-B. dendrobatidis bacteria, are thought to influence the B. dendrobatidis infection outcome for the amphibian. Yet how anti-B. dendrobatidis bacterial species vary among amphibian species and populations is unknown. We determined the distribution of anti-B. dendrobatidis bacterial species among three salamander species (n = 61) sampled at three localities. We identified 50 unique anti-B. dendrobatidis bacterial species and found that all of the tested salamanders were negative for B. dendrobatidis. Five anti-B. dendrobatidis bacterial species were commonly detected, suggesting a stable, functional association with these salamanders. The number of anti-B. dendrobatidis bacteria per individual varied among localities but not among co-occurring salamander species, demonstrating that environment is more influential than host factors in structuring the anti-B. dendrobatidis bacterial community. These anti-B. dendrobatidis bacteria may serve a protective function for their salamander hosts.

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Most of the Dominant Members of Amphibian Skin Bacterial Communities Can Be Readily Cultured

Applied and Environmental Microbiology ◽

10.1128/aem.01486-15 ◽

2015 ◽

Vol 81 (19) ◽

pp. 6589-6600 ◽

Cited By ~ 25

Author(s):

Jenifer B. Walke ◽

Matthew H. Becker ◽

Myra C. Hughey ◽

Meredith C. Swartwout ◽

Roderick V. Jensen ◽

...

Keyword(s):

Bacterial Community ◽

Batrachochytrium Dendrobatidis ◽

Amplicon Sequencing ◽

Reference Database ◽

Rrna Gene ◽

Amphibian Skin ◽

Amphibian Species ◽

Content Type ◽

Average Percentage ◽

Culture Independent

ABSTRACTCurrently, 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 pathogenBatrachochytrium dendrobatidis, and thus there is great interest in using these symbionts as probiotics for the conservation of amphibians threatened byB. 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.

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The effect of captivity on the skin microbial symbionts in three Atelopus species from the lowlands of Colombia and Ecuador

PeerJ ◽

10.7717/peerj.3594 ◽

2017 ◽

Vol 5 ◽

pp. e3594 ◽

Cited By ~ 5

Author(s):

Sandra V. Flechas ◽

Ailin Blasco-Zúñiga ◽

Andrés Merino-Viteri ◽

Valeria Ramírez-Castañeda ◽

Miryan Rivera ◽

...

Keyword(s):

Bacterial Community ◽

Batrachochytrium Dendrobatidis ◽

Ex Situ ◽

Rrna Gene ◽

Natural Habitats ◽

Amphibian Species ◽

Skin Bacteria ◽

In Captivity ◽

In The Wild ◽

Microbial Symbionts

Many amphibian species are at risk of extinction in their natural habitats due to the presence of the fungal pathogen Batrachochytrium dendrobatidis (Bd). For the most highly endangered species, captive assurance colonies have been established as an emergency measure to avoid extinction. Experimental research has suggested that symbiotic microorganisms in the skin of amphibians play a key role against Bd. While previous studies have addressed the effects of captivity on the cutaneous bacterial community, it remains poorly studied whether and how captive conditions affect the proportion of beneficial bacteria or their anti-Bd performance on amphibian hosts. In this study we sampled three amphibian species of the highly threatened genus, Atelopus, that remain in the wild but are also part of ex situ breeding programs in Colombia and Ecuador. Our goals were to (1) estimate the diversity of culturable bacterial assemblages in these three species of Atelopus, (2) describe the effect of captivity on the composition of skin microbiota, and (3) examine how captivity affects the bacterial ability to inhibit Bd growth. Using challenge assays we tested each bacterial isolate against Bd, and through sequencing of the 16S rRNA gene, we identified species from thirteen genera of bacteria that inhibited Bd growth. Surprisingly, we did not detect a reduction in skin bacteria diversity in captive frogs. Moreover, we found that frogs in captivity still harbor bacteria with anti-Bd activity. Although the scope of our study is limited to a few species and to the culturable portion of the bacterial community, our results indicate that captive programs do not necessarily change bacterial communities of the toad skins in a way that impedes the control of Bd in case of an eventual reintroduction.

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The Effect of Aquatic and Terrestrial Environmental Factors on the Interaction Between Grand Teton Boreal Toads and a Lethal Fungal Pathogen

The UW National Parks Service Research Station Annual Reports ◽

10.13001/uwnpsrc.2008.3723 ◽

2008 ◽

Vol 31 ◽

pp. 125-131

Author(s):

Peter Murphy ◽

Sophie St-Hilaire ◽

Charles Peterson

Keyword(s):

Fungal Pathogen ◽

Batrachochytrium Dendrobatidis ◽

Chytrid Fungus ◽

Population Declines ◽

Amphibian Skin ◽

Amphibian Species ◽

American Bullfrog ◽

The North ◽

S 100 ◽

Boreal Toads

Batrachochytrium dendrobatidis (Bd), the chytrid fungus which infects keratinized amphibian skin and causes the lethal disease chytridiomycosis, has been linked to population declines and extinctions worldwide (Lips et al. 2006). Amphibians infected with Bd may suffer a variety of outcomes. Individuals of some species have been killed by :S 100 Bd zoospores, while other species, such as the North American bullfrog Rana catesbiana, are highly resistant (Daszak et al. 2004). Within an amphibian species, populations may also respond differently to Bd, with some declining et al. remaining stable (Kriger and Hero 2006). Divergent outcomes among species and populations with respect to Bd may arise from at least three factors, or their interaction.

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Impact of Substratum Surface on Microbial Community Structure and Treatment Performance in Biological Aerated Filters

Applied and Environmental Microbiology ◽

10.1128/aem.03001-13 ◽

2013 ◽

Vol 80 (1) ◽

pp. 177-183 ◽

Cited By ~ 10

Author(s):

Lavane Kim ◽

Eulyn Pagaling ◽

Yi Y. Zuo ◽

Tao Yan

Keyword(s):

Microbial Community ◽

Microbial Communities ◽

Bacterial Species ◽

Community Analysis ◽

Microbial Community Analysis ◽

Rrna Gene ◽

Content Type ◽

Property Change ◽

And Control ◽

Start Ups

ABSTRACTThe impact of substratum surface property change on biofilm community structure was investigated using laboratory biological aerated filter (BAF) reactors and molecular microbial community analysis. Two substratum surfaces that differed in surface properties were created via surface coating and used to develop biofilms in test (modified surface) and control (original surface) BAF reactors. Microbial community analysis by 16S rRNA gene-based PCR-denaturing gradient gel electrophoresis (DGGE) showed that the surface property change consistently resulted in distinct profiles of microbial populations during replicate reactor start-ups. Pyrosequencing of the bar-coded 16S rRNA gene amplicons surveyed more than 90% of the microbial diversity in the microbial communities and identified 72 unique bacterial species within 19 bacterial orders. Among the 19 orders of bacteria detected,BurkholderialesandRhodocyclalesof theBetaproteobacteriaclass were numerically dominant and accounted for 90.5 to 97.4% of the sequence reads, and their relative abundances in the test and control BAF reactors were different in consistent patterns during the two reactor start-ups. Three of the five dominant bacterial species also showed consistent relative abundance changes between the test and control BAF reactors. The different biofilm microbial communities led to different treatment efficiencies, with consistently higher total organic carbon (TOC) removal in the test reactor than in the control reactor. Further understanding of how surface properties affect biofilm microbial communities and functional performance would enable the rational design of new generations of substrata for the improvement of biofilm-based biological treatment processes.

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Establishement of a national monitoring program based on environmental DNA for amphibians and the chytrid fungus Batrachochytrium dendrobatidis

ARPHA Conference Abstracts ◽

10.3897/aca.4.e65362 ◽

2021 ◽

Vol 4 ◽

Author(s):

Omneya Osman ◽

Johan Andersson ◽

Tomas Larsson ◽

Mats Töpel ◽

Alexander Eiler

Keyword(s):

Batrachochytrium Dendrobatidis ◽

National Level ◽

Monitoring Program ◽

12S Rrna ◽

Rrna Gene ◽

Chytrid Fungus ◽

Rana Arvalis ◽

Amphibian Species ◽

National Monitoring ◽

Wide Range

National monitoring programs provide the basis for evaluating the integrity of ecosystems, their responses to disturbances, and the success of actions taken to conserve or recover biodiversity. In this study, we successfully established a national program for the invasive chytrid fungus Batrachochytrium dendrobatidis (Bd) based on dual TaqMan assays. Amphibian diversity based on metabarcoding of the mitochondrial 12S rRNA gene was also performed. Assays were optimized for sensitive detection of target species from a wide range of amphibian ponds with variable potential of inhibitions for eDNA based detection. An amphibian mock community of 5 species was used to validate the metabarcoding approach while internal standards were used in the case of the dual TaqMan assays. First sampling of over 170 ponds in Norway resulted in Bd detection in 12 environmental samples and one swab sample taken over multiple years indicating the establishment of Bd in Norway. Five amphibian species Bufo bufo, Lissotriton vulgaris, Triturus cristatus, Rana arvalis and Rana temporaria as predicted from data in long-term citizen science reporting systems were widely detected in the collected eDNA samples. Our large scale-monitoring program indicates a low risk of a Bd outbreak and amphibian decline caused by chytridiomycosis but continued monitoring is recommended in the future. These findings indicate that eDNA is an effective method to detect invasive species, and to monitor endangered amphibian species. Still, several shortcomings (such as PCR inhibitors and sample volume) were identified that need to be addressed to improve eDNA-based monitoring at the national level.

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Nocardioides donggukensis sp. nov. and Hyunsoonleella aquatilis sp. nov., isolated from Jeongbang Waterfall on Jeju Island

INTERNATIONAL JOURNAL OF SYSTEMATIC AND EVOLUTIONARY MICROBIOLOGY ◽

10.1099/ijsem.0.005176 ◽

2021 ◽

Vol 71 (12) ◽

Author(s):

Inhyup Kim ◽

Geeta Chhetri ◽

Jiyoun Kim ◽

Minchung Kang ◽

Yoonseop So ◽

...

Keyword(s):

Type Strain ◽

Type Species ◽

Bacterial Species ◽

Jeju Island ◽

Nucleotide Identity ◽

Rrna Gene ◽

Average Nucleotide Identity ◽

Phenotypic Data ◽

Content Type ◽

Link Type

Two bacterial strains, designated MJB4T and SJ7T, were isolated from water samples collected from Jeongbang Falls on Jeju Island, Republic of Korea. Phylogenetic analysis of 16S rRNA gene sequences indicated that the two strains belonged to the genera Nocardioides and Hyunsoonleella , owing to their high similarities to Nocardioides jensenii DSM 29641T (97.5 %) and Hyunsoonleella rubra FA042 T (96.3 %), respectively. These values are much lower than the gold standard for bacterial species (98.7 %). The average nucleotide identity values between strains MJB4T, SJ7T and the reference strains, Nocardioides jensenii DSM 29641T, Nocardioides daejeonensis MJ31T and Hyunsoonleella flava T58T were 77.2, 75.9 and 75.4 %, respectively. Strains MJB4T and SJ7T and the type strains of the species involved in system incidence have average nucleotide identity and average amino acid threshold values of 60.1–82.6 % for the species boundary (95–96 %), which confirms that strains MJB4T and SJ7T represent two new species of genus Nocardioides and Hyunsoonleella , respectively. Based on phylogenetic and phenotypic data, strains MJB4T and SJ7T are considered to represent novel species of the genus Nocardioides and Hyunsoonleella , respectively, for which the names Nocardioides donggukensis sp. nov. (type strain MJB4T=KACC 21724T=NBRC 114402T) and Hyunsoonleella aquatilis sp. nov., (type strain SJ7T=KACC 21715T=NBRC 114486T) have been proposed.

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Pseudoalteromonas ostreae sp. nov., a new bacterial species harboured by the flat oyster Ostrea edulis

INTERNATIONAL JOURNAL OF SYSTEMATIC AND EVOLUTIONARY MICROBIOLOGY ◽

10.1099/ijsem.0.005070 ◽

2021 ◽

Vol 71 (11) ◽

Author(s):

Héléna Cuny ◽

Clément Offret ◽

Amine M. Boukerb ◽

Leila Parizadeh ◽

Olivier Lesouhaitier ◽

...

Keyword(s):

Type Species ◽

Bacterial Species ◽

Acid Methyl Ester ◽

Phenotypic Traits ◽

Rrna Gene ◽

Ostrea Edulis ◽

Content Type ◽

Link Type ◽

A Genome ◽

Flat Oyster

Three bacterial strains, named hOe-66T, hOe-124 and hOe-125, were isolated from the haemolymph of different specimens of the flat oyster Ostrea edulis collected in Concarneau bay (Finistère, France). These strains were characterized by a polyphasic approach, including (i) whole genome analyses with 16S rRNA gene sequence alignment and pangenome analysis, determination of the G+C content, average nucleotide identity (ANI), and in silico DNA–DNA hybridization (isDDH), and (ii) fatty acid methyl ester and other phenotypic analyses. Strains hOe-66T, hOe-124 and hOe-125 were closely related to both type strains Pseudoalteromonas rhizosphaerae RA15T and Pseudoalteromonas neustonica PAMC 28425T with less than 93.3% ANI and 52.3% isDDH values. Regarding their phenotypic traits, the three strains were Gram-negative, 1–2 µm rod-shaped, aerobic, motile and non-spore-forming bacteria. Cells grew optimally at 25 °C in 2.5% NaCl and at 7–8 pH. The most abundant fatty acids were summed feature 3 (C16:1 ω7c/C16:1 ω6c), C16:0 and C17:1 ω8c. The strains carried a genome average size of 4.64 Mb and a G+C content of 40.28 mol%. The genetic and phenotypic results suggested that strains hOe-66T, hOe-124 and hOe-125 belong to a new species of the genus Pseudoalteromonas . In this context, we propose the name Pseudoalteromonas ostreae sp. nov. The type strain is hOe-66T (=CECT 30303T=CIP 111911T).

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Transition of Bacterial Diversity and Composition in Tongue Microbiota during the First Two Years of Life

mSphere ◽

10.1128/msphere.00187-19 ◽

2019 ◽

Vol 4 (3) ◽

Cited By ~ 1

Author(s):

Shinya Kageyama ◽

Mikari Asakawa ◽

Toru Takeshita ◽

Yukari Ihara ◽

Shunsuke Kanno ◽

...

Keyword(s):

Oral Cavity ◽

Bacterial Diversity ◽

Bacterial Species ◽

16S Rrna Gene Sequencing ◽

Rrna Gene ◽

Oral Microbiota ◽

Bacterial Composition ◽

Content Type ◽

Operational Taxonomic Units ◽

Rrna Gene Sequencing

ABSTRACTNewborns are constantly exposed to various microbes from birth; hence, diverse commensal bacteria colonize the oral cavity. However, how or when these bacteria construct a complex and stable ecosystem remains unclear. This prospective cohort study examined the temporal changes in bacterial diversity and composition in tongue microbiota during infancy. We longitudinally collected a total of 464 tongue swab samples from 8 infants (age of <6 months at baseline) for approximately 2 years. We also collected samples from 32 children (aged 0 to 2 years) and 73 adults (aged 20 to 29 years) cross-sectionally as control groups. Bacterial diversities and compositions were determined by 16S rRNA gene sequencing. The tongue bacterial diversity in infancy, measured as the number of observed operational taxonomic units (OTUs), rapidly increased and nearly reached the same level as that in adults by around 80 weeks. The overall tongue bacterial composition in the transitional phase, 80 to 120 weeks, was more similar to that of adults than to that of the early exponential phase (EEP), 10 to 29 weeks, according to analysis of similarities. Dominant OTUs in the EEP corresponding toStreptococcus perorisandStreptococcus lactariusexponentially decreased immediately after EEP, around 30 to 49 weeks, whereas several OTUs corresponding toGranulicatella adiacens,Actinomyces odontolyticus, andFusobacterium periodonticumreciprocally increased during the same period. These results suggest that a drastic compositional shift of tongue microbiota occurs before the age of 1 year, and then bacterial diversity and overall bacterial composition reach levels comparable to those in adults by the age of 2 years.IMPORTANCEEvaluating the development of oral microbiota during infancy is important for understanding the subsequent colonization of bacterial species and the process of formation of mature microbiota in the oral cavity. We examined tongue microbiota longitudinally collected from 8 infants and found that drastic compositional shifts in tongue microbiota occur before the age of 1 year, and then bacterial diversity and overall bacterial composition reach levels comparable to those in adults by the age of 2 years. These results may be helpful for preventing the development of various diseases associated with oral microbiota throughout life.

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Phylogenetic investigation of skin sloughing rates in frogs: relationships with skin characteristics and disease-driven declines

Proceedings of The Royal Society B Biological Sciences ◽

10.1098/rspb.2018.2378 ◽

2019 ◽

Vol 286 (1896) ◽

pp. 20182378 ◽

Cited By ~ 4

Author(s):

Michel E. B. Ohmer ◽

Rebecca L. Cramp ◽

Craig R. White ◽

Peter S. Harlow ◽

Michael S. McFadden ◽

...

Keyword(s):

Batrachochytrium Dendrobatidis ◽

Phylogenetic Signal ◽

Interspecific Variation ◽

Phylogenetic Position ◽

Turnover Rates ◽

Amphibian Skin ◽

Amphibian Species ◽

Wide Range ◽

Skin Morphology ◽

And Function

Amphibian skin is highly variable in structure and function across anurans, and plays an important role in physiological homeostasis and immune defence. For example, skin sloughing has been shown to reduce pathogen loads on the skin, such as the lethal fungus Batrachochytrium dendrobatidis ( Bd ), but interspecific variation in sloughing frequency is largely unknown. Using phylogenetic linear mixed models, we assessed the relationship between skin turnover rate, skin morphology, ecological traits and overall evidence of Bd -driven declines. We examined skin sloughing rates in 21 frog species from three continents, as well as structural skin characteristics measured from preserved specimens. We found that sloughing rate varies significantly with phylogenetic group, but was not associated with evidence of Bd -driven declines, or other skin characteristics examined. This is the first comparison of sloughing rate across a wide range of amphibian species, and creates the first database of amphibian sloughing behaviour. Given the strong phylogenetic signal observed in sloughing rate, approximate sloughing rates of related species may be predicted based on phylogenetic position. While not related to available evidence of declines, understanding variation in sloughing rate may help explain differences in the severity of infection in genera with relatively slow skin turnover rates (e.g. Atelopus ).

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Recovery and resiliency of skin microbial communities on the southern leopard frog (Lithobates sphenocephalus) following two biotic disturbances

Animal Microbiome ◽

10.1186/s42523-020-00053-5 ◽

2020 ◽

Vol 2 (1) ◽

Author(s):

Denita M. Weeks ◽

Matthew J. Parris ◽

Shawn P. Brown

Keyword(s):

Microbial Communities ◽

Fungal Pathogen ◽

Bacterial Species ◽

Leopard Frog ◽

Multiple Time ◽

Population Declines ◽

Community Interactions ◽

Amphibian Species ◽

Functional Relationships ◽

Species Specific

Abstract Background 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. Results 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. Conclusions 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.

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