AMPHIBIAWEB

The Amphibian Skin Microbiome


By Sofia Prado-Irwin and Alicia Bird
August 5, 2015
Addendum by Ann Chang

Outline:

  1. Introduction
  2. Ecology of the Amphibian Host-Microbiome Relationship
  3. Skin Microbiome and Disease
  4. Conservation Applications
  5. Characterizing the Microbiome
  6. Addendum
  7. Literature Cited

I. Introduction

One of the fastest-growing fields of biology today is the study of host-microbiome interactions, and with good reason; recent studies using high-throughput DNA sequencing and analysis technologies have shown that microbial communities living in association with animal hosts play a significant role in many aspects of host life history, including development, physiology, behavior, and health (Rosenberg et al. 2011, Spor et al. 2011, McFall-Ngai et al. 2013, Kueneman et al. 2013).

In humans for example, many diseases strongly correlate to a specific gut microbial community structure, distinct from that seen in healthy individuals (Cho & Blaser 2012, Cenit et al. 2014). Additionally, gut microbiomes of certain healthy individuals have been shown to actually combat disease, leading to innovative health treatments involving gut microbiota transplants (Aroniadis & Brandt 2013, Ding & Schloss 2014). The mechanisms of such microbe-facilitated diseases and treatments are not always fully understood, but new discoveries are occurring constantly through research using germ-free model organisms, human observations, clinical trials, and more (Cryan et al. 2011, Spor et al. 2011, Cenit et al. 2014).

Such promising research in the field of human health and medicine has led ecologists to start investigating the role of the skin microbiome in the health of other taxa as well, including amphibians. In amphibian research and conservation, the skin-associated microbiome has thus emerged as an exciting and promising new area of inquiry, with potential applications to conservation and disease management, particularly as it relates to the fungal pathogen Batrachochytrium dendrobatidis (Bd). This field is quite new, so much remains to be learned about the host-microbiome relationship, but recent studies have made great strides in understanding skin microbiome dynamics and developing conservation tools.

II. Ecology of the Amphibian Host-Microbiome Relationship

Relationships between microbial communities and their hosts can be highly complex. A wide range of factors can influence these interactions, from host traits such as genetics, life history, and behavior to broader effects of habitat and exposure (Ding & Schloss 2014). Additionally, the skin of different amphibian species and individuals may vary by the presence and types of anti-microbial peptides, mucosal secretions, and levels of skin sloughing, which can all affect the establishment and maintenance of resident microbial communities (Meyer et al. 2012, Walke et al. 2014). Researchers are currently working to understand the basic biology governing the host-microbiome relationship in order to confidently and effectively harness microbiome science in conservation efforts.

Several studies in the past few years have aimed to characterize variation in microbiota across different amphibian species and populations. Researchers have found that host species identity is one of the greatest factors influencing the amphibian skin microbiome; conspecific individuals exhibit microbial communities that are more similar to one another than to those present on cohabiting individuals of different species. Habitat has also been shown to influence the microbiome. Within one species, members of the same population have microbiomes that are more similar to one another than to individuals of different populations. These results suggest that the skin of different amphibian species selects for a relatively specific microbiome, but is also colonized and influenced by environmental reservoirs (McKenzie et al. 2012, Kueneman et al. 2013, Walke et al. 2014).

Rana cascadae by William Flaxington Rana yavapaiensis by Richard Sage Eleutherodactylus coqui by Dante Fenolio
Rana cascadae by William Flaxington Rana yavapaiensis by Richard Sage Eleutherodactylus coqui by Dante Fenolio

Additionally, a distinct microbiome signature across life stage has been identified in several species. In the metamorphic frog Rana cascadae, adults and subadults have a similar microbiomes, but they are significantly distinct from microbiomes of tadpoles (Kueneman et al. 2013). A recent study on the direct-developing frog Eleutherodactylus coqui showed a similar result, with juvenile frogs exhibiting a microbiome distinct from that of the adults (Longo et al. 2015). Combined, these data suggest an ontogenetic pattern in microbiome variation. The reason for this difference remains unclear, but it is known that as individuals age their immune systems change, which could lead to the cultivation of a specific skin microbiome between life stages (Longo et al. 2015).

Seasonality has also been shown to impact the microbiome. Longo et al. (2015) observed significant seasonal changes in skin microbial communities of adult Rana (Lithobates) yavapaiensis, a metamorphic frog occurring in temperate North America. The authors found that skin microbial communities increase in diversity from summer to winter, which they attribute to possible temperature-mediated changes in host immune function and/or bacterial growth. Interestingly, pathogens such as Batrachochytrium dendrobatidis also show seasonal variation, which may interact with the microbiome as well (Walke et al. 2015).

While these whole-microbiome approaches have shown very interesting and important patterns, researchers are also investigating the individual bacterial taxa that are driving these differences. Evidence from other host taxa shows that the gut microbiome contains endemic bacteria that have coevolved mutualistically with the host, and perform important symbiotic functions (Engel et al. 2012, Wylie et al. 2012). Of course, the skin is a very different environment from the gut and experiences different environmental pressures, but bacterial species that beneficially affect host health or development may colonize it and form coevolving communities. In order to fully understand the structure and function of the skin microbiome, it is important to identify and research these taxa.

To that end, several studies have identified bacterial species that are present on amphibian skin but absent or rare in the environment (Loudon et al. 2014a, Walke et al. 2014). Researchers suggest several explanations for this finding. First, the bacteria that are specific to amphibians may actually be present at low abundance in the environment and thus undetectable in environmental samples, but abundant on amphibians, selectively colonizing amphibian skin. It is also possible that such taxa are unique to amphibians, and are maintained throughout host generations through vertical or horizontal transmission, forming coevolutionary symbiotic relationships (Walke et al. 2014). Interestingly, not all studies have found such amphibian-specific bacteria. In their study comparing Plethodon jordani to environmental substrates, Fitzpatrick & Allison (2014) found no bacterial species that were specific to the salamanders. However, some bacterial species exhibited salamander-adapted genotypes - these bacteria were found in both environmental and salamander samples, but the sequences of the salamander-associated strains were distinct from sequences found in the environment. Thus despite their somewhat contrasting results, the data from these studies and others support a selective recruitment model of symbiotic community assembly in which the salamander skin environment selects for a specific community of microbes (Fitzpatrick & Allison 2014), with a number of potentially coevolved species present on the amphibian skin as well (Walke et al. 2014). The role of these microbes in development and health is an active area of research.

III. Skin Microbiome and Disease

Research on amphibian skin microbiomes is greatly motivated by the desire for a deeper understanding of the interaction between the microbiome and amphibian disease. Of particular focus is the skin disease chytridiomycosis, caused by the fungal pathogen Batrachochytrium dendrobatidis (Bd). (See more details on Bd and chytridiomycosis). The degree of susceptibility to Bd varies greatly between species, and is attributable to multiple factors including host physiology, environmental conditions, and the skin microbial communities (Bletz et al. 2013, Bielby et al. 2015). Of these factors, skin microbial communities have come to the forefront of research on Bd susceptibility due to the role symbiotic bacteria play in host resistance.

Chytrid Spore by Wikimedia Commons Rana muscosa by Sam McNally Chytrid-infected frog by Forrest Brem
SEM of Bd spore from CSIRO Rana muscosa tadpoles by Sam McNally A Chytrid-infected frog by Forrest Brem, Wikimedia Commons

The importance of the skin microbiome in mitigating amphibian disease has been demonstrated in several ways. The most common approach to date has been culture-based studies, which have identified many species of bacteria present on the skin of various amphibian species that can inhibit the growth of Bd (Woodhams et al. 2007, Harris et al. 2009a). One common example is the bacteria Janthinobacterium lividum, which produces violacein, an antifungal metabolite that is deadly to Bd when present at a high enough concentration (Brucker et al. 2008). While culture-based studies have helped identify Bd resistant bacteria, lab trials have further elucidated the role such bacteria play in host susceptibility. Such studies have demonstrated the effectiveness of symbiotic bacteria at resisting disease by inoculating susceptible species with antifungal bacteria, resulting in lower levels of infection (Becker et al. 2009, Harris et al. 2009b). Such knowledge gained in the lab can be applied to field studies, to help us understand Bd trends we observe in nature. For example, Bresciano et al. (2015) found that in high elevation areas where Bd prevalence was high, animals were able to persist with infections. These stable populations of amphibians living with Bd had several species of antifungal bacteria that had been identified in culture-based studies living on their skin. Another study found that several populations of two highly susceptible species Rana muscosa and Rana sierrae were able to persist in the presence of the pathogen primarily due to the fact that those populations included a higher proportion of individuals that possessed antifungal bacteria (Lam et al. 2010). While a lot of progress has been made by focusing on specific symbiotic antifungal bacterial species, the interactions between microorganisms are critical to the overall function of the microbiome (Loudon et al. 2014b, Woodhams et al. 2014). Thus to truly understand host-microbiome disease dynamics, we must develop a greater understanding of how the entire community of microorganisms living on amphibian skin contributes to Bd susceptibility.

One study (Becker & Harris 2010) highlighted the importance of the whole-microbiome approach by showing that when cleared of most of their resident bacteria prior to Bd exposure, Plethodon cinereus was significantly less resistant to infection. It is clear that altering the microbiome can have significant health consequences for the host. The first study to evaluate the entire amphibian skin microbiome in relation to Bd in the field looked at several populations of the endangered frog Rana sierrae that had persisted through a Bd outbreak (Jani & Briggs 2014). They found found that Bd infection causes a significant shift in the skin microbiome. Not only does microbial community composition differ between healthy and infected frogs, Bd infection load also correlates with the degree of change in the microbiome, such that the more infected the frogs were, the more their microbiomes diverged from those of healthy non-infected frogs (Jani & Briggs 2014). Longo et al. (2015) also found that individual Eleutherodactylus coqui with high intensity Bd infections had reduced microbiome diversity compared to healthy frogs. Taken together, these studies suggest that Bd infection may cause microbial dysbiosis, or disruption of the healthy microbiome. However, because these studies were observational and not experimental, it is not completely clear that Bd causes these changes, although it is certainly suggestive.

Interestingly, one recent study showed a different pattern. In a susceptibility trial using Atelopus zeteki, Becker et al. (2014) found that the bacterial communities present on frogs before the trial began predicted survival of each individual, i.e. the microbiomes of frogs that survived were significantly different from frogs that eventually died, even before they were infected. This evidence suggests that the resident microbial communities actually helped the surviving frogs clear the Bd infection, and were not themselves significantly changed by Bd.

It is clear from these studies that Bd and resident skin microbes interact with one another in various ways, with both influencing the other’s abundance and presence. The mechanisms of these interactions are still unclear, and further studies closely monitoring microbial changes through time during infection, across a range of amphibian species, will certainly lead to a greater understanding of this phenomenon.

IV. Conservation Applications

As the study of the amphibian skin microbiome is relatively new, few studies have been conducted concerning conservation applications of this research, but the future possibilities are promising. The microbiome can be a useful tool throughout the conservation process, from identifying populations of concern through captive breeding. While there are many factors influencing amphibian declines (See more at Declines), conservation strategies relating to the skin microbiome have thus far focused on combatting Bd.

So far, the most well-researched approach to conserving species using the skin-associated bacteria is bioaugmentation. The goal of bioaugmentation is to enhance the host’s ability to resist Bd using naturally occurring symbiotic bacteria. When a large proportion of individuals are protected from Bd, community or 'herd' immunity allows the entire population to persist. The population is be protected in several ways, either by a keystone anti-Bd microbe, an abundant anti-Bd microbe, or highly diverse microbiome. The ultimate goal of probiotic therapy is to increase the proportion of protected individuals in the population, thus allowing the population to persist with Bd (Bletz et al 2013).

To do this, researchers must identify resident skin bacteria that inhibit Bd growth, culture these bacteria, and innoculate individuals with this culture to instigate recruitment of the beneficial bacteria on the skin. A number of studies have identified bacterial species that actively inhibit Bd in culture (Woodhams et al. 2007, Lauer et al. 2008, Lam et al. 2010) and some have been further used in inoculation trials, with mixed results.

Plethodon cinereus by John White J. lividum by Wikimedia Commons Atelopus zeteki by JOhn White
Plethodon cinereus from John White Janthinobacterium lividum bacterium, Wikimedia Commons Atelopus zeteki by John White

One of the most well-researched of these bacteria is Janthinobacterium lividum, a relatively common cutaneous bacteria that has been successfully used as a probiotic. Lab inoculations with J. lividum significantly lowered Bd infection rates in several species including Rana muscosa and Plethodon cinereus (Becker et al. 2009, Harris et al. 2009b), but other species such as Atelopus zeteki exhibited no such benefit (Becker et al. 2015). Such differential success is likely due to a number of factors, including differences between host immune systems, environments, and resident microbes on the skin (Bletz et al. 2013, Woodhams 2014). Skin microbial communities are highly diverse and complex, and the bacteria present in these communities interact with one another in many different ways (Loudon et al. 2014b, Woodhams et al 2014). Probiotic species are no exception; Bd-inhibiting bacteria may only be able to colonize certain microbial communities, and may thus not be effective in every species or in every scenario. Additionally, probiotic trials have yet to be successfully implemented in the field. In fact, a recent study from Küng et al. (2014) showed that skin microbiomes of frogs tested in a semi-natural mesocosm were highly resistant to colonization, suggesting that probiotic approaches in the field may require strategies more complex than inoculation alone. Despite these complexities, probiotic bacteria are an important and promising tool in the fight against amphibian disease.

While bioaugmentation studies show significant potential, most focus on one or a few species of bacteria. With the advent of microbiomics, researchers are beginning to take a whole-microbiome approach to conservation, and have discovered several interesting patterns. For example, one study (Becker et al. 2014) showed that the microbial community is predictive of individual susceptibility. In this trial, the microbiomes of individuals that succumbed to Bd were distinct from individuals that cleared infection, and these differences were evident even before the trial began, showing that the resident microbes had a very strong effect on disease outcome. These results suggest that researchers may be able to use microbiome profiles to identify individuals or populations in the field that are at higher risk of disease, leading to more accurate conservation targets.

Studies in the field have revealed important patterns as well. As mentioned previously, researchers have shown that the skin microbial communities shift across different seasons and different stages of development (Kueneman et al. 2013, Longo et al. 2014). Interestingly, Bd dynamics also change seasonally, and juvenile amphibians are often more susceptible to the fungus (Longo et al. 2014). Such information can inform future bioaugmentation efforts in the field, allowing researchers to time treatments seasonally and developmentally for most effective results.

Preventing species decline and extinction in the field is the best case scenario, but this is not always possible for every species or population. In such cases, captive breeding programs are often used. The captive environment is different from the wild in many respects, and can affect animals in a number of ways, leading to complications when reintroduced. To date, most research on the effects of captivity have focused on genetics and behavior (Bloxam & Tonge 1995, Snyder et al. 1996), but several recent studies on amphibians have begun to investigate the effects of captivity on the microbiome. These studies have shown very clear differences between captive and wild individuals, with captive individuals exhibiting a reduction in microbiome diversity in two studies (Antwis et al. 2014, Loudon et al. 2014a), and an increase in diversity in a third (Becker et al. 2014). Interestingly, animals housed with an environmental substrate (soil) maintained a microbial more similar to that in the wild, while those housed with sterile media showed decreased species diversity and evenness (Loudon et al. 2014a). Diet has also been shown to affect the microbiome in captivity, and such differences may impact an animal’s ability to resist pathogens or limit their suitability for reintroduction (Antwis et al. 2014). As such, factors such as substrate and diet should be taken into consideration when developing animal husbandry plans for captive conservation programs.

While data are still limited, these studies show that incorporating microbiome considerations in conservation research and planning may enhance the success of such programs, and should be implemented in future amphibian conservation work.

V. Characterizing the Microbiome

Early studies of the amphibian microbiome used culture-based techniques, in which researchers would isolate bacteria from the skin of amphibians and grow them on agar or other media. The bacteria were then sequenced (16S rRNA gene) and/or used in experimental trials (Woodhams et al. 2007, Lauer et al. 2008). With recent technological developments, it is now possible to sequence the entire bacterial community, not just those that grow on culture. Such an approach gives a much more comprehensive view of the microbiome, as it has been estimated that only 1% of bacterial species can be cultured. Currently, the microbiome is generally characterized using high-throughput DNA sequencing techniques, most commonly using an Illumina or 454 Life Sciences sequencing platform. Most studies focus on the bacterial microbiome, sequencing a region or regions of the bacterial 16S rRNA gene. Resulting sequences are then classified into operational taxonomic units (OTUs) and compared to a reference database (e.g. Greengenes) for identification (Caporaso et al. 2011). Microbiome samples are then compared using a number of analyses to evaluate phylogenetic community differences, community richness and relative abundance, and other measures of diversity.

Vi. Addendum by Ann Chang

Most amphibians are tropical, yet the majority of studies on beneficial cutaneous bacterial have been conducted on amphibians in the temperate zone. Because previous studies showed that both species and locality are factors determining success of Bd-inhibiting bacteria, Fleches et al. (2012) addressed this disparity by studying Colombian frogs: Atelopus spurrelli, A. aff. limosus, and A. elegans. Specimens were swabbed for culturable bacteria, which were tested for their ability to inhibit Bd growth. From 148 bacterial morphotypes obtained, 12 caused anti-Bd activity. Consistent with other studies, the bacterial species that inhibited Bd growth came from six genera: Pseudomonas, Acinetobacter, Stenoptophomonas, Comamonas, Chryseobacterium and Elizabethkingia. The 12 species were further split into 38 strains with the three strains of highest anti-Bd activity coming from two Pseudomonas.

An Antifungal Isolate Database has been established to help researchers and managers select the appropriate bacterial strain for bioaugmentation, predict how the isolate will function, and aid in the search during whole genome sequencing for genes with antifungal capabilities (Woodhams et al. 2015). Two files can be downloaded: (1) a FASTA file that include the bacterial isolates’ 16S rRNA gene sequences and (2) a text file with metadata that includes host species (n = 37), host life stage, geographic region (12 countries from 5 continents), and the taxonomic identity (~2000) and antifungal capacity of the isolate.

While identification of antifungal isolates is important, mounting evidence suggests complex community microbiome and Bd interactions. Community structure is altered by Bd infections (Jani and Briggs 2014). Whole microbiome communities may have beneficial properties for resistance to Bd (e.g. Walke et al. 2015). This interaction is important to understand as captivity -- one of the main methods of combatting Bd spread -- is also known to significantly change the cutaneous microbiome (Becker et al. 2014).

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Recent Scientific Publications

AmphibiaWeb maintains a list of recent scientific publications on amphibian declines and amphibian conservation.

This list is compiled and updated monthly by Professor Tim Halliday (formerly DAPTF International Director) (t.r.halliday@open.ac.uk).

Please cite as (MLA compatible): Prado-Irwin, Sofia, and Alicia Bird. "The Amphibian Skin Microbiome." Microbiome of Amphibian Skin. AmphibiaWeb: The Regents of University of California. Web. Accessed [date]. .