Introduction

All animals and plants harbor microbial communities that frequently have crucial roles in maintaining hosts’ health [1]. The skin-associated bacterial communities of amphibians play an important role in host defense against pathogens in synergy with innate and adaptive immune system [2]. Low levels of infection risk in amphibians have been associated with various features of the microbiome, including bacterial species richness [3], specific microbial community assemblages [4], and the presence of microbes that produce metabolites that inhibit pathogens growth [5]. A specific pathogen of global importance is the fungus Batrachochytrium dendrobatidis (Bd), which has been a major factor in the global decline and extinction of amphibian populations and species [6]. Of 1 240 amphibian species sampled worldwide up to 2013, 516 (42%) were infected with Bd [7]. Different amphibian species and populations within species frequently present variations in their vulnerability to Bd infection [8].

Although there are multiple factors that might have a role in amphibian resistance to Bd, including host genetics, immunology [9], skin peptides [10], and environmental factors [11], bacterial communities living on amphibians’ skin also have a crucial role in host’s resistance [2]. Several bacteria found on amphibian’s skin are known to produce chemical compounds that are able to inhibit growth of Bd and to reduce amphibian mortality under laboratory conditions [12]. Janthinobacterium lividum, for example, was successfully used to mitigate Bd infection in Rana muscosa [13], and Pedobacter cryoconitis reduced Bd load infection in the same frog species [14]. Up to 2015, there were about 255 anti-Bd bacterial operational units (OTUs) identified from the skin of 37 species of amphibians that had been deposited in a reference database [15].

Amphibians skin bacteria are not a random assemblage of environmental bacteria [16] maintaining a particular structure across amphibian species, populations, and individuals [17]. Amphibians differ in physical and chemical properties of their skin, producing different suites of antimicrobial peptides [18], mucins and glycoproteins [19], and synthesized alkaloids [20], and according to some authors, these differences can shape the cutaneous bacteria community [21].

The amphibian assemblages of skin bacteria can change seasonally in tropical and temperate zones with marked variations throughout the year in environmental features, such as temperature and precipitation [3, 21]. Adults individuals of Lithobates yavapaiensis sampled in winter presented a high infection of Bd that coincided with a high OTU richness [3]. Seasonal differences in skin bacteria and Bd infection in amphibians are a result of variation in environmental conditions such as temperature, since some host-pathogen responses are temperature-dependent [22]. The optimal immune response of amphibians depends on specific temperature ranges [23], and in cool temperatures, these immune responses could be ineffective [3], leaving amphibians unable to eliminate Bd, and some authors have proposed that microbial colonization during winter evolved as a response to fight Bd infections [3].

There is an information paucity of how similar skin-associated bacterial communities are among wild co-occurring amphibian species. There is also limited information on the role of specific microbial assemblages in the resistance to Bd in wild amphibian populations, as well as on the importance of environmental conditions on the efficiency of anti-Bd bacterial skin symbionts. Characterizing these patterns is important to understand evolutionary and ecological processes structuring functionally microbial skin assemblages, which may help in the design of conservation strategies [24].

In the central region of the trans-Mexican volcanic belt, several amphibian species have been reported to have Bd infection, including the facultative neotenic salamander Ambystoma rivulare and the semi-aquatic frog Lithobates spectabilis [25]. Both species occur at the same range but not share habitats [25]. The goal of the present study was to evaluate if two populations of these co-occurring species (L. spectabilis and A. rivulare) presented different skin bacterial communities and if taxonomic composition and structure of these communities influenced host resistance to Bd. Additionally, we evaluated the effect of seasonality on this Bd resistance.

Considering that different amphibian species present different levels of susceptibility to Bd infection and that skin-associated bacteria have the potential to influence this susceptibility, we hypothesized that skin bacterial composition and structure of L. spectabilis and A. rivulare were different and that bacterial species richness and relative abundance of inhibitor bacteria were negatively related to Bd load, an indicator of the severity of disease symptoms of the host. We also hypothesized that environmental conditions associated with seasons modulate the relationship between bacterial community and Bd infection.

Methods

Field Site

Our study was conducted at the core zone of the Monarch Butterfly Biosphere Reserve (MBBR), located in the trans-Mexican volcanic belt on the border of Michoacán state and state of Mexico. The reserve’s core zones (13,551 ha) are located above 3000 m and the main vegetation cover consists of fir forests. Climate is humid-cold with a mean annual temperature of 14°C (range = −1.5°C in December to 19.9°C in May) and a mean rainfall of 700 to 1250 mm3.

We sampled six independent streams (Table 1), each surrounded by fir forest and alpine grasslands in six different periods between 2016 and 2017 (three samplings surveys in the rainy and three in the dry season). Streams were separated by a distance of at least 1 km. Altitude at the streams ranged from 3000 to 3200 m. Ambystoma rivulare was sampled in Arroyo del Ejido Garatachía, Arroyo la Mesa, and Arroyo del Llano el Establo; meanwhile, L. spectabilis was sampled in Arroyo Oyamel, Arroyo La Calera, and Arroyo Jesús el Nazareno [25].

Table 1 Geographic location of the sampled streams in the core zone of the Monarch Butterfly Biosphere Reserve (MBBR)
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Sampling Sites

Along each of the sampled streams, a 100 × 100 m plot was positioned at random. Each transect was 50 m wide from stream banks in a straight line towards uplands. Four people visually searched each transect for 1 hour. Sampling was carried out at daylight (09:00–10:00) and at night (20:00–21:00). To determine the presence of Bd and to collect skin bacteria, we took skin samples of A. rivulare and L. spectabilis according to the methods of Hyatt et al. [26]. All samples are from postmetamorphic adults in the case of L. spectabilis and adult individuals in the case of A. rivulare. We rinsed each individual with 60 ml of sterile water before swabbing to remove transient microbes, using fresh gloves, and then we took each sample with a sterile rayon-tipped swab, in the ventral and inguinal regions of each collected individual [27]. We standardized the number of swabs per individual and body region and used new gloves to avoid sample cross-contamination [26]. Each swab was placed and stored in vials with alcohol (70%) at −20°C. Before DNA extraction, swabs were dried to evaporate residual ethanol. To prevent Bd dispersal, sampling material was disinfected with alcohol (70%) and chlorine bleach (6%), after sampling was completed on each plot.

DNA Extraction

We used swab samples to analyze skin bacterial communities and determine the presence of Bd in wild co-occurring populations of the salamander A. rivulare and the frog L. spectabilis. For DNA extraction of both Bd and bacterial communities, we used Prep Man® Ultra (Applied Biosystems). Tubes were centrifuged for 3 min at 13 000×g, incubated for 10 min at 100°C. The supernatant was then aspirated from swabs and placed in a sterile 1.5-ml tube. For Bd testing, we used Real-Time PCR analysis (qPCR) on an Applied Biosystems Step One Plus TM Real-Time PCR System, following the methodology used in Nava-González et al. [25]. The Bd strain used for standards was GPL1 isolated from Pseudoeurycea leprosa from the National Park La Malinche, México, and cultured at the Instituto de Biología, UNAM. For PCR reactions and amplification parameters, we used the methods of Boyle, Boyle, and Olsen [28], and each sample was run in triplicate.

To measure Bd intensity infection (Bd load), we calculated zoospore equivalents (ZE) by multiplying the raw genomic output by 80 as DNA extracts were diluted 80 times through extraction and qPCR [29]. A result was considered positive when the sample amplified (0.1 ZE) before cycle 39 for at least two times; the mean of the two positive samples was used to calculate ZE.

Skin Bacterial Community Metagenomics

Bacterial 16S rRNA amplicon taxonomic fingerprinting was performed amplifying V3 variable region of the 16S rRNA gene with the primer set V3-338f and V3-533r and sequenced in an Illumina MiniSeq at CIAD Mazatlán Unit, Genomic Services, Mexico. Reads were assembled with Flash v.1.2.7 software [30], and VSEARCH [31] was used for further processing, obtaining a relative abundance matrix of bacterial OTUs0.03 (Operational Taxonomic Units clustered at 97% identity) that was normalized using the metagenome Seq method [32]. Taxonomic annotation was performed using VSEARCH against RDP database v11.5 [33].

Data Analysis

The Bd load and bacterial community were analyzed using two independent and complementary methods. For the first, we used univariate analysis and for the second multivariate analysis. The univariate analysis aimed to evaluate the role of the skin bacterial community in the restraining of Bd infection. We used the following parameters, Bacterial Shannon Index (BSI), expected richness (Chao 1), and observed species richness (OSR), that were calculated from the OTUs matrix with the phyloseq package of R software [32]. We normalized the data. The percentage of taxa of inhibitory bacteria (TIB) and the percentage of relative abundance of these taxa (AIB) registered in the two sampled species of amphibians were calculated. Each of the bacteria taxa registered was searched in a reference database of antifungal amphibian skin-associated bacteria [15], and articles published after this review [34,35,36,37]. We also reported skin-associated bacteria that have not been tested for Bd-inhibitory capacity and bacteria that did not show Bd-inhibitory capacity. The number of sampled bacteria taxa listed in the database was used to calculate the percentage of taxa that matched isolates previously shown to inhibit growth of Bd. This is a rough estimate of the Bd-inhibitory potential of the OTUs observed in this study, and several taxa were previously shown to contain isolates with both Bd-inhibitory and Bd-facilitating capacities, or no effect on Bd. Thus, the percentage of inhibitory bacteria taxa (IBT) and the percentage of relative abundance of inhibitory bacteria should be considered predictive rather than descriptive measures.

The percentage of this inhibitory taxa relative abundance was also calculated in relation to the total bacterial relative abundance of the samples. To explore the relationship between total bacterial community and Bd load in the two amphibian species (A. rivulare and L. spectabilis) and the two different seasons (rainy vs. dry), we used generalized linear models (GLMs) with an ANCOVA design. We developed one model for each of the parameters of the total bacterial community (observed taxa richness, Chao 1, and Shannon Index) as the continuous explanatory variables and the amphibian species (A. rivulare and L. spectabilis) and the seasons (dry and rainy) were the categorical variables and Bd load was the response variable. We then developed separately a model for the percentage of IBT and the percentage of relative AIB and Bd load in the two amphibian species and the two different seasons using also GLMs with an ANCOVA design in which the response variable was Bd load and the parameters of inhibitory bacteria were the continuous explanatory variables and the amphibian species and the seasons were the categorical variables. We developed saturated models considering the main effect of the factors and the two and three interactions among the factors. When a term was not significant, the model was reduced to use only significant terms. For GLMs, we used a Poisson error and a logarithmic link function. For each relationship, we obtained a model of the form y = mx + b, where m is the slope and b the ordinate. When significant, differences between slopes were tested among species and season. The regression lines presented in the results were obtained from the GLMs. We conducted GLMs analysis with the R statistical software R 3.6.

The multivariate methods were used to compare the structure composition of bacterial communities in different categories of Bd load (ZE= 0, ZE0.01>100, ZE>100) among the two different amphibian species and seasons. For metagenomic data, vegan R library v2.4-6 was used to generate a non-parametric multidimensional scaling plot (NMDS) with OTUs0.03 matrix, and a statistical analysis was performed with the ANOSIM function to evaluate groups using the Bray-Curtis distance and 999 permutations.

Results

We sampled a total of 52 individuals, 27 of A. rivulare (13 samples from Arroyo del Ejido Garatachía, 13 samples from Arroyo la Mesa, and 1 sample from Arroyo del Llano el Establo) and 25 of L. spectabilis (10 samples from Arroyo Oyamel, 8 samples from Arroyo La Calera, and 7 samples from Arroyo Jesús el Nazareno).

Regarding Bd infection, we found that 17 (63%) individuals of A. rivulare from the 27 sampled were Bd positive (95% binomial confidence interval: p ± CI = 0.80 ± 0.15). From the 25 L. spectabilis individuals sampled, we found that 20 (80%) were Bd positive (95% binomial confidence interval: p ± CI = 0.63 ± 0.18). Of these Bd positive individuals, 21 were registered in the rainy season (10 A. rivulare, 11 L. spectabilis), and 16 in the dry season (7 A. rivulare, 9 L. spectabilis). Overall, we registered 5837 skin-associated bacterial OTUs, 3361 in A. rivulare, and 4729 in L. spectabilis. Of these bacterial OTUs, 29 phyla, 52 classes, 105 orders, 207 families, and 622 genera were annotated. Of the 52 classes registered, four classes represented about 86 and 96% of the total abundance for A. rivulare and L. spectabilis. The Class Gammaproteobacteria was the most abundant for both species with ~50% of total abundance (Fig. 1). Of the annotated bacterial OTUs, we registered 516 genera that have not been tested for Bd-inhibitory capacity, 33 genera with a non-significant Bd-inhibitory capacity, and 73 inhibitory Bd bacterial genera, representing on average about 55% of the total relative abundance of bacterial genera (54% in the rainy season and 61% in the dry season for A. rivulare; 53% in the rainy season and 57% in the dry season for L. spectabilis). Of these identified inhibitory genera, Pseudomonas, Stenotrophomonas, Microbacterium, Variovorax, and Massilia were the most abundant taxa in both species, A. rivulare and L. spectabilis (Table 2, Fig. 2). We observed that high bacterial species richness and Chao1 were associated with low Bd loads (e.g., ZE = 1.75), while at low species richness, we found Bd loads as high as ZE = 853. This negative relationship was significant, and the GLMs indicated no differences in terms of infection prevalence neither between species (A. rivulare and L. spectabilis) nor seasons (rainy and dry), and the interactions between these factors (Table 3, Fig. 3 a and b). Contrastingly, Shannon index did not explain any of the variations of Bd loads. In relation to analysis of Bd inhibitory bacteria, we also found a negative relationship in the case of relative abundance (percentage); at high levels of inhibitory bacterial relative abundance, low Bd loads were registered. This pattern was independent of amphibian species and season (Table 3, Fig. 3c). Percentage of inhibitory bacterial taxa did not show a significant relationship with Bd loads.

Fig. 1
figure 1

Relative abundance of taxonomic classes registered in skin samples of A. rivulare and L. spectabilis in the Monarch Butterfly Biosphere Reserve during the rainy and dry season

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Table 2 Abundances (%) of the genus registered in skin samples of A. rivulare and L. spectabilis in the Monarch Butterfly Biosphere Reserve during the rainy and dry season
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Fig. 2
figure 2

Rank-abundance curves for the community of genus of bacteria in two amphibian species in the Monarch Butterfly Biosphere Reserve, Mexico. Ambystoma rivulare in the upper and L. spectabilis in the bottom charts during the rainy season (a and c) and the dry season (b and d)

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Table 3 Statistics for the GLMs to evaluate the response of Bd load to total bacterial community and the inhibitory bacteria in the skin of wild populations of Ambystoma rivulare and Lithobates spectabilis in the Monarch Butterfly Reserve, Mexico
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Fig. 3
figure 3

Relationship among Bd load and the bacterial community attributes. a Observed species richness. b Expected species richness. c Abundance of inhibitor bacteria. The trend lines and 0.95 CI were obtained from the GLM Poisson model. We included the effect of species and season; however, these were not different. Species are distinguished in blue for A. rivulare and red for L. spectabilis, respectively

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Microbial profiles are separated by host species, and for L. spectabilis, there was a separation for rain and dry season (Fig. 4). Although ANOSIM values do not stress a significant difference for the 12 analyzed groups (3 Bd load categories, 2 host species, 2 seasons), there was a tendency for the bacterial community of A. rivulare to group in the rainy season (Fig. 4). We observed a more dispersed community in the dry season for this species. For L. spectabilis, a clear group of high Bd load in the dry season was observed (Fig. 4).

Fig. 4
figure 4

NMDS analysis of microbial OTUs0.03 profiles separated by host species and seasons with a total of 12 analyzed groups (3 Bd load categories, 2 host species, 2 seasons). We observe in red colors L. spectabilis and in blue colors Ambystoma rivulare. In triangles high Bd load, in squares low Bd load, and in circles no Bd load. Microbial community seems to structure depending on both species and season, and Bd presence seems to be correlated with certain bacterial community and Bd absence with the dispersion of the bacterial diversity in both amphibian species

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Discussion

Although the understanding of the dynamics between Bd infection and skin microbial ecology in amphibians is rapidly growing [3, 27], the number of field studies on the modulation of this relationship by Bd inhibitory bacteria, bacterial community structure, and seasonal effects is still limited. In this study, we address some aspects of these knowledge gaps by assessing the relation between structure of skin bacterial communities, number and relative abundance of Bd inhibitory bacteria, and seasonal influences on Bd infection load of two co-occurring amphibian species. Our results suggest that richness of the skin bacterial community diminishes the intensity of Bd infection in both the salamander and the frog species studied. We also found that relative abundance of Bd inhibitory bacteria affects the intensity of Bd infection. Our results are congruent with studies in similar habitats. Burkart et al. [38] described that a Bd-resistant amphibian, Gastrotheca excubitor, harbored more isolates of cultivable anti-Bd bacteria compared with G. nebulanastes with a weaker ability to inhibit Bd. Catenazzi et al. [39] assayed the antifungal abilities of 133 bacterial isolates from 26 frog species of the Peruvian Andes, and their results showed that the highest proportion and inhibition strengths of anti-Bd isolates were found in two non-susceptible species G. excubitor and Hypsiboas gladiator.

It is relevant to consider that our results of Bd inhibitory bacteria must be considered predictive rather than descriptive since our bacteria database-searching approach was based on taxa and not sequence matching. Besides, according to the literature, the antifungal nature of bacterial species can change between in vitro and in vivo conditions [40]. From all published studies about the ability of bacteria to inhibit Bd, only 9.2% were experimental trials in vitro, compared with experimental trials on amphibians in vivo that comprised 24.2% of the publications [41]. The OTUs identified in our study could show variation in the Bd-inhibition capacity. Besides, most of bacterial OTUs found in this study have not been yet tested for Bd-inhibitory capacity.

The number of identified skin-associated bacterial OTUs for each of the studied species, 3361 for A. rivulare and 4729 for L. spectabilis, falls within the range reported in other amphibian species (e.g., 2667 for Craugastor fitzingeri to 10,192 for Alytes obstetricans) [16, 17, 42], although these variations in reported numbers might be at least partially due to method biases. We registered a significant negative relationship between infection load (number of Bd zoospores) and skin bacterial community richness independent of host species and seasons. Several studies have reported negative relationships between skin bacterial community richness and Bd load in amphibians [e.g., 3]. Skin bacterial community richness but not diversity was negatively correlated with Bd load. Richness can be more sensitive to rare OTUs since all OTUs are counted as either present or absent.

Considering the most abundant bacterial genera for each of the two host species, only four that ranked among the 10 most abundant genera were shared. Additionally, NMDS plots showed little overlap in bacterial community structure, with different dispersion between the two host species. In L. spectabilis, the seasonal (rain vs. dry) skin bacterial community structure was also evident in the NMDS plot (Fig. 4). Several studies [16, 17, 42] have also reported differences in composition and structure of the skin-associated bacterial communities within assemblages of amphibian species. Differences in composition and structure of skin microbial communities among amphibian species may be associated with host traits, such as chemical composition of the skin mucus [19], host behavior, skin shedding rate, and diet [42]. Although we cannot be certain, differences in phylogenetic position (host species are included in two distantly related orders, Caudata and Anura) might be the cause, at least partially, of differences in skin bacterial communities between A. rivulare and L. spectabilis. Rebollar et al. [16] suggest that amphibian species that are phylogenetically closely related might have similar skin chemical conditions, and therefore similarities in bacterial communities.

Additionally, although the studied populations of the two species are co-occurring in the same range, adult stages present differences in life cycle, habitat use, and traits associated with differences in bacterial communities among host species, and such as diet and behavior may contribute to the microbial differences registered in the two species. Ambystoma rivulare is an aquatic salamander with a completely aquatic life cycle [25]. This species is associated with slow-flow streams in pine or pine-oak forests and undergoes metamorphosis, but the adults remain in the water [43]. Diet composition in this salamander is markedly dominated by ostracods. On the other side, L. spectabilis is a semiaquatic frog with a life cycle associated with water, at least during the mating season during which riparian habitats are used [25]. This frog is rarely seen in purely aquatic habitats, and this species uses rock microhabitats and occasionally pool, sand, plant, river, and anthropogenic substrate microhabitats [44]. Although this species is a dietary generalist that feeds on invertebrates, predation on other anurans has been recorded. Finally, if the environmental bacterial source in both types of habitat is different, it would be reasonable to expect differences in the skin bacterial communities between the two host species.

Notwithstanding differences in skin bacterial communities, the most abundant bacterial genus in the two host species was Pseudomonas spp. This bacterial genus is dominant in the skin-associated bacterial communities of several Bd non-susceptible amphibian species [16]. The second and third most dominant genus in the skin bacterial community of L. spectabilis and A. rivulare, respectively, was Stenotrophomonas spp. Both Pseudomonas and Stenotrophomonas genera have been reported to inhibit the intensity of Bd infection [31]. At the level of phyla, microbial skin communities in both host species were dominated by the same three phyla (Proteobacteria, Actinobacteria, and Bacteroidetes). The two dominant bacterial genera, Pseudomonas and Streptrophomonas belong to Proteobacteria, the phylum that dominates skin bacterial communities in amphibians [45] and that comprise about 80% of bacteria with known anti-Bd effects [13]. Of the bacterial genera registered, 73 (55% of total bacterial relative abundance) are considered to be inhibitory of the intensity of Bd infection [13]. Additionally, Pseudomonas, an inhibitory bacterium genus, was the most abundant in both species. Intensity of Bd infection measured as Bd load ranged for both species between 1.75 and 853 ZE (average ZE = 152 ± 35). Longo et al. [46] consider Bd infection to be low in Eleutherodactylus coqui when load is less than 100 Bd zoozpores, and Jani and Briggs [27] consider a Bd load of 10,000 Bd cells to be the threshold at which Rana sierrae succumbs to chytridiomycosis. Therefore, we can assume that in general, Bd infection intensity in both of our studied species ranged from low to moderate.

It is also interesting to highlight the finding of two important bacteria genus in L. spectabilis. These bacteria were not always found in amphibian’s skin: Pedobacter in rainy season and Janthinobacterium in dry season. Pedobacter species have shown Bd-inhibitory effects in Plethodon cinereus and Hemidactylium scutatum [47]. This bacterium genus is characterized by having a high metabolic diversity, what allows it to be dominant in aerobic communities in aquatic and terrestrial environments, and another important characteristic is that Pedobacter thrive over other bacteria genera in changing microclimates [48]. In the case of Janthinobacterium, J. lividum is recognized as a mutualistic partner with salamanders, such as Plethodon cinereus and Hemidactylium scutatum [49]. However, J. lividum is not present naturally on the skin of many amphibians [47]. In fact, this genus is limited to some habitats. For example, J. lividum has been found in many temperate amphibian communities, is extremely rare in low elevation cloud forests [39], and uncommon in tropical regions [39]. This bacterium grows at low temperatures, such as high elevation streams [50] and produces a metabolite, violacein, an agent of chemical defense with broad bioactivity profile including antibacterial, antiviral, and antitumoral activity, and its application has been proposed as a bio-augmentation or probiotic treatment to control chytridiomycosis [47].

We registered a tendency of higher percentage of relative abundance of Bd inhibitory bacteria in the dry season (A. rivulare: rainy season 54% vs. 61% dry season; L. spectabilis: rainy season 53% vs. 57% dry season). In the dry season, we also registered for both host species a smaller number of Bd positive individuals (A. rivulare 10 in rainy season vs. 7 in the dry season; L. spectabilis 11 in rainy season vs. 9 in dry season). A lower Bd load was also registered during the dry season in our study site in the assemblage of amphibians, including the two host species of our study [25]. These findings suggest that environmental conditions such as low temperature (< 7°C) and low humidity (< 50 mm of rain), as well as the higher relative abundance of inhibitory bacteria, might be causal factors in the decrease of Bd load in the dry season. Additionally, we also registered a higher bacterial richness in the dry season for A. rivulare, but not for L. spectabilis. Longo et al. [3] registered a higher skin bacterial richness and higher Bd load in Lithobates yavapaiensis during the winter. Therefore, our results suggest that there might be a synergic effect of temperature, humidity, bacterial richness, and relative abundance of inhibitory bacteria that results in a lower intensity of Bd infection during the dry season.

Although 63 % of sampled individuals of A. rivulare and 80 % of L. spectabilis were Bd positive, no dead or apparently sick individuals were registered during the field surveys. The relatively high percentages of infected individuals of both host species and the low to moderate intensity of Bd load level that apparently do not kill or sicken infected individuals suggest that both host species are able to survive and thrive under a dynamic relation with enzootic infections of Bd. Relative abundance of Bd inhibitory bacterial genera in the skin of both host species might be an important biological defense that limits infection intensity of Bd regardless of host species and seasons. Our study sets a precedent in the amphibian’s microbial ecology studies in Mexico, and it is also suitable for future comparisons with other similar high-altitude studies to reveal epidemiology dynamics of Bd and its relationship with associated bacteria.