and Prevention, Convention Center, Santa Fe, New Mexico. Information: Medical Support Systems, AHA Epidemiology. Conference Abstract Submissions, At the genus level, we found in Santa Ana, Puerto Rico, a high dominance new niches as was first reported by a large mammalian study (Ley et al., ), .. Harrell, F. E. (). 23, – doi: /mec Cargill, Charles W., Cypress Avenue, Chino, .. ley, Pennsylvania Ewing, George , Santa Fe, New Mexico Eyman.

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Ecology of Amphibian-Microbial Symbioses. Rhinella marina is ly toad native to South America that has been introduced in the Antilles, likely carrying high loads of microorganisms, potentially impacting local community diversity. The amphibian skin is involved in pathogen defense and its microbiota has satna relatively well studied, however, research focusing on the cane toad microbiota is lacking. We hypothesize that the skin microbial communities will differ between toads inhabiting different geographical regions in Central America santz the Caribbean.

To test our hypothesis, we compared the microbiota of three populations of R. Separate swab samples were collected from the dorsal and ventral 12150 resulting in 42 samples. We found significant differences in the structure of the microbial communities between Puerto Rico and Costa Rica.

We detected as much as 35 different phyla; however, communities were dominated by Proteobacteria, Bacteroidetes, Firmicutes, and Actinobacteria. Alpha diversity and richness were significantly higher in toads from Puerto Rico and betadiversity revealed significant differences between the microbiota samples from the two countries.

This is the first report of Niabella associated with the amphibian skin. These results provide insights into the habitat-induced microbial changes facing this amphibian species. The differences in the microbial diversity in Puerto Rican toads compared to those in Costa Rica provide additional evidence of the geographically induced patterns in the amphibian skin microbiome, and highlight the importance of discussing the ef tradeoffs in the colonization of new ecosystems.

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In the last 30 years amphibians have undergone massive population declines Whittaker et al. This phenomenon is attributed to climate change, habitat loss, pollution, and the presence of emerging infectious diseases, among other causes Whitfield et al. It is suggested that the appearance of these emerging diseases is due to the introduction of exotic pathogens, such as Batrachochytrium dendrobatidis Bd Longcore et al. Pathogen spread has also been attributed to human trafficking of amphibian species Bacigalupe et al.

Due to the increase of infectious diseases, introduced species represent a constant threat to local fauna Schloegel et al. Problems with introduced amphibians and reptiles have occurred worldwide, as in the case of the bullfrog Lithobates catesbeianus in the western areas of the United States, the Caribbean, and in South America Young et al.

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In Puerto Rico, a decline of several native amphibian species has been documented, and among other possible factors is the introduction of the pathogen Bddrought, and habitat loss Burrowes et al. In addition, Puerto Rico has a great number of introduced species maintaining a constant threat to the native fauna, including six species of frogs Joglar et al. The cane toad is one of such species, introduced in Puerto Rico in the early 20th century aiming at controlling a beetle infestation in sugarcane plantations, successfully halting the damage Tyler, ; Thomas, The cane toad has, in fact, a broad geographic distribution.

Many of lsy introductions have been made with the aim of controlling agricultural pests, but have had little proven success. The cane toad has become a constant threat and the Invasive Species Specialist group of the Union for Conservation of Nature IUCN has declared it pey of the most damaging invasive species in the world Lowe et al.

Recent taxonomic changes subdivided this species into R. When introducing an exotic species, either accidentally saanta intentionally, the potential pathogens that can be loaded are generally not analyzed, because molecular microbiological essays are never performed.

It has been documented that the cane toad can carry Salmonella species that can affect other native species Eanta et al. These pathogens can be a severe problem to local fauna since invasive species are difficult to control and eliminate. Furthermore, some frog species are much less susceptible to death from particular pathogens and may act as carriers; for example, the cane toad is less susceptible to Bd but can still carry it as asymptomatic infections Lips et al.

It is now possible to study the diversity of microbial communities in any habitat or species through next-generation sequencing, an approach that has allowed researchers to characterize the patterns of changes in the microbiota, revealing possible pathogens and symbionts associated with a given host Rebollar et al. One such sana is the resistance of some frogs to pathogens, likely due to the presence of beneficial bacteria in their skin Harris et al. Culture-independent techniques have shown differences in bacterial diversity depending on the degree of Bd infection among the same amphibian species Rebollar et al.


Variations in sanra skin microbiota of species across different geographies have been attributed to fw factors, including: Similarly, amphibian bacterial communities have been compared between families in temperate and tropical regions Belden et al. To bridge this knowledge gap, this work represents the first report comparing the microbial communities of R. We hypothesize that there will be differences in the skin microbial communities between the dorsal and ventral sides of toads, and between the three sampling locations in its native Lye Rica and exotic Puerto Rico ranges.

Here, we identify the differences between microbial communities of toads in Puerto Rico and Costa Rica, define the unique taxa for each santw, and define which bacterial groups compose the core microbiome of this species. Here we applied the Holdridge classification system Holdridge, that considers tropical altitudinal sants to be in a range of 0— m.

A total te 21 Cane toads were collected using disposable nitrile gloves. Each toad was washed for 7 s using 50 ml of sterile distilled water to reduce transient surface bacteria Madison et al. Sterile swabs were santaa 10 times in the ventral and the dorsal area of toad, yielding two samples per individual. This study was exempt from IACUC protocol review since animals were collected without interfering with its environment.

After the brief sterile skin swabbing in situtoads were released immediately in their natural environment.

A total of 42 swab samples were obtained from the ventral and dorsal skin surfaces of toads, 20 from Puerto Rico and 22 from Costa Rica. For each individual toad, we measured the following parameters: Environmental variables including temperature, humidity, and precipitation were obtained from nearby meteorological stations in both countries.

A first quality control analyses using FastQC Andrews, revealed that only forward reads were useful for downstream analyses. Analyses were done in two ways: The resulting OTUs underwent rarefication to mitigate bias due to different sequence depth per sample.

Values in the mapping file were also collapsed by grouping dorsal and ventral samples into one sample. The data analyses were performed using a rarefaction level of sequences per sample when considering all 42 samples dorsal and ventral swabsand of 32, sequences when collapsing dorsal and ventral samples in individuals, to avoid the bias caused by differences in sequence depth.

This core diversity workflow does an extensive diversity analyses including alpha rarefaction diversity analyses such as the Chao 1 abundance-based richness estimator and the phylogenetic diversity PD metric of Faith, both computed in QIIME. Chao 1 values represent the estimated true species richness of a sample and are calculated with the script for alpha rarefaction in QIIME that in turn implements the Chao 1 abundance-based estimator Chao, It also calculates the PD metric of Faith, which does not take abundance into account but rather branch lengths of the phylogenies connecting all species to each community Faith, Beta diversity analysis was performed as a non-metric multidimensional scaling plot NMDS using the Bray—Curtis distance metric and calculating stress values using the R packages Phyloseq McMurdie and Holmes,vegan Oksanen et al.

The significantly different phyla as determined by ANOVA, as well as the selected genus-level OTUs significantly associated with each location, were visualized as boxplots combining R packages ggplot2 Wickham,RColorBrewer Neuwirth,and scales Wickham, A heatmap of the significantly different taxa FDR-adjusted p -values for the two metadata categories location and country was built using heatmap.

Data normalization was done through DESeq2 negative binomial Wald normalization for visualization purposes due to differences in the numbers of individuals per sample. Significant differences of alpha diversity were calculated using a non-parametric two-sample t -test using Monte-Carlo permutations using the QIIME Caporaso et al.

Significance tests were computed for each group comparison with the Chao1 abundance-based estimator, the alpha PD metric of Faith, and the Shannon index, for the sample dataset. Same significance tests on alpha PD and Chao 1 were used on the sample dataset. We performed Analyses of Variance tests using the aov function in R R Development Core Team, on the abundance values at each taxonomic Phyla, using the -biom-derived data matrices from QIIME L2 tablecomparing the relative abundance of each Phyla in the three sampling locations.

Boxplots of the significant changes at the phyla level were plotted with ggplot2 Wickham, and RColorBrewer Neuwirth,using a normalized table of values, by running the R interface package of DESeq2 for table normalization, DESeq outputs negative values for lower abundant OTUs as a result of its log transformation.

A total of 5, good quality sequences were employed in the analyses. Environmental measurements in the collection sites were very similar across the three locations, confirming that these sites have the same tropical environmental conditions in both countries.


We found no significant differences between the microbial community structure in dorsal and ventral samples in any of the three locations Figure 1. We found a total of 35 assigned phyla, with 6 of these dominating across all the samples: Overall, at the genus level we found a dominance of Niabella and Pseudomonas across all samples Figure 1B.

The PD was nearly identical between ventral and dorsal swab samples at each of the three locations: Santa Ana dorsal vs. As the analyses of the 42 samples did not show significant differences, we collapsed the dorsal and ventral samples considering now 21 samples, one per individual. Microbiota diversity in dorsal and ventral swab samples among toads in Puerto Rico and Costa Rica. A Taxonomic bar plots showing bacterial phyla among ventral and dorsal samples.

B Taxonomic bar plots at the genus level. Hence, considering the 21 individuals, microbial communities in the samples from Puerto Rico were clearly grouped together as shown by NMDS based on the relative dissimilarities of the samples Bray—Curtis with a stress value of 0.

Costa Rican samples show a close aggregation with Puerto Rican samples, especially those from Turrialba Figure 2. As discussed before, the dominating phyla were Proteobacteria, Bacteroidetes, Actinobacteria, and Firmicutes Figure 3. Taxonomic profiles at the phyla-level A,B and genus-level C,D.

Significantly different phyla among the two countries. Abundances were normalized through DESeq2 negative binomial Wald normalization. Tables representing the sant abundance values for each sample at the phyla and genus levels can be found in the Supplementary Tables S5S6. Rarefaction analyses were based on 32, sequences per sample type.

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Taxonomic profile of core OTUs. Significantly different taxa between countries resulted in 20 OTUs, most remarkably an abundance in Niabella and Flavobacteriaceae in Puerto Rico, and a dominance of Halomonas in Costa Rica Figures 78. Capitalizing on advances in next-generation sequencing, several recent studies on amphibian skin microbiota have lye the importance of cutaneous microbes for host disease resistance Kueneman et al. This is the first report of the microbiota of the successful toad colonist R.

Given that we had a small sample number at each location and only two countries were compared, we will limit the discussion to geographical differences and the possible effects of habitat and environment.

Overall, many genera found in this study correspond to previous reports in other bufonids. In fact, PseudomonasSphingobacteriumand Bacteroides were the most common genera found in the western toad, Anaxyrus boreas Kueneman et al.

All these genera, except Pantoeawere represented in the Rhinella microbiota. Microbial symbioses have been considered a foundational principle for the invasive success of several different species. Microbiomes enhance the capability of species to adapt to new niches as was first reported by a large mammalian study Ley et al. We found that alpha diversity measures were significantly higher in Puerto Rico where R. Interestingly, a similar pattern was found in plant bacterial communities, where native plants shown key have lower microbial species diversity and increased abundance of pathogens compared to their invasive counterparts Coats and Rumpho, The high diversity in the Puerto Rican samples may be related to a number of factors including environmental or genetic factors associated with different populations ve seen in other amphibians Kueneman et al.

A higher diversity in the Puerto Rican frogs those in the native range may provide the host with a plethora of antimicrobial peptides, and the capacity to use resources more efficiently than communities with low species richness in the native range.

Like plant roots, the toad skin surface is in close contact sanga the environment, mainly with soil and water; therefore, it would not be surprising to find microbial lej in frog skin to have similar patterns as those of plants in introduced environments.

Interestingly, statistical tests on beta diversity confirm significant differences between toad microbes in the two geographies, similar to the separation between microbiota of frogs from tropical and temperate zones Belden et al. Although the impact of host factors on the skin microbiota is acceptable, it is still poorly understood fd environmental factors influence the biogeographic patterns of microbial communities in amphibians, which may be due to precipitation or even nitrogen deposition in these tropical ecosystems Hietz et al.

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