Amphibian tolerance to arsenic: microbiome-mediated insights

Abstract

Amphibians are often recognized as bioindicators of healthy ecosystems. The persistence of amphibian populations in heavily contaminated environments provides an excellent opportunity to investigate rapid vertebrate adaptations to harmful contaminants. Using a combination of culture-based challenge assays and a skin permeability assay, we tested whether the skin-associated microbiota may confer adaptive tolerance to tropical amphibians in regions heavily contaminated with arsenic, thus supporting the adaptive microbiome principle and immune interactions of the amphibian mucus. At lower arsenic concentrations (1 and 5 mM As³⁺), we found a significantly higher number of bacterial isolates tolerant to arsenic from amphibians sampled at an arsenic contaminated region (TES) than from amphibians sampled at an arsenic free region (JN). Strikingly, none of the bacterial isolates from our arsenic free region tolerated high concentrations of arsenic. In our skin permeability experiment, where we tested whether a subset of arsenic-tolerant bacterial isolates could reduce skin permeability to arsenic, we found that isolates known to tolerate high concentrations of arsenic significantly reduced amphibian skin permeability to this metalloid. This pattern did not hold true for bacterial isolates with low arsenic tolerance. Our results describe a pattern of environmental selection of arsenic-tolerant skin bacteria capable of protecting amphibians from intoxication, which helps explain the persistence of amphibian populations in water bodies heavily contaminated with arsenic.

Figure 1

Sampling regions of our focal study species. TES Tripuí Ecological Station, an area naturally contaminated by metals, located in the state of Minas Gerais (yellow). JN municipality of João Neiva, uncontaminated area, located in the state of Espírito Santo (green). Images (a–e) highlight the morphological characteristics of the five species sampled in both regions.

Figure 2

Analysis of the tolerance of bacterial isolates to different concentrations of arsenic. (A) Evaluation of the degree of tolerance of bacterial isolates in different arsenic concentrations. n: total number of tolerant isolates in the investigated conditions. TES contaminated area and JN uncontaminated area. n: total number of tolerant isolates in the investigated conditions. p-values of pairwise comparisons are shown. In the box plots, the median is depicted by a horizontal line, while the box marks the first and third quartiles. Vertical lines extend to the maximum and minimum values. (B) Evaluation of the degree of tolerance of bacterial isolates in our focal amphibian species sampled JN and TES sites. Numbers ranging from 0 to 100 represent the percentage of tolerance assessed at each of the six growth times. Colors represent arsenic concentrations to which isolates exhibited tolerance.

Figure 3

Steps involved in the construction of the apparatus for simulating exposure to arsenic and analysis of the influence of skin-associated microbiota on the permeability of this contaminant. (A) Ilustrative summary of the steps described in the methodology. Numbers 1 to 7 represent the following steps: 1—reactivation of the selected bacteria; 2—in parallel, fragments of bullfrog skin (Lithobates catesbeianus) were prepared; 3—skin fragments were placed in contact with bacterial cultures in Petri dishes and incubated at 28 °C for 24 h; 4—after incubation, skin fragments were removed from the Petri dish and separated; 5—skin fragments were tied to the open end of the test tube containing the arsenic solution (5 mM NaAsO₂ final concentration); 6—test tube containing the tied skin was exposed to Amphibian Ringer's solution (ARS); 7—test tube was suspended by a rigid black paper plate, thus limiting the contact of the ARS with the environment. (B) Method for measuring the change in conductivity due to the permeabilization of arsenic through the amphibian skin, previously treated with bacteria.

Figure 4

Qualitative analysis of the results obtained from the amphibian simulation apparatus to contaminated environments. (A) Growth chronology and tolerance profile of selected isolates at different arsenic concentrations. JN1 and JN2: isolates obtained from an uncontaminated area. TES1, TES2, and TES3: isolates obtained from contaminated area. Colored squares represent the times in which the growth rate of bacterial isolates was confirmed in the following arsenic concentrations (1 mM yellow, 5 mM orange, and 10 mM red). (B) Change of conductivity in Amphibian Ringer's solution after 24 h. The observed increase in conductivity was directly associated with diffusion of arsenic through the amphibian skin. Error bars (SE) were determined from three independent experimental trials. (C) Summary of qualitative results associated with the use of the apparatus. + represents positive bacterial growth and- negative. Values within parentheses are average ± standard deviation. Upward facing arrows indicate increased conductivity after 24 h of testing. Levels not connected by the same letter are significantly different according to aposteriori Tukey HSD test for multiple comparisons. The number of arrows is directly associated with increases of conductivity values.

Figure 5

Model proposed to suggest how the epithelial microbiota interferes in the adaptation of the anurans in contaminated environments. (A) Resilience (left) and animal susceptibility are dependent on the tolerance profile of the associated bacterial species. Amphibian species that have tolerant microbiota may not be good indicators of environmental contamination. (B) Metabolic diversity is found in bacteria associated with arsenic metabolism. 1—Biofilm reduces metal contact with animal tissue; 2—redox reactions reduce ion toxicity; 3—biotransformation reactions transform more toxic species into less toxic ones; 4—efflux of ions or molecules carrying the contaminant; 5—chelation of harmful ions to organic molecules; 6—activation of oxidative species metabolism, increasing cell protection.