Dynamic responses of gut microbiota to agricultural and wildfire ash: insights from different amphibian developmental stages

Abstract

Combustion by-products-specifically wildfire ash and rice-straw ash-are emerging contaminants in freshwater ecosystems. However, their impacts on amphibian survival and gut microbiota across various developmental stages remains largely unclear, thereby limiting evidence-based conservation strategies in fire-affected habitats. This study evaluated the effects of artificial water (control, C) and aqueous extracts of ash (AEAs) derived from wildfire ash (W) and rice straw ash (S) on the survival and gut (G) microbiota of Rana dybowskii tadpoles (T) and adult frogs (F). Exposure to wildfire ash significantly reduced tadpole survival compared to rice straw ash, whereas no significant differences were observed in adult frogs. Alpha diversity of the gut microbiota differed significantly among tadpole groups but not among adult groups. Bray-Curtis and weighted UniFrac analyses revealed significant differences in the gut microbiota of adult frogs and tadpoles across different treatment groups. Linear discriminant analysis effect size (LEfSe) identified a significant enrichment of specific bacterial genera across treatment groups. BugBase analysis indicated that in the TCG, TSG, and TWG groups, notable variations in the TCG, TSG, and TWG groups, there were notable differences in Forms-Biofilms and Potentially-Pathogenic, while in the FCG, FSG, and FWG groups, significant differences were observed in Aerobic, Gram-Positive, Potentially-Pathogenic, and Stress-Tolerant. These findings suggest that wildfire ash exhibits greater toxicity than rice straw ash to both life stages of R. dybowskii, with tadpoles being more vulnerable. By elucidating the link between ash-derived pollutants and amphibian gut health, this study underscores the growing threat of routine straw burning and intensifying wildfires to global freshwater biodiversity and advocates for ash-specific mitigation measures and microbiota-informed conservation strategies.

Keywords: Rana dybowskii; gut microbiota; rice straw; survival; wildfire ash.

Figure 1

Survival rate of R. dybowskii adults and tadpoles exposed to various types of ashes. The level of significance compared to the control was represented by “a,” “b,” and “c.” One-way ANOVA followed by Tukey's HSD test and Benjamini-Hochberg FDR correction was used to analyze the statistical difference between the control and the experiments. (A) Adults stage survival rate of rice straw ash and wildfire ash (30 d). (B) Survival rate of the tadpole stage of rice straw ash and wildfire ash (28 d).

Figure 2

Examining the influence of straw ash and wildfire ash on frogs and tadpoles' microbiota beta diversity utilizing Non-metric multidimensional scaling (NMDS) analysis. Each data dot signifies a specific sample procured from the gut, and each color represents a distinct group. Showing the 95% confidence ellipses for samples taken on six groups. (A) The Bray-Curtis dissimilarity matrix was used to perform NMDS analysis comparing adults and tadpoles in the control, rice straw ash, and wildfire ash groups. (B) NMDS based on the weighted-UniFrac distance matrix of adults and tadpole stages at control, rice straw ash, and wildfire ash groups.

Figure 3

Relative abundances of gut microbiota compositions in control, straw ash, and wildfire ash adults and tadpoles. Show only those phyla and genera taxa with relative abundances exceeding 1% in a minimum of one sample. Relative abundances of the dominant phyla (A,C) and genera (B,D) of adults and tadpoles' gut microbiota. The x-axis represents the sample names, and the y-axis represents the proportion of species in each sample. Different colored bars represent different species, and the length of the bars indicates the proportion of each species.

Figure 4

Cladogram of linear discriminant analysis effect size (LEfSe) results (from phylum to genus level) according to the different groups. Post-treatment color differentiation was classified into multiple distinct groups. The FCG/TCG group represents control samples, FSG/TSG group indicates straw ash samples, FWG/TWG group represents wildfire ash samples, and F indicates frog stage (B) and T indicates tadpole stage (A). The abundance of each group was shown by the diameter of the corresponding circle. A versatile multiclass analysis displays at least one class difference. Inner to outer circles represent taxonomic classifications from domain to genus. Depictions of phylum, class, order, family, and genus labels were present. Taxa exhibiting an LDA > 4 were illustrated.

Figure 5

Phenotype prediction of bacteria by BugBase analysis. Assessing the influence of straw ash and wildfire ash on adult (B) and tadpole (A) gut microbiota phenotypes, bacterial characteristics were examined and projected using the BugBase approach. The Kruskal-Wallis H-test was used to analyze the statistical difference between the control and the experimental groups. P is the significant difference value: *P < 0.05, **P < 0.01, ***P < 0.001.