Supplementary file 1_Assessing the individual and combined effects of ZnO nanoparticles and glyphosate on the gut microbiota of tadpoles.docx

IntroductionFreshwater co-contamination by zinc oxide nanoparticles (ZnO NPs) and glyphosate poses a rising threat to amphibians. While the gut microbiota is central to host metabolism and immunity, the individual and synergistic impacts of these pollutants on tadpole microbial homeostasis—and the resulting vulnerability—remain largely unexplored.
MethodsThis study examines the impact of ZnO NPs and glyphosate, alone and combined, on the gut microbiota of Rana dybowskii tadpoles. Treatments included exposure to ZnO NPs, glyphosate, both, or a control. Total DNA was extracted from intestinal contents samples, and the V3-V4 region of the bacterial 16S rRNA gene was amplified and sequenced on an Illumina MiSeq platform.
ResultsDiversity analyses revealed significant alterations in alpha and beta diversity indices, with the ZnO and glyphosate treatments individually and synergistically disrupting microbial communities. The gut microbiota dysbiosis index (MDI) was elevated in treated groups, indicating microbial imbalance. Dominant phyla such as Firmicutes, Proteobacteria, and Actinobacteriota exhibited significant shifts in abundance across treatments. Functional pathway predictions via Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis highlighted disruptions in metabolic processes, immune function, and disease-related pathways. Ecological assembly models suggested that deterministic processes predominantly governed microbiota changes under pollutant exposure. These findings underscore the detrimental impact of ZnO NPs and glyphosate on amphibian gut microbiota, with potential implications for amphibian health and ecosystem stability.
DiscussionBy integrating diversity metrics, a dysbiosis index, and community assembly models, we show that ZnO NPs and glyphosate can shift the tadpole gut microbiota toward an alternative functional state. This links mixture pollution to microbiota-mediated vulnerability and provides microbial biomarkers and mechanistic targets for amphibian risk assessment.