Metatranscriptomic insights into nitrogen loss mediated by multi-kingdom microbial interactions in periphytic biofilms under imidacloprid exposure

Periphytic biofilms are ubiquitous and drive biogeochemical nutrient cycling in paddy ecosystems. However, the mechanisms of multi-kingdom microbial interactions regulating nitrogen (N) cycling in the biofilms remain unexplored. This study conducted a microcosm experiment to investigate the effects of imidacloprid (IMI), a widely used neonicotinoid pesticide, on N cycling in periphytic biofilms. Throughout the 30-day experiment, 0.02 to 0.12 mg of N was lost from the water-biofilm microcosm systems, increasing with higher IMI exposure concentrations. Metatranscriptomic sequencing demonstrated 84.3%–92.2% downregulation of nitrification genes and 33.9%–143% upregulation of denitrification genes in the biofilms. These alterations in gene expression were regulated by microbial competition and cooperation within ecological clusters associated with the biofilm potential nitrification and denitrification activities, respectively. Correlation between the relative abundance of microorganisms in these clusters and gene abundance revealed multi-kingdom nitrifying and denitrifying communities, including Nitrosopumilus (Archaea) and Nitrospira (Bacteria) for nitrification, and Enterococcus (Bacteria) and Aspergillus (Fungi) for denitrification. Partial least squares path modeling indicated that the biofilm-mediated N loss was driven by promoted denitrification (total effect = -0.83) rather than suppressed nitrification (total effect = 0.52). Our findings highlight multi-kingdom microbial interactions regulating N cycling in periphytic biofilms and neonicotinoid pesticide risks.