Insect microbiomes are influenced by numerous environmental and genetic factors, including geographic location, climate, and host genotype. These factors can significantly impact insect phenotypes, such as insecticide resistance. The brown planthopper (*Nilaparvata lugens*) is a major rice pest in Asia exhibiting resistance to multiple insecticides. Its annual migration across diverse regions exposes it to varying insecticide pressures and environmental conditions, resulting in geographically varying resistance profiles. Insecticide resistance in *N. lugens* is partly attributed to the expression of cytochrome P450 (P450) genes, such as *NICYP6ER1*, a key gene mediating resistance to several insecticides. Recent studies suggest that bacterial symbionts can influence *N. lugens* insecticide resistance by regulating host detoxifying gene expression. This study aimed to explore the correlation between host genetics, microbiome composition, gene expression, and insecticide resistance in various *N. lugens* strains. Specifically, the study investigated whether host genetics or microbiome variations correlate with insecticide resistance and if specific environmental factors predict microbiome variation patterns. Nine field strains (FS) from different geographic locations and two laboratory strains (LS) were compared. The study hypothesized that insecticide susceptibility phenotypes would be associated with both microbiome variations and the expression levels of key detoxifying genes, and that certain environmental abiotic factors could predict these microbiome variations.

Existing literature demonstrates the impact of environmental factors and host genotype on microbiome composition across various insect species. Studies have shown temperature-dependent changes in microbiome composition affecting host insecticide resistance. In *N. lugens*, insecticide resistance is linked to the expression of P450 genes, including *NICYP6ER1*. Previous research also highlighted the role of bacterial symbionts in modulating *N. lugens* insecticide resistance through regulation of host detoxifying gene expression. While the impact of insecticide exposure on resistance evolution is well-studied, other environmental factors remain less explored. This study builds upon this existing research by integrating the effects of host genetics, microbiome composition, gene expression, and environmental factors to gain a comprehensive understanding of insecticide susceptibility variation in *N. lugens*.

Nine field strains (*N. lugens*) were collected from various locations in China in 2019, along with two laboratory strains. Insecticide susceptibility was assessed using a rice-seeding dip method, exposing third-instar nymphs to eleven insecticides at various concentrations. Mortality was recorded after specific durations depending on the insecticide. Transcriptome sequencing was performed on RNA extracted from pooled third-instar nymphs to analyze gene expression levels. Microbiome profiling was conducted using 16S rRNA and ITS amplicon sequencing on cDNA synthesized from extracted RNA, focusing on bacterial and fungal communities. Core microbiome taxa were defined as those with a relative abundance exceeding 0.05% and present in more than 60% of samples. Genetic background analysis was performed using inter-simple sequence repeat (ISSR) markers. Statistical analysis included LC50 calculations (Polo Plus), Bray-Curtis dissimilarity matrices (vegan), PERMANOVA, Spearman correlation (WGCNA), and network analysis (Gephi). Correlations between microbiome diversity, environmental factors (temperature, precipitation, latitude, longitude), and geographic distances were also analyzed.

Insecticide susceptibility varied significantly among the *N. lugens* strains. Clothianidin showed the highest inter-strain variability, while triflumezopyrim showed the lowest. Neonicotinoid insecticides exhibited greater susceptibility variations. The susceptibility profiles of laboratory strains were distinct from field strains. Positive correlations were observed between insecticides acting on nicotinic acetylcholine receptors, indicating cross-resistance. Transcriptome analysis revealed significant differences in gene expression among strains, with a strong correlation between *N. lugens* transcript abundances and insecticide susceptibilities. Many genes correlated with insecticide susceptibility were shared, especially among insecticides targeting the same group. The expression of *CYP6ER1*, a key P450 gene, was positively correlated with the LC50 of seven insecticides. Microbiome analysis identified 372 bacterial and 1732 fungal taxa. Inter-strain variations in bacterial and fungal communities were significant. Seven P450, two GST, and three EST genes correlated with specific bacterial taxa, suggesting that bacterial abundance could be a marker for insecticide susceptibility. The expression of several *NICYP6ER1* variants correlated with the abundances of six bacteria. The abundance of *Arsenophonus* positively correlated with *NICYP6AX1* expression. Core microbiome diversity weakly correlated with host genetic background, indicating stronger environmental influences. Fungal, but not bacterial, α-diversity significantly correlated with temperature and longitude. The abundance of several bacterial and fungal genera significantly correlated with environmental factors, some of which also correlated with detoxifying gene expression.

This study demonstrates that environmental abiotic factors are stronger predictors of *N. lugens* microbiome variation than host genotype. The observed variations in insecticide susceptibility are likely a result of complex interactions between the environment, microbiome, and host detoxification metabolism. The higher basal expression of detoxifying genes in some strains might be a consequence of chronic insecticide exposure in specific locations. The study supports the concept of an “insecticide-resistant microbiome,” where the presence or abundance of certain taxa could predict resistance. The horizontal transmission of symbionts, like *Arsenophonus*, may contribute to the spread of insecticide resistance. The lack of correlation between *Wolbachia* and detoxifying gene expression in field strains suggests that its impact on resistance may be strain-specific or dependent on host genotype. Future research should focus on the impact of climate change on microbiome diversity and its subsequent effect on insecticide resistance.

This research reveals a significant correlation between microbiome variation and insecticide susceptibility in *N. lugens* field strains. Environmental factors, rather than host genetics, are major drivers of microbiome variation and insecticide resistance. The study underscores the importance of considering microbiome-host-environment interactions in insecticide resistance management strategies. Future research should investigate the causal relationships between specific microbiome members and insecticide resistance and explore the application of microbiome manipulation for resistance management.

The study's analyses were based on *N. lugens* samples collected in a single year, potentially limiting the generalizability of the findings. The study did not fully evaluate the contribution of genetic background variations among strains to insecticide susceptibility differences. Further research is needed to establish causality between microbiome variation and insecticide resistance, rather than simply identifying correlations.