The oriental armyworm, Mythimna separata, is a major insect pest in China and other Asian countries. Unfortunately, suitable reference genes for quantitative real-time PCR (qRT-PCR) have not been previously identified in M. separata for normalizing target gene expression. In this study, we evaluated the expression stability of eight candidate genes (18S, ACT, EF1-α, GAPDH, RPS7, RPS13, RPL32 and TUB) in M. separata using the comparative ΔCt method, BestKeeper, Normfinder geNorm and ReFinder, a comprehensive software platform. The results indicated that the appropriate reference gene varied depending on the experimental conditions. We found that ACTIN, EF1-α and TUB were optimal for different developmental stages; TUB, RPS13 and EF1-α showed the most stable expresssion in different tissues; RPS13 and 18S were the best reference genes for monitoring expression under high temperature conditions; TUB, RPS13 and RPS7 exhibited the most stable expression under larval-crowding conditions; RPS7, EF1-α, RPL32 and GAPDH were the best for pesticide exposure experiments. This study provides tools for reliable normalization of qRT-PCR data and forms a foundation for functional studies of target gene expression in M. separata.

The gut microbiota of insects plays a fundamental role in modulating host physiology, including nutrition, development, and adaptability to environmental challenges. The rice water weevil, Lissorhoptrus oryzophilus Kuschel (Coleoptera: Curculionidae), is a major invasive pest of rice worldwide, yet the composition and functional profile of its gut microbial community remain poorly characterized. Here, we employed metagenome sequencing on the Illumina NovaSeq X Plus platform to explore the gut microbial diversity and predicted functions in adults of L. oryzophilus. Our results revealed a rich microbial community, comprising 26 phyla, 42 classes, 72 orders, 111 families, and 191 genera. The bacterial microbiota was overwhelmingly dominated by the phylum Proteobacteria (85.13% of total abundance). At the genus level, Pantoea (48.86%) was the most predominant taxon, followed by Wolbachia (14.57%) and Rickettsia (11.81%). KEGG analysis suggested that the gut microbiota is primarily associated with metabolic pathways such as membrane transport, carbohydrate and amino acid metabolism, cofactor and vitamin metabolism, energy metabolism, and signal transduction. eggNOG annotation further highlighted significant gene representation in amino acid and carbohydrate transport and metabolism, while CAZy annotation revealed glycosyl transferases (GTs) and glycoside hydrolases (GHs) as the dominant carbohydrate-active enzymes. This study provides the first comprehensive insight into the gut microbiome of L. oryzophilus adults, highlighting its potential role in the ecological success of this invasive pest. Our findings lay groundwork for future research aimed at developing novel microbial-based strategies for the sustainable management of L. oryzophilus.