host transcriptomics to characterize the response of the H. illucens to selenite.
Larvae body samples from the CK and Se20 groups (collected on days 7 and 14) were used for RNA extraction using the TRNzol Universal Reagent kit (TIANGEN Biotech, Beijing, China) following the manufacturers instructions. RNA concentration,purity and integrity was checked using the Bioanalyzer 5400 system (Agilent Technologies, CA, USA). Library construction and transcriptome sequencing were performed at Shenzhen Wekemo Technology Group Co., Ltd. The cDNA synthesis, adapter ligation, and PCR amplification were performed following mRNA purification. The RNA-seq library was prepared according to the manufacturers instructions for the NEB Next Ultra TM II DNA Library Kit (Beckman). The library sequences of the gut samples were obtained by DNBSEQ-T7 Platform, producing 150 bp paired reads. Raw sequencing data were quality filtered using Trimmomatic (v0.39). Clean reads were aligned to the reference genome (Hermetia_illucens. GCA_905115235.1.genome.fa.) using HISAT2 (Kim et al., 2015), and gene expression patterns were quantified in Fragments Per kb Per Million Reads (FPKM) using HTSeq-count. Differentially expressed genes (DEGs) were calculated using the DESeq2 R package (1.16.1). To eliminate any technical bias arising from unequal sequencing depth across samples (particularly the deeply re-sequenced Se12-2), gene expression counts were normalized internally via DESeq2s median-of-ratios method prior to statistical testing. Genes that exhibited |log2(fold change)|  1 and a falsediscovery-rate-adjusted p value <0.05 were considered differentially expressed. To elucidate the biological functions of the DEGs, Gene Ontology (GO) and KEGG pathway enrichment analyses were conducted, with significance established at a false discovery rate (FDR) p-value of less than 0.05(Memon et al., 2025)