Single-cell transcriptomic profiling reveals diet-dependent dynamics of glucosinolate sulfatases expression and cellular origin in the midgut of Plutella xylostella.

The diamondback moth (Plutella xylostella), a globally destructive pest, has Brassicaceae as its long-term co-evolved host and can also utilize Fabaceae as an alternative field host. The primary differential factor between these plant families is glucosinolates (GLs). Conventional transcriptome data revealed high midgut expression of glucosinolate sulfatases (GSSs) in response to glucosinolates. However, due to the inability of conventional transcriptomics to resolve cellular heterogeneity, it remained unclear which specific cell types express GSSs and whether exposure to different field hosts triggers distinct differentiation fates in these cells. To address these questions, we constructed a high-resolution atlas of 28,451 midgut cells from larvae reared on radish and pea leaves. Marker gene-based clustering identified 16 distinct cell types, including enterocytes (ECs), enteroendocrine cells (EEs), and intestinal stem cells (ISCs). ScRNA-seq and qRT-PCR analyses revealed host-dependent differential expression patterns of three GSS genes (GSS1-GSS3) among ECs, EEs, and ISCs. Notably, elevated GSS3 protein levels in radish-fed larvae were confirmed by Western blot (WB) and Immunohistochemistry (IHC), while GSS1 and GSS2 exhibited more variable expression patterns. Pseudo-time trajectory analysis further demonstrated that all three GSSs followed differentiation pathways from ISCs into EEs or ECs, but dietary conditions led to distinct differentiation trajectories. This study elucidates the diet-dependent regulatory landscape of GSSs in P. xylostella and delineates the differentiation trajectory of GSS-expressing cells.