Gut microbiota-mediated nitrogen recycling in the white Grub Holotrichia longipennis: A model for microbiome-targeted pest control.

White grubs, Holotrichia longipennis, are major agricultural pests that cause extensive crop damage. The gut microbiota plays a critical role in nitrogen metabolism, enabling larvae to thrive on nitrogen-poor diets. Shotgun metagenomic sequencing revealed a diverse gut microbiota dominated by Proteobacteria, Firmicutes, Bacteroidetes, and Actinobacteria, with Enterobacter (32%), Bacillus (20%), and Rhizobium showing clear spatial variation across gut compartments. Functional annotation (FOAM, FAPROTAX) identified bacterial genes involved in uric acid degradation (puuE, allC), urea hydrolysis (ureA, ureB, ureC), and ammonia assimilation via the GS–GOGAT/GDH pathways (gdhA, glnA, glnK, gltB, gltD), whereas nitrogen fixation genes (nifH, nifK, nifD) were absent. KEGG-based quantification (FeatureCounts, HTSeq) showed significantly higher nitrogen metabolism gene abundance in the hindgut, especially gdhA, glnA, and urease genes, which was validated by qPCR with 86-fold, 108-fold, and 34-fold upregulation, respectively. Uricolytic and ammonia-tolerant bacteria (Sporosarcina, Ureaplasma, Corynebacterium, Klebsiella) were isolated and functionally characterized, confirming their active role in nitrogen recycling, with urease assays showing higher ammonia production in the hindgut (0.155 µmol NH₃/min/mg protein). Importantly, antibiotic treatment caused a dose-dependent reduction in gut bacterial abundance and larval survival, demonstrating the essential role of symbionts in host physiology. This study provides the first comprehensive evidence that H. longipennis larvae depend on gut microbes for nitrogenous waste recycling and identifies gut nitrogen-cycling bacteria and their key metabolic pathways (urease and ammonia-assimilation systems) as actionable targets for microbiome-based pest control strategies.