Microbial zonation and functional roles in the gut of white grub (Maladera insanabilis) larvae

Maladera insanabilis, a widespread and destructive agricultural pest in India, thrives in nitrogen-deficient subsoil environments due to its dependency on gut bacteria. In particular, the hindgut is an anaerobic fermentation chamber, supporting microbial-driven nitrogen transformations essential for larval development. Despite its ecological significance, detailed studies exploring gut bacterial diversity and functional role in M. insanabilis are lacking. This study integrates metagenomics, culture-based techniques, enzymatic assays, and gene expression analyses to characterize the nitrogen-cycling potential of gut microbiota along the different gut compartments. The culture-based analysis isolated 16 aerobic and 8 anaerobic bacterial strains, predominantly from Bacillota and Pseudomonadota. High-throughput 16 S rRNA Illumina sequencing revealed 134 shared amplicon sequence variants (ASVs), with distinct bacterial assemblages, Burkholderia and Pseudomonas in the foregut, Paenibacillus in the midgut, and anaerobic genera such as Bacteroides and Desulfovibrio dominating the hindgut. Functional annotation using the KEGG database indicated that anaerobic gut bacteria are actively involved in nitrification, denitrification, and nitrogen fixation. The Enzyme assays confirmed high nitrate and nitrite reductase activity, with Burkholderia contaminans and Bacillus cepacia showing the highest activities. Michaelis-Menten kinetics and Lineweaver-Burk analysis (R² = 0.9871) showed a higher capacity (Vmax) for nitrate and nitrite reduction; a small Km indicates a high affinity for nitrate and nitrite. Gene expression studies viz., hzo, nifH, amx, nirS, and nirK revealed a significantly high expression level in the hindgut, especially under vermicompost treatment. This study provides the first comprehensive insight into nitrogen-cycling gut bacteria in M. insanabilis, highlighting their role in host nutrition and nitrogen transformation. These findings lay a foundation for future microbiome-targeted pest control strategies aimed at disrupting nutrient acquisition in soil-dwelling grubs.