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Abstract BACKGROUND Xylophagous insects, as nitrogen‐limited organisms, face severe nutritional constraints due to the inherently low nitrogen content of lignocellulosic substrates—insufficient for growth. To alleviate this limitation, they rely on gut microbiota‐mediated symbiotic nitrogen fixation and nitrogenous waste recycling. Apriona swainsoni , a model wood‐boring cerambycid, exemplifies this adaptation: under extreme nitrogen scarcity in its xylem diet. While gut symbionts are hypothesized to overcome nitrogen limitation, the underlying mechanisms remain unclear. RESULTS First, metagenomic sequencing and functional gene analysis revealed enrichment of nitrogenase and urease genes in the posterior hindgut (PHG). Metaproteomics detected the nitrogenase gene nifU but no urease proteins, identifying nitrogen fixation as the primary nitrogen limitation mitigation strategy in A. swainsoni larvae. Subsequently, in vivo / in vitro 15 N isotope tracing showed peak 15 N in the PHG (105.02% higher than the natural environment) and ~ 25‐fold greater 15 N incorporation in cultured Klebsiella oxytoca versus controls. Targeted amino acid profiling further demonstrated 15 N enrichment in both essential and non‐essential amino acids, with a spatial gradient (intestinal tissues > extra‐intestinal tissues > frass)—indicating efficient microbial conversion of nitrogen into host‐utilizable amino acids. Importantly, we identified that intestinal microbiota primarily mediate ammonia‐to‐amino acid conversion via the glutamine synthetase‐glutamate synthase (GS/GOGAT) pathway in the PHG. This is the first reported GS/GOGAT‐mediated nitrogen fixation pathway in cerambycids. CONCLUSIONS Our comprehensive analysis of gut microbial nitrogen metabolism might elucidate a set of mechanisms by which some xylophagous insects may overcome nutritional constraints in nitrogen‐deficient niches, via evolutionarily optimized host‐microbe metabolic interactions. © 2025 Society of Chemical Industry.
Zhang et al. (Tue,) studied this question.