First site-specific N-glycoproteome of the spider mite Tetranychus urticae reveals a glycan signature different from insect models and functional insights for pest management.

Protein N-glycosylation is a fundamental post-translational modification that shapes protein folding, stability, trafficking, and biological function. In arthropods, current understanding of N-glycosylation is largely derived from insect models, whereas chelicerates, including mites and ticks, remain poorly characterized despite their ecological and economic importance. Here, we present the first comprehensive, site-specific N-glycoproteomic analysis of a chelicerate arthropod, the two-spotted spider mite Tetranychus urticae, a globally important agricultural pest characterized by rapid development, high fecundity, extreme polyphagy, and exceptional resistance to chemical control. Using intact glycopeptide enrichment coupled with high-resolution Orbitrap LC-MS/MS, and following fragment-level validation of glycan compositions, we identified 2535 intact N-glycopeptides (glycoforms) corresponding to 815 N-glycosylation sites across 543 glycoproteins. The T. urticae N-glycome is strongly dominated by oligomannose-type glycans, which account for 89.2% of the total glycopeptide signal intensity. Notably, high-mannose structures (56.2%) are more abundant than paucimannose glycans, a pattern that contrasts with the canonical insect glycosylation paradigm. Fucosylation is comparatively modest, representing 13.5% of the total glycopeptide signal intensity, and difucosylated structures, common in insects, are rare and restricted to small core-difucosylated paucimannosidic glycans. Despite this overall simplicity, T. urticae retains the enzymatic capacity to synthesize hybrid- and complex-type N-glycans, including branched structures, indicating selective deployment of advanced glycan processing, consistent with this, orthologs of the relevant glycosyltransferases are present in the T. urticae genome. Comparative analysis with multiple insect species places T. urticae at the low-fucosylation end of the arthropod range, alongside a distinctive Man5/Man4 oligomannose signature. We hypothesize that these features may reflect an ancestral or lineage-restricted glycosylation phenotype associated with ametabolous development, although confirmation will require glycoproteomic sample of additional chelicerate species. Functional annotation reveals strong enrichment of N-glycoproteins involved in proteolysis, lipid transport and metabolism, lysosomal function, cuticle-associated processes, and acetylcholinesterase activity, linking glycosylation to key aspects of mite feeding biology, molting, rapid growth, and insecticide resistance. Together, the present study establishes a foundational, site-specific N-glycoproteomic resource for Acari, expands current understanding of arthropod glycan evolution, and provides a molecular framework for exploring glycosylation-dependent mechanisms underlying mite development, host-plant interactions, and resistance to chemical control.