The research team, led by scientists at Nanjing Agricultural University together with collaborators in Kenya, focused on IR75q.2, a member of the IR75 clade, in the cotton leafworm Spodoptera litura and the beet armyworm Spodoptera exigua. Both species are major agricultural pests with broad host ranges and formidable dispersal abilities, making them priority targets for novel pest management strategies. IR75q.2 had previously been characterized in a third species, the fall armyworm Spodoptera frugiperda, where it was shown to mediate avoidance of nonanoic acid. The central question of the new work was whether this ligand recognition profile, and the structural features underlying it, have been preserved across related moth lineages or whether evolutionary divergence has reshaped them.

To answer that question, the researchers assembled a multi-pronged analytical pipeline that combined phylogenetics, expression profiling, electrophysiology, structural modeling, molecular docking, and site-directed mutagenesis. Phylogenetic analysis of IR75 amino acid sequences from three Spodoptera species and two additional noctuid moths, Agrotis segetum and Helicoverpa armigera, revealed that the IR75 receptors form a highly conserved clade. Motif analysis using the MEME program identified ten conserved motifs arranged in nearly identical order across the sequences, with statistical significance values so extreme that the probability of chance occurrence fell below ten to the power of minus 170. This remarkable sequence conservation suggested that members of the IR75 clade share structural features tied to ligand recognition.

Transcriptome data added a functional dimension to this conservation story. Among all IR75 genes in the two Spodoptera species, IR75q.2 exhibited the highest expression in the antennae of both sexes, with no significant differences between males and females. Because antennae are the primary olfactory organs, this expression pattern pointed to an important role in odor detection. Sequence comparisons showed that the SlitIR75q.2 and SexiIR75q.2 proteins share 90.65 percent amino acid identity, both genes contain eleven exons, and both proteins possess three transmembrane domains. Evolutionary analysis using the PAML package revealed that while selective pressures vary across branches, all omega values were below one, indicating strong purifying selection has preserved the function of these orthologs over evolutionary time.

The functional core of the study came from experiments using the Xenopus oocyte expression system with two-electrode voltage clamp recording. The team co-expressed each IR75q.2 ortholog with its obligatory co-receptor IR8a and tested 59 odorant compounds in three mixtures. Both receptors responded primarily to just six compounds: three medium-chain fatty acids, namely octanoic acid, nonanoic acid, and decanoic acid, and their corresponding aldehydes, octanal, nonanal, and decanal. Nonanoic acid was by far the strongest ligand for both receptors, eliciting peak currents of 491.61 nanoamperes in SlitIR75q.2 and 347.70 nanoamperes in SexiIR75q.2. Dose-response assays yielded half-maximal effective concentrations of 7.596 times ten to the minus four molar for the cotton leafworm receptor and 4.052 times ten to the minus four molar for the beet armyworm receptor, confirming nearly identical sensitivities.

To probe the structural basis of this shared tuning, the researchers predicted three-dimensional structures of both receptors using AlphaFold 3.0 and docked nonanoic acid into the binding pockets using AutoDock Vina. The predicted binding energies were minus 6.04 kilocalories per mole for SlitIR75q.2 and minus 4.93 kilocalories per mole for SexiIR75q.2. In both receptors, a phenylalanine and an arginine residue within the S1 domain of the ligand-binding domain were predicted to form hydrogen bonds with the ligand: Phe285 and Arg290 in SlitIR75q.2, and Phe284 and Arg289 in SexiIR75q.2. Ten amino acid residues within four angstroms of nonanoic acid were conserved between the two receptors, underscoring the preservation of the binding pocket.

Site-directed mutagenesis put these structural predictions to a rigorous test. Substituting either Phe285 or Arg290 with alanine in SlitIR75q.2 completely abolished the response to nonanoic acid, preventing any estimate of sensitivity. In SexiIR75q.2, single alanine substitutions of Phe284 or Arg289 did not eliminate responses but increased the half-maximal effective concentration roughly four to five-fold, from 1.38 times ten to the minus four molar in the intact receptor to 6.3 and 6.2 times ten to the minus four molar in the mutants. These results establish Phe and Arg as conserved key residues for nonanoic acid recognition and suggest that subtle changes in binding-pocket chemistry can modulate ligand sensitivity across species. Docking of the remaining five ligands showed the same hydrogen-bonding anchors plus conserved hydrophobic contacts involving residues such as Ile230/229, Trp257/256, and Pro282/282.

The physiological findings translated into behavior for at least one of the two species. Electroantennogram recordings demonstrated that adults of both Spodoptera litura and Spodoptera exigua responded in a dose-dependent manner to nonanoic acid and the other five ligands, confirming that the antennal sensory system detects these compounds. In Y-tube olfactometer assays, nonanoic acid significantly repelled S. litura adults at doses of 50 and 100 micrograms. Behavioral testing of S. exigua proved unreliable because the beet armyworm adults showed extremely low locomotor activity in the olfactometer, but the electrophysiological evidence indicates the compound is detected by this species as well. Together, the behavioral and physiological data align with the earlier finding in the fall armyworm, reinforcing the idea that nonanoic acid serves as a conserved aversive cue across Spodoptera moths.

The broader evolutionary implications of the study are considerable. The ligands detected by IR75q.2 overlap partly with those sensed by certain odorant receptors, and nonanal in particular is known to attract females of S. frugiperda and S. litura through a conserved odorant receptor lineage. The authors propose that odorant receptors and ionotropic receptors do not simply duplicate one another but instead provide complementary information: the former tuned largely to neutral plant volatiles, the latter to acids, amines, and other polar ecological cues. This parallel coding may help moths discriminate chemically related compounds with different behavioral valences and broaden their detectable chemical space. At the same time, comparisons with the related IR75q.1 group, which prefers the aversive compound octanoic acid, suggest that subfunctionalization after gene duplication has shaped the ligand preferences of acid-sensing IRs in Lepidoptera. By pinpointing conserved binding residues and demonstrating functional conservation across two major pests, the study provides a molecular framework both for understanding how olfactory systems evolve and for designing targeted behavioral interventions, such as repellent formulations built around nonanoic acid analogs, that could exploit this preserved sensory channel in the fight against some of the world’s most destructive crop pests.

Subject of Research: Functional conservation of the ionotropic receptor IR75q.2 in detecting volatile acids and aldehydes across two Spodoptera moth species

Article Title: Functional conservation of IR75q.2 in the recognition of volatile acids and aldehydes in two Spodoptera species

Article References: Guo, J.-M., Wang, J.-X., He, Y., Luan, X.-P., Wei, Z.-Q., Liu, X.-L., Obiero, G. F., Yan, Q., Dong, S.-L., & Zhang, J. (2026). Functional conservation of IR75q.2 in the recognition of volatile acids and aldehydes in two Spodoptera species. Crop Health, 4(1), Article 20. https://doi.org/10.1007/s44297-026-00082-7

Image Credits: AI Generated

DOI: 10.1007/s44297-026-00082-7

Keywords: ionotropic receptors, IR75q.2, Spodoptera litura, Spodoptera exigua, nonanoic acid, insect olfaction, chemosensory receptors, Xenopus oocyte expression, molecular docking, site-directed mutagenesis, purifying selection, pest management