Endosymbiont-derived microbial volatiles mediate conspecific female attraction in the melon fruit fly Zeugodacus cucurbitae.

Microbial symbionts associated with phytophagous insects are increasingly recognised as key mediators of chemical communication and behaviour. In this study, we investigated the taxonomic diversity and attractancy potential of culturable bacterial symbionts associated with the melon fly, Zeugodacus cucurbitae (Diptera: Tephritidae), with the aim of identifying behaviourally active microbial volatiles with potential application in pest management. Bacteria were isolated from the gut, reproductive organs, and rectal glands of female melon flies from wild and laboratory-reared populations and identified using 16S rRNA gene sequencing. A total of 54 bacterial isolates, predominantly belonging to the phylum Proteobacteria, were identified, with greater taxonomic diversity observed in wild flies than in laboratory-reared populations. Behavioural screening using four-arm olfactometer assays revealed that volatiles from seven bacterial isolates elicited significant attraction of female Z. cucurbitae. Subsequent laboratory cage and field bioassays further narrowed these to four consistently attractive isolates, among which Citrobacter portucalensis and Klebsiella oxytoca showed particularly strong and female-biased attraction under field conditions. Gas chromatography-electroantennographic detection (GC-EAD) and gas chromatography-mass spectrometry (GC–MS) analyses identified 17 electrophysiologically active volatile compounds associated with the attractive bacterial isolates, including phenols, aromatic alcohols, ketones, alkanes, and indole. Behavioural assays with synthetic standards demonstrated that indole alone elicited significant attraction, with responses comparable to those induced by the corresponding bacterial volatile. In silico molecular docking further supported these findings by revealing selective and relatively strong interactions between behaviourally active volatiles and specific Z. cucurbitae odorant-binding proteins, providing molecular-level support for olfactory recognition of bacterial cues. Overall, the results demonstrate that bacterial endosymbionts of Z. cucurbitae play a critical role in mediating adult attraction through the production of behaviourally active volatiles. Exploitation of symbiont-derived semiochemicals offers a promising, environmentally sustainable avenue for developing novel lures and strengthening integrated pest management strategies for melon fly control.