Cresol Derivatives from Bacillus subtilis as Natural Oviposition Modulator of Culex quinquefasciatus: A Molecular...

Mosquitoes rely heavily on olfactory cues for locating suitable oviposition sites, with microbial communities in aquatic habitats playing a crucial role in producing volatile organic compounds (VOCs) that influence mosquito behaviour. In this study, we isolated Bacillus subtilis DHB13 from the breeding habitat of Culex quinquefasciatus, a major vector of several human diseases. The partial 16S rRNA gene sequence of the isolate has been submitted to NCBI GenBank with the accession number PV698100. The identity and resistance profile of the strain was confirmed through biochemical and antibiotic susceptibility tests. The bacterial suspension demonstrated a notable oviposition activity index (OAI) of 0.77 ± SE, with moderate variation among treatments (F(3, 8) = 3.631, p = 0.0642). Multiple comparison analysis (Tukey's test) showed that OAI values for DHB13-treated media did not differ significantly from natural habitat water but were significantly higher than the sterile control, indicating a biologically relevant attraction of gravid female mosquitoes. LC-MS analysis of the bacterial culture supernatant revealed the presence of three cresol derivatives: diisopropyl-m-cresol, 3-ethyl-p-cresol, and 6-ethyl-o-cresol. These compounds were evaluated through molecular docking against Cx. quinquefasciatus Odorant Binding Protein 1 (CxOBP1), a protein known to mediate olfactory-driven oviposition behaviour. However, mosquito olfaction involves several OBPs, receptors, and enzymes, so interaction with CxOBP1 represents only part of this complex sensory system. Molecular docking revealed strong binding of CxOBP1 with diisopropyl-m-cresol (-6.7 kcal/mol), 3-ethyl-p-cresol (-6.2 kcal/mol), and 6-ethyl-o-cresol (-5.9 kcal/mol), indicating potential oviposition attractant activity. All three ligands were found to bind within a conserved binding pocket of CxOBP1, behavioural assays confirmed the oviposition-stimulant properties of the bacterial suspension, indicating that the detected compounds mimic natural semio-chemicals such as p-cresol, previously recognized as an oviposition cue. These findings reinforce the role of microbiota in shaping mosquito reproductive behaviour through the production of volatile attractants. Moreover, they highlight the potential of using microbial VOCs as environmentally sustainable tools for mosquito surveillance and vector control. This integrative approach linking microbial ecology, chemical analysis, and mosquito behaviour provides novel insights for the development of attractant-based control strategies.