Yeasts Associated with Mosquito Larval Habitats Produce Volatile Cues That Attract Gravid Females and Show Potential as Safe Tools for Behavioural Control

Background Fungi associated with mosquito larvae can contribute to host development and may influence oviposition-site selection through the release of volatile organic compounds (VOCs). However, it remains unclear to what extent larval mycobiota reflects passive acquisition from breeding waters versus host-associated filtering. Here, we characterized fungal communities associated with larvae and breeding waters of Culex pipiens , Aedes albopictus , and the invasive Aedes koreicus in Italy, and assessed the behavioural activity and preliminary biosafety of larval habitat-derived yeasts. Results Using ITS metabarcoding coupled with culture-dependent isolation, we identified diverse fungal assemblages across mosquito species and sampling sites, with partial overlap between larval and aquatic communities and a set of recurrent yeast taxa shared among hosts and habitats. Culture-based methods recovered seven yeast species including Barnettozyma californica , Candida parapsilosis , Metschnikowia pulcherrima , Rhodotorula paludigena , Schwanniomyces vanrijiae , Sporobolomyces roseus , and Wickerhamomyces anomalus . In vitro screening on HaCaT keratinocytes, performed by co-culturing human cells with seven fungal isolates from breeding water (one per yeast species), demonstrated that B. californica , M. pulcherrima , R. paludigena , W. anomalus isolates did not induce detectable cytotoxic effects. The yeast volatilomes of the seven isolates were profiled by HS-SPME–GC–MS, revealing species-specific VOC blends dominated by alcohols and esters in Ascomycota and enriched in phenolic compounds and organic acids in Basidiomycota. Based on VOC profiles and absence of detectable cytotoxicity, a core of four selected yeasts were further evaluated in dual-choice oviposition assays with gravid Ae. albopictus . Yeast-treated oviposition substrates significantly altered egg-laying preferences, with all tested yeast species resulting in oviposition percentages greater than 50% in the treatment group. Among the tested species, W. anomalus and B. californica exhibited the strongest attractiveness, followed by R. paludigena , whereas M. pulcherrima showed a comparatively weaker effect. Conclusion Fungal communities associated with mosquito larvae mainly mirror breeding-site ecology but also exhibit signs of host-associated filtering. Notably, in addition to Culex pipiens and Aedes albopictus , this study included Aedes koreicus , for which information on mycobiota composition has so far been limited. Importantly, yeasts isolated from larval habitats emit VOC blends that can modulate oviposition behaviour in gravid Ae. albopictus . Together, these results support further investigation of selected yeast-based cues as components of environmentally compatible, oviposition-targeted mosquito management strategies, alongside expanded safety assessment and field validation.