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

Yeasts Associated with Mosquito Larval Habitats Produce Volatile Cues That Attract Gravid Females and Show Potential as Safe Tools for Behavioural Control | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Yeasts Associated with Mosquito Larval Habitats Produce Volatile Cues That Attract Gravid Females and Show Potential as Safe Tools for Behavioural Control Alessia Cappelli, Claudia Damiani, Cecilia Baldassarri, Gianmarco Rotondi, and 6 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8958698/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract 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. Mycobiota mosquitoes larvae volatile organic compounds oviposition behaviour behavioural control Full Text Additional Declarations No competing interests reported. Supplementary Files SupplementaryMaterial1.docx SupplementaryMaterial2.xlsx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8958698","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":596825716,"identity":"c703cb63-214c-44df-a08b-b58ee81eb9a8","order_by":0,"name":"Alessia Cappelli","email":"","orcid":"","institution":"University of Camerino","correspondingAuthor":false,"prefix":"","firstName":"Alessia","middleName":"","lastName":"Cappelli","suffix":""},{"id":596825717,"identity":"b0ba3b7a-c570-4830-bda2-5d19639028cf","order_by":1,"name":"Claudia Damiani","email":"","orcid":"","institution":"University of Camerino","correspondingAuthor":false,"prefix":"","firstName":"Claudia","middleName":"","lastName":"Damiani","suffix":""},{"id":596825718,"identity":"4491e62f-4c6d-4a83-92b7-9d7c9f0f0342","order_by":2,"name":"Cecilia Baldassarri","email":"","orcid":"","institution":"University of Camerino","correspondingAuthor":false,"prefix":"","firstName":"Cecilia","middleName":"","lastName":"Baldassarri","suffix":""},{"id":596825719,"identity":"8e9f4b68-438c-4ba5-b4d7-4a065973ec1c","order_by":3,"name":"Gianmarco Rotondi","email":"","orcid":"","institution":"University of Camerino","correspondingAuthor":false,"prefix":"","firstName":"Gianmarco","middleName":"","lastName":"Rotondi","suffix":""},{"id":596825720,"identity":"bd14ba5a-78eb-434b-b6ac-c734f02562a4","order_by":4,"name":"Mohammad Sameer Zubair","email":"","orcid":"","institution":"University of Camerino","correspondingAuthor":false,"prefix":"","firstName":"Mohammad","middleName":"Sameer","lastName":"Zubair","suffix":""},{"id":596825721,"identity":"922f181f-073c-4fb5-af36-65c72fa9f233","order_by":5,"name":"Dennis Fiorini","email":"","orcid":"","institution":"University of Camerino","correspondingAuthor":false,"prefix":"","firstName":"Dennis","middleName":"","lastName":"Fiorini","suffix":""},{"id":596825722,"identity":"ec8d16dc-67dc-4821-b78c-8c57f29b12c1","order_by":6,"name":"Valentina Cecarini","email":"","orcid":"","institution":"University of Camerino","correspondingAuthor":false,"prefix":"","firstName":"Valentina","middleName":"","lastName":"Cecarini","suffix":""},{"id":596825723,"identity":"5be0968b-5d47-47bc-b97d-de3cc03c8fc7","order_by":7,"name":"Consuelo Amantini","email":"","orcid":"","institution":"University of Camerino","correspondingAuthor":false,"prefix":"","firstName":"Consuelo","middleName":"","lastName":"Amantini","suffix":""},{"id":596825724,"identity":"15f136e4-d104-43b4-b832-79d866e84588","order_by":8,"name":"Guido Favia","email":"","orcid":"","institution":"University of Camerino","correspondingAuthor":false,"prefix":"","firstName":"Guido","middleName":"","lastName":"Favia","suffix":""},{"id":596825725,"identity":"674cfe91-b354-4f66-9f64-7ad09a617883","order_by":9,"name":"Irene Ricci","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA0klEQVRIiWNgGAWjYBACNijNw8DA2PiAgUECzDtArJZmA7gWvHqQtUvAmfi08EkfPvbgA8M9Gf72w23VvDss5PmnHWA8/AGfyXxp6YYzGIp5JM4ktt3mPSNhOON2An6HsfHwmEnzMCTwGEgwArW0SSQwENbC/036D1RLMUiLPBG2sEkzQLUwg7QYENbCZibZY5AA8kuz5Nw2CcONtxMbDpzBo0W+h/mZxI+KBHv+9uMPP7xtq5OXu518+EMFHi0QYIDCY2wgqGEUjIJRMApGAX4AAO5pQ2mj5jFPAAAAAElFTkSuQmCC","orcid":"","institution":"University of Camerino","correspondingAuthor":true,"prefix":"","firstName":"Irene","middleName":"","lastName":"Ricci","suffix":""}],"badges":[],"createdAt":"2026-02-24 14:39:45","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8958698/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8958698/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":106893516,"identity":"2405deb6-824a-4d5b-8970-336699fb5454","added_by":"auto","created_at":"2026-04-14 13:58:03","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1213544,"visible":true,"origin":"","legend":"","description":"","filename":"Manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8958698/v1_covered_37bc2d5d-b305-4450-b9cb-549a55283f99.pdf"},{"id":103798130,"identity":"f8e14f33-aae3-4fdc-9b9a-df9a8618e717","added_by":"auto","created_at":"2026-03-03 04:55:06","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":1318467,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryMaterial1.docx","url":"https://assets-eu.researchsquare.com/files/rs-8958698/v1/8224d60501da760cdecacaab.docx"},{"id":103798135,"identity":"2dde992c-e3f3-4a3d-a487-f5bfaeab2d24","added_by":"auto","created_at":"2026-03-03 04:55:10","extension":"xlsx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":38006,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryMaterial2.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-8958698/v1/273cc83278ee7789d0806b94.xlsx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Yeasts Associated with Mosquito Larval Habitats Produce Volatile Cues That Attract Gravid Females and Show Potential as Safe Tools for Behavioural Control","fulltext":[],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":false,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":true,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":true,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Mycobiota, mosquitoes, larvae, volatile organic compounds, oviposition behaviour, behavioural control","lastPublishedDoi":"10.21203/rs.3.rs-8958698/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8958698/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eFungi 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 \u003cem\u003eCulex pipiens\u003c/em\u003e, \u003cem\u003eAedes albopictus\u003c/em\u003e, and the invasive \u003cem\u003eAedes koreicus\u003c/em\u003e in Italy, and assessed the behavioural activity and preliminary biosafety of larval habitat-derived yeasts.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eUsing 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 \u003cem\u003eBarnettozyma californica\u003c/em\u003e, \u003cem\u003eCandida parapsilosis\u003c/em\u003e, \u003cem\u003eMetschnikowia pulcherrima\u003c/em\u003e, \u003cem\u003eRhodotorula paludigena\u003c/em\u003e, \u003cem\u003eSchwanniomyces vanrijiae\u003c/em\u003e, \u003cem\u003eSporobolomyces roseus\u003c/em\u003e, and \u003cem\u003eWickerhamomyces anomalus\u003c/em\u003e. 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 \u003cem\u003eB. californica\u003c/em\u003e, \u003cem\u003eM. pulcherrima\u003c/em\u003e, \u003cem\u003eR. paludigena\u003c/em\u003e, \u003cem\u003eW. anomalus\u003c/em\u003e isolates did not induce detectable cytotoxic effects. The yeast volatilomes of the seven isolates were profiled by HS-SPME\u0026ndash;GC\u0026ndash;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 \u003cem\u003eAe. albopictus\u003c/em\u003e. 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, \u003cem\u003eW. anomalus\u003c/em\u003e and \u003cem\u003eB. californica\u003c/em\u003e exhibited the strongest attractiveness, followed by \u003cem\u003eR. paludigena\u003c/em\u003e, whereas \u003cem\u003eM. pulcherrima\u003c/em\u003e showed a comparatively weaker effect.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e \u003cp\u003eFungal communities associated with mosquito larvae mainly mirror breeding-site ecology but also exhibit signs of host-associated filtering. Notably, in addition to \u003cem\u003eCulex pipiens\u003c/em\u003e and \u003cem\u003eAedes albopictus\u003c/em\u003e, this study included \u003cem\u003eAedes koreicus\u003c/em\u003e, 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 \u003cem\u003eAe. albopictus\u003c/em\u003e. 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.\u003c/p\u003e","manuscriptTitle":"Yeasts Associated with Mosquito Larval Habitats Produce Volatile Cues That Attract Gravid Females and Show Potential as Safe Tools for Behavioural Control","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-03-03 04:54:39","doi":"10.21203/rs.3.rs-8958698/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"ea0e3db3-d381-4ca7-a9d9-d4263aecc99b","owner":[],"postedDate":"March 3rd, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-04-14T13:56:32+00:00","versionOfRecord":[],"versionCreatedAt":"2026-03-03 04:54:39","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8958698","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8958698","identity":"rs-8958698","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}