{"paper_id":"034233e8-be46-4f88-8c8f-10a200e2b5de","body_text":"Insecticidal effectiveness of nicotine from Nicotiana tabacum against sub-species ofAnopheles gambiae sensu lato and Anopheles funestus sensu lato (Diptera: Culicidae) from the Luapula Province of Zambia | 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 Insecticidal effectiveness of nicotine from Nicotiana tabacum against sub-species ofAnopheles gambiae sensu lato and Anopheles funestus sensu lato (Diptera: Culicidae) from the Luapula Province of Zambia Nicholus C. Sande, Martin Simuunza, Kaampwe Muzandu, Danny Muzata, and 8 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8477919/v1 This work is licensed under a CC BY 4.0 License Status: Under Revision Version 1 posted 13 You are reading this latest preprint version Abstract Background: One of the main challenges affecting malaria control initiatives in sub-Saharan Africa is the swift and widespread emergence of insecticide resistance. Consequently, there is a need to explore alternative insecticides for controlling malaria vectors. This study investigated the effectiveness of tobacco extract on wild Anopheles mosquitoes during the dry season (October–November 2021) in Chebele village, Mwense District, Luapula Province, Zambia. Methods: Wild Anopheles larvae were collected using the pipetting and dipping method along the Mwense stream and reared to the F1 progeny. Wild Anopheles mosquitoes were identified using morphological taxonomic keys. Specimens belonging to the Anopheles gambiae complex and Anopheles funestus group were further identified by multiplex Polymerase Chain Reaction (PCR). The solvent extraction method was used to extract tobacco compounds from tobacco leaves. Filter papers were impregnated with clothianidin (0.132 g/ml; SumiShield® 50WG) in accordance with the World Health Organization (WHO) Standard Operating Procedure for susceptibility testing (tube tests). Clothianidin was used as a positive control, while distilled water served as a negative control. Non-blood-fed, 2- to 3-day-old female wild Anopheles mosquitoes were exposed to clothianidin (0.132 g/ml) and tobacco extract at different concentrations (25% v/v, 33.3% v/v, 50% v/v, 62.5% v/v, 71.43% v/v, and 83.3% v/v) using the World Health Organization (WHO) bottle bioassay. The insectary-reared Kisumu strain ( An. gambiae sensu stricto) was used as a reference for insecticide susceptibility tests. Results: Morphological identification of adult mosquitoes collected from 73 households showed that 98.03% were Anopheles funestus s.l. (n = 199), 1.48% were Anopheles gambiae s.l. (n = 3), and 0.49% were Culex species (n = 1). Subsequent PCR analysis of the wild-caught adult mosquitoes revealed that Anopheles funestus sensu stricto (s.s.) (n = 93; 46%) and Anopheles gambiae s.s. (n = 81; 40%) were the dominant species within the An. funestus group and the An. gambiae complex, respectively. Tobacco extract (83.3% v/v) and clothianidin (0.132 g/ml) were found to have mean knockdown rates at 80 minutes of 61.88% (95% CI: 34.03–89.74%) and 85.05% (95% CI: 72.01–98.08%), respectively. Results showed that both clothianidin and tobacco extract elicited 100% mortality in adult wild Anopheles mosquitoes and the Kisumu strain after 24 hours. The estimated LC₅₀ for tobacco extract on wild Anopheles mosquitoes was estimated to 38.77% v/v (95% CI: 9.99–55.91% v/v), as determined by probit analysis. Conclusion: The findings of this study suggest that Nicotiana tabacum leaf extract may be considered a potential bio-insecticide for controlling An. gambiae s.s. and An. funestus s.s., which are significant vectors of the malaria pathogen. Anopheles gambiae Anopheles funestus lethal concentration Nicotiana tabacum vector control Zambia Full Text Additional Declarations No competing interests reported. Cite Share Download PDF Status: Under Revision Version 1 posted Editorial decision: Revision requested 25 Mar, 2026 Reviews received at journal 25 Mar, 2026 Reviews received at journal 24 Mar, 2026 Reviews received at journal 23 Mar, 2026 Reviews received at journal 22 Mar, 2026 Reviewers agreed at journal 05 Mar, 2026 Reviewers agreed at journal 05 Mar, 2026 Reviewers agreed at journal 05 Mar, 2026 Reviewers agreed at journal 05 Mar, 2026 Reviewers invited by journal 05 Mar, 2026 Editor assigned by journal 04 Jan, 2026 Submission checks completed at journal 02 Jan, 2026 First submitted to journal 30 Dec, 2025 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-8477919\",\"acceptedTermsAndConditions\":true,\"allowDirectSubmit\":false,\"archivedVersions\":[],\"articleType\":\"Research Article\",\"associatedPublications\":[],\"authors\":[{\"id\":569445389,\"identity\":\"b17a315b-b5cb-41d5-b2da-19c1f2206064\",\"order_by\":0,\"name\":\"Nicholus C. 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Journal\",\"twitterHandle\":\"@malariajournal\",\"acdcEnabled\":true,\"dfaEnabled\":true,\"editorialSystem\":\"em\",\"reportingPortfolio\":\"BMC/SO AJ\",\"inReviewEnabled\":true,\"inReviewRevisionsEnabled\":true},\"keywords\":\"Anopheles gambiae, Anopheles funestus, lethal concentration, Nicotiana tabacum, vector control, Zambia\",\"lastPublishedDoi\":\"10.21203/rs.3.rs-8477919/v1\",\"lastPublishedDoiUrl\":\"https://doi.org/10.21203/rs.3.rs-8477919/v1\",\"license\":{\"name\":\"CC BY 4.0\",\"url\":\"https://creativecommons.org/licenses/by/4.0/\"},\"manuscriptAbstract\":\"\\u003cp\\u003e\\u003cstrong\\u003eBackground:\\u003c/strong\\u003e One of the main challenges affecting malaria control initiatives in sub-Saharan Africa is the swift and widespread emergence of insecticide resistance. Consequently, there is a need to explore alternative insecticides for controlling malaria vectors. This study investigated the effectiveness of tobacco extract on wild \\u003cem\\u003eAnopheles\\u003c/em\\u003e mosquitoes during the dry season (October–November 2021) in Chebele village, Mwense District, Luapula Province, Zambia.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eMethods:\\u003c/strong\\u003e Wild \\u003cem\\u003eAnopheles\\u003c/em\\u003e larvae were collected using the pipetting and dipping method along the Mwense stream and reared to the F1 progeny. Wild \\u003cem\\u003eAnopheles\\u003c/em\\u003e mosquitoes were identified using morphological taxonomic keys. Specimens belonging to the \\u003cem\\u003eAnopheles gambiae\\u003c/em\\u003e complex and \\u003cem\\u003eAnopheles funestus\\u003c/em\\u003e group were further identified by multiplex Polymerase Chain Reaction (PCR). The solvent extraction method was used to extract tobacco compounds from tobacco leaves.\\u003c/p\\u003e\\n\\u003cp\\u003eFilter papers were impregnated with clothianidin (0.132 g/ml; SumiShield® 50WG) in accordance with the World Health Organization (WHO) Standard Operating Procedure for susceptibility testing (tube tests). Clothianidin was used as a positive control, while distilled water served as a negative control. Non-blood-fed, 2- to 3-day-old female wild \\u003cem\\u003eAnopheles\\u003c/em\\u003e mosquitoes were exposed to clothianidin (0.132 g/ml) and tobacco extract at different concentrations (25% v/v, 33.3% v/v, 50% v/v, 62.5% v/v, 71.43% v/v, and 83.3% v/v) using the World Health Organization (WHO) bottle bioassay. The insectary-reared Kisumu strain (\\u003cem\\u003eAn. gambiae\\u003c/em\\u003e sensu stricto) was used as a reference for insecticide susceptibility tests.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eResults:\\u003c/strong\\u003e Morphological identification of adult mosquitoes collected from 73 households showed that 98.03% were \\u003cem\\u003eAnopheles funestus\\u003c/em\\u003e s.l. (n = 199), 1.48% were \\u003cem\\u003eAnopheles gambiae\\u003c/em\\u003e s.l. (n = 3), and 0.49% were \\u003cem\\u003eCulex\\u003c/em\\u003e species (n = 1). Subsequent PCR analysis of the wild-caught adult mosquitoes revealed that \\u003cem\\u003eAnopheles funestus\\u003c/em\\u003e sensu stricto (s.s.) (n = 93; 46%) and \\u003cem\\u003eAnopheles gambiae\\u003c/em\\u003es.s. (n = 81; 40%) were the dominant species within the \\u003cem\\u003eAn. funestus\\u003c/em\\u003e group and the \\u003cem\\u003eAn. gambiae\\u003c/em\\u003e complex, respectively.\\u003c/p\\u003e\\n\\u003cp\\u003eTobacco extract (83.3% v/v) and clothianidin (0.132 g/ml) were found to have mean knockdown rates at 80 minutes of 61.88% (95% CI: 34.03–89.74%) and 85.05% (95% CI: 72.01–98.08%), respectively. Results showed that both clothianidin and tobacco extract elicited 100% mortality in adult wild \\u003cem\\u003eAnopheles\\u003c/em\\u003emosquitoes and the Kisumu strain after 24 hours. The estimated LC₅₀ for tobacco extract on wild \\u003cem\\u003eAnopheles\\u003c/em\\u003e mosquitoes was estimated to 38.77% v/v (95% CI: 9.99–55.91% v/v), as determined by probit analysis.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eConclusion:\\u003c/strong\\u003eThe findings of this study suggest that \\u003cem\\u003eNicotiana tabacum\\u003c/em\\u003e leaf extract may be considered a potential bio-insecticide for controlling \\u003cem\\u003eAn. gambiae\\u003c/em\\u003e s.s. and \\u003cem\\u003eAn. funestus\\u003c/em\\u003e s.s., which are significant vectors of the malaria pathogen.\\u003c/p\\u003e\",\"manuscriptTitle\":\"Insecticidal effectiveness of nicotine from Nicotiana tabacum against sub-species ofAnopheles gambiae sensu lato and Anopheles funestus sensu lato (Diptera: Culicidae) from the Luapula Province of Zambia\",\"msid\":\"\",\"msnumber\":\"\",\"nonDraftVersions\":[{\"code\":1,\"date\":\"2026-01-07 08:05:23\",\"doi\":\"10.21203/rs.3.rs-8477919/v1\",\"editorialEvents\":[{\"type\":\"communityComments\",\"content\":0},{\"type\":\"decision\",\"content\":\"Revision requested\",\"date\":\"2026-03-25T11:45:58+00:00\",\"index\":\"\",\"fulltext\":\"\"},{\"type\":\"editorInvitedReview\",\"content\":\"\",\"date\":\"2026-03-25T06:51:30+00:00\",\"index\":\"hide\",\"fulltext\":\"\"},{\"type\":\"editorInvitedReview\",\"content\":\"\",\"date\":\"2026-03-24T21:49:22+00:00\",\"index\":\"hide\",\"fulltext\":\"\"},{\"type\":\"editorInvitedReview\",\"content\":\"\",\"date\":\"2026-03-23T09:37:28+00:00\",\"index\":\"hide\",\"fulltext\":\"\"},{\"type\":\"editorInvitedReview\",\"content\":\"\",\"date\":\"2026-03-22T12:10:42+00:00\",\"index\":\"hide\",\"fulltext\":\"\"},{\"type\":\"reviewerAgreed\",\"content\":\"49967738377235280355590209012800654571\",\"date\":\"2026-03-05T14:57:41+00:00\",\"index\":\"hide\",\"fulltext\":\"\"},{\"type\":\"reviewerAgreed\",\"content\":\"164424275036405080567654930433376910231\",\"date\":\"2026-03-05T13:22:53+00:00\",\"index\":\"hide\",\"fulltext\":\"\"},{\"type\":\"reviewerAgreed\",\"content\":\"263834323143283924040305362220966385784\",\"date\":\"2026-03-05T12:24:26+00:00\",\"index\":\"hide\",\"fulltext\":\"\"},{\"type\":\"reviewerAgreed\",\"content\":\"312102444961126157768433501955982129631\",\"date\":\"2026-03-05T12:06:23+00:00\",\"index\":\"hide\",\"fulltext\":\"\"},{\"type\":\"reviewersInvited\",\"content\":\"\",\"date\":\"2026-03-05T11:11:48+00:00\",\"index\":\"\",\"fulltext\":\"\"},{\"type\":\"editorAssigned\",\"content\":\"\",\"date\":\"2026-01-04T21:36:03+00:00\",\"index\":\"\",\"fulltext\":\"\"},{\"type\":\"checksComplete\",\"content\":\"\",\"date\":\"2026-01-02T07:52:26+00:00\",\"index\":\"\",\"fulltext\":\"\"},{\"type\":\"submitted\",\"content\":\"Malaria Journal\",\"date\":\"2025-12-30T06:11:17+00:00\",\"index\":\"\",\"fulltext\":\"\"}],\"status\":\"published\",\"journal\":{\"display\":true,\"email\":\"info@researchsquare.com\",\"identity\":\"malaria-journal\",\"isNatureJournal\":false,\"hasQc\":true,\"allowDirectSubmit\":false,\"externalIdentity\":\"malj\",\"sideBox\":\"Learn more about [Malaria Journal](http://malariajournal.biomedcentral.com/)\",\"snPcode\":\"12936\",\"submissionUrl\":\"https://submission.nature.com/new-submission/12936/3\",\"title\":\"Malaria Journal\",\"twitterHandle\":\"@malariajournal\",\"acdcEnabled\":true,\"dfaEnabled\":true,\"editorialSystem\":\"em\",\"reportingPortfolio\":\"BMC/SO AJ\",\"inReviewEnabled\":true,\"inReviewRevisionsEnabled\":true}}],\"origin\":\"\",\"ownerIdentity\":\"84680e1c-ce66-4a7b-bd3d-a636fa2166b5\",\"owner\":[],\"postedDate\":\"January 7th, 2026\",\"published\":true,\"recentEditorialEvents\":[],\"rejectedJournal\":[],\"revision\":\"\",\"amendment\":\"\",\"status\":\"in-revision\",\"subjectAreas\":[],\"tags\":[],\"updatedAt\":\"2026-03-25T11:55:31+00:00\",\"versionOfRecord\":[],\"versionCreatedAt\":\"2026-01-07 08:05:23\",\"video\":\"\",\"vorDoi\":\"\",\"vorDoiUrl\":\"\",\"workflowStages\":[]},\"version\":\"v1\",\"identity\":\"rs-8477919\",\"journalConfig\":\"researchsquare\"},\"__N_SSP\":true},\"page\":\"/article/[identity]/[[...version]]\",\"query\":{\"redirect\":\"/article/rs-8477919\",\"identity\":\"rs-8477919\",\"version\":[\"v1\"]},\"buildId\":\"XKTyCvWXoU3ODBz1xrDgd\",\"isFallback\":false,\"isExperimentalCompile\":false,\"dynamicIds\":[84888],\"gssp\":true,\"scriptLoader\":[]}","source_license":"CC-BY-4.0","license_restricted":false}