Development of oviposition attractants from supernatant of Bacillus thuringiensis var israelensis (Serotype H-14, Strain VCRC B-17) using various culture media

Development of oviposition attractants from supernatant of Bacillus thuringiensis var israelensis (Serotype H-14, Strain VCRC B-17) using various culture media | 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 Short Report Development of oviposition attractants from supernatant of Bacillus thuringiensis var israelensis (Serotype H-14, Strain VCRC B-17) using various culture media Lakshmi Thalappilly Prabhakaran, Athisaya Mary Kulandaisamy, Prabakaran Gnanasundaram This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8078323/v1 This work is licensed under a CC BY 4.0 License Status: Under Revision Version 1 posted 11 You are reading this latest preprint version Abstract objectives Mosquito-borne diseases pose a significant global health threat, with vector control being the primary method for managing these diseases. Biological control methods, particularly the use of ovitraps, bio control agents, and oviposition attractants, play a crucial role. Oviposition attractants, like organic materials such as bamboo infusion, are employed to lure gravid females for egg-laying. Objectives Bacillus thuringiensis var israelensis various culture media supernatant was assessed for oviposition response in Culex, Anopheles , and Aedes species. Materials and Methods Various media were prepared, and Bacillus thuringiensis var israelensis culture supernatant was tested for attractancy. The results, analysed through Oviposition Active Index (OAI) and Mann-Whitney U test. Results In this study, it is identified that supernatant from groundnut cake and milk media has enhanced mosquito oviposition attraction, with significantly higher OAI values in experimental setups compared to controls. Gas Chromatography-Mass Spectrometry analysis revealed the presence of volatile organic compounds potentially responsible for attractant properties. Conclusion Groundnut cake and milk media demonstrated high attractancy for Culex, Anopheles , and Aedes mosquitoes. The compounds identified contribute to the overall oviposition attraction. This study highlights promising biological control measures in mosquito vector management. Biological control Bacillus thuringiensis Culture filtrate GC MS analysis groundnut cake media mosquito oviposition attractancy Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Introduction Mosquitoes are vectors of diseases such as malaria, dengue and chikungunya that impact humans [1]. The available method to tackle the problem is by controlling the vectors [2]. The goal of vector control is to minimize pathogen transmission by preventing human from mosquito bite [3]. Conventional vector control strategies rely heavily on chemical insecticides, which have led to resistance development and environmental concerns. Another common method is the use of ovitraps (oviposition traps), create artificial breeding areas for mosquitoes to lay their eggs. Oviposition attractants can be used to monitor vector populations, especially those of the Aedes and Culex species. The addition of organic materials such as infusions made from leaves produces volatile chemicals, attracts gravid females to lay more eggs in the containers [4]. Microorganisms are also involved in the fermentation processes of the infusions. Earlier research indicates that bacteria including Trichoderma viride , Pseudomonas fluorescence , and Bacillus sp . can be used [5,6]. In the present study, the supernatant of the Bacillus thuringiensis var israelensis ( Bti) culture media was evaluated for the oviposition response among Culex, Anophels and Aedes sps . Materials and methods Preparation of NYSM Media: Nutrient Yeast Salt medium (NYSM) medium was prepared with Glucose (5 g), yeast extract (5 g), Peptone (5 g), Na Cl (5 g), Beef extract (3 g), salt solution with calcium chloride, magnesium chloride and manganese chloride. The pH was adjusted to 7.5 and autoclaved at 121°C for 15 minutes. Preparation of various media: For the preparation of 1000 ml of different media, 10 gms of Soybean flour (Medium“A”) ,Wheat bran (Medium“B”), Groundnut cake powder (GC) (Medium“C”), Rice bran (Medium“D”), Coconut cake powder (Medium“E”), Bengal gram media (Medium“F”), Coconut water (Medium“G”), Egg (Medium“H”), Milk (Medium “I”) were used. Powdered substrates (Medium A- F) were mixed in water, filtered and sterilized while liquid substrates (Medium G-H) were diluted (1:10) before autoclaving. Preparation of seed culture: A 100 L of glycerol stock ICMR-VCRC strain of Bti was inoculated into 100 ml of NYSM medium and incubated at 30°C, 180 rpm for 16 hours. The culture was expanded, then inoculated (2 % v/v) into different media and incubated for 72 hours. Spore formation was confirmed using malachite green and safranin spore staining. Oviposition attractancy study: Full-fed female Culex quinquefasciatus, Anopheles stephensi, and Aedes aegypti mosquitoes were exposed to supernatants in a 55cm 3 mosquito cage under controlled conditions (28°C±2°C,70-80RH). For screening experiment: Fifty fully fed Culex quinquefasciatus were used per trail. 10 different media supernatant (200mL) were individually exposed in enamel bowls. Tap water was placed in a separate bowl in the centre of the cage. The experiment was repeated 3 times. The bowls were placed at opposite corners of the cage and their positions were changed in each repetition. Mosquitoes were released at 16.00h and egg rafts were counted at 10.00h the following day. Final experiment: Full-fed female Culex quinquefasciatus, Anopheles stephensi, and Aedes aegypti mosquitoes were placed in cages under controlled conditions. The most effective three media (NYSM, groundnut cake and milk) of 200mL each were selected for testing. The experimental conditions remained same as in the screening test. For Aedes aegypti, a filter paper was placed around the bowl for egg collection. Each experiment was repeated three times. Comparison with para -Cresol (p-Cresol): The most effective media supernatant was compared with p-Cresol (75µg/mL) as a standard attractant. Data Analysis: The oviposition attractancy index (OAI) of different bacterial culture supernatant and p-cresol concentrations was calculated. Nt, Ns are number of eggs in test solution and control respectively. As per the study of Kramer and Mulla (1979), OAI ≥ 0.3 were attractants and OAI ≤ –0.3 were repellents [7]. The difference in the groups was evaluated using the Mann-Whitney U test. Cost comparison of the effective media was also done. Gas Chromatography- Mass Spectrometry Analysis: Lyophilized supernatants from groundnut cake and milk media were dissolved in methanol and analysed sing GC-MS (NIST Library search) at the Sophisticated Analytical Instrumentation Facility (SAIF), IIT Madras, to identify volatile compounds responsible for mosquito attraction. Results Screening of Bacillus thuringiensis var israelensis culture supernatant for oviposition attraction in Culex quinquefasciatus From the initial experiment, the egg laying was found in Milk, Groundnut cake extract, Bengal gram powder, wheat bran, coconut cake extracts and egg media as compared to water (Table.1). For Culex quinquefasciatus, the highest number of egg rafts was observed in milk (68±4) (Mean±SD), followed by p - Cresol (63±8.18) and groundnut cake media (GC) (47.67±19.09) (Figure.1). No oviposition was observed in water and NYSM media. Statistical analysis showed significant differences compared between GC and NYSM (p = 0.037) as well as between GC and p - Cresol (p=0.049) (Table.2) (Fig.4). OAI showed negative value ( -0.14) for GC media (Table.5). For Anopheles stephensi , the highest egg laying was found in GC media (1471±17.21), followed by NYSM (683.27±35.22), milk (188±70.40), p - Cresol (116±10.21) and water (80±6.24) (Figure.2). GC media and NYSM showed significantly higher egg counts compared to p- Cresol and water (p= 0.049) (Table.3) (Fig.5). OAI was 0.90 for GC and 0.24 for milk media (Table.5). For Aedes aegypti , GC media had the highest mean egg count (1186 ± 1371.38), followed by milk (483±93.29), water (102±38.79) and p- Cresol (72±30.35) with no oviposition in NYSM (Fig.3). Statistical analysis revealed significant differences between GC and p- Cresol (p=0.049) (Table.4) (Fig.6). OAI was 0.84 for GC and 0.74 for milk media (Table.5). The cost for the production of the culture supernatant from groundnut cake extract was estimated as ₹2.72 whereas milk as ₹5.00 for 1 litre. Gas chromatography- Mass Spectrometry analysis of the culture supernatant of groundnut cake and milk media Agilent Model 8890 GC System with Single Quadrupole Mass Spectrometer (5977B MSD) analyser is used for the separation and identification of volatile compounds. NIST spectral library search was also used for the compound identification as it provides reference mass spectra for GC/MS (by electron ionization) as well as gas phase retention indices for gas chromatography. GC–MS analysis of the two media, Groundnut cake extract and milk media revealed the presence of 10 compounds in groundnut cake (Table.6) and 14 volatile compounds in milk media (Table.7) for parameters such as retention time (RT), molecular formula (MF), mass- ratio (m/z) and concentration (% peak area). The total ion chromatograms (TIC) of groundnut cake media and milk media revealed distinct chemical compositions. GC media showed a significant peak at 22.253 min, while milk media exhibited a dominant peak at 8.507 min (Fig.7, 8). The mass spectral analysis of GC media identified Cyclo (L- prolyl- L- Valine) as the most probable compound at RT: 22.253 min (Fig.9). Additionally, Benzaldehyde, 3- methyl, aromatic compound was also detected. But the presence of Ticlopidine is unexpected and requires further validation. In milk media, Vitamin C, 1,11- Undecanedioic acid and 2- Methylbutyryl-L-carnitine as major compounds at RT: 8.507 min (Fig.10). Butanoic acid, 3-hydoxy-3-methyl and pentanoic acid, propyl esters were also identified. Groundnut cake media was rich in peptide derivatives and aromatic compounds while milk media contained vitamins, fatty acids and esters. Discussion Microorganisms play a crucial role in mosquito breeding sites by decomposing organic materials, leading to the production of volatile secondary metabolites [8,9,10] These microbial byproducts act as semiochemicals that attract gravid mosquitoes to suitable habitats for oviposition [11,12]. In this study Bacillus thuringieisis var israelensis (Bti) culture supernatants derived from groundnut cake and milk media were found to contain bioactive compounds that effectively attracted mosquitoes for egg laying. With respect to OAI, groundnut cake media was the most effective attractant for Aedes aegypti (OAI + 0.84) and Anopheles stephensi (OAI + 0.90). In contrast, milk media was most effective for Culex quinquefasciatus (OAI + 1.00) and Aedes aegypti (OAI + 0.74). These results suggest that different mosquito species exhibit species-specific oviposition preferences based on the chemical composition of microbial metabolites. The result of GC-MS analysis revealed the presence of various metabolites that may contribute the observed oviposition attraction. Notably, Cyclo (L- prolyl- L- Valine), Benzaldehyde, 3- methyl identified in GC media, have been previously reported as attractant by for Aedes aegypti and Culex pipens [13]. Similarly, milk media contained compounds such as Vitamin C, 1,11-Undecanedioic acid and 2-Methylbutyryl-L-carnitine. Additionally, Butanoic acid, 3hydroxy3-methyl, Pentanoic acid, propyl ester, compounds reported to attract Aedes aegypti, Aedes albopictus and Anopheles gambiae , were also detected [14,15] The specific chemical cues that attract gravid mosquitoes can contribute to the development of natural oviposition lures for mosquito surveillance and control programmes. It is possible to design effective vector management strategies that reduce mosquito population and limit the spread of mosquito borne diseases. Abbreviations ICMR – Indian Council of Medical Research Bti - Bacillus thuringiensis var israelensis CC - Coconut cake GC - Groundnut cake WB - Wheat bran g – Gram Hrs- Hours GC-MS - Gas Chromatography-Mass Spectrometry OAI - Oviposition Active Index mL- Millilitre mg- Milligram Min- Minutes NYSM - Nutrient Yeast Salt medium Nt - number of eggs in test solution Ns - number of eggs in control solution RH- Relative Humidity Rpm - Revolutions per minute SAIF - Sophisticated Analytical Instrumentation Facility. SD- Standard Deviation NIST- National Institute of Standards and Technology IIT- Indian Institute of Technology Declarations Ethics approval and consent to participate This study does not involve human or animal samples. The project under this study was done was approved by the institutional Scientific Advisory Committee (SAC) held in May 2022 and the ID number for the project is IM: 2235. Consent for publication: All authors have read, accepted and provide their consent for this publication and are not involved in any misconduct Availability of data and materials: All the raw data are available with the first author / corresponding author Competing Interests: There is no competing or conflict of interests Funding: ICMR - VCRC HRD and institute funding Authors' contributions: All authors have contributed sufficiently to qualify for authorship. L.T.P contributed to conceptualization, methodology, data curation and wrote the main manuscript text. K.A and G.P contributed to formal analysis, validation, reviewing and editing the manuscript. Acknowledgements: To Dr. Ashwani Kumar, Former Director, ICMR- VCRC, Puducherry for the approval of the research proposal and funding the project, to Mr. M. Sundharesan, Technical Officer for his technical support and to Mr. S. Paneerselvam for technical assistance. Authors also acknowledge the Head and Technical staffs of Sophisticated Analytical Instruments Facility (SAIF), Indian Institute of Technology, Madras , for the analysis of Gas Chromatography-Mass Spectrometry. References Baik LS, Carlson JR. The mosquito taste system and disease control. Proc Natl Acad Sci U S A. 2020 Dec 29;117(52):32848–56. Day J. Mosquito Oviposition Behavior and Vector Control. Insects. 2016 Nov 18; 7:65. Wilson AL, Courtenay O, Kelly-Hope LA, Scott TW, Takken W, Torr SJ, et al. The importance of vector control for the control and elimination of vector-borne diseases. PLoS Negl Trop Dis. 2020 Jan 16;14(1):e0007831. Millar JG, Chaney JD, Mulla MS. Identification of oviposition attractants for Culex quinquefasciatus from fermented Bermuda grass infusions. J Am Mosq Control Assoc. 1992 Mar;8(1):11–7. Geetha I, Paily KP, Padmanaban V, Balaraman K. Oviposition response of the mosquito, Culex quinquefasciatus to the secondary metabolite(s) of the fungus, Trichoderma viride. Mem Inst Oswaldo Cruz. 2003 Mar;98(2):223–6. Poonam S, Paily KP, Balaraman K. Oviposition attractancy of bacterial culture filtrates: response of Culex quinquefasciatus. Mem Inst Oswaldo Cruz. 2002 Apr;97(3):359–62. Hwang YS, Kramer WL, Mulla MS. Oviposition attractants and repellents of mosquitoes. J Chem Ecol. 1980 Jan 1;6(1):71–80. Chao J, Wistreich GA 1959. Microbial isolation from the midgut of Culex tarsalis. J Insect Pathol 1: 311-318. Chao J, Wistreich GA 1960. Microorganisms from the mid-gut of larval and adult Culex quinquefasciatus. J Insect Pathol 2: 220-224.Tolle MA. Mosquito-borne diseases. Curr Probl Pediatr Adolesc Health Care. 2009 Apr;39(4):97–140. Manonmani AM, Hoti SL, Balaraman K 1987. Isolation of mosquito pathogenic Bacillus thuringiensis strains from mosquito breeding habitats in Tamil Nadu. Indian J Med Res 86: 462-468. Himeidan YE, Temu EA, El Rayah EA, Munga S, Kweka EJ. Chemical Cues for Malaria Vectors Oviposition Site Selection: Challenges and Opportunities. J Insects. 2013 Nov 30;2013:e685182. Androsov GK, Skupchenko NL, Orlvskaia NV 1986. Microflora of blood-sucking mosquitoes in the taiga zone of the European part of northeastern USSR. Med Parazitoll (Mosk) 1: 21-24 Peach DAH, Gries G. Mosquito phytophagy – sources exploited, ecological function, and evolutionary transition to haematophagy. Entomol Exp Appl. 2020;168(2):120–36. Seenivasagan T, Guha L, Parashar BD, Agrawal OP, Sukumaran D. Olfaction in Asian tiger mosquito Aedes albopictus: flight orientation response to certain saturated carboxylic acids in human skin emanations. Parasitol Res. 2014 May 1;113(5):1927–32. Smallegange RC, Qiu YT, Bukovinszkiné-Kiss G, Van Loon JJA, Takken W. The effect of aliphatic carboxylic acids on olfaction-based host-seeking of the malaria mosquito Anopheles gambiae sensu stricto. J Chem Ecol. 2009 Aug;35(8):933–43. Tables Tables 1 to 7 are available in the Supplementary Files section. Additional Declarations No competing interests reported. Supplementary Files Tables.zip Cite Share Download PDF Status: Under Revision Version 1 posted Editorial decision: Revision requested 09 Feb, 2026 Reviews received at journal 25 Jan, 2026 Reviewers agreed at journal 09 Jan, 2026 Reviewers agreed at journal 10 Dec, 2025 Reviews received at journal 02 Dec, 2025 Reviewers agreed at journal 02 Dec, 2025 Reviewers invited by journal 02 Dec, 2025 Editor assigned by journal 02 Dec, 2025 Editor invited by journal 01 Dec, 2025 Submission checks completed at journal 01 Dec, 2025 First submitted to journal 01 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. 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5","display":"","copyAsset":false,"role":"figure","size":119386,"visible":true,"origin":"","legend":"\u003cp\u003eSee image above for figure legend.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-8078323/v1/c6c479d2ca750e6ea617be07.png"},{"id":97445405,"identity":"634d755e-f432-44c7-893c-d5c9e6b63090","added_by":"auto","created_at":"2025-12-04 12:43:57","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":116300,"visible":true,"origin":"","legend":"\u003cp\u003eSee image above for figure legend.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-8078323/v1/5d0e35f2f5f1de60c979d7b6.png"},{"id":97445411,"identity":"feb1e33a-6410-4641-970c-81ee211ebb43","added_by":"auto","created_at":"2025-12-04 12:43:57","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":178934,"visible":true,"origin":"","legend":"\u003cp\u003eSee image above for figure legend.\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-8078323/v1/4dc505353278e159b3085684.png"},{"id":97445407,"identity":"68daf510-5b07-4545-b08c-18802887b139","added_by":"auto","created_at":"2025-12-04 12:43:57","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":172681,"visible":true,"origin":"","legend":"\u003cp\u003eSee image above for figure legend.\u003c/p\u003e","description":"","filename":"8.png","url":"https://assets-eu.researchsquare.com/files/rs-8078323/v1/47abea7b34951f99a9e47060.png"},{"id":97668656,"identity":"31cc1154-1f4e-4a98-b394-45013ce701cf","added_by":"auto","created_at":"2025-12-08 09:25:58","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":330870,"visible":true,"origin":"","legend":"\u003cp\u003eSee image above for figure legend.\u003c/p\u003e","description":"","filename":"9.png","url":"https://assets-eu.researchsquare.com/files/rs-8078323/v1/c0a57730df47024fe600eedd.png"},{"id":97677625,"identity":"5f301cf8-4d65-48f4-b902-50290022c235","added_by":"auto","created_at":"2025-12-08 09:53:46","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3920048,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8078323/v1/7c3579d0-0c15-4127-bc2a-96e0437f4a4f.pdf"},{"id":97445404,"identity":"bfaa8785-f49e-447c-b71f-36171a4d6022","added_by":"auto","created_at":"2025-12-04 12:43:57","extension":"zip","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":813467,"visible":true,"origin":"","legend":"","description":"","filename":"Tables.zip","url":"https://assets-eu.researchsquare.com/files/rs-8078323/v1/0abae4ffb1dbf36bbd7667c2.zip"}],"financialInterests":"No competing interests reported.","formattedTitle":"Development of oviposition attractants from supernatant of Bacillus thuringiensis var israelensis (Serotype H-14, Strain VCRC B-17) using various culture media","fulltext":[{"header":"Introduction","content":"\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eMosquitoes are vectors of diseases such as malaria, dengue and chikungunya that impact humans [1]. The available method to tackle the problem is by controlling the vectors [2]. The goal of vector control is to minimize pathogen transmission by preventing human from mosquito bite [3]. Conventional vector control strategies rely heavily on chemical insecticides, which have led to resistance development and environmental concerns. Another common method is the use of ovitraps (oviposition traps), create artificial breeding areas for mosquitoes to lay their eggs. Oviposition attractants can be used to monitor vector populations, especially those of the \u003cem\u003eAedes\u003c/em\u003e and \u003cem\u003eCulex\u003c/em\u003e species. The addition of organic materials such as infusions made from leaves produces volatile chemicals, attracts gravid females to lay more eggs in the containers [4]. Microorganisms are also involved in the fermentation processes of the infusions. Earlier research indicates that bacteria including \u003cem\u003eTrichoderma viride\u003c/em\u003e, \u003cem\u003ePseudomonas fluorescence\u003c/em\u003e, and \u003cem\u003eBacillus sp\u003c/em\u003e. can be used [5,6]. In the present study, the supernatant of the \u003cem\u003eBacillus thuringiensis var israelensis\u003c/em\u003e (\u003cem\u003eBti)\u003c/em\u003e culture media was evaluated for the oviposition response among \u003cem\u003eCulex, Anophels and Aedes sps\u003c/em\u003e.\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cp\u003e\u003cstrong\u003ePreparation of NYSM Media:\u0026nbsp;\u003c/strong\u003eNutrient Yeast Salt medium (NYSM) medium was prepared with Glucose (5 g), yeast extract (5 g), Peptone (5 g), Na Cl (5 g), Beef extract (3 g), salt solution with calcium chloride, magnesium chloride and manganese chloride. The pH was adjusted to 7.5 and autoclaved at 121\u0026deg;C for 15 minutes. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePreparation of various media:\u0026nbsp;\u003c/strong\u003eFor the preparation of 1000 ml of different media, 10 gms of Soybean flour (Medium\u0026ldquo;A\u0026rdquo;) ,Wheat bran (Medium\u0026ldquo;B\u0026rdquo;), Groundnut cake powder (GC) (Medium\u0026ldquo;C\u0026rdquo;), Rice bran (Medium\u0026ldquo;D\u0026rdquo;), \u0026nbsp;Coconut cake powder (Medium\u0026ldquo;E\u0026rdquo;), Bengal gram media (Medium\u0026ldquo;F\u0026rdquo;), Coconut water (Medium\u0026ldquo;G\u0026rdquo;), Egg (Medium\u0026ldquo;H\u0026rdquo;), Milk (Medium \u0026ldquo;I\u0026rdquo;) were used. Powdered substrates (Medium A- F) were mixed in water, filtered and sterilized while liquid substrates (Medium G-H) were diluted (1:10) before autoclaving.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePreparation of seed culture:\u003c/strong\u003e A\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e100 \u003cimg width=\"9\" height=\"11\" src=\"https://myfiles.space/user_files/58895_8739fc6c57c1c19a/58895_custom_files/img1764850299.gif\" alt=\"image\"\u003eL of glycerol stock ICMR-VCRC strain of \u003cem\u003eBti\u003c/em\u003e was inoculated into 100 ml of NYSM medium and incubated at 30\u0026deg;C, 180 rpm for 16 hours. The culture was expanded, then inoculated (2 % v/v) into different media and incubated for 72 hours. Spore formation was confirmed using malachite green and safranin spore staining.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eOviposition attractancy study: \u0026nbsp;\u003c/strong\u003eFull-fed female \u003cem\u003eCulex quinquefasciatus, Anopheles stephensi, and Aedes aegypti\u003c/em\u003e mosquitoes were exposed to supernatants in a 55cm\u003csup\u003e3\u003c/sup\u003e mosquito cage under controlled conditions (28\u0026deg;C\u0026plusmn;2\u0026deg;C,70-80RH). \u0026nbsp;\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFor screening experiment:\u003c/strong\u003e\u003cem\u003e\u0026nbsp;\u003c/em\u003eFifty fully fed \u003cem\u003eCulex quinquefasciatus\u0026nbsp;\u003c/em\u003ewere used per trail. 10 different media supernatant (200mL) were individually exposed in enamel bowls. Tap water was placed in a separate bowl in the centre of the cage. The experiment was repeated 3 times. The bowls were placed at opposite corners of the cage and their positions were changed in each repetition. Mosquitoes were released at 16.00h and egg rafts were counted at 10.00h the following day. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFinal experiment: \u0026nbsp;\u003c/strong\u003e Full-fed female \u003cem\u003eCulex quinquefasciatus, Anopheles stephensi, and Aedes aegypti\u003c/em\u003e mosquitoes were placed in cages under controlled conditions. The most effective three media (NYSM, groundnut cake and milk) of 200mL each were selected for testing. The experimental conditions remained same as in the screening test. For \u003cem\u003eAedes aegypti,\u0026nbsp;\u003c/em\u003ea filter paper was placed around the bowl for egg collection. Each experiment was repeated three times.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eComparison with\u0026nbsp;\u003c/em\u003e\u003cem\u003epara\u003c/em\u003e-Cresol (p-Cresol): The most effective media supernatant was compared with p-Cresol (75\u0026micro;g/mL) as a standard attractant.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Analysis:\u003c/strong\u003e The oviposition attractancy index (OAI) of different bacterial culture supernatant and p-cresol concentrations was calculated. \u0026nbsp;\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cimg src=\"https://myfiles.space/user_files/58895_8739fc6c57c1c19a/58895_custom_files/img1764850357.png\" width=\"186\" height=\"75\"\u003e\u003c/p\u003e\n\u003cp\u003eNt, Ns are number of eggs in test solution and control respectively.\u003c/p\u003e\n\u003cp\u003eAs per the study of Kramer and Mulla (1979), OAI \u0026ge; 0.3 were attractants and OAI \u0026le; \u0026ndash;0.3 were repellents [7].\u003c/p\u003e\n\u003cp\u003eThe difference in the groups was evaluated using the Mann-Whitney U test. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eCost comparison of the effective media was also done.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eGas Chromatography- Mass Spectrometry Analysis:\u0026nbsp;\u003c/strong\u003eLyophilized supernatants from groundnut cake and milk media were dissolved in methanol and analysed sing GC-MS (NIST Library search) at the Sophisticated Analytical Instrumentation Facility (SAIF), IIT Madras, to identify volatile compounds responsible for mosquito attraction.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eScreening of\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003e\u003cem\u003eBacillus thuringiensis var\u0026nbsp;\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u003cem\u003eisraelensis\u003c/em\u003e\u003c/strong\u003e \u003cstrong\u003eculture supernatant for oviposition\u003cem\u003e\u0026nbsp;\u003c/em\u003eattraction in\u003cem\u003e\u0026nbsp;Culex quinquefasciatus\u0026nbsp;\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFrom the initial experiment, the egg laying was found in Milk, Groundnut cake extract, Bengal gram powder, wheat bran, coconut cake extracts and egg media as compared to water (Table.1).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eFor\u003cem\u003e\u0026nbsp;Culex quinquefasciatus,\u0026nbsp;\u003c/em\u003ethe highest number of egg rafts was observed in milk (68\u0026plusmn;4) (Mean\u0026plusmn;SD), followed by \u003cem\u003ep\u003c/em\u003e- Cresol (63\u0026plusmn;8.18) and groundnut cake media (GC) (47.67\u0026plusmn;19.09) (Figure.1). No oviposition was observed in water and NYSM media. Statistical analysis showed significant differences compared between GC and NYSM (p = 0.037) as well as between GC and \u003cem\u003ep\u003c/em\u003e- Cresol (p=0.049) (Table.2) (Fig.4). OAI showed negative value ( -0.14) for GC media\u0026nbsp;(Table.5).\u003c/p\u003e\n\u003cp\u003eFor \u003cem\u003eAnopheles stephensi\u003cstrong\u003e,\u0026nbsp;\u003c/strong\u003e\u003c/em\u003ethe highest egg laying was found in GC media (1471\u0026plusmn;17.21), followed by NYSM (683.27\u0026plusmn;35.22), milk (188\u0026plusmn;70.40), \u003cem\u003ep\u003c/em\u003e- Cresol (116\u0026plusmn;10.21) and water (80\u0026plusmn;6.24) (Figure.2). GC media and NYSM showed significantly higher egg counts compared to p- Cresol and water (p= 0.049) (Table.3) (Fig.5). OAI was 0.90 for GC and 0.24 for milk media\u0026nbsp;(Table.5).\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFor \u003cem\u003eAedes aegypti\u003c/em\u003e, GC media had the highest mean egg count (1186 \u0026plusmn; 1371.38), followed by milk (483\u0026plusmn;93.29), water (102\u0026plusmn;38.79) and p- Cresol (72\u0026plusmn;30.35) with no oviposition in NYSM (Fig.3). Statistical analysis revealed significant differences between GC and\u003cem\u003e\u0026nbsp;\u003c/em\u003ep- Cresol (p=0.049) (Table.4) (Fig.6). OAI was 0.84 for GC and 0.74 for milk media (Table.5).\u003c/p\u003e\n\u003cp\u003eThe cost for the production of the culture supernatant from groundnut cake extract was estimated as ₹2.72 whereas milk as ₹5.00\u0026nbsp;for 1 litre.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eGas chromatography- Mass Spectrometry analysis of the culture supernatant of groundnut cake and milk media\u003c/strong\u003e \u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAgilent Model 8890 GC System with Single Quadrupole Mass Spectrometer (5977B MSD) analyser is used for the separation and identification of volatile compounds. NIST spectral library search was also used for the compound identification as it provides reference mass spectra for GC/MS (by electron ionization) as well as gas phase retention indices for gas chromatography. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eGC\u0026ndash;MS analysis of the two media, Groundnut cake extract and milk media revealed the presence of 10 compounds in groundnut cake (Table.6) and 14 volatile compounds in milk media (Table.7) for parameters such as retention time (RT), molecular formula (MF), mass- ratio (m/z) and concentration (% peak area).\u003c/p\u003e\n\u003cp\u003eThe total ion chromatograms (TIC) of groundnut cake media and milk media revealed distinct chemical compositions. GC media showed a significant peak at 22.253 min, while milk media exhibited a dominant peak at 8.507 min (Fig.7, 8).\u003c/p\u003e\n\u003cp\u003eThe mass spectral analysis of GC media identified Cyclo (L- prolyl- L- Valine) as the most probable compound at RT: 22.253 min (Fig.9). Additionally, Benzaldehyde, 3- methyl, aromatic compound was also detected. But the presence of Ticlopidine is unexpected and requires further validation.\u003csup\u003e\u0026nbsp;\u0026nbsp;\u003c/sup\u003e\u003c/p\u003e\n\u003cp\u003eIn milk media, Vitamin C, 1,11- Undecanedioic acid and 2- Methylbutyryl-L-carnitine as major compounds at RT: 8.507 min (Fig.10). \u0026nbsp;Butanoic acid, 3-hydoxy-3-methyl and pentanoic acid, propyl esters were also identified. Groundnut cake media was rich in peptide derivatives and aromatic compounds while milk media contained vitamins, fatty acids and esters.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eMicroorganisms play a crucial role in mosquito breeding sites by decomposing organic materials, leading to the production of volatile secondary metabolites [8,9,10] These microbial byproducts act as semiochemicals that attract gravid mosquitoes to suitable habitats for oviposition [11,12]. In this study \u003cem\u003eBacillus thuringieisis var israelensis\u003c/em\u003e (Bti) culture supernatants derived from groundnut cake and milk media were found to contain bioactive compounds that effectively attracted mosquitoes for egg laying.\u003c/p\u003e\u003cp\u003eWith respect to OAI, groundnut cake media was the most effective attractant for \u003cem\u003eAedes aegypti\u003c/em\u003e (OAI\u0026thinsp;+\u0026thinsp;0.84) and \u003cem\u003eAnopheles stephensi\u003c/em\u003e (OAI\u0026thinsp;+\u0026thinsp;0.90). In contrast, milk media was most effective for \u003cem\u003eCulex quinquefasciatus\u003c/em\u003e (OAI\u0026thinsp;+\u0026thinsp;1.00) and \u003cem\u003eAedes aegypti\u003c/em\u003e (OAI\u0026thinsp;+\u0026thinsp;0.74). These results suggest that different mosquito species exhibit species-specific oviposition preferences based on the chemical composition of microbial metabolites.\u003c/p\u003e\u003cp\u003eThe result of GC-MS analysis revealed the presence of various metabolites that may contribute the observed oviposition attraction. Notably, Cyclo (L- prolyl- L- Valine), Benzaldehyde, 3- methyl identified in GC media, have been previously reported as attractant by for \u003cem\u003eAedes aegypti\u003c/em\u003e and \u003cem\u003eCulex pipens\u003c/em\u003e [13]. Similarly, milk media contained compounds such as Vitamin C, 1,11-Undecanedioic acid and 2-Methylbutyryl-L-carnitine. Additionally, Butanoic acid, 3hydroxy3-methyl, Pentanoic acid, propyl ester, compounds reported to attract \u003cem\u003eAedes aegypti, Aedes albopictus\u003c/em\u003e and \u003cem\u003eAnopheles gambiae\u003c/em\u003e, were also detected [14,15]\u003c/p\u003e\u003cp\u003eThe specific chemical cues that attract gravid mosquitoes can contribute to the development of natural oviposition lures for mosquito surveillance and control programmes. It is possible to design effective vector management strategies that reduce mosquito population and limit the spread of mosquito borne diseases.\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eICMR\u003cstrong\u003e\u0026nbsp;\u0026ndash; \u0026nbsp;Indian Council of Medical Research\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBti\u003cem\u003e- Bacillus thuringiensis var israelensis\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eCC - Coconut cake\u003c/p\u003e\n\u003cp\u003eGC - Groundnut cake\u003c/p\u003e\n\u003cp\u003eWB - Wheat bran\u003c/p\u003e\n\u003cp\u003eg \u0026ndash; Gram\u003c/p\u003e\n\u003cp\u003eHrs- Hours\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eGC-MS - Gas Chromatography-Mass Spectrometry\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eOAI - Oviposition Active Index\u0026nbsp;\u003c/p\u003e\n\u003cp\u003emL- Millilitre\u003c/p\u003e\n\u003cp\u003emg- Milligram\u003c/p\u003e\n\u003cp\u003eMin- Minutes\u003c/p\u003e\n\u003cp\u003eNYSM - \u0026nbsp;Nutrient Yeast Salt medium\u003c/p\u003e\n\u003cp\u003eNt - number of eggs in test solution\u003c/p\u003e\n\u003cp\u003eNs - number of eggs in control solution\u003c/p\u003e\n\u003cp\u003eRH- Relative Humidity\u003c/p\u003e\n\u003cp\u003eRpm - Revolutions per minute\u003c/p\u003e\n\u003cp\u003eSAIF - Sophisticated Analytical Instrumentation Facility.\u003c/p\u003e\n\u003cp\u003eSD- Standard Deviation\u003c/p\u003e\n\u003cp\u003eNIST- National Institute of Standards and Technology\u003c/p\u003e\n\u003cp\u003eIIT- Indian Institute of Technology\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study does not involve human or animal samples. The project under this study was done was approved by the institutional Scientific Advisory Committee (SAC) held in May 2022 and the ID number for the project is IM: 2235.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors have read, accepted and provide their consent for this publication and are not involved in any misconduct\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll the raw data are available with the first author / corresponding author\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interests:\u003c/strong\u003e\u003cstrong\u003e\u003cbr\u003e\u003c/strong\u003eThere is no competing or conflict of interests\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eICMR - VCRC HRD and institute funding\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026apos; contributions:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors have contributed sufficiently to qualify for authorship.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eL.T.P contributed to conceptualization, methodology, data curation and wrote the main manuscript text.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eK.A and G.P contributed to formal analysis, validation, reviewing and editing the manuscript.\u003cstrong\u003e\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements: \u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo Dr. Ashwani Kumar, Former Director, ICMR- VCRC, Puducherry for the approval of the research proposal and funding the project, to Mr. M. Sundharesan, Technical Officer for his technical support and to Mr. S. Paneerselvam for technical assistance. Authors also acknowledge the Head and Technical staffs of\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eSophisticated Analytical Instruments Facility (SAIF), Indian Institute of Technology, Madras\u003cstrong\u003e,\u003c/strong\u003e for the analysis of Gas Chromatography-Mass Spectrometry.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eBaik LS, Carlson JR. The mosquito taste system and disease control. Proc Natl Acad Sci U S A. 2020 Dec 29;117(52):32848\u0026ndash;56. \u0026nbsp; \u0026nbsp;\u003c/li\u003e\n \u003cli\u003eDay J. Mosquito Oviposition Behavior and Vector Control. Insects. 2016 Nov 18; 7:65. \u0026nbsp;\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eWilson AL, Courtenay O, Kelly-Hope LA, Scott TW, Takken W, Torr SJ, et al. The importance of vector control for the control and elimination of vector-borne diseases. PLoS Negl Trop Dis. 2020 Jan 16;14(1):e0007831. \u0026nbsp;\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eMillar JG, Chaney JD, Mulla MS. Identification of oviposition attractants for Culex quinquefasciatus from fermented Bermuda grass infusions. J Am Mosq Control Assoc. 1992 Mar;8(1):11\u0026ndash;7. \u0026nbsp;\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eGeetha I, Paily KP, Padmanaban V, Balaraman K. Oviposition response of the mosquito, Culex quinquefasciatus to the secondary metabolite(s) of the fungus, Trichoderma viride.\u0026nbsp;Mem Inst Oswaldo Cruz. 2003 Mar;98(2):223\u0026ndash;6. \u0026nbsp;\u0026nbsp;\u003c/li\u003e\n \u003cli\u003ePoonam S, Paily KP, Balaraman K. Oviposition attractancy of bacterial culture filtrates: response of Culex quinquefasciatus. Mem Inst Oswaldo Cruz. 2002 Apr;97(3):359\u0026ndash;62. \u0026nbsp; \u0026nbsp;\u003c/li\u003e\n \u003cli\u003eHwang YS, Kramer WL, Mulla MS. Oviposition attractants and repellents of mosquitoes. J Chem Ecol. 1980 Jan 1;6(1):71\u0026ndash;80. \u0026nbsp;\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eChao J, Wistreich GA 1959. Microbial isolation from the midgut of Culex tarsalis. J Insect Pathol 1: 311-318. \u0026nbsp;\u003c/li\u003e\n \u003cli\u003eChao J, Wistreich GA 1960. Microorganisms from the mid-gut of larval and adult Culex quinquefasciatus. J Insect Pathol 2: 220-224.Tolle MA. Mosquito-borne diseases. Curr Probl Pediatr Adolesc Health Care. 2009 Apr;39(4):97\u0026ndash;140. \u0026nbsp;\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eManonmani AM, Hoti SL, Balaraman K 1987. Isolation of mosquito pathogenic Bacillus thuringiensis\u0026nbsp;strains from mosquito breeding habitats in Tamil Nadu. Indian J Med Res 86: 462-468.\u003c/li\u003e\n \u003cli\u003eHimeidan YE, Temu EA, El Rayah EA, Munga S, Kweka EJ. Chemical Cues for Malaria Vectors Oviposition Site Selection: Challenges and Opportunities. J Insects. 2013 Nov 30;2013:e685182. \u0026nbsp;\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eAndrosov GK, Skupchenko NL, Orlvskaia NV 1986. Microflora of blood-sucking mosquitoes in the taiga zone of the European part of northeastern USSR. Med Parazitoll (Mosk) 1: 21-24\u003c/li\u003e\n \u003cli\u003ePeach DAH, Gries G. Mosquito phytophagy \u0026ndash; sources exploited, ecological function, and evolutionary transition to haematophagy. Entomol Exp Appl. 2020;168(2):120\u0026ndash;36. \u0026nbsp;\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eSeenivasagan T, Guha L, Parashar BD, Agrawal OP, Sukumaran D. Olfaction in Asian tiger mosquito Aedes albopictus: flight orientation response to certain saturated carboxylic acids in human skin emanations. Parasitol Res. 2014 May 1;113(5):1927\u0026ndash;32. \u0026nbsp;\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eSmallegange RC, Qiu YT, Bukovinszkin\u0026eacute;-Kiss G, Van Loon JJA, Takken W. The effect of aliphatic carboxylic acids on olfaction-based host-seeking of the malaria mosquito Anopheles gambiae sensu stricto. J Chem Ecol. 2009 Aug;35(8):933\u0026ndash;43. \u0026nbsp;\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003eTables 1 to 7 are available in the Supplementary Files section.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bmc-research-notes","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"resn","sideBox":"Learn more about [BMC Research Notes](http://bmcresnotes.biomedcentral.com)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/resn/default.aspx","title":"BMC Research Notes","twitterHandle":"@BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Biological control, Bacillus thuringiensis, Culture filtrate, GC,MS analysis, groundnut cake media, mosquito oviposition attractancy","lastPublishedDoi":"10.21203/rs.3.rs-8078323/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8078323/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cb\u003eobjectives\u003c/b\u003e\u003c/p\u003e\u003cp\u003eMosquito-borne diseases pose a significant global health threat, with vector control being the primary method for managing these diseases. Biological control methods, particularly the use of ovitraps, bio control agents, and oviposition attractants, play a crucial role. Oviposition attractants, like organic materials such as bamboo infusion, are employed to lure gravid females for egg-laying.\u003c/p\u003e\u003cp\u003e\u003cb\u003eObjectives\u003c/b\u003e\u003c/p\u003e\u003cp\u003e\u003cem\u003eBacillus thuringiensis\u003c/em\u003e var \u003cem\u003eisraelensis\u003c/em\u003e various culture media supernatant was assessed for oviposition response in \u003cem\u003eCulex, Anopheles\u003c/em\u003e, and \u003cem\u003eAedes\u003c/em\u003e species.\u003c/p\u003e\u003cp\u003e\u003cb\u003eMaterials and Methods\u003c/b\u003e\u003c/p\u003e\u003cp\u003eVarious media were prepared, and \u003cem\u003eBacillus thuringiensis var israelensis\u003c/em\u003e culture supernatant was tested for attractancy. The results, analysed through Oviposition Active Index (OAI) and Mann-Whitney U test.\u003c/p\u003e\u003cp\u003e\u003cb\u003eResults\u003c/b\u003e\u003c/p\u003e\u003cp\u003eIn this study, it is identified that supernatant from groundnut cake and milk media has enhanced mosquito oviposition attraction, with significantly higher OAI values in experimental setups compared to controls. Gas Chromatography-Mass Spectrometry analysis revealed the presence of volatile organic compounds potentially responsible for attractant properties.\u003c/p\u003e\u003cp\u003e\u003cb\u003eConclusion\u003c/b\u003e\u003c/p\u003e\u003cp\u003eGroundnut cake and milk media demonstrated high attractancy for \u003cem\u003eCulex, Anopheles\u003c/em\u003e, and \u003cem\u003eAedes\u003c/em\u003e mosquitoes. The compounds identified contribute to the overall oviposition attraction. This study highlights promising biological control measures in mosquito vector management.\u003c/p\u003e","manuscriptTitle":"Development of oviposition attractants from supernatant of Bacillus thuringiensis var israelensis (Serotype H-14, Strain VCRC B-17) using various culture media","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-12-04 12:43:52","doi":"10.21203/rs.3.rs-8078323/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-02-09T11:26:51+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-01-25T10:14:08+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"166528971801989733925196799721395997665","date":"2026-01-09T16:37:17+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"286131364410842530428828106846189199229","date":"2025-12-10T16:00:17+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-12-02T16:52:32+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"258124826270947304183276599240302191349","date":"2025-12-02T07:52:21+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-12-02T07:44:44+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-12-02T07:40:15+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-12-01T16:20:24+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-12-01T15:37:59+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Research Notes","date":"2025-12-01T15:02:35+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-research-notes","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"resn","sideBox":"Learn more about [BMC Research Notes](http://bmcresnotes.biomedcentral.com)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/resn/default.aspx","title":"BMC Research Notes","twitterHandle":"@BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"1f7bc077-762c-4651-b7fe-25257a484a62","owner":[],"postedDate":"December 4th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"in-revision","subjectAreas":[],"tags":[],"updatedAt":"2026-04-23T06:56:00+00:00","versionOfRecord":[],"versionCreatedAt":"2025-12-04 12:43:52","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8078323","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8078323","identity":"rs-8078323","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}