Effects of a caffeine-based diet on insecticide resistance and longevity in infected Anopheles coluzzii

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Koella, Benjamin Koudou Guibéhi, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7427747/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 Alkaloids such as caffeine can be toxic for insects. However, although mosquitoes feed on many plants with nectar containing alkaloids, their impact on the vectorial capacity of mosquitoes is not known, in particular in the face of increasing resistance to insecticides. We assessed with the mosquito Anopheles coluzzii how caffeine affects several measures of resistance to deltamethrin – the rate at which mosquitoes are knocked-down during exposure, the mortality within 24 hours of exposure, and the longevity following exposure – and we compared these traits for mosquitoes that were uninfected or infected by the malaria parasite Plasmodium falciparum . The mosquitoes were fed throughout their lives on a 10% sugar solution supplemented with 0, 50, or 200 ppm caffeine. Three or four days after emergence, they were given an infected or an uninfected blood meal. Another three days later, they were exposed to deltamethrin or to a sham and checked for knock-down during the exposure and death within the next 48 hours. We monitored the surviving mosquitoes for longevity and assessed their infection status when they died. The rate of mosquitoes knocked down by the insecticide increased with higher caffeine concentrations, but neither the infection status nor its interaction with caffeine concentration influenced the knockdown rate. Similarly, caffeine increased the mortality of insecticide-exposed mosquitoes within 48 hours after exposure. The mortality was highest if mosquitoes had fed on infected blood but harbored no parasites, and lowest if they had not fed on infected blood. The longevity, once the mosquitoes had survived the first 48 hours, was not affected by the concentration of caffeine or by any of the combination of caffeine with infection status or insecticide, but, the mosquitoes that had been exposed to the insecticide lived longer than unexposed ones, in particular if they had fed on infected blood but were not infected. Overall, our experiment highlights that the level of resistance to an insecticide is affected by complex interactions between the mosquito’s diet and infection by malaria. Entomology caffeine malaria infection insecticide resistance Figures Figure 1 Figure 2 Figure 3 Introduction Anopheles gambiae , the main vector of malaria in Africa, lives on average less than 10 days in natural situations, but it takes 11 to 14 days for the malaria parasite to develop into infectious stages [ 1 ] [ 2 ] [ 3 ] [ 4 ]. Thus, most infected mosquitoes do not survive long enough to transmit malaria, and transmission is very sensitive to changes in the life-span [ 5 ] [ 6 ] [ 7 ]. Indeed, this is one of the reasons why targeting adults with insecticides (either through residual indoor spraying or on insecticide-treated bed nets) to shorten the mosquitoes’ life-span has become the main method of vector control [ 8 ] [ 9 ]. However, increasing mosquito resistance is reducing the effectiveness of insecticides and therefore posing a significant public health threat [ 10 ] [ 11 ] [ 12 ]. Nevertheless, insecticide-based vector control considerably decreased malaria incidence in many parts of Africa despite increasing resistance of the local malaria vectors ( [ 13 ] [ 14 ] [ 14 ] [ 15 ] ). One of the reasons for the discrepancy between measures of resistance and their epidemiological consequences is that the impact of genes conferring resistance depends on environmental parameters like the quantity of food [ 16 ] and infection by parasites [ 17 ] including malaria [ 18 ]. An important aspect of a mosquito’s diet is its nectar meal [ 19 ], and variation of the quality of nectar among plant species influences longevity [ 20 ] and resistance to insecticides [ 21 ]. Sugars are the main biochemical components of nectar [ 22 ]. This variation is partly due to the concentrations of the types of sugar in the nectar [ 23 ] [ 24 ] [ 25 ]. An additional impact of the nectar can be through its toxic compounds like alkaloids [ 26 ] [ 27 ] [ 28 ]. One alkaloid is caffeine, which occurs in the fruit and nectar of many plant species including Coffea , Citrus, Cocoa , tea plants [ 29 ] [ 30 ] [ 31 ] [ 32 ] [ 33 ] [ 34 ]. Although caffeine can act as an insecticide because of its neurotoxic properties [ 35 ] [ 36 ] [ 37 ] [ 38 ], not much is known about how it, on its own or in conjunction with malaria, affects insecticide-resistance. We therefore assessed how caffeine (in concentrations commonly found in nectar) interacts with the infectious status of mosquitoes to influence their response to insecticide, measured as the rate of knock-down during exposure, the mortality within 48 hours of exposure and the longevity following exposure. Methods The experiment was run at the Institut de Recherches en Science de la Santé de Bobo Dioulasso (IRSS) in Burkina Faso. We used the strain ‘vallée du Kou’ of the mosquito Anopheles coluzzi , which has metabolic resistance mediated by cytochrome p450 and target-site resistance mediated by the gene kdr. The mosquitoes were maintained in a biosafety room at 27 ± 2°C, 70 ± 5% relative humidity and 12:12 light-dark cycle. Experimental design The experiment was run in three blocks, each one with the blood of a different gametocytic child as a source of malaria parasites. Within each block, we reared larvae in 40 to 50 trays containing between 250 and 300 larvae in 1 l of tap water and fed them daily with Tetramin baby fish food according to their age. We fed them daily with Tetramin baby fish food according to their age (day of hatching: 14 mg per tray; 1 day old: 21 mg; 2 days old: 29 mg; 3 days old: 58 mg; 4 days old: 115 mg, 5 days old or older: 216 mg). Adult males were discarded, and females were given continual access to a 10% glucose solution supplemented with 0, 50 or 200 ppm of caffeine and were given the opportunity to feed on infected or uninfected blood three or four days after emergence. (See below for the method of infection.) Each treatment was replicated in four cups of 55 to 65 mosquitoes. The females that did not take a full blood meal were discarded, leaving between 18 and 30 mosquitoes per cup). 72 hours later they were exposed to 0.25% deltamethrin or to a sham for 1 hour with the WHO bioassay kit [ 39 ]. The mosquitoes of each cup were tested together. During the exposure to the insecticide, we counted the number of mosquitoes that were knocked down, and 48 hours after the exposure we counted the number of dead mosquitoes. The survivors were maintained for at most 30 days with continual access to their diet, and their mortality was assessed every day. Once they had died, the mosquitoes that had fed on infected blood were tested for the presence of malaria parasites with standard PCR-methods [ 40 ]; within each block 100 haphazardly selected mosquitoes that had fed on uninfected blood were also tested for malaria to ensure the absence of parasites. Infection status was classified as ‘Uninfected’ for those fed on an uninfected blood, ‘Harbored parasite’ for those fed on infected blood and have been harbored parasite and ‘No harbored parasite’ for those fed on infected blood but not harboring any parasites. Experimental infection For each block, 200 five- to 15-year-old schoolchildren were recruited in Nasso village, located 13 km from Bobo-Dioulasso, Burkina Faso. Female mosquitoes were exposed to blood samples from naturally P. falciparum gametocyte-infected from these children using a direct membrane feeding assay (DMFA) as described previously [ 41 , 42 ] [ 43 ] [ 41 ]. Briefly, thick blood smears were taken from each volunteer, air-dried, Giemsa-stained and examined by microscopy for the presence of P. falciparum at the IRSS laboratory in Bobo-Dioulasso. Asexual trophozoite parasite stages were counted against 200 leucocytes, while infectious gametocyte stages were counted against 1000 leukocytes. Children with asexual parasitaemia of > 1000 parasites per microlitre (estimated based on an average of 8000 leucocytes ml − 1 ) were treated in accordance with national guidelines. Asymptomatic P. falciparum gametocyte-positive children were recruited for the study. Blood from gametocyte isolates was collected by venipuncture in heparinized tubes. Three distinct parasite isolates (named hereafter A, B and C), with respective gametocytaemia of 56, 72 and 128 gametocytes µl − 1 of blood, were used for the experimental infections. DMFA was performed by replacing donor plasma with an equivalent volume of AB + serum from malaria-naïve European donors. After centrifugation (Eppendorf, centrifuge 5702 R) at 37°C at 12,000 rpm for 5 min. A horizontal line marking the upper limit of the plasma phase was drawn on the tube. The plasma was aspirated with a pipette, and AB + serum was introduced up to the marked line. For each gametocyte carrier, half of the blood sample was transferred into 1.5 mL Eppendorf tubes and inactivated at 45°C for 20 minutes at 900 rpm using a compact thermomixer [ 43 ]. The mosquitoes were starved of sugar solution for 24 h and then fed on infected or uninfected blood via membrane filters for 1 hour. Data analysis The statistical analyses were performed with R version R-4.4.2 [ 44 ]. We found the significance of the effects with the function Anova (package car) [ 45 ], using a type 3 SS if the interactions were significant and a type 2 if they were not. We analysed the knock-down proportion and 48-hour mortality using GLMs. For mortality, we included diet, infection status, insecticide and their interactions as fixed factors. For the knock-down analysis, we replaced infection status with blood type. Since only 0.13% of mosquitoes in the group not exposed to insecticide were knocked down, compared to 59.62% in the insecticide-exposed group, we focused solely on the mosquitoes that were exposed to insecticide. In both models, blocks (source of blood) and cups were included as random effects. In the analysis of longevity we included only the mosquitoes that had survived the first 48 hours after being exposed to the insecticide to avoid confounding impacts on longevity and on mortality. 196 mosquitoes lived longer than 30 days and were right-censored. We used a coxme model that included the diet, the infection status, the exposure to insecticide, and all interactions as fixed factors and the block (source of blood) and cups as random factors. Results Of the 1632 mosquitoes exposed to insecticide, 59.6% were knocked down within 1 hour of the exposure. The knock-down rate increased with caffeine concentration from 41.4% (95% c.i: 24.26%-60.9%) at 0 ppm, 61.4% (41.7%-78.0%) at 50 ppm to 73.7% (53.7%-87.1%) at 200 ppm, (𝝌²=38.43, df = 1, p < 0.001). However, neither the type of the blood ingested (𝝌²=0.55, df = 1, p = 0.459) nor the interaction between the caffeine concentration and the type of blood ingested (𝝌²=1.80, df = 1, p = 0.408) had an effect on the rate of knock-down ( Fig. 1 ). Of the 1,559 mosquitoes exposed to insecticide, 53.2% (95% CI: 50.7–55.6%) died within 48 hours, compared to 9.4% (95% CI: 8.0–11.0%) of the 1,471 mosquitoes exposed to the sham. Insecticide exposure significantly increased 48-hour post-exposure mortality, (𝝌² = 260.79, df = 1,p < 0.001). Caffeine diet alone did not influence the mortality rate, (𝝌²=0.81, df = 2, p = 0.668). Howether, there was a combined effect of caffeine and insecticide exposure: Without insecticide, mortality was relatively low, ranging from 7.0% ( 5.1%- 9.7%) at 200 ppm to 10.2% (7.9% − 13.1%) at 0 ppm. In the presence of insecticide, mortality increased significantly, reaching 43.6% (39.3% − 48.0%) at 0 ppm, 50.4% ( 46.1% − 54.6%) at 50 ppm and 64.8% (60.7% − 68.7%) at 200 ppm, (𝝌²=17.76, df = 2, p < 0.001). Mortality varied according to infection status. Infected mosquitoes harboring parasites had a lower mortality rate with 23.9% (21.3–26.7%), while infected mosquitoes that have not harbouring any parasites had a higher mortality of 47.4% (43.4–51.4%). Uninfected mosquitoes had an intermediate mortality of 30.8% (28.5–33.2%), (𝝌²=17.50, df = 2, p < 0.001). A significant interaction was detected between insecticide exposure and infection status. Mortality increased across all groups after insecticide exposure, and the magnitude varied: 33.8 percentage points in those infected and harboring parasite [ 8.5% (6.4–11.3%) to 42.3% (37.7–46.9%) ], 54.4 in those wich infected but did not harboring parasite [14.2% (10.3–19.2%) to 68.6%(63.7–73.1%)], and 43.5 in uninfected mosquitoes [8.4% (6.6–10.7%) to 51.9%(48.4–55.5%)], (𝝌² = 7.25, df = 2, p = 0.026). There was no significant interaction between the caffeine-based diet and infection status (𝝌²=8.87, df = 4, p = 0.064), but a significant three-way interaction between infection status, insecticide exposure, and caffeine concentration influenced mosquito mortality (𝝌²=9.85, df = 4, p = 0.043). At 0 ppm caffeine, mortality after insecticide exposure was highest in those which have been infected but don’t harboring parasite [63.7% (54.5%-72.0%) vs. infected and harboring parasite 37.7% (30.4%-45.7%) and uninfected 37.4% (31.4%-43.9%)]. At 200 ppm, caffeine reduced mortality in non-exposed mosquitoes infected which did not harboring any parasite 2.5% (0.7%-8.6%), but mortality after exposure remained highest in this group 77.7% (69.5%-84.2%) vs. uninfected 68.5% (62.7%-73.8%) and those infected harboring parasite 47.3%(39.3%-55.3%), ( Fig. 2 ) . The average longevity of the 2,063 mosquitoes that survived the first 48 hours was 18.6 days. It was not significantly affected by caffeine concentration (𝝌2 = 1.46, df = 2, p = 0.48), nor by any of its interactions (𝝌2 0.17), (Fig. 3). Longevity was about 1 day longer among insecticide-exposed mosquitoes than among unexposed ones (𝝌2 = 4.49, df = 1, p = 0.034). The effect of exposure depended on whether the mosquitoes were infected by malaria. In infected mosquitoes harboring parasites, the average longevity was 18.3 (17.5–19.1) days without exposure to the insecticide and 17.7 days (16.5–18.8) with exposure. In infected mosquitoes which didn’t harbor parasites, the average longevity was 17.5 days (16.3–18.6) without insecticide exposure and 19.6 days (17.9–21.9) with insecticide exposure. In uninfected mosquitoes, average longevity was 18.7 days (18.0-19.3) without insecticide and 20.0 days (19.0-20.9) with insecticide exposure (interaction exposure * infection status: 𝝌2 = 6.06, df = 2, p = 0.048), (Fig. 4). Make sure the figures are formatted the same. Same titles, same shading, ….. Discussion Our study examined how caffeine and mosquito infection status influence the phenotype of resistance and the longevity of survivors after exposure to insecticide. Mortality increased as caffeine concentration rose in the presence of insecticide exposure. Our work shows that increasing the concentration of caffeine increases the mortality rate of mosquitoes after exposure to the insecticide. Caffeine, an alkaloid, may enhance insecticide efficacy in Anopheles gambiae by acting as a neurotoxin and inducing oxidative stress [ 46 ] [ 47 ] [ 48 ]. Since exposure to the insecticide also induces ROS production via the activity of cytochrome P450. The combination of insecticide detoxification and caffeine-induced oxidative stress overloads the physiological defenses impairing detoxification enzyme efficiency and reducing resistance to insecticide, thereby increasing mortality. Our findings align with (Cissé et al., unpublished work), we observed increasing mortality of mosquitoes with rising caffeine concentration. Previous studies on Aedes larvae similarly demonstrated dose-dependent toxicity of caffeine [ 49 ] [ 50 ]. The decrease in mosquito resistance with increasing caffeine concentration could also be explained by a repellent effect of the molecule [ 51 ], leading to smaller meals and lower energy reserves. As caffeine concentration increases, the repellent effect becomes stronger, reducing meal intake and making mosquitoes more sensitive to the insecticide. Infected mosquitoes that did not harbor any parasites and were exposed to insecticide were more susceptible to insecticide compared to other mosquitoes The infected mosquitoes which did not harbor any parasites may have mounted an immune response against the infection, even though no parasites were ultimately detected [ 52 ] [ 53 ] [ 54 ]. [ 55 ] [ 56 ]. Additionally, the immune response is often linked to an increased production of reactive oxygen species (ROS) [ 57 ] [ 58 ] [ 59 ] which may amplify the cytotoxic effects of the insecticide, thereby increasing mortality [ 60 ]. The high mortality rate could, therefore, result from the combined effects of the immune response cost and the oxidative stress induced by the insecticide toxicity. Caffeine alone had no effect on the longevity of the survivors The absence of an effect of caffeine on mosquito longevity was unexpected, given that caffeine is both a neurotoxic and pro-oxidant compound, and especially since it significantly affected mosquito resistance within 48 hours after insecticide exposure. Our result contrasts with several studies that have reported an effect of caffeine on various mosquito life-history traits. For instance, it has been shown to increase the locomotion of larva stage [ 61 ], and on the other hand, it would lead to a reduction of fertility in the species Aedes albopictus [ 62 ]. Other studies have shown that a caffeine-based diet reduces the longevity of Aedes albopictus at the same concentrations used in our study [ 51 ]. In addition, a reduction of longevity was observed in some flies species Musca domestica [ 63 ], Drosophila prosaltans et D. melanogaster [ 64 ] [ 65 ] due to their caffeine-based diet. Caffeine increases the lifespan of bees [ 66 ]. Insecticide exposure increased the general longevity of mosquitoes The improved survival of mosquitoes exposed to insecticide can be attributed to selective sorting that favors larger individuals, which likely benefit from energy reserves accumulated during their larval stage [ 16 ] [ 67 ]. Larger mosquitoes tend to possess a higher teneral reserve, which may enhance their longevity [ 68 ]. Mosquitoes exposed to Plasmodium falciparum but not infected show high mortality within 48 hours of exposure to the insecticide, but survivors live longer than those in other groups. This phenomenon could be explained by the fact that exposure to gametocytes without actual infection would trigger an immune or metabolic response, enhancing their long-term survival after the elimination of the weakest individuals. Our study has several limitations. First, the mosquitoes used originated from a single laboratory strain known for its high level of insecticide resistance and were reared under controlled conditions. This may limit the generalizability of our findings to wild Anopheles gambiae populations, which may exhibit different ecological and physiological characteristics. Second, the detection of Plasmodium DNA in some mosquitoes does not confirm the presence of viable or developing parasites, as PCR may amplify viable or non-viable DNA. Finally, since only one insecticide (deltamethrin) was tested, the results cannot be extrapolated to other classes of insecticides, to which resistant mosquitoes might respond differently. Conclusion Our study shows that a caffeine-based diet increases mosquito mortality within 48 hours following insecticide exposure, but does not affect the longevity of survivors. Moreover, infection status did not interact with the caffeine diet to influence either mortality 48 hours after exposure or longevity of the survivors. The results highlight the potential of caffeine as a complementary vector control tool, capable of enhancing mosquito susceptibility to insecticides. Caffeine could be incorporated into Attractive Targeted Sugar Baits (ATSBs) to improve their effectiveness. This study underlines the complexity relationship between diet, the phenotype of resistance and infection status of mosquitoes. Declarations Ethics statement Ethical approval was given by the Centre Muraz Institutional Ethics Committee and the National Ethics Committee of Burkina Faso, N° A010-2023/ CEIRES/IRSS, 1 Avril 2023. Before we tested the children for malaria, their legal guardians provided written consent on behalf of the children. Before we obtained the blood from the three children, further informed consent was obtained from their legal guardians. 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Antioxid Redox Signal 14:943–955 Bahia AC, Oliveira JHM, Kubota MS, Araújo HRC, Lima JBP, Ríos-Velásquez CM, Lacerda MVG, Oliveira PL, Traub-Csekö YM, Pimenta PFP (2013) The role of reactive oxygen species in Anopheles aquasalis response to Plasmodium vivax infection. PLoS ONE 8:e57014 Mangum LC, Borazjani A, Stokes JV, Matthews AT, Lee JH, Chambers JE, Ross MK (2015) Organochlorine insecticides induce NADPH oxidase-dependent reactive oxygen species in human monocytic cells via phospholipase A2/arachidonic acid. Chem Res Toxicol 28:570–584 Ishay JS, Paniry VA (1979) Effects of caffeine and various xanthines on hornets and bees. Psychopharmacology 65:299–309 Abernathy HA, Boyce RM, Reiskind MH (2023) Exploring the effects of caffeine on Aedes albopictus (Diptera: Culicidae) survival and fecundity. J Med Entomol 60:837–841 Srinivasan A, Kesavan PC (1979) Effect of caffeine on longevity and reproduction of the housefly. Toxicol Lett 3:101–105 Itoyama MM, Bicudo HE, Manzato AJ (1998) The development of resistance to caffeine in Drosophila prosaltans: productivity and longevity after ten generations of treatment. Cytobios 96:81–93 Suh HJ, Shin B, Han S-H, Woo MJ, Hong K-B (2017) Behavioral changes and survival in Drosophila melanogaster: Effects of ascorbic acid, taurine, and caffeine. Biol Pharm Bull 40:1873–1882 Bernklau E, Bjostad L, Hogeboom A, Carlisle A, H, S A (2019) Dietary phytochemicals, honey bee longevity and pathogen tolerance. Insects 10:14 Barreaux AMG, Barreaux P, Thievent K, Koella JC (2016) Larval environment influences vector competence of the malaria mosquito Anopheles gambiae. MalariaWorld J 7:8 (1990) Metabolic relationship between female body size, reserves, and fecundity of Aedes aegypti. J Insect Physiol 36:165–172 Additional Declarations The authors declare no competing interests. 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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-7427747","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":503777190,"identity":"3c0b8554-42c7-4e30-b449-27065be72e95","order_by":0,"name":"Khadidiatou Cissé-Niambélé","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA4ElEQVRIiWNgGAWjYBACgxsMDBIJFQwM/CBeQgERWgxngLScYWCQbABpMSBCi7EEUAtjC9C6A2BLidBiJt388MbDhjo54/OrEz88MGCQ5xc7gF+LjcwxY4vEHYeNzW683SwBdJjhzNkJBLRIJJhJJJ45kLjtxtkNIC0JBrcJaDGTSP8mkdhWV795xtnNP4jSYiyRA7SljTnBgL93G3G2GM7IKbZIOHPYcMYN3m0WCQYShP1icCN9480fFXXy/P1nNwMZNvL80gS0IIAEWKUEscpBgP8AKapHwSgYBaNgJAEAGflHxdK7+fgAAAAASUVORK5CYII=","orcid":"https://orcid.org/0000-0003-1523-0274","institution":"Institute of Biology, University of Neuchâtel, Switzerland","correspondingAuthor":true,"prefix":"","firstName":"Khadidiatou","middleName":"","lastName":"Cissé-Niambélé","suffix":""},{"id":503777191,"identity":"7ce5a3fd-ee3d-49cd-bf60-b7c3e1bab57d","order_by":1,"name":"Jacob C. Koella","email":"","orcid":"","institution":"Institute of Biology, University of Neuchâtel, Switzerland","correspondingAuthor":false,"prefix":"","firstName":"Jacob","middleName":"C.","lastName":"Koella","suffix":""},{"id":503777192,"identity":"44ddf50c-3ac3-453b-8f3e-c21bc7dac4c9","order_by":2,"name":"Benjamin Koudou Guibéhi","email":"","orcid":"","institution":"Centre Suisse de Recherches Scientifiques en Côte d’Ivoire, Abidjan, Côte d’Ivoire","correspondingAuthor":false,"prefix":"","firstName":"Benjamin","middleName":"Koudou","lastName":"Guibéhi","suffix":""},{"id":503779923,"identity":"1797b21d-8a0f-4bb2-8959-cf3980ac6f96","order_by":3,"name":"Hien Francois","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Hien","middleName":"","lastName":"Francois","suffix":""}],"badges":[],"createdAt":"2025-08-21 15:39:07","currentVersionCode":1,"declarations":{"humanSubjects":false,"vertebrateSubjects":false,"conflictsOfInterestStatement":false,"humanSubjectEthicalGuidelines":false,"humanSubjectConsent":false,"humanSubjectClinicalTrial":false,"humanSubjectCaseReport":false,"vertebrateSubjectEthicalGuidelines":false},"doi":"10.21203/rs.3.rs-7427747/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7427747/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":89660212,"identity":"22c2c3ad-7647-4ccf-8693-f3fd8602b602","added_by":"auto","created_at":"2025-08-22 11:03:48","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":200498,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eProportion of mosquitoes knocked-down one hour after exposure to insecticide as a function of the concentration of caffeine and their infection status. \u003c/strong\u003eThe error bars represent the 95% confidence intervals of the proportions.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-7427747/v1/37ea790f854f97e95b98a9a5.png"},{"id":89660206,"identity":"34c8163f-2b37-4023-81ff-599d9a978876","added_by":"auto","created_at":"2025-08-22 11:03:48","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":409581,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMortality within 48 hours of exposure as a function of the exposure to the insecticide, concentration of caffeine and the infection status of mosquitoes. \u003c/strong\u003eThe error bars representthe 95% confidence intervals of the proportions.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-7427747/v1/a4422d25c3b9378e4be3a4cb.png"},{"id":89660214,"identity":"4ad9073c-95b9-4414-bb7b-4e60b9ceb0da","added_by":"auto","created_at":"2025-08-22 11:03:48","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":388336,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eLongevity Starting from 48 hours after exposure) as a function of the exposure to the insecticide, Effects of different concentrations of caffeine and the infection status of mosquitoes. \u003c/strong\u003eThe rectangles represent the longevities between the 25th and the 75th percentiles, the horizontal lines within the rectangles denote the medians. The vertical lines span 1.5 times above the 75th percentiles and below 25th percentiles and the dots show outliers that are beyond this range.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-7427747/v1/3bccc0a5e9c2271e4b76ace8.png"},{"id":89662063,"identity":"d25f0241-6e25-4bf9-8e75-ef465fed3f40","added_by":"auto","created_at":"2025-08-22 11:19:48","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1451935,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7427747/v1/8a9efa21-dcf8-490c-ae57-edfd4aa2c0d4.pdf"}],"financialInterests":"The authors declare no competing interests.","formattedTitle":"\u003cp\u003eEffects of a caffeine-based diet on insecticide resistance and longevity in infected \u003cem\u003eAnopheles coluzzii\u003c/em\u003e\u003c/p\u003e","fulltext":[{"header":"Introduction","content":"\u003cp\u003e\u003cem\u003eAnopheles gambiae\u003c/em\u003e, the main vector of malaria in Africa, lives on average less than 10 days in natural situations, but it takes 11 to 14 days for the malaria parasite to develop into infectious stages [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e] [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e] [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e] [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Thus, most infected mosquitoes do not survive long enough to transmit malaria, and transmission is very sensitive to changes in the life-span [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e] [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e] [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Indeed, this is one of the reasons why targeting adults with insecticides (either through residual indoor spraying or on insecticide-treated bed nets) to shorten the mosquitoes\u0026rsquo; life-span has become the main method of vector control [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e] [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. However, increasing mosquito resistance is reducing the effectiveness of insecticides and therefore posing a significant public health threat [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e] [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e] [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eNevertheless, insecticide-based vector control considerably decreased malaria incidence in many parts of Africa despite increasing resistance of the local malaria vectors ( [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e] [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e] [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e] [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e] ). One of the reasons for the discrepancy between measures of resistance and their epidemiological consequences is that the impact of genes conferring resistance depends on environmental parameters like the quantity of food [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e] and infection by parasites [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e] including malaria [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eAn important aspect of a mosquito\u0026rsquo;s diet is its nectar meal [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e], and variation of the quality of nectar among plant species influences longevity [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e] and resistance to insecticides [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Sugars are the main biochemical components of nectar [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. This variation is partly due to the concentrations of the types of sugar in the nectar [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e] [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e] [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. An additional impact of the nectar can be through its toxic compounds like alkaloids [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e] [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e] [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. One alkaloid is caffeine, which occurs in the fruit and nectar of many plant species including \u003cem\u003eCoffea\u003c/em\u003e, \u003cem\u003eCitrus, Cocoa\u003c/em\u003e, tea plants [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e] [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e] [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e] [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e] [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e] [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. Although caffeine can act as an insecticide because of its neurotoxic properties [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e] [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e] [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e] [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e], not much is known about how it, on its own or in conjunction with malaria, affects insecticide-resistance.\u003c/p\u003e\u003cp\u003eWe therefore assessed how caffeine (in concentrations commonly found in nectar) interacts with the infectious status of mosquitoes to influence their response to insecticide, measured as the rate of knock-down during exposure, the mortality within 48 hours of exposure and the longevity following exposure.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003eThe experiment was run at the Institut de Recherches en Science de la Sant\u0026eacute; de Bobo Dioulasso (IRSS) in Burkina Faso. We used the strain \u0026lsquo;vall\u0026eacute;e du Kou\u0026rsquo; of the mosquito \u003cem\u003eAnopheles coluzzi\u003c/em\u003e, which has metabolic resistance mediated by cytochrome p450 and target-site resistance mediated by the gene kdr. The mosquitoes were maintained in a biosafety room at 27\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u0026deg;C, 70\u0026thinsp;\u0026plusmn;\u0026thinsp;5% relative humidity and 12:12 light-dark cycle.\u003c/p\u003e\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eExperimental design\u003c/h2\u003e\u003cp\u003eThe experiment was run in three blocks, each one with the blood of a different gametocytic child as a source of malaria parasites. Within each block, we reared larvae in 40 to 50 trays containing between 250 and 300 larvae in 1 l of tap water and fed them daily with Tetramin baby fish food according to their age. We fed them daily with Tetramin baby fish food according to their age (day of hatching: 14 mg per tray; 1 day old: 21 mg; 2 days old: 29 mg; 3 days old: 58 mg; 4 days old: 115 mg, 5 days old or older: 216 mg). Adult males were discarded, and females were given continual access to a 10% glucose solution supplemented with 0, 50 or 200 ppm of caffeine and were given the opportunity to feed on infected or uninfected blood three or four days after emergence. (See below for the method of infection.) Each treatment was replicated in four cups of 55 to 65 mosquitoes. The females that did not take a full blood meal were discarded, leaving between 18 and 30 mosquitoes per cup). 72 hours later they were exposed to 0.25% deltamethrin or to a sham for 1 hour with the WHO bioassay kit [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. The mosquitoes of each cup were tested together.\u003c/p\u003e\u003cp\u003eDuring the exposure to the insecticide, we counted the number of mosquitoes that were knocked down, and 48 hours after the exposure we counted the number of dead mosquitoes. The survivors were maintained for at most 30 days with continual access to their diet, and their mortality was assessed every day. Once they had died, the mosquitoes that had fed on infected blood were tested for the presence of malaria parasites with standard PCR-methods [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]; within each block 100 haphazardly selected mosquitoes that had fed on uninfected blood were also tested for malaria to ensure the absence of parasites. Infection status was classified as \u0026lsquo;Uninfected\u0026rsquo; for those fed on an uninfected blood, \u0026lsquo;Harbored parasite\u0026rsquo; for those fed on infected blood and have been harbored parasite and \u0026lsquo;No harbored parasite\u0026rsquo; for those fed on infected blood but not harboring any parasites.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eExperimental infection\u003c/h3\u003e\n\u003cp\u003eFor each block, 200 five- to 15-year-old schoolchildren were recruited in Nasso village, located 13 km from Bobo-Dioulasso, Burkina Faso.\u003c/p\u003e\u003cp\u003eFemale mosquitoes were exposed to blood samples from naturally \u003cem\u003eP. falciparum\u003c/em\u003e gametocyte-infected from these children using a direct membrane feeding assay (DMFA) as described previously [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e] [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e] [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eBriefly, thick blood smears were taken from each volunteer, air-dried, Giemsa-stained and examined by microscopy for the presence of \u003cem\u003eP. falciparum\u003c/em\u003e at the IRSS laboratory in Bobo-Dioulasso. Asexual trophozoite parasite stages were counted against 200 leucocytes, while infectious gametocyte stages were counted against 1000 leukocytes. Children with asexual parasitaemia of \u0026gt;\u0026thinsp;1000 parasites per microlitre (estimated based on an average of 8000 leucocytes ml\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) were treated in accordance with national guidelines. Asymptomatic \u003cem\u003eP. falciparum\u003c/em\u003e gametocyte-positive children were recruited for the study. Blood from gametocyte isolates was collected by venipuncture in heparinized tubes. Three distinct parasite isolates (named hereafter A, B and C), with respective gametocytaemia of 56, 72 and 128 gametocytes \u0026micro;l\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e of blood, were used for the experimental infections. DMFA was performed by replacing donor plasma with an equivalent volume of AB\u0026thinsp;+\u0026thinsp;serum from malaria-na\u0026iuml;ve European donors. After centrifugation (Eppendorf, centrifuge 5702 R) at 37\u0026deg;C at 12,000 rpm for 5 min. A horizontal line marking the upper limit of the plasma phase was drawn on the tube. The plasma was aspirated with a pipette, and AB\u0026thinsp;+\u0026thinsp;serum was introduced up to the marked line.\u003c/p\u003e\u003cp\u003eFor each gametocyte carrier, half of the blood sample was transferred into 1.5 mL Eppendorf tubes and inactivated at 45\u0026deg;C for 20 minutes at 900 rpm using a compact thermomixer [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. The mosquitoes were starved of sugar solution for 24 h and then fed on infected or uninfected blood via membrane filters for 1 hour.\u003c/p\u003e\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\u003ch2\u003eData analysis\u003c/h2\u003e\u003cp\u003eThe statistical analyses were performed with R version R-4.4.2 [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. We found the significance of the effects with the function Anova (package car) [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e], using a type 3 SS if the interactions were significant and a type 2 if they were not.\u003c/p\u003e\u003cp\u003eWe analysed the knock-down proportion and 48-hour mortality using GLMs. For mortality, we included diet, infection status, insecticide and their interactions as fixed factors. For the knock-down analysis, we replaced infection status with blood type. Since only 0.13% of mosquitoes in the group not exposed to insecticide were knocked down, compared to 59.62% in the insecticide-exposed group, we focused solely on the mosquitoes that were exposed to insecticide. In both models, blocks (source of blood) and cups were included as random effects.\u003c/p\u003e\u003cp\u003eIn the analysis of longevity we included only the mosquitoes that had survived the first 48 hours after being exposed to the insecticide to avoid confounding impacts on longevity and on mortality. 196 mosquitoes lived longer than 30 days and were right-censored. We used a coxme model that included the diet, the infection status, the exposure to insecticide, and all interactions as fixed factors and the block (source of blood) and cups as random factors.\u003c/p\u003e\u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003eOf the 1632 mosquitoes exposed to insecticide, 59.6% were knocked down within 1 hour of the exposure. The knock-down rate increased with caffeine concentration from 41.4% (95% c.i: 24.26%-60.9%) at 0 ppm, 61.4% (41.7%-78.0%) at 50 ppm to 73.7% (53.7%-87.1%) at 200 ppm, (\u0026#120652;\u0026sup2;=38.43, df\u0026thinsp;=\u0026thinsp;1, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). However, neither the type of the blood ingested (\u0026#120652;\u0026sup2;=0.55, df\u0026thinsp;=\u0026thinsp;1, p\u0026thinsp;=\u0026thinsp;0.459) nor the interaction between the caffeine concentration and the type of blood ingested (\u0026#120652;\u0026sup2;=1.80, df\u0026thinsp;=\u0026thinsp;1, p\u0026thinsp;=\u0026thinsp;0.408) had an effect on the rate of knock-down \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e\u003cb\u003e).\u003c/b\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eOf the 1,559 mosquitoes exposed to insecticide, 53.2% (95% CI: 50.7\u0026ndash;55.6%) died within 48 hours, compared to 9.4% (95% CI: 8.0\u0026ndash;11.0%) of the 1,471 mosquitoes exposed to the sham. Insecticide exposure significantly increased 48-hour post-exposure mortality, (\u0026#120652;\u0026sup2; = 260.79, df\u0026thinsp;=\u0026thinsp;1,p\u0026thinsp;\u0026lt;\u0026thinsp;0.001).\u003c/p\u003e\u003cp\u003eCaffeine diet alone did not influence the mortality rate, (\u0026#120652;\u0026sup2;=0.81, df\u0026thinsp;=\u0026thinsp;2, p\u0026thinsp;=\u0026thinsp;0.668). Howether, there was a combined effect of caffeine and insecticide exposure: Without insecticide, mortality was relatively low, ranging from 7.0% ( 5.1%- 9.7%) at 200 ppm to 10.2% (7.9% \u0026minus;\u0026thinsp;13.1%) at 0 ppm. In the presence of insecticide, mortality increased significantly, reaching 43.6% (39.3% \u0026minus;\u0026thinsp;48.0%) at 0 ppm, 50.4% ( 46.1% \u0026minus;\u0026thinsp;54.6%) at 50 ppm and 64.8% (60.7% \u0026minus;\u0026thinsp;68.7%) at 200 ppm, (\u0026#120652;\u0026sup2;=17.76, df\u0026thinsp;=\u0026thinsp;2, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001).\u003c/p\u003e\u003cp\u003eMortality varied according to infection status. Infected mosquitoes harboring parasites had a lower mortality rate with 23.9% (21.3\u0026ndash;26.7%), while infected mosquitoes that have not harbouring any parasites had a higher mortality of 47.4% (43.4\u0026ndash;51.4%). Uninfected mosquitoes had an intermediate mortality of 30.8% (28.5\u0026ndash;33.2%), (\u0026#120652;\u0026sup2;=17.50, df\u0026thinsp;=\u0026thinsp;2, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001).\u003c/p\u003e\u003cp\u003eA significant interaction was detected between insecticide exposure and infection status. Mortality increased across all groups after insecticide exposure, and the magnitude varied: 33.8 percentage points in those infected and harboring parasite [ 8.5% (6.4\u0026ndash;11.3%) to 42.3% (37.7\u0026ndash;46.9%) ], 54.4 in those wich infected but did not harboring parasite [14.2% (10.3\u0026ndash;19.2%) to 68.6%(63.7\u0026ndash;73.1%)], and 43.5 in uninfected mosquitoes [8.4% (6.6\u0026ndash;10.7%) to 51.9%(48.4\u0026ndash;55.5%)], (\u0026#120652;\u0026sup2; = 7.25, df\u0026thinsp;=\u0026thinsp;2, p\u0026thinsp;=\u0026thinsp;0.026).\u003c/p\u003e\u003cp\u003eThere was no significant interaction between the caffeine-based diet and infection status (\u0026#120652;\u0026sup2;=8.87, df\u0026thinsp;=\u0026thinsp;4, p\u0026thinsp;=\u0026thinsp;0.064), but a significant three-way interaction between infection status, insecticide exposure, and caffeine concentration influenced mosquito mortality (\u0026#120652;\u0026sup2;=9.85, df\u0026thinsp;=\u0026thinsp;4, p\u0026thinsp;=\u0026thinsp;0.043). At 0 ppm caffeine, mortality after insecticide exposure was highest in those which have been infected but don\u0026rsquo;t harboring parasite [63.7% (54.5%-72.0%) vs. infected and harboring parasite 37.7% (30.4%-45.7%) and uninfected 37.4% (31.4%-43.9%)]. At 200 ppm, caffeine reduced mortality in non-exposed mosquitoes infected which did not harboring any parasite 2.5% (0.7%-8.6%), but mortality after exposure remained highest in this group 77.7% (69.5%-84.2%) vs. uninfected 68.5% (62.7%-73.8%) and those infected harboring parasite 47.3%(39.3%-55.3%), \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e\u003cb\u003e)\u003c/b\u003e.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eThe average longevity of the 2,063 mosquitoes that survived the first 48 hours was 18.6 days. It was not significantly affected by caffeine concentration (\u0026#120652;2\u0026thinsp;=\u0026thinsp;1.46, df\u0026thinsp;=\u0026thinsp;2, p\u0026thinsp;=\u0026thinsp;0.48), nor by any of its interactions (\u0026#120652;2\u0026thinsp;\u0026lt;\u0026thinsp;6.4, df\u0026thinsp;=\u0026thinsp;2\u0026ndash;4, p\u0026thinsp;\u0026gt;\u0026thinsp;0.17), \u003cb\u003e(Fig.\u0026nbsp;3).\u003c/b\u003e\u003c/p\u003e\u003cp\u003eLongevity was about 1 day longer among insecticide-exposed mosquitoes than among unexposed ones (\u0026#120652;2\u0026thinsp;=\u0026thinsp;4.49, df\u0026thinsp;=\u0026thinsp;1, p\u0026thinsp;=\u0026thinsp;0.034). The effect of exposure depended on whether the mosquitoes were infected by malaria. In infected mosquitoes harboring parasites, the average longevity was 18.3 (17.5\u0026ndash;19.1) days without exposure to the insecticide and 17.7 days (16.5\u0026ndash;18.8) with exposure. In infected mosquitoes which didn\u0026rsquo;t harbor parasites, the average longevity was 17.5 days (16.3\u0026ndash;18.6) without insecticide exposure and 19.6 days (17.9\u0026ndash;21.9) with insecticide exposure. In uninfected mosquitoes, average longevity was 18.7 days (18.0-19.3) without insecticide and 20.0 days (19.0-20.9) with insecticide exposure (interaction exposure * infection status: \u0026#120652;2\u0026thinsp;=\u0026thinsp;6.06, df\u0026thinsp;=\u0026thinsp;2, p\u0026thinsp;=\u0026thinsp;0.048), \u003cb\u003e(Fig.\u0026nbsp;4).\u003c/b\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eMake sure the figures are formatted the same. Same titles, same shading, \u0026hellip;..\u003c/b\u003e\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eOur study examined how caffeine and mosquito infection status influence the phenotype of resistance and the longevity of survivors after exposure to insecticide.\u003c/p\u003e\u003cp\u003e\u003cb\u003eMortality increased as caffeine concentration rose in the presence of insecticide exposure.\u003c/b\u003e\u003c/p\u003e\u003cp\u003eOur work shows that increasing the concentration of caffeine increases the mortality rate of mosquitoes after exposure to the insecticide. Caffeine, an alkaloid, may enhance insecticide efficacy in \u003cem\u003eAnopheles gambiae\u003c/em\u003e by acting as a neurotoxin and inducing oxidative stress [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e] [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e] [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]. Since exposure to the insecticide also induces ROS production via the activity of cytochrome P450. The combination of insecticide detoxification and caffeine-induced oxidative stress overloads the physiological defenses impairing detoxification enzyme efficiency and reducing resistance to insecticide, thereby increasing mortality.\u003c/p\u003e\u003cp\u003eOur findings align with (Ciss\u0026eacute; et al., unpublished work), we observed increasing mortality of mosquitoes with rising caffeine concentration. Previous studies on \u003cem\u003eAedes\u003c/em\u003e larvae similarly demonstrated dose-dependent toxicity of caffeine [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e] [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eThe decrease in mosquito resistance with increasing caffeine concentration could also be explained by a repellent effect of the molecule [\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e], leading to smaller meals and lower energy reserves. As caffeine concentration increases, the repellent effect becomes stronger, reducing meal intake and making mosquitoes more sensitive to the insecticide.\u003c/p\u003e\u003cp\u003e\u003cb\u003eInfected mosquitoes that did not harbor any parasites and were exposed to insecticide were more susceptible to insecticide compared to other mosquitoes\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe infected mosquitoes which did not harbor any parasites may have mounted an immune response against the infection, even though no parasites were ultimately detected [\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e] [\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e] [\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e]. [\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e] [\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e]. Additionally, the immune response is often linked to an increased production of reactive oxygen species (ROS) [\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e] [\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e] [\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e] which may amplify the cytotoxic effects of the insecticide, thereby increasing mortality [\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e]. The high mortality rate could, therefore, result from the combined effects of the immune response cost and the oxidative stress induced by the insecticide toxicity.\u003c/p\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003eCaffeine alone had no effect on the longevity of the survivors\u003c/h2\u003e\u003cp\u003eThe absence of an effect of caffeine on mosquito longevity was unexpected, given that caffeine is both a neurotoxic and pro-oxidant compound, and especially since it significantly affected mosquito resistance within 48 hours after insecticide exposure. Our result contrasts with several studies that have reported an effect of caffeine on various mosquito life-history traits. For instance, it has been shown to increase the locomotion of larva stage [\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e], and on the other hand, it would lead to a reduction of fertility in the species \u003cem\u003eAedes albopictus\u003c/em\u003e [\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eOther studies have shown that a caffeine-based diet reduces the longevity of \u003cem\u003eAedes albopictus\u003c/em\u003e at the same concentrations used in our study [\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e]. In addition, a reduction of longevity was observed in some flies species Musca domestica [\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e], Drosophila prosaltans et D. melanogaster [\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e] [\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e] due to their caffeine-based diet. Caffeine increases the lifespan of bees [\u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e].\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eInsecticide exposure increased the general longevity of mosquitoes\u003c/h3\u003e\n\u003cp\u003eThe improved survival of mosquitoes exposed to insecticide can be attributed to selective sorting that favors larger individuals, which likely benefit from energy reserves accumulated during their larval stage [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e] [\u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e]. Larger mosquitoes tend to possess a higher teneral reserve, which may enhance their longevity [\u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e].\u003c/p\u003e\u003cp\u003e\u003cb\u003eMosquitoes exposed to\u003c/b\u003e \u003cb\u003ePlasmodium falciparum\u003c/b\u003e \u003cb\u003ebut not infected show high mortality within 48 hours of exposure to the insecticide, but survivors live longer than those in other groups.\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThis phenomenon could be explained by the fact that exposure to gametocytes without actual infection would trigger an immune or metabolic response, enhancing their long-term survival after the elimination of the weakest individuals.\u003c/p\u003e\u003cp\u003eOur study has several limitations. First, the mosquitoes used originated from a single laboratory strain known for its high level of insecticide resistance and were reared under controlled conditions. This may limit the generalizability of our findings to wild \u003cem\u003eAnopheles gambiae\u003c/em\u003e populations, which may exhibit different ecological and physiological characteristics. Second, the detection of \u003cem\u003ePlasmodium\u003c/em\u003e DNA in some mosquitoes does not confirm the presence of viable or developing parasites, as PCR may amplify viable or non-viable DNA. Finally, since only one insecticide (deltamethrin) was tested, the results cannot be extrapolated to other classes of insecticides, to which resistant mosquitoes might respond differently.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eOur study shows that a caffeine-based diet increases mosquito mortality within 48 hours following insecticide exposure, but does not affect the longevity of survivors. Moreover, infection status did not interact with the caffeine diet to influence either mortality 48 hours after exposure or longevity of the survivors.\u003c/p\u003e\u003cp\u003eThe results highlight the potential of caffeine as a complementary vector control tool, capable of enhancing mosquito susceptibility to insecticides. Caffeine could be incorporated into Attractive Targeted Sugar Baits (ATSBs) to improve their effectiveness.\u003c/p\u003e\u003cp\u003eThis study underlines the complexity relationship between diet, the phenotype of resistance and infection status of mosquitoes.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003eEthics statement\u003c/h2\u003e\u003cp\u003eEthical approval was given by the Centre Muraz Institutional Ethics Committee and the National Ethics Committee of Burkina Faso, N\u0026deg; A010-2023/ CEIRES/IRSS, 1 Avril 2023. Before we tested the children for malaria, their legal guardians provided written consent on behalf of the children. Before we obtained the blood from the three children, further informed consent was obtained from their legal guardians.\u003c/p\u003e\u003c/p\u003e\u003c/div\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eTaye B, Lelisa K, Emana D, Asale A, Yewhalaw D (2016) Seasonal Dynamics, Longevity, and Biting Activity of Anopheline Mosquitoes in Southwestern Ethiopia. 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J Insect Physiol 36:165\u0026ndash;172\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"University of Neuchâtel","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"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":"caffeine, malaria infection, insecticide resistance","lastPublishedDoi":"10.21203/rs.3.rs-7427747/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7427747/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eAlkaloids such as caffeine can be toxic for insects. However, although mosquitoes feed on many plants with nectar containing alkaloids, their impact on the vectorial capacity of mosquitoes is not known, in particular in the face of increasing resistance to insecticides. We assessed with the mosquito \u003cem\u003eAnopheles coluzzii\u003c/em\u003e how caffeine affects several measures of resistance to deltamethrin \u0026ndash; the rate at which mosquitoes are knocked-down during exposure, the mortality within 24 hours of exposure, and the longevity following exposure \u0026ndash; and we compared these traits for mosquitoes that were uninfected or infected by the malaria parasite \u003cem\u003ePlasmodium falciparum\u003c/em\u003e. The mosquitoes were fed throughout their lives on a 10% sugar solution supplemented with 0, 50, or 200 ppm caffeine. Three or four days after emergence, they were given an infected or an uninfected blood meal. Another three days later, they were exposed to deltamethrin or to a sham and checked for knock-down during the exposure and death within the next 48 hours. We monitored the surviving mosquitoes for longevity and assessed their infection status when they died. The rate of mosquitoes knocked down by the insecticide increased with higher caffeine concentrations, but neither the infection status nor its interaction with caffeine concentration influenced the knockdown rate. Similarly, caffeine increased the mortality of insecticide-exposed mosquitoes within 48 hours after exposure. The mortality was highest if mosquitoes had fed on infected blood but harbored no parasites, and lowest if they had not fed on infected blood. The longevity, once the mosquitoes had survived the first 48 hours, was not affected by the concentration of caffeine or by any of the combination of caffeine with infection status or insecticide, but, the mosquitoes that had been exposed to the insecticide lived longer than unexposed ones, in particular if they had fed on infected blood but were not infected.\u003c/p\u003e\u003cp\u003eOverall, our experiment highlights that the level of resistance to an insecticide is affected by complex interactions between the mosquito\u0026rsquo;s diet and infection by malaria.\u003c/p\u003e","manuscriptTitle":"Effects of a caffeine-based diet on insecticide resistance and longevity in infected Anopheles coluzzii","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-08-22 11:03:43","doi":"10.21203/rs.3.rs-7427747/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":"afd3baa4-7698-4a62-a3a9-c82d19118e25","owner":[],"postedDate":"August 22nd, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":53522705,"name":"Entomology"}],"tags":[],"updatedAt":"2025-10-08T12:53:30+00:00","versionOfRecord":[],"versionCreatedAt":"2025-08-22 11:03:43","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7427747","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7427747","identity":"rs-7427747","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}