{"paper_id":"953ae5db-72a6-4a6a-abc3-1b33498fa7ba","body_text":"Maternal and cross-stage effects of Metarhizium fungal infection on the malaria mosquito \nAnopheles coluzzii life-history\nIssiaka SARE1,2,3,4 , Mafalda VIANA5,Abel MILLOGO1,2,7, Doubé Lucien LAMY1,2,7, Assita \nTENETAO1,2,7, Athanase BADOLO 3, Florencia DJIGMA 4, Abdoulaye DIABATE* 1,2, \nFrancesco BALDINI5,6 ,and Etienne BILGO*1,2\n \nAffiliations :\n        1- Institut de Recherche en Sciences de la Santé, Direction Régionale de l’Ouest Bobo-     \nDioulasso 01, BP 545, Burkina Faso\n2-Institut National de Santé Publique / Centre Muraz, Bobo Dioulasso, Burkina Faso\n3-Laboratoire d’Entomologie Fondamentale et Appliquée (LEFA), Université Joseph Ki-\nZerbo, Ouagadougou 03 BP 7021, Burkina Faso\n4-Laboratoire de Biologie Moléculaire et de Génétique (LABIOGENE), Ecole Doctorale \nSciences et Technologie, Université Joseph Ki-Zerbo ; Centre de Recherche Biomoléculaire \nPiétro Annigoni (CERBA), Ouagadougou 01, BP, 364, Burkina Faso\n5- School of Biodiversity One Health and Veterinary Medicine, University of Glasgow, \nGlasgow, G12 8QQ, UK\n6- Ifakara Health Institute, Environmental Health, and Ecological Sciences Department, \nMorogoro, United Republic of Tanzania.\n7-Université Nazi Boni, 01 BP 1091.Bobo Dioulasso 01, Burkina Faso\n*Corresponding Authors: bilgo02@yahoo.fr and npiediab@gmail.com\n \nAbstract\nBackground\nEntomopathogenic fungi of the Metarhizium genus are widely used as biocontrol agents against \nharmful insects. These fungi are cost-effective and eco-friendly for vector control, providing \nan alternative to synthetic chemical insecticides. They have great potential as larvicides against \nmalaria vectors, but their impacts on mosquito fitness have not been fully measured.  This study \nevaluated the effect of Metarhizium fungal strains, locally isolated in Burkina Faso, on the \nlarval survival of the mosquito Anopheles coluzzii, the life history of the emerging adults and \non the maternal effects of exposed females.\nMethods\n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted May 14, 2025. ; https://doi.org/10.1101/2025.05.14.654141doi: bioRxiv preprint \n\n   We assessed the efficacy of Metarhizium pingshaense (strains S10 and S26) conidia against \nAn. coluzzii larvae. First, larvae were reared in the presence of fungal spores and the survival \nof the larvae and adults that emerged, their wing length, oviposition rate and blood feeding \nbehaviour were measured. Additionally, we assessed the efficacy of fungal strains S10 and S26 \nconidia against An. coluzzii adults by spraying them with spore suspensions and assessing the \nsurvival of their larval offspring. Survival data was analyzed using Cox proportional hazards \nmodel, while other life history traits using generalized linear mixed models.\nResults\nThe fungal suspension applied to the water in which the larvae were reared caused mortality at \nthe pupae stage.  Only a small number of larvae emerged to reach adulthood. Furthermore, at \nthe adult stage, these mosquitoes exhibited reduced survival compared to the control. However, \nbody size and blood-feeding behavior were not affected by the treatment. When fungi was \napplied to adult females, the number of eggs layed was more abundant in infected group \ncompared to controls, however a lower proportion of larvae successfully developed into adults.\nConclusion\n The results of this study demonstrate the potential of Metarhizium pingshaense conidia for \nmosquito larval control. The identified cross-stage and maternal effects showed additional \nvirulent effects of Metarhizium, thus reinforcing the evidence that this biocontrol agent should \nbe part of an integrated vector management. Future work should focus on the molecular \nmechanism of the fungal infection at the larval stage to improve formulation or genetically \nengineer the conidia of these strains to make them more virulent. \nKeywords: Metarhizium pingshaense, Anopheles coluzzii, fitness, Malaria, Burkina Faso\nIntroduction\nThe widespread use of insecticide-treated nets at the turn of the century was associated with a \nmarked reduction in malaria mortality[1]. However, in recent years, the decline in malaria cases \nhas stalled, threatening current malaria control efforts[2]. The increasing spread of insecticide \nresistance in malaria vector populations is among the factors contributing to the slowdown of \nthe control efforts [3,4]. Indeed, the vast majority of vector control tools still rely on insecticide-\n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted May 14, 2025. ; https://doi.org/10.1101/2025.05.14.654141doi: bioRxiv preprint \n\nbased interventions, such as long-lasting insecticidal nets (LLINs) and Indoor Residual \nSpraying (IRS) so new and complementary tools will be needed for effective control. One tool \ngaining traction against malaria vectors are larvicides, or more broadly, tools that target the \nlarval stage of mosquitoes [5]. Since larvicides are typically applied to larval breeding sites in \nthe environment, traditional chemicals, while widely used, pose significant ecological risks due \nto their toxicity to non-target organisms and potential environmental persistence[6]. This has \nled to an increasing demand for alternative biocontrol solutions that are both selective and \nbiodegradable. Among these, entomopathogenic fungi have emerged as a promising class of \nbio-insecticides with a narrower spectrum of action and reduced environmental impact[7]. \nThese fungi, such as Metarhizium anisopliae and Beauveria bassiana, have demonstrated their \nability to be specific to infect and kill larvae of major mosquito genera, \nincluding Anopheles, Aedes, and Culex [8]. Their mode of action involves the production of \nvirulence factors and active metabolites that facilitate host invasion and ultimately lead to \nmortality. These metabolites also play a role in insect defense mechanisms against pathogens, \nfurther influencing host-pathogen interactions[9]. Experimental studies have provided \nsubstantial evidence supporting the efficacy of entomopathogenic fungi (EPF) in adult \nmosquito control, highlighting their potential as biopesticides in integrated vector \nmanagement[10]. Metarhizium could also be effective for larval mosquito control because its \nspores can persist in aquatic environments, potentially infecting larvae through contact with \ncontaminated surfaces or by ingestion, disrupting their development and increasing mortality.\nMany laboratory and semi-field investigations have demonstrated that entomopathogenic \nfungal species such as Metarhizium anisopliae (ICIPE-30) and Beauveria bassiana (IMI-\n391510) exhibit significant larvicidal activity against major malaria vectors, \nincluding Anopheles stephensi and Anopheles gambiae [11]. These fungi act as natural \nbiological control agents, infecting mosquito larvae primarily through direct contact with \nconidiospores present in the aquatic environment. Upon contact, the fungal spores adhere to \nthe larval cuticle and germinate, forming specialized structures called appressoria, which \nfacilitate penetration of the cuticle[12]. Once inside the host, the fungus proliferates within the \nhemocoel, disrupting physiological processes and leading to systemic infection[13,14]. As the \nfungal hyphae spread, they deplete larval energy reserves, produce toxic secondary \nmetabolites, and compromise immune defenses, ultimately causing mortality[15]. The speed \nand efficacy of fungal infection depend on environmental factors such as temperature, \nhumidity, and the larval developmental stage[12,16]. Additionally, some studies suggest that \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted May 14, 2025. ; https://doi.org/10.1101/2025.05.14.654141doi: bioRxiv preprint \n\nfungal infections may weaken larvae, making them more susceptible to other stressors, such as \npredation and chemical insecticides[17,18]. These findings highlight the potential \nof Metarhizium anisopliae  as promising candidates for integrated vector management \nstrategies targeting malaria-transmitting mosquitoes. Further studies have revealed that EPF \ntarget critical physiological systems within mosquito larvae. Histopathological analyses \nindicate that fungal infection disrupts the integrity of the cuticle, allowing fungal hyphae to \ninvade internal tissues such as the alimentary and respiratory tracts. This invasion not only \nimpairs nutrient absorption and metabolic functions but also leads to systemic mycosis, \nultimately resulting in larval death[11,19] . Additionally, certain isolates of B. bassiana have \ndemonstrated efficacy against culicine mosquito larvae (Culex spp.), suggesting a broader \nspectrum of activity across different vector species [20].\nBeyond their direct lethal effects, EPF exhibit sublethal impacts that may further contribute to \nmosquito population suppression. Infected larvae often experience developmental delays, \nreduced pupation rates, and compromised adult emergence, all of which can disrupt population \ndynamics and transmission potential[21,22]. These multifaceted mechanisms position EPF as \na promising alternative to conventional larvicides, particularly in the context of insecticide \nresistance and environmental sustainability. By offering an eco-friendly, target-specific, and \npotentially self-propagating solution, entomopathogenic fungi represent a valuable component \nof integrated vector management strategies aimed at reducing malaria transmission in endemic \nregions. These different studies conducted in the laboratory give satisfactory results on the \nability of entomopathogens to control mosquito larvae. Our previous studies have shown that \nMetarhizium strains S10 and S26 two strains of fungi isolated in Burkina Faso in west Africa \ncan reduce the survival of adult mosquitoes [23,24]. However, the ability of these two strains \nof Metarhizium to control mosquito larvae and the implications for the general fitness of the \nmosquito derived from the fungal suspension remain unknown. The aims of the present study \nwere to investigate the potential use of the fungi Metarhizium pingshanse strains S10 and S26 \nto control both effect on larvae and subsequent emerging adults. This study evaluated the \neffects of larval exposure to entomopathogenic fungi on mosquito survival, development time, \nand adult emergence. We further assessed key adult life history traits, including feeding \npropensity, fecundity, and wing size as a proxy for fitness. The aim was to determine the \ncumulative impact of fungal exposure on mosquito biology and potential vectorial capacity.\n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted May 14, 2025. ; https://doi.org/10.1101/2025.05.14.654141doi: bioRxiv preprint \n\nMethodology\nWe conducted two experiments. In the first we tested the cross-stage effect of infecting larvae \nwith Metarhizium (Figure 1A). In the second, we exposed adults to Metarhizium and monitors \nthe progeny life history traits.\nFungal suspension preparation \nUsing a sterile spatula, small fragments of fungal mycelium and conidia were carefully \ncollected by gently scraping the surface of the fungal culture in a Petri dish under aseptic \nconditions to prevent contamination[23,25]. The harvested material was then suspended in a \nsterile aqueous solution containing 0.05% Tween 80, a non-ionic surfactant commonly used to \nimprove conidial dispersion and prevent clumping. This suspension was vortexed for several \nminutes to ensure a homogeneous distribution of conidia.\nTo determine the conidial concentration, an aliquot of the suspension was subjected to \nquantification using a Neubauer hemocytometer under a phase-contrast microscope. The \ndesired concentration of 10 7 conidia/mL was achieved by serial dilution or concentration \nadjustment, ensuring a standardized inoculum density for bioassays.\nOnce prepared, the conidial suspension was introduced into larval breeding trays containing \nthird and fourth instar mosquito larvae. The trays were maintained under controlled \nenvironmental conditions, including temperature (27 ± 2°C), relative humidity (75 ± 5%), and \na 12:12 h light-dark photoperiod, to mimic natural breeding habitats. The exposure period was \nstandardized to allow sufficient fungal attachment and germination on larval cuticles, \nfacilitating infection. This experimental setup enabled the assessment of fungal pathogenicity \nand virulence against mosquito larvae under laboratory conditions.\nMosquito rearing \nThe mosquito strain of Anopheles coluzzii used in the experiment was the 11th generation of a \nline that originated from the Vallée du Kou and was established in the laboratory at Institut de \nRecherche en Sciences de la Santé (IRSS), Bobo Dioulasso, Burkina Faso. The colony was \nmaintained at 27 ± 2°C, relative humidity of 70 ± 5% and photoperiod of 12L:12D.This colony \nis known to have almost fixed 1014F Kdr allele [26]. The larvae were kept in plastic trays filled \nwith tap water and fed at all stages with Tetra-min®. All emerged mosquitoes had access to 6% \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted May 14, 2025. ; https://doi.org/10.1101/2025.05.14.654141doi: bioRxiv preprint \n\nglucose and female mosquitoes were fed on rabbit blood. Eggs were laid on wet filter paper in \nthe cages and transferred to the larval trays. \nLarval survival after fungal infection\nA total of ten plastic trays were used in the experiment, with each tray containing 30 L4 larvae. \nThis setup was replicated three times, resulting in a total of 780 larvae. The larvae were exposed \nto the fungal solution and remained in the treated environment until they reached the pupal \nstage. Each tray contained a total volume of 50 mL, composed  of 49 mL of tap water and 1 \nmL of a fungal suspension at an initial concentration of 10 7conidia/mL. The addition of the \nfungal solution to the water led to a final concentration of 2×105 conidia/mL in each tray. Then, \nwe monitored and recorded larval survival rates throughout their development, from the larval \nstage to adulthood.\nSurvival of emerging adults\nThe adult mosquitoes that successfully emerged from the treated larval suspension were \ncarefully collected and transferred to designated rearing cages( ~ 30 mosquitoes per cage) \nunder controlled conditions. Inside the cages, they were provided with a continuous supply of \na 5% glucose solution to ensure proper feeding. Mortality was monitored daily, with dead \nmosquitoes being systematically removed from the cages for up to five days post-emergence. \nThis allowed for the assessment of delayed mortality effects potentially caused by fungal \nexposure during the larval stage.\nMosquitoes wing size measuring after fungal infection \nThe left wings of the adult mosquitoes were carefully dissected using fine-tipped forceps and \na sterile needle to ensure precision and minimize structural damage. Each excised wing was \nthen mounted onto a microscope slide with a coverslip for detailed morphometric analysis, \nfollowing the methodology described by[27].\nHigh-resolution digital images of the mounted wings were captured using a Nikon SMZ1500 \nstereomicroscope (Nikon, Japan) equipped with an integrated camera. The imaging process \nwas conducted at a magnification of 11.25X to ensure accurate measurement of wing \ndimensions. Wing length was determined by measuring the linear distance from the distal wing \ntip to the alular notch, a standard landmark for wing morphometry in mosquitoes.\n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted May 14, 2025. ; https://doi.org/10.1101/2025.05.14.654141doi: bioRxiv preprint \n\nA total of 225 mosquitoes were analyzed for this experiment, with the study conducted in three \nindependent replicates to ensure statistical robustness and reproducibility of the findings.\nFungus infection effect on female mosquito fecundity and fertility\nFour experimental cages, each containing approximately 100 mosquitoes (50 males and 50 \nfemales), were used to assess the impact of fungal exposure on mosquito reproductive success. \nThe mosquitoes, aged 3–5 days post-emergence, originated from either the fungal-treated larval \nsuspension or the untreated control group. To ensure sufficient blood intake for egg \ndevelopment, all mosquitoes were offered a rabbit blood meal twice, thereby maximizing the \nlikelihood of successful feeding.\nFollowing blood feeding, engorged females were individually transferred to oviposition cups \nto facilitate precise monitoring of egg-laying behavior. The total number of eggs laid per female \nwas recorded to evaluate fecundity. A total of 205 females were analyzed for this experiment.\nTo assess egg viability, the collected eggs were submerged in 50 mL of tap water, and their \nhatching success was systematically evaluated. This step allowed for the determination of \npotential carryover effects of fungal exposure on mosquito reproductive output and offspring \ndevelopment.\nEffect of fungal infection of adult mosquitoes on their offspring (G1)\nFor each treatment, approximately 100 blood-fed female mosquitoes were exposed to fungal \nsuspensions (S10 and S26) in 10 independent replicates. Prior to fungal application, \nmosquitoes were temporarily immobilized by chilling in a freezer at –4°C for 15 seconds, \nensuring minimal stress while facilitating uniform exposure to fungal spores. Immobilized \nmosquitoes were then transferred onto a Petri dish lined with sterile filter paper to maintain \naseptic conditions and prevent contamination during the spraying process.\nMosquitoes were sprayed with a 1 mL fungal suspension containing M. pingshaense conidia at \na standardized concentration of 1×10 7 spores/mL, formulated in 0.01% (v/v) aqueous Tween \n80. This formulation was applied using an Ami pulvérisateur (Zhejiang, China), a precision \nspray device designed to ensure consistent and homogeneous deposition of conidia on \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted May 14, 2025. ; https://doi.org/10.1101/2025.05.14.654141doi: bioRxiv preprint \n\nmosquito cuticles. Control mosquitoes were sprayed with 1 mL of 0.01% (v/v) aqueous Tween \n80 alone, serving as a negative control to account for any effects of the spraying \nprocedure.After treatment, mosquitoes were transferred to holding cups (7 cm diameter × 9 cm \nheight) and maintained under controlled environmental conditions: temperature of 25°C, \nrelative humidity of 70 ± 10%, and a 12:12-hour light-dark cycle. To sustain physiological \nactivity, mosquitoes were provided a 5% glucose solution soaked in a cotton ball. Egg-laying \nbehavior was monitored, and the number of fourth-stage larvae (L4) and the number of larvae \nsuccessfully emerging as adults were recorded.\n Statistical analysis\n Larval survival(figure 2) rate was analyzed using a binomial Generalized Linear Model. \nTreatment (3 levels: Control, S10 and S26), time (4 levels: J1, J2, J3 and J4) and their \ninteraction were included as fixed effects. A Cox proportional hazard models from the R \npackage “survival”, was developed to determine the impact of fungal solution on the survival \nof adults (Figure 3 A). For this, adult survival was the response variable with treatment was \nincluded as fixed effect and the random effect of ‘replicate’ was incorporated as a frailty \nfunction[28,29]. To understand the impact of the treatment on different entomological \nparameters of the adult mosquitoes, separate generalized linear mixed models (GLMMs) with \nnegative binomial family distribution were developed with the following response variables: \ni)feeding proportion (Figure 3B); ii) number of eggs (Figure 3C ); iii) Wing size (Figure 3 \nD&E), vi) hatched larvae (Figure 4A); v) generation G1(first offsprings from infected \nmothers) (Figure 4B) using the R package ‘glmmTMB’. Other parameters such as larval \nabundance was modelled using a negative binomial family distributed to account for the full \ndispersal in the data, while wing size was modelled following a Gaussian distributed GLMM \nwith R package “lme4”. All these models were fitted with treatment as fixed effect and replicate \nas random effect. For all models described above we performed model selection using stepwise \nremoval of terms, followed by likelihood ratio tests. The best model retained only significant \nterms that improved model goodness. Model performance diagnostics (i.e., residuals and \ndispersion) were evaluated for all models using the R package ‘DHARma’. All statistical \nanalysis were performed using R version 4.1.2.\n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted May 14, 2025. ; https://doi.org/10.1101/2025.05.14.654141doi: bioRxiv preprint \n\nResults\nExposure of larvae to fungi increases mortality compared to controls\nThere was a significant treatment × time interaction effect on larval survival rate ( 𝜒2\n6=17.79, \np=0.006); At J1, J2 and J3(days post exposure), larval survival (L4 stage) rate was close to \n100% regardless of treatment; however, at J4, larval survival rate in the control treatment batch \nwas still close to 100% while it was down to approx. 70% in both the S10 and S26 fungal \ntreatment batches (Figure2).\n  \nFungal exposure at larval stage reduces survival of emerging adults and increases their \nfecundity\nWe monitored the survival of the 103 adults that emerged after being exposed to fungi or \ncontrol at the larval stage for 5 days after emergence. We found that fungal exposure decreased \nadult mosquitoes’ survival (X2 = 26.361, df =2, p< 0.001) (Figure 3A). \nFemale adults were blood fed to determine the impact of infection on feeding behavior and \nfecundity. We analysed a total of 205 female mosquitoes and found that fungal treatment during \nthe larval stage did not influence the proportion of blood fed females (X2= 96.434 , df = 2, p= \n0.131) (Figure 3B).\nAfter removing non-blood fed mosquitoes, we monitored egg-laying in 150 fed mosquitoes \nindividually and found that fungal treatment during the larval stage increased the number of \neggs laid compared to controls by 1.27 times (X2= 96.434, df = 2, p<0.001) (Figure 3C). \nLarval fungal exposure does not alter the body size of emerged adults compared to \ncontrol\nThe increased number of eggs laid by adults emerging from larval fungal infection suggests \nthat infection might have resulted in larger adults, which is associated with increased fecundity \n[30]. To test this hypothesis, we measured mosquito wing lengths as a proxy of their body size \nbetween control and fungal infection treatments. Surprisingly, we found that fungal treatment \nat the larval stage had no effect on mosquito size (X 2=1.0292, df =2, p=0.5977) (Figure 3D), \nsuggesting that the fecundity effect is mediated by the direct effect of the fungi, not on selection \nof larger individuals. Overall, females were larger than males (X2= 3.8963, df =1, p= 0.04839) \n(Figure 3E).\n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted May 14, 2025. ; https://doi.org/10.1101/2025.05.14.654141doi: bioRxiv preprint \n\nMaternal fungal exposure increases the abundance of larval progeny but decreases the \nnumber of emerging adults compared to control\nAs we found cross-stages effects of fungal exposure, where treatment of larvae decreased adult \nsurvival and increased fecundity, we tested if infection at the maternal stage could also affect \nthe progeny by infecting adult females and measuring larval and adult abundance of the \nprogeny.  Out of the total 266 female mosquitoes treated, we found that maternal fungal \ntreatment significantly increased the number of larvae (L4) compared to controls. Indeed, the \nnumber of mosquitoes larvae treated with both S26 and S10 was 1.44 and 1.47 times higher, \nrespectively, than the untreated control ( X2= 654.57,df =2, p<0.001) (Figure 4A). However, \nthe number of larvae that became adults was two times lower in the groups treated with the \nfungi than in the untreated ones (X2= 642.49, df =2, p<0.001) (Figure 4B). \nDiscussion\nEfficient management of mosquito breeding sites would be a powerful complementary \ntool for the control of malaria. This could involve the use of natural enemies, including \nentomopathogens such as Metharizium. This study confirmed previous findings that \nMetharizium can be effective at targeting malaria mosquito larval stages[11,31] and added \nnovel insights into the cross- stage and generation effects. We found that although larvae that \nare exposed to fungi have a lower survival rate, those that survive as emerged adults generally \nhad increased mortality but their fecundity was increased in females. Together our findings \nshow there are some trade-offs of the impact of Metarhizium exposure on different life-history \ntraits. \nWe found that infecting adults mosquitoes increase the number of larvae produced in the \ntreated population compared to the control population. This suggests that the infected \nindividuals are under enormous pressure to survive and they are forced to invest in early \nreproduction. This finding is in line with studies  [32–34] that reported the entomopathogenic \nfungi Beauveria bassiana and Metarhizium anisopliae significantly reduce fecundity and egg \nviability in various hosts, thereby compromising their offspring. Their impact goes beyond \ndirect mortality by exerting reproductive pressure, enhancing their potential as biological \ncontrol agents. However, we noted that a very small proportion of these larvae reach the adult \nstage in the next generation. Since the larval density was standardized across treatments, we \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted May 14, 2025. ; https://doi.org/10.1101/2025.05.14.654141doi: bioRxiv preprint \n\nbelieve the increased mortality cannot be explain by density pressure. Instead, a plausible \nexplanation could be that the eggs were immature, so those that hatched did not possess the \nnecessary biological components for proper development. Also, regarding the infection at \nlarval stage the results show how certain using of entomopathogenic fungus can improve the \nability of spores to spread on a water surface and infect their hosts. We found high mortality \nof the larvae in both treatments at the pupal stage. This suggests that the larvae ingested a lethal \ndose of the fungus as food during their larval stage similar results have been reported by [35–\n39]. That studies reported that high pupal-stage mortality suggests larvae ingested lethal fungal \ndoses during development.\nAdults from larvae exposed to fungi solution had a relatively low survival rate compared to \ncontrol group. We hypothesize  that the spore had already passed through the larval cuticle \nbefore moulting into the adult stage[20,40,41], so these spores expressed their toxins once in \nthe adult mosquito's haemolymph or that, even in the larval stage, the fungus had already passed \nthrough the mosquito's haemolymph. Indeed, as An. coluzzii larvae have different rates of \nfiltration and ingestion on the surface and the spore can infect by ingestion or contact [16]; \nHowever, for those  larvae that survived the  infection, it is possible that the spore was still on \nthe surface of the host and that during molting the spore was shed or that there was no contact \nbetween the larva and the spore[40,41]. Molting has been reported to be an important factor in \nthe resistance of arthropods to fungal infection, particularly in arthropods with short ecdysis \nintervals [29]. \nIn a context where the multiplicity of mosquito breeding sites is a real bottleneck for malaria \ncontrol programs. The use of fungi in larval breeding sites could help to reduce mosquito \npopulations at both the larval and adult stages[42].\nObservation of the size and sex of treated and untreated individuals showed no significant \ndifference. This is consistent with previous studies showing similar wing size measurements \nbetween untreated and treated individuals with the fungi, but they show males mosquitoes \nWere bigger than female’s [27][43]. This suggests that infection at the larval stage does not \ninfluence the size of individuals according to their treatment status; however, females appear \nlarger than males emerging from the  fungal solution [16,19,38]. Based on the data collected \nregarding larval exposure to entomopathogenic fungi, the results can be interpreted as follows: \ninfected larvae tend to occur at lower densities within their habitat, which may promote \nenhanced individual growth and, consequently, lead to higher egg production at the adult stage. \nHowever, this hypothesis is not supported by body size measurements, which did not reveal \nsignificant differences between infected and control individuals. This suggests that the \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted May 14, 2025. ; https://doi.org/10.1101/2025.05.14.654141doi: bioRxiv preprint \n\nobserved increase in fecundity is not mediated by body size variation but rather by a \nphysiological response triggered by the infection. It is plausible that the physiological stress \ninduced by fungal exposure prompts females to invest more heavily in early reproduction, \npossibly as a compensatory mechanism in anticipation of reduced lifespan. This interpretation \nis consistent with observations made in infected adults, where a higher number of eggs laid \nwas also recorded, reinforcing the hypothesis of increased reproductive effort in infected or \nstressed females.These findings confirm that entomopathogenic fungi and their effective \neffects are possible candidates to  swap synthetic insecticides for controlling larvae, pupae and \nadult mosquitoes [44,45].\nConclusion\nMetarhizium seems to be a promising biocontrol agent for many insects including mosquitoes. \nAlthough feeding, oviposition and mosquito size do not seem to be influenced by the fungi \ntreatment at the concentration tested, An. coluzzi lifespan seems to be greatly reduced when \nthey are infected with conidia and the fungal spores seem to also impact the survival of larvae \nand adult emergence and survival, which will likely have important consequences for vectorial \ncapacity. These findings highlight the complex trade-offs induced by fungal infection and \nsupport the integration of entomopathogens into vector control strategies as a complementary \nor alternative tool to synthetic insecticides.  \nAcknowledgements \nWe express our sincere gratitude to all the study participants for their time and contribution to \nthis study. We are grateful to Vallée du Kou community and IRSS lab technicians du  for their \nhelp in conducting lab activities. \nAuthor contributions  \nIS, FB, AB, FD, MV,AD, AM, LL, AT and EB conceived of the study. IS, EB conducted the \nexperiments. FB, MV, IS and EB analysed the data. All authors drafted the manuscript. All \nauthors read and approved the final manuscript.\nFunding \nThis work was supported by the National Institute for Health Research (NIHR) (using the UK’s \nOfficial Development Assistance (ODA) Funding) and \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted May 14, 2025. ; https://doi.org/10.1101/2025.05.14.654141doi: bioRxiv preprint \n\nWellcome Trust grant ref218771/Z/19/Z
 under the NIHR-Wellcome Partnership for Global \nHealth Research. The views expressed are those of the authors and not necessarily those of \nWellcome Trust, the NIHR or the Department of Health and Social Care’. Preliminary field \nactivities were supported by the Open Philanthropy grant. FB is supported by the Academy \nMedical Sciences Springboard Award (ref:SBF007\\100094). MV is supported by the European \nResearch Council under the European Union’s Horizon 2020 Research and Innovation \nProgramme (grant agreement no. 852957)\nEthical approval\nNot applicable \nAvailability of data and materials \nAll data for this study will be available upon request. \nAbbreviations\nB.: Beauveria \nAn.:Anopheles\nCrtl: control group\nS10: Metarhizium strain 10 in our stump library\nS26: Metarhizium strain 26 in our stump library\nCompeting interests \nThe authors declare no competing interests. \nAuthor details \n        1Institut de Recherche en Sciences de la Santé, Direction Régionale de l’Ouest Bobo-\nDioulasso 01, BP 545, Burkina Faso\n2Institut National de Santé Publique / Centre Muraz, Bobo Dioulasso, Burkina Faso\n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted May 14, 2025. ; https://doi.org/10.1101/2025.05.14.654141doi: bioRxiv preprint \n\n3Laboratoire d’Entomologie Fondamentale et Appliquée (LEFA), Université Joseph Ki-\nZerbo, Ouagadougou 03 BP 7021, Burkina Faso\n4Laboratoire de Biologie Moléculaire et de Génétique (LABIOGENE), Ecole Doctorale \nSciences et Technologie, Université Joseph Ki-Zerbo ; Centre de Recherche Biomoléculaire \nPiétro Annigoni (CERBA), Ouagadougou 01, BP, 364, Burkina Faso\n5School of Biodiversity One Health and Veterinary Medicine, University of Glasgow, \nGlasgow, G12 8QQ, UK\n6Ifakara Health Institute, Environmental Health, and Ecological Sciences Department, \nMorogoro, United Republic of Tanzania.\n7Université Nazi Boni, 01 BP 1091.Bobo Dioulasso 01, Burkina Faso\nReferences\n1. World Health Organization. World Health Organization (WHO). 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It is \nThe copyright holder for this preprintthis version posted May 14, 2025. ; https://doi.org/10.1101/2025.05.14.654141doi: bioRxiv preprint \n\n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted May 14, 2025. ; https://doi.org/10.1101/2025.05.14.654141doi: bioRxiv preprint \n\n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted May 14, 2025. ; https://doi.org/10.1101/2025.05.14.654141doi: bioRxiv preprint \n\n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted May 14, 2025. ; https://doi.org/10.1101/2025.05.14.654141doi: bioRxiv preprint","source_license":"CC-BY-4.0","license_restricted":false}