In Burkina Faso, despite considerable efforts to combat infectious diseases, the country experienced significant dengue epidemics in 2016, 2017, and 2023 [1] [2] . The rise of insecticide resistance in the Aedes aegypti mosquito has challenged traditional public health measures for controlling the disease [1] . Additionally, mosquito-resting behavior is highly variable, even within the same species, with individuals displaying endophilic (resting indoors) or exophilic (resting outdoors) tendencies after a blood meal [3] . Current interventions targeting indoor mosquitoes, such as long-lasting insecticidal nets and newly developed entomopathogenic fungi, are less effective against exophilic mosquitoes. Their effectiveness may be further threatened by shifts in mosquito behavior toward outdoor resting and feeding [4] . Horizontal sexual transmission of arhizium spp. could increase the efficacy of this fungus on both exophilic and endophilic mosquitoes and may complement other promising Aedes control strategies, such as the Sterile Insect Technique (SIT) [5] , Wolbachia-based technologies [6] and some medicinal plants against Aedes aegypti [7] . While previous studies have demonstrated the potential for Metarhizium to be sexually transmitted, the inoculum loads (~25 conidia per female mosquito) and the resulting mortality rates have been relatively low [8] . Although autodissemination of fungal spores by female Anopheles gambae s.s. has been documented, there is limited data on the use of a more virulent strain, Metarhizium pingshaense Met_S26 [9] within Aedes mosquitoes, and its ability to transfer from fungus-infected male Aedes mosquitoes to uninfected females and vice versa. This study aims to evaluate a new approach for deploying Metarhizium pingshaense fungus through sexual auto-dissemination within Aedes aegypti mosquitoes, in the context of dengue vector control. It also explores the potential of integrating this strategy with other promising control methods, such as Wolbachia and traditional SIT approaches.

The virulence of M. pingshaense Met_S26 against females of Aedes aegypti mosquitoes over 2 weeks is given in Figure 1 . The LT80 for the three concentrations of Met_S26 were 8.67 ± 0.17 days, 9.67 ± 0.93 days and 11.83 ± 0.44 day for C1, 1 × 10⁸ conidia/ml; C2, 1 × 10⁷ conidia/ml; C3, 1 × 10⁶ conidia/ml respectively. The LT80 (Lethal Time 80%) is the minimum time required to kill 80% of a population by the action of an insecticide. This sensitivity threshold is set by the World Health Organization for insecticide bioassays [11] .

As for the sexual dissemination of Metarhizium pingshaense, Figure 2 shows the sexual transmission of the fungus from infected males to uninfected females, and vice versa. This resulted in lower survival rates for uninfected females (19 ± 1.9%) when mating with fungus-infected males, and survival of 17 ± 2% for uninfected males when mating with fungus-infected females. All fungus-infected males and females used for the crossing died within 14 days. The hypothesis that uninfected females or males became infected by picking up spores from the surfaces of the mosquito cages where they were confined during cohabitation can be ruled out. During the 48-hour exposure period, both sexes (males and females) walked inside the mosquito cages, which could have facilitated the movement and spread of conidia, leading to passive transmission. However, survival remained similar to that of uninfected control mosquitoes ( Figure 2 ).

The LT80 obtained with M. pingshaense Met_S26 presents unprecedented and promising results for the safe biological control of Aedes-borne diseases. Previous studies have shown that M. pingshaense Met_S26 is specific to mosquitoes and does not harm non-target insects such as honeybees and cockroaches [10] . Most fungal-based strategies targeting mosquitoes have been developed for indoor biting species [12] . The high mortality rates observed through sexual auto-dissemination of Metarhizium pingshaense suggest a new avenue for controlling outdoor-biting Aedes mosquitoes. Information on the auto-dissemination of entomopathogenic fungi via sexual transmission in hematophagous insects is scarce in the literature. The first study addressing this topic was conducted on the tsetse fly, Glossina morsitans morsitans Westwood, infected with Metarhizium anisopliae and Beauveria bassiana in Africa. This study recorded 90% - 100% mortality in both sexes [13] . The second report involved sexual transmission of M. anisopliae in Anopheles gambiae s.l., the major malaria vector, with auto-dissemination rates of around 30% between males and females [8] . Compared to these earlier works, this current study is the first to demonstrate significantly higher mortality rates resulting from sexual transmission of Metarhizium pingshaense in Aedes aegypti mosquitoes in the field. This is a critical advancement in mosquito control strategies targeting vector populations in outdoor environments. We hypothesize that M. pingshaense Met_S26 could also be integrated with other promising Aedes vector control strategies, such as Sterile Insect Technique (SIT) or even as a stand-alone Sexually Transmitted Insecticide (STI). These combinations of strategies could provide a powerful dual approach. However, further investigations are necessary to validate these hypotheses and assess the long-term impact of these methods.

This study demonstrated that local strains of Metarhizium pingshaense Met_S26 fungus from Burkina Faso exhibit significant virulence against Aedes mosquitoes, including the primary vector Aedes aegypti that is responsible for transmitting dengue and other arboviruses. Remarkably, the virulence of Metarhizium pingshaense extends to sexually transmitted cases within Aedes aegypti, underscoring its potential as a novel biological control agent. The findings suggest that Metarhizium pingshaense could serve as an effective tool for controlling both indoor and outdoor populations of Aedes mosquitoes.

1) Institut de Recherche en Sciences de la Santé (IRSS) Direction Régionale de l’Ouest (DRO)/CNRST, Bobo-Dioulasso, Burkina Faso.

2) Institut National de Santé Publique (INSP), Centre Muraz, Bobo Dioulasso, Burkina Faso.

3) Sciences and Techniques Department, Institut du Dévelopement Rural, Université́ Nazi Boni, Bobo-Dioulasso, Burkina Faso.

This work was supported by the National Institute for Health Research (NIHR) (using the UK’s Official Development Assistance (ODA) Funding) and Wellcome Trust (Grant reference no. 218771/Z/19/Z) under the NIHR-Wellcome Partnership for Global Health Research. The views expressed are those of the authors and not necessarily those of Wellcome Trust, the NIHR or the Department of Health and Social Care.

EB designed the study. EB, FDDH, EJG, SI and SAM performed laboratory and field. EB analysed the data. EB wrote the first draft of the manuscript. All authors read and approved the final manuscript.

Supplementary Materials and Methods, data and codes are available at: https://github.com/EtienneBilgo/Bilgo-et-al-_Advances-in-Entomology . Correspondence and requests for materials should be addressed to E.B. and A.D.