Biological and Genetic Approaches in Mosquito Vector Control: Current Advances and Future Challenges

Wolbachia-based vector control

Wolbachia is an intracellular Gram-negative endosymbiotic bacterium of the class Alphaproteobacteria, maternally inherited through vertical transmission from females to their offspring. Generally, Wolbachia facilitates its own proliferation and transmission by manipulating host reproduction through feminization, male killing, parthenogenesis, or cytoplasmic incompatibility (CI) (Fig. 1D). Natural infection with this bacterium has been detected in various genera of medically important insect species. However, Wolbachia infection has never been detected in Aedes aegypti, the primary vector for dengue, Zika, and chikungunya viruses. Approximately 66% of all insect species naturally harbor different strains of Wolbachia pipientis. Research over the last two decades on Wolbachia infections has grown rapidly, and many studies have shown successful Wolbachia introduction into A. aegypti. Results from these studies have shown two major biological outcomes: (1) suppression of pathogen replication, known as “pathogen blocking,” and (2) induction of CI in the host system, which reduces mosquito fertility by manipulating reproduction (Walker et al., 2011, Utarini et al., 2021). CI occurs when uninfected females mate with Wolbachia-infected males, resulting in embryo lethality or nonviable offspring, ultimately leading to population reduction (Fig. 1D). These experimental results provide a new strategy for vector control through either population suppression or population replacement by substituting wild populations with high transmission capacity with less competent vector species (O’Neill, 2018). Under the World Mosquito Program, experimental field trials with Wolbachia-infected mosquitoes in multiple countries—including Colombia (Velez et al., 2023), Vietnam (Turner et al., 2023), Brazil (Pinto et al., 2021), Indonesia (Indriani et al., 2020), and Australia (Ogunlade et al., 2023)—have demonstrated significant reductions in arthropod-borne virus (arbovirus) transmission and long-term establishment of Wolbachia-infected mosquito populations. A recent randomized trial conducted in Yogyakarta, Indonesia, showed a 77% reduction in dengue incidence after the release of Wolbachia-infected mosquito vectors (Utarini et al., 2021). Despite these successes, several challenges remain, including temperature sensitivity of some Wolbachia strains (e.g., wMel), incomplete maternal transmission, fitness costs, and potential competition with native microbiota.

Paratransgenesis: Asaia and Serratia

Paratransgenesis is an innovative method that involves genetically modifying symbiotic microorganisms, such as bacteria, fungi, and viruses, that are already present in mosquitoes to deliver anti-pathogen molecules within the vector’s body, preventing them from spreading pathogens. These microorganisms are constantly associated with the host system and can be found in different organs, for example, the midgut, salivary glands, and reproductive organs. Unlike transgenesis, in which modification occurs directly in the mosquito genome, paratransgenesis uses these microorganisms to manipulate the host system where the growth of infectious agents takes place. These symbiotic microorganisms are genetically engineered to block the development and transmission of pathogens by expressing effector molecules (Ratcliffe et al., 2022). Asaia spp. and Serratia spp. are the most promising candidates for paratransgenesis because of several significant features: their ability to naturally colonize mosquito tissues, their ease of cultivation under laboratory conditions, their widespread presence in mosquito populations, their potential for both vertical and horizontal transmission, and ease of genetic manipulation. These qualities make them ideal vehicles for delivering anti-parasitic or antiviral proteins (Gendrin and Christophides, 2013).

Entomopathogenic fungi

Entomopathogenic fungi (EPF) naturally infect insects, including mosquito vectors, leading to morbidity and mortality. EPF cause death either by directly penetrating the host cuticle or by being ingested (conidia, blastospores), then colonizing the hemocoel and ultimately killing the host through nutrient depletion and toxin production (Gielen et al., 2024). Infection begins when fungal conidia adhere to the mosquito cuticle, generally on the tarsi or abdominal segments. After adhesion, the conidia germinate, penetrate the cuticle through mechanical pressure and enzymatic degradation (e.g., proteases and chitinases), and develop within the hemocoel. The fungus disrupts physiological activities and secretes secondary metabolites, such as destruxins, which inhibit the host immune response and lead to death within 4–10 days (Ma et al., 2024) (Fig. 1C).

Several studies have demonstrated the high efficacy of entomopathogenic fungi against mosquito vectors. For instance, exposure to Metarhizium anisopliae has been shown to cause up to 80–90% mortality in Anopheles mosquitoes within a few days under laboratory conditions (Scholte et al., 2005). Additionally, fungal infection significantly reduces mosquito longevity and vector competence, thereby lowering transmission potential (Blanford et al., 2005; Thomas and Read, 2007). Unlike bacterial or viral pathogens, EPF do not depend solely on ingestion; they are capable of infecting insecticide-resistant mosquito species, making them an attractive component of Integrated Vector Management (IVM). Several EPF species, including Beauveria, Lagenidium, Isaria, Tolypocladium, Aspergillus, Culicinomyces, and Coelomomyces, are effective biocontrol agents against mosquito vectors (Accoti et al., 2021). Beauveria bassiana and M. anisopliae are the two most frequently studied entomopathogens and are used in mosquito control programs because of their demonstrated efficacies against species such as A. gambiae, A. stephensi, and A. aegypti (Scholte et al., 2005).

Incompatible insect technique

The incompatible insect technique (IIT) is quite similar to sterile insect technique (SIT) but uses Wolbachia-induced CI in the host system, which produces nonviable offspring. IIT does not require irradiation and therefore avoids the fitness costs associated with irradiation in mosquitoes. In IIT, male mosquitoes infected with a specific Wolbachia strain are released into the wild, where they produce embryos that fail to develop due to CI when mating with uninfected females or females carrying incompatible Wolbachia strains (Pagendam et al., 2020) (Fig. 2B). Due to this convenient procedure, IIT has gained attention in various vector control programs globally, particularly in Asia, Australia, and Europe, targeting Aedes albopictus and A. aegypti (Lim et al., 2024). According to a recent study, weekly releases of Wolbachia-infected male mosquitoes in Guangzhou, China, suppressed 94% of local A. albopictus populations (Zeng et al., 2022). Moreover, combining IIT with low-dose irradiation (IIT–SIT combination) could reduce the risk of accidentally releasing Wolbachia-infected females (Bansal et al., 2024). IIT is closely linked to Wolbachia-based strategies, as both rely on CI to suppress mosquito populations. While Wolbachia-based approaches are often used for population replacement, IIT primarily focuses on population suppression through repeated male releases. In practice, these approaches are increasingly being combined with SIT to improve efficiency and reduce operational risks, particularly the unintended release of females. In addition to these methods, related genetic approaches such as release of insects carrying a dominant lethal (RIDL) have also been explored for mosquito population suppression, further expanding the range of available vector control strategies. Species specificity, nonreliance on chemical insecticides, and reduced operational expenses compared to SIT are some of the key benefits of IIT (Kittayapong et al., 2025). Moreover, some of the ecological issues related to gene drive systems are also addressed by a built-in confinement mechanism offered by the self-limiting nature of IIT (in the absence of released females) (Li et al., 2024). To implement the IIT method successfully, several factors must be considered. These include precise strain selection to ensure bidirectional incompatibility, mass production of high-quality male mosquitoes, and suitable field conditions such as temperature and humidity. Population density at the release site is also a critical factor (Aldridge et al., 2024). All these factors can significantly affect the successful implementation of IIT (CI-based suppression) for vector control. However, current studies are highly focused on developing methods based on IIT–SIT combinations and exploring new strategies for mosquito release methods in the field, considering cost-effectiveness, such as drone-based methods. At the same time, establishing the long-term epidemiological impacts of IIT on disease transmission are being predicted quantitatively by mathematical modeling (Lim et al., 2024).

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FIG. 2.

Different methods of population suppression approaches. (A) Sterile insect technique: (1) Mass production of mosquitoes in a controlled environment. (2) Sorting of males from females. (3) Sterilization of mosquitoes by ionizing radiation. (4) Mass release of sterile males in the open environment. (5) Wild-type males compete with sterile males. (6) Sterile males when mate with the wild-type females produce nonviable eggs. (B) Incompatible insect technique: (1) Mass production of mosquitoes in a controlled environment. (2) Sorting of males form females. (3) Wolbachia is artificially injected into the mosquito to make it sterile. (4) Sterile male mosquitoes are released. (5) Wolbachia-infected male mosquitoes compete with wild-type male mosquito. (6) Unidirectional and bidirectional cytoplasmic incompatibility take place and ultimately reduces the vector population. (C) Release of insects with a dominant lethal (RIDL): (1) When tetracycline is present, it binds to the tetracycline activator protein (t-TA) and renders it inactive, which ultimately prevents it from binding with the tetracycline operator (tetO) and dominant lethal gene cannot express itself. This allows the mosquito to survive in the laboratory setting where tetracycline is provided as a nutrient in food. (2) In the absence of tetracycline, the t-TA is active and binds with the tetO, allowing the dominant fatal gene to express itself, which ultimately kills the mosquito.

Sterile insect technique

The SIT was originally developed in the 1950s to suppress agricultural pests, mainly the screwworm fly, and has since been established as one of the most effective methods of insect control (Lees et al., 2021). The main principle of this method involves mass-rearing target mosquitoes in a controlled environment, separating males from females, and sterilizing the males with ionizing radiation (mainly gamma rays), which damages their reproductive cells (sperm), rendering them incapable of fertilizing eggs during mating. When these sterile males are released into the wild to mate with wild females, the resulting eggs are either nonviable or have reduced viability. Ultimately, this leads to a significant reduction in the target population size over successive generations (Lees et al., 2021) (Fig. 2A). SIT is currently being utilized to control several medically important mosquito vectors, particularly species of the Aedes and Anopheles genera.

Over the past decade, SIT and related sterile male-based suppression strategies have advanced substantially from experimental frameworks to increasingly sophisticated operational deployments across multiple countries and ecological settings. In China, landmark large-scale combined IIT–SIT field trials targeting A. albopictus achieved >90% suppression of wild populations over multiple years, demonstrating robust feasibility for urban mosquito control while highlighting the advantages of integrating Wolbachia-based incompatibility with conventional sterilization (Zheng et al., 2019). In Italy, sustained releases of sterile A. albopictus males significantly reduced egg hatch rates and local mosquito population density, further validating SIT under European field conditions (Bellini et al., 2013). In Brazil, genetically based sterile male suppression strategies targeting A. aegypti, including transgenic and sterile male–based interventions, achieved local population reductions exceeding 80% under semi-operational conditions, reinforcing the applicability of sterile male–based mosquito control in endemic tropical regions (Macedo et al., 2026). In Greece, pilot sterile male release programs against A. albopictus further demonstrated the ecological adaptability of SIT under Mediterranean environmental conditions (Balatsos et al., 2024). In the United States, field deployment of self-limiting A. aegypti strains in Florida provided additional evidence for the translational potential of sterile male–based mosquito suppression technologies within highly regulated operational frameworks (Morreale et al., 2025). Together, these deployments across China, Italy, Brazil, Greece, and the United States demonstrate that SIT has evolved beyond its classical irradiation-based foundation into a broader and increasingly field-validated mosquito suppression platform. Recent innovations, particularly combined IIT–SIT frameworks, further enhance suppression efficiency, reduce risks associated with female contamination, and partially offset fitness costs linked to irradiation alone (Zheng et al., 2019; Bansal et al., 2024). Despite these advances, broader implementation remains constrained by major operational barriers, including cost-intensive mass rearing, precise sex separation, maintenance of sterile male mating competitiveness, regulatory oversight, and the substantial infrastructure required for sustained area-wide deployment (Lees et al., 2021). To address these limitations, innovations such as genetic sexing strains, automated sex-sorting technologies, and optimized artificial diets are increasingly being developed to improve scalability, cost-effectiveness, and long-term implementation feasibility (Lees et al., 2021). Moreover, the Food and Agriculture Organization of the United Nations (FAO) and the International Atomic Energy Agency (IAEA) has actively promoted SIT as part of integrated vector management programs in multiple countries, reinforcing its growing institutional relevance in sustainable mosquito control. Despite these successes, SIT has limitations, including difficulties in large-scale mass rearing, incomplete sex separation, potential reductions in mating competitiveness of irradiated males, and high operational costs (Lees et al., 2021). Advances in automation, artificial diets, and genetic sexing strains are currently being explored to overcome these barriers.

Release of insects carrying a dominant lethal

RIDL is a transgenic mosquito suppression strategy that employs genetically engineered male mosquitoes carrying a dominant, self-limiting lethal gene designed to suppress wild vector populations through inherited offspring mortality. RIDL mosquitoes are typically reared under laboratory conditions supplemented with tetracycline, which suppresses lethal gene expression and permits colony maintenance. The RIDL mechanism and population suppression strategy are illustrated in Fig. 2C. Once released into natural environments, engineered RIDL males mate with wild females; however, in the absence of tetracycline, the lethal transgene is activated in progeny, causing death before reproductive maturity and thereby progressively reducing local mosquito population density over successive generations (Alphey et al., 2010; 2014). Unlike classical SIT, RIDL does not depend on irradiation, potentially preserving male mating competitiveness while maintaining species-specific suppression.

Over the past decade, RIDL has become one of the most operationally advanced transgenic mosquito control strategies, particularly against A. aegypti. Early open-field trials in the Cayman Islands using Oxitec’s OX513A strain demonstrated ∼80% suppression of local A. aegypti populations (Harris et al., 2012). Subsequent sustained releases in Juazeiro, Brazil, reported population suppression frequently exceeding 80–95%, providing robust large-scale field validation under endemic tropical conditions (Carvalho et al., 2015). Additional operational deployments in Panama further supported RIDL’s ecological adaptability across geographically distinct transmission settings (Gorman et al., 2016). These multinational studies collectively established RIDL as a major proof-of-concept for scalable, genetically based mosquito suppression. RIDL is considered a self-limiting strategy because its effects depend on repeated releases of engineered males rather than permanent spread through wild populations. This feature may provide greater control compared with gene drive systems, since suppression can be adjusted or stopped if needed; however, it also means that long-term success depends on continuous operational investment. RIDL offers several advantages, including species-specific targeting, reduced reliance on chemical insecticides, and potential integration with other mosquito suppression approaches such as SIT or IIT. At the same time, important challenges remain. These include the need for repeated large-scale releases, high production and implementation costs, regulatory and biosafety requirements, and ongoing public concerns regarding the release of genetically modified mosquitoes. In many low-resource settings, these economic, infrastructural, and social barriers may limit large-scale sustainability. Therefore, although RIDL has become one of the most operationally advanced transgenic mosquito suppression strategies currently available, its wider adoption will depend not only on biological effectiveness but also on affordability, regulatory acceptance, and public trust. While RIDL provides a self-limiting transgenic suppression strategy, gene drive systems represent a more self-sustaining genetic approach aimed at spreading engineered traits through wild populations.

Gene drives

One of the most powerful and highly controversial genetic technologies for vector control is the gene drive. This method has a mechanism of inheritance that completely contrasts with the conventional Mendelian process. Gene drives increase the frequency of a gene within a population so rapidly that it facilitates the spread of the gene throughout the target population (Fig. 3). This technology is being explored to modify or control vector populations either by decreasing their competence to carry disease-causing agents or by altering their reproductive systems in ways that lead to infertility or poor survival (Hefferon and Herring, 2020). Homing Endonuclease Genes (HEGs), Zinc Finger Nucleases (ZFNs), Transcription Activator-Like Effector Nucleases (TALENs), and the CRISPR–Cas9 system are a few examples of gene drive mechanisms that have been extensively studied (Fig. 3). Among these, CRISPR-based gene drives have been especially attractive and have captured the most attention due to their ease of use, effectiveness, and programmable capability (Costa et al., 2017).

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FIG. 3.

Different methods of population modification approaches using gene drive.

One of the most powerful and controversial genetic technologies for vector control is the gene drive (Burt, 2003; Hefferon and Herring, 2020). Unlike conventional Mendelian inheritance, gene drives bias gene transmission, allowing specific traits to spread rapidly through target populations (Fig. 3). These systems are being explored either to suppress mosquito populations by disrupting fertility or survival or to modify them by reducing pathogen transmission capacity (Hefferon and Herring, 2020). Several platforms, including HEGs, ZFNs, TALENs, and especially CRISPR–Cas9, have been investigated, with CRISPR-based systems attracting the greatest attention because of their efficiency and programmability (Costa et al., 2017). Experimental studies in A. gambiae have demonstrated remarkable suppression potential under controlled settings, including near-complete population collapse in caged populations (Kyrou et al., 2018). However, despite their transformative promise, gene drives remain largely experimental and have not yet reached broad field deployment comparable to SIT, IIT, or RIDL. Key concerns include resistance evolution, ecological uncertainty, unintended spread beyond target regions, and significant ethical and regulatory challenges (National Academies of Sciences, Engineering, and Medicine, 2016). Therefore, although gene drives represent a potentially powerful future tool for mosquito-borne disease control, their practical implementation currently remains limited by substantial biosafety, governance, and societal concerns.

Homing endonuclease genes

HEGs are naturally occurring autonomous genetic elements that can identify and cleave specific DNA sequences. Because of this ability, HEGs can cleave the homologous wild-type allele upon their introduction into mosquitoes. Later, this gene drive sequence can be replicated at the cleaved site through the natural replication process of mosquitoes in general, and by homology-directed repair in particular. As a result, nearly all offspring acquire this gene drive rather than the expected 50% (Oberhofer et al., 2018). Previously, HEG-based gene drives targeted genes involved in fertilization or sex determination. For example, HEGs directed against the dsx gene in A. gambiae led to female sterility and progressive population collapse (Bier, 2022). However, some limitations exist with HEGs, such as the requirement for strict sequence recognition and lower flexibility compared to CRISPR.

Zinc finger nucleases and transcription activator-like effector nucleases

In ZFNs and TALENs, specific engineered proteins are required to recognize and cleave DNA; therefore, their design and validation are required for each target site. The literature shows their use in mosquitoes for vector control; however, their application has been limited due to poor efficiency and highly complex protein designs (Gaj et al., 2013).

CRISPR–Cas9-mediated gene drive

CRISPR–Cas9 gene drives have become one of the most popular platforms for genetic control due to their versatility, which allows them to target almost any gene. Typically, two elements, the Cas9 enzyme, which functions like molecular scissors, and guide RNA (gRNA), which contains the genetic information required to target a DNA sequence, are inserted into the mosquito genome in the CRISPR-based drive system to make modifications. When these modified mosquitoes mate with unmodified (wild-type) ones, the Cas9/gRNA complex cleaves the targeted region in the gene on the wild-type chromosome. The drive-carrying chromosome then serves as a template for repair. The CRISPR–Cas9 system is sometimes referred to as a highly developed biological “search/replace” mechanism that cuts DNA and replaces the old code with a new one using gRNAs. This process of copying the drive segment is called “homing,” and it ensures that nearly 100% of offspring inherit the drive (Kistler et al., 2015). Various studies have demonstrated successful results of vector control in the laboratory using this system. For example, knocking out the fibrinogen-related protein 1 (FREP1) gene from A. gambiae mosquitoes using the CRISPR–Cas9 system may develop resistance to Plasmodium. In another study conducted in 2018, Hammond et al. reported intersex phenotypes or 100% sterilization in females of A. gambiae when the sex-differentiation controlling gene double-sex was targeted. Other successful studies on A. gambiae have also been documented, where the drive spread through a caged population within only 10 generations, leading to total population collapse (Kistler et al., 2015). Similar studies have been reported in A. aegypti to spread anti-pathogen effectors or female-specific lethal genes. Despite several promising features such as potential and specificity, gene drives are associated with significant ecological, ethical, and regulatory challenges: (1) mutations at the target site can hamper cleavage and lead to resistant alleles, ultimately blocking drive spread, (2) cleavage at nontargeted sites might result in unanticipated ecological or physiological consequences, (3) difficulty or irreversibility in recalling the drive once released, especially in highly mobile species, and (4) nontarget spread across species or unintended transport to other areas, which may threaten ecological balance (Collins, 2018). To overcome these concerns, various strategies are being explored. These include the use of molecular safeguards such as split drives, which separate Cas9 and gRNA components, and “threshold-dependent” or “daisy-chain” drives, which restrict persistence. Moreover, field trials are being designed with rigorous confinement, stakeholder participation, and phased testing frameworks. Furthermore, organizations such as WHO, the Convention on Biological Diversity, and national biosafety agencies are establishing international rules for gene drive research and deployment (Kelsey et al., 2020). However, no information is available about the release of CRISPR-based gene drives into open environments. Target Malaria, a nonprofit consortium developing gene drive technology, is pursuing laboratory research followed by limited field trials on A. gambiae in Africa and represents one of the most well-known gene drive initiatives. With rapid advancements, strong funding support, community engagement, transparent disclosure of risks, and cross-sector collaboration, CRISPR-based gene drives may eventually become feasible to implement (Kelsey et al., 2020).