Genomics and Applied Biology 2024, Vol.15, No.2, 107-119 http://bioscipublisher.com/index.php/gab 113 6.2 Electroporation and viral vectors Electroporation has emerged as a viable alternative to microinjection, particularly for non-embryonic stages. This technique uses electric pulses to create transient pores in cell membranes, allowing the uptake of nucleic acids. A study demonstrated the effectiveness of electroporation-mediated delivery in Anopheles sinensis, achieving significant gene silencing and stable transgenesis in targeted body parts (Che et al., 2020). Additionally, viral vectors such as baculovirus have been explored for their ability to transduce mosquito cells efficiently. Baculovirus vectors can deliver genes without viral propagation, resulting in high-level gene expression with minimal negative effects on cell viability (Naik et al., 2018). 6.3 Nanotechnology-based delivery systems Nanotechnology offers innovative solutions for gene delivery, leveraging the small size and unique properties of nanoparticles to enhance transfection efficiency and reduce cellular perturbation. Nanostructure electro-injection (NEI) platforms, for example, utilize localized electric fields to facilitate the entry of DNA into cells, achieving higher transfection efficiency and lower cell stress compared to traditional methods (Tay and Melosh, 2019). Polyester-based nanoparticles, such as those made from poly (lactic-co-glycolic acid) (PLGA), have also been employed to deliver CRISPR/Cas9 components, offering advantages in terms of safety, targeting specificity, and scalability (Piperno et al., 2021). 6.4 Innovations in delivery efficiency The quest for more efficient and safer delivery methods has led to the exploration of non-viral delivery platforms. These platforms aim to overcome the limitations of viral vectors, such as immunogenicity and packaging constraints. Non-viral methods, including the use of synthetic vectors like lipid nanoparticles, have shown promise in delivering CRISPR/Cas9 components with minimal off-target effects and immune activation (Wilbie et al., 2019). Additionally, advancements in physical transfection methods, such as micro and nanotechnology-based approaches, are being developed to improve the performance and applicability of gene editing tools (Fajrial et al., 2020). 7 Impacts on Mosquito Behavior and Ecology 7.1 Genetic modifications and mosquito fitness Genetic modifications in mosquitoes, particularly through CRISPR/Cas9-based gene editing, have shown significant impacts on mosquito fitness. For instance, the introduction of the kdr mutation L1014F in Anopheles gambiae increased resistance to pyrethroids and DDT but also resulted in fitness disadvantages such as increased larval mortality and reduced adult longevity and fecundity (Grigoraki et al., 2021). Similarly, gene-drive systems targeting the kynurenine hydroxylase gene in Anopheles stephensi have been developed to relieve the fitness load in females, ensuring efficient population modification (Adolfi et al., 2020). These modifications are crucial for the success of gene-drive technologies, as they must balance the benefits of pathogen resistance with the potential fitness costs to the mosquito population. Grigoraki et al. (2021) illustrates the effects of the L1014F mutation on the development, fecundity, fertility, and survival of Kisumu mosquito strains. It highlights that mosquitoes carrying the L1014F mutation (Kisumu-F/F) experience notable developmental delays, with a lower percentage of larvae reaching the pupae stage compared to wild-type mosquitoes (Kisumu-L/L). The fecundity of Kisumu-F/F females is also reduced, with fewer females laying eggs after a blood meal. However, there is no significant difference in the number of eggs laid or larvae hatched between the two strains. Additionally, Kisumu-F/F females have a shorter lifespan than the wild-type strain. Overall, the mutation introduces fitness costs, affecting multiple life history traits that could influence the success of genetically modified mosquito populations in control strategies. 7.2 Ecological risks and benefits The deployment of genetically engineered mosquitoes (GEMs) carries both ecological risks and benefits. One of the primary benefits is the potential to reduce the transmission of vector-borne diseases such as malaria and dengue by modifying mosquito populations to be refractory to pathogens (Lanzaro et al., 2021; Wang et al., 2021).
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