Engineered Escherichia coli-Derived dsRNA Identifies β-Tubulin as a Candidate RNAi Target in Mosquito Larvae

Identifying conserved RNAi targets that remain effective across mosquito species and resistance backgrounds is important for developing broadly applicable mosquito control tools. Here, an engineered Escherichia coli HT115-L4440 system was used to produce β-tubulin double-stranded RNA (dsRNA), and its larvicidal activity was evaluated in susceptible and pyrethroid-resistant strains of Culex quinquefasciatus and Aedes albopictus. Recombinant bacteria produced species-specific β-tubulin dsRNA fragments of approximately 360 bp. Immersion treatment caused high larval mortality in both species. In Cx. quinquefasciatus, mortality reached 85.00% and 89.00% in susceptible and resistant strains, respectively, compared with 8.00% and 10.00% in controls, while β-tubulin transcript abundance showed maximum reductions of 571-fold and 333-fold, respectively. In Ae. albopictus, mortality reached 91.00% and 82.00% in susceptible and resistant strains, compared with 9% and 8% in controls, with maximum reductions in β-tubulin expression of 157-fold and 337-fold, respectively. These comparable effects suggest that β-tubulin dsRNA is not compromised by conventional pyrethroid-resistance mechanisms. Transcriptomic and proteomic analyses showed that β-tubulin knockdown disrupted key cellular processes, including cytoskeletal regulation, metabolism, protein synthesis, intracellular transport, detoxification, oxidative stress, DNA replication, and autophagy. Overall, engineered bacteria-derived β-tubulin dsRNA induced consistent gene silencing and larval mortality in both susceptible and resistant mosquito larvae, supporting β-tubulin as a conserved candidate RNAi target for mosquito control.