RNA interference mediated mortality in Aedes albopictus: a challenging journey toward species-specific vector control.

BACKGROUND: Aedes albopictus is a major vector of pathogens, including arboviruses, causing thousands of deaths annually. With no effective antiviral therapies and increasing concerns about the ecological impact of chemical insecticides, species-specific strategies, such as RNA interference (RNAi), are beneficial. Thus, identifying and validating target genes that induce mortality is essential. However, RNAi efficacy in Ae. albopictus is often inconsistent, owing to multiple factors including degradation by nucleases. Therefore, molecular identification and quantification of the underlying nucleases will provide a basis for improving RNAi efficacy. METHODS: Target genes were selected from previous studies, identified in Ae. albopictus, and their corresponding long double-stranded RNAs (dsRNAs) were designed. Using U4.4. cells as a first model, cytotoxicity was assessed with the CellTiter-Glo assay and gene knockdown via RT-qPCR. Larval survival assays and RT-qPCR were then used to evaluate in vivo effects. Owing to the lack of significant larval mortality, dsRNA complex size was analyzed using dynamic light scattering and their oral uptake was visualized by fluorescence microscopy. Suspecting degradation, dsRNA stability was assessed by agarose gel electrophoresis following incubation with larval gut extracts. This prompted the identification, characterization, and validation of two putative dsRNases. Finally, transfection reagents (TRs) were tested for their ability to protect dsRNA from degradation. RESULTS: Only one of the synthesized dsRNAs targeting the inhibitor of apoptosis (IAP) significantly reduced U4.4 cell viability to 65% (uncomplexed-dsRNA) and 13% (K4-complexed dsRNA). However, all tested dsRNAs achieved significant gene knockdown in the cell-based assay. None of the dsRNAs induced significant larval mortality, because dsRNA was rapidly degraded by larval gut extracts within 4 min. Although, gene knockdown was confirmed in the gut tissue. Each of  the two identified dsRNases contained a signal peptide, catalytic residues, and substrate- and Mg2⁺-binding sites, and were highly expressed in larval guts. Of the dsRNA, 65% remained intact at 15 min when complexed with K4, but declining to 13% by 24 h. CONCLUSIONS: All target genes were significantly silenced in cells, and IAP in larval gut tissue. Although TRs improved dsRNA stability in vitro, no significant larval mortality was observed, likely due to rapid gut degradation. Therefore, effective RNAi-based control of Ae. albopictus requires identifying gut-specific essential genes and improved delivery systems.