Double-stranded RNA targeting vATPase B reveals a potential target for pest management of Henosepilachna vigintioctopunctata.

The development of genetic based techniques, specifically RNA interference (RNAi), has emerged as a powerful tool in novel pest management strategies for pestiferous coleoptera. The 28-spotted ladybird beetle, Henosepilachna vigintioctopunctata, is a dynamic foliar pest of solenaceous plants, primarily potato plants, and has quickly become one of the most important pests attacking many crops in Asian countries. In this study, we demonstrate the efficacy of dietary RNAi targeting vATPase B, which led to significant gene silencing. Downstream effects of vATPase B silencing appeared to be both time- and partial dose-dependent. Our results indicate that silencing of vATPase B caused a significant decrease in survival rate, as well as reduced the food stuffs consumption and inhibited the overall development of H. vigintioctopunctata. Furthermore, results demonstrate expression of insect melanism related genes, TH and DDC, was significantly up regulated under the dsvATPase B (RNAi molecule designed against vATPase B) treatment. The impact of oral dsvATPase B delivery on the survival of 1st, 3rd instars, and adults was investigated through bacterially expressed dsRNA. The effectiveness of RNAi-based gene silencing in H. vigintioctopunctata provides a powerful reverse genetic tool for the functional annotation of its genes. This study demonstrates that vATPase B may represent a candidate gene for RNAi-based control of H. vigintioctopunctata.

Selection of reference genes for RT-qPCR analysis in a predatory biological control agent, Coleomegilla maculata (Coleoptera: Coccinellidae).

Reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR) is a reliable technique for quantifying gene expression across various biological processes, of which requires a set of suited reference genes to normalize the expression data. Coleomegilla maculata (Coleoptera: Coccinellidae), is one of the most extensively used biological control agents in the field to manage arthropod pest species. In this study, expression profiles of 16 housekeeping genes selected from C. maculata were cloned and investigated. The performance of these candidates as endogenous controls under specific experimental conditions was evaluated by dedicated algorithms, including geNorm, Normfinder, BestKeeper, and ΔCt method. In addition, RefFinder, a comprehensive platform integrating all the above-mentioned algorithms, ranked the overall stability of these candidate genes. As a result, various sets of suitable reference genes were recommended specifically for experiments involving different tissues, developmental stages, sex, and C. maculate larvae treated with dietary double stranded RNA. This study represents the critical first step to establish a standardized RT-qPCR protocol for the functional genomics research in a ladybeetle C. maculate. Furthermore, it lays the foundation for conducting ecological risk assessment of RNAi-based gene silencing biotechnologies on non-target organisms; in this case, a key predatory biological control agent.