Evaluation of three vacuolar ATPase genes as potential RNAi target in Henosepilachna vigintioctopunctata

Abstract

Henosepilachna vigintioctopunctata, a major threat to commercial potato production, is sensitive to RNA interference (RNAi), a gene-silencing mechanism triggered by double stranded RNA (dsRNA) matching a specific target gene sequence. Vacuolar ATPases (vATPases) are ATP-driven proton pumps involved in numerous biological processes. A holoenzyme vATPase consists of at least 14 different subunits and accessory proteins. Previous results reveal that silencing vATPaseB decreases survival rate, reduces food consumption and inhibits development in H. vigintioctopunctata. In this report, we identified two other subunit genes (vATPasea and vATPased) and compared the RNAi efficacy of vATPasea and vATPased with that of vATPaseB. We found the highest expression level of HvvATPasea, HvvATPaseB and HvvATPased occurred in the hindgut, followed by those in the foregut, Malpighian tubules and midgut, and lowest in the epidermis and fat body. When the fourth-instar larvae were allowed to ingest corresponding dsRNA for three days, the relative transcript levels of HvvATPasea, HvvATPaseB and HvvATPased were significantly decreased by 79%, 83% and 73%, respectively. Knockdown of HvvATPasea, HvvATPaseB and HvvATPased caused 50%, 85% and 100% larval lethality. After the third-instar larvae had exposed to corresponding dsRNA for three days, the levels of HvvATPasea and HvvATPased significantly reduced by 91% and 96% respectively. All the treated larvae were dead. Our results demonstrate that RNAi efficiencies varied among different vATPase subunit genes and various development stages. Moreover, our data imply the feasibility of RNAi as an alternative method for controlling this critical potato pest.