Knockdown of Vacuolar ATPase Subunit G Gene Affects Larval Survival and Impaired Pupation and Adult Emergence in Henosepilachna vigintioctopunctata

The vATPase holoenzyme consists of two functional subcomplexes, the cytoplasmic (peripheral) V1 and the membrane-embedded V0. Both V1 and V0 sectors contain eight subunits, with stoichiometry of A3B3CDE3FG3H in V1 and ac8c’c”def(Voa1p) in V0 respectively. However, the function of G subunit has not been characterized in any non-Drosophilid insect species. In the present paper, we uncovered that HvvATPaseG was actively transcribed from embryo to adult in a Coleopteran pest Henosepilachna vigintioctopunctata. Its mRNA levels peaked in larval hindgut and Malpighian tubules. RNA interference (RNAi)-mediated knockdown of HvvATPaseG significantly reduced larval feeding, affected chitin biosynthesis, destroyed midgut integrity, damaged midgut peritrophic membrane, and retarded larval growth. The function of Malpighian tubules was damaged, the contents of glucose, trehalose, lipid, total soluble amino acids and protein were lowered and the fat bodies were lessened in the HvvATPaseG RNAi larvae, compared with those in the PBS- and dsegfp-fed beetles. In contrast, the amount of glycogen was dramatically increased in the HvvATPaseG depletion ladybirds. As a result, the development was arrested, pupation was inhibited and adult emergence was impaired in the HvvATPaseG hypomorphs. Our results demonstrated that G subunit plays a critical role during larval development in H. vigintioctopunctata.

Effects of Delayed Mating on the Reproductive Performance of Henosepilachna vigintioctopunctata (F.) (Coleoptera: Coccinellidae)

Henosepilachna vigintioctopunctata (F.) is a serious pest of numerous solanaceous crops in many Asian countries. The purpose of this study was to clarify the effects of delayed mating on mating success, fecundity, fertility, pre-oviposition period, oviposition period, adult longevity, and population life table parameters (including net reproductive rate, intrinsic and finite rates of increase, doubling time, and mean generation time) of H. vigintioctopunctata. Beginning three days after emergence for both sexes, mating was delayed an additional 0, 2, 4, 6, or 8 days. We compared the data when mating was delayed for males only with the data when mating was similarly delayed for females only. Reproductive and life table parameters were calculated from the two data sets and compared. The results showed that the preoviposition and oviposition period of adults was significantly reduced by delayed mating, while the preoviposition period was not significantly different in adults mated at older ages. The mating success rate, fecundity, and proportion of hatching eggs decreased with increasing mating age. Longevity was not affected by the age at mating. Mating delay also affected the life table parameters of H. vigintioctopunctata, with a similar trend observed in the net reproductive rate and intrinsic and finite rates of increase, all of which decreased gradually as the number of delay days increased. The population doubling time increased with increases in mating age. The results also showed that delayed mating was an effective measure to consider in controlling H. vigintioctopunctata. It is hoped that our data will provide a scientific basis and contribute technical guidance for forecasting and integrated management of this pest.

RNAi of vacuolar-type H+-ATPase genes causes growth delay and molting defect in Henosepilachna vigintioctopunctata

Henosepilachna vigintioctopunctata is one of the most serious insect pests to a large number of nightshades and cucurbits. RNA interference (RNAi) triggered by double-stranded RNA (dsRNA) offers a reduced risk approach to control the beetle. Identification of amenable target genes and determination of appropriate life stage for dsRNA treatment are two critical steps in order to improve RNAi efficiency. In the present paper, we identified three vATPase genes, namely HvvATPaseC, HvvATPaseE and HvvATPaseH. We found that the three transcripts were widely expressed in the eggs, first- to fourth-instar larvae, prepupae, pupae and adults. They were abundantly transcribed in the hindgut and Malpighian tubules, in contrast to the epidermis and fat body. Three days' ingestion of dsvATPaseC, dsvATPaseE and dsvATPaseH by the fourth-instar larvae significantly decreased corresponding transcript level by 90.1, 88.9 and 97.2%, greatly reduced larval fresh weight by 28.0, 29.9 and 28.0%, and caused 66.7, 100 and 78.7% larval lethality respectively. Comparably, 3 days' exposure of the third-instar larvae to dsvATPaseC significantly reduced HvvATPaseC mRNA level by 89.5%, decreased approximately 80% of the larval fresh weight, and killed 100% of the treated larvae. Therefore, the three vATPase genes, especially HvvATPaseE, are potential amenable target genes and young larvae are more susceptible to dsRNA. Our findings will enable the development of the dsRNA-based pesticide to control H. vigintioctopunctata.