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

Jie Zeng,Lin Jin,Ahmad Ali Anjum,Guo-Qing Li,Wei-Nan Kang

doi:10.3390/insects12100935

Abstract

Simple SummaryVacuolar ATPase (vATPase), a proton pump driven by ATP hydrolysis, acts as a membrane energizer to motivate the movement of ions and nutrients across the cellular membrane in insect guts and Malpighian tubules, among others. The vATPase holoenzyme contains 16 subunits. Out of these subunits, mammalian G subunit includes three isoforms (G1-G3) which are encoded by three distinctive genes. The physiological role of a specific G isoform can be compensated by others. Thus, current experimental evidence on the in vivo function of G is rather limited among eight V1 subunits. In the present paper, particular attention was paid to an insect model, Henosepilachna vigintioctopunctata ladybird, a serious defoliator of Solanaceae and Cucurbitaceae plants in many Asian countries. Given that the beetle is sensitive to RNA interference (RNAi), HvvATPaseG gene was knocked down by ingestion of its corresponding dsRNA at the fourth-instar larval stage. Silence of HvvATPaseG affected larval growth and survival, impaired pupation and adult emergence. Our results provide a basis for further functional research on the vATPase G subunit in insects and suggest new ideas for the management of H. 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.

Highlights

Vacuolar-type H+-ATPase is a proton translocating pump present in the internal membranes of all eukaryotic cells [1,2]
5 dinayWosnUelaypffutoetronrtehdeeaixnrtpeste,eercfsuittmensdpcetenitochtin.eesAcc,hosDanre.taxemcpnteetelsracintzoeoafgdttai,rositntgehrloeyf[c3dve3Aar].itTdaPIenia,nstteohttsehauelpbsruHoenslvueivtnbAGtleTphPaaapmassebeirnG,eoewnhaeydcipioddcoesumnmatoinfierdnepdtphedrsaowteeinres sigsnuibfiucnainttGlygleonweeinr tahnaonnt-hDorosesoipnhtihlied PinBsSec-tasnpdecdiesse,gHfp.-vfiegdinltaiorcvtoapeu(nFcitgatuar.eW6eDu–nFc)o.vered that HvvATPaseG transcripts were detectable from embryo to adult; its mRNA level peaked at day 1 fourth-instar larvae
According to the temporal expression patterns, we knocked down HvvATPaseG using RNA interference (RNAi) to characterize its functions during larval development in H. vigintioctopunctata

Summary

Introduction

Vacuolar-type H+-ATPase (vATPase) is a proton translocating pump present in the internal membranes of all eukaryotic cells [1,2]. The Vm can motivate transmembrane movement of Cl− through a Cl− channel and maintain pH gradients in specific organelles such as Golgi, endosomes, lysosomes or secretory vesicles in eukaryotic cells [5,6]. CO3− provides the main anions to alkalinize the luminal contents to pH above 10 [16,17,18], an appropriate alkaline condition to maintain the activities of numerous digestive enzymes in the insect midguts [19]. In insect Malpighian tubules, a high level of K+ in the lumen drives a passive movement of anions, such as Cl−, HCO3− and perhaps urate ions to form primary urine [6,14]

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Conclusion