Abstract
The corn planthopper, Peregrinus maidis, is a major pest of agronomically-important crops. Peregrinus maidis has a large geographical distribution and transmits Maize mosaic rhabdovirus (MMV) and Maize stripe tenuivirus (MSpV). The objective of this study was to develop effective RNAi methods for P. maidis. Vacuolar-ATPase (V-ATPase) is an essential enzyme for hydrolysis of ATP and for transport of protons out of cells thereby maintaining membrane ion balance, and it has been demonstrated to be an efficacious target for RNAi in other insects. In this study, two genes encoding subunits of P. maidis V-ATPase (V-ATPase B and V-ATPase D) were chosen as RNAi target genes. The open reading frames of V-ATPase B and D were generated and used for constructing dsRNA fragments. Experiments were conducted using oral delivery and microinjection of V-ATPase B and V-ATPase D dsRNA to investigate the effectiveness of RNAi in P. maidis. Real-time quantitative reverse transcriptase-PCR (qRT-PCR) analysis indicated that microinjection of V-ATPase dsRNA led to a minimum reduction of 27-fold in the normalized abundance of V-ATPase transcripts two days post injection, while ingestion of dsRNA resulted in a two-fold reduction after six days of feeding. While both methods of dsRNA delivery resulted in knockdown of target transcripts, the injection method was more rapid and effective. The reduction in V-ATPase transcript abundance resulted in observable phenotypes. Specifically, the development of nymphs injected with 200 ng of either V-ATPase B or D dsRNA was impaired, resulting in higher mortality and lower fecundity than control insects injected with GFP dsRNA. Microscopic examination of these insects revealed that female reproductive organs did not develop normally. The successful development of RNAi in P. maidis to target specific genes will enable the development of new insect control strategies and functional analysis of vital genes and genes associated with interactions between P. maidis and MMV.
Highlights
The corn planthopper, Peregrinus maidis (Hemiptera: Delphacidae), is a widely distributed and destructive insect that causes significant yield losses by feeding on important crops such as corn, sorghum, and pearl millet
Blastx analysis of open reading frame (ORF) nucleotide sequences revealed that both VATPase B and D subunits are well-conserved sequences having more than 90% identities in amino acid sequence between P. maidis and Acyrthosiphon pisum, another Hemipteran
In vitrosynthesized Double-stranded RNA (dsRNA) was used to feed or inject P. maidis nymphs and adults to investigate the relative abundance of target gene transcripts, i.e., magnitude of gene knockdown
Summary
The corn planthopper, Peregrinus maidis (Hemiptera: Delphacidae), is a widely distributed and destructive insect that causes significant yield losses by feeding on important crops such as corn, sorghum, and pearl millet. The insect has three main developmental stages: eggs, 1st–5th-stage nymphs and adults. Corn planthoppers develop into one of three winged adult forms: macropters (longwinged), koeliopters (short-winged), and brachypters (wingless), depending on the type of habitat and population density [2]. Higher population density of nymphs promotes the appearance of macropters, and the long-distance migration of macropters allows P. maidis to exploit temporary crop or weed habitats, where they are capable of surviving on multiple hosts [3]. The male and female macropters and female brachypters are common, while koeliopters occur in lower numbers in the field [3]
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