Genome-wide screening for components of smallinterfering RNA (siRNA) and micro-RNA (miRNA)pathways in the brown planthopper, Nilaparvata lugens(Hemiptera: Delphacidae)

H-J. Xu, T. Chen, X-F. Ma, J. Xue, P-L. Pan,X-C. Zhang, J-A. Cheng and C-X. Zhang

State Key Laboratory of Rice Biology and Ministry ofAgriculture Key Laboratory of Agricultural Entomology,Institute of Insect Science, Zhejiang University,Hangzhou, China

Abstract

The brown planthopper (BPH), Nilaparvata lugens, is amajor rice pest in Asia, and accumulated evidenceindicates that this species is susceptible to RNA inter-ference (RNAi); however, the mechanism underlyingRNAi and parental RNAi has not yet been determined.We comprehensively investigated the repertoire ofcore genes involved in small interfering RNA (siRNA)and micro-RNA (miRNA) pathways in the BPH by com-paring its newly assembled transcriptome and genomewith those of Drosophila melanogaster, Triboliumcastaneum and Caenorhabditis elegans. Our analysisshowed that the BPH possesses one drosha and twoDicer (DCR) genes, three dsRNA-binding motif proteingenes, two Argonaute (ago) genes, two Eri-1-likegenes (eri-1), and a Sid-1-like gene (sid-1).Additionally,we report for first time that parental RNAi might occurin this species, and siRNA pathway and Sid-1 wererequired for high efficiency of systemic RNAi triggeredby exogenous dsRNA. Furthermore, our results alsodemonstrated that the miRNA pathway was involved inBPH metamorphosis as depletion of the ago1 or dcr1gene severely impaired ecdysis. The BPH might be agood model system to study the molecular mechanism

of systemic RNAi in hemimetabolous insects, andRNAi has potential to be developed to control this pestin agricultural settings.

Keywords: Brown planthopper, RNAi pathway, sys-temic RNAi, parental RNAi.

Introduction

As an evolutionarily conserved gene-regulatory mecha-nism, RNA interference (RNAi) occurs in a wide variety ofeukaryotic organisms (Fire et al., 1998; Tijsterman et al.,2002; Meister & Tuschl, 2004; Mello & Conte, 2004). RNAiis triggered by the presence of double-stranded RNA(dsRNA), resulting in the degradation- or translation-repression of complementary single-stranded RNA (Fireet al., 1998). RNAi also plays versatile roles in gene regu-latory pathways including cell proliferation, antiviraldefence, DNA methylation, heterochromatin formation,silencing of transposable elements and DNA elimination(Grishok et al., 2001; Ambros, 2004; Baulcombe, 2004;Vastenhouw & Plasterk, 2004; Matzke & Birchler, 2005).RNAi can occur through three pathways, endogenoussmall interfering RNA (siRNA), micro-RNA (miRNA) andPIWI-interacting RNA (piRNA), according to theirbiogenesis mechanism and the type of Argonaute proteinswith which they are associated (Kim et al., 2009). LongdsRNAs are processed by the conserved RNase IIIenzyme Dicer to generate small RNAs in both the siRNAand miRNA pathways (Bernstein et al., 2001; Liu et al.,2003; Carmell & Hannon, 2004). These small RNAs thenpreferentially bind to RNA-induced silencing complexes(RISCs) for either transcriptional or post-transcriptionalsilencing in the siRNA pathway, or to miRNA ribonu-cleoparticles for translational suppression in the miRNApathway (Martinez & Tuschl, 2004). During this process,proteins that contain dsRNA-binding motifs, such as R2D2and Loquacious, facilitate dsRNA binding to the silencingcomplex, which is mainly composed of Argonaute proteins

First published online 13 August 2013.

Correspondence: Chuan-Xi Zhang, State Key Laboratory of Rice Biologyand Ministry of Agriculture Key Laboratory of Agricultural Entomology,Institute of Insect Science, Zhejiang University, Hangzhou 310058, China.e-mail: chxzhang@zju.edu.cn

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InsectMolecular

Biology

Insect Molecular Biology (2013) 22(6), 635–647 doi: 10.1111/imb.12051

© 2013 Royal Entomological Society 635

(Tabara et al., 2002; Saito et al., 2005; Parker & Barford,2006). The Argonaute proteins bound to small RNAs arethen believed to mediate target recognition. In plants andnematodes, systemic RNAi requires RNA-dependent RNApolymerases (RdRPs) and the Sid-1 transmembraneprotein (Wassenegger & Pelissier, 1998; Sijen et al., 2001;Winston et al., 2002; Feinberg & Hunter, 2003), but RdRP-like proteins have never been confirmed in insects (Jose &Hunter, 2007; Richards et al., 2008; Tomoyasu et al.,2008).

Since the first report that exogenous dsRNA silencescomplementary endogenous mRNA in Caenorhabditiselegans (Fire et al., 1998), RNAi has been very useful infunctional genomic research, and it has considerablepotential to be used to control pests in agricultural settings(Huvenne & Smagghe, 2010). RNAi has been successfullyused in many live insect species, including Coleoptera,Diptera, Dictyoptera, Hemiptera, Hymenoptera, Isoptera,Lepidoptera, Neuroptera, and Orthoptera, although silenc-ing efficiency varies between species (Bellés, 2010). Insome organisms, such as C. elegans and Triboliumcastaneum, it is feasible to induce an RNAi effect systemi-cally (Bucher et al., 2002; Tomoyasu & Denell, 2004;Grishok, 2005; May & Plasterk, 2005), but some organ-isms, such as Drosophila melanogaster and Lepidopterainsects, are refractory to robust systemic RNAi effects(Roignant et al., 2003; Terenius et al., 2011). Accordingly,further understanding of the underlying RNAi mechanism isnecessary to improve the use of systemic RNAi in insects.

The brown planthopper (BPH), Nilaparvata lugens, isthe most destructive Asian rice insect pest. This pestcauses significant reductions in rice yields throughsucking, ovipositing and virus transmission (Wang et al.,2008). Outbreaks of rice planthoppers have been closelymonitored in recent years in China and other Asian coun-tries. Because of its ability to migrate long distances, toquickly adapt to resistant rice varieties and to becomehighly resistant to pesticides, effective approaches are not

currently available for controlling this pest (Lou & Cheng,2011). Multiple studies indicated that a robust RNAiresponse was elicited in this species by delivering dsRNAeither by microinjection or by feeding (Chen et al., 2010;Liu et al., 2010; Dong et al., 2011; Li et al., 2011; Zhaet al., 2011; Wang et al., 2012; Wu et al., 2012; Xue et al.,2013). A greater understanding of the biological mecha-nism of RNAi in this species might inform methods ofexploiting RNAi to control this pest in agricultural settings.

Although RNAi has been extensively used in BPH todetermine gene function, the genes involved in the BPHRNAi pathway have not yet been identified. Using ourtranscriptome data (Xue et al., 2010) and the newlyassembled complete genome sequence of BPH, we com-prehensively investigated the repertoire of genes involvedin the BPH siRNA and miRNA pathways by comparing thesequence data with orthologues from D. melanogaster,T. castaneum and C. elegans, and for the first time experi-mentally described its native RNAi features.

Results

Genes encoding Dicer family and double-strandedRNA-binding motif proteins

Dicer ribonucleases process dsRNA to initiate gene silenc-ing, and the Drosha ribonuclease is involved in the produc-tion of mature miRNA from miRNA precursors (Lee et al.,2003, 2004). A BLAST search of the BPH genomic andtranscriptomal database revealed one drosha and twodicer genes in the BPH genome, tentatively designatedNl-drosha, Nl-dcr1 and Nl-dcr2, respectively. The openreading frames (ORFs) of Nl-dcr1, Nl-dcr2 and Nl-droshaare 5004, 4347 and 3003 nucleotides (nt) in length, whichencode 1667, 1448, and 1000 amino acid (aa) proteins(Table S1), respectively. A ScanProsite search revealedthat Nl-Dcr1 was more similar to Dm-Dcr1 in domain archi-tecture than to Ce-Dcr1 or Tc-Dcr1 because both Nl-Dcr1and Dm-Dcr1 lack one helicase domain (Fig. 1A); however,

Figure 1. Protein domain comparison and phylogenetic analysis of the Dicer protein family and its cofactors. A, Domain architecture of Dicer1 andDicer2 proteins predicted by SCANPROSITE. Nl-Dcr1 showed more similar domain architecture to Dm-Dcr1 than to Tc-Dcr1, and both Dm-Dcr1 andNl-Dcr1 lacked a helicase domain. Nl-Dcr2 was similar to Tc-Dcr2, and both of them lacked the dsRNA-binding domain (dsRBD). B, A maximumlikelihood phylogenetic tree was created using the full-length sequences of Dicer and Drosha proteins. Three groups were formed: Dicer1, Dicer2, andDrosha. Nl-Dcr1, Nl-Dcr2 and Nl-Drosha formed monophyletic groups with Tribolium orthologues, respectively. C, A phylogenetic tree was created usingthe full-length sequences of Dicer cofactor proteins. Nl-Loqs, Nl-R2D2, and Nl-Pasha clustered together with orthologs from Tribolium. Ce:Caenorhabditis elegans; Dm: Drosophila melanogaster; Nl: Nilaparvata lugens; Tc: Tribolium castaneum.

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a phylogenetic analysis based on the full-length proteinsequences demonstrated that Nl-Dcr1 was monophyleticwith Tc-Dcr1 and not Dm-Dcr1 (Fig. 1B). In contrast toNl-Dcr1, Nl-Dcr2 lacked the dsRNA-binding domain(dsRBD) and was more similar to Tc-Dcr2 than to eitherDm-Dcr2 or Ce-Dcr1 based on domain architecture(Fig. 1A) and phylogenetic analyses (Fig. 1B). Thephylogenetic analysis also showed that all Drosha proteinsformed a distinct clade, in contrast to Dcr1 and Dcr2, whichcomposed a larger group, and that Nl-Drosha was clus-tered with Tc-Drosha (Fig. 1B).

The Drosophila species contains three classes of Dicercofactors, Loquacious (Loqs), R2D2 and Pasha, whichfunction with Dcr-1, Dcr-2, and Drosha, respectively. Bysearching the genome and transcriptome database ofBPH, we identified one orthologue of Loquacious (Nl-loqs),one orthologue of R2D2 (Nl-r2d2) and one orthologue ofPasha (Nl-pasha). The ORFs of Nl-loqs, Nl-r2d2, andNl-pasha are 1386-, 1008-, and 2541-nt in length, whichencode 461-, 334-, and 846-aa proteins (Tale S1), respec-tively. These cofactors of the Dicer protein family werefound to be most similar to the orthologues of Triboliumwhen aligned with Drosophila and C. elegans (Fig. 1C).

Argonaute proteins in small interfering RNA andmicro-RNA pathways

The Argonaute proteins are key components of the RISC,characterized by PAZ and PIWI domains which functionin recognizing and binding small RNAs and supportingendonucleolytic cleavage (Lingel et al., 2003; Liu et al.,2004; Martinez & Tuschl, 2004; Song et al., 2004; Maet al., 2005; Parker et al., 2005; Rivas et al., 2005).

In BPH, two clear orthologues of Drosophila Argonautegenes, Nl-ago1 and Nl-ago2, were identified, which wereinvolved in miRNA and siRNA pathways, respectively. TheORFs of Nl-ago1 and Nl-ago2, are 2916 and 2481 nt inlength, and encode 971 and 826 aa proteins (Table S1),respectively. The BPH Ago family exhibited conservationof key metal coordination residues, Aspartic acid (D),Aspartic acid (D) and Histidine (H), in the PIWI domain(Fig. 2A). A phylogenetic analysis clearly demonstrated

that the Argonautes composed two subgroups that weredefined based on their silencing processes: siRNA andmiRNA Argonautes (Fig. 2B).

Genes encoding Eri-1-like protein

In C. elegans, several Eri proteins such as Eri-1 and Eri-6/7 negatively regulate the RNAi pathway (Kennedy et al.,2004; Fisher et al., 2008). By searching the BPH genomeand transcriptome database, we identified two eri1-likegenes, Nl-eri-1a and Nl-eri-1b, which are 771 and 2106 ntin length, and encode 256 and 701 aa proteins (Table S1),respectively; however, consistent with the Eri-1 ortholo-gues from Drosophila and Tribolium (Tomoyasu et al.,2008), Nl-Eri1 protein also lacks a SAP domain.

To investigate whether the two Eri-1-like proteinswere involved in RNAi resistance in neural tissues, weexamined the silencing effect by using dsRNA target-ing acetylcholinesterase 1 (ace1), which encodes a keyenzyme in the cholinergic system that terminates nerveimpulses by catalysing the hydrolysis of the neurotrans-mitter acetylcholine in the synaptic cleft. Five groups ofthird instar nymphs were micro-injected with dsRNAstargeting gfp, ace1, eri-1a and ace1, eri-2 and ace1, aswell as triple mixtures of eri-1a, eri-1b and ace1 genes(i.e. dsGfp, dsAce1, dsEri-1a+dsAce1, dsEri-1b+dsAce1,and dsEri-1a+dsEri-1b+dsAce1), respectively. Three daysafter injection, quantitative real-time (qRT)-PCR wascarried out to analyse the effect of dsRNA-mediatedsilencing of transcripts for each gene. In each case, theamount of targeted transcript level was greatly reduced(Fig. 3). Notably, the dsAce1 efficiently silenced the ace1gene despite the existence of functional eri-1 genes, indi-cating the feasibility of RNAi in BPH neural tissues.

A sid1-like gene

We identified a sid-1-like gene in the BPH genome whichencodes a 763-aa protein containing an amino-terminalextracellular domain followed by 11 transmembranedomains, a feature also found in its C. elegans orthologue(Winston et al., 2002). A phylogenetic analysis based on

Figure 2. Alignment and phylogenetic analysis of Argonaute proteins. A, Alignment of conserved key metal coordination residues in the PIWI domain.Conserved residues (Aspartic acid (D), Aspartic acid (D) and Histidine (H)) were indicated by stars. Argonaute proteins identified in the brownplanthopper genome were indicated by triangles. B, A phylogenetic tree was created using the full-length sequences of Argonaute proteins. Basically,Argonautes were subclassed into micro-RNA and small interfering RNA groups according to their associated silencing processes.

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the conserved transmembrane regions at the carboxylterminus of Sid-1 clearly demonstrated that the Sid-1 pro-teins could be subclassed into three groups (Fig. 4):Lepidoptera, Hymenoptera and Hemiptera/Coleoptera.C. elegans and Tribolium each carry three sid-1-likegenes; however, no orthologue has been found inDrosophila (Tomoyasu et al., 2008), indicating that thelack of systemic RNAi in Drosophila is at least in partattributable to the absence of sid-1. It is worthy to note thatone of the Tribolium sid-1 genes (Tc-silC) was closer to theLepidoptera clade, which probably diverged from two

other genes (Tc-silA and Tc-silB). In addition, Ce-taq130was more similar to the Hemiptera/Coleoptera group thanthe outgroup that included Ce-sil and Ce-Y37H2C.1(Fig. 4).

Parental RNA interference in Nilaparvata lugens

Given that the BPH possesses all the core genes requiredfor siRNA and miRNA pathways, and exhibits high sensi-tivity of RNAi, we next examined whether parental RNAioccurs in this species. In the present study, we used theDistal-less (dll) gene, a homeodomain transcription factorthat plays a major role in establishing distal limb struc-tures, as a molecular marker to answer this question.

We identified a dll orthologue in the BPH genome (Nl-dll) and injected the naïve third instar BPH with dsDll toobserve any deleterious phenotypes caused by nymphalRNAi. At 4 days after micro-injection, the dll transcriptsdecreased to 20% compared with the dsGfp group(Fig. 5A). The injected third instar nymphs were able tomoult into fifth instar, but most of them (∼80%, n = 50)showed a strong RNAi defect, reflected by the absence ofclaw in all six legs in each individual (Fig. 5B, C and D),and they died before transition into adults because ofan incapacity to climb onto the rice stem. The remainingnymphs with moderate defects were successfully moultedinto adults, with only one or two legs with missed claws.By contrast, a normal leg structure was observed in thecontrol group that was injected with dsGfp.

In the parental RNAi experiments, we injected naïvefifth instar BPH with dsDll, and the metamorphosed adultswere allowed to mate to produce offspring. Most of thethird instar nymphs from the next generation were mor-phologically similar to controls; however, some third instarnymphs (∼5%, n = 100) displayed a mild defect in clawformation (Fig. 6A), and occasionally a strong defect wasalso observed in adults (Fig. 6B). By contrast, the groupswith dsGfp injection exhibited normal leg development(Fig. 6C and D). The above results strongly indicate thatparental RNAi might occur in the BPH.

Figure 3. Double-stranded RNA (dsRNA) targetingacetylcholinesterase 1 (ace1) in the brownplanthopper. Five groups of third instar nymphs weremicro-injected with gfp, ace1, eri-1a and ace1, eri-2and ace1, and eri-1a, eri-1b and ace1 dsRNA (i.e.dsGfp, dsAce1, dsEri-1a+dsAce1, dsEri-1b+dsAce1,dsEri-1a+dsEri-1b+dsAce1), respectively. At 3 daysafter injection, quantitative real-time PCR was carriedout to analyse the effect of dsRNA-mediatedsilencing of transcripts for each gene. White column:dsGfp nymphs; Black column: dsAce1 nymphs;Column filled with dashed lines: dsEri-1a+dsAce1nymphs; Grey column: dsEri-1b+dsAce1 nymphs;Column filled with grid: dsEri-1a+ dsEri-1b+ dsAce1nymphs.

Figure 4. Phylogenetic analysis of Sid-1 proteins. A maximum likelihoodtree was created using the alignment of conserved carboxyl terminaltransmembrane regions. Sid-1-like proteins were identified inLepidoptera, Coleoptera/Hemiptera, and Hymenoptera insects. Af: Apisflorae; Ag: Aphis gossypii; Am: Apis mellifera; Ap: Acyrthosiphon pisum;Bi: Bombus impatiens; Bt: Bombus terrestris; Cf: Camponotus floridanus;Dp: Danaus plexippus; Nv: Nasonia vitripennis; Mr: Megachile rotundata;Phc: Pediculus humanus corporis.

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Small interfering RNA pathway and Sid-1 are requiredfor RNA interference

To assess whether the siRNA pathway is functional inBPH, two groups of third instar nymphs were micro-injected with dsAgo2 and dsDll (dsAgo2+dsDll), anddsGfp and dsDll (dsGfp+dsDll), respectively. Six daysafter injection, 29 out of 30 nymphs in dsAgo2+dsDllgroup displayed normal claw structure, while defects wereobserved in all the nymphs (n = 30) in the dsGfp+dsDllgroup (Fig. 7). This result indicated that gene silencingtriggered by exogenous dsRNA was mediated by thesiRNA pathway in BPH.

To assess whether Sid-1 is required for systemic RNAi,two groups of third instar nymphs were micro-injected withdsSid1 and dsDll (dsSid1+dsDll), and dsGfp and dsDll(dsGfp+dsDll), respectively. Three days after injection, theSid1 transcripts were greatly reduced in the dsSid1+dsDllgroup when compared with the dsGfp+dsDll group(Fig. 8A). Six days after injection, only three out of 33nymphs in the dsSid1+dsDll group displayed mild defects inclaw formation (Fig. 8C), while the remaining 30 nymphsshowed normal claw structure (Fig. 8B). This result indi-cated that Sid-1 is necessary for systemic RNAi in BPH.

Micro-RNA pathway is involved in brownplanthopper metamorphosis

Since accumulated data have documented that smallRNAs are involved in many biological processes, we won-dered whether they might be also play a role in BPHdevelopment. The third instar nymphs were injected witheither dsDcr1 or dsAgo1 (∼50 ng/nymph). Our results

demonstrated that the transcriptional level of dcr1 andago1 decreased significantly and all the nymphs in thedsDcr1 or dsAgo1 group died within 10 days (Fig. S1).Examination of dead nymphs revealed that either dsDcr1or dsAgo1 nymphs failed to extricate their old cuticle, andeventually died without completing ecdysis (Fig. 9A, C). Inthe control group of dsGfp injection, most nymphs suc-cessfully moulted into normal adults (Fig. 9F), and moult-ing incapability was apparently not the direct reason forthe dead nymphs (Fig. 9E).

To investigate whether BPH with miRNA pathway deple-tion could complete the nymph-adult transformation, thepenultimate nymphs (fourth instar, day one) were injectedwith either dsDcr1 or dsAgo1. In either group, ∼75%nymphs (n = 80) died before eclosion because of an inca-pability to extricate the old cuticle. The remaining 25%successfully shed their exuviae and finished nymph-adulttransformation; however, ∼80% of the surviving insectswere unable to stretch their wings well (Fig 9B, D). Accord-ingly, our data strongly indicated that the miRNA pathwaymight play a vital role in BPH metamorphosis.

Discussion

In the present study, we took advantage of the newlyassembled BPH transcriptome (Xue et al., 2010) andgenome data to comprehensively investigate the corecomponents of the siRNA and miRNA pathways in BPH.We identified three genes of the Dicer family (Nl-dcr1,Nl-dcr2 and Nl-drosha), three genes encoding dsRNA-binding motif proteins (Nl-loqs, Nl-r2d2 and NL-pasha),two Argonaute genes (Nl-ago1 and Nl-ago2), two Eri-1-like genes (Nl-eri-1a and Nl-eri-1b), and a Sid-1-like gene

Figure 5. Strong nymphal RNA interference (RNAi) effect in the brown planthopper (BPH). A, Double-stranded RNA (dsRNA)-targeting Nl-dll wasinjected into third instar BPH, the relative dll transcripts were examined at 4 days after injection. The BPH moulted to fifth instar, and most of themexerted strong RNAi toxicity, reflected by all six legs of BPH failing to develop a claw (B, C and D). Standard bar represents three independent repeats.Arrow heads indicate the claw position.

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(Nl-sid1) in the BPH genome through a comparison withorthologues from Drosophila, Tribolium and C. elegans.Based on domain architectures and phylogenetic analy-sis, we found that these core genes have a closer rela-tionship with Tribolium than with Drosophila, which mightindicate that BPH has high RNAi sensitivity as inTribolium.

Compared with orthologues from Tribolium, Drosophilaand C. elegans, the core components of the BPH RNAipathway showed some unique features. Firstly, domainanalysis reveals that a helicase domain and a dsRBD aremissing at the amino terminus of Nl-Dcr1 and carboxy-terminus of NL-Dcr2, respectively. Drosophila andTribolium possess two Dicer genes, Dm-dcr1 and Dm-dcr2,which function in miRNA and siRNA pathways, respec-tively. Notably, only one Dicer gene was identified inC. elegans that was involved in both miRNA and siRNApathways (Knight & Bass, 2001; Duchaine et al., 2006).These Dicer proteins are characterized by a typical domainorganization that includes amino-terminal DExH-Box

helicase domain(s), a PAZ (Piwi/Argonaute/Zwille) domain,tandem RNase III domains and a carboxy-terminal dsRBD(Carmell & Hannon, 2004); however, only one helicasedomain was predicted in the BPH Dicer1 in contrast totwo that exist in orthologues of Tribolium and C. elegans(Fig. 1A). We initially suspected that this was a truncatedversion of Nl-Dcr1 in the BPH genome, but 5′ rapid ampli-fication of cDNA ends experiments demonstrated thatNl-Dcr1 contained only one helicase domain (data notshown). We searched public insect sequence databasesfor Dicer1 proteins and found that all insects expressDicer1 proteins that contain two helicase domainsexcept for Anopheles gambiae, Acyrthosiphon pisum andDanaus plexippus, in addition to BPH. RNAi is effectivein An. gambiae and BPH, suggesting that the number ofhelicase domains in Dicer proteins was not the determinantfactor for RNAi efficiency.Additionally, only one dsRBD wasfound in BPH Dicer2 (Fig. 1A), which is consistent with itbeing an orthologue of Tribolium but not Drosophila.Furthermore, BPH encodes two eri-1-like genes, in con-

Figure 6. Parental RNA interference of BPH. The Nl-dll dsRNA was injected into fifth instar BPH, which were then allowed to mate to produce offspring.The third instar nymphs (A) and adults (B) from the next generation were examined for morphology alternation. Control groups of third instar nymphs (C)and adults (D) were injected with dsGfp. Defective claws and normal claws are shown by arrows and arrowheads, respectively.

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trast to only one gene being found in Tribolium and C.elegans. Eri-1 is an evolutionarily conserved proteinbearing a DEDDh-like 3′ to 5′ exonuclease domain and aSAP/SAF-box domain. Eri-1 inhibits RNAi by degrading the3′ overhangs of siRNA, and the eri1 mutant increased RNAiin certain tissues, especially those of the nervous system(Kennedy et al., 2004). Recently, it has been shown thatEri-1 expression was up-regulated in mammals when theywere challenged with high doses of siRNA (Bian et al.,2011). The presence of two eri-1-like genes in BPH stronglyindicated that BPH is refractory to RNAi in the nervoussystem; however, our results indicated that eri-1-like genesin BPH played a minor role, if any, in RNAi in neural tissues(Fig. 3), and the precise function of Eri-1 in BPH needs tobe further characterized.

In contrast to 27 Argonaute genes in C. elegans, onlyfive Argonaute genes were identified in Drosophila andTribolium (Yigit et al., 2006; Tomoyasu et al., 2008). Dis-tinct Argonaute protein interacts with small RNAs in differ-ent RNAi pathways. Ago1 and Ago2 are involved inmiRNA and siRNA pathways (Okamura et al., 2004),respectively, whereas Ago3, Piwi and Aub are believed tobe associated with piRNA (Brennecke et al., 2007; Kimet al., 2009). As expected, the BPH contains Ago1 andAgo2 associated with miRNA and siRNA pathways,respectively. In addition to that, BPH also encodes Ago3,Piwi and Aub for piRNA pathway (Table S1). The piRNAacts as the primary defence in the germline against trans-posable element mobilization (Ghildiyal & Zamore, 2009),whether piRNA-mediated genes are involved in regulation

Figure 7. Knockdown of ago2 abolished the dsDll effect. Two groups of third instar nymphs were micro-injected with dsAgo2 and dsDll (dsAgo2+dsDll),and dsGfp and dsDll (dsGfp+dsDll), respectively. Six days after injection, nymphs were collected for claw examination under microscopy. A, Nymphsinjected with dsAgo2+dsDll displayed normal claw structure (shown by arrows). B, Nymphs injected with dsGfp+dsDll displayed claw defect (shown byarrowheads).

Figure 8. Knockdown of sid-1 abolished systemic RNAi in BPH. Two groups of 3rd instar nymphs were micro-injected with dsSid1 and dsDll(dsSid1+dsDll), and dsGfp and dsDll (dsGfp+dsDll), respectively. Three days after injection, nymphs (n = 20) were collected to examine the sid-1transcripts (A). Six days after injection, 30 out of 33 nymphs showed normal claw structure (B, shown in arrows) and 3 out of 33 nymphs displayed amild defect in claw formation (C, shown in arrow heads).

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Figure 9. Deleption of dcr1 or ago1 affected ecdysis and wing stretch. A and B, the third instar nymphs (n = 60) were injected with either dsDcr1 ordsAgo1. Most of the nymphs died as a result of an inability to extricate their cuticle in both dsDcr1 (A) and dsAgo1 (B) groups. C and D, the fourth instarnymphs (n = 80) were injected with either dsDcr1 or dsAgo1. A total of 25% of the insects successfully completed nymph-adult transformation, but 80%of the survivors did not stretch their wings well in either the dsDcr1 (C) or dsAgo1 (D) group. E and F, the third instar nymphs (n = 60) were injected withdsGfp, no moulting inability was observed in dead nymphs (E), and the majority of nymphs successfully moulted into adults (F).

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for other tissue development in BPH is still under investi-gation. Additionally, no secondary Argonaute proteinswere found when BLAST was used to search the BPHgenome and transcriptome for the C. elegans secondaryAgo family, e.g. Ppw1, Ppw2, Ce-Sago and Ce-Ppw.

Systemic RNAi requires silencing signals to cross cellu-lar boundaries, and studies on systemic RNAi mechanismssuggest that transmembrane channel-mediated andendocytosis-mediated dsRNA uptake mechanismsmay play a role (Sijen et al., 2001; Saleh et al., 2006). InC. elegans, a mutant of the transmembrane channel genesid-1 resulted in complete inhibition of systemic RNAi(Winston et al., 2002; Feinberg & Hunter, 2003; Shih &Hunter, 2011), implying that this gene might play a key roleduring this process. BPH contains one sid-1-like gene whilethree genes were encoded by Tribolium and C. elegans, butnone was identified in Drosophila. By searching for insectSid-1 orthologues in public sequence databases, weidentified Sid-1-like proteins in ants (Acromyrmexechinatior, Harpegnathos saltator, Camponotus floridanusand Solenopsis invicta), bees (Apis mellifera, Bombusterrestris, Bombus impatiens and Megachile rotundata),aphids (Aphis gossypii and Acyrthosiphon pisum), wasps(Nasonia vitripennis), body louses (Pediculus humanuscorporis), butterflies (Danaus Plexippus) and silkworm(Bombyx mori) in addition to Tribolium and BPH. However,this hypothesis is not supported in Lepidoptera speciessuch as Bombyx mori, which contain three sid-1-like genesbut systemic RNAi is much less robust than Tribolium(Terenius et al., 2011). The results of the present studystrongly indicated that Sid-1 was devoted to systemic RNAiin BPH because nymphs with sid-1 knockdown failed toefficiently silence a test target gene (Fig. 8B). The neces-sity of Nl-Sid-1 for systemic RNAi in BPH contradictsphylogenetic analysis, which showed that the Nl-Sid-1was more closely related to CeTag130 (Fig. 4). SinceCeTaq130 is not necessary for the systemic RNAiresponse in C. elegans (Tomoyasu et al., 2008), themechanism for such discrepancy needs to be further inves-tigated. Currently, we are constructing a transgenicLepidoptera cell line (Sf9) by integrating the Nl-sid-1 geneinto its genome to investigate whether the NL-Sid-1improves the RNAi efficiency of Lepidoptera cell line or not.

In addition, Saleh et al. (2006) proposed that dsRNA istaken up by an active process involving receptor-mediatedendocytosis. The authors screened a subset of 23 genesof the endocytotic pathway for RNAi silencing (Saleh et al.2006). We also identified all these 23 orthologues in theBPH genome (Table S2), indicating that these compo-nents are highly conserved in insects, but species-specificdifferences in their ability to take up dsRNA should befurther studied.

Although RNAi was extensively used to determine genefunction in BPH, parental RNAi in this species has not yet

reported. We recruited a molecular marker, Distal-less (dll)gene, to test whether parental RNAi occurs in this species.In Drosophila, Dll appears to have a critical role in thedevelopment of ventral appendages, legs and antenna,which failed to develop in the absence of Dll activity(Cohen et al., 1989; Cohen & Jürgens, 1989). In addition,Dll is involved in the differentiation of the wing marginpattern (Gorfinkiel et al., 1997). In Tribolium, mutations indll result in defective and truncated legs and headappendages (Beermann et al., 2001). The defect was alsoobtained in parental RNAi experiments when pupae wereinjected with dsRNA targeting dll (Bucher et al., 2002). Inthe present study, we experimentally proved that parentalRNAi occurred in BPH, although its efficiency is muchlower than nymphal RNAi. For nymphal RNAi, almost allthe nymphs were susceptible to RNAi and severe defectsin claw formation were found (Fig. 5B, C, and D). Bycontrast, a small number of nymphs were observed withmild defects (Fig. 6A) in parental RNAi. It is worth notingthat we didn’t observe any morphological defects either onwing or antenna when BPH was challenged with dsDll. Inaddition, the fifth instar BPH with dsDll injection wascapable of producing eggs, which subsequently hatchedinto nymphs, although the embryonic lethality was notexamined in this study. Accordingly, we speculate that thesensitive tissue responding to exogenous dsRNA varies ininsects.

Multiple studies have shown that miRNA plays wide-spread roles in an organism’s development. For instancein Drosophila, the mutation of Dicer1 gene blocks theprocessing of miRNA precursors, and results in translationrepression (Lee et al., 2004). Whereas in the cockroach(Blattella germanica), silencing the expression of Dicer1inhibited metamorphosis, and the last instar nymphs wereable to moult again (Gomez-Orte & Belles, 2009). Toexplore the function of the miRNA pathway in the devel-opment of BPH, we injected the third and fourth instarnymphs with either dsDcr1 or dsAgo1. Most of the nymphscould not complete the ecdysis and died before eclosion,and the survivors didn’t stretch their wings well, which is inline with the phenomenon observed in the cockroach(Gomez-Orte & Belles, 2009); however, the mechanism(s)of BPH metamorphosis regulated by miRNA is stillunknown.

The BPH N. lugens causes serious yield losses world-wide, especially in Asia, but effective approaches arecurrently not available for controlling this pest. The multi-ple successful RNAi experiments that have been con-ducted recently may help provide a promising method forthe control of this pest in the field. We hope that thenewly assembled BPH genome will not only deepen ourunderstanding of the RNAi mechanism, but will alsoprovide the potential to control BPH in agriculturalsettings.

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Experimental procedures

Insects

The N. lugens colony was initially obtained from the ZhejiangUniversity Farm (Hangzhou, China), and was maintained in awalk-in chamber at 25 (± 1 °C) under a photoperiod of 16 hlight:8 h dark at a relative humidity of 60% (± 5%) on rice seed-lings. The same colony was used for genome sequencing andtranscriptome sequencing. A pooled RNA sample including differ-ent developmental stages, sexes and wing forms (eggs, secondinstar nymphs, fifth instar nymphs, brachypterous female adults,macropterous female adults and macropterous male adults) wasused for transcriptome analysis (Xue et al., 2010).

Bioinformatic analysis

The amino acid sequences of RNAi components from Drosophila,Tribolium or C. elegans were used as query sequences to searchsequences in the BPH transcriptome and genome database(unpublished) with TBLASTN using a cut-off E value of 10−5. Thededuced amino acid sequences were aligned with CLUSTALX andedited with GeneDoc software. MotifScan was used to predictmotifs on the website (http://prosite.expasy.org/). A phylogenetictree was constructed using a maximum likelihood method withMolecular Evolutionary Genetic Analysis software (MEGA, ver.5.1). The Transmembrane prediction was done with TMHMM(ver. 2.0) on the website (http://www.cbs.dtu.dk/services/TMHMM/).

dsDll synthesis and injection

Total RNA was extracted from BPH nymphs with Trizol(Invitrogen, Shanghai, China), the cDNA was synthesized withrandom primers (Takara, Dalian, China), and subsequently usedfor gene amplification. The dll gene fragment was amplified withprimers BPH-dll-F and BPH-dll-R (Table S3), and subsequentlyused as dsRNA template. The amplified dll gene fragment wasconfirmed by sequencing.

The dsRNA was synthesized using a MEGAscript T7 High YieldTranscription Kit (Ambion, Shanghai, China). The primers usedfor synthesizing dsRNA were BPH-dll-RNAi-F and BPH-dll-RNAi-R (Table S3), containing a T7 RdRP promoter. We used themicro-injection process described in our previous report (Wanget al., 2012). Groups of 50 third instar (day 1) or fifth instar (day 4)nymphs were injected with 50 ng and 150 ng dsRNA, respec-tively. For the third instar group, the insects were allowed to moultto adults and then we observed their morphology alternationunder microscopy. For the fifth instar group, the insects wereallowed to mate to produce offspring and then the third instarnymphs and adults from the next generation were examined formorphology alternation. In the control group, dsGfp was synthe-sized with gfp-RNAi-F and gfp-RNAi-R (Table S3), and then usedfor injection.

RNA interference targeting dcr1 and ago1

The dcr1 and ago1 gene fragments were amplified from BPHcDNA with the primer pairs BPH-Dicer1-F/BPH-Dicer1-R andBPH-ago1-F/ BPH-ago1-R, respectively. All the cloned geneswere confirmed by sequencing, and then subsequently used as

template for dsRNA synthesis. The primer pairs BPH-Dicer1-RNAi-F/BPH-Dicer1-RNAi-R and BPH-ago1-RNAi-F/BPH-ago1-RNAi-R correspond to dcr1, and ago1 for dsRNA synthesis,respectively. All the above primers are listed in Table S3.

For microinjection, groups of third instar nymphs (n = 60) wereinjected with 50 ng dsRNA each. The nymphs were reared in jarswith plenty of food, and mortality was monitored every 2 days.

RNA interference targeting ago2, sid-1 and dll

The sid-1 gene fragment was amplified from BPH cDNA with theprimer pair BPH-Sid-1-F/BPH-Sid-1-R (Table S3). The clonedgenes were confirmed by sequencing, and then subsequentlyused as template for dsRNA synthesis. The primer pairs BPH-Sid1-RNAi-F/BPH-Sid-RNAi-R and QBPH-Sid1-F/QBPH-Sid1-R(Table S3) were used for dsSid1 synthesis and quantitative real-time PCR (qRT-PCR) analysis, respectively.

To assess the function of Ago2 and Sid-1 in RNAi process,three groups of third instar nymphs (n = 80) were injected at adose of 50 ng dsRNA mixtures of dsAgo2 (25 ng) and dsDll(25 ng), dsSid1 (25 ng) and dsDll (25 ng), and dsGfp (25 ng) anddsDll (25 ng) for each nymph, respectively. Three days later, 20nymphs were collected for determining sid1 and ago2 transcrip-tional level by qRT-PCR, and the remaining nymphs were con-tinually reared for claw examination under microscopy 6 dayslater.

RNA interference targeting ace1, eri-1a, and eri-1b

Templates for eri-1a and eri-1b in vitro transcription reactionswere prepared by PCR amplification from BPH cDNA with theprimer pair BPH-Eri-1a-F/BPH-Eri-1a-R, BPH-Eri-1b-F/BPH-Eri-1b-R, respectively. The PCR products were subcloned and con-firmed by sequencing. The cloning vector containing the ORFregion of acetylcholinesterase 1 (ace1) gene was stored in ourlaboratory (Li et al., 2012). dsEri-1a, dsEri-1b and dsAce1 weresynthesized with the primer pairs BPH-Eri-1a-RNAi-F/BPH-Eri-1a-RNAi-R, BPH-Eri-1b-RNAi-F/BPH-Eri-1b-RNAi-R and BPH-Ace1-RNAi-F/BPH-Ace1-RNAi-R, respectively. All the relatedprimers are listed in Table S3.

Five groups of third instar nymphs (n = 60) were injected withdsGfp or dsAce1 at a dose of 50 ng dsRNA for each nymph. Fordouble or triple dsRNA injection, the third nymphs were injectedwith dsRNA mixtures of dsEri-1a (25 ng) and dsAce1 (25 ng), anddsEri-1b (25 ng) and dsAce1 (25 ng) as well as triple mixtures ofdsEri-1a (16.7ng), dsEri-1b (16.7ng) and dsAce1 (16.7 ng) foreach nymph, respectively. Three days later, 20 nymphs werecollected for determining ace1, eri-1a and eri-1b transcriptionallevel by qRT-PCR.

Quantitative real-time PCR analysis

To examine the RNAi effect by qRT-PCR, specific primers foreach target genes were designed using Primer 3 software (http://frodo.wi.mit.edu/primer3/input.htm). The qRT-PCR primer pairsfor dll, dcr1 and ago1 were QBPH-dll-F/QBPH-dll-R, QBPH-dcr1-F/QBPH-dcr1-R and QBPH-ago1-F/QBPH-ago1-R (Table S3),respectively. RNA was extracted from BPH at 4 days after dsRNAinjection, and treated with DNase (Promega, Shanghai, China) toremove any genomic DNA contamination. The cDNA synthesizedwith random primers was used as template for qRT-PCR (ABI

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7300). The ribosomal 18S rRNA was used for normalization. Foreach reaction, 0.4 μl of each primer (10 μM), 0.4μl Rox referenceDys and 10 SYBR Primer Ex Taq were added into a total volumeof 20μl. The PCR procedure was 95 °C for 30 s, followed by 40cycles of at 95 °C for 5 s and 60 °C for 30 s.

Statistical methods

For mortality calculation, a group of 60 heads of BPH wereconsidered a replicate. ANOVA was performed using SPSS (v13)(Tang & Zhang, 2013). Means were compared using Tukey’s testwith a significance level of 0.05.

Acknowledgements

This work was supported by National Basic ResearchProgram of China (973 Program, no. 2010CB126205) andby the National Science Foundation of China (31201509).The authors declare that they have no competing interests.

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Supporting Information

Additional Supporting Information may be found in theonline version of this article at the publisher’s web-site:

Figure S1. The mortality of BPH with dsDcr1 and dsAgo1 treatment. Thethird instar nymphs were injected with dsRNA at dose of 50 ng eachnymph. At 4 days after injection, the relative transcripts of target geneswere examined by quantitative real-time PCR (A). Mortality was calculatedevery two days (B). The third instar nymphs were injected either withdsGfp, PBS, or non-injection (Wt), and mortality was monitored every 2days. Standard bars represent three independent repeats. Significant dif-ference was examined by Tukey test (P < 0.05).

Table S1. Core RNAi components in BPH.

Table S2. Candidate genes for dsRNA uptake in BPH.

Table S3. Primers used in this study.

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