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Using transcriptome analysis of tissues of the brown planthopper (BPH), Nilaparvata lugens, we identified a gene tentatively designated NlCP21. 92 that was expressed at high levels in the integument . Spatiotemporal expression profiling with quantitative PCR and Western blotting verified its integument-specific expression and showed periodic expression during molting. The open reading frame was GC-rich and encoded a hydrophobic polypeptide . The polypeptide contained AAPA/V repeat motifs and other sequence features similar to several reported cuticular proteins but lacked an R&R consensus and other chitin-binding domains. Double-stranded RNA–mediated RNA interference of the NlCP21.92 resulted in abnormal and lethal morphological phenotypes, and transmission electron microscopy revealed the corresponding ultrastructural defects. Immunohistochemical staining demonstrated that the NlCP21.92 protein was primarily located in the procuticle. Our results suggest that NlCP21.92 is a novel ungrouped cuticular protein essential for normal endocuticle formation.

In insect eggs, the chorion has the essential function of protecting the embryo from external agents during development while allowing gas exchange for respiration. In this study, we found a novel gene, Nilaparvata lugens chorion protein (NlChP), that is involved in chorion formation in the brown planthopper, Nilaparvata lugens. NlChP was highly expressed in the follicular cells of female adult brown planthoppers. Knockdown of NlChP resulted in oocyte malformation and the inability to perform oviposition, and electron microscopy showed that the malformed oocytes had thin and rough endochorion layers compared to the control group. Liquid chromatography with tandem mass spectrometry analysis of the eggshell components revealed four unique peptides that were matched to NlChP. Our results demonstrate that NlChP is a novel chorion protein essential for egg maturation in N. lugens, a hemipteran insect with telotrophic meroistic ovaries. NlChP may be a potential target in RNA interference-based insect pest management.

Significance The cuticle, mainly composed of chitin and cuticular proteins (CPs), is a multifunctional structure of arthropods. CPs usually account for >1% of the total insect proteins encoded in the genome. Why does an insect need so many different CPs? In this study, we use comprehensive large-scale technologies to study the full complement of CPs and their functions in the brown planthopper (BPH). A total of 32 of the 140 BPH CP genes are found to be essential for nymph/adult development, egg production, or embryo development; in addition, redundant and complementary functions of CPs are revealed., Cuticle, mainly composed of chitin and cuticular proteins (CPs), is a multifunctional structure of arthropods. CPs usually account for >1% of the total insect proteins. Why does an insect encode so many different CP genes in the genome? In this study, we use comprehensive large-scale technologies to study the full complement of CPs (i.e., the CP-ome) of the brown planthopper (BPH), Nilaparvata lugens, a major rice plant pest. Eight CP families (CPR, CPF, TWDL, CPLCP, CPG, CPAP1, CPAP3, and CPAPn) including 140 proteins in BPH, in which CPAPn is a CP family that we discovered. The CPG family that was considered to be restricted to the Lepidoptera has also been identified in BPH. As reported here, CPLCP family members are characterized by three conserved sequence motifs. In addition, we identified a testis protein family with a peritrophin A domain that we named TPAP. We authenticated the real existence of 106 proteins among the 140 CPs. RNA interference (RNAi) experiments were conducted against 135 CP genes in early- and late-instar nymphs and newly emerged female adults, demonstrating that 32 CPs were essential for BPH normal development or egg production. Combined RNAi experiments suggested redundant and complementary functions of the large number of CPs. Transcriptomic data revealed that the CP genes were expressed in a tissue-specific manner, and there were four clusters of developmental expression patterns. This study gives a comprehensive understanding of the roles of CPs in an insect cuticle.

β-N-Acetylhexosaminidases (HEXs) are enzymes that can degrade the chitin oligosaccharides that are produced by the activity of chitinases on chitin in insects. Using bioinformatic methods based on genome and transcriptome databases, 11 β-N-acetylhexosaminidase genes (NlHexs) in Nilaparvata lugens were identified and characterized. Phylogenetic analysis revealed a six-grouped tree topology. The O-Linked N-acetylglucosaminidase (O-GlcNAcase) group includes NlHex11, which harbours a catalytic domain that differs from that of the other 10 NlHexs. Observations of the expression of NlHexs during different developmental stages revealed that NlHex4 is expressed with periodicity during moulting. Although the tissue-specific expression patterns of most NlHexs were nonspecific, NlHex4 was found to be expressed mainly in the female reproductive system as well as in the integument. RNA interference (RNAi) demonstrated failure to shed the old cuticle only in the nymphs treated with double-stranded RNA (dsRNA) targeting NlHex4, and these nymphs eventually died; no observable morphological abnormalities were found in insects treated with dsRNAs targeting the other 10 NlHexs. Based on this study and our previous analyses, a '5 + 1 + 3' pattern of chitinolytic enzymes is proposed, in which five chitinases, one NlHEX and three chitin deacetylases are required for moulting in N. lugens. A better understanding of chitin metabolism in the hemimetabolous insect, N. lugens, would be achieved by considering three chitinolytic enzyme families: chitinase, chitin deacetylase and β-N-acetylhexosaminidase.

The multicopper oxidase (MCO) family of enzymes includes laccases, ascorbate oxidases, bilirubin oxidases and a subgroup of metal oxidases. On the basis of a bioinformatics investigation, we identified 7 genes encoding putative multicopper oxidase proteins in the genome of the brown planthopper (BPH), Nilaparvata lugens (Hemiptera: Delphacidae). MCO1 and MCO2 are conserved, while others diverse in insects. Analysis of developmental and tissue-specific expression patterns revealed the following: NlMCO2 was mainly expressed in the integument, and its expression peaked periodically during molting; NlMCO3 was an ovary-specific MCO gene with a high expression level only at the adult stage; NlMCO4 was a salivary gland-specific MCO gene that was expressed at all developmental stages; NlMCO5 only had short-term expression in the middle of the fourth instar stage and was expressed mainly in the gut; NlMCO6 had a developmental expression pattern similar to that of NlMCO2 and was expressed in most N. lugens tissues; and NlMCO1 was expressed in most N. lugens tissues except for the testis, whereas NlMCO7 was mainly expressed in the gut and the Malpighian tube. BPHs injected with double-stranded RNA (dsRNA) targeting NlMCO2 failed to pigment and sclerotize, were colorless and soft-bodied and subsequently died in a short time. Lethal phenotypes were also observed in insects challenged by dsRNA targeting NlMCO6. However, no observable morphological or internal structural abnormality was obtained in the insects treated with dsRNA for NlMCO1, NlMCO3, NlMCO4, NlMCO5 or NlMCO7.

Wing polyphenism is an evolutionarily successful feature found in a wide range of insects. Long-winged morphs can fly, which allows them to escape adverse habitats and track changing resources, whereas short-winged morphs are flightless, but usually possess higher fecundity than the winged morphs. Studies on aphids, crickets and planthoppers have revealed that alternative wing morphs develop in response to various environmental cues, and that the response to these cues may be mediated by developmental hormones, although research in this area has yielded equivocal and conflicting results about exactly which hormones are involved. As it stands, the molecular mechanism underlying wing morph determination in insects has remained elusive. Here we show that two insulin receptors in the migratory brown planthopper Nilaparvata lugens, InR1 and InR2, have opposing roles in controlling long wing versus short wing development by regulating the activity of the forkhead transcription factor Foxo. InR1, acting via the phosphatidylinositol-3-OH kinase (PI(3)K)-protein kinase B (Akt) signalling cascade, leads to the long-winged morph if active and the short-winged morph if inactive. InR2, by contrast, functions as a negative regulator of the InR1-PI(3)K-Akt pathway: suppression of InR2 results in development of the long-winged morph. The brain-secreted ligand Ilp3 triggers development of long-winged morphs. Our findings provide the first evidence of a molecular basis for the regulation of wing polyphenism in insects, and they are also the first demonstration--to our knowledge--of binary control over alternative developmental outcomes, and thus deepen our understanding of the development and evolution of phenotypic plasticity.

Most animals are oviparous. However, the genes regulating egg shell formation remain not very clear. In this study, we found that Nilaparvata lugens Forkhead box transcription factor L2 ( Nl FoxL2) directly activated follicle cell protein 3C ( NlFcp3C ) to regulate chorion formation. NlFoxL2 and NlFcp3C had a similar expression pattern, both highly expressed in the follicular cells of female adults. Knockdown of NlFoxL2 or NlFcp3C also resulted in the same phenotypes: obesity and female infertility. RNA interference (RNAi) results suggested that NlFcp3C is a downstream gene of NlFoxL2 . Furthermore, transient expression showed that Nl FoxL2 could directly activate the NlFcp3C promoter. These results suggest that NlFcp3C is a direct target gene of Nl FoxL2. Depletion of NlFoxL2 or NlFcp3C prevented normal chorion formation. Our results first revealed the functions of Fcp3C and FoxL2 in regulation of oocyte maturation in an oviparous animal.

NlFoxL2 was aligned with FoxL2 orthologs from 16 other insect species using the ClustalX program.

Chitinases are important enzymes required for chitin degradation and reconstruction in insects. Based on a bioinformatics investigation, we identified 12 genes encoding putative chitinase-like proteins, including 10 chitinases (Cht), one imaginal disc growth factor (IDGF) and one endo-β-N-acetylglucosaminidase (ENGase) in the genome of the brown planthopper, Nilaparvata lugens (Hemiptera: Delphacidae). These 12 genes were clustered into nine different groups, with 11 in glycoside hydrolase family 18 groups (groups I-VIII) and one in the ENGase group. Developmental and tissue-specific expression pattern analysis revealed that the transcript levels of eight genes peaked periodically during moulting and were mainly expressed in the integument, except NlCht2, NlCht4, NlIDGF and NlENGase. NlCht2, NlIDGF and NlENGase were expressed at all stages with slight periodical changes and mainly expressed in the female reproductive organs in adults, whereas NlCht4 was highly expressed only at the adult stage in the male reproductive organs. Lethal phenotypes were observed in insects challenged by double-stranded RNAs for NlCht1, NlCht5, NlCht7, NlCht9 and NlCht10 during moulting, suggesting their significant roles in old cuticle degradation. NlCht1 was the most sensitive gene, inducing 50% mortality even at 0.01 ng per insect. Our results illustrate the structural and functional differences of chitinase-like family genes and provide potential targets for RNA interference-based rice planthopper management.

Chitin deacetylases (CDAs) are enzymes required for one of the pathways of chitin degradation, in which chitosan is produced by the deacetylation of chitin. Bioinformatic investigations with genomic and transcriptomic databases identified four genes encoding CDAs in Nilaparvata lugens (NlCDAs). Phylogenetic analysis showed that insect CDAs were clustered into five major groups. Group I, III and IV CDAs are found in all insect species, whereas the pupa-specific group II and gut-specific group V CDAs are not found in the plant-sap/blood-sucking hemimetabolous species from Hemiptera and Anoplura. The developmental and tissue-specific expression patterns of four NlCDAs revealed that NlCDA3 was a gut-specific CDA, with high expression at all developmental stages; NlCDA1, NlCDA2 and NlCDA4 were highly expressed in the integument and peaked periodically during every moulting, which suggests their roles in chitin turnover of the insect old cuticle. Lethal phenotypes of cuticle shedding failure and high mortality after the injection of double-stranded RNAs (dsRNAs) for NlCDA1, NlCDA2 and NlCDA4 provide further evidence for their functions associated with moulting. No observable morphological and internal structural abnormality was obtained in insects treated with dsRNA for gut-specific NlCDA3.

The brown planthopper (BPH), Nilaparvata lugens, is a major rice pest in Asia, and accumulated evidence indicates that this species is susceptible to RNA interference (RNAi); however, the mechanism underlying RNAi and parental RNAi has not yet been determined. We comprehensively investigated the repertoire of core genes involved in small interfering RNA (siRNA) and micro-RNA (miRNA) pathways in the BPH by comparing its newly assembled transcriptome and genome with those of Drosophila melanogaster, Tribolium castaneum and Caenorhabditis elegans. Our analysis showed that the BPH possesses one drosha and two Dicer (dcr) genes, three dsRNA-binding motif protein genes, two Argonaute (ago) genes, two Eri-1-like genes (eri-1), and a Sid-1-like gene (sid-1). Additionally, we report for first time that parental RNAi might occur in this species, and siRNA pathway and Sid-1 were required for high efficiency of systemic RNAi triggered by exogenous dsRNA. Furthermore, our results also demonstrated that the miRNA pathway was involved in BPH metamorphosis as depletion of the ago1 or dcr1 gene severely impaired ecdysis. The BPH might be a good model system to study the molecular mechanism of systemic RNAi in hemimetabolous insects, and RNAi has potential to be developed to control this pest in agricultural settings.

Background The brown planthopper, Nilaparvata lugens, the most destructive pest of rice, is a typical monophagous herbivore that feeds exclusively on rice sap, which migrates over long distances. Outbreaks of it have re-occurred approximately every three years in Asia. It has also been used as a model system for ecological studies and for developing effective pest management. To better understand how a monophagous sap-sucking arthropod herbivore has adapted to its exclusive host selection and to provide insights to improve pest control, we analyzed the genomes of the brown planthopper and its two endosymbionts. Results We describe the 1.14 gigabase planthopper draft genome and the genomes of two microbial endosymbionts that permit the planthopper to forage exclusively on rice fields. Only 40.8% of the 27,571 identified Nilaparvata protein coding genes have detectable shared homology with the proteomes of the other 14 arthropods included in this study, reflecting large-scale gene losses including in evolutionarily conserved gene families and biochemical pathways. These unique genomic features are functionally associated with the animal’s exclusive plant host selection. Genes missing from the insect in conserved biochemical pathways that are essential for its survival on the nutritionally imbalanced sap diet are present in the genomes of its microbial endosymbionts, which have evolved to complement the mutualistic nutritional needs of the host. Conclusions Our study reveals a series of complex adaptations of the brown planthopper involving a variety of biological processes, that result in its highly destructive impact on the exclusive host rice. All these findings highlight potential directions for effective pest control of the planthopper. Electronic supplementary material The online version of this article (doi:10.1186/s13059-014-0521-0) contains supplementary material, which is available to authorized users.