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Digestion and absorption of old cuticles during insect molting are necessary for new cuticle formation, during which complicated enzyme catalysis is essential. To date, a few carboxypeptidases, aminopeptidases and serine proteases (mostly trypsins) connected with cuticle digestion, zymogen activation and histological differentiation during the ecdysis of lepidopteran, dipteran and hymenopteran insects have been identified. However, little is known about these proteins in hemimetabolous insects. In this study, we identified 33 candidate trypsin and trypsin-like homologs, 14 metallocarboxypeptidase and 32 aminopeptidase genes in the brown planthopper Nilaparvata lugens, a hemipteran rice pest. Among the proteins encoded by these genes, 9 trypsin-like proteases, 3 metallocarboxypeptidases and 1 aminopeptidase were selected as potential procuticle hydrolases by bioinformatics analysis and in vivo validation. RNA interference targeting these genes demonstrated that 3 trypsin-like proteases (NlTrypsin-8, NlTrypsin-29 and NlTrypsin-32) genes and 1 metallocarboxypeptidase (NlCpB) gene were found to be essential for ecdysis in N. lugens; specifically, gene silencing led to incomplete cuticle degradation and arrested ecdysis, causing lethal morphological phenotype acquisition. Spatiotemporal expression profiling by quantitative PCR and western blotting revealed their specific expression in the integument and their periodic expression during each stadium, with a peak before ecdysis and eclosion. Transmission electron microscopy demonstrated corresponding ultrastructural defects after RNAi targeting, with NlCpB-silenced specimens having the most undigested old procuticles. Immunohistochemical staining revealed that NlTrypsin-8, NlTrypsin-29 and NlCpB were predominantly located in the exuvial space. This research further adds to our understanding of proteases and its potential role in insect ecdysis.

The oviduct serves as a delivery tube for mature eggs ovulated from ovaries to egg-laying sites. Oviduct secreted components play important roles in ovulation and fertilization in mammals, however, no oviduct secreted protein has been characterized in an insect to date. Here, we identified a gene highly expressed in the lateral oviduct of the adult females in the brown planthopper (BPH), Nilaparvata lugens, the most destructive rice insect pest. Western blotting and immunofluorescence analyses revealed that the gene encodes a protein that is specifically expressed in the lateral oviduct as a component of the gel-like material secreted by the oviduct epithelial cells into the lumen of the swollen part of the lateral oviducts. The protein was tentatively named N. lugens oviduct secreted protein (Nlodsp). RNA interference (RNAi) against NlOdsp transcripts caused a failure of the lateral oviducts to deliver oocytes to the common oviduct that was, by consequence, plugged by 1-2 oocytes. Moreover, although oocytes in the Nlodsp-deficient ovariole were not released to the oviduct, they continued to develop, finally resulting in the presence of several matured oocytes in an ovariole. These defects evidently declined female fecundity. Together, our results demonstrate that NlOdsp plays an essential role in egg transport through the oviduct during ovulation. This work deepens our understanding of insect reproductive system and provides a potential target gene for RNAi-based insect pest control.

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.

The brown planthopper Nilaparvata lugens is one of the most destructive insect pests in Asia, demonstrating high fertility and causing huge crop losses by sucking sap of rice as well as transmitting viruses. However, functional genomic studies on N. lugens are seriously constrained by lack of genetic tools. Here, we employed two eye pigmentation genes to generate germ-line mutations in N. lugens using the CRISPR/Cas9 (clustered regularly interspaced palindromic repeats/CRISPR-associated) system. We showed that injection of single guide RNA of the cinnabar gene of N. lugens (Nl-cn ) into pre-blastoderm eggs induced insertion and deletion (indels) in the founder generation (G 0), which were heritably transmitted to the following G1 generation, leading to bright red compound eyes and ocelli. Mutations of N. lugens white (Nl-w ) generated a high mutant rate of up to 27.3%, resulting in mosaic eyes consisting of white and lightly pigmented ommatidia in both G 0 and G1 individuals. The specificity of CRISPR/Cas9-mediated mutagenesis was further bolstered by PCR and RNA interference-based knockdown analysis. These results show that CRISPR/Cas9-mediated gene editing is achievable in a hemipteran insect, offering a valuable tool for the study of functional genomics and pest management in this planthopper species.

The cuticular hydrocarbon (CHC) biosynthetic pathways branches off from the synthesis of fatty acids. Fatty acid elongases (ELOs) are enzymes catalyzing the synthesis of long-chain fatty acids and thereby contribute to the diversification of CHCs. Based on bioinformatics analyses we identified 20 ELO genes in the brown planthopper , Nilaparvata lugens . RNA interference against these genes demonstrated that 9 NlELO genes were essential for the survival of N. lugens nymphs and adults. Indeed, knockdown of NlELOs 1, 3, 4, 7, 8, 9, 10, 12 and 18 caused lethal phenotypes with a thin and wizened body and reduced lipids in the fat body. Surface analysis by scanning electron microscopy and CHC quantification indicated that knockdown of NlELOs 2, 3, 8 and 16 additionally resulted in a smooth body surface and a decrease in CHC amounts. Therefore, we speculate that long-chain CHCs are needed for CHC attachment to the cuticle surface. CHC deficiency, in turn, resulted in increased adhesion of water droplets and secreted honeydew to the animal surface and the inability of N. lugens to survive in paddy fields with varying humidity. Our present study provides an initial comprehensive analysis of ELO gene functions in an insect, and may serve to better understand the biology of CHCs.

In the sex determination cascade, the genes dsx (doublesex) in insects, mab-3 (male abnormal 3) in nematodes, and Dmrt1 (dsx/mab-3 related transcription factor-1) in vertebrates act as the base molecular switches and play important roles. Moreover, these genes share the same conserved feature domain-DNA-binding oligomerization domain (OD1), and female-specific dsx also has a conserved oligomerization domain 2 (OD2). Although sex determination and the functions of dsx in several holometabolous insects have been well documented, sex determination and the function of dsx in hemimetabolous insects remain a mystery. In this study, four dsx homologs were unexpectedly found in the Nilaparvata lugens (brown planthopper, BPH, order Hemiptera), which also showed a different evolutionary status. We found that only one of the four homologs, Nldsx, which has three alternative splicing variants (female-specific NldsxF, male-specific NldsxM, non-sex-specific NldsxC), was required in the sexual development of N. lugens. Compared with that of holometabolous species, the dsx of N. lugens contains a less conserved OD1, while the OD2 domain of BPH was not identifiable because the common region is poorly conserved, and the female-specific region is short. RNAi-mediated knockdown of Nldsx in female BPH resulted in a larger body size with a normal abdomen and reproductive system, while no changes in fertility were noted. However, adult males with RNA interference knockdown of NldsxM in nymphs became pseudofemales, were infertile, had abnormal copulatory organs, and had impassable deferent ducts with hyperplastic walls; additionally, the pseudofemales could not produce the normal courtship signals. Our results suggest that dsx plays a critical role in male BPH somatic development and mating behavior. This is the first study to show that dsx is essential for sexual development in a hemipteran species.

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.

Chitin synthase (CHS) is an enzyme that is required for chitin formation in insect cuticles and other tissues. In this study, CHS genes from two destructive rice insect pests, the brown planthopper Nilaparvata lugens and the small brown planthopper Laodelphax striatellus, were cloned. Phylogenetic analysis showed that these genes belonged to class CHS1 of the CHS gene family. Most insects possess two CHS genes (CHS1 and CHS2); however, genome and transcriptome searches showed that N. lugens possibly possess only CHS1 in both databases. Two transcript variants (CHS1a and CHS1b) resulting from exclusively alternative splicing (exon 19a or 19b in N. lugens) were identified for each of the two rice planthopper CHS1s. Gene structure comparison using the genomes that are currently sequenced showed that the CHS1 genes in all insects except Acyrthosiphon pisum have two transcript variants. Transcription of NlCHS1a reached its highest level just after molting, whereas NlCHS1b reached its highest expression level 1–2 days before molting. Injection of the N. lugens nymphs with double-strand RNA (dsRNA) of CHS1, CHS1a and CHS1b reduced the corresponding variant transcript levels and exhibited subsequent phenotypes. Silencing of CHS1 and CHS1a resulted in elongated distal wing pads and the “wasp-waisted” or crimpled cuticle phenotypes and eventually died, whereas the phenotypes caused by injection of NlCHS1b dsRNA seem not so obvious although slightly increased mortality was observed. Our results suggest that N. lugens likely lacks CHS2 and CHS1 may be efficient target gene for RNAi-based N. lugens control.

Salivary secretions, including gel saliva and watery saliva, play crucial roles in the interaction between the insect and plant during feeding. In this study, we identified a salivary gland-specific gene encoding a salivary sheath protein (NlShp) in Nilaparvata lugens. NlShp has two alternative splicing variants; both are expressed at high levels during the nymph and adult stages. Immunohistochemical staining showed that the NlShp were synthesized in the principal gland cells of the salivary gland. LC-MS/MS and western blot analysis confirmed that NlShp was one of the components of the salivary sheath. Simultaneously knocking down the two NlShp variants by RNA interference inhibited both salivary flange and salivary sheath formation and resulted in a lethal phenotype within four days for the brown planthopper (BPH) feeding on rice plants, indicating that the salivary sheath and salivary flanges were essential for plant-associated feeding. Despite the salivary sheath deficiency, no obvious phenotype was observed in the NlShp-knockdown BPHs fed on artificial diet. The electrical penetration graph (EPG) results showed that salivary sheath-deficient BPHs exhibited a prolonged nonpenetration period, scarce sap period, and increased stylet movement on rice plants and eventually starved to death. Our results provided evidence that the interaction between the salivary sheath and host plant might be a critical step in successful BPH feeding. According to present research, we propose a salivary sheath required feeding model for piercing-sucking insects and provide a potential target for rice planthopper management.

Flightin was initially identified in Drosophila melanogaster. Previous work has shown that Drosophila flightin plays a key role in indirect flight muscle (IFM) function and has limited expression in the IFM. In this study, we demonstrated that flightin is conserved across the Pancrustacea species, including winged insects, non-winged insects, non-insect hexapods and several crustaceans. The brown planthopper (BPH), Nilaparvata lugens (Stal) (Hemiptera: Delphacidae), a long-distance migration insect with wing dimorphism, is the most destructive rice pest in Asia. We showed that flightin was one of the most differentially expressed genes in macropterous and brachypterous BPH adults. In female BPHs, flightin was expressed in the IFM of macropterous adults, no expression was detected in brachypterous ones; while in male BPHs, flightin was not only expressed in the IFM of macropterous adults, but also in the dorsal longitudinal muscle (DLM) in the basal two abdominal segments of both macropterous and brachypterous ones. RNAi and transmission electron microscopy results showed that flightin played key roles in maintaining IFM and male DLM structure, which drive wing movements in macropterous adults and the vibration of the male-specific tymbal, respectively. Using Daphnia magna as an example of a crustacean species, we observed that flightin was expressed in juvenile instars and adults, and was localized in the antenna muscles. These results illustrate the functional variations of flightin in insects and other arthropod species and provide clues as to how insects with flight apparatuses evolved from ancient pancrustaceans.

DSC1 and BSC1 are two founding members of a novel family of invertebrate voltage-gated cation channels with close structural and evolutionary relationships to voltage-gated sodium and calcium channels. In this study, we searched the published genome sequences for DSC1 orthologs. DSC1 orthologs were found in all 48 insect species, and in other invertebrate species belonging to phyla Mollusca, Cnidaria, Hemichordata and Echinodermata. However, DSC1 orthologs were not found in four arachnid species, Ixodes scapularis, Rhipicephalus microplus, Tetranychus urticae and Varroa destructor, two species in Annelida or any vertebrate species. We then cloned and sequenced NlSC1 and BmSC1 full-length cDNAs from the brown planthopper (Nilaparvata lugens) and the silkworm (Bombyx mori), respectively. NlSC1 and BmSC1 share about 50% identity with DSC1, and the expression of NlSC1 and BmSC1 transcripts was most abundant in the head and antenna in adults. All DSC1 orthologs contain a unique and conserved DEEA motif, instead of the EEEE or EEDD motif in classical calcium channels or the DEKA motif in sodium channels. Phylogenetic analyses revealed that DSC1 and its orthologs form a separate group distinct from the classical voltage-gated sodium and calcium channels and constitute a unique family of cation channels. The DSC1/BSC1-family channels could be potential targets of new and safe insecticides for pest control.

In this study, we isolated two reeler cDNAs from bacteria-challenged larval fat bodies of the silkworm, Bombyx mori. A reeler domain spanned most of the coding regions of these two cDNAs, and their expression patterns were different in B. mori larval tissues. The reeler1 gene was strongly induced by Escherichia coli K12 and Bacillus subtilis in B. mori larval hemocytes, fat bodies and midguts, but reeler2 was expressed at extremely low levels in these tissues. We focused on the reeler1 gene for functional analysis. Interference by double-stranded reeler1 RNA in vivo led to reduced nodule formation in bacteria-injected larvae, while the injection of recombinant Reeler1 promoted nodule formation in reeler1 gene-silenced larvae, indicating that Reeler1 is involved in the nodulation response. Knockdown of the reeler1 gene significantly decreased phenoloxidase activity in bacteria-challenged larval hemolymph, while injection of recombinant Reeler1 enhanced phenoloxidase activity, suggesting that Reeler1 is involved in the prophenoloxidase activation cascade. Our results provide new mechanistic evidence about the melanization cascade in the insect immunity.

The voltage-gated sodium channel mediates the rapid rising phase of action potentials in almost all excitable cells and is a molecular target of a variety of neurotoxins including pyrethroid insecticides. Most studies have focused on the expression of sodium channel genes in the adult stage, information on other developmental stages, however, is limited. In this study, we characterized the para sodium channel orthologous gene ( BmNa v ) of the silkworm Bombyx mori , a model insect of Lepidopteran species. The BmNa v gene covers a 31 kb genome region and contains 36 exons. The longest ORF contained 6258 bp and encoded 2085 amino acid residues, which shares 74%, and 77% overall amino acid sequence identities with the sodium channel proteins from Drosophila melanogaster and Blattella germanica , respectively. Using high-throughput Solexa sequence technology we conducted sequence analysis of BmNa v cDNAs from embryos, larvae, pupae and adults of the silkworm, identified alternative splicing sites and determined the frequencies of these splicing events in four developmental stages. Three optional exons, two sets of mutually exclusive exons, and one internal spliced exon were identified. One optional exon is unique to BmNa v , while the others are conserved in other insect sodium channel genes. Interestingly, the expression of the mutually exclusive exons is developmentally regulated.

We investigated variations in the gene expression of Bombyx mori following infection with a densonucleosis virus (BmDNV-Z). Two B. mori near-isogenic lines, Jingsong and Jingsong.nsd-Z.NIL, which are highly susceptible and completely resistant to BmDNV-Z, respectively, were used in this study. The infection profiles of BmDNV-Z in the midguts of the B. mori Jingsong and Jingsong.nsd-Z.NIL larvae revealed that the virus invaded the midguts of both of these strains. However, its proliferation was notably inhibited in the midgut of the resistant strain. By using the suppression subtractive hybridization method, three cDNA libraries were constructed to compare BmDNV-Z responsive gene expression between the two silkworm lines. In total, 151 differentially expressed genes were obtained. Real-time qPCR analysis confirmed that 11 genes were significantly up-regulated in the midgut of the Jingsong.nsd-Z.NIL strain following BmDNV-Z infection. Our results imply that these up-regulated genes might be involved in B. mori immune responses against BmDNV infection.