Regulatory Effects of Quercetin on Bone Homeostasis: Research Updates and Future Perspectives.

The imbalance of bone homeostasis has become a major public medical problem amid the background of an aging population, which is closely related to the occurrence of osteoporosis, osteoarthritis, and fractures. Presently, most drugs used in the clinical treatment of bone homeostasis imbalance are bisphosphonates, calcitonin, estrogen receptor modulators, and biological agents that inhibit bone resorption or parathyroid hormone analogs that promote bone formation. However, there are many adverse reactions. Therefore, it is necessary to explore potential drugs. Quercetin, as a flavonol compound with various biological activities, is widely distributed in plants. Studies have found that quercetin can regulate bone homeostasis through multiple pathways and targets. An in-depth exploration of the pharmacological mechanism of quercetin is of great significance for the development of new drugs. This review discusses the therapeutic mechanisms of quercetin on bone homeostasis, such as regulating the expression of long non-coding RNA, signaling pathways of bone metabolism, various types of programmed cell death, bone nutrients supply pathways, anti-oxidative stress, anti-inflammation, and activation of Sirtuins. We also summarize recent progress in improving quercetin bioavailability and propose some issues worth paying attention to, which may help guide future research efforts.

Potential Therapeutic Mechanism of Radix Angelicae Biseratae and Dipsaci Radix Herb Pair against Osteoarthritis: Based on Network Pharmacology and Molecular Docking.

Background: A major contributor to older disability is osteoarthritis. Radix Angelicae Biseratae (known as Duhuo in China, DH, the dried rhizome of Angelica pubescens) and Dipsaci Radix (known as Xuduan in China, XD, the dried rhizome of Dipsacus asper Wall) herb pair (DXHP) is widely used to treat osteoarthritis, but the underlying molecular mechanisms still have not been revealed. This research aimed to illustrate the therapeutic mechanism of DXHP against osteoarthritis through the techniques of network pharmacology and molecular docking. Methods: Gene targets for osteoarthritis and active ingredients for DXHP were screened based on the pharmacology public database and the gene-disease target database. The software program Cytoscape was used to visualize the active chemical target-disease gene network. The STRING biological information website was used to investigate protein interactions. On the Metascape bioinformatics website, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment were carried out. The molecular docking of the important chemicals and primary targets identified by the aforementioned screening was performed using Autodock software. Results: Twenty-six active substances from the DXHP that had strong connections to 138 osteoarthritis-related targets were screened out. According to network analysis, TNF, GAPDH, IL-6, AKT-1, IL-1B, and VEGFA are prospective therapeutic targets, while osthole, cauloside A, ammidin, angelicone, beta-sitosterol, and asperosaponin VI may be significant active components. 1705 biological processes (BP), 155 molecular functions (MF), and 89 cellular components (CC) were identified by GO analysis. KEGG analysis indicated that IL-17, NF-kappa B, HIF-1, MAPK, and AGE-RAGE signaling pathways are potentially involved. Molecular docking showed that cauloside A, osthole, and ß-sitosterol have excellent binding activity with main targets. Conclusions: This study comprehensively illuminated the active ingredients, potential targets, primary pharmacological effects, and relevant mechanisms of the DXHP in the treatment of OA. These findings provide fresh thoughts into the therapeutic mechanisms of the main active ingredients of DXHP and provide a reference for further exploration and clinical applications of DXHP.

Neuronal calcium sensor 2 is key to moulting and oocyte development in the brown planthopper, Nilaparvata lugens.

Intracellular calcium (Ca2+ ) is vital for signal transduction in many cellular events. Several Ca2+ -binding proteins mediate the transduction of intracellular calcium signals. The EF-hand motifs containing neuronal calcium sensor (NCS) proteins are mainly expressed in the nervous system, where they have important roles in the regulation of a variety of neuronal functions. NCS1 has four EF-hand motifs and well-defined neuronal development functions in a variety of eukaryotes. However, NCS2 has only been identified in invertebrates such as insects and nematodes thus far. The functions of NCS2 remain largely unknown. Here, we identified an orthologous NCS2 in the hemipteran Nilaparvata lugens. Based on qRT-PCR, this gene was found to be primarily expressed in the brain. Knockdown of NCS2 in each nymphal instar by RNA interference led to lethality and caused aggradation and disordered arrangement of lipid droplets in the ovaries and testes of adults, which were associated with the absence of mature oocytes in female ovaries and reduction of spermiation in male adults. Our findings revealed a novel function for NCS2 as a regulator in development and reproduction and suggested that this protein had an important role in modulating lipid droplet remodelling in ovary and testis of N. lugens adults.

CPR Gene Contributes to Integument Function and Ovary Development in a Rice Planthopper.

Nicotinamide adenine dinucleotide phosphate (NADPH)-cytochrome P450 reductase (CPR) is an essential enzyme that transfers electrons from NADPH to cytochrome P450 monooxygenases. CPR is involved in cuticular hydrocarbon (CHC) synthesis in insects and is vital for insect development and survival. Here, we clarify the physiological function of a CPR gene in Nilaparvata lugens, an important rice pest, by using RNA interference. CPR gene knockdown leads to the functional loss of waterproofing and water retention in the integument of female adults, which causes significantly reduced body weight and a lethal phenotype. Scanning electron microscopy shows that the lipid layer on the outermost surface of the abdominal cuticle becomes thin in dsCPR-injected adults. Furthermore, CHC profile analysis reveals that CPR knockdown significantly decreases the contents of CHCs with a carbon chain length ≥ C27 in adult females. Moreover, we find that CPR knockdown generates a deficient phenotype in ovaries with deformed oocytes and a complete failure of egg-laying. These findings suggest that CPR plays multiple functional roles in CHC biosynthesis and embryo development in insects.

A CYP380C10 gene is required for waterproofing and water retention in the insect integument.

Cuticular hydrocarbons (CHCs) are important components in the integument of insects and are required for development and survival. Insect-specific CYP4G subfamily, of the P450 enzymes, catalyze the oxidative decarbonylation step in the biosynthesis of CHCs. Here, we characterized CYP380C10 gene function in a Hemiptera rice pest, Nilaparvata lugens. We used RNA interference-mediated expression silencing to reveal that NlCYP380C10 played a key role in waterproofing and water-retention in the integument of N. lugens. Knockdown of NlCYP380C10 significantly reduced body weight and caused mortality. Scanning electron microscopy showed the loss of the lipid layer on the surface of the abdominal cuticle of the dsNlCYP380C10-injected adults. Furthermore, CHC profile analysis revealed that NlCYP380C10 knockdown significantly decreased the amounts of CHCs in adult females. This suggested that NlCYP380C10 was involved in CHC biosynthesis. Reduction of CHC content caused the loss of the intact lipid layer of the cuticle, which resulted in loss of the waterproofing and water-retention functions. This led to failure of molting and eclosion. Our findings expanded the knowledge of CHC biosynthesis in the insect integument and led to a better understanding of the functional roles of CYP450 genes involved in waterproofing and water-retention in insects.

ATPase subunits of the 26S proteasome are important for oocyte maturation in the brown planthopper.

The 26S proteasome is the major engine of protein degradation in all eukaryotic cells. Adenosine triphosphatase (ATPase) regulatory subunits (Rpts) are constituents of the proteasome that are involved in the unfolding and translocation of substrate proteins into the core particle. In this study, by using the brown planthopper Nilaparvata lugens as a model insect, we report the biological importance of Rpts in female reproduction. We identified six homologous Rpt genes (Rpt1-6) in N. lugens. These genes were detected at high transcript levels in eggs and ovaries of females but at low transcript levels in males. RNA interference-mediated knockdown of N. lugens Rpt genes significantly decreased the proteolytic activity of the proteasome and impeded the transcription of triacylglycerol lipase and vitellogenin genes in the fat bodies and ovaries of adult females and reduced the triglyceride content in the ovaries. The decrease in the proteolytic activity of the proteasome via knockdown of Rpts also downregulated the transcription of the CYP307A2 gene encoding an important rate-limiting enzyme in the 20-hydroxyecdysone biosynthetic pathway in the ovaries, reduced 20E production in adult females and impaired ovarian development and oocyte maturation, leading to the failure of egg production and egg-laying. These novel findings indicate that Rpts are required for the proteolytic activity of the proteasome, which is important for female reproductive success in N. lugens.

An MD-2-related lipid-recognition protein is required for insect reproduction and integument development.

The myeloid differentiation factor 2 (MD-2)-related lipid-recognition protein is involved in immune responses through recognizing bacteria lipopolysaccharide in mammals, arthropods and plants. However, the physiological roles of MD-2 in other biological processes are largely unknown. Here, we identified three homologue MD-2 genes (NlML1, NlML2 and NlML3) by searching the genome and transcriptome databases of the brown planthopper Nilaparvata lugens, a hemipteran insect species. Temporospatial analysis showed that the NlML1 gene was highly expressed in the fat body but much less so in the other tissues, while the NlML2 and NlML3 genes were highly expressed in the testis or digestive tract. RNA interference-mediated depletion of the NlML1 gene significantly downregulated the transcription of numerous integument protein genes. The NlML1 knockdown led to moulting failure and mortality at the nymph-adult transition phase, impaired egg laying and hatching, and reduced 20-hydroxyecdysone (20E) production in the nymphs. 20E could rescue the deficient moulting phenotypes derived from dsNlML1 RNAi. These novel findings indicate that NlML1 is required for nymphal moulting and female reproductive success as it plays an important role in regulating 20E synthesis, lipid and chitin metabolisms in N. lugens, thus contributing to our understanding of developmental and reproductive mechanisms in insects.

Proteolytic activity of the proteasome is required for female insect reproduction.

Non-ATPase regulatory subunits (Rpns) are components of the 26S proteasome involved in polyubiquitinated substrate recognition and deubiquitination in eukaryotes. Here, we identified 15 homologues sequences of Rpn and associated genes by searching the genome and transcriptome databases of the brown planthopper, Nilaparvata lugens, a hemipteran rice pest. Temporospatial analysis showed that NlRpn genes were significantly highly expressed in eggs and ovaries but were less-highly expressed in males. RNA interference-mediated depletion of NlRpn genes decreased the proteolytic activity of proteasome and impeded the transcription of lipase and vitellogenin genes in the fat bodies and ovaries in adult females, and reduced the triglyceride content in the ovaries. Decrease of the proteolytic activity of the proteasome via knockdown of NlRpns also inhibited the transcription of halloween genes, including NlCYP307A2, NlCYP306A2 and NlCYP314A1, in the 20-hydroxyecdysone (20E) biosynthetic pathway in the ovaries, reduced 20E production in adult females, and impaired ovarian development and oocyte maturation, resulting in reduced fecundity. These novel findings indicate that the proteolytic activity of the proteasome is required for female reproductive processes in N. lugens, thus furthering our understanding of the reproductive and developmental strategies in insects.

Identification and characterization of a novel rhabdovirus in green rice leafhopper, Nephotettix cincticeps.

The family Rhabdoviridae is one of the most ecological diverse groups of RNA viruses, with remarkable genome complexity and wide host range. Rhabdoviruses are discovered in many insect species, and insects, such as midges, mosquitoes, aphids and leafhoppers, act as crucial vectors for rhabdovirus transmission. Here, a novel rhabdovirus was identified in green rice leafhopper, Nephotettix cincticeps (Hemiptera: Cicadellidae), a common virus vector on rice. This virus was named as Nephotettix cincticeps negative-stranded RNA virus-1 (NcNSRV-1). The genome of NcNSRV-1 is 12,361 nucleotides in length, flanked by untranslated 3' leader and 5' trailer. The anti-sense viral genome consists of five major structural protein genes (N, P, M, G and L), which shares a typical architecture with the family Rhabdoviridae. An additional gene, P6, is interposed between G and L genes. NcNSRV-1 is phylogenetically clustered with the unclassified rhabdoviruses isolated from insects and exhibits low sequence identities with other viruses. The transcription regulatory sequences in NcNSRV-1 gene junctions were determined, revealing a unique transcription initiation sequence. In view of the phylogeny, sequence identity and genome organization, NcNSRV-1 is likely to be an insect rhabdovirus. Field surveys showed NcNSRV-1 was prevalent in the rice field populations of N. cincticeps in China.

NADPH Oxidase 5 Is Essential for Molting and Oviposition in a Rice Planthopper Nilaparvata lugens.

The brown planthopper Nilaparvata lugens is a typical monophagous insect herbivore that feeds exclusively on rice sap. This insect pest causes serious damage to rice crops throughout East Asian countries. Chemical control remains the first choice for managing N. lugens populations; however, the use of insecticides has given rise to planthopper resurgence and additional environmental risks. Nilaparvata lugens is a model insect of Hemiptera because its whole genome sequence has been elucidated and is susceptible to RNA interference. In this study, our findings revealed that a superoxide-generating gene, NADPH oxidase 5 (Nox5), is essential for molting and oviposition in a Hemipteran insect Nilaparvata lugens. Knockdown of Nox5 transcript levels by RNA interference in 2nd-5th-instar nymphs results in significantly lethal deficits in the molting transitions from nymph-nymph and nymph-adult. Nox5 knockdown leads to a reduction of hydrogen peroxide in female ovaries and failure of oviposition from the insect ovipositor into the rice leaf sheath. Here, we provide in vivo evidence demonstrating that Nox5 is a key enzyme for regulating molting and oviposition in this insect species.

Functional analysis of ecdysteroid biosynthetic enzymes of the rice planthopper, Nilaparvata lugens.

Ecdysteroids, insect steroid hormones, play key roles in regulating insect development and reproduction. Hemipteran insects require ecdysteroids for egg production; however, ecdysteroid synthesis (ecdysteroidogenesis) details have not been elucidated. We identified all known genes encoding ecdysteroidogenic enzymes in Nilaparvata lugens and clarified their necessity during nymphal and ovarian development. We confirmed that N. lugens utilized 20-hydroxyecdysone as an active hormone. Assays using heterologous expression of enzymes in Drosophila S2 cells showed conserved functions of enzymes Neverland, CYP306A2, CYP314A1 and CYP315A1, but not CYP302A1. RNA interference and rescue analysis using 20-hydroxyecdysone demonstrated that most of the genes were necessary for nymphal development. The identified N. lugens enzymes showed conserved functions and pathways for ecdysteroidogenesis. Knockdown of ecdysteroidogenic enzyme genes in newly molted females caused failure of egg production: less vitellogenic and mature eggs in ovaries, fewer laid eggs and embryonic development deficiency of laid eggs. Considering the high expressions of ecdysteroidogenic enzyme genes in adults and ovaries, ecdysteroidogenesis in ovaries was critical for N. lugens ovarian development. Our study presents initial evidence that hemipteran insects require ecdysteroidogenesis for ovarian development.

A Novel Iflavirus Was Discovered in Green Rice Leafhopper Nephotettix cincticeps and Its Proliferation Was Inhibited by Infection of Rice Dwarf Virus.

The green rice leafhopper, Nephotettix cincticeps (Hemiptera: Cicadellidae), is a key insect vector transmitting rice dwarf virus (RDV) that causes rice dwarf disease. We discovered a novel iflavirus from the transcriptomes of N. cincticeps and named it as Nephotettix cincticeps positive-stranded RNA virus-1 (NcPSRV-1). The viral genome consists of 10,524 nucleotides excluding the poly(A) tail and contains one predicted open reading frame encoding a polyprotein of 3,192 amino acids, flanked by 5' and 3' untranslated regions. NcPSRV-1 has a typical iflavirus genome arrangement and is clustered with the members of the family Iflaviridae in the phylogenetic analysis. NcPSRV-1 was detected in all tested tissues and life stages of N. cincticeps and could be transmitted horizontally and vertically. Moreover, NcPSRV-1 had high prevalence in the laboratory populations and was widely spread in field populations of N. cincticeps. NcPSRV-1 could also infect the two-striped leafhopper, Nephotettix apicalis, at a 3.33% infection rate, but was absent in the zigzag leafhopper, Recilia dorsalis, and rice Oryza sativa variety TN1. The infection of RDV altered the viral load and infection rate of NcPSRV-1 in N. cincticeps, for which it seems that RDV has an antagonistic effect on NcPSRV-1 infection in the host.

Identification of novel antimicrobial peptides from rice planthopper, Nilaparvata lugens.

In this study, two novel antibacterial peptide genes, termed lugensin A and B were identified and characterized from a rice sap-sucking hemipteran insect pest, the brown planthopper, Nilaparvata lugens. Lugensin gene expression was significantly induced by Gram-negative and Gram-positive bacterial stains under the regulation of a signal receptor, the long peptidoglycan recognition protein (PGRP-LC) in the IMD pathway. Knockdown of PGRP-LC by RNAi eliminated bacterium induced Lugensin gene expression. Lugensins had the apparent antibacterial activities against Escherichia coli K12, Bacillus subtilis and the rice bacterial brown stripe pathogen Acidovorax avenae subsp. avenae (Aaa) strain RS-1. Lugensins inhibited bacterial proliferation by disrupting the integrity of the bacterial membranes. Scanning electron microscopy revealed abnormal membrane morphology of the recombinant Lugensin-treated bacteria. Lugensins induced complete cell disruption of E. coli K12 and B. subtilis strains while formed the holes on the cell surface of Aaa RS-1 strain. Immunofluorescence showed that Lugensins localized in the cell membrane of E. coli K12 while accumulated in the cytosol of B. subtilis. Differently, Lugensins remained in both the cell membrane and the cytosol of Aaa RS-1 strain, suggesting different action modes of Lugensins to different microbes. This is the first report of the novel antibacterial peptides found in the rice sap-sucking hemipteran insect species.

Combined transcriptomic/proteomic analysis of salivary gland and secreted saliva in three planthopper species.

The planthoppers are piercing-sucking pests that continuously inject saliva into host plants using specialized stylets. However, knowledge on the constituent and function of planthopper saliva proteins was still limited. In this study, the transcriptomic and proteomic approach were adopted to characterize the composition of salivary glands and their secreted saliva in three planthoppers, respectively. Gene repertoires of salivary glands in brown planthopper (Nilaparvata lugens, BPH), white-backed planthopper (Sogatella furcifera, WBPH) and small brown planthopper (Laodelphax striatellus, SBPH) were very similar, which actively involved in protein synthesis and energy metabolism. Comparative analysis of saliva proteome was performed among three planthoppers and other reported insect species. The saliva composition in three planthoppers was diverse, with 55 saliva proteins commonly identified in more than two species. A few proteins, including serine protease, carboxylesterase, aminopeptidase N, lipophorin, elongation factor, carbonic anhydrase, and calcium binding protein were ubiquitous distributed in different insects, indicating conserved function of saliva. While, the majority of saliva proteins were specifically identified in planthoppers, which might be the evolutional adaptation of insects to different hosts. Our work gained insight into the interaction between insect and host plant through salivary approach, and provided a good resource for functional characterization of effectors. BIOLOGICAL SIGNIFICANCE: Secreted saliva from insects is attracting immense research interest on the global level due to the crucial roles in determining the compatibility between the insects and their hosts. The three planthoppers: brown planthopper (Nilaparvata lugens, BPH), small brown planthopper (Laodelphax striatellus, SBPH), and white-backed planthopper (Sogatella furcifera, WBPH) caused serious damage to rice plants throughout Asia. However, knowledge on the composition and function of their secreted saliva proteins was limited. Our study characterizes the global gene expression of salivary glands and their secreted saliva by Illumina sequencing technology and LC-MS/MS analysis, respectively. By comparative analysis, the ubiquitous and specific saliva compounds in different insects were unveiled.

Genome sequence of the small brown planthopper, Laodelphax striatellus.

Background: Laodelphax striatellus Fallén (Hemiptera: Delphacidae) is one of the most destructive rice pests. L. striatellus is different from 2 other rice planthoppers with a released genome sequence, Sogatella furcifera and Nilaparvata lugens, in many biological characteristics, such as host range, dispersal capacity, and vectoring plant viruses. Deciphering the genome of L. striatellus will further the understanding of the genetic basis of the biological differences among the 3 rice planthoppers. Findings: A total of 190 Gb of Illumina data and 32.4 Gb of Pacbio data were generated and used to assemble a high-quality L. striatellus genome sequence, which is 541 Mb in length and has a contig N50 of 118 Kb and a scaffold N50 of 1.08 Mb. Annotated repetitive elements account for 25.7% of the genome. A total of 17 736 protein-coding genes were annotated, capturing 97.6% and 98% of the BUSCO eukaryote and arthropoda genes, respectively. Compared with N. lugens and S. furcifera, L. striatellus has the smallest genome and the lowest gene number. Gene family expansion and transcriptomic analyses provided hints to the genomic basis of the differences in important traits such as host range, migratory habit, and plant virus transmission between L. striatellus and the other 2 planthoppers. Conclusions: We report a high-quality genome assembly of L. striatellus, which is an important genomic resource not only for the study of the biology of L. striatellus and its interactions with plant hosts and plant viruses, but also for comparison with other planthoppers.

Mucin-like protein, a saliva component involved in brown planthopper virulence and host adaptation.

The rice brown planthopper (BPH), Nilaparvata lugens, can rapidly adapt to new resistant rice varieties within several generations, rendering its management burdensome. However, the molecular mechanism underlying its adaptability remains unclear. In this study, we investigated the potential role of mucin-like protein (NlMul) in N. lugens virulence and adaptation to host resistance. NlMul is an important glycoprotein that constitutes both gelling and watery saliva, and specifically expressed in the salivary glands at all developmental stages except the egg period. Knocking down the expression of NlMul resulted in the secretion of short and single-branched salivary sheaths. NlMul might help BPH deal with plant resistance, and altered gene expression was observed when BPHs were transferred from a susceptible rice variety to a resistant one. The NlMul-deficient BPHs showed disordered developmental duration and a portion of these insects reared on resistant rice exhibited lethal effects. Our results uncover a saliva-mediated interaction between insect and host plant, and provide useful information in rice breeding and planthopper management.

A mitochondrial membrane protein is a target for rice ragged stunt virus in its insect vector.

Rice ragged stunt virus (RRSV; Reoviridae) is exclusively transmitted by the brown planthopper Nilaparvata lugens in a persistent-propagative manner. It is understood that RNA viral proliferation is associated with the intracellular membranes of the insect host cells. However, the molecular mechanisms of the interaction between the RRSV proliferation and the intracellular membranes remain essentially unknown. It will be of great interest to determine whether RRSV protein(s) directly interact with intracellular membrane components of its host cells. In this study, we identified a RRSV nonstructural protein Pns10 interacting with a host oligomycin-sensitivity conferral protein (OSCP) using yeast two-hybrid system. The interaction between RRSV Pns10 and N. lugens OSCP was verified by a glutathione S-transferase pull-down assay. Confocal miscopy revealed colocalization of these two proteins in the cytoplasm of the salivary gland cells during the viral infection. The virions were further detected in the mitochondria under confocal miscopy and transmission electron microscopy combined with western blotting assay. This is the first observation that RRSV protein has a direct link with mitochondria. Suppressing OSCP gene expression by RNA interference notably decreased the viral loads in RRSV-infected insects. These findings revealed novel aspects of a viral protein in targeting the host mitochondrial membrane and provide insights concerning the mitochondrial membrane protein-based virus proliferation mode in the insect vector.

Screening and Functional Analyses of Nilaparvata lugens Salivary Proteome.

Most phloem-feeding insects secrete gelling and watery saliva during the feeding process. However, the functions of salivary proteins are poorly understood. In this study, our purpose was to reveal the components and functions of saliva in a rice sap-sucking insect pest, Nilaparvata lugens. The accomplishment of the whole genome and transcriptome sequencing in N. lugens would be helpful for elucidating the gene information and expression specificity of the salivary proteins. In this study, we have, for the first time, identified the abundant protein components from gelling and watery saliva in a monophagous sap-sucking insect species through shotgun proteomic detection combined with the genomic and transcriptomic analysis. Eight unknown secreted proteins were limited to N. lugens, indicating species-specific saliva components. A group of annexin-like proteins first identified in the secreted saliva displayed different domain structure and expression specificity with typical insect annexins. Nineteen genes encoding five annexin-like proteins, six salivaps (salivary glands-specific proteins with unknown function), seven putative enzymes, and a mucin-like protein showed salivary gland-specific expression pattern, suggesting their importance in the physiological mechanisms of salivary gland and saliva in this insect species. RNA interference revealed that salivap-3 is a key protein factor in forming the salivary sheath, while annexin-like5 and carbonic anhydrase are indispensable for N. lugens survival. These novel findings will greatly help to clarify the detailed functions of salivary proteins in the physiological process of N. lugens and elucidate the interaction mechanisms between N. lugens and the rice plant, which could provide important targets for the future management of rice pests.

A salivary sheath protein essential for the interaction of the brown planthopper with rice plants.

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.

Genomic and transcriptomic insights into the cytochrome P450 monooxygenase gene repertoire in the rice pest brown planthopper, Nilaparvata lugens.

The cytochrome P450 monooxygenase (P450) gene family is one of the most abundant eukaryotic gene families that encode detoxification enzymes. In this study, we identified an abundance of P450 gene repertoire through genome- and transcriptome-wide analysis in the brown planthopper (Nilaparvata lugens), the most destructive rice pest in Asia. Detailed gene information including the exon-intron organization, size, transcription orientation and distribution in the genome revealed that many P450 loci were closely situated on the same scaffold, indicating frequent occurrence of gene duplications. Insecticide-response expression profiling revealed that imidacloprid significantly increased NlCYP6CS1v2, NLCYP4CE1v2, NlCYP4DE1, NlCYP417A1v2 and NlCYP439A1 expression; while triazophos and deltamethrin notably enhanced NlCYP303A1 expression. Expression analysis at the developmental stage showed the egg-, nymph-, male- and female-specific expression patterns of N. lugens P450 genes. These novel findings will be helpful for clarifying the P450 functions in physiological processes including development, reproduction and insecticide resistance in this insect species.