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.

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.

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.

Two insulin receptors determine alternative wing morphs in planthoppers.

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.

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.

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.

Genomic insights into the serine protease gene family and expression profile analysis in the planthopper, Nilaparvata lugens.

BACKGROUND: The brown planthopper (Nilaparvata lugens) is one of the most destructive rice plant pests in Asia. N. lugens causes extensive damage to rice by sucking rice phloem sap, which results in hopper burn (complete death of the rice plants). Despite its importance, little is known about the digestion, development and defense mechanisms of this hemimetabolous insect pest. In this study, we aim to identify the serine protease (SP) and serine protease homolog (SPH) genes, which form a large family in eukaryotes, due to the potential for multiple physiological roles. Having a fully sequenced genome for N. lugens allows us to perform in-depth analysis of the gene structures, reveal the evolutionary relationships and predict the physiological functions of SP genes. RESULTS: The genome- and transcriptome-wide analysis identified 90 putative SP (65) and SPH (25) genes in N. lugens. Detailed gene information regarding the exon-intron organization, size, distribution and transcription orientation in the genome revealed that many SP/SPH loci are closely situated on the same scaffold, indicating the frequent occurrence of gene duplications in this large gene family. The gene expression profiles revealed new findings with regard to how SPs/SPHs respond to bacterial infections as well as their tissue-, development- and sex-specific expressions. CONCLUSIONS: Our findings provide comprehensive gene sequence resources and expression profiles of the N. lugens SP and SPH genes, which give insights into clarifying the potentially functional roles of these genes in the biological processes including development, digestion, reproduction and immunity.

The genome- and transcriptome-wide analysis of innate immunity in the brown planthopper, Nilaparvata lugens.

BACKGROUND: The brown planthopper (Nilaparvata lugens) is one of the most serious rice plant pests in Asia. N. lugens causes extensive rice damage by sucking rice phloem sap, which results in stunted plant growth and the transmission of plant viruses. Despite the importance of this insect pest, little is known about the immunological mechanisms occurring in this hemimetabolous insect species. RESULTS: In this study, we performed a genome- and transcriptome-wide analysis aiming at the immune-related genes. The transcriptome datasets include the N. lugens intestine, the developmental stage, wing formation, and sex-specific expression information that provided useful gene expression sequence data for the genome-wide analysis. As a result, we identified a large number of genes encoding N. lugens pattern recognition proteins, modulation proteins in the prophenoloxidase (proPO) activating cascade, immune effectors, and the signal transduction molecules involved in the immune pathways, including the Toll, Immune deficiency (Imd) and Janus kinase signal transducers and activators of transcription (JAK-STAT) pathways. The genome scale analysis revealed detailed information of the gene structure, distribution and transcription orientations in scaffolds. A comparison of the genome-available hemimetabolous and metabolous insect species indicate the differences in the immune-related gene constitution. We investigated the gene expression profiles with regards to how they responded to bacterial infections and tissue, as well as development and sex expression specificity. CONCLUSIONS: The genome- and transcriptome-wide analysis of immune-related genes including pattern recognition and modulation molecules, immune effectors, and the signal transduction molecules involved in the immune pathways is an important step in determining the overall architecture and functional network of the immune components in N. lugens. Our findings provide the comprehensive gene sequence resource and expression profiles of the immune-related genes of N. lugens, which could facilitate the understanding of the innate immune mechanisms in the hemimetabolous insect species. These data give insight into clarifying the potential functional roles of the immune-related genes involved in the biological processes of development, reproduction, and virus transmission in N. lugens.

Dynamic interactions between Bombyx mori nucleopolyhedrovirus and its host cells revealed by transcriptome analysis.

Although microarray and expressed sequence tag (EST)-based approaches have been used to profile gene expression during baculovirus infection, the response of host genes to baculovirus infection and the interaction between baculovirus and its host remain largely unknown. To determine the host response to Bombyx mori nucleopolyhedrovirus infection and the dynamic interaction between the virus and its host, eight digital gene expression libraries were examined in a Bm5 cell line before infection and at 1.5, 3, 6, 12, 24, 48, and 96 h postinfection. Gene set enrichment analysis of differentially expressed genes at each time point following infection showed that gene sets including cytoskeleton, transcription, translation, energy metabolism, iron ion metabolism, and the ubiquitin-proteasome pathway were altered after viral infection. In addition, a time course depicting protein-protein interaction networks between the baculovirus and the host were constructed and revealed that viral proteins interact with a multitude of cellular machineries, such as the proteasome, cytoskeleton, and spliceosome. Several viral proteins, including IE2, CG30, PE38, and PK-1/2, were predicted to play key roles in mediating virus-host interactions. Based on these results, we tested the role of the ubiquitin-proteasome pathway and iron ion metabolism in the viral infection cycle. Treatment with a proteasome inhibitor and deferoxamine mesylate in vitro and in vivo confirmed that these pathways regulate viral infection. Taken together, these findings provide new insights into the interaction between the baculovirus and its host and identify molecular mechanisms that can be used to block viral infection and improve baculovirus expression systems.

De novo intestine-specific transcriptome of the brown planthopper Nilaparvata lugens revealed potential functions in digestion, detoxification and immune response.

The brown planthopper (Nilaparvata lugens, BPH) is the most serious rice plant pests in Asia. In this study, we performed transcriptome-wide analysis on BPH intestine. We obtained more than 26 million sequencing reads that were then assembled into 53,553 unigenes with a mean size of 388 bp. Based on similarity search with the nucleotide sequences available at NCBI, BPH intestine-specific transcriptome analysis identified 21,405 sequences. Assembled sequences were annotated with gene description, gene ontology and clusters of orthologous group terms. The digestion-, defense- and xenobiotic metabolism-related genes were abundantly detected in the transcripts from BPH intestine. Many novel genes including 33 digestion-related genes, 25 immune responsive genes and 27 detoxification-related genes are first reported here. We investigated the gene expression patterns at the transcript levels in different tissues by quantitative real-time PCR analysis, which revealed that some genes had intestine-specific expression, implicating their potential significance for BPH management.

Global analysis of the transcriptional response of whitefly to tomato yellow leaf curl China virus reveals the relationship of coevolved adaptations.

The begomoviruses are the largest and most economically important group of plant viruses transmitted exclusively by the whitefly Bemisia tabaci in a circulative, persistent manner. The circulation of the viruses within the insect vectors involves complex interactions between virus and vector components; however, the molecular mechanisms of these interactions remain largely unknown. Here we investigated the transcriptional response of the invasive B. tabaci Middle East-Asia Minor 1 species to Tomato yellow leaf curl China virus (TYLCCNV) using Illumina sequencing technology. Results showed that 1,606 genes involved in 157 biochemical pathways were differentially expressed in the viruliferous whiteflies. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis indicated that TYLCCNV can perturb the cell cycle and primary metabolism in the whitefly, which explains the negative effect of this virus on the longevity and fecundity of B. tabaci. Our data also demonstrated that TYLCCNV can activate whitefly immune responses, such as autophagy and antimicrobial peptide production, which might lead to a gradual decrease of viral particles within the body of the viruliferous whitefly. Furthermore, PCR results showed that TYLCCNV can invade the ovary and fat body tissues of the whitefly, and Lysotracker and Western blot analyses revealed that the invasion of TYLCCNV induced autophagy in both the ovary and fat body tissues. Surprisingly, TYLCCNV also suppressed the whitefly immune responses by downregulating the expression of genes involved in Toll-like signaling and mitogen-activated protein kinase (MAPK) pathways. Taken together, these results reveal the relationship of coevolved adaptations between begomoviruses and whiteflies and will provide a road map for future investigations into the complex interactions between plant viruses and their insect vectors.

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.

ODV-associated proteins of the Pieris rapae granulovirus.

Alphabaculovirus (lepidopteran-specific nucleopolyhedroviruses, NPV) and Betabaculovirus (granuloviruses, GV) are two main genera of the family Baculoviridae. The virion proteomes of Alphabaculovirus have been well studied; however, the Betabaculovirus virion compositions remain unclear. Pieris rapae granulovirus (PrGV) can kill larvae of P. rapae, a worldwide and important pest of mustard family crops. In this study, the occlusion-derived virus (ODV)-associated proteins of PrGV were identified using three mass spectrometry (MS) approaches. The MS analyses demonstrated that 47 proteins were present in PrGV-ODV. Of the 47 PrGV-ODV proteins, 33 have homologues identified previously in other baculovirus ODV/BVs, whereas 14 (P10, Pr21, Pr29, Pr35, Pr42, Pr54, P45/48, Pr83, Pr84, Pr89, Pr92, Pr111, Pr114 and FGF3) were newly identified ODV proteins. Seven of the 14 newly identified ODV proteins are specific to Betabaculovirus, including Pr35, Pr42, Pr54, Pr83, Pr84, Pr111 and Pr114. Furthermore, the data derived from these MS approaches were validated by immunoblotting analysis using antisera prepared from 11 randomly selected recombinant PrGV-ODV proteins (including 5 Betabaculovirus-unique proteins). Comparison analyses revealed the similar and different compositions between Betabaculovirus and Alphabaculovirus virions, which deepen our understanding of the baculovirus virion structure and provide helpful information on Betabaculovirus--host interaction studies.

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.

De novo characterization of a whitefly transcriptome and analysis of its gene expression during development.

BACKGROUND: Whitefly (Bemisia tabaci) causes extensive crop damage throughout the world by feeding directly on plants and by vectoring hundreds of species of begomoviruses. Yet little is understood about its genes involved in development, insecticide resistance, host range plasticity and virus transmission. RESULTS: To facilitate research on whitefly, we present a method for de novo assembly of whitefly transcriptome using short read sequencing technology (Illumina). In a single run, we produced more than 43 million sequencing reads. These reads were assembled into 168,900 unique sequences (mean size = 266 bp) which represent more than 10-fold of all the whitefly sequences deposited in the GenBank (as of March 2010). Based on similarity search with known proteins, these analyses identified 27,290 sequences with a cut-off E-value above 10-5. Assembled sequences were annotated with gene descriptions, gene ontology and clusters of orthologous group terms. In addition, we investigated the transcriptome changes during whitefly development using a tag-based digital gene expression (DGE) system. We obtained a sequencing depth of over 2.5 million tags per sample and identified a large number of genes associated with specific developmental stages and insecticide resistance. CONCLUSION: Our data provides the most comprehensive sequence resource available for whitefly study and demonstrates that the Illumina sequencing allows de novo transcriptome assembly and gene expression analysis in a species lacking genome information. We anticipate that next generation sequencing technologies hold great potential for the study of the transcriptome in other non-model organisms.

Gene expression profiling of resistant and susceptible Bombyx mori strains reveals nucleopolyhedrovirus-associated variations in host gene transcript levels.

We investigated variations in the gene expression of Bombyx mori following infection with a nucleopolyhedrovirus (BmNPV). Two B. mori strains, KN and 306, which are highly resistant and susceptible to BmNPV infection, respectively, were used in this study. The infection profiles of BmNPV in the B. mori KN and 306 larvae revealed that the virus invaded the midguts of both these strains. However, its proliferation was notably inhibited in the midgut of the resistant strain. By using the suppression subtractive hybridization method, two cDNA libraries were constructed in order to compare the BmNPV responsive gene expressions between the two silkworm lines. In total, 62 differentially expressed genes were obtained. Real-time qPCR analysis confirmed that eight genes were significantly up-regulated in the midgut of the KN strain following BmNPV infection. Our results imply that these up-regulated genes may be involved in the B. mori immune response against BmNPV infection.

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.

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.