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The damage caused by the white-back planthopper (WBPH, Sogatella furcifera ) and brown planthopper (BPH, Nilaparvata lugens ), as well as southern rice black-streaked dwarf virus (SRBSDV), considerably decreases the grain yield of rice. Identification of rice germplasms with sufficient resistance to planthoppers and SRBSDV is essential to the breeding and deployment of resistant varieties and, hence, the control of the pests and disease. In this study, 318 rice accessions were evaluated for their reactions to the infestation of both BPH and WBPH at the seedling stage using the standard seed-box screening test method; insect quantification was further conducted at the end of the tillering and grain-filling stages in field trials. Accessions HN12-239 and HN12-328 were resistant to both BPH and WBPH at all tested stages. Field trials were conducted to identify resistance in the collection to SRBSDV based on the virus infection rate under artificial inoculation. Rathu Heenati (RHT) and HN12-239 were moderately resistant to SRBSDV. In addition, we found that WBPH did not penetrate stems with stylets but did do more probing bouts and xylem sap ingestion when feeding on HN12-239 than the susceptible control rice Taichung Native 1. The resistance of rice accessions HN12-239, HN12-328, and RHT to BPH, WBPH, and/or SRBSDV should be valuable to the development of resistant rice varieties., Competing Interests: The author(s) declare no conflict of interest.

Background: The brown planthopper (Nilaparvata lugens Stål)is a notorious rice pest in many areas of Asia. Study on the molecular mechanisms underlying its development and reproduction will provide scientific basis for effective control. SPARC (Secreted Protein, Acidic and Rich in Cysteine) is one of structural component of the extracellular matrix, which influences a diverse array of biological functions. In this study, the gene for SPARC was identified and functionally analysed from N.lugens., Results: The result showed that the NlSPARC mRNA was highly expressed in fat body, hemolymph and early embryo. The mortality increased significantly when NlSPARC was downregulated after RNA interference (RNAi) in 3 ~ 4 th instar nymphs. Downregulation of NlSPARC in adults significantly reduced the number of eggs and offspring, as well as the transcription level of NlSPARC in newly hatched nymphs and survival rate in progeny. The observation with microanatomy on individuals after NlSPARC RNAi showed smaller and less abundant fat body than that in control. No obvious morphological abnormalities in the nymphal development and no differences in development of internal reproductive organ were observed when compared with control., Conclusion: NlSPARC is required for oviposition and nymphal development mainly through regulating the tissue of fat body in N.lugens. NlSPARC could be a new candidate target for controlling the rapid propagation of N.lugens population. Our results also demonstrated that the effect of NlSPARC RNAi can transfer to the next generation in N.lugens., (© 2022. The Author(s).)

The brown planthopper (BPH), Nilaparvata lugens (Stål), is one of the most destructive pests in rice-growing regions of Asia. Extensive studies have suggested that SWI/SNF chromatin remodeling ATPase Brahma (BRM) plays multiple roles in the insect model Drosophila. Yet much less is known about the physiological properties for NlBRM. In the present study, the cloned full-length cDNA of NlBRM was 5637 bp and contained an ORF of 5292 bp encoding a 194.53 kD protein. The spatiotemporal dynamics of NlBRM was investigated by qPCR, which showed that it was abundantly expressed in the egg and ovary. Then significant downregulation of NlBRM by dsRNA injection had a relatively greater impact on female survival than male. Moreover, the number of oviposition marks of the NlBRM-RNAi females were declined by 61.11% - 73.33% compared with the controls during the subsequent 5 days after dsRNA injection. Meanwhile, the number of newly hatched BPH nymphs also decreased correspondingly by 93.56% - 100%. Phenotypic analysis revealed that none of normally banana-shaped eggs were discernable in the ovaries of NlBRM-deficient females, where mRNA expression of N. lugens vitellogenin gene was also reduced. Our results demonstrated that NlBRM played a crucial role in ovarian development and fecundity of BPH, likely by regulating the vitellogenin gene in vivo, which could be as a promising target for parental RNAi-based control of this serious rice pest., (Copyright © 2022. Published by Elsevier Inc.)

Viral pathogens are a major threat to stable crop production. Using a backcross strategy, we find that integrating a dominant brown planthopper (BPH) resistance gene Bph3 into a high-yield and BPH-susceptible indica rice variety significantly enhances BPH resistance. However, when Bph3 -carrying backcross lines are infested with BPH, these BPH-resistant lines exhibit sterile characteristics, displaying panicle enclosure and failure of seed production at their mature stage. As we suspected, BPH-mediated viral infections could cause the observed sterile symptoms, and we characterized rice-infecting viruses using deep metatranscriptomic sequencing. Our analyses revealed eight novel virus species and five known viruses, including a highly divergent virus clustered within a currently unclassified family. Additionally, we characterized rice plant antiviral responses using small RNA sequencing. The results revealed abundant virus-derived small interfering RNAs in sterile rice plants, providing evidence for Dicer-like and Argonaute-mediated immune responses in rice plants. Together, our results provide insights into the diversity of viruses in rice plants, and our findings suggest that multiple virus infections occur in rice plants.

Alternative splicing of protein coding genes plays a profound role in phenotypic variation for many eukaryotic organisms. The development of high-throughput sequencing and bioinformatics algorithms provides the possibility of genome-wide identification of alternative splicing events in eukaryotes. However, for the brown planthopper Nilaparvata lugens, a destructive pest of rice crops, whole-genome distribution of alternative splicing events and the role of alternative splicing in the phenotypic plasticity of virulence have not previously been estimated. Here, we developed an analysis pipeline to identify alternative splicing events in the genome of N. lugens. Differential expression analysis and functional annotation were performed on datasets related to different virulence phenotypes. In total, 27,880 alternative splicing events corresponding to 9,787 multi-exon genes were detected in N. lugens. Among them, specifically expressed alternative splicing transcripts in the virulent Mudgo population were enriched in metabolic process categories, while transcripts in the avirulent TN1 population were enriched in regulator activity categories. In addition, genes encoding odorant receptor, secreted saliva protein and xenobiotic metabolic P450 monooxygenase showed different splicing patterns between Mudgo population and TN1 population. Host change experiment also revealed that an isoform of a P450 gene could be specially induced by the stimulation of resistant rice variety Mudgo. This research pioneered a genome-wide study of alternative gene splicing in the rice brown planthopper. Differences in alternative splicing between virulent and avirulent populations indicated that alternative splicing might play an important role in the formation of virulence phenotypes in N. lugens., (© The Author(s) 2021. Published by Oxford University Press on behalf of Entomological Society of America. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Rice production and sustainability are challenged by its most dreadful pest, the brown planthopper (Nilaparvata lugens Stål, BPH). Therefore, the studies on rice-BPH interactions and their underlying mechanisms are of high interest. The rice ontogenetic defense, such as the role of microRNAs (miRNAs) has mostly been investigated against the pathogens, with only a few reports existing against the insect infestations. Thus, revealing the involvement of rice miRNAs in response to BPH infestations will be beneficial in understanding these complex interactions. In this study, the small RNA profiling of the IR56 rice in response to separate BPH infestations of varied virulence levels identified the BPH-responsive miRNAs and revealed the differential transcript abundance of several miRNAs during a compatible and incompatible rice-BPH interaction. The miRNA sequence analysis identified 218 known and 28 novel miRNAs distributed in 54 miRNA families. Additionally, 138 and 140 numbers of differentially expressed (DE) miRNAs were identified during the compatible and incompatible interaction, respectively. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis revealed the target gene candidates of DE miRNAs (including osa-miR2871a-3p, osa-miR172a, osa-miR166a-5p, osa-miR2120, and osa-miR1859) that might be involved in the IR56 rice defense responses against BPH infestation. Conversely, osa-miR530-5p, osa-miR812s, osa-miR2118g, osa-miR156l-5p, osa-miR435 and two of the novel miRNAs, including novel_16 and novel_52 might negatively modulate the IR56 rice defense. The expressional validation of the selected miRNAs and their targets further supported the IR56 rice defense regulatory network. Based on our results, we have proposed a conceptual model depicting the miRNA defense regulatory network in the IR56 rice against BPH infestation. The findings from the study add further insights into the molecular mechanisms of rice-BPH interactions and will be helpful for the future researches.

Background: The application of RNA interference (RNAi) technique in controlling agricultural insect pests has been receiving much attention since the discovery of RNAi. The brown planthopper (BPH) Nilaparvata lugens, a notorious pest of rice, has evolved a high level of resistance to many kinds of insecticides. Tyrosine hydroxylase (Th) is an indispensable survival gene in holometabolous insects, playing key roles in cuticle tanning and immunity. In this study, we investigated whether Th could be used as a potential target in controlling N. lugens., Results: Here, we demonstrated that NlTh had a periodical expression pattern during molting with the highest level observed in epidermis. Dysfunction of NlTH by dsNlTh microinjection or 3-IT feeding similarly caused rapid death of N. lugens. Compared with dsGFP control BPHs, dsNlTh injected BPHs (i) had cuticle pigmentation and sclerotizaton defects; (ii) had less endocuticle lamella in tergum integument; (iii) showed higher mortality during the molting process as a result of defective cuticle shedding; (iv) showed feeding disorders indicated by a low number of probe wound dots on rice; (v) had more vulnerable cuticle., Conclusion: This study demonstrated that TH orthologues play a conservative and crucial role for exocuticle tanning in both holometabolous and hemimetabolous insects, and NlTh could be targeted for RNAi-mediated BPH control. The rapid lethal phenotype of NlTH dysfunction BPHs partly induced by cuticle formation defects. © 2020 Society of Chemical Industry., (© 2020 Society of Chemical Industry.)

The Bph1 gene was the first reported brown planthopper (BPH, Nilaparvata lugens) resistance gene in Mudgo rice and was widely used as a commercial cultivar for controlling BPH infestations. However, rapid adaptations of BPH on the Mudgo rice resulted in its resistance breakdown and the emergence of virulent BPH populations. Thus, specific BPH populations and rice varieties can serve as good model systems for studying the roles of different bio-compounds and proteins in the insect-plant interactions. Although our understandings have been improved on the complexity of BPH and rice interactions, the underlying molecular mechanisms remain largely unknown. Here we analyzed the feeding performances and the transcriptomic responses of two BPH populations (Mugdo-BPH and TN1-BPH) during compatible (Mudog-BPH feeding on Mudgo rice) and incompatible (TN1-BPH feeding on Mudgo rice) interactions. The electrical penetration graph (EPG) results indicated that the BPH feeding and performances during the incompatible interaction are significantly affected in terms of decreased honeydew, loss of weight, decreased phloem sap ingestion (N4 waveform), but increased non-penetration (NP waveform) phase. Abundance of glucose and trehalose was reduced in BPH during the incompatible interaction. Transcriptomic surveys of insects in both interactions revealed that genes involved in cuticle formation, detoxification, metabolite transport, digestion, RNA processing, lipid or fatty acid metabolism, and proteolysis were significantly down-regulated during the incompatible interaction, whereas genes involved in insulin signaling were significantly upregulated. Knockdown of four genes, including the sugar transporter NlST45, the serine and arginine-rich protein NlSRp54, the cytochrome P450 gene NlCYP6AY1, and the cuticle protein NlCPR70 through RNA-interference revealed thess genes are important for BPH survival. Overall, the results of this study will be helpful for the future researches on BPH virulence shifts.

This study reports the identification of splice variants for the calcium/calmodulin-dependent protein kinase II ( CaMKII ) gene from Nilaparvata lugens , Laodelphax striatellus , and Sogatella furcifera . CaMKII is a multifunctional serine/threonine protein kinase that transduces Ca 2+ signals in cells to control a range of cellular processes in the nervous system and muscular tissue. Sequence analysis showed that CaMKII was 99.0% identical at the amino acid level among three rice planthoppers, with the exception of a variable region located in the association domain. Four kinds of 20-81 amino acid "inserts" were found in the variable region. The phylogenetic tree of the deduced amino acid sequences showed that the NlCaMKII isoforms were more closely related to the LsCaMKII isoforms and were slightly distinct from SfCaMKII. CaMKII-E was the dominant type among the five main isoforms. CaMKII genes were constitutively expressed in various nymphal and adult stages and in tested tissues with the predominant transcription occurring in the head. There was no major tissue specificity of isoform expression, but the expression pattern and relative abundance of isoforms varied when compared with the RT-PCR between tissues. In addition, RNAi in N. lugens with dsRNA at a concentration of 200 ng nymph -1 induced a mortality of 77.7% on the 10th day and a reduction in the mRNA expression level of 67.2%. Unlike the holometabolous insect Helicoverpa armigera , the knockdown of NlCaMKII did not suppress the expression of 20E response genes, such as ECR , USP1 , and HR3 , in N. lugens . These results indicate that the role of CaMKII in hemimetabolous insects may be different from that in holometabolous insects.

Ommochrome is the major source for eye coloration of all insect species so far examined. Phenoxazinone synthetase (PHS) has always been regarded as the terminal step enzyme for ommochrome formation, which is encoded by cardinal or karmoisin genes. Our previous study indicated that the karmoisin ortholog gene (Nl-karmoisin) product in the brown planthopper (BPH) was a monocarboxylate transporter, while not a PHS. Here, based on full-length complementary DNA, the cardinal ortholog gene in BPH (Nl-cardinal) product was predicted to be a haem peroxidase rather than a PHS. We suggest for the first time that neither karmoisin nor cardinal encodes the PHS, but whether PHS participates in BPH eye pigmentation needs further research. Nymphal RNA interference (RNAi) experiments showed that knockdown Nl-cardinal transcript led the BPH ocelli and compound eye to color change from brown to red, while knockdown Nl-karmoisin only made the ocelli present the red phenotype. Notably, not only the Nl-cardinal transcript, dscd injection (Nl-cardinal targeting double-stranded DNA (dsRNA)) also significantly reduced the Nl-karmoisin transcript by 33.7%, while dska (Nl-karmoisin targeting dsRNA) injection did not significantly change the Nl-cardinal transcript. Considering the above RNAi and quantitative real-time polymerase chain reaction results, we propose that Nl-cardinal plays a more important role in ommochrome synthesis than Nl-karmoisin, and it may be an upstream gene of Nl-karmoisin. The present study suggested that both karmoisin and cardinal ortholog genes play a role in ommochrome synthesis in a hemimetabolous insect., (© 2017 Institute of Zoology, Chinese Academy of Sciences.)

The brown planthopper (BPH), Nilaparvata lugens (Stål) (Hemiptera: Delphacidae), is a destructive rice pest of Asia. Currently, one important monitoring method of BPH is through black light trapping. However, two sibling species of BPH, Nilaparvata bakeri (Muri) and Nilaparvata muiri China, can also be trapped by black light, and these species feed only on gramineous weeds rather than on rice. Therefore, the accurate identification of Nilaparvata species is crucial for N. lugens forecasting and management. The traditional morphological identification method is not feasible for subadults and damaged specimens. Furthermore, this error-prone morphological identification method is time and labor intensive, with the need for expertise and experience. Here, we established a direct multiplex polymerase chain reaction (dmPCR) assay using crude tissue fluid as a template, omitting purified DNA extraction. The crude tissue fluid can be obtained by grinding specimens without any biological reagent but only using distilled water. This dmPCR assay, using three pairs of diagnostic primers, is based on internal transcribed spacers (ITS). Each primer pair amplifies a species-specific fragment of a different size, which were easily and reliably separated in a 2% agarose gel. Furthermore, the dmPCR was verified to be applicable to damaged tissue specimens, such as head, thorax, or abdomen. In conclusion, this dmPCR assay is a novel, time-saving, cost-effective, and easy-to-apply molecular diagnostic method for the identification of the above three sibling species, N. lugens, N. bakeri, and N. muiri.

The conserved mitogen-activated protein kinase (MAPK) cascades play vital roles in plant defense responses against pathogens and insects. In the current study, the expression profiles of 17 OsMPK s were determined in the TN1 and IR56 rice varieties under the infestation of brown planthopper (BPH), one of the most destructive hemimetabolous rice pests. The virulent IR56 BPH population (IR56-BPH) and the avirulent TN1 BPH population (TN-BPH) were used to reveal the roles of OsMPK s in the compatible (IR56-BPH infested on the TN1 and IR56 rice varieties, and TN1-BPH infested on the TN1 rice variety) and the incompatible (TN1-BPH infested on the IR56 rice variety) interaction. The statistical analysis revealed that rice variety, BPH population type, and infestation period have significant effects on the transcription of OsMPK s. Out of these genes, five OsMPK s ( OsMPK1 , OsMPK3 , OsMPK7 , OsMPK14 , and OsMPK16 ) were found to exhibit upregulated expression only during incompatible interaction. Six OsMPK s ( OsMPK4 , OsMPK5 , OsMPK8 , OsMPK9 , OsMPK12 , and OsMPK13 ) were associated with both incompatible and compatible interactions. The transcription analysis of salicylic acid, jasmonic acid, and ethylene phytohormone signaling genes revealed their roles during the rice⁻BPH interactions. The upregulated expression of OsC4H , OsCHS , and OsCHI in the incompatible interaction implied the potential defense regulatory roles of phenylpropanoids. In both varieties, the elevated transcript accumulations of OsGST and OsSOD , and the increased enzyme activities of POD, SOD, and GST at 1 day post-infestation (dpi), but not at 3 dpi, indicated that reactive oxygen species (ROS) signaling might be an early event in rice⁻BPH interactions. Furthermore, upregulated transcription of OsLecRK3 and OsLecRK4 was found only during an incompatible interaction, suggesting their involvement in the BPH resistance response in the IR56 rice variety. Lastly, based on the findings of this study, we have proposed a model of interactions of IR56 rice with TN1-BPH and IR56-BPH that depicts the resistance and susceptibility reactions, respectively.

In the present paper, four cDNAs encoding the alpha and gamma subunits of elongation factor 1 (EF-1) were cloned and sequenced from Nilaparvata lugens, named NlEF-1α, NlEF-1γ, and its yeast-like symbiont (YLS), named YsEF-1α and YsEF-1γ, respectively. Comparisons with sequences from other species indicated a greater conservation for EF-1α than for EF-1γ. NlEF-1α has two identical copies. The deduced amino acid sequence homology of NlEF-1α and NlEF-1γ is 96 and 64%, respectively, compared with Homalodisca vitripennis and Locusta migratoria. The deduced amino acid sequence homology of YsEF-1α and YsEF-1γ is 96 and 74%, respectively, compared with Metarhizium anisopliae and Ophiocordyceps sinensis. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) analysis revealed that the expression level of NlEF-1α and NlEF-1γ mRNA in hemolymph, ovary, fat body and salivary glands were higher than the midgut and leg tissue. YsEF-1α and YsEF-1γ was highly expressed in fat body. The expression level of NlEF-1α was higher than that of NlEF-1γ. Through RNA interference (RNAi) of the two genes, the mortality of nymph reached 92.2% at the 11th day after treatment and the ovarian development was severely hindered. The RT-qPCR analysis verified the correlation between mortality, sterility and the down-regulation of the target genes. The expression and synthesis of vitellogenin (Vg) protein in insects injected with NlEF-1α and NlEF-1γ double-stranded RNA (dsRNA) was significantly lower than control groups. Attempts to knockdown the YsEF-1 genes in the YLS was unsuccessful. However, the phenotype of N. lugens injected with YsEF-1α dsRNA was the same as that injected with NlEF-1α dsRNA, possibly due to the high similarity (up to 71.9%) in the nucleotide sequences between NlEF-1α and YsEF-1α. We demonstrated that partial silencing of NlEF-1α and NlEF-1γ genes caused lethal and sterility effect on N. lugens. NlEF-1γ shares low identity with that of other insects and therefore it could be a potential target for RNAi-based pest management.

Nilaparvata lugens is a typical phloem feeder. Rice phloem is high in simple sugars and very low in essential amino acids. Nilaparvata lugens harbors an ascomycete Entomomyces delphacidicola that hypothetically biosynthesizes several amino acids to meet the nutrition requirement of the planthopper. Among these amino acids, here, we focused on arginine biosynthesis. A complete cDNA of an E. delphacidicola gene, arginine-succinate lyase, EdArg4, the last step in arginine biosynthesis, was obtained. RNAi-mediated suppression of EdArg4 reduced arginine content in the hemolymph, and decreased the expression of several arginine biosynthesis genes. Silencing of EdArg4 delayed nymphal development and led to nymphal lethality. About 20% of the EdArg4 RNAi surviving adults were deformed. The most obvious defect was wider and larger abdomen. The EdArg4 RNAi-treated planthoppers had thickened wings and enlarged antennae, legs, and anal tubes and a few adults did not normally emerge. Arginine deficiency in the EdArg4 RNAi planthoppers repressed nitric oxide signaling, determined at the transcriptional level. We infer that E. delphacidicola biosynthesizes essential arginine to compensate for nutrition deficiency in N. lugens., (© 2017 Wiley Periodicals, Inc.)

Twenty-nine cDNAs encoding Ras-like family small GTPases (RSGs) were cloned and sequenced from Nilaparvata lugens. Twenty-eight proteins are described here: 3 from Rho, 2 from Ras, 9 from Arf and 14 from Rabs. These RSGs from N.lugens have five conserved G-loop motifs and displayed a higher degree of sequence conservation with orthologues from insects. RT-qPCR analysis revealed NlRSGs expressed at all life stages and the highest expression was observed in hemolymph, gut or wing for most of NlRSGs. RNAi demonstrated that eighteen NlRSGs play a crucial role in nymphal development. Nymphs with silenced NlRSGs failed to molt, eclosion or development arrest. The qRT-PCR analysis verified the correlation between mortality and the down-regulation of the target genes. The expression level of nuclear receptors, Kr-h1, Hr3, FTZ-F1 and E93 involved in 20E and JH signal pathway was impacted in nymphs with silenced twelve NlRSGs individually. The expression of two halloween genes, Cyp314a1 and Cyp315a1 involved in ecdysone synthesis, decreased in nymphs with silenced NlSar1 or NlArf1. Cyp307a1 increased in nymphs with silenced NlArf6. In N.lugens with silenced NlSRβ, NlSar1 and NlRab2 at 9th day individually, 0.0% eclosion rate and almost 100.0% mortality was demonstrated. Further analysis showed NlSRβ could be served as a candidate target for dsRNA-based pesticides for N.lugens control.

Nilaparvata lugens (Stål) (Hemiptera: Delphacidae) is a major rice pest that harbors an endosymbiont ascomycete fungus, Entomomyces delphacidicola str. NLU (also known as yeast-like symbiont, YLS). Driving by demand of novel population management tactics (e.g. RNAi), the importance of YLS has been studied and revealed, which greatly boosts the interest of molecular level studies related to YLS. The current study focuses on reference genes for RT-qPCR studies related to YLS. Eight previously unreported YLS genes were cloned, and their expressions were evaluated for N. lugens samples of different developmental stages and sexes, and under different nutritional conditions and temperatures. Expression stabilities were analyzed by BestKeeper, geNorm, NormFinder, ΔCt method and RefFinder. Furthermore, the selected reference genes for RT-qPCR of YLS genes were validated using targeted YLS genes that respond to different nutritional conditions (amino acid deprivation) and RNAi. The results suggest that ylsRPS15p/ylsACT are the most suitable reference genes for temporal gene expression profiling, while ylsTUB/ylsACT and ylsRPS15e/ylsGADPH are the most suitable reference gene choices for evaluating nutrition and temperature effects. Validation studies demonstrated the advantage of using endogenous YLS reference genes for YLS studies., Competing Interests: The authors declare no competing financial interests.

Histidine is an essential amino acid assumed to be synthesized by an obligatory yeast-like symbiont (Entomomyces delphacidicola str. NLU) in Nilaparvata lugens, an important rice pest. The adenosine-triphosphate phosphoribosyltransferase (ATP-PRTase) facilities the committed first step of the histidine biosynthesis pathway. In the current study, a putative ATP-PRTase was cloned and verified to be of E. delphacidicola origin (EdePRTase). The expression of the gene was spatial and temporal universal with a profile that matched the distribution of the fungal symbiont. RNA interference aided the knockdown of the EdePRTase-suppressed EdePRTase expression by 32-48 %. Hemolymph histidine level was also reduced followed by significant reduction of adult body weight. However, other performance characters including nymph development, survival, and adult sex ratio were not adversely affected by the knockdown. Furthermore, forced histidine exposure (through injection or feeding) significantly inhibited the EdePRTase mRNA levels at higher concentrations, but significantly increased EdePRTase expression levels at lower concentrations (feeding only). The significance of these findings support that the EdePRTase is from symbiont E. delphacidicola, and its involvement in histidine biosynthesis of N. lugens was discussed. The results provide a better understanding of EdePRTase and the encoded functional ATP-PRTase enzyme regulation in N. lugens and insects in general.

Ran (RanGTPase) in insects participates in the 20-hydroxyecdysone signal transduction pathway in which downstream genes, FTZ-F1, Krüppel-homolog 1 (Kr-h1) and vitellogenin, are involved. A putative Ran gene (NlRan) was cloned from Nilaparvata lugens, a destructive phloem-feeding pest of rice. NlRan has the typical Ran primary structure features that are conserved in insects. NlRan showed higher mRNA abundance immediately after molting and peaked in newly emerged female adults. Among the examined tissues ovary had the highest transcript level, followed by fat body, midgut and integument, and legs. Three days after dsNlRan injection the NlRan mRNA abundance in the third-, fourth-, and fifth-instar nymphs was decreased by 94.3%, 98.4% and 97.0%, respectively. NlFTZ-F1 expression levels in treated third- and fourth-instar nymphs were reduced by 89.3% and 23.8%, respectively. In contrast, NlKr-h1 mRNA levels were up-regulated by 67.5 and 1.5 folds, respectively. NlRan knockdown significantly decreased the body weights, delayed development, and killed >85% of the nymphs at day seven. Two apparent phenotypic defects were observed: (1) Extended body form, and failed to molt; (2) The cuticle at the notum was split open but cannot completely shed off. The newly emerged female adults from dsNlRan injected fifth-instar nymphs showed lower levels of NlRan and vitellogenin, lower weight gain and honeydew excretion comparing with the blank control, and no offspring. Those results suggest that NlRan encodes a functional protein that was involved in development and reproduction. The study established proof of concept that NlRan could serve as a target for dsRNA-based pesticides for N. lugens control.

The brown planthopper, Nilaparvata lugens (Stål), is a destructive insect pest on rice throughout Asia. As a visible genetic marker, red eye mutant colony of brown planthopper is a valuable material. Here, we established the near-isogenic lines, NIL-BB and NIL-rr, through mating red eye females to brown eye brothers for eight successive generations. Biological experiments showed that NIL-BB had big fitness cost; however, NIL-rr had comparable survival and fertility parameters with BB, a normal laboratory brown planthopper strain. Significantly lower number eggs per female and egg hatchability were the key factors resulting in big fitness cost of NIL-BB. The population trend indexes of BB, NIL-rr, and NIL-BB were 52.18, 43.80, and 4.19, respectively. Real-time PCR study suggested that the poorer fertility of NIL-BB was not mediated by the differential expression of genes relating to oogenesis. The stronger fitness of NIL-rr compared with NIL-BB may be caused by the eye mutant gene or its closely linked genes having stronger compensation ability for reproduction. The comparable fitness of NIL-rr with BB indicated that NIL-rr may be used in field research. The NIL-BB strain with significantly declined fecundity and survival ability can be used as study model for the signal pathways relating to fecundity., (© The Authors 2015. Published by Oxford University Press on behalf of Entomological Society of America. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

The brown planthopper Nilaparvata lugens is a serious phloem-feeding pest of rice in China. The current study focuses on a saccharopine dehydrogenase (SDH) that catalyzes the penultimate reaction in biosynthesis of the amino acid lysine (Lys), which plays a role in insect growth and carnitine production (as a substrate). The protein, provisionally designated as NlylsSDH [a SDH derived from yeast-like symbiont (YLS) in N. lugens], had a higher transcript level in abdomens, compared with heads, wings, legs and thoraces, which agrees with YLS distribution in N. lugens. Ingestion of Nlylssdh targeted double-stranded RNA (dsNlylssdh) for 5, 10 and 15 days decreased the mRNA abundance in the hoppers by 47, 70 and 31%, respectively, comparing with those ingesting normal or dsegfp diets. Nlylssdh knockdown slightly decreased the body weights, significantly delayed the development of females, and killed approximately 30% of the nymphs. Moreover, some surviving adults showed two apparent phenotypic defects: wing deformation and nymphal cuticles remained on tips of the legs and abdomens. The brachypterours/macropterours and sex ratios (female/male) of the adults on the dsRNA diet were lowered compared with the adults on diets without dsRNA. These results suggest that Nlylssdh encodes a functional SDH protein. The adverse effect of Nlylssdh knockdown on N. lugens implies the importance of Lys in hopper development. This study provides a proof of concept example that Nlylssdh could serve as a possible dsRNA-based pesticide for planthopper control.

Nilaparvata lugens harbors yeast-like symbionts (YLSs). In present paper, a genome-wide analysis found 115 genes from Ni. lugens and 90 genes from YLSs that were involved in the metabolic degradation of 20 proteinogenic amino acids. These 205 genes encoded for 77 enzymes. Accordingly, the degradation pathways for the 20 amino acids were manually constructed. It is postulated that Ni. lugens can independently degrade fourteen amino acids (threonine, alanine, glycine, serine, aspartate, asparagine, phenylalanine, tyrosine, glutamate, glutamine, proline, histidine, leucine and lysine). Ni. lugens and YLSs enzymes may work collaboratively to break down tryptophan, cysteine, arginine, isoleucine, methionine and valine. We cloned a lysine-ketoglutarate reductase/saccharopine dehydrogenase gene (Nllkr/sdh) that encoded a bifunctional enzyme catalyzing the first two steps of lysine catabolism. Nllkr/sdh is widely expressed in the first through fifth instar nymphs and adults, and is highly expressed in the fat body, ovary and gut in adults. Ingestion of dsNllkr/sdh by nymphs successfully knocked down the target gene, and caused nymphal/adult mortality, shortened nymphal development stage and reduced adult fresh weight. Moreover, Nllkr/sdh knockdown resulted in three defects: wings were shortened and thickened; cuticles were stretched and thinned; and old nymphal cuticles remained on the tips of legs and abdomen and were not completely shed. These data indicate that impaired lysine degradation negatively affects the survival and development of Ni. lugens.

A red-eye colony was established in our laboratory in brown planthopper (BPH), Nilaparvata lugens (Stål), a major rice pest in Asia. Except for the red-eye phenotype, no other differences were observed between the wild-type (brown eye) and the mutant-type (red eye) in external characters. Genetic analysis revealed that the red-eye phenotype was controlled by a single autosomal recessive allele. Biological studies found that egg production and egg viability in the red-eye mutant colony were not significantly different from those in the wild-type BPH. Biochemical analysis and electronic microscopy examination revealed that the red-eye mutants contained decreased levels of both xanthommatin (brown) and pteridine (red) and reduced number of pigment granules. Thus, the changes of amount and ratio of the two pigments is the biochemical basis of this red-eye mutation. Our results indicate that the red-eye mutant gene (red) might be involved in one common gene locus shared by the two pigments in pigment transportation, pigment granule formation or some other processes., (© 2013 Institute of Zoology, Chinese Academy of Sciences.)

Nilaparvata lugens is a serious phloem-feeding pest of rice throughout Asia. Rice phloem sap can meet its nutrition requirement for sugars but not for some essential amino acids such as isoleucine, leucine, methionine, phenylalanine, tryptophan, lysine, arginine and histidine. N. lugens harbours yeast-like symbionts in mycetocytes formed by abdominal fat body cells. Removal of the symbionts results in negative physiological effects, suggesting that the symbionts play a pivotal role in the nitrogen metabolism. In the present paper, 521 mRNA expressed sequence tags (ESTs) encoding 126 enzymes that were involved in amino acid biosynthesis were identified based on a transcriptome data, reverse transcription (RT)-PCR and rapid amplification of cDNA ends. Similarity analysis, codon usage bias, along with tissue-biased expression and phylogenetic analysis of a subset of ESTs, suggest that 437 ESTs out of the 521 originate from symbionts, and the remaining 84 mRNA fragments come from N. lugens. Accordingly, the biosynthesis pathways for 20 amino acids were manually constructed. It is postulated that both N. lugens and its symbiont can independently assimilate ammonia and biosynthesize seven non-essential amino acids: glutamate; glutamine; aspartate; asparagine; alanine; serine; and glycine. N. lugens and symbiont enzymes may work collaboratively to catalyse the biosynthesis of proline, methionine, valine, leucine, isoleucine, phenylalanine and tyrosine. We infer from this that symbionts function in the biosynthesis of lysine, arginine, tryptophan, threonine, histidine and cysteine. Our data support the previously proposed hypothesis, i.e. the yeast-like symbionts compensate for, at least partially, the amino acid needs of N. lugens., (© 2013 The Royal Entomological Society.)