Amelogenin domain‐containing NlChP38 is necessary for normal ovulation in the brown planthopper

The brown planthopper (BPH), Nilaparvata lugens, is a major threat to rice production. The eggshell plays an important role in insect reproduction. The constituents and formation process of BPH eggshells remains largely unknown. Here, we report a novel eggshell‐associated protein, NlChP38, containing an amelogenin domain, that is essential for normal ovulation in the BPH. NlChP38 is specifically expressed in the follicular cells from egg chambers at both RNA and protein levels. RNA interference of NlChP38 resulted in oocytes with loose and thin eggshell structure and caused ovulation difficulties. Immunofluorescence localization showed NlChP38 is deposited between follicular cells and oocytes during late choriogenesis. These results indicate that NlChP38 plays an important role in eggshell formation and could be a potential target for RNA interference control of the BPH.     

Immunodetection of a brown planthopper (Nilaparvata lugens Stål) salivary catalase‐like protein into tissues of rice,Oryza sativa

Saliva plays an important role in host plant–phloem‐feeding insect molecular interactions. To better elucidate the role of insect saliva, a series of experiments were conducted to establish if catalase from the salivary glands of the brown planthopper (BPH; Nilaparvata lugens Stål) was secreted into rice host plant tissue during feeding. Catalase is the main enzyme that decomposes hydrogen peroxide (H₂O₂) at high concentrations. H₂O₂ is a part of the free radicals system that mediates important physiological roles including signalling and defence. Previous studies have suggested that H₂O₂ is involved in the rice endogenous response to BPH feeding. If, the BPH secretes catalase into host plant tissue this will counter the effects of H₂O₂, from detoxification to interfering with plant signalling and defence mechanisms. When BPHs were fed on a hopper‐resistant rice variety for 24 h, catalase activity in the salivary glands increased 3.5‐fold compared with hoppers fed on a susceptible rice variety. Further supporting evidence of the effects of BPH catalase was demonstrated by immunodetection analyses where results from two independent sources: BPH‐infested rice tissue and BPH‐probed artificial diets, suggest that the BPH secretes catalase‐like protein during feeding. The possible physiological roles of BPH‐secreted catalase are discussed.     

Cycle affects imidacloprid efficiency by mediating cytochrome P450 expression in the brown planthopper Nilaparvata lugens

Circadian clocks influence most behaviours and physiological activities in animals, including daily fluctuations in metabolism. However, how the clock gene cycle influences insects' responses to pesticides has rarely been reported. Here, we provide evidence that cycle affects imidacloprid efficacy by mediating the expression of cytochrome P450 genes in the brown planthopper (BPH) Nilaparvata lugens, a serious insect pest of rice. Survival bioassays showed that the susceptibility of BPH adults to imidacloprid differed significantly between the two time points tested [Zeitgeber Time 8 (ZT8) and ZT4]. After cloning the cycle gene in the BPH (Nlcycle), we found that Nlcycle was expressed at higher levels in the fat body and midgut, and its expression was rhythmic with two peaks. Knockdown of Nlcycle affected the expression levels and rhythms of cytochrome P450 genes as well as susceptibility to imidacloprid. The survival rates of BPH adults after treatment with imidacloprid did not significantly differ between ZT4 and ZT8 after double‐stranded Nlcycle treatment. These findings can be used to improve pesticide use and increase pesticide efficiency in the field.     

Feeding‐based RNA interference of a trehalose phosphate synthase gene in the brown planthopper,Nilaparvata lugens

The brown planthopper, Nilaparvata lugens, is the most devastating rice insect pest to have given rise to an outbreak in recent years. RNA interference (RNAi) is a technological breakthrough that has been developed as a powerful tool for studying gene function and for the highly targeted control of insect pests. Here, we examined the effects of using a feeding‐based RNAi technique to target the gene trehalose phosphate synthase (TPS) in N. lugens. The full‐length cDNA of N. lugens TPS (NlTPS) is 3235 bp and has an open reading frame of 2424 bp, encoding a protein of 807 amino acids. NlTPS was expressed in the fat body, midgut and ovary. Quantitative real‐time PCR (qRT‐PCR) analysis revealed that NlTPS mRNA is expressed continuously with little change during the life of the insect. Efficient silencing of the TPS gene through double‐stranded RNA (dsRNA) feeding led to rapid and significant reduction levels of TPS mRNA and enzymatic activity. Additionally, the development of N. lugens larvae that had been fed with the dsRNA was disturbed, resulting in lethality, and the cumulative survival rates dropped to 75.56, 64.44, 55.56 and 40.00% after continuous ingestion of 0.5 µg/µl dsRNA for 2, 4, 7 and 10 days, respectively. These values were significantly lower than those of the insects in the control group, suggesting that NlTPS dsRNA may be useful as a means of insect pest control.     

Identification and functional analysis of a novel chorion protein essential for egg maturation in the brown planthopper

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

Molecular characterization of insulin‐like peptides in the brown planthopper,Nilaparvata lugens (Hemiptera: Delphacidae)

Insulin‐like peptides (ILPs) including insulin, insulin‐like growth factor (IGF) and relaxin are evolutionarily conserved hormones in metazoans, and they are involved in diverse physiological processes. The migratory brown planthopper (BPH), Nilaparvata lugens, encodes four ILP genes (Nlilp1, Nlilp2, Nlilp3 and Nlilp4) but their physiological roles are largely unknown. Sequence analysis showed that NlILP1 contained a relaxin‐specific G protein‐coupled receptor‐binding motif and a variant motif of cysteine residues, and NlILP2 and NlILP4 resembled vertebrate IGFs. RNA interference (RNAi)‐mediated gene silencing showed that depletion of each of Nlilp1, 2 and 3 significantly delayed the developmental duration of nymphs, and this effect could be exacerbated by double or triple gene depletion. Depletion of Nlilp1, Nlilp2 or Nlilp3 induces the accumulation of glucose, trehalose and glycogen, which is contradictory to depletion of the insulin receptor (NlInR1) in the BPH. Depletion of Nlilp1 significantly enhanced starvation resistance in both females and males although its extent was smaller than NlInR1 depletion. A parental RNAi assay showed that depletion of each of Nlilp1–4 dramatically impaired female fecundity. These findings indicate that NlILP1–4 have redundant and distinct roles in physiological processes in the BPH, thereby enhancing our understanding of the contribution of each NlILP to the ecological success of this species in natural habitats.     

Identification of promoter polymorphisms in the cytochrome P450 CYP6AY1 linked with insecticide resistance in the brown planthopper,Nilaparvata lugens

Imidacloprid resistance in the brown planthopper, Nilaparvata lugens, is primarily the result of the over‐expression of cytochrome P450 monooxygenases. Here, a field‐collected strain of N. lugens was shown to be highly resistant to both imidacloprid and buprofezin. Insecticide exposure and quantitative real‐time PCR revealed that its resistance was mainly associated with a cytochrome P450 gene, CYP6AY1. CYP6AY1 is known to metabolize imidacloprid but its effect on buprofezin is unclear. In the 5′‐untranslated region of CYP6AY1, a novel alternative splicing was detected. After a 1990‐bp promoter region was cloned, its basal luciferase activity was assessed. Furthermore, genotyping studies identified 12 variations in the promoter region that discriminated between the field‐collected and control strain. Finally, survival bioassays revealed a single nucleotide polymorphism and an insertion‐deletion polymorphism linked to buprofezin and imidacloprid resistance. Mutagenesis of these sites enhanced the promoter activity of CYP6AY1. These results suggest that promoter polymorphisms may affect P450‐mediated multiple insecticide resistance of pests.