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.     

Overexpression of a cytochrome P450 monooxygenase, CYP6ER1, is associated with resistance to imidacloprid in the brown planthopper, Nilaparvata lugens

The brown planthopper, Nilaparvata lugens, is an economically significant pest of rice throughout Asia and has evolved resistance to many insecticides including the neonicotinoid imidacloprid. The resistance of field populations of N. lugens to imidacloprid has been attributed to enhanced detoxification by cytochrome P450 monooxygenases (P450s), although, to date, the causative P450(s) has (have) not been identified. In the present study, biochemical assays using the model substrate 7-ethoxycoumarin showed enhanced P450 activity in several resistant N. lugens field strains when compared with a susceptible reference strain. Thirty three cDNA sequences encoding tentative unique P450s were identified from two recent sequencing projects and by degenerate PCR. The mRNA expression level of 32 of these was examined in susceptible, moderately resistant and highly resistant N. lugens strains using quantitative real-time PCR. A single P450 gene (CYP6ER1) was highly overexpressed in all resistant strains (up to 40-fold) and the level of expression observed in the different N. lugens strains was significantly correlated with the resistance phenotype. These results provide strong evidence for a role of CYP6ER1 in the resistance of N. lugens to imidacloprid.     

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.     

Molecular cloning and immunolocalization of a diuretic hormone receptor in rice brown planthopper (Nilaparvata lugens)

RNA extracted from guts of rice brown planthopper, Nilaparvata lugens, was used to clone cDNA predicted to encode a diuretic hormone receptor (DHR). The DHR, a member of the calcitonin/secretin/corticotropin-releasing factor family of G-protein-coupled receptors, contains seven transmembrane domains and a large N-terminal extracellular domain potentially involved in hormone binding. The N-terminal domain was expressed as a recombinant protein, purified and used to raise antibodies. Anti-DHR IgG bound specifically to Malpighian tubules in immunolocalization experiments using dissected guts, and to a putative DHR polypeptide from N. lugens gut on Western blots. Anti-DHR IgG delivered orally to insects was not detected in the haemolymph, and showed no binding to gut or tubules, confirming that DHR N-terminal hormone-binding domain is not exposed to the gut lumen.     

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.     

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.     

Molecular characterization of the amplified carboxylesterase gene associated with organophosphorus insecticide resistance in the brown planthopper, Nilaparvata lugens

Widespread resistance to organophosphorus insecticides (OPs) in Nilaparvata lugens is associated with elevation of carboxylesterase activity. A cDNA encoding a carboxylesterase, Nl-EST1, has been isolated from an OP-resistant Sri Lankan strain of N. lugens. The full-length cDNA codes for a 547-amino acid protein with high homology to other esterases/lipases. Nl-EST1 has an N-terminal hydrophobic signal peptide sequence of 24 amino acids which suggests that the mature protein is secreted from cells expressing it. The nucleotide sequence of the homologue of Nl-EST1 in an OP-susceptible, low esterase Sri Lankan strain of N. lugens is identical to Nl-EST1. Southern analysis of genomic DNA from the Sri Lankan OP-resistant and susceptible strains suggests that Nl-EST1 is amplified in the resistant strain. Therefore, resistance to OPs in the Sri Lankan strain is through amplification of a gene identical to that found in the susceptible strain.     

Selectivity of lynx proteins on insect nicotinic acetylcholine receptors in the brown planthopper,Nilaparvata lugens

Neuronal nicotinic acetylcholine receptors (nAChRs) are major excitatory neurotransmitter receptors in both vertebrates and invertebrates. Two lynx proteins (Nl‐lynx1 and Nl‐lynx2) have been identified in the brown planthopper, Nilaparvata lugens, which act as modulators on insect nAChRs. In the present study, two lynx proteins were found to act on the triplet receptor Nlα1/Nlα2/β2 expressed in Xenopus oocytes, increasing agonist‐evoked macroscopic currents, but not changing agonist sensitivity and desensitization properties. Nl‐lynx1 and Nl‐lynx2 increased I_max (maximum responses) of acetylcholine to 4.85‐fold and 2.40‐fold of that of Nlα1/Nlα2/β2 alone, and they also increased I_max of imidacloprid to 2.57‐fold and 1.25‐fold. Although, on another triplet nAChRs Nlα3/Nlα8/β2, Nl‐lynx2 increased I_max of acetylcholine and imidacloprid to 3.63‐fold and 2.16‐fold, Nl‐lynx1 had no effects on I_max of either acetylcholine or imidacloprid. The results demonstrate the selectivity of lynx proteins for different insect nAChR subtypes. This selectivity was also identified in native N. Lugens. Co‐immunoprecipitation was found between Nlα1/Nlα2‐containing receptors and both Nl‐lynx1 and Nl‐lynx2, but was only found between Nlα3/Nlα8‐containing receptors and Nl‐lynx2. When the previously identified Nlα1^Y151S and Nlα3^Y151S mutations were included (Nlα1^Y151S/Nlα2/β2 and Nlα3^Y151S/Nlα8/β2), the increase in I_max of imidacloprid, but not acetylcholine, caused by co‐expression of Nl‐lynx1 and Nl‐lynx2 was more noticeable than that of their wildtype counterparts. Taken together, these data suggest that two modulators, Nl‐lynx1 and Nl‐lynx2, might serve as an influencing factor in target site insensitivity in N. lugens, such as Y151S mutation.