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.     

Comparison of esterase gene amplification, gene expression and esterase activity in insecticide susceptible and resistant strains of the brown planthopper, Nilaparvata lugens (Stål)

Organophosphorus and carbamate insecticide resistance in Nilaparvata lugens is based on amplification of a carboxylesterase gene, Nl-EST1. An identical gene occurs in susceptible insects. Quantitative real-time PCR was used to demonstrate that Nl-EST1 is amplified 3-7-fold in the genome of resistant compared to susceptible planthoppers. Expression levels were similar to amplification levels, with 1-15-fold more Nl-EST1 mRNA in individual insects and 5-11-fold more Nl-EST1 mRNA in mass whole body homogenates of resistant females compared to susceptibles. These values corresponded to an 8-10-fold increase in esterase activity in the head and thorax of individual resistant insects. Although amplification, expression and activity levels of Nl-EST1 in resistant N. lugens were similar, the correlation between esterase activity and Nl-EST1 mRNA levels in resistant individuals was not linear.     

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.     

Pharmacological characterization of cis-nitromethylene neonicotinoids in relation to imidacloprid binding sites in the brown planthopper, Nilaparvata lugens

Neonicotinoid insecticides, such as imidacloprid, are selective agonists of the insect nicotinic acetylcholine receptors (nAChRs) and extensively used in areas of crop protection and animal health to control a variety of insect pest species. Here we describe that two cis‐nitromethylene neonicotinoids (IPPA152002 and IPPA152004), recently synthesized in our laboratory, discriminated between the high and low affinity imidacloprid binding sites in the brown planthopper, Nilaparvata lugens, a major insect pest of rice crops in many parts of Asia. [³H]imidacloprid has two binding sites with different affinities (Kd value of 0.0035 ± 0.0006 nM for the high‐affinity site and 1.47 ± 0.22 nM for the low‐affinity site). Although the cis‐nitromethylene neonicotinoids showed low displacement ability (Ki values of 0.15 ± 0.03 µM and 0.42 ± 0.07 µM for IPPA152002 and IPPA152004, respectively) against [³H]imidacloprid binding, low concentrations (0.01 µM) of IPPA152002 completely inhibited [³H]imidacloprid binding at its high‐affinity site. In Xenopus oocytes co‐injected with cRNA encoding Nlα1 and rat β2 subunits, obvious inward currents were detected in response to applications of IPPA152002 and IPPA152004, although the agonist potency is reduced to that of imidacloprid. The previously identified Y151S mutation in Nlα1 showed significant effects on the agonist potency of IPPA152002 and IPPA152004, such as a 75.8% and 70.6% reduction in Imax, and a 2.4‐ and 2.1‐fold increase in EC₅₀. This data clearly shows that the two newly described cis‐nitromethylene neonicotinoids act on insect nAChRs and like imidacloprid, discriminated between high and low affinity binding sites in N. lugens native nAChRs. These compounds may be useful tools to further elucidate the pharmacology and nature of neonicotinoid binding sites.     

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.     

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.     

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.