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.     

Point mutations in acetylcholinesterase 1 associated with chlorpyrifos resistance in the brown planthopper,Nilaparvata lugens Stål

Insecticide resistance frequently results from target‐site insensitivity, such as point mutations in acetylcholinesterases (AChEs) for resistance to organophosphates and carbamates. From a field‐originated population of Nilaparvata lugens, a major rice pest, a resistant population (R9) was obtained by nine‐generation continuous selection with chlorpyrifos. From the same field population, a relatively susceptible population (S9) was also constructed through rearing without any insecticides. Compared to the susceptible strain, Sus [medium lethal dose (LC₅₀) = 0.012 mg/l], R9 had a resistance ratio (RR) of 253.08‐fold, whereas the RR of S9 was only 2.25‐fold. Piperonyl butoxide and triphenyl phosphate synergized chlorpyrifos in R9 less than three‐fold, indicating other important mechanisms for high resistance. The target‐site insensitivity was supported by the key property differences of crude AChEs between R9 and S9. Compared to S9, three mutations (G119S, F331C and I332L) were detected in NlAChE1 from individuals of the R9 and field populations, but no mutation was detected in NlAChE2. G119S and F331C could decreased insecticide sensitivities in recombinant NlAChE1, whereas I332L took effect through increasing the influence of F331C on target insensitivity. F331C might be deleterious because of its influence on the catalytic efficiency of NlAChE1, whereas I332L would decrease these adverse effects and maintain the normal functions of AChEs.     

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.     

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.     

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.     

The involvement of clathrin‐mediated endocytosis and two Sid‐1‐like transmembrane proteins in double‐stranded RNA uptake in the Colorado potato beetle midgut

RNA interference (RNAi) is a powerful tool in entomology and shows promise as a crop protection strategy, but variability in its efficiency across different insect species limits its applicability. For oral uptake of the double‐stranded RNA (dsRNA), the RNAi trigger, two different mechanisms are known: systemic RNA interference deficient‐1 (Sid‐1) transmembrane channel‐mediated uptake and clathrin‐mediated endocytosis. So far, a wide range of experiments has been conducted, confirming the involvement of one of the pathways in dsRNA uptake, but never both pathways in the same species. We investigated the role of both pathways in dsRNA uptake in the Colorado potato beetle, Leptinotarsa decemlineata, known to have an efficient RNAi response. Through RNAi‐of‐RNAi experiments, we demonstrated the contribution of two different sid‐1‐like (sil) genes, silA and silC, and clathrin heavy chain and the 16kDa subunit of the vacuolar H⁺ ATPase (vha16), elements of the endocytic pathway, to the RNAi response. Furthermore, the sid‐1‐like genes were examined through phylogenetic and hydrophobicity analysis. This article reports for the first time on the involvement of two pathways in dsRNA uptake in an insect species and stresses the importance of evaluating both pathways through a well‐devised reporter system in any future experiments on cellular dsRNA uptake.     

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.     

Two sphingomyelin synthase homologues regulate body weight and sphingomyelin synthesis in female brown planthopper,N. lugens (Stål)

Although sphingomyelins known to be are lipid constituents of the plasma membrane in vertebrates, much remains obscure about the metabolism of sphingomyelins in insects. With ultra performance liquid chromatography‐time‐of‐flight‐tandem mass spectrometry analysis, we revealed for the first time that sphingomyelins are abundant in Nilaparvata lugens (Stål), the brown planthopper (BPH), and their biosynthesis is carried out by sphingomyelin synthase‐like protein 2 (SMSL2), which is homologous to sphingomyelin synthase‐related protein (SMSr). Unlike other insect species, high concentrations of sphingomyelins rather than ceramide phosphoethanolamines exist in the BPH. Two putative genes, which are homologous to SMSr, are named Nilaparvata lugens SMS‐like 1 (NlSMSL1) and 2 (NlSMSL2). Knockdowns of both NlSMSL2 and NlSMSL1 were conducted but only the first decreased concentrations of sphingomyelins in the BPH, indicating that NlSMSL2 plays a role in the biosynthesis of sphingomyelins. Real‐time quantitative PCR analysis revealed both NlSMSL1 and NlSMSL2 are highly expressed in BPH adults, with NlSMSL1 specifically highly expressed in reproductive organs (ovaries and testes) whereas NlSMSL2 was highly expressed in the malpighian tubules. The knockdown of NlSMSL1 or NlSMSL2 increased BPH female body weight but not that of males, suggesting sex‐specific roles for SMSLs in influencing BPH body weight. The results suggest that NlSMSL2 catalyses the synthesis of sphingomyelins and maintains female BPH body weight through alteration of sphingolipid content.