Yueliang Zhang

Cross-resistance and possible mechanisms of chlorpyrifos resistance in Laodelphax striatellus (Fallén).

BACKGROUND Laodelphax striatellus (Fallén) is a major pest of cultivated rice and is commonly controlled in China with the organophosphate insecticides. To develop a better resistance management strategy, a chlorpyrifos-resistant strain of L. striatellus was selected in the laboratory, and its cross-resistance to other insecticides and possible mechanisms of the chlorpyrifos resistance were investigated. RESULTS After 25 generations of selection with chlorpyrifos, the selected strain of L. striatellus developed 188-fold resistance to chlorpyrifos in comparison with the susceptible strain, and showed 14- and 1.6-fold cross-resistance to dichlorvos and thiamethoxam respectively. There was no apparent cross-resistance to abamectin. Chlorpyrifos was synergised by the inhibitor triphenyl phosphate; the carboxylesterase synergistic ratio was 3.8 for the selected strain, but only 0.92 for the susceptible strain. The carboxylesterase activity of the selected strain was approximately 4 times that of the susceptible strain, whereas there was no significant change in the activities of alkaline phosphatase, acid phosphatase, glutathione S-transferase and cytochrome P450 monooxygenase between the strains. The Michaelis constant of acetylcholinesterase, maximum velocity of acetylcholinesterase and median inhibitory concentration of chlorpyrifos-oxon on acetylcholinesterase were 1.7, 2.5 and 5 times higher respectively in the selected strain. CONCLUSION The high cross-resistance to the organophosphate dichlorvos in the chlorpyrifos-resistant strain suggests that other non-organophosphate insecticides would be necessary to counter resistance, should it arise in the field. Enhanced activities of carboxylesterase and the acetylcholinesterase insensitivity appear to be important mechanisms for chlorpyrifos resistance in L. striatellus.

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.

An antennae‐enriched carboxylesterase from Spodoptera exigua displays degradation activity in both plant volatiles and female sex pheromones

Carboxyl/cholinesterase (CCE) is a large gene family of diverse functions, but in insects its function with respect to catabolism of sex pheromone components and plant volatiles is not well understood. In the present study, we cloned and functionally characterized one putative odorant‐degrading enzyme (ODE) of the CCE family, SexiCXE14, from Spodoptera exigua. The tissue‐temporal expression pattern revealed that the mRNA level of SexiCXE14 is antennae‐enriched, sex equivalent and peaks at 3 days after moth eclosion. Functional study using the recombinant enzyme determined that SexiCXE14 has high degrading activity (Vmax) to host plant volatiles, suggesting its role in degradation of these volatiles. In addition, SexiCXE14 may also play a role in the degradation of sex pheromone components, as the Vmax and affinity parameter (Km) values with the sex pheromones are similar to those of reported pheromone degrading enzymes (PDEs). Further analysis of the relationship between substrate structure and enzymatic activity demonstrated that carbon chain length is a major influential factor, while the number of double bonds also affects the enzymatic activity. In addition, SexiCXE14 displays lower activity at acidic pH levels (pH 5.0) than in neutral conditions (pH 6.5). By characterizing this new ODE the present study provides insights in understanding of the high sensitivity of the moth olfactory system.

Functional characterization of sex pheromone receptors in the purple stem borer, Sesamia inferens (Walker)

The sex pheromone communication system in moths is highly species‐specific and extremely sensitive, and pheromone receptors (PRs) are thought to be the most important factors in males. In the present study, three full‐length cDNAs encoding PRs were characterized from Sesamia inferens antennae. These three PRs were all male‐specific in expression, but their relative expression levels were very different; SinfOR29 was 17‐ to 23‐fold higher than the other two PRs. Phylogenetic and motif pattern analyses showed that these three PRs were allocated to different PR subfamilies with different motif patterns. Functional analysis using the heterologous expression system of Xenopus oocytes demonstrated that SinfOR29 specifically and sensitively responded to the major pheromone component, Z11‐16:OAc [concentration for 50% of maximal effect (EC50) = 3.431 × 10−7 M], while SinfOR21 responded robustly to a minor pheromone component Z11‐16:OH (EC50 = 1.087 × 10−6 M). SinfOR27, however, displayed no response to any of the three pheromone components, but, interestingly, it was sensitive to a non‐sex pheromone component Z9,E12‐14:OAc (EC50 = 1.522 × 10−6 M). Our results provide insight into the molecular mechanisms of specificity and sensitivity of the sex pheromone communication system in moths.

Knockdown of NADPH-cytochrome P450 reductase results in reduced resistance to buprofezin in the small brown planthopper, Laodelphax striatellus (fallen).

NADPH-cytochrome P450 reductase (CPR) plays an important role in cytochrome P450 function, and CPR knockdown in several insects leads to increased susceptibility to insecticides. However, a putative CPR gene has not yet been fully characterized in the small brown planthopper Laodelphax striatellus, a notorious agricultural pest in rice that causes serious damage by transmitting rice stripe and rice black-streaked dwarf viruses. The objective of this study was to clone the cDNA and to knock down the expression of the gene that encodes L. striatellus CPR (LsCPR) to further determine whether P450s are involved in the resistance of L. striatellus to buprofezin. First, the full-length cDNA of LsCPR was cloned and found to contain an open reading frame (ORF) encoding a polypeptide of 679 amino acids with a calculated molecular mass and isoelectric point of 76.92kDa and 5.37, respectively. The deduced amino acid sequence shares high identity with the CPRs of other insects (98%, 97%, 75% and 68% for Sogatella furcifera, Nilaparvata lugens, Cimex lectularius and Anopheles gambiae, respectively) and possesses the characteristic features of classical CPRs, such as an N-terminal membrane anchor and conserved domains for flavin mononucleotide (FMN), flavin adenine dinucleotide (FAD) and nicotinamide adenine dinucleotide phosphate (NADPH) binding. Phylogenetic analysis revealed that LsCPR is located in a branch along with the CPRs of other hemipteran insects. LsCPR mRNA was detectable in all examined body parts and developmental stages of L. striatellus, as determined by real-time quantitative PCR (qPCR), and transcripts were most abundant in the adult abdomen and in first-instar nymphs and adults. Ingestion of 200μg/mL of LsCPR double-stranded RNA (dsLsCPR) by the planthopper for 5days significantly reduced the transcription level of LsCPR. Moreover, silencing of LsCPR caused increased susceptibility to buprofezin in a buprofezin-resistant (YN-BPF) strain but not in a susceptible (YN) strain. These data further suggested that the P450-mediated metabolic detoxification of xenobiotics might be an important mechanism for buprofezin resistance in L. striatellus.

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.

Constitutive overexpression of cytochrome P450 monooxygenase genes contributes to chlorantraniliprole resistance in Chilo suppressalis (Walker): P450s mediate chlorantraniliprole resistance in C. suppressalis

BACKGROUND The rice striped stem borer (SSB), Chilo suppressalis (Walker), which is one of the most economically important phytophagous pests, has developed resistance to multiple insecticides. The resistance of SSB against chlorantraniliprole has been investigated in detail. However, the mechanism of its metabolic resistance has rarely been studied. RESULTS A field population from Wuhu City, China was used to establish chlorantraniliprole resistant and susceptible strains (WHR and WHS) by laboratory continuous selection. Enzyme activities data suggested the potential involvement of cytochrome P450 monooxygenase in WHR. CYP6CV5, CYP9A68, CYP321F3, and CYP324A12 were significantly overexpressed in WHR (from 4.48 to 44.88-fold). These four P450 genes were expressed in the late developmental stages of WHR; however, they were almost absent during the egg stage. In addition, their expressions were much more sensitive to chlorantraniliprole induction in WHR than in WHS. Injection of individual and mixture dsRNAs reduced the expression of the four target genes (55.2-73.2% and 43.2-50.2%, respectively) and caused significant larvae mortality (55.1-65.1% and 88.2%, respectively). CONCLUSION Multiple overexpressed P450 genes were potentially associated with chlorantraniliprole resistance, as confirmed by the RNA interference (RNAi) assay. Our findings suggested that metabolic resistance to chlorantraniliprole might be mediated by P450s.

Resistance to cycloxaprid in Laodelphax striatellus is associated with altered expression of nicotinic acetylcholine receptor subunits

BACKGROUND Cycloxaprid is a new oxabridged cis-configuration neonicotinoid insecticide, the resistance development potential and underlying resistance mechanism of which were investigated in the small brown planthopper, Laodelphax striatellus (Fallén), an important agricultural pest of rice. RESULTS A cycloxaprid-resistant strain (YN-CPD) only achieved 10-fold higher resistance, in contrast to 106-fold higher resistance to buprofezin and 332-fold higher resistance to chlorpyrifos achieved after exposure to similar selection pressure, and the cycloxaprid selected line showed no cross-resistance to the buprofezin and chlorpyrifos-selected resistance strains. Moreover, we identified 10 nicotinic acetylcholine receptor (nAChR) subunits from the transcriptome of L. striatellus, and six segments had open reading frames (ORFs). While we did not find mutations in the nAChR genes of L. striatellus, subunits Lsα1 and Lsβ1 exhibited, respectively, 9.60-fold and 3.36-fold higher expression in the resistant strain, while Lsα8 exhibited 0.44-fold lower expression. Suppression of Lsα1 through ingestion of dsLsα1 led to an increase in susceptibility to cycloxaprid. CONCLUSION The findings indicate that resistance to cycloxaprid develops slowly compared with resistance to other chemicals and without cross-resistance to chlorpyrifos or buprofezin; over-expressed Lsα1 is associated with low cycloxaprid resistance levels, but the importance of over-expressed Lsβ1 and reduced expression of Lsα8 could not be excluded.

Differential Expression of P450 Genes and nAChR Subunits Associated With Imidacloprid Resistance in Laodelphax striatellus (Hemiptera: Delphacidae)

Abstract Imidacloprid is a key insecticide used for controlling sucking insect pests, including the small brown planthopper (Laodelphax striatellus, Fallén) (Hemiptera: Delphacidae), an important agricultural pest of rice. A strain of L. striatellus (YN-ILR) developed 21-fold resistance when selected with imidacloprid on a susceptible YN strain. An in vitro study on piperonyl butoxide synergism indicated that enhanced detoxification mediated by cytochrome P450s contributed to imidacloprid resistance to some extent, and multiple P450 genes showed altered expression in the imidacloprid-resistantYN-ILR strain compared with the susceptibleYN strain (CYP425B1-CYP6BD10 had 1.51- to 11.45-fold higher expression, CYP4CE2-CYP4DD1V2 had 0.12- to 0.57-fold lower expression). While there were no mutations in target nicotinic acetylcholine receptor (nAChR) genes, subunits of Lsα1, Lsβ1, and Lsβ3 in the YN-ILR strain showed 3.86-, 4.39-, and 2.59-fold higher expression and Lsa8 displayed 0.38-fold lower expression than the YN strain. Moreover, 21-fold moderate imidacloprid resistance in individuals of L. striatellus did not produce a fitness cost. The findings suggest that L. striatellus has the capacity to develop resistance to imidacloprid through P450 detoxification and potential target nAChR expression changes, and moderate imidacloprid resistance was not associated with a fitness cost.

Resistance monitoring and cross-resistance role of CYP6CW1 between buprofezin and pymetrozine in field populations of Laodelphax striatellus (Fallén)

Monitoring resistance and investigating insecticide resistance mechanisms are necessary for controlling the small brown planthopper, Laodelphax striatellus. The susceptibility to four common insecticides of L. striatellus collected from Jiangsu, Anhui, Zhejiang and Jilin provinces of China in 2015 was monitored. The results showed that all field populations remained susceptible to chlorpyrifos and thiamethoxam with resistance ratios (RRs) of 2.3- to 9.5 and 1.6- to 3.3, respectively, while the insects had developed moderate pymetrozine resistance with RRs of 18.7 to 34.5. Resistance against buprofezin had developed to an alarmingly high level in three southeastern provinces of China with RRs of 108.8 to 156.1, but in Jilin it had an RR of only 26.6. Moreover, in line with both the buprofezin and pymetrozine resistance levels, we found LsCYP6CW1 to be over-expressed in all field L. striatellus populations, which indicated that it might be important for cross-resistance between buprofezin and pymetrozine. RNA interference (RNAi) ingestion resulted in the effective suppression of LsCYP6CW1 expression, and significantly increased susceptibility to both buprofezin and pymetrozine compared with the control, which further confirmed that overexpression of LsCYP6CW1 was involved in the cross-resistance to buprofezin and pymetrozine in field L. Striatellus populations.