BACKGROUND The brown planthopper, Nilaparvata lugens, is considered the most destructive pest of rice in many Asian countries including China. Use of pymetrozine in insect resistance management (IRM) has been one strategy to control this pest. In this study, we reported the status of pymetrozine resistance in Nilaparvata lugens (Stål) collected from China over the period 2017–2021 and selected a strain of N. lugens resistant to pymetrozine and evaluated the cross‐resistance, inheritance and fitness costs of the resistance. RESULTS Monitoring data (2017–2021) showed that field populations of N. lugens in China developed moderate‐ to high‐level pymetrozine resistance during these 5 years. By continuous selection with pymetrozine in the lab, the pymetrozine selected N. lugens strain (Pym‐R98) developed a 225.2‐fold resistance compared to a susceptible strain. The Pym‐R98 strain showed high cross‐resistance to dinotefuran (66.6‐fold) and low cross‐resistance to nitenpyram (5.2‐fold) and sulfoxaflor (5.8‐fold). Inheritance pattern analysis of Pym‐R93 revealed that resistance to pymetrozine was polygenic, autosomal and incompletely dominant. Fitness costs of pymetrozine resistance were present in Pym‐R90 and WA2020 strains with a relative fitness of 0.72 and 0.60, respectively. The developmental duration of Pym‐R90 and WA2020 was significantly longer and hatchability was significantly lower compared to pymetrozine‐susceptible strain (Pym‐S). CONCLUSIONS N. lugens has developed high level of resistance to pymetrozine. Pymetrozine‐resistance brown planthopper had cross‐resistance with some of neonicotinoids such as dinotefuran, nitenpyram and sulfoxaflor. The autosomal, incompletely dominant and polygenic resistance to pymetrozine in N. lugens and the fitness costs associated with this resistance can be exploited in IRM strategies to preserve the lifetime of pymetrozine for control of N. lugens in China. © 2022 Society of Chemical Industry.

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BACKGROUND: The brown planthopper (Nilaparvata lugens) is one of the major rice insect pests in Asia. Recently, high levels of insecticide resistance have been frequently reported and cytochrome P450 monooxygenase (P450)-mediated metabolic detoxification is a common resistance mechanism in N. lugens. However, there has been no persuasive genetic method to prove the role of P450s in insecticide resistance in N. lugens.RESULTS: Here, CRISPR/Cas9 system was used to disrupt the P450 gene NlCYP6CS1 to elucidate its role in insecticide resistance in field populations of N. lugens. We successfully constructed a homozygous strain (Nl6CS1-KO) with a 5-bp deletion and 1-bp insertion mutation of NlCYP6CS1. Compared with a background resistant strain (Nl-R), the susceptibility of knockout strain Nl6CS1-KO to imidacloprid, nitenpyram, thiamethoxam, dinotefuran, and pymetrozine was increased by 2.3-, 3.4-, 7.0-, 4.2and 3.9-fold, respectively, but not significantly changed to triflumezopyrim, chlorpyrifos and buprofezin. Life table analysis demonstrated that the Nl6CS1-KO strain resembled the Nl-R strain in terms of egg and nymph developmental duration and adult lifespan, but differed from the Nl-R strain in the survival rate of eggs and nymphs, reproduction, and body weight.CONCLUSIONS: Our study demonstrates the effect of functional deletion of NlCYP6CS1 on multiple insecticide resistance in N. lugens. For the first time, we applied CRISPR/Cas9 system to reveal the mechanism of insecticide resistance in N. lugens, which may shed light on similar studies in other hemipteran insects.

To explore the effects of pymetrozine on the reproductive behavior of N. lugens, we established a bioassay method to accurately evaluate the toxicity of pymetrozine in N. lugens and establish the level of pymetrozine resistance of N. lugens in the field. In this study, pymetrozine’s effects on the fecundity of N. lugens were evaluated using the topical application method and rice-seedling dipping method. Moreover, the resistance of N. lugens to pymetrozine in a pymetrozine-resistant strain (Pym-R) and two field populations (YZ21 and QS21) was determined using the rice-seedling dipping method and fecundity assay methods. The results showed that treatment of N. lugens third-instar nymphs with LC15, LC50, and LC85 doses of pymetrozine resulted in significantly reduced male fertility and female fecundity. In addition, N. lugens adults treated with pymetrozine using the rice-seedling dipping and topical application method also exhibited significantly in-hibited male courtship, fertility, and female receptivity. Using the rice-stem dipping method, pymetrozine resistance was shown to be at high levels in Pym-R (194.6-fold), YZ21 (205.9-fold), and QS21 (212.8-fold), with LC50 values of 522.520 mg/L (Pym-R), 552.962 mg/L (YZ21), and 571.315 (QS21) mg/L. However, when using the rice-seedling dipping or topical application fe-cundity assay method, Pym-R (EC50: 14.370 mg/L, RR=12.4-fold; ED50: 0.560 ng/adult, RR=10.8-fold), YZ21 (EC50: 12.890 mg/L, RR=11.2-fold; ED50: 0.280 ng/adult; RR=5.4-fold), and QS21 (EC50: 13.70 mg/L, RR=11.9-fold) exhibited moderate or low levels of resistance to pymet-rozine. Our studies show that pymetrozine can significantly inhibit the fecundity of N. lugens. The fecundity assay results showed that N. lugens only developed low to moderate levels of resistance to pymetrozine, indicating that pymetrozine can still achieve effective control on the next gen-eration of N. lugens populations.

On the basis of the inhibition effects of pymetrozine on the reproductive behavior of N. lugens, we established a bioassay method to accurately evaluate the toxicity of pymetrozine in N. lugens and clarified the level of pymetrozine resistance of N. lugens in the field. In this study, pymetrozine’s effects on the fecundity of N. lugens were evaluated using the topical application method and rice-seedling-dipping method. Moreover, the resistance of N. lugens to pymetrozine in a pymetrozine-resistant strain (Pym-R) and two field populations (YZ21 and QS21) was determined using the rice-seedling-dipping method and fecundity assay methods. The results showed that treatment of N. lugens third-instar nymphs with LC15, LC50, and LC85 doses of pymetrozine resulted in a significantly reduced fecundity of N. lugens. In addition, N. lugens adults treated with pymetrozine, using the rice-seedling-dipping and topical application method, also exhibited a significantly inhibited fecundity. Using the rice-stem-dipping method, pymetrozine resistance levels were shown to be high in Pym-R (194.6-fold), YZ21 (205.9-fold), and QS21 (212.8-fold), with LC50 values of 522.520 mg/L (Pym-R), 552.962 mg/L (YZ21), and 571.315 (QS21) mg/L. However, when using the rice-seedling-dipping or topical application fecundity assay method, Pym-R (EC50: 14.370 mg/L, RR = 12.4-fold; ED50: 0.560 ng/adult, RR = 10.8-fold), YZ21 (EC50: 12.890 mg/L, RR = 11.2-fold; ED50: 0.280 ng/adult; RR = 5.4-fold), and QS21 (EC50: 13.700 mg/L, RR = 11.9-fold) exhibited moderate or low levels of resistance to pymetrozine. Our studies show that pymetrozine can significantly inhibit the fecundity of N. lugens. The fecundity assay results showed that N. lugens only developed low to moderate levels of resistance to pymetrozine, indicating that pymetrozine can still achieve effective control on the next generation of N. lugens populations.