小菜蛾Plutella xylostella（L.）具有世代周期短、繁殖力强、抗药性产生快等特点，是世界上为害十字花科作物最严重的害虫之一，对上百种杀虫成分产生了不同程度的抗药性。溴虫腈（chlorfenapyr）属于吡咯类杀虫剂，经昆虫体内氧化酶代谢为有毒物质，该代谢物能够作用于细胞线粒体，对氧化磷酸化进行解偶联。溴虫腈具有低毒、持久和速效的特点，被广泛应用于鳞翅目和螨类害虫的防治。然而，由于不合理的使用，中国和巴西已有小菜蛾种群对溴虫腈演化出高水平抗性，使抗性治理形势更加严峻。

目前，害虫溴虫腈抗性特征和抗性机理的研究报道较少，亟需在具备较高抗性的材料中开展相关研究，为溴虫腈的抗性治理提供依据。本文将选育的小菜蛾溴虫腈抗性种群TH-R与室内敏感品系IPP-S多代回交，获得抗溴虫腈近等基因系TH-BC5F2，明确了其抗性遗传方式、交互抗性和生化机理。对TH-R品系的溴虫腈抗性基因座进行BSA定位，通过对SNP分离比的统计分析确定了抗性连锁区间。研究结果有助于针对性地制订小菜蛾溴虫腈抗性治理方案，为探明小菜蛾溴虫腈抗性机理提供重要线索。

1. 小菜蛾溴虫腈抗性品系筛选和近等基因系构建

实验室前期采集自通海的小菜蛾种群（TH20）对溴虫腈有较高抗性，其致死中浓度LC50为290.1 mg/L，与室内敏感品系IPP-S相比，抗性达371倍。在室内用300 mg/L的溴虫腈对TH20连续筛选7代，获得溴虫腈抗性品系TH-R，其抗性倍数达436倍。通过药剂筛选辅助的多代回交策略，将TH-R品系的溴虫腈抗性基因逐代渗入敏感品系IPP-S，获得与IPP-S品系具有98.4%相同遗传背景的近等基因系TH-BC5F2。

2. 小菜蛾抗溴虫腈近等基因系的抗性特征及生化机理

与初始品系IPP-S相比，近等基因系TH-BC5F2对溴虫腈具备275倍的高水平抗性，对多杀菌素存在9.25倍交互抗性，对阿维菌素为4.16倍低水平交互抗性，对毒死蜱、茚虫威、氯虫苯甲酰胺、高效氯氰菊酯、溴虫氟苯双酰胺没有抗性。TH-BC5F2与敏感品系IPP-S正反交的F1代对溴虫腈的LC50分别为106.3 mg/L和83.40 mg/L，且二者95%置信区间基本重叠，显性度分别为0.75和0.66，表明其遗传方式为常染色体基因控制的偏显性遗传。BC回交代的LD-P线在死亡率为50%处存在明显的平台，适合性检验结果符合单基因假设，证明TH-BC5F2小菜蛾品系对溴虫腈的抗性受单基因控制。增效实验显示，酶抑制剂PBO、DEF、DEM在TH-BC5F2品系中对溴虫腈的毒力增效1.03-1.45倍，在敏感品系IPP-S中的增效系数为1.37-1.72倍。解毒代谢酶活性测定的实验中，TH-BC5F2品系的解毒代谢酶（MFO、GST、EST）活力相对于IPP-S敏感品系在1.1-2.2倍之间，无显著提高，说明TH-BC5F2的溴虫腈抗性可能与解毒代谢增强无关。

3. 溴虫腈抗性基因座的初步定位

根据两相定位法构建抗性基因交换型群体，将溴虫腈高抗品系TH-R与野生型品系Roth进行单对杂交，F1家系分别与IPP-S敏感个体回交获得BC代。经20 mg/L溴虫腈处理BC群体，存活个体组成BC-R池，未经处理个体混合为对照池BC-CK。通过高通量测序和BSA分析，将溴虫腈抗性连锁区域定位于PxChr16的7.5-8.5 Mb。为了进一步缩小QTL范围，在BC-R与BC-CK组中分别利用PCR扩增选定的SNP区域并进行直接测序，通过分离比差异显著的SNP标记将范围缩小至PxChr16的7.8-8.0 Mbp区域。基因注释分析发现该区间包含19个基因，主要涉及代谢、运输、生长类基因或通路。

Diamondback moth, Plutella xylostella (L.) (Lepidoptera: Plutellidae), is one of the most destructive insect pests of cruciferous vegetables in the world. To date, P. xylostella have been developed resistance to more than 100 active ingredients, due to its short generation time, high productivity, and particularly the extensive selection pressure in the field. Chlorfenapyr, a new halogenated pyrrole compound, is a pro-insecticide, and oxidative removal of the N-ethoxymethyl group of chlorfenapyr by mixed function oxidases leads to a toxic form that interrupts the conversion of ADP to ATP in cell mitochondria. Chlorfenapyr has good insecticidal activity and low mammalian toxicity, and widely using for several mites and insect pests control. However, chlorfenapyr-resistant populations from China and Brazil seriously threatened the useful life of the chemical and the management of field P. xylostella.

At present, there are few studies focus on the characteristics of and mechanisms of resistance to chlorfenapyr. It is therefore necessary to perform the relevant research in chlorfenapyr-resistant insect species, aim to provide important information for chlorfenapyr resistance management. Here, through multiple rounds of backcrossing (the field-derived TH-R strain crossed with the susceptible IPP-S strain) and chlorfenapyr selection, we produced a chlorfenapyr-resistant near-isogenic line, TH-BC5F2, and the inheritance mode, cross-resistance pattern and biochemical mechanism of resistance were then determinated. Using a bulked segregant analysis (BSA) study and a subsequently SNP assistant validation analysis, the preliminary mapping of chlorfenapyr resistance locus of TH-R strain was performed. The results will be helpful to formulate of the resistance management strategy of chlorfenapyr, and provide an important clue to decode the chlorfenapyr resistance mechanism in P. xylostella.

1. Selection of a field chlorfenapyr-resistant population and construction of the near-isogenic Plutella xylostella strain

The sensitivity of P. xylostella collected from Tonghai in 2020 to chlorfenapyr was determined by leaf dipping method, with a 290.1 mg/L LC50. Compared with the susceptible IPP-S strain, the resistance ratio (RR) to chlorfenapyr of TH20 was 371-fold. The TH20 population was continuously selected by 300 mg/L for 7 generations, and a chlorfenapyr-resistant strain TH-R was generated, with 436-fold resistance. Using the field-derived TH-R strain and the susceptible IPP-S strain as parents, the resistance loci were introgressed into IPP-S strain, and the obtained TH-BC5F2 strain showed 98.4% genetic similarity to the susceptible parental strain.

2. Characterisation of the TH-BC5F2 strain of P. xylostella to chlorfenapyr.

Compared with the original IPP-S strain, the near isogenic TH-BC5F2 strain showed 275-fold resistance to chlorfenapyr, and 9.25-fold of resistance to spinosad, and a low level of resistance (4.16-fold) to abamectin. There was no cross-resistance to indoxacarb, chlorantraniliprole, broflanilide, chlorpyrifos and beta-cypermethrin. The LC50 for chlorfenapyr of F1 and F1׳ progeny were 106.3 mg/L and 83.40 mg/L, respectively. The 95% confidence interval of the LC50s were overlapped. The dominance values of F1 and F1׳ progeny were 0.75 and 0.66, respectively, indicating that the chlorfenapyr resistance in TH-BC5F2 was autosomal incomplete dominant inheritance. In the log dosage-probit mortality curve of the backcross progeny to chlorfebapyr there was a distinct plateau correronding to 50% mortality. The χ2 test of the goodness of fit between the observations and predictions under the assumption of monofactorial inheritance accept the single gene hypothesis. Synergistic experiments showed that the PBO, DEF and DEM had slight effects on the toxicity of chlorfenapyr (1.03- to 1.45-fold) in TH-BC5F2 strain, similar to that in IPP-S strain with 1.37- to 1.72-fold SRs. Compared with the IPP-S strain, metabolic enzyme activities of TH-BC5F2 were increased by 1.1- to 2.2-fold. Enzyme activity analysis results are consistent with those from the synergism experiment, which indicates that metabolic detoxification is not involved in chlorfenapyr resistance in TH-BC5F2.

3. Preliminary mapping of chlorfenapyr resistance loci

Firstly, the high-resistant chlorfenapyr strain TH-R was single-pair crossed with the wild-type Roth strain, and then the F1 families were backcrossed with IPP-S individual to generate BC progeny. For each BC progeny, the survivals, after treated with 20 mg/L chlorfenapyr, were pooled to a BC-R group, while the others without treatment formed a control group, BC-CK. Through high-throughput sequencing and BSA analysis, we found that the chlorfenapyr resistance was linked to a region from 7.5 to 8.5 Mbp on chromosome 16 (PxChr16). In order to further narrow the loci region, we used direct-sequencing to genotype the individual resistant and susceptible larvae from BC-R and BC-CK groups for SNPs at eight marker sites within the mapping region. Through analysis of the segregation ratio of each SNP marker in BC-R and BC-CK groups, the chlorfenapyr resistance loci were narrowed to a range from 7.8 to 8.0 Mbp on PxChr16. By using gene annotation and KEEG analysis, we found that the linked region contains 19 genes, which mainly involvs in the pathways of metabolism, transportation and growth.

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