澳门地区褐家鼠对溴鼠灵的抗性检测及其Vkorc1基因多态性分析

doi:10.16829/j.slxb.150579

兽类学报 ›› 2022, Vol. 42 ›› Issue (6): 698-704.DOI: 10.16829/j.slxb.150579

澳门地区褐家鼠对溴鼠灵的抗性检测及其Vkorc1基因多态性分析

郭敏¹(), 梁捷¹, 何向阳¹, 欧伟新², 彭定雄², 麦展昭², 黄海涛¹^,³(), 张礼标¹()

^1.广东省科学院动物研究所，广东省动物保护与资源利用重点实验室，广东省野生动物保护与利用公共实验室，广州 510260
^2.澳门市政署，澳门特别行政区 999078
^3.广东科建生物技术有限公司，广州 510260

收稿日期:2021-06-22 接受日期:2022-03-10 出版日期:2022-11-30 发布日期:2022-12-02
通讯作者: 黄海涛,张礼标
作者简介:郭敏 (1983- )，男，主要从事动物生态及行为机制研究.E-mail: guomin5208@163.com
基金资助:
广东省科学院建设国内一流研究机构行动专项资金项目(2021GDASYL-20210103052);广东省科技计划项目(2021B1212110003)

An investigation of brodifacoum resistance and Vkorc1 gene polymorphism in the Rattus norvegicus from Macao

Min GUO¹(), Jie LIANG¹, Xiangyang HE¹, Weixin OU², Dingxiong PENG², Zhanzhao MAI², Haitao HUANG¹^,³(), Libiao ZHANG¹()

^1.Guangdong Key Laboratory of Animal Conservation and Resource Utilization, Guangdong Public Laboratory of Wild Animal Conservation and Utilization, Institute of Zoology, Guangdong Academy of Sciences, Guangzhou 510260, China
^2.The Municipal Affairs Bureau, Macao 999078, China
^3.Guangdong Kejian Biotechnology Co. , Ltd, Guangzhou 510260, China

Received:2021-06-22 Accepted:2022-03-10 Online:2022-11-30 Published:2022-12-02
Contact: Haitao HUANG,Libiao ZHANG

摘要/Abstract

摘要：

第二代抗凝血灭鼠剂因比第一代抗凝血灭鼠剂有更好的灭杀效果和安全性而被广泛使用，但长期使用同样也存在引发鼠类抗性的隐患。近年，学者已发现了第二代抗凝血灭鼠剂溴敌隆 (bromadiolone) 和鼠得克 (difenacoum) 的抗性鼠。溴鼠灵 (brodifacoum) 是一种目前在国内广泛使用且毒性极强的第二代抗凝血灭鼠剂，能引发鼠类凝血功能障碍和细胞毒性，但对于溴鼠灵是否已驱动鼠类发生抗性进化尚不清楚。澳门地区从1995年开始持续使用溴鼠灵，为研究鼠类对溴鼠灵的抗药性进化提供了良好模型。本研究于2019年10—12月在澳门地区共捕获61只褐家鼠，对其中44只开展了LFP实验 (0.005%溴鼠灵)，实验鼠的平均摄毒量为 (15.28 ± 1.40) mg/kg，在7天内全部死亡。此外，对全部61只褐家鼠的维生素K环氧化物还原酶复合体亚单位1 (vitamin K-epoxide reductas complex 1, Vkorc1) 的基因序列测定表明，在Vkorc1基因外显子的已知抗药关联位点处未检测到非同义核苷酸突变，但检测到2个同义核苷酸突变 [第68位氨基酸：H (CAC)-H (CAT)，突变发生率为100%；第82位氨基酸：I (ATA)-I (ATT)，突变发生率为32.72%]。该研究表明，使用溴鼠灵25年后，澳门地区褐家鼠虽没有产生群体抗药性，但仍需持续监测抗药性的发生和发展。

关键词: 澳门地区, 褐家鼠, 抗药性, 溴鼠灵, Vkorc1基因

Abstract:

The 2nd-generation anticoagulant rodenticides (ARs) are widely used for their higher eradicative rate and safety than the 1st-generation ARs. However, long-term use of the 2nd generation ARs could cause AR resistance in rats. Recently, researchers have reported resistance to the 2nd generation ARs bromadiolone and difenacoum in rats. Brodifacoum is a widely used 2nd-generation ARs of high toxicity that causes coagulation dysfunction and cytotoxicity in rats, but at present, the evolution of rat resistance against brodifacoum remains unclear. Macao has continuously used brodifacoum since 1995, allowing us to explore the development of rat resistance against brodifacoum. In this study, a total of 61 rats (Rattus norvegicus) were captured in Macao from October to December 2019. Among them, 44 rats were chosen to perform the LFP test (with 0.005% brodifacoum). The results showed that these rats consumed (15.28 ± 1.40) mg/kg (mean ± SE) brodifacoum on average, resulting in all of them dying within 7 days. Furthermore, we detected the nucleotide polymorphism of vitamin K-epoxide reductase complex 1 (Vkorc1) in all the 61 captured rats, and found none of the previously identified AR-resistant nonsynonymous mutations in Vkorc1 genes, but did detect 2 synonymous mutations [at amino acid locus 68: H (CAC)-H (CAT), mutation rate: 100%; and amino acid locus 82: I (ATA)-I (ATT), mutation rate: 32.72%]. The fact that none of the rats were brodifacoum-resistant suggests that no resistance propagated in R. norvegicus population after 25 years of use of brodifacoum in Macao. Nevertheless, persistent surveillance on the development of rat resistance to brodifacoum in the region is still needed.

Key words: Macao, Rattus norvegicus, Anticoagulant rodenticides resistance, Brodifacoum, Vkorc1 gene

中图分类号:

Q494

郭敏, 梁捷, 何向阳, 欧伟新, 彭定雄, 麦展昭, 黄海涛, 张礼标. 澳门地区褐家鼠对溴鼠灵的抗性检测及其Vkorc1基因多态性分析[J]. 兽类学报, 2022, 42(6): 698-704.

Min GUO, Jie LIANG, Xiangyang HE, Weixin OU, Dingxiong PENG, Zhanzhao MAI, Haitao HUANG, Libiao ZHANG. An investigation of brodifacoum resistance and Vkorc1 gene polymorphism in the Rattus norvegicus from Macao[J]. ACTA THERIOLOGICA SINICA, 2022, 42(6): 698-704.

图/表 2

图1 澳门地区不同地点、性别褐家鼠的摄毒量.图中圆点代表实验鼠个体.纵坐标表示个体在食毒期内的单位体重摄毒剂量 (mg/kg).通过Wilcoxon方法检验按地点或性别分组的褐家鼠组间平均摄毒量差异的显著性

Fig. 1 Variations in brodifacoum intake among R.norvegicus grouped by gender and living site from Macao. Solid dots denote rat individuals. The Y-axis shows individual brodifacoum intake per weight (mg/kg) after LFP test. Significance of the difference between genders or living sites was detected by Wilcoxon method

表1 Vkorc1基因片段阳性扩增子测序成功的褐家鼠个体数

Table 1 Number of R. norvegicus successfully sequenced after positive amplification of Vkorc1 gene fragments

引物对 Primer pair	引物名称 Primer name	单向引物上成功测序的褐家鼠个体数（只） Number of rats successfully sequenced with single primer	一对引物上均成功测序的褐家鼠个体数（只） Number of rats successfully sequenced with a pair of primers	两对引物上均成功测序的褐家鼠个体数（只） Number of rats successfully sequenced with two pairs of primers
第一对引物Primer pair 1	rVKOR-S1	57	55	12
第一对引物Primer pair 1	rVKOR-AS1	55	55
第二对引物Primer pair 2	rVKORC1-S2	15	13
第二对引物Primer pair 2	rVKORC1-AS2	18	13

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澳门地区褐家鼠对溴鼠灵的抗性检测及其Vkorc1基因多态性分析

An investigation of brodifacoum resistance and Vkorc1 gene polymorphism in the Rattus norvegicus from Macao

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