Obaid Muhammad Kashif, Ren Qiaoyun, Luo Jin, Wang Jinming, Rashid Muhammad, Zeb Jehan, Zan Xiaoqing, Lan Xinting, Liu Guangyuan, Luo Jianxun, Yin Hong, Chen Ze, Guan Guiquan
State Key Laboratory for Animal Disease Control and Prevention, Key Laboratory of Veterinary Parasitology of Gansu Province, Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Lanzhou, Gansu, China.
State Key Laboratory for Animal Disease Control and Prevention, Key Laboratory of Veterinary Parasitology of Gansu Province, Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Lanzhou, Gansu, China; Hebei Key Laboratory of Animal Physiology, Biochemistry and Molecular Biology, Hebei Collaborative Innovation Center for Eco-Environment, Ministry of Education Key Laboratory of Molecular and Cellular Biology, College of Life Sciences, Hebei Normal University, Shijiazhuang 050024, China.
Vet Parasitol. 2025 Feb;334:110407. doi: 10.1016/j.vetpar.2025.110407. Epub 2025 Jan 27.
Southern cattle tick, Rhipicephalus (R) microplus, is a major challenge in transmitting Babesia spp., Theileria spp., and Anaplasma spp., worldwide. Mitigation of this tick and associated pathogens is crucial for animal and human-health. In present study, laboratory-based larval packet tests (LPTs) were performed to assess fipronil efficacy against R. microplus ticks collected from four Chinese localities, Nanning, Guangshui, Macheng, and Tengchong. Additionally, we reported first molecular characterization of the full-length gamma-aminobutyric acid (GABA)-gated chloride channel gene of R. microplus ticks from China and Pakistan (Khyber Pakhtunkhwa and Punjab). For LPTs bioassay, fipronil's concentrations (5-ppm, 25-ppm, 50-ppm, 75-ppm, 100-ppm) in acetone and DNase-free water were prepared. The highest average mortality rate of larvae was observed in Nanning (4.34-98.13 %) while lowest in Tengchong (5.70-82.29 %) via triplicate LPTs. The recorded LC values were 33.2 ppm, 35.7 ppm, 49.7 ppm, and 55.4 ppm, respectively, for the four localities (Guangshui, Macheng, Nanning, and Tengchong) while the LC values were 722 ppm, 827 ppm, 949 ppm, and 1342.3 ppm, respectively. The resistance factors (RR) were 13.83, 14.88, 20.71, and 23.08, indicating the development of level-II resistance in the tick populations from these regions respectively. Molecularly, consensus sequences of R. microplus GABA-gene from China and Pakistan resulted 99.24-99.52 % and 99.10-99.39 % identities with dieldrin-susceptible NRFS (GQ398111.1) and dieldrin-resistant (GQ398112.1) R. microplus ticks, respectively. Findings revealed 21 single nucleotide polymorphisms (SNPs) [8-non-synonymous (NS) and 13-synonymous (S)], including specifically 10-SNPs from Nanning, 9-SNPs from Macheng, 7-SNPs from Guangshui, 10-SNPs from Tengchong, and 9-SNPs from Pakistani R. microplus full-length GABA gene. Explicitly, we obtained one NS-SNP in TM3 (T-871-G; F-291-V) and two NS-SNPs in TM4 (A-1438-G; N-480-D and A-1439-G; N-480-G) regions of Chinese and Pakistani, while one NS-SNP in TM2 (A-763-G; T-255-A) region of Pakistani R. microplus ticks was documented. Coexistence among SNPs in TM3 (T-871-G; F-291-V), TM4 (A-1439-G; N-480-G) and one linker SNP (A-1378-G; S-460-G) were also recorded. Presence of SNPs and their coexistence suggest the possible tri-dimensional structural modification in GABA-Cl channels that might interfere with binding of fipronil, resulting in development of resistance. This comprehensive research will provide a basis for governmental and pharmaceutical industries for development of effective tick control-strategies as well as to manage the effectiveness and functionality of the available acaricides.
南方牛蜱，微小扇头蜱（Rhipicephalus (R) microplus），是全球传播巴贝斯虫属、泰勒虫属和无形体属病原体的主要挑战。减轻这种蜱虫及相关病原体对动物和人类健康至关重要。在本研究中，进行了基于实验室的幼虫包囊试验（LPTs），以评估氟虫腈对从中国四个地方（南宁、广水、麻城和腾冲）采集的微小扇头蜱的疗效。此外，我们首次报道了来自中国和巴基斯坦（开伯尔-普赫图赫瓦省和旁遮普省）的微小扇头蜱全长γ-氨基丁酸（GABA）门控氯离子通道基因的分子特征。对于LPTs生物测定，制备了丙酮和无DNase水中氟虫腈的浓度（5 ppm、25 ppm、50 ppm、75 ppm、100 ppm）。通过一式三份的LPTs观察到，南宁幼虫的平均死亡率最高（4.34 - 98.13%），而腾冲最低（5.70 - 82.29%）。四个地方（广水、麻城、南宁和腾冲）记录的LC值分别为33.2 ppm、35.7 ppm、49.7 ppm和55.4 ppm，而LC值分别为722 ppm、827 ppm、949 ppm和1342.3 ppm。抗性因子（RR）分别为13.83、14.88、20.71和23.08，表明这些地区蜱虫种群中出现了二级抗性。在分子层面，来自中国和巴基斯坦的微小扇头蜱GABA基因的共有序列与狄氏剂敏感的NRFS（GQ398111.1）和狄氏剂抗性的（GQ398112.1）微小扇头蜱的同一性分别为99.24 - 99.52%和99.10 - 99.39%。研究结果揭示了21个单核苷酸多态性（SNP）[8个非同义（NS）和13个同义（S）]，具体包括来自南宁的10个SNP、来自麻城的9个SNP、来自广水的7个SNP、来自腾冲的10个SNP以及来自巴基斯坦微小扇头蜱全长GABA基因的9个SNP。明确地说，我们在中国和巴基斯坦微小扇头蜱的TM3（T - 871 - G；F - 291 - V）区域获得了1个NS - SNP，在TM4（A - 1438 - G；N - 480 - D和A - 1439 - G；N - 480 - G）区域获得了2个NS - SNP，而在巴基斯坦微小扇头蜱的TM2（A - 763 - G；T - 255 - A）区域记录了1个NS - SNP。还记录了TM3（T - 871 - G；F - 291 - V）、TM4（A - 1439 - G；N - 480 - G）中的SNP以及1个连接子SNP（A - 1378 - G；S - 460 - G）之间的共存情况。SNP的存在及其共存表明GABA - Cl通道可能存在三维结构修饰，这可能会干扰氟虫腈的结合，从而导致抗性的产生。这项全面的研究将为政府和制药行业制定有效的蜱虫控制策略以及管理现有杀螨剂的有效性和功能提供依据。
