Evaluation of fipronil efficacy and first molecular report of gamma-aminobutyric acid (GABA) gated chloride channel gene of Rhipicephalus microplus ticks in China and Pakistan.

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.