Genotypic detection of synthetic pyrethroid and organophosphate resistance-associated mutations in Rhipicephalus (Boophilus) microplus infesting ruminants across three agro-ecological zones in Pakistan

Acaricides are used to control ticks; however, their irrational use may lead to resistance, making tick control more difficult. This study aimed to determine the spatial distribution of ticks and detect genetic mutations associated with resistance to organophosphates (OPs) and synthetic pyrethroids (SPs) in Rhipicephalus (Boophilus) microplus ticks in Punjab, Pakistan. Ticks were collected from livestock farms across three agroecological zones in Punjab. They were identified morphologically and confirmed through molecular assays. The polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) technique was used to detect the mutation at G1120A in the acetylcholinesterase gene using the EcoRI restriction enzyme. To detect mutation at T2134A of voltage-gated sodium channel gene, allele-specific PCR was performed. A total of 1,003 ixodid ticks were collected representing seven species: Hyalomma anatolicum (457; 45.56%), R. (B.) microplus (236; 23.52%), H. excavatum (139; 13.85%), H. dromedarii (76; 7.57%), H. rufipes (49; 4.88%), R. (B.) annulatus (37; 3.68%), and R. (B.) decoloratus (9; 0.89%). Of the 236 R. (B.) microplus ticks, 70.76% (167/236) and 32.62% (77/236) exhibited resistance (AB or BB) to SPs and OPs, respectively. Among SP-resistant populations, 69.46% (116/167) were heterozygotes. Similarly, 31.16% (24/77) were heterozygotes among OP-resistant R. (B.) microplus. Resistance to OPs was significantly different among zones (p < 0.041), while a no significant difference was observed for SP resistance. This study highlights that the control of R. (B.) microplus remains challenging because of resistance-associated mutations in multiple acaricide target genes, which may compromise the efficacy of commonly used acaricides. These findings provide important evidence to support the development of targeted and evidence-based tick management strategies.