title: Multiple mutations in the acetylcholinesterase 3 gene associated with organophosphate resistance in Rhipicephalus (Boophilus) microplus ticks from Punjab, India.
abstract: The organophosphate (OP) resistance status in Rhipicephalus (Boophilus) microplus ticks collected from seventeen districts located in the northwestern Indian state, Punjab were characterized using three data sets (bioassay, biochemical and molecular assays). Adult immersion test (AIT) was adopted and the resistance factors (RF) for the field isolates were determined. Resistance to malathion was detected in 12 isolates among which 11 showed level I resistance status while level II status was recorded in one isolate (RF of 5.35). To understand the possible mechanism of resistance development, acetylcholinesterase (AChE) activity and gene sequences of the AChE3 were analyzed. A significantly (P<0.001) higher level of percent uninhibited AChE activity was recorded in all field isolates (36.36±0.46-43.77±1.21) in comparison to the susceptible population (29.39±0.40). The AChE activity was positively correlated with RF against malathion with a correlation coefficient (r) of 0.359. Analysis of nucleotides and their deduced amino acids sequences of partial AChE3 gene revealed the presence of six amino acid substitutions (I48L, I54V, V71A, I77M, S79P and R86Q). Three novel amino acid substitutions (V71A, I77M and S79P) in partial AChE3 gene were also identified in some of the isolates which may possibly have a role in OP resistance development. The PCR-RFLP assay with HaeIII revealed the presence of restriction site corresponding to R86Q mutation in all the field isolates along with an additional restriction site in seven field isolates corresponding to V71A mutation. The results of the study indicate the involvement of both insensitive AChE and higher percent uninhibited AChE activity as the possible mechanism in these field isolates.
authors.affiliation: Department of Veterinary Parasitology, College of Veterinary Science, Guru Angad Dev Veterinary and Animal Sciences University, Ludhiana 141004, India. Electronic address: jyoti76vet@gmail.com.
authors.lastname: Jyoti
authors.affiliation: Department of Veterinary Parasitology, College of Veterinary Science, Guru Angad Dev Veterinary and Animal Sciences University, Ludhiana 141004, India.
authors.forename: Nirbhay Kumar
authors.initials: NK
authors.lastname: Singh
authors.affiliation: Department of Veterinary Parasitology, College of Veterinary Science, Guru Angad Dev Veterinary and Animal Sciences University, Ludhiana 141004, India.
authors.forename: Harkirat
authors.initials: H
authors.lastname: Singh
authors.affiliation: School of Animal Biotechnology, Guru Angad Dev Veterinary and Animal Sciences University, Ludhiana 141004, India.
authors.forename: Niraj Kumar
authors.initials: NK
authors.lastname: Singh
authors.affiliation: Department of Veterinary Parasitology, College of Veterinary Science, Guru Angad Dev Veterinary and Animal Sciences University, Ludhiana 141004, India.
authors.forename: Shitanshu S
authors.initials: SS
authors.lastname: Rath
mesh_terms: D056810:Acaricides / Q000494:pharmacology*; D000110:Acetylcholinesterase / Q000235:genetics*; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001681:Biological Assay; D004305:Dose-Response Relationship, Drug; D005260:Female; D007194:India; D007305:Insecticide Resistance / Q000235:genetics*; D007928:Lethal Dose 50; D008294:Malathion / Q000494:pharmacology; D008969:Molecular Sequence Data; D009154:Mutation; D010755:Organophosphates / Q000494:pharmacology*; D016133:Polymerase Chain Reaction; D012150:Polymorphism, Restriction Fragment Length; D048494:Rhipicephalus / Q000187:drug effects / Q000201:enzymology / Q000235:genetics*; D016415:Sequence Alignment
chemical_list: D056810:Acaricides; D010755:Organophosphates; D000110:Acetylcholinesterase; D008294:Malathion
keywords: Acetylcholinesterase; India; OP resistance; Punjab; Rhipicephalus (Boophilus) microplus