Mutation Report for: R86Q_boomi-ACHE3

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.

Mutations I48L, I54V, R86Q, V137I, I492M, and T548A were identified previously in BmAChE3, a gene encoding acetylcholinesterase, from the organophosphate (OP) acaricide-resistant San Rommn strain of Rhipicephalus (Boophilus) microplus. Recombinant BmAChE3 acetylcholinesterase containing the R86Q mutation was shown to exhibit nearly 20-fold reduction in the rate of phosphorylation by paraoxon relative to the wild-type sequence. In addition, the R86Q mutation was present in resistant laboratory strains at elevated frequency compared with OP-susceptible strains but was insufficient to alone generate the OP-resistant phenotype (J. Med. Entomol. 44: 1013-1018). Here, we developed assays to genotype the remaining five mutations and evaluated frequency of all six BmAChE3 mutations in individual R. microplus ticks from laboratory and Mexican field-collected strains. We found a substantial number of individuals in known OP-susceptible strains that seemed to be homozygous for each of the mutations surveyed, the exception being I48L, which was infrequent in all strains, leading us to conclude that none of the mutations alone were responsible for generation of phenotypic resistance to OP acaricide.

Mutations were identified in the cDNA sequence encoding the acetylcholinesterase BmAChE3 in strains of Rhipicephalus (Boophilus) microplus (Canestrini) resistant or susceptible to organophosphate (OP) acaricide. The mutation that occurred most frequently in the OP-resistant San Roman strain resulted in a substitution of glutamine (Q) for arginine (R) at position 86 in BmAChE3 (position 66 in mature BmAChE). Clones containing the mutant and wild-type cDNA sequences were expressed in the baculovirus system. Enzyme kinetics of recombinant BmAChE3 containing or lacking the R86Q mutation demonstrated that the R86Q mutation increased substrate affinity and conferred insensitivity to paraoxon inhibition. This is the first demonstration of a mutation in a gene encoding an ixodid acetylcholinesterase resulting in OP insensitivity. A restriction fragment length polymorphism assay was developed and used to diagnose the frequency of the R86Q mutation in BmAChE3 genomic DNA from seven laboratory-colonized strains. Use of the R86Q diagnostic assay detected an increased frequency of the R86Q mutation in OP-resistant tick strains compared with that of OP-susceptible strains; however, the R86Q mutation was also present in OP-susceptible strains at unexpectedly high frequency. Because the R86Q mutation generates an OP-resistant enzyme in vitro and it is present at an elevated frequency in laboratory strains selected for OP resistance, we conclude that the data are consistent with a potential role for BmAChE3 in development of OP resistance; however, because the R86Q mutation has a high frequency in susceptible strains, the R86Q mutation alone is insufficient to generate the OP-resistant phenotype at the organismal level. There are likely to be additional mutations in BmAChE3, mutations in additional acetylcholinesterase genes, or additional resistance mechanisms (e.g., oxidative metabolism) that contribute to expression of the OP-resistant phenotype.