Study of the molecular nature of resistance to bifenazate in a <i>Tetranychus urticae</i> Koch Laboratory Strain

Abstract BACKGROUND The two-spotted spider mite, Tetranychus urticae Koch, is a major agricultural pest with a rapid propensity for developing acaricide resistance. Bifenazate targets mitochondrial cytochrome b (CYTB). While the G126S mutation is frequently associated with resistance, its independent role remains unclear as it often occurs with other substitutions. This study explores the molecular basis of bifenazate resistance in a Russian laboratory strain derived from a St. Petersburg greenhouse population. RESULTS Disruptive selection with increasing bifenazate concentrations generated resistant and susceptible isofemale lines. AlphaFold2 structural modeling of CYTB indicated that G126S causes a steric clash, leading to conformational destabilization, whereas other reported mutations primarily affect the ligand-binding pocket. Oxford Nanopore sequencing revealed a very low initial frequency of the G126S allele (&lt;1%; 226/35,895 reads) in the unselected population. After one year of stepwise selection (0.00005–0.031% a.i.), the mutant allele frequency surged to 90% (7,272/8,056 reads). No other known resistance-associated mutations were found in the analyzed cytb fragment. CONCLUSION We report the first identification of the G126S mutation in a Russian T. urticae population and demonstrate its rapid fixation under bifenazate selection. Within this genetic background, G126S alone appears sufficient to confer high-level resistance, emphasizing the population-specific nature of resistance evolution and the critical need for local monitoring.