Identification and CRISPR-Cas9 validation of a novel β-adrenergic-like octopamine receptor mutation associated with amitraz resistance in Varroa destructor.

Varroa destructor is widely recognized as a significant contributor to colony collapse disorder. Chemical acaricides, such as amitraz, have been extensively used for Varroa control due to their selectivity within beehives. However, the increasing number of cases of amitraz resistance across global V. destructor populations poses a significant challenge. In this study, we conducted a comprehensive molecular screening of the β-adrenergic-like octopamine receptor (Octβ2R), the target-site of amitraz, across 66 Turkish and 63 Belgian V. destructor populations. Although previously reported amitraz resistance mutations were not detected, the screening revealed a novel Y337F mutation located within transmembrane 7 (TM7) of Octβ2R in Turkish Varroa populations. Notably, this mutation was identified in the last residue of the highly conserved NPxxY motif associated with the activation of G-protein coupled receptors (GPCR). Among the 66 Varroa samples from Türkiye, twenty harbored the Y337F mutation, with eight samples exhibiting fixation of the mutation. Subsequent bioassays revealed over 8-fold resistance to amitraz in populations that contain the Y337F mutation. Genotyping of mites after exposure to 10 mg a.i./l amitraz demonstrated that all surviving mites were homozygous for the Y337F mutation, whereas dead mites carried susceptible alleles, providing genetic linkage between mutation and phenotype. Further, we used CRISPR-Cas9 editing to introduce the Y337F mutation in the orthologous Octβ2R of the model organism Tetranychus urticae. Crispants exhibited over threefold resistance to amitraz. In conclusion, this study identified and validated a novel amitraz resistance mutation. Additional research is required to further evaluate the phenotypic strength of Y337F in the context of operational resistance with current treatment strategies.