N318L Blocks the Interaction of Fluralaner but Not Broflanilide or Fipronil with the Insect GABA Receptor In Vivo

Fluralaner is a novel insecticide targeting the ionotropic GABA receptor (GABAR) subunit, RDL. A recent study revealed that N316L, a substitution of asparagine (N) with leucine (L), in the second transmembrane (M2)-spanning region reduced the antagonist action of fluralaner on the housefly Musca domestica RDL (MdRDL) in vitro. To verify the impact of N316L in vivo, the corresponding mutation (N318L) in the fruitfly Drosophila melanogaster RDL (DmRDL) was constructed using CRISPR/Cas9 genome editing. The homozygous DmRDL^N318L mutant showed a 9.87-fold resistance to fluralaner compared with w¹¹¹⁸ while still being highly sensitive to broflanilide and fipronil, which is consistent with those findings observed in the electrophysiology assays of the homomeric DmRDL^WT or DmRDL^N318L channel. Moreover, DmRDL^N318L led to malformed ovaries, stunted eggs, and sterility in homozygous females. These results highlighted N318 as a molecular site for fluralaner in vivo and in vitro and might elucidate the resistance mechanisms of insects against fluralaner.