N318L Blocks the\nInteraction of Fluralaner but Not\nBroflanilide or Fipronil with the Insect GABA Receptor <i>In\nVivo</i>

Fluralaner is a novel insecticide\ntargeting the ionotropic GABA\nreceptor (GABAR) subunit, RDL. A recent study revealed that N316L,\na substitution of asparagine (N) with leucine (L), in the second transmembrane\n(M2)-spanning region reduced the antagonist action of fluralaner on\nthe housefly Musca domestica RDL (<i>Md</i>RDL) <i>in vitro</i>. To verify the impact of\nN316L <i>in vivo</i>, the corresponding mutation (N318L)\nin the fruitfly Drosophila melanogaster RDL (<i>Dm</i>RDL) was constructed using CRISPR/Cas9 genome\nediting. The homozygous <i>Dm</i>RDL<sup>N318L</sup> mutant\nshowed a 9.87-fold resistance to fluralaner compared with <i>w</i><sup>1118</sup> while still being highly sensitive to broflanilide\nand fipronil, which is consistent with those findings observed in\nthe electrophysiology assays of the homomeric <i>Dm</i>RDL<sup>WT</sup> or <i>Dm</i>RDL<sup>N318L</sup> channel. Moreover, <i>Dm</i>RDL<sup>N318L</sup> led to malformed ovaries, stunted\neggs, and sterility in homozygous females. These results highlighted\nN318 as a molecular site for fluralaner <i>in vivo</i> and <i>in vitro</i> and might elucidate the resistance mechanisms of\ninsects against fluralaner.