N318L Blocks the Interaction of Fluralaner but Not Broflanilide or Fipronil withGABA Receptor

What is it about?

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 DmRDLN318L 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 DmRDLWT or DmRDLN318L channel. Moreover, DmRDLN318L 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.

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Why is it important?

the effect of RDLN318L in vivo on the sensitivity to fluralaner was assessed using genome-edited D. melanogaster. Homozygous DmRDLN318L mutants were resistant to fluralaner while still being sensitive to broflanilide and fipronil. Meanwhile, electrophysiological assays also showed that N318L did not affect the antagonism of DMBF to the homomeric DmRDL channel in vitro. It is also worth noting that homozygous DmRDLN318L females were sterile with malformed eggs, indicating fitness costs associated with the DmRDLN318L mutation. These results highlight N318 in M2 as potentially being a target site for fluralaner action and could help us understand possible mechanisms underlying insect resistance to isoxazoline and meta-diamide insecticides.

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