A 110-bp ssODN (single-strand oligodeoxynucleotide) was synthesized (GenScript, Nanjing, China) as the donor template to replace the targeted genomic region.
After growth for 6 hours at 37°C, cells were collected, and MpNaam was purified using glutathione resin(GenScript, L00206).
The purified sample was shipped to GenScript to generate the rabbit polyclonal antibodies.
Total protein extracts (10 g) were separated by SDS-PAGE (8 to 16%; GenScript, M00658) and transferred to polyvinylidene difluoride membranes (Millipore, ISEQ00010) using the eBlot L1 wet protein transfer system (GenScript).
Hundreds of neurotoxic insecticides are currently in use. However, only a few direct targets have been identified. Here, using Drosophila and the insecticide flonicamid, we identified nicotinamidase (Naam) as a previous unidentified molecular target for an insecticide. Naam is expressed in chordotonal stretch-receptor neurons, and inhibition of Naam by a metabolite of flonicamid, TFNA-AM (4-trifluoromethylnicotinamide), induces accumulation of substrate nicotinamide and greatly inhibits negative geotaxis. Engineered flies harboring a point mutation in the active site show insecticide resistance and defects in gravity sensing. Bees are resistant to flonicamid because of a gene duplication, resulting in the gener... More
Hundreds of neurotoxic insecticides are currently in use. However, only a few direct targets have been identified. Here, using Drosophila and the insecticide flonicamid, we identified nicotinamidase (Naam) as a previous unidentified molecular target for an insecticide. Naam is expressed in chordotonal stretch-receptor neurons, and inhibition of Naam by a metabolite of flonicamid, TFNA-AM (4-trifluoromethylnicotinamide), induces accumulation of substrate nicotinamide and greatly inhibits negative geotaxis. Engineered flies harboring a point mutation in the active site show insecticide resistance and defects in gravity sensing. Bees are resistant to flonicamid because of a gene duplication, resulting in the generation of a TFNA-AM-insensitive Naam. Our results, in combination with the absence of genes encoding Naam in vertebrate genomes, suggest that TFNA-AM and potential species-specific Naam inhibitors could be developed as novel insecticides, anthelmintics, and antimicrobials for agriculture and human health.