Immunodetection of Truncated Forms of the α6 Subunit of the nAChR in the Brain of Spinosad Resistant Ceratitis capitata Phenotypes

SIMPLE SUMMARY: The insecticide spinosad is widely used for the control of the Mediterranean fruit fly (medfly), Ceratitis capitata, in citrus crops in Spain. However, the sustainable use of this insecticide is compromised by the detection of spinosad resistant alleles in field populations, which may lead to control failures in the future. Mutations, generating premature stop codons at the gene coding for the target alpha 6 subunit of the nicotinic acetylcholine receptor of C. capitata (Ccα6), have been associated with spinosad resistance in both laboratory strains and field populations. In this work, we showed that full-length transcripts from individuals carrying wild-type isoforms of the gene are translated into Ccα6 proteins that locate in the membrane of the brain cells, while truncated transcripts from spinosad resistant strains could be translated into truncated Ccα6 that, for the most part, are unable to reach their expected functional destination in the membrane. We proposed that the difference of location of Ccα6 observed in spinosad resistant strains is probably determining its resistant phenotype. In addition, we provide a tool for immunodetection of truncated forms of Ccα6, that can be useful for resistance management programs. ABSTRACT: The α6 subunit of the nicotinic acetylcholine receptor (nAChR) has been proposed as the target for spinosad in insects. Point mutations that result in premature stop codons in the α6 gene of Ceratitis capitata flies have been previously associated with spinosad resistance, but it is unknown if these transcripts are translated and if so, what is the location of the putative truncated proteins. In this work, we produced a specific antibody against C. capitata α6 (Ccα6) and validated it by ELISA, Western blotting and immunofluorescence assays in brain tissues. The antibody detects both wild-type and truncated forms of Ccα6 in vivo, and the protein is located in the cell membrane of the brain of wild-type spinosad sensitive flies. On the contrary, the shortened transcripts present in resistant flies generate putative truncated proteins that, for the most part, fail to reach their final destination in the membrane of the cells and remain in the cytoplasm. The differences observed in the locations of wild-type and truncated α6 proteins are proposed to determine the susceptibility or resistance to spinosad.