Multiple sodium channel genes and their differential genotype at the L-to-F kdr locus in the mosquitoes, Culex quinquefasciatus

A single mutation of leucine to phenylalanine (L-to-F), termed kdr mutationin, in the sodium channel gene has been clearly demonstrated to be associated with resistance to pyrethroid and DDT in many insect species. Studies revealed that the L-to-F mutation resulted from a substitution of cytosine to thymine (C-to-T) on sodium channel gene of house flies might undergo posttranscriptional regulation. To further understand the molecular basis of the posttranscriptional regulation of L-to-F mutation and insecticide resistance development, genomic organization, gene expression patterns, and the allelic expression at the L-to-F site of the sodium channel gene were characterized between susceptible and resistant mosquitoes. Multiple copies of the sodium channel gene were revealed by Southern blot analysis. The result was further confirmed by polymerase chain reaction (PCR) analysis with primers designed for amplifying fragments of sodium channel genomic DNA, where the L-to-F mutation resides. In addition, the PCR analysis indicated one of the sodium channel genomic DNA fragment that lack the intron near the L-to-F site. A strong correlation of the frequency of kdr allele (T) in the genomic DNA level with the kdr allelic expression at the RNA level and the levels of resistance was identified only from the DNA fragment with the intron, but not that without intron. Nevertheless, a single sodium channel transcript was identified by Northern blot analysis. Taking together, these results suggested a posttranscriptional regulation mechanism in the formation of a single functional transcript of multiple sodium channel copies and, in turn, in the development of resistance in mosquitoes.