Transcriptional Overexpression of Cytochrome P450 Gene(s) CYP6B8/CYP6B28, Is Associated with Cypermethrin Survivorship of Field-Collected Bollworm Males (Helicoverpa zea)

The bollworm, Helicoverpa zea, is a major agricultural pest especially in cotton, corn and grain sorghum, and currently dominates the heliothine complex in the cotton belt. Pyrethroid applications, widely used and effective against H. zea, exert high selection pressure on bollworm populations leading to resistance. We investigated if the transcriptional overexpression of cytochrome P450 genes, CYP6B8/CYP6B28 and CYP6B9, was associated with survivorship to cypermethrin in field collected males, using semi-quantitative reverse transcription PCR (Semi-Q RT-PCR) and qPCR. The relative levels of expression (as the ratio of the band intensities of the CYP6B gene(s) product(s) over the corresponding actin RT-PCR products) were compared between resistant and susceptible insects.  In RT-PCR analyses performed with insects collected in 2010, relative levels of expression of CYP6B8/6B28 with respect to actin analyzed from males surviving 5 µg cypermethrin/vial ranged from 0.89 to 3.25 and for those collected in 2006 ranged from 1.21 to 1.48.  We had previously found that the expression of both CYP transcripts was significantly higher in H. zea males surviving high dosages of cypermethrin in comparison to susceptible males (from a factor of 3.7 to 34.9 for CYP6B8/CYP6B28 and from 5.6 to 39.6 for CYP6B9) when transcripts were analyzed by qPCR. In summary, analysis of individual males collected across ecological regions over years in Texas showed that overexpression of cytochrome P450 CYP6B8/CYP6B28 and CYP6B9 genes is a mechanism for survivorship to pyrethroids in H. zea. Our discovery will allow for greater focus on increased metabolism studies at the population level. However, we have previously identified mutations in the sodium channel in resistant males, indicating that at least two mechanisms for pyrethroid resistance, target site insensitivity and oxidative metabolism are present in field collected insects.  Understanding mechanisms responsible for resistance will greatly improve the ability to monitor resistance and make better control recommendations.