|The Southern house mosquito, Culex quinquefasciatus, is a vector of several human diseases including West Nile fever and St. Louis encephalitis, owing to its blood feeding behavior whereby the female takes multiple blood meals. Vector control of mosquitoes has been a critical part of the current global strategy to control mosquito-associated diseases. Insecticides, especially pyrethroids, are important components in the vector-control effort. The successful management of mosquitoes, however, is negatively impacted by the development of resistance to insecticides within mosquito populations and the lack of the knowledge on the molecular basis of blood feeding behavior. The goals of our research, thus, were to gain a better understanding of insecticide resistance in Cx. quinquefasciatus and to identify the genes that may be involved in preparing the female for the taking of a blood meal with four specific objectives: 1) Characterize the genes up-regulated in insecticide-resistant mosquitoes, 2) Determine the genes that are differentially-expressed in response to insecticide treatment, 3) Investigate the cytochrome P450 detoxification genes in resistance, 4) Examine the changes in the gene expression profiles of newly-eclosed females as they become old enough to take a blood meal and also in response to blood feeding.
RNA-Seq was used to investigate the gene expression profiles of a highly permethrin-resistant strain of Cx. quinquefasciatus, and to investigate the changes in the gene expression levels after exposure to permethrin. Overall, we identified multiple genes up-regulated in insecticide-resistant mosquitoes, including genes involved in detoxification, regulation, and proteases. We further identified that detoxification genes and proteases were up-regulated in response to permethrin exposure, while serum storage proteins were down-regulated, suggesting that fourth instar Cx. quinquefasciatus delay development to the pupal stage in response to insecticide challenge, possibly in response to a cessation of feeding as a means of behavioral resistance in order to reduce oral exposure to permethrin. To better understand which detoxification genes identified in Cx. quinquefasciatus were important in other mosquito species, we investigated the gene expression profiles of the cytochrome P450 genes in a pyrethroid-resistant field strain of the yellow fever mosquito, Aedes aegypti using qRT-PCR. We identified that multiple cytochrome P450 genes were up-regulated, and functional studies revealed that two of the up-regulated P450s, CYP4D4 and CYP4J15v1 could confer resistance to permethrin when over-expressed in a GAL4:UAS enhancer trap Drosophila melanogaster system. These cytochrome P450s were likely involved in the permethrin resistance response of mosquitoes.
Examination of the genes in response to the blood feeding was conducted by determining the temporal changes in gene expression from newly eclosed female adults to those capable of taking a blood meal using RNA-Seq techniques. We found that while no females would freely take a blood meal prior to 48 h post-eclosion, the main gene expression changes in newly-eclosed females occurred within the initial 12-24 h post-eclosion, including genes encoding salivary proteins, odorant-binding proteins, proteases, and cuticular proteins. A smaller second peak of up-regulated genes was identified at 48 – 60 h post-eclosion, which coincided with the onset of the maximal time-to-mating for Cx. quinquefasciatus. Taken together, these results suggested that the genes needed for blood feeding in Cx. quinquefasciatus are primarily up-regualted within 12-24 h post-eclosion, while other later genes may be up-regulated in response to mating.