| The eastern salt marsh mosquito, Aedes sollicitans (Walker), is an important vector of eastern equine encephalitis as well as several other mosquitoes-borne encephalitides. Metabolism of xenobiotics and endogenous compounds by the cytochrome P450 monooxygenases can lead to insecticide resistance, physiological regulation and evolutionary adaptations. In vivo toxicities of carbaryl, resmethrin and temephos to Ae. sollicitans larvae were determined in two mosquito populations. P450 sequences potentially involved in insecticide detoxification were isolated and the mRNA expression pattern was observed under different conditions to approach the potential functions of these P450 sequences.; Both larvae and adults of the Ae. sollicitans population from Ocean County, New Jersey, displayed increased tolerance to temephos, but not to carbaryl and resmethrin, when compared to the population from Middlesex County. Administration of the synergists piperonyl butoxide (PBO) and S,S,S-tributyl phosphotrithiate (DEF) revealed that P450s are the major enzymes detoxifying carbaryl and resmethrin, whereas esterases are the major enzymes detoxifying temephos.; For larvae raised in pure water, low salinity (1%-3.5%) promoted the larval survival but high salinity (5%) deteriorated the larval survival under temephos treatment. High salinity also caused reduction in larval body size and 5% salinity alone caused mortality for the larvae raised in pure water. Cross-interaction of salinity and temephos was observed. Salinity did not affect the temephos toxicity to larvae raised in salt water.; Using a pair of degenerate primers based on two conserved regions of insect CYP6 cytochrome P450s, 13 cytochrome P450 fragments were isolated from Ae. sollicitans. Based on these fragments, two novel full-length cytochrome P450s, CYP6BB1 and CYP6P10, and one-partial length P450, CYP6Z5, were identified using the rapid amplification of cDNA ends (RACE) method. Both CYP6BB1 and CYP6P10 have at least five allelic sequences. CYP6BB1V1-CYP6BB1V5 and CYP6P10V1-CYP6P10V5, respectively. The CYP6BB1 allelic cDNAs ranged in length from 1571 to 1656 nucleotides with a common open reading frame of 1518 nucleotides encoding five allelic amino acid sequences with 506 residues. The CYP6P10 allelic cDNAs ranged in length from 2023 to 2041 nucleotides with a common open reading frame of 1521 nucleotides encoding five allelic amino acid sequences with 507 residues. CYP6Z5 also has five allelic cDNA sequences, CYP6Z5V1-CYP6Z5V5, encoding 5 partial allelic amino acid sequences with 130 residues. All the alleles are typical microsomal P450s sharing signature sequences with other P450s. The deduced amino acid sequences of CYP6BB1 alleles shared the highest sequence identity, 56%, with CYP6P4 and 51%-54% identities with several other CYP6P members. The deduced amino acid sequences of CYP6P10 alleles shared the highest identity, 65%, with CYP6P4 and 59%-61% identities with other CYP6P P450s. The CYP6Z5 amino acid sequences shared the highest identity, 64%, with CYP6Z2.; Quantitative real-time expression analysis showed that CYP6BB1 was significantly over-expressed in adult mosquitoes but not in larvae in the Ocean County population. Expression analysis also showed that neither CYP6BB1 nor CYP6P10 were larva-, adult- or sex-specific. Blood feeding significantly increased the CYP6BB1 mRNA expression. Expression of CYP6P10 appeared not to be significantly different between the two populations or after a blood meal. The real function(s) of CYP6BB1 have to be confirmed by recombinant experiments to define the substrate specificities.; This is the first P450 study in Ae. sollicitans and the first CYP6 P450 sequences isolated from Ae. sollicitans and their expression patterns in two populations, among different life stages and after blood feeding. Understanding the importance of P450-mediated metabolism is hindered not only by the limited number of P450 genes identified in insects, but also by... |
