Molecular cloning and xenobiotic induction of seven novel cytochrome P450 monooxygenases in Aedes albopictus

Abstract

Cytochrome P450 monooxygenase (P450) is a superfamily of enzymes that is important in metabolism of endogenous and exogenous compounds. In insects, these enzymes confer resistance to insecticides through its metabolic activities. Members of P450 from family 6 in insects are known to play a role in such function. In this study, we have isolated seven novel family 6 P450 from Aedes albopictus (Skuse) (Diptera: Culicidae), a vector of dengue and chikungunya fever. Induction profile of these seven genes was studied using several insecticides and xenobiotics. It was found that deltamethrin and permethrin did not induce expression of any genes. Another insecticide, temephos, inhibited expression of CYP6P15 for fivefold and twofold for CYP6N29, CYP6Y7, and CYP6Z18. In addition, copper II sulfate induced expression of CYP6M17 and CYP6N28 for up to sixfold. Benzothiazole (BZT), a tire leachate induced the expression of CYP6M17 by fourfold, CYP6N28 by sevenfold, but inhibited the expression of CYP6P15 for threefold and CYP6Y7 for twofold. Meanwhile, piperonyl butoxide (PBO) induced the expression CYP6N28 (twofold), while it inhibited the expression of CYP6P15 (fivefold) and CYP6Y7 (twofold). Remarkably, all seven genes were induced two- to eightfold by acetone in larval stage, but not adult stage. Expression of CYP6N28 was twofold higher, while expression of CYP6P15 was 15-fold lower in adult than larva. The other five P450s were not differentially expressed between the larvae and adult. This finding showed that acetone can be a good inducer of P450 in Ae. albopictus. On the other hand, temephos can act as good suppressor of P450, which may affect its own bioefficacy because it needs to be bioactivated by P450. To the best of our knowledge, this is the first report on acetone-inducible P450 in insects. Further study is needed to characterize the mechanisms involved in acetone induction in P450.

Keywords: acetone; cytochrome P450 monooxygenase; induction.

Fig. 1.

Inferred tree generated by MEGA4 ( Tamura et al. 2007 ) from sequences of isolated family 6 P450 together with selected P450 genes from Nelson (2009) . The evolutionary history was inferred using the neighbor-joining method ( Saitou and Nei 1987 ). The bootstrap consensus tree inferred from 10,000 replicates is taken to represent the evolutionary history of the taxa analyzed ( Felsenstein 1985 ). Branches corresponding to partitions reproduced in <50% bootstrap replicates collapsed. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (10,000 replicates) are shown next to the branches ( Felsenstein 1985 ). The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the Poisson correction method ( Zuckerkandl and Pauling 1965 ) and are in the units of the number of amino acid substitutions per site. The analysis involved 30 amino acid sequences. All positions containing gaps and missing data were eliminated. There were a total of 296 positions in the final data set.

Fig. 2.

Expression level of seven family 6 P450 genes in female adult relative to larva. Positive value indicates higher expression in adult than larva and vice versa. Error bars represent SE; asterisks represent significant difference in expression level ( P  < 0.05).

Fig. 3.

Expression ratio of seven P450 after induction by BZT, CuSO ₄ , temephos, deltamethrin, and permethrin in relation to its respective calibrator. Positive value indicates overexpression, while negative value indicates underexpression. Error bars represent SE; asterisk indicates statistically significant differential expression ( P  < 0.05).

Fig. 4.

Mean expression ± SE of family 6 P450 genes in Ae. albopictus after induction by acetone. Positive value indicates overexpression and negative value indicates otherwise. Error bars represent SE; asterisks represent significant differential expression ( P  < 0.05). Relative expressions of both larvae and adult are incomparable because calibrator samples used were different.