Zhimou Wen

Insect cytochromes P450: diversity, insecticide resistance and tolerance to plant toxins.

In the last decade, studies of individual insect P450s have blossomed. This new information has furthered our understanding of P450 diversity, insecticide resistance and tolerance to plant toxins. Insect P450s can be adult specific, larval specific or life stage independent. Similarly, insect P450s vary as to the tissues where they are expressed and in their response to inducers. Insect P450s can now be rapidly sequenced using degenerate PCR primers. Given the huge diversity represented by the Class Insecta, this technique will provide vast amounts of new information about insect P450s and the evolution of the P450 gene superfamily. CYP6D1 is responsible for monooxygenase-mediated resistance to pyrethroid insecticides in the house fly. CYP6D1 is ubiquitously expressed in adults with 10-fold higher levels found in the resistant strain compared to susceptible strains. CYP6D1 is on autosome 1 in house fly. The high level of expression found in the resistant strain is due to genes on autosomes 1 and 2. Whether or not the different CYP6D1 alleles found in resistant and susceptible strains have any role in resistance remains to be elucidated. The CYP6B gene subfamily is involved in the metabolism of host plant toxins (i.e. furanocoumarins). CYP6B gene transcripts in two Papilio (swallowtail) species have been shown to be induced by host plant toxins and in turn to metabolize these toxins. CYP6B P450s play a critical role in allowing Papilio to adapt to furanocoumarin-containing host plants. Similarities in structural and promoter regions of the CYP6B genes suggest that they are derived from a common ancestral gene. Although the P450 monooxygenases of insects are important for the metabolism of hormones and phermones, no individual P450 has yet been shown to metabolize an endogenous compound. Advances in this area are critical because they will provide important new information about insect physiology, biochemistry and development.

Genetic and biochemical mechanisms limiting fipronil toxicity in the LPR strain of house fly, Musca domestica

Fipronil is a new insecticide which exerts its toxic action by interacting with the insect GABA-gated chloride channel. Previous studies have shown that cyclodiene-resistant insects have low to moderate levels of cross-resistance to fipronil, while other resistant strains are usually susceptible. In contrast, we recently found a strain (LPR) of house fly (Musca domestica L) with 15-fold cross-resistance to fipronil that was not associated with cyclodiene resistance. Fipronil cross-resistance in LPR was inherited as an intermediately dominant, autosomal, multigenic trait. [ 14 C]Fipronil was observed to penetrate into LPR flies more slowly than into susceptible flies. S,S,S-tributylphosphorotrithioate and diethyl maleate pretreatment did not reduce the level of fipronil cross-resistance, while piperonyl butoxide resulted in a slight decrease. These results indicate that decreased penetration and monooxygenase-mediated detoxification may be mechanisms contributing to fipronil cross-resistance in the LPR strain.

Cytochrome P450 CYP6L1 is specifically expressed in the reproductive tissues of adult male German cockroaches, Blattella germanica (L.)

A full-length cDNA encoding a new cytochrome P450, CYP6L1, was cloned from German cockroaches, Blattella germanica. CYP6L1 has an open reading frame of 1509 nucleotides with a deduced protein of 503 amino acids and molecular mass of 57 Kd. CYP6L1 is most similar to CYP6H1, a putative ecdysone 20-hydroxylase from Locusta migratoria. CYP6L1 mRNA was not detected in embryos nor nymphs, nor in adult females. CYP6L1 mRNA was detected only in the testes and accessory glands of male adult German cockroaches. Given that the testes and accessory glands are the most important components of the reproductive system in male insects, the expression of CYP6L1 mRNA exclusively in these tissues strongly suggests that CYP6L1 has a role in reproduction. Possible substrates for CYP6L1 are discussed.

Cloning of two novel P450 cDNAs from German cockroaches, Blattella germanica (L.): CYP6K1 and CYP6J1

Two novel P450 cDNAs, CYP6K1 and CYP6J1, were isolated from German cockroaches, Blattella germanica (L). Both CYP6K1 and CYP6J1 are typical microsomal P450s and their deduced amino acid sequences share a number of common characteristics with other members of the P450 superfamily. Both CYP6K1 and CYP6J1 showed the highest per cent identity (based on the deduced amino acid sequence) to CYP6L1 from B. germanica and CYP6H1, a putative ecdysone 20‐hydroxylase from Locusta migratoria. Using a CYP6K1 probe, two mRNA signals (~2.5 and ~2.1 kb) were detected in all life stages. Both signals were just detectable in the eggs and became stronger in later instars. The strongest signals were detected in the fifth and sixth instars as well as in adults. These two bands were also detected in the abdomens and in the remainder of bodies of both male and female adults. Southern blots suggest the two mRNA bands detected in the Northern blot might be a result of alternative splicing. No signal could be detected at any life stage using the CYP6J1 probe, suggesting that CYP6J1 was expressed at a low level.