Molecular cloning, nucleotide sequences and gene expression of new cytochrome P450s (CYP6A24, CYP6D3v2) from the pyrethroid resistant housefly, Musca domestica L. (Diptera: Muscidae).

Abstract

Nine partial sequences characteristic of cytochrome P450 (CYP) were obtained by RT-PCR using degenerate primers designed from conserved regions of the CYP6 family. The sequences were CYP6A1, CYP6D1 and seven new CYPs. Full amino acid sequences of CYP6D1 and two new CYP isoforms, CYP6A24 and CYP6D3v2 were analyzed. The amino acid sequence of CYP6D1 in the pyrethroid resistant strains was identical with that of a susceptible strain, CS, already reported. Northern blot analysis indicated that CYP6D1, CYP6A24 and CYP6D3v2 were overexpressed in all pyrethroid resistant strains when compared with the susceptible strain. In conclusion, pyrethroid resistant strains in Japan show simultaneous overexpression of several CYP isoforms.

Transcription profiling of a recently colonised pyrethroid resistant Anopheles gambiae strain from Ghana

“…The most similar P450s to CYP6Z2 as retrieved by BLAST [32] found in other insect species are CYP6D3 in Musca domestica (44% amino sequence identity) and CYP6D4 in Drosophila melanogaster (43% amino sequence identity). Intriguingly CYP6D3 is over-expressed in several housefly strains resistant to permethrin [33] while deletion of CYP6D4 in D. melanogaster did not alter the toxicity of several insecticides including pyrethroids [34]. P450s most similar to CYP6M2 identified using BLAST [32] in A. gambiae are the CYP6N3 in Aedes albopictus (54% amino sequence identity), CYP6BB1 in the eastern saltmarsh mosquito, Ochlerotatus sollicitans , (49% amino sequence identity), and CYP6A5 in M. domestica (49% amino sequence identity).…”

The Anopheles gambiae detoxification chip: A highly specific microarray to study metabolic-based insecticide resistance in malaria vectors

“…Many studies have implicated P450s in metabolic resistance to pyrethroids in insects (18,19), and the gene CYP6Z1 has been shown to be significantly overexpressed in the permethrinresistant RSP strain of An. gambiae with a quantitative RT-PCR ratio of 3.5-fold (20).…”

Identification of potential detoxification enzyme genes in Leptinotarsa decemlineata (Say) and study of their expression in insects reared on different plants

“…Messenger RNA (0.2 mg) from the beetles kept on potato plants was used for the first strand cDNA synthesis using a BD SMART TM RACE cDNA Amplification Kit (BD Biosciences, Mississauga, ON). Degenerate primers (Table 1) were designed based on the consensus sequence of heme-binding domain (FxxGxxxCxG) of P450s from other insect species in the GenBank database (Kamiya et al 2001;Rongnoparut et al 2003). The degenerate RACE was conducted for 35 cycles at 948C for 30 s, 50Á 658C (58C below the Tm of each primer) for 30 s and 728C for 2 min using a single degenerate primer and the universal primer from the RACE kit.…”

“…This allows them to colonize plant species otherwise toxic to them (Ahammad-Sahib et al 1994;Scharf et al 1997;Kamiya et al 2001;Daborn et al 2002). The abilities largely rely on three kinds of detoxification enzymes: cytochromes P450 (P450), glutathione S-transferases (GST) and esterases (Wadleigh et al 1988;Danielson et al 1997).…”

“…Therefore, the use of the microarray approach to study the gene expression of insect detoxification enzymes has to start with gene isolation. The conserved heme-binding domain in P450s makes it possible to use degenerate PCR for amplification of P450 ESTs (Shen et al 1993;Wachenfeldt and Johnson 1995;Kamiya et al 2001). Since an insect genome may contain fewer than 200 of the potential detoxification enzyme genes, a low-density microarray approach would be ideal to monitor expression of these genes.…”