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Co-up-regulation of three P450 genes in response to permethrin exposure in permethrin resistant house flies, Musca domestica.

Zhu F, Li T, Zhang L, Liu N - BMC Physiol. (2008)

Bottom Line: Comparison of the deduced protein sequences of these three P450s from resistant ALHF and susceptible aabys and CS house flies revealed identical protein sequences.Genetic linkage analysis located CYP4D4v2 and CYP6A38 on autosome 5, corresponding to the linkage of P450-mediated resistance in ALHF, whereas CYP4G2 was located on autosome 3, where the major insecticide resistance factor(s) for ALHF had been mapped but no P450 genes reported prior to this study.Our study provides the first direct evidence that multiple P450 genes are co-up-regulated in permethrin resistant house flies through the induction mechanism, which increases overall expression levels of P450 genes in resistant house flies.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Entomology and Plant Pathology, Auburn University, Auburn, AL 36849, USA. fangzhudy@uky.edu

ABSTRACT

Background: Insects may use various biochemical pathways to enable them to tolerate the lethal action of insecticides. For example, increased cytochrome P450 detoxification is known to play an important role in many insect species. Both constitutively increased expression (overexpression) and induction of P450s are thought to be responsible for increased levels of detoxification of insecticides. However, unlike constitutively overexpressed P450 genes, whose expression association with insecticide resistance has been extensively studied, the induction of P450s is less well characterized in insecticide resistance. The current study focuses on the characterization of individual P450 genes that are induced in response to permethrin treatment in permethrin resistant house flies.

Results: The expression of 3 P450 genes, CYP4D4v2, CYP4G2, and CYP6A38, was co-up-regulated by permethrin treatment in permethrin resistant ALHF house flies in a time and dose-dependent manner. Comparison of the deduced protein sequences of these three P450s from resistant ALHF and susceptible aabys and CS house flies revealed identical protein sequences. Genetic linkage analysis located CYP4D4v2 and CYP6A38 on autosome 5, corresponding to the linkage of P450-mediated resistance in ALHF, whereas CYP4G2 was located on autosome 3, where the major insecticide resistance factor(s) for ALHF had been mapped but no P450 genes reported prior to this study.

Conclusion: Our study provides the first direct evidence that multiple P450 genes are co-up-regulated in permethrin resistant house flies through the induction mechanism, which increases overall expression levels of P450 genes in resistant house flies. Taken together with the significant induction of CYP4D4v2, CYP4G2, and CYP6A38 expression by permethrin only in permethrin resistant house flies and the correlation of the linkage of the genes with resistance and/or P450-mediated resistance in resistant ALHF house flies, this study sheds new light on the functional importance of P450 genes in response to insecticide treatment, detoxification of insecticides, the adaptation of insects to their environment, and the evolution of insecticide resistance.

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Graphic representation of CYP4G2, CYP4D4v2, and CYP6A38, showing locations and sequences of SNP-specific primers for SNP determinations and genetic linkage analyses of three P450 genes. A: CYP4G2. B: CYP4D4v2. C: CYP6A38.
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Figure 7: Graphic representation of CYP4G2, CYP4D4v2, and CYP6A38, showing locations and sequences of SNP-specific primers for SNP determinations and genetic linkage analyses of three P450 genes. A: CYP4G2. B: CYP4D4v2. C: CYP6A38.

Mentions: We previously demonstrated that permethrin resistance in ALHF house flies was linked to autosomes 1, 2, 3, and 5, with major factors on autosomes 3 and 5 [26]. We also demonstrated that the P450-mediated resistance in ALHF was predominantly linked to autosome 5 [26]. To determine whether there is a causal link between the P450 genes and insecticide resistance, we examined the genetic linkage of CYP4D4v2, CYP4G2, and CYP6A38 with 5 back-cross (BC1) house fly lines derived from crosses of ALHF and a susceptible morphological marker strain, aabys, by allele specific single nucleotide polymorphism (SNP) determination. Sequence comparisons of the three genes between ALHF and aabys revealed several nucleotide polymorphisms in the coding regions of CYP4D4v2 and CYP4G2, while no nucleotide polymorphisms were identified in the coding region of CYP6A38. We therefore cloned a ~700 bp 5' flanking region of CYP6A38 in order to genetically map the CYP6A38 gene. Comparison of the nucleotide sequence of the 5' flanking region of CYP6A38 uncovered several nucleotide polymorphisms between ALHF and aabys (data not shown), so the nucleotide polymorphisms, C to T, C to T, and G to T, in CYP4D4v2, CYP4G2, and CYP6A38, respectively, in ALHF relative to aabys (Fig. 7), were used to determine the linkage of P450 genes relative to the recessive morphological markers in the aabys strain. The SNP determination reactions were conducted for each of the genes using a specific primer (Fig. 7) designed according to the sequences immediately upstream of the nucleotide polymorphism in order to distinguish the single nucleotide polymorphism for the P450 allele in each house fly strain or line. Our results showed that the BC1 lines with the genotypes of ac/ac, +/ar, +/bwb, +/ye, +/sw (A2345), +/ac, ar/ar, +/bwb, +/ye, +/sw (A1345), +/ac, +/ar, bwb/bwb, +/ye, +/sw (A1245), and +/ac, +/ar, +/bwb, ye/ye, +/sw (A1235) were heterozygous for CYP4D4v2 and CYP6A38, where as the BC1 line with the genotype of +/ac, +/ar, +/bwb, +/ye, sw/sw (A1234) was homozygous for both the CYP4D4v2 and CYP6A38 alleles from aabys (Table 2). These results strongly indicate that both CYP4D4v2 and CYP6A38 are located on autosome 5 in house flies. The BC1 lines of A2345, A1345, A1234, and A1235 were heterozygous for CYP4G2, whereas the A1245 line was homozygous for the CYP4G2 allele from aabys (Table 2), indicating that CYP4G2 is located on autosome 3 in house flies.


Co-up-regulation of three P450 genes in response to permethrin exposure in permethrin resistant house flies, Musca domestica.

Zhu F, Li T, Zhang L, Liu N - BMC Physiol. (2008)

Graphic representation of CYP4G2, CYP4D4v2, and CYP6A38, showing locations and sequences of SNP-specific primers for SNP determinations and genetic linkage analyses of three P450 genes. A: CYP4G2. B: CYP4D4v2. C: CYP6A38.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
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Figure 7: Graphic representation of CYP4G2, CYP4D4v2, and CYP6A38, showing locations and sequences of SNP-specific primers for SNP determinations and genetic linkage analyses of three P450 genes. A: CYP4G2. B: CYP4D4v2. C: CYP6A38.
Mentions: We previously demonstrated that permethrin resistance in ALHF house flies was linked to autosomes 1, 2, 3, and 5, with major factors on autosomes 3 and 5 [26]. We also demonstrated that the P450-mediated resistance in ALHF was predominantly linked to autosome 5 [26]. To determine whether there is a causal link between the P450 genes and insecticide resistance, we examined the genetic linkage of CYP4D4v2, CYP4G2, and CYP6A38 with 5 back-cross (BC1) house fly lines derived from crosses of ALHF and a susceptible morphological marker strain, aabys, by allele specific single nucleotide polymorphism (SNP) determination. Sequence comparisons of the three genes between ALHF and aabys revealed several nucleotide polymorphisms in the coding regions of CYP4D4v2 and CYP4G2, while no nucleotide polymorphisms were identified in the coding region of CYP6A38. We therefore cloned a ~700 bp 5' flanking region of CYP6A38 in order to genetically map the CYP6A38 gene. Comparison of the nucleotide sequence of the 5' flanking region of CYP6A38 uncovered several nucleotide polymorphisms between ALHF and aabys (data not shown), so the nucleotide polymorphisms, C to T, C to T, and G to T, in CYP4D4v2, CYP4G2, and CYP6A38, respectively, in ALHF relative to aabys (Fig. 7), were used to determine the linkage of P450 genes relative to the recessive morphological markers in the aabys strain. The SNP determination reactions were conducted for each of the genes using a specific primer (Fig. 7) designed according to the sequences immediately upstream of the nucleotide polymorphism in order to distinguish the single nucleotide polymorphism for the P450 allele in each house fly strain or line. Our results showed that the BC1 lines with the genotypes of ac/ac, +/ar, +/bwb, +/ye, +/sw (A2345), +/ac, ar/ar, +/bwb, +/ye, +/sw (A1345), +/ac, +/ar, bwb/bwb, +/ye, +/sw (A1245), and +/ac, +/ar, +/bwb, ye/ye, +/sw (A1235) were heterozygous for CYP4D4v2 and CYP6A38, where as the BC1 line with the genotype of +/ac, +/ar, +/bwb, +/ye, sw/sw (A1234) was homozygous for both the CYP4D4v2 and CYP6A38 alleles from aabys (Table 2). These results strongly indicate that both CYP4D4v2 and CYP6A38 are located on autosome 5 in house flies. The BC1 lines of A2345, A1345, A1234, and A1235 were heterozygous for CYP4G2, whereas the A1245 line was homozygous for the CYP4G2 allele from aabys (Table 2), indicating that CYP4G2 is located on autosome 3 in house flies.

Bottom Line: Comparison of the deduced protein sequences of these three P450s from resistant ALHF and susceptible aabys and CS house flies revealed identical protein sequences.Genetic linkage analysis located CYP4D4v2 and CYP6A38 on autosome 5, corresponding to the linkage of P450-mediated resistance in ALHF, whereas CYP4G2 was located on autosome 3, where the major insecticide resistance factor(s) for ALHF had been mapped but no P450 genes reported prior to this study.Our study provides the first direct evidence that multiple P450 genes are co-up-regulated in permethrin resistant house flies through the induction mechanism, which increases overall expression levels of P450 genes in resistant house flies.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Entomology and Plant Pathology, Auburn University, Auburn, AL 36849, USA. fangzhudy@uky.edu

ABSTRACT

Background: Insects may use various biochemical pathways to enable them to tolerate the lethal action of insecticides. For example, increased cytochrome P450 detoxification is known to play an important role in many insect species. Both constitutively increased expression (overexpression) and induction of P450s are thought to be responsible for increased levels of detoxification of insecticides. However, unlike constitutively overexpressed P450 genes, whose expression association with insecticide resistance has been extensively studied, the induction of P450s is less well characterized in insecticide resistance. The current study focuses on the characterization of individual P450 genes that are induced in response to permethrin treatment in permethrin resistant house flies.

Results: The expression of 3 P450 genes, CYP4D4v2, CYP4G2, and CYP6A38, was co-up-regulated by permethrin treatment in permethrin resistant ALHF house flies in a time and dose-dependent manner. Comparison of the deduced protein sequences of these three P450s from resistant ALHF and susceptible aabys and CS house flies revealed identical protein sequences. Genetic linkage analysis located CYP4D4v2 and CYP6A38 on autosome 5, corresponding to the linkage of P450-mediated resistance in ALHF, whereas CYP4G2 was located on autosome 3, where the major insecticide resistance factor(s) for ALHF had been mapped but no P450 genes reported prior to this study.

Conclusion: Our study provides the first direct evidence that multiple P450 genes are co-up-regulated in permethrin resistant house flies through the induction mechanism, which increases overall expression levels of P450 genes in resistant house flies. Taken together with the significant induction of CYP4D4v2, CYP4G2, and CYP6A38 expression by permethrin only in permethrin resistant house flies and the correlation of the linkage of the genes with resistance and/or P450-mediated resistance in resistant ALHF house flies, this study sheds new light on the functional importance of P450 genes in response to insecticide treatment, detoxification of insecticides, the adaptation of insects to their environment, and the evolution of insecticide resistance.

Show MeSH