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Cytochrome P450 Confers Single or Multiple Factorial Resistance?
B. Y. Sathish Kumar and V. A. Vijayan
Department of Studies in Zoology,
University of Mysore, Manasagangothri,
Mysore - 570 000. India.
Most insecticide resistance phenomenon are
genetically determined and mediated by a group of microsomal enzymes called
Cytochrome monooxygenases or Cyps. The cytochrome P450 monooxygenases
are found from bacteria to mammals. In insects, these enzymes play key
roles in process ranging from host plant utilization to xenobiotic resistance1.
The P450 monooxygenases are involved in the metabolism of all class of
insecticides, leading to an activation of the molecules or more generally
to detoxification2, 3. The principle of P450 enzyme activation is oxidation
of substrate with release of H2O molecule in presence
of a reductase (NADPH). The reaction occurs as follows:
Substrate(S) + (NADPH + H+) +
O2 --> S(O) + NADP+ + H2O
Cytochrome b5 can also stimulate this reaction. The key protein of this enzymatic system responsible for the specificity of the reaction is cytochrome P450. P450 stands for the absorption peak of this protein at 450 nm when reduced and saturated with CO. The genes for the cytochrome P450s are large families involved in a wide variety of metabolic functions. All gene members of the P450 super family are designated with a Cyp prefix, followed by a numeral for the family, a letter for the subfamily, and a numeral for the individual gene. There are eighty-three genes in D. melanogaster that putatively encode functional P450s. These have been classified into twenty-five different families, but more than 50% belong to either the CYP4 or CYP6 families4.
The involvement of P450 enzymes in metabolic resistance by insects to insecticides has been worked out2, 3. A direct way to show the intervention of P450 in resistance to insecticides is to compare the NADPH-dependent metabolism of insecticide in resistant and susceptible strains. In the case of DDT-resistant strain RDDTR of Drosophila, the NADPH-dependent metabolism of DDT is ten times higher than that of a susceptible strain5. Using various PCR methods, P450's were found to be constitutively overproduced in resistant strains: Cyp6A1 in Rutgers strain of housefly6, Cyp6D1 in Learn PyrR strain of housefly7, Cyp6B2 in Helicoverpa8 and Cyp9A1 in Heliothis virescens9. Although the involvement of P450 in insect resistance is documented, the upregulation of this metabolic enzyme associated with resistance remains less understood1, 10, 11.
A group led by ffrench-Constant, have employed D. melanogaster as a model insect to dissect the genetic basis of metabolic insecticide resistance11, 12. Employing microarray analysis of all P450's in Drosophila melanogaster, Daborn et al., (2002) showed that DDT-R, a gene conferring resistance to DDT, to be associated with over transcription of a single cytochrome P450 gene, Cyp6g1. They have employed Hikone-R (a resistance strain established from field collection in the early 1960s) and WC2 (a field collected DDT-resistant strain) relative to Canton-S (a susceptible reference strain). DDT-R locus maps to right arm of chromosome II at 64.5cM and over transcription of this gene alone confers resistance to DDT (used for vector control), neonicotinoid nicotinic acetylcholine receptor agonist (imidacloprid and nitenpyram), and a novel insect growth regulator (lufenuron). They also sequenced the first intron of Cyp6g1 in the same resistant and susceptible strains to determine the relatedness of the DDT-R alleles using PCR, which detect the presence of the transposon and based on the length of the product generated, showed insertion to present in all 20 resistant alleles examined. They demonstrated by transgenic experiments that over transcription of Cyp6g1 alone is both necessary and sufficient for P450 - mediated DDT resistance.
Recently Pedra et al., 13 by Genome-wide micro array analysis (Affymetrix array) have demonstrated that multiple cytochromes P450 are over expressed and potentially contribute to the DDT resistance phenotype. They employed (1) the laboratory DDT - selected Rst(2)DDT91-R, (2) the isochromosomal DDT - resistant field isolate RST(2)DDTWisconsin and (3) the DDT - susceptible Cantons-S lines of Drosophila melanogaster, there are four detoxification enzyme gene constitutively over transcribed in both RST(2)DDTWisconsin and Rst(2)DDT91-R (Cyp6g1, Cyp12d1, Cyp6a2, Cyp6w1). The relative transcript expression of Cyp6a2 in Rst(2)DDT91-R was 255- and 9.1- fold greater than Canton-S and Rst(2)DDTWisconsin. To examine whether the position of genes over expressed in DDT-resistant Drosophila were random or clustered together in a region. The cytological position of each differentially transcribed probe set revealed that the transcripts were widely distributed across all chromosomes except for its moderate representation in the right arm of the second chromosome. Thus reported that, multiple cytochromes P450 are over expressed and potentially contribute to the DDT resistance phenotype. For the discrepancies found between their findings and those of Daborn et al., (2002); Pedra et al., (2004) listed the differences to be underlying at statistical methodologies, array technology employed, age or gender employed.
As far as now, the debate of single or multiple factorial influences in resistance development is still open. P450 appear to be a prime candidate to work on far sorting this issue. Answer to this question will not only unravel a chapter on evolution of insecticidal resistance but it will also help in designing accurate methodologies for monitoring resistance alleles of P450 and their spread in wild population of agricultural pests or vectors of diseases.
REFERENCES
1. Feyereisen, R. Ann. Rev. Entomology. 1999, 44, 507-533.
2. Hodgson, E. Comp. Insect Physiol. Biochem. Physiol. 1985, 11, 225-321.
3. Agosin, M. Comp. Insect Physiol. Biochem. Physiol. 1985, 12, 647-712.
4. Tijet, N., Helvig, C. and Feyereisen, R. Gene. 2001, 189-198.
5. Cauny, A., Pralavorio, M., Pauron, D., Berge, J. -B., Fournier, D., Blais, C., Lafont, R., Salaun, J. P., Weissbart, D., Larrooque, C., and Lange, R. Pestic. Biochem. Physiol. 1990, 37, 293- 302.
6. Carino, F. A.,Koener, J. F., Plapp, F. W. and Feyereisen, R. ACS Symp. Ser. 1994, 505, 31-40.
7. Scott, J. G., Sridhar, P. and Liu, N. Archs Insect Biochem. Physiol. 1996, 31, 313-323.
8. Xiao-Ping, W and Hobbs, A. A. Insect Biochem. Molec. Biol. 1995, 1001-1009.
9. Rose, R. L., Goh, D., Thompson, D. M., Verma, K. D., Heckel, D. G., Gahan, L. J., Roe, R. M., and Hodgson, E. Insect Biochem. Molec. Biol. 1997, 27, 605-615.
10. Dunkov, B. C., Mocelin, G., Shotkoski, F., Ffrench-Constant, R. H. & Feyereisen, R. DNA Cell Biol. 1997, 16, 1345-1356.
11. Daborn, P. J., Yen, J. L., Bogwitz, M. R., Le Goff, G., Feil, E., Jeffers, S., Tijet, N., Perry, T., Heckel, D., Batterham, P., Feyereisen, R., Wilson, T. G., Ffrench-Constant, R. H. Science, 2002, 297, 2253-2256.
12. ffrench-Constant, R. T. Roush, F. Carino, in Molecular Approaches to pure and Applied Entomology, (Eds. Whitten, M. J., Oakeshott, J. G.) Springer-Verlag, Berlin, 1992, 1-37.
13. Pedra, J. H. F., McIntyre, L. M., Scharf, M. E., and Pittendrigh B. R. Proc. Natl. Acad. Sci. USA, 2004, 101, 7034-7039.
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