161 Amplification of a xanthine dehydrogenase gene is associated with insecticide mistance in the common house mosquito Cula qni~nefarciaars. MICHAEL ...
526s Biochemical Society Transactions (1997) 25 161 Amplification of a xanthine dehydrogenase gene is associated with insecticide mistance in the common house mosquito Culaqni~nefarciaars
MICHAEL COLEMAN and JANET HEMINGWAY. School of Pure and Applied Biology, University of Wales Cardiff, PO Box 915, Cardiff CFl3TL., United Kingdom. Organ~pho~phorug insecticide resistance in the mosquito Cukx quinquefasciatm (Say), the major vector of filariasis, is due to the elevation of carboxylesteraseactivity [l]. The underlying molecular basis of the enzymes elevation is gene amplification. The esterases occur on amplified sectionsof DNA (ampticons) which contain up to 40 kb of DNA. There can be up to 250 copies of the amplicon per genome in the resistant insects, whereas the esterases occur in single copy form in the insecticide susceptible insects. Two major phenotypes exist which involve either co-amplification of esterases E s W ' and EstP2' or amplification of variants of Estel[2]. Amplification of Esta.2' and EstP2' is most prevalent, being found in >95% of resistant Culex populations world-wide [1,3]. Its' predominance in C. quinquefmiatus populations is not simply due to two esterases being better than one, as recently it has been shown that EstPl is co-amplified with E W in some strains [4,5]. There does, however, appear to be a significant advantage to the Esta2' \ EstP2' phenotype over the other elevated esterases, as in the field this phenotype out-competes the others and capidly replaces them. The elevated Esta2' and EstP2' enzymes occur in complete linkage disequilibrium as they are on the same amplicon. Nucleotide sequence data suggests that this amplification event has occurred only once in C. quinquefmciatus,and then spread rapidly to all continents over the last 20 years. The amplicon is larger than the two genes which are -3kb each separated by -2.7kb of noncoding DNA [1,2]. Sequencing out from the 3' end of Esta2' a new open reading frame was detected within 1kb of the end of the Esta2' gene which had a high level of homology to xanthine dehydrogenasefrom other species [ 5 ] . The two genes were in a tail to tail orientation. We have continued to sequence out from the 3' end of the Esta2' gene over >7kb of DNA. The nucleotide sequence over this stretch of DNA continues to code for an open reading h e . This open reading frame has 600h homology to xanthine dehydrogenase from Lhawphila mekmogaster, and has greater homology to this gene than to any other in the database.. A Southern blot of the resistant Pel RR strain of C quinquejiasciatus which has the Esta.2' \ EstP2' amplicon and the insecticide susceptible Pel SS strain, which contains no amplified esterases, was probed with the 3' end of the xanthine dehydrogenase gene. This demonstrated that the 1 1 1 length putative xanthine dehydrogenase gene in the Pel RR strain was within the boundaries of the insecticide resistance-associated amplicon. In contrast, when blots of genomic digests of strains containing the Estpl', EstPl* and Esta3 amplimns were probed there was no evidence of amplification of xanthine dehydrogenase in these strains. Hence this third gene on the E s W ' \ EstP2' amplicon may have some influence on the fitness of the insects which carry the gene. A polyclonal antiserum to D. melamgaster xanthine dehydrogenase was kindly provided by Prof. Chovnik. This antiserum cross-reacted well with the xanthine dehydrogenase of C. quinquejaxiatus, and was used to determine whether the amplified xanthine dehydrogenase in this species was expressed.
Preliminary analysis suggests that the amplified xanthine dehydrogenase gene is probably expressed in the insecticide resistant C. quinquefaxiatus strains, although its activity levels vary with the life stage of the insect. What if any part does this amplified xanthine dehydrogenase play in the fitness of the insects which carry the amplicon on which it is situated? In Drosophila xanthine dehydrogenase can protect against free radical attack [6]. The larvae of mosquitoes are susceptible to oxygen free radicals produced by herbicides. such as paraquat, in &heirenvironment. Some commonly used pesticides also induce the production of reactive oxygen species, causing oxidative tissue damage, which contributes to their toxic manifestations[7]. P r e l i studies on the sub lethal effects of paraquat in C. quinquefasciatus suggest that the insecticide resistant Pel RR strain is better equipped to deal with this toxicant than is the susceptible Pel SS strain. These strains were both originally selected from the same parental colony [8] and differ only in the presence of the amplicon in the resistant strain. As neither of the amplified esterases recognise paraquat as a substrate or an inhibitor, other fktors on the amplicon may be afFecting the fitness of individuals in the presence of paraquat. Given that in Drosophilo xanthine dehydrogenase null insects are more susceptibleto the effects of paraquat than insects which express the enzyme, it is possible that the amplified xanthine dehydrogenase in C. quinquefasciatus is contributing to the reduced toxicity of this compound to the Pel RR strain. Thus the dominance of the Esta2' \ EstP2' phenotype may in part be due to the presence of the xanthine dehydrogenase gene alongside the esterase genes in this amplicon.
Acknowledgements This work is hnded by a Wellcome Trust Prize Studentship in Ecotoxicology. ReferenceS. 1 . Vaughan, A. and Hemingway, J. (1995) Journal of Biological Chemistry. 270,17044-17049. 2. Villani, F. White, G.B. Curtis C.F. and Miles S.J. (1983) Bulletin of Entomological Research. 73, 153-170. 3. Vaughan, A. Rodriguez, M. and Hemingway, J. (1995) Biochemical Journal, 305,651-658. 4. Vaughan, A. (1995) PhD thesis, University of London. 5. Small, G.J. (1996) PhD thesis, University of London. 6. Humphreys, J.M. Hillker, A.J. and Phillips, J.P. (1993) Genome, 36,162-165. 7. Bagchi, D. Bagchi,M. Hassoun, E.A. and Stohs, S.J. (1995) Toxicology, 104, 129-140. 8. Amin, A. M. Peins, H.T.R.(1990) Med. Vet. Entomology, 4, 269-279.
