Molecular Mechanisms of Insecticide Resistance - ACS Publications

Chapter 17 Esterase Genes Conferring Insecticide Resistance in Aphids

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L. M . Field and A. L. Devonshire AFRC Institute of Arable Crops Research, Rothamsted Experimental Station, Harpenden, Herts, AL5 2JQ, United Kingdom

The amplified esterase genes responsible for insecticide resistance in the aphid Myzus persicae were remarkably conserved in standard laboratory clones and in samples collected from field populations throughout the U K during 1990. Only two restriction patterns were found, each associated with the presence of a different amplified esterase (E4 or FE4). The E4 and FE4 D N A sequences in the field aphids contained 5methylcytosine (5 mC) at the same sites as in standard clones and although there were no examples of complete loss of methylation and esterase gene transcription, as previously found in revertant clones, there was evidence for partial loss of 5 mC and expression. An apparently identical esterase gene amplification and elevated enzyme (FE4) were found in a resistant Greek population of a closely related species, Myzus nicotianae.

The extensive use of chemicals to control insect pests worldwide has resulted in the development of resistance in many diverse insect species. At least 20 different aphid species have been shown by bioassay to be resistant to one or more insecticides, although evidence for the biochemical nature of the resistance is available for only a few (7). Aphis gossypii has become resistant to pirimicarb by developing a mutant, less sensitive form of the target protein, acetylcholinesterase, whereas Myzus persicae resists a range of insecticides by detoxification via increased carboxylesterase activity. Resistant Phorodon humuli also have increased esterase activity, although its role in resistance has not been established biochemically. The tobacco-feeding form of M. persicae which was recently classified as a new species, Myzus nicotianae (2), also resists organophosphorous insecticides by increased carboxylesterase activity (J). The molecular genetic basis of increased esterase activity in aphids is known only

0097-6156/92/0505-0209$06.00/D © 1992 American Chemical Society

In Molecular Mechanisms of Insecticide Resistance; Mullin, C., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1992.

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MOLECULAR MECHANISMS OF INSECTICIDE RESISTANCE

in the case of M. persicae (4) and this paper reviews the evidence for amplification and transcriptional control of esterase genes in this species and reports recent studies on the structure and methylation of esterase genomic sequences including homologous genes in Af. nicotianae.


Insecticide Resistance and Amplified Esterase Genes in Myzus persicae A positive correlation between insecticide resistance and total carboxylesterase activity was first demonstrated in M persicae over 20 years ago (5), and it was subsequently shown that only a single esterase is responsible and that the increased activity results from more esterase protein (6). Two forms of the esterase can confer resistance, E4 (a protein of c 65 kDa) present in resistant aphids with an Al,3 chromosome translocation or FE4 (c 66 kDa) found in aphids of apparently normal karyotype (7). An observed doubling in esterase content through a series of seven progressively more resistant M persicae clones led Devonshire & Sawicki in 1979 (8) to propose gene amplification as the basis of the elevated enzyme synthesis. This was confirmed in 1989 when an E4 cDNA (pMp 24) was used to demonstrate increased esterase gene copy number in resistant aphids with either E4 or FE4 enzyme (4). Furthermore, the binding of the probe to EcoRI digested aphid D N A showed that amplified esterase sequences were present on an 8kb fragment in translocated aphids with E4 and a 4 kb fragment in aphids with FE4. This has now been demonstrated for many resistant clones from widely diverse origins and six examples are shown in Figure 1. In addition to the 4 kb and 8 kb fragments containing amplified FE4 and E4 sequences, there are other fainter bands in Figure 1, presumably resulting from binding of the E4 cDNA to non-amplified E4-related sequences. There is an apparent heterozygosity for the 10 and 15 kb fragments in translocated aphids (Figure 1, B samples) compared with homozygosity of the 10 kb fragment in aphids of normal karyotype (A samples). This marked conservation of restriction patterns between diverse aphid clones suggests that there is little polymorphism at the amplified E4 and FE4 loci. The 4 kb and 8 kb EcoRI genomic fragments have been cloned and analyzed by restriction mapping using various cDNAs as probes (Field, L M and Devonshire, A L> Monograph of SCI meeting "Resistance '91" 1991, in press). This showed that E4 and FE4 genomic sequences are very similar and that the two genes probably differ only at the 3' end, where there are both qualitative and quantitative variations. The findings are summarized in Figure 2B where the maps explain observed differences in Mspl digests in different aphid clones (see next section). Instability of Resistance in M . persicae Clones In the absence of selection pressure, extremely resistant M persicae clones with the A l , 3 translocation can spontaneously lose resistance and elevated E4 between successive parthenogenic generations (9). Using E4 cDNA to probe



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Esterase Genes Conferring Insecticide Resistance 211

Figure 1. Binding of an E4 cDNA (pMp24)to Southern blots of EcoRldigested D N A (10 fig) from 6 different insecticide-resistant M persicae clones with either A . Normal karyotype and FE4 enzyme or B. Translocated karyotype and E4 enzyme. Clones A l and A2 were established with aphids collected from peach trees in Italy and France respectively, whilst A3 originated on field potatoes in the U K The three B clones originated in the UK, two in glasshouses ( B l and B3) and the third (B2) in a sugar beet field. Arrows indicate sizes of the major fragments in kilobases.



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nucleic acids from such "revertant" clones has shown that they lose elevated E4 mRNA but retain the amplified E4 genes with no major D N A rearrangement (70). Thus the basis of reversion is a change in expression of the amplified E4 genes. So far the only difference observed between the expressed and silent E4 sequences is a change in the presence of 5-methylcytosine (5 mC) (10), a fifth base that is known to affect gene transcription in vertebrates (11). The presence of 5 mC in aphid esterase D N A sequences can be demonstrated by comparing thefragmentsproduced when aphid D N A is cut with Mspl or HpaU and probed with E4 cDNA. Both Mspl and HpaU cut C C G G sites but only Mspl will cut if the internal cytosine is methylated (i.e. is a 5 mC). Thus a difference in banding pattern between the 2 enzyme digests, as shown in Figure 2A for R! and R aphids indicates the presence of 5 mC, in both FE4 and E4 sequences whereas the identical Mspl and Hpall digests of D N A from the revertant aphid clone (Rev) show that in the silent E4 D N A sequences the C C G G sites are not methylated. This correlation holds for a large number of aphid clones. It is very surprising in the light of many studies with vertebrate genes where D N A methylation has been shown to correlate with gene inactivation and demethylation is a necessary prerequisite for transcription to occur (11). The banding patterns in Figure 2A can be explained by the restriction maps in Figure 2B. Thus the Mspl digest of Ri (FE4 genes) reveals amplified esterase sequences on 2.8 and 1.8 kbfragments,whereas R and Rev both with amplified E4 genes have 2.8 and 2.2 kb bands. When the D N A is methylated in R and R , the small fragments are absent in the Hpall digests indicating that at least two of the Mspl sites in both E4 and FE4 sequences must contain 5 mC; in the revertant the 3 Mspl sites in the E4 sequences must lack methylation. Since the 2.8 kb fragment is common to both genes the E4 cDNA probe will have equal homology to this region and the difference in intensity between Rj and R reflects differences in gene copy number. However, the homology of the E4 cDNA to fragments downstream of the Smal site, where the 2 genes differ, should be greater for the E4 gene (2.2 kb fragment) than for the FE4 (1.8 kb fragment), and this can be seen clearly in Figure 2A. To avoid this complication in studies of esterase genes in aphids from field and glasshouse populations, we have subcloned the genomic fragments shown in Figure 2B to use as probes. 3

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3

3

Esterase Genes in U K Field Populations of M. persicae When revertant aphid clones are subjected to insecticide treatment, resistance can be re-selected very quickly (72), and therefore it is important to know to what extent revertants occur in the field. We have used the characteristic Mspl and HpaU banding patterns to identify the type of amplified esterase genes present, and their methylation, in samples of M. persicae taken from populations in the U K during 1990, and have made preliminary attempts to relate this to the expression of the genes as judged by immunoassay (73). Individual aphids were placed on potato leaves in small plastic boxes,



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Figure 2. A . Binding of an E4 cDNA (pMp24) to Southern blots of Mspl (M)- or Hpall (H)-restricted DNA from resistant Af. persicae clones with expressed FE4 (Rj) or E4 (R ) genes and a revertant clone (Rev) with unexpressed E4 genes. Arrows indicate sizes of the major fragments in kilobases. B. Restriction maps of the 4 kb and 8 kb EcoRI fragments in Rj and R aphids containing amplified FE4 and E4 sequences respectively. E = EcoRI C = Clal M = Mspl K = Kpnl S = Smal H = Hindm Restriction at the M sites in 2B gives the Mspl fragments seen in 2A (see text for details). 3

3


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D N A was extracted from all of their first generation clonal offspring (c 20 aphids) and pairs of Mspl and Hpall digests were probed with a mixture of the esterase genomic sequences described above. The aphids used to establish these single generation "miniclones were from diverse populations. In the examples shown in Figure 3, samples 5 and 6 were collected from a potato field in Scotland and the remainder originated in England; 1-3 and 9 from field crops of potatoes, cauliflowers, sugar beet and fodder beet respectively, sample 4 from sugar beet in a glasshouse and samples 7 and 8 from insect suction traps. In all, 84 samples were analysed; most (75), had amplified esterase genes, 25 with E4, as judged by the presence of 2.8 and 2.2 kb Mspl fragments, (e.g. samples 1-4, Figure 3) and 50 with FE4, having 2.8 kb and 1.8 kb Mspl fragments (e.g. samples 5-8, Figure 3). There were no cases of aphids with both 2.2 and 1.8 kb bands, suggesting that amplified E4 and FE4 genes do not co-exist in an individual insect. In the majority (66) of samples with amplified esterase sequences the small fragments were absent in the Hpall digests (i.e. like samples 1, 2 and 5, 6 Figure 3) indicating methylation of the C C G G sites discussed above. In all such cases the immunoassay showed correspondingly high esterase activity. However, some samples retained the small fragments in the Hpall tracks even though most of their amplified esterase sequences appeared on larger fragments (e.g. samples 3, 7 and 8 Figure 3); this suggests that both E4 and FE4 D N A sequences can be "partially" methylated at the 3 C C G G sites, perhaps by differential methylation between cells, tissues and/or embryos, or perhaps between individuals within the miniclones. This partial methylation correlated with enzyme concentrations less than expected from the amount of probe binding although this could not be quantified accurately; thus both E4 and FE4 genes seem to be partially silenced in these aphids. This is particularly interesting for aphids with amplified FE4 genes since reversion has not been observed in aphid clones with elevated FE4. The miniclone established from a glasshouse sample (sample 4 Figure 3) was clearly a revertant with E4 genes as judged by the 2.8 and 2.2 kb fragments in the Hpall digest and by the presence of an esterase concentration typical of susceptible aphids even though the amount of probe binding suggests a high degree of E4 gene amplification. This is consistent with our previous finding that glasshouse aphids with extremely high levels of resistance and amplified E4 genes frequently revert, although changes in their D N A methylation were not studied (74). None of the samples from field populations showed this complete loss of 5 mC, but this may be more likely to occur as extremely resistant aphid variants become more common in the field. Nine of the samples did not have amplified esterase D N A or elevated enzyme (e.g. sample 9, Figure 3) and were deemed to be susceptible types. In all 9 clones the 2.8 kb band was present in both Mspl and Hpall digests but neither the 2.2 nor the 1.8 was detected, showing that esterase sequences in susceptible field aphids are unmethylated (as they are in susceptible clones, 10) and suggesting that the original unamplified esterase gene and/or flanking D N A in susceptible aphids may differ from the amplified E4 and FE4 D N A sequences.


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All of the field aphids with amplified esterase sequences had one of the two restriction patterns found in resistant clones, again supporting the view that there is little polymorphism at these amplified loci.


Esterases and their DNA Sequences in M. nicotianae Blackman (2), using multivariate morphometric techniques, showed that the tobacco-adapted form of Af. persicae should be considered as a separate species, Af. nicotianae. He also showed that M. nicotianae populations in the USA frequently had a heterozygous Al,3 chromosome translocation, apparently the same as that present in Af. persicae. He suggested that this had arisen independently since the separation of the two species, occurring either just after or at the same time as the acquisition of insecticide resistance by M. nicotianae. This suggested that M. nicotianae might have the same insecticide resistance mechanism as Af. persicae, a hypothesis supported by recent publications on the esterases of both species in the USA (3). We have examined insecticide resistant fundatrigeniae from peaches and summer populations of aphids from tobacco in Greece, identified as Af. nicotianae of normal karyotype (Blackman, Pers Comm). Gel electrophoresis patterns of individual M. nicotianae (samples 2-8, Figure 4) were indistinguishable from that of a non-translocated very resistant M. persicae clone (800 F,sample 1, Figure 4) from Ferrara (7), showing the same range of non-amplified esterases (El/2, E5, E6 and E7) and similar levels of the elevated esterase, FE4. DNA probing with E4 and FE4 genomic sequences (Figure 4B) showed that the Af. nicotianae contained homologous amplified esterase sequences on fragments consistent with the presence of the same FE4 genes as found in M. persicae, i.e. 2.8 and 1.8 kb bands in Mspl digests. Furthermore, the Hpall digests gave a dominant 12 kb band showing that the esterase sequences in M. nicotianae are methylated in the same way as in resistant M. persicae. Thus the molecular genetic basis of insecticide resistance appears to be the same in both species. It is possible that the insecticide resistant M. nicotianae in this study evolved from resistant M. persicae and therefore the amplified esterase genes were present before the divergence of the two species. Alternatively, insecticide resistance in the Greek population may have evolved after they separated from M. persicae, as suggested by Blackman (2) for the translocated USA populations, in which case two independent amplification events must have occurred. Since the amplified sequences in M. persicae and M. nicotianae appear to be identical it would suggest that aphids are somehow "predisposed" for a particular amplification event. If so, then the same amplification event could also have occurred more than once in Af. persicae which would explain the observed, conserved sequences in clones of widely different origin. This would be in contrast to the situation for insecticide resistant culicine mosquitoes where there is evidence that a single esterase B2 gene amplification event occurred and that this was subsequently spread worldwide



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Figure 3. Binding of a mixture of esterase genomic fragments (see Figure 2) to Southern blots of D N A (2 pg) extracted from 9 Af. persicae Miniclones and digested with either Mspl (M) or Hpall (H). Arrows indicate sizes of major fragments in kilobases. Lower line gives the level of esterase in the clone as judged by immunoassay. M

H

Figure 4. A . Polyacrylamide gel electrophoresis of individual aphid homogenates stained for esterase activity using 1-naphthyl acetate (d). Sample 1 is a nontranslocated resistant Af. persicae standard (Ferrara) and samples 2-8 are Af. nicotianae. B. Binding of mixed esterase genomic fragments (see Figure 2) to Southern blots of D N A (2/ig) extracted from 3 resistant M. nicotianae clones (of normal karyotype) and digested with Mspl (M) or Hpall (H). Indicated are the sizes of major fragments in kilobases.


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by migration (75). The example of gene amplification in mosquitoes is the only other confirmed case of insecticide resistance arising from esterase gene amplification, although there is circumstantial evidence in several other species. As in Af. persicae, resistant mosquitoes can show amplification of various esterase alleles but there is no evidence for transcriptional control of the genes nor for methylation of the D N A sequences. Similarities and differences between aphid and mosquito resistance are discussed fully in a recent review of gene amplification and insecticide resistance (76). Acknowledgements We thank Sara Smith and Mary Stribley for rearing and biochemical characterisation of aphid clones. George Michalopoulos kindly supplied the Greek Af. nicotianae aphids, the identity of which was confirmed by Roger Blackman. Literature cited 1. Devonshire, A. L.; In Aphids, their Biology, Natural Enemies and Control, Volume C; Editors, A . K. Minks and P. Harrewijn; Elsevier Science Publishers B.V., Amsterdam, 1989, pp 123-39. 2. Blackman, R. L. Bull. ent. Res. 1987, 77, 713-30. 3. Abdel-Aal, Y.A.I.; Wolff, M . A.; Roe, R. M . ; Lampert, E. P. Pestic. Biochem. Physiol. 1990, 38, 255-66. 4. Field, L. M., Devonshire, A. L.; Forde, B. G. Biochem. J. 1988, 251, 30912. 5. Needham, P. H.; Sawicki, R. M . Nature 1971, 230, 126-7. 6. Devonshire, A. L.; Moores, G. D. Pestic. Biochem.Physiol.1982, 18, 23546. 7. Devonshire, A . L. Pestic.Sci.1989, 26, 375-82. 8. Devonshire, A . L.; Sawicki, R. M . Nature 1979, 270, 140-1. 9. Sawicki, R. M.; Devonshire, A. L.; Payne, R.W.; Petzing, S. M . Pestic. Sci. 1980, 11, 33-42. 10. Field, L. M.; Devonshire, A. L.; ffrench-Constant, R. H . ; Forde, B. G. FEBS Letts. 1989, 243, 323-7. 11. Holliday, R.Sci.Amer. 1989 (June), 40-8. 12. ffrench-Constant, R. H.; Devonshire, A. L.; White, R. P. Pestic. Biochem. Physiol. 1988, 30, 1-10. 13. Devonshire, A . L.; Moores, G. D. Bull. ent. Res. 1986, 76, 97-107. 14. Field, L. M.; Devonshire, A.L.; ffrench-Constant, R. H . ; Forde, B. G. Pestic. Biochem. Physiol. 1989, 34, 174-8. 15. Raymond, M.; Callaghan, A.; Fort, P.; Pasteur, N. Nature 1991, 350, 151-3. 16. Devonshire, A . L.; Field, L. M . Annu. Rev. Entomol. 1991, 36, 1-23. R E C E I V E D November 25,

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Molecular Mechanisms of Insecticide Resistance - ACS Publications

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