Molecular Genetics and Evolution of Pesticide Resistance - American

Chapter 8

Expression of Amplified Esterase Genes in Insecticide-Resistant Myzus persicae (Sulzer) 1

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Linda M . Field , Caroline A. Hick , Alan L. Devonshire , Naghmy Javed , Jennifer M . Spence , and Roger L. Blackman Downloaded by UNIV MASSACHUSETTS AMHERST on October 8, 2012 | http://pubs.acs.org Publication Date: September 27, 1996 | doi: 10.1021/bk-1996-0645.ch008

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Biological and Ecological Chemistry Department, Institute of Arable Crops Research-Rothamsted, Harpenden, Hertfordshire, AL5 2JQ, United Kingdom Department of Entomology, Natural History Museum, London SW7 5BD, United Kingdom 2

Insecticide resistance in Myzus persicae resultsfromamplification of genes encoding insecticide-detoxifying esterases and from differential transcription of the amplified genes which may be mediated by changes in DNA methylation. Methylation is usually stable in resistant aphid clones and can be inherited during sexual reproduction. However, when a sudden loss of methylation occurs within a clone, it is accompanied by silencing of the amplified genes. When reselected with insecticides some recovery of expression can occur but this is not accompanied by methylation. Some aphid clones have amplified esterase genes arranged as tandem repeats at a single locus, whereas others have arrays dispersed around the genome. Thus, although resistance in M. persicae is dependent primarily on gene amplification, this may be modulated by other molecular and genetic factors. There are now two well established examples of insecticide resistance resulting from the amplification of genes encoding insecticide detoxifying carboxylesterases ( i ) . In the mosquito, Culexpipiens quinquefasciatus, resistant insects with more than 250 copies of the B l esterase gene have been reported (2). In the aphid, Myzus persicae, variation i n both copy number (3) and transcription (4) of amplified esterase E4 genes can affect the resistance status of an individual insect, the latter being associated with changes i n the presence of 5-methylcytosine i n and around the amplified genes. In Culex there is no evidence for changes in expression of the esterase genes (5). Here we bring together the current knowledge of D N A amplification, E4 gene expression and changes i n D N A methylation during both asexual and sexual reproduction i n Af. persicae and attempt to assess the relative contributions these genetic phenomena make to insecticide resistance. 0097-6156/96/0645-0072$15.00/0 © 1996 American Chemical Society

In Molecular Genetics and Evolution of Pesticide Resistance; Brown, T.; ACS Symposium Series; American Chemical Society: Washington, DC, 1996.

Amplified Esterase Genes in Resistant Myzus persicae 73

8. FIELD ET AL.

Myzus persicae

Quantification of Amplified Esterase Sequences i n It is now more than seven years since the cloning of a c D N A , encoding the E4 esterase (carboxylester hydrolase, EC3.1.1.1), and the identification of amplified esterase genes were reported in insecticide-resistant M. persicae (3). A t that time it was noted that although the esterase gene copy number increased with increasing levels of resistance, there was not the difference between susceptible and extremely resistant R aphids which would be expected from their relative amounts of esterase enzyme. Figure 1 summarises the situation. Immunoassays of the E4 enzyme in R aphids (A) can be accurately quantified in a microplate reader (6) and are in line with enzyme titration studies using radiolabelled ligands which showed an approximately 60-fold increase i n esterase protein compared with susceptible aphids (7). This is reflected i n dot blots of poly A R N A probed with E4 c D N A (Figure IB), which show c. 4-fold increases between S and R , R and R , and R and R , in line with their levels of esterase enzyme. However, probing of D N A dot blots (Figure 1C) shows much smaller differences between the aphid clones with only a c. 8-fold increase between susceptible and R . This has been attributed to non-specific binding of the probe to related sequences (3), but even in Southern blots of D N A digested with restriction enzymes, the binding to E4 fragments was difficult to quantify because of the band diversity and uncertainty regarding the extent of homology of the probe to amplified and susceptible esterase sequences. Recent attempts to quantify the E4 gene copy number in an R aphid clone, 794J, have used a cloned EcdSl/Kpnl fragment from the amplified E4 gene (known to be present in the D N A of susceptible aphids) to probe EcoW/Kpnl digests of susceptible and 794J aphid D N A (Field, Devonshire and Tyler-Smith, Biochem. J. in press). This showed only 5-10 times more esterase sequences in 794J aphids, in which the amplification is known to be heterozygous at a single locus, suggesting the presence of a single array of 10-22 copies of the E4 gene (assuming two copies per diploid genome of susceptible aphids). In the same study, analyses of the repeat units (amplicons) containing the E 4 genes, using pulsed-field gel electrophoresis, confirmed the presence of only c. 12 copies of the E4 gene, each on c. 24 kb amplicons, arranged as a tandem array of direct repeats. This is consistent with in situ hybridisation studies (8) and with crossing experiments (Blackman, Spence, Field, Javed and Devonshire, Heredity, in press) which confirm that amplified E4 genes are at a single heterozygous locus. Thus in the R aphid clone, 794J, the increase in E4 copy number is not sufficient to account for the levels of E4 enzyme synthesized.

Downloaded by UNIV MASSACHUSETTS AMHERST on October 8, 2012 | http://pubs.acs.org Publication Date: September 27, 1996 | doi: 10.1021/bk-1996-0645.ch008

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Methylation of amplified sequences in

M. persicae

The presence of 5-methylcytosine in the amplified esterase genes and their flanking D N A has been detected in M. persicae using an Mspl/HpaU diagnostic assay (4,9). This showed that esterase sequences in susceptible aphids are unmethylated, in line with most of the aphid genome. However, amplified expressed E4 genes in resistant aphids contain 5-methylcytosine, which is not



MOLECULAR GENETICS AND EVOLUTION OF PESTICIDE RESISTANCE

Figure 1. A : Immunoassay of the E 4 enzyme present i n susceptible (S, 64 light-coloured wells) and highly resistant (R , 32 dark wells) aphids and binding of an E4 c D N A probe to 2-fold serial dilutions of B : poly A R N A and C : genomic D N A extracted from susceptible and increasingly resistant (Rx-R ) aphids. 3

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8. FIELD ET AL.


present i n aphids that have lost resistance and are not expressing their amplified genes (revertants). These results were surprising since it was generally thought that insect D N A does not contain significant amounts of 5-methylcytosine (10) and in most studies of vertebrate gene expression the presence of D N A methylation prevents transcription (11). We have recently monitored changes in D N A methylation during the loss of resistance in an aphid clone established i n 1991 from a U K glasshouse population (Hick, Field and Devonshire, Insect Biochem. & Mol Biol, i n press) and shown a concomitant loss of E4 gene transcription and 5-methylcytosine, as the aphids' progeny changed from R to susceptible levels of E 4 activity over three generations. When these revertant aphids were reselected for resistance with insecticide treatments, their E4 esterase content increased to R levels without an accompanying change i n D N A methylation. Thus there is a complex interaction between esterase copy number, D N A methylation and expression which is summarised in Table 1. 3

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Table 1. Summary of molecular changes during loss and reselection of resistance in Af. persicae Resistance Status

Gene Amplification

DNA Methylation

Expression ( E levels)

Susceptible*

-(2x)

-

S(lx)

Resistant

+ (cl2x)

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R (60x)

Revertant

+ (cl2x)

-

S(lx)

Reselected

+ (cl2x)

-

Ri/2 (4-160

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*susceptible included for comparison, assumed diploid for esterase gene These data suggest that resistance occurs primarily by E4 gene amplification, but with an increase in transcription, possibly as the result of D N A methylation. During reversion the methylation is lost and transcription of the amplified genes diminishes drastically. After reselection the amplified genes are again expressed but not re-methylated. It is interesting that we have not been able to reselect aphids to produce R levels of E4 and it may be that the lack of methylation limits the E4 levels to reflect the gene copy number (i.e. 12 copies giving Ry levels). This would support the view that it is the methylation of the amplified genes which is involved in their overexpression. 3

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Inheritance of D N A methylation during sexual reproduction The presence of 5-methylcytosine in the amplified genes of Af. persicae indicates that, even if methylation is not common in the aphid genome, the aphid must have the enzyme systems necessary for both de novo methylation and its maintenance during asexual cell growth and development. It should be noted that the loss of methylation discussed earlier is a rare event and normally



MOLECULAR GENETICS AND EVOLUTION OF PESTTCKDE RESISTANCE

Figure 2. Binding of cloned esterase genomic sequences to Mspl (M) and HpaTL (H) digests of D N A extracted from resistant aphid clones F r R and 800F, and the offspring of 2 generations of sexual crosses.



8. FIELD ET AL.


resistance, E4 gene expression and the presence of 5-methylcytosine are stably inherited in asexual reproduction. So are patterns of D N A methylation also stable during sexual reproduction, i.e. can they be maintained through meiosis? We have recently been studying the inheritance of amplified esterase genes through the sexual phase of M . persicae (Blackman, Spence, Field, Javed & Devonshire, Heredity, In press) and were able to monitor the methylation patterns in successive sexual generations. Figure 2 shows the Mspl and HpaU esterase restriction fragments obtained over 3 generations. The presence of 2.8 and 1.8 kb bands in Mspl digests shows the presence of amplified genes (see ref 2); which encode F E 4 , an insecticide-detoxifying esterase very closely related to E4 (11). When these small bands are absent in HpaU digests and replaced by larger fragments it shows that the amplified sequences are methylated (9). The two parental clones F r R and 800F (Figure 2A) have amplified F E 4 genes, but whereas the 800F sequences are fully methylated (i.e. they have no 2.8 and 1.8 kb bands in the HpaU digest), the F r R sequences show "partial" methylation, with only some of the sites cut by HpaU. When 800F was crossed with susceptible (DS) aphids or selfed all amplified sequences inherited by the F l clones were fully methylated (e.g. Figure 2C) and when one of these ( U N ) was selfed, the methylation was again inherited (Figure 2E). For the FrR/susceptible cross a few aphids lost methylation completely (e.g. 6 D Figure 2B) but most maintained the "partial" state (e.g. 5 N of Figure 2B) which was again inherited during selfing (Figure 2D). When 5 N was crossed with 6D the esterase genes in the offspring showed varying degrees of methylation (Figure 2F). Thus D N A methylation can be stable during sexual reproduction even if only some of the sites are methylated. Future Prospects This work has identified a so-called "partial" state of D N A methylation (9) where only some of the amplified esterase sequences in an aphid contain 5-methylcuytosine. Until recently it has only been possible to characterise methylation in homogenates of whole aphids since the Mspl/HpaU technique requires enough D N A for two restriction digests. However, we have now developed a PCR-based assay (Field, Crick and Devonshire, in preparation) to diagnose the type of amplified gene present (E4 or FE4) and the presence of 5-methylcytosine in very small amounts of aphid D N A (< 0.001 aphid). This now opens the possibility of studying D N A methylation in individual embryos and tissues during aphid development and reproduction. Although amplified E4 genes are usually at a single heterozygous locus, in situ hybridization has shown that there are at least two other sites where amplified E4 genes can be found, and F E 4 genes can also occur at several sites (8). This raises the possibility of an involvement of transposable elements i n the development of insecticide resistance in M. persicae as has been suggested for mosquitoes (13)> and this possibility will be explored further. We now have aphid clones established from the offspring of the crosses


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MOLECULAR GENETICS AND EVOLUTION OF PESTKTDE RESISTANCE

having single sites of amplified genes and this creates the opportunity to establish the gene copy number, amplicon size and structure, D N A methylation and esterase expression at each of the loci. Thus a detailed picture of the genetic changes underlying resistance i n this major agricultural pest can be achieved.


Acknowledgments I A C R receives grant-aided support from the Biotechnology and Biological Sciences Research (BBSRC) Council of the United Kingdom and this work was supported by a B B S R C Linked Research Groups grant. Literature Cited 1. 2.

3. 4. 5. 6. 7. 8. 9.

10. 11. 12. 13.

Devonshire, A.L.; Field, L.M. Ann. Rev. Entomol. 1991, 36, 1-23. Mouchès, C.; Pasteur, N.; Bergé, J.B.; Hyrie, O.; Raymond, M.; De Saint Vincent, B.R.; De Silvestri, M.; Georghiou, G.P. Science 1986, 233, 778-780 Field, L.M.; Devonshire, A.L.; Forde, B.G. Biochem. J. 1988, 251, 309-312. Field, L.M.; Devonshire, A.L.;ffrench-Constant,R.H.; Forde, B.G. FEBS Letts1989.243, 323-327. Raymond, M ; Poulin, E.; Boiroux, V.; Dupont,E.; Pasteur, N. Heredity 1993, 70, 301-307. Devonshire, A.L.; Moores, G.D.; ffrench-Constant, R.H. Bull. Ent. Res. 1986, 76, 97-107. Devonshire, A.L.; Sawicki, R.M. Nature, 1979, 270, 140-141. Blackman, R.L.; Spence, J.M.; Field, L.M.; Devonshire, A.L. Heredity, 1995, 75, 297-302. Field, L.M.; Devonshire, A.L. In Molecular Mechanisms of Insecticide Resistance; Mullin, C.A. and Scott, J.G. Eds; ACS Symposium Series; American Chemical Society, Washington DC, 1992, pp. 209-217. Adams, R.L.P. Biochem. J. 1990, 265, 309-320. Eden, S.; Cedar, H . Current Opinions in Genetics and Development, 1994, 4, 255-259. Field, L.M.; Williamson, M.S.; Moores, G.D.; Devonshire, A.L. Biochem. J. 1993, 294, 569-574. Mouchès, G.; Agarwal, M.; Campbell, K. In Insecticides: Mechanisms of Action and Resistance; Intercept Ltd Andover, Hants, UK, 1992; pp. 345-353.


Molecular Genetics and Evolution of Pesticide Resistance - American

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