Insect Molecular Biology (1994) 3(3), 143-148

The peach-potato aphid Myzus persicae and the tobacco aphid Myzus nicotianae have the same esterase-based mechanisms of insecticide resistance

L. M. Field, N. Javed,' M. F. Stribley and A. L. Devonshire Institute of Arable Crops Research, Rothams fed Experimental Station, Harpenden, Herts., and 'The Natural History Museum, London

Abstract

Biochemical and molecular studies have established that in the peach-potato aphid, M y z u s persicae, insec- ticide resistance is conferred by amplification of genes encoding the insecticide-detoxifying esterases E4 or FE4. Here we report that two insecticide-resistant clones of the closely related tobacco aphid M y z u s nicotianae have elevated esterases indistinguishable from E4 and FE4 and amplified esterase DNA se- quences, and flanking regions, with identical restric- tion maps to the M. persicae genes. Furthermore, the DNA sequences of c. 630 bp fragments of the E4 and FE4 genes of M. persicae are different from each other but identical to the fragment from corresponding M. nicotianae clones. The existence of apparently ident- ical insecticide resistance genes in the two species can be best explained by the selection of the amplified genes in M. persicae, transfer to hybrids of M. persicae and M . nicotianae by sexual reproduction and sub- sequent spread through M . nicofianae populations.

Keywords : M y z u s persicae, M y z u s nicotianae, ester- ase gene amplification, insecticide resistance.

Introduction

Needham & Sawicki (1 971) first established that insecti- cide resistance in the peach-potato aphid, Myzuspersicae, correlated with total carboxylesterase activity in aphid homogenates. Since then it has been shown that the overproduction of a single esterase confers resistance by increasing the hydrolysis and sequestration of insecticides (Devonshire & Moores, 1982), and that two slightly differ-

Received 16 February 1994; accepted 22 April 1994. Correspondence: Dr Linda Field, Institute of Arable Crops Research, Rothamsted Experimental Station, Harpenden, Herts. AL5 2JQ.

ent forms of elevated esterase (E4 and FE4) can occur (Devonshire ef a/., 1983).

The molecular genetic mechanism underlying the elev- ated esterases has been shown to be amplification of esterase structural genes (Field et al., 1988) and cDNAs and genomic sequences of E4 and FE4 genes have been cloned and characterized (Field eta/., 1993). Blackman et al. (1 978) demonstrated that insecticide resistance in M. persicae can be genetically linked with a heterozygous Al,3 chromosome translocation, and biochemical and mol- ecular studies have shown a correlation between the presence of amplified E4 genes and the chromosome translocation, whilst amplified FE4 genes are present in resistant aphids of apparently normal karyotype (Field et al., 1988).

M. persicae is a member of a complex of closely related (sibling) species, and within this complex a tobacco- feeding form, which can be distinguished from M. persicae using multivariate morphometrics, has been designated to be a very closely related but distinct species, Myzus nico- tianae (Blackman, 1987). The two species also show differences in their allozymes of glutamate oxaloacetate transaminase (Blackman & Spence, 1992).

Like most aphid species, M. persicae and M. nicotianae spend the summer as asexual clones on herbaceous secondary hosts and, depending on factors such as cli- mate or host availability, either continue as such overwinter or go through a sexual stage on their primary host peach, Prunus persicae (Blackman, 1974). Although the two species are able to interbreed under laboratory conditions, they can maintain discrete identities even in regions where they overwinter on the same primary host trees (Blackman & Spence, 1992), and they have also been reported to co- exist in glasshouses on a non-tobacco secondary host (Boiteau & Lowery, 1989). The mechanism for this partial or total reproductive isolation is unknown, but Blackman & Spence (1 992) have suggested either prezygotic isolation by behavioural/phenological factors or reduced viability of the F1 hybrids.

As in M. persicae (Takada, 1979), both red and green colour morphs of M. nicotianae are found, and it has been proposed that the red form arose as a mutation of the original green form and subsequently increased rapidly in numbers relative to the green, possibly as the result of

143

144 L. M. Fieldet al.

some selective advantage (Blackman, 1987). Reed & Semtner (1991) have suggested that the selective advan- tage of the red morph lies in an increased ability to survive, develop and reproduce at temperatures above 25”C, which field populations of M. nicotianae will often encounter. However, Blackman (1 987) has put forward the hypothesis that in North American populations the original green form recently underwent rapid genetic change, developing re- sistance to organophosphorus insecticides followed by, or concomitant with, the acquisition of a heterozygous chromosome translocation indistinguishable from that in M. persicae, and then a subsequent mutation produced the red form. This implies that it might be resistance to insecti- cides which gives the red aphids a selective advantage. However, in a study of M. nicotianae from North Carolina, although those with the translocation were more resistant to insecticides, and all red morphs examined carried the translocation, resistant green morphs were also detected (Harlow & Lampert, 1990; Harlow eta/., 1991).

The presence of elevated esterase has been used to monitor insecticide resistance in M. persicae for more than 20 years (Needham & Sawicki, 1971; Devonshire, 1989). This has involved a range of biochemical techniques in- cluding an EWFE4-specific immunoassay which can be done on large numbers of individual aphids (ffrench- Constant & Devonshire, 1988). For M. nicotianae, variation in total esterase activity has been measured in individuals using a microplate assay (Abdel-Aal et a/., 1990) and an elevated esterase of the same mobility (by electrofocusing) as the E4 of M. persicae has been identified (Abdel-Aal et a/., 1990, 1992).

If the increased esterase activity of M. nicotianae is due to overproduction of the enzyme, as in M. persicae, it raises the question of whether the molecular basis is the same in both species, i.e. the same or a similar gene amplification. It has been suggested that the A1,3 translocation in M.

FE4

nicotianae, although apparently identical to that of M. persicae, arose independently (Blackman, 1987), which, given the linkage between this and insecticide resistance, suggests that resistance also evolved independently in the two species. However, our preliminary study of a non- translocated resistant population of M. nicotianae from Greece showed an elevated esterase apparently identical biochemically to FE4 and the presence of amplified ester- ase DNA, hybridizing strongly to an M. persicae DNA probe and giving the Mspl restriction fragments as the amplified FE4 genes in M. persicae (Field & Devonshire, 1992). Here we report a more detailed comparative study of M. persicae and M. nicotianae elevated esterases and their amplified genes in aphids of both translocated and normal karyotype.

Results

Polyacrylamide gel electrophoresis of aphid homogenates, stained for esterase activity, shows that the M. nicotianae clone of normal karyotype (926B) has an identical banding pattern to that of the M. persicae clone of normal karyotype (800F), including an elevated FE4 band of equivalent intensity (Fig. 1). Similarly, the translocated M. nicotianae (934L) has an elevated esterase corresponding to the E4 band of the translocated M. persicae clone (TIV), but its intensity is slightly less, and there is an E5 band as found in M, persicaeof normal karyotype (Devonshire, 1989). Like- wise, the E4 and FE4 of both species are indistinguishable in their reaction with E4 antiserum (data not shown).

Previous gene mapping and sequencing of cDNAs and cloned genomic sequences had determined the structure of the amplified E4 and FE4 genes in M. persicae, giving the position of exons and introns relative to restriction sites within the gene (Field ef a/., 1993). However, further work has shown an additional small intron at the 5‘ end of the E4 gene, giving seven introns and eight exons (see Fig. 2). We

E4 €5

Figure 1. Polyacrylarnide gel electrophoresis of aphid homogenates, stained for esterase activity. A = M. persicae with elevated FE4 (8OOF). 6 = M. nicotianae of normal karyotype (9266). C = M. persicae with elevated €4 (TlV). D L M. nicotianae with the A l , 3 chromosome transiocation (934L).

Insecticide resistance in Myzus spp. 145

B c---.)

A 7

S S'H' K CECM K M CX'MP 6' E H K I 1

1 2 3 45 6 7 8

C c-)

S K H' C ECM K M HB'E C M A XPA'H'X B H K FE4 I II I 1 " ' " I "

I

I k b H

Figure 2. Restriction maps of amplified esterase genes and flanking DNA in M. pefsicae E4 from clone T1 V and FE4 from clone 800F. Boxed regions show the location of the esterase gene, solid bars are exons (1-8 for E4) and the hatched region is the putative 5 ' end of FE4. t-) indicates regions cloned and used as probes (A, B and C). Restriction sites: A = A w l , A' = Apal, 8 = BarnHI, 8' = Bg/Il, C = C/al, E = EC~RI, H = /-/;ndlll, H' = K = Kpnl, M = Msp/, P = Pstl, S = Smal, S' = Sall. X = Xbol, X ' = Xbal

Table 1. Sizes of restriction fragments (kb) from Mpersicae and M. nicotianae DNA, digested with enzymes shown (for key to letters see legend to Fig. 2), and probed with cloned genornic DNA fragments A for 5' end, or B with C for the 3' end (see Fiq. 2).

Enzymes ~~ ~ ~~ ~~ ~~

Species Clone Probe K K+A K i A ' K r B K+B' K+C K+E K+H K+H' K+M K i P K t X K t X '

8OOF A 7.0 7.0 7.0 7.0 7.0 1.5.0.8 1.8 7.0 3.3 1.5 7.0 7.0 7.0 9268 B+C 11.5 4.2 5.4 7.5 2.1 3 1 2.4 2.0 5.5 3.1 5.0 4.6 6.1

A 3 0 3.0 3.0 3.0 3.0 1.5.0.8 1.8 3.0 3.0 1.2 3.0 3.0 3.0

M. persicae

M persicae M. nicotianae 1 i i4"L B+C 10.0 10.0 10.0 10.0 4.8 3.4 6.3 7.0 10.0 3.8 4.2 10.0 3.6

have now extended the restriction mapping to the DNA flanking the amplified genes using single and double digests and the probes indicated (Fig. 2). The restriction sites in the flanking DNA are clearly different between T1 V and 800F aphids, and only one or other of these two maps was obtained for fourteen other M. persicae clones accord- ing to whether they had amplified E4 or FE4 genes (data not shown).

The restriction fragments from M. persicae and M. nico- tianae DNA digested with combinations of enzymes and probed with E4iFE4 genomic sequences are shown in Table 1. Both translocated clones (T1 V for M. persjcae and 934L for M. nicotianae) have exactly the same amplified fragments, which differ from those present in both clones of normal karyotype (800F for M. persicae and 9268 for M. nicotianae), which are identical to each other. Thus the M. nicotianae clone 9268 has amplified esterase genes and flanking DNA indistinguishable from M. persicae FE4 genes, and clone 934L has amplified sequences the same as E4 genes and their flanking DNA.

From the known sequences of E4 and FE4 cDNAs (Field et a/., 1993) and the position of the introns we designed PCR primers to amplify a c. 630 bp product from the 3' border of exon 6 to 100 bases into exon 8 and encompass- ing introns 6 and 7 and exon 7 (see Fig. 2). The primers

amplified fragments of the same size from both of the M. persicaeand both of the-M. nicotianaeaphid DNAs, and the products of all four reactions were cloned and sequenced in both strands; the sequences are given in Fig. 3. For M. persicae the sequences of two independent PCR products were verified by comparison with sequences of the same regions in genomic DNA (data not shown) cloned by fragment enrichment (Field eta/., 1993). For the M. nicotia- nae aphids the sequences were confirmed in three inde- pendent clones of the PCR products.

The PCR products from M. persicae clones T1V and 800F have the same exon sequences as those reported previously for the corresponding E4 and FE4 cDNAs from 794J and 800F aphids respectively (Field ef a/., 1993); intron 6 (78 bp) is identical for both genes but intron 7 (316 bp for E4 in T1V and 312 bp for FE4 in 800F) shows considerable variation with fifty-six base substitutions and four and eight deleted bases in E4 and FE4 respectively. The 626 bp product of the E4 gene shows c. 90% homology with the 622 bp product from the FE4 gene.

The PCR product from DNA of clone 934L has an identical sequence to that from clone TIV, and similarly those from clones 800F and 9268 are identical. Thus these regions of the amplified esterase genes in M. nicotianae show 100°/o homology with E4 and FE4 sequences of M.

146

TIV 9341, 800F 926B

T IV 934L 800F 926B

TIV 934L 800F 926B

TIV 934L 800F 926B

TIV 934L 800F 9268

TIV 934L 800F 926B

T IV 934L 800F 926B

TIV 934L 800F 926B

T IV 934L 800F 926B

1. M. Fieldet al.

Figure 3. Sequences of PCR products from the end of exon 6, spanning introns 6 and 7 and exon 7, and 100 bases of exan 8. Upper case = exon sequence; lower case = intron sequence. Dashes indicate identical nucleotides and [ 1 indicate gaps introduced to maintain alignment. Underlined regions are positions of oligonucleotides A and B used for sequencing.

Insecticide resistance in Myzus spp. 147

persicae and there can be little doubt that M. nicotianae has the same amplified E4 or FE4 genes as those found in M. persica e.

Discussion

Since the tobacco-feeding form of M. persicae was desig- nated as M. nicofianae in 1987, several publications have addressed the nature of its resistance to insecticides (Abdel-Aal, 1990; Abdel-Aal ef a/., 1990, 1992; Harlow & Lampert, 1990). These have shown clearly that increased esterase activity is implicated but, due to different experi- mental approaches, there has been some ambiguity as to whether this was the same as in the putative progenitor species, M. persicae. However, the present paper shows unequivocally that identical genes are amplified in both species, the FE4 form in aphids of normal karyotype and the E4 gene in aphids with the common Al,3 translocation.

How then did both forms of this resistance mechanism evolve identically in the two karyotypes of each species? The view that the translocation evolved independently in M. nicotianae would support the view that the amplification occurred independently, but it seems highly unlikely that exactly the same amplification events, including the same flanking DNA, arose for both E4 and FE4 genes on two separate occasions. It is much more likely that the amplifi- cations occurred in one species (probably M. persicae) and then the genes were transferred to the other by sexual reproduction between the two species. Certainly in Greece, from whence the 926B clone originated, migration to the peach primary host for sexual reproduction is cornmon-place. Even very limited interbreeding could transfer the amplified genes to some viable hybrid off- spring, in which case the selective advantage of the hybrids, in the presence of insecticide treatments, would enable the amplified genes to be maintained, and they could subsequently spread through M. nicotianae popu- lations. This raises the question of whether M. nicofianae should be considered as a distinct species.

The data presented here also provide further insight into the relationship between the amplified E4 and FE4 genes. Previous cDNA sequence data had shown that both forms of amplified gene in M. persjcae are very similar in their coding regions (Field et a1.,1993); however, the DNA se- quence within intron 7 shows considerable variation be- tween the two genes, suggesting that either they arose by amplification of two slightly different single copy genes or that they have diverged since amplification occurred. Pre- liminary analysis of the same part of an unamplified ester- ase gene from susceptible aphids has given a combination of E4 and FE4-like sequences (unpublished data).

The identical sequences within each set of five cloned PCR products (i.e. three from M. nicofianae and two from M. persicae for each of E4 and FE4) suggest that there is

little, if any, heterogeneity of sequence between the ampli- fied gene copies. This is further supporied by the finding that the TIV sequence is identical to that of the same PCR product from another aphid clone (794J) with amplified E4 genes (unpublished data).

Experimental Procedures

Aphid clones

The insecticide resistant M. persicae clone with normal karyotype and elevated FE4 enzyme (800f) was originally established from a holocyclic population on peaches in Italy (Devonshire et a/., 1983) and the resistant clone with the A1,3 translocation and elevated E4 enzyme (T1 V) came from sugar beet in the east of England (Sawicki eta/., 1980). Previous work had established that 800F contains amplified FE4 genes and TlV has amplified E4 genes (Field eta/., 1988). These clones are typical, with respect to esterases and karyotype, of many clones of M. persicae from around the world (unpublished).

The resistant M. nicotianaeclone, 934L, was established from a sample of the red morph with the translocation and elevated esterase activity (Duplin Red strain of Abdel-Aal, 1990). This was originally collected from tobacco in North Carolina and was a kind gift from Y. A. I. Abdel-Aal. G. Michalopoulos generously supplied the resistant M. nicotianae green morph of normal karyotype from a Greek population on tobacco, from which we established clone 9268. The identity and karyotype of both M. nicotianae clones was confirmed by R. L. Blackman (personal communication).

All four aphid clones were reared on Chinese cabbage plants at 20°C with a 16 h light, 8 h dark regime.

Analysis of aphid esterase enzymes

Homogenates of individual aphids were electrophoresed in poly- acrylamide gels and stained for esterase activity using l-naphthyl acetate as described by Devonshire & Moores (1 982).

Identification of esterase restriction fragments in aphid DNA

DNA (5 pg), prepared from 800F, TlV, 9341. and 9268 aphid clones, was digested with one or more restriction enzymes, electrophoresed in agarose gels, Southern blotted and probed according to the protocol of Field eta/. (1 988). The probes were the cloned E4 and FE4 genomic sequences as described by Field & Devonshire (1991), and as indicated in Fig. 2 (probes A, Band C).

Cloning and sequencing of esterase genomic sequences generated by PCR

The known sequences of E4 and FE4 cDNAs (Field eta/., 1993) were used to design oligonucleotide primers to give a c. 630 bp PCR product between 3’ border of exon 6 and 100 bases into exon 8 (see Fig. 2). DNA (500 ng) from each of the aphid clones was mixed with 250 ng of each primer, in a total volume of 32.5411, and 8 p! of dNTPs (1.25 rnM stock) and 4.5 pl of 1 Ox Taq DNA polym- erase buffer were added. The tubes were heated to 94°C for 5 rnin and then held at 80°C while 3 units of Taq DNA polymerase

148 L. M. Field et al.

(Boehringer Mannheirn) were added in 5pl of 1 x buffer. The PCR proceeded for thirty cycles (94°C for 1 min, 45'C for 1.5 min, and 72°C for 2 min) and the reaction products were checked by electrophoresis in 1% agarose gels. The PCR products were ligated into PCR-ScriptTM SK(+) plasmid DNA in the presence of Srfl enzyme, and then used to transform XL1 Blue Supercompe- tent cells, using the Stratagene PCR-ScriptTM SK( +) cloning kit. Transformed cells were grown on agar plates containing selective antibiotic (ampicillin) and colonies containing esterase sequences were identified by probing colony blots (Sambrook ef al., 1989) with E4 and FE4 genomic sequences (a mixture of probes Band C, see Fig. 2). Positive clones were used to prepare pDNAs (Sam- brook et a/., 1989) which were then sequenced by the dideoxy method using a Pharrnacia T7 sequencing kit. M13 forward and reverse primers which hybridize to the vector were used to se- quence in from each end of the cloned fragments and two oligo- nucleotide primers one at the 3' border of the exon 7 (Oligo A) and one at the 5' border of exon 8 (Oligo 8) (see Fig. 3) were used to complete the sequence of the fragments in both directions.

Acknowledgements

This work was supported in part by MAFF, and by an AFRC Link grant (N.J.) with R. L. Blackman of the Natural History Museum, whom we thank for helpful discussions, critical reading of the manuscript and identification of the aphid clones.

The Resistance Group at Rothamsted is a member of the European Network for Insect Genetics in Medicine and Agriculture (ENIGMA).

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