Establishing the resistance of Myzus persicae (Sulzer) by molecular methods
ABSTRACT In two years of investigating resistance of the peach-potato aphid Myzus persicae (Sulzer) by molecular methods, several types of resistance were established in the majority of individuals from peach and tobacco in Serbia and Montenegro. Most of the tested individuals had the FE4 gene, which encodes production of FE4 esterase. The gene responsible for kdr (knock-down resistance) was found in the majority of individuals, but in the heterozygous state, while resistance based on formation of modified acetlycholinesterase (MACE) was least represented. Also, tests showed aphids from tobacco to be more sensitive to insecticide action than aphids from peach. Three tests were used in these investigations, e.g., the PCR - esterase, PCR - kdr, and RFLP - PCR tests, each for a single type of resistance.
- SourceAvailable from: nih.gov[show abstract] [hide abstract]
ABSTRACT: The peach-potato aphid Myzus persicae (Sulzer) can resist a wide range of insecticides, but until recently (1990) the only mechanism identified was the increased production of carboxylesterases (E4 or FE4), which cause enhanced degradation and sequestration of insecticidal esters. We have now identified two forms of target-site resistance involving changes in the acetylcholinesterase (AChE) and sodium channel (kdr) genes. Biochemical and DNA diagnostic methods can be used to identify all three mechanisms in individual aphids, and thereby establish their spatial distributions and temporal dynamics. Amplified genes underlie the increased production of esterases but their expression is modulated by DNA methylation. Amplification of the E4 gene is in strong linkage disequilibrium with the kdr mechanism. This may reflect strong insecticidal selection favouring aphids with multiple mechanisms, tight chromosomal linkage and/or the prominence of parthenogenesis in many M. persicae populations. The decreased fitness of resistant aphids under winter conditions may be a consequence of the altered sodium-channel gene affecting behaviour and/or the perception of external stimuli.01/1998;
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ABSTRACT: The amplification of genes encoding the esterases E4 and FE4 is a widespread mechanism of insecticide resistance in the peach-potato aphid, Myzus persicae (Sulzer). We present evidence that in susceptible aphids the two genes are adjacent to each other in a head-to-tail arrangement with E4 upstream of FE4 and with approx. 19 kb of intervening sequence. There are also at least two other closely related sequences which might come from other members of an esterase gene family, in line with reports of other insect gene families encoding detoxifying enzymes. The close identity between E4 and FE4 genes indicates a recent duplication and divergence. The subsequent amplifications giving multiple copies of either E4 or FE4 must have involved two separate events, each probably occurring once and then being selected by insecticide exposure and spread by migration. The cloning of sequences upstream of the FE4 gene suggest, by comparison with E4, that the two genes are regulated in different ways. FE4 has sequences corresponding to a conventional promoter (TATA box and CAP site) that are not present in E4; on the other hand, FE4 lacks the CpG island present 5' of E4 genes that may control expression through changes in DNA methylation. The differences are likely to have occurred by the duplication event that gave rise to E4 and FE4 leading to different 5' sequences.Biochemical Journal 03/1998; 330 ( Pt 1):169-73. · 4.65 Impact Factor
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ABSTRACT: In northern Greece, insecticides have been used intensively against aphid populations of the Myzus persicae group, both on its primary host peach, on which annual sexual reproduction occurs, and on secondary host field crops such as tobacco, on which reproduction is entirely parthenogenetic. This has resulted in the selection of high levels of resistance based on the amplification of two genes encoding insecticide-degrading esterases, E4 and FE4. We have used fluorescence in situ hybridization (FISH) to study variation in the number and distribution of loci with amplified esterase genes in clones established from field populations of M. persicae- group aphids sampled on various crops in northern Greece. All clones collected from peach, as well as most of the clones (74% ) from tobacco and other secondary host plants, had amplified FE4 genes and were of normal karyotype. Amplicon clusters containing FE4 occurred at multiple sites which varied in number, zygosity and distribution between clones. Most loci were on autosome 1, which also had the only site that was consistently occupied by amplified FE4, situated near subtelomeric repetitive DNA. Possibly this was the original site of FE4 gene amplification, and the location of the single-copy 'wild type' esterase genes. The rest of the clones from tobacco and other secondary hosts (26% ) had amplified E4 genes, and all those analysed by FISH had an amplicon cluster on autosome 3T at a site close to the breakpoint of an A1,3 translocation, confirming the close linkage of this translocation with, and its probable involvement in, E4-based resistance. Three translocated clones collected on capsicum at one site had both E4 and FE4 amplified, the first time that both esterase gene types have been found together in individual aphids from field populations.Keywords: aphid chromosomes, FISH, gene amplification, insecticide resistance, multiple lociHeredity 01/1999; 82(2):180-186. · 4.11 Impact Factor
The peach-potato aphid Myzus persicae (Sulzer) is
a very significant pest of peach, tobacco, vegetables,
and flowers. Two subspecies have in recent years
been established in Europe, viz., Myzus persicae
persicae (Sulzer), whose primary host is peach, and
Myzus persicae nicotianae (Blackman), which is
adapted to tobacco, but the mechanisms of resis-
tance are the same in both subspecies (Eastop
and Blackman, 2005). Myzus persicae has been
subjected to intensive treatment with insecticides,
owing to its pronounced polyphagy, the large num-
ber of generations it produces annually, its capacity
for parthenogenesis, and the existence of wingless
and winged forms of it that are significant vectors
of viruses. This constant pressure of insecticides
has led to the development of resistance to many
Two mechanisms of resistance are present in
Myzus persicae: increased detoxication of insecti-
cides; and changes in sensitivity of the action sites.
These two mechanisms provide resistance to a wide
range of insecticides with different modes of action.
Increased detoxication of insecticides occurs due
to increased production of two related esterases
designated E4 and FE4, as the genes encoding their
production are also referred to (Field et al., 1989).
Changes of action site sensitivity are involved in two
other types of resistance. They are a consequence
of point mutations of genes causing amino acid
substitutions in protein synthesis. The first type
of resistance results from formation of modified
acetylcholinesterase or MACE, which occurs due
to substitution of phenylalanine for serine near the
catalytic part of the enzyme. The second type is
knock-down resistance, which occurs due to substi-
tution of phenylalanine for leucine, which results in
mutations in Na-channels of the nerve synapse.
Esterase E4 and FE4 account for more than one
percent of total proteins in the body of the aphid and
perform hydrolysis of esters of insecticides and their
capture before they reach the target in the nervous
system. Increased activity of carboxylesterases is
a consequence of amplification of their structural
genes (Devonshire and Sawicki, 1979; cited
Establishing thE rEsistancE of Myzus persicae (sulzEr) by molEcular mEthods
ANDjA VUčETIć1, OlIVERA PETROVIć-OBRADOVIć1,
j. MARgARITOPOUlOS2 and P. SkOURAS2
1Faculty of Agriculture, University of Belgrade, 11000 Belgrade-Zemun, Serbia
2Laboratory of Entomology and Agricultural Zoology, Department of Crop Production and Agricultural Environment,
University of Thessaly, 380 Volos, greece
Abstract — In two years of investigating resistance of the peach-potato aphid Myzus persicae (Sulzer) by molecular
methods, several types of resistance were established in the majority of individuals from peach and tobacco in Serbia
and Montenegro. Most of the tested individuals had the FE4 gene, which encodes production of FE4 esterase. The gene
responsible for kdr (knock-down resistance) was found in the majority of individuals, but in the heterozygous state, while
resistance based on formation of modified acetlycholinesterase (MACE) was least represented. Also, tests showed aphids
from tobacco to be more sensitive to insecticide action than aphids from peach. Three tests were used in these investiga-
tions, e.g., the PCR - esterase, PCR - kdr, and RFlP - PCR tests, each for a single type of resistance.
Key words: Myzus persicae persicae, M. persicae nicotianae, resistance, esterase, kdr, MACE, Serbia, Montenegro
Arch. Biol. Sci., Belgrade, 60 (3), 493-499, 2008 DOI:10.2298/ABS0803493V
ANDjA VUčETIć ET Al.
by Devonshire et al., 1998). This creates high
resistance to organophosphates and pyrethroids and
somewhat lower resistance to carbamates (Foster
et al., 2000).
Another form of resistance is based on modified
insensitive acetylcholinesterase (AChE) as the tar-
get of organophosphates and carbamates, which in
Myzus persicae was first detected in 1990 (Moores
et al., 1994). Acetylcholinesterase is a member of the
serine hydrolase family of enzymes and is respon-
sible for hydrolysis of the neurotransmitter acetyl-
choline (ACh) in nerve synapses. Organophosphates
and carbamates act by inhibiting acetylcholinester-
ase, which causes repetitive discharges on postsyn-
aptic nerves, leading to insensitivity of the nervous
system and possible death of the organism (javed
et al., 2003). Modified AChE (MACE) creates high
resistance to pyrimicarbs and triazamatimes, as well
as to triazolimes, as was later established (Dewar
et al., 1984).
The last discovered resistance mechanism
is "knock-down" resistance (kdr), which creates
resistance to pyrethroids and DDT (Martinez-
Torres et al., 1999). It applies to changes in
structure of the protein sheath of sodium channels
in the nerve membrane. This form of resistance is
referred to as kdr (knock-down resistance) because
the instantaneous paralyzing effect of pyrethroids
and DDT is called a knock-down. Resistance based
on increased esterase production is of secondary
significance in relation to kdr where pyrethroids are
Biochemical methods of establishing resistance
revealed decreased sensitivity of Myzus persicae
populations to the most often employed groups of
insecticides and indicated that resistance is devel-
oped to a certain extent in populations from Serbia
and Montenegro (Vučetić et al., 2007).
The purpose of the present investigations was
to use molecular methods to detect the existence
of genes responsible for the development of resis-
tance and thereby establish the level of resistance
of M. persicae to the most often employed groups
of insecticides and obtain a more complete picture
of the state of resistance of this aphid in Serbia and
MATERIAl AND METHODS
Samples were taken from a number of localities
on the territory of Serbia and Montenegro during
the years 2004 and 2005. Peach orchards, individual
peach trees growing in yards and alongside roads,
and tobacco fields were covered in the sampling.
leaves attacked by leaf aphids were brought into
the Entomological laboratory of the Faculty of
Agriculture in Zemun, where the aphids were deter-
mined. One female from each sample was left in a
phytotron for rearing under controlled conditions,
while a greater number of females were put in a
freezer or fixed in alcohol. Molecular investigations
were carried out in the laboratory of Entomology
and Agricultural Zoology, University of Thessaly,
Department of Crop Production in Volos, greece.
Molecular testing of the resistance level was per-
formed on living individuals of Myzus persicae, ones
fixed in alcohol (75 or 96%), or frozen specimens.
Extraction of DNA was carried out by the
methods of Sannucks and Hales (1996) and
Martinez-Torres et al. (1997). Testing of resis-
tance by molecular methods involved establishing
the site in the insect's genome responsible for one
of the three existing types of resistance by means of
PCR (the polymerase chain reaction).
The PCR-esterase test was used to establish
whether genes (E4 and FE4) responsible for resis-
tance exist in the aphid's genome. Results were con-
firmed by horizontal electrophoresis in 1% agarose
gel, by staining with ethidium bromide, and in the
presence of an appropriate marker. Aphids with an
amplified E4 gene always give a PCR product with
a value of 572 bp, whereas aphids with an amplified
FE4 gene give one with a value of 865 bp, which
show up as clear lines and are easy to distinguish
in agarose gel. Sensitive aphids either give no result
of any kind after electrophoresis in agarose gel or
else give two pale lines indicating small amounts
of amplified genes, one with a value of 572 bp,
the other with a value of 865 bp (Field and
RESISTANCE OF MyZUS PErSiCAE
The PCR - kdr (knock-down resistance) test was
used to establish existence in the aphid's genome of
a mutation responsible for creation of the knock-
down type of resistance, i.e., to establish whether
mutation leading to the amino acid substitution
responsible for changes in the structure of Na-chan-
nels in the nerve membrane occurred in the genome.
The test was carried out according to the procedure
of Martinez-Torres et al. (1999). Results were
confirmed by horizontal electrophoresis in 1% aga-
rose gel, by staining with ethidium bromide, and in
the presence of an appropriate marker. Fragments
with a value of 300 bp indicate resistance, whereas
fragments with a value of 600 bp indicate sensitivity.
If both lines appear in a sample, this means that the
gene in it is found in the heterozygous state (RS).
The RFlP (restriction fragment length poly-
morphism) - PCR test was used to establish the site
in the genome responsible for changes in the struc-
ture of acetylcholinesterase. Based on identification
of the site of the point mutation that leads to amino
acid substitution and creation of modified AChE, it
also indicates whether the mutation is in the homo-
zygous or the heterozygous state. By means of PCR
with the aid of appropriate primers, an amplified
DNA segment measuring 1269 bp is obtained which
constitutes 2/3 of full length of the DNA sequence
that encodes AChE. In the wild type, further diges-
tive restriction enzyme action yields three fragments
with values of 435, 780, and 54 bp, whereas the 780
and 54 fragments are fused in the type in which
mutation occurred (S431F). Restriction enzymes
belong to the nuclease group, enzymes that cut the
phosphoester bonds of polynucleotide chains. They
are endonucleases whose activity is strictly confined
to a specific sequence of nucleotide pairs in DNA
(Marinković et al., 1991). Isolation of restriction
enzymes made controlled fragmentation of chromo-
The protocol for this method was devised by
Cassanelli et al. (2004), and the results are read
in vertical electrophoresis in polyacrylamide gel and
staining with silver nitrate. DNA fragments of differ-
ent size will be arranged differently, and on the basis
of their position in the presence of an appropriate
marker, it can be clearly seen whether the enzyme
cut DNA and if so whether the sites cut by it are in
the homozygous or heterozygous state.
RESUlTS AND DISCUSSION
Aphids from tobacco growing at 33 locali-
ties were tested. In aphids from three samples
(Mladenovac 1, čoka 8, and Bački Petrovac, Serbia),
amplification of the FE4 and E4 genes occurred,
which indicates sensitivity. The FE4 gene was found
in all other samples. The gene responsible for kdr
was not found in three samples (Bački Petrovac,
Mladenovac 2, and čoka 1), whereas it was found in
the heterozygous state in the remaining 30 samples.
As for resistance based on modified acetylcholines-
terase, the test showed that only one sample (Senta
1) possessed this gene, but in the heterozygous state,
whereas its presence was not established in the other
tested aphids (Table 1).
Table 2 presents the results of tests performed on
peach. Aphids from 20 localities were tested.
Amplification of the FE4 gene occurred in all of
the tested aphids. The gene responsible for kdr was
found in the homozygous state in aphids from one
sample (Podgorica 2), whereas it was found in the
heterozygous state in the other samples. The pres-
ence of MACE resistance was not established in any
of the tested samples.
Analyzing the results of all tests, we are able to
conclude that resistance based on increased pro-
duction of carboxylesterase (here caused by only
the single gene FE4) is dominant. It is followed by
kdr, which was not registered in only three samples.
Modified acetylcholinesterase turned out to be the
rarest form of resistance. However, in view of the
fact that a certain number of individuals possessed
the gene encoding its production, the possibility
exists that it will undergo expansion as a result of
selection of such individuals.
It was established by means of the PCR - ester-
ase test that amplification of the FE4 and E4 genes
occurred (horizontal electrophoresis yielded pale
lines measuring 865 and 572 bp) in three samples
from tobacco, and it can be asserted that the aphids
in question are sensitive, since it was demonstrated
ANDjA VUčETIć ET Al.
by Blackman et al. (1996, 1998) and by Field
and Devonshire (1998) that sensitive aphids
are carriers of both genes. All of the other samples
had an amplified FE4 gene. According to the data of
Spence and Blackman (1997), the existence of
genes E4 and FE4 is closely linked with the life cycle
of aphids. The FE4 gene is usually present where
a sexual generation exists on peach. In greece,
aphids from tobacco in peach-growing regions can
overwinter as eggs on peach, but there too a cer-
tain number of aphids overwinter anholocyclically
(Margaritopoulos et al., 2002).
The PCR - kdr test showed that three samples
from tobacco did not have the resistance gene,
while one of the tested samples from peach had RR
resistance. Aphids with the gene in the homozygous
state (RR) were from a peach orchard where inten-
sive measures of protection are employed. All of the
kanjiža - Mali Pesak
kanjiža - M.Pesak2
kanjiža - M. Pijace
kanjiža - M.Pijace5
kanjiža - M.Pijace6
Šid - kukujevci
Šid - Vašica
Podgorica - Sukuruć
Podgorica – Vranj1
Podgorica – Vranj3
Podgorica - Tuzi
Table 1. Results of molecular tests of resistance performed on
aphids from tobacco (Myzus persicae nicotianae).
Table 2. Results of molecular tests of resistance performed on
aphids from peach (Myzus persicae persicae).
Belgrade - Radmilovac 2 FE4RS-
Stara Pazova FE4 RS-
Bela CrkvaFE4 RS-
Podgorica 1FE4 RS-
Belgrade - Radmilovac FE4RSSS
Belgrade - Brestovik1 FE4 RSSS
Belgrade - Brestovik2FE4 RSSS
Belgrade - Ritopek FE4 RSSS
Belgrade - Ritopek2FE4 RS SS
Belgrade - Vinča1 FE4RS SS
Belgrade - Vinča2FE4RS SS
Belgrade - Vinča3FE4RS SS
Belgrade - galenikaFE4RS SS
Bela CrkvaFE4RS SS
RESISTANCE OF MyZUS PErSiCAE
other tested aphids had this gene, but in the hetero-
zygous state. Changes in the Na-channels of nerve
membranes that lead to this form of resistance affect
behavior of the aphid and reduce its capacity for
survival (Foster et al., 1996, 1997, 1999), so that
a cold winter effectively eliminates highly resistant
(RR) aphids. Inasmuch as winters in our country are
long and cold, it is understandable why the number
of RR aphids is small.
MACE resistance turned out to be the rarest
form of resistance. In certain other investigations,
ones where resistance was monitored over a longer
period of years, this form of resistance proved to
be the most unstable. In the 10-year investigations
of Foster et al. (2002), it turned out that 68% of
the tested samples had this form of resistance in
1996, but the percentage dropped precipitously in
the years to follow (only 7% had it in 1997). The
cause of this is not known, but studies have shown
that it is closely linked with increased production of
carboxylesterase and that it occurs most frequently
in R2 and R3 aphids (Anstead et al., 2004). In
investigations where biochemical methods were
used to establish the level of resistance of Myzus
persicae from peach and tobacco, not one aphid with
the R3 level of resistance was found in our country
(Vučetić et al., 2007). That is possibly the reason
why this form of resistance is not widespread among
the tested populations.
Comparing the samples from tobacco and
peach, we can say that no very significant differ-
ences exist. Nevertheless, the esterase test showed
three samples from tobacco to be sensitive. Also, the
kdr test revealed three sensitive samples, again from
tobacco, one of which exhibited sensitivity to both
tests. No sample from peach was sensitive, and one
had RR resistance in relation to the kdr test. Even
though the differences are slight, they indicate that
aphids from tobacco are somewhat more sensitive
than aphids from peach. In view of the fact that pro-
tective measures, especially in large peach orchards,
are more intensive than in the case of tobacco, it is
understandable why aphids from tobacco are more
sensitive to insecticides than are aphids from peach
Such a state of resistance, where one or two
mechanisms are dominant and a third does not
occur or else occurs very rarely, is normal, since
few individuals manifest multiple resistance, i.e.,
are characterized by development of all resistance
mechanisms (li and Han, 2004).
Molecular methods of establishing resistance
have shown us that large percentages of M. persicae
individuals from both peach and tobacco are car-
riers of genes responsible for the development of
resistance, with the result that both subspecies show
reduced sensitivity to the most often employed
groups of insecticides. However, populations of M.
persicae in Serbia and Montenegro still do not belong
to the category of extremely resistant aphids.
Acknowledgments — The present study was supported by the
Serbian Ministry of Science (grant 143006B).
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RESISTANCE OF MyZUS PErSiCAE
утврђивање резистентности Myzus persicae (sulzEr) молекуларним методама
анђа вучетић1, оливера поповић-обрадовић1,
j. MARgARITOPOUlOS2 и P. SkOURAS2
1Пољопривредни факултет, Универзитет у Београду, 11000 београд, Србија
2Laboratory of Entomology and Agricultural Zoology, Department of Crop Production and Agricultural
Environment,University of Thessaly, 380 Volos, greece
у двогодишњим истраживањима резистент-
ности зелене бресквине ваши, Myzus persicae
(Sulzer) молекуларним методама, установљено је
да је код већине тестираних јединки са брескве
и дувана у Србији и Црној Гори, утврђен неки
од типова резистентности. већина тестираних
јединки имала је FE4 ген који кодира продукци-
ју FE4 карбоксилестеразе. Ген одговоран за kdr
(knock-down резистентност) нађен је код већине
јединки, али у хетерозиготном стању док је рези-
стентност која се заснива на стварању модифи-
коване ацетилхолинестеразе (MACE) најмање
заступљена. такође, тестови су показали да су
ваши са дувана осетљивије на дејство инсекти-
цида од вашију са брескве. у овим истражива-
њима коришћена су три теста: PCR-естераза
тест, PCR–kdr и RFlP–PCR, сваки за по један тип
у овим истражива-