Pest Management Science

Pest Manag Sci 64:964970 (2008)

Toxicological and biochemical response

to azinphos-methyl in Cydia pomonella L.
(Lepidoptera: Tortricidae) among orchards
from the Argentinian Patagonia
1 Liliana Anguiano,1 Ana Pechen de DAngelo,1 Liliana Cichon,
2
Jimena Soleno,
2

Daro Fernandez
and Cristina Montagna3
1 Departamento

Argentina
de Qumica, Universidad Nacional del Comahue, Buenos Aires 1400, (8300) Neuquen,
Ruta 22, Km 1192, Allen, Ro Negro, Argentina
3 Escuela Superior de Salud y Ambiente, Universidad Nacional del Comahue, Buenos Aires 1400, (8300) Neuquen,
Argentina
2 INTA

Abstract
BACKGROUND: Azinphos-methyl is the main insecticide used to control codling moth on apple and pears in
Northern Patagonia. The aim of this study was to evaluate the toxicological and biochemical response of diapausing
larvae of codling moth in orchards subjected to different insecticide selection pressure.
RESULTS: Dosemortality assays with azinphos-methyl in diapausing larvae of Cydia pomonella L. showed
significant differences between the LD95 from a population collected in one untreated orchard (2.52 g moth1 )
compared with that in a laboratory-susceptible population (0.33 g moth1 ). Toxicity to azinphos-methyl in field
populations of diapausing larvae collected during 20032005 was evaluated by topical application of a discriminating
dose (2.5 g moth1 ) that was obtained from larvae collected in the untreated orchard (field reference strain).
Significantly lower mortality (37.7184.21%) was observed in three out of eight field populations compared with
that in the field reference strain. Most of the field populations showed higher esterase activity than that determined
in both the laboratory susceptible and the field reference strains. Moreover, there was a high association between
esterase activity and mortality (R2 = 0.64) among the field populations. On the other hand, a poor correlation
was observed between glutathione S-transferase activity and mortality (R2 = 0.33) among larvae collected from
different orchards.
CONCLUSIONS: All the field populations evaluated exhibited some degree of azinphos-methyl tolerance in
relation to the laboratory susceptible strain. Biochemical results demonstrated that esterases are at least one of
the principal mechanisms involved in tolerance to this insecticide.
2008 Society of Chemical Industry

Keywords: codling moth; Cydia pomonella; resistance; azinphos-methyl; esterases; glutathione S-transferases

1 INTRODUCTION
The codling moth Cydia pomonella L. (Lepidoptera: Tortricidae) is a major pest of apples
and pears throughout the world. Organophosphorus insecticides, particularly azinphos-methyl,
have been extensively used for control of codling
moth in the United States,1,2 Chile,3,4 the Czech
Republic5 and Australia.6 Consequently, resistance to this compound,2,3,4,7 9 phosalone5,10 and
parathion has occurred.11 Field-exposed populations
of codling moth have also developed resistance to
pyrethroids,2,12 insect growth inhibitors,10,13,14 insect
growth regulators3,10,15 and biological products.16
Moreover, field populations of codling moth resistant to azinphos-methyl have shown cross-resistance

to other conventionally neurotoxic insecticides8 and

insect growth inhibitors.5 Resistance to insecticides
in C. pomonella is expressed in both adults and
neonates,2 as well as in diapausing larvae.12 Detoxifying enzymes3,4,12,17,18 in addition to target insensibility
have been implicated as insecticide resistance mechanisms in codling moths from field and laboratoryselected populations.19 21 Bioassays on adult moths
are successfully utilised for monitoring resistance in
codling moth.7,8 More recently, it has been demonstrated that diapausing larvae can also be used
for detection of resistance in field collections.12
Besides doseresponse bioassays, discriminating doses
have been used since 1993 to detect field resistant
populations in adults2,11 and diapausing larvae.3,10,22

Correspondence to: Cristina Montagna, Escuela Superior de Salud y Ambiente, Universidad Nacional del Comahue, Buenos Aires 1400, (8300)
Argentina
Neuquen,
E-mail: mmontagn@uncoma.edu.ar
(Received 3 August 2007; revised version received 3 December 2007; accepted 22 December 2007)
Published online 31 March 2008; DOI: 10.1002/ps.1582

2008 Society of Chemical Industry. Pest Manag Sci 1526498X/2008/$30.00

Resistance to azinphos-methyl in Patagonian C. pomonella

This methodology has the advantage of detecting

resistance even in those cases where the number of
organisms available is small and resistance frequencies
are low.22 In addition to toxicological assays, biochemical assays can also be used to monitor resistance.
Indeed, several authors have shown that the mechanisms of resistance remain active during diapause.12,23
The Ro Negro and Neuquen Valley situated in
Northern Patagonia is the most important apple and
pear growing region in Argentina. Extensive insecticide
usage has been the practice for many years for
controlling C. pomonella and other fruit tree pests.
In this region, codling moth has three generations per
year, and pest control programmes usually include
several applications of broad-spectrum insecticides in
each growing season. Insecticides are applied using
air-blast sprayers beginning in October and ending in
late February or March.
Pyrethroids were introduced in 1982 and have
been widely used for almost 20 years. Pyrethroid
applications were alternated with organophosphate
compounds such as azinphos-methyl or the carbamate
carbaryl. Since the growing season 19931994, many
farmers have reported control failures when using
pyrethroids, especially esfenvalerate. A few years
later, azinphos-methyl was primarily recommended
for codling moth management along with other
insecticides with different modes of action, such
as insect growth regulators.24 Alternative methods
like mating disruption, organic (diatomaceous earth)
and microbial insecticides have also been introduced
among big commercial orchards. In spite of a trend to
introduce integrated pest management approaches in
the region, small farmers still continue with the use of
only broad-spectrum insecticides.
In view of the intense use of azinphos-methyl, the
objective of this study was to monitor the toxicological
and biochemical response to this insecticide in
diapausing larvae of codling moth.

2 MATERIALS AND METHODS

2.1 Chemicals
The organophosphate azinphos-methyl (97.8% pure)
was purchased from AccuStandard Inc., New Haven,
CT. Reduced glutathione (GSH), 1-chloro-2,4dinitrobenzene (CDNB), -naphthyl acetate (-NA),
-naphthol (-N), 1,5-bis(4-allyldimethylammoniumphenyl)pentan-3-one dibromide (BW284C5), Fast
Garnet GBC salt, Triton X-100 and bovine serum
albumin were purchased from Sigma Chemical
Co., USA.
2.2 Insects
Diapausing larvae from untreated, organic and
conventionally managed orchards were collected using
corrugated paper strips during 2003, 2004 and 2005.
Larvae were then transferred to clean corrugated paper
and stored at 3 1 C with a 12:12 h light:dark
photoperiod in order to satisfy the chill requirement.
Pest Manag Sci 64:964970 (2008)
DOI: 10.1002/ps

Diapause termination was induced by exposing the

larvae for 1 day to a 16:8 h light:dark photoperiod at
22 1 C.3,22,25 The names of the larvae populations
given in the text correspond to the locality of
collection, i.e. Cinco Saltos (38 49 S, 68 04 W), Allen
(38 59 S, 67 49 W), Centenario (38 48 S, 68 08 W),
General Roca (39 02 S, 67 38 W), Guerrico (39 01 S,
67 44 W), Valle Azul (39 09 S, 67 08 W), Villa
Regina (39 06 S, 67 04 W) and Vista Alegre (38 45 S,
68 11 W). Larvae populations from different orchards
at the same locality were designated by subscript.
Larval populations from Cinco Saltos and Vista Alegre
were evaluated in three and two different seasons
respectively. Larvae collected at the locality of Cinco
Saltos from an orchard controlled by cardboard tree
bands (untreated) since 1998 were considered as a
field reference population. The laboratory susceptible
population was established in 1991 by collection of
diapausing larvae from an abandoned orchard, and
had been maintained since then without pesticide
exposure. Before toxicological and biochemical assays
were performed, diapausing larvae from both the
laboratory and the field were transferred to the postchilling conditions (25 C, 70% RH and 16:8 h
light:dark photoperiod) for 24 h. The age of diapausing
larvae from the laboratory was 125 2 days old (from
the day of ecdysis into the fifth instar to the end of the
chilling period).
2.3 Toxicological assays
Toxicological assays on diapausing larvae were
performed using a discriminating dose of azinphosmethyl determined by a doseresponse bioassay
with larvae collected from the Cinco Saltos orchard
controlled by cardboard tree bands. A quantity of
1 L of the discriminating dose was applied to the
dorsum of each larva using a Hamilton microsyringe.
Experiments were conducted on 35 groups of
20 larvae placed in petri dishes according to the
available insects. Two or three batches of larvae
treated with acetone were used as control. Larvae
were subsequently placed under controlled conditions
(25 C, 70% RH and 16:8 h light:dark photoperiod)
during 48 h. Larvae were considered dead if they did
not respond as the controls after a brush touch. Before
scoring mortality, larvae were removed (most of them
had spun a new cocoon) and placed in the cap of
the corresponding petri dish. Larvae were considered
moribund if they displayed one of the following
after a brush touch: they continued lying on their
side or in the dorsal position, they were unable to
move in a coordinated manner, they showed repeated
body contractions or they moved backwards and
forwards without displacement. Categories of dead
and moribund were combined to assess percentage
mortality.
2.4 Biochemical assays
Non-specific esterase activities were assayed in diapausing larvae using -naphthyl acetate as substrate.26
965

 et al.
Jimena Soleno

Five larvae were homogenised in 1 mL of ice-cold

0.1 M sodium phosphate buffer (pH 6.5) containing
5.35 g L1 Triton X-100. Each sample was centrifuged
at 10 000 g for 10 min at 4 C, and the supernatant
was used as the enzyme source. Aliquots (200 L)
of diluted supernatant were incubated at 25 C for
15 min with 400 L of the substrate solution containing distilled water, 0.1M sodium phosphate buffer
(pH 6.5) plus 5.35 g L1 Triton X-100, a final concentration of 2 mM -naphthyl acetate and 0.002 mM
BW284C5 (an acetylcholinesterase inhibitor). A quantity of 400 L of freshly prepared 2.5 mM Fast Garnet
GBC salt was added, and absorbencies were recorded
10 min later at 550 nm in a UV/visible spectrophotometer (Shimadzu, Kyoto, Japan). Absorbency values
were transformed into mol of -naphthol from an naphthol standard curve. According to the available
larvae, between 5 and 17 enzyme preparations were
obtained from each population.
Glutathione S-transferase (GST) activities were
assayed by the method of Habig et al.27 Batches of
five larvae from each population were homogenised in
1 mL of 66 mM ice-cold phosphate buffer containing
25 mM sucrose (pH 7.0). Each sample was centrifuged
at 16 000 g for 20 min at 4 C, and the supernatants
were used with no further purification. Glutathione
S-transferase activities were measured using CDNB
(0.5 mM in acetonitrile) as substrate. The reaction
mixture in a final volume of 3 mL consisted of
2.78 mL of 0.1 M phosphate buffer (pH 6.5), 40 L
of the enzyme source, 30 L of CDNB and 150 L
of 2.5 mM reduced glutathione. Absorbency was
recorded continuously at 340 nm for 2 min in the
UV/visible spectrophotometer. Rate measurements
were corrected for the non-enzymatic reaction and
transformed into mol of CDNB conjugates using the
extinction coefficient 9.6 mM cm1 . Eight different
enzyme preparations from each population were
evaluated.
Protein was assayed by the method of Lowry
et al.28 Absorbency was measured at 750 nm and
transformed into protein concentration from a bovine
serum albumin standard curve.
2.5 Statistical analysis
LD50 and LD95 values for both the laboratory
susceptible and the field reference populations were
calculated by probit analysis using a BASIC program.
These values were considered significantly different
if their confidence limits did not overlap. Control
mortality was corrected by the same program. For
the larvae evaluation, 2 values were calculated in
comparison with azinphos-methyl mortality of the
same year collection of the Cinco Saltos population.
Correction for control mortality, which was always
below 10%, was made by Abbotts method.29 Mean
esterase and glutathione S-transferase activities were
analysed by Students t-test. The relationship between
enzyme activities (esterase and GST) and percentage
966

of azinphos-methyl mortality was evaluated by linear

regression.

3 RESULTS
3.1 Toxicological assays on diapausing larvae
The azinphos-methyl responses of diapausing larvae
from both a laboratory (susceptible) and an untreated
orchard (Cinco Saltos) population are presented in
Fig. 1. The data from both doseresponse bioassays
showed a good fit to the probit model. The
LD95 (0.33 g larva1 ) of the laboratory population
was about eightfold lower than that observed in
the Cinco Saltos population (2.52 g larva1 ). The
slope from the laboratory population was very steep
(6.2 1.5) compared with that from the Cinco Saltos
population (3.1 0.3), and thus they exhibited only
fourfold difference at the LC50 level. The LC50
as well as the LC95 values from both populations
were significantly different according to their 95%
confidence limits. Because of the substantial difference
at the LC95 between these two strains, larvae from
Cinco Saltos were considered as the field reference
strain among the field populations. Indeed, it was
the only population that had not been recently
subjected to chemical or biological control (5 years)
at the time of these assays. Table 1 shows the
azinphos-methyl response of diapausing larva from
field populations subjected to different insecticide
selection pressure. Assays performed during 2003
showed that the mortality at the discriminating dose of
azinphos-methyl (2.5 g larva1 ) in diapausing larvae
collected at the locality of Cinco Saltos (96.55%) was
significantly higher than the mortality observed on
larvae collected in two orchards at Allen (38.33 and
84.21%). On the other hand, the mortality registered
on the population from Centenario (92.85%) did
not differ statistically from that exhibited by the
reference strain. Field populations from General Roca,
Guerrico and Valle Azul evaluated for azinphos-methyl
response during 2004 were found to be significantly
more susceptible (98.18, 98.21 and 98.24% mortality)
than that from Cinco Saltos (91.66%). Among
the populations collected during 2004, only larvae
from Vista Alegre (60.34%) showed significantly
lower mortality than larvae from Cinco Saltos at
the discriminating dose of azinphos-methyl. Larvae
collected during 2005 from the same orchard at
Vista Alegre showed not only significantly lower
mortality (37.71%) than that recorded in the Cinco
Saltos population (91.25%) but also significantly
lower mortality than that observed during the 2004
collection. Finally, larvae from Cinco Saltos collected
during 2003, 2004 and 2005 did not show statistical
differences in mortality.
3.2 Biochemical assays
Means from esterase and GST activities across
diapausing larvae from a laboratory susceptible
population and field populations are shown in Table 2.
Pest Manag Sci 64:964970 (2008)
DOI: 10.1002/ps

Resistance to azinphos-methyl in Patagonian C. pomonella

7
Laboratory
Cinco Saltos

PROBIT

80
60

40
20

% MORTALITY

95

3
0.1
1
DOSE (mg per larva1)

10

Figure 1. Dosagemortality regression lines for azinphos-methyl

against diapausing larvae of Cydia pomonella from the laboratory
(susceptible) and from an organic orchard.

Table 1. Response of diapausing larvae at discriminating dose of

azinphos-methyl (2.5 g larva1 ) from an organic and conventionally
managed orchards

Azinphos-methyl
Populationa
Cinco Saltos (u)

Allen1 (t)
Allen2 (t)
Centenario (t)
General Roca (t)
Guerrico (t)
Valle Azul (o)
Villa Regina (t)
Vista Alegre (t)

Year of
collection

nb

Mortality (%)c

2003
2004
2005
2003
2003
2003
2004
2004
2004
2005
2004
2005

61
96
80
60
57
56
55
56
57
60
80
55

96.55
91.66
91.25
38.33
84.21
92.85
98.18
98.21
98.24
90.00
60.34
37.71

610.84
26.18
2.30
4.09
4.17
4.27
0.12
74.56
286.78

Letters in parentheses indicate type of orchard management:

u, untreated; o, organic; t, treated with conventional and nonconventional insecticides).
b Total number of insects treated after subtraction of those larvae that
had escaped.
c Percentage mortalities of larvae at each site of collection were
compared with that from Cinco Saltos according to the sampled
period. Significance thresholds: P < 0.05; P < 0.01, P < 0.001,
using the 2 test.

in larval populations collected in 2004 from Guerrico

(0.045 0.012), Valle Azul (0.062 0.0088) and
Vista Alegre (0.13 0.037) compared with that in
the reference field population. Larvae collections
during 2005 from both Vista Alegre and Villa Regina
exhibited the highest esterase activities (0.20 0.10
and 0.18 0.036 respectively) recorded in this study.
It is worth noting the very high variability in esterase
activity recorded among larvae from Vista Alegre.
Diapausing larvae collected in 2003 from Allen1 ,
Allen2 and Centenario displayed significantly higher
mean GST activities (mol CDNB conjugated
min1 mg1 protein: 0.24 0.040, 0.27 0.074 and
0.27 0.057 respectively) than that from Cinco Saltos
(0.077 0.029). Among the populations collected
during 2004, only the mean GST activity in larvae
from Vista Alegre (0.21 0.034) was significantly
higher than that from Cinco Saltos (0.096 0.033).
There were no significant differences in GST activities
between larvae from the laboratory and larvae from
Cinco Saltos collected during 2003 and 2004.
Figure 2 shows the relationship between enzyme
activity and percentage of mortality among the field
populations. The results show a high correlation
between esterase activity and mortality (R2 = 0.64)
and a poor association between GST activity and
mortality (R2 = 0.33).

4 DISCUSSION
Two of eight field populations (25%) that were
collected during 20032005 displayed no significant
difference in mortality to azinphos-methyl to that
in the Cinco Saltos population. Furthermore, three
populations, including the one from an organic
orchard, registered significantly lower mortalities than
that of the field reference population. However,
larvae from the untreated orchard were found to
Table 2. Esterase and GST activities of diapausing larvae from a
laboratory susceptible and field populations

Activity (mol min1 mg1

protein) ( SD)a
Locality

Mean esterase activities (mol -N min1 mg1

protein) recorded from field populations collected
in 2003 were all significantly different. The lowest
activities were registered for larvae collected from
both Centenario (0.018 0.0072) and Cinco Saltos
(0.029 0.0058). Mean esterase activities determined
in larvae from Allen1 (0.14 0.019) and Allen2
(0.14 0.026) were about fivefold higher than that
observed in larvae from Cinco Saltos. No significant
differences in esterase activity were recorded in the
Cinco Saltos population during 2003 and 2004;
however, both were significantly lower than the activity
recorded in the laboratory strain (0.040 0.0049).
Significantly higher esterase activities were determined
Pest Manag Sci 64:964970 (2008)
DOI: 10.1002/ps

Laboratory
Cinco Saltos
Allen1
Allen2
Centenario
General Roca
Guerrico
Valle Azul
Villa Regina
Vista Alegre

Year of
collection

Esterase

GSTb

2004
2003
2004
2003
2003
2003
2004
2004
2004
2005
2004
2005

0.040 (0.0049)d
0.029 (0.0058)a
0.024 (0.0062)ab
0.14 (0.019)c
0.14 (0.026)c
0.018 (0.0072)b
0.019 (0.0069)b
0.045 (0.012)d
0.062 (0.0088)
0.18 (0.036)c
0.13 (0.037)c
0.20 (0.10)c

0.080 (0.017)a
0.077 (0.029)a
0.096 (0.033)a
0.24 (0.040)b
0.27 (0.074)b
0.27 (0.057)b
0.10 (0.045)a
0.077 (0.041)a
0.091 (0.033)a
nd
0.21 (0.034)b
nd

Means in a column followed by the same letter are not significantly

different ( = 0.01, Students t-test).
b nd = not determined.

967

 et al.
Jimena Soleno

ESTERASE ACTIVITY
(mmol min1 mg protein1)

R2 = 0.639
0.25
0.20
0.15
0.10
0.05

15

B
GST ACTIVITY
(mmol min1 mg protein1)

0.30

30

45
60
% MORTALITY

75

90

30

45
60
% MORTALITY

75

90

R2 = 0.326

0.25
0.20
0.15
0.10
0.05

15

Figure 2. Correlation of esterases (A) and GST (B) with

azinphos-methyl mortality in diapausing larvae of Cydia pomonella.

be almost eightfold more tolerant at the LD95 level

than the susceptible larvae from the laboratory,
indicating that all of the field populations evaluated
exhibited some degree of tolerance. Insecticide
selection pressure substantially varies among orchards
from the valley. Farmers who obtain the main profit
from fruit exportation have introduced integrated
pest management approaches because of increasing
regulation demands. On the other hand, small farmers
still continue with the use of only broad-spectrum
insecticides including, in some cases, pyrethroids.
L and Fernandez D, unpubPrevious work (Cichon
lished) recorded no significant differences in mortality
of adult moths among three field populations collected during the season 20002001 (74.6594.78%)
and the laboratory susceptible population (80.50%)
exposed to a diagnostic dose of azinphos-methyl
(0.36 g moth1 ). However, the authors found significantly lower mortality (2.2030.71%) to a diagnostic
dose of esfenvalerate (0.045 g moth1 ) in the field
populations than that observed in the susceptible
laboratory population (78.55%). The most tolerant
population also showed cross-resistance to lambdacyhalothrin (diagnostic dose 0.073 g moth1 ). In
addition, the authors showed that esfenvalerate resistance was completely overcome in all three populations
by pretreatment with piperonyl butoxide, suggesting the role of mixed-function oxidases (MFO) in
968

pyrethroid detoxification. Azinphos-methyl susceptibility at that time was probably due to a prevailing
insecticide activation by MFO over detoxification
mechanisms.
Synergism of pyrethroids by organophosphorus
insecticides has been reported in resistant populations

of Helicoverpa armigera (Hubner)

which have shown
enhanced activities of esterases30 or mixed-function
oxidases.31 On the other hand, the joint action of
some pyrethroids and organophosphates produces
potentiation or antagonism in this species.32
In general, the field populations that showed
significantly lower mortalities to azinphos-methyl
compared with that in the field reference strain were
highly associated with higher esterase activities and to a
lesser extent with GST activities. Furthermore, larvae
from Allen2 and Centenario exhibited the highest GST
activities, but the former showed significantly lower
mortality to azinphos-methyl in relation to the field
reference population. The fact that the population
from Allen2 showed almost eightfold higher esterase
activity than that observed in the population from
Centenario is an additional support for the hypothesis
of the prevalent role of esterases in the observed
insecticide tolerance.
It has been demonstrated that, in addition to the
active catalytic hydrolysis of carboxyl acid esters
(which are rarely found within organophosphates),
carboxyl esterases are phosphorylated by many oxons,
thus protecting the AChE from inhibition.33 Field
populations of codling moth resistant to azinphosmethyl have been associated with esterases in
synergism studies with DEF (esterase inhibitor).
However, the authors observed no differences in
enzyme activity between the susceptible and the
resistant population using p-nitrophenyl acetate as the
substrate.9 Increased esterase activity as a mechanism
of resistance to methyl-azinphos has also been found
in other tortricid field populations.34,35 On the other
hand, azinphos-methyl resistance in field populations
of C. pomonella was attributed to GST,4 or to
both GST and MFO,3 associated with a decreased
esterase activity to novel substrates. Further, a possible
structural alteration of AChE has also been suggested
as responsible for resistance to this compound.9
Insect growth inhibitors and insect growth regulators are applied by some farmers as a part of an
integrated pest management programme. To protect the efficacy of these products, azinphos-methyl
must be used carefully. Experience in other countries has demonstrated that this compound shows
cross-resistance to diflubenzuron36 and tebufenozide
in another tortricid pest.37 41
In conclusion, codling moth populations from the
Ro Negro and Neuquen Valley showed different
degrees of tolerance associated with increased esterase
activities. To extend the effectiveness of azinphosmethyl and other organophosphates in the Ro Negro
and Neuquen Valley, a regional integrated pest
management programme must be implemented.
Pest Manag Sci 64:964970 (2008)
DOI: 10.1002/ps

Resistance to azinphos-methyl in Patagonian C. pomonella

ACKNOWLEDGEMENTS
The authors gratefully acknowledge Dr Benot
Sauphanor for valuable suggestions on a previous
version of the manuscript. This study was supported by
a grant from the Universidad Nacional del Comahue
Project I 940.
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