ISSN 1022-7954, Russian Journal of Genetics, 2008, Vol. 44, No. 5, pp. 553–558. © Pleiades Publishing, Inc., 2008.

Original Russian Text © G.V. Ben’kovskaya, M.B. Udalov, E.K. Khusnutdinova, 2008, published in Genetika, 2008, Vol. 44, No. 5, pp. 638–644.

GENERAL
GENETICS

The Genetic Base and Phenotypic Manifestations of Colorado

Potato Beetle Resistance to Organophosphorus Insecticides
G. V. Ben’kovskaya, M. B. Udalov, and E. K. Khusnutdinova
Institute of Biochemistry and Genetics, Ufa Research Center, Russian Academy of Sciences, Ufa, 450054 Russia
e-mail: bengal2@yandex.ru
Received March 23, 2007

Abstract—A high level of resistance to organophosphorus insecticides was recorded in a local population of
the Colorado potato beetle (Ufa district, Bashkortostan). Toxicological analysis demonstrated that the fraction
of susceptible individuals did not exceed 0.025. DNA analysis of the polymorphism of acetylcholinesterase
gene (AChE) according to the presence of mutation 980A>G in the sample of Colorado potato beetle imagoes
demonstrated the absence of wild-type (sensitive, SS) homozygotes and prevalence (0.79) of the homozygotes
with mutant resistant type (RR). The phenetic analysis of the variation of the integument pattern phenes dem-
onstrated statistically significant differences between the imago phenotypes in the ratio of SS and RR genotypes.
DOI: 10.1134/S1022795408050074

INTRODUCTION of Colorado potato beetle (Leptinotarsa decemlineata

Insecticide resistance of insect pests is among the
most important negative side effects of their use. Resis-
tance, determined as an inherited change in susceptibil-
ity to chemical compounds in pest populations [1], is a
natural consequence of the severe selective pressure
resulting from repeated treatment with high concentra-
tion of insecticides. Resistance assessment by the stan-
dard methods used in toxicology (determination of
resistance index RI) involves comparison of the dose
(concentration) lethal for 50% of individuals (in a sam-
ple from the studied population), LD50 (LC50), with the
same characteristic for the reference population taken
as a susceptibility standard [2].
Due to genetic heterogeneity of natural populations
of insect pests and ticks, both sensitive and resistant
individuals can be met among the 50% of survived indi-
viduals; this resistance can be determined by various
mechanisms. A five–sixfold excess over the LC50 value
is sufficient to indicate the risk of presence in the pop-
ulation of the individuals with genetically based resis-
tance. These can be carriers of the mutations causing a
decreased susceptibility of insecticide targets [3, 4];
they can include the individuals heterozygous at many
adaptive loci with a successful combination of such loci
that had coadapted during the existence of a population
(species) [5]. Detection of the presence of resistant
individuals by toxicological methods includes applica-
tion of diagnostic dose (concentration) of insecticide
(DD or DC) calculated as doubled LD95 or LC95 [2].
Numerous works have recently appeared that deter-
mined particular genetic bases of insect resistance to
pyrethroid, cyclodiene, organophosphorus, orga-
nochlorine, and carbamate insecticides [3, 6, 7]. The
attempts to detect association between the phenotypes
Say) and the genetic component of resistance [8–10]
caused by a high level of phenetic polymorphism
reflected in the variation of imago integument patterns
so far have resulted only in a statement of the fact of
changes in population phenetic structure caused by
insecticide stress [11]. In this work, we attempted not
only to detect one of the mutations known to cause the
resistance to organophosphorus insecticides (OPI) by
the example of Colorado potato beetle local population,
but also to clarify the correlation between the genetic
and phenetic markers of this resistance type.
MATERIALS AND METHODS
The overwintered imagoes of Colorado potato bee-
tle (N = 98) collected in the local population of Ufa dis-
trict, Bashkortostan, were used as the object of molec-
ular genetic studies. In addition, imagoes from several
other districts of Bashkortostan were used in toxicolog-
ical and phenetic analyses.
The pattern phenes (Fig. 1) of the vertex (five varia-
tions [9, 10]), occiput (three variations [10]), pronotum
(nine variations [8]) and elytra (five variations [9, 10])
were used for analyzing phenetic polymorphism.
The following preparations were used for toxicolog-
ical assessment of the susceptibility of Colorado potato
beetle imagoes: Decis (active substance is delta-
methrine, a pyrethroid), Karbofos (active substance,
malathion, an OPI), Volaton (active substance, phoxim,
an OPI), Actara (active substance, thiametoxam, a neo-
nicotinoid), and Mospilan (active substance, acetami-
prid, a neonicotinoid). The experiments were con-
ducted with the samples of overwintered imagoes from
natural local populations. Insecticides were applied

553
554 BEN’KOVSKAYA et al.

(a) (b)

o á å + N –

m ¯

(c)

(d)
1 2 3

W W/V V
4 5 6

Z Y
7 8 9
Fig. 1. Phenes of the patterns of the regions of Colorado potato beetle: variations of (a) vertex, (b) occiput, (c) pronotum, and (d)
elytra phenes (see [10] for details).

topically [12] as working solutions at a dose of rax. The working solutions of insecticides were pre-
1 µl/individual. The solutions were applied with an pared using 96% ethanol. The control insects were
MSh-1 microsyringe onto the ventral part of the protho- treated with ethanol. After treatment, the insects were
placed into petri dishes in ten replicates taking ten indi-
viduals per replicate [2]. The treated beetles were fed
(‡) clean potato leaves. The experiments were conducted at
a temperature of 25°C. The mortality was recorded
T yr M et Ser Ser Glu A rg A la after 3 days. The total mortality rate in all experimental
5' T A C ATG T CC A G T G A A CG C G C A 3' variants was calculated with the correction for control
using the Abbot equation [13]. The LC50 and LC95 val-
5' T A C ATG T CC GG T G A A CG C G C A 3'
ues were calculated by probit analysis in the Miller–
T yr M et Ser Gly Gl u A rg A la Tainter modification [14].
DNA was isolated from the legs of Colorado potato
(b) beetle [15] by guanidine thiocyanate–phenol–chloro-
293 form extraction [16]. DNA was amplified by PCR in a
Tertsik (Moscow, Russia) thermocycler. The amplifica-
3
......................................... ............................. tion mode and primer sequences were used as described
SS ......................................... AT ............................. in [17]. The primers were synthesized by Sintol (Mos-
206 2 4 cow, Russia).
126 The point missense mutations were detected in the
genome of Colorado potato beetle by bidirectional PCR
3 1
......................................... G ............................. amplification of specific alleles (bi-PASA) [18]. This
RR ......................................... C ............................. method makes it possible to identify both the sensitive
4 and resistant homozygotes SS and RR (Fig. 2b) and the
293 heterozygotes SR [17]. After PCR and electrophoresis,
the susceptible homozygotes (wild-type alleles, SS)
Fig. 2. Identification of mutant alleles of AChE gene. were detectable according to the presence of 206-bp
(a) Comparison of nucleotide and amino acid sequences: DNA fragment; resistant homozygotes RR, to the pres-
upper sequence, wild type and lower, mutant type; vertical ence of 126-bp fragment; and heterozygotes, to the
frame shows the mutant triplet [25]. (b) Scheme of bi-PASA presence of both fragments.
for detection of the 980A>G mutation of AChE gene
(according to [18]): dashed lines, native DNA strands; solid The parameters of survival λ and relative fitness ω
lines, amplification products (the length is shown in bp);
arrows 1 and 2, allele-specific primers and arrows 3 and 4, were calculated as described in [19]. Statistical signifi-
allele-nonspecific primers with the directions of further cance was estimated using Student’s test; correlation
elongation. between qualitative characters, using Pearson’s χ2 test

RUSSIAN JOURNAL OF GENETICS Vol. 44 No. 5 2008

 THE GENETIC BASE AND PHENOTYPIC MANIFESTATIONS 555

Table 1. Insecticide susceptibility of Colorado potato beetle imagoes in local populations of Bashkortostan (data of 2005)

Mortality rate (with correction for control, %) after treatment with DC of preparations
District
Decis Karbofos Actara Mospilan

Ufa 67.0 ± 6.9 2.5 ± 0.1 95.0 ± 1.2 79.0 ± 7.5

Burzyan 94.7 ± 3.4 42.1 ± 1.1 95.0 ± 2.6 84.2 ± 8.1

Sterlibashevo 89.5 ± 5.3 26.3 ± 1.6 No data No data

Karmaskaly 10.0 ± 0.5 0.0 ± 0.0 100 40.0 ± 2.0

Ilishevsky 50.0 ± 4.9 0.0 90.0 ± 5.5 33.3 ± 4.6

Aurgazy 50.0 ± 1.9 22.2 ± 0.8 94.4 ± 3.5 88.9 ± 4.1

Arkhangel'sk 70.0 ± 5.2 45.0 ± 3.8 No data No data

Kirgiz-Miyaki 31.6 ± 1.8 15.8 ± 0.7 100 73.7 ± 5.7

[20]. All computations were done using Microsoft
Excel 2002, (1985–2001, Microsoft).
RESULTS AND DISCUSSION
All the studies on phenotypic polymorphism of
Colorado potato beetle paid special attention to high
frequency of variations 3, 6, and 9 of the pronotum pat-
tern phene [8, 10, 21] in resistant populations. On the
other hand, we have demonstrated that the individuals
with variations 3, 6, and 9 belong to the phenotypes dif-
ferent with respect to general melanization of integu-
ments. Variation 9 occurs in the individuals belonging
to achromatic type (phenotype A); variation 6, in the indi-
viduals with intermediate melanization (phenotype I), and
variation 3, in melanized individuals, or melanists (phe-
notype M) [10]. Taking this into account, we compared
the results of toxicological, molecular biological, and
phenetic analyses.
We assessed the susceptibility of Colorado potato
beetle to OPI using Karbofos (malathion). This prepa-
ration, which is not in the official list of the insecticides
recommended for controlling Colorado potato beetle
on potato, was nonetheless actively used in individual
farms in the 1980s, as it was among the most available
insecticides. The toxicological assessment of local pop-
ulations to the previously determined Karbofos diag-
nostic concentration (DC = 1.0%) was conducted in
2003 through 2005. Comparison of the results of anal-
ogous experiments conducted in the early 1990s
allowed us to evaluate the trends and rates of the pro-
cesses occurring in the population.
The imago mortality rate caused by DC with the
correction for control shows the percentage of individ-
uals in the population still susceptible to the insecticide.
Only resistant individuals are able to survive. This, DC
is a sort of genetic characteristic of the local population
analyzed, which allows the genotypes of susceptible
and resistant individuals of Colorado potato beetle to be
detected.
The results of toxicological experiments are listed in
Table 1. In the eight examined regions, application of
Karbofos at DC resulted in 50% mortality rate, which
corresponds to the stage of multiple resistance develop-
ment [22]. No Karbofos-caused mortality was recorded
in certain local populations. The mortality rate for Ufa
district in 2005 amounted to 2.5%, i.e., the number of
sensitive individuals in this population did not exceed
25 per 1000.
It is known that one of the main mechanisms render-
ing insects resistant to OPI is a decrease in the suscep-
tibility of mutant acetylcholinesterase (AChE) to insec-
ticides [3]. On molecular level, this results from nucle-
otide mutations detected in the genotypes of resistant
individuals, and, consequently, amino acid mutations of
AChE. The resistance of Colorado potato beetle AChE
to OPI is connected with the transition 980A>G, which
results in the substitution of serine with glycine at posi-
tion 291 (Fig. 2a) [23, 24].
To determine the distribution of the frequencies of
AChE gene alleles, we conducted DNA typing of the
samples of Colorado potato beetle from the local popu-
lation of Ufa district (Bashkortostan). This population
was chosen as a model, as it displayed a high level of
multiple resistance not only to Karbofos, but also to

RUSSIAN JOURNAL OF GENETICS Vol. 44 No. 5 2008

556 BEN’KOVSKAYA et al.
SR RR RR SR RR RR RR
293 bp
206 bp
126 bp
Fig. 3. Electrophoretic patterns of AChE gene fragment of
Colorado potato beetle obtained by bi-PASA: RR, resistant
homozygotes (126-bp fragment), SR, heterozygotes (206-
and 126-bp fragments), and 293-bp fragment, the product of
nonspecific PCR with two external primers.
Decis (a synthetic pyrethroid insecticide) and neonico-
tinoid insecticides [25]. Analysis of the genotyping data
for the imagoes on the presence of the mutation in
AChE gene has demonstrated that the SS individuals
(wild-type genotype determining the native level of
susceptibility to OPI) were undetectable in the model
local population (Fig. 3). The fraction of individuals
carrying the homozygous genotype RR, corresponding
to the presence of mutation, constituted 0.79 in this
population; the heterozygotes SR, 0.21.
Evaluation of association between the phenetic
polymorphism of imagoes in the melanization degree
of their integument patterns [10] and the genetic poly-
morphism according to AChE mutation has demon-
strated an uneven distribution of the mutant allele fre-
quencies in the phenotypes in question. In the type A
group (achromatic type), amounting in this sample to
Table 2. Differentiation of survival of Colorado potato beetle imago phenotypes under the action of Volaton (data of 1993)
Mortality rate
Variant
(with correction for control, %)
Control
Sublethal concentrations (0.0025–0.02%)
LC50 0.025–0.032%
Selective concentrations (0.04–0.08%)
Lethal concentrations (0.1–0.4%)
RUSSIAN JOURNAL OF GENETICS
0.15, the fraction of heterozygotes SR is 0.43 and
homozygotes RR, 0.57. The melanized group displays
the ratio drastically shifted towards the homozygotes
RR, their fraction constitutes 0.85 versus 0.15 of SR. As
for the individuals with the intermediate phenotype,
these fractions are 0.82 and 0.18, respectively. Testing
the null hypothesis using the Student test for k > 60 and
5% significance level has confirmed a nonrandom char-
acter of this shift in the distribution of SR- and RR gen-
otypes in phenotypes I and M.
Calculations of the proportions of the mutant and
normal alleles in the sample analyzed according to
Hardy–Weinberg have demonstrated that only 11 indi-
viduals per 1000 will display genotype SS at the estab-
lished distribution; thus, we could fail to detect the only
SS individual with the sample size of N = 98. In our
view, such a low concentration of wild-type allele in the
sample studied is explained by the fact that the geno-
typing was conducted at the imaginal stage, whereas
the susceptible individuals were eliminated from the
population under the action of insecticides at earlier
developmental stages. It should be taken into account
that despite a high development synchronization of the
Colorado potato beetle first summer generation, part of the
larvae that outpaced the rest in hatching by 1.5–2 weeks
could already complete their larval stage and go into
soil for pupation by the time of insecticide treatment of
potato plants (the second- and third-instar larvae are
usually present on the plants during the treatment). Pre-
sumably, this possibility to wait through the contact
with insecticide underlies the preservation of suscepti-
ble individuals in the population and maintenance of a
certain heterozygosity level.
In the toxicological experiment with the sample of
first generation imagoes (totally, 1039 individuals),
topical treatment with Volaton at concentrations of
0.0025 to 0.4% with respect to the active substance (the
Survival of phenotypes λ
A I M
8.3 ± 0.78 1.00 0.56 0.77
10.9–18.2 0.84 0.88 0.37
32.7–54.5 0.6 0.58 0.47
59.9–67.3 0.43 0.38 0.25
86.4–99.7 0.18 0.03 0.03
Vol. 44 No. 5 2008
 THE GENETIC BASE AND PHENOTYPIC MANIFESTATIONS 557

range of sublethal to diagnostic concentrations) Table 3. Changes in the adaptation of Colorado potato beetle
allowed us to assess the adaptation of each phenotype, imagoes to the action of high doses of organophosphorus insec-
characterized by the survival rate under the action of ticides (Volaton, Actellic, and Karbofos) during 1993–2006
insecticide and without it (in the control variant). The
overall sample was phenetically analyzed to calculate λ, proportions ω (phenotype A)
survival index λ [18] for each phenotype. Phenotype
1993 2006 1993 2006
The results of this experiment (Table 2) demonstrate
that the susceptibility to insecticides of the M type indi- A 0.18 0.83 1 1
viduals is higher than that of the A type. We assume that
the pressure of insecticide treatments as a selection fac- I 0.03 0.81 0.17 0.97
tor leads to gradual substitution of the individuals with
RR genotype for the remaining genotypes within the M 0.03 0.73 0.17 0.88
phenotypes M and I in this local population. Presum-
ably, this phenomenon can be widespread, as confirmed
by 24–27-fold increase in λ values (Table 3) for the the epicuticle. Consequently, spaces of achromatic cuti-
phenotypes M and I over the last 13 years. cle can appear in the integument pigment patterns as
The high number of the heterozygotes with muta- well as some spots and parts of bands can disappear.
tion of AChE gene among the achromatic type results
from the presence of a component of the constitutive It is necessary to test the heritability of these traits
resistance in these particular individuals. Presumably, and to clarify their biochemical basis and ontogenetic
this component is connected with an increased density determination to verify the proposed hypothesis that the
of integuments (endocuticle) and gut epithelium. Possi- density of insect integuments and melanization inten-
bly, the constitutive resistance maintains the existence sity are independently inherited polygenic traits.
of SR phenotypes in population, appearing in the
repeatedly recorded association between pronotum pat-
tern phene variations 3, 6, and 9 [9, 10, 26] and a notice- ACKNOWLEDGMENTS
able level of the resistance to insecticides. A complex
pattern of resistance inheritance, which is confirmed by The work was supported by the Russian Foundation for
the recorded sexual dimorphism in distribution of the
frequencies of pronotum pattern variants in the carriers Basic Research (grant no. 05-04-97916-r_agidel'_a).
of SR and RR genotypes (Fig. 4), demonstrates that sev-
eral, possibly, independent gene complexes contribute 0.3
to this trait. Calculations of the Chuprov–Pearson con-
tingency coefficient K demonstrate a weak yet statisti- SR
cally significant (K = 0.258 and χ2 = 12.06 at α = 0.5%) 0.2
correlation between the AChE genotypes and the phe-
notypes marked with pronotum pattern variations 3, 6,
and 9 (we designated this trait the integument density). 0.1
A similar level of correlation (K = 0.211) between the
AChE genotypes and the phenotypes corresponding to
the melanization intensity were found as well as 0
between the phenotypes separated according to the 0.3
integument density and melanization intensity
RR
(K = 0.238).
The integument density of Colorado potato beetle is 0.2
determined by several components; of them, the protein
matrix structure and cuticle sclerotization intensity are
the most significant. The procuticle is the layer formed 0.1
by chitin bound to sclerotized protein matrix [27]. This
is a complex process, where production of quinones
from tyrosine derivatives, first and foremost, DOPA, 0
1 2 3 4 5 6 7 8 9
dopamine, and several other catecholamines, plays a
significant role. Here, the degree of integument mela-
nization can indicate the intensity of deposition of epi- Fig. 4. Distribution of the frequencies of pronotum pattern
cuticle polyphenolic layer. An increase in the density of phene among heterozygous (SR) and homozygous (RR)
Colorado potato beetle individuals according to the pres-
endocuticle protein matrix or its structural modifica- ence of AChE gene mutation: light columns, males; gray
tions leading to thickening of integuments can appear columns, females; the abscissa, numbers of phene varia-
phenotypically as a less intensive melanin deposition in tions; and the ordinate, the frequencies of variations.

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558 BEN’KOVSKAYA et al.
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