Vol.3, No.4B, 8-19 (2013) Open Journal of Animal Sciences
http://dx.doi.org/10.4236/ojas.2013.34A2002
Effect of sublethal doses of the insecticide
imidacloprid on adaptive traits of Drosophila
melanogaster: Response to treatment over and after
15 consecutive generations
Thais de França Patarro1, Antonio José Manzato2, Lílian Madi-Ravazzi1,
Hermione E. M. de Campos Bicudo1*
1Departmento de Biologia, Universidade Estadual Paulista-UNESP, Instituto de Biociências, Letras e Ciências Exatas-IBILCE, São
José do Rio Preto, Brazil; *Corresponding Author:
2Departmento de Computação, Universidade Estadual Paulista-UNESP, Instituto de Biociências, Letras e Ciências Exatas-IBILCE,
São José do Rio Preto, Brazil
Received 3 September 2013; revised 8 October 2013; accepted 22 October 2013
Copyright © 2013 Thais de França Patarro et al. This is an open access article distributed under the Creative Commons Attribution
License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
ABSTRACT
A sublethal dose of Imidacloprid, considered
actually as the most widely used insecticide
against biting and sucking insects, was admin-
istered to Dro sophila mel anoga ster fo r dete cting
effects on biological traits. The choice of this
species as organism-model potentially opens
the possibility to explore more deeply the proc-
esses involved in those effects because, among
other reasons, there is a large accumulation of
biological knowledge on this species and be-
cause it propitiates multiple approaches in
laboratory and nature. The flies were treated
along 15 consecutive generations. F1 parents
were randomly taken among virgin flies from the
stocks, but the parents of the successive gen-
erations were the firs t 15 couples e merged in th e
previous one. The number of progeny (produc-
tivity) and the duration of the emergence period
were analyzed in every generation revealing in-
secticide toxicity in 12 of the 15 generations.
The observation of an increase in the number of
progeny over the generations, which occurred in
both control and treated experiments (although
maintaining higher productivity in the control),
suggested an effect of the use of the first 15
emerged couples in successive generations. A
comparative analysis of the mortality of the F15
adult flies exposed to imidacloprid by contact,
which involved flies from the control, treatment
and from the stocks that originated the experi-
ments, reinforced this idea, indicating a genetic
interplay of the emergence speed with produc-
tivity and adult tolerance to the insecticide, a
subject that may be better explored in another
study. Toxicity was also observed for the traits
longevity, viability during development from egg
to adult and oviposition rate. Considering the
present intensive use of imidacloprid, the harm-
ful effects observed in these important biologi-
cal characteristics may be considered able to
decrease the adaptive value of D. melanogaster
populations exposing them at risk of decline.
Keywords: Productivity; Longevity; Emergence
Time Period; Egg-Adult Viability; Oviposition;
Tolerance
1. INTRODUCTION
Imidacloprid {1-[(6-chloro-3-pyridinyl)methyl]-N-nitro-
2-imidazolidinimine, which is registered in more than a
hundred countries in the world, is considered actually the
most widely used insecticide for killing sucking and bit-
ing insects [1]. Currently, it is intensively used as seed
treatment in citrus, cotton, fruits, grapes, potatoes, rice
soybeans, sugarcane, tobacco and vegetables [2].
Several studies have provided strong evidence that, in
addition to direct mortality, imidacloprid impacts popu-
lations through sublethal effects. The exposure to sub-
lethal doses of imidacloprid causes deleterious effects on
biological traits of target and non-target organisms. De-
crease of the progeny number is one of the most fre-
quent effects in different organisms, but the decreases of

T. de França Patarro et al. / Open Journal of Animal Sc iences 3 (2013) 8-19 9
survival rate and longevity have also been observed in
some cases [3,4]. In honeybees (Apis mellifera), pro-
bably the more intensively studied non-target organism,
imidacloprid is considered one of the causes (or the main
cause) of bee populations decline occurring since 1990
[5-8]. In addition, this insecticide has been assigned to a
bee malady termed Colony Collapse Disorder (CCD) [9].
Imidacloprid is a neonicotinoid that, similarly to nico-
tine, acts as an agonist at the postsynaptic acetylcholine
receptor (nAChR) [10]. Like other neonicotinoids, imi-
dacloprid causes persistent activation of the receptors
leading to hyperexcitation and death [11]. It is effective
on contact and via stomach action [12,8]. The wide use
of the neonicotinoids is due to that they were considered
to kill insects by paralyzing nerves but to have low toxic-
ity for other animals. However, data in literature have
shown that they affect organisms other than insects. For
example, significant adverse effects of this insecticide
have been reported in aquatic invertebrates (freshwater
and estuarine/marine), being ascribed, at least partially,
to its high solubility in water and moderate persistence
[13]. In mammals, high doses as well low doses and long
exposures were associated with degenerative changes in
several organs and other health problems [14].
We used Drosophila melanogaster as organism-model
to study how sublethal doses of imidacloprid affect some
biological traits. The importance of using Drosophila is
that it metabolizes toxic compounds in a way very simi-
lar to humans and its biological characteristics favor
many possibilities for methodological approaches, in
laboratory and nature. Specifically D. melanogaster, with
a great amount of biological information accumulated in
more than a hundred years of studies, may favor the un-
derstanding of new observations foreseeing the possibil-
ity of a deeper study about the findings. This species has
been successfully used as an organism-model for analy-
sis of normal and pathological mechanisms involved
with essential human biological processes, including
metabolism, development and physiology [15,16].
The treatment in 15 successive generations (nine
months of duration) was used to study the impact of the
insecticide on a set of biological traits that are very im-
portant to preserve the continuity of the species. The re-
sults in D. melanogaster confirmed the harmful effects of
imidacloprid and raised interesting questions to be an-
swered in future works.
2. MATERIALS AND METHODS
2.1. Strain Origin and Culture in the
Laboratory
The strain used was Drosophila melanogaster from
São José do Rio Preto, State of São Paulo, Brazil, col-
lected approximately two years before the beginning of
the study, and kept in the Department of Biology of the
IBILCE-UNESP. The original mass crosses for preparing
the stocks involved more than 20 couples collected in
nature. Stocks and experiments were maintained in ba-
nana-agar culture medium, at 20˚C ± 1˚C.
2.2. Experiments
Imidacloprid (Confidor 700WG, Bayer, 70% imida-
cloprid) was administered orally to the flies, being added
to the culture medium at 5 μg/mL concentration (value
corrected for the percentage contained in the product).
This is a sublethal dose, since data unpublished, obtained
by L. M. Ravazzi, one of the authors of this paper, for
adult flies from the same D. melanogaster strain, showed
LC50 value = 49.27 μg/ml water.
The present study involved 15 successive generations.
In each of them three replicates were prepared for control
and three for treatment. Each generation started with the
15 virgin couples first emerged in the previous genera-
tion, except the first generation that started with 15 virgin
males and females taken randomly from the stocks. Most
flies used in F2 to F15 were from the first and second
days of emergence, but in some cases, flies from the third
day were used to complete the 15 couples.
2.3. Adaptive T rait s Analy zed
Flies treated with imidacloprid and controls were com-
paratively analyzed as to their effects on the following
traits: 1) productivity (number of progeny); 2) duration
of the emergence period (the time elapsed between the
emergence of the first and last fly); 3) longevity; 4) ovi-
position rate; 5) viability from egg to adult stages and 6)
mortality of adult flies.
Productivity and duration of the emergence period
were evaluated in each of the fifteen generations while
the other traits were analyzed in the generation F15, ex-
cept longevity that was analyzed in F12.
2.3.1. Productivity and Duration of the
Emergence Period
The number of progeny (productivity) was computed
daily in the control (C) and treated (T) replicates, sepa-
rately by sex, from the beginning to the end of the adults’
emergence period, in every generation till the 15th.
2.3.2. Longevity
Twenty five virgin, recently emerged males and fe-
males taken from F12 generation in T and C groups were
transferred to tubes containing culture medium with and
without the insecticide, respectively. F12 was chosen for
this analysis in order to decrease the amount of simulta-
neous work that would be done in F15. Dead flies were

T. de F r ança Patarro et al. / Open Journal of Animal Sciences 3 (2013) 8-19
10
computed daily, separately by sex and treatment, till the
last one had died. Half-life (the time required for mortal-
ity of half of the total number of flies) and mean longev-
ity of the flies were computed.
2.3.3. Oviposition Rate
Ten virgin, three days aged males and females from
each experimental groups C and T, and also from the
stock that originated the experiments (S), were separately
put to cross in tubes containing culture medium (without
insecticide for C and S, and with it for T flies). The cou-
ples were left in the tubes for 8 hours and then the fe-
males were individually transferred to empty, clean tubes
containing a transparent plastic tea spoon full of agar-
sugar culture medium (prepared with 0.5 g agar-agar dis-
solved in 100 mL of hot water and addition of 2.5 g
sugar). Twenty-two hours latter, the spoons were re-
moved for counting the eggs (in a stereomicroscope) and
a second set of spoons was included in the same vials.
The eggs in this second set of spoons were counted again
after 22 h, totalizing 44 h observation.
2.3.4. Viability Egg-Adult
The eggs obtained in the study of the oviposition rate
were used in the analysis of viability egg-adult. After
being computed for oviposition rate, the eggs from T, C
and S flies were put for development in the respective
culture medium and the percentage of adults obtained in
relation to the initial number of eggs gave the viability
egg-adult. Males and females were computed separately.
2.3.5. Mort ality of Adult Flies Exposed to
Imidacloprid by Contact
This experiment was done in order to detect possible
changes in the degree of tolerance of the adults after 15
generations of treatment. Adult flies from C and T groups
taken in F15, and S flies were put in contact with the
insecticide at 10 mg/mL water concentration) imbibed in
pieces of filter paper introduced in the vials. This analy-
sis intended also to detect the existence of interference of
the method used (selection of the first 15 couples), in the
results. Ninety couples, three days old (virgin males and
females), from each experimental group, were put in
contact with the imidacloprid. Strips of filter paper were
impregnated with the aqueous solution of the insecticide,
put to air dry and placed into the tubes (9.0 cm × 6.5 cm),
covering their interior. Females and males were placed
separately in these tubes. The count of dead flies was
done at 24 and 48 hours after exposing the flies to the
impregnated paper. For making sure that the flies didn’t
die due to desiccation or hunger, a piece of cotton im-
bibed in aqueous solution of glucose (0.8%) was fixed in
the upper part of the tube.
2.4. Statistical Analysis
Statistical analysis of the data involved exploratory
analysis, Student’s t-test, non-parametric test of Kruskal-
Wallis, χ2 for comparison of several proportions, Tukey
test for multiple comparisons of proportions two by two
(involving the Tukey’s angular transformation). and the
Z test with normal approximation. The statistical meth-
ods used were based on [17] and [18]. The software was
the Minitab Release Package 14.
3. RESULTS
3.1. Characteristics Analyzed in the 15
Generations
3.1.1. Productivity
The descriptive statistics (Table 1) shows, for each
generation, the data on the mean number of progeny and
standard error, separately for males and females, in C and
T experiments. The total number of progeny produced in
both experiments was 16,620 flies.
The smallest productivity was obtained in the first
generation. The mean values for C and T replicates of
this generation were 98.3 and 84.7, respectively, while
the mean for F2 to F15 was 219.21 for C and 163.46 for
T. The mean of the means of the progeny in the 15 gen-
erations was: for males, C = 104.23 and T = 79.93; for
females, C = 106.44 and T= 78.23; and for males plus
females, C= 211.15 and T = 158.21.
Student’s t-test for comparison between C and T ex-
periments relative to the mean productivity of males,
females and their sum, in each generation (Table 2),
showed that the differences between C and T were sig-
nificant for males in the generations F4 (t = 3.37, P =
0.003), F9 (t = 3.05, P = 0.039 and F12 T = 17.17, P =
0.00+ [P value close to zero]). For females, the significant
differences were detected in F6 (t = 3.14, P = 0.003) and
F12 (t = 5.23, P = 0.001), and for the mean total progeny
(males plus females), in F1 (t = 3.04, P = 0.039, F4 (t =
2.75, P = 0.048), F6 (t = 2.80, P = 0.049), F9 (t = 2.82, P
= 0.047) and F12 (t = 9.31, P = 0.000). In every case of
significant difference, values in C were greater than in T.
However, in the light of the numbers, male progeny was
greater in C than in T in 12 of the 15 generations, female
progeny, in 10, and males plus females in 12.
Boxplot of data (Figure 1) showed the wide variation
of the progeny number among replicates, mainly for C
group. To better visualize the difference between C and
T over generations the results were submitted to the
statistical method of smoothing 4253H (Figure 2). With
this method, significant differences on progeny number
were found for males in F3 to F12, for females in F5
and F9, and for males plus females, in F6 to F11. Ex-
cept in F11, the productivity was lower in T experi-

T. de F r ança Patarro et al. / Open Journal of Animal Sciences 3 (2013) 8-19
11
Tabl e 1. Mean and standard error of data on productivity (number of progeny) separately for males, females and males plus females
(Total) in the control (C) and imidacloprid treated (T) experiments of the 15 generations.
Males Females Total
Generations
Mean ± s.e. Mean ± s.e. Mean ± s.e.
C 48.0  2.6 43.7  4.7 98.3  0.3
F1
T 41.7  4.2 43.0  3.1 84.7  4.5
C 125.3  19.9 115.3  15.7 240.7  35.4
F2
T 84.0  10.1 78.3  3.2 162.3  12.3
C 65.3  2.0 58.3  11.6 123.7  13.2
F3
T 62.3  8.8 64.3  6.4 126.7  14.2
C 92.3
 8.6 101.3  14.0 194.0  22.9
F4
T 62.0
 2.5 69.0  1.7 131.0  1.2
C 89.0  7.0 86.7  6.4 175.7  13.2
F5
T 76.0  1.5 78.0  14.4 154.0  15.8
C 136.3  23.2 136.7  20.2 273.0  42.8
F6
T 80.0  7.2 67.3  9.0 147.3  13.2
C 75.0  2.3 84.0  8.1 159.0  8.1
F7
T 67.3  7.3 64.3  4.9 131.7  11.9
C 122.0  13.8 110.7  11.9 232.7  25.7
F8
T 89.0  4.7 95.7  10.5 184.7  15.0
C 124.0  11.4 126.3  4.1 250.3  13.2
F9
T 85.0  5.9 86.7  19.7 171.7  24.6
C 102.3  17.6 102.0  17.1 204.3  33.3
F10
T 59.3  12.4 53.3  7.8 112.7  19.3
C 66.0
 2.1 60.3  2.8 126.3  1.2
F11
T 86.7  11.9 87.3  13.0 174.0  22.3
C 195.0
 1.5 194.0  13.5 389.0  12.6
F12
T 110.7  4.7 109.0  9.0 219.7  13.1
C 154.7  8.5 169.3  18.2 324.0  26.6
F13
T 119.3
 16.3 119.3  35.4 238.7  50.4
C 59.3  10.4 60.7  17.4 120.0  26.2
F14
T 78.7
 7.4 71.3  15.2 150.3  21.1
C 109.0  16.5 147.3  28.9 256.3  44.5
F15
T 97.0  7.5 86.7  3.3 183.7  10.0
C 104.23 106.44 211.15
Mean
T 79.93 78.23 158.21

T. de F r ança Patarro et al. / Open Journal of Animal Sciences 3 (2013) 8-19
12
Ta ble 2. Differences of means on productivity (number of progeny) between control (C) and imidacloprid treated (T) experiments,
separately for males, females and males plus females (Total), using normal data.
Difference of Means
Males Females Total
Generation C-T t P C-T t P C-T t P
F1 6.3 1.28 0.250 0.7 0.12 0.925 13.7 3.04 0.039
F2 41.3 1.85 0.168 37.0 2.31 0.055 78.3 2.09 0.100
F3 3.0 0.33 0.678 −6.0 −0.45 0.756 −3.0 −0.15 0.852
F4 30.3 3.37 0.003 32.3 2.30 0.057 63.0 2.75 0.048
F5 13.0 1.81 0.182 8.7 0.55 0.659 21.7 1.05 0.389
F6 56.3 2.32 0.058 69.3 3.14 0.003 125.7 2.80 0.049
F7 7.7 1.00 0.392 19.7 2.08 0.102 27.3 1.90 0.112
F8 33.0 2.26 0.058 15.0 0.94 0.400 48.0 1.61 0.123
F9 39.0 3.05 0.039 39.7 1.97 0.115 78.7 2.82 0.047
F10 43.0 2.00 0.096 48.7 2.59 0.056 91.7 2.38 0.057
F11 −20.7 −1.72 0.150 −27.0 −2.03 0.123 −47.7 −2.13 0.100
F12 84.3 17.17 0.00+ 85.0 5.23 0.001 169.3 9.31 0.00+
F13 35.3 1.93 0.183 50.0 1.26 0.278 85.3 1.50 0.221
F14 −19.3 −1.52 0.200 −10.7 −0.46 0.723 −30.3 −0.90 0.398
F15 12.0 0.66 0.552 60.7 2.08 0.100 72.7 1.59 0.223
Figure 1. Boxplot for productivity (total number of progeny) of
the replicates in the experiments control (C1, C2 and C3) and
imidacloprid treated (T1, T2 and T3).
ments. The smoothing data also allowed visualizing more
clearly the increase in the number of offspring that oc-
curred in C and T, over generations (Table 3).
3.1.2. Duration of the Emergence Period
The time, in days, elapsed between the emergence of
the first fly and the last one was also evaluated in each
replicate of the fifteen generations of C and T experi-
ments (Figure 3). Seen in the light of the numbers, nine
of the fifteen generations showed mean emergence dura-
tion of C replicates greater than that from T. Student’s
t-test showed significant difference between them only in
F6 (t = 6.01; P = 0.027) and F8 (t = 3.77; P = 0.033).
(a)
(b)
Figure 2. Graphs for mean productivity in the groups
control and imidacloprid treated, in the generations F1 to
F15. (a): Normal data; (b): Smoothed data.

T. de F r ança Patarro et al. / Open Journal of Animal Sciences 3 (2013) 8-19 13
Ta ble 3. Differences of means on productivity (number of progeny) between control (C) and imidacloprid treated (T) experiments,
separately for males, females and males plus females (Total), using smoothed data.
Males Females Total
Generation C-T t P C-T t P C-T t P
F1 7.4 1.2 0.18 −3.6 −1.07 0.191 11.5 1.12 0.191
F2 13.8 2.3 0.05 10.9 1.55 0.13 26.2 1.84 0.07
F3 17.8 2.5 0.04 20.1 2.00 0.055 36.6 2.04 0.085
F4 19.5 2.4 0.02 24.0 2.26 0.052 42.2 2.16 0.058
F5 21.6 2.4 0.02 25.9 2.49 0.03 45.7 2.26 0.056
F6 26.2 2.6 0.03 28.0 2.73 0.028 51.9 2.53 0.041
F7 30.3 2.8 0.03 29.0 3.2 0.024 57.4 2.92 0.027
F8 30.7 2.9 0.03 28.9 3.54 0.012 58 3.07 0.025
F9 28.6 2.7 0.03 28.2 3.00 0.026 56.2 2.96 0.031
F10 26.6 2.7 0.03 29.6 2.35 0.051 57.4 2.74 0.032
F11 25.7 2.7 0.03 34.1 2.12 0.061 61.3 2.44 0.034
F12 24.6 2.6 0.03 38.2 2.18 0.06 63.5 2.31 0.057
F13 21.1 2.2 0.06 41.0 2.36 0.062 62.4 2.16 0.059
F14 15.6 1.3 0.11 42.6 2.46 0.046 58.9 1.79 0.085
F15 12.6 0.7 0.18 43.0 2.28 0.055 60.1 1.52 0.089
Figure 3. Means of emergence time duration of progeny (in
days) from the control and imidacloprid treated experiments, in
each of the 15 generations.
However, among the other seven non-significant genera-
tions with mean duration of emergence period greater in
C, the difference from T varied from two to six days. The
mean of the replicate means for all generations was
13.66 days for C and 11.54 days for T (Tables 4 and 5).
Due to the similarity of profiles in the graphs of num-
ber of progeny and emergence time duration, the coeffi-
cient of correlation was calculated for each replicate us-
ing Pearson’s coefficient of linear correlation and sig-
nificance test for P = 0.00+. Except in the replicate num-
ber 3 of T, the two characteristics showed high correla-
tion: control P = 0.95; treated P = 0.85 (Figure 4).
Ta bl e 4 . Duration (in days) of the emergence period computed
from the first to the last fly emerged in the replicates from ex-
periments control (C1, C2, C3) and imidacloprid treated (T1,
T2, T3), in the 15 generations.
Emergence Time (days)
GenerationC1C2C3 Mean T1 T2 T3Mean
F1 7787.33 5 7 8 6.67
F2 16 91613.67 16 15 1615.67
F3 8798.00 6 11 119.33
F4 1081210.00 8 8 9 8.33
F5 1181411.00 8 13 7 9.33
F6 16131615.00 9 9 9 9.00
F7 1411911.33 12 9 9 10.00
F8 17131715.67 11 10 8 9.67
F9 17131214.00 15 10 9 11.33
F10 19101514.67 10 9 8 9.00
F11 1112 810.33 10 12 1311.67
F12 282524 25.67 21 19 1217.33
F13 261923 22.67 31 22 1522.67
F14 6 11119.33 13 10 1011.00
F15 25101215.67 13 14 8 11.67

T. de F r ança Patarro et al. / Open Journal of Animal Sciences 3 (2013) 8-19
14
Table 5. Student’s t-test for comparison on duration of emer-
gence time of progeny from control and imidacloprid treated
experiments, in the 15 generations.
Generation t P
F1 0.71 0.549
F2 0.85 0.485
F3 0.76 0.529
F4 1.39 0.299
F5 0.66 0.557
F6 6.01 0.027*
F7 0.76 0.505
F8 3.77 0.033*
F9 1.11 0.347
F10 2.13 0.167
F11 0.89 0.437
F12 2.80 0.108
F13 0.00 0.999
F14 0.86 0.453
F15 0.79 0.511
*= significant values.
(a)
(b)
Figure 4. Dispersion graphs between total productivity
and mean emergence time for control (a) and imidaclo-
prid treated (b) experiments (Pearson’s linear correlation
r = 0.95 and r = 0.85, respectively).
3.2. Characteristics Analyzed in a Single
Generation
3.2.1. Longevity
The time elapsed between the emergence and the death
of the adult flies was studied for virgin males and fe-
males from F12, in C and T groups. Longevity graphs
(Figures 5 and 6), showed that, in C experiments, half
life was approximately 58 days for females and 57 days
for males while, in T, they were about 50 and 48 days,
respectively Mean longevity values for C were 54.32
days for females and 56.84 days for males while for T,
they were 50.52 and 48.92, respectively. Thus, in the T
experiments, the mean longevity was reduced in 7.92
days for males and 3.80 days for females.
3.2.2. Oviposition Rate
Egg numbers were counted at the first and second 22h
periods after flies from groups C, T and from S (stock)
were put in contact with the specific culture medium
(Figure 7). Due to the variability of the standard error,
(a)
(b)
Figure 5. Graphs for comparison of longevity between
males and females from F12 generation, in the control (a)
and imidacloprid treated (b) groups. Horizontal line at
0.5 proportion indicates half-life value.

T. de F r ança Patarro et al. / Open Journal of Animal Sciences 3 (2013) 8-19 15
(a)
(b)
Figure 6. Graphs for comparison of longevity between control
and imidacloprid treated flies, from the F12 generation, for
males (a) and females (b). Horizontal line at 0.5 proportion
indicates half-life value.
Figure 7. Distribution graph of the oviposition rate, computed
at 22 and 44h after the beginning of the experiment, for flies
from the control (C), the imidacloprid treated experiment (T)
and the stock (S).
the non-parametric test Kruskal-Wallis for comparison of
the experimental groups and pairwise comparisons be-
tween groups were used in the analyses.
Kuskal-Wallis statistics (H) and P values for compare-
son among groups, at 22 and 44 h showed significant
differences in both counts: at 22 h (H = 18.94; P = 0.000)
and at 44 h (H = 13.68, P = 0.001). Pairwise comparisons
showed that the oviposition proportion at 22 h was
higher for flies from T than from C (q = 3.376), higher
for flies from T than from S (q = 5.855) and did not dif-
fer between C and S (q = 2.478). Thus, at 22h, T > C = S.
At 44 h, the oviposition proportion was higher in C than
in S (q = 4.993) and also higher in T than in S (q = 3.771)
but it did not differ between C and T (q = 1.221).Thus, at
44 h, T = C > S. In every case, critic q value was 3.134.
In the total time of observation, C flies laid 246 eggs, T
flies 381 and S flies 23. The differences in the oviposi-
tion rate between the first and second 22 h periods, in
each group, was evaluated using Student’s t-test for
paired data and showed significant differences only for C,
being the oviposition rate at the second 22 h period
higher than that at the first 22 h (t = 4.24; P = 0.002) (Ta-
ble 6).
3.2.3. Viability Egg-Adult
The viability egg-adult was analyzed for C, T and S
flies, using the eggs collected in the tests for oviposition
rate (Tabl e 7). The comparisons between groups relative
to the proportion of males, females and males plus fe-
males obtained from egg samples showed significant
differences for females (χ2 = 22.26, P = 0.00+), for males
(χ2 = 10.42, P = 0.027) and for total progeny (χ2 = 30.95,
P = 0.00+). χ2 analysis was also used for pairwise com-
parisons of groups as to the difference of proportion of
males, females and total of flies obtained from the eggs.
The result was the same in every comparison, that is,
viability egg-adult in T was smaller than in C and S,
which did not differ from each other. Thus, T < C = S.
(for females: T × S, q = 4.559, T × C, q = 5.583 and C ×
S, q = 1.705; for males and for males plus females T × S,
q = 4.95, T × C, q = 4.26, C × S, q = 1.95. In every case,
critical q was 3.682.
Z statistics for comparison of the mortality rate be-
tween adult females and males in each group (C, T and S)
showed significant differences in C, in the second 24 h
(Z = 2.51, P = 0.012) and in the total 48h exposure (Z =
2.32, P = 0.020), and in T (in the 48h, Z = 2.72, P =
0.007). In both groups, mortality rate of males was
greater than that of females. However, considering the
numbers, the male mortality was higher than that of fe-
males in the three exposition times of S flies (1st 24, 2nd
24 and total 48h) and also in the 2nd 24h exposure of T.
3.2.4. Mort ality of Adults
Data on mortality were obtained for adult flies from C,
T and S exposed to imidacloprid and evaluated after the
first and second 24 h exposition, separately for females,
males and females plus males (Table 8). Comparison
among groups could not be done for females and for
males at the first 24 h due to the low frequency of dead
lies (less than five). For females, in the second 24 h and f

T. de F r ança Patarro et al. / Open Journal of Animal Sciences 3 (2013) 8-19
16
Table 6. Difference of oviposition values in comparisons of the control (C), imidacloprid treated (T) and flies from the stock (S) as to
the number of eggs laid at 22 and 44 h, including residual squares.
22 h 44 h
Comparisons Ri - Rj SE q qcritical ConclusionRi - Rj SE q qcritical Conclusion
T × C 94 27.84 3.376 3.134 T > C 34 27.84 1.221 3.134 C = T
T × S 163 27.84 5.855 3.134 T > S 105 27.84 3.771 3.134 T > S
C × S 69 27.84 2.478 3.134 C = S 139 27.84 4.993 3.134 C > S
Table 7. Multiple pairwise comparisons for data on egg to adult
viability of flies from the control (C), imidacloprid treated (T)
and stock (S).
Experiments
Sex
T C S
Conclusions
Females
Proportions 0.07 0.17 0.24
Comparisons q qcritical
T × S 4.559 3.682 S > T
T × C 5.583 3.682 C > T
C × S 1.705 3.682 C = S
Males
Proportions 0.08 0.08 0.16
Comparisons q qcritical
C × T 4.26 3.682 C > T
C × S 1.95 3.682 C = S
S × T 4.95 3.682 S > T
Total
Proportions 0.15 0.32 0.33
Comparisons q qcritical
C × T 4.26 3.682 C > T
C × S 1.95 3.682 C = S
S × T 4.95 3.682 S > T
total 48 h the comparison showed significant difference
among groups (χ2 = 9.603, P = 0.008 and χ2 = 18.412, P =
0.0009, respectively), while, for males, significant dif-
ferences were obtained only for total 48h (χ2 = 11.498, P
= 0.00492). For females plus males the differences
among groups were significant in the two evaluations
(for second 24 h: χ2 = 12.009, P = 0.004 and for total 48
h (χ2 = 40.5689, P = 0.00001). Pairwise comparisons of
groups showed that for females at the second 24 h and
total 48 h the mortality of S flies was greater than that of
T and C, which did not differ from each other (thus, S >T
= C). At the second 24 h, q values were = 3.749 for S × C,
3.401 for S × T and 0.349 for T × C. For males, in the
only significant counting (total 48 h), pairwise compare-
sons also showed the sequence S > T = C (q values, in S
× C = 4.174, in S × T = 3.937 and in T × C = 0.237).
Pairwise comparisons of the mortality degree for fe-
males + males also showed the sequence S > T = C, in
the three periods of analysis (for mortality rate at the first
24 h, q values for S × T = 4.559, for S × T = 4.174 and
for T × C = 0.385; at the second 24h, q values for S × T =
4.193, for S × C = 3.987, and for T × C = 0.206; and in
the total 48 h, q for S × T , for S × C = 6.275, and for C ×
T = 0.000. In every sex comparison critical q = 3.377.
Thus, for all mortality comparisons S >T = C.
4. DISCUSSION
A sublethal dose of the insecticide imidacloprid (0.5
μg/mL culture medium) administered to Drosophila
melanogaster affected the traits productivity, duration of
the emergence period, viability of adults, longevity, vi-
ability during the development from egg to adults, and
oviposition. Except oviposition that was affected in a
different way, all the traits had their values reduced, re-
vealing the toxicity of imidacloprid on Drosophila biol-
ogy. These traits are among the ones that in the literature
have been named life history traits because they are
shaped by natural selection in the organism’s lifetime,
resulting in a variety of strategies for survival and re-
production [19,20]. Harmful effects on the life history
traits such as these due to the insecticide imidacloprid
(that is intensively used and thus practically continuously
present in the environment) can put in risk these strate-
gies and consequently the survival of the populations.
Relative to the productivity, 12 of the 15 generations
showed lower numbers of progeny in the treated experi-
ments (T), although only in four generations the differ-
ences between control (C) and T were significant. Con-
sidering the 15 generations, the mean decrease of the
males plus females in T was about 25% in relation to C.
However, the proportion of males and females produced
in both C and T did not differ, suggesting equivalence of
susceptibility of sexes to the insecticide in this trait.

T. de F r ança Patarro et al. / Open Journal of Animal Sciences 3 (2013) 8-19 17
Table 8. Mortality (number and percentage) of females and males during 48 h to 10 μg/ml imidacloprid, computed in the first and the
second 24 h and in the total 48 h. Ninety males and ninety females were used for tests in control (C) and imidacloprid treated (T)
groups, taken in the F15 generation, and also from the stocks which originated the experiments (S).
Females Males
Group 1st 24h 2nd 24h Total 1st 24h 2nd 24h Total
C 3 (3.4%) 8 (8.9%) 11 (12.2%) 3 (3.4%) 20 (22.3%) 23 (25.56%)
T 1 (1.2%) 9 (10.0%) 10 (11.1%) 6 (6.7%) 18 (20.0%) 24 (26.7%)
S 9(10.0%) 21 (23.4%) 30 (33.3%) 13 (14.4%) 29 (32.2%) 42 (46.7%)
The duration of the emergence period was also ana-
lyzed in every generation. In the light of the numbers, the
mean emergence period of C flies was greater than that
of T, in nine of the 15 generations, although significant
results were observed only in two of them. Taking into
account all the generations, we observed for T a mean
emergence period 2.13 days (15.5%) shorter than that for
C, a difference that may not be neglected considering the
mean value of 13.6 days for the duration of emergence in
the control experiments. The correlation between the
number of progeny and the emergence time duration also
opened a new problem to be studied: is the decrease of
emergence days due to a greater mortality of the larvae
with longer developmental time and consequently with
longer exposure to the insecticide?
The longevity (or lifespan) of D. melanogaster, in
laboratory, is known since long ago as being about eight
weeks. The influence of the genetic background and the
environmental variation in their expression have also
been demonstrated in laboratory tests as well by the sig-
nificant variation in longevity observed within and among
natural populations [21]. Our measures of longevity in C
experiments showed values included in the known limits,
while the treated flies showed mean longevity decrease
of about four and eight days for females and males, re-
spectively, showing that, for longevity, imidacloprid was
more toxic for males than for females.
The viability egg-adult of the imidacloprid treated flies
differed significantly between T and C experiments, with
T producing lower percentage of adults than C and S,
which did not differ from each other. In the study of this
and also the traits oviposition and tolerance, comparisons
of C and T flies with flies from the stock (that had been
normally maintained in the laboratory during the time of
the study) were performed in order to detect possible
effects of using the 15 first couples for producing the
consecutive generations. Thus, for viability egg to adult
this effect apparently does not occur. The lower viability
of the treated flies during development may be an im-
portant factor to explain the lower productivity observed
under the imidacloprid effect. The harmful effect on the
viability egg-adult of Drosophila has also been described
in experiments with other insecticides [22].
Oviposition was affected by imidacloprid in a different
way. It provoked a decrease of the pre-oviposition time.
At 22 h, T flies had laid 55% of the total number of eggs
counted in the complete period of analysis (44 h), a per-
centage significantly higher than the 15% laid by C flies
in the same period. Since in S flies the egg-laying time
did not differ significantly from C, the anticipation of
oviposition in that period apparently was not influenced
by the method of preparing the consecutive generations.
However, at 44 h, while S remained in a slow pace, T
and C oviposition rates equaled due to the significant in-
crease of the egg number in C. Recently, [4] also re-
ported a shortening of the pre-oviposition period in the
mirid bug Apoligus lucorum treated with imidacloprid,
but parallel to this shortening they observed an increase
of the embryogenesis time that doesn’t seem to occur in
our study in Drosophila because we detected decrease in
the duration of the emergence period of the treated flies.
The meaning and the mechanism of these changes in
oviposition remain to be understood.
The development of resistance to imidacloprid has
been reported in many insect species, including field
populations and laboratory selected strains (in honeybee,
[9]; in the whitefly Bemisia tabaci [23,24]; in the brown
planthoper Lugaparvata lugens [25] and in the potato
beetle Leptinitarsa decemlineata [26]. In the present
study, although we had not selected the flies for resis-
tance with the usual method (selecting the more resistant
flies in each generation to prepare the next), during 15
successive generations the parental adults and the larvae
of the T experiments were fed exclusively with the cul-
ture medium containing imidacloprid. We expected that,
if some tolerance had developed, the number of progeny
would increase in T experiments over the generations.
The smoothing technique applied to the productivity data
revealed more clearly, in graph, a tendency for this in-
crease. However, the increase occurred in C and T ex-
periments, which allowed hypothesizing that the use of
the first 15 emerged couples to prepare the consecutive
generations was revealing an interaction between emer-
gence speed and productivity. In situations like this, the
characteristics that seem to be interdependent must be
necessarily considered genetically correlated [27].

T. de F r ança Patarro et al. / Open Journal of Animal Sciences 3 (2013) 8-19
18
A similar idea resulted from the study of mortality of
adult flies exposed to the imidacloprid, in which we
compared flies from C and T experiments, and also flies
from the stock (S). The mortality of flies from S was
higher than that of C and T, which, in turn, did not differ
from each other. These results indicated that the toler-
ance in C and T, as also observed in the productivity in-
crease is, in some way, related with the selection for
emergence speed. Previous studies on emergence time in
Drosophila suggested that this trait can be correlated
with fecundity [28] and with stress resistance [29] rein-
forcing the present hypothesis.
In summary, the present work showed harmful effects
of the insecticide imidacloprid on Drosophila, affecting a
set of important components of organisms’ fitness. Be-
yond this focus on the biological danger due to the ex-
tensive presence of this neonicotinoid in the environment,
the analysis of different biological traits allowed propos-
ing a relationship of cause and effect among some of
them (for example, the viability decrease during devel-
opment from egg to adult and the productivity decrease,
in the treated experiments). There are also other ques-
tions such as the possible shortening of the emergence
period due to a preferential mortality of the larvae de-
layed in development and consequently exposed longer
to the insecticide; another question is what the conse-
quences of the pre-oviposition time decrease are—Are
these eggs viable? Do they develop earlier?
Furthermore, the results suggested the interplay of the
trait developmental speed of flies with other characteris-
tics such as progeny number and development of toler-
ance. This aspect also deserves further studies, for Dro-
sophila melanogaster is a very propitious model.
The clarifying of the mentioned questions will be im-
portant, considering, on one side, the need to increase the
knowledge on the effects of imidacloprid considered
currently the insecticide of prevalent use in the world,
and, on the other, the still poor knowledge of the mecha-
nisms underlying the adaptive traits.
5. ACKNOWLEDGEMENTS
Research supported by FAPESP (Scientific initiation fellowship,
Thais de França Patarro).
REFERENCES
[1] Yamamoto, I. and Casida, J.E. (1999) Nicotinoid insecti-
cides and the nicotinic acetylcholine receptor. Springer
Verlag, Berlin.
[2] Bayer Crop Science (2013) Imidacloprid.
http://www.bayergroupindia.com/imidacloprid.html
[3] Bao, H., Liu, S., Gu, J., Wang, X., Liang X. and Liu, Z.
(2009) Sublethal effects of four insecticides on the re-
production and wing formation of brown planthopper,
Nilaparvata lugens. Pest Management Science, 65, 170-
174. http://dx.doi.org/10.1002/ps.1664
[4] Tan, Y., Biondi, A., Desneux, N. and Gao, X. (2012) As-
sessment of physiological sublethal effects of imidaclo-
prid on the mirid bug Apolygus lucorum (Meyer-Dür).
Ecotoxicology, 21, 1989-1997.
http://dx.doi.org/10.1007/s10646-012-0933-0
[5] Blacquière, T., Smagghe, G., Van Gestel, C.A.M. and
Mommaerts, V. (2012) Neonicotinoids in bees: A review
on concentrations, side-effects and risk assessment.
Ecotoxicology, 21, 973-992.
http://dx.doi.org/10.1007/s10646-012-0863-x
[6] Bortolotti, L., Montanari, R., Marcelino, J., Medrzycki, P.,
Maini, S. and Porrini, C., (2003) Effects of sub-lethal
imidacloprid doses on the homing rate and foraging ac-
tivity of honey bees. Bulletin of Insectology, 56, 63-67.
[7] Henry, M., Béguin, M., Requier, F., Rollin, O., Odoux, J.,
Aupinel, P., Aptel, J., Tchamitchian, S. and Decourtye, A.
(2012) A Common Pesticide Decreases Foraging Success
and Survival in Honey Bees. Science, 336, 348-350.
http://dx.doi.org/10.1126/science.1215039
[8] Tapparo, A., Marton, D., Giorio, C., Zanella, A., Solda,
L., Marzaro, M., Vivan, L. and Girolami,V. (2012) As-
sessment of the environmental exposure of honeybees to
particulate matter containing neonicotinoid insecticides
coming from corn coated seeds. Environmental Science &
Technology, 46, 2592-2599.
http://dx.doi.org/10.1021/es2035152
[9] Johnson, R.M., Ellis, M.D., Mullin, C.A. and Frazier, M.,
2010. Pesticides and honey bee toxicity—USA. Apidolo-
gie, 41, 312-331.
http://dx.doi.org/10.1051/apido/2010018
[10] Tomizawa, M. and Casida, J.E. (2003) Selective toxicity
of neonicotinoids attributable to specificity of insect and
mammalian nicotinic receptors. Annual Review of Ento-
mology, 48, 339-364.
http://dx.doi.org/10.1146/annurev.ento.48.091801.112731
[11] Jeschke, P. and Nauen, R. (2008) Neonicotinoids—From
zero to hero in insecticide chemistry. Pest Management
Science, 64, 1084-1098.
http://dx.doi.org/10.1002/ps.1631
[12] Magalhães, L.C., Hunt, T.E. and Siegfried, B.D. (2008)
Development of methods to evaluate susceptibility of
soybean aphid to imidacloprid and thiamethoxam at lethal
and sublethal concentrations. Entomologia Experimen-
talis et Applicata, 128, 330-336.
http://dx.doi.org/10.1111/j.1570-7458.2008.00718.x
[13] Sarkar, M.A., Biswas, P.K., Roy, S., Kole, R.K. and
Chowdhury, A. (1999) Effect of pH and type of formula-
tion on the persistence of lmidacloprid in water. Bulletin
of Environmental Contamination and Toxicology, 63,
604-609. http://dx.doi.org/10.1007/s001289901023
[14] Anatra-Cordone, M. and Durkin, P. (2005) Human Health
and Ecological Risk Assessment. Final Report, USDA/
Forest Service/ Forest Health Protection, New York.
[15] Niwa, R. and Niwa, Y. S. (2011) The fruit fly Drosophila
melanogaster as a model system to study cholesterol me-
tabolism and homeostasis. Cholesterol, 2011, 1-6.
http://dx.doi.org/10.1155/2011/176802

T. de F r ança Patarro et al. / Open Journal of Animal Sciences 3 (2013) 8-19
19
[16] Wolf, M.J. and Rockman, H.A. (2008) Drosophila
melanogaster as a model system for the genetics of post-
natal cardiac function. Drug Discovery Today, 5, 117-
123.
[17] Zar, J.H. (1999) Biostatistical Analysis. Prentice Hall,
New Jersey.
[18] Moore, D.A. (2004) Estatística Básica e Sua Prática.
Livros Técnicos e Científicos S.A., Brasil.
[19] Stearns, S.C. (1977) The evolution of life history traits: a
critique of the theory and a review of the data. Annual
Review of Ecology, Evolution, and Systematics, 8, 145-
171.
http://dx.doi.org/10.1146/annurev.es.08.110177.001045
[20] Fabian, D. and Flatt, T. (2012) Life History Evolution.
Nature Education Knowledge Project. 3, 24.
http://www.nature.com/scitable/knowledge/library/life-his
tory-evolution-68245673
[21] Paaby, A.B. and Schmidt, P.S. (2009) Dissecting the ge-
netics of longevity in Drosophila melanogaster. Fly, 3,
29-38. http://dx.doi.org/10.4161/fly.3.1.7771
[22] Karataş, A., Bahçeci, Z. and Başpinar, E. (2011) The ef-
fect of diazinon on egg fertility and development in Dro-
sophila melanogaster. Turkish Journal of Biology, 35,
95-101.
[23] Sethi, A., Bons, M.S. and Dilawari, V.K. (2008) Realized
heritability and genetic analysis of insecticide resistance
in whitefly, Bemisia tabaci (Genn.). Journal of Entomol-
ogy, 5, 1-9. http://dx.doi.org/10.3923/je.2008.1.9
[24] Liu, Z. and Han, Z. (2006) Fitness costs of laboratory-
selected imidacloprid resistance in the brown planthopper,
Nilaparvata lugens Stål. Pest Management Science, 62,
279-282. http://dx.doi.org/10.1002/ps.1169
[25] Wen, Y., Liu, Z., Bao, H. and Han, Z. (2009) Imidacloprid
resistance and its mechanisms in field populations of
brown planthopper, Nilaparvata lugens Stål in China.
Pesticide Biochemistry and Physiology, 94, 36-42.
http://dx.doi.org/10.1016/j.pestbp.2009.02.009
[26] Alyokhin, A., Dively, G., Patterson, M., Castaldo, C.,
Rogers, D., Mahoney, M. and Wollam J. (2006) Resis-
tance and cross-resistance to imidacloprid and thiameth-
oxam in the Colorado potato beetle Leptinotarsa Decem-
lineata. Pest Management Science, 63, 32-41.
http://dx.doi.org/10.1002/ps.1305
[27] Fuller, R.C., Baer, C.F. and Travis, J. (2005) How and
When Selection Experiments Might Actually Be Useful.
Integrative and Comparative Biology, 45, 391-404.
http://dx.doi.org/10.1093/icb/45.3.391
[28] Nunney, L. (1996) The response to selection for fast lar-
val development in Drosophila melanogaster and its ef-
fect on adult weight: An example of a fitness trade-off.
Evolution, 50, 1193-1204.
http://dx.doi.org/10.2307/2410660
[29] Sørensen, J.G. and Loeschcke, V. (2004) Effects of rela-
tive emergence time on heat stress resistance traits, lon-
gevity and hsp70 expression level in Drosophila melano-
gaster. Journal of Thermal Biology, 29, 195-203.
http://dx.doi.org/10.1016/j.jtherbio.2004.02.004