Vol.3, No.4, 343-353 (2013) Open Journal of Animal Sciences
http://dx.doi.org/10.4236/ojas.2013.34051
Evaluation of the toxic effect of insecticide
chlorantraniliprole on the silkworm Bombyx mori
(Lepidoptera: Bombycidae)
Roxelle Ethienne Ferreira Munhoz1, Thaís Souto Bignotto1, Naiara Climas Pereira1,
Cláudia Regina das Neves Saez1, Rafaela Bespalhuk1, Verônica Aureliana Fassina1,
Graziele Milani Pessini1, Mayarha Patrícia Dequigiovanni Baggio2,
Lucinéia de Fátima Chasko Ribeiro2, Rose Meire Costa Brancalhão2, Shunsuke Mizuno3,
Willian Shigeaki Aita3, Maria Aparecida Fernandez1*
1Departamento de Biotecnologia, Genética e Biologia Celular, Universidade Estadual de Maringá, Maringá, Brasil;
*Corresponding Author:
2Universidade Estadual do Oeste do Paraná, Cascavel, Brasil
3Fiação de Seda Bratac S. A. Bastos, São Paulo, Brasil
Received 10 September 2013; revised 15 October 2013; accepted 21 October 2013
Copyright © 2013 Roxelle Ethienne Ferreira Munhoz 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
The silkworm Bombyx mori feeds exclusiv ely on
mulberry leaves and is highly sensitive to pesti-
cides in general. Although mulberry plantations
are free of agrochemicals, pesticide drift can
occur. Chlorantraniliprole, a novel insecticide of
the anthranilic diamides class, has been used to
control pests in field crops. In this study, we
investigated the biological effects of different
concentrations of chlorantraniliprole on B. mori
silkworm commercial Brazilian hybrids. To eva-
luate the toxicity of chlorantraniliprole, bio- as-
says were carried out and data on the lethal
concentrations, symptomatology, morphology
and variables of silk production were collected.
Results indicated that B. mori is extremely sen-
sitive to chlorantraniliprole, even in low con-
centrations. The highest silkworm mortality
rates were observed in the two highest chloran-
traniliprole concentrations, 0.2 and 0.1 ppm. Al-
though lower chlorantraniliprole concentrations
did not cause death of all the silkworm larvae,
various symptoms of toxicity were observed:
feeding cessation, regurgitation, late develop-
ment and incomplete ecdysis. Such symptoms
reflect the morphological changes we observed
in the midgut epithelium, which affected nutrient
uptake and metabolism, and even the produc-
tion of cocoons. Exposed larvae also produced
thin-shelled cocoons, which constitutes a seri-
ous economic problem because this type of
cocoon is not useful for the silk industry. The
results provided herein confirm the toxicity of
chlorantraniliprole in silkworm larvae. There-
fore, we strongly suggest that, competent au-
thorities of the National Health Surveillance
Agency, in pesticide management should take
measures to reduce or eliminate the use of chlo-
rantraniliprole in areas nearest to silkworm cul-
tivation.
Keywords: Silkworm, Pesticide Drift; Mortality;
Symptomatology; Chlorantraniliprole
1. INTRODUCTION
The silkworm Bombyx mori L. (Lepidoptera: Bomby-
cidae), an insect originally from China, was domesticated
5000 years ago for silk production [1]. Silkworms are no
longer found in nature and are totally dependent on man
to survive. Bombyx mori most likely evolved from B.
mandarina, a wild silkworm species with similar charac-
teristics and reproduction to B. mori [2]. Silkworms are
herbivorous and feed exclusively on fresh mulberry
leaves during the larval phase [3].
The practice of sericulture includes mulberry cultiva-
tion, egg production, larvae creation and maintenance
until cocoon production and, finally, silk yard production
by the industrial sector. Brazil’s weather is favorable for

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344
cultivating both mulberry and silkworms, which makes
sericulture a good alternative for this country, especially
for small farmers.
Bombyx mori is highly susceptible to insecticides, and
the application of insecticides near sericulture projects is
not advisable because the silkworms can be harmed by
chemicals on the leaves, either through consumption of
contaminated leaves or through other contact with the
insecticides [4]. Hence, mulberry plantations intended
for silkworms are free of pesticides. However, the com-
plete elimination of pesticide drift is impossible and in-
secticides that are employed in other crops, especially
those that are applied through aerial spraying, can dam-
age sericulture activity.
Chlorantraniliprole is a novel insecticide belonging to
a potent class of anthranilic diamides that is usually safe
for mammals [5] and is commonly used in rice, coffee,
sugar cane, apple and peach crops. Chlorantraniliprole
eliminates Lepidoptera insects [6], as well as other pest
orders such as Coleoptera, Diptera, Isoptera and Hemip-
tera [7]. This insecticide, once ingested, acts as a selec-
tive agonist for ryanodine receptors, a class of intracel-
lular calcium channels. Ryanodine receptors play an im-
portant role in muscle function because the contrac-
tion of muscle cells requires exact liberation of calcium
from intracellular deposits in the cytoplasm. Chloran-
traniliprole causes unregulated Ca2+ release from the
sarcoplasmic reticulum of muscle cells [8], which im-
pedes the insect’s ability to regulate muscle function and
leads to permanent muscle contraction. Poisoning symp-
toms include rapid cessation of feeding, lethargy, regur-
gitation, muscle paralysis and, ultimately, insect death
[9-11].
The effects of chlorantraniliprole in different insects
have been well-studied in many taxa: Choristoneura ro-
saceana [12], termites Reticulitermes flavipes [13], ho-
ney bees Apis mellifera and bumble bees Bombus ter-
restris [8] and Leptinotarsa decemlineata [14]. How-
ever, little information is described about the effects of
chlorantraniliprole on B. mori [11]. Although this insec-
ticide is not used in mulberry fields, it can cause loss of
cocoon production in Brazilian silkworm farms near ar-
eas where chlorantraniliprole is used. Therefore, in this
study, we investigated the effects of different concentra-
tions of chlorantraniliprole on commercial Brazilian hy-
brids of the B. mori silkworm.
Recently, big crops such as sugarcane are using new
kinds of insecticide to do the control of Diatrea sac-
charalis larvae in Paraná State, Brazil. The areas of
mulberry plantation, which are close to the sugarcane
crops, are affected by aerial spraying of the insecticide,
which results in B. mori larvae death. Studies made by
the agriculture secretary of Paraná State show a loss of
cocoons equal to 8,626 kilograms. This State is the big-
gest producer of cocoons from Brazil, responsible for
92% of the total production in this country and due to
this recent problem with cloranthraliniprole insecticide.
This work was developed with the intention of describing
the symptomatology of the intoxication in B. mori larvae
that were exposed to this insecticide. The description of
this symptom induced in laboratory can help the com-
prehension of the toxic effect caused by cloranthralini-
prole in the larvae, which has a commercial interest for
several countries. Those data can further help the Brazil-
ian sericulture, as the results can assist either the farmer
who works with sericulture or the company behind all
the production.
2. MATERIAL AND METHODS
2.1. Bombyx mori Culture
The bioassays were carried out at the Fiação de Seda
BRATAC laboratory at Bastos city, São Paulo, Brazil
with the commercial hybrids of B. mori from this com-
pany. Silkworm larvae were reared under hygienic conp-
ditions and in controlled environments. Rearing rooms
were adjusted to 28˚C ± 0.5˚C and 90% ± 5 % of relative
humidity (RH) for the creation of 1st instar silkworm
larvae, while 2nd instar larvae were reared at 27˚C ±
0.5˚C and with 85% ± 5 % of RH. Larvae of the 3rd, 4th
and 5th instars were reared at 25 to 26˚C ± 0.5˚C and with
RH of 70% ± 5 %. Artificial white lighting was employed
in the rearing rooms to submit silkworm larvae to four-
teen-hour daily of photoperiod with 10 hours of dark.
During the experiments, larval mortality and symptoms
of exposure were recorded.
2.2. Lethal Concentration
Treatments consisted of three replicates, with 20 silk-
worm larvae each. The following chlorantraniliprole
concentrations, in ppm (parts per million), were used: 0.2,
0.1, 0.05, 0.025, 0.0125, 6.25 × 10−3, 3.13 ×10−3 and 1.57
× 10−3. Control groups consisted of silkworm larvae that
were fed exclusively on mulberry leaves free of chloran-
traniliprole.
Silkworm larvae were exposed once to mulberry
leaves containing chlorantraniliprole at different days
and different instars, depending on the treatment, as fol-
lows: 1st day of the 1st instar, 2nd day of the 1st instar, 1st
day of the 2nd instar, 3rd day of the 2nd instar, 1st day of
the 3rd instar, 3rd day of the 3rd instar, 1st day of the 4th
instar, 3rd day of the 4th instar, 1st day of the 5th instar and
5th day of the 5th instar. Besides, bioassays also included
5th instar larvae that were exposed twice to chloran- tra-
niliprole: on the 1st and 2nd days of the 5th instar and on
the 5th and 6th days of the 5th instar.
For the lethal concentration bioassays, mulberry leaves
were immersed in aqueous solutions containing dif-

R. E. F. Munhoz et al. / Open Journal of Animal Sciences 3 (2013) 343-353 345
ferent concentrations of chlorantraniliprole, as mentioned
above. Leaves were dried at room temperature and then
offered to B. mori larvae. Each replicate included 20
silkworm larvae that were fed once a day in petri-dishes
with different amounts of mulberry leaves containing
chlorantraniliprole, as follows: larvae exposed to the
insecticide in the 1st or 2nd days of the 1st instar were fed
with 7 cm2 of mulberry leaves, while larvae exposed in
the 1st or 3rd days of the 2nd instar were fed with 16 cm2;
larvae in the 1st or 3rd days of the 3rd instar were fed with
10 g of mulberry leaves and those in the 1st or 3rd days of
the 4th instar and in the 1st or 5th days of the 5th instar
were fed with 30 g of mulberry leaves. Treatments in-
volving silkworm larvae exposed twice to chloran- trani-
liprole were fed with 30 g of mulberry leaves when ex-
posure occurred in the 1st and 2nd days of the 5th instar
and with 100 g of mulberry leaves when exposure oc-
curred in the 5th and 6th days of the 5th instar. Before and
after chlorantraniliprole exposure, larvae were fed in
abundance with mulberry leaves free of chlorantrani-
liprole.
Mulberry trees used in the bioassays have been grown
in the experimental field of the company Fiação de Seda
BRATAC, Bastos city in São Paulo state, Brazil. Mul-
berry leaves used for silkworm feeding were taken from
75-days mulberry trees. Silkworm larvae of the 1st, 2nd
and 3rd instars were fed with young mulberry leaves tak-
en from the top part of the principal branche, while old
leaves were offered for 4th and 5th silkworm larvae.
2.3. Symptomatology
After chlorantraniliprole applications, symptoms re-
lated to insecticide exposure were registered immediately
after the first day of exposure: feeding cessation, regur-
gitation, late development and irregular ecdysis. These
symptoms were classified according to the intensity,
where 0 represents the absence of symptoms and 5 re-
presents the highest level of the symptom. In addition,
the following productive variables were collected: mean
number of normal cocoons (NC), mean weight of normal
cocoon (NCW), mean number of thin-shelled cocoons
(TSC), mean weight of thin-shelled cocoons (TSCW)
and mean number of live pupae (LP). Although no meas-
urements were taken to distinguish thin-shelled cocoons
from normal cocoons, thin-shelled cocoons can be visu-
ally and unambiguously differentiated from normal co-
coons since their shells are thinner and translucent.
2.4. Morphological Analysis
The effects of chlorantraniliprole in B. mori midgut
tissue were evaluated. Larvae of 5th instar were exposed
to two different concentrations of this insecticide: 0.2
ppm and 0.1 ppm, in addition to the control group. On
the 4th day after exposure to chlorantraniliprole, larvae
were anesthetized and dissected. Segments of the midgut
wall were removed and fixed in DuBosq Brasil, dehy-
drated in ethanol and preserved in paraffin. Sequential
cuts were made on an Olympus CUT4055 microtome at
thicknesses ranging between 5 and 7 µm. Sections were
stained with hematoxylin-eosin for general tissue analy-
sis. Images were obtained with on Olympus BX 60 pho-
tomicroscope.
2.5. Statistical Analysis
The experimental delineation was completely ran-
domized with three replicates per treatment (total 108
treatments). Twenty silkworm larvae were used per rep-
licate. The data were compared with an analysis of vari-
ance, followed by the Scott-Knott means test (0.05%)
using the statistical software SISVAR 5.3 [15].
3. RESULTS AND DISCUSSION
This insecticide is frequently applied by spray in sug-
arcane plantations next at silkworm producers in south of
Brazil, causing damaging and lost of cocoon production.
In the last years, Brazilian silkworm producers have been
reporting great losses in cocoon production. Apparently,
these losses are related to the period of time when insec-
ticides are aerial sprayed in crops near silkworm farms.
Recently, chlorantraniliprole has been employed in crops
to eliminate Lepidoptera insects, especially sugarcane
crops. In this study, we performed bioassays to verify if
the symptoms caused by chlorantraniliprole in silkworm
larvae are similar to those observed by producers in
silkworm farms. To evaluate the toxicity and symptoma-
tology of chlorantraniliprole in Brazilian hybrids of B.
mori, we exposed groups of silkworm larvae to different
concentrations of chlorantraniliprole.
3.1. Lethal Chlorantraniliprole
Concentration
We analyzed the mortality of silkworm larvae after
they were exposed to eight different concentrations of
chlorantraniliprole (Table 1). The two highest chloran-
traniliprole concentrations, 0.2 ppm and 0.1 ppm, re-
sulted in 100% larval mortality in most silkworm larvae
instars, though not when exposure occurred on the 1st and
3rd day of the 2nd instar. Complete larval mortality was
also observed in the groups exposed to 0.05 ppm in the
5th day of the 5th instar and in the 5th and 6th days of the
5th instar. Silkworm larvae that were exposed to a con-
centration of 0.025 ppm on the 5th and 6th days of the 5th
instar also had a high mortality rate of 98.33%. Overall,
exposures of silkworm larvae on the 5th and 6th days of
the 5th instar to the four highest chlorantraniliprole con-
centrations resulted in a 100% rate of larval mortality,

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346
Table 1. Bombyx mori larvae mortality considering instar, day of exposure and concentration of the insecticide chlorantraniliprole.
Instar Day of exposure Chlorantraniliprole concentration (ppm) Mortality (%)
1st instar 1st day 0.2 100.00 a
1st instar 1st day 0.1 100.00 a
1st instar 1st day 0.05 80.00 b
1st instar 1st day 0.025 0.00 f
1st instar 1st day 0.0125 0.00 f
1st instar 1st day 6.25 × 10−3 0.00 f
1st instar 1st day 3.13 × 10−3 0.00 f
1st instar 1st day 1.57 × 10−3 0.00 f
1st instar 1st day Control group 0.00 f
1st instar 2nd day 0.2 100.00 a
1st instar 2nd day 0.1 100.00 a
1st instar 2nd day 0.05 96.66 a
1st instar 2nd day 0.025 0.00 f
1st instar 2nd day 0.0125 0.00 f
1st instar 2nd day 6.25 × 10−3 0.00 f
1st instar 2nd day 3.13 × 10−3 0.00 f
1st instar 2nd day 1.57 × 10−3 0.00 f
1st instar 2nd day Control group 0.00 f
2nd instar 1st day 0.2 100.00 a
2nd instar 1st day 0.1 85.00 b
2nd instar 1st day 0.05 0.00 f
2nd instar 1st day 0.025 0.00 f
2nd instar 1st day 0.0125 0.00 f
2nd instar 1st day 6.25 × 10−3 0.00 f
2nd instar 1st day 3.13 × 10−3 0.00 f
2nd instar 1st day 1.57 × 10−3 0.00 f
2nd instar 1st day Control group 0.00 f
2nd instar 3rd day 0.2 88.33 b
2nd instar 3rd day 0.1 55.00 c
2nd instar 3rd day 0.05 0.00 f
2nd instar 3rd day 0.025 0.00 f
2nd instar 3rd day 0.0125 0.00 f
2nd instar 3rd day 6.25 × 10−3 0.00 f
2nd instar 3rd day 3.13 × 10−3 0.00 f
2nd instar 3rd day 1.57 × 10−3 0.00 f
2nd instar 3rd day Control group 0.00 f
3rd instar 1st day 0.2 100.00 a
3rd instar 1st day 0.1 100.00 a
3rd instar 1st day 0.05 5.00 f
3rd instar 1st day 0.025 0.00 f
3rd instar 1st day 0.0125 0.00 f
3rd instar 1st day 6.25 × 10−3 0.00 f
3rd instar 1st day 3.13 × 10−3 0.00 f

R. E. F. Munhoz et al. / Open Journal of Animal Sciences 3 (2013) 343-353 347
Continued
3rd instar 1st day 1.57 × 10−3 0.00 f
3rd instar 1st day Control group 0.00 f
3rd instar 3rd day 0.2 100.00 a
3rd instar 3rd day 0.1 98.33 a
3rd instar 3rd day 0.05 33.33 d
3rd instar 3rd day 0.025 13.33 e
3rd instar 3rd day 0.0125 0.00 f
3rd instar 3rd day 6.25 × 10−3 0.00 f
3rd instar 3rd day 3.13 × 10−3 0.00 f
3rd instar 3rd day 1.57 × 10−3 0.00 f
3rd instar 3rd day Control group 0.00 f
4th instar 1st day 0.2 100.00 a
4th instar 1st day 0.1 100.00 a
4th instar 1st day 0.05 3.33 f
4th instar 1st day 0.025 1.66 f
4th instar 1st day 0.0125 3.33 f
4th instar 1st day 6.25 × 10−3 6.66 f
4th instar 1st day 3.13 × 10−3 1.66 f
4th instar 1st day 1.57 × 10−3 3.33 f
4th instar 1st day Control group 0.00 f
4th instar 3rd day 0.2 100.00 a
4th instar 3rd day 0.1 100.00 a
4th instar 3rd day 0.05 15.00 e
4th instar 3rd day 0.025 5.00 f
4th instar 3rd day 0.0125 0.00 f
4th instar 3rd day 6.25 × 10−3 5.00 f
4th instar 3rd day 3.13 × 10−3 1.66 f
4th instar 3rd day 1.57 × 10−3 3.33 f
4th instar 3rd day Control group 1.66 f
5th instar 1st day 0.2 100.00 a
5th instar 1st day 0.1 100.00 a
5th instar 1st day 0.05 13.33 e
5th instar 1st day 0.025 3.33 f
5th instar 1st day 0.0125 3.33 f
5th instar 1st day 6.25 × 10−3 1.66 f
5th instar 1st day 3.13 × 10−3 3.33 f
5th instar 1st day 1.57 × 10−3 1.66 f
5th instar 1st day Control group 0.00 f
5th instar 5th day 0.2 100.00 a
5th instar 5th day 0.1 100.00 a
5th instar 5th day 0.05 100.00 a
5th instar 5th day 0.025 56.66 c
5th instar 5th day 0.0125 1.66 f
5th instar 5th day 6.25 × 10−3 0.00 f

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348
Continued
5th instar 5th day 3.13 × 10−3 1.66 f
5th instar 5th day 1.57 × 10−3 0.00 f
5th instar 5th day Control group 0.00 f
5th instar 1st - 2nd days 0.2 100.00 a
5th instar 1st - 2nd days 0.1 100.00 a
5th instar 1st - 2nd days 0.05 56.66 c
5th instar 1st - 2nd days 0.025 3.33 f
5th instar 1st - 2nd days 0.0125 1.66 f
5th instar 1st - 2nd days 6.25 × 10−3 0.00 f
5th instar 1st - 2nd days 3.13 × 10−3 0.00 f
5th instar 1st - 2nd days 1.57 × 10−3 0.00 f
5th instar 1st - 2nd days Control group 0.00 f
5th instar 5th - 6th days 0.2 100.00 a
5th instar 5th - 6th days 0.1 100.00 a
5th instar 5th - 6th days 0.05 100.00 a
5th instar 5th - 6th days 0.025 98.33 a
5th instar 5th - 6th days 0.0125 0.00 f
5th instar 5th - 6th days 6.25 × 10−3 5.00 f
5th instar 5th - 6th days 3.13 × 10−3 0.00 f
5th instar 5th - 6th days 1.57 × 10−3 3.33 f
5th instar 5th - 6th days Control group 0.00 f
Overall mean 28.34
VC (%) 23.78
(a-f) in the columns are different by the Scott-Knott test (P < 0.05).
which indicates that larvae are more susceptible to the
detrimental effects of this insecticide under such condi-
tions.
Exposure to chlorantraniliprole at a concentration of
0.05 ppm resulted in variable rates of mortality, as seen
in Table 1. The highest silkworm larvae mortality
(96.66%) occurred when the insecticide was applied
once on the 2nd day of the 1st instar, on the 5th day of the
5th instar or twice on the 5th and 6th days of the 5th instar.
High mortality (80%) was also observed when exposure
occurred on the 1st day of the 1st instar. On the other hand,
the lowest rates of larval mortality were observed follow-
ing exposure on the 1st or 3rd days of the 2nd instar, the 1st
day of the 3rd instar or on the 1st day of the 4th instar.
Few larvae died when they were exposed to a concen-
tration of 0.05 ppm of chlorantraniliprole in the early
days of the instar. This finding can be explained by the
fact that, once ecdysis occurs, silkworm larvae are le-
thargic and eat less than in the last days of each instar.
When chlorantraniliprole exposure occurred in the last
days of an instar, the larvae likely consumed more con-
taminated leaves and therefore suffered a higher mortal-
ity rate.
The other chlorantraniliprole concentrations (0.025,
0.0125, 6.25 × 10−3, 3.13 × 10−3 and 1.57 × 10−3 ppm)
resulted in lower or no silkworm larval mortality. An
exception occurred when larvae were exposed to chlor-
antraniliprole at a concentration of 0.025 ppm on the 5th
day of the 5th instar or on the 5th and 6th days of the 5th
instar, which resulted in 56.66% and 98.33% larval mor-
tality rates, respectively. These lower concentrations are
not generally fatal to B. mori larvae, but they may cause
damage to silkworms, in the end days of the last instar
like discussed above.
High concentrations of chlorantraniliprole, such as 0.2
ppm and 0.1 ppm, caused 100% larval mortality in most
of the larval instars, except when the insecticide was
applied in the 1st day of the 2nd instar, in this case only
for 0.1 ppm, or in the 3rd day of the 2nd instar (Table 1 ).
Although lower concentrations of this insecticide cause
little or no larval mortality, they still result in damage
such as cessation of feeding and production of a thin-
shelled cocoon, which is economically not viable. Hence,
our results demonstrated that Brazilian hybrids of the B.
mori silkworm are extremely sensitive to chloran- trani-
liprole. In experiments conducted in China was de-
tected a high sensitivity to chlorantraniliprole in the B.
mori silkworm and identified a threshold value of LC50 at

R. E. F. Munhoz et al. / Open Journal of Animal Sciences 3 (2013) 343-353 349
0.02045 ppm of chlorantraniliprole working with 3rd in-
star larvae [11].
We were not able to detect a median lethal concentra-
tion unique, LC50, for all treatments with the chloran-
traniliprole concentrations used in this study. The results
of our bioassays have demonstrated that three treatments
had LC50 was well defined, such as, 2nd instar in 3rd day
at concentration of 0.1 ppm, 5th instar in 5th day at con-
centration 0.025 ppm and in the treatment with two ap-
plications of insecticide, in 5th instar in the 1rst - 2nd days
at concentrations of 0.05 ppm. However, high concentra-
tions of this insecticide were responsible for 100% larval
mortality. The lethal concentrations (LC) for all treat-
ments are 0.2 ppm of chlorantraniliprole and 0.1 ppm and
0.5 ppm causes high mortality in all treatments (Table 1).
The value of LC50 presented by [11] is similar our
results but with larvae in 5th instar instead with larvae 3rd
instar, probably due to genetic differences of silkworms
and climatic conditions.
The value of LC50 presented by [11] is similar our re-
sults but with larvae in 5th instar instead with larvae 3rd
instar, probably due to genetic differences of silkworms
and climatic conditions.
3.2. Symptomatology Analysis
The concentrations under 0.1 ppm of the insecticide do
not cause mortality, but the symptoms were common in
the sub-lethal chlorantraniliprole concentrations and re-
sult in lost productivity. Between the symptoms in sub-
lethal concentrations are important the difficult of reach
the cocoon stage. In most treatments, we observed near
complete feeding cessation by silkworm larvae, as well
as regurgitation. So, the symptoms included feeding ces-
sation, regurgitation, late development and irregular ec-
dysis (incomplete larvae ecdysis; Figures 1(A) and (B)).
Another common symptom at sublethal concentrations
was the presence of rosary-shaped excrements (Figure
1(C)), which may have been due to the peristaltic move-
ment of the intestine being compromised by the muscle
myofibrils.
In addition to feeding cessation, regurgitation and ro-
sary-shaped excrements, we observed the production of
thin-shelled cocoons by silkworm larvae (Figure 1(D)).
This kind of cocoon has very thin shell which is not use-
ful for spinning. Hence, sericulture companies do not
accept thin-shelled cocoons and silkworm producers
have to assume for financial loss. The symptoms varied
in relation the insecticide’s concentration and day of ex-
posure of life circle. For the farmers do not is interesting
lost productivity either for lethal doses or not lethal, be
cause the symptoms are very damaging for the produc-
tion.
Figure 1. Symptomatology observed in Bombyx mori Brazilian silkworm hybrids exposed to chlorantraniliprole: incomplete
ecdysis (A and B), rosary-shaped excrements (C), and thin-shelled cocoons (D).

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350
We observed symptoms similar to those found by
[11]. They also reported that symptoms emerged very
quickly, in agreement with our observations. The ob-
served symptoms are most likely related to the unregu-
lated calcium release, which controls muscle myofibril
activity [9], and alterations in feeding lead to changes in
the conversion of ingested and digested food, which may
eventually cause abnormalities in subsequent develop-
ment [16].
We also evaluated productive variables of silkworm
cocoons and pupae. Analyses were conducted only for
treatments in which exposure to chlorantraniliprole oc-
curred in 4th and 5th silkworm instars; treatments with
concentrations equal to 0.2 ppm and 0.1 ppm were ex-
cluded from the analysis because they were lethal, caus-
ing 100% of larval mortality. Table 2 illustrates the data
obtained for the following variables: mean number of
normal cocoons (NC), mean weight of normal cocoon
(NCW), mean number of thin-shelled cocoons (TSC),
mean weight of thin-shelled cocoons (TSCW) and mean
number of live pupae (LP).
In general, there was little variation among treatments.
There was no statistical variation among treatments in
which chlorantraniliprole exposure occurred in the 4th
larval.
When the larvae were exposed during later stages of
the 5th instar, there was greater statistical variation,
mainly in the NC, NCW and LP variables. After expo-
sure on the 1st day of the 5th instar, there were no signifi-
cant differences and only modest variation in the variable
NCW. Low values of NCW were observed at chloran-
traniliprole concentrations of 0.025 ppm and 0.0125 ppm,
with values equal to 2.23 and 2.26 g, respectively.
When exposure to chlorantraniliprole occurred on the
5th day of the 5th instar, the response was variable in each
variable except TSCW. A chlorantraniliprole concentra-
tion of 0.05 ppm resulted in the production of only
thin-shelled cocoons by the silkworms, so the variables
NC, NCW and LP could not be assessed. The NC, NCW,
TSC and LP for the groups exposed to 0.025 ppm were
significantly different from the other chlorantraniliprole
concentrations, and their values were 6.66 cocoons, 2.13
g, 7.66 cocoons and 8.66 pupae, respectively.
Treatments with chlorantraniliprole also involved two
exposures of this insecticide at different ages of the 5th
instar. When chlorantraniliprole was applied twice in the
5th instar, on the 1st and 2nd days, there was no variation
among NCW, TSC and, consequently, TSCW in the
treatments. The NC and the LP, had the lowest values in
the treatment with chlorantraniliprole at a concentration
of 0.05 ppm, reaching values of 8.33 cocoons and 11.33
pupae, respectively.
Finally, our experiment also evaluated exposure to
chlorantraniliprole twice on the 5th and 6th days of the 5th
instar. At a concentration of 0.05 ppm, there was total
mortality, and no further data are available. NC, NCW
and LP varied at the chlorantraniliprole concentration of
0.025 ppm and had the overall lowest values at this con-
centration: 0.33 cocoons, 0.70 g and 0.33 pupae, respec-
tively. It is important to note that the TSCW was less
than half of the NCW for most of the treatments. More-
over, the NC decreased significantly when high concen-
trations of chlorantraniliprole were applied on the 5th day
of the 5th instar.
3.3. Morphological Alterations
The morphological features of the columnar epithelial
cells in the midgut of B. mori larvae were altered by
chlorantraniliprole exposure. The apical columnar cell
surface exhibited protrusions in addition to the con- spi-
cuous microvilli related to apocrine vesicles. Some of
these vesicles contained cell nuclei (Figures 2(B) and
(D); black arrows).
Cell fragments enclosed by a membrane with mor-
phology similar to apoptotic bodies were observed along
the midgut epithelium. The term apoptotic body is often
used during the process of death by classical apoptosis to
define the membrane-bound portion of the cell contain-
ing nuclear and cytoplasmic residue from fragmentation
during apoptosis [17]. The presence of the nucleus in the
structures observed in light microscopy was confirmed to
be the result of the death of epithelial cells of the midgut.
Columnar cells are most abundant in the midgut epithe-
lium of insects and are responsible for processing food,
secreting digestive enzymes and absorbing the final di-
gestion products [18].
Their malfunction affects the entire metabolism, in-
cluding silk production.
The regenerative cells presented in the midgut epithe-
lium underwent hypertrophy and hyperplasia (Figure
2(D)), changing their appearance. The regenerative cells
are responsible for cell renewal [18]. It is assumed that
these cells are the precursors of others cells types, in-
cluding columnar cells [18,19]. However, the regenera-
tion of columnar cells did not occur due to changes and
hypertrophy resulting from exposure.
We also observed the intestinal wall and its layers of
longitudinal and circular muscles, which work in peri-
stalsis. These muscles were disorganized, possibly due to
the disarray of their myofibrils (Figures 2(B) and (D);
MM). Figure 2(A) shows normal muscle control for
comparison. The midgut is responsible for the food di-
gestion and absorption of necessary nutrients for the de-
velopment and maintenance of normal metabolic func-
tion of the insect. Thus, the microscopic changes ob-
served in response to chlorantraniliprole at concentra-
tions of 0.2 ppm and 0.1 ppm harmed the development of
their normal organic functions and was responsible for

R. E. F. Munhoz et al. / Open Journal of Animal Sciences 3 (2013) 343-353 351
Tab le 2. Productive evaluation1 of silkworm Bombyx mori cocoons and pupae exposed to several concentrations of chloran- tranili-
prole insecticide.
Instar DE [] (ppm) NC NCW TSC TSCW LP
4th instar 1st 0.05 19.00 a 2.46 a 0.33 b 0.97 a 19.33 a
4th instar 1st 0.025 19.00 a 2.65 a 0.33 b 0.98 a 19.66 a
4th instar 1st 0.0125 19.33 a 2.55 a 0.00 b 0.00 a 19.33 a
4th instar 1st 6.25 × 10−3 19.66 a 2.41 a 0.33 b 1.40 a 18.66 a
4th instar 1st 3.13 × 10−3 19.00 a 2.48 a 0.33 b 0.73 a 19.66 a
4th instar 1st 1.57 × 10−3 19.33 a 2.47 a 0.66 b 0.83 a 19.33 a
4th instar 1st Control group 19.66 a 2.47 a 0.33 b 1.00 a 20.00 a
4th instar 3rd 0.05 17.33 a 2.37 a 0.33 b 0.80 a 17.00 a
4th instar 3rd 0.025 18.66 a 2.37 a 0.66 b 1.71 a 19.00 a
4th instar 3rd 0.0125 19.33 a 2.31 a 0.0 0b 0.00 a 20.00 a
4th instar 3rd 6.25 × 10−3 17.66 a 2.30 a 0.33 b 0.68 a 19.00 a
4th instar 3rd 3.13 × 10−3 19.00 a 2.29 a 0.33 b 0.82 a 19.66 a
4th instar 3rd 1.57 × 10−3 18.66 a 2.40 a 0.0 0b 0.00 a 19.33 a
4th instar 3rd Control group 19.33 a 2.46 a 0.66 b 1.57 a 19.66 a
5th instar 1st 0.05 16.66 a 2.35 a 0.00 b 0.00 a 17.33 a
5th instar 1st 0.025 16.66 a 2.23 b 0.66 b 1.74 a 19.33 a
5th instar 1st 0.0125 18.66 a 2.26 b 0.33 b 0.99 a 19.33 a
5th instar 1st 6.25 × 10−3 19.33 a 2.29 a 0.00 b 0.00 a 19.66 a
5th instar 1st 3.13 × 10−3 19.00 a 2.30 a 0.33 b 0.79 a 19.33 a
5th instar 1st 1.57 × 10−3 17.00 a 2.29 a 1.00 b 1.53 a 19.66 a
5th instar 1st Control group 19.33 a 2.41 a 0.66b 1.46 a 20.00 a
5th instar 5th 0.05 -* -
* 0.66 b 1.40 a -*
5th instar 5th 0.025 6.66 b 2.13 b 7.66 a 2.17 a 8.66 d
5th instar 5th 0.0125 16.66 a 2.11 b 0.00 b 0.00 a 19.66 a
5th instar 5th 6.25 × 10−3 19.66 a 2.16 b 0.66 b 1.03 a 20.00 a
5th instar 5th 3.13 × 10−3 18.66 a 2.27 a 0.66 b 0.64 a 20.00 a
5th instar 5th 1.57 × 10−3 18.33 a 2.16 b 0.33 b 0.64 a 20.00 a
5th instar 5th Control group 18.33 a 2.20 b 0.00 b 0.00 a 20.00 a
5th instar 1st - 2nd 0.05 8.33 b 1.85 b 0.33 b 0.60 a 11.33 c
5th instar 1st - 2nd 0.025 17.33 a 2.07 b 0.66 b 1.00 a 16.00 b
5th instar 1st - 2nd 0.0125 18.00 a 2.17 b 0.00 b 0.00 a 19.00 a
5th instar 1st - 2nd 6.25 × 10−3 18.00 a 2.20 b 0.00 b 0.00 a 20.00 a
5th instar 1st - 2nd 3.13 × 10−3 17.33 a 2.08 b 0.00 b 0.00 a 20.00 a
5th instar 1st - 2nd 1.57 × 10−3 18.66 a 2.12 b 0.00 b 0.00 a 20.00 a
5th instar 1st - 2nd Control group 16.33 a 1.99 b 0.00 b 0.00 a 20.00 a
5th instar 5th - 6th 0.05 -* -
* - * - * - *
5th instar 5th - 6th 0.025 0.33 c** 0.70 c** 0.00 b** 0.00 a** 0.33 e**
5th instar 5th - 6th 0.0125 17.00 a 2.03 b 0.33 b 0.60 a 20.00 a

R. E. F. Munhoz et al. / Open Journal of Animal Sciences 3 (2013) 343-353
352
Continued
5th instar 5th - 6th 6.25 × 10−3 16.00 a 2.27 b 2.33 b 1.53 a 19.00 a
5th instar 5th - 6th 3.13 × 10−3 19.33 a 2.02 b 0.00 b 0.00 a 20.00 a
5th instar 5th - 6th 1.57 × 10−3 18.00 a 2.05b 0.00 b 0.00 a 19.33 a
5th instar 5th - 6th Control group 17.00 a 1.98 b 0.00 b 0.00 a 20.00 a
1Productive evaluation: DE—Day of exposure to chlorantraniliprole, []—Chlorantraniliprole concentration (ppm), NC—mean number of normal cocoons,
NCW—mean weight of normal cocoon, TSC—mean number of thin shell cocoons, TSCW—mean weight of thin shell cocoons weight, LP—mean number of
alive pupae. Means followed by distinct letters (a-d) in the columns are different by the Scott-Knott test (P < 0.05); *Missing plots; **Value of one experimental
plot.
Figure 2. Bombyx mori 5th instar larvae midgut epithelial cells. (A) Morphology of
the control group midgut epithelium showing columnar cell nuclei (white arrow), a
goblet cell lumen (★), microvilli (hollow arrow), muscle layer (MM) and hemo-
coel (He). (B), (C) and (D) Morphologic changes to the midgut epithelium caused
by chlorantraniliprole: (B) The nucleus of the columnar cells (white arrow) in the
dilated cellular apex and an abundance of hypertrophic regenerative cells (black
arrows) in the basal region of epithelium. (C) Part of the cytoplasm of columnar
cells is released by budding, culminating in the expulsion of the cell nucleus to the
intestinal lumen (Lu), as seen in detail in (D). Hematoxilin-eosin stain.
many of the symptoms such as irregular development,
rosary-shaped excrements, and even the production of
thin-shelled cocoons.
4. CONCLUSIONS
Aerial application of insecticides to agricultural fields
may cause damage to silkworm production when the
chemicals drift to nearby mulberry plantations that pro-
vide food for the silkworms. Several authors have re-
ported disturbances to the silkworm life cycle and co-
coon production due to different types of pesticides
[16,20,21].
Although most of the studies reported insecticides
that belong to classes which do not include chloran-
traniliprole, which is an anthranilic diamide, the symp-
toms of intoxication are very similar to those we ob-
served. Chlorantraniliprole is damaging to Brazilian silk-
worm hybrids, even in low concentrations, as revealed
by our study.
Hence, actions should be taken to avoid this damage.
Previous report proposed that authorities in pesticide
management should take measures to reduce the influ-
ence of chlorantraniliprole on silkworm rearing and for-
bid its use in sericulture areas [11]. Although chloran-
traniliprole is not used in mulberry plantations, it can
injure silkworms whenever this insecticide is applied
incorrectly near mulberry fields and drifts into them.

R. E. F. Munhoz et al. / Open Journal of Animal Sciences 3 (2013) 343-353 353
5. ACKNOWLEDGEMENTS
This work was supported by CAPES, CNPq, FINEP/Fundação Arau-
cária and Secretaria de Estado da Ciência, Tecnologia e Ensino Superior—
FUNDO PARANÁ.
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