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Chitosan Efficacy against the Cotton Leaf Worm Spodoptera littoralis (Boisd.)
(Lepidoptera: Noctuidae)

Article  in  Egyptian journal of pest control · December 2014

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 Egyptian Journal of Biological Pest Control, 24(2), 2014, 461-464

Chitosan Efficacy against the Cotton Leaf Worm

Spodoptera littoralis (Boisd.) (Lepidoptera: Noctuidae)

Sayed*, R. M.; A. A, Khidr** and Hemat Z. Moustafa**

*
Natural Product Dept., National Center of Radiation Research and Technology, Atomic Energy Authority, Cairo, Egypt.
**
Plant Protection Research Institute, Agricultural Research Center, Dokki, Giza, Egypt.
(Received: October 28, 2014 and Accepted: December 2, 2014)

ABSTRACT

Chitosan is a derivative of chitin naturally occurring compound and became of great interest not only for its medical
utilities but also as an alternative to chemical insecticides. Insecticidal activity of chitosan was investigated against the
4th larval instar of the cotton leaf worm Spodoptera littoralis (Boisd.) under laboratory conditions. Results showed that
the percent mortality increased with increasing chitosan concentration. LC 50 was 734.923ppm. Marked changes were
recognized in some enzymatic activity when chitosan was applied at the LC50 level. Significant decrease was recorded in
the levels of protease, invertase, phenoloxidase, chitinase and acetylcholinesterase activities. On the other hand,
acetylcholine concentration increased after chitosan application. These findings suggest that chitosan caused
physiological disturbance in S. littoralis larvae and may be used in IPM programs for controlling this pest.

Key words: Spodoptera littoralis, Chitosan, Physiological disturbance, Enzymatic activities.

INTRODUCTION
The Egyptian cotton leaf worm, Spodoptera
littoralis (Boisd.) (Lepidoptera: Noctuidae) is an
economically important polyphagous pest, that
causes considerable destruction to numerous
important crops in Egypt (Rawi et al., 2011).
Chemical insecticides are the most effective
control means for this pest, but this approach has
become less attractive due to its side effects;
environmental pollution, insect resistance,
disturbance in natural balance between pests and their
natural enemies and high cost of pesticides (Gamil
et al., 2011). The ultimate aim of recent researches
focuses on development and evaluation of various
alternative control strategies to reduce dependence
on synthetic pesticides. Recently, attention has been
focused on using natural materials (Badawy and
El-Aswad, 2012).
Chitosan is derived by deacetylation of chitin, the
second most abundant natural biopolymer isolated
from crustaceans such as crab and shrimp as well as
from fungi (Kurita et al., 2000). Chitosan may serve
as a good alternative because, at its relatively low
dosage levels used for controlling pests, it can be
considered non-toxic to vertebrates and humans,
biodegradable and it may possess insecticidal and
microbicidal properties (Rabea et al., 2003 and
Badawy et al., 2005).
The present study aimed to throw light on
insecticidal effect of chitosan on the 4th larval instar
of S. littoralis and on some physiological activities of
the pest.
MATERIALS AND METHODS
Rearing of Spodoptera littoralis
[
A culture of the cotton leaf worm, S. littoralis,
used in this study, was obtained from a standard
laboratory colony, reared at Cotton Leaf Worm Dept.,
Plant Protection Research Institute; Agricultural
Research Center (ARC), Giza, Egypt. The pest was
fed on castor bean leaves and maintained under
laboratory conditions of 27±2 ºC and R.H. 70±5%
according to the method described by El-Defrawi et
al. (1964).
Chitosan
Chitosan was obtained from the Faculty of
Science, Cairo University, Giza, Egypt.
Toxicity assay
A serial of concentrations was prepared from the
stock solution based on ppm of chitosan (w/v)
dissolved in water for the tested larvae. Castor bean
leaves were dipped in each concentration for 30
seconds then left to dry at room temperature for an
hour. The 4th larval instar was left for feeding on the
treated leaves in glass jars covered with muslin.
Larvae were tested in three replicates (40 larvae/
replicate). Mortality percentages were recorded 3
days after larval feeding and were corrected
according to Abbott’s formula in each case (Abbott,
1925). The LC50 values were determined using
Finney (1971).
Biochemical assay
Preparation of insect homogenates
Fourth instar larvae of S. littoralis were treated at
the LC50 level of chitosan. Batches of the treated alive
larvae were collected, weighted and counted, two
days post treatment. The larvae were mechanically
homogenized in 10 volumes (w/v) of 0.1 M phosphate
buffer, pH7 for 2 minutes, using a Teflon
homogenizer surrounded with a jacket crushed ice.
The homogenates were then centrifuged at 8000 rpm
for 15 min at 5°C, using cooling centrifuge. The
resulted supernatant was used to determine the
462

activities of protease, chitinase, phenoloxidase, test (1955) to estimate significant differences among
invertase, acetylcholinesterase (A.Ch.E.) and treatments. The 5% level of probability was used in
acetylcholine (A.Ch.). all statistical tests.
Protease activity
Protease activity was measured as described by RESULTS AND DISCUSSION
Trachell et al. (1972), with some modifications by
Larval mortality and efficacy of chitosan against the
measuring the increase in free amino acids split from
4th instar larvae of S. littoralis are shown in table (1).
substrate protein (albumin), during one hour
Data obtained indicated that the larval mortality
incubation at 30°C adopted by Lee and Takabashi
increased with increasing chitosan concentration.
(1966) method.
Mortality percentages ranged between 27.08 and
Chitinase activity 92.19% at the concentration rate of 312.5 and 5000
Depending on N-acetylglucoseamine produced as ppm, respectively.
a result of chitin digestion by chitinase, the colloidal Presented data indicated that chitosan showed
chitin was prepared (Bade and Stinson, 1981) and the insecticidal efficacy against the 4th instar larvae of S.
reaction mixture was prepared according to Ishaaya Littoralis larvae which agreed with the finding of
and Casida (1974). N-acetylglucoseamine was Zhang et al. (2003) who declared that chitosan was
determined by the sensitive method of Waterhoused active against lepidopterous and homopterous
et al. (1961). insects; causing up to 80% mortality. Insecticidal
Phenoloxidase activity activity of chitosan to Plutella xylostella L. was
Phenoloxidase activity was determined according higher than that to Spodoptera exigua Hübner, while
to the modification of Ishaaya (1971). Enzyme the mortality of six types of aphids ranged between
reaction was initiated by adding catechol solution and 60–80%.
after 1 min, the optical density was determined at 405 Enzymatic activity in larvae homogenate of S.
nm. littoralis after the treatment with LC50 of chitosan is
Invertase activity summarized in table (2). Protease, invertase and
Invertase activity was determined according to the phenoloxidase activities showed a significant
procedure described by Ishaaya and Swirski (1976), decrease compared to control. Likewise; chitinase
using 4% sucrose as a substrate. The reaction was activity ecreased without significance and remained
stopped by adding dinitrosalicylic acid for each tube at a level lower than that of the control.
in boiling water for 5 min. The enzyme activity was Previous results were coincided with Abd
expressed as µg glucose released /min/gm fresh EL-Mageed and Shalaby (2011) who found that the
weight. chemical insecticide (Kingbo) caused a significant
Acetylcholinesterase (A.Ch.E.) and Acetylcholine
(A.Ch.) activity Table (1): Susceptibility of the 4th instar larvae of the
Acetylcholinesterase (A.Ch.E.) activity was cotton leaf worm, S. littoralis to chitosan toxicity
Chitosan Observed mortality Corrected
measured according to the method described by
Conc. (ppm) (%) after 3 days mortality (%)
Simpson et al. (1964), using acetylcholine bromide
control 4 0
(AchBr) as a substrate. The decrease in AchBr from
312.5 30 27.08
hydrolysis by AchE was read at 515 nm. For ACh 625 47.5 44.27
determination, the same method of AChE, except 1250 67.5 66.15
incubation of the insect homogenate with the 2500 82.5 81.77
substrate was followed. 5000 92.5 92.19
Statistical Analysis Slope 1.727
Data were statistically evaluated by analysis of LC50 734.923
variance (F), followed by Duncan's multiple range LC90 4049.595

Table (2): Changes in protease, invertase, phenoloxidase and chitinase activities of the total body tissue
homogenate of the 4th larval instar of S. littoralis treated with the LC50 of chitosan
Protease Activity Invertase Activity Phenoloxidase Activity Chitinase Activity
Treatment
µg alanine/min/g.b.wt µg glucose/min/g.b.wt unitsx103/min/g.b.wt µgglucose/min/g.b.wt
Control 14.33±0.72a 2705±78.6 a 4661±106.4 a 823.33±20.2
b b
LC50 of chitosan 8.06±0.67 1825±27.3 3221.3±61.5 b 809.67±8.64
F value 41.43** 111.735*** 137.18*** 0.384ns
***
Values represent the mean ± S.E of ten larvae for each group. Statistically significant at p<0.05.
a&b Statistically significant at p<0.05 with reference to control. Ns: not significant at p<0.05.

Table (3): Changes in acetylcholine (A.Ch.) and
acetylcholinesterase (A.Ch.E.) concentrations in
the total body tissue homogenate of 4th instar
larvae of S. littoralis treated by LC50 of chitosan
Acetylcholine Conc. Acetylcholinesterase
Treatment
µg chBr/min/g.b.wt Activity µg ch/g.b.wt
Control 47.27±1.9 20.66±1.3b
LC50 of chitosan 40.47±1.5 28.78±1.1 a
F value 7.38ns 21.391**
Values represent the mean ± S.E of ten larvae for each group.
a&b Statistically significant at p<0.05 with reference to control.
***Statistically significant at p<0.05, Ns: not significant at p<0.05.
decrease in chitinase activity of S. littoralis larvae.
Also, Al-shannaf et al. (2012) carried out field
experiments against Helicoverpa armigera larvae and
found that Chlorfluazuron, pyriproxyfen and Dipel
DF significantly decreased the invertase activity after
3 and 7 days post treatment.
Changes in total body tissue activity of
acetylcholinesterase (A.Ch.E) and acetylcholine
(A.Ch.) of the 4th imstar larvae of S. littoralis are
shown in table (3). Data proved that treatment
implemented by the LC50 of chitosan; significantly
declined (A.Ch.E.) activity. In contrast, acetylcholine
concentration showed increase after chitosan
treatment, in comparison with the control.
Obtained results are in line with those reported
by Abd EL-Mageed and Shalaby (2011) who reported
that five new insecticide mixtures: chlorosan,
feroban, cygron, engeo, and kingbo caused a
significant increase in acetyl cholinesterase activity
of S. littoralis larvae as compared to the control. In
this field of study, Rabea et al. (2010) found that
oxymatrine and chlorfluazuron inhibited A.Ch.E in
Apis mellifera L. under laboratory conditions.
As described in other studies, the mechanism
could include repellency, disruption of feeding
physiology or a chronic toxicity possibly related to
the insecticidal action (Ross and Brown, 1982).
Kauss et al., (1989) clarified that the activity of
chitosan is mediated through the interaction of its
molecule with negatively-charged phospholipids in
the cells which lead to changes in the cell membrane
permeability then death. Kaku et al., (1997) stated
that the activity of chitosan created disturbance in
chitinase and glucanase activities. Furthermore, the
response to chitosan include generation of reactive
oxygen species (ROS) (Kuchitsu et al., 1995) which
cumulatively, known as oxidative stress, causing
cellular, DNA, RNA and protein damage and
contributes to physiology of ageing, then cell death
(Patel et al., 1999).
The above results expressed that chitosan had
insecticidal activity against S. littoralis and had
destructive effect on some important enzymes
463
(reduction of protease and invertase), hormonal
defense (inhibition of phenoloxidase) and affected
nerve impulses by increase acetylcholinesterase, and
reduction of acetylcholine concentration.
Subsequently, it could be concluded that chitosan
may form a contribution in IPM program of S.
littoralis control strategy.
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