RABEA, E.I., BADAWY, M.E.I., STEURBAUT, W ... - Insects.ugent.be

Comm. Appl. Biol. Sci, Ghent University, 70/4, 2005 823
INSECTICIDAL AND GROWTH INHIBITION EFFECTS
OF CHITOSAN DERIVATIVES CONTAINING AN N-ALKYL
GROUP ON THE COTTON LEAFWORM
SPODOPTERA LITTORALIS
E.I. RABEA 1 , M.E.I. BADAWY 1 , W. STEURBAUT 2 , T.M. ROGGE 3 ,
C.V. STEVENS 3 & G. SMAGGHE 2
1 Department of Pesticide Chemistry, Faculty of Agriculture, Alexandria University, Egypt,
2 Department of Crop Protection; 3 Department of Organic Chemistry; Faculty of Bioscience
Engineering, Ghent University, Coupure links 653, B-9000 Gent, Belgium
E-mail: entsar_ibrahim@yahoo.com
INTRODUCTION
Deacetylation of chitin, the second most abundant biopolymer isolated from
insects, crustaceans such as crab and shrimp as well as from fungi, leads to
poly-(1,4)-D-glucosamine or the so called chitosan with excellent biodegradable
and biocompatible characteristics (Kurita et al., 2000). Chitosan has
become of great interest not only as an underutilized resource, but also as a
new functional material with high potential in various fields. N-alkyl chitosan
derivatives were prepared by introducing alkyl groups into the amine
groups of chitosan (Muzzarelli et al., 1983 and 1990). In this study, a series
of chitosan derivatives containing an alkyl group, N-alkyl chitosan (NAC)
derivatives, were synthesized using a reductive alkylation reaction to examine
their insecticidal and growth inhibition activities against the larvae of
the cotton leafworm Spodoptera littoralis (Boisduval) (Lepidoptera: Noctuidae).
MATERIALS AND METHODS
Materials
Chitosan of low molecular weight (made from crab shells, 85% degree of
deacetylation, DDA) and aldehydes were purchased from Sigma-Aldrich Co.
(Bornem, Belgium). Soybean-wheat germ insect artificial diet was purchased
from Stonefly Ind. (Bryan, TX, USA). NAC derivatives were synthesized according
to Kim et al., (1997). All materials were used without further purification.
Methods
Insecticidal feeding and Growth-inhibitory bioassay against S. littoralis
Third-instar larvae of S. littoralis were selected from a laboratory colony reared
on artificial diet under controlled conditions at 25±2°C, 70±5% RH and a
16h light photoperiod (Smagghe et al., 2001). Chitosan and synthesized derivatives
were tested at 5 g kg -1 in artificial diet, with 30 larvae per product
each. After 7 days of continuous feeding on treated artificial diet, mortality

824
was scored and compared with the control that had been exposed to a diet
treated with solvent only (water and 1% acetic acid in water) (Smagghe et al.,
2000).
The growth inhibitory activity against S. littoralis was measured by a feeding
trial using artificial diet based on larval weight gain through 5 days of feeding
on artificial diet incorporated with the most active compounds with a
growth-inhibitory effect, namely N-(propyl) chitosan, N-(undecanyl) chitosan
and N-(3-phenylpropyl) chitosan. Growth inhibition percentage was calculated
from this equation:
Growth inhibition (%) = [(CL-TL)/CL] x100
Where CL is the larval weight gained in the control and TL is the larval weight
gained in the treatment.
RESULTS AND DISCUSSION
Insecticidal activity of NAC derivatives against S. littoralis
The NAC derivatives had a better insecticidal activity than chitosan itself
(Table 1). This might be explained by the increased lipophilicity of the derivatives
which can lead to improved membrane permeability. The result shows
that the most active compound was N-(3-phenylbutyl) chitosan (17) with
50% mortality, followed by N-(tridecanyl) chitosan (13) and N-(2-phenylethyl)
chitosan (14) with 47% and 37%, respectively. N-pentyl chitosan (2), N-
(ethylbutyl) chitosan (7), N-(2-nonenyl) chitosan (10) and N-(3-phenylpropyl)
chitosan (16) were the moderately active compounds. N-(nonyl) chitosan (9)
was the least active in these derivatives. As exemplified with compounds 2
and 3, branching of the side chains led to a slight decrease in the insecticidal
activity, but the activity was still relatively high. The activity was further
increased when a double bond was introduced in the side chain (compound
9 vs. 10), but overall the activity was still adequate. The exchange of
the hydrogen atom in the long chain with a phenyl ring led to high toxicity
toward larvae of S. littoralis (see compounds 14, 15, 16, 17). In addition, our
results demonstrated that the activity was elevated with an increase of the
chain length. From our results it can be concluded that chitosan has low
insecticidal activity against larvae of S. littoralis but chemical modification of
it led to a moderate increase of the activity especially for N-(3-phenylbutyl)
chitosan (17) and N-(tridecanyl) chitosan (13).
In addition, the growth inhibitory activity of N-(propyl) chitosan (1), N-
(undecanyl) chitosan (12) and N-(3-phenyl propyl) chitosan (16) at 5 g kg -1 on
S. littoralis are shown in Table 2. The tested compounds inhibited the larval
growth in a time-dependant manner from the first day of feeding on the treated
diet. As shown in Table 2, the maximum inhibition of growth was observed
on the 4 th day. The development of larvae reared on a treated diet was
shown in Fig. 1. There was a 2-3 fold reduction in weight gain and length in
larvae fed with treated diet. Typically in intoxicated larvae, the normal ecdysis
process was affected with symptoms of inhibition of feeding and weight
gain and the larvae were very small compared to the control. An incomplete
shedding of the old cuticle and the old head capsule covering the new white

Comm. Appl. Biol. Sci, Ghent University, 70/4, 2005 825
capsule were observed. Inhibition of growth and antifeedant effects was observed
(Fig. 1). However, the targets and mechanism of action in insects for
these chitosan derivatives remain unknown so far. As described in the literature,
the mechanisms could include repellency, disruption of feeding physiology,
or a chronic toxicity possibly related to the insecticidal action (Ross
and Brown, 1982).
Table 1. Insecticidal activity (%) of NAC derivatives at 5 g kg -1 against third-instar
larvae of S. littoralis by feeding on artificial diet. Data are expressed as mean percentages
± SEM based on 3 replicates per tested compound, n=30.
OH
OH
O
O O
HO
O
NH HO
O
NH
H 2 C
R AC
n
Compound R (DS) Mortality (%)
± SE
1 CH3CH2 0.05 26.7±6.7
2 CH3(CH2)3 0.28 30±5.8
3 CH3CH(CH3)CH2 0.04 20±9.9
4 HOCH2(CH2)3 0.02 10±0.0
5 CH3CH(CH3)(CH2)2 0.17 16.7±3.3
6 cyclohexyl 0.25 16.7±12.0
7 CH3CH(C2H5)CH2 0.15 33.3±3.3
8 CH3(CH2)5 0.23 13.3±8.8
9 CH3(CH2)7 0.04 6.7±6.7
10 CH3(CH2)5CH=CH 0.22 30±15.3
11 CH3(CH2)8 0.22 33.3±3.3
12 CH3(CH2)9 0.06 30±0.0
13 CH3(CH2)11 0.09 46.7±8.8
14 (C6H5)CH2 0.10 36.7±13.3
15 (C6H5)2CH 0.32 33.3±6.7
16 (C6H5)CH2CH2 0.14 33.3±6.7
17 CH3CH(C6H5)CH2 0.37 50±0.0
a Degree of substitution
Table 2. Growth inhibition activity (%) of N-(propyl) chitosan, N-(undecanyl) chitosan
and N-(3-phenylpropyl) chitosan at 5 g kg -1 on S. littoralis during 5 days of feeding on
treated diet. Data are expressed as mean percentages ± SEM based on 3 replicates per
tested compound; n=30.
Compound
Time (day)
1 2 3 4 5
1 57.04±0.02 61.09±0.04 66.75±0.05 75.86±0.04 70.05±0.16
12 51.46±0.004 65.35±0.04 63.67±0.04 65.94±0.02 59.46±0.14
16 44.25±0.02 55.27±0.03 60.41±0.03 64.73±0.04 59.53±0.15

826
450
400
350
Contol
Compound 1
Compound 12
(A)
300
Compound 16
Weight of larvae (mg)
250
200
150
100
50
0
0 1 2 3 4 5
Time (day)
Control
(1)
(12)
Larval length
(mm±SEM )
(26.8±0.10)
(12.4±0.08)
(13.2±0.05)
Figure 1. Development of S. littoralis reared
on artificial diet containing 5 g kg -1 of N-
(propyl) chitosan (1), N-(undecanyl) chitosan
(12) and N-(3-phenylpropyl) chitosan (16). A:
Weight of larvae (mg) of control and treatments
were critically measured on several
days of treatment and data are expressed as
mean percentages ± SEM based on 3 replicates
per tested compound; n=30. B: Photograph
of larvae grown on a diet without
treatment showing normal growth and on a
diet containing 5 g kg -1 of treatments showing
stunted growth.
(16)
(13.8±0.10)
(B)
REFERENCES
KIM C.H., CHO J.W. & CHUN H.J. (1997). Synthesis of chitosan derivatives with quaternary
ammonium salt and their antibacterial activity. Polym Bull 38:387-393.
KURITA K., KOJIMA T., NISHIYAMA Y. & SHIMOJOH M. (2000). Synthesis and some properties
of nonnatural amino polysaccharides: Branched chitin and chitosan. Macromol
33:4711-4716.
MUZZARELLI R., TARSI R., FILLIPINI O., GIOVANETTI E., BIANGINI G. & VARALDO P.E. (1990).
Antimicrob. Agents Chemother. 34:2019-2023.
MUZZARELLI R.A.A., TANFANI F., EMANUELLI M. & MARIOTTI S. (1983). The characterization
of N-methyl, N-ethyl, N-propyl, N-butyl and N-hexyl chitosans, novel film-forming
polymers. J Membrane Sci 18:295-308.

Comm. Appl. Biol. Sci, Ghent University, 70/4, 2005 827
ROSS D.C. & BROWN T.M. (1982). Inhibation of larval growth in Spodoptera frugiperda
by sublethal dietary concentrations of insecticides. J Agric Food Chem 30:193-196.
SMAGGHE G., CARTON B., DECOMBEL L. & TIRRY L. (2001). Significance of absorption,
oxidation, and binding to toxicity of four ecdysone agonists in multi-resistant cotton
leafworm. Arch Insect Biochemistry & Physiology 46:127-139.
SMAGGHE G., MEDINA P., SCHUYESMANS S., TIRRY L. & VINUELA E. (2000). Insecticide
resistant monitoring of tebufenozide for managing Spodoptera exigua (Hübner
[1808]). Bol San Veg Plagas 26:475-481.

828