48 

ISJ 16: 48-59, 2019                                                                 ISSN 1824-307X 

 
 

RESEARCH REPORT 

 
Immunological and antioxidant responses of larval Helicoverpa armigera 
(Lepidoptera: Noctuidae) to gibberellic acid in the diet 
 
D Shayegan, JJ Sendi*, A Sahragard, A Zibaee 
 
Department of Plant Protection, Faculty of Agricultural Sciences, University of Guilan, Rasht, Iran 

 
 

   Accepted March 8, 2019 

 
Abstract 

Gibberellic acid (GA3) is usually used as a plant growth regulator to enhance the quality and 
quantity of crop yield. Moreover, GA3 may also affect insects and other herbivores that rely on a 
particular plant for their living. Hence, in the present study the defensive mechanisms of Helicoverpa 
armigera (Hübner) fed on the diet contating GA3 have been considered. The larvae fed with GA3 
significantly exhibited cellular and humoral inhibitory responses such as, reduced nodule formation 
and phenoloxidase activity. Antioxidant system was also affected; the lowest activity of peroxidase, 
catalase, superoxide dismutase, ascorbate peroxidise, glucose-6-phosphate dehydrogenase, and 
glutathione S-transferases was observed in control larvae while the highest activity was found in those 
larvae that were provided with a diet containing 800 µg/g GA3. The metabolites like triglyceride, 
glycogen, and cholesterol were reduced compared to the control. It is concluded that the use of plant 
growth regulators like GA3 not only does benefit plants for their growth and yield, but also can 
somehow help plants to withstand the impact of herbivores. Hence, studies covering direct field 
collected insects from crop plants treated with PGRs would be beneficial for further studies.  
 
Key Words: cellular immunity; detoxifying enzymes; oxidative stress; phenoloxidase 

 

 
Introduction 

 
Helicoverpa armigera (Hübner) (Lepidoptera: 

Noctuidae), is a cosmopolitan insect pest of various 
crops such as tomato, soybean, pea, cowpea, and 
chickpea (Sharma, 2005). Gibberellic acid (GA3) as 
a plant growth regulator is being used in certain 
crops to improve the quality and quantity of the 
yield. For example, tomato crop (Gelmesa et al., 
2012; Ning and Subroto, 2018), soybean, cowpea 
(Leite et al., 2003; Emongor, 2007), chickpea, and 
pea (Bora and Sarma, 2006; Mazid, 2014).  

In addition, the adverse impacts of GA3 use on 
insects have also been worked out in some studies. 
For example, a reduction in fecundity and fertility 
and shortening of the longevity has been reported in 
Bactrocera cucurbitae (Coquillett) (Diptera: 
Tephritidae) (Kaur and Rup, 2002; Kaur and Rup, 
2003a). Abdellaoui et al. (2009) demonstrated that 
the use of GA3 in the diet led to higher mortality in 
Spodoptera littoralis F. (Lepidoptera, Noctuidae) and 
also in Locusta migratoria L. (Orthoptera, Acrididae). 
___________________________________________________________________________ 

 
Corresponding author: 

Jalal Jalali Sendi 

Department of Plant Protection 
Faculty of Agricultural Sciences 
University of Guilan 

Rasht, Iran 
E-mail: jjalali@guilan.ac.ir 

 
 

The effects were particularly evident in discrepancy 
occurred in exuviations. The damages were also 
traced in target tissues of foregut and gastric caeca 
of L. migratoria (Abdellaoui et al., 2009). A decrease 
in most of the stored energy by GA3 has been 
reported in the hemolymph of Galleria mellonella L. 
(Lepidoptera: Pyralidae) (Uckan et al., 2011). 
Altuntaş et al. (2014) showed undulating titre in 
haemolymph free amino acids in G. mellonella 
larvae with different concentrations of GA3 used. 
Gibberellic acid also significantly reduced proteins, 
carbohydrates, and lipids in the ovaries of L. 
migratoria, as well as the amounts of ecdysteroid in 
ovaries and freshly laid eggs. In addition, GA3 
significantly reduced both fecundity and fertility in L. 
migratoria (Abdellaoui et al., 2015). 

To the best of our knowledge, there is no report 
so far considering GA3 on H. armigera and 
specifically looking into the mode of action of this 
beneficial compound. Therefore, various aspects of 
its effect on H. armigera were investigated 
including life table parameters (Shayegan et al., in 
press) and digestion (Shayegan et al., in press), and 
here we report its effect on immunity and antioxidant 
system which has remained unexplored in the 
studied insects. 

Immune responses in insects under the effect 
of various stresses including xenobiotics are depicted 



49 

 
 
Fig. 1 The total hemocyte numbers in the sixth instar H. armigera larvae fed on control and different 
concentrations of gibberellic acid (C= 0 µg/g diet - C800 = 800 µg/g diet). Statistical differences have been done 
within each treatments and marked by various letters (at p< 0.05, Tukey’s test) 
 
 
 
 
 
in the of form cellular and humoral responses. 
Cellular responses often involve hemocytes 
(particularly granulocytes and plasmatocytes) that 
are directly responsible for processes such as 
phagocytosis and nodule formation against foreign 
bodies entering hemolymph. The humoral 
responses include antimicrobial peptides and 
prophenoloxidase system that are responsible for 
eliminating the pathogens (Beckage, 2011). 
Antioxidants are compounds that inhibit oxidation 
and removes reactive oxygen species (ROS) which 
can damage the cells of any organism (Felton and 
Summers, 1995; Akbar et al., 2012). Similarly, 
detoxifying enzymes like general esterases and 
glutathione S-transferase react against external 
stimulants and are also considered as a part of 
insect defense mechanism (Grant and Matsumura, 
1989; Hemingway and Karunaratne, 1998; Zibaee 
and Bandani, 2010; Valizadeh et al., 2013). 

 There are some works which explore the effect 
of GA3 on insects, including L. migratoria and G. 
mellonella (Uckan et al., 2011; Altuntaş et al., 2014; 
Abdellaoui et al., 2015). It is a common belief that 
plant growth regulators (PGRs) like GA3 benefit 
plants in terms of their growth and yield. This 
compound can also somehow help plants to 
withstand the pressures implemented through 
herbivores. Our main aim in the present study was 
to explore the mechanisms indulged in reduction of 
insect immunity and make them susceptible to 
various environmental pressures.  
 
Materials and Methods 
 
Insect rearing and experimental conditions 

The larvae of Helicoverpa armigera (Hübner) 
were collected from tomato farms at the University 
of Guilan in the city of Rasht in northern Iran 

(37°16′51″N 49°34′59″E). They were individually 
reared on artificial diet containing cowpea (204 g), 
yeast (30 g), wheat germ (30 g), ascorbic acid (3.5 
g), sorbic acid (1.3 g), formalin (2.7 mL), refined 
sunflower oil (4 mL), agar (14 g), and distilled water 
(600 mL) (Shorey and Hale, 1965). The insects 
were reared on this diet for two generations and the 
third generation was provided with the same diet 
including gibberellic acid (Suvchem, Mumbai, 
Maharashtra, INDIA). The concentrations included 
100, 200, 400, and 800 µg/g diet (denoted as C100, 
C200, C400, and C800, respectively). The rearing 
jars included transparent plastic containers (10×5×5 
cm) maintained at 26 ± 1 °C, 65 ± 5% RH, and a 
photoperiod of 16:8 hours (L:D). The adult moths of 
both sexes were released into cages made of 
transparent PVC (14×9×20 cm), enclosed at the top 
with a fine-mesh net. A cotton wool soaked in 10% 
honey solution was provided to the moths. The 
hatched larvae were directly collected from the 
rearing boxes using a soft brush. They were reared 
on artificial diet including various concentrations of 
GA3. The desired concentrations of GA3 were 
made in distilled water and then were mixed with the 
diet. The controls received only equal amounts of 
distilled water in the diet. Thirty first instar larvae 
were individually reared on the diet containing 
different concentrations of GA3. They were 
maintained on their respective diet up to the sixth 
instar larval stage. Similarly, the controls were 
provided with their own diet. Totally, 150 larvae 
were used in these experiments. 
 
Total and differential hemocyte count (THC and 
DHC) 

The hemolymph of 48 hours sixth instar larvae 
(in all concentrations and controls) were collected 
from the first abdominal proleg. For THC a Neubauer 



50 

 
 
Fig. 2 Percentage of granulocytes and plasmatocytes in the sixth instar H. armigera larvae fed on control and 
different concentrations of gibberellic acid (C= 0 µg/g diet - C800 = 800 µg/g diet). Statistical differences have 
been done within each treatments and marked by various letters (at p< 0.05, Tukey’s test) 
 
 
 
 
 
hemocytometer (HBG, Germany) was used. For this 
purpose, 10 µL hemolymph was mixed with 290 µL 
of anticoagulant solution (0.017 M EDTA, 0.041 M 
Citric acid, 0.098 M NaOH, 0.186 M NaCl, pH 4.5) 
(Gupta,1979; Amaral et al., 2010). The DHC was 
counted using heat fixed larvae (larvae were 
immersed in hot distilled water (60 °C) for five min), 
after drying with blotting paper, the first abdominal 
proleg was excised and a drop of hemolymph was 
released on a clean slide and a smear was made 
using another slide. The air-dried smears were 
stained with 1 to 10 diluted stock Giemsa (Merck, 
Germany) and after 14 min the slides were washed 
by distilled water. The smears were dipped for 5 sec 
in saturated lithium carbonate (LiCO3) for 
differentiation of cytoplasm and nucleus and then 
washed again in distilled water for a few minutes, 
dried at room temperature and then permanent slide 
was prepared in Canada balsam (Merck, Germany). 
The cells were identified based on the 
morphological characteristics observed under a 
microscope (Leica light-microscope) (Baghban et 
al., 2018). Two hundred cells were randomly 
counted from four corners and central part of each 
slide (Wu et al., 2016). Totally, 800 cells from four 
individuals were counted and percentage of each 
cell type was calculated. The number of cells in 
controls was also simultaneously recorded.  

 
Immune response assay  

The latex beads were used to stimulate the 
immune system (Borges et al., 2008) of H. armigera 
in order to explore the interaction of gibberellic acid 
and its immunity response (i.e. nodulation process). 
Therefore, the larvae fed on GA3-mixed diet and 
control were injected with latex beads (10 distillate 

latex beads with distilled water using 10 μL Hamilton 
syringe). The THC and DHC were counted at 3, 6, 
12, 24, and 48-hour time intervals post injection as 
the method described above. Effect of latex beads 
on nodulation response was counted at 3, 6, 12, 24, 
and 48-hour time intervals post injection. For this 
purpose the hemolymph was collected from each 
larvae, then samples in four replicates were poured 
into the hemocytometer, and then the number of 
nodules were counted (Franssens et al., 2006, 
Seyedtalebi, 2017). 

 
Phenoloxidase activity assay  

For measuring phenoloxidase activity, 10 μL of 
hemolymph and 90 μL of ice-cold sterile phosphate 
buffered saline (PBS) (0.13 M NaCl, 2.68 mM KCl, 
8.1 mM Na2HPO4 and 1.47 mM KH2PO4, pH 7.4, 
autoclaved) were used. The L-DOPA (3,4- 
dihydroxyphenylalanine) (10 mM, Sigma-Aldrich 
Co., USA) was used as the substrate for assaying 
this enzyme using the method designed by Catalán 
et al. (2012), with some modifications. For this 
purpose, samples were centrifuged at 5,000 g at 4 
°C for 5 min. Then 50 μL of hemolymph-buffer 
supernatant was added to 150 μL of L-DOPA. The 
activity measurement of phenoloxidase was read at 
490 nm during the linear phase of the reaction. 
Calculation of specific activity was based on dividing 
absorbance with protein content in hemolymph 
using a microplate reader (Awareness Technology 
Inc., Florida, USA) (Khosravi et al., 2014; Baghban 
et al., 2018). Protein contents were quantified by the 
method of Lowry (1951) (using the manufacturer’s 
procedure, Biochem Co., Iran). To compute the 
specific activity of all enzymes, the absorbance 
values were divided by protein content. 



51 

 
 
Fig. 3 The effects of different concentrations of gibberellic acid (C= 0 µg/g diet - C800 = 800 µg/g diet) on total 
hemocyte numbers in the sixth instar H. armigera larvae after injection of latex beads. Statistical differences have 
been done within each time intervals and marked by various letters (at p< 0.05, Tukey’s test) 
 
 
 
 
 
The antioxidant defense assays 
Sample Preparation 

Nine larvae for every treated diet were 
randomly selected and the whole larvae were 
homogenized by a hand homogenizer in PBS (10 
mM). Then, the homogenate was centrifuged at 
28.600xg for 20 min at 4 °C. Supernatants were 
used to determine enzymes activities of H. armigera 
larvae. Protein contents were quantified by the 
method of Lowry (1951) (using the manufacturer’s 
procedure, Biochem Co., Iran). To obtain the 
specific activity of all enzymes, the absorbance 
values were divided by protein content. 
 
Peroxidase assay 

The method described by Addy and Goodman 
(1972) was used for determination of the activity of 
this enzyme. Hence, 3 mL of pyrogallol buffer (0.05 
M pyrogallol was mixed with 0.1 M PBS (pH 7.0)) 
were mixed with 500 μL of 1% H2O2. Then, 50 µL of 
sample was added. The absorbance was recorded 
every 30 sec for two min at 430 nm. Finally, the 
activity was calculated by an extinction coefficient of 
oxidized pyrogallol (4.5 liters/mol). 
 
Superoxide dismutase assay 

According to the method of McCord and 
Fridovich (1969), 80 µL of samples were added to 
500 µL of reaction solution (70 µM of nitroblue 
tetrazolium and 125 µM of xanthine dissolved in 
PBS, and were mixed with 20 µL of xanthine 
oxidase solution containing 10 mg of bovine serum 
albumin). Then, 100 µL of xanthine oxidase (5.87 
U/mL) (dissolved in 2 mL of PBS) was added to the 
reaction mixture and the reaction was initiated in 
darkness at 28 °C for 20 min. Absorbance was read 
at 560 nm. 

Catalase assay 
Catalase assay was performed following the 

method of Wang et al. (2001) with slight 
modification. The reaction mixture containing 50 µL 
of the sample and 500 µL of Hydrogen peroxide 
(1%) was incubated at 28 °C for 10 min. The 
absorbance was read at 240 nm. 
 
Ascorbate peroxidase assay 

For ascorbate peroxidase assay, 750 μL of 
sample was added to 250 µL PBS (67 mM, pH 7). 
Then, 70 µL of ascorbic acid (2.5 mM) and 200 µL 
of hydrogen peroxide (30 mM) were added to the 
reaction mixture. The absorbance was read at 290 
nm for 5 min (Asada, 1984). 
 
Glucose-6-phosphate dehydrogenase assay 

According to the method of Balinsky and 
Bernstein (1963), 100 μL of Tris-HCl (2-amino-2-
(hydroxymethyl) propane-1,3-diol; hydrochloride) 
(100 mM, pH 8.2) was mixed with 30 μL of MgCl2 
(0.1 M), 50 μL of NADP (0.2 mM), 50 μL of water, 
50 μL of the sample, and 100 μL of glucose-6-
phosphate dehydrogenase (6 mM). The absorbance 
of the reaction mixture was read at 340 nm. 
 
The detoxifying enzymes assay 
Sample Preparation 

Nine larvae for every treated diet were 
randomly selected and the whole larvae were 
homogenized by a hand homogenizer in PBS (10 
mM) and the homogenate was centrifuged at 13000 
rpm for 20 min at 4 °C. Supernatants were used for 
determining enzymes activities of H. armigera 
larvae. Protein contents were quantified by the 
method of Lowry (1951) (using the manufacturer’s 
procedure, Biochem Co., Iran). To compute the 



52 

 
 
Fig. 4 The effects of different concentrations of gibberellic acid (C= 0 µg/g diet - C800 = 800 µg/g diet) on 
percentage of granulocytes in the sixth instar H. armigera larvae after injection of latex beads. Statistical 
differences have been done within each time intervals and marked by various letters (at p< 0.05, Tukey’s test) 
 
 
 
 
 
specific activity of all enzymes, the absorbance 
values were divided by protein content. 
 
Esterase activity assay 

Esterase activity was measured by α-naphthyl 
acetate and β-naphthyl acetate (10 mM) as 
substrates, separately. Twenty μL of each substrate, 
50 μL fast blue RR salt (1 mM), and 20 μL of 
enzyme solution were mixed and incubated for 1 
min and then the absorbance was recorded at 450 
nm (Han et al., 1998). 
 
Glutathione S-transferase assay 

Twenty μL of CDNB (1-chloro-2, 4- 
dinitrobenzene, 20 mM) and DCNB (1, 2-dichloro-4-
nitro-benzene, 40 mM) were separately added to 10 
μL of the sample and 1 mM glutathione. The GST 
activity was read at 340 nm/min/mg protein after 5 
min (Oppenoorth et al., 1979). 
 
Amount of protein, glycogen, triglyceride, 
cholesterol, and glucose  
Protein  

Protein contents of the treated-larvae and 
control were assayed according to the method of 
Lowry et al. (1951) (recommended by Biochem Co., 
Iran).  
 
Glycogen  

For glycogen assay, the fat body of the control 
and treated-larvae were plunged in tubes contained 
1mL of lysis buffer (30% KOH with Na2SO4), at first. 
Then the samples of tubes were boiled for 20-30 
min. Then the tubes were shaken and cooled in ice. 
For precipitation of glycogen from the samples, 2 
mL of 95% EtOH was added and then was shaken 

again and cooled in ice for 30 min. Then the 
samples were centrifuged done at 22,000 g for 30 
min. The pellets (containing glycogen) were 
transported to new tubes and 1 mL of distilled water 
was added to the tubes and was shaken. The 
samples were mixed with 5% phenol and incubated 
in ice bath for 30 min. The absorbance of samples 
was read at 492 nm. A glycogen standard curve 
was used for calculating the glycogen concentration 
(Chun and Yin, 1998). 
 
Triglyceride  

A diagnostic kit (Pars Azmoon Co., Iran) was 
used for triglyceride assay. The method was based 
on the method of Fossati and Prencipe (1982), the 
reaction mixture included 10 µL of sample and 70 
µL of reagent containing phosphate buffer saline (50 
mM, pH 7.2), 4-chlorophenol (4 mM), adenosine 
triphosphate (2 mM), Mg2+ (15 mM), glycerokinase 
(0.4 kU/L), peroxidase (2 kU/L), lipoprotein lipase (2 
kU/L), 4-aminoantipyrine (0.5 mM), and glycerol-3-
phosphate-oxidase (0.5 kU/L). The reaction was 
done for 15 min at 25 °C. The samples and reagent 
absorbance were read at 546 nm. To obtain of the 
amount of triglyceride, the following equation was 
used: 
 
Mg/dl= (OD of sample)/ (OD of standard) × 0.01126 
 
Cholesterol  

Cholesterol was measured based on 
Richmond’s method (Richmond, 1973) and by 
utilizing a total cholesterol assay kit (Pars Azmoon 
Co., Iran). This method is based on hydrolyzing 
cholesterol esters with cholesterol oxidase, 
cholesterol esterase and peroxidase. 



53 

 
 
Fig. 5 The effects of different concentrations of gibberellic acid (C= 0 µg/g diet - C800 = 800 µg/g diet) on 
percentage of plasmatocytes in the sixth instar H. armigera larvae after injection of latex beads. The Statistical 
differences have been done within each time intervals and marked by various letters (at p< 0.05, Tukey’s test) 
 
 
 
 
 
Glucose  

Glucose assay was performed by a diagnostic 
kit (Pars Azmoon Co., Tehran, Iran) based on the 
procedure of Siegert (1987). Whole body (100 µL) 
added to 500 µL of 0.3 N perchloric acid and 
centrifuged at 12000g for 10 min. For determination 
of glucose concentrations, the supernatants were 
used.  
 
Statistical analysis 

The data obtained are presented as means ± 
standard error. All data were tested for normality 
(Kolmogorov-Smirnov test) and homogeneity of 
variance (Bartlett's test). The data were compared 
by one-way analysis of variance (ANOVA). 
Differences between the treatments were 
determined by Tukey’s family error rate by Minitab® 
statistical software. The statistical differences were 
considered at a probability less than 5%. 
 
Results 
 
The reaction of cellular immunity of H. armigera 
against GA3  

Effects of GA3 on total hemocyte count (THC) 
are shown in figure 1 (F= 14.98; df= 4, 19; p< 0.05). 
As depicted in figure 1 the lowest THC are observed 
in those larvae that were reared on C800 diet. 
Differential hemocyte counts (DHC) in the highest 
concentration of GA3 resulted in significant reduction 
of granulocytes (Fig. 2) (F= 11.47; df= 4, 19; p< 0.05). 
This reduction was also evident (56.4% and 57.2%) 
for C400 and C800 diets, respectively. However, 
amount of 67.2% was recorded for granulocytes in 
control. Percentage of plasmatocytes in any of the 
treatments did not significantly alter (Fig. 2). 

Figure 3 demonstrates the effects of GA3-diet 
treated diet on THC following injection of latex 
beads. A significant decrease in THC was detected 
six hours (F= 13.62; df= 4, 19; p< 0.05) and 12 
hours (F= 32.01; df, 19= 4; p< 0.05) after injection of 
latex beads to the larvae fed on C400 and C800 
diets, while there were no significant changes in 
these treatments at 3, 24, and 48 h after injection. 
Percentage of granulocytes in C200, C400 and, 
C800 treatments did not change significantly after 
three hours, but significantly decreased after six 
hours (Fig. 4) (F= 57.69; df= 4, 19; p< 0.05). 
Percentage of these cells was lower than control in 
all treatments after 12 hours (F= 49.79; df= 4, 19; p< 
0.05), while after 24 hours the percentage of cells 
was significantly lower only in C200 and C800 
treatments (F= 4.86; df= 4, 19; p< 0.05). After 48 
hours, the percentage of granulocytes did not 
change significantly in different treatments (Fig. 4). 
On the other hand, after the latex beads infusion 
into larvae reared on different GA3 treatments, 
significant changes were observed in the 
percentage of plasmatocytes (Fig. 5). After three 
hours, all treatments except C800 showed no 
significant differences with the control (F= 3.99; df= 
4, 19; p< 0.05), while after six hours, the control 
showed lower levels than treatments above C100 
(F= 5.34; df= 4, 19; p< 0.05). Twelve hours after the 
latex beads injection, the percentage of 
plasmatocytes in the control larvae was 
significantly higher than those in the larvae fed 
with GA3 diet (F= 6.28; df= 4, 19; p< 0.05). After 
24 and 48 hours, these cells were lower only in 
C400 and C800 treatments (F= 9.23; df= 4, 19; p< 
0.05 and F= 39.72; df= 4, 19; p< 0.05, 
respectively). 



54 

 

 
 
Fig. 6 The effects of different concentrations of gibberellic acid (C= 0 µg/g diet - C800 = 800 µg/g diet) on nodule 
formation in the sixth instar Helicoverpa armigera larvae after injection of latex beads. Statistical differences have 
been done within each time intervals and marked by various letters (at p< 0.05, Tukey’s test) 
 
 
 
 
 

The larvae fed on the diet containing GA3 
significantly inhibited the nodule formation after 
injection of latex beads (Fig. 6). In all time intervals 
except for 48 hours, the number of nodules in the 
larvae fed by C400 and C800 diet was lower than 
control (Fig. 6). In all treatments, 24 and 48 hours 
after injection the phenoloxidase activity was 
enhanced. The larvae feeding on the highest 
concentration of GA3 (C400 and C800) 
demonstrated the lowest phenoloxidase activity in 
all time intervals (Fig. 7). 
 
Antioxidant systems of H. armigera against GA3  

Feeding on the diet containing GA3 caused 
different effects on antioxidant enzymes of H. armigera 
(Table 1). The lowest activity of peroxidase and 
catalase was observed in the control larvae (0.063 and 
0.794 U/ mg protein, respectively), while the highest 
activity was found in those larvae feeding on C800 diet 
(0.120 and 1.892 U/ mg protein respectively) (F= 
26.48; df= 4, 44; p< 0.05 and F= 14.08; df= 4, 44; p< 
0.05, respectively). The activity of superoxide dismutase, 
ascorbate peroxidase and glucose-6-phosphate 
dehydrogenase was the lowest in the control larvae, 
but it increased in GA3-diet fed larvae in a dose-
dependent manner (F= 148.03; df= 4, 44; p< 0.05, F= 
615.48; df= 4, 44; p< 0.05 and F= 36.23; df= 4, 44; p< 
0.05, respectively) (Table 1).  
 
The reaction of the detoxifying enzymes of H. 
armigera against GA3 

The larvae bred on higher concentrations of 
GA3 depicted a statistically significant raise in 

esterase activities only when β naphtyl acetate was 
used as a substrate (F= 40.06; df= 4, 44; p< 0.05) 
(Table 2). In the case of glutathione S-transferase, 
enzymatic activities of larvae fed on C800 diet were 
significantly higher than those of control.  
 

Effect of GA3 on protein, glycogen, triglyceride, 
cholesterol, and glucose content of H. armigera 

Effect of GA3 on macromolecules in the body of 
H. armigera larvae are shown in Table 3. No 
significant differences were found in the amount of 
protein and glucose, while the lowest amount of 
triglyceride was observed in larvae fed on GA3 diet 
compared to the control (F= 32.40; df= 4, 44; p< 
0.05). The larvae fed on C800 had the lowest amount 
of glycogen and cholesterol (F= 11.21; df= 4, 44; p< 
0.05 and F= 43.48; df= 4, 44; p< 0.05, respectively). 

 

Discussion 
 

Exogenous compounds could affect the number 
of circulating hemocytes (Perez and Fontanetti, 
2011). Our results indicate that feeding on diet 
containing gibberellic acid affect the number of 
circulating hemocytes, especially granulocytes. It is 
inferred from the reducing number of circulating 
hemocytes, that the hemocytes might have migrated 
to damaged tissues in order to phagocytize the 
remains of the damaged tissues (Pipe and Coles, 
1995). We have noticed damages to the midgut 
under the effect of GA3 feeding (unpublished data). 
Reports by Abdellaoui et al. (2009; 2013) also 
reported damages implicated on the digestive 
system, which support our own results. The THC and 



55 

 
 
Fig. 7 The effects of different concentrations of gibberellic acid (C= 0 µg/g diet - C800 = 800 µg/g diet) on the 
phenoloxidase activity in the sixth instar H. armigera larvae after injection of latex beads. Statistical differences 
have been done within each time intervals and marked by various letters (at p< 0.05, Tukey’s test) 
 
 
 
DHC (granulocytes and plasmatocytes) reduction 
have been shown in G. mellonella under the effect 
of abscisic acid, another PGR (Er and Keskin, 
2016). Paradoxically, there is a report showing an 
increased THC in G. mellonella larvae fed on diet 
treated with gibberellic acid. However, DHC in 
granulocytes and plasmatocytes did not change 
significantly (Altuntaş et al., 2012).  

Insects show cellular immune defense by 
phagocytosis, encapsulation, or nodulation against 
invaders (Er and Keskin, 2016). In order to confirm 
the hypothesis that GA3 weakens the immune 
system, we undertook the experiment of injecting 
latex beads. A trend of reducing hemocytes 
confirms that the immunity of our insect has been 

affected by feeding on GA3 treated-diet. The 
decrease in insect's ability to confront foreign bodies 
is similar to the reports by Altuntas et al. (2012) 
when using GA3 in the diet of G. mellonella. The 
mode of action of GA3 is rather variable and one of 
the possible modes could be that this hormone 
might act the way juvenile hormone (JH) does to the 
insect (Kaur and Rup, 2002; Abdellaoui et al., 
2015). Structural similarity of PGRs to insect 
indigenous JH might reflect similar mode of action. 
Several studies have shown that juvenile hormone 
and their analogues reduce the ability of insect 
larvae to form nodules by inhibiting cell proliferation 
(Rantala et al., 2003; Franssens et al., 2006; Zibaee 
et al., 2012). 

 
 
 
Table 1 Antioxidant enzyme activities of sixth instar H. armigera larvae provided with different concentrations of 
gibberellic acid in their diet 
 

Enzymes 
Specific activity of enzymes (U/ mg protein) 

C C100 C200 C400 C800 

Peroxidase 0.063±0.003
c
 0.097±0.004

b
 0.111±0.002

a,b
 0.108±0.002

a,b
 0.120±0.006

a
 

Superoxide dismutase 0.073±0.002
d
 0.292±0.033

c
 0.408±0.011

b
 0.430±0.005

b
 0.606±0.003

a
 

Catalase 0.794±0.140
c 

0.965±0.115
b,c

 1.643±0.119
a 

1.366±0.089
a,b

 1.892±0.136
a
 

Ascorbate peroxidase 0.232±0.014
d
 0.732±0.038

c
 1.207±0.018

b
 1.234±0.032

b
 1.944±0.013

a
 

Glucose-6-phosphate 
dehydrogenase 

0.199±0.003
d
 0.269±0.004

c
 0.366±0.010

b
 0.383±0.003

b
 0.502±0.021

a
 

 
Means with the same letters in a row are not significantly different at p < 0.05 (Tukey’s test) 



56 

Table 2 Detoxifying enzyme activities of sixth instar H. armigera larvae provided with different concentrations of 
gibberellic acid in their diet 
 

Enzymes 

Enzyme activity (OD/ min) 

C C100 C200 C400 C800 

General esterases (α-naphtyl acetate) 0.054±0.007
a
 0.053±0.001

a
 0.055±0.002

a
 0.055±0.005

a
 0.056±0.004

a
 

General esterases (β-naphtyl acetate) 0.077±0.004
c
 0.101±0.007

b
 0.095±0.012

b,c 
0.127±0.004

a
 0.138±0.001

a
 

Glutathione S-transferases (CDNB) 0.163±0.008
c
 0.173±0.013

c
 0.193±0.020

c
 0.303±0.012

b
 0.450±0.017

a
 

Glutathione S-transferases (DCNB) 0.270±0.017
b
 0.320±0.028

b
 0.486±0.044

b
 0.486±0.046

b
 1.273±0.093

a
 

 
The activity of glutathione S-transferase using CDNB (1-chloro-2, 4- dinitrobenzene) and DCNB (1, 2-dichloro-4-
nitro-benzene) as reagents and the activity of general esterases using α-naphtyl acetate and β-naphtyl acetate as 
substrates. Means with the same letters in a row are not significantly different at p < 0.05 (Tukey’s test) 
 
 
 
 
 

Humoral immunity in insects is considered as 
complementary to cellular immunity by production of 
antimicrobial peptides and phenoloxidases (Lavine 
and Strand, 2002; Beckage, 2011). The 
phenoloxidases are also a complementary part of 
cellular immunity in the last stage of encapsulation 
and nodulation processes, forming melanisation of 
both. The precursors of phenoloxidases are the 
prophenoloxidases which are maintained in 
hemocytes (mostly oenocytoids) (Lavine and 
Strand, 2002; Beckage, 2011). We assume that the 
reduced activity of phenoloxidases might be due to 
reduction in cell number. Although while counting 
DHC we did not count the oenocytoids and instead 
focused on immunocytes (i.e. granulocytes and 
plasmatocytes). However, THC reduction gives a 
clear picture of what was expected under the 
impression of GA3. The similarity in structure of 
GA3 to insect JH brings it to the mind that the 
inhibition of phenoloxidase activity is the same as 
what JH does. The JH has been shown to have 
inhibitory effect on phenoloxidase activity (Hiruma 
and Riddiford, 1988; Rolff and Siva-Jothy, 2002; 
Rantala et al., 2003). 

The antioxidant system enables the insect to 
inhibit oxidation reactions that produce free radicals 
damaging the cells (Dkhil et al., 2015). Increasing 
activity of peroxidase, catalase, superoxide 
dismutase, ascorbate peroxidise, and glucose-6-
phosphate dehydrogenase can be explained since 
this insect has faced exogenous stress, here GA3 
(Lyakhovich et al., 2006, Altuntas, 2015; Sezer and 
Ozalp, 2015).  

Glutathione S-transferase is a detoxifying and 
antioxidant enzyme that eliminates lipid peroxidation 
products or hydroperoxides of cells (Dubovskiy et 
al., 2008). This enzyme also plays an important role 
in detoxification by increasing the solubility of toxic 
substances (Grant and Matsumura, 1989). 
Esterases are important detoxifying enzymes which 
hydrolyse esoteric bonds in synthetic chemicals 
(Hemingway and Karunaratne, 1998). Increasing 

activity of glutathione S-transferases and esterases 
is an important indication of the unpleasant effects 
of GA3 on the insect. Mirhaghparast et al. (2016) 
reported increased activity of these two enzymes in 
C. suppressalis larvae under the influence of 
pyriproxyfen. The detoxifying enzymes and 
oxidative defense system work in coordination to 
increase adaptation of insects by deactivating and 
eliminating harmful compounds (Felton and 
Summers, 1995; Hemingway and Karunaratne, 
1998; Büyükgüzel et al., 2010; Aslanturk et al., 
2011).  

In assessing the effect of gibberellic acid on 
protein and glucose in the insect body no significant 
changes were observed. Contrary to our report 
Uckan et al. (2011) showed higher protein levels in 
all concentrations (50 to 2000 ppm) of GA3 in the 
larvae of G. mellonella. However, hemolymph free 
amino acids in G. mellonella larvae after using GA3 
undulated among dosages (Altuntaş et al., 2014). 
Also, carbohydrate levels of larval G. mellonella 
decreased significantly after GA3 use. Moreover, 
the amount of glycogen in Zaprionus paravittiger 
(Godbole and Vaidya) (Diptera: Drosophilidae) and 
B. cucurbitae was reduced after using gibberellic 
acid (Rup et al., 1998; Kaur and Rup, 2003b). 
Triglycerides and cholesterol reduction was similar 
to what has been reported in other insects (Rup et 
al., 1998; Uckan et al., 2008). The lower level of 
total lipid, as well as triglyceride lipid biosynthesis in 
the experimental larvae, emphasizes the need of 
this macromolecule for energy demands due to 
induced stress by GA3. Furthermore, the reactive 
oxygen species also cause cell death, mutation, and 
even death through degradation on macromolecules 
such as lipids (Ryter et al., 2007). 
 
Conclusion 

 
The PGRs can be used to increase the crop 

yield quality and quantity. However, we should be 
cautious about the interaction between the GA3, 



57 

Table 3 Amount of protein, glycogen, triglyceride, cholesterol, and glucose in sixth instar H. armigera larvae 
provided with different concentrations of gibberellic acid in their diet 
 

Macromolecules 
(mg/ml) 

Treatments 

C C100 C200 C400 C800 

Protein 3.560±0.061
a
 3.681±0.046

a
 3.656±0.070

a
 3.808±0.066

a
 3.712±0.063

a
 

Glycogen 0.251±0.004
a,b 

0.242±0.007
a,b

 0.232±0.012
b,c 

0.270±0.004
a
 0.207±0.001

c 

Triglyceride 0.171±0.001
a
 0.166±0.001

b 
0.166±0.005

b
 0.158±0.009

c
 0.154±0.001

c
 

Cholesterol 1.260±0.018
b
 1.454±0.048

a
 1.107±0.047

b,c
 0.990±0.043

c
 0.763±0.032

d
 

Glucose 0.172±0.003
a
 0.165±0.002

a
 0.171±0.002

a
 0.165±0.002

a
 0.163±0.001

a
 

 
Means with the same letters in a row are not significantly different at p < 0.05 (Tukey’s test) 
 
 
 
 
 
insect, and the crop plant. In the present and 
previous reports we demonstrated this effect which 
aimed at digestion, reproduction, and now the 
immunity. Further studies concerning insects 
collected directly from treated and untreated crops 
would help for planning on the use of various 
PGRs. 
 
Acknowledgements 

The authors express their sincere gratitude to 
the Vice chancellor in research affairs, University of 
Guilan for providing financial in the form of grant 
(1987). 
 
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