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ISJ 20: 10-20, 2023                                                                 ISSN 1824-307X 

 
 

RESEARCH REPORT 

 
Induction of antioxidant and detoxifying systems of Chilo suppressalis Walker after 
exposure to entomopathogenic fungi 

 
M Shahriari1, A Zibaee1*, MF Dinan2, A Armand1, M Tabari3, H Hoda3 

 

1Department of Plant Protection, Faculty of Agricultural Sciences, University of Guilan, Rasht, Iran 
2Department of Medical Entomology and Vector Control, School of Public Health and Health Sciences Research 
Centre, Mazandaran University of Medical Sciences, Sari, Iran 
3Rice Research Institute of Iran, Mazandaran Branch, Agricultural Research, Education and Extension 
Organization (AREEO), Amol, Iran 
 
 
This is an open access article published under the CC BY license                                                        Accepted January 10, 2023 

 
Abstract 

The current study aimed to determine antioxidant and detoxifying responses of Chilo suppressalis 
Walker (Lepidoptera: Crambidae) to Beauveria bassiana (Strains BBRR1, BBAL1, BBLN1, BBLN2), 
Metarhizium anisopliae and Hirsutella subulata. The interactions of insect humoral immune responses 
with the entered conidia of entomopathogenic fungi in addition to nodule formation and melanization 
caused the production of several reactive oxygenate species (ROS), such as hydrogen peroxidase 
(H2O2), hydroperoxides (ROOH), superoxide radicals (O2−), and hydroxyl radical (OH−). The highest 
activity of catalase was recorded by BBRR1 and BBAL1, treatment after 48 to 96 h while the larvae 
treated by BBRR1 showed the highest peroxidase activity. Both ascorbate peroxidase and glucose-6-
phosphate dehydrogenase showed the highest activity in the larvae treated by BBRR1 after 48-96 h. 
The highest concentration of Malondialdehyde (MDA) reported in the larvae treated by BBRR1, 
BBAL1 and BBLN1, after 48 hours. The highest activity of general esterases was recorded in the 
larvae treated by BBRR1 after 48-96 hours. Similar results were recorded in the activity of glutathione-
S-transferase but the enzyme had also the highest activity in the larvae treated by BBAL1 and BBLN2 
after 48 hours. The larvae treated by BBRR1 and BBLN1 showed the highest activity of acid 
phosphatase (ACP) after 72 and 96 hours while the highest activity of alkaline phosphatase (ALP) was 
obtained in the larvae only treated by BBRR1 after 48-96 hours. The results clearly revealed that 
BBRR1 significantly and severely induced antioxidant and detoxifying systems of C. suppressalis 
larvae implying on virulence and immune induction of BBRR1 against the larvae. 

 
Key Words: entomopathogen; antioxidant; Chilo Suppressalis; detoxification 
 

 
Introduction 

 
Entomopathogenic fungi are among the most 

successful microbial agents to combat insect pests 
that are exist in most aquatic and terrestrial habitats. 
They cause enzootic and epidemics in insect 
populations and serve as one of the alternatives to 
chemical insecticides in agroecosystems because of 
their adaptations and interactions with 
environmental factors such as pH, salinity, organic 
and inorganic materials as well as imposition of 
ecological and physiological costs on host insects 
(Lacey et al., 2015; Zibaee and Ramzi, 2018). 
_________________________________________ 

 

Corresponding author: 
Arash Zibaee 
Department of Plant Protection 
Faculty of Agricultural Sciences 
Box 41635-1314, Rasht, Iran 
E-mail: arash.zibaee@gmx.com; 
arash.zibaee@guilan.ac.ir 

 
 

Moreover, entomopathogenic fungi are the unique 
microbial agents that infect their host by penetrating 
thorough integument rather than bacteria or viruses 
that require to be ingested. Therefore, insect 
exposure to the fungal conidia in the contaminated 
environment will cause the onset of infection 
(Zibaee and Ramzi, 2018). Pathogenicity by 
entomopathogenic fungi initiates following 
attachment of conidia to the insect cuticle, passing 
through by germination, and proliferating after 
arrival into host hemocoel. Eventually, the entered 
fungal cells acquire yeast-like forms known as 
hyphal body that sequentially utilize food resources 
of host body and cause cellular death by producing 
secondary metabolites (Brownbridge et al., 2001). 

Cell death imposed to different tissues of 
insects following penetration of fungal hyphae and 
the production of secondary metabolites causes 
lipid peroxidation and production of reactive oxygen 



11 

 
 
 

 
 

 
 

Fig. 1 Changes in superoxide dismutase, catalase and peroxidase activities of C. suppressalis larvae treatments 
by different isolates of entomopathogenic fungi. Different letters indicate statistical differences at p < 0.05 in each 
time interval. B. bassiana includes BBRR1, BBAL1, BBLN1 and BBLN2 strains as well as one isolate of M. 
anisopliae (MASA) and H. subulata (HSLA) 



12 

 
 

 
 
Fig. 2 Changes in ascorbate peroxidase and glucose-6-phosphate dehydrogenase activities of C. suppressalis 

larvae treatments by different isolates of entomopathogenic fungi. Different letters indicate statistical differences 
at p < 0.05 in each time interval. B. bassiana includes BBRR1, BBAL1, BBLN1 and BBLN2 strains as well as one 
isolate of M. anisopliae (MASA) and H. subulata (HSLA) 
 
 
 
 
 
species (ROS) (Gulpov et al., 2003; Karthi et al., 
2018; Shamakhi et al., 2020). These molecules are 
involved in cell signaling and stimulate the genes 
engaged in host defense. The increased 
concentrations of ROS inflict damages to cell 
structure under a series of reactions under oxidative 
stress (Dalton et al., 1999; Kamata and Hirata, 
1999; Shamakhi et al., 2019, 2020). Moreover, the 
interactions of insect humoral immune responses 
with the entered conidia of entomopathogenic fungi 
mainly nodule formation and melanization caused 
the production of several (ROS), such as hydrogen 
peroxidase (H2O2), hydroperoxides (ROOH), 
superoxide radicals (O2−), and hydroxyl radical 
(OH−). These molecules act bilaterally to disable the 
pathogenic infliction of invading organism and to 

impose some harmful effects on insect tissues 
including dissociation of cell membrane, apoptosis 
and DNA damage that led to suppress developmental 
and reproductive disorders (Robinson and Badwey, 
1994; Whitten and Ratcliffe, 1999; Monaghan et al., 

2009). Therefore, insects have recruited several 
components of antioxidant systems to regulate ROS 
level within body. These enzymatic and non-
enzymatic molecules include catalase (CAT), 
peroxidase (POD), superoxide dismutase (SOD), 
asocorbate peroxidase (APOX), glucose-6-
phosphate dehydrogenase (GPDH), ascorbic acid, 
thiols, and α-tocopherol (Wang et al., 2001; 
Dubovskiy et al., 2008; Shamakhi et al., 2020). 

There are several reports to imply induction of 
detoxifying system of insects after microbial 



13 

infections that may trigger insect resistance to 
chemical insecticides (Sokolova and Sundukov, 
1999; Xia et al., 2000, 2001; Zibaee et al., 2009). 

Glutathione-S-transferase (GST) and general 
esterases (ESTs) are the multifunctional and the 
important enzymes in detoxification of xenobiotics in 
insects. GST conjugates the reduced glutathione to 
the electrophilic center of xenobiotics and it may 
engage in both detoxification and cellular 
antioxidant responses (Substrate, 2002; Li et al., 
2007). ESTs catalyze the esters of higher fatty acids 
within metabolic process of insects and xenobiotic 
hydrolyzation (Li et al., 2007). Nevertheless, these 
enzymes have been reported to engage in 
degradation of toxic molecules synthesized during 
fungal penetration (Serebrov et al., 2006; Dubovskiy 
et al., 2008; Zibaee et al., 2009).  

Understanding the physiological interaction of 
entomopathogenic fungi with their hosts is critical to 
success of agricultural pest control. These 
interactions can vary depending on the fungal 
virulence and the host's immune as well as 
biochemical responses. In the previous study, the 
cellular and humoral immunity of Chilo suppressalis 

Walker (Lepidoptera: Crambidae) to the different 
entomopathogenic fungi was investigated by 
evaluating the changes in hemocyte count and the 
expression of antimicrobial peptide genes at 
different time intervals (Shahriari et al., 2021a). 
These studies are of interest because C. 
suppressalis is an economically important rice pest 
in north of Iran. The larvae severely fed on stems 
causing “dead heart” and “white head” of rice plants. 
Such deficiencies significantly decrease rice yield of 
imposed field requiring insecticide treatment. Hence 
the current study was done to determine the effects 
of four isolates of Beauveria bassiana, one isolate of 
Metarhizium anisopliae and Hirsutella subulata on 
the antioxidant and detoxification system of C. 
suppressalis to elucidate other aspects of fungi-

insect interactions.  
 
Materials and Methods 
 
Insect rearing 

The field collected adults were transferred to 
the laboratory and allowed to lay eggs on rice 
leaves of Hashemi variety. The hatched larvae were 
gently transferred on rice stems and kept in sterile 
containers at 28 ± 2 °C, 80% relative humidity (RH) 
and 16h light:8h dark (LD 16:8). Fresh stems were 
provided daily until the fourth instars. Two 
generations were reared on laboratory to have a 
cohort with the least environmental stresses (Zibaee 
and Malagoli, 2014).  
 
Entomopathogenic fungi culture 

Four isolates of B. bassiana including BBRR1, 
BBAL1, BBLN1 and BBLN2 in addition to one 
isolate of M. anisopliae (MASA) and H. subulata 
(HSLA) were selected based on the results of our 
previous studies, and cultured on Potato dextrose 
agar at 25 ± 2 °C. After 14 days, conidia were gently 
gathered and added onto a 0.01% solution of 
Tween 80 (Sigma Aldrich, USA) to prepared 
desirable concentration.  
 

Larval treatment and sample preparations 
The fourth instar larvae of C. suppressalis were 

randomly immersed into 105 conidia/mL 
concentration of each fungal isolate (each 
containing 30 specimens in the three replicates of 
10) while the control larvae were dipped in Tween-
80 (0.02%) alone. After 24, 48, 72 and 96 h of post-
treatment, the total bodies of the larvae were 
separately homogenized in distilled buffer by a glass 
pestle in a 1.5 ml of microtubes then centrifuged at 
20000 ×g, 4 °C for 20 min. The supernatants were 

stored at -20 °C and used in the biochemical 
experiments. 
 
Antioxidant enzymes 
Catalase (EC 1.11.1.6) 

Briefly, 50 μL of enzyme sample was added 
into 500 μL of hydrogen peroxide (1%) and 
incubated at 28 °C for 10 min. Then, the 
absorbance was read at 240 nm (Wang et al., 

2001). 
 
Superoxide dismutase (EC 1.15.1.1) 

The method of McCord and Fridovich (1969) 
was used to measure SOD activity. Briefly, 50 μL of 
enzyme sample was added into 500 μL of a solution 
containing 70 μM of NBT, 125 μM of xanthine, 
dissolved in PBS (20 mM, pH 7.1). Then, 100 μL of 
xanthine oxidase solution added by 10 mg of bovine 
serum albumin was mixed with the earlier medium. 
Finally, 100 μL of xanthine oxidase (5.87 units/ml) 
was added and incubated at darkness, 28 °C for 20 
min. At the end of incubation time, the absorbance 
was read at 560 nm. 
 
Peroxidase (EC 1.11.1.x) 

The reaction mixture contained 50 μL of 
enzyme sample, 250 μL of buffered pyrogallol (0.05 
M pyrogallol in 0.1 M phosphate buffer [pH 7.0]) and 
250 μL of H2O2 (1%). The absorbance was 
continuously recorded every 30 s for 2 min at 430 
nm (Addy and Goodman, 1972).  
 
Ascorbate peroxidase (EC 1.11.1.11) 

The enzyme assay was done by preparing a 
reaction mixture containing 50 μL of sample, 150 μL 
potassium phosphate buffer (67 mM, pH 7.0), 70 μL 
ascorbic acid (2.5 mM) and 200 μL H2O2 (30 mM). 
The absorbance was read every 40 s for 5 min at 
290 nm (Asada, 1992).  
 
Glucose-6-phosphate dehydrogenase (EC 1.1.1.49) 

The enzyme assay was done by mixing 100 μL 
Tris-HCl (100 mM, pH 8.2), 50 μL NADP (0.2 mM) 
and 30 μL MgCl2 (0.1 M). After 1 min, 50 μL water, 
50 μL of the sample and 100 μL GPDH (6 mM) was 
added to the earlier mixture. Finally, the absorbance 
was read at 340 nm after 5 min (Balinsky and 
Bernstein, 1963).  
 
Malondialdehyde (MDA)  

The MDA concentration was measured by 
mixing 100 μL of 20% trichloroacetic acid and 50 μL 
of supernatant. The mixture was centrifuged at 
15,000 g for 10 min at 4 °C. Then, 100 μL of 0.8% 

TBA reagent was added into the gained supernatant 
 



14 

 
 
Fig. 3 Changes in malondialdehyde content of C. suppressalis larvae treatments by different isolates of 
entomopathogenic fungi. Different letters indicate statistical differences at p < 0.05 in each time interval. B. 
bassiana includes BBRR1, BBAL1, BBLN1 and BBLN2 strains as well as one isolate of M. anisopliae (MASA) and 
H. subulata (HSLA) 
 
 
 
 
 
and incubated at 100 °C for 60 min before to read 
absorbance at 535 nm. The MDA concentration was 
calculated using a molar extinction (Bar-Or et al., 
2001).  
 
Detoxifying enzymes 
General esterase (EC 3.1.1.1.) 

Seventy-five microliters of the two substrates, 
α-naphthyl acetate and β-naphthyl acetate (10 mm) 
was separately added into 75 μL fast blue RR salt (1 
mm). Then, 50 μL of the enzyme solution was 
added to the reaction mixture and the absorbance 
was read at 450 nm w at intervals of 10 s for 1 min 
(Han et al., 1995). 
 
Glutathione-S-transferase (EC 2.5.1.18) 

Twenty microliters of CDNB (20 mm) or DCNB 
(40 mm) were separately was added into 100 μL of 
enzyme solution. Then the absorbance was read at 
340 nm at intervals of 9 s in 1 min (Oppenoorth et 
al., 1979). 
 
Alkaline- (EC 3.1.3.1) and acid (EC 3.1.3.2) 
phosphatase  

The enzyme assay was done based on the 
method of Bessey (1954). The buffered substrate, 
p-nitrophenol phosphate in Tris-HCl (20 mM) pH 8 
and (pH 5) was separately used to assay alkaline- 
and acid phosphatase, respectively. Twenty 
microliter of the enzyme assay was added into the 
buffered substrate separately and incubated for 5 
min before reading the absorbance at 450 nm.  

Protein Assay 

The protein content of the samples in both 
control and fungal treated was assayed by the 
method of Lowry et al. (1951) (recommended by 
Ziest Chem. Co., Tehran-Iran). 
 
Statistical Analyses 

The date of antioxidant and detoxification 
experiments were compared by one-way analysis of 
variance (ANOVA) using Tukey’s test (SAS 9.3 
2010). The statistical differences were marked by 
different letters at a probability less than 5%. 
 
Results and Discussion 

 
Exposure of C. suppressalis larvae to the six 

native isolates of B. bassiana, M. anisopliae and H. 
subulata significantly enhanced antioxidant and 
detoxifying activities in the fourth instar larvae C. 
suppressalis. The activities of SOD, CAT and POD 
in the larvae treated by BBRR1, BBAL1 and BBLN1 
significantly increased at 48 and 72 h of post-
treatment while no significant difference was 
observed between fungal treatments and control 
larvae after 24 h (Pr>F: 52.88; df= 6; P<0.0001, Fig. 
1). Moreover, the activities of SOD, CAT and POX 
in the larvae significantly elevated at 96 h post-
treatment for all isolates (Pr>F: 71.23; df= 6; 
P<0.0001, Fig. 1). Entomopathogens and their 
toxins have been recognized as one of the 
important exogenous resources that generated 
free radicals (Jia et al., 2016; Karthi et al., 2018; 



15 

 
 

 
 
Fig. 4 Changes in general esterase activity of C. suppressalis larvae treatments by different isolates of 
entomopathogenic fungi. Different letters indicate statistical differences at p < 0.05 in each time interval. B. 
bassiana includes BBRR1, BBAL1, BBLN1 and BBLN2 strains as well as one isolate of M. anisopliae (MASA) and 
H. subulata (HSLA) 
 
 
 
 
 
Shamakhi et al., 2020). Overproduction of ROSs by 
biological and chemical toxic compounds, apart 
from its nature of origin, resulted in oxidative stress 
that eventualy leads to lipid peroxidation and 
damage of DNA (Dubovskiy et al., 2008). Several 
reports have shown involvements of SOD, CAT, 
POD, GPDH, APOX and nonenzymatic components 
(e.g., MDA and α-tocopherol) in the antioxidant 
defenses of insects (Dubovskiy et al., 2008; Rahimi 
et al., 2018; Shahriari et al., 2019; Shamakhi et al., 
2019, 2020). However, there are few reports 
regarding the effects of entomopathogenic fungi on 
antioxidant induction of insect pests. SOD has an 
important role to catalyze O2- radicals into H2O2. 

Then, H2O2 changed to H2O and oxygen by the 
activities of CAT and POD (Dubovskiy et al., 2008). 
Because the activities of CAT and POD 
significantly increased in the larvae treated by the 
all six isolates, it seems that metabolites produced 
from fungal conidia may induce contents of 
superoxide radicals and hydrogen peroxide which 
subsequently elevated activation of the antioxidant 
enzymes. Moreover, the enhanced activities of 
CAT and POD may be attributed to the higher 
activity of SOD which generates hydrogen 
peroxides in the larvae of C. suppressalis. Similar 
results were reported regarding the effects of B. 
bassiana on C. suppressalis larvae (Shamakhi et 

 
 



16 

 
 

 
 
Fig. 5 Changes in glutathione S-transferase activity of C. suppressalis larvae treatments by different isolates of 
entomopathogenic fungi. Different letters indicate statistical differences at p < 0.05 in each time interval. B. 
bassiana includes BBRR1, BBAL1, BBLN1 and BBLN2 strains as well as one isolate of M. anisopliae (MASA) and 
H. subulata (HSLA) 
 
 
 
 
 
al., 2020). Karthi et al. (2018) demonstrated that 
infection of Spodoptera litura (Fabricius) 
(Lepidoptera; Noctuidae) by Aspergillus flavus 
conidia caused the higher activities of SOD, CAT 
and POD. Jia et al. (2016) reported that M. 
anisopliae caused the higher activities of SOD, 
CAT, and POD in Locusta migratoria (Meyen) 
(Orthoptera: Acrididae). The highest activity of 
antioxidant enzymes in C. suppressalis larvae was 
observed in the larvae treated by BBRR1, BBAL1 
and BBLN1. In our previous study, BBRR1, BBAL1 
and BBLN1 showed the highest virulence in the 
shortest time against the larvae of C. suppressalis 
(Shahriari et al., 2021b). Moreover, the authors 

demonstrated the highest activities of the 
extracellular enzymes in BBAL1, BBRR1 and 

BBLN1 therefore the conidia of these isolates pass 
through the cuticle of larvae in a faster time than 
other isolates (Shahriari et al., 2021b). These 
differences may be correlated to the presence of 
different molecules in the cell membrane of conidia 
and production of toxins by the isolates which 
impose oxidative stress in the larvae. 

Exposure of BBRR1, BBAL1, BBLN1 and 
BBLN2 on C. suppressalis larvae increased the 
activities of APOX and GPDH after 48 and 72 h so 
that these enzymes showed the highest activities 
compared to control at 96 h post-treatment (Pr>F: 
65.23; df= 6; P<0.0001, Fig. 2). GPDH and APOX 
are the two antioxidant enzymes which engaged in 
detoxification of pro-oxidant compounds within 
insects (Asada, 1992). APOX eliminates H2O2 in 



17 

 
 

 
 
Fig. 6 Changes in acid- and alkaline activities of C. suppressalis larvae treatments by different isolates of 
entomopathogenic fungi. Different letters indicate statistical differences at p < 0.05 in each time interval. B. 
bassiana includes BBRR1, BBAL1, BBLN1 and BBLN2 strains as well as one isolate of M. anisopliae (MASA) and 
H. subulata (HSLA) 
 
 
 
 
 
chloroplasts, cytoplasm, and mitochondria, while 
GPDH involved in deleting oxidant compounds in 
cytosol via transduction of NADPH to NADP+ 
(Asada, 1992). Similar to our results, the larvae of 
Galleria mellonella L. (Lepidoptera: Pyralidae) 
infected by Bacillus thuringiensis showed also the 
higher activities of APOX and GPDH compared to 
control (Dubovskiy et al., 2008). Shamakhi et al. 
(2020) demonstrated significant higher activities of 
APOX and GPDH in C. suppressalis injected with B. 
bassiana conidia. The higher GPDH activity led to 

elevation of NADPH synthesis to remove products 
of APX activity. Moreover, NADPH reduces the toxic 
effects via transporting electrons to free radicals 
(Barbehenn, 2002; Shamakhi et al., 2020). 

Content of MDA in the larvae treated by 
BBRR1, BBAL1 and BBLN1 were significantly 
induced after 48 h while other isolates caused the 
MDA elevation in the larvae of C. suppressalis after 
72 and 96 h (Pr>F: 67.23; df= 6; P<0.0001, Fig. 3). 
MDA is an oxidative stress indicator to show 
enhancement in radical oxidative stresses. The 



18 

increased content of MDA indicated occurrence of 
oxidative stress following lipid peroxidation (Rahimi 
et al. 2018). On the other hand, ROSs led to 

catalysis of polyunsaturated acids, generation of 
MDA and elevation of toxic compounds within cells 
(Wang et al., 2001). Results of our study 
demonstrated that lipid peroxidation was higher in 
the larvae treated by fungal isolates than control so 
it can be concluded that conidia proliferation and 
secretion of secondary metabolites increased lipid 
peroxidation in hemocoel that leads to cytotoxicity. 
Some research reported a direct correlation 
between fungal infection and lipid peroxidation in 
insects. For example, the induction of MDA under 
mycoses showed in C. suppressalis larvae injected 
by B. bassiana (Shamakhi et al., 2020), as well as 
S. litura larvae infected by A. flavus (Karthi et al., 
2018). 

Activity of general esterase significantly 
increased after 72 and 96 h for all isolates except 
for BBRR1, BBAL1 and BBLN1, while no significant 
difference was observed among isolates and control 
after 24 h (Pr>F: 49.73; df= 6; P<0.0001, Fig. 4). 
Similar results were obtained for GSTs activity in 
which the exposure of all isolates led to enhanced 
activity of GSTs at 72 and 96 h of larval post-
treatment (Pr>F: 49.18; df= 6; P<0.0001, Fig. 5). In 
spite of efforts to develop chemical and biological 
pesticides, insects are also looking for ways to 
neutralize the effects of these pesticides. ESTs and 
GSTs are the two important groups of detoxifying 
enzymes that involve in the breakdown and 
neutralization of toxic compounds entering insect 
body (Zhu et al., 2011). Findings of our study 
support results of earlier investigations regarding 
the effect of fungal conidia on detoxifying enzymes 
of insects (Xia et al., 2000, 2001; Serebov et al., 
2001, 2006; Dubovskiy et al., 2008; Zibaee et al., 
2009; Fan et al., 2013; Petlamul et al., 2019). In our 
study, treatment of C. suppressalis larvae by conidia 

of the entomopathogenic fungi increased activities 
of ESTs and GSTs especially in the larvae treated 
by BBRR1, BBAL1 and BBLN1. This could be a 
nonspecific response to integument damage. In fact, 
tissue damage is the most common feature of 
infection by entomopathogenic fungi causing 
secretion of detoxifying enzymes which could be the 
usual response of larvae to infection irrespective of 
the agent nature (Xia et al., 2001; Serebov et al., 
2006; Zibaee et al., 2009). 

ACP activity in the larvae treated by all isolates 
significantly increased at 96 h of post-treatment 
while no significant difference was observed among 
isolates and control after 24 and 48 h (Pr>F: 34.12; 
df= 6; P<0.0001, Fig. 6). Activity of ALP significantly 
induced after 96 h for all isolates except for BBRR1, 
BBAL1 and BBLN1 isolates but no significant 
difference was observed among isolates and control 
after 24 h of treatment (Pr>F: 47.26 df= 6; 
P<0.0001, Fig. 6). ACP and ALP are the two 
hydrolytic enzymes responsible for removing 
phosphate groups from some molecules like 
proteins, nucleotides and alkaloids in acidic and 
alkaline conditions, respectively (Ramzi et al., 
2014). These enzymes also are able to support, 
modulate and accelerate phagocytosis (Karthi et al., 
2018). In the present study, the activities of ACP 

and ALP significantly increased in the larvae treated 
by all isolates. The higher phosphatase activity was 
found in the larvae exposed to conidia of BBRR1, 
BBAL1 and BBLN1. Our findings are parallel to the 
research of Karthi et al. (2018), who demonstrated 
ACP and ALP activities increased in S. litura after 
exposure to A. flavus. Similarly, B. bassiana and M. 
anisopliae significantly increased the activities of 
ACP and ALP in S. littoralis (Mirhaghparast et al., 
2013). In contrast, Shaurub et al. (2020) reported 
that the LD50 of B. bassiana did not affect ALP 
activity in the 4th-instar larvae of S. litura. These 
findings are expected because enzyme activity is 
dependent on several factors, such as pathogen 
strain, dose, host insect and etc. 
 

Conclusions 

 
Results of our research reported the induction 

of antioxidant system in the larvae of C. 
suppressalis exposed to several isolates of 
entomopathogenic fungi that may be the result of 
ROSs production after larval immune responses 
and damages to tissues caused by 
entomopathogenic fungi infection. Also, larval 
infection by the isolates increased the activities of 
detoxifying enzymes. Therefore, it may be 
concluded that the antioxidant and detoxifying 
systems of C. suppressalis is a part of larval 
defense mechanisms against fungal infection in 
addition to humoral or cellular responses which 
have been previously reported. 
 

Acknowledgments 

The research leading to these results received 
funding from the Iran National Science Foundation 
(INSF) under grant agreement of No. 96005511. 

 
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