Skip to main content

Aspergillus flavus induced oxidative stress and immunosuppressive activity in Spodoptera litura as well as safety for mammals



In the last few decades, considerable attention has been paid to entomopathogenic fungi as biocontrol agents, however little is known about their mode of action and safety. This study aimed to investigate the toxicity of Aspergillus flavus in insect Spodoptera litura by analyzing the effect of fungal extract on antioxidant and cellular immune defense. In antioxidant defense, the lipid peroxidation (Malondialdehyde content) and antioxidant enzymes activities (Catalase, Ascorbate peroxidase, Superoxide dismutase) were examined. In cellular immune defense, effect of A. flavus extract was analyzed on haemocytes using Scanning Electron Microscopy (SEM). Furthermore, mammalian toxicity was analyzed with respect to DNA damage induced in treated rat relative to control by comet assay using different tissues of rat (blood, liver, and kidney).


Ethyl acetate extract of A. flavus was administrated to the larvae of S.litura using artificial diet method having concentration 1340.84 μg/ml (LC50 of fungus). The effect was observed using haemolymph of insect larvae for different time intervals (24, 48, 72 and 96). In particular, Malondialdehyde content and antioxidant enzymes activities were found to be significantly (p ≤ 0.05) increased in treated larvae as compared to control. A. flavus ethyl acetate extract also exhibit negative impact on haemocytes having major role in cellular immune defense. Various deformities were observed in different haemocytes like cytoplasmic leakage and surface abnormalities etc. Genotoxicity on rat was assessed using different tissues of rat (blood, liver, and kidney) by comet assay. Non-significant effect of A. flavus extract was found in all the tissues (blood, liver, and kidney).


Overall the study provides important information regarding the oxidative stress causing potential and immunosuppressant nature of A. flavus against S. litura and its non toxicity to mammals (rat), mammals (rat), suggesting it an environment friendly pest management agent.

Peer Review reports


Food security and environment safety are the major concerns in ever expanding human population on the earth planet. Each and every year insect pests cause a severe damage in agricultural field which cost billions of dollars annually to farmers. To reduce the crop damage due to insect pests, the reliance on chemical pesticides has increased. However, irrespective use of chemical pesticides such as Endosulfan, Benzene hexachloride, Aldicarb, and Fenobucarb in agricultural field raised several types of issues include their non-biodegradable nature, development of insecticide resistance and adverse effects on human health as well as environmental concerns [1]. Overall, synthetic pesticides wreak havoc on the environment, threatening biodiversity and human survival [2]. Thus, there is a need to explore alternative ecofriendly strategies for pest’s management which protect and strengthen natural ecosystems rather than contaminate.

Biological control using fungi is one of the most promising technique, due to their unique mechanism of action while infection, low cost, specificity and safety to ecosystem [3, 4]. Entomopathogenic fungi are natural pest controlling agents manifesting immense significance to be used as mycoinsecticides against wide range of insect pests. Among entomopathogenic fungi Beauveria bassiana and Metarhizium anisopliae have been extensively explored as insecticidal agents and their formulations are also commercially available. Moreover various other fungal spp. viz. Aspergillus sp., Alternaria sp. and Nomuraea sp. have also been disclosed to be entomopathogenic [5,6,7,8]. Different Aspergillus spp. viz. A. ochraceus, A. kanagawaensis, A. sulphureus, A. flavus and A. ochraceus were found to be pathogenic against several insects such as Aedes fluviatilis (Lutz), Culex quinquefasciatus (Say), Anopheles gambiae (Giles), Oligonychus coffeae (Nietner) [9,10,11,12]. The insecticidal activity of fungi could be attributed to different secondary metabolites produced by them. Mycotoxins viz. aflatoxins, ochratoxins, fumonisins, zearalenone, have great importance in agriculture for pest management [13]. Various fungal secondary metabolites like avermectins, destruxins, pantherine, ibotenic acid, and tricholomic acid were found to be highly active against insects [14].

Although various studies have been done on the role of fungi as an insect pathogenic agents but many of them failed to address the mode of action. In order to discover the insecticidal potential, the effect on antioxidant and cellular immune defense in insects would be evaluated. Insects possess antioxidant and cellular immune defense system to ward of infection. Antioxidant defense system comprises various antioxidants enzymes which are catalase (CAT), glutathione-S-transferases (GSTs), peroxidase (POX), and superoxide dismutase (SOD). All these play an important role in protecting cells and maintaining homeostasis by removing oxidative stress [15]. Various xenobiotics incite the production of reactive oxygen species (ROS) cause the oxidative stress which ultimately induces oxidative damage, cytotoxicity or immunotoxicity and an increase in insects’ mortality [16]. Cellular immune defense in insects is accomplished through haemocytes. They consists the mixture of cells having different morphological and biological functions and help in providing defense against parasites, pathogens and other foreign bodies enter in the hemocoel [17,18,19,20]. Change in number and configuration of haemocytes ultimately affect the immunity and health of insects [21]. So, these parameters are significant while estimating the stress caused by xenobiotics.

On the basis of aforementioned discussion, the study examines the toxicity of ethyl acetate extract of A. flavus on insect Spodoptera litura (Fabricius), one of the major polyphagous pests, by analyzing the effect on antioxidant and cellular immune defense of insect.

However, if secondary metabolites of fungi are found to deter insects then it would be equitably important to detect whether these metabolites have any mammalian toxicity. As, various chronic diseases have been associated with pesticides exposures, including reproductive or developmental disorders, neurological disorders, cancer etc. Epidemiological studies suggested that occupationally exposed populations to pesticides like pesticide applicators, pesticide manufacturing workers and field workers have developed the high risk of cancer which is due to genomic damage [22,23,24]. So it is necessary to check genotoxicity of the agent which can be used as pesticide in order to decipher its effects on other non-target species. Various genotoxic markers are chromosomal abbretions (CA), sister chromatid exchange (SCE), micronuclei (MN), comet assay (CO). Comet assay or SCGE is one of the finest techniques for qualitative and quantitative analysis of DNA damage and repair. It was extensively explored in mammal and human cell studies [25,26,27] and successfully applied on the cells of various animal groups [28]. So in the present study this technique was used to assess the genotoxicity of A. flavus on mammals using rat as an animal model to confirm its safety on mammals.


Toxicity test of fungus and LC50 value against S.litura

The entomopathogenic fungus, A. flavus was tested at different concentrations for toxicity against larvae of S. litura. The mortality percentages were proportional to extract concentration as shown in Table 1. Different concentrations of the extract caused 16.66–56.66% mortality as compared to 10% in control. The concentrations ranging between 500 and 2000 μg/ml resulted in a significantly higher mortality with respect to control (F = 8.38, p ≤ 0.01) (Table 1). The LC50 value of ethyl acetate extract of A. flavus as calculated by probit analysis using SPSS software was found to be 1340.84 μg/ml.

Table 1 Mortality of S. litura larvae fed on diet supplemented with different concentrations of A. flavus

Effect on malondialdehyde (MDA) content and antioxidant enzymes

Larvae treated with ethyl acetate extract of A. flavus showed hike in level of lipid peroxidation as indicated by MDA content which significantly increased in all treated groups as compared to control in haemolymph of S. litura (Student’s t-test) except for 24 h group in which non-significant increase was observed. MDA content was maximum at 96 h (8.76 ± 0.16nmoles/ml) which was significantly higher from control (5.75 ± 0.13nmoles/ml) (t = 14.80, p ≤ 0.01). One Way ANOVA reveals the effect of duration was found to be significant (F = 76.14, p ≤ 0.01). Further Tukey’s test reveals the significant difference between 48, 72 and 96 h exposure groups (Fig. 1a).

Fig. 1
figure 1

(a-d): Malondialdehyde (MDA) content (a), Catalase (CAT) activity (b), Ascorbate peroxidase (APOX) activity (c) and Superoxide dismutase (SOD) activity (d) in haemolymph of S.litura after treatment with ethyl acetate extract of A.flavus for different time intervals. EG = Exposed group. Bars represent mean ± S.E. *Ascribes the significant difference between exposed group and control group (t-test, p ≤ 0.05). Different letters a, b, c, d are significantly different (Tukey’s test, p ≤ 0.05) and signify the effect of duration

Statistically significant (t test, p ≤ 0.05) increase in activities of all enzymes with respect to control of the exposed S.litura larvae was observed. Figure 2b reflected the significant (t test, p ≤ 0.05) hike in catalase (CAT) activity of treated larvae as compared to control. A measured value of CAT was 3 fold higher than control one, for 96 h exposure time group. The difference between all exposure time groups was statistically significant (ANOVA) and significant difference observed between 48 h, 72 h and 96 h groups (Tukey’s test) (Fig. 1b).

Fig. 2
figure 2

Microphotographs (a-d) showing Group-1 haemocytes (a). Normal haemocytes (Group-1) (b-d). Various deformities observed in haemocytes after treatment with ethyl acetate extract of A. flavus (b-c). Cell perforation (d). Surface abnormalities and Cytoplasmic leakage. Microphotographs (e-f) showing Group-2 haemocytes (e). Normal haemocyte; (f). Strumae and surface abnormalities in haemocytes after treatment with ethyl acetate extract of A.flavus. Microphotographs (g-h) showing Group-3 haemocytes (g). Normal haemocytes (h) surface abnormalities in haemocytes after treatment with ethyl acetate extract of A.flavus

An upsurge in Ascorbate peroxidase (APOX) activity was noticed in haemolymph of S. litura after treatment with A.flavus ethyl acetate extract. The level of enzyme activity was found to be 67.77 ± 0.34, 82.20 ± 3.23, 87.42 ± 0.56, 125.80 ± 4.59μmole/ml following treatment up to 24 h, 48 h, 72 h and 96 h respectively. Highest activity was found in 96 h exposure group where almost 3.74 fold increase was observed in treated groups as compared to control (t = 19.60, p ≤ 0.01). Time dependent significant effect was observed (ANOVA) (F = 76.81, p ≤ 0.01) however significant changes were observed between 24 h and 48 h, 72 h and 96 h exposure groups (Tukey’s test) (Fig. 1c).

Superoxide dismutase (SOD) activity also found to be increase in larvae fed with diet amended with ethyl acetate extract of A. flavus (Fig. 1d). Significant (t test, p ≤ 0.05) rise in SOD activity was occurred in all exposure groups as compared to control however highest activity was obtained at 72 h and 96 h exposure groups where activity increased from 28.19 ± 0.17 μmol/ml (control) to 41.54 ± 0.87 μmol/ml (exposed group) and 28.33 ± 0.18 μmol/ml (control) to 51.68 ± 0.88 μmol/ml (exposed group) respectively. With increase in time duration the enzyme activity was significantly increased (ANOVA) (F = 202.57, p ≤ 0.01). Significant difference observed between 48 h, 72 h and 96 h exposure groups (Tukey’s test) (Fig. 1d).

Effect on haemocytes

The scanning electron microscopy studies revealed that the haemocytes of S.litura were changed very apparently after treatment with A.flavus ethyl acetate extract (Fig. 2). After 96 h various morphological deformities were observed in different haemocytes. As compared to normal haemocytes (Group-1) treated ones showed cell perforation and cytoplasmic leakage (Fig. 2a-d). Similarly, normal haemocytes of Group-2 not shown any deformity but treated ones showed strumae and surface abnormalities (Fig. 2e-f). Group − 3 haemocytes showed surface abnormalities after treatment with A.flavus (2 g-h). Overall SEM studies revealed that morphology of haemocytes was highly disrupted after treatment with ethyl acetate extract of A.flavus for 96 h which might be leads to cytotoxicity.

Relative to the control, the percentage of haemocytes with various deformities were found to be significantly increased in treated larvae due to the toxic effects of fungal extract. After 96 h of feeding, the percentage of cells having various deformities were 78.33% as compared to 8.33% in control (Fig. 3).

Fig. 3
figure 3

The percentage of cells showing various deformities

Mammalian toxicity

Comet assay was conducted to assess genotoxicity on rat using parameters, Tail length, % Tail DNA, Tail Moment and Olive Tail Moment. The obtained data in Tables 234 showed that there were no significant differences in all the parameters after 24 and 96 h of rat’s administration with A.flavus extract at dose level of 100 mg/kg body weight and 200 mg/kg body weight relative to control (ANOVA). In case of rat blood non-significant increase was observed for all the parameters except in case of tail length of 96 h group where significant increase was observed from 14.78 ± 0.13 (control) to 15.51 ± 0.11 (200 mg/kg b.wt). In all other groups non-significant increase was observed after treatment with different concentrations of A. flavus (Table 2). Similarly in rat liver non-significant effect was obtained for all the parameters after treatment with both concentrations ethyl acetate extract of A. flavus (ANOVA) (Table 3). In rat kidney all parameters showed non-significant increase except tail length of 24 h group and %tail DNA of 96 h group where significant increase was observed (Table 4). Effect of duration was also found to be non-significant in all the tissues as revealed by student’s t-test. Overall non-significant effect was observed. Photomicrographs showing DNA damage in different tissues of rat after treatment with A. flavus fungal extracts are shown in Fig. 4.

Table 2 Effect of different concentrations of ethyl acetate extract of A. flavus on different parameters of comet assay in rat blood
Table 3 Effect of different concentrations of ethyl acetate extract of A. flavus on different parameters of comet assay in rat liver
Table 4 Effect of different concentrations of ethyl acetate extract of A. flavus on different parameters of comet assay in rat kidney
Fig. 4
figure 4

Photomicrographs showing DNA extracted from (a, b) rat blood cells (a) Control; (b) After treatment with A.flavus ethyl acetate extract (c, d) rat kidney cells (a) Control; (b) After treatment with A.flavus ethyl acetate extract (e, f) rat liver cells (a) Control; (b) After treatment with A.flavus ethyl acetate extract


In the last few decades, entomopathogenic fungi have attracted considerable attention as biocontrol agents in sustainable agriculture. They are known to have several advantages as compared to synthetic pesticides. To date, several studies have indicated the fungal agents as insect pathogens [5, 6, 29, 30]. So, in the present study secondary metabolites of A. flavus were extracted using ethyl acetate and tested for their toxicity on insect, Spodoptera litura. Ethyl acetate extract was found to exhibit negative impact on larval survival. Previously various species of Aspergillus are found to be entomopathogenic like A.flavus, A.oryzae, A.tamarii and A.versicolor, A.parasititus etc. [5, 6, 31, 32]. Two strains of A. flavus named as A. flavus NRRL and A. flavus AF36 are commercially available as biocontrol agents [33, 34]. Their active ingredients are being used in various pesticides which also help in reducing aflatoxin contamination in crops.

To decipher the mode of action the effect on antioxidant and cellular immune response was studied. Insects have evolved multiple defense mechanisms including antioxidant and cellular immune defense, to respond to pathogens. Antioxidant defense systems in insects include various antioxidant enzymes viz. Catalase (CAT), Ascorbate peroxidase (APOX), Superoxide dismutase (SOD) and Glutathione-S-Transferase (GST). All work co-coordinately to maintain the state of dynamic equilibrium in organism, keeping ROS low level to prevent the cells from damage [35, 36]. Increase in malondialdehyde (MDA) content and antioxidant enzymes activity is an important indicator of oxidative stress. MDA is the product formed during lipid peroxidation (LP) which cause DNA damage and cell death [37]. So, in this study, the MDA content and activities of antioxidant enzymes were determined in Spodoptera litura larvae at different times, after treatment with ethyl acetate extract of A.flavus, in order to speculate A.flavus induced oxidative stress and effect on antioxidant defense. Results showed significant increase in MDA level in response to fungal extract as compared to control. At 96 h value increased 1.5 times as compared to control. This finding is in corroboration with the finding of Karthi et al. [12] revealing the toxic effects of Aspergillus flavus spores on S.litura.

The activities of antioxidant enzymes are also found to be significantly increased after exposure to A.flavus extract, showing influence of fungus on antioxidant defense of insect. CAT and APOX activities increased 3.00 and 3.74 fold respectively, however, SOD activity was found to be increased 1.82 fold as compared to control, at 96 h, in haemolymph of S. litura. This might be due to activation of host response after toxicity induced by A.flavus, in which enzymes activities were remarkably accelerated to metabolize the ROS, reaching the maximum value at 96 h. Similar increase in antioxidant enzymes activities [superoxide dismutase (SOD), catalase (CAT), peroxidases (POX)] was observed by Karthi et al. [12] under the influence of A. flavus spores. Scarce reports recorded the effect of fungal agents on protective enzymes activities. Ding et al. [38] observed the increase in protective enzymes activity in Xylotrechus rusticus (Linnaeus) under the influence of B. bassiana. M. anisopliae also found to alter the antioxidant and detoxifying enzymes activities in Periplaneta americana (Linnaeus) and Locusta migratoria (Linnaeus) [39, 40]. Chaurasia et al. [41] observed variable activity of antioxidant enzymes in P. americana under the influence of entomopathogenic fungus Hirsutella thompsonii. However alteration in enzymes activities due to different chemicals and other stress factors in insects has been reported by many studies [42,43,44,45,46].

Cellular response in insect immune system acts as an important barrier to the infection process [47, 48]. Haemocytes types and their specific responses while insect–pathogen interaction act as a good indicators of insect defense reactions [49, 50]. There are different types of haemocytes which have been morphologically and functionally characterized in various insects [51,52,53]. Multifunctional role of haemocytes are phagocytosis, encapsulation, cell agglutination, detoxification etc. Change in number and configuration was observed in haemocytes under different stresses which ultimately affect the health of insects. Consequently these cells have been used to ascertain the cytogenetic damage by toxic chemicals [21, 54].

So, in our investigation effect on cellular immune response was also judged by analyzing effect on haemocytes by scanning electron microscopy. SEM results showed various cellular deformities in different haemocytes like cell perforation, cytoplasmic leakage, strumae and surface abnormalities after treatment with ethyl acetate extract of A.flavus as compared to control. The results are in corroboration with study of Fan et al. [55] which observed the cell perforation and rupturing with cytoplasmic leakage in haemocytes of Bombyx mori (Linnaeus) after treatment with destruxin A. There are very few studies which reveal the abnormalities of insect haemocytes using SEM, however technique has been used by various researchers to observe and characterize the different types of haemocytes in insects [17, 56] and to observe spores accumulation in insect’s body after fungal infection [5, 6, 57]. Recently Duan et al. [58] observed the infection of B. bassiana to Leptinotarsa decemlineata (Say) via scanning electron microscopy. However the morphological changes observed in present study were demonstrated by various workers due to entomopathogenic fungi and insecticides under light microscopy [59,60,61].

Mammalian toxicity of A.flavus was also carried out by assessing DNA damage in blood, liver and kidney of rat. Non-significant effect was observed in all the tissues of fungal extracts treated rats as compared to control. Similarly negligible toxicity of azadirachtin, a neem biopesticides was earlier reported in rats [62] and human [63]. There are few other botanical extracts which were also checked for their toxicity on rat such as Cassia senna, Caesalpinia gilliesii, Thespesia populnea, Chrysanthemum frutescens, Euonymus japonicus, Bauhinia purpurea, and Cassia fistula extracts [64], Dichaetanthera africana extract [65]. Toxicity of fungal extracts on mammals was not checked previously however, Sprando et al. [66] reported the safety of bacterial species Paenibacillus Alvei to rat.

Moreover, the insecticidal activity of the fungal extract is attributed mainly to various secondary metabolites found in it. Our previous study reported the presence of various phenolic compounds viz. gallic acid, caffeic acid, quercetin and kaempferol by UHPLC in A. flavus extract. So different results observed of A.flavus in insects and rat may be attributed to the presence of these phenolics. Phenolic compounds act as antioxidants in mammals [67, 68] and pro-oxidants in insects [67, 69,70,71] which oxidized after consumption by insects and the reaction products formed are responsible for causing oxidative stress [69]. The other thing regarding its safety and non toxicity on mammals is that it’s a non-aflatoxigenic Aspergillus flavus, the strain does not produce aflatoxin as proved in our previous study by LC-MS [29]. Previously various non-aflatoxin fungal strains of A. flavus are being used to prevent aflatoxin contamination in crops and to control pests [33, 34]. So, on the basis of mammalian toxicity and aflatoxins detection studies, the fungus could be considered safe and can be used as a biocontrol agent by further field trials.


Overall, the study highlights the adverse effect of A. flavus on the physiology of S.litura which might be due to the negative impact on antioxidant and cellular immune defense of an insect. The study helps to identify the insect defenses that could be manipulated to accelerate host death in biological control scenario. The study also showed its safety for mammals as it showed negligible toxicity on rat, suggesting it to be used as bio-pesticide after further field studies.


Insects rearing

Spodoptera litura (Lepidoptera) eggs were obtained from the cauliflower fields around Amritsar (India). After hatching of eggs larvae were fed on castor leaf. Subsequent generations of culture were maintained in laboratory at 25 ± 2 °C temperature, 65 ± 5% relative humidity and 12:12 (D: L) photoperiod [72].

Fungal culture isolation, production and identification

Fungus was isolated from the surface of dead insect (Spodoptera litura) and named as IL (insect larva) fungus. The cause of death was due to natural mould formation. Mycelium fragments were picked directly from the surface of dead insect and point inoculated on Potato Dextrose Agar (PDA) plates supplemented with ampicillin (200 mg/ml). Plates were incubated at 30 °C. After 2–3 days of incubation mycelium regeneration was transferred to new PDA plates for purification purpose. Purified culture was maintained on PDA and stored at 4 °C [29].

The production was carried out in 50 ml malt extract (malt extract = 20 g/l, dextrose = 20 g/l,peptone = 1 g/l, pH = 5.5) broth in 250 ml Erlenmeyer flask by inoculating one plug (1 cm square) taken from the periphery of an actively growing culture. The flasks were incubated at 30 °C and 250 rpm for 10 days. After 10 days extraction was carried out twice using ethyl acetate at 120 rpm and 40 °C. The extracts were concentrated by using rotavapor and dissolved in 1 ml DMSO and stored at 4 °C.

The IL fungus was identified as Aspergillus flavus on morphological (Fig. S1: Morphology of A.flavus showing, Hyphae and Conidiophore under SEM) and molecular basis as indicated in our previous study [29] by using ITS1 and ITS4 primer to amplify ITS1–5.8S- rDNA- ITS2 region. Amplified ITS region was Purified and sequenced at first base sequencing (Malaysia). The sequence similarity was matched with other available databases retrieved from NCBI using BLAST [73]. Phylogenetic Analysis was done using sequences obtained after blast which were aligned using ClustalW program. MEGA 7 software was used to construct a phylogeny tree. Evolutionary history was inferred by Neighbor-joining method and Kimura-2-parameter model [29]. The sequence obtained was submitted to GeneBank under the accession number MF680839 [29] and the final name of fungus is Aspergillus flavus isolate IL (MF680839) [29].

Toxicity test of fungus and LC50 value against S.litura

Toxicity of fungus was tested by checking mortality rate. For this different concentrations (125, 250, 500, 1000 and 2000 μg/ml) of fungal extract were made in 0.5% DMSO and added in artificial diet. The Second instar larvae (6 days old) were reared on fungal extract amended diets as well as with control diet (0.5% DMSO) at controlled temperature 25 ± 2 °C and relative humidity 70 ± 5% conditions. The experiment was replicated six times with five larvae per replication. Each larva was put in separate container (4 × 6 cm) and the diet was changed daily till pupation. Dead larvae were checked daily till pupation. The total numbers of dead larvae were counted. The toxic effect of fungal extract on S.litura was calculated using the probit analysis LC50 (lethal concentration) determination method.

Effect on malondialdehyde (MDA) content and antioxidant enzymes activity

To evaluate the effect of fungal extracts on lipid peroxidation and antioxidant enzymes, the third instar larvae (12 days old) were fed with fungal extracts supplemented diet having concentration 1340.84 μg/ml (LC50 of fungus). The MDA content and enzymes activities [Superoxide dismutase (SOD), catalase (CAT), Ascorbate peroxidase (APOX)] were analyzed in haemolymph of third instar (12 days) larvae.

Larvae were divided into two groups, treatment and control. Treatment group was treated with LC50 of fungus at controlled temperature 25 ± 2 °C and relative humidity 65 ± 5%. The second group was treated with control diet (0.5% DMSO) at same conditions of temperature and relative humidity. The effect of fungal extract has been recorded after different time intervals (24 h, 48 h, 72 h and 96 h) in lipid peroxidation and enzyme activities. The experiment was replicated three times. For each treatment and control there are 10 larvae per replication were taken.

Tissue collection

Haemolymph was collected by cutting proleg with microscissor from 10 different larvae fed with same concentration and then it was pooled. Pooled haemolymph (10%) was mixed with PBS (Phosphate Buffer Saline pH 7.0) containing 0.01%phenylthiourea and centrifuged for 20 min at 10000 g, 4 °C and supernatant obtained was used for enzyme activities studies.

The extraction procedure was same for lipid peroxidation and all enzymes.

Malondialdehyde (MDA) content

MDA content was measured according to Jain and Levine [74] with slight modifications. MDA content as an indicator of lipid peroxidation was determined after incubation of 0.5 ml of sample (supernatant) at 95 °C with Trichloroacetic acid (TCA) (20% w/v), Thiobarbituric acid (TBA) (1% w/v). Absorbance was taken at 532 nm against the blank. MDA content was expressed as nanomole/ml by using 1.56 × 105 M− 1 cm− 1 extinction coefficient.

Catalase (CAT) activity

Enzyme activity was estimated according to methodology given by Aebi [75] with slight modifications. 0.1 ml of supernatant was added into 2.9 ml of H2O2 in a cuvette. Decrease in absorbance was read at 240 nm for 5 min at 1 min interval (25 °C). The enzyme activity was expressed as μM/ml (haemolymph).

Ascorbate peroxidase (APOX) activity

The enzyme activity was calculated according to methodology given by Asada [76] with slight modifications. 0.1 ml of sample, 0.6 ml extraction buffer and 0.125 ml of 0.3%H2O2 were taken in cuvette. The decrease in absorbance was recorded at 290 nm for 5 min at 30s interval (25 °C). The enzyme activity was expressed as μM/ml (haemolymph).

Superoxide dismutase (SOD) activity

The enzyme activity was calculated according to methodology given by Kono [77] with slight modifications. 0.05 ml sample, 1.5 ml sodium carbonate buffer, 0.5 ml NBT (Nitroblue tetrazolium), 0.1 ml TritonX-100, 0.1 ml hydroxylamine hydrochloride were taken in cuvette and increase in absorbance was recorded at 540 nm. The enzyme activity was expressed as μM/ml (haemolymph).

Effect on haemocytes

Haemolymph was collected and pooled from 10 larvae fed with same concentration. Effect on haemocytes was studied by scanning electron microscopy (SEM).

Scanning electron microscopy (SEM)

SEM was done according to methodology of Wang et al. [78] with slight modifications. Haemolymph was bled on termanox discs after cutting proleg of larvae. It was allowed to dry and fixed with 2.5% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.2) for two hours. After this sequential dehydration was done by using graded series of ethanol i.e. 25% followed by 50, 70, 90% and at the end with absolute (100%) alcohol. Then discs were placed in dry chamber for proper drying. At the end silver coating was done by mounting samples on aluminium stubs and haemocytes were observed under SEM at magnification of 10.00KX operated at 10KV after 96 h of treatment with fungal extracts.

Mammalian toxicity study

Sexually mature male wistar albino rats having weight 120 ± 20 g were used in study. Animals were reared on commercial pellet diet and water adds libitum and housed in cages at particular temperature (25 ± 2 °C) and humidity conditions (50–60%). The rats were brought from private source. All experiments were performed according to guidelines provided of Institutional Animal Ethics Committee (IAEC) of Guru Nanak Dev University, Amritsar, Punjab (India). The application for permission for animal experiments was submitted to the CPCSEA, New Delhi and after approval of Institutional Animal Ethics Committee (IAEC) got Registration number: 226/PO/Re/S/2000/CPCSEA. The animals were acclimatized 5 days before experiments. Two concentrations 100 mg/kg b.wt and 200 mg/kg b.wt of fungal extracts were selected for experiments and effects were studied after 24 h and 96 h of exposure. The A.flavus fungal extract dissolved in 0.5%DMSO was injected intraperitoneally to the rat. The experiment was replicated thrice and DNA damage was assessed by comet assay according to methodology given by Ahuja and Saran [79] in different tissues viz. blood, liver and kidney. Rats were sacrificed using gas inhalation method. Blood samples (1 ml) were taken directly from heart and used as such after adding anticoagulant however two tissues i.e. liver, kidney were homogenized in PBS and centrifuged at 10,000 g for 10 min. Cell suspension was taken and used for DNA damage study.

Statistical analysis

To study the effect of duration one way analysis of variance (ANOVA) with Tukey’s test was performed and to study the effect of treatment student’s t-test was applied.

Availability of data and materials

All data generated or analyzed during this study are included in this article and its additional files.





Ascorbate peroxidase


Superoxide dismutase


Glutathione- S-Transferase


  1. Nicholson GM. Fighting the global pest problem: preface to the special Toxicon issue on insecticidal toxins and their potential for insect pest control. Toxicon. 2007;49(4):413–22.

    Article  CAS  PubMed  Google Scholar 

  2. Aktar W, Sengupta D, Chowdhury A. Impact of pesticides use in agriculture: their benefits and hazards. Interdiscip Toxicol. 2009;2(1):1–2.

    Article  PubMed  PubMed Central  Google Scholar 

  3. Castillo MA, Moya P, Hernández E, Primo-Yufera E. Susceptibility of Ceratitis capitata Wiedemann (Diptera: Tephritidae) to entomopathogenic fungi and their extracts. Biol Control. 2000;19(3):274–82.

    Article  Google Scholar 

  4. Charnley AK, Collins SA. Entomopathogenic Fungi and their role in Pest control. Environ Microbial Relationships. 2007;4:159–65.

    Article  Google Scholar 

  5. Bawin T, Seye F, Boukraa S, Zimmer JY, Raharimalala FN, Zune Q, et al. Production of two entomopathogenic Aspergillus species and insecticidal activity against the mosquito Culex quinquefasciatus compared to Metarhizium anisopliae. Biocontrol Sci Tech. 2016a;26(5):617–29.

    Article  Google Scholar 

  6. Bawin T, Seye F, Boukraa S, Zimmer JY, Raharimalala FN, Ndiaye M, et al. Histopathological effects of Aspergillus clavatus (Ascomycota: Trichocomaceae) on larvae of the southern house mosquito, Culex quinquefasciatus (Diptera: Culicidae). Fungal Biol. 2016b;120(4):489–99.

    Article  PubMed  Google Scholar 

  7. Devi PV. Conidia production of the entomopathogenic fungus Nomuraea rileyi and its evaluation for control of Spodoptera litura (fab) on Ricinus communis. J Invertebr Pathol. 1994;63(2):145–50.

    Article  Google Scholar 

  8. Kaur HP, Singh B, Kaur A, Kaur S. Antifeedent and toxic activity of endophytic Alternaria alternata against tobacco caterpillar Spodoptera litura. J Pest Sci. 2013;86(3):543–50.

    Article  Google Scholar 

  9. Lage AM, Moraes GL, Costa MZ. The entomopathogenic potential of Aspergillus spp. in mosquitoes vectors of tropical diseases. J Basic Microbiol. 2001;41(1):45–9.<45::AID-JOBM45>3.0.CO;2-5.

    Article  Google Scholar 

  10. Mazid S, Rajkhowa RC, Kalita JC. Pathogenicity of Aspergillus Niger and Aspergillus flavus on red spider mite (Oligonychus coffeae Nietner), a serious pest of tea. J Entomol Zool Stud. 2015;3(3):11–3.

    Google Scholar 

  11. Seye F, Faye O, Ndiaye M, Njie E, Marie AJ. Pathogenicity of the fungus, Aspergillus clavatus, isolated from the locust, Oedaleus senegalensis, against larvae of the mosquitoes Aedes aegypti, Anopheles gambiae and Culex quinquefasciatus. J Insect Sci. 2009;9(53):1–7.

    Article  PubMed  Google Scholar 

  12. Karthi S, Vaideki K, Shivakumar MS, Ponsankar A, Thanigaivel A, Chellappandian M, et al. Effect of Aspergillus flavus on the mortality and activity of antioxidant enzymes of Spodoptera litura fab.(Lepidoptera: Noctuidae) larvae. Pestic Biochem Physiol. 2018;149:54–60.

    Article  CAS  PubMed  Google Scholar 

  13. Huffman J, Gerber R, Du L. Recent advancements in the biosynthetic mechanisms for polyketide-derived mycotoxins. Biopolymers. 2010;93(9):764–76.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Busi S, Rajkumari J, Hnamte S. Feeding deterrence, acute toxicity and sublethal growth effects of kojic acid isolated from Aspergillus funiculosus. Nat Prod J. 2014;4(1):18–22.

    CAS  Google Scholar 

  15. Rudneva II. Antioxidant system of Black Sea animals in early development. Comp Biochem Physiol C Pharmacol Toxicol Endocrinol. 1999;122(2):265–71.

    Article  CAS  PubMed  Google Scholar 

  16. Freeman BA, Crapo JD. Biology of disease: free radicals and tissue injury. Lab Invest. 1982;47(5):412–26.

    CAS  PubMed  Google Scholar 

  17. Falleiros ÂMF, Bombonato MTS, Gregório EA. Ultrastructural and quantitative studies of hemocytes in the sugarcane borer, Diatraea saccharalis (Lepidoptera: Pyralidae). Braz Arch Biol Technol. 2003;46(2):287–94.

    Article  Google Scholar 

  18. Ratcliffe NA, Gagen SJ. Cellular defense reactions of insect hemocytes in vivo: nodule formation and development in Galleria mellonella and Pieris brassicae larvae. J Invert Pathol. 1976;28(3):373–82.

    Article  Google Scholar 

  19. Ratcliffe NA, Rowley AF, Fitzgerald SW, Rhodes CP. Invertebrate immunity: basic concepts and recent advances. Int Rev Cytol. 1985;97:183–350.

    Article  CAS  Google Scholar 

  20. Lackie AM. Haemocyte behaviour. Adv Insect Physiol. 1988;21:85–178.

    Article  CAS  Google Scholar 

  21. Gayfullina LR, Saltykova ES, Nikolenko AG. Cellular immune reactions participating in resistance formation of Colorado beetle (Leptinotarsa decemlineata say) larvae and imago to a biopreparation for potato. Resistant Pest Manage Newsl. 2006;15:22–4.

    Google Scholar 

  22. Gangemi S, Miozzi E, Teodoro M, Briguglio G, De Luca A, Alibrando C, et al. Occupational exposure to pesticides as a possible risk factor for the development of chronic diseases in humans. Mol Med Rep. 2016;14(5):4475–88.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Rodgers KM, Udesky JO, Rudel RA, Brody JG. Environmental chemicals and breast cancer: an updated review of epidemiological literature informed by biological mechanisms. Environ Res. 2018;160:152–82.

    Article  CAS  PubMed  Google Scholar 

  24. Curl CL, Spivak M, Phinney R, Montrose L. Synthetic pesticides and health in vulnerable populations: agricultural workers. Curr Environ Health Rep. 2020;7(1):13–29.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Burlinson B, Tice RR, Speit G, Agurell E, Brendler-Schwaab SY, Collins AR, et al. Fourth international workgroup on genotoxicity testing: results of the in vivo comet assay workgroup. Mutat Res-Gen Tox En. 2007;627(1):31–5.

    Article  CAS  Google Scholar 

  26. Collins AR, Azqueta A. DNA repair as a biomarker in human biomonitoring studies; further applications of the comet assay. Mutat RES-Fund Mol M. 2012;736(1–2):122–9.

    Article  CAS  Google Scholar 

  27. Collins A, Koppen G, Valdiglesias V, Dusinska M, Kruszewski M, Møller P, et al. The comet assay as a tool for human biomonitoring studies: the ComNet project. Mutat Res-Rev Mutat. 2014;759:27–39.

    Article  CAS  Google Scholar 

  28. Woźniak K, Blasiak J. In vitro genotoxicity of lead acetate: induction of single and double DNA strand breaks and DNA–protein cross-links. Mutat Res. 2003;535(2):127–39.

    Article  PubMed  Google Scholar 

  29. Kaur M, Chadha P, Kaur S, Kaur A, Kaur R, Yadav AK, et al. Evaluation of genotoxic and cytotoxic effects of ethyl acetate extract of Aspergillus flavus on Spodoptera litura. J Appl Microbiol. 2019;126(3):881–93.

    Article  CAS  PubMed  Google Scholar 

  30. Jaber S, Mercier A, Knio K, Brun S, Kambris Z. Isolation of fungi from dead arthropods and identification of a new mosquito natural pathogen. Parasites Vectors. 2016;9(1):491–5.

    Article  PubMed  PubMed Central  Google Scholar 

  31. Senthilkumar N, Murugesan S, Babu DS. Metabolite profiling of the extracts of endophytic Fungi of Entomopathogenic significance, Aspergillus flavus and Nigrospora sphaerica isolated from tropical tree species of India, Tectona grandis L. J Agri Life Sci. 2014;1(1):108–14.

    Google Scholar 

  32. Bhan S, Mohan L, Srivastava CN. Synergistic larvicidal potential of Temephos and entomopathogenic fungus, Aspergillus flavus against filarial vector, Culex quinquefaciatus (say). Int J Mosq Res. 2015;2(2):33–7.

    Google Scholar 

  33. U.S. Environmental Protection Agency. Biopesticide registration action document Aspergillus flavus AF36. 2003;Available at: 006456.pdf. Accessed 20 Oct 2009.

    Google Scholar 

  34. U.S. Environmental Protection Agency. Biopesticide registration action document Aspergillus flavus (NRRL 21882). 2004;Available at: brad_006500.pdf. Accessed 20 Oct 2009.

    Google Scholar 

  35. Zhang Y, Wang Z, Huang D. Interrelation between carboxylesterase and glutathione-S-transferase in Apriona germari larvae and secondary metabolites of poplar trees. Sci Silvae Sinicae. 2001;37(3):106–11.

    CAS  Google Scholar 

  36. Gao XR, Cui YL, Xu FH, Li RF. The effects SOD scavenging O2−· as CAT existing. J Heibei Normal Univ (Natur. Sci.). 1995;4:59–62.

    Google Scholar 

  37. Ayala A, Muñoz MF, Argüelles S. Lipid peroxidation: production, metabolism, and signaling mechanisms of malondialdehyde and 4-hydroxy-2-nonenal. Oxidative Med Cell Longev. 2014;2014:11–9.

    Article  Google Scholar 

  38. Ding JN, Zhang HH, Chi DF. Effects of a pathogenic Beauveria bassiana (Hypocreales: Cordycipitaceae) strain on detoxifying and protective enzyme activities in Xylotrechus rusticus (Coleoptera: Cerambycidae) larvae. Fla Entomol. 2015;98(4):1148–56.

    Article  CAS  Google Scholar 

  39. Mutyala NB, Vadlamani P. Induced oxidative stress by Metarhizium anisopliae spp. instigates changes in lipid peroxidation and ultra structure in Periplaneta Americana. Afr J Microbiol Res. 2013;7(38):4629–37.

    Article  Google Scholar 

  40. Jia M, Cao G, Li Y, Tu X, Wang G, Nong X, et al. Biochemical basis of synergism between pathogenic fungus Metarhizium anisopliae and insecticide chlorantraniliprole in Locusta migratoria (Meyen). Sci Rep. 2016;6(1):28424–34.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Chaurasia A, Lone Y, Wani O, Gupta US. Effect of certain entomopathogenic fungi on oxidative stress and mortality of Periplaneta americana. J Entomol Zool Stu. 2016;4(1):234–9.

    Google Scholar 

  42. Hyrsl P, Buyukguzel E, Buyukguzel K. The effects of boric acid-induced oxidative stress on antioxidant enzymes and survivorship in Galleria mellonella. Arch Insect Physiol. 2007;66:23–31.

    Article  CAS  Google Scholar 

  43. Barata C, Lekumberri I, Vila-Escalé M, Prat N, Porte C. Trace metal concentration, antioxidant enzyme activities and susceptibility to oxidative stress in the tricoptera larvae Hydropsyche exocellata from the Llobregat river basin (NE Spain). Aquat Toxicol. 2005;74(1):3–19.

    Article  CAS  PubMed  Google Scholar 

  44. Aslanturk A, Kalender S, Uzunhisarcikli M, Kalender Y. Effects of methidathion on antioxidant enzyme activities and malondialdehyde level in midgut tissues of Lymantriadispar (lepidoptera) larvae. J Entomol Res Soc. 2011;13(3):27–38.

    Google Scholar 

  45. Karthi S, Sankari R, Shivakumar MS. Ultraviolet-B light induced oxidative stress: effects on antioxidant response of Spodoptera litura. Photoch Photobiol B. 2014;135:1–6.

    Article  CAS  Google Scholar 

  46. Ali A, Rashid MA, Huang QY, Lei CL. Influence of UV-A radiation on oxidative stress and antioxidant enzymes in Mythimna separata (Lepidoptera: Noctuidae). Environ Sci Pollut Res. 2017;24(9):8392–8.

    Article  CAS  Google Scholar 

  47. Hoffmann JA. Innate immunity of insects. Curr Opin Immunol. 1995;7(1):4–10.

    Article  CAS  PubMed  Google Scholar 

  48. Hoffmann JA. The immune response of drosophila. Nature. 2003;426(6962):33–8.

    Article  CAS  PubMed  Google Scholar 

  49. Gillespie JP, Burnett C, Charnley AK. The immune response of the desert locust Schistocerca gregaria during mycosis of the entomopathogenic fungus, Metarhizium anisopliae var acridum. J Insect Physiol. 2000;46(4):429–37.

    Article  CAS  PubMed  Google Scholar 

  50. da Silva C, Dunphy GB, Rau ME. Interaction of hemocytes and prophenoloxidase system of fifth instar nymphs of Acheta domesticus with bacteria. Dev Comp Immunol. 2000;24(4):367–79.

    Article  PubMed  Google Scholar 

  51. Giulianini PG, Bertolo F, Battistella S, Amirante GA. Ultrastructure of the hemocytes of Cetonischema aeruginosa larvae (Coleoptera, Scarabaeidae): involvement of both granulocytes and oenocytoids in in vivo phagocytosis. Tissue Cell. 2003;35(4):243–51.

    Article  CAS  PubMed  Google Scholar 

  52. Costa SC, Ribeiro C, Girard PA, Zumbihl R, Brehélin M. Modes of phagocytosis of gram-positive and gram-negative bacteria by Spodoptera littoralis granular haemocytes. J Insect Physiol. 2005;51(1):39–46.

    Article  CAS  PubMed  Google Scholar 

  53. Lavine MD, Strand MR. Insect hemocytes and their role in immunity. Insect Biochem Mol Bio. 2002;32(10):1295–309.

    Article  CAS  Google Scholar 

  54. Begum R, Gohain R. Detoxication of PP'DDT by the hemocytes of the fifth instar Philosamia ricini Boisd. J Environ Biol. 1996;17(2):149–55.

    CAS  Google Scholar 

  55. Fan JQ, Chen XR, Hu QB. Effects of destruxin a on hemocytes morphology of Bombyx mori. J Integr Agric. 2013;12(6):1042–8.

    Article  Google Scholar 

  56. Silva JEB, Boleli IC, Simões ZLP. Hemocyte types and total and differential counts in unparasitized and parasitized Anastrepha obliqua (Diptera, Tephritidae) larvae. Braz J Biol. 2002;62(4A):689–99.

    Article  CAS  PubMed  Google Scholar 

  57. Baggio MV, Ferreira MDC, Monteiro AC, Maldonado Junior W, Lemos MVF. Pathogenicity of Aspergillus westerdijkiae to females and oothecae of Periplaneta Americana. Ciência Rural. 2016;46(1):20–5.

    Article  CAS  Google Scholar 

  58. Duan Y, Wu H, Ma Z, Yang L, Ma D. Scanning electron microscopy and histopathological observations of Beauveria bassiana infection of Colorado potato beetle larvae. Microb Pathog. 2017;111:435–9.

    Article  PubMed  Google Scholar 

  59. Ferrarese R, Brivio M, Congiu T, Falabella P, Grimaldi A, Mastore M, et al. Early suppression of immune response in Heliothis virescens larvae by the endophagous parasitoid Toxoneuron nigriceps. Invert Surviv J. 2005;2(1):60–8.

    Google Scholar 

  60. Habeeb SM, El-Hag HA. Ultrastructural changes in hemocyte cells of hard tick (Hyalomma dromedarii: Ixodidae): a model of Bacillus thuringiensis var. thuringiensis H14;-endotoxin mode of action. Am-Euras J Agric Environ Sci. 2008;3:829–36.

    Google Scholar 

  61. Kaur HP, Singh B, Thakur A, Kaur A, Kaur S. Studies on immunomodulatory effect of endophytic fungus Alternaria alternata on Spodoptera litura. J Asia-Pacific Entomol. 2015;18(1):67–75.

    Article  CAS  Google Scholar 

  62. Raizada RB, Srivastava MK, Kaushal RA, Singh RP. Azadirachtin, a neem biopesticide: subchronic toxicity assessment in rats. Food Chem Toxicol. 2001;39(5):477–83.

    Article  CAS  PubMed  Google Scholar 

  63. Boeke SJ, Boersma MG, Alink GM, van Loon JJ, van Huis A, Dicke M, et al. Safety evaluation of neem (Azadirachta indica) derived pesticides. J Ethnopharmacol. 2004;94(1):25–41.

    Article  CAS  PubMed  Google Scholar 

  64. Derbalah AS. Efficacy of some botanical extracts against Trogoderma granarium in wheat grains with toxicity evaluation. Sci World J. 2012;2012:1–9.

    Article  Google Scholar 

  65. Kognou ALM, Tchamgoue AD, Tchokouaha LRY, Ngima D, Nthenge-Ngumbau PVTF, Tchinda AT, et al. Acute and sub-chronic toxicity studies of Dichaetanthera africana (hook. F.) Jacq. Fel.(Melastomataceae) stem bark ethanol extract. J Appl Pharm Sci. 2018;8(6):147–55.

    Article  CAS  Google Scholar 

  66. Sprando RL, Black T, Olejnik N, Keltner Z, Topping V, Ferguson M, et al. Assessing the effect of oral exposure to Paenibacillus alvei, a potential biocontrol agent, in male, non-pregnant, pregnant animals and the developing rat fetus. Food Chem Toxicol. 2017;103:203–13.

    Article  CAS  PubMed  Google Scholar 

  67. Grace SC. Phenolics as antioxidants. Antioxidants and reactive oxygen species in plants; 2005. p. 141–68.

    Google Scholar 

  68. Swallah MS, Sun H, Affoh R, Fu H, Yu H. Antioxidant potential overviews of secondary metabolites (polyphenols) in fruits. Int J Food Sci. 2020;2020:1–9.

    Article  CAS  Google Scholar 

  69. Felton GW, Summers CB. Antioxidant systems in insects. Arch Insect Biochem Physiol. 1995;29(2):187–97.

    Article  CAS  PubMed  Google Scholar 

  70. Summers CB, Felton GW. Prooxidant effects of phenolic acids on the generalist herbivore Helicoverpa zea (Lepidoptera: Noctuidae): potential mode of action for phenolic compounds in plant anti-herbivore chemistry. Insect Biochem Mol Biol. 1994;24(9):943–53.

    Article  CAS  Google Scholar 

  71. Sambangi P, Rani PU. Physiological effects of resveratrol and coumaric acid on two major groundnut pests and their egg parasitoid behavior. Arch Insect Biochem Physiol. 2016;91(4):230–45.

    Article  CAS  PubMed  Google Scholar 

  72. Kaur M, Chadha P, Kaur S, Kaur A, Kaur R, Yadav AK, et al. Schizophyllum commune induced genotoxic and cytotoxic effects in Spodoptera litura. Sci Rep. 2018;8(1):1–12.

    Google Scholar 

  73. Sharma M, Chadha BS, Kaur M, Ghatora SK, Saini HS. Molecular characterization of multiple xylanase producing thermophilic/thermotolerant fungi isolated from composting materials. Lett Appl Microbiol. 2008;46(5):526–35.

    Article  CAS  PubMed  Google Scholar 

  74. Jain SK, Levine SN. Elevated lipid peroxidation and vitamin E-quinone levels in heart ventricles of streptozotocin-treated diabetic rats. Free Radic Biol Med. 1995;18(2):337–41.

    Article  CAS  PubMed  Google Scholar 

  75. Aebi H. Catalase in vitro. Method Enzymol. 1984;105:121–6.

    Article  CAS  Google Scholar 

  76. Asada K. Chloroplasts: formation of active oxygen and its scavenging. Method Enzymol. 1984;105:422–9.

    Article  CAS  Google Scholar 

  77. Kono Y. Generation of superoxide radical during autoxidation of hydroxylamine and an assay for superoxide dismutase. Arch Biochem Biophs. 1978;86(1):189–95.

    Article  Google Scholar 

  78. Wang Y, Hu M, Chiang MWL, Shin PKS, Cheung SG. Characterization of subpopulations and immune-related parameters of hemocytes in the green-lipped mussel Perna viridis. Fish Shellfish Immun. 2012;32(3):381–90.

    Article  CAS  Google Scholar 

  79. Ahuja YR, Saran R. Potential of single cell gel electrophoresis assay (comet assay) in heavy ion radiation biology. Int J Hum Genet. 2001;1(2):151–6.

    Article  Google Scholar 

Download references


The grants received from UGC (University Grant Commission), New Delhi, India under SAP (Special Assistance Program) and UPE schemes are thankfully acknowledged.


This work was supported by Grant from UGC (University Grant Commission) – SAP (Special Assistance Program) and UPE grants.

Grant number of SAP- No.F.4–4/2016/DRS-I (SAP-II).

Funding bodies had no roles in study design and collection, analysis, and interpretation of data, or preparation of the manuscript.

Author information

Authors and Affiliations



PC, AK and SK designed the study and analyzed the content. MK performed the experiments and analyzed the content related to it. All authors have read and approved the manuscript.

Corresponding author

Correspondence to Pooja Chadha.

Ethics declarations

Ethics approval and consent to participate

This article contains studies involving animals. Rats were used after getting permission from animal ethical committee.

Consent for publication

Not applicable.

Competing interests

All authors declare that they have no conflict of interest.

Additional information

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary Information

Additional file 1: Figure S1.

Morphology of A. flavus: Hyphae and Conidiophore under SEM.

Rights and permissions

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit The Creative Commons Public Domain Dedication waiver ( applies to the data made available in this article, unless otherwise stated in a credit line to the data.

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Kaur, M., Chadha, P., Kaur, S. et al. Aspergillus flavus induced oxidative stress and immunosuppressive activity in Spodoptera litura as well as safety for mammals. BMC Microbiol 21, 180 (2021).

Download citation

  • Received:

  • Accepted:

  • Published:

  • DOI: