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±2⁰C temperature, 65±5% relative humidity and 12:12 (D: L) photoperiod .
Fungal culture isolation, production and identification
Fungus was isolated from the surface of dead insect (Kaur et al. 2019). The production was carried out in 50ml malt extract (malt extract=20g/l, dextrose=20g/l,peptone = 1g/l, pH=5.5) broth in 250ml Erlenmeyer flask by inoculating one plug (1 cm square) taken from the periphery of an actively growing culture. The flasks were incubated at 30ºC and 250rpm for 10 days. After 10 days extraction was carried out twice using ethyl acetate at 120rpm and 40ºC. The extracts were concentrated by using rotavapor and dissolved in 1ml DMSO and stored at 4⁰C.
The 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  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 . The sequence was submitted to GeneBank under the accession number MF680839 .
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 2000µg/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±2⁰C and relative humidity 70±5% conditions. The experiment was replicated six times with five larvae per replication. Each larva was put in separate container (4×6cm) 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 1340.84µg/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 (12days) larvae.
Larvae were divided into two groups, treatment and control. Treatment group was treated with LC50 of fungus at controlled temperature 25±2⁰C 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 (24hr, 48hr, 72hr and 96hr) 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.
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 10000g, 4ºC 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  with slight modifications. MDA content as an indicator of lipid peroxidation was determined after incubation of 0.5ml 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-1cm-1 extinction coefficient.
Catalase (CAT) activity:
Enzyme activity was estimated according to methodology given by Aebi  with slight modifications. 0.1ml of supernatant was added into 2.9ml of H2O2 in a cuvette. Decrease in absorbance was read at 240nm for 5min at 1min interval (25⁰C). The enzyme activity was expressed as μM/ml (haemolymph).
Ascorbate peroxidase (APOX) activity:
The enzyme activity was calculated according to methodology given by Asada  with slight modifications. 0.1ml of sample, 0.6ml extraction buffer and 0.125ml of 0.3%H2O2 were taken in cuvette. The decrease in absorbance was recorded at 290nm for 5min at 30sec interval (25⁰C). The enzyme activity was expressed as μM/ml (haemolymph).
Superoxide dismutase (SOD) activity:
The enzyme activity was calculated according to methodology given by Kono  with slight modifications. 0.05ml sample, 1.5ml sodium carbonate buffer, 0.5ml NBT (Nitroblue tetrazolium), 0.1ml TritonX-100, 0.1ml hydroxylamine hydrochloride were taken in cuvette and increase in absorbance was recorded at 540nm. 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.  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 0.1M 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 96hr of treatment with fungal extracts.
Mammalian toxicity study:
Sexually mature male wistar albino rats having weight 120±20gm were used in study. Animals were reared on commercial pellet diet and water adds libitum and housed in cages at particular temperature (25±2⁰C) 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 5days before experiments. Two concentrations 100mg/kg b.wt and 200mg/kg b.wt of fungal extracts were selected for experiments and effects were studied after 24hr and 96hr 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  in different tissues viz. blood, liver and kidney. Rats were sacrificed using gas inhalation method. Blood samples (1ml) 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 10,000g for 10min. Cell suspension was taken and used for DNA damage study.
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