Rearing of Spodoptera litura:
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 [80].
Fungal culture isolation, production and identification:
Endophytic fungus was isolated from leaves of Aloe vera collected from Amritsar (India). The leaves were thoroughly washed with distilled water, followed by sterilization with 70% ethanol (2 min), 5% sodium hypochlorite solution (5 min) and finally rinsed with sterile distilled water. Sterilized samples were cut into small pieces and they were placed on water agar plates having ampicillin (200mg/ml) as antibacterial agent and incubated at 30⁰C. After emergence of hyphae, the hyphae tips were picked and cultured on PDA (potato dextrose agar) plates. Then the culture was purified and maintained on PDA for further studies [81].
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 Schizophyllum commune on morphological (Fig S1: Morphology of S. commune showing hyphae and clamp connection, characterstics of basidiomycetes under SEM) and molecular basis as indicated in our previous study [79] by using ITS1 and ITS4 primers 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 [82]. The sequence was deposited into GeneBank under accession number: MF680077.
Toxicity test:
On the basis of bioassay studies the LC50 value of ethyl acetate extract of S. commune was found to be 276.542µg/ml [79]. This concentration is selected for, to analyze its effect on antioxidant and detoxification enzymes and to decipher various morphological changes in haemocytes.
Antioxidant enzyme activities:
To evaluate the effect of fungal extracts on antioxidant enzymes, the third instar larvae (12 days old) were fed with fungal extracts supplemented diet having concentration 276.54µg/ml. The enzyme activities [Superoxide dismutase (SOD), catalase (CAT), Ascorbate peroxidase (APOX) and Glutathione-S-Transferase (GST)] were analyzed in haemolymph and midgut of third instar (12days) larvae.
Larvae were divided into two groups, treatment and control. Treatment group was exposed 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 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 10000g, 4ºC and supernatant obtained was used for enzyme activities studies. Similarly midgut tissue was also taken after dissection with microscissor from 10 different larvae fed with same concentration and homogenate (10% w/v) was prepared by homogenizing larval midguts (100 mg in 1 ml) in PBS. Afterwards, homogenate was centrifuged in PBS for 20 min at 10000g, 4ºC and supernatant obtained was used for enzyme activities studies.
The extraction procedure was same for all enzymes.
Catalase (CAT) activity:
Enzyme activity was estimated according to methodology followed by Aebi [83] 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) and μM/mg (midgut) weight.
Ascorbate peroxidase (APOX) activity:
The enzyme activity was calculated according to methodology inroduced by Asada [84] with slight modifications. 0.1ml of sample, 0.6ml extraction buffer (50mM potassium phosphate buffer pH 7.0) 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) and μM/mg (midgut) weight.
Superoxide dismutase (SOD) activity:
The enzyme activity was calculated according to methodology followed by Kono [85] with slight modifications. 0.05ml sample, 1.5ml extraction buffer (50mM sodium carbonate buffer pH10.0), 0.5ml of 96µM NBT (Nitroblue tetrazolium), 0.1ml TritonX-100, 0.1ml of 20mM hydroxylamine hydrochloride were taken in cuvette and increase in absorbance was recorded at 540nm. The enzyme activity was expressed as μM/ml (haemolymph) and μM/mg (midgut) weight.
Glutathione- S-transferase (GST) activity:
GST activity was estimated using the method of Habig et al. [86] with minor modifications. 50µl of 10mM CDNB (1- chloro-2, 4-dinitrobenzene), 100µl GSH (Reduced glutathione), 50µl of sample and 0.2ml of 0.1M sodium phosphate buffer containing PTU (phenylthiourea) were incubated at 25⁰C and absorbance change was recorded at 340nm for 5min at 1min interval. The enzyme activity was expressed as μM/ml (haemolymph) and μM/mg (midgut) weight.
Effect on morphology of haemocytes:
To study the morphological alterations in haemocytes, the methodology of Wang et al. [87] with slight modifications was followed. Haemolymph of insects exposed for 96 hours 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 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. The percentage of cells showing various deformities were also calculated in treatment and control group after 96hr exposure to S.commune ethyl acetate extract.
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.