Fungal culture
The pre-maintained pure culture of B. bassiana and M. anisopliae was obtained from the Insect pathology lab (Institute of Plant Protection) MNS-University of Agriculture Multan (Punjab) Pakistan. The collected culture was purified by using the single spore method described by (Zhang et al., 2013; Noman et al., 2018).
Preparation of liquid culture for the production of proteins
Different types of liquid fermentations were found effective for the mass production of many fungi. B. bassiana and M. anisopliae were found effectively mass-produced using liquid culture for commercial production (Mascarin et al., 2015; Jaronski and Mascarin, 2017). Different liquid solutions were prepared to evaluate protein production in liquid media. The primary ingredients used to prepare liquid media were Mycological Peptone, Fructose, Glucose, Yeast Extract, and Dextrose. The effect of glucose level on the production of blastospores was well studied (Kobori et al., 2015). Different liquid mediums were made according to (Ortiz-Urquiza et al., 2010) for the production of fungal proteins in liquid culture. Along with these liquid mediums, the Czapek Dox medium was also prepared for effective protein production (Table 1.1).
The primary solutions were prepared using 250 Erlenmeyer glass bottles. Fresh spore/conidial suspensions were calculated at 1×107 and inoculated to a liquid medium before autoclaving the liquid medium. The inoculated flasks were placed in a shaking incubator at 110 rpm for 4 days at a temperature of 25°C described by (Ortiz-Urquiza et al., 2010).
Precipitation of proteins using ammonium sulfate [(NH4)2SO4]
Protein solubility in the liquid medium was affected by the concentrations of ammonium sulfate. This (NH4)2SO4 possesses high ionic strength compared to other ions, is highly soluble, and is cheaper. (NH4)2SO4 has properties to stabilize protein structure due to high ionic strength (Doung and Gabelli, 2014). The liquid medium was filtered to make cell-free culture and the crude secreted proteins were precipitated at 95% Ammonium Sulfate described by (Wingfield, 2016). Further centrifugation at 10000 g to remove supernatant and pallets was used for protein analysis.
Analysis of proteins using SDS-PAGE
The Mini-Protean II (Gel Electrophoresis Unit) was used for SDS-PAGE analysis for protein analysis. The resolving gel (12%) and stacking gel (4%) were prepared. The 1X running buffer was prepared using Tris-Base (30.3g), Glycine 144.0g, SDS (10.0g) in a 1000mL final volume. 4X sample buffer was also prepared using 125 mM Tris-HCl (pH 6.8) (5mL), 20% Glycerol (8mL), 4% SDS (8mL), 10% β-Marceptoethanol (4mL), and 0.5 mg/ml Bromophenol Blue (15mL) in a final volume of 40mL. Samples were loaded before making them reduced through PCR. The gel was run providing electricity (120V, 70mA) for 45 minutes until the dye front reached the bottom of the apparatus. After running, the gel was removed gently and placed into a small bucket. Staining was done using a staining solution (150 mL ethanol, 50 mL Glacial Acetic Acid, 1 g of Coomassie Brilliant Blue R-250 in a final volume of 500mL. 20-30 minutes were found enough for staining with gentle shaking. Destaining was done using Ethanol 300mL, Glacial Acetic Acid 100 mL in DH2O. After destaining, the gel was kept for documentation.
Insect culture
B. zonata were taken from the lab stock colony of Insect Rearing lab at Institute of Plant Protection, MNS-University of Agriculture Multan. The colony was maintained and augmented yearly by adding infested fruits of mango, guava, and other commodities. The rearing conditions were set at a temperature of 25±2°C, relative humidity of 55-60%, with a photoperiod of 16:8. The primary diet given to the insect was banana crumbs along with an artificial diet described by (Aslam et al., 2019). The primary ingredients of the artificial diet were 200 mL sterile water, 18.8 g sugar, and 5 g of baker’s yeast. Baker’s Yeast 5g and 5mL Protein hydrolysate were also found well in egg hatchability of B. zonata demonstrated by (Shinwari et al., 2015).
Application to B. zonata larvae
Mycoproteins (crude) proteins from B. bassiana and M. anisopliae were tested against larvae using diet tests (Quesada-Moraga et al., 2006). The diet test of larvae was done in standardized square Petri plates. Newly molted 2nd instar larvae were exposed to an artificial feed containing protein. Ten larvae were exposed to one Petri plate with different concentrations of 2, 4, 6, 8, and 10 μl/g per plate along with untreated control. The treatments were replicated 3 times under a completely randomized block design. The experimental conditions were provided with Temperatures 25±2°C and 60±5 RH at 16: 8 light and dark photoperiods. The data about larval mortality was recorded after 3, 5, and 7 post-application intervals.
Application to B. zonata pupae
The activity of mycoproteins from B. bassiana and M. anisopliae was evaluated when 3-day old pupae were treated with different concentrations of proteins. Before treating the pupae, fine sterile sand was taken in square Petri plates to incubate treated pupae for adult emergence. There were five different concentrations of proteins (10, 15, 20, 25, and 30 µl). The treatments were replicated 4 times in a completely randomized design. The data about adult emergence was recorded at 4, 7, and 10 days post-application intervals.
Application to B. zonata adult
The insecticidal activity of mycoproteins from B. bassiana and M. anisopliae were evaluated against 3-day old adults of B. zonata using a diet test (Ortiz-Urquiza et al., 2010). Five pairs of adult B. zonata 3 days old (♀ and ♂) were placed in plastic rearing cages (9cm×3.5cm×3.4cm). There were six different concentrations of proteins (5, 10, 15, 20, 25, and 30µl/g along with untreated control. The treatments were replicated 5 times in a completely randomized block design. Experimental conditions were maintained at 25±2°C and RH of 60±5 at 16:8 light and dark photoperiods.