Insect collection: Podontia quatuordecimpunctata larva of different ages (2, 3, 6, 8, 10 and 12 days) and adults were collected from leaves of an infested hog plum tree during April-June from Fatehpur village near the Chittagong University campus, Bangladesh. Until dissection, insects were kept on or in close proximity to plant host tissues where insects were actively feeding.
Gut dissection and fluid collection: Insects were dissected on the same day of collection. After immobilizing on ice for around half an h, the larva were kept abdomen side up on a petri dish which was on ice and dissected to remove the entire, intact guts. The multiple guts of different day’s larva and adult were taken separately in micro centrifuge tubes. Dissected guts were cut into small pieces, homogenized by vortexing to ensure fluid extraction and centrifuged at 10,000 rpm for 10 min at 4°C. The resulting supernatants were transferred to new 1.5 mL micro centrifuge tubes and stored at -20°C until use. All the biochemical analyses with this supernatant were carried out in triplicates.
Plate assay for cellulolytic activity: A modified substrate-agar plate assay (Teather and Wood, 1982) was used to assay endo-β-1,4-D-glucanase activity qualitatively in the gut fluid of eight day’s larva. To observe the clear zone formation around the sample well against a red-staining background on agar plates, 1% CMC and 3% agar were mixed with 0.1 M Na-acetate buffer, pH 5.3 and poured in petri dishes to solidify the gel. 220 μg of gut protein in water was loaded in the hole punched in the center of plate. After overnight incubation at 37°C, the plates were stained with 1% Congo red (Sigma-Aldrich) for 10-15 min before de-staining with 1.0 M NaCl solution for 15-20 min for several times.
Measurement of endo-β-1,4-D-glucanase activity: Endo-β-1,4-D-glucanase activity was quantified as the rate of production of reducing sugars from the substrate, carboxymethyl cellulose (BDH chemical Ltd pool England), using a modified 3,5-dinitrosalicylic acid (DNSA) assay method. Gut fluids (33 μg of protein) of each respective day’s larva and adult mentioned above and 235 μL of 1% CMC suspended in 0.1 M Na-acetate buffer, pH 5.3 were mixed separately and incubated for 30 min at 37°C. The reactions were stopped by adding 450 μL DNSA solution followed by heating in boiling water for 10 min. After adding 40% Rochelle salt (MARK), the reaction mixers were cooled at room temperature for 5 min and centrifuged at 10,000 rpm for 5 min. Supernatants were transferred to new microcentrifuge tubes to measure absorbance at 540 nm on a UV spectrophotometer (Shimadzu). A blank was prepared with substrate and DNSA solution before adding gut juice so that enzymes present in the juice could not hydrolyze CMC and the subsequent process same as described above. One unit of cellulolytic activity was defined as the amount of enzyme required to produce 1 μmol of reducing sugar per min at 37°C and pH 5.3. A standard curve of absorbance against amount of glucose (50-300 μg) was constructed to enable calculation of the amount of reducing sugar (glucose equivalents) released during endo-β-1,4-D-glucanase assays (Fig. 1). In all following experiments, enzyme activity was measured using this method and the extract of 8 days larvae was used.
Effect of temperature and pH on enzyme activity: The effects of temperature and pH on enzyme activity of gut fluids were estimated at eleven temperatures and nine pH values. The effect of temperature on endo-β-1,4-D-glucanase activity was examined by incubating the reaction mixture (in 0.1 M Na-acetate buffer pH 5.3) over a temperature range of 20 to 70°C (using 1% CMC as substrate). The thermal stability of endo-β-1,4-D-glucanase was determined by pre-incubation of the sample at 20, 25, 30, 35, 40, 45, 50 and 55°C for 30 min, followed by measurement of activity under the standard test conditions as mentioned before. Activity at optimum temperature was taken as 100. Optimal pH for the enzyme activity was determined by measuring the hydrolysis of CMC in a series of buffers at various pH values ranging from pH 2 to 10. The buffers used were KCl-HCl buffer 0.1 M pH 2.0, K-hydrogen pthelate HCl buffer (0.1 M) pH 3, Na-acetate buffer (0.1 M) pH 4 and 5, phosphate buffer (0.1 M) pH 6 and 7, tris-HCl buffer (0.1 M) pH 8, glycine-NaOH buffer (0.1 M) pH 9 and 10.The pH at which activity was highest was considered as 100%.
Zymography: Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) were performed with some alterations to detect activity of endo-β-1,4-D-glucanase through zymogram techniques. For the CMC zymogram, SDS-10% PAGE resolving gel was prepared by adding 0.2% CMC slowly to prevent aggregation. After dissolving CMC, ammonium persulfate (APS) and tetramethylethylenediamine (TEMED) were added and gels were incubated at 45°C for 2 h or until polymerization. Defoliator gut fluids (8 μg) of different aged larva and adult were solubilized in 1 volume of sample buffer (50 mM Tris-HCl pH 6.8, 2% SDS, 10% glycerol, 1% β-mercaptoethanol, 0.01% bromophenol blue) and loaded onto the gel. Proteins in samples were separated at a constant 100 V at 4°C for approximately 4 h or until dye reached the bottom of the gel. After electrophoresis, the gel was washed with 50 mL wash buffer (0.1 M Na-succinate pH 5.8 and 0.1% β-mercaptoethanol) at room temperature for five times (each was for 30 min) but last wash was at 30°C to allow for enzyme activity against the substrate. Then, the remaining CMC in the gel was stained with 0.1% Congo red for 30 min and destained by washing in a solution of 1 M NaCl at room temperature to reveal zones of clearing where CMC had been degraded by enzyme. For improving visualization of clearing area, 100 μL of glacial acetic acid was added after destaining the gel. Zymography was also carried out with 8 days’ larval gut protein after heating the solubilized sample at 45 and 55°C for 30 min.
Measurement of protein in gut fluid of P. quatuordecimpunctata: The concentrations of protein in samples of gut fluid were measured using FCR method. In brief, different aliquots of standard Bovine Serum Albumin (BSA) solution (250 μg mL-1) were pipetted out in different tubes along with the aliquots of samples in separate tubes. Equal amount of alkaline copper sulphate was added in each tube and then allowed to stand for 15 min. Equal amount of Folin-Ciocalteu reagent was added in each tube and the tubes were left for 30 min. Blue color was formed which was measured at 650 nm against a proper blank where no protein solution was used. The protein concentration of the extract was calculated from the standard curve.