Theleaves of A. latifolium Lam. were used for the study(Pradeepkumar et al. 2018).
Isolation of bioactive compound composition from the leaves of A. latifolium
Bioactivity guided isolation of the compound composition and its phytochemical characterization were described in our previous study(Pradeepkumar et al. 2019b). The bioactivity was determined based on the antibacterial activity against gut bacteria isolated from O. rhinoceros larvae in our previous study(Pradeepkumar et al. 2018).
In vitrostudy of antibacterial activity
Antibacterial activity was evaluated by agar well diffusion methodusing 24 h grown gut bacteria of strainsgram +veBacillus cereus andgram-veStenotrophomonas maltophilia (Pradeepkumar et al. 2018). The plates were incubated at 37°C for 48 h after adding 100 µL samples in the wells. The diameter of clear zone was measured against test culture.
Larvicidal activity of the compound composition
The larvae of O. rhinoceros maintained on sterilized cow dung(Mini and Prabhu 1986)were used for the experiment. Third instar larvae (body weight 8.5 ± 1 g) were used for testing larvicidal activity. The compound composition thoroughly mixed with sterilized cow dung after dissolving it in 25 mL of distilled water with 0.02 % Tween 80, at various concentrations (1, 2, 3, 4, 5 mg/100 g), was provided to larvae as food. The controls were fed on cow dung mixed with appropriate concentration of Tween 80. The larvae were reared in plastic containers, each group including six larvae. The body weight and mortality of larvae were recorded for three weeks. The medium was changed once in every three days.
Haemolymph sample preparation for enzyme studies
Haemolymph from both the control and treated larvae were collected, by cutting its 3rd prothoracic leg and gently squeezing its body, stored in sterile eppendorf tubes in a deep freezer. Circulating haemocytes were separated from the haemolymph by centrifugation at 14,000 g at 4°C for 15 min.
Bioassay of the level of 20-hydroxyecdysone (20E) in haemolymph
20E level in haemolymph was assayed by the method described byPorcheron et al. (1989) using a commercial ELISA kit (Cayman Chemical, USA) as explained in our previous study(Pradeepkumar et al. 2019a). Pure acetylcholinesterase from the electric eel (Electrophorus electricus)has been covalently coupled to a 20-hydroxyecdysone-6-carboxymethoxime derivative and the conjugate used as a tracer. Immunological reaction was performed in a 96-well microtiter plate precoated with secondary antibody.
Assay of cathepsin D
The assay of cathepsin D was done following the method ofMycek (1970)using 100 µL haemolymph as the test sample. One unit of cathepsin activity has been arbitrarily defined as the amount of enzyme in the test solution that caused the absorbancy of the supernatant to increase by 1.0 in 1.0 min.
Assay of phenol oxidase
Activity of phenoloxidase was estimated by the method ofLerch (1987). One unit of enzyme activity is defined as the amount of enzyme which catalyses the oxidation of one µmol of L-DOPA/ min at 30°C. This corresponds to the absorption change of 0.6/min at 475 nm. Specific activity is expressed as enzyme units/ mL haemolymph.
Total haemocyte count (THC)
THC was done by using Neubauer haemocytometer.Haemolymph is collected from both the control and treated larvae as explained earlier, diluted tenfold using WBC diluting fluid and stored in deep freezer. 100 µL of diluted haemolymph was immediately placed over haemocytometer and kept undisturbed for 3 min. The haemocytes in the four corner ruled squares were counted under a light microscope at 400X magnification. The number of circulating haemocytes per cubic millimeter was calculated. Replicates of the experiments were performed to confirm the results obtained.
Differential haemocyte count (DHC)
A drop of haemolymph was placed on clean microscopic glass slide and a thin smear was made by drawing the second slide across the first one at 45° angle. The smear was air dried and stained using Giemsa stain for 20 min. The slides were then washed in running water to remove excess stain then air-dried. Cells were observed, differential count was done and microphotographed using Labomed digital camera (400X). One hundred cells per slide were analyzed. The haemocytes were identified by their distinguishing characters as described byMathur (2002) and Wigglesworth (1972). The percentage of different types of haemocytes in both the treated and control samples were calculated.
Detection of apoptosis of haemocytes by flow cytometry
Apoptosing cells were quantified using FITC Annexin V cell apoptosis assay kit (Invitrogen, Thermo Fischer Scientific). The percentage of apoptotic haemocytes were assessed by flow cytometry(Shapiro 2003). Haemocytes were fixed in 70 % ethanol before staining. Washed the cells twice with cold PBS and then resuspended in binding buffer. 100 μL of the FITC Annexin V followed by propidium iodide were added to the samples. The tubes were mixed thoroughly by vortexing at a medium speed for 3 to 5 sec followed by incubation for 20 min at room temperature in the dark. The cell populations were distinguished using a flow cytometer with 488 nm line of an argon-ion laser for excitation.
Cell cycle analysis
Cell cycle analysis was done by using standard kit (Muse® Cell Cycle Assay Kit, Merck Millipore). Haemocytes were separated from the haemolymph asdescribed earlier. Appropriate volume of PBS was added to each tube and the contents were mixed by gently vortexing. The cells were centrifuged at 3,000 g for 5 min. The supernatant was discarded without disturbing the cell pellet, leaving approximately 50 µL of PBS. Resuspended the cell pellet in the residual PBS by gently vortexing. The resuspended cells were added drop wise into the tube containing 1 mL of ice cold 70 % ethanol. Caped and freezed the tube at – 20 ºC.After overnight incubation, the samples were centrifuged at 3,000 g for 5 min at room temperature. The supernatant was removed and 250 µL PBS was added to the pellet. Again centrifuged and discarded the supernatant. 250 µL of cell cycle reagent was added to the cell pellet.This was incubated at dark for 30 min. After this it was analyzed using a flow cytometer. Gating was performed with reference to untreated control cells and samples were analyzed. Quantitative measurement of the percentage of the cells in G0/G1, S, and G2/M phases of cell cycle was done on Muse Cell Analyzer. The experiments were performed in triplicate.
The statistical significance of data for control and treated groups was assessed by analysis of variance (ANOVA) using SPSS 13 for Windows. Statistical significance was accepted when P≤0.05. Post-hoc testing was carried out using Duncan’s new multiple range test (DMRT). LD50 values were calculated by probit analysis.