Laboratory mass culture of N. lugens
The N. lugens culture is maintained in the laboratory of the SPKCES, M.S University, Alwarkurichi without any prior exposure to insecticide. These insects were maintained on O. sativa (IR 20) L. seedlings (nine to eleven days after germination (DAG) for first to third instar nymph; 21 DAG for late third instar nymph to adult) in acrylic cages.
Glasshouse experiment
The glass house experiments were conducted for CO2 studies. The chambers (1 m × 0.5 m × 1.0 m) were sustained at 25±2 °C and maximum 60% relative humidity. Daily daylight was supplemented by nine 400 W halide bulbs, positioned 0.3 m above the chambers (16-L: 8-D photoperiod). The level of CO2 inside the chamber controlled by using an infrared gas analyzer and 12 chambers (six-ambient CO2 at 350 ppm; six-elevated CO2 at 725 ppm) were set to circulate air from an external source. Concentration of CO2 was continuously monitored by the infra-red gas analyzer.
Experiment on the rice plant
Two rice varieties ‘IR 20- susceptible, conventional, and ADT 46- resistant conventional, semi-dwarf long grain were used for this study. The rice varieties were grown in a soil, six seedlings per pot were sown in and exposed to ambient and elevated CO2, watered twice a week with tap water to ensure saturation. After germination, five pots per rice variety were used. In total, the experiment consisted of 30 pots per analysis and each pot contain two rice plants. Plants were randomly owed to glasshouse chambers of the same CO2 treatment after watering. Every week, the placement of the pots within the chambers were re- arranged to provide uniform experimental conditions.
Effect of elevated CO2 on rice plant
For the biomass experiments, the rice plants were removed from the chambers after 5 weeks (36 days) and harvested for dry and wet weights measurement. The leaves were collected and counted randomly from each treatment. The collected plants were individually oven dried at 80 °C for 7 d, to measure their dry weight percentage. The nitrogen content was analyzed by using Kjehldahl procedure and carbon content was analyzed by using a CHN analyzer (Model Vario EL III).
Percentage of emergence, root and shoot weight length and ratio estimation
The experiment was carried as described previously under ‘experiment on the rice plant’ in completely randomized design with alternate-day watering. Observations were recorded every day from the first day of germination after four days of sowing; ‘the first true-leaf arose after six days’. Seedlings were counted, cleaned, and upper-part and root fresh weight (mg/plant) as well as the root length (cm/plant) and the greater leaf length (cm) were determined. Percentage of emergence (PE) was calculated according to the formula 151.
‘Where: ‘n’ is the number of seeds that had germinated on day D and D is the number of days counted from the beginning of germination’.
Estimation of defense enzymes
Ambient and elevated CO2 grown rice plants under glasshouse condition were examined. Five replications were sustained in each treatment; each replicate contain of five pots and in each pot four plants were maintained. For biochemical assays the leaf tissue was taken from the fifteenth day (15, 20, 25, 30, 35, 40 and 45d) after grown in ambient and elevated CO2 condition.
Estimation of peroxidase (PO) activity
Hammerschmidt et al. 52 protocol was followed for estimating the PO activity. Konn weight (1 g) of the rice leaves was homogenized in 2 ml of 0.1M sodium phosphate buffer (pH 6.5) and centrifuged at 10,000 × g for 25 min at 4 ºC. The upperpart of the supernatant was used as an enzyme source. The enzyme extract (100µl) was taken along with 1.5ml of pyrogallol (0.05M). To initiate the response, 100ml of hydrogen peroxide (1%) (v/v) was supplemented to the sample cuvette and the absorbance was read at 420nm (Lambda 25, UV/Vis spectrometer, PerkinElmer). The enzyme activity was expressed as change in absorbance min-1 g-1 fresh tissue.
Estimation of Superoxide dismutase (SOD) activity
Giannopolitis and Reis53 procedure was following to determine the SOD activity. Known quantity (1g) of the plant leaves was homogenized in 2 ml of 0.1M sodium phosphate buffer (pH 6.5) and centrifuged at 10,000 × g for 25 min at 4 ºC. The enzyme extract (100µl) was taken with nitro blue tetrazolium (NBT) in a reaction medium containing 50 mM potassium phosphate (pH 7.8), 14 mM methionine, EDTA 0.1 µM, NBT 75 µM and riboflavin 2 µM. The samples were illuminated for 7 min under 20 W. The spectrophotometer analysis was done in 560nm, where one unit of SOD was measured as the amount of enzyme able to inhibit by 50% the photoreduction of NBT under the experimental conditions. The SOD activity was expressed in U mg-1 of protein.
Biology and reproduction of N. lugens
Immature and mature insect of the N. lugens were fed on rice plants grown with ambient and elevated CO2. Daily mortality and number of eggs laid by insects were noted every day 54,55.
To estimate the effects of CO2 treatment on percentage of egg hatched, 5 pairs of newly hatched brachypterous males and females were caged on 20-day-old caged plants. Each treatment was replicated for five times. The total number of nymphs that emerged denoted the number of viable eggs produced by the females. At the end of nymphal emergence, unhatched eggs were recorded by separating leaf sheaths under stereo microscope 56. Average lifetime number of eggs laid by the female and average daily eggs laid by the female was analyzed by analysis of variance (ANOVA) using Minitap® 16 statistical software package (Minitap, State College, PA).
Food utilization of BPH
To determine the intake of ingested and assimilated food, newly hatched BPH females that had been starved for three hours were evaluated individually on a microbalance. Each BPH was placed within a sealed parafilm sachet on the stem of twenty-five-day-old test plants. After 24 h, the BPH female and excreta weighed. The following formula (2) was used to estimate the food utlilization44.
“where: IW = initial weight of test insect, FW-finial weight of test insect, IC-initial weight of control insect FC = final weight of control insect; and food ingested = food assimilated + weight of excreta”. There were four replications for each treatment including the control and the experiments were repeated three times for accuracy.
Population growth index
The population growth index was estimated by following method. Twenty-five-day-old caged rice plants of control and CO2 treated were infested with five pairs of BPH per experimental cage. Each treatment was replicated five times. Nymphs and adults were counted 30 days after infestation 57.
Mature and immature insects life span were analyzed using a log-rank χ2 test of equality over strata (PROC LIFE Table) along with formula (3) with Minitap®16 statistical software package (Minitab® 17, State College, PA).
Statistical analysis
Biology and nutritional indices were recorded as the mean of four replications and normalized by arcsine-square root transformation of percentages. The transformed percentages were undergone to analysis of variance (ANOVA). Differences between the five treatments were determined by Tukey’s–Kramer HSD test (P=0.05) by using Minitab 17® software package. Population growth index data’s were analyzed using a log-rank χ2 test (PROC LIFE Table) with Minitab 17® statistical software package (Minitab, State College, PA).