Experimental studies were initiated by the development of 3 types of cultures, i.e., plant culture of cauliflower and cabbage cultivars, P. xylostella culture, and D. insulare culture. Insect rearing jars made up of transparent plastic sheets and muslin cloth were used. Different temperature regimes were maintained according to the need of experimental studies, and to acclimatize with the abiotic and biotic conditions, insects’ cultures (P. xylostella and D. insulare) were reared for 3 generations under similar conditions before performing a certain experimental study.
Plant culture of cauliflower and cabbage cultivars
Five cultivars, each of cauliflower (i.e., Smilla, White marble, Cashmere, White magic, and TSX-C22) and cabbage (i.e., Quisor, Stonehead, Baseball, Asha, and Delight ball), were selected and grown from seeds into seedling trays in glasshouse. Peltracom® growing media were utilized to grow plants. After 5 weeks, plants were transplanted into plastic pots having 7.5cm diameter until hardening stage. At the age of 45–50 days, plants were utilized for P. xylostella and D. insulare rearing and further experimental studies.
Plutella xylostella culture
Larvae and pupae of P. xylostella were collected from cauliflower/cabbage fields at Rawalpindi (Punjab, Pakistan) sites. Collected larvae were brought into insect growth chamber (25 °C, 65±5% RH, and 16L: 8D hours) and released on fresh and untreated potted plants of cauliflower/cabbage and placed in rearing jars, whereas collected pupae were placed singly in glass vials, provided with honey solution (20%) soaked cotton plugs. Plants were replaced (where needed) until the pupation of feeding larvae, and then, developed pupae were shifted to glass vials for adult emergence. To obtain the moth’s progeny of homogenous age and acclimatize with the host plant cultivars as well as abiotic controlled conditions, separate plant cultivars were placed in separate rearing jars and mated P. xylostella were released for 6 h to oviposit. Emerged larvae were fed on relevant plant cultivars, and developed pupae were collected in marked glass vials singly. Emerging moths were used for further rearing and research studies.
Diadegma insulare culture
Parasitized larvae and pupae of P. xylostella were collected from cauliflower/cabbage crops at Rawalpindi (Punjab, Pakistan) sites. Collected larvae were brought into insect growth chamber (25 °C, 65±5% RH, and 16L: 8D hours) and released on fresh and untreated potted plants of cauliflower/cabbage and placed in rearing jars whereas collected pupae were placed singly in glass vials. From this field collection, emerged adults of D. insulare were identified and released into rearing jars for mating, provided with honey solution (20%) soaked cotton swabs in petri plates. To obtain the parasitoid’s progeny of homogenous age and acclimatize with the host plant cultivars as well as abiotic controlled conditions, nine potted plants from each cultivar were placed in separate rearing jars along with ten third instar larvae of P. xylostella on each plant. After that, 10 pairs of mated D. insulare were released for parasitization in each rearing jar for 24 h. Plants were replaced (where needed) until the pupation of all the larvae, and then, developed parasitized pupae were harvested and kept in marked glass vials for adult emergence. Emerging parasitoids were used for further rearing and research studies.
Parasitism and sex ratio of D. insulare
Twenty potted plants of each cultivar of cauliflower and cabbage were used singly in separate rearing jars, and each plant was infested with 10 newly molted 3rd instar larvae of P. xylostella. One pair of 1-day-old mated D. insulare was released for 24 h on 10 plants of each cultivar whereas the remaining 10 plants received herbivores only and served as control (without parasitoids). Insects were observed daily, and the number of surviving larvae was recorded until pupation. Afterwards, pupae were harvested and kept in the labeled glass vials provided with honey solution (20%) soaked cotton plugs for emerging adults and placed under required controlled conditions. After adults’ emergence, parasitism (%) was recorded and corrected by using control mortality. Females and males of parasitoid were identified on the basis of ovipositor and counted for sex ratio determination. Similar experimental studies were performed under 3 different temperature regimes (i.e., 19, 23, and 27 °C) along with 65±5% RH and photoperiod of 16L: 8D hours in SANYO incubator, MIR-254 (Netherlands).
Biochemical analysis
Plant leaves from each cultivar were collected, washed with distilled water, and oven dried at 65 °C for 48 h. After the achievement of constant weight, samples were ground and subjected to different tissue nutrient analyses, following recommended procedures. Kjeldahl’s (1883) method was followed for the determination of total nitrogen (N) contents, whereas potassium (K) contents were assessed by flame photometer (Knudsen et al. 1982) and phosphorus (P) contents were analyzed by spectrophotometer (Anderson and Ingram 1993).
Statistical analysis
Parasitism (%) was determined using the following equation:
$$ \mathrm{Parasitism}\ \left(\%\right)=\left\{\left({P}_{\mathrm{Di}}\div {L}_{\mathrm{t}}\right)\times 100\right\}+{M}_{\mathrm{C}} $$
whereas corrected mortalities (MC) were obtained using the Schneider-Orelli (1947) equation:
$$ {M}_{\mathrm{c}}=\left\{\frac{\left(\mathrm{treatment}\ \mathrm{mortality}\%-\mathrm{control}\ \mathrm{mortality}\%\right)}{\left(100-\mathrm{control}\ \mathrm{mortality}\%\right)}\right\}\times 100 $$
Sex ratio was determined using the following equation:
$$ \mathrm{Sex}\ \mathrm{ratio}=\frac{\mathrm{number}\ \mathrm{of}\ \mathrm{emerged}\ \mathrm{females}}{\mathrm{total}\ \mathrm{number}\ \mathrm{of}\ \mathrm{emerged}\ D. insulare} $$
Completely randomized design was followed in factorial manner for experimental layout and analysis of variance for parasitism and sex ratio in offspring of the parasitoid. All the data were subjected to arcsine transformation prior to ANOVA. Means of significant treatments were compared using Tukey’s HSD test. Analysis of the collected data was computed using Statistix 8.1 software (McGraw-Hill 2008).