Starter Culture and Population Homogenization:
Pink bollworm Pectinophora gossypiella infested fruiting bodies (flowers, squares and bolls) were collected from three different locations of districts Faisalabad (Fig. 1 a, b and c). These locations were: 1) Young wala, farmer’s fields (31°26ʹ05ʺ N, 73°03ʹ45ʺ E), Ayub Agricultural Research Institute, field area (31°24ʹ11ʺ N, 73°02ʹ54ʺ E), and Jhipal chak no. 73 field area (31°21ʹ37ʺ N, 72°52ʹ16ʺ E) where different Bt-cotton varieties were sown. The infested samples were brought in the pink bollworm rearing laboratory, Department of Entomology, University of Agriculture, Faisalabad.
The larvae from all infested materials were isolated and reared on 10~20 days old freshly picked green cotton bolls Naik et al. (2021). The bolls were washed with tap water, blotted dried, and pricked with sterilized surgical needle prior to provide field collected larvae of P. gossypiella Fabrick et al. (2015). Pricking of the green bolls was done to facilitate the entry of larvae inside the bolls. Pupae of the same age were collected in glass petri-dish lined with a layer of paper towel. The petri-dish containing pupae was placed in adult chambers. Glass Chimneys were set as chamber for adults. The top and bottom of the adults chambers were lined with a layer of oviposition substrate Sabry and Abdel-Aziz (2013) as, and 10% sucrose solution was provided as adult diet. In each chamber fifteen moth pairs (1:1 male to female) of P. gossypiella were released which were identified at larval and pupal stages of during development Dharajothi et al. (2010).
Preparation of standard laboratory larval diet and experiment layout
The standard laboratory diet (control treatment/T7) was prepared by performing the subsequent steps as
- Agar-agar 20g (table 1, serial No. 5) was dissolved in 500 ml distilled water (table 1, serial No. 15) in liter 1000 ml glass beaker. The mixture is then boiled for 3~4 minutes in an oven with continuous stirring and heating at 30 seconds intervals to avoid flocculation.
- The remaining amount of distilled water, 230 ml (table 1, serial No. 15) was added in blending jug (electric blending machine).
- The ingredients (table 1, serial No. 3 to 14 excluding agar-agar) were accurately weighed, and added to distilled water mentioned at step No 2.
- The hot boiling mixture of agar-agar prepared at step No. 1 was poured in the blender contents mentioned at step No. 3.
- The final mixture was blended continuously for 2~3 minutes with intermittent pausing.
- The final uniform mixture (standard laboratory diet) was poured in glass petri-dishes previously sterilized, let the diet to settle at room temperature for 30 minutes.
- Once the diet in petri-dishes solidified, was sliced into pieces of different sizes according to the requirement of the experiment (usually 0.50cm3) with the help of a knife.
- The test diets for the treatments (T1 and T2) were prepared by following the same protocol as for standard laboratory diet (T7) (Fig. 2, a, b, f). The difference is the only that wheat germ meal in standard laboratory diet (T7) was replaced with cottonseed meal and okra seed meal (table 1, serial No. 1 and 2) for test diets in T1 and T2, respectively.
- The sliced diet was presented to the larvae of P. gossypiella in plastic cups (Fig. 3) Wu et al. (2008).
Prior to larval diet preparation, stock solution of decavitamin was prepared by weighing all the constituents (table 2). The ingredients were added in a glass vial containing distilled (solvent). The final mixture of decavitamin was made by vigorous shaking for 2~3 minutes. The mixture of decavitamin was stored at 20ºC in a refrigerator for further use.
The seed sprouts of cotton and okra to be used as test larval diets (treatment T3 and T4) were prepared by following predefined procedure Vanderzant et al. (1956) (Fig. 2, c). The seeds of cotton (FH-301) and okra (Sabz Pari) were obtained from Punjab Seed Corporation, Ayub Agricultural Research Institute, Faisalabad, for preparing their sprouts. Cotton bolls (FH-301), 10~20 days old were picked from the field area. The bolls washed and disinfected with formalin (0.1%) by dipping in for 2~3 minutes to avoid secondary infection. The traces of formalin were removed by subsequent 2~3 washings and dried at room temperature Fand et al. (2020). These bolls were dried with paper towel and left for five minutes then offered to the larvae as diet (treatment,T5) (Fig. 2, d). Similarly, okra fruit was sliced in (1/2ʺ) pieces on a paper to remove mucilaginous material as the larvae may not stick in and become immobile in the corresponding treatment (T6) (Fig. 2, e). The detail of the different diets according as per treatments was tabulated (table 3).
First instar larvae were released in plastic cups according to the treatments, labelled and placed in larval rearing chamber with controlled conditions maintained at 27±0.5°C (Dharajothi et al. 2016; Shrinivas et al. 2019), 60±5% relative humidity, and 12:12 (L: D) photoperiod Reyaz et al. (2018) (Fig 3).
The diets in treatments T1, T2 and T7 were sliced into 0.50 cm3 and 2~3 pieces were placed in each cup as larval diet. The diets in all treatments were refreshed after 48 hours except T5 (Freshly picked green cotton bolls) until the completion of the larval development (Fig. 4, b). Those larvae pupated on the same day were separated from those still in larval phase and placed in petri-dish lined with a layer of tissue paper to avoid trauma (Fig. 4, c). Adults emerging from pupae were shifted in adult chambers (glass chimneys cages), provided with 10% honey solution as adult food (Hariprasad, 1999; Wu et al. 2006) (Fig. 4, d). The adults were held at 27±2°C, 60-70% relative humidity, and 14:10 (L: D) photoperiod (Wu et al. 2013; Malthankar and Gujar, 2014; Zinzuvadiya et al. 2017; Fand et al. 2020). The egg receptacles were removed from adult chambers (Fig. 4, a), incubated in transparent plastic boxes (12.5 x 4.5 x 2.5 cm) with air tight lids, kept at 28±1°C Muralimohan et al. (2009).
The experiment was executed following completely randomized design (CRD) with five replication (one larvae/cup to avoid canabolism) and seven treatments. For adults, fifteen moth pairs (1:1 male and female) were released in each adult chamber (glass chimney cage) that represented on replication.
Data collection and statistical analysis
Data regarding a number of biological characteristics like larval longevity, larval weight, pupal weight, pupae formation, pupal duration, adult’s emergence, male and female life span was documented. The parameters of fecundity, egg viability, larval mortality, pupation and deformed pupation in the decedents were also recorded. The collected data was statistically analyzed using statistical software Statistica to check the significance of the results at 5% probability and means±S.E of each parameter were compared following Tukey’s HSD.