Larval collection
The larvae of S. exigua were collected from Faisalabad, Bahawalpur, and Multan to develop laboratory colonies in 2015 and 2016. The field-collected populations were assigned as Faisalabad (FSD), Bahawalpur (BWP), and Multan (MLT). The collected larvae were placed in glass vials provided with semi-synthetic diet and transported to the Insect Biodiversity and Biosystematics Laboratory, Department of Entomology, University of Agriculture Faisalabad. The susceptible laboratory population was obtained from the Nuclear Institute of Agriculture and Biotechnology (NIAB), Faisalabad.
Artificial diet
The field and susceptible populations were reared on artificial diet as reported by (Shorey and Hale, 1965; Hamed and Nadeem, 2008)
Rearing procedure
Larvae were reared at 25 ± 5 °C and 75 ± 5% RH under 16 h/ 8 h L/D cycle on the artificial diet and allowed to pupate. Pupae were collected and placed in a Petri plate. The pupae were disinfected in a 0.2% sodium hypochlorite solution. Petri plates containing pupae were placed in plastic containers until adult emergence. Adults were kept in open plastic containers covered with white netting. Adult diet (10% (v/v) honey solution) was provided by soaking the cotton wool pad in the honey solution and placed on the top of the netting cage. Every 48 h, egg-laden netting was replaced, and this process was repeated until egg production decreased or no further eggs were required. The netting was cut into small squares, and the netting containing eggs was surface sterilized in 0.05% sodium hypochlorite solution before being placed in a 250-ml round plastic cups sealed with plastic lids. The cups were placed in a controlled environment room until eggs hatching. From the first (F1) generation neonates, three larval instars (L1), (L2), and (L3) were used for bioassays.
Bacillus thuringiensis toxin
Bt Cry1Ac toxin was obtained from Genralpest Biotech Research Co. Ltd, Beijing, China and was stored at − 20 °C. It had been expressed as crystalline inclusions in Escherichia coli, with the protoxins purified by sonication and successive washes with 0.5 M NaCl and water as described by Sayyed et al. (2008). The toxin was freshly prepared in distilled water for diet incorporation.
Diet incorporates bioassays
Diet incorporation method was used for conducting bioassay, which is similar to the methods established for the baseline susceptibility of Helicoverpa armigera to B. thuringiensis toxins (Wei et al. 2017). Seven concentrations (0.25, 0.50, 1, 2, 4, 8, 16 μg ml−1) of Cry1Ac and a control were prepared. The artificial diet preparation was similar to that as described earlier except for the exclusion of ampicillin and a 10% reduction in a distilled water. Approximately 5 ml of the diet containing a toxin concentration was placed into a small aerated cup. Four replications were used for each bioassay. All bioassays were carried out under controlled environment in a growth chamber at 25 ± 2 °C, 65 ± 10% (RH) with a 16:8 (light/dark) cycle.
Data statistical analysis
The molt inhibitory and mortality rates were recorded after 7 days of bioassay. Molt inhibition was recorded when the larvae were unable to molt to the next larval instar. These counts were considered dead larvae. The mortality rate was recorded when larvae failed to respond with a gentle touch of a fine brush and considered as dead. The molt inhibition and mortality data were corrected from control mortality by Abbott formula (Abbott 1925), where needed. Probit analyses were done with PoloPlus (LeOra Software, 2003). The LC50 and MIC50 values, with their related fiducial limits at 95%, were assessed. The resistance ratios were determined by dividing LC50/MIC50 of field with susceptible laboratory population. LC50 and MIC50 values were considered significantly different when they did not overlap each other with their respective 95% fiducial limits.