Larvae of the cotton leaf worm, S. littoralis, originally collected from cotton fields located in Kafer Elsheikh Governorate, Egypt, were reared on the artificial diet described by Kranthi (2005) at 27 ± 1 °C, 70% RH, and a photoperiod of 14:10 (L:D) h. After pupation, the pupae were collected and kept in glass jars until adult emergence. The adults were allowed to feed on 10% sugar solution and lay eggs in the same jars. The eggs were collected on tissue paper and kept in small jars along with a wetted cotton piece as a source of moist for hatching.
Preparation of B. thuringiensis Cry1C toxin
Cry1C toxin was cultured and purified as described by Abdullah et al. (2009) and modified by Moussa (2009). The bacterial cells were inoculated in a 5-ml culture tube for overnight. The overnight culture was then subcultured in a 1-L flask to grow further for 3–5 days in T3 medium (3.0 g Trypton, 2.0 g Tryptose, 1.5 g yeast extract, 0.0005 g MnCl2, 8.9 g NaH2PO4). The growth was harvested at 5200 rpm for 10 min at 4 °C. The pellets were collected and washed in (50 mM EDTA) buffer for 4–6 times/each with 10,000 rpm for 10 min at 4 °C. The obtained pellet was re-suspended in 5 ml of (50 mM Tris, HCl, 5 mM EDTA, pH 7.0) buffer. These inclusion bodies were examined by 10% SDS-PAGE gel electrophoresis. The protein concentration was determined using the Bradford method according to Bradford (1976). The inclusion bodies were then aliquoted in 1.5 ml Eppendroff tubes and stored at − 20 °C.
Bioassay of Cry1C toxin
Bioassay Cry1C toxin was conducted using diet incorporation method. Five different concentrations of Cry1C toxin in water were prepared, and 4 replicates for each concentration were represented. Moreover, the control replicates were treated by dH2O instead of Cry1C toxin. Twenty newly hatched neonates were placed onto the surface of the diet in each replicate, using a thin brush and then kept under laboratory conditions. The larval mortality was recorded 7 days after treatment, and the median lethal concentration LC50 was calculated according to Finney (1971).
Selection of S. littoralis neonates for resistance was initiated by transferring of 500 neonates onto the surface of artificial diet incorporating Cry1C toxin for 4 subsequent days with care always taken to obtain about 75% mortality or higher. The survived larvae were then transferred to feed on toxin-free diet until pupation. The emerged adults were allowed to feed on 10% sugar solution. The laid eggs were collected and kept in plastic cups along with wetted cotton piece until hatching. The above selection procedure was repeated on the newly hatched neonates of the second generation, and this regular work was performed at every generation until 12 generations. The bioassay was conducted at F1, F3, F6, F9, and F12 in order to calculate the LC50. Resistance ratios were calculated by dividing the LC50 of selected generation by the LC50 of F1.
Preparation of gut extract
Gut extract was performed referring to the method described by Moussa (2009). Ten 4th instar larvae were dissected on ice, and their guts were pooled in 300 μl dH2O in microcentrifuge tubes. An amount of 1.5 phenyl methane sulfonyl fluriode (PMSF) was added to inhibit proteinase enzymes. The guts were grinded gently then centrifuged at 14,000 rpm for 15 min at 4 °C. The supernatant was carefully transferred into sterilized Eppendorf tubes and kept at − 20 °C for further use.
Digestion and processing of Cry1C toxin
In order to compare the processing of Cry1C toxin in the midgut of susceptible (F1) and resistant line (F12) of the cotton leaf worm, an amount of 50 μl gut extract sample was mixed with 10 μg of Cry1C toxin in microcentrifuge tube, and the volume was completed to 25 μl, using universal buffer (11.5 mM boric acid, 7.8 nM citric acid, 18.7 mM Na2HPO4, and 68.9 mM NaOH, PH 9.75) (Frugoni 1957). Samples were incubated at room temperature at different time intervals, viz., 5, 15, 30 min, and 1 h with Cry1C toxin. Toxin processing was terminated by heating the sample at 100 °C for 5 min. The samples were cooled down at room temperature, and sample buffer was added. The samples were again boiled for 5 min for protein digestion (Moussa 2009). Finally, the samples were analyzed using SDS-PAGE (4% stalking gel and 10% separating gel).