Rearing of Sitotroga cerealella (Olivier)
The eggs of Angoumois grain moth, S. cerealella (Olivier), were used as a natural food source for mass production of the green lacewing, C. carnea. In order to maintain S. cerealella pest under laboratory condition, its mother colony was kindly provided by the Stored Grain Department at the Plant Protection Research Institute. To obtain a mass production of S. cerealella eggs, the insect culture was maintained according to the method described by Hassan (1995) with some modifications where a bulk of soft wheat grains were brought to the laboratory and were sterilized at 120 °C for 2 h. About 2–3 kg of sterilized wheat grains was poured into sterilized and clean metal trays (70 × 50 cm size). These trays were kept horizontally on the bench and infested with 1 g of S. cerealella eggs/kg of wheat grains in each tray and immediately transferred into large cages for 10–12 days. The trays were then taken and kept vertically in ovipositor cages for 25–30 days till adult emergency and egg production. The deposited S. cerealella eggs were collected in small jars in 2-day intervals and were sieved in order to remove all the insect scales.
Rearing of predator, C. carnea
The starter culture of the green lacewing, C. carnea, was established by collecting the adult stage from cotton field, insecticide-free and transferred immediately to the laboratory. This culture was maintained under lab condition for several generations before starting the experiments. The adults of C. carnea were taken from original culture and were mated in plastic boxes. A number of ten pairs of adults were placed in plastic boxes (22 × 13 × 10 cm) and were covered with black muslin cloth for egg laying. A semi-artificial diet solution was prepared according to Morrison (1985). The adults were provided with droplets of mixed yeast and sugar on muslin cloth. The deposited eggs were collected daily and kept in glass jars until hatching. The hatched larvae were reared on S. cerealella eggs as mentioned above.
Rearing of the aphid, Rhopalosiphum maidis (Fitch)
In order to obtain enough culture of aphid that fed for several generations on Bt corn plants, a number of 20–30 individuals of R. maidis were released on Bt corn which contained a synthetic Cry2Ab/1Ac gene encoding a nearly full length Cry2Ab protein. Both transgenic and non-transgenic corn plants were sown and grown in the greenhouses. Three months after cultivation, the nymphs of GCLA were collected from the infested corn leaves for predator feeding assay.
Predator feeding assay
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A.
Feeding effect of aphid reared on Bt transgenic lines on the development of C. carnea
In order to study the influence of feeding C. carnea on hosts that fed on transgenic line, the infested leaves with R. maidis individuals of Bt corn and non-Bt corn greenhouses were collected and transferred to the lab for predator assay. Twenty individuals of the second instar larvae of C. carnea were kept individually in culture tubes; each tube was considered as one replicate. Fifty aphids were collected from the infested corn leaves then introduced to each larva daily till pupation. Daily observations were performed till adult stage. In each assay, the larval duration of the C. carnea, larval mortality percentage, pupation percentage, pupal duration, percentage of the emerged adult, percentage of assayed larvae survived till adult emergence and the number of aphid individuals eaten by each larva were recorded.
Also, to study the effect of Cry2Ab and Cry1Ac toxins on either egg hatchability or larval feeding of C. carnea, we undertook the following experiments:
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B.
Bacillus thuringiensis Cry1Ac and Cry2Ab toxin preparation
BtCry1Ac toxin was cultured and partially purified as described earlier by Abdullah et al. (2009). Additionally, protein concentration was determined by Bio-Rad protein assay based on the Bradford method using bovine serum albumin (BSA) as a standard (Bradford, 1976). The BtCry2Ab protoxin was partially purified according to methods described by Moussa et al. (2016). BtCry2Ab was inoculated in a 5-ml Loria broth (LB) culture tube and incubated overnight at 150 rpm shaking at 37 °C. The bacterial cell was sub-cultured in a bigger flask of LB media and kept to grow under the previous condition for 3–4 days. The cells were pelleted down using 5200 rpm centrifugation at 4 °C for 10 min. The pellet was re-suspended in lysis buffer (50 mM Tris; pH 8.0; 50 mM EDTA; 15% sucrose; 10 μg/ml of lysozyme).
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C.
BtCry1Ac and Cry2Ab effects on egg hatchability of C. carnea
To study the effect of Bt Cry1Ac and Cry2Ab toxins on egg hatchability of C. carnea, 20 eggs of C. carnea were sprayed with different concentrations of the abovementioned toxins and left for hatching. Three replicates were used per each dose. In both toxins, three different concentrations were used viz. 4.0, 8.0, and 10.0 μg/ml in comparison to the recommended dose of chemical insecticide cyper-methrine (2 μg/μl) which was used as a positive control treatment. The negative control treatment sprayed with distilled water (dH2O) was also considered. The sprayed eggs were incubated at 27 ± 1 °C for hatching and the eggs were daily observed till day fifth. The hatchability percentage of C. carnea was calculated and recorded.
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D.
Feeding effect of S. cerealella eggs sprayed with BtCry1Ac/2Ab toxins on C. carnea
To determine the feeding effect of S. cerealella eggs treated with Bt Cry1Ac and Cry2Ab toxins on green lacewing predator compared to the effect feeding of GCLA reared on Bt corn, the partially purified BtCry1Ac and BtCry2Ab toxins were sprayed on eggs of S. cerealella pest at various concentrations which ranged from 4 to 10 μg toxin/ml dH2O. Similarly, the negative and positive control treatments were also deliberated, but Bt toxins in this experiment were replaced with dH2O (as a negative control) and cypermethrin compound (as a positive control). In each concentration, three replicates of 20 green lacewings on the first day of the second larval instar were released on the treated eggs with a fine brush and then kept at the abovementioned laboratory condition for 4 days and examined 4 days after treatment. The experiments were repeated several times in order to obtain accurate data. The experiments were accomplished at 26 ± 1 °C and 65–70% RH.
Data analysis
One-way analysis of variance (ANOVA) was applied by the Duncan multiple range test for comparison of means at P < 0.05, Student’s t test, and depletion rates, which were computerized according to IBM-SPSS, 2011). Additionally, Abbott’s formula (Abbott, 1925) was implemented to correct the larval mortality percentage.