Two transgenic Bt cotton hybrids, MRC 6301 Bt (event Mon 831) and Ankur 3028 BG-II (event 15895), approved by Genetic Engineering Approval Committee (GEAC) in India and one non-Bt variety, LH 2108, recommended by Punjab Agricultural University, Ludhiana, were used for the experiments. MRC 6301 Bt plants express Cry1Ac gene from B. thuringiensis targeting bollworms, while Ankur 3028 BG-II plants express Cry1Ac and Cry2Ab genes targeting bollworms and leaf-feeding insects (Navarro and Hautea 2014). Seeds were individually sown in earthen pots (12 l), filled with humus rich soil and plants were raised for rearing of herbivores and further experimentation.
Insect materials (preys)
Plants raised in pots were used for rearing of sucking insect pests P. solenopsis, B. tabaci and A. biguttula biguttula in separate insect proof screen cages (1.5 × 1.5 × 1.5 m) under field conditions. The insects were initially collected from field-grown eggplant, Solanum melongena L., and okra, Abelmoschus esculentus (L.) Moench, crops. Different screen cages were also used for Bollgard, Bollgard II and non-Bt cultivars to prevent insects from moving between cultivars. The plants of the respective cotton cultivar/variety were changed from time to time for continuous supply of fresh food to the insects and availability of their culture throughout the study period. The cultures of all insects used as preys in bioassays were maintained for multiple generations.
The culture of C. zastrowi sillemi was maintained on eggs of Corcyra cephalonica (Stainton) (Lepidoptera: Pyralidae), as a factitious host, in the Biocontrol Laboratory at Entomological Research Farm, Punjab Agricultural University, Ludhiana. Field-collected C. zastrowi sillemi adults were released in specially designed wooden oviposition cage (50 × 30 × 20 cm; Amar Chand & Company, India) with a sliding roof plank, covered with black muslin cloth. Standard adult diet (honey 1 g, sucrose 5 mg, protein 5 g, yeast 1 g and distilled water 40 ml) was provided twice daily as droplets on Perspex sheet strips by the help of a fine camel hair brush (Sattar and Abro 2011). The stalked eggs laid by the females on the roof plank were destalked after 24 h by the help of a sterilised razor blade. These eggs were transferred to individual plastic vials (4 × 3 cm) by the help of a soft camel hair brush. After hatching, fresh eggs of C. cephalonica were provided daily in each individual vial to the Chrysoperla larva till pupation. The adults emerging from the cocoons were collected individually and transferred again to the oviposition cages. The newly emerged adults were provided by a nutritional diet as described earlier. The culture of C. zastrowi sillemi was used for further experimentations.
Tri-trophic bioassay with C. zastrowi sillemi
Development, survival and body weight
All bioassays were conducted in an environmental chamber at 27 ± 2 °C and 65 ± 5% RH (Macro Scientific Works Ltd., India). Leaves from the upper third portion of 70-day-old Bollgard or Bollgard II or non-Bt plants were detached and placed in separate plastic jars (20 × 10 cm) lined with a muslin cloth for aeration. Similar sized mealybug nymphs (n = 50), whitefly adults (n = 50) and leafhopper nymphs (n = 50), from the stock cultures reared on Bollgard or Bollgard II or non-Bt leaves were collected and released in the respective jars. A soft camel hair brush was used for collection and release of mealybug nymphs, while an aspirator (Rescholar Equipment, India) was used to collect whitefly adults and leafhopper nymphs. The collected insects were released in separate plastic containers, with a hole in the screw cap. The cotton leaf was placed in each container before release of insects. The petiole of detached leaves was wrapped with water-soaked cotton swab so as to keep the leaves fresh and turgid for a longer period of time. A newly hatched single Chrysoperla larva was introduced in each plastic container for feeding. The hole was then plugged by a cotton wool. Each prey treatment (P. solenopsis, B. tabaci and A. biguttula biguttula), reared on Bollgard, Bollgard II or non-Bt leaves, was conducted simultaneously. The experiment was initiated with 30 Chrysoperla larvae for each treatment (one larva/replication). Chrysoperla larvae were inspected twice daily, and life-table parameters (development and mortality) were recorded. Last (3rd) instar Chrysoperla larvae, in each prey-predator combination, were weighed at the end of bioassay, using an electronic balance. The cocoons were collected, weighed and kept in separate glass vials individually to record the pupal period and percentages of adults’ emergence. After emergence from cocoons, adults were provided by an adult diet as described earlier and their longevity was recorded (Additional file 1).
An experiment on predatory potential was conducted in an environmental chamber set at 27 ± 2 °C and 65 ± 5% RH (Macro Scientific Works Ltd., India). In no-choice tests, 2nd instar mealybug nymphs (n = 30) reared on Bollgard or Bollgard II or non-Bt leaves were released by the help of a soft camel hair brush on respective leaves in separate plastic jars (20 × 10 cm) lined with muslin cloth for aeration (10 replications for each set). A newly hatched single Chrysoperla larva was introduced in each jar for feeding. The number of mealybugs consumed were counted daily and replaced with new sets till pupation. The mean consumption of mealybugs per day was worked out for each larval instar.
Cry toxin expression in tri-trophic pathway (cotton leaves, prey and predator)
To confirm Cry1Ac and Cry2Ab expression in bioassays, leaf samples were collected from the upper third portion of 70-day-old Bollgard (Cry1Ac) or Bollgard II (Cry 1Ac and Cry2Ab) or non-Bt plants. Leaf discs measuring 20 mm per replication were placed in separate 1.5 ml centrifuge tubes, and Cry protein measurements, using enzyme-linked immunosorbent assays (ELISA; see below), were made. To quantify Bt toxin in P. solenopsis nymphs, B. tabaci adults and A. biguttula biguttula nymphs, protein was extracted from each prey herbivore, reared either on Bollgard or Bollgard II or non-Bt leaves. Similarly, to assess the potential transfer of Cry1Ac and Cry2Ab proteins via food chain, C. zastrowi sillemi larvae fed on prey herbivores reared on Bollgard or Bollgard II or non-Bt leaves were collected and assayed, using ELISA. For all ELISA samples, five replications were maintained.
Bt protein concentrations in cotton leaves, insect prey and predator were measured, using sandwich ELISA, using quantiplate kits (Wu et al. 2014). The concentration of Cry1Ac protein was measured, using Cry 1Ab/Cry1Ac kit (AP 003 QT V50) and Cry2Ab using Cry2A kit (AP 005 QT BC V50) of ENVIROLOGIX 500 Riverside Industrial Parkway Portland, ME, USA. Before analysis, all insects (preys and predator) were washed in phosphate-buffered saline with Tween-20 buffer to remove any protein from their outer surface. Leaf samples weighing 20 mg were homogenised in 0.5 ml buffer solution and diluted (1: 10 and 1: 50) for Cry1Ac and Cry2Ab, respectively. Quantities (mg/replication) of material and buffer dilutions used from different prey herbivores and predator were ≈20 mg P. solenopsis (in batch) homogenised in 0.5 ml buffer (no dilution), ≈20 mg B. tabaci (in batch) homogenised in 0.5 ml buffer (no dilution), ≈20 mg A. biguttula biguttula (in batch) homogenised in 0.5 ml buffer (no dilution) and 15 ± 2 mg C. zastrowi sillemi homogenised in 0.5 ml buffer (no dilution). All the samples in buffer were ground by hands, using a plastic pestle. For every sample, a fresh pestle was used to avoid any possible cross contamination of the individual sample. After vortexing for 3 h on a vortex shaker (Spinix; Tarson Products Ltd., India), centrifugation for 1 min (at 10,000 rpm in microcentrifuge) (Eppendorf AG 5415D, Germany) and appropriate dilution of supernatants, ELISA was performed according to manufacturer’s protocol. The absorbance of each well solution was recorded at 450 nm by using micro filter plate reader (Thermo Electron Corporation, China). The optical density (OD) value of each calibrator and corresponding concentrations of Cry1Ac and Cry2Ab (standards provided in the kit) were used to prepare the standard curve. The proteins’ concentration of each sample was determined by finding its OD value and the corresponding concentration level in the linear curve, using regression analysis. The results from standard curve were multiplied by the dilution factor incurred during extraction. To determine the dilution factor, the volume in milliliter of extraction solution was divided by weight of samples in grams. For leaf samples, protein concentrations in μg g−1 fresh weight were calculated by multiplying these results by (1:10 or 1:50) dilutions made for Cry1Ac and Cry2Ab, respectively.
The data on the life parameters (larval period, pupal period and adult longevity) and weight (larval and cocoon weight) were subjected to one-way ANOVA and Tukey’s multiple range test. Data are presented as mean ± standard error. Data on Chrysoperla larval and pupal survivals were analysed and presented in the form of Wald Chi-square χ2 test and P values. Data on feeding performance were subjected to two-way ANOVA and Tukey’s multiple range tests. All statistical tests were carried out using IBM SPSS 22.0 for Windows (IBM Corporation, Armonk, New York, USA).