Insects
Propylea japonica adults were collected from the research farm of Cotton Research Institute, Chinese Academy of Agricultural Sciences (CRI-CAAS) Anyang, (36° N, 114° E, HSL: 69 m) Henan, China, and were reared on the pea aphid (Acyrthosiphon pisum [Harris]) in acrylic rearing chamber (45 × 45 × 25 cm) in the laboratory under controlled conditions. The pea aphid was reared on potted broad bean, Vicia faba seedlings. Young larvae (˂ 12 h-old) of P. japonica were used in the experiment.
Bio-insecticidal compounds
The bio-insecticidal compounds used in this study were as follows: Bt protein, Vip3Aa (95–98% pure) and boric acid (99.5%). Bt protein Vip3Aa was purchased from Envirotest-China (an agent for EnviroLogix, Portland, ME). A Vip3A clone in Escherichia coli was used as a source of Vip3Aa toxin production. The methods used to express and purified the Vip3Aa toxin generally, followed procedure described by Chen et al. (2002). The purity of Vip3Aa was estimated, using SDS–polyacrylamide gel electrophoresis. Activated protein was stored at − 80 °C in the laboratory. Boric acid was purchased from Sigma-Aldrich. The median effective concentration (EC50) of Vip3Aa was determined about 500 ng/g, using bioassay previously described by Ali et al. (2017). Briefly, newly emerged first instar larvae of a susceptible strain of Helicoverpa armigera (Hübner) (Lepidoptera: Noctuidae) were exposed to various concentrations of Vip3Aa protein ranging from 100 to 500 ng/g synthetic diet for 7 days and EC50 (Effective pathogenic concentration that results in 50% weight reduction in the tested insect compared to that of the control) was estimated comparing larval weights to control treatment.
Synthetic diet-based rearing and dietary exposure test
The semi-solid synthetic diet previously used to rear the larvae of Harmonia axyridis (Pallas) and P. japonica was used to rear and expose P. japonica to elevated concentration of Bt protein (Ali et al. 2016). Diet was freshly prepared using beef, beef liver, egg yolk, yeast extract, honey, sucrose, royal jelly, ascorbic acid, vitamins powder and preservative in distilled water, and ingredients were thoroughly mixed in a blender to achieve semi-solid consistency. Bt protein Vip3Aa was added. Boric acid added diet served as positive control while pure synthetic diet served as negative control. Diet was stored at − 20 °C.
In order to expose P. japonica to Bt protein, young larvae (˂ 12 h-old) were individually reared in 8-ml centrifuge tubes having 2 holes for ventilation and fed on synthetic diet containing Vip3Aa, boric acid as positive control and pure synthetic diet as the negative control. The nominal concentration of Vip3Aa protein incorporated in the diet was 200 μg/g of fresh diet whereas the boric acid: a stomach poison was used as a positive control to indicate the validity of experiment to test effects of toxin incorporated into diet. The nominal concentration of boric acid incorporated into the fresh synthetic diet was 50 µg/g (Ali et al. 2017). Every day fresh diet was provided twice as small droplets to the immature P. japonica with the help of a syringe during larval development until larvae pupated or died in all treatment.
Seventy young larvae of P. japonica were reared in each treatment and biological parameters like survival, pupation, larval development and 7-day larval weights were recorded. Emerged adults were weighed (within 12 h), sexed, and then stored at − 80 °C to perform ELISA test. The adult weights were compared separately based on their sex. The bioassay was considered terminated when the larvae in all treatments either developed into adults or died.
Stability of Vip3Aa in synthetic diet
Stability of Vip3Aa was estimated in five samples (20–30 mg) drawn from the diet before and after exposure to the ladybird beetle, using double-antibody sandwich enzyme-linked immunosorbent assays (DAS-ELISA) as previously described by Ali et al. (2017). Briefly, all insects were washed in Phosphate-buffered saline Tween (PBST) to remove Vip3Aa toxin from their body surface. For Vip3Aa protein extraction, insects were weighted and mixed with PBST at a ratio 1:100 (mg sample: μl buffer) in 5-ml centrifuge tubes. Subsequently, samples were fully ground using glass grinding tube and pestle. After centrifugation at 4.66 × 103g for 10 min at 4 °C, the supernatants obtained were diluted to measureable concentration and ELISA (EnviroLogix Portland, Maine, USA) was performed according to the manufacturer’s instructions. The optical density was measured at wavelength 450 nm and calibrated to a range of standards made from purified toxin to estimate Bt toxin.
Detection of Vip3Aa in the emerged adults
Uptake of Vip3Aa was determined in 5 insects randomly selected from the insects reared in each treatment by using ELISA technique as described above.
Bioactivity verification of Vip3Aa
Larvae of Bt-susceptible strain of H. armigera already maintained in the laboratory were used as test insect to verify the bioactivity of Vip3Aa. The supernatant used for the ELISA was diluted to the extent of the estimated EC50 (500 ng/g fresh diet) and then incorporated into the synthetic diet used to rear the larvae of H. armigera (Zhou et al. 1981). Newly emerged first instar larvae of H. armigera were individually kept into multi-well culture plates covered with a thick cloth and lid to prevent escape. Sixty H. armigera larvae were fed on synthetic diet containing supernatant for 7 days and larvae were weighed to assess bioactivity in terms of reduced larval weight compared to the control treatment (pure synthetic diet).
Data analyses
Pupation and adult emergence rates of P. japonica were analyzed using chi-square test, while larval development times, the male and female adult weights were analyzed using Mann–Whitney U test as data followed non-normal distribution. Larval weights of H. armigera reared on pure synthetic diet and synthetic diet containing supernatant were compared using student’s t test. Similarly, concentrations of Vip3Aa in the fresh diet and diet exposed for 12 h feeding were analyzed using student’s t test. All the statistical analyses were performed using SPSS ver. 20 software package for the Windows 2007 (SPSS, Inc., Chicago, IL, USA).