Evaluating the effects of Bt protein Vip3Aa on immature

Background: Genetically modified (GM) crops have become a common tool for managing insect pests of economic importance. However, before their commercialization, bio‐safety evaluation on non‐target arthropods such as pollina‐ tors and biological control agents is a pre‐requisite to save services rendered by them in agro‐ecosystem. Results: In the present experiment, the potential adverse effects of Bt protein (Vip3Aa) were studied on the life table parameters (pupation and emergence rates, 7‐day larval weights, larval development time, and adult weight) of a predatory beetle, Propylea japonica (Thunberg) (Coleoptera: Coccinellidae) in the laboratory. P. japonica was exposed to Bt protein incorporated in synthetic diet at the rate of 200 μg/g of fresh weight of diet. Temporal stabil‐ ity, bioactivity and the uptake of insecticidal protein were confirmed by enzyme‐linked immunosorbent assay and a sensitive insect bioassay. The results indicated that pupation and adult emergence rates did not differ significantly from that of negative control (pure synthetic diet). Similarly, the fresh weight of male and female adults emerged in Vip3Aa treatment (2.87 mg and 3.86 mg, respectively) did not vary significantly from that of the negative control treatment (2.66 mg, 3.75 mg). However, the time taken by the larvae to complete their development (12.70 days) was significantly extended than that of the negative control (10.21 days). In contrast, the tested life‐table parameters of P. japonica were adversely affected when fed on synthetic diet containing boric acid (positive control) indicating experi‐ mental fitness to detect adverse effects. Conclusions: On the basis of the results, it is suggested that GM crops expressing Bt protein (Vip3Aa) are safe for non‐target insects such as P. japonica and are compatible with other biological programs for the integrated manage‐ ment of insect pests.


Background
Extensive use of insecticides not only increases resistance in insect pests but also reduces the bio-diversity of non-target arthropods (NTA) (Jafar et al. 2013). To overcome these problems, scientists have modified the crops genetically using the genes of Bacillus thuringiensis (Berliner) with the aim to provide enhanced resistance in plants against insect pests without affecting NTA. Bt synthesizes potent insecticidal proteins, such as the Cry proteins during sporulation, commonly used in GM crops (Palma et al. 2014). In addition, Bt secretes numerous vegetative insecticidal proteins (Vip) during its vegetative phase. These proteins have also shown a broad-spectrum activity against Lepidoptera without sharing target binding sites with Cry proteins in the target insect midgut. Therefore, these Bt proteins are considered a good complement to Cry proteins (Chakrabarty et al. 2020). The Page 2 of 6 Ali et al. Egyptian Journal of Biological Pest Control (2021) 31:145 genes coding for expressing various Cry and Vip3 proteins have been incorporated genetically in GM plants to protect them from insect pests of economic importance (Kumar et al. 2020). GM crops are now being opted as a significant tool to manage the insect pests of economic concerns worldwide with less number of broad-spectrum chemical applications (Klümper and Qaim 2014), and these GM crops are generally considered safe to NTA (Yin et al. 2020). However, there is a possibility that GM crops might have lethal or sub-lethal effects on the fitness of NTA found abundantly in cropping ecosystems, and playing a substantial role in the suppression of insect pests without or with little human assistance. Thus, it is considered imperative to evaluate GM crops for their potential adverse effects on NTA before their release for wide commercial cultivation in the field (Romeis et al. 2008).
The Japanese ladybird, Propylea japonica (Thunberg) (Coleoptera: Coccinellidae), is a common predatory coccinellid found in a wide range of natural and agricultural habitats of the world including China, Japan, Korea and Pakistan, and prefers to feed on insect pests that belonging to Aphidoidea, Psylloidea, Coccoidea, and mites. It is a successful predator both in arid and semi-arid regions because of its tolerance to high temperature and capability to reproduce under nutritionally poor quality prey and pollen supplements. P. japonica also remains active throughout the year, without undergoing aestivation, and completes 3-4 and 7-8 generations in Northern and Southern China, respectively. Both stages of the beetle, larvae and adults are predacious with a very high rate of daily prey consumption and, thus, play a considerable role in suppression of economic pests (Luo et al. 2014). Many studies demonstrated that the Bt proteins do not pose any adverse effect on the NTAs. However, few studies reflect deleterious effects of Bt proteins on some NTAs (Liu et al. 2016).
Non-target arthropods such as P. japonica are usually exposed to plant expressed Bt proteins when feeding on prey and pollens in GM crops. Therefore, it is needed that bio-safety of Bt protein such as Vip3Aa must be evaluated to ensure safety of GM crops expressing Vip3Aa. P. japonica being a sensitive and easy to be reared in the laboratory is considered as the model test insect for studying the potential adverse impacts of GM crops and pesticides before their registration (Li et al. 2017). It is also being mass reared on natural and factitious prey under controlled laboratory conditions to be used in inoculative and augmentative biological control programs (Mirkhalilzadeh et al. 2013). Zhang et al. (2013) reported 400 ng concentration of Cry2Aa in P. japonica collected from the GM rice and also assessed the ecological effects of two Bt cotton cultivars, ZMS and ZMKCKC, expressing Cry1Ac/Cry2Ab and Cry1Ac/EPSPS, respectively, on P. japonica through its herbivorous prey, Aphis gossypii (Glov.) and found that GM cotton cultivars had no detrimental effects on the biology of P. japonica. In the present study, potential adverse effects of Vip3Aa protein which transgene exhibited into some GM crops were being evaluated on the larval development, mortality and adult weight of P. japonica.

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 (EC 50 ) 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 EC 50 (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 Page 3 of 6 Ali et al. Egyptian Journal of Biological Pest Control (2021) 31:145 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 × 10 3 g 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 EC 50 (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).

Impact of Vip3Aa on the life table parameters of P. japonica
The results indicated that about 57-60% larvae successfully completed their development and successfully pupated in the negative control and Vip3Aa treatments, which did not differ significantly (Fig. 1). Similarly emergence rate was above 85% among the pupated larvae in both treatments. Likewise, the male and female fresh weights did not vary significantly from that of the negative control (Table 1). However, the time taken by the larvae to complete larval development was significantly prolonged in the Vip3Aa treatment than that of the negative control (Table 1). In contrast, positive control when larvae were nourished on synthetic diet containing boric acid all the larvae died during their earlier developmental instars. Therefore, this treatment was excluded from analysis (Table 1). Death of larvae in their earlier Page 4 of 6 Ali et al. Egyptian Journal of Biological Pest Control (2021) 31:145 developmental instar in positive control indicated the sensitivity of the bioassay to detect any adverse effect of insecticide incorporated in diet on the test organism.

Vip3Aa stability
The reliability of test's results also depends on whether the protein in question is biologically active and remains stable during diet preparation and diet storage. The ingestion factor also plays a crucial role in deciding the biosafety of protein to non-target arthropods. The results of the present study showed that Vip3Aa remained stable when incorporated into the synthetic diet during diet preparation and storage. The mean concentrations of Vip3Aa protein detected in the fresh diet and in diet after 12 h. of feeding exposure were 136.83 and 113.14 µg/g, respectively. This difference in the degradation rate (17%) was non-significant (student's t test; t = 1.06; df = 6; p = 0.33) (Fig. 2a). Consequently, the protein remained stable during the diet preparation and feeding exposure to avoid false negative results.

Bioactivity verification and detection of Vip3Aa in emerged adults
Bioactivity of the Vip3Aa was confirmed by exposing the young larvae of susceptible H. armigera to the protein. The results indicated that when susceptible newly emerged first instar larvae of H. armigera were fed for 7 days on the diets containing supernatants extracted from fresh and exposed synthetic diets containing Vip3Aa protein, the mean fresh larval weight (1.41 g) of H. armigera was reduced significantly than the control (4.87 g) (student's t test; t = 6.59, df = 91, p = 0.00) (Fig. 2b). In the present study, average concentration of ingested Vip3Aa protein in the emerged adults of P. japonica was also measured, using DAS-ELISA which was 178.56 ng/g of fresh body weight, whereas no Vip3Aa was detected in the emerged adults in the control treatment.

Discussion
Genetically modified crops producing Bt endotoxins are planted worldwide to control the target insect pests particularly those belonging to the order Lepidoptera (e.g., Helicoverpa zea, H. armigera). However, their prolific increase in plantation expose the NTAs including pollinators, predators and parasitoids to the pathogenic concentration that may prove lethal at the highest trophic level and may reduce their population as well as their services as pollinator and biological control agents (Harwood et al. 2005). Therefore, it is considered obligatory to test the effects of GM crops on NTAs before issuing approval for their commercial plantation in the field.   et al. Egyptian Journal of Biological Pest Control (2021) 31:145 For this purpose, a project was designed to evaluate the potential adverse effects of various Bt proteins on NTAs and this experiment was the part of that project which emphasized to assess the direct effects of Vip3Aa protein on life table parameters of P. japonica an abundant ladybird in China and neighboring countries. Many previous studies highlighted no adverse effects of Bt proteins on the NTA, However, few studies also exhibit mild adverse effects of Bt proteins on the NTA. These studies indicated mild side effects, when the NTAs were fed on the preys reared on the Bt crops. For instance, developmental delays were observed when Chrysoperla carnea (Steph.) were fed on the Spodoptera littoralis (Boisd.) larvae reared on the Bt maize expressing Cry1Ab (Dutton et al. 2002). Similarly, developmental delays were observed when Chrysoperla sinica (Tjeder) were fed on Bemisia tabaci (Genn.) reared on Bt cotton producing Cry1Ac (Guo et al. 2004). However, there are also some studies that differed from these findings and hinted that Bt proteins had none of the apparent adverse effects on the survival of green lace wing larvae (Wang et al. 2012) or other NTAs when administered through direct incorporation into the synthetic diet or indirectly through prey (Tian et al. 2018 The results of the experiment seem to be over-estimated rather than under-estimated because quantity of protein administered via diet (200 µg/g of fresh diet) to Japanese beetle was 10 times higher than that quantity which may be possibly ingested by the beetle consuming pollens and preys in the GM crops. For instance, Vip3Aa concentration measured in MIR 162 (Corn) varied from 17.35-24.62 µg/g to 50.41-56.56 µg/g in the whole plant and leaves, respectively (Raybould and Vlachos 2011). Ladybird beetle may possibly consume 20% of the highest expressed concentration in plants while consuming pollen and prey.

Conclusions
It is concluded that immature P. japonica was found insensitive to Vip3Aa and mild adverse effects observed during development at much higher concentration are not possible in field conditions where protein ingestion level is potentially very low, while consuming pollen and preys in a Bt-crop. It is further concluded on the bases of results of the present experiment that Bt crops expressing b 7th day larval weight of H. armigera (g) Series1 Fig. 2 a Concentration of Vip3Aa (µg/g) detected in fresh and exposed diets. b 7-day post-treatments larval weight of Helicoverpa armigera larvae fed diet with and without Vip3Aa Page 6 of 6 Ali et al. Egyptian Journal of Biological Pest Control (2021) 31:145 Vip3Aa are not harmful to the non-target organisms and can be integrated in the biological pest control programs as an integrated pest management strategy.