Evaluation of Cry1Ac and Cry2Aa toxin binding to two important

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Agricultural and Environmental Chemistry

Evaluation of Cry1Ac and Cry2Aa toxin binding to two important beneficial cotton field insects, Harmonia axyridis and Orius similis Yong Wang, Dabo Li, Hao Zhou, Hui Liu, Lin Niu, Lihua Wang, and Weihua Ma J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b02634 • Publication Date (Web): 30 Jul 2018 Downloaded from http://pubs.acs.org on August 7, 2018

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Journal of Agricultural and Food Chemistry

Abstract: Transgenic

crops

expressing

Cry

toxins

are

effective

and

considered

environmentally friendly alternatives to synthetic pesticides, but assessment of environmental risks of their application on non-target organisms is ongoing. The main risk is the transfer of Cry toxins to natural enemies through the food chain. There is reported evidence supporting that Cry toxins can be detected in the body and gut of some natural enemy insects. Considering that binding of Cry toxins to insect proteins is an essential step in the intoxication process, this work was conducted to evaluate interactions between Cry1Ac and Cry2Aa toxins with proteins from larvae/nymphs and adults of two important predatory natural enemies in cotton fields, Harmonia axyridis and Orius similis. Results support absence of Cry1Ac or Cry2Aa binding proteins in immature stages of H. axyridis and O. similis, as well as in imaginal stage of H. axyridis. One same binding band about 70 kDa was found in imaginal total protein of O. similis when probed with the two Cry proteins, with the best match to Hsc70 of O. sauteri in the Uniprot database. However, non-specific binding was verified by following competitive binding assays between the two Cry proteins and imaginal total protein of O. similis. From these results, we may infer that Cry1Ac and Cry2Aa have no likely detrimental effects on H. axyridis and O. similis. Keywords: Cry protein; binding potentiality; natural enemy; Bt cotton; risk assessment

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1. INTRODUCTION Transgenic crops producing Bacillus thuringiensis (Bt) toxins are widely used for pest control, and the area devoted to these crops has increased from 1.70 million hectares to 185 million hectares in the past 21 years (1996 to 2016) 1. However, the potential risks and benefits of Bt crops are still being scrutinized. One of the main concerns with Bt crop usage is their potential impacts on non-target predatory insects, which show important ecological functions 2. Consequently, the potential unintended effects of Cry toxins and their combinations on the environment are tested for ecological risks assessment 3, 4. Many studies have tested the potential adverse effects of Bt toxins on non-target organisms in the past 30 years 5. In general, most recent studies reported that Cry toxins did not have negative effects or negligible harmful effects on non-target organisms and biodiversity 5-10. Both Bt maize and rice are being evaluated for future commercial production in China. Recently, a meta-analysis of Bt rice and maize supported that they posed negligible risks to non-target arthropods 2, 11. In contrast, other reports documented contradictory results on the unintended effects of Bt toxins on several organism categories, even on the same species 5, 10-15. For example, larvae of the ladybeetle Adalia bipunctata exhibited significantly higher mortality when exposed to lepidopteran-active Cry1Ab toxin in laboratory ecotoxicity testing 12. Detrimental effects were reported for the ladybeetle Propylaea japonica when they fed on Bt crop reared prey 13, while no


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significant effects of Cry toxins were reported in other ladybeetle species, including Harmonia axyridis, P. japonica and Coleomegilla maculata 10, 16, 17. In addition, an

increasing number of Cry toxins are shown to have inter-order pesticidal activity, such as Cry2Aa been active against species in Lepidoptera, Diptera and Hemiptera 18, supporting the need for further risk assessment studies. Most studies regarding the risk assessment of Bt crops on natural enemies mainly focus on biology, ecology and behavior levels by bitrophic, tritrophic or artificial diet experiments. Several factors, such as environmental conditions, the prey qualities, feeding preferences and the physiological states of insect species may affect results and lead to inconsistencies 4, 5. In addressing this issue, Rodrigo-Simón et al. suggested a novel approach for determining the potential impact of Cry toxins on natural enemies based on the critical importance of binding to midgut receptors for susceptibility to Cry toxins, i.e., the binding potentiality evaluation of Cry toxins to non-target organisms 19. Binding potentiality evaluation provides a premise for the adverse effects of Cry toxins on non-target organisms; if no specific binding proteins are detected in an organism, low or no toxicity of Bt toxins would be expected 18, 20, 21. Only a few binding potentiality studies have been performed to date to evaluate risks of Cry toxins to non-target organisms 6-9, 19-21. The ladybeetle, H. axyridis, and the predatory flower bug, Orius similis, are two important predators in cotton fields

3, 10, 11, 22

. Accumulation of Cry toxins through the

food chain or after direct feeding on Bt pollen/anther was shown in these two predators



4, 5, 11, 14

. Moreover, several articles reported that Cry toxins were also absorbed and

bioaccumulated in the body of H. axyridis

10, 23, 24

, indicating potential increase of

environmental risks of Cry toxins to these species. In the current study, we investigated the binding potentiality of Cry1Ac and Cry2Aa to these two important predators. Experiments were carried out to evaluate the binding potentiality of Cry1Ac and Cry2Aa in larvae and adults of H. axyridis and nymphs and adults of O. similis, and to identify the candidate Cry1Ac/Cry2Aa binding proteins. The results support lack of specific binding interactions between Cry1Ac or Cry2Aa toxins with proteins from H. axyridis and O. similis and support safety of Bt crops producing these proteins to the tested predatory insects. 2. MATERIALS AND METHODS 2.1 Insects Adults of H. axyridis and O. similis were collected from greenhouse populations at Huazhong Agricultural University, Wuhan, Hubei Province (China), and reared on pea aphids (Acyrthosiphon pisum) under controlled conditions (24±1 ℃, 60-70% relative humidity; 16L: 8D) for multiple generations. During the oviposition period, eggs were collected daily and placed in Petri dishes with a moisturizing filter paper. Newly hatched larvae of H. axyridis and nymphs of O. similis were reared on pea aphids (A. pisum) individually (one natural enemy per dish) in Petri dishes until pupation or adult emergence. Fourth and fifth larvae/nymphs and adults of both natural enemies were collected and immediately frozen in liquid nitrogen for experimentation.


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2.2 Protein and BBMV preparation Total protein from whole bodies rather than midgut proteins were used based on previous detection of Cry1Ac and Cry1F toxins in the gut and internally of H. axyridis 23, 24

. The frozen natural enemies were grounded in liquid nitrogen, to which a 1/25

volume of protease inhibitor (1 mM PMSF) and a final concentration of 50 µg/mL RNase and 250 µg/mL DNase were added. The samples were incubated for 15 min at room temperature, and then the homogenate was centrifuged at 15,000 x g for 60 min at 4 ℃, and the supernatant was collected and stored at -80 ℃. Brush border membrane vesicles (BBMV) from the beet armyworm, Spodoptera exigua, were used as a positive control to verify the correctness of the system adopted by us, for these BBMV were readily and specifically bind to Cry toxins prepared from midguts of fourth instar S. exigua larvae

9, 25

and

26, 27

. Briefly, the pooled

dissected midguts were homogenized in nine volume of MET buffer A (0.3 M Mannitol, 5 mM EGTA, 17 mM Tris-HCl pH 7.5), then one volume of 24 mM MgCl2 was added. The homogenate was incubated on ice for 15 min, later centrifuged at 2,500 x g for 15 min at 4 ℃. This step was repeated three times, and all collected supernatants were pooled and centrifuged again (30 min, 30,000 x g, 4 ℃). The resulting pellet was re-suspended in ice-cold buffer (10 mM HEPES, 130 mM KCl, 10% glycerol, pH 7.5) containing 1 mM PMSF. Protein concentrations were determined by the Bradford method according to the manufacturer’s instructions (Bradford protein assay kit, Beyotime Biotechnology, China).



2.3. Ligand blotting analysis Ligand blotting analysis was performed as described previously 8. Activated Cry1Ac and Cry2Aa toxins were purchased from Envirologix Inc. (Portland, ME, USA). The total protein samples from H. axyridis and O. similis and the BBMV from the midgut of S. exigua larvae were mixed with 2 x SDS protein loading buffer (100 mM Tris-HCl, pH 6.8, 200 mM dithiothreitol, 4% SDS, 0.2% bromphenol blue, and 20% glycerol), heat denatured at 100 ℃ for 5 min, and then resolved by 10% SDS-PAGE, the loading volumes for each lane were present in Fig. 1 and 2. After electrophoresis, separated proteins were transferred to a polyvinylidene difluoride (PVDF) membrane (Bio-Rad, California, USA). After transfer, the PVDF membrane was blocked with 5% (w/v) skim milk in PBS with 0.1% Tween-20 (PBST) for 2 h. After incubation with 2 µg/ml of activated Cry1Ac or Cry2Aa proteins in blocking buffer (4 ℃overnight), filters were washed thrice (10 minutes per wash) with PBST buffer (PBS/0.1 % BSA/0.1 % Tween-20) and then the PVDF membrane was probed using rat polyclonal antiserum against Cry1Ac or Cry2Aa (1:7,000 dilution) in blocking buffer for 1 h. After washing as before, the membrane was incubated with HRP-conjugated anti-rabbit IgG (1:3,500 dilution) in PBST for 1 h. The antisera were detected with DAB substrate (Sigma-Aldrich, St. Louis, MO). 2.4. Mass spectrometric analysis by Capillary-HPLC/MS Mass spectrometric identification of toxin binding proteins was performed using the method described previously 26. After ligand blotting, the protein bands bound to


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Cry1Ac and Cry2Aa were cut and rinsed in decolorizing solution (200-400 µl, 30% acetonitrile/100 mM NH4HCO3), and then freeze dried. Later, the bands were incubated with 100 mM dithiothreitol at 56 ℃ for 30 min, followed by incubation with 200 mM indole-3-acetic acid for 20 min in the dark and with 100 mM NH4HCO3 for 15 min at room temperature. Finally, the bands were treated with 100% acetonitrile for 5 min, freeze dried, and digested with 2.5-10 ng/µg trypsin for 24 h at 37 ℃ Samples were analyzed by capillary high-performance liquid chromatography/mass spectrometric analysis (Capillary-HPLC/MS) at Shanghai Life Science Research Institute (Chinese Academy of Sciences, Shanghai, China). The mass spectrometry results were queried to the Uniprot database using the Mascot 2.2 software. 2.5. Competitive binding assays with biotinylated Cry toxins Activated Cry proteins were biotinylated using the EZ-Link sulfo-N-hydroxysuccinimide liquid chromatography biotinylation kit (Pierce, FL, USA) according to the manufacturer’s instructions. The competitive binding assays were performed as our previous studies 8, 9. Binding assays in 100 µl binding reactions included 20 µg of S. exigua BBMV or total protein samples from larvae/nymphs or adults of H. axyridia and O. silimis, and 0.1 µg of biotinylated Cry1Ac/Cry2Aa in binding buffer (PBS plus 0.1% BSA). After incubation for 1 h at room temperature reactions were terminated by centrifugation for 10 min at 15,000 x g at 4 °C. Pellets were washed with 0.5 ml of ice-cold binding buffer thrice. After the final centrifugation, pellets were solubilized in 10 µl of SDS sample buffer, heat denatured at 100 °C for 5



min, and resolved by 10% SDS-PAGE. After electrophoresis, proteins were electrotransferred to PVDF filters, which were blocked for 1 h at room temperature in PBS buffer (135 mM NaCl, 2 mM KCl, 10 mM Na2HPO4, 1.7 mM KH2PO4, pH 7.5) containing 3% bovine serum albumin (BSA) and 0.1% Tween 20. Filters were probed with streptavidin-HRP conjugate (1:10,000) for 1 h at room temperature, and then washed consecutively for 10 min with washing buffer for 1 h. Then filters were developed using the ECL chemiluminescence detection kit (Thermo Fisher Scientiﬁc, Waltham, USA). The same protocol was followed for competitive assays, except that a 30-fold excess of unlabeled competitor toxin was included in the binding reactions. 3. RESULTS AND DISCUSSION 3.1 Binding of Cry1Ac and Cry2Aa toxins to total protein extracts from H. axyridia and O. similis Toxicity of Cry proteins is dependent on binding to proteins in the insect 18. Consequently, evaluating the binding potentiality of Cry toxins to proteins of non-target organisms was suggested to be a novel approach to determine the potential impact of Cry toxins on non-target organisms 19. Until now, only limited studies have reported the binding properties of Cry toxins to proteins in non-target insects. For example, the Cry1Ac toxin was reported to be absorbed and bioaccumulate not only in the gut of H. axyridia, but also in the body 23, 24. Consequently, we were interested in testing accumulation of Cry1Ac and Cry2Aa in total protein extracts of H. axyridia and O. similis. Since larvae/ nymphs and adult stages can be exposed to toxins directly or


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indirectly 3, 11, we used total protein extracts from larvae/ nymphs and adults in order to get more accurate and complete results. Gut BBMV of S. exigua were used as a positive control for specific binding of Cry toxins 9, 25. The results of testing Cry toxins binding to total protein extracts from H. axyridia are shown in Fig. 1. Negative controls (insect protein only) showed no evidence of background signals for Cry1Ac toxin in H. axyridia and S. exigua (Fig. 1A and 1B, lanes 4 and 6, respectively). Ligand blot analysis exhibited two binding protein bands, approximately 110 and 120 kDa in size, recognized by Cry1Ac in S. exigua BBMV (Fig. 1A and B, lanes 5). In contrast, no Cry1Ac binding protein was observed for both larval and imaginal total protein extracts of H. axyridia (Fig. 1A for larvae, Fig. 1B for adults, lanes 1-3 showed different sample size). For Cry2Aa toxin, there were three binding bands of about 35, 90 and 120 kDa in S. exigua BBMV (Fig. 1C, lane 5). One protein band bound by Cry2Aa was observed in larval total protein extracts of H. axyridia (Fig. 1C, lanes 1-3). However, there was also a corresponding band in the negative control of H. axyridia larval total protein extracts only (Fig. 1C, lane 4). Similar results were also observed in imaginal total protein extracts of H. axyridia probed with Cry2Aa (Fig. 1D, lanes 1-4). All these results suggested that these bands represent unspecific interactions between a H. axyridia protein and the streptavidin used for detection. Results from testing binding of Cry proteins to total protein extracts from O. similis are shown in Fig. 2. No Cry1Ac or Cry2Aa binding proteins were detected in nymphal total protein extracts of O. similis (Fig. 2A, lanes 1-3 and Fig. 2C, lanes 1-3), while one



protein band of about 70 kDa in imaginal total protein extracts was recognized by both Cry1Ac and Cry2Aa (Fig. 2B, lanes 1-3 and Fig. 2D, lanes 1-3). Using similar ligand blotting analyses, we previously reported lack of Cry1Ac and Cry2Ab binding protein bands in the gut BBMV from the honeybee Apis mellifera, the plant bug, Adelphocoris suturalis, and the pollinating beetle, Haptoncus luteolus 6-8. Similarly, no protein binding Cry1Ac, Cry2Aa or Cry1Ca was detected in the brown planthopper Nilaparvata lugens and the pond wolf spider, Pardosa pseudoannulata 9. 3.2 Mass spectrometric analysis by Capillary-HPLC/MS The detected Cry1Ac and Cry2Aa binding band in O. silimis was identified by ESI MS/MS (Table 1) to match heat shock proteins (Hsc70 and Hsp90) of O. sauteri in the Uniprot database. These Hsps play roles in an extraordinary variety of normal cellular processes, including protein transport, posttranslational modification, molecular chaperones, and signal transduction 28. The binding affinity of Hsps with Cry toxins were previously reported in Helicoverpa armigera 27, Chilo suppressalis 29, Anthonomus grandis 30 and Ostrinia furnacalis 31, but unlikely to act as a receptor of Cry toxins 31. It was suggested that Hsps potentially function in protection against Cry intoxication. For example, hsp70 and hsp90 were up-regulated as response to Cry toxins in Ostrinia nubilalis 32, Cnaphalocrocis medinalis 33, and Pardosa pseudoannulata 34. And, it was reported that Galleria mellonella larvae were more resistant to infection by Bt when larvae were exposed to heat shock, which leading to an increased protein level of Hsp90 35

. But, the suppressed expression of Hsps response to Cry toxins was also found in


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Plutella xylostella 28, H. armigera 36, Choristoneura fumiferana 37 and Trichoplusia ni TnH5 cells 38. Hence, the interaction between Hsps and Cry toxin might be complex, which may need more analysis in future. 3.3 Competitive binding assays of Cry1Ac and Cry2Aa to imaginal total protein of O. silimis To test whether the detected interactions between Cry toxins and the O. silimis Hsp protein were specific, we conducted competitive binding assays. As a control, binding of Cry1Ac or Cry2Aa to BBMV from S. exigua was displaced by excess of unlabeled toxin, which supported specific binding (Fig. 3A and 3B). In contrast, binding of labeled Cry1Ac or Cry2Aa to imaginal total protein extracts of O. silimis was not reduced in the presence of unlabeled homologous competitors, supporting non-specific binding (Fig. 3A and 3B). Specific binding is necessary but not sufficient to determine toxicity

18, 20

. Slight

binding of biotin-labeled Cry toxins detected in the gut BBMV of several natural enemies and pollinators was found to be non-specific. Examples include non-specific binding of Cry1Ab and Cry1Ac to larvae of the green lacewing Chrysoperla carnea 19, and non-specific binding of Cry1Ac and Cry2Ab to adult honey bee A. mellifera 7, 8, the plant bug A. suturalis and the pollinating beetle, H. luteolus 6. These non-specific interactions explain the lack of observed adverse effects 6-9, 19. In conclusion, though ligand blotting and competitive binding analysis, we demonstrated no specific binding of Cry1Ac or Cry2Aa to total protein extracts from



larval and imaginal H. axyridis, and nymphal and imaginal O. similis total protein extracts. In imaginal total protein extracts of O. similis, we detected one protein of about 70 kDa binding Cry1Ac and Cry2Aa matching to Hsc70 and Hsp90 of O. sauteri, yet this binding interaction was non-specific. Based on the importance of specific binding for toxicity of Cry toxins, evidence from the present study suggests that Cry1Ac and Cry2Aa have no likely adverse effects on two important predatory natural enemies in cotton fields, H. axyridis and O. similis. This information further deepens our understanding of the safety of transgenic Bt crops and provides a reference for integrated pest management. ACKNOWLEDGEMENTS We would like to thank Prof. Juan Luis Jurat-Fuentes from University of Tennessee for comments and modifications of the manuscript. This work was supported by the Ministry of Agriculture of China (Grant No. 2016ZX08011002) and the Scientific Research Project of Hubei Education Department (Grant No. Q20162707). CONFLICT OF INTEREST The authors declare no conflict of interest. REFERENCES 1.

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FIGURE CAPTIONS Figure 1. Binding of Cry1Ac and Cry2Aa toxins to total protein extracts from both larvae and adults of H. axyridis. A and B, larval or imaginal total protein extracts of H. axyridis probed with Cry1Ac. C and D, larval or imaginal total protein extracts of H. axyridis probed with Cry2Aa. In A and C, the loading quantities were 0.68, 1.36 and 2.04 µg for lanes 1-3; 1.0 µg for lane 4; 1.5 µg for lane 5 and 6, respectively. In B and D, the loading quantities were 0.62, 1.24 and 1.86 µg for lanes 1-3; 0.9 µg for lane 4; 1.5 µg for lane 5 and 6, respectively. Figure 2. Binding of Cry1Ac and Cry2Aa toxins and total protein extracts from both larvae and adults of O. similis. A and B, larval or imaginal total protein extracts of O. similis probed with Cry1Ac. C and D, larval or imaginal total protein extracts of O. similis probed with Cry2Aa. In A and C, the loading quantities were 0.45, 0.9 and 1.35 µg for lanes 1-3; 0.9 µg for lane 4; 1.5 µg for lane 5 and 6, respectively. In B and D, the loading quantities were 0.36, 0.72 and 1.44 µg for lanes 1-3; 1.08 µg for lane 4; 1.5 µg for lane 5 and 6, respectively. Figure 3. Competitive binding between Cry1Ac (A) and Cry2Aa (B) to total protein extracts from O. similis adults. Twenty micrograms of S. exigua BBMV protein or imaginal total protein extracts of O. similis were used along with 0.1 micrograms of biotinylated Cry proteins. A 30-fold excess of unlabeled Cry1Ac or Cry2Aa was used as competitor. Toxin binding to S. exigua BBMV was used as a positive control.



Figure 1


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Figure 2



Figure 3


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TABLES Table 1. Identification of Cry1Ac and Cry2Aa binding proteins in imaginal total protein extracts of O. similis using Uniprot database and Mascot 2.2 software.

Accession No.

Cover

MW

Percent

(KDa)

tr|A0A060A9H7 14.81%

71.6

PI

Protein Name

Species

5.38

Heat shock cognate

O. sauteri

protein 70 tr|A0A060A5H6 3.46%

83.0

4.92

Heat shock protein 90


O. sauteri

Evaluation of Cry1Ac and Cry2Aa toxin binding to two important

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