Safety Assessment of Bacillus thuringiensis Insecticidal Proteins

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Agricultural and Environmental Chemistry

Safety assessment of Bacillus thuringiensis (Bt) insecticidal proteins Cry1C and Cry2A with a zebrafish embryotoxicity test Yan-jie Gao, Hao-Jun Zhu, Yi Chen, Yun-he Li, Yu-fa Peng, and Xiu-Ping Chen J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b01070 • Publication Date (Web): 13 Apr 2018 Downloaded from http://pubs.acs.org on April 15, 2018

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Journal of Agricultural and Food Chemistry

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Safety assessment of Bacillus thuringiensis (Bt) insecticidal

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proteins Cry1C and Cry2A with a zebrafish embryotoxicity test

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Yan-Jie Gao†, Hao-Jun Zhu†,‡, Yi Chen†,§, Yun-He Li†, Yu-Fa Peng†, Xiu-Ping Chen†,*

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†

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Plant Protection, Chinese Academy of Agricultural Sciences, Beijing 100193, China

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‡

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Ministry of Agriculture, Freshwater Fisheries Research Center, Chinese Academy of

The State Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of

Key Laboratory of Freshwater Fisheries and Germplasm Resources Utilization,

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Fishery Sciences, Wuxi 214081, China

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§

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Switzerland

Agroscope, Research Division Agroecology and Environment, 8046 Zurich,

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address:

Yan-Jie

Gao:

[email protected];

Hao-Jun

Zhu:

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Email

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[email protected]; Yi Chen: [email protected]; Yun-He Li: [email protected];

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Yu-Fa Peng: [email protected]; Xiu-Ping Chen: [email protected].

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*Corresponding author: Xiu-Ping Chen

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No. 2 West Yuanmingyuan Road, Haidian District, Beijing, China

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Tel: +86-10-62815947; Fax: +86-10-62896114;

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E-mail: [email protected]

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ACS Paragon Plus Environment


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ABSTRACT

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Because of the large-scale planting of transgenic Bt crops, fish would be exposed to

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freely soluble Bt insecticidal protein(s) that are released from Bt crop tissues into

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adjacent bodies of water or by way of direct feeding on deposited plant material. To

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assess the safety of two Bt proteins Cry1C and Cry2A to fish, we used zebrafish as a

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representative species and exposed their embryos to 0.1, 1, and 10 mg/L of the two

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Cry proteins until 132 hours post-fertilization, and then several developmental,

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biochemical, and molecular parameters were evaluated. Chlorpyrifos (CPF), a known

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toxicant to aquatic organisms, was used as a positive control. Although CPF exposure

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resulted in significant developmental, biochemical, and molecular changes in the

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zebrafish embryos, there were almost no significant differences after Cry1C or Cry2A

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exposure. Thus, we conclude that zebrafish embryos are not sensitive to Cry1C and

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Cry2A insecticidal proteins at test concentration.

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KEYWORDS: Bt crops, Cry1C, Cry2A, nontarget effect, aquatic organism, fish,

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zebrafish embryo test

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INTRODUCTION

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Since the development of transgenic crops that express Bacillus thuringiensis (Bt)

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insecticidal proteins, their potential to cause adverse effects on the environment has

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drawn much attention. The environmental risk assessment of Bt crops has focused on

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terrestrial ecosystems, whereas their potential effects on aquatic ecosystems have been

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relatively ignored.1-2 Studies have, however, confirmed that particulate organic matter

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from Bt crops (e.g., pollen, crop dust, detritus) can be deposited in adjacent bodies of

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water or transported along water courses to downstream bodies of water, therefore

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exposing non-target aquatic organisms to the Bt insecticidal proteins1,3-5. Field studies

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have shown that Bt corn releases detectable amounts of Bt protein into natural water

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environments5, with a maximum of 130 ng/L during the flowering season6. Some

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studies have assessed the effects of Bt crop products on aquatic organisms such as

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caddisflies Helicopsyche borealis (Trichoptera: Helicopsychidae) and Lepidostoma

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liba (Trichoptera: Lepidostomatidae)3,4,7, water flea Daphnia magna (Cladocera:

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Pulicidae)8-12, benthic amphipod Hyalella azteca (Amphipoda: Hyalellidae) and

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scraping snail Gyraulus (Pulmonata: Planorbidae)4, crane fly Tipula abdominalis

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(Diptera:

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Chlorellaceae)14, and African clawed frog Xenopus laevis (Anura: Pipidae)15-16. Most

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of these studies showed that Bt crops are safe with respect to aquatic organisms, but a

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few studies had conflicting conclusions. For example, Rosi-Marshall et al. found

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lower growth rates and higher mortality of stream caddisflies in the Bt treatment when

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compared with non-Bt treatment.3 Bøhn et al. reported that Bt protein or Bt crop

Tipulidae)7,13,

green

alga

Chlorella

pyrenoidosa

3


(Chlorococcale:


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tissues significantly reduced the fitness of D. magna.10-12

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Fish constitute almost half of the total number of vertebrates in the world, they live

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in almost all conceivable aquatic habitats, and they occupy an important position in

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aquatic ecosystems.17 They eat excess aquatic plants, algae, and the pupae of insect

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pests; help to balance water pH; and are an indicator of water quality.18,19 Fish may be

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exposed to Bt protein through two routes: (1) exposure to freely soluble proteins, such

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as proteins released from Bt crop tissues into an adjacent body of water; or (2)

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exposure to proteins in deposited plant material via direct feeding.20,21 For the safety

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assessment of Bt crops with respect to fish, the findings have been inconsistent. Some

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studies showed that a diet consisting of Bt crops has no obviously adverse effects on

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fish growth.22-25 One study even showed that Bt crops promote fish growth26, whereas

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another indicated the opposite27.

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The zebrafish (Danio rerio) is a model organism that is widely used in

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developmental, molecular, and toxicology studies because of its rapid development,

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large number of offspring, easy maintenance, transparency of embryos, high

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sensitivity to external contaminants, and low maintenance costs.28,29 The International

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Organization for Standardization30 (ISO) and the Organization for Economic

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Co-operation and Development31 (OECD) have listed zebrafish as the standard

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organism for testing chemicals and water quality. The early stage of zebrafish

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development, i.e., the embryo, is more sensitive to toxin exposure than D. magna and

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goldfish embryos, and zebrafish embryos are typically used in a 96-h acute toxicity

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test32. In addition, acute toxicity in zebrafish embryos correlates very well with acute 4


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toxicity in adults.29

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In the present study, we assessed the safety of two Bt insecticidal proteins, Cry1C

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and Cry2A, in a zebrafish embryotoxicity test. These Cry proteins have been

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expressed in rice33,34 and other plants 35,36 for the control of lepidopteran pests, such as

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Plutella xylostella (Linnaeus) (Lepidoptera: Plutellidae), Chilo suppressalis (Walker)

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(Lepidoptera: Crambidae), and Scirpophaga incertulas (Walker) (Lepidoptera:

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Pyralidae). In addition, biochemical and molecular experiments were conducted to

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evaluate the potential toxicity of the two Cry proteins on this species.

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MATERIALS AND METHODS

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Zebrafish maintenance and embryo collection. Adult zebrafish (Danio rerio, AB

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strain) were purchased from the Center of National Zebrafish Resources of China

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(Wuhan, Hubei province, China). They were cultured in an aquarium recirculation

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system at ambient temperature (28 ± 1°C) under 14-h light/10-h dark cycles according

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to standard procedures37. The fish were fed twice a day with freshly hatched brine

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shrimp (Artemia salina). To generate embryos, male and female fish were placed in a

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breeding tank, with a sex ratio of 2:1 males/females overnight. Spawning was

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triggered after the light was turned on the next morning and was completed within 30

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min. Embryos were collected and rinsed three times with distilled water. An optical

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microscope (SZ2-ILST, OLYMPUS; Tokyo, Japan) was used to examine the quality

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and developmental stage of the embryos. Embryos that had developed normally and 5



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reached the blastula stage were selected for subsequent experiments according to the

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standardized staging38.

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Stability and bioactivity analysis of Cry proteins in E3 medium. Purified Cry

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protein was dissolved in 70 mL E3 culture medium [5 mmol/L NaCl, 0.33 mmol/L

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CaCl2, 0.33 mmol/L MgSO4·7H2O, 0.17 mmol/L KCl; All of the chemicals were

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purchased from Sigma Chemical Company (St. Louis, MO, USA)] at the different

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concentrations (0, 0.1, 1, 10 mg/L) and referred to as mixed E3 medium (MEM). After

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a 24-h incubation at 28°C, the MEMs were collected, and the actual Cry amount in

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each MEM was quantified using enzyme-linked immunosorbent assay (ELISA, see

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below).

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Bt-susceptible C. suppressalis larvae were used as sensitive insects to assess the

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bioactivity of the Cry1C and Cry2A proteins dissolved in the above-prepared MEMs

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as previously described.9 Each MEM was mixed with the artificial diet of C.

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suppressalis at a 1:2 (w/w) ratio of MEM to diet. E3 medium with no Cry protein

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served as the blank control. To avoid degradation of the Cry proteins during

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preparation of the diet, the MEM was mixed into the diet only after the diet

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temperature was < 60°C. Once the diet mixture had solidified, it was cut into slices

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and placed in Petri dishes (9 cm in diameter, 1 cm in height), together with 10 neonate

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larvae of C. suppressalis. The Petri dishes were then sealed with Parafilm. Four

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replicates were tested for each treatment. After 7 days, the mortality and weight of

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each C. suppressalis larva were recorded.

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Zebrafish embryo toxicity assay. Normal zebrafish embryos at 2 hours

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post-fertilization (hpf) were randomly distributed among the wells of a 24-well plate

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containing 1 mL of each of the different test solutions. Twenty individuals were tested

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for each of the five following solutions: (1) E3 medium alone, used as the blank

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control; (2) E3 medium containing 2 mg/L CPF (Sigma; St. Louis, MO, USA), which

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was used as the positive control (its working concentration was determined based on

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preliminary experiments, data not shown); (3) E3 medium containing 0.1, 1, and 10

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mg/L Cry1C or either Cry2A. The above assays were independently replicated three

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times, and thus the total number of embryos used in this study was 600. The exposure

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solution was renewed daily, and the mortality and malformation percentages of each

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test group were calculated every 12 h. Mortality was identified by coagulation of the

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embryos during the first 24 hpf and by the disappearance of a heart beat after 24 hpf.

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At 60, 72, and 84 hpf, the hatched larvae were counted; the body lengths and

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malformation of the larval zebrafish in all of the test groups were photographed and

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measured at 132 hpf via digital microscope (vhx-2000, Keyence; Osaka, Japan). All

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living larvae were anesthetized with 40 µg/mL tricaine (Sigma; St. Louis, MO, USA)

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and collected at the end of the experiment and then stored at –70°C for subsequent

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analysis of mRNA expression and determination of enzyme activities and ELISA.

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Gene expression analysis. To further study the effects of Cry proteins on zebrafish

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embryos, we also analyzed the mRNA expression level of the following five genes:

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B-cell lymphoma-2 (bcl2), catalase (cat), cyclo-oxygen-ase 1 (cox1), glutathione

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S-transferase pi 2 (gstp2), and superoxide dismutase 2 (sod2), they are closely related 7



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to stress responses or apoptosis. Total RNA from 30 homogenized zebrafish larvae

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from each treatment was extracted by using RNAprep Pure Kit for Tissue (Tiangen

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Biotech Co., Ltd; Beijing, China). The concentration of each sample was determined

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by absorbance at 260 nm using a Nanodrop 2000 spectrophotometer (Thermo

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Electron Corporation; Waltham, MA, USA), and RNA quality was quantified using 1%

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agarose formaldehyde gel electrophoresis and the spectrophotometric 260/280 nm

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ratio. First-strand cDNA was synthesized using the RT reagent Kit with oligo dT

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primers and gDNA Eraser (Tiangen; Beijing, China). Real-time quantitative PCR

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(qPCR) reactions were performed with the ABI 7500 q-PCR system (ABI; Carlsbad,

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CA, USA). The 20-µL qPCR reactions consisted of 10 µL of 2× SYBR Green PCR

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super Mix (Tiangen; Beijing, China), 0.6 µL of 10 mM each of forward and reverse

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primers (Table 1), 1 µL of cDNA template (500 ng), 0.4 µL of 50× ROX reference dye,

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and 7.4 µL of RNase-free water. The thermal cycle was performed as follows: initial

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denaturation for 15 min at 95°C followed by 40 cycles of 95°C for 15 s and 56°C for

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30 s. The relative gene expression of the treatment groups was analyzed by the 2–△△Ct

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method and reported as fold change over the control.

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Determination of enzyme activities and malondialdehyde (MDA) content. The

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activities of the antioxidant-related enzymes superoxide dismutase (SOD) and catalase

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(CAT), as well as the level of MDA in the zebrafish larvae after treatment, were

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determined using commercial kits purchased from Nanjing Jiancheng Bioengineering

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Institute (Nanjing, Jiangsu province, China). All samples were homogenized in 0.8%

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physiological saline at a ratio of 1:9 (w/v) and centrifuged at 2500 × g at 4°C for 10 8


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min. The supernatants were collected and immediately used for analysis following the

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manufacturer’s instructions. Optical density values were read with a microplate

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spectrophotometer (PowerWave XS2, BioTek; Winooski, VT, USA), and enzyme

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activities and MDA content were calculated by calibration against a range of

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standards provided with the kits.

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Determination of Cry protein content by ELISA. The Cry1C and Cry2A protein

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content in C. suppressalis diet, zebrafish larvae after exposure until 132 hpf and E3

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culture medium after a 24-h incubation were measured by ELISA using a Cry1C or

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Cry2A detection kit (Quanti-plate kit; EnviroLogix Inc.; Portland, OR, USA). To

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remove Bt toxins from the outer surface, larvae were washed in phosphate-buffered

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saline/Tween-20 (PBST, provided with the kit) before analysis. For Cry protein

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extraction, larvae and diet were weighted and mixed with fresh PBST at a ratio of

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1:10 to 1:100 (mg sample/µL PBST) in a 1.5 mL centrifuge tube. The samples were

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then homogenized using an electric grinding rod. This step was not necessary for the

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liquid samples. After centrifugation and appropriate dilution of the supernatants,

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ELISA was performed according to the manufacturer’s instructions. Optical density

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values were read as described above, and Cry1C and Cry2A concentrations were

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calculated based on the standard curve provided with the kit.

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Statistical analysis. All data are expressed as the mean ± standard error (SE)

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unless otherwise indicated. The survival rates of C. suppressalis and D. rerio between

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each treatment and the control were compared using the Chi-square test. Body

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weights of C. suppressalis were compared by a one-way ANOVA with Duncan’s 9



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multiple-range test. The effects of Cry1C, Cry2A or CPF treatment and the blank

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control on D. rerio survival and malformation were analyzed using the Kaplan-Meier

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procedure and the log rank test. Statistical comparisons were made among the

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treatment and control groups using a one-way ANOVA with Tukey’s honest

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significant difference (HSD) test for the following parameters: Cry content in E3

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medium and diet, hatching rate of eggs, body length, Cry concentrations in fish larvae,

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mRNA expression of specific genes, SOD and CAT enzyme activity, and MDA level.

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Differences were considered significant at p < 0.05.

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RESULTS

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Degradation and bioactivity of Cry protein in E3 medium. The mean actual

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concentrations of the two Cry proteins when mixed in E3 medium (0.005, 0.12, and

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1.76 mg/L for Cry1C; 0.001, 0.02, and 0.23 mg/L for Cry2A) as measured by ELISA

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were significantly lower than their nominal concentrations (0.1, 1, 10 mg/L; Table 2).

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The sensitive-insect bioassay showed that the survival rate of C. suppressalis exposed

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to medium containing 10 mg/L Cry1C was significantly lower than that of the blank

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control group (χ2 = 18.46, df = 1, p < 0.01; Table 2). In contrast, the survival rates of C.

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suppressalis exposed to 0.1 and 1 mg/L Cry1C and to all three Cry2A treatments were

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not significantly changed when compared with the blank control. Meanwhile, the

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body weights of the C. suppressalis larvae exposed to the three Cry1C concentrations

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(one-way ANOVA with Duncan’s test; F = 79, df = 3, p < 0.05) or to the two higher

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Cry2A concentrations (one-way ANOVA with Duncan’s test; F =184.13, df = 3, p
0.05). When compared with exposure to the blank

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control, the body length of the hatched D. rerio larvae was not significantly affected

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by Cry proteins (one-way ANOVA with Tukey’s HSD test; F = 11.86, df = 4, p > 0.05

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for Cry1C assay; F = 7.42, df = 4, p > 0.05 for Cry2A assay; Table 3), although it was

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significantly shortened by 2 mg/L CPF, the positive control (one-way ANOVA with

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Tukey’s HSD test; p < 0.05).

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When compared with the blank control and the Cry1C and Cry2A treatment groups,

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the survival rate was significantly reduced when D. rerio embryos were cultured in

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medium containing 2 mg/L CPF (Kaplan-Meier and log rank test; χ2 = 21.19, p < 0.05

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in the Cry1C assay; χ2 = 19.12, p < 0.05 in the Cry2A assay; Figure 1). In contrast,

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there were no significant differences between the survival rates of zebrafish embryos

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in the control group and the 0.1 mg/L, 1 mg/L, and 10 mg/L Cry1C and Cry2A

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treatment groups (Kaplan-Meier and log rank test; all p > 0.05; Figure 1).

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Embryo malformations were observed very infrequently based on analyses of

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spinal curvature and pericardialites (data not shown) in any of the Cry protein

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treatment groups or the blank control during the entire exposure period (Table 3 and

11



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Figure 1). Embryos exposed to 2 mg/L CPF did not show toxicity at 84 hpf, whereas

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the malformation percentage increased remarkably between 84 and 96 hpf, with the

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maximum percentage of malformation reaching 100% at 132 hpf (Figure 1).

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To determine the bioaccumulation of the two Cry proteins, we also measured

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Cry1C and Cry2A concentrations by ELISA in zebrafish after exposure until 132 hpf.

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In all Cry test groups, the Cry proteins were detectable and their concentrations

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increased with increasing amounts of added Cry protein, whereas no Cry protein was

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detected in any of the three control replicates (Figure 2).

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mRNA expression levels of genes related to oxidative stress or apoptosis.

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Levels of sod2, cat, and bcl2 mRNA were significantly lower (one-way ANOVA with

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Tukey’s HSD test; all p < 0.05; Figure 3A, 3B and 3E), whereas cox1 and gstp2

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mRNA expression levels were significantly elevated by CPF exposure (Tukey’s HSD

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test; all p < 0.05; Figure 3C and 3D); however, no significant differences were

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observed among the blank control and the three Cry1C testing groups (Tukey’s HSD

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test; all p > 0.05), except that the sod2 mRNA expression in the three Cry1C testing

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groups was significantly lower than that of the blank control (F = 31.81, df = 4, all p
0.05).

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DISCUSSION

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In the present study, we used a zebrafish embryotoxicity test to assess the safety of

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two Bt insecticidal proteins, Cry1C and Cry2A, with respect to fish. Almost none of

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the characterized developmental, biochemical, and molecular parameters of the

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zebrafish embryos were significantly different among the blank control and Cry1C or

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Cry2A treatment groups, but these parameters were significantly changed by the

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positive control, CPF. Overall, our bioassays revealed that zebrafish embryos were not

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adversely affected by exposure to Cry1C or Cry2A at test concentration.

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Some researchers have proposed that the biological activity of a test substance

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should be confirmed before risk assessment of transgenic crops or their byproducts is

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initiated.20,21,39 Specifically, use of a validated ELISA to fully characterize the test

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substance should be paired with a sensitive insect bioassay because the concentration

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of protein detected by ELISA does not necessarily correlate with bioactivity of the

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protein.20 In the present study, before we conducted our toxicity assessment, we first

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studied whether the Cry1C and Cry2A proteins have insecticidal activity after they

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were dissolved in a water-based medium. Our results showed that the weights of

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target insect C. suppressalis larvae fed on a diet containing Cry1C or Cry2A for 7 13



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days were significantly reduced. The EC50 value (toxin concentration resulting in 50%

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weight reduction compared with the control) was estimated to be 2.2 and 4.8 ng/g diet

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for Cry1C and Cry2A, respectively. This result was lower than those reported results,

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however, it proved the insecticidal activity of the two Cry proteins dissolved in E3

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medium.40,41 Moreover, we also detected the concentration of the two Cry proteins in

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zebrafish at the end of the experiment. Cry proteins were detectable in all Cry test

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groups, and their concentrations increased with increasing amounts of added Cry

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protein, thus confirming that our test species were exposed to fully bio-active Cry

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proteins.

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The ELISA results showed that the mean concentrations of the two Cry proteins in

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E3 medium measured by ELISA were significantly lower than their nominal

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concentration. This may be attributed to the rapid degradation of any freely soluble

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proteins.21 Carstens et al. calculated that aquatic organisms in a US EPA standard

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pond or a EU static ditch would be exposed to a maximum concentration of

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22.5–1,125 µg/L or 0.67–33 µg/L of Bt protein, respectively, under worse-case

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assumptions.20 In fact, the maximum Bt protein concentration that has been reported

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in a natural aquatic environment is < 130 ng/L for Bt corn.5,6 For Bt rice, the

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maximum reported level is even lower, at 30 ng/L. 42-44 Thus, the maximum Cry1C

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and Cry2A levels used in our study, i.e., 10 mg/L, with detected levels of 1.76 and

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0.23 mg/L, respectively, were at least 10-fold greater than those zebrafish would

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encounter in the natural aquatic environment, which meets the requirement of a Tier-1

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test system for the environmental safety assessment of Bt crops.45 14


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Chlorpyrifos is a broad-spectrum organophosphorothioate insecticide, and it was

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selected as a positive control in our study. Aquatic invertebrates, particularly

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crustaceans and insect larvae, are sensitive to CPF exposure. Fish appear to be less

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sensitive, with LC50 values generally between 1 and 100 mg/L and no-observed-effect

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concentrations of ~0.5 mg/L.46 In our study, 2 mg/L CPF significantly reduced the

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survival rate and body length and enhanced the malformation of the zebrafish larvae.

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This is consistent with the reported toxicity study of CPF relative to zebrafish

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embryos.47-49 However, none of the above developmental parameters were

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significantly changed when embryos were cultured in E3 alone or in medium

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containing three concentrations of Cry1C or Cry2A proteins, demonstrating that

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Cry1C and Cry2A are safe for zebrafish from a developmental perspective.

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Some substances, although they do not lead to significant developmental alterations,

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do induce significant changes in some physiological and biochemical indices.50,51

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Researchers have found that Cry protein can change the transcripts encoding

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antioxidant enzymes in targeted pest insect.52 Furthermore, it was reported that crystal

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proteins from Bacillus thuringiensis Bt9875 induced apoptosis of human acute

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myelogenous leukemia cells HL-60.53 Therefore, we also examined biochemical

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parameters such as sod2, cat, cox1, gstp2, and bcl2 mRNA expression level; SOD and

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CAT enzymatic activity; and MDA level in the exposed zebrafish embryos. All of

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these parameters are closely related to the oxidative stress, except that bcl2 is an

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anti-apoptosis gene.54-58 CPF exposure led to significant changes in the expression of

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all five mRNAs, in SOD and CAT enzymatic activities, and in MDA levels. The 15



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changes in sod2, cat, cox1, gstp2, and bcl2 mRNA expression in zebrafish after CPF

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exposure have been previously reported.47, 56-58 SOD, CAT, and MDA results have

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been less consistent, however, as SOD and CAT activity and the MDA level are

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significantly enhanced by low-level exposure of zebrafish embryos to CPF56, whereas

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parameters related to oxidative stress were decreased with increasing amounts of

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added CPF in other studies

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levels and the SOD and CAT activity were significantly lower in zebrafish embryos

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exposed to CPF relative to those exposed to the blank control. This may have been

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induced by the high level of CPF exposure (2 mg/L), which could have overwhelmed

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the detoxification ability of the zebrafish, resulting in superabundant reactive oxygen

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species that thus inhibited the mRNA expression of sod2, cat, and bcl2 and reduced

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SOD and CAT enzymatic activities. It is, however, worth noting that none of the

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above molecular parameters showed significant changes among the blank control and

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Cry1C or Cry2A treatment groups, except that the sod2 and, in some cases, cat mRNA

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levels in the Cry testing groups were significantly lower than those of the blank

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control. However, this difference disappeared at the biochemical level, i.e., with

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respect to SOD and CAT activity. Hence, the above biochemical and molecular results

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further confirmed that Cry1C and Cry2A are safe for zebrafish.

47,58

. In the present study, the sod2, cat, and bcl2 mRNA

337

For the safety assessment of Bt crops relative to fish, researchers have fed flounder

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and rockfish22, carp23, salmon24,27, and zebrafish25,26 diets containing Bt crop material.

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Some of these studies showed that that a diet containing Bt soya or maize has no

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obviously adverse effects on fish growth22-25, some even showed that Bt crops 16


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promote fish growth26, and some found that they inhibit fish growth27. Our results

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indicate that Bt proteins, especially Cry1C and Cry2A, have no adverse effects on the

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development of zebrafish. It was reported that Cry proteins showed zebrafish-embryo

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toxicity and developmental delay after exposure to the concentrations of 25, 50, 100

345

and 150 mg/L for 96-h.59 We speculated that this phenomenon maybe induced by high

346

levels of Bt protein; or the negative effects were not induced by Bt itself but by some

347

other factors, because they used the strains of B. thuringiensis instead of purified Bt

348

proteins in their study. Based on the known spectrum of Cry1C and Cry2A activity

349

against lepidopteran species33,34 and the phylogenetic distances between fish and

350

target species, D. rerio was not expected to be susceptible to Cry1C. Evidence

351

indicates that exposure to purified Cry1C or Cry2A protein does not harm the

352

following non-target organisms: water flea D. magna 9, green alga C. pyrenoidosa14,

353

mice (Rodentia: Muridae)60, honeybee Apis mellifera (Hymenoptera: Apidae)40,

354

springtail Folsomia candida (Collembola: Isotomidae)41, and fruit fly Drosophila

355

melanogaster (Diptera: Drosophilidae)61. The results of all aforementioned studies are

356

consistent with ours.

357

Ours is the first study to assess the effects of purified Bt proteins on the fitness of

358

zebrafish embryos. Almost none of the investigated developmental, biochemical or

359

molecular parameters of the zebrafish embryos were significantly altered by exposure

360

to Cry1C or Cry2A. The results demonstrated that Cry1C and Cry2A proteins have no

361

deleterious effect on the zebrafish embryos at doses tested.

362 17



363

ABBREVIATIONS USED

364

bcl2, B-cell lymphoma-2; Bt, Bacillus thuringiensis; CAT, catalase; CPF, Chlorpyrifos;

365

cox1, cyclo-oxygen-ase 1; gstp2, glutathione S-transferase pi 2; hpf, hours

366

post-fertilization; MDA, malondialdehyde; SOD, superoxide dismutase

367

368

AUTHOR INFORMATION

369

Corresponding Author

370

*X.-P. C.: Tel: +86-10-62815947; Fax: +86-10-62896114;

371

E-mail: [email protected]

372

Funding

373

This study was supported by the National GMO New Variety Breeding Program of the

374

People’s Republic of China (2016ZX08011-001).

375

376

Notes

377

The authors declare no competing financial interest.

378 379

REFERENCES

380

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381

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Cry1Ab protein from an herbivore reduces anti-oxidant enzyme activities in two spider

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immunotoxicity. Fish Shellfish Immunol. 2015, 43 (2), 405–414.

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57. Yu, K.; Li, G.; Feng, W.; Liu, L.; Zhang, J.; Wu, W.; Xu, L.; Yan, Y. Chlorpyrifos is

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estrogenic and alters embryonic hatching, cell proliferation and apoptosis in zebrafish. Chem.

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chlorpyrifos on zebrafish embryos. China Envir. Sci. 2016, 36 (3), 927–934 (In Chinese with

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English abstract).

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59. Grisolia, C. K.; Oliveira, R.; Domingues, I.; Oliveira-Filho, E. C.; Monerat, R. G.; Soares, A.

549

M. Genotoxic evaluation of different δ-endotoxins from Bacillus thuringiensis on zebrafish

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adults and development in early life stages. Mutat. Res. 2009, 672 (2), 119–123.

551

60. Cao, S. S.; He, X. Y.; Xu, W. T.; Ran, W. J.; Liang, L.; Luo, Y. B.; Yuan, Y. F.; Zhang, N.;

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Zhou, X.; Huang, K. L. Safety assessment of Cry1C protein from genetically modified rice

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according to the national standards of PR China for a new food resource. Regul. Toxicol.

554

Pharmacol. 2010, 58 (3), 474–481.

555 556

61. Haller, S.; Romeis J.; Meissle, M. Effects of purified or plant-produced Cry proteins on Drosophila melanogaster (Diptera: Drosophilidae) larvae. Sci. Rep. 2017, 7, 11172.

557 26


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558

Figure Captions

559

Figure 1. Survival and malformation rates when zebrafish embryos were exposed

560

to E3 medium alone (blank control) or to medium containing 0.1, 1, or 10 mg/L

561

Cry1C or Cry2A protein or 2 mg/L CPF (positive control) for the indicated time.

562

Values are the mean ± SE from three replicates. Data were analyzed with the

563

Kaplan-Meier procedure and log rank test. An asterisk indicates a significant

564

difference relative to the blank control (p < 0.05).

565 566

Figure 2. ELISA results for the concentration of Cry1C or Cry2A protein

567

detected in zebrafish larvae after exposure until 132 hpf to different

568

concentrations of the two Cry proteins. Values are the mean ± SE from three

569

replicates. Different letters above bars indicate significant differences among groups

570

(one-way ANOVA with Tukey’s HSD test; p < 0.05).

571 572

Figure 3. mRNA expression levels of genes related to oxidative stress or apoptosis

573

in zebrafish larvae after exposure to E3 medium alone (blank control) or to

574

medium containing 0.1, 1, or 10 mg/L Cry1C protein or 2 mg/L CPF (positive

575

control) until 132 hpf. The following genes were analyzed: sod2 (A), cat (B), cox1

576

(C), gstp2 (D), and bcl2 (E). Values are the mean ± SE from three replicates. Different

577

letters above bars indicate significant differences among groups (one-way ANOVA

578

with Tukey’s HSD test; p < 0.05).

579 27



580

Figure 4 mRNA expression levels of genes related to oxidative stress or apoptosis

581

in zebrafish larvae after exposure to E3 medium alone (blank control) or to

582

medium containing 0.1, 1, or 10 mg/L Cry2A protein or 2 mg/L CPF (positive

583

control) until 132 hpf. The following genes were analyzed: sod2 (A), cat (B), cox1

584

(C), gstp2 (D), and bcl2 (E). Values are the mean ± SE from three replicates. Different

585

letters above bars indicate significant differences among groups (one-way ANOVA

586

with Tukey’s HSD test; p < 0.05).

587 588

Figure 5. Activities of SOD (A) and CAT (B) and MDA content (C) in zebrafish

589

larvae after exposure to E3 medium alone (blank control) or to medium

590

containing 0.1, 1, or 10 mg/L Cry1C protein or 2 mg/L CPF (positive control)

591

until 132 hpf. Values are the mean ± SE from three replicates. An asterisk indicates a

592

significant difference from other groups (one-way ANOVA with Tukey’s HSD test; p

593

< 0.05).

594 595

Figure 6 Activities of SOD (A) and CAT (B) and MDA content (C) in zebrafish

596

larvae after exposure to E3 medium alone (blank control) or to medium

597

containing 0.1, 1, or 10 mg/L Cry2A protein or 2 mg/L CPF (positive control)

598

until 132 hpf. Values are the mean ± SE from three replicates. An asterisk indicates a

599

significant difference from other groups (one-way ANOVA with Tukey’s HSD test; p

600

< 0.05).

601 28


Page 28 of 36

Page 29 of 36


602

Table 1. Primer Sequences for Genes Tested in Zebrafish Embryos Exposed to

603

Cry Proteins or CPF.

primer sequence (5’–3’) gene

GenBank accession

forward

reverse

number

bcl2

CGCAACGCAGCTTTCTAAC

GCATCCCAACCTCCATTTT

AY695820.1

cat

CGGACATGGTTTGGGATTT

TGCCCTGATTAGTCTTGTAGTGG

NM_130912.2

cox1

AGCCCAGTCCGAATGTTGT

AATAAGCCTCCCAGTTCAAGTAA

AY028584.1

gstp2

ACAGGACTGGATGAAGGGTGA

GCTTTATGTATTTCTGGCGAAGA

NM_001020513.1

sod2

GGAGGCCATAAAGCGTGAC

CAGACATCTATCCCAAGCAGTG

NM_199976.1

β-actin

TGAATCCCAAAGCCAACAGA

GGAAGAGCGTAACCCTCATAGA

AF057040.1

604

bcl2, B-cell lymphoma-2; cat, catalase; cox1, cyclo-oxygen-ase 1; gstp2, glutathione

605

S-transferase pi 2; sod2, superoxide dismutase 2.

606

29



Page 30 of 36

607

Table 2. Determination of the Effects of Cry1C and Cry2A Proteins Dissolved in

608

E3 Medium on Chilo suppressalis Larvae

Cry content in E3

Cry content in

survival rate

body weight

treatment medium (µg/L)

a

a

b

a,c

diet (ng/g)

(%, n)

0.00 a

0.00 a

100.0 (40)

4.36 ± 0.15 a

5.24 ± 0.30 a

0.30 ± 0.01 a

100.0 (40)

3.16 ± 0.17 b

1 mg/L

120.93 ± 6.71 b

10.68 ± 0.17 b

97.5 (39)

1.54 ± 0.08 c

10 mg/L

1760.33 ± 96.54 c

255.81 ± 4.91 c

62.5 (25)*

0.10 ± 0.01 d

0.00 a

0.00 a

100.0 (40)

4.36 ± 0.15 a

0.1 mg/L

0.86 ± 0.32 a

0.34 ± 0.03 a

100.0 (40)

3.90 ± 0.13 a

1 mg/L

15.62 ± 0.93 b

6.11 ± 0.47 b

97.5 (39)

2.55 ± 0.14 b

10 mg/L

226.07 ± 2.95 c

23.79 ± 1.61 c

95.0 (38)

0.44 ± 0.05 c

Blank control 0.1 mg/L

(mg)

Cry1C

Blank control

Cry2A

609

a

610

10 larvae per treatment group).

611

b

612

c

613

ANOVA with Tukey’s HSD test; p < 0.05).

614

*Denotes a significant difference between a toxin treatment and the control.

Values represent the mean ± SE; four independent replicates were carried out (n = 4,

Chi-square test with Bonferroni correction (adjusted α = 0.0083).

Different letters within the same column indicate significant differences (one-way

615

30


Page 31 of 36


616

Table 3. Developmental Effects of Cry1C, Cry2A, or CPF Exposure on Zebrafish

617

Embryos hatching rate

body length

survival rate

malformation

a

treatment

(%)

Cry1C

Cry2A

b,c

(mm)

c, d

(%)

d,e

rate (%)

d,e

Blank control

98.25 ± 1.75

4.20 ± 0.04

96.67 ± 1.67

1.67 ± 2.89

CPF (2 mg/L)

96.58 ± 1.71

3.89 ± 0.05*

61.67 ± 1.67*

100 ± 0.00*

Cry1C (0.1 mg/L)

96.58 ± 1.71

4.12 ± 0.03

98.33 ± 1.67

1.67 ± 2.89

Cry1C (1 mg/L)

96.39 ± 1.81

4.13 ± 0.03

95.00 ± 2.89

0.00 ± 0.00

Cry1C (10 mg/L)

100.00 ± 0.00

4.13 ± 0.03

96.67 ± 1.67

1.73 ± 3.00

Blank control

96.97 ± 3.03

4.19 ± 0.06

94.63 ± 2.69

1.50 ± 1.50

CPF (2 mg/L)

98.33 ± 1.67

3.90 ± 0.07*

63.90 ± 4.55*

100 ± 0.00*

Cry2A (0.1 mg/L)

96.66 ± 1.68

4.21 ± 0.04

95.00 ± 2.89

3.30 ± 1.66

Cry2A (1 mg/L)

100.00 ± 0.00

4.18 ± 0.03

96.73 ± 1.63

3.40 ± 1.70

Cry2A (10 mg/L)

100.00 ± 0.00

4.21 ± 0.04

91.83 ± 1.59

3.50 ± 1.76

618

a

619

protein at the indicated concentrations, or to CPF as a positive control until 132 hpf;

620

three independent replicates were carried out (n = 3, 20 embryos per treatment group).

621

b

622

c

623

d

624

e

625

= 0.0125).

626

*Denotes a significant difference between a toxin treatment and the control.

Embryos were exposed to E3 medium alone (blank control), to Cry1C or Cry2A

Parameters were detected after exposure for 84 hpf.

Differences were analyzed by one-way ANOVA with Tukey’s HSD test. Parameters were detected after exposure until 132 hpf.

Differences were analyzed by Chi-square test with Bonferroni correction (adjusted α

627 31



628

Figure Graphics

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Figure 1

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Figure 2

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Figure 3

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639

Figure 4

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Figure 5

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Figure 6

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653

GRAPHIC FOR TABLE OF CONTENT

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Safety Assessment of Bacillus thuringiensis Insecticidal Proteins

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