Effects of Bt cabbage pollen on the honeybee Apis mellifera L

Abstract

Honeybees may be exposed to insecticidal proteins from transgenic plants via pollen during their foraging activity. Assessing effects of such exposures on honeybees is an essential part of the risk assessment process for transgenic Bacillus thuringiensis (Bt) cabbage. Feeding trials were conducted in a laboratory setting to test for possible effects of Cry1Ba3 cabbage pollen on Italian-derived honeybees Apis mellifera L. Newly emerged A. mellifera were fed transgenic pollen, activated Cry1Ba3 toxin, pure sugar syrup (60% w/v sucrose solution), and non-transgenic cabbage pollen, respectively. Then the effects on survival, pollen consumption, weight, detoxification enzyme activity and midgut enzyme activity of A. mellifera were monitored. The results showed that there were no significant differences in survival, pollen consumption, weight, detoxification enzyme activity among all treatments. No significant differences in the activities of total proteolytic enzyme, active alkaline trypsin-like enzyme and weak alkaline trypsin-like enzyme were observed among all treatments. These results indicate that the side-effects of the Cry1Ba3 cabbage pollen on A. mellifera L. are unlikely.

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SCIentIFIC RepoRts | (2018) 8:482 | DOI:10.1038/s41598-017-18883-w
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Eects of Bt cabbage pollen on the
honeybee Apis mellifera L
Dengxia Yi1, Zhiyuan Fang2 & Limei Yang2
Honeybees may be exposed to insecticidal proteins from transgenic plants via pollen during their
foraging activity. Assessing eects of such exposures on honeybees is an essential part of the risk
assessment process for transgenic Bacillus thuringiensis (Bt) cabbage. Feeding trials were conducted in
a laboratory setting to test for possible eects of Cry1Ba3 cabbage pollen on Italian-derived honeybees
Apis mellifera L. Newly emerged A. mellifera were fed transgenic pollen, activated Cry1Ba3 toxin, pure
sugar syrup (60% w/v sucrose solution), and non-transgenic cabbage pollen, respectively. Then the
eects on survival, pollen consumption, weight, detoxication enzyme activity and midgut enzyme
activity of A. mellifera were monitored. The results showed that there were no signicant dierences
in survival, pollen consumption, weight, detoxication enzyme activity among all treatments. No
signicant dierences in the activities of total proteolytic enzyme, active alkaline trypsin-like enzyme
and weak alkaline trypsin-like enzyme were observed among all treatments. These results indicate that
the side-eects of the Cry1Ba3 cabbage pollen on A. mellifera L. are unlikely.
Genetic engineering has been successfully applied in many crop breeding programs, and 2 billion hectares of
transgenic crops were successfully cultivated globally from 1996 to 20151. e planting of transgenic crops has
increased resistance by the target pests, but reduced the use of chemical pesticides, and produced great economic
and social benets1,2. However, the worldwide planting of transgenic crops has triggered concerns about their
potential eects on non-target organisms3–5, such as honeybees. Honeybees are economically valuable pollinators
that are essential for seed production of many crops and wild plants. ey can also maintain ecological balance via
pollination. Honeybees may be exposed to insecticidal proteins in the pollen from transgenic plants, therefore,
they are considered as an important non-target organism in the biosafety assessment of transgenic crops3,6.
Multiple studies have been conducted to evaluate the eects of transgenic products on honeybees6–13. Adult
Apis mellifera L. fed on transgenic corn pollen (containing cry1Ab) mixed thoroughly into sugar syrup showed
no signicant dierences in survival and hypopharyngeal gland growth compared with controls aer 10 days14.
No signicant dierences were detected in the pollen consumption and hypopharyngeal gland weight of A. mel-
lifera and Apis cerana cerana worker honeybees fed on sugar syrup containing the Cry1Ah toxin compared with
the control15, and transgenic Cry1Ah maize pollen did not aect the midgut communities in larvae and adult
honeybees16. In addition to the growth and survival rate of honeybees, pupal dry weight17, longevity and food
consumption rate18–20, mortality21,22, ight activity23, foraging activity and learning performance10,20, cap rate and
emergence rate24, as well as feeding behaviour25 have also been studied, and overall these results indicate that Bt
toxins have no negative eect on honeybees.
Midgut and detoxication enzymes are important parameters that should be evaluated as part of the risk
assessment necessary for the commercialization of Bacillus thuringiensis (Bt) transgenic crops11,26. Midgut
enzymes play an important role in the digestion process of pollen ingested by honeybees27. e total midgut
proteolytic enzyme activity is directly related to the ability to digest protein-rich pollen and may be used to
assess digestion28. Furthermore, midgut protein digestion is associated with the development of hypopharyngeal
gland and the production of extractable proteins occurs in the hypopharyngeal gland when honeybees are fed on
pollen28. Sagili et al.28 reported that honeybees fed on pollen containing 1% soybean trypsin inhibitor had signif-
icantly reduced midgut proteolytic enzyme activity. Detoxication enzymes, such as α-naphthylacetate esterase,
glutathione-S-transferase, and acetylcholinesterase, catalyze metabolic reactions that convert foreign compounds
into forms that can be excreted from the body26.
1Institute of Animal Science, Chinese Academy of Agricultural Sciences, Beijing, 100193, China. 2Key Laboratory
of Biology and Improvement of Horticultural Crops, Ministry of Agriculture, Institute of Vegetables and Flowers,
Chinese Academy of Agricultural Sciences, Beijing, 100081, China. Correspondence and requests for materials
should be addressed to D.Y. (email: yidengxia@163.com) or L.Y. (email: yanglimeicaas@163.com)
Received: 24 April 2017
Accepted: 19 December 2017
Published: xx xx xxxx
OPEN
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The Bt cry1Ba3 gene was cloned by the Institute of Plant Protection, Chinese Academy of Agriculture
Sciences29. We previously incorporated a synthetic cry1Ba3 gene into the genome of an elite inbred cabbage line
A21–3 via Agrobacterium tumefaciens-mediated transformation method to produce fertile transgenic plants29.
Insect bioassays indicated that expressing cry1Ba3 in transgenic cabbage plants eectively controlled both sus-
ceptible and Cry1Ac-resistant Plutella xylostella larvae in the laboratory29. A healthy honeybee hive relies on
landscapes with ample and nutritious sources of pollen yielding owers. Cabbage’s owers are bright, yellow,
fragrant and attractive to honeybees. It was reported that pollen from Brassicaceae plants, including cabbages,
made up about 4.89%-12.62% of all pollen collected by honeybees during the blooming period30. Honeybees
could be easily exposed to insecticidal proteins from Bt cabbage owers during foraging. It is important to assess
the non-target eects of transgenic cabbage pollen on honeybees before its commercial release. e objective of
this study was, therefore, to examine the eects of Cry1Ba3 cabbage pollen on the survival, pollen consumption,
weight, and enzyme activities of worker honeybees (A. mellifera).
Results
Concentration of Cry1Ba3 protein in Bt cabbage pollen. e content of Cry1Ba3 protein in trans-
genic cabbage pollen was 778.5 ± 16.22 ng/g (mean ± SE). is pollen was used in the following experiments.
Survival, pollen consumption and body weight. e survival rate of A. mellifera did not signicantly
dier among the honeybees that were fed on Bt-C1, Bt-C2, non-Bt, and sugar syrup at any time point during
the 21 days of the experiment (Fig.1; Table1). e average survival rate on day 21 for the honeybees exposed to
Bt-C1, Bt-C2, non-Bt and sugar syrup was 68.7%, 64.6%, 66.3% and 66.0%, respectively and there were no sig-
nicant dierences among all treatments (F = 0.66, df = 23, P = 0.59; Fig.1). Moreover, no signicant dierences
were found in the pollen consumption of A. Mellifera fed on Bt-C1 and Bt-C2 compared with the control groups
at any time point (Table2). e body weight of A. Mellifera also did not dier signicantly among Bt-C1, Bt-C2,
non-Bt pollen and sugar syrup on days 7 (F = 1.14, df = 23, P = 0.36; Table3), 14 (F = 2.05, df = 23, P = 0.14;
Table3) and 21 (F = 1.93, df = 23, P = 0.16; Table3).
Assessment of detoxification enzyme activity. After 7 days of feeding, the activities of
α-naphthylacetate esterase (F = 0.63, df = 11, P = 0.62), glutathione-S-transferase (F = 0.70, df = 11, P = 0.58),
and acetylcholinesterase (F = 0.13, df = 11, P = 0.94) in A. Mellifera fed on Bt-C1 and Bt-C2 were not signicantly
dierent from the control groups fed on non-Bt pollen or sugar syrup (Table4).
Assessment of midgut enzyme activity. e values of midgut enzyme activity in honeybees fed on dif-
ferent foods are shown in Fig.2. No signicant dierences in total proteolytic enzyme activity (F = 0.17, df = 11,
P = 0.91; Fig.2), active alkaline trypsin-like enzyme activity (F = 2.26, df = 11, P = 0.16; Fig.2) and weak alkaline
trypsin-like enzyme activity (F = 2.13, df = 11, P = 0.17; Fig.2) were observed among all treatments, respectively.
However, honeybees that were fed on Bt-C1 or Bt-C2 showed slightly lower values of chymotrypsin-like enzyme
activity (F = 3.79, df = 11, P = 0.059; Fig.2). But considering that the sample size is small (n = 30), the lack of
statistical signicance is marginal.
Discussion
In this study, the eects of Cry1Ba3 cabbage pollen on survival, pollen consumption, weight, detoxication
enzyme activity and midgut enzyme activity of A. mellifera were evaluated. e results suggest that transgenic
Bt cabbage pollen carries no risk for A. Mellifera and are consistent with previous reports6–13. Although a slight
decrease in the value of chymotrypsin-like enzyme activity was observed, the potential side eects of Cry1Ba3
cabbage pollen on the honeybee A. mellifera were limited (F = 3.79, df = 11, P = 0.059; Fig.2). e midgut enzyme
activity (total proteolytic enzyme, active alkaline trypsin-like enzyme weak alkaline trypsin-like enzyme, and
chymotrypsin-like enzyme), which are directly related to digestive capacity of protein-rich pollen, could be sensi-
tive indicator for assessing the development of honeybee hypopharyngeal gland28. A signicantly decrease in the
value of chymotrypsin-like enzyme activity may imply that the development of honeybee hypopharyngeal gland
were inuenced when fed on transgenic pollen.
Figure 1. Survival of A. Mellifera fed with Bt-C1, Bt-C2, non-Bt pollen and pure sugar syrup for 21 days. e
percentage of the initial number of honeybees surviving at each day aer the start of treatment is shown.
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Days
Survival rate (mean ± SE)
Fdf PBt-C1 Bt-C2 non-Bt sugar syrup
1 1.000 ± 0.000 1.000 ± 0.000 1.000 ± 0.000 0.997 ± 0.001 1 23 0.41
2 0.990 ± 0.002 1.000 ± 0.000 1.000 ± 0.000 0.997 ± 0.001 2.86 23 0.06
3 0.990 ± 0.002 0.990 ± 0.003 0.980 ± 0.004 0.983 ± 0.003 0.47 23 0.70
4 0.967 ± 0.003 0.963 ± 0.003 0.963 ± 0.003 0.973 ± 0.002 0.65 23 0.60
5 0.933 ± 0.003 0.923 ± 0.003 0.917 ± 0.004 0.927 ± 0.002 0.88 23 0.47
6 0.917 ± 0.003 0.903 ± 0.004 0.887 ± 0.002 0.893 ± 0.004 2.79 23 0.07
7 0.870 ± 0.003 0.880 ± 0.003 0.877 ± 0.003 0.887 ± 0.003 0.72 23 0.50
8 0.843 ± 0.004 0.863 ± 0.005 0.870 ± 0.003 0.847 ± 0.003 1.74 23 0.19
9 0.840 ± 0.003 0.820 ± 0.004 0.837 ± 0.004 0.813 ± 0.003 2.24 23 0.11
10 0.827 ± 0.003 0.807 ± 0.003 0.817 ± 0.004 0.813 ± 0.003 1.11 23 0.37
11 0.790 ± 0.003 0.803 ± 0.004 0.800 ± 0.003 0.807 ± 0.002 0.88 23 0.47
12 0.763 ± 0.006 0.803 ± 0.004 0.790 ± 0.002 0.777 ± 0.004 2.96 23 0.06
13 0.753 ± 0.003 0.763 ± 0.005 0.767 ± 0.002 0.770 ± 0.003 0.68 23 0.58
14 0.750 ± 0.003 0.753 ± 0.007 0.763 ± 0.001 0.737 ± 0.006 0.90 23 0.46
15 0.737 ± 0.003 0.753 ± 0.007 0.750 ± 0.003 0.727 ± 0.005 1.17 23 0.35
16 0.720 ± 0.002 0.743 ± 0.007 0.730 ± 0.003 0.713 ± 0.003 1.43 23 0.26
17 0.713 ± 0.003 0.703 ± 0.003 0.727 ± 0.003 0.700 ± 0.003 2.46 23 0.09
18 0.703 ± 0.004 0.677 ± 0.005 0.717 ± 0.004 0.697 ± 0.003 2.84 23 0.06
19 0.703 ± 0.004 0.663 ± 0.006 0.703 ± 0.006 0.687 ± 0.005 2.16 23 0.12
20 0.697 ± 0.006 0.657 ± 0.007 0.673 ± 0.009 0.663 ± 0.006 1.00 23 0.41
21 0.687 ± 0.008 0.647 ± 0.0009 0.663 ± 0.010 0.660 ± 0.006 0.66 23 0.59
Table 1. Mean survival rate of A. Mellifera subjected to chronic exposure to Bt-C1, Bt-C2, non-Bt pollen and
pure sugar syrup during a 21-day oral exposure.
Days
Pollen consumption (mg) per bee (mean ± SE)
Fdf PBt-C1 Bt-C2 non-Bt sugar syrup
1–3 9.81 ± 0.35 9.22 ± 0.34 9.75 ± 0.32 9.18 ± 0.33 0.17 23 0.91
4–6 9.40 ± 0.37 9.65 ± 0.37 9.53 ± 0.38 8.88 ± 0.28 0.16 23 0.92
7–9 8.00 ± 0.31 7.90 ± 0.36 7.55 ± 0.35 7.41 ± 0.34 0.11 23 0.95
10–12 6.93 ± 0.30 7.91 ± 0.31 7.02 ± 0.28 6.59 ± 0.35 0.56 23 0.65
13–15 5.57 ± 0.41 4.49 ± 0.25 4.92 ± 0.30 5.26 ± 0.29 0.35 23 0.79
16–18 4.45 ± 0.25 3.25 ± 0.20 3.50 ± 0.33 3.41 ± 0.18 0.81 23 0.50
19–21 1.85 ± 0.15 1.85 ± 0.22 1.73 ± 0.20 1.48 ± 0.15 0.15 23 0.93
Sum 46.01 ± 0.49 44.27 ± 0.74 44.00 ± 0.58 42.20 ± 0.88 0.85 23 0.48
Table 2. Mean three-day cumulative quantify of food consumed (±SE) by A. Mellifera subjected to chronic
exposure to Bt-C1, Bt-C2, non-Bt pollen and pure sugar syrup during a 21-day oral exposure.
Days
Body weight (mg, mean ± SE)
Fdf PBt-C1 Bt-C2 non-Bt sugar syrup
7 94.59 ± 0.99 92.07 ± 1.67 97.12 ± 1.30 88.53 ± 1.54 1.14 23 0.36
14 128.38 ± 1.82 138.07 ± 1.04 135.27 ± 0.95 132.52 ± 0.52 2.05 23 0.14
21 117.59 ± 1.16 107.43 ± 1.25 112.93 ± 0.87 111.95 ± 1.53 1.93 23 0.16
Table 3. Body weight of A. Mellifera fed with Bt-C1, Bt-C2, non-Bt pollen and pure sugar syrup for 21 days.
Detoxication enzyme
Enzyme activity (mmol·L−1·mg−1·min−1)
Fdf PBt-C1 Bt-C2 non-Bt sugar syrup
Acetylcholinesterase 0.055 ± 0.001 0.052 ± 0.002 0.052 ± 0.003 0.053 ± 0.003 0.13 11 0.94
Glutathione-S-transferase 0.015 ± 0.000 0.016 ± 0.001 0.014 ± 0.001 0.013 ± 0.001 0.70 11 0.58
α-naphthylacetate esterase 0.034 ± 0.002 0.040 ± 0.003 0.037 ± 0.003 0.035 ± 0.001 0.63 11 0.62
Table 4. e activities of three detoxication enzymes in A. Mellifera fed with Bt-C1, Bt-C2, non-Bt pollen and
pure sugar syrup for 7 days.
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In the present study, A. mellifera exposed to Bt cabbage pollen or Bt toxin showed slightly lower values of
chymotrypsin-like enzyme activity, although the corresponding activity was not signicantly lower than the con-
trol groups (F = 3.79, df = 11, P = 0.059; Fig.2). In assessing of chymotrypsin-like enzyme activity, ten in total
honeybees from each treatment were used in each replicate and three replicates were undertaken. e sample
size was 30 (n = 30). In the experimental protocols reported by Han et al.11, laboratory studies to measure mid-
gut enzyme activity tested 120 honeybees. eir results showed no side eects of CCRI41 cotton pollen on total
midgut proteolytic enzyme activity of honeybees when they were subjected to chronic exposure to the transgenic
CCRI41 cotton pollen. Our ndings were consistent with the report, thus our results can be considered as valid
and reliable. Taking the previous protocols11 into consideration, the sample size used in our study was relatively
small. To ensure the precision, a large sample size would be required in further studies.
In this study, both Bt cabbage pollen and Bt toxin were used. e higher concentration of Cry1Ba3 toxin
(Bt-C2) used is unlikely to be encountered by honeybees in the eld and hence represents a worst case scenario.
e lower concentration of Cry1Ba3 toxin (Bt-C1) represents a value closer to that in the eld if it is expressed in
the pollen. It must be noted that our study was conducted in a laboratory setting, and the results are preliminary.
Some other parameters including foraging activity, learning performance, the time of rst ight and the duration
of ight activity should be investigated in future studies. In addition to laboratory feeding of the honeybees, eld
studies are also important for understanding the eects of transgenic plants on non-target organisms31,32. Future
research should be conducted on the joint eects of transgenic Bt cabbage on honeybees in the eld.
Materials and Methods
Cabbage pollen. e transgenic cabbage inbred line A21–3 containing the synthetic Bt cry1Ba3 gene was
produced by Yi et al.29. Non-transgenic A21–3 was used as the control. Bt and control cabbages were planted in
a greenhouse belonging to the Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences.
Routine management was carried out and pesticide applications were avoided. Bt and control cabbage pollen were
collected using a multi-point eld sampling method at the stage of full bloom. Samples were stored at −80 °C in a
refrigerator until they were used for experiments.
Quantitative detection of Cry1Ba3 protein in Bt cabbage pollen. Quantitative determination of
Cry1Ba3 protein in transgenic cabbage pollen was performed using an enzyme-liked immunosorbent assay (ELISA)
kit provided by the State Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant Protection,
Chinese Academy of Agricultural Sciences. ELISA was carried out according to a previously published method29,33.
Cry1Ba3 was puried from transgenic pollen using the following procedure. e pollen was homogenized in 2 ml
extraction phosphate buered saline with tween-20 (PBST; 8.0 g NaCl, 2.7 g Na2HPO4·12H2O, 0.4 g NaH2PO4·2H2O,
dissolved in 1000 ml water, pH = 7.4). en the sample was washed with 2 ml PBST and kept in a 10 ml centrifuge
tube at 4 °C overnight to extract the insecticidal protein. e tube was then centrifuged at 5000 rpm for 20 min. e
insecticidal protein content in the supernatant was quantied using the ELISA kit as described by Yi et al.29.
Honyebees and treatments. Worker honeybees (A. mellifera) were provided by the Institute of Apicultural
Research, Chinese Academy of Agricultural Sciences. Brood frames were placed in an incubator (34 ± 1 °C,
60 ± 5% relative humidity, darkness) aer the cells were capped at approximately 9 d. Newly emerged honeybees
(less than 12 h old) were assigned randomly to wooden cages (9 cm × 9 cm × 10 cm) with mesh on two sides. Each
cage was tted with a gravity feeder. Four dierent treatments were applied with six replications per treatment and
50 honeybees per cage. e rst treatment was transgenic pollen, which was mixed thoroughly into sugar syrup
(60% w/v sucrose solution) at a concentrations of 13 mg/mL (Bt-C1). Activated Cry1Ba3 toxin, provided by the
Figure 2. e activities of total proteolytic enzyme (n = 30), active alkaline trypsin-like enzyme (n = 30), weak
alkaline trypsin-like enzyme (n = 30) and chymotrypsin-like enzyme (n = 30) in A. Mellifera fed with Bt-C1, Bt-
C2, non-Bt pollen and pure sugar syrup for 7 days.
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Institute of Plant Protection, Chinese Academy of Agricultural Sciences, was mixed thoroughly into sugar syrup
(60% w/v sucrose solution) at a concentrations of 10 µg/mL (Bt-C2). is high-concentration Cry1Ba3 toxin
treatment represents the worst case scenario. Pure sugar syrup (60% w/v sucrose solution) and non-transgenic
cabbage pollen were used as controls.
Pollen consumption. For each cage, 2 g of corresponding food was supplied. is food was weighed and
replaced with fresh food every 3 days for 21 days. e cages were kept in an incubator (34 ± 1 °C, 60 ± 5% relative
humidity, darkness).
Survival and weight. e honeybees were exposed to the dierent treatments described above for 21 days34.
e number of surviving honeybees in each cage was recorded daily at 5:00 pm. Honeybees were considered dead
when they remained completely immobile and the dead honeybees were removed from cages every day20. e
body weight of ten randomly selected honeybees for each treatment was recorded on days 7, 14 and 2134.
Measurement of detoxication enzyme activity. In each replicate, ten 7-day-old honeybees in total taken
from each treatment were used for the measurement of detoxication enzyme activity. e honeybees were placed
in a pre-cooled glass homogenizer and then 0.1 mol/L phosphate buer (containing 0.1% Triton X-100, pH 8.0) was
added (1:10, w/v). e mixtures were homogenized in an ice bath and then centrifuged at 10,000 × g for 30 min at
4 °C. e supernatant was analyzed to estimate detoxication enzyme activity. ree replicates were undertaken
per treatment. e activities of glutathione-S-transferase, acetylcholinesterase and α-naphthyl acetate esterase were
measured using the procedures previously described by Booth et al.35, Ellman36 and van Asperern37, respectively.
Measurement of midgut enzyme activity. In each replicate, ten 7-day-old honeybees in total were ran-
domly chosen from each treatment. e honeybees were dissected in an ice bath and ushed using pre-cooled
NaCl (0.15 mol/L). e midguts were isolated immediately, placed in a glass homogenizer, and homogenized in an
ice bath aer adding 1 mL of 0.15 mol/L NaCl. e extract was then centrifuged at 15,000 × g for 15 min at 4 °C. e
supernatant was analyzed to estimate the midgut enzyme activity. ree replicates were undertaken per treatment.
Total proteolytic enzyme activity in the midgut was measured as previously described38. Azocasein was used as
the substrate for the proteolysis reaction and the absorption was measured at 440 nm using an 8452 A type ultravi-
olet spectrophotometer. e measurement of tryptase activity included assessing active alkaline trypsin-like and
weak alkaline trypsin-like enzymes. Specic substrates were used to distinguish between distinct protease classes:
Nα-Benzoyl-DL-arginine 4-nitroanilide hydrochloride was used to measure the active alkaline trypsin-like activ-
it y, Nα-p-tosyl-L-Arg methyl ester was used to measure the weak alkaline trypsin-like activity, and Trichlorpyr
butoxyethyl ester was used to measure the chymotrypsin-like enzyme activity. e absorption was measured at
406 nm, 248 nm, and 256 nm, respectively.
Statistical analyses. Survival was tested using the Kaplan-Meier estimator. Signicant dierences among all
treatments for pollen consumption, weight and enzyme activity were evaluated using one-way analysis of variance
(ANOVA). If signicant dierences were found (P < 0.05), multiple comparison procedures were performed with
Duncans multiple-range test.
Data availability. The datasets analysed during the current study are available in the [Supplementary
Dataset] repository.
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Acknowledgements
We thank Dr. Pingli Dai (Apicultural Research, Chinese Academy of Agricultural Sciences) for providing the
honeybees. We are grateful to Prof. Jie Zhang (Institute of Plant Protection, Chinese Academy of Agricultural
Sciences) for sending us the Bt gene and ELISA kit. is work was supported by the National High Technology
Research and Development Program of China (2012AA100101), the Key Projects in the National Science &
Technology Pillar Program during the Twelh Five-Year Plan Period (2012BAD02B01), the Agricultural Science
and Technology Innovation Program (ASTIP-IAS10, CAAS-ASTIP-2013-IVFCAAS) of Chinese Academy
of Agricultural Sciences, the Modern Agricultural Industry Technology Research System (CARS-34, CARS-
25-B-01), and the Fundamental Research Funds for Central Non-prot Scientic Institution (2016ywf-yb-10).
Author Contributions
L.Y. and Z.F. designed the study and revised the manuscript; D.Y. performed the experiments, analyzed the data
and wrote the manuscript. All authors have read and approved the nal manuscript.
Additional Information
Supplementary information accompanies this paper at https://doi.org/10.1038/s41598-017-18883-w.
Competing Interests: e authors declare that they have no competing interests.
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