Consumption of Bt Maize Pollen Containing Cry1Ie Does Not Negatively Affect Propylea japonica (Thunberg) (Coleoptera: Coccinellidae)

Propylea japonica (Thunberg) (Coleoptera: Coccinellidae) are prevalent predators and pollen feeders in East Asian maize fields. They are therefore indirectly (via prey) and directly (via pollen) exposed to Cry proteins within Bt-transgenic maize fields. The effects of Cry1Ie-producing transgenic maize pollen on the fitness of P. japonica was assessed using two dietary-exposure experiments in the laboratory. In the first experiment, survival, larval developmental time, adult fresh weight, and fecundity did not differ between ladybirds consuming Bt or non-Bt maize pollen. In the second experiment, none of the tested lethal and sublethal parameters of P. japonica were negatively affected when fed a rapeseed pollen-based diet containing Cry1Ie protein at 200 μg/g dry weight of diet. In contrast, the larval developmental time, adult fresh weight, and fecundity of P. japonica were significantly adversely affected when fed diet containing the positive control compound E-64. In both experiments, the bioactivity of the Cry1Ie protein in the food sources was confirmed by bioassays with a Cry1Ie-sensitive lepidopteran species. These results indicated that P. japonica are not affected by the consumption of Cry1Ie-expressing maize pollen and are not sensitive to the Cry1Ie protein, suggesting that the growing of Bt maize expressing Cry1Ie protein will pose a negligible risk to P. japonica.


Introduction
Maize (Zea mays L.) is one of the most important cereal crops in the world and plays a decisive role in food and feed production. In China, it is mainly grown for food, feed, and ethanol production [1]. However, the yield of maize can be reduced heavily by insect pests. The most important maize pest in China is the Asia maize borer, Ostrinia furnacalis Guenée (Lepidoptera: Crambidae). The Asia maize borer was estimated to cause approximately 10% of yield loss each year and more than 30% in years of heavy infestations [2]. Table Parameters When fed with maize pollen, over 73% of the P. japonica larvae developed to adults, and the pupation rates and eclosion rates did not significantly differ between the Bt and the non-Bt maize pollen treatments (both P > 0.05) ( Table 1). Larval development time (days to pupa) and adult fresh weight were not significantly affected, either, by feeding on Bt pollen (development time: U = 2008.5, P = 0.63; t = 0.38 df = 61; adult weight: P = 0.71 for females and t = −1.09, df = 49, P = 0.28 for males). Similarly, the fecundity of P. japonica females was not significantly affected by feeding on Bt pollen (t = −0.93, df = 43, P = 0.36) ( Table 1).

Bioactivity of Cry1Ie Protein in Maize Pollen
The mean (±SE) weight of the Cry1Ie-sensitive Chilo suppressalis Walker (Lepidoptera: Crambidae) larvae was significantly reduced when fed on an artificial diet containing fresh Bt maize pollen for seven days (0.22 ± 0.01 mg) compared to larvae fed on fresh control maize pollen (4.14 ± 0.29 mg) (t = 13.6, df = 57, P < 0.001). C. suppressalis larval weight was slightly, but significantly, higher when fed Bt maize pollen that had been exposed to P. japonica for two days (0.26 ± 0.01 mg) as compared to freshly prepared Bt maize pollen (0.22 ± 0.01 mg) (t = −2.12, df = 57, P = 0.038). Table Parameters Pair-wise comparisons revealed that the treatment containing Cry1Ie protein did not differ significantly from the untreated (negative) control for any of the P. japonica test parameters including pupation rate, ecolosion rate, development time, adult fresh weight (male/female), and total fecundity (total number of eggs laid per female) (all P > 0.05) ( Table 2). In contrast, the larval development time of P. japonica was significantly prolonged (U = 35.0, P < 0.001), and the mean weight of the freshly emerged adults was significantly reduced when feeding on diet containing E-64 (Dunnett test; P < 0.001 for females, and P = 0.002 for males). No significant difference was detected for pupation rate, eclosion rate, and the survival rate of P. japonica between the untreated control treatment and the E-64 treatment (all P > 0.05) ( Table 2). Each toxin treatment was compared to the control. An asterisk denotes a significant difference between a toxin treatment and the control; a χ 2 test with Bonferroni correction (adjusted α = 0.025); b Mann-Whitney U-test with Bonferroni correction (adjusted α = 0.025); c Dunnett test. * An asterisk denotes a significant difference between a toxin treatment and the control.

Bioactivity of Cry1Ie Protein in Rapeseed Pollen
Sensitive-insect bioassays indicated that the mean (±SE) weight of C. suppressalis larvae was significantly reduced when fed an artificial diet containing Cry1Ie (0.26 ± 0.02 mg) for seven days compared to those fed untreated control diet (0.50 ± 0.06 mg) (t = 4.02, df = 52, P < 0.001). No statistical differences was detected for the mean weight of C. suppressalis larvae when fed a Cry1Ie-containing diet that had been freshly prepared (0.26 ± 0.02 mg) compared to a diet that had been exposed to P. japonica for two days (0.28 ± 0.02 mg) (t = −0.58, df = 55, P = 0.57).

Discussion
The cry1Ie gene has been identified from Bacillus thuringiensis isolate Btc007 and is a relatively new gene used for plant transformation [23]. It was found that a transgenic maize line expressing cry1Ie was highly resistant against the stem borer O. furnacalis [24]. In addition, it appears that the Cry1Ie protein has no cross-resistance with other Lepidoptera-active insecticidal proteins such as Cry1Ab, Cry1Ac, Cry1Ah, or Cry1F [25][26][27], making it a suitable candidate gene for developing stacked events for improved pest control. However, our knowledge regarding the potential effects of the Cry1Ie protein on non-target beneficial arthropods is still limited.
Our bioassays revealed no adverse effects of Bt maize pollen containing Cry1Ie on the fitness of P. japonica. In our feeding experiment, the ladybirds were continually fed on Bt maize pollen for more than four weeks, while the pollen shedding period of maize normally lasts for 5-8 days with a maximum of 14 days [28,29]. In addition, our previous study had confirmed that P. japonica ingested much larger amounts of maize pollen under laboratory, confined conditions when compared to the field situation [30]. Consequently, the ladybirds in our laboratory bioassays were exposed to Cry1Ie protein at much higher levels than in the field. In addition, the bioactivity of Cry1Ie in the pollen samples used in the experiments was confirmed by sensitive insect bioassays using larvae of C. suppressalis. This demonstrates that P. japonica was exposed to a constant and elevated level of active Cry1Ie protein.
To further confirm that P. japonica is not sensitive to Cry1Ie, a second experiment was conducted where the ladybirds were directly fed purified Cry1Ie protein at a very high dose of 200 µg/g dry weight of diet using a validated test system [31]. No detrimental effects were detected on the tested lethal and sublethal life table parameters. The positive control (E-64) treatment, in contrast, caused a significant prolongation of the larval development time, a lower adult fresh weight, and a decrease in fecundity. The results demonstrate that P. japonica indeed ingested Cry1Ie protein and that the experimental system used in the current study was able to detect adverse effects, if present. Furthermore, the bioactivity of Cry1Ie protein was confirmed by sensitive insect bioassays with the same batch of Cry protein, suggesting that the test insects were exposed to bioactive Cry1Ie protein during the duration of the bioassay. In addition, the Cry1Ie protein concentration in this experiment was approximately 40 times of the concentration in the Cry1Ie-transgenic maize pollen (5 µg/g fresh weight) [32]. Therefore, we can conclude that P. japonica is not sensitive to Cry1Ie at a level much higher than they may encounter in Bt maize fields.
Because of the ecological importance as a natural enemy and its availability and amenability for laboratory studies, P. japonica has been selected as representative species to support the risk assessment of IRGE crops [33]. Our previous study indicated that P. japonica larvae are not sensitive to Cry1Ab, Cry1Ac, and Cry1F proteins [31], which are produced in different Bt-transgenic crops, including maize, cotton, and rice. In addition, feeding experiments with Bt rice pollen showed that the fitness of P. japonica was not adversely affected by consumption of pollen containing Cry1Ab, Cry1C, or Cry2A [34,35]. Similarly, ingestion of Bt maize pollen containing Cry1Ab/2Aj or Cry1Ac had no detrimental effect on P. japonica larvae [30]. Although a previous study reported that consumption of Cry1Ah-containing maize pollen affected the activity of some gut enzymes of P. japonica [36], no effect was detected on the growth or development of P. japonica larvae when fed Cry1Ah-containing maize pollen [37]. Similarly, a tritrophic studies showed that the development of P. japonica was not affected when fed with Nilaparvata lugens (Stål) (Hemiptera: Delphacidae) that had been reared on Cry1Ab-contained Bt rice [38]. The current data complement our knowledge on the toxicity of Cry proteins to P. japonica. Overall, the available results demonstrate that this important predatory ladybird species is not sensitive to Cry proteins that are widely used for plant transformation to control lepidopteran pests or might be used in the future.
To our knowledge, the current study is the first to assess the potential effects of Bt maize producing Cry1Ie protein on a predatory insect in the laboratory. So far, the non-target insect assessment concerning Cry1Ie has only focused on the survival of Chinese honey bees, Aphis cerana cerana (Hymenoptera: Apidae) [39], the diversity of the midgut bacteria of the worker bees, Apis mellifera ligustica (Hymenoptera: Apidae) [40], and the survival, pollen consumption, and olfactory learning of young adult honey bees, A. mellifera [41]. None of those studies has revealed any adverse effects of Cry1Ie consumption. In addition, two studies reported that Cry1Ie-expressing maize had no significant effects on the arthropod community in maize fields [42,43].
In summary, the present study shows that the consumption of Bt maize pollen expressing Cry1Ie does not negatively affect the fitness of P. japonica and that the ladybirds are not sensitive to Cry1Ie at 200 µg/g diet that is significantly higher than they may encounter in the Bt maize fields. Therefore, we conclude that growing of Cry1Ie expressing maize should pose a negligible risk to P. japonica.

Insects
Specimens of P. japonica were collected from an experiment maize field of the Institute of Plant Protection, Chinese Academy of Agricultural Sciences (CAAS), near Langfang City, Hebei Province, China (39.5 • N, 116.7 • E) in 2015. A colony was subsequently maintained in the laboratory without introduction of field-collected insects for over two generations. Both larvae and adults of P. japonica were reared on soybean seedlings infested with Aphis glycines Matsumura (Hemiptera: Aphididae). The aphids were replaced daily, ensuring ad libitum food for the developing P. japonica. Newly hatched P. japonica larvae (<12 h after emergence) were used for the experiments. A Bt-susceptible strain of C. suppressalis was maintained on an artificial diet for over 80 generations in the laboratory [44]. All insects were reared in a climatic chamber at 26 ± 1 • C, 75% ± 5% RH and a 16:8 h light: dark photoperiod.

Maize Plants and Pollen Collection
The transgenic maize line IE09S034 and the corresponding non-transformed near isoline, Z31 (Zong31), were used in the experiment. During maize anthesis in late July 2014, maize pollen was collected daily by shaking the maize tassels in a plastic bag. The collected pollen was air dried at room temperature for 48 h and subsequently passed through a sieve with a mesh size of 0.2 mm to remove anthers and contaminants. Pollen collected from each maize line was pooled and stored at −60 • C until further use.

Feeding System for P. japonica
The pollen-based diet used in present study was developed and validated in previous studies and has been successfully used to assess the potential effects of Bt rice and Bt maize pollen or purified Cry proteins (mixed into a rapeseed pollen-based diet) on P. japonica [30,31,35]. The P. japonica larvae were individually confined in Petri dishes (6.0 cm diameter, 1.5 cm height). Larvae were fed with pollen on the first day of each instar and then provided with a mixture of pollen and soybean aphids until they had developed into the next instar. For adults, single pairs of P. japonica were fed with pollen or with a combination of pollen and soybean aphids every alternate day in the same Petri dishes. The pollen were directly sprinkled on the bottom of the dish, and the aphids were provided on 1-cm segments of heavily infested soybean seedlings. Pollen was replaced every other day and the aphids were replaced daily. In addition, an open 2-mL centrifuge tube containing solidified 1% agar solution was added to each dish as a water source. All of the food elements were provided ad libitum. For adults, several folded paper tapes (0.6 cm width, 10 cm length) served as oviposition substrates. Maize pollen was used in the first experiment, and rapeseed pollen was used in the second experiment as described in the following sections.

Bt Maize Pollen Experiment
Using the feeding system described above, an experiment was conducted in which P. japonica were fed Bt or non-Bt (control) maize pollen. There were two treatments with 76 neonate P. japonica per treatment: (i) IE09S034 maize pollen containing Cry1Ie; and (ii) Z31 maize pollen (control). Larval survival, pupation rate, eclosion rate, and development time were recorded based on daily observations. When adults emerged (<12 h), they were individually weighted using an electronic balance (CPA224S; Sartorius AG; readability = 0.1 mg, repeatability <± 0.1 mg). Subsequently, the sex of the freshly emerged P. japonica adults was determined, and randomly selected pairs were continuously fed with Bt or non-Bt maize pollen and soybean aphids as described above. A total of 20-25 pairs of adult P. japonica were tested for each treatment. Survival and total fecundity (number of eggs laid per female) were recorded based on daily observation. This experiment was terminated after 19 days.
To determine the bioactivity of the Bt protein in the maize pollen during the experiment, three subsamples of Bt maize pollen and control pollen were taken before and after the two days feeding exposure. The samples were stored at −60 • C until further use.

Purified Cry Protein Experiment
The test system used for the purified Cry protein bioassay was the same as described by Zhang et al. [31]. Newly hatched larvae of P. japonica were tested for each of three treatment: (i) rapeseed pollen containing Cry1Ie protein at 200 µg/g dry weight (DW) of pollen; (ii) rapeseed pollen containing E-64 protein at 400 µg/g DW of pollen (positive control); and (iii) rapeseed pollen (negative control). The Cry1Ie protein concentration in the pollen diet was approximately 40 times that of the concentration in the Cry1Ie-transgenic maize pollen (5 µg/g fresh weight) [32]. E-64 served as a positive control since it is known to be (i) toxic to P. japonica at 400 µg/g DW, (ii) a gut-active compound like the Cry1Ie protein, and (iii) stable during the test duration [31,35]. Rapeseed pollen was used because it has been confirmed to be a highly nutritional food source for P. japonica and is commercially available. The pollen-based diets were prepared before the beginning of the experiment and stored at −20 • C until used. Soybean aphids were provided as a supplement dietary in each treatment as described above. Diets were replaced every two days to avoid the degradation of the test compounds.
The experiment was started with 82-84 neonate P. japonica per treatment. Larval development, survival, pupation rate, and eclosion rate were recorded based on daily observations. Adult P. japonica were weighted within 12 h of emergence. Subsequently, the sex of the freshly emerged adults was determined, and randomly selected pairs were individually kept in the dishes as described above. Thirty-one pairs of adults were test in the control treatment, and 30 pairs of adults were test in the Cry1Ie protein treatments, while only 21 pairs of adults were test in the E-64 protein treatments because of the low eclosion rates. The eggs laid by each female were recorded based on daily observation. After 20 days, the experiment was terminated.
To determine the bioactivity of Bt proteins in the rapeseed pollen-based diet during the experiment, three subsamples were taken from the pollen-based diet before and after it had been exposed to P. japonica for 2 days. The samples were stored at −60 • C until further use.

Sensitive-Insect Bioassay
The bioactivity of the Cry1Ie protein in Bt maize pollen and the rapeseed pollen-based diet before and after exposure to P. japonica for two days was determined with a sensitive-insect bioassay that used C. suppressalis larvae. 300 mg pollen and 4.6 g artificial diet of C. suppressalis larvae were weighted separately using an electronic balance. Subsequently, the pollen was thoroughly incorporated into the artificial diet for C. suppressalis larvae. The Z31 maize pollen and the control rapeseed pollen-based diet served as control treatments. The artificial diets were cut into slices and placed in Petri dishes (9 cm diameter, 1 cm height) with neonate larvae of C. suppressalis (one slice and one larvae per dish). Subsequently, the Petri dishes were sealed with Parafilm and reinforced with surgical tape. Each treatment was represented by 30 replicate dishes. After seven days, the C. suppressalis larvae were weighted.

Data Analyses
Pair-wise statistical comparisons were made between the Bt maize pollen and non-Bt pollen treatments in the first experiment, and between the Cry1Ie or E-64 treatments and the control in the second experiment. Chi-square tests were used to compare pupation rates and eclosion rates. Mann-Whitney U-tests were used to compare larval developmental times because such data did not satisfy the assumptions for parametric analyses (normal distribution of residuals and homogeneity of error variances).
Data on adult fresh weight and total fecundity were compared using Student's t-test in the maize pollen experiment. Dunnett tests were conducted to compare adult weight and total fecundity in the Cry protein bioassay. In addition, Student's t-test was carried out to compare the weights of C. suppressalis larvae that were fed with artificial diets containing different pollen treatments.
All statistical analyses were conducted using the software package SPSS (version22; SPSS, Inc., Chicago, IL, USA).