Ecotoxicology DOI 10.1007/s10646-014-1301-z
Influence of transgenic rice expressing a fused Cry1Ab/1Ac protein on frogs in paddy fields Jia-Mei Wang • Xiu-Ping Chen • Yu-Yong Liang Hao-Jun Zhu • Jia-Tong Ding • Yu-Fa Peng
•
Accepted: 5 August 2014 Ó Springer Science+Business Media New York 2014
Abstract As genetic engineering in plants is increasingly used to control agricultural pests, it is important to determine whether such transgenic plants adversely affect nontarget organisms within and around cultivated fields. The cry1Ab/1Ac fusion gene from Bacillus thuringiensis (Bt) has insecticidal activity and has been introduced into rice line Minghui 63 (MH63). We evaluated the effect of transgenic cry1Ab/1Ac rice (Huahui 1, HH1) on paddy frogs by comparing HH1 and MH63 rice paddies with and without pesticide treatment. The density of tadpoles in rice fields was surveyed at regular intervals, and Cry1Ab/1Ac protein levels were determined in tissues of tadpoles and froglets collected from the paddy fields. In addition, Rana nigromaculata froglets were raised in purse nets placed within these experimental plots. The survival, body weight, feeding habits, and histological characteristics of the digestive tract of these froglets were analyzed. We found that the tadpole density was significantly decreased immediately after pesticide application, and the weight of R. nigromaculata froglets of pesticide groups was J.-M. Wang X.-P. Chen (&) H.-J. Zhu Y.-F. Peng (&) State Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, No. 2 West Yuanmingyuan Road, Haidian, Beijing 100193, China e-mail: [email protected] Y.-F. Peng e-mail: [email protected] J.-M. Wang H.-J. Zhu J.-T. Ding College of Animal Science and Technology, Yangzhou University, Yangzhou 225009, China Y.-Y. Liang Institute of Plant Protection, Jiangxi Academy of Agricultural Sciences, Nanchang 330200, China
significantly reduced compared with no pesticide treatment, but we found no differences between Bt and non-Bt rice groups. Moreover, no Cry1Ab/1Ac protein was detected in tissue samples collected from 192 tadpoles and froglets representing all four experimental groups. In addition, R. nigromaculata froglets raised in purse seines fed primarily on stem borer and non-target insects, and showed no obvious abnormality in the microstructure of their digestive tracts. Based on these results, we conclude that cultivation of transgenic cry1Ab/1Ac rice does not adversely affect paddy frogs. Keywords Transgenic rice Bt protein Frog Safety assessment Non-target effect
Introduction In recent years, genetic engineering techniques have been used to introduce exogenous genes into crop plants, with the goal of controlling pests and improving crop yields (Betz et al. 2000; High et al. 2004). During 17 years of commercialization, the total area devoted to cultivating transgenic crops has increased 100-fold, from 1.7 million ha in 1996 to 175 million ha in 2013 (James 2013). Approximately 40 % of transgenic crops express an insecticidal protein, which can greatly reduce the need to apply pesticides (Qaim 2009; Brookes and Barfoot 2013). Agricultural scientists in China have developed numerous transgenic lines of rice to control pests such as Chilo suppressalis Walker (Lepidoptera: Crambidae) and Scirpophaga incertulas Walker (Lepidoptera: Pyralidae). A number of transgenic rice lines expressing insecticidal genes from the bacterium Bacillus thuringiensis (Bt) have been developed (Chen et al. 2011; Li et al. 2014).
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In 2000, the cry1Ab/1Ac fusion gene from Bt was transformed into the elite rice indica restorer line Minghui 63 (MH63), generating Bt rice line Huahui 1 (HH1) (Tu et al. 2000). Field tests have shown that HH1 is resistant to stem borers and leaf folders (Tu et al. 2000; Wang et al., 2010). In August 2009, China’s Ministry of Agriculture issued a safety certificate for this Bt rice line, but it has not been approved for commercial cultivation (Jia 2010; Lu 2010). Detractors argue that the planting of Bt rice may lead to non-target effects. Research regarding the non-target effects of Bt rice has primarily focused on terrestrial organisms such as insect herbivores (Akhtar et al. 2010; Mannakkara et al. 2013; Lu et al. 2014), their natural enemies (Tian et al. 2010; Wang et al. 2012), economically important insects (Yao et al. 2008), soil (Wu et al. 2004) and soil microorganisms (Liu et al. 2008). A comprehensive examination of 217 river biotopes neighboring fields of Bt corn revealed corn byproducts in 86 % of these sites 6 months after harvest, with 12.9 % of biotopes having corn detritus containing Cry1Ab protein (95 ± 73 ng per g dry weight) (Tank et al. 2010). Similarly, Wang et al. (2013) found that Bt rice releases detectable amounts of Bt protein into irrigation water. To date, only a few studies have assessed the effects of Bt crops on aquatic organisms such as Daphnia magna (Bøhn et al. 2008, 2010; Raybould and Vlachos 2011) and caddisflies (Rosi-Marshall et al. 2007; Jensen et al. 2010). Frogs are commonly found in rice fields and play an extremely important role in maintaining the biodiversity and stability of the paddy-field ecosystem. In recent decades, however, the heavy use of pesticides has contaminated water bodies and led to sharp declines in frog populations (Hayes et al. 2006; Blaustein et al. 2011). Rice growth differs from that of dry-land crops, as rice requires water during most stages of development. Frogs might be affected by Bt rice in two ways. First, the cultivation of Bt rice may lead to the release of Bt proteins into the water through exudation from roots, pollen dispersal, and disposal of postharvest detritus (Rosi-Marshall et al. 2007; Viktorov 2011; Carstens et al. 2012). The relatively high
permeability of frog skin makes it likely that frogs would be exposed to any Bt proteins accumulated in water. Second, Bt rice effectively reduces the population of target insects (Wang et al. 2010; Han et al. 2011), which may dramatically alter the composition of dominant insect species in a rice field (Lu et al. 2010). It is unclear whether this could affect the frogs’ diet, development, or digestive tract microstructure. Thus, it is important to assess the potential non-target effects of Bt rice on frogs in rice paddies. To our knowledge, there are no publications regarding the effect of transgenic crops on amphibian species of frogs. Results from this study provide important information concerning the environmental safety of genetically modified strains of rice.
Materials and methods Experimental rice fields and planting Transgenic cry1Ab/1Ac insect-resistant rice (HH1) and the parental non-transformed control strain (MH63) were cultivated at a scientific research base at Jiangxi Academy of Agricultural Sciences (28.22° N, 115.54° E), where transgenic rice had never been planted. HH1 and MH63 rice were planted on June 20 of 2012 and 2013. Test paddy fields were separated and managed in accordance with regulations of the Agricultural GMO Safety Management of China. Rice fields were divided into four treatment groups: HH1 without pesticides (T group), MH63 without pesticides (N group), HH1 with pesticides (T? group), and MH63 with pesticides (N? group). Three 300-m2 experimental plots were assigned to each treatment group, and each plot had an independent water inlet and outlet system. Chemical prevention and treatment procedures for pesticide groups during 2012 and 2013 are shown in Table 1. One day after pesticides were applied on August 2, 2013, heavy rains flooded the rice field, thereby linking water systems of the different treatment groups.
Table 1 Chemical control and treatment procedures for T? and N? groups Date of control
Pesticides used
Control goal
2012-07-03
Butachlor, imidacloprid, buprofezin, triazophos, chlorpyrifos
Weeds, rice stem borers
2012-08-14
Imidacloprid, triazophos, chlorpyrifos
Rice stem borers
2012-09-05
Thiamethoxam, endosulfan
Rice planthopper
2013-07-02
Pymetrozine, Thiamethoxam, Isocarbophos, emamectin benzoate
Rice leaf folder, stem borers, planthopper
2013-08-02a
Bacterium bacillus, chlorpyrifos, thiamethoxam
Rice leaf folder, stem borers, planthopper
2013-09-02
Chlorpyrifos, pymetrozine
Rice leaf folder, planthopper
a
Rainfall occurred the day after pesticides were applied
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The effects of Bt rice on paddy frogs
Sensitive insect bioassay of Bt rice To confirm the bioactivity of the test substance, rice leaf and stem samples were collected to detect insecticidal activity using a sensitive insect bioassay. Five fresh leaves or stems were cut into 2-cm lengths and placed in 35-mm cultured dishes with 10 newly hatched C. suppressalis larvae. Four replicates were performed for each treatment group. The dishes were sealed, and the C. suppressalis larvae were cultured in a lighted incubator at 25 °C and 70 % humidity. After 48 h, dead insects were counted to calculate mortality. Survey of tadpole density in rice fields Tadpole density was surveyed at regular intervals in 2012 and 2013. A five-point survey method was used as described (Li et al. 2010). The number of tadpoles was counted in five regions (1 9 1 m) of each experimental plot (four corners and one central region), for a total of 15 1-m2 regions per treatment group. Raising of Rana nigromaculata froglets in purse seines Nylon nets (60-mm mesh size, 2 9 2-m bottom, 1.5-m height) were placed in each experimental plot. The bottom of each net was buried in 20–30 cm of soil, and rice was subsequently planted in that soil. In early August 2013, healthy young R. nigromaculata frogs of uniform size and weight (*2.5 g) were purchased (Changnan Frog Farms, Nanchang, China). Three froglets were placed into each purse seine. Froglets were not fed during the experiment but were allowed to eat freely. Before the rice was harvested in October 2013, the frogs were collected. Surviving frogs in each purse seine were counted and weighed. In addition, the stomach contents of each frog were collected and photographed. Analysis of R. nigromaculata tissue slices Gastric and intestinal R. nigromaculata tissues were collected, fixed in 4 % neutral formalin, embedded in paraffin, sectioned, and stained with hematoxylin and eosin. The samples were analyzed and photographed using a microscope (BX63, Olympus, Japan). Frogs were treated in accordance with the NIH Guide for the Care and Use of Laboratory Animals (National Research Council 1996). Determination of Cry1Ab/1Ac content by ELISA Tadpoles and frogs were collected from test rice fields on different dates. Cry1Ab/1Ac levels in Bt rice roots, stems, leaves, rhizosphere soil, and frog tissues (*0.2 g each)
were determined using a Bt-Cry1Ab/1Ac protein kit (QuantiPlate, EnviroLogix, Portland, USA) with a detection limit of 0.1 ng/g. Before analysis, all the samples were washed in phosphate-buffered saline/Tween-20 (provided with the kit) to remove Bt toxin from their outer surfaces, and then lyophilized, homogenized in 1 ml phosphatebuffered saline/Tween-20 using a micro pestle and mortar on ice. After centrifugation and appropriate dilution of the supernatants, the ELISA detection was performed following manufacturer’s protocol. Optical density values were read using a microplate spectrophotometer (PowerWave XS2, BioTek, USA). A standard curve derived from purified Cry1Ab/1Ac samples was used to calculate Cry1Ab/ 1Ac levels. Data analysis All data represent mean ± standard deviation (SD) unless indicated otherwise. The mortality of C. suppressalis that were fed Bt or non-Bt rice was analyzed by V2 test. Oneway analysis of variance followed by Dunn’s multiple comparison test was used to analyze differences in tadpole density and R. nigromaculata weight among the four treatment groups. Differences were considered significant at p \ 0.05.
Results Level of Bt protein and insecticidal activity in rice tissues Cry1Ab/1Ac was detected by ELISA in the roots, stems, and leaves of HH1 rice at levels [2 lg/g. In contrast, Cry1Ab/1Ac was not detected in the MH63 parental strain or in the HH1 rhizosphere soil (Table 2). When the target insect C. suppressalis was incubated with rice stems or leaves, the insecticidal activity of HH1 stems and leaves was significantly higher than that of MH63 tissues (V2, p \ 0.05; Table 2), indicating that our test Bt rice had high bioactivity for the target insect. Tadpole density in rice paddies To assess the effects of Bt rice on frog populations, we surveyed tadpole density at regular intervals in 2012 and 2013. On July 3, 2012, pesticides were first applied to the T? and N? paddies. On July 5, 2012, field investigations revealed that T? and N? rice paddies (0.07 ± 0.11 and 0.13 ± 0.12 tadpoles/m2, respectively) had significantly fewer tadpoles than paddies not treated with pesticides (T and N groups, 1.00 ± 0.35 and 0.87 ± 0.12 tadpoles/m2, respectively). In some T? and N? fields, no living tadpoles
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J.-M. Wang et al. Table 2 Bt protein levels and insecticidal activity of rice tissues Rice tissues
Cry1Ab/1Ac protein levels (lg/g fresh weight)
Mortality of C. suppressalis larvae
HH1
MH63
HH1
MH63
Statistics
Roots
2.40 ± 0.89
N
–
–
–
Stems
2.91 ± 0.50
N
67.50 % (27/40)a
0.00 % (0/40)b
V2 = 40.76; p = 0.000
Leaves
3.78 ± 0.84
N
62.50 % (25/40)a
0.00 % (0/40)b
V2 = 36.36; p = 0.000
Rhizosphere soil
N
N
–
–
–
N not detected, — analysis not performed a, b
Different lowercase letters in the same row indicate a statistically significant difference (p \ 0.05)
Table 3 Tadpole density in treatment groups on different survey dates Group
Treatment T
T?
N
N?
2012-07-05
1.00 ± 0.35a
0.07 ± 0.11b
0.87 ± 0.12a
0.13 ± 0.12b
2012-08-05 2013-05-231
0.53 ± 0.50 1.27 ± 1.50
0.53 ± 0.61 1.20 ± 1.11
0.60 ± 0.60 1.47 ± 1.14
0.53 ± 0.50 1.13 ± 1.63
2013-07-04
0.13 ± 0.23a
0.00 ± 0.00b
0.20 ± 0.20a
0.00 ± 0.00b
0
0
0
0
2013-08-10
2
Data indicate tadpoles/m
2
T HH1 without pesticides, T? HH1 with pesticides, N MH63 without pesticides, N? MH63 with pesticides a, b
Different lowercase letters in the same row indicate a statistically significant difference (p \ 0.05)
1
Before rice seeding
2
Rainfall occurred the day after pesticides were applied
were found. Moreover, the surface water in T? and N? paddy fields was clear, with only a small amount of plankton, whereas plankton, tadpoles, and tadpole feces were abundant in T and N fields. Furthermore, tadpole density was similar between the T and N fields (Table 3). Subsequent surveys performed on August 5, 2012 (33 days after pesticide application on July 3) and May 23, 2013 ([8 months after last pesticide application on September 5, 2012) revealed no significant differences in tadpole density among the four treatment groups. These two surveys found 0.5–1 tadpoles/m2 and 1–1.5 tadpoles/m2, respectively (Table 3). On July 2 and August 2, 2013, pesticides were again applied to the T? and N? paddies. On July 4, 2013, field surveys revealed that there were no living tadpoles in the T? and N? paddies and that there were 0.0–0.5 tadpoles/m2 in the T and N fields. The final survey on August 10, 2013 (8 days after pesticide application on August 2), found no living tadpoles in any of the treatment groups (Table 3). This likely resulted from heavy rainfall on August 3, which flooded the rice field and circulated pesticides to all experimental paddies. These data suggested that there was an immediate decline in tadpole density as a result of pesticide application but that tadpole densities recovered fully over time,
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Table 4 Survival and body weight of R. nigromaculata raised in purse seines Group
Number of frogs raised
Initial body weight (g)
Number of frogs harvested
Final body weight (g)
T
9
2.59 ± 0.45
6
6.44 ± 2.37a
9
2.59 ± 0.33
8
2.52 ± 1.33b
9
2.52 ± 0.50
6
7.74 ± 1.66a
9
2.50 ± 0.30
9
2.57 ± 0.79b
?
T
N ?
N
T HH1 without pesticides, T? HH1 with pesticides, N MH63 without pesticides, N? MH63 with pesticides a, b
Different lowercase letters in the same column indicate a statistically significant difference (p \ 0.05)
likely due to pesticide degradation and consequent loss of toxicity. Moreover, the data indicated that the introduction of Bt genes into rice did not affect tadpole density over the course of 1 year. Body weight and feeding habits of R. nigromaculata raised in purse seines To assess the effects of Bt rice on the development and feeding habits of paddy frogs, R. nigromaculata froglets
The effects of Bt rice on paddy frogs
Fig. 1 Gastric contents of R. nigromaculata from different treatment groups. A–D are T, T?, N, and N? groups, respectively. (a) Hymenoptera (b) Orthoptera (c) spider (d) paddy (e) stem borer (f) bee (g) rice leaf (h) nematode
were raised in purse seines for almost 2 months (August to October 2013). Froglets were collected from purse seines before the rice was harvested. Characteristics of froglets collected are shown in Table 4. Froglets raised in T? and N? fields were significantly lighter than those raised in T and N fields (p \ 0.05), but no significant difference in body weight was observed between rice strains (T and N groups). These results indicated that the introduction of Bt genes into rice had no adverse effect on the growth of R. nigromaculata froglets. Examination of the gastric contents of the froglets showed that spiders, Hymenoptera, and other non-target insects accounted for a large proportion of the diet in the T group, whereas Lepidoptera larvae accounted for a large
proportion of the N group’s diet (Fig. 1). In contrast, fewer gastric contents were obtained from the T? and N? groups compared with the T and N groups, and some frogs from the pesticide-treated fields ate rice grain (Fig. 1B.d) and leaves (Fig. 1D.g) due to the lack of food. Histological structure of R. nigromaculata stomach and intestinal tissues To assess the effect of Bt rice on the microstructure of the frog digestive tract, stomach and intestinal tissues from frogs collected from purse seines were sectioned and examined microscopically. Very few tissue abnormalities were found (Figs. 2, 3). The frog stomach is composed
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Fig. 2 Histological structure of the R. nigromaculata stomach. A–D are T, T?, N, and N? groups, respectively. MUL muscularis mucosa, SM serosa, GG gastric gland, SCEp simple ciliated columnar epithelium. Scale bar 200 lm
(from inside out) of mucosa, submucosa, muscular layer, and adventitia. The gastric mucosa protruded into the gastric cavity and a number of prominent mucosal folds were formed. The gastric mucosal epithelium is a simple columnar epithelium, and these cells had long, elliptical nuclei and large numbers of gastric pits on the mucosal surfaces. In addition, the mucous layer contained a large number of gastric glands, which consisted of simple tubular glands that were evenly distributed throughout the lamina propria (Fig. 2a, c). The gastric tunica muscularis was thicker in frogs raised in T and N fields than in frogs raised in T? and N? fields. In addition, the gastric mucosa protruded further into the gastric cavity of frogs in the T? and N? groups than in those in the T and N groups (Fig. 2b, d). Samples of intestinal tissues were taken from the rectum. Small folds were observed in the intestinal tract, and the mucosal epithelium consisted of columnar epithelial cells and goblet cells. No cellular differences were found among the four treatment groups (Fig. 3). These results demonstrated that there was no obvious difference in the microstructure of the digestive tract between the T and N groups.
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Cry1Ab/1Ac levels in frog tissues To determine whether Bt protein accumulates in frog tissues in a natural environment, ELISA was used to measure Cry1Ab/1Ac levels in intestine, stomach, heart, liver, and muscle from tadpoles and froglets collected from all four test groups. A total of 77 tadpoles and 115 froglets were tested, but no Cry1Ab/1Ac protein was detected (Table 5), suggesting that Bt protein did not accumulate in frogs.
Discussion Most transgenic Bt rice is highly resistant to target insects and can effectively control lepidopterous pests (Tu et al. 2000; Han et al. 2006, 2007, 2011). In our experiment, HH1 exhibited high resistance ([60 %) to the target insect C. suppressalis compared with control plants (0 %) under laboratory conditions, which confirmed the bioactivity (Romeis et al. 2011) of the test rice line. Outbreaks of mirid bugs (Heteroptera: Miridae) in multiple crop plants correlate with wide-scale adoption of
The effects of Bt rice on paddy frogs
Fig. 3 Histological structure of the R. nigromaculata intestine. A–D are T, T?, N, and N? groups, respectively. MUL muscularis mucosa, GC goblet cell, Ad adventitia. Scale bar 40 lm
Table 5 Cry1Ab/1Ac levels in tissues of frogs collected from rice field Sampling time point
Developmental stage
Number of animals
Tissues
Protein expressiona
2012-07-05 2012-08-05
Tadpoles Tadpoles
20 27
Intestines, muscle Intestines, muscle
0/20 0/27
2013-05-23
Tadpoles
30
Intestines, stomach
0/30
2013-07-05
Froglets
23
Intestine, stomach, heart, liver
0/23
2013-08-10
Froglets
69
Intestines, stomach
0/69
2013-09-08
Froglets
23
Intestines, stomach
0/23
a
Number of protein-positive animals/total animals
Bt cotton in China (Lu et al. 2010), suggesting that nontarget insects thrive when target insect populations are reduced. Similarly, planting of Bt rice may affect nontarget arthropod populations in rice fields by decreasing the number of C. suppressalis, Cnaphalocrocis medinalis, and other lepidopterous target insects (Tu et al. 2000; Han et al. 2006, 2007, 2011). This in turn could affect the diet of resident R. nigromaculata. In the current study, we set artificial fences to restrict the movement of R. nigromaculata so that their stomach contents generally reflected the distribution of insects in the different rice fields. Froglets
raised in Bt rice fields fed primarily on spiders and other non-target insects, whereas froglets raised in non-Bt rice fields ate primarily stem borers. This result is consistent with reports discussed above (Tu et al. 2000; Han et al. 2006, 2007, 2011). Different frog species have different feeding habits. R. nigromaculata eat mainly Nematoda in Protocoelomata, Oligochaeta in Annelida, Shoe Cephalopoda in Mollusca, and Crustacea, Myriapoda, Arachnoidea, and Hexapeda in Arthropoda. They may also unintentionally swallow grass leaves, grass seeds, rice grains, duckweed, and sand. The
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beneficial coefficient (used to measure the impact of beneficial insects or animals on crops or pests) for R. nigromaculata is high, but changing habitats and other forces may affect this coefficient (Chen 2011). In our experiment, R. nigromaculata in Bt rice fields fed on spiders and nontarget insects, thereby reducing their beneficial coefficient. It has been shown that R. nigromaculata of different weights and sizes consume different prey (Hirai 2002). Here, R. nigromaculata raised in the presence of pesticides (T? and N? groups) were smaller than frogs not exposed to pesticides, and were more inclined to consume small prey, such as nematodes. In contrast, R. nigromaculata in the T and N groups fed on larger prey, such as spiders. Cry1Ab/1Ac specifically binds receptors that localize to gut epithelial cells of target insects. This results in perforation of the cell membrane, cell swelling and lysis, and ultimately death of the target insect (Hofmann et al. 1988). In the present study, R. nigromaculata in the N and T groups consumed different foods, but this did not affect the histology of stomach or intestinal tissues. Compared with non-pesticide groups (T and N), R. nigromaculata in the pesticide groups (T? and N?) exhibited undeveloped gastric tunica muscularis and prominent protrusions of the gastric mucosa into the gastric cavity. These histological differences may have resulted from the lack of food caused by pesticide spraying (i.e., inducing a fasting state), as hunger can affect the histology of gastrointestinal tissues (Tam and Avenant-Oldewage 2009; Zeng et al. 2012). Alternatively, these differences may have resulted from pesticide toxicity. Pesticides also affected the density of aquatic tadpoles. This study used organophosphorus pesticides such as trichloride, endosulfan, and chlorpyrifos, which are highly toxic to aquatic organisms and likely caused a large number of tadpole deaths within a short period (Sparling and Fellers 2007; Jones et al. 2009). The planting of transgenic cry1Ab/ 1Ac rice did not adversely affect tadpole density, but further studies are required to determine whether there are any long-term effects. Our results indicate that transgenic cry1Ab/1Ac rice did not affect tadpole density or frog body weight, whereas the application of pesticides quickly killed tadpoles and reduced the body weight of surviving froglets. Previous studies concerning the effects of Bt gene products on non-target aquatic organisms have yielded inconsistent results. Adverse effects were observed when D. magna (Diplostraca: Daphniidae) were fed flour from Bt maize containing cry1Ab (Bøhn et al. 2008, 2010) or when two species of caddisflies (Trichoptera) were fed Bt maize tissue or pollen (Rosi-Marshall et al. 2007). However, Jensen et al. (2010) could not confirm the Trichoptera results. In addition, Raybould and Vlachos (2011) exposed D. magna to the Bt vegetative insecticidal protein Vip3Aa20 and observed no effect on survival or fecundity
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compared with controls, but they did observe a small reduction in growth. Some researchers have proposed that the adverse effects observed in D. magna and larval of Trichoptera are caused by factors other than the Cry protein (Ricroch et al. 2010; Romeis et al. 2013). Bt proteins expressed by transgenic crops may be passed down the food chain, but concentrations are low at higher trophic levels (Garcı´a et al. 2010; Li and Romeis 2010). Frogs are high trophic–level organisms in farmland ecosystems and may consume prey that contain some target proteins (transgenic rice ? target or non-target pests ? frogs). Moreover, the relatively high permeability of frog skin makes it likely that frogs would be exposed to Bt proteins accumulated in water. However, we did not detect any Cry1Ab/1Ac protein in frog tissues collected at different time points, suggesting that Bt proteins do not accumulate in frogs. In addition, we fed Xenopus laevis froglets diets containing HH1 rice grain under laboratory conditions. Their survival rate, body and organ weight, and microstructure of the digestive tract were detected, and no adverse effects were observed (Zhu et al., unpublished data). So, we conclude that transgenic cry1Ab/1Ac rice line HH1 does not adversely affect paddy frogs and that planting Bt rice will promote the survival and growth of frogs compared with pesticide spraying of non-Bt rice. Acknowledgments We thank Professor Yongjun Lin (Huazhong Agricultural University, Wuhan, China) for kindly providing transgenic rice seeds. We also thank Professor Fajun Chen (Nanjing Agricultural University, Nanjing, China) for his constructive comments on the early draft of this manuscript. This work was supported by the National GMO New Variety Breeding Program of the PRC (2012ZX08011-002 and 2014ZX08011-001). Conflict of interest of interest.
The authors declare that they have no conflict
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Influence of transgenic rice expressing a fused Cry1Ab/1Ac protein on frogs in paddy fields Jia-Mei Wang • Xiu-Ping Chen • Yu-Yong Liang Hao-Jun Zhu • Jia-Tong Ding • Yu-Fa Peng
•
Accepted: 5 August 2014 Ó Springer Science+Business Media New York 2014
Abstract As genetic engineering in plants is increasingly used to control agricultural pests, it is important to determine whether such transgenic plants adversely affect nontarget organisms within and around cultivated fields. The cry1Ab/1Ac fusion gene from Bacillus thuringiensis (Bt) has insecticidal activity and has been introduced into rice line Minghui 63 (MH63). We evaluated the effect of transgenic cry1Ab/1Ac rice (Huahui 1, HH1) on paddy frogs by comparing HH1 and MH63 rice paddies with and without pesticide treatment. The density of tadpoles in rice fields was surveyed at regular intervals, and Cry1Ab/1Ac protein levels were determined in tissues of tadpoles and froglets collected from the paddy fields. In addition, Rana nigromaculata froglets were raised in purse nets placed within these experimental plots. The survival, body weight, feeding habits, and histological characteristics of the digestive tract of these froglets were analyzed. We found that the tadpole density was significantly decreased immediately after pesticide application, and the weight of R. nigromaculata froglets of pesticide groups was J.-M. Wang X.-P. Chen (&) H.-J. Zhu Y.-F. Peng (&) State Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, No. 2 West Yuanmingyuan Road, Haidian, Beijing 100193, China e-mail: [email protected] Y.-F. Peng e-mail: [email protected] J.-M. Wang H.-J. Zhu J.-T. Ding College of Animal Science and Technology, Yangzhou University, Yangzhou 225009, China Y.-Y. Liang Institute of Plant Protection, Jiangxi Academy of Agricultural Sciences, Nanchang 330200, China
significantly reduced compared with no pesticide treatment, but we found no differences between Bt and non-Bt rice groups. Moreover, no Cry1Ab/1Ac protein was detected in tissue samples collected from 192 tadpoles and froglets representing all four experimental groups. In addition, R. nigromaculata froglets raised in purse seines fed primarily on stem borer and non-target insects, and showed no obvious abnormality in the microstructure of their digestive tracts. Based on these results, we conclude that cultivation of transgenic cry1Ab/1Ac rice does not adversely affect paddy frogs. Keywords Transgenic rice Bt protein Frog Safety assessment Non-target effect
Introduction In recent years, genetic engineering techniques have been used to introduce exogenous genes into crop plants, with the goal of controlling pests and improving crop yields (Betz et al. 2000; High et al. 2004). During 17 years of commercialization, the total area devoted to cultivating transgenic crops has increased 100-fold, from 1.7 million ha in 1996 to 175 million ha in 2013 (James 2013). Approximately 40 % of transgenic crops express an insecticidal protein, which can greatly reduce the need to apply pesticides (Qaim 2009; Brookes and Barfoot 2013). Agricultural scientists in China have developed numerous transgenic lines of rice to control pests such as Chilo suppressalis Walker (Lepidoptera: Crambidae) and Scirpophaga incertulas Walker (Lepidoptera: Pyralidae). A number of transgenic rice lines expressing insecticidal genes from the bacterium Bacillus thuringiensis (Bt) have been developed (Chen et al. 2011; Li et al. 2014).
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In 2000, the cry1Ab/1Ac fusion gene from Bt was transformed into the elite rice indica restorer line Minghui 63 (MH63), generating Bt rice line Huahui 1 (HH1) (Tu et al. 2000). Field tests have shown that HH1 is resistant to stem borers and leaf folders (Tu et al. 2000; Wang et al., 2010). In August 2009, China’s Ministry of Agriculture issued a safety certificate for this Bt rice line, but it has not been approved for commercial cultivation (Jia 2010; Lu 2010). Detractors argue that the planting of Bt rice may lead to non-target effects. Research regarding the non-target effects of Bt rice has primarily focused on terrestrial organisms such as insect herbivores (Akhtar et al. 2010; Mannakkara et al. 2013; Lu et al. 2014), their natural enemies (Tian et al. 2010; Wang et al. 2012), economically important insects (Yao et al. 2008), soil (Wu et al. 2004) and soil microorganisms (Liu et al. 2008). A comprehensive examination of 217 river biotopes neighboring fields of Bt corn revealed corn byproducts in 86 % of these sites 6 months after harvest, with 12.9 % of biotopes having corn detritus containing Cry1Ab protein (95 ± 73 ng per g dry weight) (Tank et al. 2010). Similarly, Wang et al. (2013) found that Bt rice releases detectable amounts of Bt protein into irrigation water. To date, only a few studies have assessed the effects of Bt crops on aquatic organisms such as Daphnia magna (Bøhn et al. 2008, 2010; Raybould and Vlachos 2011) and caddisflies (Rosi-Marshall et al. 2007; Jensen et al. 2010). Frogs are commonly found in rice fields and play an extremely important role in maintaining the biodiversity and stability of the paddy-field ecosystem. In recent decades, however, the heavy use of pesticides has contaminated water bodies and led to sharp declines in frog populations (Hayes et al. 2006; Blaustein et al. 2011). Rice growth differs from that of dry-land crops, as rice requires water during most stages of development. Frogs might be affected by Bt rice in two ways. First, the cultivation of Bt rice may lead to the release of Bt proteins into the water through exudation from roots, pollen dispersal, and disposal of postharvest detritus (Rosi-Marshall et al. 2007; Viktorov 2011; Carstens et al. 2012). The relatively high
permeability of frog skin makes it likely that frogs would be exposed to any Bt proteins accumulated in water. Second, Bt rice effectively reduces the population of target insects (Wang et al. 2010; Han et al. 2011), which may dramatically alter the composition of dominant insect species in a rice field (Lu et al. 2010). It is unclear whether this could affect the frogs’ diet, development, or digestive tract microstructure. Thus, it is important to assess the potential non-target effects of Bt rice on frogs in rice paddies. To our knowledge, there are no publications regarding the effect of transgenic crops on amphibian species of frogs. Results from this study provide important information concerning the environmental safety of genetically modified strains of rice.
Materials and methods Experimental rice fields and planting Transgenic cry1Ab/1Ac insect-resistant rice (HH1) and the parental non-transformed control strain (MH63) were cultivated at a scientific research base at Jiangxi Academy of Agricultural Sciences (28.22° N, 115.54° E), where transgenic rice had never been planted. HH1 and MH63 rice were planted on June 20 of 2012 and 2013. Test paddy fields were separated and managed in accordance with regulations of the Agricultural GMO Safety Management of China. Rice fields were divided into four treatment groups: HH1 without pesticides (T group), MH63 without pesticides (N group), HH1 with pesticides (T? group), and MH63 with pesticides (N? group). Three 300-m2 experimental plots were assigned to each treatment group, and each plot had an independent water inlet and outlet system. Chemical prevention and treatment procedures for pesticide groups during 2012 and 2013 are shown in Table 1. One day after pesticides were applied on August 2, 2013, heavy rains flooded the rice field, thereby linking water systems of the different treatment groups.
Table 1 Chemical control and treatment procedures for T? and N? groups Date of control
Pesticides used
Control goal
2012-07-03
Butachlor, imidacloprid, buprofezin, triazophos, chlorpyrifos
Weeds, rice stem borers
2012-08-14
Imidacloprid, triazophos, chlorpyrifos
Rice stem borers
2012-09-05
Thiamethoxam, endosulfan
Rice planthopper
2013-07-02
Pymetrozine, Thiamethoxam, Isocarbophos, emamectin benzoate
Rice leaf folder, stem borers, planthopper
2013-08-02a
Bacterium bacillus, chlorpyrifos, thiamethoxam
Rice leaf folder, stem borers, planthopper
2013-09-02
Chlorpyrifos, pymetrozine
Rice leaf folder, planthopper
a
Rainfall occurred the day after pesticides were applied
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The effects of Bt rice on paddy frogs
Sensitive insect bioassay of Bt rice To confirm the bioactivity of the test substance, rice leaf and stem samples were collected to detect insecticidal activity using a sensitive insect bioassay. Five fresh leaves or stems were cut into 2-cm lengths and placed in 35-mm cultured dishes with 10 newly hatched C. suppressalis larvae. Four replicates were performed for each treatment group. The dishes were sealed, and the C. suppressalis larvae were cultured in a lighted incubator at 25 °C and 70 % humidity. After 48 h, dead insects were counted to calculate mortality. Survey of tadpole density in rice fields Tadpole density was surveyed at regular intervals in 2012 and 2013. A five-point survey method was used as described (Li et al. 2010). The number of tadpoles was counted in five regions (1 9 1 m) of each experimental plot (four corners and one central region), for a total of 15 1-m2 regions per treatment group. Raising of Rana nigromaculata froglets in purse seines Nylon nets (60-mm mesh size, 2 9 2-m bottom, 1.5-m height) were placed in each experimental plot. The bottom of each net was buried in 20–30 cm of soil, and rice was subsequently planted in that soil. In early August 2013, healthy young R. nigromaculata frogs of uniform size and weight (*2.5 g) were purchased (Changnan Frog Farms, Nanchang, China). Three froglets were placed into each purse seine. Froglets were not fed during the experiment but were allowed to eat freely. Before the rice was harvested in October 2013, the frogs were collected. Surviving frogs in each purse seine were counted and weighed. In addition, the stomach contents of each frog were collected and photographed. Analysis of R. nigromaculata tissue slices Gastric and intestinal R. nigromaculata tissues were collected, fixed in 4 % neutral formalin, embedded in paraffin, sectioned, and stained with hematoxylin and eosin. The samples were analyzed and photographed using a microscope (BX63, Olympus, Japan). Frogs were treated in accordance with the NIH Guide for the Care and Use of Laboratory Animals (National Research Council 1996). Determination of Cry1Ab/1Ac content by ELISA Tadpoles and frogs were collected from test rice fields on different dates. Cry1Ab/1Ac levels in Bt rice roots, stems, leaves, rhizosphere soil, and frog tissues (*0.2 g each)
were determined using a Bt-Cry1Ab/1Ac protein kit (QuantiPlate, EnviroLogix, Portland, USA) with a detection limit of 0.1 ng/g. Before analysis, all the samples were washed in phosphate-buffered saline/Tween-20 (provided with the kit) to remove Bt toxin from their outer surfaces, and then lyophilized, homogenized in 1 ml phosphatebuffered saline/Tween-20 using a micro pestle and mortar on ice. After centrifugation and appropriate dilution of the supernatants, the ELISA detection was performed following manufacturer’s protocol. Optical density values were read using a microplate spectrophotometer (PowerWave XS2, BioTek, USA). A standard curve derived from purified Cry1Ab/1Ac samples was used to calculate Cry1Ab/ 1Ac levels. Data analysis All data represent mean ± standard deviation (SD) unless indicated otherwise. The mortality of C. suppressalis that were fed Bt or non-Bt rice was analyzed by V2 test. Oneway analysis of variance followed by Dunn’s multiple comparison test was used to analyze differences in tadpole density and R. nigromaculata weight among the four treatment groups. Differences were considered significant at p \ 0.05.
Results Level of Bt protein and insecticidal activity in rice tissues Cry1Ab/1Ac was detected by ELISA in the roots, stems, and leaves of HH1 rice at levels [2 lg/g. In contrast, Cry1Ab/1Ac was not detected in the MH63 parental strain or in the HH1 rhizosphere soil (Table 2). When the target insect C. suppressalis was incubated with rice stems or leaves, the insecticidal activity of HH1 stems and leaves was significantly higher than that of MH63 tissues (V2, p \ 0.05; Table 2), indicating that our test Bt rice had high bioactivity for the target insect. Tadpole density in rice paddies To assess the effects of Bt rice on frog populations, we surveyed tadpole density at regular intervals in 2012 and 2013. On July 3, 2012, pesticides were first applied to the T? and N? paddies. On July 5, 2012, field investigations revealed that T? and N? rice paddies (0.07 ± 0.11 and 0.13 ± 0.12 tadpoles/m2, respectively) had significantly fewer tadpoles than paddies not treated with pesticides (T and N groups, 1.00 ± 0.35 and 0.87 ± 0.12 tadpoles/m2, respectively). In some T? and N? fields, no living tadpoles
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J.-M. Wang et al. Table 2 Bt protein levels and insecticidal activity of rice tissues Rice tissues
Cry1Ab/1Ac protein levels (lg/g fresh weight)
Mortality of C. suppressalis larvae
HH1
MH63
HH1
MH63
Statistics
Roots
2.40 ± 0.89
N
–
–
–
Stems
2.91 ± 0.50
N
67.50 % (27/40)a
0.00 % (0/40)b
V2 = 40.76; p = 0.000
Leaves
3.78 ± 0.84
N
62.50 % (25/40)a
0.00 % (0/40)b
V2 = 36.36; p = 0.000
Rhizosphere soil
N
N
–
–
–
N not detected, — analysis not performed a, b
Different lowercase letters in the same row indicate a statistically significant difference (p \ 0.05)
Table 3 Tadpole density in treatment groups on different survey dates Group
Treatment T
T?
N
N?
2012-07-05
1.00 ± 0.35a
0.07 ± 0.11b
0.87 ± 0.12a
0.13 ± 0.12b
2012-08-05 2013-05-231
0.53 ± 0.50 1.27 ± 1.50
0.53 ± 0.61 1.20 ± 1.11
0.60 ± 0.60 1.47 ± 1.14
0.53 ± 0.50 1.13 ± 1.63
2013-07-04
0.13 ± 0.23a
0.00 ± 0.00b
0.20 ± 0.20a
0.00 ± 0.00b
0
0
0
0
2013-08-10
2
Data indicate tadpoles/m
2
T HH1 without pesticides, T? HH1 with pesticides, N MH63 without pesticides, N? MH63 with pesticides a, b
Different lowercase letters in the same row indicate a statistically significant difference (p \ 0.05)
1
Before rice seeding
2
Rainfall occurred the day after pesticides were applied
were found. Moreover, the surface water in T? and N? paddy fields was clear, with only a small amount of plankton, whereas plankton, tadpoles, and tadpole feces were abundant in T and N fields. Furthermore, tadpole density was similar between the T and N fields (Table 3). Subsequent surveys performed on August 5, 2012 (33 days after pesticide application on July 3) and May 23, 2013 ([8 months after last pesticide application on September 5, 2012) revealed no significant differences in tadpole density among the four treatment groups. These two surveys found 0.5–1 tadpoles/m2 and 1–1.5 tadpoles/m2, respectively (Table 3). On July 2 and August 2, 2013, pesticides were again applied to the T? and N? paddies. On July 4, 2013, field surveys revealed that there were no living tadpoles in the T? and N? paddies and that there were 0.0–0.5 tadpoles/m2 in the T and N fields. The final survey on August 10, 2013 (8 days after pesticide application on August 2), found no living tadpoles in any of the treatment groups (Table 3). This likely resulted from heavy rainfall on August 3, which flooded the rice field and circulated pesticides to all experimental paddies. These data suggested that there was an immediate decline in tadpole density as a result of pesticide application but that tadpole densities recovered fully over time,
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Table 4 Survival and body weight of R. nigromaculata raised in purse seines Group
Number of frogs raised
Initial body weight (g)
Number of frogs harvested
Final body weight (g)
T
9
2.59 ± 0.45
6
6.44 ± 2.37a
9
2.59 ± 0.33
8
2.52 ± 1.33b
9
2.52 ± 0.50
6
7.74 ± 1.66a
9
2.50 ± 0.30
9
2.57 ± 0.79b
?
T
N ?
N
T HH1 without pesticides, T? HH1 with pesticides, N MH63 without pesticides, N? MH63 with pesticides a, b
Different lowercase letters in the same column indicate a statistically significant difference (p \ 0.05)
likely due to pesticide degradation and consequent loss of toxicity. Moreover, the data indicated that the introduction of Bt genes into rice did not affect tadpole density over the course of 1 year. Body weight and feeding habits of R. nigromaculata raised in purse seines To assess the effects of Bt rice on the development and feeding habits of paddy frogs, R. nigromaculata froglets
The effects of Bt rice on paddy frogs
Fig. 1 Gastric contents of R. nigromaculata from different treatment groups. A–D are T, T?, N, and N? groups, respectively. (a) Hymenoptera (b) Orthoptera (c) spider (d) paddy (e) stem borer (f) bee (g) rice leaf (h) nematode
were raised in purse seines for almost 2 months (August to October 2013). Froglets were collected from purse seines before the rice was harvested. Characteristics of froglets collected are shown in Table 4. Froglets raised in T? and N? fields were significantly lighter than those raised in T and N fields (p \ 0.05), but no significant difference in body weight was observed between rice strains (T and N groups). These results indicated that the introduction of Bt genes into rice had no adverse effect on the growth of R. nigromaculata froglets. Examination of the gastric contents of the froglets showed that spiders, Hymenoptera, and other non-target insects accounted for a large proportion of the diet in the T group, whereas Lepidoptera larvae accounted for a large
proportion of the N group’s diet (Fig. 1). In contrast, fewer gastric contents were obtained from the T? and N? groups compared with the T and N groups, and some frogs from the pesticide-treated fields ate rice grain (Fig. 1B.d) and leaves (Fig. 1D.g) due to the lack of food. Histological structure of R. nigromaculata stomach and intestinal tissues To assess the effect of Bt rice on the microstructure of the frog digestive tract, stomach and intestinal tissues from frogs collected from purse seines were sectioned and examined microscopically. Very few tissue abnormalities were found (Figs. 2, 3). The frog stomach is composed
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Fig. 2 Histological structure of the R. nigromaculata stomach. A–D are T, T?, N, and N? groups, respectively. MUL muscularis mucosa, SM serosa, GG gastric gland, SCEp simple ciliated columnar epithelium. Scale bar 200 lm
(from inside out) of mucosa, submucosa, muscular layer, and adventitia. The gastric mucosa protruded into the gastric cavity and a number of prominent mucosal folds were formed. The gastric mucosal epithelium is a simple columnar epithelium, and these cells had long, elliptical nuclei and large numbers of gastric pits on the mucosal surfaces. In addition, the mucous layer contained a large number of gastric glands, which consisted of simple tubular glands that were evenly distributed throughout the lamina propria (Fig. 2a, c). The gastric tunica muscularis was thicker in frogs raised in T and N fields than in frogs raised in T? and N? fields. In addition, the gastric mucosa protruded further into the gastric cavity of frogs in the T? and N? groups than in those in the T and N groups (Fig. 2b, d). Samples of intestinal tissues were taken from the rectum. Small folds were observed in the intestinal tract, and the mucosal epithelium consisted of columnar epithelial cells and goblet cells. No cellular differences were found among the four treatment groups (Fig. 3). These results demonstrated that there was no obvious difference in the microstructure of the digestive tract between the T and N groups.
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Cry1Ab/1Ac levels in frog tissues To determine whether Bt protein accumulates in frog tissues in a natural environment, ELISA was used to measure Cry1Ab/1Ac levels in intestine, stomach, heart, liver, and muscle from tadpoles and froglets collected from all four test groups. A total of 77 tadpoles and 115 froglets were tested, but no Cry1Ab/1Ac protein was detected (Table 5), suggesting that Bt protein did not accumulate in frogs.
Discussion Most transgenic Bt rice is highly resistant to target insects and can effectively control lepidopterous pests (Tu et al. 2000; Han et al. 2006, 2007, 2011). In our experiment, HH1 exhibited high resistance ([60 %) to the target insect C. suppressalis compared with control plants (0 %) under laboratory conditions, which confirmed the bioactivity (Romeis et al. 2011) of the test rice line. Outbreaks of mirid bugs (Heteroptera: Miridae) in multiple crop plants correlate with wide-scale adoption of
The effects of Bt rice on paddy frogs
Fig. 3 Histological structure of the R. nigromaculata intestine. A–D are T, T?, N, and N? groups, respectively. MUL muscularis mucosa, GC goblet cell, Ad adventitia. Scale bar 40 lm
Table 5 Cry1Ab/1Ac levels in tissues of frogs collected from rice field Sampling time point
Developmental stage
Number of animals
Tissues
Protein expressiona
2012-07-05 2012-08-05
Tadpoles Tadpoles
20 27
Intestines, muscle Intestines, muscle
0/20 0/27
2013-05-23
Tadpoles
30
Intestines, stomach
0/30
2013-07-05
Froglets
23
Intestine, stomach, heart, liver
0/23
2013-08-10
Froglets
69
Intestines, stomach
0/69
2013-09-08
Froglets
23
Intestines, stomach
0/23
a
Number of protein-positive animals/total animals
Bt cotton in China (Lu et al. 2010), suggesting that nontarget insects thrive when target insect populations are reduced. Similarly, planting of Bt rice may affect nontarget arthropod populations in rice fields by decreasing the number of C. suppressalis, Cnaphalocrocis medinalis, and other lepidopterous target insects (Tu et al. 2000; Han et al. 2006, 2007, 2011). This in turn could affect the diet of resident R. nigromaculata. In the current study, we set artificial fences to restrict the movement of R. nigromaculata so that their stomach contents generally reflected the distribution of insects in the different rice fields. Froglets
raised in Bt rice fields fed primarily on spiders and other non-target insects, whereas froglets raised in non-Bt rice fields ate primarily stem borers. This result is consistent with reports discussed above (Tu et al. 2000; Han et al. 2006, 2007, 2011). Different frog species have different feeding habits. R. nigromaculata eat mainly Nematoda in Protocoelomata, Oligochaeta in Annelida, Shoe Cephalopoda in Mollusca, and Crustacea, Myriapoda, Arachnoidea, and Hexapeda in Arthropoda. They may also unintentionally swallow grass leaves, grass seeds, rice grains, duckweed, and sand. The
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beneficial coefficient (used to measure the impact of beneficial insects or animals on crops or pests) for R. nigromaculata is high, but changing habitats and other forces may affect this coefficient (Chen 2011). In our experiment, R. nigromaculata in Bt rice fields fed on spiders and nontarget insects, thereby reducing their beneficial coefficient. It has been shown that R. nigromaculata of different weights and sizes consume different prey (Hirai 2002). Here, R. nigromaculata raised in the presence of pesticides (T? and N? groups) were smaller than frogs not exposed to pesticides, and were more inclined to consume small prey, such as nematodes. In contrast, R. nigromaculata in the T and N groups fed on larger prey, such as spiders. Cry1Ab/1Ac specifically binds receptors that localize to gut epithelial cells of target insects. This results in perforation of the cell membrane, cell swelling and lysis, and ultimately death of the target insect (Hofmann et al. 1988). In the present study, R. nigromaculata in the N and T groups consumed different foods, but this did not affect the histology of stomach or intestinal tissues. Compared with non-pesticide groups (T and N), R. nigromaculata in the pesticide groups (T? and N?) exhibited undeveloped gastric tunica muscularis and prominent protrusions of the gastric mucosa into the gastric cavity. These histological differences may have resulted from the lack of food caused by pesticide spraying (i.e., inducing a fasting state), as hunger can affect the histology of gastrointestinal tissues (Tam and Avenant-Oldewage 2009; Zeng et al. 2012). Alternatively, these differences may have resulted from pesticide toxicity. Pesticides also affected the density of aquatic tadpoles. This study used organophosphorus pesticides such as trichloride, endosulfan, and chlorpyrifos, which are highly toxic to aquatic organisms and likely caused a large number of tadpole deaths within a short period (Sparling and Fellers 2007; Jones et al. 2009). The planting of transgenic cry1Ab/ 1Ac rice did not adversely affect tadpole density, but further studies are required to determine whether there are any long-term effects. Our results indicate that transgenic cry1Ab/1Ac rice did not affect tadpole density or frog body weight, whereas the application of pesticides quickly killed tadpoles and reduced the body weight of surviving froglets. Previous studies concerning the effects of Bt gene products on non-target aquatic organisms have yielded inconsistent results. Adverse effects were observed when D. magna (Diplostraca: Daphniidae) were fed flour from Bt maize containing cry1Ab (Bøhn et al. 2008, 2010) or when two species of caddisflies (Trichoptera) were fed Bt maize tissue or pollen (Rosi-Marshall et al. 2007). However, Jensen et al. (2010) could not confirm the Trichoptera results. In addition, Raybould and Vlachos (2011) exposed D. magna to the Bt vegetative insecticidal protein Vip3Aa20 and observed no effect on survival or fecundity
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compared with controls, but they did observe a small reduction in growth. Some researchers have proposed that the adverse effects observed in D. magna and larval of Trichoptera are caused by factors other than the Cry protein (Ricroch et al. 2010; Romeis et al. 2013). Bt proteins expressed by transgenic crops may be passed down the food chain, but concentrations are low at higher trophic levels (Garcı´a et al. 2010; Li and Romeis 2010). Frogs are high trophic–level organisms in farmland ecosystems and may consume prey that contain some target proteins (transgenic rice ? target or non-target pests ? frogs). Moreover, the relatively high permeability of frog skin makes it likely that frogs would be exposed to Bt proteins accumulated in water. However, we did not detect any Cry1Ab/1Ac protein in frog tissues collected at different time points, suggesting that Bt proteins do not accumulate in frogs. In addition, we fed Xenopus laevis froglets diets containing HH1 rice grain under laboratory conditions. Their survival rate, body and organ weight, and microstructure of the digestive tract were detected, and no adverse effects were observed (Zhu et al., unpublished data). So, we conclude that transgenic cry1Ab/1Ac rice line HH1 does not adversely affect paddy frogs and that planting Bt rice will promote the survival and growth of frogs compared with pesticide spraying of non-Bt rice. Acknowledgments We thank Professor Yongjun Lin (Huazhong Agricultural University, Wuhan, China) for kindly providing transgenic rice seeds. We also thank Professor Fajun Chen (Nanjing Agricultural University, Nanjing, China) for his constructive comments on the early draft of this manuscript. This work was supported by the National GMO New Variety Breeding Program of the PRC (2012ZX08011-002 and 2014ZX08011-001). Conflict of interest of interest.
The authors declare that they have no conflict
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