Effects Of Transgenic Bt Rice On Several Typical Aquatic Organisms

The production of insecticidal crystal proteins in Bacillus thuringiensis (Bt) normally coincides with sporulation, resulting in the appearance of parasporal crystalline inclusions within the mother cell. These toxins have strict specificity to target insects providing a feasible chemical control method. At present, several genetically modified plants containing Bt genes have been commercial cultivated to protect the crop from a particular pest. The Bt proteins in genetically modified crops could conceivably enter aquatic systems from agricultural fields through multiple routes such as root exudates, pollen dispersion and crop byproducts, therefore, it is extremely necessary to evaluate the potential risk of the Bt protein to the aquatic organisms in the water environment. The Chlorella pyrenoidosa, Daphnia magna, and amphibian species of frogs are common aquatic organisms in paddy fields, representing the primary producer, primary consumer and secondary consumer in the food chain. The objective of this study is to evaluate the environmental safety of transgenic Bt rice cultivation on the above three aquatic organisms by a series of indoor and outdoor experimental research, using the Cry1Ca purified protein, crylCa gene insect-resistant rice (T1C-19), crylAb/Ac gene insect-resistant rice (HH1) and their isoline (MH63) as the research material. The results are as follows:1) Effects of purified Cry1Ca protien on the growth of Chlorella pyrenoidosa:The effect of purified Cry1Ca insecticidal protein on the growth of Chlorella pyrenoidosa was studied through a three generation toxicity test. The C. pyrenoidosa medium with a density of5.4x105cell/mL was subcultured for three generations by adding Cry1Ca protein at levels of0,10,100 and1000μg/L, and cell numbers determined daily. To explore the distribution of Cry1Ca in C. pyrenoidosa and the culture medium, Cry1Ca was added to algae with a high density of4.8x106cell/ml at1000μg/L, and daily Cry1Ca content in C. pyrenoidosa and the culture medium were determined by enzyme-linked immunosorbent assays (ELISA). Our results showed that:①Growth curves of C. pyrenoidosa exposed to10,100and1000μg/L of Cry1Ca almost overlapped with the blank control, and there were no statistically significant differences among the four treatments from day0to day7, regardless of generation.②The content of Cry1Ca in the culture medium and in C. pyrenoidosa sharply decreased under high concentration exposure with high initial C. pyrenoidosa cells.2) Effects of straw Ieachates from Cry1Ca-expressing transgenic rice on the growth of chlorella pyrenoidosa:To assess the effects of purified Bt Cry1Ca and leachates extracted from Cry1Ca-expressing transgenic rice (T1C-19) straw on the microalga Chlorella pyrenoidosa, we added purified Cry1Ca (10,100, and1000μg/l) and five concentrations of diluted extracts (5,10,20,40, and80%) from T1C-19and the non-transformed control strain MH63into the medium of C. pyrenoidosa. We determined the ability of C. pyrenoidosa to absorb the Bt protein and the effects of straw leachate on algal growth and enzymatic activity. We found that①The order of C. pyrenoidosa growth rates for the TIC-19leachate concentration groups was5%>10%>20%>control>40%>80%and for the MH63concentration groups was5%>10%>control>20%>40%>80%, but there were no statistical differences between the20%TIC-19or20%MH63leachate treatment and the medium control on day8;②After7days of culture, CrylCa could be detected in C. pyrenoidosa treated with different concentrations of T1C-19straw leachate; however, the protein was almost undetectable in the T1C-19culture medium and the MH63control.3) Effects of Cry1Ca-expressing transgenic rice on the growth and reproduction of Daphnia magna:Daphnia magna was exposed to500μg/L,250μg/L and125μg/L Cry1C protein to survey the effect of Cry1C protein on the Daphnia magna. At the same time. Daphnia magna were fed by the transgenic rice flour and its isoline rice flour as food source to assessment the safety of Bt rice. The results show that:①Cry1C protein had no significant influence on the mortality, body weight, body length and body width of D. magna in7-day exposure test;②The high concentration of500g/L Cry1Ca protein has adverse effects on the growth and reproduction of D. magna in21-day exposure test, but this adverse effect remains to be further confirmed.③The growth and reproductive performance have no significant differences between TIC-19and MH63groups of the first generation (Fo), and the second generation (F1) of D. magna fed by the rice flour.4) Effects of Cry1Ca-expressing transgenic rice on the growth of Xenopus Laevis tadpoles and larvae:By adding Cry1Ca pure protein and rice straw leaching solution into water and feeding Xenopus Laevis larvae feed containing T1C-19rice, the experiment was designed to ensure whether CrylCa-expressing transgenic rice effect the growth and development.We found that:①There were no significant differences among1C-100g/L,1C-10g/L and control groups in terms of survival rate, body weight, body length, time to completed metamorphosis, viscera weight and enzymatic activity in liver (p>0.05).②Although the groups of adding straw leachate were significant difference with the control group in some detection index (p<0.05), but there was no significant difference between T1C-19and MH63groups.③There was no significant effect on the mortality rate, body weight, daily gain weight, liver weight and body fat of the Xenopus laevis froglets between T1C-19and MH63groups after90days of feeding the diets containing TIC-19rice grain, and there were also no abnormal pathological changes in the organs of the stomach, intestines, liver, kidney and gonads.5) Influence of transgenic rice expressing a fused CrylAb/1Ac protein on frogs in paddy fields:Genetic engineering techniques have been used to control agricultural pests. It is important to determine, however, whether such transgenic plants adversely affect non-target organisms within and around cultivated fields. The crylAb/Ac fusion gene from Bacillus thuringiensis (Bt) has insecticidal activity and has been introduced into the rice line Minghui63(MH63). To evaluate the effect of transgenic crylAb/1Ac rice (Huahui1, HH1) on paddy frogs, we established four treatment groups:HH1without pesticides, MH63without pesticides, HH1with pesticides, and MH63with pesticides. The density of tadpoles in the rice fields was surveyed at regular intervals. 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. Levels of Cryl Ab/1Ac were also determined in tissues of tadpoles and frogs collected from the paddy fields. We found that①the introduction of Bt gene into rice did not affect tadpole density or the weight of Rana nigromaculata froglets. In contrast, application of pesticides resulted in the rapid death of tadpoles and affected the growth of froglets.②R. nigromaculata froglets raised in transgenic rice fields primarily fed on spiders and other non-target insects, but this did not affect the microstructure of their digestive tracts.③No Cry1Ab/1Ac was detected in tissue samples collected from these paddy frogs.The planting of insect-resistant protein expressing transgenic rice had no negative effect on the three kinds of typical aquatic organisms(chlorella, large fleas and frogs), but the problems about Bt protein residual and cumulative effect need to be researched further.