Investigating the presence of transgenic crop constructs in DNA of organisms of aquatic ecosystems

PhD (Environmental Sciences), North-West University, Potchefstroom CampusGenetically modified Bt crops, which produce proteins toxic to certain agricultural pests, have been cultivated for two decades. In that time, several hundred papers examining their interaction with terrestrial ecosystems have been published, while fewer than 50 have been published which deal with aquatic ecosystems. Aquatic organisms are exposed to Bt crop mostly through deposition of crop detritus due to wind, rain, and runoff. Bt transgenes and proteins are released into the water, present in the crop detritus, and present in the food chain as organisms consume Bt plant material and/or other organisms which have consumed the plant material. Interactions between Bt crops and adjacent ecosystems may be positive, due to the replacement of other, more harmful pesticides. Questions have been raised, however about potential effects on non-target organisms, for horizontal gene transfer (HGT) of transgenes to bacteria, and potential effects on ecosystem services and biogeochemical cycling have also been raised. In this study, DNA-based methods were used to better understand the interactions between Bt maize MON810, and the aquatic environment of the Vaalharts Irrigation Scheme, a farming area in the North-West Province with a high rate of Bt maize adoption. The irrigation system comprises a variety of aquatic environments, including dams, rivers, and canals. Macroinvertebrates and water samples were collected from sites spread through the irrigation system. Samples were also collected from the Tshiombo Irrigation System in Venda, which was used as a control site since no Bt maize was grown in that area at the time of sampling. Macroinvertebrates were identified according to morphology. Microorganisms (bacteria, yeast and fungi) were cultured from the water samples on a variety of media. Following DNA isolation, a PCR-based approach using MON810 primers (CM01 and CM02 and Hsp70 and cry1Ab primers being the most successful of the batch) was used to detect transgene DNA in the DNA isolated from the aquatic organisms. Positive results were detected in 7 macroinvertebrates, 56 bacteria isolates, and 20 yeast and fungi isolates. In the case of the macroinvertebrates, this was taken as an indication of exposure to Bt plant material, most likely through diet. In microorganisms, the presence of transgene sequences was seen as a potential occurrence of HGT. A selection of bacterial isolates was chosen for whole genome sequencing (Illumina MiSeq). These bacteria were identified as Aeromonas veronii, A. salmonicida, Arthrbacter sp., Pseudomonas mendocina, P. protegens, Massilia sp., and Serratia fonticola. It was hoped that detection of transgene fragments in the assembled genomes of the selected organisms would provide information regarding the genomic context of the insertion sites and any genes which had been interrupted due to recombination with transgene fragments. However, after scrutinising the genomes and the sequencing reads using a mapping based approach (Daisy and a BWA-MEM), traces of transgene DNA could not be detected in the isolates' draft assemblies. This may be due to a lack of sequencing coverage in some areas of the genome, or possibly due to loss of the sequences over time. Though HGT was not detected in this study, there is still a need to take the possibility of HGT of transgene constructs borne by genetically modified plants seriously. This study has contributed towards filling the knowledge gap regarding the interaction of Bt crops and aquatic environments by providing information on exposure of aquatic macroinvertebrates to Bt crops, and by surveying the microbial community for potential HGT of transgenic DNA. A workflow which could be used in future studies detecting transfer of short DNA fragments was developed. Recommendations regarding the monitoring of Bt crops and aquatic ecosystems more generally have been made, as well as suggestions for how HGT of transgenic fragments might be detected in a high-throughput, culture-free method in future.Doctora