Recipient Organization
RICE UNIVERSITY
PO BOX 1892
HOUSTON,TX 77251
Performing Department
BioSciences at Rice
Non Technical Summary
Understanding the dispersal and non-target impact of genetically engineered (GE) organisms and their byproducts is a critical component of the safe and responsible use of transgenic technology in the environment. However, we currently lack the ability to track the movement and non-target effects of GE organisms and their byproducts in the environment, even though previous work has demonstrated that GE organisms can escape, or be unintentionally transported, from their intended range. For example, GE crop byproducts can disperse off of agricultural fields to be transported throughout river networks, and these byproducts can have non-target effects on aquatic organisms.As one example, insect-resistant GE maize is now planted throughout North America and globally, with >80% of maize in the US planted in Bt or stacked varieties. Bt maize is one of the more common insect-resistant GE maize varieties, which expresses the insecticidal Cry1Ab protein from Bacillus thuringiensis to resist crop damage by the European corn borer (Ostrinia nubilalis), a moth in the family Crambidae (Lepidoptera). The Cry1Ab protein is expressed throughout the tissues of Bt maize, and after maize is harvested, the protein remains detectable in detrital material for up to seven months. The potential effects of Bt maize detritus on non-target terrestrial organisms and ecosystems is of great interest to stake holders, federal agencies, and the general public. In addition, Bt maize detritus enters, is processed, and can be transported within streams and rivers flowing adjacent to crop fields. Laboratory trials suggested that consumption of Bt maize detritus may affect stream-dwelling invertebrates, but may be dependent on duration of leaching of submerged detritus. Thus, the potential effects of Bt maize on aquatic ecosystems have attracted a great deal of attention, despite only a handful of studies exploring this topic.Our multidisciplinary team, with training in evolutionary biology, entomology, stream ecology, and biogeochemistry, has developed a novel approach to address multiple program areas in the Biotechnology Risk Assessment Research Grants Program (BRAG), including methods to monitor dispersal of GE-organisms (Area #2) and assessing impacts of GE-organisms and their byproducts on non-target organisms (Area #4). Our novel approach uses evolution as a monitoring tool in freshwater ecosystems.We will apply this novel monitoring tool to an aquatic keystone species, the caddisfly (order Trichoptera), as our focal organism, and its response to the Cry-proteins produced by Bt-maize that have been shown to leach into aquatic environments as a pseudo-persistent contaminant. We will measure predicted changes across the caddisfly genome in response to the non-target effects of Cry-proteins as the metric to assess the incursion and impact of GE-organisms and their by-products in natural systems. We are confident that this novel approach will allow us to assess the effect of Cry-proteins in freshwater ecosystems across the agricultural Midwest, uniquely disentangling it from other environmental stressors (e.g., habitat degradation, eutrophication), and pioneer a novel method for the detection of GE-organisms and their impact on natural systems.Previous lab experiments have demonstrated that Cry-proteins have non-target effects on caddisflies, which are closely related to the Cry-proteins' targeted pests from the insect order Lepidoptera. Specifically, the target organisms for Cry-proteins are most often pest caterpillars in the insect order Lepidoptera, which includes moths and butterflies, but the closest relative or sister group to these targets is the insect order Trichoptera, which encompasses the aquatic keystone species, the caddisflies. Therefore, based on their evolutionary relationships, Cry-proteins are predicted to act as a selective pressure in agriculturally-impacted freshwaters, just as it has acted on terrestrial agricultural Lepidopteran pests. This evolutionary linkage highlights the general approach of this proposal using evolution as a monitoring tool in an applied environmental context. Here, by informing our approach using the evolutionary relationships between intended targets of Bt-Cry and potential non-target organisms, we can zero in on the species groups of greatest concern.Our overarching hypothesis is that Cry-proteins will generate unique genomic signatures of selection in genes associated with Cry-protein resistance in aquatic caddisflies just as it has done in its closely related terrestrial counterparts and that we can use that signature across the genome as a tool for GE detection in nature. Thus, we will use an experimental genomics approach to monitor the potential impact of Cry-proteins on keystone taxa common in agricultural streams.
Animal Health Component
50%
Research Effort Categories
Basic
50%
Applied
50%
Developmental
(N/A)
Goals / Objectives
Understanding the dispersal and non-target impact of genetically engineered (GE) organisms and their byproducts is a critical component of the safe and responsible use of transgenic technology in the environment. However, we currently lack the ability to track the movement and non-target effects of GE organisms and their byproducts in the environment, even though previous work has demonstrated that GE organisms can escape, or be unintentionally transported, from their intended range. Our multidisciplinary team, with training in evolutionary biology, entomology, stream ecology, and biogeochemistry, has developed a novel approach to address multiple program areas in the Biotechnology Risk Assessment Research Grants Program (BRAG), including methods to monitor dispersal of GE-organisms (Area #2) and assessing impacts of GE-organisms and their byproducts on non-target organisms (Area #4). Our novel approach uses evolution as a monitoring tool in freshwater ecosystems.We will apply this novel monitoring tool to an aquatic keystone species, the caddisfly (order Trichoptera), as our focal organism, and its response to the Cry-proteins produced by Bt-maize that have been shown to leach into aquatic environments as a pseudo-persistent contaminant. We will measure predicted changes across the caddisfly genome in response to the non-target effects of Cry-proteins as the metric to assess the incursion and impact of GE-organisms and their by-products in natural systems. We are confident that this novel approach will allow us to assess the effect of Cry-proteins in freshwater ecosystems across the agricultural Midwest, uniquely disentangling it from other environmental stressors (e.g., habitat degradation, eutrophication), and pioneer a novel method for the detection of GE-organisms and their impact on natural systems.Our overarching hypothesis is that Cry-proteins will generate unique genomic signatures of selection in genes associated with Cry-protein resistance in aquatic caddisflies just as it has done in its closely related terrestrial counterparts and that we can use that signature across the genome as a tool for GE detection in nature. Thus, we will use an experimental genomics approach to monitor the potential impact of Cry-proteins on keystone taxa common in agricultural streams. To address the overarching hypothesis, our proposed research will address five interrelated Objectives. Objective 1: Perform laboratory mesocosm and controlled field experiments comparing the fitness of caddisflies under variable Bt-Cry exposure. Objective 2: Compare fitness differences between populations of caddisflies from streams across the Midwestern U.S. with variable influence from Bt-maize.Objective 3: Compare fitness differences between populations of caddisflies from streams across the Midwestern U.S. in historic museum samples collected prior to widespread use of Cry proteins to contemporary specimens (Obj. 2).Objective 4: Develop a panel of genomic regions associated with resistance to Cry-proteins based on a literature review on resistance mechanisms identified for agricultural pests. Objective 5: Sequence the genome of caddisflies from experiments (Obj. 1), field samples (Obj. 2), and museums specimens (Obj. 3) and apply evolutionary genomics tools to test for genomic regions associated with the potential impacts of Cry-proteins on freshwater ecosystems.
Project Methods
Our general plan includes three levels of comparison: experiments, field sampling, and museum studies. For each objective, we will then sequence groups of individuals that were exposed to Bt-Cry and those that were not; and apply a similar set of genomic and computational tools to compare regions of the genome that exhibit strong differentiation and thus strong association with survival and/or resistance to Bt-Cry. For Objective 1, we will challenge populations of caddisflies with varying levels of Bt-Cry that naturally leaches out of Bt-maize tissue. We will perform experiments at two levels of environmental complexity: the indoor Artificial Stream Mesocosm (ASM) Facility and the outdoor Linked Experimental Ecosystem Facility (LEEF), both housed at Notre Dame. For Objective 2, we will sample real-world stream populations in the field, targeting populations with varying exposure to Bt-Cry ranging from no exposure to high-exposure in agricultural steams draining maize fields. For Objective 3, we will sample museum specimens preserved prior to the widespread use of Bt maize in the Midwestern U.S. to capture the temporal changes associated with Bt-Cry exposure (see Letter of Support; Notre Dame Museum of Biodiversity). For Objective 4, we will produce a literature review of the genes associated with Bt-resistance across invertebrates (see Table 1; preliminary scan for this review). This review and synthesis will result in a stand-alone publication, but will also provide guidance for our interpretation of results. Using the caddisflies from each of these objectives, for Objective 5, we will use a common set of genetic and computational tools to compare genetic differences from organisms from the control/no Bt-Cry exposure with experimental/high Bt-Cry exposure. Concordant changes observed across the genome between experiments, spatial samples in nature, and temporal samples from museum specimens will isolate and identify the genes or gene regions associated with the impacts of Cry-proteins on caddisfly populations. While we focus on the filtering hydropsychid caddisfly, Hydropsyche betteni, as our exemplar, we remain flexible to the natural conditions in the field, and can adapt our strategy to other caddisfly species, if they prove to be more amenable to our proposed work. Overall, the goal of this proposal is that all objectives are interrelated and synergistic. To test for concordant genomic patterns across experiments for the same genes or gene regions, we will compare genetic differentiation (FST) for individual SNPs across comparisons to determine the extent to which divergence between populations under no Cry and Bt-Cry exposure consistently involves the same SNPs (i.e., parallelism). These analyses will result in diagnostic gene regions to be surveyed in novel populations to assess the direct influence of Bt-Cry on this aquatic keystone species.The results will be evaluated based on our ability to properly perform the experiments and genetic sequencing. Any resulting answer will be informative, including no concordance, which would imply little effect of Bt-Cry or non-parallel changes across experiments, to high concordance, where the same genomic regions exhibt strong differentiation across all Objecitves and provide a signature of evolutionary change that can be used to detect an influence of Cry on aquatic species. Key milestones are included in the Table below and include the systematic completion of each of the Objectives in the propopsal, as well as the presentation of our results in national scientific conferences, published in the peer-reviewd literature, and communicated clearly to the public and other stakeholders.General timeline for the proposed research.ObjectiveYear 1Year 2Year 31. ExperimentsXX2. Field samplingXX3. Museum surveysXXXX4. Review: Target genesXX5. Genomic test of Bt-CryXXXX*Publishing our work*XXX