2013 Annual Report
1a.Objectives (from AD-416):
The overall goal of this proposal is to determine the likelihood of exposure to and toxicity of interference RNA to a corn-based arthropod food web. Specifically, this research will establish which species are at risk through consuming dsRNA containing corn tissue under field conditions, and whether dsRNAs are transferred to higher trophic levels via consuming herbivorous prey. The research will establish crucial infrastructure that can be used to establish risk of both existing RNAi-based GM crops as well as future constructs.
1. Use PCR-based gut content analysis to establish trophic linkages to corn within an arthropod community.
2. Establish whether dsRNA passes to higher trophic levels (predators and parasitoids) via consuming herbivorous prey and which natural enemies are exposed.
3. Sequence the genomes of key taxa from corn to determine whether sequence homologies exist that place these organisms at risk from crop-produced dsRNA.
1b.Approach (from AD-416):
1) qPCR-based gut analysis will be applied to the corn arthropod community to generate a taxon-specific consumption index of corn tissue that is based on the frequency of consumption and the quantity of DNA consumed. A preliminary description of the corn arthropod community has produced gut-DNA extractions from 5,672 arthropods collected from corn fields of eastern South Dakota. We have developed and published primer sets that amplify a 141 bp fragment of the corn COI gene that is useful in determining which arthropods have consumed corn tissue through gut analysis..
2)Laboratory based assays will be used to administer a range of putative insecticidal RNAi that have been identified in the literature to three pests with different feeding ecologies: Tetranychus urticae, Spodoptera frugiperda and Rhopalosiphum padi. These pests will be fed to a suite of predators, each with their own feeding ecologies (e.g, Coleomegilla maculata, Orius insidiosus, and Chrysoperla carnea). The presence of the interference RNA molecules in the various insects will be assessed using targeted PCR. If interference RNA is found to be present in the predators, then an exposure analysis of key herbivores and their predator communities will be conducted using PCR-based gut analysis..
3)Of those species that are exposed to the RNAi-containing corn or prey, specific indicator taxa will be selected from key functional groups and their genomes will be sequenced and placed into a searchable database. Sequence homologies between current and future RNAi targets can be screened against this database for targeting non-target toxicity assays.
Hiring for this project was delayed (and continues to be delayed) by the federal sequestration and internal challenges for filling vacancies within the USDA. Despite these hurdles, the post-doctoral and undergraduate assistant positions were filled, and research is underway (the GS-7 technician position remains to be filled). To describe the arthropod food web associated with corn, we identified 10 farmers producing field corn without the use of insecticides or GM traits that might affect insect communities. As of 7/29/2013, our research team has visited each of these sites twice. From each site, 50 plants, seven soil cores, and five soil surface samples (collected using quadrats) were randomly selected and all arthropods were frozen at -20° C for later gut analysis. A third sampling is planned for late August. Poecilus chalcites and Coleomegilla maculata were collected and are in culture for conducting the laboratory toxicity assays involving prey-fed dsRNAs. All milestones laid out in the project timeline have been successfully met.