RNA interference (RNAi) has previously been shown to be effective in western corn rootworm (WCR, Diabrotica virgifera virgifera LeConte) larvae via oral delivery of synthetic double-stranded RNA (dsRNA) in an artificial diet bioassay, as well as by ingestion of transgenic corn plant tissues engineered to express dsRNA. Although the RNAi machinery components appear to be conserved in Coleopteran insects, the key steps in this process have not been reported for WCR. Here we characterized the sequence of events that result in mortality after ingestion of a dsRNA designed against WCR larvae. We selected the Snf7 ortholog (DvSnf7) as the target mRNA, which encodes an essential protein involved in intracellular trafficking. Our results showed that dsRNAs greater than or equal to approximately 60 base-pairs (bp) are required for biological activity in artificial diet bioassays. Additionally, 240 bp dsRNAs containing a single 21 bp match to the target sequence were also efficacious, whereas 21 bp short interfering (si) RNAs matching the target sequence were not. This result was further investigated in WCR midgut tissues: uptake of 240 bp dsRNA was evident in WCR midgut cells while a 21 bp siRNA was not, supporting the size-activity relationship established in diet bioassays. DvSnf7 suppression was observed in a time-dependent manner with suppression at the mRNA level preceding suppression at the protein level when a 240 bp dsRNA was fed to WCR larvae. DvSnf7 suppression was shown to spread to tissues beyond the midgut within 24 h after dsRNA ingestion. These events (dsRNA uptake, target mRNA and protein suppression, systemic spreading, growth inhibition and eventual mortality) comprise the overall mechanism of action by which DvSnf7 dsRNA affects WCR via oral delivery and provides insights as to how targeted dsRNAs in general are active against insects.

The sequence specificity of the endogenous RNA interference pathway allows targeted suppression of genes essential for insect survival and enables the development of durable and efficacious insecticidal products having a low likelihood to adversely impact non-target organisms. The spectrum of insecticidal activity of a 240 nucleotide (nt) dsRNA targeting the Snf7 ortholog in Western Corn Rootworm (WCR; Diabrotica virgifera virgifera) was characterized by selecting and testing insects based upon their phylogenetic relatedness to WCR. Insect species, representing 10 families and 4 Orders, were evaluated in subchronic or chronic diet bioassays that measured potential lethal and sublethal effects. When a specific species could not be tested in diet bioassays, the ortholog to the WCR Snf7 gene (DvSnf7) was cloned and corresponding dsRNAs were tested against WCR and Colorado potato beetle (Leptinotarsa decemlineata); model systems known to be sensitive to ingested dsRNA. Bioassay results demonstrate that the spectrum of activity for DvSnf7 is narrow and activity is only evident in a subset of beetles within the Galerucinae subfamily of Chrysomelidae (>90 % identity with WCR Snf7 240 nt). This approach allowed for evaluating the relationship between minimum shared nt sequence length and activity. A shared sequence length of ≥21 nt was required for efficacy against WCR (containing 221 potential 21-nt matches) and all active orthologs contained at least three 21 nt matches. These results also suggest that WCR resistance to DvSnf7 dsRNA due to single nucleotide polymorphisms in the target sequence of 240 nt is highly unlikely.Electronic supplementary materialThe online version of this article (doi:10.1007/s11248-013-9716-5) contains supplementary material, which is available to authorized users.

MON 87411 maize, which expresses DvSnf7 RNA, was developed to provide an additional mode of action to confer protection against corn rootworm (Diabrotica spp.). A critical step in the registration of a genetically engineered crop with an insecticidal trait is performing an ecological risk assessment to evaluate the potential for adverse ecological effects. For MON 87411, an assessment plan was developed that met specific protection goals by characterizing the routes and levels of exposure, and testing representative functional taxa that would be directly or indirectly exposed in the environment. The potential for toxicity of DvSnf7 RNA was evaluated with a harmonized battery of non-target organisms (NTOs) that included invertebrate predators, parasitoids, pollinators, soil biota as well as aquatic and terrestrial vertebrate species. Laboratory tests evaluated ecologically relevant endpoints such as survival, growth, development, and reproduction and were of sufficient duration to assess the potential for adverse effects. No adverse effects were observed with any species tested at, or above, the maximum expected environmental concentration (MEEC). All margins of exposure for NTOs were >10-fold the MEEC. Therefore, it is reasonable to conclude that exposure to DvSnf7 RNA, both directly and indirectly, is safe for NTOs at the expected field exposure levels.

Determining the rate of biodegradation of double-stranded RNA (dsRNA) in the environment is an essential element of a comprehensive risk assessment of an RNA-based agricultural product. This information is used during problem formulation to define relevant routes and durations of environmental exposure for in planta-expressed dsRNA. Although exposure to biotechnology-derived crops expressing dsRNA traits in the aquatic environment is predicted to be minimal, little is known regarding the fate of dsRNA in these environments. To assess exposure to aquatic environments, a study was conducted to measure the rate of biodegradation of DvSnf7 dsRNA in aerobic water-sediment systems. Aquatic systems containing natural water and sediments that varied in physical and chemical characteristics were treated with dsRNA by applying DvSnf7 dsRNA directly to the water column. In the present study, DvSnf7 dsRNA dissipated rapidly from the water phase and was undetectable within 7 d as measured by QuantiGene (Affymetrix) and a sensitive insect bioassay in these diverse systems. Degradation kinetics estimated a half-life (time to 50% dissipation [DT50]) of less than 3 d and a time to 90% dissipation of approximately 4 d. Further analysis indicated that DvSnf7 dsRNA had DT50 values of less than 6 d in both sediment-free systems containing natural water and systems with only sediment. Taken together, the results of the present study indicate that dsRNA-based agricultural products rapidly degrade and consequently are unlikely to persist in aquatic environments. Environ Toxicol Chem 2017;36:727-734. © 2016 SETAC.

The spectrum of insecticidal activity of Cry51Aa2.834_16 protein targeting hemipteran and thysanopteran insect pests in cotton was characterized by selecting and screening multiple pest and non-pest species, based on representation of ecological functional groups, taxonomic relatedness (e.g. relationship to species where activity was observed), and availability for effective testing. Seven invertebrate orders, comprising 12 families and 17 representative species were screened for susceptibility to Cry51Aa2.834_16 protein and/or the ability of the protein to protect against feeding damage in laboratory, controlled environments (e.g. greenhouse/growth chamber), and/or field studies when present in cotton plants. The screening results presented for Cry51Aa2.834_16 demonstrate selective and limited activity within three insect orders. Other than Orius insidiosus, no activity was observed for Cry51Aa2.834_16 against several groups of arthropods that perform key ecological roles in some agricultural ecosystems (e.g. pollinators, decomposers, and natural enemies).