Sequence-Activity Relationships for the Snf7 Insecticidal dsRNA in Chrysomelidae.

The responsiveness of insects to oral delivery of insecticidal dsRNA has been shown to be dependent on dsRNA length and sequence match. Previous work with the western corn rootworm (WCR, Diabrotica virgifera virgifera; Coleoptera: Chrysomelidae) demonstrated that at least one ≥21 nt match must be present in the DvSnf7 dsRNA of approximately ≥60 base-pairs (bp) for activity. Further data is needed on the activity of <21 nt matches along with characterization of relationship between activity and the number of ≥21 nt matches. To characterize the sequence-activity relationship for insecticidal dsRNA further, the activity of orthologous Snf7 dsRNAs with 19, 20, and 21 nt contiguous matches against WCR was compared. Neither 19 nor 20 nt sequence matches were active, supporting that a ≥21 nt sequence match is required for activity. The relationship between the number of 21 nt matches with activity of Snf7 dsRNA orthologs from several Chrysomelid species was characterized using WCR and Colorado potato beetle (CPB, Leptinotarsa decemlineata; Coleoptera Chrysomelidae). For WCR, there was a strong relationship between an increasing number of 21 nt matches and increased activity (i.e., lower LC50 values). A similar relationship was observed for CPB with an exception for a single ortholog, which may be related to the exceptionally high rate of polymorphisms in CPB. Overall, these results demonstrate a general relationship between the number of 21 nt matches and activity, and this relationship could be used to inform a testing and assessment plan for an ecological risk assessment for an insecticidal dsRNA.

Environmental Fate and Dissipation of Applied dsRNA in Soil, Aquatic Systems, and Plants.

Two primary use patterns exist for dsRNA-based products for crop protection: in planta produced dsRNA such as in a genetically engineered (GE) crop; and topically applied dsRNA such as a spray application. To enable effective environmental risk assessments for these products, dsRNA must be successfully measured in relevant environmental compartments (soil, sediment, surface water) to provide information on potential exposure. This perspective reviews results from numerous environmental fate and degradation studies with topically applied unformulated dsRNAs to demonstrate the high lability of these molecules and low potential for persistence in the environment. Additionally, we report on results of a pilot study of topically applied dsRNA on soybean plants demonstrating similar rapid degradation under field conditions. Microbial degradation of nucleic acids in environmental compartments has been shown to be a key driver for this lack of persistence. In fact, the instability of dsRNA in the environment has posed a challenge for the development of commercial topically-applied products. Formulations or other approaches that mitigate environmental degradation may lead to development of commercially successful products but may change the known degradation kinetics of dsRNAs. The formulation of these products and the resultant impacts on the stability of the dsRNA in environmental compartments will need to be addressed using problem formulation and product formulation testing may be required on a case by case basis to ensure an effective risk assessment.

Characterization of the Activity Spectrum of MON 88702 and the Plant-Incorporated Protectant Cry51Aa2.834_16.

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).

Ecological risk assessment for DvSnf7 RNA: A plant-incorporated protectant with targeted activity against western corn rootworm.

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.

No impact of DvSnf7 RNA on honey bee (Apis mellifera L.) adults and larvae in dietary feeding tests.

The honey bee (Apis mellifera L.) is the most important managed pollinator species worldwide and plays a critical role in the pollination of a diverse range of economically important crops. This species is important to agriculture and historically has been used as a surrogate species for pollinators to evaluate the potential adverse effects for conventional, biological, and microbial pesticides, as well as for genetically engineered plants that produce pesticidal products. As part of the ecological risk assessment of MON 87411 maize, which expresses a double-stranded RNA targeting the Snf7 ortholog (DvSnf7) in western corn rootworm (Diabrotica virgifera virgifera), dietary feeding studies with honey bee larvae and adults were conducted. Based on the mode of action of the DvSnf7 RNA in western corn rootworm, the present studies were designed to be of sufficient duration to evaluate the potential for adverse effects on larval survival and development through emergence and adult survival to a significant portion of the adult stage. Testing was conducted at concentrations of DvSnf7 RNA that greatly exceeded environmentally relevant exposure levels based on expression levels in maize pollen. No adverse effects were observed in either larval or adult honey bees at these high exposure levels, providing a large margin of safety between environmental exposure levels and no-observed-adverse-effect levels.

Independent action between DvSnf7 RNA and Cry3Bb1 protein in southern corn rootworm, Diabrotica undecimpunctata howardi and Colorado potato beetle, Leptinotarsa decemlineata.

In recent years, corn rootworm (CRW)-resistant maize events producing two or more CRW-active Bt proteins have been commercialized to enhance efficacy against the target pest(s) by providing multiple modes of action (MoA). The maize hybrid MON 87411 has been developed that produces the CRW-active Cry3Bb1 Bt protein (hereafter Cry3Bb1) and expresses a RNAi-mediated MoA that also targets CRW. As part of an environmental risk assessment for MON 87411, the potential for an interaction between the CRW-active DvSnf7 RNA (hereafter DvSnf7) and Cry3Bb1 was assessed in 12-day diet incorporation bioassays with the southern corn rootworm (SCR, Diabrotica undecimpunctata howardi). The potential for an interaction between DvSnf7 and Cry3Bb1 was evaluated with two established experimental approaches. The first approach evaluated each substance alone and in combination over three different response levels. For all three response levels, observed responses were shown to be additive and not significantly different from predicted responses under the assumption of independent action. The second approach evaluated the potential for a fixed sub-lethal concentration of Cry3Bb1 to decrease the median lethal concentration (LC50) of DvSnf7 and vice-versa. With this approach, the LC50 value of DvSnf7 was not altered by a sub-lethal concentration of Cry3Bb1 and vice-versa. In addition, the potential for an interaction between the Cry3Bb1 and DvSnf7 was tested with Colorado potato beetle (CPB, Leptinotarsa decemlineata), which is sensitive to Cry3Bb1 but not DvSnf7. CPB assays also demonstrated that DvSnf7 does not alter the activity of Cry3Bb1. The results from this study provide multiple lines of evidence that DvSnf7 and Cry3Bb1 produced in MON 87411 have independent action.

Characterization of the spectrum of insecticidal activity of a double-stranded RNA with targeted activity against Western Corn Rootworm (Diabrotica virgifera virgifera LeConte).

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.

Characterizing the mechanism of action of double-stranded RNA activity against western corn rootworm (Diabrotica virgifera virgifera LeConte).

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.

Genetically modified crops and aquatic ecosystems: considerations for environmental risk assessment and non-target organism testing.

Environmental risk assessments (ERA) support regulatory decisions for the commercial cultivation of genetically modified (GM) crops. The ERA for terrestrial agroecosystems is well-developed, whereas guidance for ERA of GM crops in aquatic ecosystems is not as well-defined. The purpose of this document is to demonstrate how comprehensive problem formulation can be used to develop a conceptual model and to identify potential exposure pathways, using Bacillus thuringiensis (Bt) maize as a case study. Within problem formulation, the insecticidal trait, the crop, the receiving environment, and protection goals were characterized, and a conceptual model was developed to identify routes through which aquatic organisms may be exposed to insecticidal proteins in maize tissue. Following a tiered approach for exposure assessment, worst-case exposures were estimated using standardized models, and factors mitigating exposure were described. Based on exposure estimates, shredders were identified as the functional group most likely to be exposed to insecticidal proteins. However, even using worst-case assumptions, the exposure of shredders to Bt maize was low and studies supporting the current risk assessments were deemed adequate. Determining if early tier toxicity studies are necessary to inform the risk assessment for a specific GM crop should be done on a case by case basis, and should be guided by thorough problem formulation and exposure assessment. The processes used to develop the Bt maize case study are intended to serve as a model for performing risk assessments on future traits and crops.

Development and survival of Orius insidiosus (Say) nymphs on encapsulated bee pollen-based diet in a Tier-I toxicity assay.

The insidious flower bug, Orius insidiosus (Say) (Heteroptera: Anthocoridae) is an important surrogate species for assessing potential effects of plant-incorporated protectants (PIPs) on nontarget heterotrophic predators. In this study, a continuous dietary exposure system was optimized by assessing the effect of diet composition and age on the survival and development of nymphs of O. insidiosus. Greater than 85% control survival and an acceptable rate of development from nymph hatching to adult was achieved using 5-d-old nymphs at test initiation and a bee pollen-based diet supplemented with 25% Ephestia eggs. There was an unacceptable level of mortality (>40%) and/or a significantly prolonged development time when nymphs were <5 d old at test initiation. When 5-d-old nymphs were fed a bee pollen diet containing 25% Ephestia eggs and 100 µg/g potassium arsenate, time-dependent mortality was observed with a median lethal time (LT50) of 4.4 d and 100% mortality was observed after 10 d of feeding, indicating the effectiveness of the test system to detect adverse effects by dietary exposure. It is recommended that well-defined 5-d-old nymphs and an encapsulated bee pollen-based diet containing 25% ground Ephestia eggs be used in a Tier-I dietary feeding exposure assay for detecting potential effects of PIPs on O. insidiosus nymphs.

Effects of salinity on native estuarine fish species in South Florida.

Variable and high salinities have been identified as key stressors in Florida Bay. The Comprehensive Everglades Restoration Plan (CERP) includes water redistribution projects that are intended to restore natural freshwater flows to northeastern Florida Bay. The present salinity regimes in the area, which span from hypo- to hypersaline, will be altered as a result of these actions. This research examined biological performance measures (i.e., growth and survival) of estuarine fish under varying salinity regimes that will occur as a result of the restoration of freshwater flow to the Bay. A series of acute and subchronic studies were conducted to determine the effects of salinity changes on various life stages (embryo/larval, juvenile, adult) of four native estuarine fish (Cyprinodon variegatus, Floridichthys carpio, Poecilia latipinna, and Gambusia holbrooki). Fish were exposed to a range of salinity concentrations (freshwater to hypersaline) based on current salinity profiles in the study areas. Growth (length, weight), abnormalities, and survival were measured. Salinity exposures included both rapid and gradual change events. Results show adverse effects of acute, abrupt salinity changes on fish survival and development due to salinity stress.