Cry75Aa (Mpp75Aa) Insecticidal Proteins for Controlling the Western Corn Rootworm, Diabrotica virgifera virgifera, (Coleoptera: Chrysomelidae), Isolated from the Insect Pathogenic Bacteria Brevibacillus laterosporus.

This study describes three closely related proteins, cloned from Brevibacillus laterosporus strains, that are lethal upon feeding to Diabrotica virgifera virgifera LeConte, the western corn rootworm (WCR). Mpp75Aa1, Mpp75Aa2 and Mpp75Aa3 were toxic to WCR larvae when fed purified protein. Transgenic plants expressing each mMpp75Aa protein were protected from feeding damage and showed significant reduction in adult emergence from infested plants by both susceptible and Cry3Bb1 and Cry34Ab1/Cry35Ab1-resistant WCR. These results demonstrate that proteins from B. laterosporus are as efficacious as the well-known Bacillus thuringiensis (Bt) insecticidal proteins in controlling major insect pests such as WCR. The deployment of transgenic maize expressing mMpp75Aa along with other active molecules lacking cross-resistance have the potential to be a useful tool for control of WCR populations resistant to current Bt traits.IMPORTANCE Insects feeding on roots of crops can damage the plant roots resulting in yield loss due to poor water and nutrient uptake and plant lodging. In maize the western corn rootworm (WCR) can cause severe damage to the roots resulting in significant economic loss for farmers. Genetically modified (GM) expressing Bacillus thuringiensis (Bt) insect control proteins, has provided a solution for control of these pests. In recent years populations of WCR resistant to the Bt proteins in commercial GM maize have emerged. There is a need to develop new insecticidal traits for the control of WCR populations resistant to current commercial traits. New proteins with commercial level efficacy on WCR from sources other than Bt are becoming more critical. The Mpp75Aa proteins, from B. laterosporus, when expressed in maize, are efficacious against the resistant populations of WCR and have the potential to provide solutions for control of resistant WCR.

Bacillus thuringiensis Cry1Da_7 and Cry1B.868 Protein Interactions with Novel Receptors Allow Control of Resistant Fall Armyworms, Spodoptera frugiperda (J.E. Smith).

Two new modified Bacillus thuringiensis (Bt) proteins, Cry1Da_7 and Cry1B.868, with activity against fall armyworms (FAW), Spodoptera frugiperda (J.E. Smith), were evaluated for their potential to bind new insect receptors compared to proteins currently deployed as plant-incorporated protectants (PIPs) in row crops. Results from resistant insect bioassays, disabled insecticidal protein (DIP) bioassays, and cell-based assays using insect cells expressing individual receptors demonstrate that receptor utilizations of the newly modified Cry1Da_7 and Cry1B.868 proteins are distinct from each other and from those of commercially available Bt proteins such as Cry1F, Cry1A.105, Cry2Ab, and Vip3A. Accordingly, these two proteins target different insect proteins in FAW midgut cells and when pyramided together should provide durability in the field against this economically important pest.IMPORTANCE There is increased concern with the development of resistance to insecticidal proteins currently expressed in crop plants, especially against high-resistance-risk pests such as fall armyworm (FAW), Spodoptera frugiperda, a maize pest that already has developed resistance to Bacillus thuringiensis (Bt) proteins such as Cry1F. Lepidopteran-specific proteins that bind new insect receptors will be critical in managing current Cry1F-resistant FAW and delaying future resistance development. Results from resistant insect assays, disabled insecticidal protein (DIP) bioassays, and cell-based assays using insect cells expressing individual receptors demonstrate that target receptors of the Cry1Da_7 and Cry1B.868 proteins are different from each other and from those of commercially available Bt proteins such as Cry1F, Cry1A.105, Cry2Ab, and Vip3A. Therefore, pyramiding these two new proteins in maize will provide durable control of this economically important pest in production agriculture.

The sequence, structural, and functional diversity within a protein family and implications for specificity and safety: The case for ETX_MTX2 insecticidal proteins.

The need for sustainable insect pest control is driving the investigation and discovery of insecticidal proteins outside of the typical 3-domain Cry protein family from Bacillus thuringiensis (Bt). Examples include Cry35 and Cry51 that belong to protein families (Toxin_10, ETX_MTX2) sharing a common ß-pore forming structure and function with known mammalian toxins such as epsilon toxin (ETX). Although ß-pore forming proteins are related to mammalian toxins, there are key differences in sequence and structure that lead to organism specificity that is useful in the weight-of-evidence approach for safety assessment. Despite low overall amino acid sequence identity among ETX_MTX2 proteins, sequence and structural similarities are found in the tail region responsible for the shared oligomerization and pore formation functions (causing the "relatedness"). Conversely, most of the sequence and structural diversity is located in the head region that is likely responsible for differential receptor binding and target species specificity (e.g., insecticidal vs. mammalian). Therefore, inclusion of a domain-based protein characterization approach that includes bioinformatic and functional comparisons of conserved and diverse domains will enhance the overall weight of evidence safety assessment of proteins including recently reported Cry51 protein variants (Cry51Aa1, Cry51Aa2, and Cry51Aa2.834_16).

Testing the joint effects hypothesis of elemental defense using Spodoptera exigua.

Metal hyperaccumulation may be an elemental defense, in which high concentrations of a metal in plant tissues decrease herbivore survival or growth rate. The Joint Effects Hypothesis suggests that a combination of metals, or a combination of a metal with an organic compound, may have an enhanced defensive effect. The enhancement may be additive or synergistic: in either case the concentration of a particular metal necessary to provide a defensive benefit for the plant is lowered. We tested the Joint Effects Hypothesis using Spodoptera exigua (beet armyworm) neonates fed artificial diets. Metal + metal experiments utilized diets amended with metal pairs, using four metals commonly hyperaccumulated by plants (Co, Cu, Ni, and Zn). We also conducted metal + organic compound experiments, pairing each metal with nicotine, mustard seed powder, or tannic acid. We tested for joint effects using both lethal (LC20 levels) and sublethal concentrations (10-25 % reduced larval weight) of the chemicals tested. For all experiments, either additive or synergistic effects were found. Of the metal + metal pairs tested, three (Co + Cu, Cu + Zn, and Ni + Zn) were synergistic in lethal concentration tests and only Co + Cu was synergistic in sublethal tests. For metal + organic combination lethal tests, synergism occurred for all combinations except for Co or Ni + nicotine, Ni + mustard seed powder, and Zn + nicotine. For sublethal tests, Zn + all three organic chemicals, Co + mustard seed powder or tannic acid, and Cu + nicotine, were synergistic. These results support the Joint Effects Hypothesis, suggesting that metals combined with other metals or organic compounds may be more effective against herbivores than individual metals.

Larvae of Colorado potato beetle (Leptinotarsa decemlineata Say) resistant to double-stranded RNA (dsRNA) remain susceptible to small-molecule pesticides.

BACKGROUND: Implementation of resistance management tools is crucial for the continued efficacy of insect control technologies. An important aspect of insect resistance management (IRM) is the combined or sequential use of different modes-of-action to reduce selection pressure and delay evolution of resistance. This is especially important for insect pests with established ability to develop resistance to insecticides, such as the Colorado potato beetle (Leptinotarsa decemlineata, CPB). A new class of insecticides, based on double-stranded RNA (dsRNA) activating the gene silencing RNA-interference (RNAi) pathway, are currently under review for regulatory approval and commercial use in the USA against CPB. However, there is no information available on the potential for cross-resistance between RNAi insecticides and other classes of insecticides used against CPB. Herein, we aim to fill this knowledge gap by capitalizing on the availability of a CPB strain highly resistant to dsRNAs and test its susceptibility to diverse small-molecule insecticide classes compared to reference dsRNA-susceptible CPB strains. RESULTS: Differences in activity were observed among the four insecticides tested, with abamectin demonstrating highest activity against all three strains of CPB. However, no differences were observed among the dsRNA-resistant and susceptible CPB strains for any of the tested compounds. Overall, these results demonstrate lack of cross-resistance to commonly used chemical insecticides in the dsRNA-resistant strain of CPB. CONCLUSION: These data support the use of these different insecticide classes along with RNAi-based insecticides as part of an effective insect resistance management framework aimed at delaying resistance in CPB. © 2023 Society of Chemical Industry.

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.

Exploring lower limits of plant elemental defense by cobalt, copper, nickel, and zinc.

Elemental defense is a relatively newly recognized phenomenon in which plants use elements present in their tissue to reduce damage by herbivores or pathogens. In the present study, neonates of the generalist herbivore, Spodoptera exigua (Hübner) (Lepidoptera: Noctuidae), were fed artificial diets amended with varying concentrations of Co, Cu, Ni, and Zn that are hyperaccumulated by plants to determine minimum lethal concentrations (MLC) and minimum sublethal concentrations (MSC) for each metal. MLC values (dry mass) for Co (45 µg/g), Ni (230 µg/g), and Zn (280 µg/g) were below published minimum hyperaccumulator levels. MSC levels (dry mass) for Co (15 µg/g), Ni (140 µg/g), and Zn (200 µg/g) were at concentrations lower than published minimum accumulator levels. Furthermore, both MLC and MSC values for Zn were within normal tissue concentrations. These results indicate that elemental defense for Co, Ni, and Zn may be effective at concentrations lower than hyperaccumulator levels and so may be more widespread than previously believed.

Field-evolved resistance: assessing the problem and ways to move forward.

"Field-evolved resistance" is defined as a "genetically based decrease in susceptibility of a population to a toxin caused by exposure to the toxin in the field." The key component of "field-evolved" resistance is that it does confer decreased susceptibility to an insecticide in the field. Another key component is that the decrease in susceptibility to the insecticide is because of previous exposure of the target insect to the toxin in the field. Several studies have reported field-evolved resistance to crops engineered to express proteins from the bacterium, Bacillus thuringiensis (Bt). However, there has not been a consistent standard in the application of the definition of field-evolved resistance for Bt crops. The inconsistency in applying the definition arises from differences in the methods used to detect resistance, the ecology of the interaction between the pest and the Bt crop, and the effective dose the pest encounters while feeding on the Bt crop. Using case studies of reported resistance to Bt crops, it is demonstrated resistance does not come in a single form, and that in most cases, resistance can still be managed.

Effects of SmartStax® and SmartStax® PRO maize on western corn rootworm (Diabrotica virgifera virgifera LeConte) larval feeding injury and adult life history parameters.

Field-evolved resistance of the western corn rootworm (WCR), Diabrotica virgifera virgifera LeConte, to Bacillus thuringiensis Berliner (Bt) proteins Cry3Bb1 and Cry34/35Ab1 (now classified as Gpp34Ab1/Tpp35Ab1) expressed in the pyramid SmartStax® has been documented in areas of the United States (U.S.) Corn Belt. SmartStax® PRO is a recently registered rootworm-active pyramid containing the same Bt proteins expressed in SmartStax® plus DvSnf7 dsRNA. Little to no published data is available comparing efficacy of the technologies or potential effects of dietary exposure on adult WCR fitness. Therefore, experiments were conducted to compare effects of adult WCR dietary exposure to SmartStax® and SmartStax® PRO on life history parameters and efficacy of the technologies in the field with both Bt-susceptible and Bt-resistant WCR populations. WCR life history parameters evaluated included adult longevity, head capsule width, egg production, and egg viability. Results of small-plot field trials indicated that both technologies provided a high level of root protection when a Bt-susceptible WCR population was present. Root protection was reduced on SmartStax® but maintained on SmartStax® PRO when WCR Bt resistance occurred. Lifetime egg production was the key life history parameter that was significantly reduced when either Bt-susceptible or Bt-resistant adult WCR were fed SmartStax® or SmartStax® PRO diet. A potential fitness advantage was apparent as egg production was significantly higher in the Bt-resistant than Bt-susceptible population. The similar response by the Bt-susceptible WCR population to SmartStax® and SmartStax® PRO indicates that results were caused by sublethal dietary exposure to Bt proteins. Adult size (males < females) and egg viability (high: >95%) were not significantly different among treatments but longevity results were inconsistent between years. Collectively, the field efficacy and life history parameter data expand existing knowledge of SmartStax® and SmartStax® PRO technologies, which will inform practical WCR resistance management programs.

Association of Cry1Ac toxin resistance in Helicoverpa zea (Boddie) with increased alkaline phosphatase levels in the midgut lumen.

Resistance to Bacillus thuringiensis Cry1Ac toxin was characterized in a population of Helicoverpa zea larvae previously shown not to have an alteration in toxin binding as the primary resistance mechanism to this toxin. Cry1Ac-selected larvae (AR1) were resistant to protoxins and toxins of Cry1Ab, Cry1Ac, and the corresponding modified proteins lacking helix α-1 (Cry1AbMod and Cry1AcMod). When comparing brush border membrane vesicles (BBMVs) prepared from susceptible (LC) and AR1 larval midguts, there were only negligible differences in overall Cry1Ac toxin binding, though AR1 had 18% reversible binding, in contrast to LC, in which all binding was irreversible. However, no differences were detected in Cry1Ac-induced pore formation activity in BBMVs from both strains. Enzymatic activities of two putative Cry1Ac receptors (aminopeptidase N [APN] and alkaline phosphatase [ALP]) were significantly reduced (2-fold and 3-fold, respectively) in BBMVs from AR1 compared to LC larvae. These reductions corresponded to reduced protein levels in midgut luminal contents only in the case of ALP, with an almost 10-fold increase in specific ALP activity in midgut fluids from AR1 compared to LC larvae. Partially purified H. zea ALP bound Cry1Ac toxin in ligand blots and competed with Cry1Ac toxin for BBMV binding. Based on these results, we suggest the existence of at least one mechanism of resistance to Cry1A toxins in H. zea involving binding of Cry1Ac toxin to an ALP receptor in the larval midgut lumen of resistant larvae.

Characterizing the sublethal effects of SmartStax PRO dietary exposure on life history traits of the western corn rootworm, Diabrotica virgifera virgifera LeConte.

The western corn rootworm (WCR), Diabrotica virgifera virgifera LeConte, is an economically important pest of field corn (Zea mays L.) across the United States (U.S.) Corn Belt. Repeated use of transgenic hybrids expressing Bacillus thuringiensis (Bt) proteins has selected for field-evolved resistance to all current rootworm-active Bt proteins. The newest product available for WCR management is SmartStax® PRO, a rootworm-active pyramid containing Cry3Bb1, Cry34/35Ab1 [now reclassified as Gpp34Ab1/Tpp35Ab1] and a new mode of action, DvSnf7 dsRNA. Understanding the fitness of adult WCR after dietary exposure to SmartStax® PRO will identify potential impacts on WCR population dynamics and inform efforts to optimize resistance management strategies. Therefore, the objective of the present study was to characterize the effect of SmartStax® PRO dietary exposure on WCR life history traits. Adult WCR were collected during 2018 and 2019 from emergence tents placed over replicated field plots of SmartStax® PRO or non-rootworm Bt corn at a site with a history of rootworm-Bt trait use and suspected resistance to Cry3Bb1 and Cry34/35Ab1. Adult survival was reduced by 97.1-99.7% in SmartStax® PRO plots relative to the non-rootworm Bt corn plots during the study. Individual male/female pairs were fed different diets of ear tissue to simulate lifetime or adult exposure. Life history parameters measured included adult longevity, adult head capsule width, lifetime female egg production, and egg viability. Results indicate that lifetime or adult exposure to SmartStax® PRO significantly reduced adult longevity and lifetime egg production. Larval exposure to SmartStax® PRO significantly reduced WCR adult size. Results from this study collectively suggest that SmartStax® PRO may negatively impact WCR life history traits, which may lead to reduced population growth when deployed in an area with WCR resistance to Bt traits.

Safety assessment of Mpp75Aa1.1, a new ETX_MTX2 protein from Brevibacillus laterosporus that controls western corn rootworm.

The recently discovered insecticidal protein Mpp75Aa1.1 from Brevibacillus laterosporus is a member of the ETX_MTX family of beta-pore forming proteins (ß-PFPs) expressed in genetically modified (GM) maize to control western corn rootworm (WCR; Diabrotica virgifera virgifera LeConte). In this manuscript, bioinformatic analysis establishes that although Mpp75Aa1.1 shares varying degrees of similarity to members of the ETX_MTX2 protein family, it is unlikely to have any allergenic, toxic, or otherwise adverse biological effects. The safety of Mpp75Aa1.1 is further supported by a weight of evidence approach including evaluation of the history of safe use (HOSU) of ETX_MTX2 proteins and Breviballus laterosporus. Comparisons between purified Mpp75Aa1.1 protein and a poly-histidine-tagged (His-tagged) variant of the Mpp75Aa1.1 protein demonstrate that both forms of the protein are heat labile at temperatures at or above 55°C, degraded by gastrointestinal proteases within 0.5 min, and have no adverse effects in acute mouse oral toxicity studies at a dose level of 1920 or 2120 mg/kg body weight. These results support the use of His-tagged proteins as suitable surrogates for assessing the safety of their non-tagged parent proteins. Taken together, we report that Mpp75Aa1.1 is the first ETX-MTX2 insecticidal protein from B. laterosporus and displays a similar safety profile as typical Cry proteins from Bacillus thuringiensis.

Structural and functional characterization of Mpp75Aa1.1, a putative beta-pore forming protein from Brevibacillus laterosporus active against the western corn rootworm.

The western corn rootworm (WCR), Diabrotica virgifera virgifera LeConte, is a major corn pest of significant economic importance in the United States. The continuous need to control this corn maize pest and the development of field-evolved resistance toward all existing transgenic maize (Zea mays L.) expressing Bacillus thuringiensis (Bt) insecticidal proteins against WCR has prompted the development of new insect-protected crops expressing distinct structural classes of insecticidal proteins. In this current study, we describe the crystal structure and functional characterization of Mpp75Aa1.1, which represents the first corn rootworm (CRW) active insecticidal protein member of the ETX_MTX2 sub-family of beta-pore forming proteins (ß-PFPs), and provides new and effective protection against WCR feeding. The Mpp75Aa1.1 crystal structure was solved at 1.94 Å resolution. The Mpp75Aa1.1 is processed at its carboxyl-terminus by WCR midgut proteases, forms an oligomer, and specifically interacts with putative membrane-associated binding partners on the midgut apical microvilli to cause cellular tissue damage resulting in insect death. Alanine substitution of the surface-exposed amino acids W206, Y212, and G217 within the Mpp75Aa1.1 putative receptor binding domain I demonstrates that at least these three amino acids are required for WCR activity. The distinctive spatial arrangement of these amino acids suggests that they are part of a receptor binding epitope, which may be unique to Mpp75Aa1.1 and not present in other ETX_MTX2 proteins that do not have WCR activity. Overall, this work establishes that Mpp75Aa1.1 shares a mode of action consistent with traditional WCR-active Bt proteins despite significant structural differences.

Bacillus thuringiensis chimeric proteins Cry1A.2 and Cry1B.2 to control soybean lepidopteran pests: New domain combinations enhance insecticidal spectrum of activity and novel receptor contributions.

Two new chimeric Bacillus thuringiensis (Bt) proteins, Cry1A.2 and Cry1B.2, were constructed using specific domains, which provide insecticidal activity against key lepidopteran soybean pests while minimizing receptor overlaps between themselves, current, and soon to be commercialized plant incorporated protectants (PIP's) in soybean. Results from insect diet bioassays demonstrate that the recombinant Cry1A.2 and Cry1B.2 are toxic to soybean looper (SBL) Chrysodeixis includens Walker, velvetbean caterpillar (VBC) Anticarsia gemmatalis Hubner, southern armyworm (SAW) Spodoptera eridania, and black armyworm (BLAW) Spodoptera cosmioides with LC50 values < 3,448 ng/cm2. Cry1B.2 is of moderate activity with significant mortality and stunting at > 3,448 ng/cm2, while Cry1A.2 lacks toxicity against old-world bollworm (OWB) Helicoverpa armigera. Results from disabled insecticidal protein (DIP) bioassays suggest that receptor utilization of Cry1A.2 and Cry1B.2 proteins are distinct from each other and from current, and yet to be commercially available, Bt proteins in soy such as Cry1Ac, Cry1A.105, Cry1F.842, Cry2Ab2 and Vip3A. However, as Cry1A.2 contains a domain common to at least one commercial soybean Bt protein, resistance to this common domain in a current commercial soybean Bt protein could possibly confer at least partial cross resistance to Cry1A2. Therefore, Cry1A.2 and Cry1B.2 should provide two new tools for controlling many of the major soybean insect pests described above.

Structural and functional insights into the first Bacillus thuringiensis vegetative insecticidal protein of the Vpb4 fold, active against western corn rootworm.

The western corn rootworm (WCR), Diabrotica virgifera virgifera LeConte, is a major maize pest in the United States causing significant economic loss. The emergence of field-evolved resistant WCR to Bacillus thuringiensis (Bt) traits has prompted the need to discover and deploy new insecticidal proteins in transgenic maize. In the current study we determined the crystal structure and mode of action (MOA) of the Vpb4Da2 protein (formerly known as Vip4Da2) from Bt, the first identified insecticidal Vpb4 protein with commercial level control against WCR. The Vpb4Da2 structure exhibits a six-domain architecture mainly comprised of antiparallel ß-sheets organized into ß-sandwich layers. The amino-terminal domains 1-3 of the protein share structural homology with the protective antigen (PA) PA14 domain and encompass a long ß-pore forming loop as in the clostridial binary-toxB module. Domains 5 and 6 at the carboxyl-terminal half of Vpb4Da2 are unique as this extension is not observed in PA or any other structurally-related protein other than Vpb4 homologs. These unique Vpb4 domains adopt the topologies of carbohydrate-binding modules known to participate in receptor-recognition. Functional assessment of Vpb4Da2 suggests that domains 4-6 comprise the WCR receptor binding region and are key in conferring the observed insecticidal activity against WCR. The current structural analysis was complemented by in vitro and in vivo characterizations, including immuno-histochemistry, demonstrating that Vpb4Da2 follows a MOA that is consistent with well-characterized 3-domain Bt insecticidal proteins despite significant structural differences.

Selection for high levels of resistance to double-stranded RNA (dsRNA) in Colorado potato beetle (Leptinotarsa decemlineata Say) using non-transgenic foliar delivery.

Insecticidal double-stranded RNAs (dsRNAs) silence expression of vital genes by activating the RNA interference (RNAi) mechanism in insect cells. Despite high commercial interest in insecticidal dsRNA, information on resistance to dsRNA is scarce, particularly for dsRNA products with non-transgenic delivery (ex. foliar/topical application) nearing regulatory review. We report the development of the CEAS 300 population of Colorado potato beetle (Leptinotarsa decemlineata Say) (Coleoptera: Chrysomelidae) with > 11,100-fold resistance to a dsRNA targeting the V-ATPase subunit A gene after nine episodes of selection using non-transgenic delivery by foliar coating. Resistance was associated with lack of target gene down-regulation in CEAS 300 larvae and cross-resistance to another dsRNA target (COPI ß; Coatomer subunit beta). In contrast, CEAS 300 larvae showed very low (~ 4-fold) reduced susceptibility to the Cry3Aa insecticidal protein from Bacillus thuringiensis. Resistance to dsRNA in CEAS 300 is transmitted as an autosomal recessive trait and is polygenic. These data represent the first documented case of resistance in an insect pest with high pesticide resistance potential using dsRNA delivered through non-transgenic techniques. Information on the genetics of resistance and availability of dsRNA-resistant L. decemlineata guide the design of resistance management tools and allow research to identify resistance alleles and estimate resistance risks.

Toxicity of seven Bacillus thuringiensis Cry proteins against Cylas puncticollis and Cylas brunneus (Coleoptera: Brentidae) using a novel artificial diet.

"Sweetpotato weevils" Cylas puncticollis (Boheman) and Cylas brunneus F. (Coleoptera: Brentidae) are the most important biological threat to sweetpotato, Ipomoea batatas L. (Lam), productivity in sub-Saharan Africa. Sweetpotato weevil control is difficult due to their cryptic feeding behavior. Expression of Cylas-active Bacillus thuringiensis (Bt) Cry proteins in sweetpotato could provide an effective control strategy. Unfortunately, Bt Cry proteins with relatively high toxicity against Cylas spp. have not been identified, partly because no published methodology for screening Bt Cry proteins against Cylas spp. in artificial diet exists. Therefore, the initial aim of this study was to develop an artificial diet for conducting bioassays with Cylas spp. and then to determine Bt Cry protein efficacy against C. puncticollis and C. brunneus by using this artificial diet. Five diets varying in their composition were evaluated. The highest survival rates for sweetpotato weevil larvae were observed for diet E that contained the highest amount of sweetpotato powder and supported weevil development from first instar to adulthood, similar to sweetpotato storage roots. Seven coleopteran-active Bt Cry proteins were incorporated into diet E and toxicity data were generated against neonate C. puncticollis and second-instar C. brunneus. All Bt Cry proteins tested had toxicity greater than the untreated control. Cry7Aa1, ET33/34, and Cry3Ca1 had LC50 values below 1 microg/g diet against both species. This study demonstrates the feasibility of using an artificial diet bioassay for screening Bt Cry proteins against sweetpotato weevil larvae and identifies candidate Bt Cry proteins for use in transforming sweetpotato varieties potentially conferring field resistance against these pests.

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.