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We identified a single diet formulation that can be used for three Diabrotica species including southern (SCR), western (WCR), and northern corn rootworm (NCR) by evaluating the performance of these pests on specialized diets (F9800B diet for SCR, WCRMO-2 diet for WCR, and NCRMO-1 diet for NCR) and a larval diet (F9772 diet) widely used for lepidopteran species. After 10 days of rearing, the WCRMO-2 diet yielded better or equal larval growth and development of all three rootworm species compared to other diets. For SCR larvae, the WCRMO-2 diet outperformed other diets. Larval fresh weight, percent molt to 2nd instar, and percent molt to 3rd instar on the WCRMO-2 diet were 12-fold, 2.7-fold, and 14-fold increases, respectively compared to that of the F9800B diet. Significantly more SCR larvae survived on the WCRMO-2 diet (98.9%) than on the F9800B diet (90.6%). The WCRMO-2 diet supported WCR and NCR larvae equal to the NCRMO-1 diet and better than other diets. The F9772 diet was the worst diet of all examined species. The availability of a universal diet (the WCRMO-2 diet) for the three Diabrotica species would facilitate research programs to monitor resistance development and develop new control tactics targeting these important pests.

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BACKGROUND: Transgenic crops producing insecticidal toxins from the bacterium Bacillus thuringiensis (Bt) have been used to manage insect pests for nearly 30 years. Dose of a Bt crop is key to assessing the risk of resistance evolution because it affects the heritability of resistance traits. Western corn rootworm (Diabrotica virgifera virgifera, LeConte), a major pest of maize, has evolved resistance to all commercially available Bt traits targeting it, and threatens resistance to future transgenic traits. Past research shows the dose of Bt maize targeting western corn rootworm can be confounded by larval density-dependent mortality. We conducted a 2-year field study at two locations to quantify larval density-dependent mortality in Bt and non-Bt maize. We used these results to calculate dose for our method and compared it to three previously published methods. Additionally, adult emergence and root injury were analyzed for predicting initial egg density. RESULTS: Increased pest density caused greater proportions of larvae to die in Bt maize than in non-Bt maize. All methods for calculating dose produced values less than high-dose, and stochastic variation had the greatest impact on dose at high and low pest densities. Our method for calculating dose did not produce values positively correlated with pest density while the three other methods did. CONCLUSION: To achieve the most accurate calculation of dose for transgenic maize targeting western corn rootworm, density-dependent mortality should be taken into account for both transgenic and non-transgenic maize and assessed at moderate pest densities. © 2024 Society of Chemical Industry.

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The selection of oviposition sites by female moths is crucial in shaping their progeny performance and survival, and consequently in determining insect fitness. Selecting suitable plants that promote the performance of the progeny is referred to as the Preference-Performance hypothesis (or 'mother-knows-best'). While root infestation generally reduces the performance of leaf herbivores, little is known about its impact on female oviposition. We investigated whether maize root infestation by the Western corn rootworm (WCR) affects the oviposition preference and larval performance of the European corn borer (ECB). ECB females used leaf volatiles to select healthy plants over WCR-infested plants. Undecane, a compound absent from the volatile bouquet of healthy plants, was the sole compound to be upregulated upon root infestation and acted as a repellent for first oviposition. ECB larvae yet performed better on plants infested below-ground than on healthy plants, suggesting an example of 'bad motherhood'. The increased ECB performance on WCR-infested plants was mirrored by an increased leaf consumption, and no changes in the plant primary or secondary metabolism were detected. Understanding plant-mediated interactions between above- and below-ground herbivores may help to predict oviposition decisions, and ultimately, to manage pest outbreaks in the field.

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Certain soil insects, such as the root-damaging larvae of the maize pest Diabrotica virgifera virgifera (Coleoptera: Chrysomelidae), are increasingly difficult to control because of recent bans of some insecticides. An alternative and safer approach may be the development of biopesticides based on entomotoxic defense proteins of higher fungi. Many of these potentially interesting proteins are protease inhibitors, and some have been shown to adversely affect insects. We examined the effects of the cysteine protease inhibitors macrocypin 1, 3, and 4 from Macrolepiota procera, clitocypin from Clitocybe nebularis, and cocaprin 1 and the serine protease inhibitor cospin 1 from Coprinopsis cinerea on D. v. virgifera. We confirmed the inhibition by mycocypins of the cysteine catalytic-type proteolytic activities in gut extracts of larvae and adults. The inhibition of pGlu-Phe-Leu-hydrolyzing activity was stronger than that of Z-Phe-Arg-hydrolyzing activity. Mycocypins and cospin resisted long-term proteolytic digestion, whereas cocaprin 1 was digested. Bioassays with overlaid artificial diet revealed no effects of proteins on neonatal mortality or stunting, and no effects on adult mortality. Immersion of eggs in protein solutions had little effect on egg hatching or mortality of hatching neonates. Microscopic analysis of the peritrophic matrix and apical surface of the midguts revealed the similarity between larvae of D. v. virgifera and the chrysomelid Leptinotarsa decemlineata, which are sensitive to these inhibitors. The resistance of D. v. virgifera to fungal protease inhibitors is likely due to effective adaptation of digestive enzyme expression to dietary protease inhibitors. We continue to study unique protein complexes of higher fungi for the development of new approaches to pest control.

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The northern corn rootworm, Diabrotica barberi Smith & Lawrence (Coleoptera: Chrysomelidae) is a major pest of maize in the United States Corn Belt. Recently, resistance to Bacillus thuringiensis (Bt) maize was reported in North Dakota and increased use of Bt maize hybrids could facilitate resistance evolution in other maize-producing states. In this study, susceptibility to Bt proteins was evaluated in wild D. barberi populations from 8 fields collected in 5 different states (Minnesota, Missouri, Nebraska, Iowa, and North Dakota). Field populations were compared to a susceptible D. barberi colony in seedling and diet toxicity assays conducted with 3 concentrations of Cry3Bb1 (0.4, 4.0, and 40.0 µg/cm2) and Gpp34/Tpp35Ab1 (previously called Cry34/35Ab1; 1.4, 14.0, and 140.0 µg/cm2). The 2019 population from Meeker Co., Minnesota (MN-2019), exhibited the lowest mortality to Cry3Bb1 and also had nominally lowest mortality to Gpp34/Tpp35Ab1 at the highest concentrations tested in diet toxicity assays. Percent second instar was also highest for larvae of the Minnesota population surviving Cry3Bb1. In seedling assays, MN and IA-2018 populations exhibited the highest proportion survival and dry weight to both proteins expressed in corn. No significant differences in mortality, percent second instar, and dry weight were observed at the highest concentration for both proteins among the populations collected in in 2020. Most D. barberi populations were still highly susceptible to Cry3Bb1 and Gpp34/Tpp35Ab1 proteins based on diet and seedling assays, but resistance appears to be developing in some D. barberi populations. Now that methods are available, resistance monitoring may also be needed for D. barberi in some regions.

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OBJECTIVE: The Western corn rootworm (WCR), Diabrotica virgifera virgifera, is a highly adaptable insect pest that has evolved resistance to a variety of control strategies, including insecticides. Therefore, it is interesting to examine how housekeeping proteins in WCR have been changed under WCR-controlling strategies. In this study, we focused on one of such proteins in WCR, a ubiquitous enzyme 5'-triphosphate nucleotidohydrolase (dUTPase). In the thymidine synthetic pathway, dUTPase hydrolyzes deoxyuridine triphosphate (dUTP) and supplies the substrate, deoxyuridine monophosphate, for the thymidylate synthase (TS). It decreases the cellular content of uracil, reducing uracil misincorporation into DNA. Suppressing the dUTPase activity, therefore, contributes to thymineless death. In this study, we investigated the enzymatic properties of dUTPase. RESULTS: The WCR dUTPase gene (DUT) was synthesized with the addition of His-tag corresponding DNA sequence and then cloned and expressed in Escherichia coli, and the protein product was purified. The product of WCR DUT hydrolyzed dUTP and was designated as dUTPase. WCR dUTPase did not hydrolyze dATP, dTTP, dCTP, or dGTP. WCR dUTPase was analyzed via size-exclusion chromatography and exhibited a molecular weight corresponding to that of trimer. The present format can be interpreted as nuclear trimer type. Possible isomers will be examined once transcriptome analyses are conducted.

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Western corn rootworm, Diabrotica virgifera virgifera (LeConte) (Coleoptera: Chrysomelidae), is the most serious economic pest of maize, Zea mays (L.) (Poales: Poaceae), in the U.S. Corn Belt and also threatens production in Europe. Traditional management options have repeatedly failed over time as western corn rootworm rapidly develops resistance to insecticides, transgenic maize and even crop rotation. Traits that improve host plant resistance and tolerance are highly sought after by plant breeders for crop protection and pest management. However, maize resistance to western corn rootworm appears to be highly complex and despite over 75 yr of breeding efforts, there are no naturally resistant hybrids available commercially. Using phenotypic data from field and greenhouse experiments on a highly diverse collection of 282 inbred lines, we screened and genetically mapped western corn rootworm-related traits to identify genetic loci which may be useful for future breeding or genetic engineering efforts. Our results confirmed that western corn rootworm resistance is complex with relatively low heritability due in part to strong genotype by environment impacts and the inherent difficulties of phenotyping below ground root traits. The results of the Genome Wide Associated Study identified 29 loci that are potentially associated with resistance to western corn rootworm. Of these loci, 16 overlap with those found in previous transcription or mapping studies indicating a higher likelihood they are truly involved in maize western corn rootworm resistance. Taken together with previous studies, these results indicate that breeding for natural western corn rootworm resistance will likely require the stacking of multiple small effect loci.

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Western corn rootworm (WCR), a major pest of corn, has been reared in laboratories since the 1960s. While established rearing methods are appropriate for maintaining WCR colonies, they are not optimal for performing germline transformation or CRISPR/Cas9-based genome editing. Here we report the development of an optimized rearing system for use in WCR functional genomics research, specifically the development of a system that facilitates the collection of preblastoderm embryos for microinjection as well as gathering large larvae and pupae for downstream phenotypic screening. Further, transgenic-based experiments require stable and well-defined survival rates and the ability to manipulate insects at every life stage. In our system, the WCR life cycle (egg to adult) takes approximately 42 days, with most individuals eclosing between 41 and 45 days post oviposition. Over the course of one year, our overall survival rate was 67%. We used this data to establish a quality control system for more accurately monitoring colony health. Herein, we also offer detailed descriptions for setting up single-pair crosses and conducting phenotypic screens to identify transgenic progeny. This study provides a model for the development of new rearing systems and the establishment of highly controlled processes for specialized purposes.

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Western corn rootworm (WCR) is one of the most devastating corn rootworm species in North America because of its ability to cause severe production loss and grain quality damage. To control the loss, it is important to identify the infection of WCR at an early stage. Because the root system is the earliest feeding source of the WCR at the larvae stage, assessing the direct damage in the root system is crucial to achieving early detection. Most of the current methods still necessitate uprooting the entire plant, which could cause permanent destruction and a loss of the original root's structural information. To measure the root damages caused by WCR non-destructively, this study utilized MISIRoot, a minimally invasive and in situ automatic plant root phenotyping robot to collect not only high-resolution images but also 3D positions of the roots without uprooting. To identify roots in the images and to study how the damages were distributed in different types of roots, a deep convolution neural network model was trained to differentiate the relatively thick and thin roots. In addition, a color camera was used to capture the above-ground morphological features, such as the leaf color, plant height, and side-view leaf area. To check if the plant shoot had any visible symptoms in the inoculated group compared to the control group, several vegetation indices were calculated based on the RGB color. Additionally, the shoot morphological features were fed into a PLS-DA model to differentiate the two groups. Results showed that none of the above-ground features or models output a statistically significant difference between the two groups at the 95% confidence level. On the contrary, many of the root structural features measured using MISIRoot could successfully differentiate the two groups with the smallest t-test p-value of 1.5791 × 10-6. The promising outcomes were solid proof of the effectiveness of MISIRoot as a potential solution for identifying WCR infestations before the plant shoot showed significant symptoms.

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Foliar-applied insecticides are commonly used for adult western corn rootworm (WCR), Diabrotica virgifera virgifera LeConte (Coleoptera: Chrysomelidae), control in Nebraska but little efficacy data is available. Anecdotal reports of reduced efficacy in areas of northeast Nebraska led to the conduct of this study. Objectives were to (i) evaluate the efficacy of commercial applications of commonly used formulated insecticides (bifenthrin, lambda-cyhalothrin, chlorpyrifos, or tank mixes) for WCR control in 7 northeast Nebraska counties during 2019 and 2020 and (ii) conduct adult WCR concentration-response vial bioassays with bifenthrin, chlorpyrifos, and dimethoate active ingredients on a subset of field populations. Whole plant counts (WPC) were used to measure WCR densities in insecticide-treated and untreated maize fields before and after insecticide application. Field control was excellent with organophosphate/pyrethroid tank mixes as proportional change in mean WPC of treated fields was significantly reduced (>0.90) versus untreated fields where little change in WPC occurred. The exception was one treated Boone County field where proportional reduction in WPC was ≤0.78. Bioassays revealed LC50s and resistance ratios of most populations exposed to bifenthrin and dimethoate were not significantly different than the susceptible control. Most populations exhibited a low level of chlorpyrifos resistance when compared to the susceptible control. Field and lab data suggest the local onset of practical WCR field-evolved resistance to bifenthrin in Boone County and chlorpyrifos in Boone and Colfax counties. Results of this study will increase our understanding of WCR resistance evolution, serve as a comprehensive baseline for future research, and inform WCR management programs.

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IPD072Aa from Pseudomonas chlororaphis is a new insecticidal protein that has been shown to have high activity against western corn rootworm (WCR). IPD072 has no sequence signatures or predicted structural motifs with any known protein revealing little insight into its mode of action using bioinformatic tools. As many bacterially derived insecticidal proteins are known to act through mechanisms that lead to death of midgut cells, we evaluated whether IPD072Aa also acts by targeting the cells of WCR midgut. IPD072Aa exhibits specific binding to brush border membrane vesicles (BBMVs) prepared from WCR guts. The binding was found to occur at binding sites that are different than those recognized by Cry3A or Cry34Ab1/Cry35Ab1, proteins expressed by current maize traits that target WCR. Using fluorescence confocal microscopy, immuno-detection of IPD072Aa in longitudinal sections from whole WCR larvae that were fed IPD072Aa revealed the association of the protein with the cells that line the gut. High-resolution scanning electron microscopy of similar whole larval sections revealed the disruption of the gut lining resulting from cell death caused by IPD072Aa exposure. These data show that the insecticidal activity of IPD072Aa results from specific targeting and killing of rootworm midgut cells. IMPORTANCE Transgenic traits targeting WCR based on insecticidal proteins from Bacillus thuringiensis have proven effective in protecting maize yield in North America. High adoption has led to WCR populations that are resistant to the trait proteins. Four proteins have been developed into commercial traits, but they represent only two modes of action due to cross-resistance among three. New proteins suited for trait development are needed. IPD072Aa, identified from the bacterium Pseudomonas chlororaphis, was shown to be effective in protecting transgenic maize against WCR. To be useful, IPD072Aa must work through binding to different receptors than those utilized by current traits to reduce risk of cross-resistance and understanding its mechanism of toxicity could aid in countering resistance development. Our results show that IPD072Aa binds to receptors in WCR gut that are different than those utilized by current commercial traits and its targeted killing of midgut cells results in larval death.

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MAIN CONCLUSION: Domestication affected the abundances and diversity of maize root volatiles more than northward spread and modern breeding, and herbivore preference for roots was correlated with volatile diversity and herbivore resistance. Studies show that herbivore defenses in crops are mediated by domestication, spread, and breeding, among other human-driven processes. They also show that those processes affected chemical communication between crop plants and herbivores. We hypothesized that (i) preference of the herbivore (Diabrotica virgifera virgifera) larvae for embryonic roots of maize (Zea mays mays) would increase and (ii) root volatile diversity would decrease with the crop's domestication, northward spread to present-day USA, and modern breeding. We used Balsas teosinte (Zea mays parviglumis), Mexican and USA landrace maizes, and US inbred maize lines to test these hypotheses. We found that herbivore preference and volatile diversity increased with maize domestication and northward spread but decreased with modern breeding. Additionally, we found that the abundances of single volatiles did not consistently increase or decrease with maize domestication, spread, and breeding; rather, volatiles grouped per their abundances were differentially affected by those processes, and domestication had the greatest effects. Altogether, our results suggested that: the herbivore's preference for maize roots is correlated with volatile diversity and herbivore resistance; changes in abundances of individual volatiles are evident at the level of volatile groups; and maize domestication, but not spread and breeding, affected the abundances of some green leaf volatiles and sesquiterpenes/sesquiterpenoids. In part, we discussed our results in the context of herbivore defense evolution when resources for plant growth and defense vary across environments. We suggested that variability in relative abundance of volatiles may be associated with their local, functional relevance across wild and agricultural environments.

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The Western corn rootworm (WCR; Diabrotica virgifera virgifera) is an economically important belowground pest of maize. Belowground feeding by WCR is damaging because it weakens the roots system, diminishes nutrient uptake, and creates entry points for fungal and bacterial pathogens and increases lodging, all of which can significantly suppress maize yields. Previously, it was demonstrated that belowground herbivory can trigger plant defense responses in the roots and the shoots, thereby impacting intraplant communication. Although several aspects of maize-WCR interactions have been reported, co-transcriptomic remodeling in the plant and insect are yet to be explored. We used a maize genotype, Mp708, that is resistant to a large guild of herbivore pests to study the underlying plant defense signaling network between below and aboveground tissues. We also evaluated WCR compensatory transcriptome responses. Using RNA-seq, we profiled the transcriptome of roots and leaves that interacted with WCR infestation up to 5 days post infestation (dpi). Our results suggest that Mp708 shoots and roots had elevated constitutive and WCR-feeding induced expression of genes related to jasmonic acid and ethylene pathways, respectively, before and after WCR feeding for 1 and 5 days. Similarly, extended feeding by WCR for 5 days in Mp708 roots suppressed many genes involved in the benzoxazinoid pathway, which is a major group of indole-derived secondary metabolites that provides resistance to several insect pests in maize. Furthermore, extended feeding by WCR on Mp708 roots revealed several genes that were downregulated in WCR, which include genes related to proteolysis, neuropeptide signaling pathway, defense response, drug catabolic process, and hormone metabolic process. These findings indicate a dynamic transcriptomic dialog between WCR and WCR-infested maize plants.

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The western corn rootworm (WCR), Diabrotica virgifera virgifera LeConte (Coleoptera: Chrysomelidae), is an economically important pest of corn (maize) in North America and Europe. Current management practices for WCR involve transgenic expression of insecticidal proteins to minimize larval feeding damage to corn roots. The evolution of resistant WCR populations to transgenic corn expressing insecticidal proteins (e.g. Cry3Bb1, Gpp34Ab1/Tpp35Ab1) necessitates efforts to discover and deploy new modes of action for WCR control. Here, we tested the hypothesis that the addition of short peptides selected for binding to the WCR gut would restore insecticidal activity of Cry3Bb1 to resistant insects. Phage display technology coupled with deep sequencing was used to identify peptides selected for binding to WCR brush border membrane vesicles and to recombinant putative receptors aminopeptidase and cadherin. The binding and specificity of selected peptides was confirmed by ELISA and pull-down assays, and candidate gut surface binding partners were identified. Although production of 284 novel Cry3Bb1 variants with these peptides did not restore activity against resistant WCR in artificial diet bioassays, 112 variants were active against susceptible insects. These results provided insights for the mechanism of Cry3Bb1 activity and toward engineering a new mode-of-action via receptor re-targeting in the context of protein structure and function.

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As agricultural biotechnology continues to develop solutions for addressing crop pests through newly expressed proteins from novel source organisms, with different modes or sites of action and/or different spectra of activity, the safety of these proteins will be assessed. The results of hazard-identification and characterization studies for the insecticidal protein IPD079Ea, which is derived from a fern (Ophioglossum pendulum) and active against the maize pest western corn rootworm (Diabrotica virgifera virgifera, Coleoptera: Chrysomelidae) are provided. Collectively these results indicate that IPD079Ea is unlikely to present a hazard to human or animal health and support the safety of genetically modified maize expressing IPD079Ea.

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Adenanthera pavonina, an underutilized tropical tree, is being promoted as an alternative food source for meeting the nutritional needs of human and animals. In this study, we have shown that trypsin inhibitors as one of the predominant proteins in the seeds of A. pavonina. DE-52 column chromatography resulted in the identification of four peaks with trypsin inhibitor activity. SDS-PAGE and immunoblot analyses revealed DE-52 peaks A and B were enriched in 17 and 15 kDa proteins and these proteins cross-reacted against soybean trypsin inhibitor antibodies. Simulated gastric fluid digestion revealed that the 15-17 kDa proteins are resistant to pepsin digestion. Roasting the seeds lowered the trypsin inhibitor activity while boiling intact seeds elevated the enzyme activity. However, the trypsin inhibitor activity was completely abolished when the seeds were boiled without their seed coats. Immunohistochemical detection and confocal microscopy demonstrated that trypsin inhibitors were localized in the cell cytosol.

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Western corn rootworm (Diabrotica virgifera virgifera LeConte) is a major pest of corn in both North America and Europe and as such presents significant challenges for farmers. IPD079Ea protein is encoded by the ipd079Ea gene from Ophioglossum pendulum (a species of fern) and was found to have activity against western corn rootworm in multiple corn events transformed to express the IPD079Ea protein. In chronic laboratory hazard studies, IPD079Ea protein was fed to eleven species in the order Coleoptera and four species in the order Lepidoptera to assess the spectrum of activity. Activity was observed on certain species of the Chrysomelidae and Coccinellidae families, with western corn rootworm as the most sensitive insect tested. No adverse effects on mortality or other sublethal endpoints were observed on any species within Lepidoptera. Overall, IPD079Ea protein appears not to have broad insecticidal properties and has potential value as an effective trait to control western corn rootworm in agricultural systems.

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Diabrocite corn rootworms are one of the most economically significant pests of maize in the United States and Europe and an emerging model for insect-plant interactions. Genome sizes of several species in the genus Diabrotica were estimated using flow cytometry along with that of Acalymma vittatum as an outgroup. Genome sizes ranged between 1.56 and 1.64 gigabase pairs and between 2.26 and 2.59 Gb, respectively, for the Diabrotica subgroups fucata and virgifera; the Acalymma vittatum genome size was around 1.65 Gb. This result indicated that a substantial increase in genome size occurred in the ancestor of the virgifera group. Further analysis of the fucata group and the virgifera group genome sequencing reads indicated that the genome size difference between the Diabrotica subgroups could be attributed to a higher content of transposable elements, mostly miniature inverted-transposable elements and gypsy-like long terminal repeat retroelements.

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Sequences derived from a novel toursvirus were identified from pooled genomic short read data from U.S. populations of southern corn rootworm (SCR, Diabrotica undecimpunctata howardi Barber) and northern corn rootworm (NCR, Diabrotica barberi Smith & Lawrence). Most viral sequences were identified from the SCR genomic dataset. As proteins encoded by toursvirus sequences from SCR and NCR were almost identical, the contig sets from SCR and NCR were combined to generate 26 contigs. A total of 108,176 bp were assembled from these contigs, with 120 putative toursviral ORFs identified indicating that most of the viral genome had been recovered. These ORFs included all 40 genes that are common to members of the Ascoviridae. Two genes typically present in Ascoviridae (ATP binding cassette transport system permeases and Baculovirus repeated open reading frame), were not detected. There was evidence for transposon insertion in viral sequences at different sites in the two host species. Phylogenetic analyses based on a concatenated set of 45 translated protein sequences clustered toursviruses into a distinct clade. Based on the combined evidence, we propose taxonomic separation of toursviruses from Ascoviridae.

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The western corn rootworm (WCR), Diabrotica virgifera virgifera LeConte, is considered one of the most economically important pests of maize (Zea mays L.) in the United States (U.S.) Corn Belt with costs of management and yield losses exceeding USD ~1-2 billion annually. WCR management has proven challenging given the ability of this insect to evolve resistance to multiple management strategies including synthetic insecticides, cultural practices, and plant-incorporated protectants, generating a constant need to develop new management tools. One of the most recent developments is maize expressing double-stranded hairpin RNA structures targeting housekeeping genes, which triggers an RNA interference (RNAi) response and eventually leads to insect death. Following the first description of in planta RNAi in 2007, traits targeting multiple genes have been explored. In June 2017, the U.S. Environmental Protection Agency approved the first in planta RNAi product against insects for commercial use. This product expresses a dsRNA targeting the WCR snf7 gene in combination with Bt proteins (Cry3Bb1 and Cry34Ab1/Cry35Ab1) to improve trait durability and will be introduced for commercial use in 2022.