RNAi for Western Corn Rootworm Management: Lessons Learned, Challenges, and Future Directions

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.

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 β-sheets organized into β-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 β-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.

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 (β-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.

A Promising Predator-In-First Strategy to Control Western Corn Rootworm Population in Maize Fields

Western Corn Rootworm is a pest of maize that mostly damages roots. Many alternative strategies have been explored to control this species, with little or non-lasting success, and it remains a threat to maize production worldwide. Gaeolaelaps aculeifer, a soil-dwelling predatory mite that inhabits the first few centimeters of the soil, showed high predatory potential against WCR larvae in the laboratory. In this study, we explored the efficiency of G. aculeifer against WCR in more realistic contexts. First, we infested maize plants isolated in pots in a greenhouse with WCR, and tested the impact of different densities of mites on plant protection. Using standard indicators of WCR population presence and impact, we confirmed that G. aculeifer has the potential to control WCR at densities starting from 100 mites/plant. Then, considering that the release of a large amount of biocontrol agents at WCR emergence might be too costly and constraining for large-scale implementation, we tested the efficiency of a predator-in-first strategy in a maize field infested by WCR. The goal was to introduce fewer G. aculeifer combined with Aleuroglyphus ovatus eggs as an alternative food source in order to let the mite population grow in the field and reach sufficient density at the critical stage for protection. This strategy gave comparable results to pesticide on all indicators examined in our field trial, highlighting the potential to sustainably manage this pest.

Soybean Foliage Consumption Reduces Adult Western Corn Rootworm (Diabrotica virgifera virgifera)(Coleoptera: Chrysomelidae) Survival and Stimulates Flight

Western corn rootworm, Diabrotica virgifera virgifera LeConte, biology is tied to the continuous availability of its host (corn, Zea mays L.). Annual rotation of corn with a nonhost, like soybean (Glycine max (L.) Merrill) was a reliable tactic to manage western corn rootworm. Behavioral resistance to annual crop rotation (rotation resistance) allowed some eastern U.S. Corn Belt populations to circumvent rotation by laying eggs in soybean and in cornfields. When active in soybean, rotation-resistant adults commonly consume foliage, in spite of detrimental effects on beetle survival. Rotation-resistant beetle activity in soybean is enabled by the expression of certain proteinases and an adapted gut microbiota that provide limited protection from soybean antiherbivore defenses. We investigated the effects of corn and soybean herbivory on rotation-resistant female survival and initiation of flight using mortality assays and wind tunnel flight tests. Among field-collected females tested with mortality assays, beetles from collection sites in a cornfield survived longer than those from collection sites in a soybean field. However, reduced survival due to soybean herbivory could be restored by consuming corn tissues. Field-collected beetles that fed on a soybean tissue laboratory diet or only water were more likely to fly in a wind tunnel than corn-feeding beetles. Regardless of collection site and laboratory diet, 90.5% of beetles that flew oriented their flights upwind. Diet-related changes in the probability of flight provide a proximate mechanism for interfield movement that facilitates restorative feeding and the survival of females previously engaged in soybean herbivory. Rotation-resistant western corn rootworm females feeding on soybean tissues experience reduced survival in mortality assays and display increased flight probability (which may facilitate flight back to a cornfield where consumption of host tissues improves survival potential and facilitates maturation of eggs). The consequences of soybean herbivory provide a proximal mechanism for behavioral resistance to crop rotation. Increased egg-laying probability while feeding on soybean tissues, facilitation of egg maturation while feeding on corn tissues, and interfield movement are previously documented consequences.

Up-regulation of apoptotic- and cell survival-related gene pathways following exposures of western corn rootworm to B. thuringiensis crystalline pesticidal proteins in transgenic maize roots

Background Resistance of pest insect species to insecticides, including B. thuringiensis (Bt) pesticidal proteins expressed by transgenic plants, is a threat to global food security. Despite the western corn rootworm, Diabrotica virgifera virgifera, being a major pest of maize and having populations showing increasing levels of resistance to hybrids expressing Bt pesticidal proteins, the cell mechanisms leading to mortality are not fully understood. Results Twenty unique RNA-seq libraries from the Bt susceptible D. v. virgifera inbred line Ped12, representing all growth stages and a range of different adult and larval exposures, were assembled into a reference transcriptome. Ten-day exposures of Ped12 larvae to transgenic Bt Cry3Bb1 and Gpp34/Tpp35Ab1 maize roots showed significant differential expression of 1055 and 1374 transcripts, respectively, compared to cohorts on non-Bt maize. Among these, 696 were differentially expressed in both Cry3Bb1 and Gpp34/Tpp35Ab1 maize exposures. Differentially-expressed transcripts encoded protein domains putatively involved in detoxification, metabolism, binding, and transport, were, in part, shared among transcripts that changed significantly following exposures to the entomopathogens Heterorhabditis bacteriophora and Metarhizium anisopliae. Differentially expressed transcripts in common between Bt and entomopathogen treatments encode proteins in general stress response pathways, including putative Bt binding receptors from the ATP binding cassette transporter superfamily. Putative caspases, pro- and anti-apoptotic factors, as well as endoplasmic reticulum (ER) stress-response factors were identified among transcripts uniquely up-regulated following exposure to either Bt protein. Conclusions Our study suggests that the up-regulation of genes involved in ER stress management and apoptotic progression may be important in determining cell fate following exposure of susceptible D. v. virgifera larvae to Bt maize roots. This study provides novel insights into insect response to Bt intoxication, and a possible framework for future investigations of resistance mechanisms.

Characterization of Thermal and Time Exposure to Improve Artificial Diet for Western Corn Rootworm Larvae

The western corn rootworm (WCR), Diabrotica virgifera LeConte, is the most serious pest of maize in the United States. In pursuit of developing a diet free of antibiotics for WCR, we characterized effects of thermal exposure (50–141 °C) and length of exposure on quality of WCRMO-2 diet measured by life history parameters of larvae (weight, molting, and survival) reared on WCRMO-2 diet. Our results indicated that temperatures had non-linear effects on performance of WCRMO-2 diet, and no impacts were observed on the length of time exposure. The optimum temperature of diet processing was 60 °C for a duration less than 30 min. A significant decline in development was observed in larvae reared on WCRMO-2 diet pretreated above 75 °C. Exposing WCRMO-2 diet to high temperatures (110–141 °C) even if constrained for brief duration (0.9–2.3 s) caused 2-fold reduction in larval weight and significant delays in larval molting but no difference in survival for 10 days compared with the control diet prepared at 65 °C for 10 min. These findings provide insights into the effects of thermal exposure in insect diet processing.

Predation capacity of soil-dwelling predatory mites on two major maize pests

The western corn rootworm Diabrotica virgifera virgifera (WCR), and the wireworm Agriotes sordidus (WW), whose eggs and first instar larvae develop in the first few centimeters of soil, are major crop pests. As soil-dwelling predatory mites are known as potential biocontrol agents against many pests, we investigated the predation capacity of Stratiolaelaps scimitus, Gaeolaelaps aculeifer and Macrocheles robustulus on immature stages of WCR and WW in a laboratory setting. While eggs of WCR and WW were never consumed, all three predator species attacked both WCR and WW first instar larvae. While these results need to be confirmed in natural conditions, our work identifies the early larval stage instead of the egg stage as the most vulnerable stage for control against WCR and WW with soil-dwelling predatory mites.