Diabrotica Research Articles

Enhanced metabolism and selection of pyrethroid-resistant western corn rootworms (Diabrotica virgifera virgifera LeConte)

Western corn rootworm (WCR) pyrethroid resistance has been previously reported in the United States (US) western Corn Belt, and cross-resistance and synergism studies suggested that both target site insensitivity and enhanced metabolism may be conferring WCR resistance to pyrethroids. The present study aimed to investigate the potential mechanisms of WCR pyrethroid resistance and to estimate the heritability of the resistance trait. Biochemical assays using model substrates and spectrophotometry revealed 2–4-fold higher activity of P450s and esterases in pyrethroid-resistant WCR populations, whereas the biological activity of glutathione S-transferase was similar between populations tested. No mutation in the voltage-gated sodium channel was detected in pyrethroid-resistant WCR individuals by sequencing PCR products containing the para-homologous L1014, T929, and M918 amino acid positions that are commonly associated with target site mutations in other pyrethroid-resistant insects. A pilot estimation of pyrethroid resistance heritability obtained during laboratory selection of a WCR population suggested a major genetic component of the resistance trait and predicted a 10-fold increase in WCR bifenthrin resistance within ~7 generations of insecticide lethal exposure. Results support earlier indirect evidence that enhanced metabolism may be contributing to WCR resistance to pyrethroids and illustrates the potential of WCR pyrethroid resistance evolution.

Evaluation of Seed Treatment and Soil-Applied Insecticides Against Larval Western and Northern Corn Rootworm, 2020

Evaluation of Seed Treatment and Soil-Applied Insecticides Against Larval Western and Northern Corn Rootworm, 2020

Efficacy of chlorantraniliprole on southern corn rootworm in peanut

Efficacy of chlorantraniliprole on southern corn rootworm in peanut

Performance of Seed Treatments and Soil-Applied Insecticides Against Larval Western and Northern Corn Rootworm, 2019

Performance of Seed Treatments and Soil-Applied Insecticides Against Larval Western and Northern Corn Rootworm, 2019

Qtl analysis of western corn rootworm resistance traits in maize IBM population grown in continuous maize

The western corn rootworm (Diabrotica virgifera virgifera LeConte; WCR) is the most destructive maize pest in the USA, and one of the most dangerous pests in Europe. In just over a decade from the introduction it became one of the most serious pests in Croatia. Native resistant cultivars could be an efficient strategy for WCR control. Although research in WCR host plant resistance has continuously been conducted for over 70 years, no resistant maize hybrid is commercially available today. The aim of this study was to evaluate resistance traits in the maize IBM intermated recombinant inbred lines (IRILs), and detect QTLs associated with resistance to WCR herbivory. Total of 207 recombinant inbred lines were phenotyped for standard WCR resistance traits. Several associated QTLs were detected on chromosomes 1, 5, 6, 8, 9, and 10. QTLs on chromosomes 1 (6.41 % variance explained), and 6 (9.26 % variance explained) were mapped in regions harboring genes connected with plant defense against herbivory. Detected QTLs provide relevant guidelines for future research on WCR resistance in maize.

Investigation of <i>Agrobacterium</i>-mediated Transient dsRNA Expression in Tobacco

The Agrobacterium tumefaciens mediated gene transfer is widely used to generate genetic transformation of plants and transient assay of temporal exogenous gene expression. Syringe infiltration system into tobacco (Nicotiana benthamiana) leaves is a powerful tool for transient expression of target protein to study protein localization, protein-protein binding and protein production. However, the protocol and technical information of transient gene expression, especially double strand RNA (dsRNA), in tobacco using Agrobacterium is not well known. Recently, dsRNA is crucial for insecticidal effect on destructive agronomic pest such as Corn rootworm. In this study, we investigated the factor influencing the dsRNA expression efficiency of syringe agro-infiltration in tobacco. To search the best combination for dsRNA transient expression in tobacco, applied two Agrobacterium cell lines and three plant vector systems. The efficiency of dsRNA expression has estimated by real-time PCR and digital PCR. As a result, pHellsgate12 vector constructs showed the most effective accumulation of dsRNA in the cell. These results indicated that the efficiency of dsRNA expression was depending on the kind of vector rather than Agrobacterium cells. In summary, the optimized combination of transient dsRNA expression system in tobacco might be useful to in vivo dsRNA expression for functional study and risk assessment of dsRNA.

RNA-Based Biocontrols—A New Paradigm in Crop Protection

Modern agribusiness plays a vital role in safeguarding and improving the production, quality, and quantity of food, feed, fiber, and fuel. Growing concerns over the impact of chemical pesticides on health and the environment have stimulated the industry to search for alternative and greener solutions. Over the last years, the RNA interference (RNAi) process has been identified as a very promising new approach to complement the arsenal of foliar spray, soil, or seed treatments applied as chemical and biological pest control agents, and of plant-incorporated protectants (PIPs). RNA-based active ingredients (AIs) possess a unique mode of action and can be implemented via both genetic modification (GM) and biocontrol approaches. RNA-based AIs promise to deliver the selectivity and sustainability desired in future crop protection agents. This is due to their utilization of a natural process to exert control and their high level of selectivity, which leads to reduced risk for non-target organisms (NTOs). This review discusses the advantages and limitations of RNA-based solutions in crop protection and recent research progress toward RNA-based biocontrols against the Colorado potato beetle (CPB), corn rootworm (CRW), and soy stink bug (SSB). Many challenges still exist on the road to the implementation of a broad range of RNA-based products and their widespread use and application. Despite these challenges, it can be expected that RNA-based AIs will become valuable new tools complementing the current arsenal of crop-protection solutions.

Control of western corn rootworm via RNAi traits in maize: lethal and sublethal effects of Sec23 dsRNA.

RNA interference (RNAi) triggered by maize plants expressing RNA hairpins against specific western corn rootworm (WCR) transcripts have proven to be effective at controlling this pest. To provide robust crop protection, mRNA transcripts targeted by double-stranded RNA must be sensitive to knockdown and encode essential proteins. Using WCR adult feeding assays, we identified Sec23 as a highly lethal RNAi target. Sec23 encodes a coatomer protein, a component of the coat protein (COPII) complex that mediates ER-Golgi transport. The lethality detected in WCR adults was also observed in early instar larvae, the life stage causing most of the crop damage, suggesting that WCR adults can serve as an alternative to larvae for dsRNA screening. Surprisingly, over 85% transcript inhibition resulted in less than 40% protein knockdown, suggesting that complete protein knockdown is not necessary for Sec23 RNAi-mediated mortality. The efficacy of Sec23 dsRNA for rootworm control was confirmed in planta; T0 maize events carrying rootworm Sec23 hairpin transgenes showed high levels of root protection in greenhouse assays. A reduction in larval survival and weight were observed in the offspring of WCR females exposed to Sec23 dsRNA LC25 in diet bioassays. We describe Sec23 as RNAi target for in planta rootworm control. High mortality in exposed adult and larvae and moderate sublethal effects in the offspring of females exposed to Sec23 dsRNA LC25 , suggest the potential for field application of this RNAi trait and the need to factor in responses to sublethal exposure into insect resistance management programs. © 2019 Society of Chemical Industry.

Structural and functional insights into the Diabrotica virgifera virgifera ATP-binding cassette transporter gene family

BackgroundThe western corn rootworm, Diabrotica virgifera virgifera, is a pervasive pest of maize in North America and Europe, which has adapted to current pest management strategies. In advance of an assembled and annotated D. v. virgifera genome, we developed transcriptomic resources to use in identifying candidate genes likely to be involved in the evolution of resistance, starting with members of the ATP-binding cassette (ABC) transporter family.ResultsIn this study, 65 putative D. v. virgifera ABC (DvvABC) transporters were identified within a combined transcriptome assembly generated from embryonic, larval, adult male, and adult female RNA-sequence libraries. Phylogenetic analysis placed the deduced amino-acid sequences of the DvvABC transporters into eight subfamilies (A to H). To supplement our sequence data with functional analysis, we identified orthologs of Tribolium castaneum ABC genes which had previously been shown to exhibit overt RNA interference (RNAi) phenotypes. We identified eight such D. v. virgifera genes, and found that they were functionally similar to their T. castaneum counterparts. Interestingly, depletion of DvvABCB_39715 and DvvABCG_3712 transcripts in adult females produced detrimental reproductive and developmental phenotypes, demonstrating the potential of these genes as targets for RNAi-mediated insect control tactics.ConclusionsBy combining sequence data from four libraries covering three distinct life stages, we have produced a relatively comprehensive de novo transcriptome assembly for D. v. virgifera. Moreover, we have identified 65 members of the ABC transporter family and provided the first insights into the developmental and physiological roles of ABC transporters in this pest species.

Esterase activity and heat shock protein levels in western corn rootworm beetles (Diabrotica virgifera virgifera LeConte, Coleoptera: Chrysomelidae) after pesticide exposure

The aim of this study was to investigate how the exposure of the western corn rootworm beetle (WCR) to dimethoate might affect their ability to detoxify and Hsps response. The activity of the esterases: acetylcholinesterase (AChE) and carboxylesterase (CbE) and the level of Hsp90, Hsp70 and Hsp40 was assayed in adult individuals 1 h and 24 h after a topical pesticide application. A slight increase in the esterase activity 1 h after pesticide exposure was followed by a significant decrease in AChE after 24 h. The ELISA method revealed differences in the Hsps concentrations that were dependent on the protein family. Western Blotting data confirmed these results. The esterase activity and Hsps response are discussed.

Development of an improved and accessible diet for western corn rootworm larvae using response surface modeling

The western corn rootworm (WCR), Diabrotica virgifera virgifera LeConte, is an important pest of maize (Zea mays L.). Published WCR diets contain corn root powder, which is not available for purchase, thereby limiting the practical use of diets containing this ingredient. We applied response surface modeling combined with mixture designs to formulate a WCR diet that does not require corn root powder. We developed the new formulation by systematically exploring eight protein ingredients from animal, plant, and yeast sources based on simultaneous evaluation of three life history parameters (weight, molting, and survival). This formulation (WCRMO-2) without corn root powder supported approximately 97% of larval survival and successful molting. Larval weight gain after 10 days of feeding on WCRMO-2 was 4-fold greater than that of larvae feeding on the current best published WCR diet. Additionally, there was no significant difference in these larval performance traits when larvae were reared on WCRMO-2 and the best proprietary WCR diet. A commercial version of WCRMO-2 was tested and found to perform comparably for these traits. These improvements met our goal of a diet comprised of available ingredients that supports performance of WCR larvae equal to or better than publicly available formulations and proprietary formulations.

Pest categorisation of Diabrotica virgifera zeae.

The EFSA Panel on Plant Health performed a pest categorisation of Diabrotica virgifera zeae (Coleoptera: Chrysomelidae), the Mexican corn rootworm, for the EU. This is one of two subspecies of D. virgifera which occurs in Central America, Mexico and central southern parts of the USA (Texas, Oklahoma and New Mexico). The preferred larval host is maize (Zea mays) roots, although larvae can feed on the roots of sorghum and other grass species. Adults feed on the leaves, silks, immature seeds of maize, and pollen of up to 63 plant genera. Eggs are laid in the soil of maize fields in late summer/early autumn and hatch in late spring. Adults are found in and near maize fields from May until frosts appear later in the year. D. virgifera zeae is univoltine except where maize is grown continuously when there can be multiple overlapping generations each year. In the Americas, D. virgifera zeae is considered a key maize pest. D. virgifera zeae is regulated by Directive 2000/29/EC (Annex IAI). A general prohibition of soil from most third countries prevents the entry of immature stages of D. virgifera zeae. However, adults could be carried on sweetcorn or green maize. Maize is grown widely across the EU, but establishment may be limited to warmer parts of southern EU. Should it establish in the EU, impact on maize yields is anticipated. Phytosanitary measures are available to inhibit entry of this pest. D. virgifera zeae satisfies the criteria, which are within the remit of EFSA to assess for it to be regarded as a potential Union quarantine pest. D. virgifera zeae does not meet the criteria of occurring in the EU, nor plants for planting being the principal means of spread, for it to be regarded as a potential Union regulated non‐quarantine pest.

Pest categorisation of Diabrotica barberi.

The EFSA Panel on Plant Health performed a pest categorisation of Diabrotica barberi (Coleoptera: Chrysomelidae), the northern corn rootworm, for the EU. D. barberi is a univoltine species occurring in mid‐western and eastern USA and Canada, where it reproduces on maize (Zea mays), the preferred larval host. A small proportion of individuals can develop to a lesser extent on spelt (Triticum spelta), rice (Oryza sativa), millet (Panicum miliaceum) and a few North American wild grasses. Eggs are laid in the soil of maize fields, where they overwinter and can enter a diapause which can extend for more than one winter. Larvae hatch in late spring and early summer. Adult emergence peaks in the summer to feed on maize tassels, silks and ear tips. Adults abandon maize fields looking for other feeding hosts and return to maize for oviposition during late summer and autumn. D. barberi is considered a key pest of maize, together with other rootworm species of the same genus. D. barberi is regulated in the EU by Directive 2000/29/EC (Annex IAI). Within this Directive, a general prohibition of soil from most third countries prevents the entry of D. barberi larvae. However, adults carried on sweetcorn or green maize are potential pathways for entry into the EU. Climatic conditions and the wide availability of maize provide conditions to support establishment in the EU. Following establishment, impact on maize yields is anticipated. Phytosanitary measures are available to inhibit entry of this pest. D. barberi satisfies the criteria, which are within the remit of EFSA to assess for it to be regarded as a potential Union quarantine pest. D. barberi does not meet the criteria of occurring in the EU nor plants for planting being the principal means of spread for it to be regarded as a potential Union regulated non‐quarantine pest.

Using Flight Mills to Measure Flight Propensity and Performance of Western Corn Rootworm, Diabrotica virgifera virgifera (LeConte).

The western corn rootworm, Diabrotica virgifera virgifera (LeConte) (Coleoptera: Chrysomelidae), is an economically important pest of corn in the northern United States. Some populations have developed resistance to management strategies including transgenic corn that produces insecticidal toxins derived from the bacterium Bacillus thuringiensis (Bt). Knowledge of western corn rootworm dispersal is of critical importance for models of resistance evolution, spread, and mitigation. Flight behavior of an insect, especially over a long distance, is inherently difficult to observe and characterize. Flight mills provide a means to directly test developmental and physiological impacts and consequences of flight in the laboratory that cannot be obtained in field studies. In this study, flight mills were used to measure the timing of flight activity, total number of flights, and the distance, duration, and speed of flights taken by female rootworms during a 22-h test period. Sixteen flight mills were housed in an environmental chamber with programmable lighting, temperature, and humidity control. The flight mill described is of a typical design, where a flight arm is free to rotate about a central pivot. Rotation is caused by flight of an insect tethered to one end of the flight arm, and each rotation is recorded by a sensor with a time-stamp. Raw data are compiled by software, which are subsequently processed to provide summary statistics for flight parameters of interest. The most difficult task for any flight mill study is attachment of the tether to the insect with an adhesive, and the method used must be tailored to each species. The attachment must be strong enough to hold the insect in a rigid orientation and to prevent detachment during movement, while not interfering with natural wing motion during flight. The attachment process requires dexterity, finesse, and speed, making video footage of the process for rootworms of value.

Plant defense resistance in natural enemies of a specialist insect herbivore

Plants defend themselves against herbivores through the production of toxic and deterrent metabolites. Adapted herbivores can tolerate and sometimes sequester these metabolites, allowing them to feed on defended plants and become toxic to their own enemies. Can herbivore natural enemies overcome sequestered plant defense metabolites to prey on adapted herbivores? To address this question, we studied how entomopathogenic nematodes cope with benzoxazinoid defense metabolites that are produced by grasses and sequestered by a specialist maize herbivore, the western corn rootworm. We find that nematodes from US maize fields in regions in which the western corn rootworm was present over the last 50 y are behaviorally and metabolically resistant to sequestered benzoxazinoids and more infective toward the western corn rootworm than nematodes from other parts of the world. Exposure of a benzoxazinoid-susceptible nematode strain to the western corn rootworm for 5 generations results in higher behavioral and metabolic resistance and benzoxazinoid-dependent infectivity toward the western corn rootworm. Thus, herbivores that are exposed to a plant defense sequestering herbivore can evolve both behavioral and metabolic resistance to plant defense metabolites, and these traits are associated with higher infectivity toward a defense sequestering herbivore. We conclude that plant defense metabolites that are transferred through adapted herbivores may result in the evolution of resistance in herbivore natural enemies. Our study also identifies plant defense resistance as a potential target for the improvement of biological control agents.

Survey of bacteria associated with western corn rootworm life stages reveals no difference between insects reared in different soils

Western corn rootworm (Diabrotica virgifera virgifera LeConte) is a serious pest of maize (Zea mays L.) in North America and parts of Europe. With most of its life cycle spent in the soil feeding on maize root tissues, this insect is likely to encounter and interact with a wide range of soil and rhizosphere microbes. Our knowledge of the role of microbes in pest management and plant health remains woefully incomplete, yet that knowledge could play an important role in effective pest management strategies. For this study, insects were reared on maize in soils from different locations. Insects from two different laboratory colonies (a diapausing and a non-diapausing colony) were sampled at each life stage to determine the possible core bacteriome. Additionally, soil was sampled at each life stage and resulting bacteria were identified to determine the possible contribution of soil to the rootworm bacteriome, if any. We analyzed the V4 hypervariable region of bacterial 16S rRNA genes with Illumina MiSeq to survey the different species of bacteria associated with the insects and the soils. The bacterial community associated with insects was significantly different from that in the soil. Some differences appear to exist between insects from non-diapausing and diapausing colonies while no significant differences in community composition existed between the insects reared on different soils. Despite differences in the bacteria present in immature stages and in male and female adults, there is a possible core bacteriome of approximately 16 operational taxonomic units (i.e., present across all life stages). This research may provide insights into Bt resistance development, improved nutrition in artificial rearing systems, and new management strategies.

Optimizing Egg Recovery From Wild Northern Corn Rootworm Beetles (Coleoptera: Chrysomelidae).

The northern corn rootworm, Diabrotica barberi Smith & Lawrence (Coleoptera: Chrysomelidae), is one of the most important insect pests in the U.S. Corn Belt. Efforts to obtain eggs from wild northern corn rootworm populations using techniques developed for other rootworm species have been unsuccessful due to lack of oviposition. In 2016, we evaluated four oviposition media in choice tests within each of three female densities in 30.5 × 30.5 × 30.5 cm BugDorm cages. The number of eggs laid per female was significantly affected by female density and the interaction of female density × oviposition media, but oviposition was relatively poor in all oviposition media (1.2 eggs per female when averaging the three female densities and all oviposition media). Single females were also evaluated in nonchoice assays in 6 cm × 6 cm × 8 cm clear plastic boxes and averaged up to 108 eggs per female depending on the oviposition media. In 2017, the cumulative number of eggs laid per female in boxes with one female was not significantly different from the number of eggs laid per female in boxes with 3 females. In 2018, the cumulative number of eggs laid per female was not significantly different between female densities of 1, 3, 5, or 10 females per box. Total egg production per box therefore increased as female density increased. More than 27,000 wild northern corn rootworm eggs were collected from just 190 females when collected relatively early in the field season. We now have an efficient and robust system for obtaining eggs from wild northern corn rootworm females.

Entomopathogenic nematodes increase predation success by inducing cadaver volatiles that attract healthy herbivores.

Herbivore natural enemies protect plants by regulating herbivore populations. Whether they can alter the behavior of their prey to increase predation success is unknown. We investigate if and how infection by the entomopathogenic nematode Heterorhabditis bacteriophora changes the behavior of healthy larvae of the western corn rootworm (Diabrotica virgifera), a major pest of maize. We found that nematode-infected rootworm cadavers are attractive to rootworm larvae, and that this behavior increases nematode reproductive success. Nematode-infected rootworms release distinct volatile bouquets, including the unusual volatile butylated hydroxytoluene (BHT). BHT alone attracts rootworms, and increases nematode reproductive success. A screen of different nematode and herbivore species shows that attraction of healthy hosts to nematode-infected cadavers is widespread and likely involves species-specific volatile cues. This study reveals a new facet of the biology of herbivore natural enemies that boosts their predation success by increasing the probability of host encounters.

Field efficacy of soil insecticides on pyrethroid-resistant western corn rootworms (Diabrotica virgifera virgifera LeConte).

Field-evolved pyrethroid resistance has been confirmed in western corn rootworm (WCR) populations collected from the United States (US) western Corn Belt. Resistance levels of WCR adults estimated in lab bioassays were confirmed to significantly reduce the efficacy of foliar-applied bifenthrin. The objective of the present study was to investigate the impact of WCR pyrethroid resistance levels on the performance of common soil-applied insecticide formulations (23.4% tefluthrin, 17.15% bifenthrin, and 0.1% cyfluthrin + 2.0% tebupirimphos). Field trials were conducted in 2016 and 2017 in three Nebraska, US, counties (Saunders, Clay, and Keith) where distinct levels of WCR susceptibility to pyrethroids (susceptible, moderately resistant, and highly resistant) had been previously reported in adult and larval bioassays. All soil insecticide treatments effectively protected maize roots from a pyrethroid-susceptible WCR population at Saunders. In contrast, the efficacy of bifenthrin and tefluthrin soil insecticides was significantly reduced at Clay and Keith, where pyrethroid-resistant WCR populations were reported. At Keith, where an additional failure of the cyfluthrin + tebupirimphos soil insecticide was observed, WCR laboratory dose-response bioassays showed a consistent ∼5-fold resistance level to the active ingredients bifenthrin, tefluthrin, and cyfluthrin. The efficacy of common soil insecticides used in the US for WCR management was significantly reduced in populations exhibiting relatively low levels of WCR pyrethroid resistance. Using a multitactical approach to manage WCR within an integrated pest management framework may mitigate resistance evolution and prolong the usefulness of WCR insecticides within the system. © 2019 Society of Chemical Industry.

Field-evolved resistance by western corn rootworm to Cry34/35Ab1 and other Bacillus thuringiensis traits in transgenic maize.

Transgenic crops producing insecticidal toxins from the bacterium Bacillus thuringiensis (Bt) are widely planted to manage agricultural insect pests. However, widespread adoption of Bt crops has led to the evolution of Bt resistance. The western corn rootworm, Diabrotica virgifera virgifera, is among the most serious pests of maize in the midwestern United States and is currently managed with Bt maize. To date, there is evidence of field-evolved resistance to all Bt toxins used to manage this pest. While western corn rootworm resistance to Cry3Bb1, and the closely related mCry3A and eCry3.1Ab traits, is widely distributed within the Midwest, fewer cases of Cry34/35Ab1 resistance have been observed, and planting of Cry34/35Ab1 maize is one of the methods used to manage Cry3-resistant rootworm. We found that fields with high levels of root injury to Cry34/35Ab1 maize by western corn rootworm were associated with Cry34/35Ab1-resistant western corn rootworm. Additionally, a population not associated with high levels of root injury was found to be resistant to Cry34/35Ab1. In all cases, populations that were resistant to Cry34/35Ab1 also were resistant to Cry3 traits. Western corn rootworm resistance to Cry34/35Ab1 has continued to persist in the agricultural landscape and has likely increased. The presence of rootworm populations with resistance to all available Bt traits threatens the utility of current and future transgenic technologies to manage this pest. Decreased reliance on Cry34/35Ab1 and better use of integrated pest management will be essential to preserve Bt susceptibility in western corn rootworm. © 2019 Society of Chemical Industry.