Development of a nondiapausing strain of northern corn rootworm with rearing techniques for both diapausing and nondiapausing strains

The northern corn rootworm, Diabrotica barberi Smith & Lawrence, has a univoltine life cycle that typically produces one generation a year. When rearing the northern corn rootworm in the laboratory, in order to break diapause, it is necessary to expose eggs to a five month cold period before raising the temperature. By selective breeding of the small fraction of eggs that hatched without cold within 19–32 days post oviposition, we were able to develop a non-diapausing colony of the northern corn rootworm within five generations of selection. Through selection, the percentages of adult emergence from egg hatch without exposure to cold treatment significantly increased from 0.52% ± 0.07 at generation zero to 29.0% ± 2.47 at generation eight. During this process, we developed an improved method for laboratory rearing of both the newly developed non-diapausing strain as well as the diapausing strain. The development of the non-diapausing colony along with the improvements to the rearing system will allow researchers to produce up to six generations of the northern corn rootworm per year, which would facilitate research and advance our knowledge of this pest at an accelerated rate.

Baseline Susceptibility of a Laboratory Strain of Northern Corn Rootworm, Diabrotica barberi (Coleoptera: Chrysomelidae) to Bacillus thuringiensis Traits in Seedling, Single Plant, and Diet-Toxicity Assays

The northern corn rootworm (NCR), Diabrotica barberi Smith & Lawrence, is an economic pest of maize in the U.S. Corn Belt. The objective of this study was to determine the baseline susceptibility of a laboratory NCR strain to Bt proteins eCry3.1Ab, mCry3A, Cry3Bb1, and Cry34/35Ab1 using seedling, single plant, and diet-toxicity assays. Plant assays were performed in greenhouse using corn hybrids expressing one of the Bt proteins and each respective near-isoline. Diet-toxicity assays, consisting of Bt proteins overlaid onto artificial diet were also conducted. In both plant assays, significantly more larvae survived Cry34/35Ab1-expressing corn compared with all other Bt-expressing corn, and larvae that survived eCry3.1Ab-expressing corn had significantly smaller head capsule widths compared with larvae that survived Cry34/35Ab1-expressing corn. In seedling assays, larvae surviving eCry3.1Ab-expressing corn also had significantly smaller head capsule widths compared with larvae that survived mCry3A-expressing corn. Additionally, larvae that survived mCry3A-expressing corn weighed significantly more than larvae surviving eCry3.1Ab- and Cry34/35Ab1-expressing corn. In single plant assays, no significant differences in larval dry weight was observed between any of the Bt-expressing corn. In diet assays, LC50s ranged from 0.14 (eCry3.1Ab) to 10.6 µg/cm2 (Cry34/35Ab1), EC50s ranged from 0.12 (Cry34/35Ab1) to 1.57 µg/cm2 (mCry3A), IC50s ranged from 0.08 (eCry3.1Ab) to 2.41 µg/cm2 (Cry34/35Ab1), and MIC50s ranged from 2.52 (eCry3.1Ab) to 14.2 µg/cm2 (mCry3A). These results establish the toxicity of four Bt proteins to a laboratory diapausing NCR strain established prior to the introduction of Bt traits and are important for monitoring resistance evolution in NCR field populations.

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.

Partial characterization of Bt resistance and the bacteriome of western corn rootworm

Western corn rootworm (Diabrotica virgifera virgifera LeConte) and northern corn rootworm (Diabrotica barberi (Smith and Lawrence)) are major pests of maize in the USA. These pests have been managed with a variety of tactics over the last century. Both Diabrotica spp. have adapted to crop rotation in different ways in certain regions of the USA as well as to some of the insecticides targeted at them. D. v. virgifera has adapted to more of the chemical control measures and transgenic control methods. Discussed in this review are the challenges associated with managing both species, and how current management strategies might be combined and implemented to help manage damage from these species. Also, we discuss the potential for new technologies, such as RNA interference, to be used in the future.

Development and improvement of artificial diets for larvae of Diabrotica species using multidimensional design space techniques

The western corn rootworm (Coleoptera: Chrysomelidae; Diabrotica virgifera virgifera LeConte) and the northern corn rootworm (Coleoptera: Chrysomelidae; Diabrotica barberi Smith and Lawrence) are highly adaptive insect pests and have developed resistance to most possible management tactics in some regions. Transgenic maize hybrids, the latest control tools, which express insecticidal crystalline toxins from Bacillus thuringiensis (Bt) Berliner, have also faltered due to physiological adaptation of western corn rootworm populations. The United States Environmental Protection Agency has mandated insect resistance management plans for corn rootworms that have been directed toward monitoring the development of resistance to each of the Bt toxins. Toxicity bioassays using artificial diet have proven to be valuable for monitoring resistance to Bt toxins in corn rootworm populations. Currently, several proprietary diet formulations for western corn rootworm larvae that have been developed by each of the maize seed companies are used by industry and public researchers in toxicity bioassays to detect decreases in susceptibility of western corn rootworm populations to Bt proteins. No artificial diet has been developed specifically for northern corn rootworm larvae. Since differences in diet formulations can lead to different results in diet toxicity assays, it is impossible to make diet comparisons between toxicity assays using different artificial diets from individual companies and it may not be possible to determine an accurate picture of the test population's phenotype as it relates to Bt resistance. We report new diet formulations that support improved weight gain, larval development, and survival compared with current diets used in western corn rootworm and northern corn rootworm bioassays. The new formulations were created by using response surface modeling coupled with n-dimensional mixture designs to identify and optimize key ingredients based on integrated evaluation of several life history parameters (i.e., weight, molting, survival) while limiting contamination. The new rootworm formulations supported approximately 97% larval survival and molting and increased larval weight gain after 10 days of feedings compared to other diets used in western corn rootworm and northern corn rootworm bioassays rearing. These new formulations provide a standardized growth medium for western corn rootworm and northern corn rootworm larvae that will facilitate toxicity test comparisons conducted by different working groups and meets all regulatory requirements. We developed an improved diet formulation (WCRMO-1) for western corn rootworm (Chapter 2), which was the optimization of diet ingredients in the only published diet for western corn rootworm larvae. This formulation was also compatible for use with all current Bt proteins targeting western corn rootworm larvae. However, this formulation contains corn root powder, which is not available for purchase, limiting the practical use of the diets. We demonstrated that essential growth factors in corn roots that assist in western corn rootworm growth can be extracted suggesting substituting corn root powder with compounds identified from the extract may be possible (Chapter 3). An improved and accessible diet for western corn rootworm (WCRMO-2) that is comparable with all current diets for western corn rootworm larvae and without corn root powder was created by exploring and characterizing protein ingredients from plant, animal and yeast products (Chapter 4). Lastly, we developed the first artificial diet (NCRMO-1) for northern corn rootworm (Chapter 5) comprised of available ingredients that supports performance of northern corn rootworm larvae better than that of publicly available rootworm formulations.