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.