An improved bioassay for the testing of entomopathogenic nematode virulence to the western corn rootworm (Diabrotica virgifera virgifera) (Coleoptera: Chrysomelidae): with focus on neonate insect assessments

Authors: GEISERT, RYAN - University Of Missouri; HUYNH, MAN - University Of Missouri; Pereira, Adriano; Shapiro Ilan, David; Hibbard, Bruce

Submitted to: Journal of Economic Entomology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 2/21/2023
Publication Date: 6/14/2023
Citation: Geisert, R.W., Huynh, M.P., Pereira, A.E., Shapiro Ilan, D.I., Hibbard, B.E. 2023. An improved bioassay for the testing of entomopathogenic nematode virulence to the western corn rootworm (Diabrotica virgifera virgifera) (Coleoptera: Chrysomelidae): with focus on neonate insect assessments. Journal of Economic Entomology. 116(3): 726-732. https://doi.org/10.1093/jee/toad052.
DOI: https://doi.org/10.1093/jee/toad052

Interpretive Summary: Insect bioassays involving newly hatched larval insects can be limited by the insects need for food. When testing natural enemies such as entomopathogenic nematodes it has been necessary to either work with older larvae or to provide corn roots as a food source, but larvae can hide inside roots. The recent development of an improved diet for western corn rootworm larvae provides a new bioassay type for evaluating entomopathogenic nematodes that also makes the testing arena easy to observe. Here, we evaluated four species of entomopathogenic nematodes against neonate western corn rootworm in diet bioassays. Nematode inoculation levels were 0, 15, 30, 60, and 120 nematodes per larvae. Percentage mortality increased for each species as the rate of inoculation increased. The diet-based bioassays were shown to be an effective method for nematode exposure to insect pests. The assays provided adequate moisture to keep nematode from desiccating while also allowing freedom of movement around the arenas. Both rootworm larvae and nematodes were contained within the assay arenas. The addition of nematodes did not cause any significant deterioration of the diet within the three-day period of testing. This new technique will allow for quick, easy, and accurate screening of new nematode species and strains in insect pests such as rootworms.

Technical Abstract: Bioassays involving newly hatched larval insects can be limited by the larvae’s feeding state. Assays attempting to monitor mortality effects can be negatively affected by starvation effects on the larvae. Neonate western corn rootworms have significant reductions in viability if not provided food within 24 h post hatch. The recent development of an improved artificial diet for western corn rootworm larvae provides a new bioassay type for evaluating entomopathogenic nematodes that also makes the testing arena easy to observe. Here, we evaluated four species of entomopathogenic nematodes including Heterorhabditis bacteriophora Poinar, Steinernema carpocapsae (Weiser), Steinernema diaprepesi Nguyen & Duncan, and Steinernema rarum (de Doucet) against neonate western corn rootworm, Diabrotica virgifera virgifera LeConte, in 96-well plate diet bioassays. Nematode inoculation levels were 0, 15, 30, 60, and 120 nematodes per larva. Percentage mortality increased for each species as the rate of inoculation increased. Overall, H. bacteriophora and S. carpocapsae caused the greatest amount of larval mortality. The diet-based bioassays were shown to be an effective method for nematode exposure to insect pests. The assays provided adequate moisture to keep nematode from desiccating while also allowing freedom of movement around the arenas. Both rootworm larvae and nematodes were contained within the assay arenas. The addition of nematodes did not cause any significant deterioration of the diet within the three-day period of testing. Overall, the diet bioassays worked well as a measure of entomopathogenic nematode virulence.