Sequestration and activation of plant toxins protect the western corn rootworm from enemies at multiple trophic levels

Highly adapted herbivores can phenocopy two-component systems by stabilizing, sequestering and reactivating plant toxins. However, whether these traits protect herbivores against their enemies is poorly understood. We demonstrate that the western corn rootworm Diabrotica virgifera virgifera, the most damaging maize pest on the planet, specifically accumulates the root-derived benzoxazinoid glucosides HDMBOA-Glc and MBOA-Glc. MBOA-Glc is produced by D. virgifera through stabilization of the benzoxazinoid breakdown product MBOA by N-glycosylation. The larvae can hydrolyze HDMBOA-Glc, but not MBOA-Glc, to produce toxic MBOA upon predator attack. Accumulation of benzoxazinoids renders D. virgifera highly resistant to nematodes which inject and feed on entomopathogenic symbiotic bacteria. While HDMBOA-Glc and MBOA reduce the growth and infectivity of both the nematodes and the bacteria, MBOA-Glc repels infective juvenile nematodes. Our results illustrate how herbivores combine stabilized and reactivated plant toxins to defend themselves against a deadly symbiosis between the third and the fourth trophic level enemies.