Dual herbivore attack and herbivore density affect metabolic profiles of Brassica nigra leaves

Plant responses to dual herbivore attack are increasingly studied, but effects on the metabolome have largely been restricted to volatile metabolites and defence-related non-volatile metabolites. However, plants subjected to stress, such as herbivory, undergo major changes in both primary and secondary metabolism. Using a naturally occurring system, we investigated metabolome-wide effects of single or dual herbivory on Brassica nigra plants by Brevicoryne brassicae aphids and Pieris brassicae caterpillars, while also considering the effect of aphid density. Metabolomic analysis of leaf material showed that single and dual herbivory had strong effects on the plant metabolome, with caterpillar feeding having the strongest influence. Additionally, aphid-density-dependent effects were found in both the single and dual infestation scenarios. Multivariate analysis revealed treatment-specific metabolomic profiles, and effects were largely driven by alterations in the glucosinolate and sugar pools. Our work shows that analysing the plant metabolome as a single entity rather than as individual metabolites provides new insights into the subcellular processes underlying plant defence against multiple herbivore attackers. These processes appear to be importantly influenced by insect density.