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. Research has increasingly shifted from studying the effects of single attackers to multiple insect herbivore attackers on a plant, and we have already gained important knowledge how insect behaviour and plant responses are affected. However, work on how the plant metabolome is affected by dual attack is largely restricted to the volatile metabolome or defence‐related pools of non‐volatile metabolites, even though herbivory leads to major changes in both primary and secondary metabolism. Here, we investigated how dual herbivore attack will reconfigure plant metabolism as a whole, and showed that both single and dual attack by aphids and caterpillars affect plant metabolome, with caterpillars having the strongest effects. Additionally, we show that aphid density is an important modulating factor, affecting metabolome during both single and dual attack. Multivariate analysis revealed treatment‐specific metabolic profiles, with alterations in the glucosinolate and sugar pools driving the effects.