Abstract
Plant responses to leaf-mining insects are still poorly studied, especially in woody species. Robinia pseudoacacia is widely cultivated in Ukraine, where it is not native. There, the trees are facing increasing damage from the likewise introduced leaf miner species Parectopa robiniella and Macrosaccus robiniella. We examined the effects of infestation by these species on foliar carbon (C) and nitrogen (N) content and on metabolic fingerprints of trees of different age and at different locations, expecting context dependency in induction responses. Uninfested or miner-infested leaflets were collected from infested trees of three age classes at one location and from trees of one age class at four distinct locations. C and N content were mostly determined by location, with minor effects of tree age and herbivory, for N in interaction. Among the 3,121 metabolic features detected with UHPLC-QTOF-MS/MS, 1,087 were quantitatively modulated by herbivory in at least one age–location combination. Metabolic responses to the two leaf miner species partially overlapped. The magnitude and direction of metabolic shifts varied with both tree age and location. The differences in leaf biochemistry and in responses to herbivory between tree age classes and locations may reflect physiological constraints, past environmental conditions, age-dependent defense strategies, resource availabilities and allocation as well as local edaphic and microclimatic conditions, but potentially also different damage levels. That the leaflets of R. pseudoacacia exhibited context-dependent biochemical responses to the leaf miners highlights the importance of accounting for plant ontogenetic stage and local environmental heterogeneity when evaluating plant–leaf miner interactions. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s12870-026-08364-6.