Within-plant variation in chemical defence of Erysimum cheiranthoides does not explain Plutella xylostella feeding preference

O_LIPlants invest a substantial fraction of their resources into defence against herbivores, with the highest levels of defence often allocated only to the most valuable tissues. Plants in the genus Erysimum (Brassicaceae) evolved the ability to produce novel cardenolides in addition to ancestrally conserved glucosinolates. While plants co-express both defences, differences in tissue-specific expression might represent an effective cost saving strategy. C_LIO_LILarvae of the glucosinolate-resistant diamondback moth Plutella xylostella occasionally feed on Erysimum cheiranthoides but tend to avoid younger leaves. Here, we predicted that caterpillar feeding preference is shaped by variation in cardenolide levels, thus we quantified within-plant variation in defensive and nutritional traits of vegetative or early reproductive plants and performed feeding assays to evaluate the relative importance of cardenolides. C_LIO_LIIn accordance with optimal defence theory (ODT), youngest leaves contained the most nutrients and the highest levels of cardenolides, glucosinolates, and trichomes, with more extreme within-plant differences found in reproductive plants. Caterpillars consistently avoided the well-defended youngest leaves both on whole plants and with detached leaf discs. Surprisingly, neither experimental addition (by external application) nor removal (by CRISPR-Cas9 knockout) of cardenolides significantly affected caterpillar feeding preference. C_LIO_LIPhysical and chemical defences, including cardenolides, co-vary within E. cheiranthoides to maximise defence of youngest leaves. While P. xylostella clearly responds to some of these traits, the prominent cardenolide defence appears to lack potency against this specialist herbivore. Nonetheless, the careful regulation and re-mobilization of cardenolides to younger leaves during plant development suggests an important role for plant functioning. C_LI One-sentence summaryDistribution of ancestral and novel defensive chemicals Erysimum cheiranthoides vary within plant according to relative leaf value and leaf age, but in isolation, chemical defences are insufficient to affect feeding preference of Plutella xylostella larvae.