Different fates of metabolites and small variation in chemical composition characterise frass chemistry in a specialist caterpillar

Specialist and generalist insect herbivores evolved different strategies to process host-plant metabolites. We explored frass composition in Laothoe populi (L.) caterpillars (Lepidoptera: Sphingidae), a specialist moth species that is closely associated with plants of the Salicaceae (Mirb.) family. We fed the caterpillars with leaves from three willow species (Salix caprea L., S. purpurea L. and S. viminalis L.), one willow hybrid (S. x rubens Schrank) and one poplar species (Populus tremula L.). Using untargeted metabolomics, we compared the chemical composition and variation among leaf and frass samples. We quantified the chemical variation using either a simple index based on the presence and concentration of metabolites (i.e., Bray-Curtis) or an index that additionally accounts for chemical structural-compositional similarity (CSCS) among metabolites. Due to the high degree of dietary specialisation, we expected low host-specific variation in frass composition among the caterpillars. Based on literature, we further hypothesised that ingested salicinoids will be largely modified in the caterpillar gut while flavonoids will be often passively excreted. Finally, we compared chemical variation among the samples when quantified with Bray-Curtis or CSCS metrics. As expected, we found relatively low host-specific variation in the chemical composition of caterpillar frass. Our results further suggest that flavonoids were largely passively excreted by the caterpillars, while salicinoids were metabolised in the gut. Finally, we found that chemical composition measures based on Bray-Curtis overestimated the differences in chemical composition between frass and leaves, suggesting that for these type of samples CSCS measures are better suited to reflect metabolic changes more realistically.