Sequestration and functional diversification of cyanogenic glucosides in the life cycle of Heliconius melpomene

butterflies are highly specialized in , laying eggs and feeding as larvae only on these plants. Interestingly, butterflies and plants both contain cyanogenic glucosides (CNglcs). While feeding on specific species, m larvae are able to sequester simple cyclopentenyl CNglcs, the most common CNglcs in this plant genus. Yet, aromatic, aliphatic, and modified CNglcs have been reported in species and they were never tested for sequestration by heliconiine larvae. As other cyanogenic lepidopterans, also biosynthesize the aliphatic CNglcs linamarin and lotaustralin, and their toxicity does not rely exclusively on sequestration. Although the genes encoding the enzymes in the CNglc biosynthesis have not yet been fully biochemically characterized in butterflies, the cytochromes P450 CYP405A4, CYP405A5, CYP405A6 and CYP332A1 are hypothesized to be involved in this pathway in . In this study, we determine how the CNglc composition and expression of the putative P450s involved in the biosynthesis of these compounds vary at different development stages of butterflies. We also established which kind of CNglcs larvae can sequestered from . By analysing the chemical composition of the haemolymph from larvae fed with different diets, we observed that is able to sequestered prunasin, an aromatic CNglcs, from . They were also able to sequester amygdalin, gynocardin, [C/C]linamarin and [C/C]lotaustralin painted on the plant leaves. The CNglc tetraphyllin B-sulphate from was not detected in the larval haemolymph, suggesting that such modified CNglcs cannot be sequestered by . Although pupae and virgin adults contain dihydrogynocardin resulting from larval sequestration, this compound was metabolized during adulthood, and not used as nuptial gift or transferred to the offspring. Thus, we speculate that dihydrogynocardin was catabolized to recycle nitrogen and glucose, and/or to produce fitness signals during courtship and calling. Mature adults had a higher concentration of CNglcs than any other developmental stages due to intense biosynthesis of linamarin and lotaustralin. All were expressed in adults, whereas larvae mostly expressed . Our results shed light on the importance of CNglcs in Heliconius biology and for their coevolution with