Heliconius butterflies are highly specialized in Passiflora plants, laying eggs and feeding as larvae only on them. Interestingly, both Heliconius butterflies and Passiflora plants contain cyanogenic glucosides (CNglcs). While feeding on specific Passiflora species, Heliconius melpomene 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 Passiflora species and they were never tested for sequestration by heliconiine larvae. As other cyanogenic lepidopterans, H. melpomene 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 biochemically characterized in butterflies, the cytochromes P450 CYP405A4, CYP405A5, CYP405A6 and CYP332A1 have been hypothesized to be involved in this pathway in H. melpomene. 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 developmental stages of Heliconius butterflies. We also establish which kind of CNglcs H. melpomene larvae can sequester from Passiflora. By analysing the chemical composition of the haemolymph from larvae fed with different Passiflora diets, we show that H. melpomene is able to sequestered prunasin, an aromatic CNglcs, from P. platyloba. They are also able to sequester amygdalin, gynocardin, [C¹³/C¹⁴]linamarin and [C¹³/C¹⁴]lotaustralin painted on the plant leaves. The CNglc tetraphyllin B-sulphate from P. caerulea is not detected in the larval haemolymph, suggesting that such modified CNglcs cannot be sequestered by Heliconius. 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 is catabolized to recycle nitrogen and glucose, and/or to produce fitness signals during courtship. Mature adults have a higher concentration of CNglcs than any other developmental stages due to increased de novo biosynthesis of linamarin and lotaustralin. Accordingly, all CYP405As are expressed in adults, whereas larvae mostly express CYP405A4. Our results shed light on the importance of CNglcs for Heliconius biology and their coevolution with Passiflora.

Heliconius蝴蝶高度专注于西番莲植物，产卵并以幼虫的形式只在它们身上进食。有趣的是，袖蝶属蝴蝶和西番莲植物含有氰苷（CNglcs）。在以特定的西番莲物种为食时，Heliconius melpomene幼虫能够隔离简单的环戊烯基 CNglcs，这是该植物属中最常见的 CNglcs。然而，在西番莲物种中已经报道了芳香族、脂肪族和修饰的 CNglcs，并且它们从未被 heliconiine 幼虫进行隔离测试。与其他含氰鳞翅目昆虫一样，H. melpomene还生物合成脂肪族 CNglcs 亚麻苦苷和 lotaustralin，它们的毒性不仅仅依赖于螯合。尽管编码 CNglc 生物合成酶的基因尚未在蝴蝶中进行生化表征，但已假设细胞色素 P450 CYP405A4、CYP405A5、CYP405A6 和 CYP332A1 参与了美洲红花蛾中的这一途径。在这项研究中，我们确定了在Heliconius蝴蝶的不同发育阶段，参与这些化合物生物合成的假定 P450s 的 CNglc 组成和表达如何变化。我们还确定了哪种 CNglcs H. melpomene幼虫可以从西番莲中隔离. 通过分析用不同西番莲饲料喂养的幼虫的血淋巴的化学成分，我们表明H. melpomene能够隔离P. platyloba 的prunasin，一种芳香的CNglcs。它们还能够隔离涂在植物叶子上的苦杏仁苷、gynocardin、[C ¹³ /C ¹⁴ ]亚麻苦苷和[C ¹³ /C ¹⁴ ]lotaustralin。从CNglc tetraphyllin B-硫酸盐P.翠在幼虫血淋巴中未检测，这表明这种修饰的CNglcs不能由被隔离袖蝶属. 虽然蛹和处女成虫含有幼虫螯合产生的二氢雌雄同心素，但这种化合物在成年期被代谢，并没有用作结婚礼物或转移给后代。因此，我们推测 dihydrogynocardin 被分解代谢以回收氮和葡萄糖，和/或在求偶期间产生健康信号。由于亚麻苦苷和 lotaustralin 的从头生物合成增加，成熟的成年人具有比任何其他发育阶段更高的 CNglcs 浓度。因此，所有CYP4 05A 都在成虫中表达，而幼虫主要表达CYP405A4。我们的结果阐明了 CNglcs 对Heliconius生物学及其与西番莲共同进化的重要性。