Additional file S2 Alignments of BGDs and GBAs from Spatial separation of the cyanogenic β-glucosidase ZfBGD2 and cyanogenic glucosides in the haemolymph of <i>Zygaena</i> larvae facilitates cyanide release

Low molecular weight compounds are typically used by insects and plants for defence against predators. They are often stored as inactive β-glucosides and kept separate from activating β-glucosidases. When the two components are mixed, the β-glucosides are hydrolysed releasing toxic aglucones. Cyanogenic plants contain cyanogenic glucosides and release hydrogen cyanide due to such a well-characterized two-component system. Some Arthropods are also cyanogenic, but comparatively little is known about their system. Here, we identify a specific β-glucosidase (<i>ZfBGD2)</i> involved in cyanogenesis from larvae of <i>Zygaena filipendulae</i> (Lepidoptera, Zygaenidae), and analyse the spatial organization of cyanide release in this specialized insect. High levels of <i>ZfBGD2</i> mRNA and protein were found in haemocytes by transcriptomic and proteomic profiling. Heterologous expression in insect cells showed that ZfBGD2 hydrolyses linamarin and lotaustralin, the two cyanogenic glucosides present in <i>Z. filipendulae</i>. Linamarin and lotaustralin as well as cyanide release were found exclusively in the haemoplasma. Phylogenetic analyses revealed that <i>ZfBGD2</i> clusters with other insect β-glucosidases, and correspondingly, the ability to hydrolyse cyanogenic glucosides catalysed by a specific β-glucosidase evolved convergently in insects and plants. The spatial separation of the β-glucosidase ZfBGD2 and its cyanogenic substrates within the haemolymph provides the basis for cyanide release in <i>Z. filipendulae</i>. This spatial separation is similar to the compartmentalization of the two components found in cyanogenic plant species, and illustrates one similarity in cyanide-based defence in these two kingdoms of life.