Additional file S3 from Spatial separation of the cyanogenic β-glucosidase ZfBGD2 and cyanogenic glucosides in the haemolymph of Zygaena 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 (ZfBGD2) involved in cyanogenesis from larvae of Zygaena filipendulae (Lepidoptera, Zygaenidae), and analyse the spatial organization of cyanide release in this specialized insect. High levels of ZfBGD2 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 Z. filipendulae. Linamarin and lotaustralin as well as cyanide release were found exclusively in the haemoplasma. Phylogenetic analyses revealed that ZfBGD2 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 Z. filipendulae. 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.低分子量化合物（low molecular weight compounds）通常被昆虫与植物用以抵御捕食者。这类物质常以无活性的β-葡萄糖苷（β-glucosides）形式储存，并与具有激活活性的β-葡萄糖苷酶（β-glucosidases）相互分隔。当两种组分混合时，β-葡萄糖苷会被水解，释放出有毒的苷元（aglucones）。生氰植物（cyanogenic plants）含有生氰葡萄糖苷（cyanogenic glucosides），并通过这种特征明确的双组分系统（two-component system）释放氰化氢（hydrogen cyanide）。部分节肢动物（arthropods）同样具备生氰特性，但目前对其相关系统的研究仍相对匮乏。本研究从Zygaena filipendulae（鳞翅目Lepidoptera，斑蛾科Zygaenidae）的幼虫体内，鉴定出一种参与生氰作用（cyanogenesis）的特异性β-葡萄糖苷酶ZfBGD2，并分析了这种特化昆虫体内氰化物释放的空间分布模式。通过转录组学（transcriptomic）与蛋白质组学（proteomic）分析，研究人员在血淋巴细胞（haemocytes）中检测到了高水平的ZfBGD2 mRNA与蛋白产物。昆虫细胞中的异源表达（heterologous expression）实验证实，ZfBGD2可水解Z. filipendulae体内存在的两种生氰葡萄糖苷：亚麻苦苷（linamarin）与百脉根苷（lotaustralin）。实验发现，亚麻苦苷、百脉根苷以及氰化物的释放仅存在于血淋巴浆（haemoplasma）中。系统发育分析（phylogenetic analyses）显示，ZfBGD2与其他昆虫β-葡萄糖苷酶聚为进化支；相应地，由特异性β-葡萄糖苷酶催化水解生氰葡萄糖苷的能力，在昆虫与植物中经历了趋同演化。β-葡萄糖苷酶ZfBGD2与其生氰底物在血淋巴（haemolymph）中的空间分隔，构成了Z. filipendulae体内氰化物释放的分子基础。这种空间分隔模式与生氰植物物种中两种组分的区室化机制高度相似，也印证了动物与植物这两个生物界在基于氰化物的防御策略上存在的共通之处。