Intraspecific Venom Variation between Tetrastichum brontispae strains

The venom apparatus is a conserved organ in parasitoid wasps that shows adaptations correlated with life-style diversification. Recent venom analyses from selected species reveal considerable complexity and high diversity in venom composition existing not only between closely related species but even between strains and individuals, which may partly determine the potential for parasitoid adaptation. However, the investigations have paid little attention to secondary venom components that also have significant functions in parasitism, and the data in regard to full and accurate quantity of venom compositions at the protein level is not available. Using a combination of transcriptomic and label-free quantitative proteomic, we here explored the venom components of the endoparasitoid Tetrastichus brontispae (Eulophidae), a species devoid of polydnavirus, and provided an in-depth comparison of the venom proteomes between its two closely related strains, Tb-On and Tb-Bl. Results showed that approximately 1505 venom proteins were identified in the venom apparatus of T. brontispae, consistent with the classical venom protein characteristics, including enzymes, protease inhibitors, binding proteins and some immune related proteins. The venom extracts also contained novel venom proteins, such as kynurenine-oxoglutarate transaminase, 4-coumarate CoA ligase and venom protein r-like protein. Comparative venom proteomes revealed that significant quantitative and qualitative changes in venom composition occurred when Tb-Bl strain, with an invasive beetle Brontispae longissima pupa as its habitual host, was exposed to another invasive beetle Octodonta nipae pupa as host consecutively for two years; although the most abundant venom proteins were shared between them. These significantly differentially expressed proteins were mainly enriched in fatty acid biosynthesis and melanotic encapsulation response by enrichment analyses. Furthermore, most of the significantly enriched proteins presented strikingly increased levels or were exclusively identified in the Tb-On strain. These combined results indicated that virulence factors in Tb-On strain might be linked to lipid metabolism or more venom is required to inhibit O. nipae pupa’s melanotic encapsulation upon its parasitism. Altogether, our data reveal that venom composition can quickly evolve and respond to host selection, mainly through rapid changes in regulation of protein abundance and/or the emergence of multigenic families by gene duplication. Our data additionally provide invaluable data for further functional analysis of parasitoid venoms.

毒液装置（venom apparatus）是寄生蜂中一类保守的器官，其演化出的适应性特征与生活史多样化密切相关。近期针对部分物种类群的毒液组学分析显示，毒液组分不仅在近缘物种间存在显著复杂性与高度多样性，甚至在品系与个体之间亦存在差异，这或许在一定程度上决定了寄生蜂的适应性演化潜力。然而，现有研究对次要毒液组分（secondary venom components）的关注较少——尽管这类组分在寄生过程中同样发挥关键功能，且目前仍缺乏蛋白质组层面上完整且精准的毒液组分定量数据。本研究结合转录组学（transcriptomics）与无标记定量蛋白质组学（label-free quantitative proteomics）技术，对无多DNA病毒（polydnavirus）的内寄生蜂（endoparasitoid）椰心叶甲啮小蜂（Tetrastichus brontispae，隶属于姬小蜂科Eulophidae）的毒液组分展开探究，并对其两个近缘品系Tb-On与Tb-Bl的毒液蛋白质组（venom proteomes）进行深度比较分析。结果显示，在椰心叶甲啮小蜂的毒液装置中共鉴定出约1505种毒液蛋白，其组分符合经典毒液蛋白特征，涵盖酶类、蛋白酶抑制剂、结合蛋白以及部分免疫相关蛋白。毒液提取物中还包含多种新型毒液蛋白，例如犬尿氨酸-酮戊二酸转氨酶（kynurenine-oxoglutarate transaminase）、4-香豆酸辅酶A连接酶（4-coumarate CoA ligase）以及类毒液蛋白r（venom protein r-like protein）。比较蛋白质组学分析发现，当原本以入侵性甲虫椰心叶甲（Brontispae longissima）蛹为常规寄主的Tb-Bl品系，连续两年以另一入侵性甲虫八角鸭甲（Octodonta nipae）蛹作为寄主时，其毒液组分在质与量上均发生显著变化；尽管二者共享丰度最高的毒液蛋白。富集分析显示，这些显著差异表达的蛋白（differentially expressed proteins）主要富集于脂肪酸生物合成（fatty acid biosynthesis）与黑化包囊响应（melanotic encapsulation response）通路。此外，多数显著富集的蛋白在Tb-On品系中呈现表达量显著上调，或仅在该品系中被鉴定到。上述综合结果表明，Tb-On品系的毒力因子（virulence factors）或许与脂代谢相关，或需要更多毒液蛋白来抑制八角鸭甲蛹在被寄生时的黑化包囊反应。综上，本研究数据揭示毒液组分可通过调控蛋白丰度快速变化，或通过基因复制（gene duplication）产生多基因家族等方式，快速演化并响应寄主选择。本研究同时为寄生蜂毒液的后续功能分析提供了极具价值的参考数据。