List of 490 TC numbers analyzed in ShinyGO.

Exocrine glands have evolved several times independently in Coleoptera to produce defensive chemical compounds with repellent, antimicrobial, or toxic effects. Research on such glands had focused on morphological or chemical ecology methods. However, modern genetic approaches were missing to better understand this biological process. With the rise of the red flour beetle, Tribolium castaneum, as a model for studies of development and pest biology, molecular genetic tools are now available to also study the safe generation of toxic compounds in defensive stink glands. Using the RNA-interference-based, genome-wide, phenotypic screen “iBeetle” and the re-analysis of gland-specific transcriptomics based on a significantly improved genome annotation, we could identify 490 genes being involved in odoriferous stink gland function. In the iBeetle screen, 247 genes were identified, of which we present here 178 genes identified during iBeetle’s 3rd phase, while the transcriptomics analyses identified 249 genes, with six genes being identified in both functional genomics approaches. Of these 490 genes, only about 40% of these genes have molecularly characterized homologs in the vinegar fly, while for 213 genes no fly homologs were recognized and for 13 genes no gene ontology at all was identified. This highlights the importance of genome-wide gene identification in tissues that have not been previously analyzed to recognize potentially new gene functions. Gene ontology analysis revealed “SNARE interactions in vesicular transport”, “Lysosome”, “Pancreatic secretion”, and “MAPK signaling pathway – fly” as key pathways. Additionally, many of the genes are encoding enzymes, transcription factors, transporters, or are involved in membrane trafficking. As the phenoloxidase responsible for generating the toxic para-benzoquinones in the stink glands of the beetle, we could identify laccase2, which is expressed in the last secretory cell in contact with the cuticle-lined vesicular organelle, where the toxic compounds are safely produced before being released into the gland reservoir.

鞘翅目（Coleoptera）昆虫的外分泌腺已多次独立演化出合成具有驱避、抗菌或毒性防御功能的化学物质的能力。过往针对此类腺体的研究多采用形态学或化学生态学手段。然而，目前仍缺乏现代遗传学研究方法以深入解析这一生物学过程。随着赤拟谷盗（Tribolium castaneum）作为发育生物学与害虫生物学研究模式生物的兴起，如今已可借助分子遗传学工具探究防御性臭腺中有毒化合物的安全合成机制。本研究依托基于RNA干扰（RNA interference）的全基因组表型筛选平台"iBeetle"，以及基于显著优化后的基因组注释对腺特异性转录组的重新分析，成功鉴定出490个与臭腺功能相关的基因。在"iBeetle"筛选中，共鉴定出247个相关基因，其中本文报道了该筛选第三阶段所获得的178个基因；转录组分析则鉴定出249个基因，两类功能基因组学方法共同鉴定到6个重叠基因。在这490个基因中，仅约40%的基因在醋蝇（vinegar fly）中存在经分子表征的同源基因；另有213个基因未在醋蝇中找到同源基因，13个基因完全未获得基因本体（gene ontology, GO）注释。这凸显了在未被前人研究的组织中开展全基因组基因鉴定，以发掘潜在新基因功能的重要性。基因本体（gene ontology, GO）分析显示，"SNARE互作与囊泡运输（SNARE interactions in vesicular transport）"、"溶酶体（Lysosome）"、"胰腺分泌（Pancreatic secretion）"以及"果蝇MAPK信号通路（MAPK signaling pathway – fly）"为关键调控通路。此外，大量相关基因编码酶类、转录因子、转运蛋白，或参与膜泡运输过程。作为负责在甲虫臭腺中合成毒性对苯醌的酚氧化酶，我们成功鉴定出漆酶2（laccase2）：该基因在与表皮包被的囊泡细胞器接触的最末分泌细胞中表达，毒性化合物正是在此处安全合成，随后被释放至腺储囊中。