Collaborative Research: The Molecular Identification and Action of Bursicon, the Insect Cuticle Sclerotizing Hormone

昆虫在蜕皮（蜕皮）过程中蜕去旧皮肤后，一种叫做囊囊素的昆虫激素对新皮肤（角质层）的硬化至关重要。滑囊的定时释放和作用对所有昆虫，很可能是所有节肢动物的生存至关重要。法氏囊蛋白于1935年被发现，是一种已知的多肽，但其分子结构尚未确定，是昆虫神经内分泌学中尚未解决的主要问题。Honegger实验室纯化了这种激素，并从一些昆虫身上获得了法氏囊的部分氨基酸序列。最近，他们能够利用这些序列在果蝇的基因组中识别出一个特殊的基因序列CG13419。本合作项目的主要目的是确定CG13419基因产物是否是功能性法囊激素，这是了解法囊作用的重要一步。真核细胞基因表达和果蝇基因过表达的分子技术将与“结扎蝇”生物测定相结合，使用相关的苍蝇（石棺蝇）来测试法氏囊活性。如果显示出生物活性法氏囊，该序列将用于曼都卡蛾的cDNA筛选，曼都卡蛾是一种经过充分研究的昆虫系统，其中调节内分泌的复杂激素相互作用已被阐明。从重组蛋白和从保守序列延伸合成的肽段中，将产生新的抗体来鉴定大脑中的神经分泌细胞，并阐明曼杜卡粘液释放的时间和空间激活。这项工作的结果将为理解大脑和内分泌系统如何共同协调导致周期性角质层硬化的多种生化事件提供潜在的突破。了解这一关键激素可能为农业开发害虫控制和管理甲壳类水产养殖提供新的策略。此外，两所院校的合作还包括多学科的博士后培训。

An insect hormone called bursicon is essential for hardening of the new skin (cuticle) after the insect has shed its old one during molting (ecdysis). The timed release and actions of bursicon are critically important for the survival of all insects and, most likely, all arthropods. Although bursicon was discovered in 1935 and is known to be a peptide, its molecular structure has not been established, and remains a major unsolved problem in insect neuroendocrinology. The Honegger lab has purified the hormone and obtained partial amino acid sequences for bursicon from some insects. Recently they were able to use these sequences to identify a particular gene sequence, CG13419, in the genome of the fruitfly Drosophila. The primary aim of this collaborative project is to determine whether the CG13419 gene product is the functional bursicon hormone, as an important step to understanding the actions of bursicon. Molecular techniques for gene expression in eukaryotic cells and overexpression in Drosophila will be combined with the 'ligated fly' bioassay using the related blowfly (Sarcophaga) to test for bursicon activity. If bioactive bursicon is shown, the sequence will be used for cDNA screening in the moth Manduca, a well-studied insect system in which the complex hormonal interactions regulating ecdysis have been illuminated. From the recombinant proteins and synthesized peptides from conserved sequence stretches, new antibodies will be produced to identify neurosecretory cells in the brain, and to clarify the temporal and spatial activation of bursicon release in Manduca. Results from this work will provide a potential breakthrough for understanding how the brain and endocrine systems together orchestrate multiple biochemical events leading to periodic cuticular hardening. Knowledge about this key hormone may provide novel strategies for agriculture in developing insect pest controls, and in managing crustacean aquaculture. In addition, this collaboration between two institutions includes multi-disciplinary postdoctoral training.