Cellular Structure and Biology of the Helicosporidia: A Unique and Unknown Group of Invertebrate Pathogens

Helicosporidia是一种独特的病原体组合，从一群不同的无脊椎动物中分离出来，只有一个命名的物种——helicosporidium parasiticum。在形态学上，这一组的定义是产生一个不寻常的包囊期，包囊期包括三个卵形细胞和一个细长的丝状细胞。这些生物一度被认为是原生动物或真菌，但自1931年以来一直未被分类。本项目概述的实验利用三个研究小组的专业知识来探索这种新型病原体的结构和功能。由于缺乏这种生物体的生物学信息，本项目采用的方法将提供形态学、生活史和分子数据的结合。概述的目标包括对螺旋孢子虫体外发育周期的完整分析，重点是营养细胞的超微结构和复制以及感染性囊肿的形态发生。初步检查显示，细孢子虫具有许多独特的细胞学特征，如由营养型和囊肿型两种表型组成的细胞膜，包围细胞质的细胞核，许多未定义的细胞器和鱼叉状的丝状细胞。高分辨率光学显微镜和电子显微镜的结合将提供关于这种生物的表面、壁结构和内部形态的组成和结构的详细见解。在受控的体外条件下，延时显微镜研究将提供该病原体发育周期的详细信息。其次，对螺旋孢子虫的体内感染过程、发育和宿主范围进行了研究。在整个研究过程中，细胞追踪技术将被优化，以分析这种病原体通过中肠屏障的进入及其随后在血液中的发展。与许多昆虫疾病不同，螺蛳虫虽然从水生环境中分离出来，但可以感染广泛的陆生昆虫宿主。为了比较，我们将使用蚊子和鳞翅目幼虫来记录体内细胞周期。迄今为止，由rDNA和两种蛋白质（肌动蛋白和微管蛋白）产生的序列数据表明，螺蛳藻属于绿藻，因此，它代表了昆虫致病性藻类的第一个记录。值得注意的是，螺旋孢子虫在一组具有完全不同形态和生活史特征的生物中筑巢，这允许进行比较研究，可以阐明为实现寄生而修改的基因和结构。分子研究的方向是产生螺旋孢子虫选定基因的序列信息和EST文库。本研究的主要目的是分析螺旋孢子虫属、原茧虫属和其他Trebouxiaphyceae成员之间的关系，以便更好地了解与寄生进化相关的形态、分子和遗传变化。此外，结合形态学（超微结构）特征分析该序列数据将确定该生物的分类地位和系统发育。该项目将为两名研究生和两名本科生研究助理提供支持和培训。目标是利用该小组的综合才能，为学生提供分子系统学，组织学和病理学的跨学科培训。这些技能对于微生物的发现、表征和降解至关重要，可以应用于广泛的科学学科，如新的害虫控制技术、新的蛋白质和小分子发现等。

The Helicosporidia are a unique assemblage of pathogens isolated from a diverse groupof invertebrates with only one named species-Helicosporidium parasiticum. Morphologically,this group is defined by the production of an unusual cyst stage that encloses three ovoid cells and a single elongate, filamentous cell. These organisms were at one time considered to be protozoa or fungi but have been unclassified since 1931. Experiments outlined in this projectutilize the expertise of three research teams to explore the structure and function of this novelpathogen. The approach taken in this project, dictated by the lack of biological information onthis organism, will provide a combination of morphology, life history, and molecular data. Theoutlined objectives include a complete analysis of the in vitro developmental cycle of theHelicosporidium sp. with an emphasis on the ultrastructure and replication of vegetative cells andthe morphogenesis of the infectious cyst. Preliminary examination has revealed thatHelicosporidia possess many unique cytological features, such as a pellicle that is formed by bothvegetative and cyst phenotypes, nuclei that enclose the cytosol, a number of undefined cellorganelles, and a harpoon-like filamentous cell. The combination of high-resolution light andelectron microscopy will provide detailed insight on the composition and structure of the surface,wall structure, and internal morphology of this organism. Under controlled in vitro conditions,time-lapse microscope studies will provide details on the developmental cycle of this pathogen.Secondly, the in vivo infection process, development, and host range of the Helicosporidium spwill be examined. Throughout the course of this study, cell-tracking technologies will beoptimized to analyze the ingress of this pathogen through the midgut barrier and its subsequentdevelopment in the hemocoel. Unlike many insect diseases, Helicosporidium sp., althoughisolated from an aquatic environment, can infect a wide range of terrestrial insect hosts. Forcomparative purposes, both mosquito and lepidopteran larvae will be used to document the invivo cell cycle. To date, sequence data generated on rDNA and two proteins, actin and tubulin,have demonstrated that Helicosporida belongs within the Chlorophyta and, as such, it representsthe first record of an insect-pathogenic alga. It should be noted that Helicosporidia nestshierarchically within a group of organisms with radically different morphologies and life historytraits, allowing for comparative studies that can shed light on genes and structures that weremodified to achieve parasitism. Molecular studies will be directed at generating sequenceinformation on select genes from the Helicosporidium sp. and on EST libraries. A primary goal isanalyze the relationship between the genera Helicosporidium, Prototheca, and other members ofthe Trebouxiaphyceae in order to gain a better understanding of the morphological, molecular,and genetic changes that are correlated with the evolution of parasitism. Furthermore, analysis ofthis sequence data in conjunction with morphological (ultrastructural) features will define thetaxonomic status and phylogeny of this organism. This project will provide support and trainingfor two graduate students and two undergraduate research assistants. The goal is to leverage thecombined talents of this group to provide students interdisciplinary training in molecular systematics,histology, and pathology. These skills are crucial for the discovery, characterization, andevaluation of microbials for applications to a broad range of scientific disciplines such as newpest control technologies, novel protein and small molecule discovery, etc.