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

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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.