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

University Of Florida, Gainesville FL

Investigators

Abstract

The Helicosporidia are a unique assemblage of pathogens isolated from a diverse group of 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 project utilize the expertise of three research teams to explore the structure and function of this novel pathogen. The approach taken in this project, dictated by the lack of biological information on this organism, will provide a combination of morphology, life history, and molecular data. The outlined objectives include a complete analysis of the in vitro developmental cycle of the Helicosporidium sp. with an emphasis on the ultrastructure and replication of vegetative cells and the morphogenesis of the infectious cyst. Preliminary examination has revealed that Helicosporidia possess many unique cytological features, such as a pellicle that is formed by both vegetative and cyst phenotypes, nuclei that enclose the cytosol, a number of undefined cell organelles, and a harpoon-like filamentous cell. The combination of high-resolution light and electron 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 sp will be examined. Throughout the course of this study, cell-tracking technologies will be optimized to analyze the ingress of this pathogen through the midgut barrier and its subsequent development in the hemocoel. Unlike many insect diseases, Helicosporidium sp., although isolated from an aquatic environment, can infect a wide range of terrestrial insect hosts. For comparative purposes, both mosquito and lepidopteran larvae will be used to document the in vivo 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 represents the first record of an insect-pathogenic alga. It should be noted that Helicosporidia nests hierarchically within a group of organisms with radically different morphologies and life history traits, allowing for comparative studies that can shed light on genes and structures that were modified to achieve parasitism. Molecular studies will be directed at generating sequence information on select genes from the Helicosporidium sp. and on EST libraries. A primary goal is analyze the relationship between the genera Helicosporidium, Prototheca, and other members of the 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 of this sequence data in conjunction with morphological (ultrastructural) features will define the taxonomic status and phylogeny of this organism. This project will provide support and training for two graduate students and two undergraduate research assistants. The goal is to leverage the combined talents of this group to provide students interdisciplinary training in molecular systematics, histology, and pathology. These skills are crucial for the discovery, characterization, and evaluation of microbials for applications to a broad range of scientific disciplines such as new pest control technologies, novel protein and small molecule discovery, etc.