Student presentation

 

In Vitro Development of Helicosporidium

 

M. Botts, S. Shapiro, J. Becnel, and D. Boucias

 

Department of Entomology and Nematology, University of Florida, Gainesville, Florida 32601

 

The protist Helicosporidum sp. is a entomopathogenic algae that is characterized by a infectious cyst stage that contains an elongate filamentous cell and 3 ovoid cells.  This infectious cyst dehisces within the midgut lumen penetrates column epithelium gaining ingress into the hemocoel. Within the nutrient rich hemolymph this pathogen undergoes multiple cycles od vegetative replication. The resulting in vivo cells can be harvested and cultured in vitro.  This in vitro growth is characterized by the production of vegetative cells that undergo a 2-4 cell asporogenous division.  Cell division and daughter cell wall formation occurs within the mother cell.  Initial in vitro growth leads to production of fully differentiated cysts that are infectious per os to insects. Successive transfers of these cultures results in a decline in cyst production with a concomitant selection of vegetative cell growth.  Multiply-passaged cultures are characterized by growth the formation of nonmotile adherent cells that cluster together via production of extracellular mucilage (palmelloid cell phenotype).  Attempts to produce cysts from palmelloid cultures have failed. In vitro we have analyzed the morphogenesis of the different cell phenotypes. In vitro produced cysts partitioned from vegetative cells using Ludox gradients can be readily dehisced using filter sterilized insect digestive fluid.  Released filamentous cells have been purified and observed in vitro.  The filamentous cells go through a period of regeneration that is characterized by the thickening of the anterior portion of the filament reorganization of the nuclear material. DAPI staining has revealed nuclear division followed by deposition of daughter cell wall material. The parental filament cell wall eventual ruptures along its horizontal axis and releasing oval-shaped daughter cells.  The time table for this regeneration is as follows: initial 24 hour period results in thickening of the of the anterior filament cell; 24-48 h nuclear division initiated; and by 72 daughter cells are released from filament cell.  Daughter cells then elongate and divide into spherical shaped vegetative cells that undergo autosporogeny.  Typically the vegetative cells will produce four cells per mother cell. The daughter cells will be released and undergo additional cycles of vegetative growth. After multiple cycles a portion of the vegetative cells differentiate into the specialized cyst stage of Helicosporidium.