We have used the vital fluorescent dye, FM4-64, as a marker of membrane development during zoospore formation in living zoosporangia of Allomyces macrogynus. Membrane development was visualized and documented using standard epifluorescence and laser scanning confocal microscopy. Video-enhanced light microscopy and transmission electron microscopy, using cryopreparation methods, were also employed in this study. In the first 10-12 min after the induction of zoospore formation, only the plasma membrane labelled with FM4- 64. During this time, nuclei were strictly located in the cortical cytoplasm with their associated centrosomes positioned immediately adjacent to the plasma membrane (Lowry and Roberson, 1997). Between 12 and 20 min post- induction, increased fluorescence appeared along regions of the plasma membrane adjacent to the nuclei. From these sites, membranes (i.e. cleavage elements) extended laterally within the cortex and then, in conjunction with nuclear migration, rapidly elongated into the sporangial cytoplasm. By 25-35 min post-induction, cleavage elements had ramified throughout the cytoplasm forming a complex, interconnected membranous network. Transmission electron microscopy revealed that cleavage elements were paired membrane sheets with a lumen consisting of an electron opaque, granular matrix. Cleavage elements developed into a highly ordered network by 35-40 min post-induction, which fully delimited zoospore initials into polyhedral-shaped cells. Zoospore discharge occurred between 40 and 50 min post-induction. Our results have shown that cleavage elements undergo four stages of development during zoospore formation in A. macrogynus: (i) development of membrane initials, (ii) cortical extension, (iii) cytoplasmic elongation and ramification and (iv) zoospore initial delimitation.
- Allomyces macrogynus
- Botanical microscopy
- Transmission electron microscopy
- Video-enhanced light microscopy
- Zoospore formation
ASJC Scopus subject areas
- Pathology and Forensic Medicine