WORKSHOPS ON OPPORTUNISTIC PROTlSTS
81s
Brachiola aZgerae Sporoplasms ANN CALI and PETER M. TAKVORIAN Department of Biological Sciences, Rutgers University,Newark. New Jersey, 07102, USA, The microsporidia are obligate intracellular protozoan parasites known to Infect every major animal group. Although much information on
microsporidh life cycles, developmental stages, and spore morphology
exists, relatively little is known about the infective sporoplasm stage. The lack of information may be due to the difficulty of identifying sporoplasmsin host tissue and their rapid degeneration when extracellular,espocialiythose in thedigestive tract. The few ultrastructural descriptions of sporoplasms report that they are generally cells containing a nucleus (or diplokaryon), with cytoplasm containing ribosomes, variable amounts of endoplasmicreticulum, vesicles and covered by a plasmalemma [1,5.7-9]. The genus Brachiola is the newest genus in the family Nosematidae [3 3. The characteristics they share include: diplokaryotic nuclei, lack of plasmodial stages, and development in direct contact with the host cell cytoplasm. The unique features of this genus are thermophilia (0 350 C), thickened plasmalemma in the proliferative phase, and extensive plasmalemmal ornamentation (variable in form). Brachiola algerae formerly Nosema algerae. was considered an “out group among the Nosemu species” based on phylogenetic molecular taxonomy [2 1. Since it also has electron dense secretions on the parasite surface (thickened plasmalemma and omamentation) from sporoplasm to spore and has been shown to grow at 370C, it was renamed [7]. TheB. algerae sporoplasm, along with its’ proliferative stages, has been described as having a thickened membrane by one-hour post extrusion in vivo [1,4] and in vitro [7]. This study demonstrate-s the nature of the sporoplasm membrane at the time of extrusion and the presence of a multilayered network inside the cytoplasm underlying the plasmalemma. This network has multiple attachment sites to both the plasmalemma and the polar tube.
for several minutes after extrusion (Fig. 1). They are o v a and measure approximately 4 m in diameter with the PT deeply embedded (Fig. 2.3). Electron microscopic examination of germinating sporesat approximately3 minutes post activation (PA) revealed the presence of binucleated (diplokaryotic) sporoplasms enclosed by a thin plasmalemma These sporopIasms were frmly attached to their FT by a unique structure, which underlies the plasmalemma This structure is a many layered network with multiple cross connections to itself, the FT,and the plasmalemma (Fig. 4).
MATERIALS AND METHODS Brachiola algerae spores were treated with germination media[6] and observed by phase contract microscopy utilizing a high speed (200 frarnes/sec) video recorder, a digital camera (Spot- RT), or fmed and processed for transmission electron microscopy (TEM). B . algerae were fied at 1,2,3,30, and 60 minutes post activation (PA). TEM observations were made with a Philips Tecnai 12. The 30 and 60 minute PA were placed in RK13 cell culture [7].
RESULTS AND DISCUSSION The high speed video (HSV) provided detailed and sequential visualization of the activated spore, including the polar tube extrusion process and emergence of the sporoplasm, which takes less than one second in total. The spores began germinating within 15 seconds of application of the sporoplasm from the FT.The sporoplasms remain fnmly attached to their VT
Fig. 1-3. Spores in germinationmedium for approximately one(1) to three minutes (2-3) and viewed by phase contrast microscopy. 1. Sporoplasm (arrowhead)still Corresponding author: A. Cali. Telephone:973-353-5364;Fax: 973-353-1007;Email:
[email protected]
connected to its PT and spore shell. Note them attachmentat sporoplasm indentation 2. Note the presence of three sporoplasms(arrowheads), each is still attached to its polar tube. 3. Enlargement of a sporoplasm with the polar tube embedded in it.
82s
.I.EUKARYOT. MICROBIOL., 2001
Fig. 4. E M of longitudinal section of polar tube attached to MIN in the sporoplasm. Fixed at three minutes PA. Note the presence of a thin membrane. plasmalemma, (arrow) surrounding the MIN and connected to it.
This multilayered interlacing network (MIN) containing eleccron dense material, dissipates within 30 minutes of sporoplasm extrusion. Remnants of this organelle appear as membranes and vesicular structures (Fig. 5). These are reminiscent of similar material in other sporoplasms[5,8-10]. The loss of this unique structure, the MIN, correlates with the formationof theplasmalemmal thickening and surface structures typical for thisorganism. Exnacellular sporoplasms, observed in cell culture media, confirm the source of the membrane thickening as parasite derived and not of host origin (Fig. 6). Thus, suggesting that the MIN provides both a structural anchor for the I T attachment and the secretory material necessary for formation of the thick membrane. ACKNOWLEDGMENTS The Rutgers Newark EM facility, F. Macaluso and M. Cammer of the Albert Einstein imaging facility for the high-speed video. and the NIH Grants # A131788, GM60067, & NCRR 1S10-RR13959.
LJTERATURE CITED 1. Avery, S. W. & Anthony, D. W. 1983. Ultrastructural study of early development of Nosemu algerae in Anopheles albimanus. J. Invert. Puthoi., 42:87-95. 2. Baker, M. D., Vossbrinck, C. R., Maddox, J. V. & Undeen, A. H. 1994. Phylogeneticrelationships among Vuirimorphu and Nosema species ( M i a s p a ) based on ribosomal RNA sequence data. J. Inverf.Pothol., 64:100-106. 3. Cali, A.. Takvorian, P. M.. Lewin, S., Rendel. M., Sian, C. S.. Wittner. M., Tanowitz, H. B.. Keohane, E. & Weiss, L. M. 1998. Bruchiolu vesiculurum, N. G., N. Sp., a new microsporidiurn associated with AIDS and myositis. J . Euk.
Microbiol., 45:240-251. 4. Chioralia, G., Trammer, T., Maier, W. A. & Seitz, H. M. 1998. Morphologic changes in Nosemu aigerue (Microspora) during extrusion. Purusif.Reseurch, 84:123-131. 5. Ishihara, R. 1968. Some observations on the fine structure of sporoplasm discharged from spores of a microsporidian, Nosemu bombycis. J.Invert. Puthol., 12:245-258. 6. LeiLch, G. L., He, Q., Wallace, S. & Visvesvara, G. S. 1993. Inhibition of the spore polar filament extrusion of the microsporidiurn. Encephulitozoon hellem, isolated from an AIDS patient. J . Euk. Microbiol., 40:711-717. 7. Lowman, P. M., Takvorian. P. M. & Cali, A. 2000. The effects of elevated temperature and various time-temperature combinations on the development of Bruchiolu (Nosem) ulgerae N. Comb. in mammalian cell culture. J . Euk. Microbiol., 41:221-234. 8. Vavra, J. & Larsson, 1.I. R. 1999. Structure of themicrosporidia. In: Wittner. M. & Weiss, L.M. (ed.),The microsporidia and microsporidiosis, ASM Press, Washington, D. C., p. 7-84. 9. Weidner, E. 1972. Ultrastructural study of microsporidian invasion into cells. Z. Puraritenk, 40:227-242.
Fig, 5.6. TEM ofsporoplasms in RK13 cell cultures fixed at 30 minutes. Note& presence of the thickened and omimented plasmalemma (arrowheads) typical of the genus Bruchiolu and the lack of MIN with remnant vesiculation remaining. 4. Sporoplasm in host e l l . 5. Extracellular sporoplam.
10. Weidner, E., Byrd, W., Scarborough, A., Pleishinger, J. & Sibley, D. 1984. Microsporidian spore discharge and the transfer of polaroplast organellemgnbrane into plasma membrane. J. Protozool.,31:195-198.
