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Extracellular Survival of an Intracellular Parasite (Spraguea lophii, Microsporea) Author(s): Earl Weidner and Ann Findley Source: Biological Bulletin, Vol. 197, No. 2, Centennial Issue: October, 1899-1999 (Oct., 1999), pp. 270-271 Published by: Marine Biological Laboratory Stable URL: http://www.jstor.org/stable/1542645 . Accessed: 25/06/2014 03:02 Your use of the JSTOR archive indicates your acceptance of the Terms & Conditions of Use, available at . http://www.jstor.org/page/info/about/policies/terms.jsp . JSTOR is a not-for-profit service that helps scholars, researchers, and students discover, use, and build upon a wide range of content in a trusted digital archive. We use information technology and tools to increase productivity and facilitate new forms of scholarship. For more information about JSTOR, please contact [email protected]. . Marine Biological Laboratory is collaborating with JSTOR to digitize, preserve and extend access to Biological Bulletin. http://www.jstor.org This content downloaded from 62.122.77.28 on Wed, 25 Jun 2014 03:02:54 AM All use subject to JSTOR Terms and Conditions

Centennial Issue: October, 1899-1999 || Extracellular Survival of an Intracellular Parasite (Spraguea lophii, Microsporea)

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Page 1: Centennial Issue: October, 1899-1999 || Extracellular Survival of an Intracellular Parasite (Spraguea lophii, Microsporea)

Extracellular Survival of an Intracellular Parasite (Spraguea lophii, Microsporea)Author(s): Earl Weidner and Ann FindleySource: Biological Bulletin, Vol. 197, No. 2, Centennial Issue: October, 1899-1999 (Oct., 1999),pp. 270-271Published by: Marine Biological LaboratoryStable URL: http://www.jstor.org/stable/1542645 .

Accessed: 25/06/2014 03:02

Your use of the JSTOR archive indicates your acceptance of the Terms & Conditions of Use, available at .http://www.jstor.org/page/info/about/policies/terms.jsp

.JSTOR is a not-for-profit service that helps scholars, researchers, and students discover, use, and build upon a wide range ofcontent in a trusted digital archive. We use information technology and tools to increase productivity and facilitate new formsof scholarship. For more information about JSTOR, please contact [email protected].

.

Marine Biological Laboratory is collaborating with JSTOR to digitize, preserve and extend access toBiological Bulletin.

http://www.jstor.org

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Page 2: Centennial Issue: October, 1899-1999 || Extracellular Survival of an Intracellular Parasite (Spraguea lophii, Microsporea)

REPORTS FROM THE MBL GENERAL SCIENTIFIC MEETINGS REPORTS FROM THE MBL GENERAL SCIENTIFIC MEETINGS

for their many helpful and well considered suggestions made in the final preparation of this manuscript.

Literature Cited

1. Silver, R. B. 1989. Dev. Biol. 131: 11-26. 2. Silver, R. B. 1996. Cell Calcium 20: 161-179. 3. Silver, R. B. 1995. Biol. Bull. 189: 203-204. 4. Silver, R. B., R. D. Cole, and W. Z. Cande. 1980. Cell 19:

505-516. 5. Bansal, V. S., and P. W. Majerus. 1990. Annu. Rev. Cell Biol. 6:

41-67. 6. Chow, S. C., and M. Jondel. 1990. J. Biol. Chem. 265: 902-907. 7. Silver, R. B., D. E. Strongin, L. R. Hurwitz, and A. P. Reeves. 1997.

Biol. Bull. 193: 236-237. 8. Silver, R. B. 1999. FASEB J. (in press). 9. Silver, R. B. 1999. Science (accepted).

10. Silver, R. B., L. A. King, and A. F. Wise. 1998. Biol. Bull. 195: 209-210.

11. Samuelsson, B. 1983. Science 220: 568-575. 12. Silver, R. B. 1986. Methods Enzymol. 134: 200-217.

for their many helpful and well considered suggestions made in the final preparation of this manuscript.

Literature Cited

1. Silver, R. B. 1989. Dev. Biol. 131: 11-26. 2. Silver, R. B. 1996. Cell Calcium 20: 161-179. 3. Silver, R. B. 1995. Biol. Bull. 189: 203-204. 4. Silver, R. B., R. D. Cole, and W. Z. Cande. 1980. Cell 19:

505-516. 5. Bansal, V. S., and P. W. Majerus. 1990. Annu. Rev. Cell Biol. 6:

41-67. 6. Chow, S. C., and M. Jondel. 1990. J. Biol. Chem. 265: 902-907. 7. Silver, R. B., D. E. Strongin, L. R. Hurwitz, and A. P. Reeves. 1997.

Biol. Bull. 193: 236-237. 8. Silver, R. B. 1999. FASEB J. (in press). 9. Silver, R. B. 1999. Science (accepted).

10. Silver, R. B., L. A. King, and A. F. Wise. 1998. Biol. Bull. 195: 209-210.

11. Samuelsson, B. 1983. Science 220: 568-575. 12. Silver, R. B. 1986. Methods Enzymol. 134: 200-217.

13. Silver, R. B. 1986. Proc. Natl. Acad. Sci. U.S.A. 83: 4302-4306. 14. Reynolds, L. J., L. L. Hughes, L. Yu, and E. A. Dennis. 1994.

Anal. Biochem. 217: 25-32. 15. Kurioka, S., and M. Matsuda. 1976. Anal. Biochem. 75: 281-289. 16. Keen, J. H., W. H. Habig, and W. B. Jakoby. 1976. J. Biol. Chem.

251: 6183-6188. 17. Pabst, M. J., W. H. Habig, and W. B. Jakoby. 1976. J. Biol. Chem.

249: 7140-7147. 18. Racker, E. 1955. J. Biol. Chem. 190: 855-865. 19. Silver, R. B., M. S. Saft, A. R. Taylor, and R. D. Cole. 1983.

J. Biol. Chem. 258: 13287-13291. 20. Silver, R. B. 1997. Pp 83.1-20 in Cells: A Laboratory Manual. D. L.

Spector, R. D. Goldman, and L. Leinwand, eds. CSHL Press. 21. Dennis, E. A. 1995. J. Biol. Chem. 269: 13057-13060. 22. Leslie, C. C. 1977. J. Biol. Chem. 272: 16709-16712. 23. Gupta, N., M. J. Gresser, and A. W. Ford-Hutchinson. 1998.

Biochim. Biophys. Acta 139: 157-168. 24. Oesch, F., and C. R. Wolf. 1989. Biochem. Pharmacol. 38: 353-

359. 25. Silver, R. B., J. B. Oblak, G. S. Jeun, J. Sung, and T. Dutta. 1994.

Biol. Bull. 187: 242-244.

13. Silver, R. B. 1986. Proc. Natl. Acad. Sci. U.S.A. 83: 4302-4306. 14. Reynolds, L. J., L. L. Hughes, L. Yu, and E. A. Dennis. 1994.

Anal. Biochem. 217: 25-32. 15. Kurioka, S., and M. Matsuda. 1976. Anal. Biochem. 75: 281-289. 16. Keen, J. H., W. H. Habig, and W. B. Jakoby. 1976. J. Biol. Chem.

251: 6183-6188. 17. Pabst, M. J., W. H. Habig, and W. B. Jakoby. 1976. J. Biol. Chem.

249: 7140-7147. 18. Racker, E. 1955. J. Biol. Chem. 190: 855-865. 19. Silver, R. B., M. S. Saft, A. R. Taylor, and R. D. Cole. 1983.

J. Biol. Chem. 258: 13287-13291. 20. Silver, R. B. 1997. Pp 83.1-20 in Cells: A Laboratory Manual. D. L.

Spector, R. D. Goldman, and L. Leinwand, eds. CSHL Press. 21. Dennis, E. A. 1995. J. Biol. Chem. 269: 13057-13060. 22. Leslie, C. C. 1977. J. Biol. Chem. 272: 16709-16712. 23. Gupta, N., M. J. Gresser, and A. W. Ford-Hutchinson. 1998.

Biochim. Biophys. Acta 139: 157-168. 24. Oesch, F., and C. R. Wolf. 1989. Biochem. Pharmacol. 38: 353-

359. 25. Silver, R. B., J. B. Oblak, G. S. Jeun, J. Sung, and T. Dutta. 1994.

Biol. Bull. 187: 242-244.

Reference: Biol. Bull. 197: 270-271. (October 1999)

Extracellular Survival of an Intracellular Parasite (Spraguea lophii, Microsporea) Earl Weidner and Ann Findley' (Biology, Louisiana State University, Baton Rouge, Louisiana)

Reference: Biol. Bull. 197: 270-271. (October 1999)

Extracellular Survival of an Intracellular Parasite (Spraguea lophii, Microsporea) Earl Weidner and Ann Findley' (Biology, Louisiana State University, Baton Rouge, Louisiana)

Microsporeans are intracellular parasites; they are located di-

rectly in host cell cytoplasm with only a plasma membrane as an interface (1). Microsporeans have an infective spore stage that

discharges the sporoplasm from a long, fine tube. The spore has but one plasma membrane which is left behind within the spore ghost during discharge. The extruded sporoplasm is surrounded by a membrane, but this structure is derived from the extrusion

apparatus within the spore. Since microsporeans have not been cultured or maintained extracellularly for more than short periods, it became an objective of this study to: (a) develop a simple protocol for isolating pure populations of discharged sporoplasms; and (b) develop a procedure for maintaining pure populations of extruded microsporean sporoplasms in culture for 24 h for further biochemical investigations.

To initiate spore discharge, spores of Spraguea lophii were incubated in 0.1 M HEPES buffer at pH 7.0 (with 50 nM Ca+ +) for 1 h. Subsequently, 10-100 I l1 of spore suspension were transferred into a thin pool on a glass coverslip. These spores were triggered to discharge by the addition of 1-2 ,ul of filtered (0.45 ,um pore size) mammalian or fish mucus onto the spore film, followed a few seconds later by the addition of 1-5 ,ul of 0.1 M HEPES buffer (pH 10). After several minutes, most of the spores had discharged sporoplasms that were attached to the cover glass surface. The unfired and discharged spore ghosts were removed from the sur- face by rapid washes with 0.1% concanavalin A (Con A) made up in HEPES (pH 7.0). The sporoplasms were transferred to a Me- dium 199 enriched with 5 mM ATP pH 7.2 (1).

The support medium that was tested at first included vitamins A

Microsporeans are intracellular parasites; they are located di-

rectly in host cell cytoplasm with only a plasma membrane as an interface (1). Microsporeans have an infective spore stage that

discharges the sporoplasm from a long, fine tube. The spore has but one plasma membrane which is left behind within the spore ghost during discharge. The extruded sporoplasm is surrounded by a membrane, but this structure is derived from the extrusion

apparatus within the spore. Since microsporeans have not been cultured or maintained extracellularly for more than short periods, it became an objective of this study to: (a) develop a simple protocol for isolating pure populations of discharged sporoplasms; and (b) develop a procedure for maintaining pure populations of extruded microsporean sporoplasms in culture for 24 h for further biochemical investigations.

To initiate spore discharge, spores of Spraguea lophii were incubated in 0.1 M HEPES buffer at pH 7.0 (with 50 nM Ca+ +) for 1 h. Subsequently, 10-100 I l1 of spore suspension were transferred into a thin pool on a glass coverslip. These spores were triggered to discharge by the addition of 1-2 ,ul of filtered (0.45 ,um pore size) mammalian or fish mucus onto the spore film, followed a few seconds later by the addition of 1-5 ,ul of 0.1 M HEPES buffer (pH 10). After several minutes, most of the spores had discharged sporoplasms that were attached to the cover glass surface. The unfired and discharged spore ghosts were removed from the sur- face by rapid washes with 0.1% concanavalin A (Con A) made up in HEPES (pH 7.0). The sporoplasms were transferred to a Me- dium 199 enriched with 5 mM ATP pH 7.2 (1).

The support medium that was tested at first included vitamins A

1 Biology, Northeastern Louisiana University, Monroe, Louisiana. 1 Biology, Northeastern Louisiana University, Monroe, Louisiana.

and C (1 nM), L-phosphatidycholine (0.1 mg/ml), glucose (0.01%), 5% bovine serum albumen, 5% fetal calf serum, cofactors NAD and Co-A (1 nM) and 5 pM concentrations of ATP and GTP. The cells were maintained at 15?C and 20?C. Although the sporo- plasms showed some stability in this medium, the cells lost much

and C (1 nM), L-phosphatidycholine (0.1 mg/ml), glucose (0.01%), 5% bovine serum albumen, 5% fetal calf serum, cofactors NAD and Co-A (1 nM) and 5 pM concentrations of ATP and GTP. The cells were maintained at 15?C and 20?C. Although the sporo- plasms showed some stability in this medium, the cells lost much

Figure 1. Spraguea lophii sporoplasms after 24 h in Medium 199 with ATP supplement and 10% Xenopus oocyte cytosol. (A) Sporoplasms (ar- row) frequently fuse or attach to one another. (B) Sporoplasms (arrows) also attach or fuse with other elements in medium. Bar scale represents 4 um.

Figure 1. Spraguea lophii sporoplasms after 24 h in Medium 199 with ATP supplement and 10% Xenopus oocyte cytosol. (A) Sporoplasms (ar- row) frequently fuse or attach to one another. (B) Sporoplasms (arrows) also attach or fuse with other elements in medium. Bar scale represents 4 um.

270 270

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Page 3: Centennial Issue: October, 1899-1999 || Extracellular Survival of an Intracellular Parasite (Spraguea lophii, Microsporea)

CELL AND DEVELOPMENTAL BIOLOGY CELL AND DEVELOPMENTAL BIOLOGY

of their cytosol and frequently fused during the first 6 h of incubation. When the support medium was suspended onto a 2%-5% gelatin matrix, the cells incubated within it developed a vacuolated cytoplasm. However, the sporoplasms appeared to have a more robust stability when they were added to Medium 199 made up in 0.15 M potassium phosphate buffer with 5 mM ATP, and were supplemented with 10%-30% Xenopus oocyte cytosol (Fig. 1). After 12-24 h, these sporoplasms retained cytoplasm and did not vacuolate, although the sporoplasms appeared to still attach to each other or fuse. There was no evidence of nuclear division

during 24 h of incubations.

Microsporean sporoplasms were clearly stabilized in Medium 199 (pH 7.1-7.2) with ATP (5 mM) on a 2% gelatin substrate onto which was added 0.01-0.02 mM cholesterol with no Xenopus cytosol. There was no evidence of vacuolation or nuclear division. However, after 12 h, the sporoplasms remained segregated and retained their cytoplasmic matrix. This significant positive effect of cholesterol addition to the medium indicates that the sporoplasm outer envelope may be devoid of cholesterol. Insertion of choles- terol into plasma membrane is an essential component of eu-

of their cytosol and frequently fused during the first 6 h of incubation. When the support medium was suspended onto a 2%-5% gelatin matrix, the cells incubated within it developed a vacuolated cytoplasm. However, the sporoplasms appeared to have a more robust stability when they were added to Medium 199 made up in 0.15 M potassium phosphate buffer with 5 mM ATP, and were supplemented with 10%-30% Xenopus oocyte cytosol (Fig. 1). After 12-24 h, these sporoplasms retained cytoplasm and did not vacuolate, although the sporoplasms appeared to still attach to each other or fuse. There was no evidence of nuclear division

during 24 h of incubations.

Microsporean sporoplasms were clearly stabilized in Medium 199 (pH 7.1-7.2) with ATP (5 mM) on a 2% gelatin substrate onto which was added 0.01-0.02 mM cholesterol with no Xenopus cytosol. There was no evidence of vacuolation or nuclear division. However, after 12 h, the sporoplasms remained segregated and retained their cytoplasmic matrix. This significant positive effect of cholesterol addition to the medium indicates that the sporoplasm outer envelope may be devoid of cholesterol. Insertion of choles- terol into plasma membrane is an essential component of eu-

karyote plasma membranes; it affects membrane fluidity and re- duces the permeability of membranes (2). Because newly extruded sporoplasms acquire an outer membrane that is believed to be derived from the extrusion apparatus (and is not the original plasma membrane of the spore), we expect that this second-hand membrane may lack a cholesterol component. This may account for the leaky condition of discharged sporoplasms when they are first entering into extracellular environs. Other primitive cells, such as mycoplasmas, also have an outer membrane that requires an external source of cholesterol from the outside environs for any level of stability. Once the cholesterol is acquired, these cells begin to regulate their internal milieu.

Literature Cited

1. Weidner, E., A. Findley, V. Dolgikh, and J. Sokolova. 1999. Pp. 172-195 in The Microsporidia and Microsporidiosis. American Society of Microbiology, Washington, D.C.

2. Dahl, J. 1993. Pp. 167-188 in Subcellular Biochemistry. Vol. 20. Plenum, New York.

karyote plasma membranes; it affects membrane fluidity and re- duces the permeability of membranes (2). Because newly extruded sporoplasms acquire an outer membrane that is believed to be derived from the extrusion apparatus (and is not the original plasma membrane of the spore), we expect that this second-hand membrane may lack a cholesterol component. This may account for the leaky condition of discharged sporoplasms when they are first entering into extracellular environs. Other primitive cells, such as mycoplasmas, also have an outer membrane that requires an external source of cholesterol from the outside environs for any level of stability. Once the cholesterol is acquired, these cells begin to regulate their internal milieu.

Literature Cited

1. Weidner, E., A. Findley, V. Dolgikh, and J. Sokolova. 1999. Pp. 172-195 in The Microsporidia and Microsporidiosis. American Society of Microbiology, Washington, D.C.

2. Dahl, J. 1993. Pp. 167-188 in Subcellular Biochemistry. Vol. 20. Plenum, New York.

Reference: Biol. Bull. 197: 271-273. (October 1999)

Intense Concanavalin A Staining and Apoptosis of Peripheral Flagellated Cells in Larvae of the Marine Sponge Microciona prolifera: Significance in Relation to Morphogenesis Jane C. Kaltenbachl, William J. Kuhns2, Tracy L. Simpson3, and Max M. Burger4

(Marine Biological Laboratory, Woods Hole, Massachusetts 02543)

Reference: Biol. Bull. 197: 271-273. (October 1999)

Intense Concanavalin A Staining and Apoptosis of Peripheral Flagellated Cells in Larvae of the Marine Sponge Microciona prolifera: Significance in Relation to Morphogenesis Jane C. Kaltenbachl, William J. Kuhns2, Tracy L. Simpson3, and Max M. Burger4

(Marine Biological Laboratory, Woods Hole, Massachusetts 02543)

Free-swimming larvae are released from adult Microciona

sponges during a brief period in late June and early July. The larvae are covered by a layer of flagellated epithelial cells, which

disappear within 24 h, at about the time of larval settlement, to a substrate such as rocks, shells, etc. (1, 2). The fate of the flagellated cells has long been discussed. As early as 1892, the inversion of these cells to form choanocytes was proposed for some species of

sponge (3). However, more recent evidence (e.g., electron micros-

copy and autoradiography) indicates that, in certain species includ-

ing Microciona prolifera, flagellated cells do not differentiate into other cell types but, near the time of settlement, are engulfed by large phagocytic cells presumed to be archaeocytes (4, 5).

The present study addresses the fate of peripheral flagellated cells in Microciona larvae with methods other than those used in

previous reports. We used lectin-based histochemical staining of surface sugars, as well as terminal UDP, nick-end labeling, com-

monly known as the TUNEL assay, and DNA gel electrophoresis, to define apoptosis.

Lectins, which have binding sites for specific sugars, can be

Mount Holyoke College, South Hadley, Massachusetts. 2 Hospital for Sick Children, Toronto, Canada. 3 University of Hartford, Hartford, Connecticut. 4 Friedrich Miescher Institute, Basel, Switzerland.

Free-swimming larvae are released from adult Microciona

sponges during a brief period in late June and early July. The larvae are covered by a layer of flagellated epithelial cells, which

disappear within 24 h, at about the time of larval settlement, to a substrate such as rocks, shells, etc. (1, 2). The fate of the flagellated cells has long been discussed. As early as 1892, the inversion of these cells to form choanocytes was proposed for some species of

sponge (3). However, more recent evidence (e.g., electron micros-

copy and autoradiography) indicates that, in certain species includ-

ing Microciona prolifera, flagellated cells do not differentiate into other cell types but, near the time of settlement, are engulfed by large phagocytic cells presumed to be archaeocytes (4, 5).

The present study addresses the fate of peripheral flagellated cells in Microciona larvae with methods other than those used in

previous reports. We used lectin-based histochemical staining of surface sugars, as well as terminal UDP, nick-end labeling, com-

monly known as the TUNEL assay, and DNA gel electrophoresis, to define apoptosis.

Lectins, which have binding sites for specific sugars, can be

Mount Holyoke College, South Hadley, Massachusetts. 2 Hospital for Sick Children, Toronto, Canada. 3 University of Hartford, Hartford, Connecticut. 4 Friedrich Miescher Institute, Basel, Switzerland.

conjugated to markers, such as horseradish peroxidase (HRP), and used as probes to localize sites of terminal sugar residues of the

glycans of membrane glycoproteins (6). To this end, larvae were fixed in 10% formalin, embedded in paraffin, and sectioned (5 jLm). The sections were treated with H202 to block endogenous peroxidase, and with bovine serum albumin to block non-specific staining. Sections were then incubated with HRP lectins (Table I). A brown color was developed with 3,3'diaminobenzidine (DAB)- H202 to indicate sites of specific sugars in the larvae. Control

Table I

Lectins and their specific affinities

Lectin Sugar

Concanavalin A (Con A) a-Mannose Wheat Germ Agglutinin (WGA) N-Acetyl-Glucosamine

(GlcNAc) Soybean Agglutinin (SBA) and N-Acetyl-Galactosamine

Dolichos biflorus Agglutinin (DBA) (GalNAc) Peanut Agglutinin (PNA) 3-Galactose Ulex europaeus Agglutinin a-L-Fucose

(UEA-1)

conjugated to markers, such as horseradish peroxidase (HRP), and used as probes to localize sites of terminal sugar residues of the

glycans of membrane glycoproteins (6). To this end, larvae were fixed in 10% formalin, embedded in paraffin, and sectioned (5 jLm). The sections were treated with H202 to block endogenous peroxidase, and with bovine serum albumin to block non-specific staining. Sections were then incubated with HRP lectins (Table I). A brown color was developed with 3,3'diaminobenzidine (DAB)- H202 to indicate sites of specific sugars in the larvae. Control

Table I

Lectins and their specific affinities

Lectin Sugar

Concanavalin A (Con A) a-Mannose Wheat Germ Agglutinin (WGA) N-Acetyl-Glucosamine

(GlcNAc) Soybean Agglutinin (SBA) and N-Acetyl-Galactosamine

Dolichos biflorus Agglutinin (DBA) (GalNAc) Peanut Agglutinin (PNA) 3-Galactose Ulex europaeus Agglutinin a-L-Fucose

(UEA-1)

271 271

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