- Email: [email protected]
Malaria, Microtubules and Merozoite Invasion
Comment 3 Parija, S.C. et al. (1997) J. Clin. Microbiol. 35, 1571–1574 4 de Colmenares, M. et al. (1995) Am. J. Trop. Med. Hyg. 52, 427–428 5 Zheng, H. et al. (1987) Am. J. Trop. Med. Hyg. 31, 554–560
6 Freillij, H.L. and Corral, R.S. (1987) J. Clin. Microbiol. 25, 133–137 7 Katzin, A.M. et al. (1991) Am. J. Trop. Med. Hyg. 45, 453–462 8 Lieshout, L. Van et al. (1991) Am. J. Trop. Med. Hyg. 44, 323–328
Subash Chandra Parija is at the Department of Microbiology, B.P. Koirala Institute of Health Sciences, Dharan, Nepal. Tel: +977 25 20802, Fax: +977 25 20251, e-mail: [email protected]
Malaria, Microtubules and Merozoite Invasion M. Hommel and J. Schrével The recent paper by Bejon et al.1 shows how little we really know about the molecular mechanisms involved in red blood cell invasion by malaria merozoites and, in particular, how drugs which interact with microtubule polymerization may inhibit this event. Merozoites, being short-lived, need to make contact with a new host erythrocyte in a matter of minutes after their release from a mature schizont; they can do this either by attaching to one of the red blood cells already in close proximity to the schizont (as may be the case in a cytoadherent ‘rosette’)2 or by random contact in the turmoil of the peripheral bloodflow. After the initial contact, the merozoite reorients and brings its apical end into intimate contact with the red blood cell membrane, then either ‘propels itself into the red cell’1 or, after first inducing the formation of a vacuole of the red blood cell membrane by injecting the content of its apical organelles (rhoptries, micronemes and, probably, dense granules)3 into the cell membrane, it forms an annular tight junction and then slides into the vacuole by ‘zipper’ action4. The interaction between the merozoite surface and the red blood cell membrane involves, of course, a number of receptors and ligands on both cells (see recent review by Holder5), where the major merozoite surface protein-1 (MSP-1) probably plays the most important role. In 1989, an interesting 60 kDa molecule had been described (termed MCP-1 or merozoite capping protein-1)6, which appeared to be responsible for the tight junction and, therefore, the forward movement of the merozoite into the vacuole; one could perhaps speculate that, if there is a renewed interest in invasion mechanisms, this ‘forgotten’ molecule may deserve further studies. Whatever the mechanism of entry, merozoite motility is important and, by 6
analogy with other apicomplexans, this is achieved either by a torsion/contraction of the network of subpellicular microtubules and/or by an actin-based contractile system, hence a process involving both microtubules and microfilaments. It would thus seem highly logical to expect that drugs known to interfere with tubulin or actin would inhibit invasion, but credible experiments to confirm interaction have, for a variety of reasons, proven very difficult to design with malaria merozoites. Plasmodium spp are known to possess some of the genes required for driving ‘motors’ based on microtubules and microfilaments: a and b tubulin, a and b actin, and at least one type of myosin have been found, but the presence of the usual microtubule-associated protein ‘motors’ (eg. dynein, kinesin or dynamin) remains to be confirmed7. Even once all the relevant genes have been catalogued, as is sure to happen in the foreseeable future in the course of the Malaria Genome Programme, any detailed analysis of stage-specific gene expression of these molecules or, more importantly, their state of phosphorylation or their posttranslational modifications, will remain extremely difficult to perform on malaria merozoites, in view of the technical difficulty in obtaining material for biochemical studies. Most merozoite invasion experiments are based on a short-term in vitro culture of malaria parasites, where schizonts (usually synchronized to a narrow window of developmental stages) are allowed to mature and reinvade new erythrocytes; the efficiency of re-invasion is measured by the number of ring-stage trophozoites present in the culture a few hours later. This represents a slippage from procedures using purified, cell-free merozoites, where the result of invasion inhibition was easier to interpret (eg. as was per-
Copyright © 1998, Elsevier Science Ltd All rights reserved 0169–4758/98/$19.00 PII: S0169-4758(97)01160-5
formed with Plasmodium knowlesi in the mid-1970s)8. The deviation in technology has transformed an invasion inhibition assay into a parasite growth assay, but this was necessary in order to study P. falciparum, a parasite for which the preparation of purified, live, cellfree merozoites is a very demanding technique9. Those who perform experiments with cultures of P. falciparum, usually use laboratory isolates of parasites maintained in human erythrocytes in various degrees of ‘freshness’, ie. from blood in transfusion packs maintained in ACD/CPD for 1–5 weeks, then in culture medium at 37°C for a number of days. We must not lose sight of the fact that, although such ‘preserved’ red blood cells may be adequate for transfusion or indeed for growing malaria parasites in vitro, they present, from the cell biologist’s point of view, important defects in the membrane structure, including a substantial loss of membrane cholesterol, changes in lectin-binding characteristics and a decrease in the level of phosphorylation of membrane proteins; any of these alterations may affect experimental studies and could explain some of the discrepancies reported between different publications. Microtubules are dynamic polymers involved in a wide variety of cellular functions. The difficulty with in vitro invasion inhibition experiments with P. falciparum is that antimicrotubular drugs may theoretically act on at least four different sites: cell divisions in the schizont, abnormal development of merozoites (‘dysgenesis’), erythrocyte membrane rigidity and merozoite motility. In order to prove that a particular drug is acting on one site rather than on another, conclusions have to be reached by a process of elimination rather than by direct experimental analysis. The effects on cell division have been demonstrated for a variety of drugs Parasitology Today, vol. 14, no. 1, 1998
Comment known to act on microtubules, including colchicine10, vinblastine11, nocodazole10, trifluralin12, Taxol ®13 and Taxotere ®14,15; all interfere with mitosis, by blocking spindle formation either by depolymerization or by enhancement of the polymerization of tubulin. Researchers have focused on this site of action, as this is the most likely target for an eventual use of the drugs as antimalarials. From a more fundamental point of view, the study of mitosis in parasites treated with antimicrotubular drugs can help to dissect the precise molecular mechanisms of mitosis during schizogony and there are suggestions that this event may not be controlled by the same checkpoints than the cell cycle of other eukaryotic cells16. The action of drugs on the red blood cell is more speculative, since a human red cell lacks both a and b tubulin, but instead possesses a network of spectrin fibers which represent the major component of its cytoskeleton. Some studies have reported that antimicrotubular drugs were capable of increasing red blood cell rigidity, in some cases accompanied by a morphological change from a biconcave disk to a sphaerocyte, which sometimes are reversible (ie. cells treated with colchicine become sphaerocytes and revert to a biconcave disk after treatment with cAMP)17. Whether this effect can be assigned to an interaction with spectrin polymerization, as has been stated in early literature17, is not certain, but a correlation between increased rigidity and merozoite invasion inhibition has been reported, and in one study, this effect was also reversible with cAMP18. This is in contrast to the observed lack of effect of colchicine on human red blood cells reported by Bejon et al.1 where the drug did not induce the formation of sphaerocytes or reduce invasion of merozoites in pre-treated red blood cells. In a different study, Taxotere ® also had an effect on the erythrocyte membrane, in direct correlation with the degree of invasion inhibition observed14. Different evidence in support of the view that red cell rigidity inhibits or hinders invasion comes from studies using either re-sealed erythrocytes where the spectrin has been crosslinked19 or red blood cells from cases of ovalocytosis20 and elliptocytosis21, two genetic disorders of red blood cells, the latter directly linked to a defect of a-spectrin; in all such studies the in vitro infection with P. falciparum was considerably reduced, sometimes entirely inhibited. Parasitology Today, vol. 14, no. 1, 1998
Another approach for understanding what happens during merozoite invasion is to look at closely related Protozoa, using models where it is easier to obtain purified, viable merozoites. Among Apicomplexa, in which the invasive stages have a comparable structure and, presumably face similar problems with cell invasion, Toxoplasma has been studied most. Here, in contrast to Plasmodium, the host cell can actually be a phagocytic cell or at the least a cell normally capable of endocytosis. Until recently, invasion by Toxoplasma merozoites was described as the following sequence: tight junction between the apical end of the merozoite and the host cell; triggering of endocytosis by the release of the content of apical organelles (as in Plasmodium, but surprisingly by molecules which do not appear to have much sequence homology, despite their close phylogenetic proximity); forward movement of the merozoite, which seemed to be pushed into the endocytic vacuole by a movement of the tight junction around its body, apparently by microfilaments on the cytoplasmic side of the host cell membrane. This classical sequence has recently been challenged by the description of merozoite entry into cytochalasin D-resistant host cells, a study which incriminates merozoite actin as the prime mover22. Unfortunately, none of the reported studies of Toxoplasma (or other apicomplexans) has so far investigated the effects of antimicrotubular drugs of merozoite motility.
References 1 Bejon, P.A. et al. (1997) Parasitology 114, 1–6 2 Wahlgren, M., Carlson, J. and Udomsangpetch, R. (1987) Parasitol. Today 5, 183–185 3 Perkins, M.E. (1992) Parasitol. Today 8, 28–32 4 Miller, L.H. et al. (1979) J. Exp. Med. 149, 172–184 5 Holder, A.A. (1994) Parasitology 108, S5–S18 6 Klotz, F.N. et al. (1989) Mol. Biochem. Parasitol. 36, 177–186 7 Reddy, G.R. (1995) Parasitol. Today 11, 37–42 8 Butcher, G.A. et al. (1978) Immunology 34, 77–86 9 Mrema, J.E.K. et al. (1982) Exp. Parasitol. 54, 285–295 10 Dieckmann-Schuppert, A. and Franklin, R.M. (1989) Cell. Biol. Int. Rep. 13, 411–418 11 Usanga, E.A., O’Brien, E. and Luzzatto, L. (1986) FEBS Lett. 20, 23–27 12 Callahan, H.L. et al. (1996) Antimicrob. Agents Chemother. 40, 947–952 13 Pouvelle, B. et al. (1994) J. Clin. Invest. 94, 413–417 14 Schrével, J. et al. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 8472–8476 15 Sinou, V., Grellier, P. and Schrével, J. (1996) Antimicrob. Agents Chemother. 40, 358–361 16 Sinou, V. et al. J. Euk. Microbiol. (in press) 17 Yawata, Y. et al. (1976) J. Lab. Clin. Med. 88, 555–562 18 McColm, A.A., Hommel, M. and Trigg, P.I. (1980) Mol. Biochem. Parasitol. 1, 119–127 19 Mohandas, N. et al. (1984) Blood 63, 1385–1392 20 Facer, C.A. (1995) Parasitol. Res. 81, 52–57 21 Dluzewski, A.R. et al. (1983) Br. J. Haematol. 55, 629–637 22 Dobrowolski, J.M. and Sibley, L.D. (1996) Cell 84, 933–939
Marcel Hommel is at the Liverpool School of Tropical Medicine, Liverpool, UK L3 5QA. Joseph Schrével is at the Muséum National d’Histoire Naturelle, ERS156 CNRS, 75231 Paris, France. Tel: +44 151 708 9393, Fax: +44 151 708 8733, e-mail: [email protected] Note. See Letters, this issue.
Websites worth visiting The International Leishmania Network has a website at http://www.bdt.org.br/bdt/leishnet. It includes useful information about epidemiology, cryobanks, available monoclonal antibodies, the genome project etc and has a link to the American Type Culture Collection. Multilateral Initiative on Malaria in Africa The Task Force on Malaria Research Capability Strengthening in Africa invites applications for research-based capability strengthening grants in Africa. Application forms [from Fabio Zicker ([email protected]) or at http://www.who. ch/programmes/tdr] must be received by 15 January 1998. SciCentral: Gateway to the Best Science and Engineering Online Resources This directory at http:// www.scicentral.com/ provides many links to resources in all fields of science but is highly recommended here because it also tells you how to subscribe to the various discussion groups monitored for ParaSite. It includes e-mail addresses and Web pages of academic institutions in the USA. A Malaria Research and Reference Reagent Repository is being established by The National Institute of Allergy and Infectious Diseases (NIAID). Details will be available on the NIAID home page at http://www.niaid.nih.gov. For information about submitting and acquiring reagents contact Dr Michael Gottlieb (e-mail: [email protected]). The Science Guide at http://www.scienceguide.com is another directory designed to help scientists find information on the Internet. It too has details of jobs and grants and a directory of newsgroups, and in addition has links to on-line journals. PROTIST (formerly Archiv für Protistenkunde) will publish substantial and novel findings in any area of research on protists. Michael Melkonian ([email protected]) is the Editor-in-Chief. Instructions to Authors can be found at http://mother.biolan.uni-koeln. de/institute/botanik/protist/.
7
6 Freillij, H.L. and Corral, R.S. (1987) J. Clin. Microbiol. 25, 133–137 7 Katzin, A.M. et al. (1991) Am. J. Trop. Med. Hyg. 45, 453–462 8 Lieshout, L. Van et al. (1991) Am. J. Trop. Med. Hyg. 44, 323–328
Subash Chandra Parija is at the Department of Microbiology, B.P. Koirala Institute of Health Sciences, Dharan, Nepal. Tel: +977 25 20802, Fax: +977 25 20251, e-mail: [email protected]
Malaria, Microtubules and Merozoite Invasion M. Hommel and J. Schrével The recent paper by Bejon et al.1 shows how little we really know about the molecular mechanisms involved in red blood cell invasion by malaria merozoites and, in particular, how drugs which interact with microtubule polymerization may inhibit this event. Merozoites, being short-lived, need to make contact with a new host erythrocyte in a matter of minutes after their release from a mature schizont; they can do this either by attaching to one of the red blood cells already in close proximity to the schizont (as may be the case in a cytoadherent ‘rosette’)2 or by random contact in the turmoil of the peripheral bloodflow. After the initial contact, the merozoite reorients and brings its apical end into intimate contact with the red blood cell membrane, then either ‘propels itself into the red cell’1 or, after first inducing the formation of a vacuole of the red blood cell membrane by injecting the content of its apical organelles (rhoptries, micronemes and, probably, dense granules)3 into the cell membrane, it forms an annular tight junction and then slides into the vacuole by ‘zipper’ action4. The interaction between the merozoite surface and the red blood cell membrane involves, of course, a number of receptors and ligands on both cells (see recent review by Holder5), where the major merozoite surface protein-1 (MSP-1) probably plays the most important role. In 1989, an interesting 60 kDa molecule had been described (termed MCP-1 or merozoite capping protein-1)6, which appeared to be responsible for the tight junction and, therefore, the forward movement of the merozoite into the vacuole; one could perhaps speculate that, if there is a renewed interest in invasion mechanisms, this ‘forgotten’ molecule may deserve further studies. Whatever the mechanism of entry, merozoite motility is important and, by 6
analogy with other apicomplexans, this is achieved either by a torsion/contraction of the network of subpellicular microtubules and/or by an actin-based contractile system, hence a process involving both microtubules and microfilaments. It would thus seem highly logical to expect that drugs known to interfere with tubulin or actin would inhibit invasion, but credible experiments to confirm interaction have, for a variety of reasons, proven very difficult to design with malaria merozoites. Plasmodium spp are known to possess some of the genes required for driving ‘motors’ based on microtubules and microfilaments: a and b tubulin, a and b actin, and at least one type of myosin have been found, but the presence of the usual microtubule-associated protein ‘motors’ (eg. dynein, kinesin or dynamin) remains to be confirmed7. Even once all the relevant genes have been catalogued, as is sure to happen in the foreseeable future in the course of the Malaria Genome Programme, any detailed analysis of stage-specific gene expression of these molecules or, more importantly, their state of phosphorylation or their posttranslational modifications, will remain extremely difficult to perform on malaria merozoites, in view of the technical difficulty in obtaining material for biochemical studies. Most merozoite invasion experiments are based on a short-term in vitro culture of malaria parasites, where schizonts (usually synchronized to a narrow window of developmental stages) are allowed to mature and reinvade new erythrocytes; the efficiency of re-invasion is measured by the number of ring-stage trophozoites present in the culture a few hours later. This represents a slippage from procedures using purified, cell-free merozoites, where the result of invasion inhibition was easier to interpret (eg. as was per-
Copyright © 1998, Elsevier Science Ltd All rights reserved 0169–4758/98/$19.00 PII: S0169-4758(97)01160-5
formed with Plasmodium knowlesi in the mid-1970s)8. The deviation in technology has transformed an invasion inhibition assay into a parasite growth assay, but this was necessary in order to study P. falciparum, a parasite for which the preparation of purified, live, cellfree merozoites is a very demanding technique9. Those who perform experiments with cultures of P. falciparum, usually use laboratory isolates of parasites maintained in human erythrocytes in various degrees of ‘freshness’, ie. from blood in transfusion packs maintained in ACD/CPD for 1–5 weeks, then in culture medium at 37°C for a number of days. We must not lose sight of the fact that, although such ‘preserved’ red blood cells may be adequate for transfusion or indeed for growing malaria parasites in vitro, they present, from the cell biologist’s point of view, important defects in the membrane structure, including a substantial loss of membrane cholesterol, changes in lectin-binding characteristics and a decrease in the level of phosphorylation of membrane proteins; any of these alterations may affect experimental studies and could explain some of the discrepancies reported between different publications. Microtubules are dynamic polymers involved in a wide variety of cellular functions. The difficulty with in vitro invasion inhibition experiments with P. falciparum is that antimicrotubular drugs may theoretically act on at least four different sites: cell divisions in the schizont, abnormal development of merozoites (‘dysgenesis’), erythrocyte membrane rigidity and merozoite motility. In order to prove that a particular drug is acting on one site rather than on another, conclusions have to be reached by a process of elimination rather than by direct experimental analysis. The effects on cell division have been demonstrated for a variety of drugs Parasitology Today, vol. 14, no. 1, 1998
Comment known to act on microtubules, including colchicine10, vinblastine11, nocodazole10, trifluralin12, Taxol ®13 and Taxotere ®14,15; all interfere with mitosis, by blocking spindle formation either by depolymerization or by enhancement of the polymerization of tubulin. Researchers have focused on this site of action, as this is the most likely target for an eventual use of the drugs as antimalarials. From a more fundamental point of view, the study of mitosis in parasites treated with antimicrotubular drugs can help to dissect the precise molecular mechanisms of mitosis during schizogony and there are suggestions that this event may not be controlled by the same checkpoints than the cell cycle of other eukaryotic cells16. The action of drugs on the red blood cell is more speculative, since a human red cell lacks both a and b tubulin, but instead possesses a network of spectrin fibers which represent the major component of its cytoskeleton. Some studies have reported that antimicrotubular drugs were capable of increasing red blood cell rigidity, in some cases accompanied by a morphological change from a biconcave disk to a sphaerocyte, which sometimes are reversible (ie. cells treated with colchicine become sphaerocytes and revert to a biconcave disk after treatment with cAMP)17. Whether this effect can be assigned to an interaction with spectrin polymerization, as has been stated in early literature17, is not certain, but a correlation between increased rigidity and merozoite invasion inhibition has been reported, and in one study, this effect was also reversible with cAMP18. This is in contrast to the observed lack of effect of colchicine on human red blood cells reported by Bejon et al.1 where the drug did not induce the formation of sphaerocytes or reduce invasion of merozoites in pre-treated red blood cells. In a different study, Taxotere ® also had an effect on the erythrocyte membrane, in direct correlation with the degree of invasion inhibition observed14. Different evidence in support of the view that red cell rigidity inhibits or hinders invasion comes from studies using either re-sealed erythrocytes where the spectrin has been crosslinked19 or red blood cells from cases of ovalocytosis20 and elliptocytosis21, two genetic disorders of red blood cells, the latter directly linked to a defect of a-spectrin; in all such studies the in vitro infection with P. falciparum was considerably reduced, sometimes entirely inhibited. Parasitology Today, vol. 14, no. 1, 1998
Another approach for understanding what happens during merozoite invasion is to look at closely related Protozoa, using models where it is easier to obtain purified, viable merozoites. Among Apicomplexa, in which the invasive stages have a comparable structure and, presumably face similar problems with cell invasion, Toxoplasma has been studied most. Here, in contrast to Plasmodium, the host cell can actually be a phagocytic cell or at the least a cell normally capable of endocytosis. Until recently, invasion by Toxoplasma merozoites was described as the following sequence: tight junction between the apical end of the merozoite and the host cell; triggering of endocytosis by the release of the content of apical organelles (as in Plasmodium, but surprisingly by molecules which do not appear to have much sequence homology, despite their close phylogenetic proximity); forward movement of the merozoite, which seemed to be pushed into the endocytic vacuole by a movement of the tight junction around its body, apparently by microfilaments on the cytoplasmic side of the host cell membrane. This classical sequence has recently been challenged by the description of merozoite entry into cytochalasin D-resistant host cells, a study which incriminates merozoite actin as the prime mover22. Unfortunately, none of the reported studies of Toxoplasma (or other apicomplexans) has so far investigated the effects of antimicrotubular drugs of merozoite motility.
References 1 Bejon, P.A. et al. (1997) Parasitology 114, 1–6 2 Wahlgren, M., Carlson, J. and Udomsangpetch, R. (1987) Parasitol. Today 5, 183–185 3 Perkins, M.E. (1992) Parasitol. Today 8, 28–32 4 Miller, L.H. et al. (1979) J. Exp. Med. 149, 172–184 5 Holder, A.A. (1994) Parasitology 108, S5–S18 6 Klotz, F.N. et al. (1989) Mol. Biochem. Parasitol. 36, 177–186 7 Reddy, G.R. (1995) Parasitol. Today 11, 37–42 8 Butcher, G.A. et al. (1978) Immunology 34, 77–86 9 Mrema, J.E.K. et al. (1982) Exp. Parasitol. 54, 285–295 10 Dieckmann-Schuppert, A. and Franklin, R.M. (1989) Cell. Biol. Int. Rep. 13, 411–418 11 Usanga, E.A., O’Brien, E. and Luzzatto, L. (1986) FEBS Lett. 20, 23–27 12 Callahan, H.L. et al. (1996) Antimicrob. Agents Chemother. 40, 947–952 13 Pouvelle, B. et al. (1994) J. Clin. Invest. 94, 413–417 14 Schrével, J. et al. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 8472–8476 15 Sinou, V., Grellier, P. and Schrével, J. (1996) Antimicrob. Agents Chemother. 40, 358–361 16 Sinou, V. et al. J. Euk. Microbiol. (in press) 17 Yawata, Y. et al. (1976) J. Lab. Clin. Med. 88, 555–562 18 McColm, A.A., Hommel, M. and Trigg, P.I. (1980) Mol. Biochem. Parasitol. 1, 119–127 19 Mohandas, N. et al. (1984) Blood 63, 1385–1392 20 Facer, C.A. (1995) Parasitol. Res. 81, 52–57 21 Dluzewski, A.R. et al. (1983) Br. J. Haematol. 55, 629–637 22 Dobrowolski, J.M. and Sibley, L.D. (1996) Cell 84, 933–939
Marcel Hommel is at the Liverpool School of Tropical Medicine, Liverpool, UK L3 5QA. Joseph Schrével is at the Muséum National d’Histoire Naturelle, ERS156 CNRS, 75231 Paris, France. Tel: +44 151 708 9393, Fax: +44 151 708 8733, e-mail: [email protected] Note. See Letters, this issue.
Websites worth visiting The International Leishmania Network has a website at http://www.bdt.org.br/bdt/leishnet. It includes useful information about epidemiology, cryobanks, available monoclonal antibodies, the genome project etc and has a link to the American Type Culture Collection. Multilateral Initiative on Malaria in Africa The Task Force on Malaria Research Capability Strengthening in Africa invites applications for research-based capability strengthening grants in Africa. Application forms [from Fabio Zicker ([email protected]) or at http://www.who. ch/programmes/tdr] must be received by 15 January 1998. SciCentral: Gateway to the Best Science and Engineering Online Resources This directory at http:// www.scicentral.com/ provides many links to resources in all fields of science but is highly recommended here because it also tells you how to subscribe to the various discussion groups monitored for ParaSite. It includes e-mail addresses and Web pages of academic institutions in the USA. A Malaria Research and Reference Reagent Repository is being established by The National Institute of Allergy and Infectious Diseases (NIAID). Details will be available on the NIAID home page at http://www.niaid.nih.gov. For information about submitting and acquiring reagents contact Dr Michael Gottlieb (e-mail: [email protected]). The Science Guide at http://www.scienceguide.com is another directory designed to help scientists find information on the Internet. It too has details of jobs and grants and a directory of newsgroups, and in addition has links to on-line journals. PROTIST (formerly Archiv für Protistenkunde) will publish substantial and novel findings in any area of research on protists. Michael Melkonian ([email protected]) is the Editor-in-Chief. Instructions to Authors can be found at http://mother.biolan.uni-koeln. de/institute/botanik/protist/.
7