Erschienen in: Protist ; 151 (2000), 3. - S. 225-238 https://dx.doi.org/10.1078/1434-4610-00021 Maullinia ectocarpii gen. et sp. nov. (Plasmodiophorea), an Intracellular Parasite in Ectocarpus siliculosus (Ectocarpales, Phaeophyceae) and other Filamentous Brown Algae Ingo Maiera,1, Elisa Parodib, Renato Westermeierc, and Dieter G. Müllera a Fachbereich Biologie, Universität Konstanz, D-78457 Konstanz, Germany b Instituto Argentino de Oceanografía, UNS-CONICET, 8000 Bahia Blanca, Argentina c Facultad de Pesquerias y Oceanografia, Universidad Austral de Chile, Casilla 1327, Puerto Montt, Chile An obligate intracellular parasite infecting Ectocarpus spp. and other filamentous marine brown algae is described. The pathogen forms an unwalled multinucleate syncytium (plasmodium) within the host cell cytoplasm and causes hypertrophy. Cruciform nuclear divisions occur during early de- velopment. Mature plasmodia become transformed into single sporangia, filling the host cell com- pletely, and then cleave into several hundred spores. The spores are motile with two unequal, whiplash-type flagella inserted subapically and also show amoeboid movement. Upon settlement, cysts with chitinous walls are formed. Infection of host cells is accomplished by means of an adheso- rium and a stachel apparatus penetrating the host cell wall, and injection of the cyst content into the host cell cytoplasm. The parasite is characterized by features specific for the plasmodiophorids and is described as a new genus and species, Maullinia ectocarpii. Introduction Filamentous brown algae are important members in made on living algal hosts and their parasites. As many benthic marine communities. They have been has been stated by Andrews (1976), “pathological frequently reported to be attacked by various investigations by algologists have been more the re- pathogens, including viruses (Müller et al. 1998) and sult of coincidence than design”. eukaryotic parasites of different phylogenetic lin- During a study on algal epiphytes on a mariculture eages: oomycetes, chytrids and hyphochytrids (An- plantation in Chile, we encountered specimens of drews 1976; Küpper and Müller 1999; Müller et al. the marine brown alga Ectocarpus siliculosus (Dill- 1999; Porter 1986; Sparrow 1960). Although there is wyn) Lyngbye which displayed symptoms of infec- good reason to speculate that, besides herbivory, tion by a parasite of unknown identity. We report parasitism may play an important role in the popula- here our observations on field and cultured material tion dynamics, few laboratory studies have been which show that the pathogen is a new taxon in the Plasmodiophorea with a potentially wide host range within the brown algae. The Plasmodiophorea have 1 Corresponding author; fax 49 7531 88 2966 recently been included into the Protoctista (Dylewski e-mail [email protected] 1990) or into the kingdom Protozoa (Protista) (Barr Konstanzer Online-Publikations-System (KOPS) URL: http://nbn-resolving.de/urn:nbn:de:bsz:352-0-310311 226 1992; Braselton 1995; Cavalier-Smith 1993a, b; fusely by spores from plurilocular sporangia and Corliss 1994). Small subunit rRNA gene sequence tended to overgrow the parasite. Intense attention comparison of Plasmodiophora brassicae Woronin was necessary to maintain the parasite in culture. (Castlebury and Domier 1998) also corroborates Therefore, we started a first infection experiment placement among the protists, with no close rela- using, as a new host, a cultured strain of E. fascicu- tionship to true fungi, Mycetozoa, Rhizopoda, or latus Harvey which was completely sterile due to a Chromista. Here we follow Buczacki (1983) and systemic virus infection. Filaments of the material Braselton (1995) in using the informal term “plas- were incubated with selected parasite sporangia modiophorids” which does not imply a certain taxo- from the raw culture. Within several weeks, the para- nomic rank. The group includes obligate endopara- site appeared on the new host. This new host- sites of higher plants, algae and oomycetes. During pathogen system was stable and therefore chosen its trophic phases, the thallus occurs as an un- for major parts of further work. It was successively walled, syncytical protoplast referred to as a plas- improved by elimination of all contaminants until it modium. Eventually it develops into a sporangium or arrived at a triphasic state consisting of the host E. a sporosorus composed of aggregates of resting fasciculatus carrying its latent virus and the parasite. spores. The terminology used here in respect to the life cycle corresponds to that suggested by Karling Development and Structure of the Parasite (1981) and Braselton (1995). The parasite propagated and new infections were established by zoospores (Figs. 3, 4), which were Results produced in large numbers. They were pyriform, 4.6 ± 0.7 µm in length and 2.3 ± 0.4 µm in diameter. Field Material The two unequal, whiplash-type flagella are inserted subapically on the broader part of the cell. The long The majority of E. siliculosus filaments collected at flagellum (19.3 ± 1.7 µm) undergoes undulating La Pasada, Chile, appeared morphologically normal movement and is oriented backward, whereas the and formed plurilocular zoidangia. The habit of the short flagellum (8.2 ± 0.6 µm) points forward at an thalli and the occasional presence of unilocular spo- oblique angle to the cell axis. Both flagella possess rangia indicated that they were sporophytes. About short hairtips. At some time after release and upon 15% of the samples were sterile and showed contact with a surface, the spores showed amoe- abortive sporangia characteristic for viral infection boid movement by means of lobose pseudopodia of (Müller et al. 1998). A still smaller proportion of thalli variable shape,which were rapidly extended and (about 1%) formed few plurilocular sporangia, but withdrawn (Figs. 5–8). Often, flagella were still pre- also clusters of relatively large lateral appendages sent at this stage. After settlement, the cell encysted along the filament axes (Figs. 1, 2). These oblong to and assumed an ellipsoid to kidney-shaped mor- sub-spherical vesicles were mostly free of pigments. phology. Cysts were 5.8 ± 0.7 µm in length and They were predominantly found sessile on filament 2.5 ± 0.2 µm in diameter. cells or on terminal cells of the main axis and lateral Eventually, a pointed, “beak”- or “stiletto”-shap- branches of the thallus, rarely intercalary. Release of ed infection apparatus (“adhesorium”, Aist & Wil- numerous non-pigmented zoospores from apical liams 1971; Keskin and Fuchs 1969) was formed at openings showed the parasitic, sporangial nature of one end of the cell (Figs. 9–11). The adhesorium is the observed structures. Intense screening of field 5–7 µm long and 0.8–1.2 µm in diameter. It is con- material revealed several cases where symptoms of nected to the cell body by a narrow neck portion of virus infection co-existed with parasite sporangia on about 0.8 µm in length. The cysts become attached the same host filament. The parasite has also been to the host cell wall by the tip of the adhesorium, identified on a microslide prepared of E. siliculosus while the cell body is lifted off the algal filament. In collected at Flinders, Westport Bay, Victoria, Aus- microscopic preparations, the cells easily became tralia in September 1988. At that time, release of detached. Spores retained in sporangia also en- zoospores had been recorded, but no further stud- cysted and produced adhesoria, as well as spores ies were carried out. trapped in mucilage at the aperture of sporangia, adhering to a parasite sporangium or settled onto Establishment of Laboratory Cultures glass cover slips without contact to host cells (Fig. 13). At the tip of adhesoria attached to coverslips, In a host-parasite co-culture established from se- minute hairpoint filaments of 1.2–1.4 µm in length or lected field material, Ectocarpus reproduced pro- occasionally a tuft of small projections have been Figure 1. Filaments of E. siliculosus infected by Maullinia ectocarpii. Parasitized cells appear as pale, hypertrophied lateral appendages (arrowheads). Scale bar = 100 µm. Figure 2. Group of infected cells at higher magnification. Scale bar = 100 µm. Figures 3 and 4. Zoospores of Maullinia fixed in OsO4 vapour. Phase contrast. Scale bar = 5 µm. Figures 5–8. Spore showing amoeboid movement with pseudopodia (arrowheads) on a glass slide. Microflash ex- posures taken within a few seconds. Scale bar = 5 µm. Figure 9 and 10. Cysts with adhesorium (arrowhead) attached to a host filament and injecting its cytoplasmic con- tent (arrow). Scale bars = 5 µm. Figures 11–12. Fluorescent chitin labeling of cyst wall (arrowhead). Scale bar = 5 µm. Figure 13. Cyst with adhesorium on coverslip, enlarged vacuole (arrow) and hairpoint tip on adhesorium (arrow- head). Scale bar = 5 µm. Figures 14 and 15. Electron micrographs of adhesoria in cysts before extrusion in longitudinal and cross-section, showing the Rohr (r), Stachel (s), plug (p), and cyst wall (w). Scale bars = 0.5 µm. 228 Figures 16 and 17. Early infection stages of M. ectocarpii in E. siliculosus, with parasite (arrowheads) and host nuclei (n) visualized by DAPI staining. The plasmodia contain from one to several nuclei in interphase. Scale bar = 10 µm. Figures 18 and 19. A later stage of development, showing host nucleus (n) and plasmodium with many parasite nuclei in interphase (arrowheads). Scale bar = 10 µm. Figures 20 and 21. Late stage in plasmodium development with numerous parasite nuclei in interphase (arrowheads). Below a freshly infected cell with a single parasite nucleus (arrowhead) and a host cell nucleus (n). Scale bar = 10 µm. Figures 22 and 23. Metaphase plates in plasmodium, with chromosomes in a circular arrangement, leaving a hole in the center (arrowheads), DAPI staining. Scale bar = 10 µm. Figure 24. As Figs. 22 and 23, haematoxylin staining. Scale bar = 10 µm. Figures 25 and 26. Cruciform metaphase configurations (arrowheads) in young plasmodia stained by haematoxylin, with host cell nucleus (n) containing a prominent nucleolus (nl).