758 the Ultrastructure of an Alloparasitic Red Alga Choreocolax

PHYCOLOGIA 12(3/4) 1973 The ultrastructure of an alloparasitic red alga Choreocolax polysiphoniae I PAUL KUGRENS Department of Botany and Plant Pathology, Colorado State University, Fort Collins, Colorado 80521, U.S.A. AND JOHN A. WEST Department of Botany, University of California, Berkeley, California 94720, U.S.A. Accepted June 18, 1973 An alloparasite, Choreocolax polysipiloniae, apparently represents one of the most evolved parasitic red algae. Chlo�oplasts are highly redu�ed and consist of dOl!ble membrane limited organelles lacking any inter nal thylako!� developmen!. The unInucleate cells have thick walls, an absence of starch in cortical cells and larg� quantIties of starch In meduII ary cells. Host-para�ite connections are made by typical red algal pit con . nectIOns. G.eneral effects of t�e InfectIOn on the host .Include cell hypertrophy, decrease in floridean starch granules, dispersed cytoplasmiC matrIces, and contorsJOn of chloroplasts. Phycologia, 12(3/4): 175-186, 1973 Introduction of the host, Cryptopleura. Her decision was The paraSItIc red algae constitute a unique based on the similarity in reproductive struc 1?irou of organisms about which surprisingly tures between the host and parasite, and she � suggested bacteria as causal agents for such lIttle IS known, although their distinctive nature . has been recognized since the late nineteenth proliferatIons. Chemin (1937) also indicated century. There are approximately 40 genera, that bacteria might be causal agents since bac unknown numbers of species, and all are ex teria were isolated from surface-sterilized thalli clusively florideophycean, belonging to all of Callocolax neglectus. Observations on Lobo orders except the Nemaliales. col�x deformans Howe also suggest that bac An observation that has impressed many in tena may be involved in its formation (Chiang, vesti ators is the close relationship in repro 1970; Hollenberg and Abbott, 1966; Kugrens, � . ductIve and vegetatIve structure exhibited by 1971� McBride, Kugrens, and West, 1973). the parasites and their hosts which are also The Idea that parasitic red algae are galls of host tissue has been disregarded by most in red algae. About 90% of the parasitic red . algae are taxonomically related to their hosts vestIgators but definitive evidence to the con trary has not been presented. (Dawson, 1966) and placed in the same fami � li es. These have been classified as adelphopara .Stud.ies on t e parasitic red algae have been . pnmanly descnptive and morphological (Adey SItes "Yhereas those that are not closely related to theIr hosts are known as alloparasites (Feld and Sperapani, 1971; Fan, 1961; Sturch, mann and Feldmann, 1958). 1923), and, consequently, little information is This close relationship in many instances available on their cytology or other aspects of has led several investigators to draw different their biology. In fact, we do not know whether they a re g�nuine Parasites since parasitism has c clusions about the nature of red algal para . �)ll been ImplIed stnctly, from morphological evi SItes. Grubb (1925) considered Gonimophyl dence such as reduced thallus size, nature of lum bufJhamiian outgrowth or proliferation attachment to the host plant, and some visible loss of pigmentation. Therefore, we have under IPortions of this manuscript constituted a part of a taken investigations on nine genera of red dissertation submitted by P. Kugrens to the Graduate algae considered to be parasitic. The ultrastruc Division at the University of California Berkeley in � partial satisfaction of the requirements for the degree tural. s udies are of primary importance in de of Doctor of Philosophy. termmmg the presence or absence of bacteria 175 PHYCOLOGIA, VOL. 12(3/4), 1973 KUGRENS AND WEST: Choreocolax ultrastructure Abbreviations used in figures:AC, axial cell; C, chloroplast; CW, cell wall; D, dictyosome; FS, floridean starch; M, mitochondrion; N, nucleus; Nu, nucleolus; PP, pit connection. Figures 1-4 are light micrographs, and Figures 5-17 are electron micrographs. FIG. 1. Habit photograph of Choreoeolax; note the hypertrophied cells of Polysiphonia; X 100. FIG. 2. Chore oeolax growing on basal portions of Polysiphonia that was collected in January; note the rhizoids (r); X 10. FIG. 3. Cross section of Choreocolax attached to Polysiphonia; compare the cells that are not infected with infected and displaced Po/ysiphonia cells (P); note the intrusive growth of Choreoeo/ax (Ch) cells, and the absence of a distinct vacuole in some of the displaced pericentral cells (P*); arrow indicates a nuclear region in a cell packed with starch; X 400. FIG. 4. Cross section of Choreoeolax showing displaced Polysiphonia cells (P) and a pit connection between the host and parasitic cells (black arrow); note the cortical cells with large amounts of starch; nuclei are visible in areas devoid of starch granules (white arrow); X500. FIG. 5. Longitudinal section showing a portion of Choreoco/ax protruding from the host; the walls of Chore ocolax are thick and undifferentiated structurally, except for an electron dense thickening near the thallus surface (arrow); X 1900. FIG. 6. Nuclear pores; X30,000. FIG. 7. Double membrane limited body (C) adja cent to mitochondrion; note the lack of thylakoid differentiation in this chloroplast; X 40,500. FIG. 8. Corti cal cell showing the acentric nucleolus, mitochondria, and lack of starch grains; X 13,500. 176 PHYCOLOGIA, VOL. 12(3/4), 1973 KUGRENS AND WEST: Choreocolax ultrastructure cw 177 PHYCOLOGIA, VOL. 12(3/4),1973 KUGRENS AND WEST: Choreocolax ultrastructure and the nature of the parasites' cytoplasmic integrity through numerous secondary pit con constituents, such as chloroplasts. nections. When reproductive, the tetrasporangia In the present paper we describe the struc and spermatia occur in the outer cell layers, ture of Choreocolax polysiphoniae, an allo but the carposporophyte is deeply embedded parasite, and presumably one of the most re within the thallus. duced forms. It occurs on both the Atlantic Choreocolax and its hosts are seasonal, dis and Pacific coasts of the United States and is appearing from the flora during November and commonly found growing on Polysiphonia, al December and reappearing in mid-January. though other hosts in the Rhodomelaceae are Perennation is accomplished through growth of also utilized. Choreocolax on persistent prostrate portions of Polysiphonia (Fig. 2) during the winter, thus Materials and Methods only the upright portions of the host can be Thalli of Choreocolax polysiphoniae growing considered truly seasonal. on Polysiphonia spp. were collected from Dux At the light microscope level it can be seen bury Reef in Marin County, California. Ma that cells are arranged in an orderly fashion terial was fixed for 1112 hours in 5 % glutaral within the thallus, however, new Choreocolax dehyde in 0.1 M cacodylate buffer containing thalli that develop in culture on Polysiphonia 3.5% w/v NaCl. All fixations were carried lack this organized cellular pattern. Infected out on ice. The remainder of the fixation was host cells are separated from one another and identical to that described in a previous paper raised upward as Choreocolax expands in size (Kugrens and West, 1972). (Fig. 3, 4). Choreocolax develops on one side Sections of araldite or epon-embedded ma of the host, many cells growing intrusively terial (Spurr, 1969) were post-stained with among the host tissue (Fig. 3). The cells of lead citrate (Reynolds, 1963) for 10 minutes Choreocolax, particularly the medullary cells, and 15 minutes, respectively. are filled with granular material presumably Thick sections of material (0.5/Lm-1.01'.m) floridean starch. The position of the nuclei can were cut with glass knives and stained with be inferred from areas in the cell where starch toluidine blue for observations with a Zeiss is absent (Fig. 3, 4). As the cells mature, they GFL light microscope. enlarge and fill with starch, whereas the outer Choreocolax polysiphoniae and its host, most layer of cells the cortical cells are small Polysiphonia, could be maintained in the la and free of starch, presumably indicative of boratory for as long as seven months when meristematic cells. placed in Provasoli's enriched sea water Primary effects on the host can be discerned medium (Provasoli, 1968) and kept at 15 C at the light microscopic level (Fig. 3). The in an illumination of about 1000 lux. Fixations cells undergo hypertrophy and are capable of could be carried out from cultured material dividing in any plane, probably as a wounding since new infections of host tissue were com reaction in response to the parasite. This divi mon. Attempted culturing of excised thalli was sion of cells obscures the earlier symmetrical unsuccessful and spores could not be isolated pattern of pericentral cells present in non from the thallus to observe possible germina infected tissue. A secondary effect can be seen tion. In fact, few thalli were found to be re in heavily infected cells. There is a lack of a productive. central vacuole (Fig. 3) and the chloroplasts Results are scattered throughout the lightly staining Thalli of Choreocolax are small (up to cytoplasm unlike the situation in non-infected 250/Lm in diam), white, and lack branches cells or lightly infected cells, where the chloro (Fig. 1). Hosts include several species of Poly plasts are situated within a peripheral cyto siphonia, and occasionally Pterosiphonia bi plasm surrounding a large central vacuole. pinnata and Pterosiphonia dendroidea, the Intimate connections between the parasite thalli occurring on the prostrate portions in the and host appear to be made via secondary pit latter. The thallus is multiaxial, and retains its connections (Fig. 4). From light microscopic FIG. 9. Choreocolax "rhizoidal" cells growing between two host cells (P); X 6000. FIG. 10. Medullary cells of Choreocolax; cells are filled with starch, surrounded by very thick walls, and have nuclei that are displaced by starch grains; arrow indicates a pit plug between two cells; X41 00.

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