Macrocysts in the life cycle of the Dictyosteliaceae. II. Germination of the macrocysts

Macrocyst germination was demonstrated in the five species of the Dictyosteliaceae known to produce these structures. The morphological changes that occurred during germination appeared to be identical in all of the strains examined, showing the following stages: (1) swelling of the dark, contracted content of the dormant cysts, (2) gradual loss of color and reappearance of cells within what previously appeared as a homogeneous protoplasmic mass, and (3) rupture of the heavy cellulosic cyst wall to liberate the myxamoebae. The age of the macrocyst appeared to be the most critical factor in determining whether or not germination would occur, since the cysts in many of the strains needed to age for several weeks or months before germination could be demonstrated. In Dictyostelium mucoroides strain DM-7, upon which the current study was centered, light was necessary to stimulate germination of young macrocysts-a requirement that gradually diminished as the cysts aged. The rate of germination and the temperature permitting germination were also age dependent: older macrocysts germinated more rapidly and at considerably higher temperatures than did young cysts. Although light was not essential for germination in every strain, the results obtained with strain DM-7 seem to be generally applicable to the germination process. MACROCYSTS are multicellular post-aggregative encystment structures produced by certain strains of the cellular slime molds (Dictyosteliaceae) under appropriate environmental conditions (Blaskovics and Raper, 1957; Nickerson and Raper, in press). Their formation is signalled by the deposition of a loose primary wall which delimits the incipient macrocyst(s) soon after the myxamoebae have moved into aggregation centers. As this initial wall is deposited, a large cell appears in the center of the developing macrocyst as reported by Dengler, Filosa, and Shao (1970) and since confirmed in an independent EM study by Erdos, Nickerson, and Raper (1972). This cell enlarges, ingests the peripheral myxamoebae, and encloses each in a vacuole. The process of phagocytosis continues, progressing outward from the center, until all of the myxamoebae have been ingested by the giant cell. A heavy secondary wall rich in cellulose is then laid down just outside the plasma membrane of the giant cell. (Under the phase contrast microscope, the macrocysts during this stage of development appear to consist of closely packed seemingly firm-walled cells, termed "endocytes" by Blaskovics and Raper in 1 Received for publication 14 June 1972. This paper represents a part of a thesis offered by the senior author in partial fulfillment of the requirements for the Ph.D. degree, University of Wisconsin, Madison. The work was supported by research grants GB-8624 from the National Science Foundation and AI-049 15 from the National Institutes of Health, U.S. Public Health Service. 1957). Some days later the cyst slowly develops a brownish coloration. As it darkens, the ingested amoebae are digested losing their cellular identity so that the content of the cyst appears homogeneous within the heavy macrocyst wall. In its fully mature and dormant state, the cyst content shrinks somewhat and loses contact with the heavy wall. It is now tightly bounded by an elastic tertiary wall, which instead of being thin, as reported by Blaskovics and Raper (1957), is now seen to be comparatively thick. Blaskovics and Raper (1957) reported a 7.2% germination of 10-day-old endocyte-filled macrocysts following incubation for 22 days at 15 C. Treating the cysts with cellulase, alternately freezing and thawing, and mechanically breaking them were all ineffective in increasing the rate or percentage of germination. However, better germination did occur in sixto eight-week-old macrocysts. Unfortunately, the sequence of changes between the time when the cyst content appeared homogeneous and the rupture of the walls to liberate the myxamoebae could not be determined. The purpose of this study was threefold: (1) to establish the sequence of morphological events that occur during the transition from the homogeneous condition of the mature dormant macrocyst to the liberation of myxamoebae at germination; (2) to identify a strain in which the process of germination could be studied advantageously; and (3) to use this strain to examine carefully the