ALKALINE EARTH ELEMENTS AND ZOOSPORE RELEASE AND DEVELOPMENT IN PROTOSIPHON BOTRYOIDES

O'KELLEY, JOSEPH C., and WALTER R. HERNDON. (U. Alabama, University.) Alkaline earth elements and zoospore release and development in Protosiphon botryoides. Amer. Jour. Bot. 48(9): 796-802. Illus. 1961.-Cells of Protosiphon botryoides Klebs from depleted nutrient medium containing Ca were washed and resuspended in fresh complete medium with Ca; or in media with a Sr, Ba or Na replacement, respectively, for Ca; or in an equivalent CaCl2 solution or deionized water. Zoospore release was observed in these media upon illumination following, a 12-hr dark period. Free zoospores were less abundant in Sr-, Baand Na-replacement media than in the Ca medium. Zoospore production and release also were depressed in solutions of only CaClU and in deionized water. In the Sr and Ba media, zoospores were formed but not released from the parent cell, as a rule; some zoospores were released in mass within a gelatinous vesicle which did not liquefy and set the zoospores free; these zoospores lost motility and continued development in Sr, producing characteristic, spheroidal clusters of aplanospores. In the Na medium, protoplasmic cleavage preceding zoospore formation was severely inhibited. A study of the reversibility of Sr inhibition of the zoospore-release mechanism revealed evidence of reversion 12 hr after replacement of Sr by Ca. Walls of cells produced in Ca are rich in ruthenium red-positive materials, whereas cells produced under conditions of Sr replacement lack these materials. The significance of these findings in relation to the Ca requirement of other algal species is discussed. ALTHOUGH Krauss (1958) listed calcium as an element considered to be required bv all algae, there had been considerable disagreement previously regarding a calcium requirement for Chlorella (see, for example, Hopkins and Wann, 1926; Trelease and Selsam, 1939; Noack, Pirson and Stegmann, 1940; Scott, 1944; Manuel, 1944; Myers, 1944; Hutner and Provasoli, 1951; Walker, 1953). This is apparently because the calcium level required for normal growth is low, and its essentiality difficult to demonstrate. In a number of species of algae studied, however, the calcium requirement has been sufficiently high to allow for its ready demonstration (Molisch, 1895; Benecke, 1898; Fraink, 1904; Richter, 1906; Maertens, 1914; Pringsheim, 1926, 1930; Waren, 1926; Chu, 1949). In still other species, a lower calcium requirement has been demonstrated by the use of citrate (Hutner, 1948; Hutner and Provasoli, 1951). At the present, it seems that more can be learned about the metabolic functions of calcium in the algae from studies of species or strains with a relatively high requirement for this element than from studies of organisms like Chlorella. However, few reports have been made of developmental and physiological changes resulting from, or accompanving, changes in nutrient-media calcium levels in species with higher requirements. Tiffany (1924) observed degeneration of colonies or filaments of 1 Received for publication March 5, 1961. This study was supported by research grant A-3680 Nutr. from the National Institute of Arthritis and Metabolic Diseases, Public Health Service. 2 Present address: Department of Botanv, University of Tennessee, Knoxville, Tenn. Spirogyra, Cladophora and Cylindrocapsa when deprived of calcium. Waren ( 1933) related cell division and also certain features of cell-wall and sheath formation to calcium nutrition in Micrasterias. and, more recently, calcium was discovered to be involved in the production of motile cells in Protosiphon (O'Kelley and Herndon, 1959). Since it has been observed that Protosiphon grown in media in which Sr replaces Ca fails to produce free-swimminig cells after an interval in such media, it appeared desirable to determine if a more immediate effect of replacement of calcium by strontium or other alkaline earth elements could be demonstrated. Further, it is important to determine whether or not inhibition of zoospore formation or release is readily reversible and whether there are demonstrable morphological or chemical changes in the cells. This paper attempts to answer these questions and reports specificallv on effects of calcium and other alkaline earth elements, strontium and barium, upon release and fate of zoospores in Protosiphon. MATERIALS AND METHODS-The Protosiphon strain used is culture LB99 of the Culture Collection of Algae of the Department of Botany of Indiana University (Starr, 1960). Composition of the basal medium used for culture of the organism and for zoospore-release experiments, a medium slightlv modified from that suggested by Bold (1949), is given in Table 1. Modifications of this basal medium (referred to subsequently as the Camedium) included substitution of an equivalent of SrCl2 for the CaCl2 (Sr-medium), and equivalent substitution of BaCI2 (Ba-medium) and NaCl (Na. medium). These media. the CaCl. solution. aind the