THE EFFECTS OF REPLACEMENT OF CALCIUM BY STRONTIUM ON THE REPRODUCTION OF CHLOROCOCCUM ECHINOZYGOTUM

In liquid inorganic axenic cultures of Chlorococcum echinozygotum containing 20 ppm CaC12, which was optimal for growth, motile cells were released in abundance; gametic fusion occurred commonly and the resulting zygospores reached a maximum of 31% of the total cell population. In a Sr-replacement medium, less growth, not quite equivalent to that with 5 ppm CaCl2, occurred, and more than 99% of the cells were non-motile vegetative cells. Old Sr-replacement cultures contained much-enlarged vegetative cells which, upon transfer to fresh Sr-medium, produced entrapped motile cells equivalent in number to those produced and released upon transfer to fresh Ca-medium. Release of these entrapped cells, after their production in Sr, was induced in 4 hr by transferring them to Ca (25 ppm CaCl2). PREVIOUS studies with Protosiphon botryoides Klebs (O'Kelley and Herndon, 1961; O'Kelley and Deason, 1962) demonstrated that a deficiency of Ca, NO3 or S04 ions caused a decrease in production of free-swimming zoospores. Techniques used in these studies did not separate the processes of protoplasmic cleavage and zoosporogenesis from zoospore release. However, observations indicated Ca as a major ion influencing zoospore release, while NO3 and S04 appeared to affect mainly protoplasmic cleavage and zoosporogenesis. With Chlorococcum echinozygotum Starr, another organism that can utilize Sr in place of Ca, at least in part, it is possible by procedures described herein to separate the processes involved in motile cell production from motile cell release. Thus, Ca and other ion effects on release can be studied, uncomplicated by the effects of these ions on motile cell formation. This paper describes the results obtained with C. echinozygotum using these procedures. MATERIALS AND METHODS-The cultural strain of Chlorococcum echinozygotum used in this investigation was No. 118 from the Indiana University Culture Collection of Algae (Starr, 1960). The basal medium containing Ca was that of Deason and Bold (1960), except that in the growth studies it was modified by lowering the CaC12 level to 20 mg/liter; Hancock (1960) determined this concentration optimal for the growth of this organism. Other media contained a Sr replacement for Ca, or a Na replacement. Stock cultures were maintained in Pyrex test 1 Received for publication March 4, 1964. 2 Present address: Mineral Nutrition Laboratory, Soil and Water Conservation Research Division, Agricultural Research Service, USDA, Beltsville, Maryland. This study was supported in part by research granit AM 03680-04A1 from the National Institute of Arthritis and Metabolic Diseases, Ptublic Health Service. tubes as slants containing basal nmedium and agar. From these stock cultures aseptic transfers of cells were made to 500-ml Pyrex Erlenmeyer flasks containing 200 ml of either Caor Srmedium. These cultures were placed under 300 ft-c illumination on a 12-hr dark-12-hr light cycle at a temperature of 21-23 C. After the cultures had reached the maximum stationary phase of growth, inoculations were made to experimental media and vessels. These experimental cultures were subject to the same conditions of light and temperature as the stock cultures. Some experimental cells were maintained in hanging-drop preparations under similar environmental conditions. For the growth measurements in Ca and in Sr, inoculations were made to experimental vessels so that the initial optical density was 0.02, as determined by a Colenman Jr. Spectrophotometer at 400 my. Method for determining production and release of motile cells-Cultures used to supply cells to be immersed in fresh media were in the maximum stationary phase of growth, i.e., the value of the velocity coefficient of the rate of growth was zero. Just before the dark period, the cells were centrifuged, the old culture medium decanted, and the cells washed twice in the experimental medium to be employed. The cells were theni placed in a small Petri dish and covered to prevent entrance of light. At the beginning of the light period, the following morning the dish was uncoveied and allowed to remain undisturbed until maxirnal zoospore production was reached-i.e., in about 2-3 hr. The cells were stirred vigorously to insure thorough mixing and were then pipetted to a haemacytometer. Motile cells which were swimming freely in the medium were counted and expressed as pereentages of total cells. Total counts involved about 200 cells. Counts were analyzed by the t-test for significant differences between mean values (Lacey. 19.533).