CONTROL OF SEXUALITY IN GOLENKINIA

Sperm formation can be induced in strains 929 and 930 of the oogamous genus Golenkinia by nutrient depletion, specifically the deprivation of either nitrogen or phosphorus, but not by a decrease in total molarity or by a change in pH. Nitrogen (or phosphorus) concentration plays a controlling role in the presexual vegetative growth and the actual period of induction. During the latter, the rate of gametogenesis is inversely proportional to the nitrogen level and can be completely stopped by nitrogen added as many as 8 hr after resuspension in a medium lacking nitrogen. White light of an intensity of 10,000 ergs per cm2 per second is required dturing the first 9 hr of resuspension in an inducing medium although a lower intensity is sufficient if given for longer periods. Sexuality in Golenkinia responds most strongly to red or blue light, indicating that photosynthesis is important in this process. Fertilization in strain 929 requires at least 0.001 M calcium. No evidence was found for the action of hormones either in sexual induction or in chemotactic sperm attraction. GOLENKINIA is a genus of unicellular green algae which belongs to the green algal order Chlorococcales. It reproduces sexually by the formation of eggs and sperm. Although the morphology of this process has been studied previously (Korshikov, 1937; Iyengar and Balakrishnan, 1956; Starr, 1963), nothing is known about the physiological basis. Golenkinia is one of relatively few unicellular oogamous green algae and therefore seemed an excellent candidate for a study of sexual induction and fertilization. Further, the study would extend the knowledge of sexual regulation in the algae to an organism outside the order Volvocales, to which most previous research has been restricted. The research reported here concentrated on four questions. (1) Is nitrogen depletion as important in gamete induction in Golenkinia as in Chlamydomonas reinhardi (Sager and Granick, 1954), C. eugametos (Trainor, 1959), C. chlamydogama (Trainor, 1958), C. sp. 24 (Tsubo, 1957), Cylindracystis (Pringsheim, 1918), and in one of the rare instances of recorded research on a member of the Chlorococcales, Scenedesmus (Trainor and Burg, 1965)? (2) Is light required for sexual induction in Golenkinia? It was found to be necessary in Chlamydomonas by Smith (1948), Lewin (1956), Trainor (1958), F6rster 1 Received for publication 20 October 1967. This study represents a portion of a dissertation presented by the senior author to the Graduate School of the University of California, Berkeley, in partial fulfillment of the requirements for the degree Doctor of Philosophy. The work was supported in part by a Pre-doctoral Traineeship granted to the first author by the National Aeronautics and Space Administration and by a research grant to the second author from the National Science Foundation. 2 Present address: Department of Biology, City College, City University of New York, New York, N. Y. 10031. (1957), and Stifter (1959). There is definite agreement about the fact of the light requirement in Chlamydomonas; however, the roles assigned to light vary greatly. (3) Is sexuality effected by extracellular products acting as inducing hormones as in Volvox aureus (Darden, 1966)? (4) Is a chemotactic hormone involved in the fertilization process in Golenkinia? Spermattractant substances have been demonstrated in several genera of green algae: Chlorogonium and Sphaeroplea (Pascher, 1931a, b), Oedogonium (Hoffman, 1960), and Chlamydomonas moewusii var. rotunda (Tsubo, 1957, 1961a). The most recent reviews of the research in these subjects are those of Lewin (1954), Coleman (1962), Lang (1965), and Machlis and Rawitscher-Kunkel (1967). MATERIAL AND METHODS-Material-Golenkinia strains 929 and 930 were used in this study. Their origin, morphology, vegetative growth characteristics and nutrition are discussed by Ellis and Machlis (1968). Sexual reproduction is accomplished by the differentiation of a vegetative cell into either eight sperm or one egg. The biflagellate sperm possess a chloroplast, one or two contractile vacuoles, and lack an eyespot. After release, they are motile for about 8 hr and may be involved in fertilization. If not, the end of the period of motility is recognizable by a pronounced swelling and sluggishness, followed by lysis and death. The egg is morphologically indistinguishable from a vegetative cell. Zygotes mature in 3-4 weeks, forming thick-walled, resistant zygospores which are approximately 20 , in diam. Germination of the zygospore has not been achieved. Starr (1963) states that, although both strains are capable of sperm production, fertilization and zygospore produc-