The Chlamydomonas zygospore is formed after gametic cell fusion by a progression of ultrastructural and physiological changes, including the synthesis of a protective and elaborately sculptured secondary wall (Figure 1). Assembly of the secondary wall occurs within the confines of a transient primary zygote wall, synthesized immediately after gamete fusion, and assembled beneath the residual gamete walls. Upon completion of secondary wall assembly, the primary zygote wall is shed from the maturing zygospore (Figure 2). This primary zygote wall, composed primarily of a β-1,3 glucan (callose), may be important in the regulation of secondary zygospore wall formation.

A temperature-sensitive mutant strain of C. monoica, zym30, has been isolated that shows marked abnormalities in both primary and secondary wall ultrastructure. The degree of wall formation in the mutant strain is inversely proportional to temperature. At 25° C most zygotes fail to synthesize either the primary or the secondary wall. At 15° C both primary and secondary wall assembly occurs to some extent and as many as 50% of the zygotes develop a “pseudo-wildtype” phenotype (Figure 3). Although release of the primary wall rarely occurs in the zym30 strain (even at permissive temperatures), those walls that are released appear thinner than the walls shed from wildtype zygospores, as viewed by phase contrast microscopy. Fluorescence images incorporating aniline blue, a fluorochrome specific for β-1,3 glucans, show decreased fluorescence suggesting reduced callose synthesis (Figure 4).

Several permanent, ultrastructurally distinct layers comprise the secondary wall, the assembly of which may be controlled, in part, by the primary wall. One or more of these secondary wall layers contain the highly durable, autofluorescent biopolymer, sporopollenin. Autofluorescence of zym30 zygospores produced at the permissive temperature is enhanced relative to wildtype zygospores. This correlates with ultrastructural changes in the primary wall and overproduction and/or misassembly of the surface secondary wall layer (Figure 5). Work is in progress to determine whether the environmental resistance, and/or viability of the zym30 “pseudowildtype” zygospores are reduced relative to wildtype zygospores.