UBA-1 in C. elegans Development Figure 2. Defects in uba-1 males. A) Male tail defects. Shown are DIC photomicrographs of the male tail from wild type or uba-1(it129) animals grown at the indicated temperature. Bracket indicates the fan and sensory rays. Arrow indicates the spicules. B) Reproductive success of male mating. Graph indicates the mean number of male and hermaphrodite progeny produced by wild type hermaphrodites (N = 6) mated to wild type or uba- 1(it129) males grown at 15uC. Only the first 500 progeny were counted for wild type. Matings combined one hermaphrodite with five males, or three hermaphrodites with 12 males (high #) for 24 h. Percent outcross is calculated by multiplying the number of male progeny by two, then dividing by the total number of progeny. C) Sperm transfer. Arrow indicates fluorescently-labeled sperm from wild type or uba-1(it129) males localized within the spermatheca of unlabeled hermaphrodites after mating. D–F, male-specific paralysis. D) Young adult uba-1(it129) male. Arrow indicates normal sinusoidal curve of tail. E) Older adult uba-1(it129) male. Arrow indicates flaccid posture of tail. F) Dead uba-1(it129) male (arrow) and two uba-1(it129) hermaphrodites. doi:10.1371/journal.pgen.1000131.g002 temperature, so the production of outcross progeny was used as an indicator of successful mating. Mating to wild-type males produced males and hermaphrodites in the expected 1:1 ratio, indicating that all of the offspring in the measured time interval resulted from fertilization by male sperm (Figure 2B, WT). In contrast, mating with uba-1(it129) males produced an average of only 56 male vs. 236 hermaphrodite progeny (Figure 2B, uba-1), suggesting that the number of outcross progeny is reduced. The same data could be explained if the nullo-X sperm, which produce male progeny, are less competent for fertilization than the X- bearing sperm that produce hermaphrodites. This explanation seems unlikely, because the percentage of male progeny is elevated if the density of males for mating is increased (Figure 2B, uba-1, high #). To eliminate conclusively this possibility, uba-1(it129) males were mated to fem-1(hc17) adult hermaphrodites that lack sperm. Only outcross progeny are produced in this experiment and, although the numbers were low, males and hermaphrodites were observed in a ratio of 1:1 (data not shown). Therefore, the protruding spicule phenotype observed in uba-1(it129) males at the permissive temperature decreases the successful transfer of sperm for fertilization. Reproductive success was also characterized in the same manner for uba-1(it129) males shifted to the restrictive temperature at L3. No outcross progeny were observed from matings to either wild-type or fem-1(hc17) hermaphrodites. This failure might arise from the inability of sperm to fertilize the oocytes (as is true for hermaphrodite sperm at 25uC), or might be a consequence of the severe morphological defects in the male copulatory apparatus that occur at the restrictive temperature. A direct assessment of sperm transfer was performed to discriminate between the two possibilities. Males from him-5(e1490) strains that are wild-type or mutant for uba-1(it129) were raised at both 15uC and 25uC, stained with a fluorescent dye, then mated to fem-1(hc17) hermaphrodites that lacked sperm. Wild-type males reared at either temperature and mutant males reared at 15uC were successful in mating 50–70% of the time, as revealed by the presence of labeled sperm in the fem-1(hc17) hermaphrodites (Figure 2C, first two panels). In contrast, uba-1(it129) males raised at 25uC successfully transferred sperm in only two out of 10 instances. Although the efficiency of mating is reduced at 25uC, defects in the male copulatory structures arising from the uba- 1(it129) mutation do not completely abrogate sperm transfer (Figure 2C, third panel). However, even those relatively rare successful matings do not give rise to outcross progeny, indicating that uba-1(it129) sperm from males are incapable of fertilization at the restrictive temperature. An additional, sex-specific phenotype was observed in uba- 1(it129) males: a late onset, progressive paralysis in two-thirds of the animals (Figure 2, D–F). The paralysis initiates at the posterior of the male and proceeds anteriorly as the worm ages, culminating in a completely paralyzed animal with a significantly shortened lifespan. Progressive paralysis is restricted to males, as uba-1(it129) PLoS Genetics | www.plosgenetics.org 4 July 2008 | Volume 4 | Issue 7 | e1000131