Figure 1. Defects in uba-1 hermaphrodites. A) Number of viable and inviable progeny produced by wild type (WT) or uba-1(it129) hermaphrodites at 15uC or 25uC. Shown are mean values and standard deviations (N = 6) of total progeny. B) DAPI staining of uba-1(it129) embryos from adults shifted to 25uC. C) Sperm-specific sterility. Number of viable progeny produced at 25uC by wild type or uba-1(it129) hermaphrodites, either unmated (self) or mated with wild-type males (cross). Shown are mean values and standard deviations (N = 6) of progeny produced in 48 h. D) Body length. Mean body length and standard deviations (N = 20) of age-synchronized adult hermaphrodites. doi:10.1371/journal.pgen.1000131.g001 phenotype is observed at 25uC, with the uba-1(it129) mutants being 16% shorter than the wild type adults. In the course of generating heterozygous strains for phenotypic characterization, we observed strong maternal effect rescue of the early developmental defects. Homozygous uba-1(it129) progeny derived from +/uba-1(it129) hermaphrodites reared at the restrictive temperature exhibited little embryonic or larval lethality (Table 2). Maternal rescue was not complete for all phenotypes; although the homozygous hermaphrodites developed normally into adulthood, sperm-specific sterility was still observed in these animals. We also tested for paternal rescue by mating uba-1(it129)/ + heterozygous males to uba-1(it129) homozygous hermaphrodites. Again, embryonic and larval lethality (though not sperm sterility) were largely rescued (Table 2). Because the presence of a single wild-type copy of uba-1 in either the hermaphrodite or male parent effectively suppresses embryonic and larval lethality in homozy- gous mutant progeny, it suggests that the maternal or paternal contribution of UBA-1 protein is sufficient to allow somatic development to proceed normally until adulthood. Male-Specific Phenotypes To facilitate the phenotypic characterization of males, we constructed a uba-1(it129) him-5(e1490) strain [the him-5(e1490) mutation produces males via nondisjunction of the X chromo- some] [37]. Temperature-shift experiments were performed with age-synchronized populations, and the same phenotypes were UBA-1 in C. elegans Development Table 2. Maternal and paternal rescue of lethality. Rescue Hermaphrodite genotype Lethality Male genotype (predicted) Maternal uba-1(it129)/+ none (self-fertile) Paternal uba-1(it129)/uba-1(it129) uba-1(it129)/+ 3.8% (25%) 7.2% (50%) Data are from five (maternal) or six (paternal) hermaphrodites. Mean total progeny numbers with s.d. are 123611 (maternal) and 73635 (paternal). doi:10.1371/journal.pgen.1000131.t002 observed in males as above: embryonic and larval lethality and a reduction in body size (data not shown). Sperm-specific sterility of mutant males was assessed by crossing to fem-1(hc17) hermaphro- dites, which lack sperm but produce oocytes that can be fertilized by mating. Experiments described below indicate that mating was successful but no cross-progeny were produced, demonstrating that male sperm are incapable of fertilization. Thus, the same array of defects are produced by the uba-1(it129) mutation in males and hermaphrodites. We also observed additional phenotypes in uba-1(it129) males. The most conspicuous phenotype in the adult was constitutive protraction of the spicules (Figure 2A, first vs. second panel). These structures are part of the reproductive apparatus of the male tail, and are normally extended only during insertion into the vulva for sperm transfer. A defect in spicule retraction was apparent in adult males at both the permissive and restrictive temperatures. Constitutively protracted spicules were observed in approximately one-third of uba-1(it129) males reared at 15uC and nearly all of those reared at 25uC. In some cases the spicules, gubernaculum, and surrounding tissues were everted, suggestive of structural defects in the integrity of the male reproductive tract as well. Additional abnormalities in the male copulatory apparatus were observed in animals reared at the restrictive temperature. The tail of the wild-type male possesses a cuticular fan containing nine pairs of sensory rays (Figure 2A, first panel), which are involved in mate detection and the behavioral responses necessary for locating the hermaphrodite vulva. The size of the fan is greatly diminished in uba-1(it129) homozygous males raised at 25uC, which results in shortening of the tail tip and sensory rays as well (Figure 2A, third panel). The number of rays is not affected, and other male reproductive structures appear superficially normal by light microscopy. The shortened fan phenotype is semi-dominant: the fan expanse in heterozygous +/uba-1(it129) males is less than in wild-type but greater than in homozygous animals (data not shown). Therefore, proper formation of the male copulatory structures appears to be quite sensitive to the dosage of UBA-1 protein. The male tail structures are critical for mating behavior and sperm transfer, so aberrations in the fan or in spicule function might adversely affect male reproductive success. Sperm from wild-type males take precedence over hermaphrodite sperm such that only outcross progeny are produced until the male sperm are depleted, at which time the production of self progeny continues [38]. Male sperm produce male and hermaphrodite progeny in equal numbers, while hermaphrodite sperm produce exclusively hermaphrodite progeny. Therefore, the number of outcross progeny, an indicator of male reproductive success, can be readily calculated by determining the number of males produced. Reproductive success was ascertained for homozygous uba- 1(it129) males grown at the permissive temperature. Some of these animals have protruding spicules, which might be predicted to impair sperm transfer. The fertility of uba-1(it129) hermaphrodites at 15uC demonstrates that sperm function is normal at this PLoS Genetics | www.plosgenetics.org 3 July 2008 | Volume 4 | Issue 7 | e1000131