hermaphrodites exhibit normal motility and lifespan (Figure 2F). The phenotype is not a consequence of aberrant somatic development but instead occurs post-developmentally, since delaying the temperature shift until adulthood still results in paralysis. Therefore, the uba-1 gene product is required for the maintenance of neuromuscular function in the adult male. Sperm-Specific Defect of uba-1 Mutation Sperm development in C. elegans has been described in detail [39,40], which allows the identification of specific cytological and functional defects in the developmental program that occur as a consequence of mutation. Normal spermatogenesis initiates from a mitotically dividing population of germ line stem cells. Primary spermatocytes separate from a syncytial cytoplasmic core and undergo a coordinated program of meiosis and differentiation. The two meiotic divisions give rise to four haploid spermatids with highly condensed nuclei. These small round cells separate from a larger residual body, which contains components not needed for subsequent steps in development. Activation by an extracellular signal converts the immotile spermatids into mature crawling spermatozoa capable of fertilization, and several compounds that promote activation in vitro have been identified [41–43]. Activation in hermaphrodites occurs in the spermatheca, where the mature spermatozoa are stored. Activation of male spermatids occurs at the time of insemination, and the male spermatozoa crawl from the uterus into the spermatheca. Fertilization takes place within the spermatheca as the oocyte squeezes into this chamber of hermaphrodite reproductive tract, and the newly formed zygote then passes into the uterus. Most of the spermatozoa are dislodged and must crawl back into the spermatheca to await the next oocyte. Sperm-specific sterility caused by the uba-1(it129) mutation was characterized in greater detail, beginning with the early events leading to spermatid formation. DAPI staining of L4 and young adult hermaphrodites and males revealed no differences in meiotic progression, the number of sperm produced, or (for hermaphro- dites) their initial localization to the spermathecae (Figure 3, A–B and data not shown). Activation of spermatids was normal in vivo and in vitro and produced crawling spermatozoa with no discernible defect in pseudopod movement or cell motility (Figure 3C). Since motility and localization appear normal and yet no zygotes are formed, the uba-1(it129) mutation produces mature spermatozoa that are nonetheless incapable of fertilization. A secondary defect in sperm function was detected later in adult hermaphrodites. Spermatozoa are displaced from the spermatheca into the uterus by each passing oocyte, and must return to the spermatheca and await the next egg. Fertilization efficiency is essentially 100% in wild type animals, with nearly every sperm being utilized for reproduction [38]. Thus, the number of sperm in the spermatheca decreases in concordance with an increase in the number of progeny produced. Because uba-1(it129) spermatozoa are motile but incapable of fertilization, one might predict that numbers within the spermatheca would remain high throughout oocyte production. Instead, the opposite phenomenon was observed, as sperm counts declined more rapidly in uba-1(it129) hermaphrodites than in wild type (Figure 3B). Furthermore, significant numbers of spermatozoa were detected in the uterus instead of the spermatheca (Figure 3A, uba-1). These cells are swept from the spermatheca by the unfertilized oocyte but are unable to return, and instead are expelled through the vulva when oocytes are deposited. Thus, although sperm motility and localization initially appear normal, these processes are clearly impaired in older animals. This observation may indicate a defect in the maintenance of sperm quality over time, which adversely impacts either motility or sperm-spermatheca interaction. UBA-1 in C. elegans Development Figure 3. Sperm defects. A) Sperm localization in the hermaphrodite reproductive tract. Wild type and uba-1(it129) adults at 25uC were fixed and stained with DAPI to count sperm nuclei. Arrowheads, location of spermathecae; small arrows, sperm displaced into the uterus. B) Summary of sperm localization data. Shown are mean values and standard deviations per hermaphrodite (N = 6). T0, before egg-laying commences; T1, after 1–2 ovulations; T2, 8 h post-T1; T3, 8 h post-T2. C) In vitro activation. Spermatids from wild type and uba-1(it129) males at 25uC were activated with monensin. Arrow indicates pseudopod of crawling spermatozoon. doi:10.1371/journal.pgen.1000131.g003 Identification of it129 as uba-1 The identity of the it129 allele was determined through a combination of genetic and physical mapping strategies (Figure 4A). Three-factor crosses placed this allele on chromosome IV between elt-1 and dpy-20 (F. Fell and S. Ward, pers. comm., and our own results; mapping data available at www.wormbase. org). Single nucleotide polymorphisms that overlap restriction sites (snip-SNPs) were analyzed in recombinant lines [44]. Strains containing the deficiency eDf19 or mDf7 failed to complement it129, further limiting its position to the overlapping 310 kb interval. A total of 80 candidate genes within the interval were available from a large-scale RNAi feeding library [35]. All were tested for the ability to replicate two of the it129 phenotypes: F1 embryonic lethality of treated adult hermaphrodites, and tail deformation in adult males treated as larvae. Only one of the plasmids tested reproduced both phenotypes. That plasmid contains a fragment of the gene encoding the ubiquitin-activating enzyme E1, which in C. elegans is known as uba-1. Complementation tests confirmed that it129 is an allele of uba-1. The Gene Knockout Consortium (http://celeganskoconsortium. omrf.org) has generated a deletion allele, uba-1(ok1374), that removes much of the third and fourth exons and is predicted to be a mutation (Figure 4B). Mutants homozygous for uba- 1(ok1374) exhibit embryonic lethality or early larval arrest, so ok1374/it129 animals were obtained from crosses at the permissive temperature to allow recovery of viable lines. Complementation between the two alleles was tested by temperature shift at various developmental stages as described above. The identical pheno- types reported for it129 homozygotes were observed for ok1374/ it129 heterozygotes: embryonic lethality, larval lethality, sperm- specific sterility, defects in male tail formation, and male-specific PLoS Genetics | www.plosgenetics.org 5 July 2008 | Volume 4 | Issue 7 | e1000131