Successful fertilization requires that sperm are activated prior to contacting an egg. In C. elegans, this activation process, called spermiogenesis, transforms round immobile spermatids into motile, fertilization-competent spermatozoa. Spermiogenesis in males uses the TRY-5 protease pathway, whereas in hermaphrodites it is controlled by the SPE-8 pathway. Here we describe the characterization of two new spermiogenesis-defective mutants. We show that spe-43 is a new component of the SPE-8 pathway; spe-43 hermaphrodites are self-sterile, while spe-43 males show wild-type fertility. Consistent with other members of the spe-8 class, spe-43 hermaphrodite sperm can be trans-activated by male seminal fluid.When spe-43 spermatids are exposed to Pronaseto activate the sperm in vitro, they form long rigid spikes radiating outward from the cell periphery instead of forming a motile pseudopod confirming a defect in spermiogenesis.Using a combination of recombinant and deletion mapping and whole genome sequencing we identified a C>T mutation in F09E8.1 as the molecular lesion in spe-43(eb63). A second allele spe-43(jn5) allows us to conclude that although the spe-43 transcript exists in two isoforms, only the isoform predicted to encode a transmembrane domain and a large extracellular domain is necessary for its function in spermiogenesis.Unlike spe-43, our second mutant, as41, affects spermiogenesis in both sexes, suggesting that the mutated gene may be downstream of both the TRY-5 and SPE-8 pathways. Spermatids from as41mutants show no signs of in vitro activation in the presence of Pronase, failing to even form spikes. We are currently in the process of using a similar whole genome sequencing strategy to identify the molecular nature of as41..