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Abstract As an entomopathogenic nematode (EPN), Steinernema hermaphroditum parasitizes insect hosts and harbors symbiotic Xenorhabdus griffinae bacteria. In contrast to other Steinernematids, S. hermaphroditum has hermaphroditic genetics, offering the experimental scope found in Caenorhabditis elegans. To enable study of S. hermaphroditum, we have assembled and analyzed its reference genome. This genome assembly has five chromosomal scaffolds and 83 unassigned scaffolds totaling 90.7 Mb, with 19,426 protein-coding genes having a BUSCO completeness of 88.0%. Its autosomes show higher densities of strongly conserved genes in their centers, as in C. elegans, but repetitive elements are evenly distributed along all chromosomes, rather than with higher arm densities as in C. elegans. Either when comparing protein motif frequencies between nematode species or when analyzing gene family expansions during nematode evolution, we observed two categories of genes preferentially associated with the origin of Steinernema or S. hermaphroditum: orthologs of venom genes in S. carpocapsae or S. feltiae; and some types of chemosensory G protein-coupled receptors, despite the tendency of parasitic nematodes to have reduced numbers of chemosensory genes. Three-quarters of venom orthologs occurred in gene clusters, with the larger clusters comprising functionally diverse gene groups rather than paralogous repeats of a single venom gene. While assembling S. hermaphroditum, we coassembled bacterial genomes, finding sequence data for not only the known symbiont, X. griffinae, but also for eight other bacterial genera. All eight genera have previously been observed to be associated with Steinernema species or the EPN Heterorhabditis, and may constitute a second bacterial circle of EPNs. 

Abstract Sexual characteristics and reproductive systems are dynamic traits in many taxa, but the developmental modifications that allow change and innovation are largely unknown. A leading model for this process is the evolution of self-fertile hermaphrodites from male/female ancestors. However, these studies require direct analysis of sex determination in male/female species, as well as in the hermaphroditic species that are related to them. In Caenorhabditis nematodes, this has only become possible recently, with the discovery of new species. Here, we use gene editing to characterize major sex determination genes in Caenorhabditis nigoni, a sister to the widely studied hermaphroditic species Caenorhabditis briggsae. These 2 species are close enough to mate and form partially fertile hybrids. First, we find that tra-1 functions as the master regulator of sex in C. nigoni, in both the soma and the germ line. Surprisingly, these mutants make only sperm, in contrast to tra-1 mutants in related hermaphroditic species. Moreover, the XX mutants display a unique defect in somatic gonad development that is not seen elsewhere in the genus. Second, the fem-3 gene acts upstream of tra-1 in C. nigoni, and the mutants are females, unlike in the sister species C. briggsae, where they develop as hermaphrodites. This result points to a divergence in the role of fem-3 in the germ line of these species. Third, tra-2 encodes a transmembrane receptor that acts upstream of fem-3 in C. nigoni. Outside of the germ line, tra-2 mutations in all species cause a similar pattern of partial masculinization. However, heterozygosity for tra-2 does not alter germ cell fates in C. nigoni, as it can in sensitized backgrounds of 2 hermaphroditic species of Caenorhabditis. Finally, the epistatic relationships point to a simple, linear germline pathway in which tra-2 regulates fem-3 which regulates tra-1, unlike the more complex relationships seen in hermaphrodite germ cell development. Taking these results together, the regulation of sex determination is more robust and streamlined in the male/female species C. nigoni than in related species that make self-fertile hermaphrodites, a conclusion supported by studies of interspecies hybrids using sex determination mutations. Thus, we infer that the origin of self-fertility not only required mutations that activated the spermatogenesis program in XX germ lines, but prior to these there must have been mutations that decanalized the sex determination process, allowing for subsequent changes to germ cell fates. 

As an entomopathogenic nematode (EPN), Steinernema hermaphroditum parasitizes insect hosts and harbors symbiotic Xenorhabdus griffinae bacteria. In contrast to other Steinernematids, S. hermaphroditum has hermaphroditic genetics, offering the experimental scope found in Caenorhabditis elegans. To enable biological analysis of S. hermaphroditum, we have assembled and analyzed its reference genome. This genome assembly has five chromosomal scaffolds and 83 unassigned scaffolds totaling 90.7 Mb, with 19,426 protein-coding genes having a BUSCO completeness of 88.0%. Its autosomes show higher densities of strongly conserved genes in their centers, as in C. elegans, but repetitive elements are evenly distributed along all chromosomes, rather than with higher arm densities as in C. elegans. Either when comparing protein motif frequencies between nematode species or when analyzing gene family expansions during nematode evolution, we observed two categories of genes preferentially associated with the origin of Steinernema or S. hermaphroditum: orthologs of venom genes in S. carpocapsae or S. feltiae; and some types of chemosensory G protein-coupled receptors, despite the tendency of parasitic nematodes to have reduced numbers of chemosensory genes. Three-quarters of venom orthologs occurred in gene clusters, with the larger clusters comprising functionally diverse pathogenicity islands rather than paralogous repeats of a single venom gene. While assembling the genome of S. hermaphroditum, we coassembled bacterial genomes, finding sequence data for not only the known symbiont, X. griffinae, but also for eight other bacterial genera. All eight genera have previously been observed to be associated with Steinernema species or the EPN Heterorhabditis, and may constitute a “second bacterial circle” of EPNs. The genome assemblies of S. hermaphroditum and its associated bacteria will enable use of these organisms as a model system for both entomopathogenicity and symbiosis.