Differential Gene Expression
To examine differential expression among life-stages and adult sexes ofN. riversi , we mapped RNA sequencing reads to the super-transcriptome that merged the official gene set with the de novo transcriptome. This super-transcriptome reference contains 17,736 genes (Table 2 ), a slight reduction relative to the official gene set. Hierarchical clustering of overall expression differences (Figure S6 ) shows that the egg stage is very strongly differentiated, relative to comparisons among the larval and adult stages. Focusing on the top 25 differentially expressed genes among each pairwise contrast (Figure S7 ), both stage and sex-specific gene expression is evident. For example, some of the top genes expressed in the larval stage are involved in cuticle formation (cuticle, endocuticle and chitin binding proteins), while some of the top genes in the adult male are associated with sperm production. These patterns are reinforced by a gene set enrichment analysis (GSEA) of the associated gene ontology terms for differentially expressed genes (FDR < 0.01), with statistical enrichment determined below a nominal p -value of 0.05 (Table S8 ). The larvae to egg contrasts highlighted terms upregulated in larvae associated with extracellular space, monooxygenase activity, chitin binding and metabolism, fatty acid synthesis, biological adhesion, and muscle fibers. The adult female to larvae contrasts highlighted terms associated with in chitin metabolism and extracellular space, while terms upregulated only in the larvae additionally highlighted gene ontology terms involved in protein digestion and sugar metabolism. The contrast of the adult male and female N. riversi showed an enrichment of terms upregulated in males involving sperm production, such as the axonemal dynein complex assembly and associated terms involving the cytoskeleton and cell motility. We examined the contrast between female and male adult beetles for differential exon usage (Table S9 ), and found that among the 7,864 genes with multiple exons, 217 showed significant differences (FDR < 0.01), with the GSEA of the gene ontology terms showing significant enrichment (FDR < 0.05) of 14 GO categories, seven of which are involved in sperm structure and function.