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.