EneoC, a queen-specific terpene synthase, produces the queen pheromone in epidermal secretory cells of Embiratermes neotenicus queens

Queens of eusocial insects maintain their reproductive monopoly through queen pheromones. We recently identified the sesquiterpene alcohol (3R,6E)-nerolidol (RNERO) as the queen pheromone in the termite Embiratermes neotenicus and characterized a terpene synthase, EneoC, that produces RNERO in vitro. Here, we aimed to (i) determine whether EneoC is responsible for RNERO biosynthesis in vivo, (ii) analyze its expression and protein abundance patterns across castes in the context of terpenoid biosynthesis and endocrine regulation, (iii) estimate the relative investment into queen signaling based on EneoC protein abundance compared to other queen proteins, (iv) assess whether eggs actively produce RNERO, and (v) localize RNERO biosynthesis in queens. We report that EneoC is strongly upregulated in queens at both transcript and protein levels, and that it ranks among the most abundant queen proteins. Using RNA interference, we demonstrate that EneoC produces RNERO in vivo. We show that EneoC is exclusively expressed in queen epidermis all over the body surface, specifically within structures derived from mitochondria in class 1 secretory cells. We identify an enrichment of additional terpenoid biosynthetic enzymes in queens, including the farnesyl diphosphate synthase EneoB, expected to supply the substrate for EneoC. Both enzymes contain mitochondrial targeting sequences, suggesting their co-localization within modified mitochondria in class 1 cells. The absence of EneoC in eggs indicates that eggs acquire RNERO from queens rather than via endogenous biosynthesis. Taken together, our findings establish EneoC as a key element underlying the reproductive dominance of queens within the complex breeding system of E. neotenicus.