ABSTRACT: Queen primer pheromones regulate reproduction and social organization in termites and are thought to function as honest signals of queen fertility. In Embiratermes neotenicus, the sesquiterpene (3R,6E)-nerolidol (RNERO) serves as a queen primer pheromone. We previously identified EneoC, a queen-specific terpene synthase, as a candidate enzyme for RNERO biosynthesis. Here, we provide in vivo functional evidence that EneoC is responsible for QPP production and characterize its spatial organization. Using integrated genome and transcriptome resources, RNA interference, proteomics, immunofluorescence, and electron microscopy, we show that EneoC is strongly upregulated in mature neotenic queens at both transcript and protein levels and represents a highly abundant protein, indicating a substantial physiological investment into pheromone production. Consistent with this, RNERO signaling likely constitutes an energetically costly and thus honest signal of reproductive status. Localization analyses reveal that RNERO biosynthesis occurs in the epidermis, where cells are extensively modified into secretory class I and class III cells. Within class I cells, immunogold labeling localizes EneoC to vesicular structures consistent with mitochondria. Notably, the upstream enzyme EneoB (farnesyl diphosphate synthase) is also abundantly localized in these mitochondria, suggesting co-localization of the final biosynthetic steps and an efficient metabolic organization. In contrast, EneoC is absent from eggs, although RNERO is present, indicating queen marking rather than embryonic synthesis. Together, our findings establish EneoC as the key enzyme underlying queen pheromone production and show that this process is confined to specialized epidermal secretory cells, with pathway organization and high protein investment consistent with the evolution of honest chemical signaling.
Karel Harant 