In winter, honey bees undergo a transition to a diutinus state, during which time broodrearing declines or stops entirely, and worker bees live for up to 20 weeks. The mechanism, causes, and geographic prevalence of this transition are unknown, and can make managing honey bees in certain regions challenging. We hypothesized that the transition to overwintering is regulated by the forager pheromone, ethyl oleate, when forager bees are relegated to the hive for longer periods of time during poor weather conditions. We exposed bees of different ages and tasks to ethyl oleate in cage conditions and measured accepted markers of overwintering: hypopharyngeal gland size and protein content, fat body weight, longevity, pollen consumption, and vitellogenin gene expression. We also investigated a possible mechanism for the increased longevity seen in diutinus bees, by looking at gene expression of an immune gene, defensin. We found ethyl oleate only had a significant effect on fat body mass, and the ratio of fat body mass to hypopharyngeal gland protein synthesis in nurse bees. This indicates that ethyl oleate may affect the efficiency of metabolism of consumed protein into fat body stores, and an increased metabolic shift from hypopharyngeal gland protein synthesis to fat body production, allowing young bees to prepare for suboptimal conditions. While these findings indicate that ethyl oleate is likely not the sole cause of a transition to a diutinus state, it is possible that when concomitant with other factors such as gradual decline in brood pheromone, pollen dearth, cold temperatures, and photoperiod, ethyl oleate may contribute to the transition to overwintering, a notion worth investigating further.

When an individual honey bee (Apis mellifera) changes in a hive, its’ glandular output also changes. For example, a worker’s mandibular glands (Mdg) typically produce larval food and other pheromones but in queen-less hives it will more closely resemble a queen’s. As the biosynthesis of insect pheromones occurs in specialized tissues and involves regulatory modifications to conserved metabolic pathways, we hypothesized microRNAs (miRs), short (~22 nucleotide) RNA negative transcriptional modifiers and known regulators of metabolic pathways, would have significant roles in regulating pheromone production in honey bee Mdgs. We further hypothesized queen-less workers would have similar miR expression profiles as queens. Small RNA-Seq of the Mdg identified robust caste-specific (queen and worker) miRs profiles as well as a high number of previously undescribed miRs. Worker-expressed genes in the Mdg were enriched for predicted miR response elements (MREs) of miRs coherently up-regulated in both queens and queen-less workers with some ovary activation. In addition, novel worker-expressed genes were enriched for MREs of novel queen-expressed miRs. Predicted miR-protein-protein interaction networks identified potential regulatory hubs and detailed regulatory roles for individual miRs expressed according to caste and mating status. We also found putative worker pheromone biosynthesis genes expressed in queens are enriched for MREs of two worker-expressed miRs; ame-miR-31a-5p and ame-miR-92b-3p. Interestingly the two fatty acid synthase genes that may be expressed according to caste are predicted to contain the highest number of MREs for anti-correlated miRs of all putative pheromone biosynthesis genes. MREs for ame-miR-375-3p that are highly up-regulated in queens and queen-less workers relative to queen-right workers, are enriched in genes down-regulated in queen-less workers relative to queen-right workers. Mandibular gland RNA-sequencing of queen-less worker bees fed an inhibitor for ame-miR-375-3p identified multiple genes involved in metabolic processes, autophagy, and stress responses reverting to the expression profile of a queen-right worker.