Behavioral plasticity in honey bees is associated with differences in brain microRNA transcriptome

Abstract

Small, non-coding microRNAs (miRNAs) have been implicated in many biological processes, including the development of the nervous system. However, the roles of miRNAs in natural behavioral and neuronal plasticity are not well understood. To help address this we characterized the microRNA transcriptome in the adult worker honey bee head and investigated whether changes in microRNA expression levels in the brain are associated with division of labor among honey bees, a well-established model for socially regulated behavior. We determined that several miRNAs were downregulated in bees that specialize on brood care (nurses) relative to foragers. Additional experiments showed that this downregulation is dependent upon social context; it only occurred when nurse bees were in colonies that also contained foragers. Analyses of conservation patterns of brain-expressed miRNAs across Hymenoptera suggest a role for certain miRNAs in the evolution of the Aculeata, which includes all the eusocial hymenopteran species. Our results support the intriguing hypothesis that miRNAs are important regulators of social behavior at both developmental and evolutionary time scales.

Figure 1. Conservation of miRNAs across Insecta

(a) All identified miRNAs in the honey bee head were used as probes in BLAST searches of various representative insect species, as well as to Caenorhabditis elegans, and Mus musculus, which represented non-insect outgroups. Green highlighting indicates the miRNA is conserved. Yellow highlighting indicates similarity with a single, mid-sequence base changed. Red highlighting indicates a miRNA is not present. (b) Conservation of miRNAs that appeared honey bee-specific in eusocial as well as non-social Hymenoptera. Color coding as in (a). Note that no miRNAs that appeared honey bee-specific in (a) were conserved in Nasonia longicornis, a nonsocial wasp.

Figure 2. Relative brain expression levels of miRNAs as a function of behavioral maturation

Bees were collected from either typical (T), or young (Y) or old (O) single-cohort colonies as either nurses (N) or foragers (F). Bees from young single-cohort colonies were 7–10-days old, while bees from old single-cohort colonies were >3-weeks old. (a) Northern blot expression for five miRNAs, along with the control U6snRNA and total RNA expression. (b) Quantified miRNA expression (total area of RNA band) normalized to U6snRNA.

Figure 3. Relative brain mRNA expression levels of miRNA processing proteins as a function of behavioral maturation

Bees were the same as those depicted in Fig. 4 (independent samples t test for each colony). df = 6 for typical, young and old colonies). N = 4 per group. * indicates P < 0.05.

Figure 4. The ame-miR-2796 and PLC-epsilon loci

(a) Structure of ame-miR-2796 pre-miRNA with hairpin loop, mature sequence (red box), and miRNA* (blue box). (b) Alignment of the genomic region around ame-miR-2796 in five insect species. (c) Relative expression of ame-miR-2796 in honey bee brain, gland, and all other head tissues left in the carcass (Other). (d) The genomic architecture of the PLC-epsilon gene in Apis and Tribolium. Green boxes are exons and the red box (marked by arrow) shows the location of ame-miR-2796. (e) Predicted targeting sites for ame-miR-2796 in the PLC-epsilon transcripts of Apis and Tribolium. No targeting sites were identified in the PLC-epsilon 3′ UTR. Minimum free energy of hybridization is shown below each duplex prediction. (f) Relative expression of PLC-epsilon in honey bee brain, gland, and all other head tissue (Other). (g) Protein tree of PLC-epsilon. (h) Relative expression of PLC-epsilon in typical, young, and old honeybee colonies. (Independent samples t test. df = 6 for typical, young, and old colonies). N = 4 per group. * indicates P < 0.05.