miRNA-34 and miRNA-210 target hexamerin genes enhancing their differential expression during early brain development of honeybee (Apis mellifera) castes.

During the honeybee larval stage, queens develop larger brains than workers, with morphological differentiation appearing at the fourth larval phase (L4), just after a boost in nutritional difference both prospective females experience. The molecular promoters of this caste-specific brain development are already ongoing in previous larval phases. Transcriptomic analyses revealed a set of differentially expressed genes in the L3 brains of queens and workers, which represents the early molecular response to differential feeding females receive during larval development. Three genes of this set, hex70b, hex70c and hex110, are more highly transcribed in the brain of workers than in queens. The microRNAs miR-34, miR-210 and miR-317 are in higher levels in the queens' brain at the same phase of larval development. Here, we tested the hypothesis that the brain of workers expresses higher levels of hexamerins than that of queens during key phases of larval development and that this differential hexamerin genes expression is further enhanced by the repressing activity of miR-34, miR-210 and miR-317. Our transcriptional analyses showed that hex70b, hex70c and hex110 genes are differentially expressed in the brain of L3 and L4 larval phases of honeybee queens and workers. In silico reconstructed miRNA-mRNA interaction networks were validated using luciferase assays, which showed miR-34 and miR-210 negatively regulate hex70b and hex110 genes by directly and redundantly binding their 3'UTR (untranslated region) sequences. Taken together, our results suggest that miR-34 and miR-210 act together promoting differential brain development in honeybee castes by downregulating the expression of the putative antineurogenic hexamerin genes hex70b and hex110.

Caste-specific gene expression underlying the differential adult brain development in the honeybee Apis mellifera.

Apis mellifera adult workers feature more developed key brain regions than queens, which allows them to cope with the broad range of duties they need to perform in a colony. However, at the end of larval development, the brain of queens is largely more developed than that of workers. Major morphogenetic changes take place after metamorphosis that shift caste-specific brain development. Here, we tested the hypothesis that this phenomenon is hormonally governed and involves differential gene expression. Our molecular screening approach revealed a set of differentially expressed genes in Pp (first pharate-adult phase) brains between castes mainly coding for tissue remodelling and energy-converting proteins (e.g. hex 70a and ATPsynß). An in-depth qPCR analysis of the transcriptional behaviour during pupal and pharate-adult developmental stage in both castes and in response to artificially augmented hormone titres of 18 genes/variants revealed that: i. subtle differences in hormone titres between castes might be responsible for the differential expression of the EcR and insulin/insulin-like signalling (IIS) pathway genes; ii. the morphogenetic activity of the IIS in brain development must be mediated by ILP-2, iii. which together with the tum, mnb and caspase system, can constitute the molecular effectors of the caste-specific opposing brain developmental trajectories.