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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., (© 2021 Royal Entomological Society.)

Epitranscriptomics is an emerging field of investigation dedicated to the study of post-transcriptional RNA modifications. RNA methylations regulate RNA metabolism and processing, including changes in response to environmental cues. Although RNA modifications are conserved from bacteria to eukaryotes, there is little evidence of an epitranscriptomic pathway in insects. Here we identified genes related to RNA m 6 A (N6-methyladenine) and m 5 C (5-methylcytosine) methylation machinery in seven bee genomes (Apis mellifera, Melipona quadrifasciata, Frieseomelitta varia, Eufriesea mexicana, Bombus terrestris, Megachile rotundata and Dufourea novaeangliae). In A. mellifera, we validated the expression of methyltransferase genes and found that the global levels of m 6 A and m 5 C measured in the fat body and brain of adult workers differ significantly. Also, m 6 A levels were differed significantly mainly between the fourth larval instar of queens and workers. Moreover, we found a conserved m 5 C site in the honeybee 28S rRNA. Taken together, we confirm the existence of epitranscriptomic machinery acting in bees and open avenues for future investigations on RNA epigenetics in a wide spectrum of hymenopteran species., (© 2021 Royal Entomological Society.)

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 ATPsynβ). 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., (© 2020 Royal Entomological Society.)

Ftz-f1 is an orphan member of the nuclear hormone receptor superfamily. A 20-hydroxyecdysone pulse allows ftz-f1 gene expression, which then regulates the activity of downstream genes involved in major developmental progression events. In honeybees, the expression of genes like vitellogenin (vg), prophenoloxidase and juvenile hormone-esterase during late pharate-adult development is known to be hormonally controlled in both queens and workers by increasing juvenile hormone (JH) titres in the presence of declining levels of ecdysteroids. Since Ftz-f1 is known for mediating intracellular JH signalling, we hypothesized that ftz-f1 could mediate JH action during the pharate-adult development of honeybees, thus controlling the expression of these genes. Here, we show that ftz-f1 has caste-specific transcription profiles during this developmental period, with a peak coinciding with the increase in JH titre, and that its expression is upregulated by JH and downregulated by ecdysteroids. RNAi-mediated knock down of ftz-f1 showed that the expression of genes essential for adult development (e.g. vg and cuticular genes) depends on ftz-f1 expression. Finally, a double-repressor hypothesis-inspired vg gene knock-down experiment suggests the existence of a positive molecular loop between JH, ftz-f1 and vg., (© 2018 The Royal Entomological Society.)

Like all other insects, two key signalling pathways [Toll and immune deficiency (Imd)] regulate the induction of honey bee immune effectors that target microbial pathogens. Amongst these effectors are antimicrobial peptides (AMPs) that are presumed to be produced by the nuclear factors kappa B (NF-κB) Dorsal and Relish from the Toll and Imd pathways, respectively. Using in silico analysis, we previously proposed that the honey bee AMP defensin-1 was regulated by the Toll pathway, whereas hymenoptaecin was regulated by Imd and abaecin by both the Toll and Imd pathways. Here we use an RNA interference (RNAi) assay to determine the role of Dorsal in regulating abaecin and defensin-1. Honey bees have two dorsal genes (dorsal-1 and dorsal-2) and two splicing isoforms of dorsal-1 (dorsal-1A and dorsal-1B). Accordingly, we used both single and multiple (double or triple) isoform knockdown strategies to clarify the roles of dorsal proteins and their isoforms. Down-regulation of defensin-1 was observed for dorsal-1A and dorsal-2 knockdowns, but abaecin expression was not affected by dorsal RNAi. We conclude that defensin-1 is regulated by Dorsal (Toll pathway)., (© 2018 The Royal Entomological Society.)