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In honeybee (Apis mellifera), odorant receptors (Ors) play a crucial role in special recognition of sex pheromones in honeybee mating activities. Four candidate sex pheromone Ors (AmOr10, AmOr11, AmOr18 and AmOr170) have been identified and found to be preferentially expressed in drone antennae. However, few studies have investigated the regulation of these four drone Ors on drone mating behaviour. This study characterised the expression patterns of these Ors across different sexual developmental stages (immature and sexually mature) and different physiological statuses (flying and crawling), using both the antennae and brains of drones. qRT-PCR results indicated that the expression of four Ors were not significantly different in drone antennae between flying and crawling statuses at immature stage. However, all four Ors expression levels in brains of flying drones were significantly higher than those of crawling drones at mature stage. Moreover, only the expression level of Or170 was significantly higher in antennae of mature flying drones than crawling ones. Therefore, this study demonstrated a link between four candidate sex pheromone Ors transcriptional expression in the brains of honeybee drones and behaviour associated with sexual maturity and mating flight. In addition, Or170 might be involved in the maturation of honeybee drones' olfactory system, and in the organisation of odour-mediated mating behaviours.

Honeybees (Apis mellifera) have haplodiploid sex determination: males develop from unfertilized eggs and females develop from fertilized ones. The differences in larval food also determine the development of females. Here we compared the total somatic gene expression profiles of 2-day and 4-day-old drone, queen and worker larvae by RNA-Seq. The results from a co-expression network analysis on all expressed genes showed that 2-day-old drone and worker larvae were closer in gene expression profiles than 2-day-old queen larvae. This indicated that for young larvae (2-day-old) environmental factors such as larval diet have a greater effect on gene expression profiles than ploidy or sex determination. Drones had the most distinct gene expression profiles at the 4-day larval stage, suggesting that haploidy, or sex dramatically affects the gene expression of honeybee larvae. Drone larvae showed fewer differences in gene expression profiles at the 2-day and 4-day time points than the worker and queen larval comparisons (598 against 1190 and 1181), suggesting a different pattern of gene expression regulation during the larval development of haploid males compared to diploid females. This study indicates that early in development the queen caste has the most distinct gene expression profile, perhaps reflecting the very rapid growth and morphological specialization of this caste compared to workers and drones. Later in development the haploid male drones have the most distinct gene expression profile, perhaps reflecting the influence of ploidy or sex determination on gene expression.

Specialised castes are considered a key reason for the evolutionary and ecological success of the social insect lifestyle. The most essential caste distinction is between the fertile queen and the sterile workers. Honey bee (Apis mellifera) workers and queens are not genetically distinct, rather these different phenotypes are the result of epigenetically regulated divergent developmental pathways. This is an important phenomenon in understanding the evolution of social insect societies. Here we studied the genomic regulation of the worker and queen developmental pathways, and the robustness of the pathways by transplanting eggs or young larvae to queen cells. Queens could be successfully reared from worker larvae transplanted up to 3 days age, but queens reared from older worker larvae had decreased queen body size and weight compared to queens from transplanted eggs. Gene expression analysis showed that queens raised from worker larvae differed from queens raised from eggs in the expression of genes involved in the immune system, caste differentiation, body development and longevity. DNA methylation levels were also higher in 3-day queen larvae raised from worker larvae compared to that raised from transplanted eggs identifying a possible mechanism stabilizing the two developmental paths. We propose that environmental (nutrition and space) changes induced by the commercial rearing practice result in a suboptimal queen phenotype via epigenetic processes, which may potentially contribute to the evolution of queen-worker dimorphism. This also has potentially contributed to the global increase in honeybee colony failure rates. This article is protected by copyright. All rights reserved.

Honey bee foragers must supply their colony with a balance of pollen and nectar to sustain optimal colony development. Inter-individual behavioural variability among foragers is observed in terms of activity levels and nectar vs. pollen collection, however the causes of such variation are still open questions. Here we explored the relationship between foraging activity and foraging performance in honey bees (Apis mellifera) by using an automated behaviour monitoring system to record mass on departing the hive, trip duration, presence of pollen on the hind legs and mass upon return to the hive, during the lifelong foraging career of individual bees. In our colonies, only a subset of foragers collected pollen, and no bee exclusively foraged for pollen. A minority of very active bees (19% of the foragers) performed 50% of the colony’s total foraging trips, contributing to both pollen and nectar collection. Foraging performance (amount and rate of food collection) depended on bees’ individual experience (amount of foraging trips completed). We argue that this reveals an important vulnerability for these social bees since environmental stressors that alter the activity and reduce the lifespan of foragers may prevent bees ever achieving maximal performance, thereby seriously compromising the effectiveness of the colony foraging force.

Queen-worker caste dimorphism is a typical trait for honeybees (Apis mellifera). We previously showed a maternal effect on caste differentiation and queen development, where queens emerged from queen-cell eggs (QE) had higher quality than queens developed from worker cell eggs (WE). In this study, newly-emerged queens were reared from QE, WE, and 2-day worker larvae (2L). The thorax size and DNA methylation levels of queens were measured. We found that queens emerging from QE had significantly larger thorax length and width than WE and 2L. Epigenetic analysis showed that QE/2L comparison had the most different methylated genes (DMGs, 612) followed by WE/2L (473), and QE/WE (371). Interestingly, a great number of DMGs (42) were in genes belonging to mTOR, MAPK, Wnt, Notch, Hedgehog, FoxO, and Hippo signaling pathways that are involved in regulating caste differentiation, reproduction and longevity. This study proved that honeybee maternal effect causes epigenetic alteration regulating caste differentiation and queen development.

Whether a female honey bee (Apis mellifera) develops into a worker or a queen depends on her nutrition during development, which changes the epigenome to alter the developmental trajectory. Beekeepers typically exploit this developmental plasticity to produce queen bee by transplanting worker larvae into queen cells to be reared as queens, thus redirecting a worker developmental pathway to a queen developmental pathway. We studied the consequences of this manipulation for the queen phenotype and methylome over four generations. Queens reared from worker larvae consistently had fewer ovarioles than queens reared from eggs. Over four generations the methylomes of lines of queens reared from eggs and worker larvae diverged, accumulating increasing differences in exons of genes related to caste differentiation, growth and immunity. We discuss the consequences of these cryptic changes to the honey bee epigenome for the health and viability of honey bee stocks.

In a honey bee colony, worker bees rear a new queen by providing her with a larger cell in which to develop and a large amount of richer food (royal jelly). Royal jelly and worker jelly (fed to developing worker larvae) differ in terms of sugar, vitamin, protein and nucleotide composition. Here we examined whether workers attending queen and worker larvae are separate specialized sub-castes of the nurse bees. We collected nurse bees attending queen larvae (AQL) and worker larvae (AWL) and compared gene expression profiles of hypopharyngeal gland tissues, using Solexa/Illumina digital gene expression tag profiling (DGE). Significant differences in gene expression were found that included a disproportionate number of genes involved in glandular secretion and royal jelly synthesis. However behavioral observations showed that these were not two entirely distinct populations. Nurse workers were observed attending both worker larvae and queen larvae, and there was no evidence of a specialized group of workers that preferentially or exclusively attended developing queens. Nevertheless, AQL attended larvae more frequently compared to AWL, suggesting that nurses sampled attending queen larvae may have been the most active nurses. This study serves as another example of the relationship between differences in gene expression and behavioral specialisation in honey bees.

Cooperative brood care is diagnostic of animal societies. This is particularly true for the advanced social insects and the honey bee is the best understood of the insect societies. A brood pheromone signaling the presence of larvae in a bee colony has been characterised and well studied, but here we explored whether honey bee larvae actively signal their food needs pheromonally to workers. We show that starving honey bee larvae signal to workers via increased production of the volatile pheromone E-β-ocimene. Analysis of volatile pheromones produced by food-deprived and fed larvae with gas chromatography-mass spectrometry showed that starving larvae produced more E-β-ocimene. Behavioural analyses showed that adding E-β-ocimene to empty cells increased the number of worker visits to those cells and similarly adding E-β-ocimene to larvae increased worker visitation rate to the larvae. RNA-seq and qRT-PCR analysis identified 3 genes in the E-β-ocimene biosynthetic pathway that were upregulated in larvae following 30 minutes of starvation and these genes also upregulated in 2-day old larvae compared to 4-day old larvae (2-day old larvae produce the most E-β-ocimene). This identifies a pheromonal mechanism by which brood can beg for food from workers to influence the allocation of resources within the colony.