Fertile queens and sterile workers are alternative forms of the adult female honeybee that develop from genetically identical larvae following differential feeding with royal jelly. We show that silencing the expression of DNA methyltransferase Dnmt3, a key driver of epigenetic global reprogramming, in newly hatched larvae led to a royal jelly-like effect on the larval developmental trajectory; the majority of Dnmt3 small interfering RNA-treated individuals emerged as queens with fully developed ovaries. Our results suggest that DNA methylation in Apis is used for storing epigenetic information, that the use of that information can be differentially altered by nutritional input, and that the flexibility of epigenetic modifications underpins, profound shifts in developmental fates, with massive implications for reproductive and behavioral status.

Small chemosensory proteins (CSPs) belong to a conserved, but poorly understood, protein family found in insects and other arthropods. They exhibit both broad and restricted expression patterns during development. In this paper, we used a combination of genome annotation, transcriptional profiling and RNA interference to unravel the functional significance of a honeybee gene (csp5) belonging to the CSP family. We show that csp5 expression resembles the maternal-zygotic pattern that is characterized by the initiation of transcription in the ovary and the replacement of maternal mRNA with embryonic mRNA. Blocking the embryonic expression of csp5 with double-stranded RNA causes abnormalities in all body parts where csp5 is highly expressed. The treated embryos show a "diffuse", often grotesque morphology, and the head skeleton appears to be severely affected. They are 'unable-to-hatch' and cannot progress to the larval stages. Our findings reveal a novel, essential role for this gene family and suggest that csp5 (unable-to-hatch) is an ectodermal gene involved in embryonic integument formation. Our study confirms the utility of an RNAi approach to functional characterization of novel developmental genes uncovered by the honeybee genome project and provides a starting point for further studies on embryonic integument formation in this insect.

We examined the effects of behaviour, age and social environment on mushroom body volume in adult bees. The mushroom bodies are regions of the central brain important for sensory integration and learning. Their volume was influenced by behaviour throughout life: always larger in forager bees than age-matched nurse bees, even in old bees up to 93 days of age as adults. Mushroom body development was influenced by the social environment in the first 8 days of adult life, with different environments having markedly different effects on mushroom body size. Compared to hive-reared bees, isolation slowed mushroom body growth, but bees reared in isolation confined with a single dead bee showed a dramatic increase in mushroom body volume comparable to that seen in active foragers. Despite their precocious mushroom body development, these bees did not show improved performance in an olfactory learning test. Since simple environmental manipulations can both accelerate and delay mushroom body growth in young bees, and since mushroom body volume is sensitive to behaviour throughout life, the honey bee has great potential as a model for exploring the interactions between environment, behaviour and brain structure.

Entomologists have used a range of techniques to treat insects with neuroactive compounds, but it is not always clear whether different treatment methods are equally effective in delivering a compound to a target organ. Here, we used five different techniques to treat honeybees with 3H-octopamine (3H-OA), and analysed the distribution of the 3H radiolabelled compound within different tissues and how it changed over time. All treatment methods, including injection of the median ocellus, resulted in 3H-OA detection in all parts of the honeybee. Injection through the median ocellus was the most effective method for delivering 3H-OA to the brain. Topical application of 3H-OA dissolved in dimethylformamide (dMF) to the thorax was as effective as thoracic injections of 3H-OA in delivering 3H-OA to the brain, but topical applications to the abdomen were less so. Most of the 3H-OA applied topically remained associated with the cuticle and the tissues of the body segment to which it had been applied. For all treatment methods, 3H-OA was rapidly lost from the brain and head capsule, and accumulated in the abdomen. Our findings demonstrate the value of thoracic topical treatment with compounds dissolved in dMF as an effective non-invasive method for short-term, systemic pharmacological treatments.

The capacity of the honey bee to produce three phenotypically distinct organisms (two female castes; queens and sterile workers, and haploid male drones) from one genotype represents one of the most remarkable examples of developmental plasticity in any phylum. The queen-worker morphological and reproductive divide is environmentally controlled during post-embryonic development by differential feeding. Previous studies implicated metabolic flux acting via epigenetic regulation, in particular DNA methylation and microRNAs, in establishing distinct patterns of gene expression underlying caste-specific developmental trajectories. We produce the first genome-wide maps of chromatin structure in the honey bee at a key larval stage in which developmental canalization into queen or worker is virtually irreversible. We find extensive genome-wide differences in H3K4me3, H3K27ac, and H3K36me3, many of which correlate with caste-specific transcription. Furthermore, we identify H3K27ac as a key chromatin modification, with caste-specific regions of intronic H3K27ac directing the worker caste. These regions may harbor the first examples of caste-specific enhancer elements in the honey bee. Our results demonstrate a key role for chromatin modifications in the establishment and maintenance of caste-specific transcriptional programs in the honey bee. We show that at 96 h of larval growth, the queen-specific chromatin pattern is already established, whereas the worker determination is not, thus providing experimental support for the perceived timing of this critical point in developmental heterochrony in two types of honey bee females. In a broader context, our study provides novel data on environmentally regulated organismal plasticity and the molecular foundation of the evolutionary origins of eusociality.

In mammals, a family of TET enzymes producing oxidized forms of 5-methylcytosine (5mC) plays an important role in modulating DNA demethylation dynamics. In contrast, nothing is known about the function of a single TET orthologue present in invertebrates. Here, we show that the honeybee TET (AmTET) catalytic domain has dioxygenase activity and converts 5mC to 5-hydroxymethylcytosine (5hmC) in a HEK293T cell assay. In vivo, the levels of 5hmC are condition-dependent and relatively low, but in testes and ovaries 5hmC is present at approximately 7–10% of the total level of 5mC, which is comparable to that reported for certain mammalian cells types. AmTET is alternatively spliced and highly expressed throughout development and in adult tissues with the highest expression found in adult brains. Our findings reveal an additional level of flexible genomic modifications in the honeybee that may be important for the selection of multiple pathways controlling contrasting phenotypic outcomes in this species. In a broader context, our study extends the current, mammalian-centred attention to TET-driven DNA hydroxymethylation to an easily manageable organism with attractive and unique biology.

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Although gene duplication is seen as the main path to evolution of new functions, molecular mechanisms by which selection favours the gain versus loss of newly duplicated genes and minimizes the fixation of pseudo-genes are not well understood. Here, we investigate in detail a duplicate honeybee gene obp11 belonging to a fast evolving insect gene family encoding odorant binding proteins (OBPs). We report that obp11 is expressed only in female bees in rare antennal sensilla basiconica in contrast to its tandem partner obp10 that is expressed in the brain in both females and males (drones). Unlike all other obp genes in the honeybee, obp11 is methylated suggesting that functional diversification of obp11 and obp10 may have been driven by an epigenetic mechanism. We also show that increased methylation in drones near one donor splice site that correlates with higher abundance of a transcript variant encoding a truncated OBP11 protein is one way of controlling its contrasting expression. Our data suggest that like in mammals and plants, DNA methylation in insects may contribute to functional diversification of proteins produced from duplicated genes, in particular to their subfunctionalization by generating complementary patterns of expression.