Phenotypically distinct female castes in honey bees are defined by alternative chromatin states during larval development.

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.

Effect of age, behaviour and social environment on honey bee brain plasticity.

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.

Comparing injection, feeding and topical application methods for treatment of honeybees with octopamine.

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.

Insights into DNA hydroxymethylation in the honeybee from in-depth analyses of TET dioxygenase.

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.