The epigenetic basis of nutrition-mediated caste identity in the honeybee

Honeybees are insect pollinators that are widely known for their honey and wax production. It is estimated that nearly 10% of the value of world food production depends on the honeybee. They live in complex societies comprising tens of thousands of individuals, most of these are female 'worker' honeybees that are unable to reproduce and instead devote their short lives to finding food in flowers (nectar or pollen) and other tasks such as nursing larvae inside the hive. In addition to these sterile female bees, the hive also contains male drones and a single female bee called the queen that has a much longer life span and can reproduce.
The queen bee can lay fertilized or unfertilized eggs. Unfertilized eggs are fed nectar/pollen and become male drones while fertilized eggs can become either queen or worker bees. Whether a fertilized egg results in a larva that will become an adult worker bee or an adult queen bee depends on the type of food the larva is fed. Larvae destined to become workers are fed a diet of pollen/nectar and those destined to become queens are fed royal jelly. This differing diet is maintained over the entire lifetime of the worker or queen bee.
The ability of an individual larva to become a drone, worker or a queen cannot be because of a different set of genes: the genome of that larva has the capacity to become all 3, it is the way those same genes are switched on or off in response to the specific diet that determines such different outcomes. Epigenetics is a dynamic set of instructions that exist 'on top' of the genetic information, that encode and direct the programme of events that leads to differential gene expression and drone, worker or queen developmental outcome. Epigenetic information can be altered by environmental factors, including diet, because of the nature of the epigenetic marks on top of the DNA. These marks or modifications are ultimately derived from metabolites and therefore the diet of the honeybee. There are two families of epigenetic modifications; one type marks the actual DNA sequence, the other type marks proteins called histones that DNA tightly wraps itself around in order to fit into the nucleus of a cell. It is these epigenetic modifications that have been shown in other biological processes to allow plasticity within the genome, in order for instance to make over 200 different cell types in the human body from one genome.
To locate these marks in the genomes of worker, queen and drone bees, we will use antibodies that can specifically recognize histone proteins that carry a particular mark or modification. The genome is chopped up into small pieces and the antibody is added and binds tightly to the modified histone and therefore purifying the antibody also results in purification of the histone and the DNA sequence wrapped around it. Sequencing the DNA associated with the histones and then matching the sequence back to the whole honeybee genome sequence using a computer, allows us to identify exactly where that modified histone was. By doing this for a variety of histone modifications at different stages of drone, worker or queen larval development and adulthood, we will be able to identify where the important differences in location are and on what genes. We will also combine this approach with sequencing all the RNA in the larvae, so we will know whether the genes are switched on or off. All this will allow us to detect changes/differences in the modifications of the histone proteins between drone, queen and worker bees and help us understand how this process works.
We will also determine which of the bee genes that add or remove these histone marks are important. We will inject larvae with small RNA molecules that will switch specific genes off. After the larva pupates and hatches we will assess the effect of that gene being switched off by how similar the resulting adult is in comparison to a normal adult worker or queen bee.

Honeybees play a fundamental role in ecosystems by maintaining genetic diversity of flowering plants. Additionally they are used for commercial pollination, accounting for 9.5% of the value of world agricultural production. They live in complex societies comprising a single reproductive queen, haploid male drones and thousands of sterile female workers. These 3 distinct but genetically indistinguishable organisms or castes are established by differentially feeding larvae either nectar/pollen or royal jelly. Therefore the honeybee genome has the capacity to encode more than one organism, with dramatically different physiologies, phenotypes and behaviours.
Studies indicate that at least one epigenetic mechanism (DNA methylation) directs nutrition-mediated caste differentiation and organismal flexibility. Given conservation of both histone sequences and epigenetic machinery between honeybees and humans, and the direct mechanistic link between the epigenetic processes of DNA methylation and histone post-translational modification (PTM), it is proposed that histone PTMs are pivotal in determining developmental trajectory in response to nutrition.
We used quantitative mass spectrometry to characterise 23 histone PTMs and identified caste-specific differences. We also show that larvae treated with a recently identified naturally occurring histone deacetylase inhibitor present in royal jelly, have higher levels of histone acetylation.
We will determine the role of chromatin in the nutritional regulation of caste differentiation using ChIP-seq and RNA-seq to examine caste-specific genome-wide distributions of histone PTMs along with transcriptome profiles. Thus enabling the identification of regions in the bee genome that respond to differential nutrition and determine developmental trajectory. To further delineate the role of chromatin, we will perform a phenotype-based RNAi screen against 50 chromatin modification enzymes and score for caste phenotype after pupation.

The aims of this proposal are consistent with BBSRC strategy, and fall within the themes of Welfare of Managed Animals, Healthy and Safe Food, Ageing Research: Lifelong Health and Well Being and Increased International Collaboration. Our work addresses the crucial issue of how one genome can specify more than one distinct organism and furthermore, how this is determined by nutrition. Moreover, our model organism is the honeybee: a critical insect pollinator. We will produce models that describe aspects of development, genome plasticity, behaviour and nutrition-genome interactions. Given the importance of honeybees for normal ecosystem function and the commercial importance in agricultural pollination, there will be a variety of beneficiaries.

Policy-makers and Governmental Organisations
It is estimated that the economic value worldwide of insect pollinators, bees mainly, is 153 billion euros. This represents 9.5% of the value of world agricultural production used for human food. Recent decline in bee populations is predicted not only to result in substantial economic losses, but also an inability to fulfil current food demand. Therefore, the importance of the honeybee to economic, public and social well-being is axiomatic and evidenced by the establishment of a government funded National Bee Unit (BeeBase), which includes the Healthy Bees Plan; a scientific advisory group whose remit "is to ensure that a sound science and evidence base underpin bee health policy and operations". Fundamental questions of the type addressed by this proposal on development, physiology and behaviour will provide important clues in the management of these important pollinators for agricultural and be central in informing public policy and policy-makers (DEFRA and FERA) on honeybee health.

Commercial beekeepers and Industry
The honeybee is used extensively for commercial pollination, requiring rational management and production of queens. Understanding the role of nutrition in developmental trajectory and identifying important regulatory genes involved in this process, will potentially allow manipulation of honeybee larvae in vitro in a high throughput manner. Moreover, the collapse of US/European bee populations (Colony Collapse Disorder) is cause of extreme concern and the proposed research has the potential to underpin investigations on altered honeybee health and physiology.

Research and professional skills of the PDRA
The programme of research presented in this proposal employs state of the art high throughput "omic" approaches in order to study genomic information in honeybees. Therefore the PDRAs will be trained in the very latest research tools. In addition, the PDRAs will have the opportunity to develop a completely new skill set (bioinformatics) and greatly enhance their future career prospects. This will add to the limited pool of scientists competent in both 'wet work' and in silico analyses.

Education of the wider public
There is considerable interest in honeybees within the general population as evidenced by numerous popular science books and initiatives such as the Urban Bees (http://www.urbanbees.co.uk/) and the BBCs "Bee Part Of It" campaign (http://www.bbc.co.uk/breathingplaces/beepartofit/). Our previously published honeybee work was well publicised within the media including articles on popular sites including BBC Science & Environment (http://www.bbc.co.uk/news/science-environment-20667948), Science Daily (http://www.sciencedaily.com/releases/2012/12/121211101942.htm) and on more specialist non-scientific apiculture sites (http://apisuk.com/Bees/2013/01/research-new-genetic-factors-in-honey-bee-decline/). Therefore, we believe the dissemination of results from this proposal will also be of significant interest to the general public. In addition, we will strive to ensure our findings reach the wider community and as part of our "Pathways to Impact" we will host a public lecture.