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

蜜蜂是昆虫传粉者，以产蜜和产蜡而闻名。据估计，世界粮食产量的近10%依赖于蜜蜂。它们生活在由数以万计的个体组成的复杂社会中，其中大多数是雌性工蜂，它们无法繁殖，而是将短暂的生命投入到花朵(花蜜或花粉)中寻找食物和其他任务，如在蜂箱内哺育幼虫。除了这些不育的雌性蜜蜂外，该蜂箱还包括雄蜂和一只名为女王的雌性蜜蜂，它的寿命要长得多，可以繁殖。蜂王可以产受精卵或未受精卵。未受精卵被喂食花蜜/花粉，成为雄蜂，而受精卵可以成为蜂王或工蜂。受精卵产生的幼虫是否会变成成年工蜂或成年蜂王，取决于幼虫被喂养的食物类型。注定要成为工蚁的幼虫被喂食花粉/花蜜，而注定要成为蜂王的幼虫被喂食蜂王浆。这种不同的饮食在工蜂或蜂王的一生中都保持不变。一只幼虫之所以能够成为雄蜂、工蜂或蜂王，不可能是因为一组不同的基因：该幼虫的基因组有能力变成全3，正是这些相同的基因被打开或关闭的方式，对特定的饮食做出了反应，决定了这种不同的结果。表观遗传学是存在于遗传信息之上的一套动态指令，这些指令编码并指导导致差异基因表达和雄蜂、工蜂或蜂后发育结果的一系列事件。表观遗传信息可以被环境因素改变，包括饮食，因为DNA顶部的表观遗传标记的性质。这些标记或修饰最终来自代谢产物，因此来自蜜蜂的饮食。有两种表观遗传修饰；一种标记实际的DNA序列，另一种标记称为组蛋白的蛋白质，DNA紧密包裹自己以适应细胞核。正是这些表观遗传修饰在其他生物过程中被证明允许基因组内的可塑性，例如，为了在人体内从一个基因组产生200多种不同的细胞类型。为了在工蜂、蜂后和雄蜂的基因组中定位这些标记，我们将使用能够特异性识别带有特定标记或修饰的组蛋白的抗体。基因组被切成小块，抗体被加入并紧密结合到修饰的组蛋白上，因此纯化抗体也会导致组蛋白和包裹在它周围的DNA序列的纯化。对与组蛋白相关的DNA进行测序，然后使用计算机将该序列与整个蜜蜂基因组序列进行匹配，这使我们能够准确地识别修改后的组蛋白在哪里。通过对雄蜂、工蜂或蜂王幼虫发育和成年期不同阶段的各种组蛋白修饰进行这一操作，我们将能够确定位置上的重要差异在哪里以及在哪些基因上。我们还将把这种方法与对幼虫中的所有RNA进行测序相结合，这样我们就可以知道基因是打开还是关闭。所有这些都将使我们能够检测雄蜂、蜂王和工蜂之间组蛋白修饰的变化/差异，并帮助我们了解这一过程是如何工作的。我们还将确定添加或移除这些组蛋白标记的蜜蜂基因中哪些是重要的。我们将给幼虫注射小RNA分子，这些小分子将关闭特定的基因。在幼虫化蛹和孵化后，我们将通过所产生的成虫与正常的成年工蜂或蜂后的相似性来评估该基因被关闭的效果。

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.