Evolution and molecular basis of caste differentiation in bees

生物学中的一个基本主题是关于基因的开关（差异表达）如何创造形态和行为多样性。“多型性”（当不同的成年变体来自相同的基因组）提供了解决差异基因表达如何在整个生物体水平上创造多样性的系统。多型现象的一个主要例子发生在蚂蚁、蜜蜂和黄蜂中，其中雌性幼虫发育成两个等级之一，即蚁后或工蜂。人们已经知道，种姓之间在大小、形状、生殖和行为方面的巨大差异源于不同的基因表达。因此，种姓发展的过程（种姓分化）使我们能够研究差异基因表达如何产生多型性。然而，我们对种姓分化的遗传基础的理解还处于早期阶段。本研究以熊蜂（Bombus）为研究对象，对影响蜂王-工蜂等级分化的基因及其进化背景和microRNA（miRNAs，在基因表达调控中起重要作用的小分子RNA）的作用进行了研究。大黄蜂是这方面的优秀典范。首先，物种在幼虫种姓决定的关键阶段（分化开始时）的数量不同，这种变化的进化历史是已知的。1种（B. terrestris）在雌性幼虫中有两个关键阶段（早期和晚期），早期关键阶段是次要的（进化衍生的）;另一个（B. hypnorum）具有一个这样的阶段（仅为晚龄幼虫）;和“布谷鸟”熊蜂，如B。Vestalis没有关键阶段，因为它们已经失去了工人种姓，所有的女性都是在其他熊蜂物种的巢穴中繁殖的女王。第二，我们发现一种miRNA，miR-6001- 5 p，在以蜂王为目标的晚龄B中过表达。这些数据使我们能够为熊蜂中种姓相关基因和miR-6001- 5 p的预期差异表达模式建立一个预测框架。具体地说，如果关键阶段在进化中通过相同基因的调控而被保守，而新的关键阶段通过额外的调控途径获得，则B。terrestris和B. hypnorum应该共享晚龄幼虫之间的差异基因表达的共同模式，但不是在早期幼虫。同样，在这两个物种中，特定于工蚁幼虫的基因表达模式在杜鹃物种B中应该是不存在的。维斯塔利斯此外，如果miR-6001- 5 p的作用在进化上是保守的，那么miR-6001- 5 p及其靶基因的相似表达模式应该发生在所有三种熊蜂的蜂王晚期幼虫中。通过使用我们的表达数据来预测miR-6001- 5 p的靶点，并通过进行测定来验证这些靶点。拟议的工作将提供一个全面的了解的进化和遗传基础的变化，在一些关键阶段的种姓决定真社会性昆虫，并将阐明的作用，在种姓分化的miRNA。除了解决基因调控多型性的基本课题外，这项工作也很新颖，因为以前没有研究利用过类似的预测框架。这是及时的，因为通过下一代测序进行的基因分析现在提供了所需的工具，我们现有的数据创造了一个令人兴奋的机会，可以在理解miRNAs如何影响种姓分化方面取得快速进展。最后，这项工作符合BBSRC的战略重点，因为它可以揭示操纵工人生产的手段，以最大限度地提高商业饲养的大黄蜂蜂群提供的授粉服务园艺。

A fundamental topic in biology concerns how the switching on and off (differential expression) of genes creates morphological and behavioural diversity. 'Polyphenism' (when different adult morphs arise from the same genome) provides systems in which to address how differential gene expression creates diversity at the level of whole organisms. A major example of polyphenism occurs in the ants, bees and wasps, in which female larvae develop into one of two castes, queens or workers. It is already known that the huge differences between the castes in size, shape, reproduction and behaviour stem from differential gene expression. Hence the process by which castes develop (caste differentiation) allows us to investigate how differential gene expression generates polyphenism. However, our understanding of the genetic basis of caste differentiation is in its early stages. The genes involved, their evolutionary background and the role of microRNAs (miRNAs, small RNA molecules important in regulating gene expression) are all poorly known.In this project we will use bumble bees (Bombus) to elucidate the evolution and regulation of the genes influencing queen-worker caste differentiation. Bumble bees represent excellent models for this purpose. First, species vary in the number of critical stages of larval caste determination (when differentiation is initiated) and the evolutionary history of this variation is known. One species (B. terrestris) has two critical stages in female larvae (early- and late-instar), with the early critical stage being secondary (evolutionarily derived); another (B. hypnorum) has one such stage (late-instar larvae only); and 'cuckoo' bumble bees such as B. vestalis have no critical stages, as they have secondarily lost the worker caste and all females are queens that reproduce in nests of other Bombus species. Second, we have discovered that a miRNA, miR-6001-5p, is overexpressed in queen-destined late-instar B. terrestris larvae, suggesting that it regulates genes influencing caste.These data allow us to create a predictive framework for expected patterns of differential expression in caste-associated genes and miR-6001-5p in Bombus. Specifically, if the critical stages are conserved in evolution via regulation of the same genes, and new critical stages are gained via additional regulatory pathways, B. terrestris and B. hypnorum should share common patterns of differential gene expression among late- but not among early-instar larvae. Likewise, patterns of gene expression specific to worker-destined larvae in these two species should be absent in the cuckoo species B. vestalis. Furthermore, if the role of miR-6001-5p is evolutionarily conserved, similar patterns of expression of miR-6001-5p and its target genes should occur across queen-destined late-instar larvae of all three Bombus species.We will test these predictions by comparing profiles of differentially expressed genes across larval phenotypes, by using our expression data to predict targets of miR-6001-5p and by conducting assays to validate these targets. The proposed work will provide a comprehensive understanding of the evolutionary and genetic basis of changes in the number of critical stages of caste determination in eusocial insects and will elucidate the role of a miRNA in caste differentiation. As well as addressing the fundamental topic of the genetic regulation of polyphenism, the work is novel because no previous study has exploited a comparable predictive framework. It is timely because gene profiling by next-generation sequencing now provides the tools required and our existing data have created an exciting opportunity to make rapid headway in understanding how miRNAs affect caste differentiation. Finally the work fits BBSRC's strategic priorities because it could reveal means of manipulating worker production to maximize pollination services to horticulture provided by commercially-reared bumble bee colonies.