Evolution and molecular basis of caste differentiation in bees

生物学中的一个基本主题涉及基因的开启和关闭(差异表达)如何创造形态和行为的多样性。“多物性”(当不同的成体形态出现在同一基因组中)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。在蚂蚁、蜜蜂和黄蜂身上出现了一个多物性的主要例子，雌性幼虫发育成两个种姓、女王或工蚁中的一个。已经知道，种姓在大小、形状、繁殖和行为上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是如何产生多面性的。然而，我们对种姓分化的遗传基础的理解还处于早期阶段。所涉及的基因、它们的进化背景以及microRNAs(miRNAs，调节基因表达的小RNA分子)的作用都知之甚少。在这个项目中，我们将以熊蜂(Bombus)为例，阐明影响女王-工蜂种姓分化的基因的进化和调控。大黄蜂是实现这一目标的极佳模型。首先，物种在决定幼虫种级的关键阶段的数量上有所不同(当分化开始时)，这种变异的进化历史是已知的。一种(B.terrestris)雌幼虫有两个临界期(早龄期和晚龄期)，早期临界期是次要的(进化衍生)；另一种(B.thnorum)有一个这样的阶段(仅限晚龄期幼虫)；而‘杜鹃’大黄蜂(B.vestalis)没有临界期，因为它们第二次失去了工蜂等级，所有的雌性都是在其他熊蜂物种的巢中繁殖的蜂王。其次，我们发现miR-6001-5P在皇后注定的晚龄稻纵卷叶蜂幼虫中过表达，表明它调节影响CAST的基因。这些数据使我们能够创建一个预测框架，用于预测种姓相关基因和熊蜂miR-6001-5P的预期差异表达模式。具体地说，如果关键阶段通过调节相同的基因在进化中保持保守，而新的关键阶段通过额外的调控途径获得，那么地面芽孢杆菌和菌落芽孢杆菌应该在晚龄幼虫中共享共同的差异基因表达模式，而在早龄幼虫中则不同。同样，这两个物种中特定于工蚁幼虫的基因表达模式应该在杜鹃物种B.vestalis中缺失。此外，如果miR-6001-5p的作用在进化上是保守的，那么miR-6001-5p及其靶基因的表达模式应该在所有三个种的蜂王注定的晚龄幼虫中出现相似的模式。我们将通过比较不同幼虫表型差异表达基因的图谱，通过使用我们的表达数据来预测miR-6001-5p的靶标，并通过进行测试来验证这些靶标，来检验这些预测。这项拟议的工作将全面了解真社会昆虫种姓决定的关键阶段数量变化的进化和遗传基础，并将阐明miRNA在种姓分化中的作用。除了解决多态现象的遗传调控这一基本主题外，这项工作也是新颖的，因为之前没有任何研究利用了可比的预测框架。这是及时的，因为下一代测序的基因图谱现在提供了所需的工具，而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方法，以最大限度地为商业饲养的大黄蜂群体提供园艺授粉服务。

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.