From control to constraint: a study of reproduction in the eusocial honey bee and the solitary red mason bee Jens Van Eeckhoven Submitted in accordance with the requirements for the degree of Doctor of Philosophy The University of Leeds School of Biology March, 2020 I The candidate confirms that the work submitted is his own, except where work which has formed part of jointly-authored publications has been included. The contribution of the candidate and the other authors to this work has been explicitly indicated below. The candidate confirms that appropriate credit has been given within the thesis where reference has been made to the work of others. Jointly-authored publication: Van Eeckhoven, J., Duncan, E.J., 2020. Mating status and the evolution of eusociality: Oogenesis is independent of mating status in the solitary bee Osmia bicornis. J. Insect Physiol. 121, 104003. Contributorship (following CRediT: Allen et al., 2019): Jens Van Eeckhoven: Conceptualization, Methodology, Data acquisition, Formal analysis, Investigation, Writing - original draft, Writing - review & editing, Visualization. Elizabeth J. Duncan: Conceptualization, Resources, Writing - review & editing, Visualization, Funding acquisition, Supervision, Project administration. This copy has been supplied on the understanding that it is copyright material and that no quotation from the thesis may be published without proper acknowledgement. © 2020 The University of Leeds and Jens Van Eeckhoven II Never underestimate the simplicity, inherent to complexity © Tal, with permission — Chantal Maas; [email protected] III Acknowledgements Believe it or not, the University actually provides a template for the acknowledgements section. ‘This research has been carried out by a team which has included (name the individuals). My own contributions, fully and explicitly indicated in the thesis, have been......(please specify) The other members of the group and their contributions have been as follows: (please specify).’ We can only guess at whether these words were carefully crafted by a dispassionate bureaucrat, some cold-hearted and bitter academic, or by Hades himself who subsequently whispered them into the ears of an unsuspecting and innocent mortal. But now that I have displayed those words here, I hope to be in line with whatever University policy may or may not outline them. So now we can safely commit them to the void that is amnesia, where such unfeeling things belong. Indeed any and all acknowledgements I have ever read, both within and outside this University, have been cheesy without fail. As well they should be. So I will endeavour to do the same here. For the Odyssean nature of doing a PhD, has left me enlisting, recruiting, conscripting and consigning all of the varying modes and degrees of support known to mankind. And I am grateful to them all. And I would like to start with, Liz, the epitome of a good supervisor. She managed to get a stats and theory nerd interested in molecular biology. From doing the first stainings and showing me the pretty images that can stem from them, she slowly ‘Mr Miyagied’ me; to the point where my dry and philosophical self ended up caring about germline stem cells and molecular pathways. Even before coming to Leeds, I had heard and become aware of horror stories surrounding PhD-supervision. Being the sensitive soul that I am, I really don’t think I would have survived under another’s guidance. And while my love for science has often stood at odds with conventions and practices within academia; Liz’s level of supervision stands testament to how it can and should be done, and as such leaves me with some hope for academia still. I would also like to thank Andrew Peel and William Kunin for their co- supervision, and Amanda Bretman serving in the capacity of assessor in addition to providing an excellent and memorable journal club. Further thanks go to the team at NBAF in Sheffield: Terence Burke, Deborah Dawson, Gavin Horsburgh, Kathryn Maher, Rachel Tucker and Paul Parsons for their training, help, and advice during my time there working on microsatellites. I should also thank various unnamed academics at the University of Leeds for the use of IV laboratory equipment, and particular thanks in this regard go out to Sally Boxall for her enduring help with the slidescanner and the use of the bio-imaging facilities (facilitated by the Wellcome Trust; WT104918MA). Lastly, I’d like to thank Chantal Maas for providing exquisite front matter artwork; and the hardworking postgraduate reception team for their unfailing logistical support. Even though I started as Liz’s first student in Leeds, I’ve had the privilege of seeing her lab grow. And so I would like to thank all my labmates: Rollsie with the easy laughs (yes I will be using our ‘breadnames’ for this), Myloaf with the chillest of demeanours, Britta Bread whom should never be referred to as Bready Britt, NorNaan with the silliest of games, an at times slightly boozy Cakey Kane, and Mackenzie. I was also delighted to work with and alongside various MBiol students and wish to thank: Vanessa Barlow for opening up an inordinate amount of cocoons, as well as Emily Birch, Flora Whiting-Fawcett, Lois Gaden, and Amy Wells. A different endeavour, entailed rolling the boulder that is my conscience, onto the hill that should have been my sanity. As it turns out, it was a rather steep hill, and a rather smooth boulder as well. However, contrary to King Sisyphus, I was fortunate that a great many people, have spent a great deal of time and effort helping me repeatedly rolling that boulder back onto that stubborn hill. These people include the many friends I have made during my time here at Leeds. The OGs are of course: both bee-Tom and fly-Tom, Laurin me-Scottish-lass, Katy my bestest man-woman, Myrna my ‘Hi friend!’, Sir Nicholas, Sil who-lies-like-the-best-or-worst-of-them, and James who-actually- has-a-heart-of-gold-no-matter-how-desperately-he-tries-to-pretend-otherwise. The people I met outside of work tried to impress upon me that there is life outside of a PhD. My heartfelt thanks: Alaa, Alice, Dimi, Hannah, Jackie, Louise, Pablo, Pascale, and Pauline. Then there are my Belgian friends, who as few others know the depths of me. Your advice, laughs, compassion and unrelenting faith in me have been a balm upon my soul. For reminding me, time and again, of who I am, thank you; Gert-Jan, Sebastjen, Nathalie, Elyssa, Lars, Sharon; I could not have done this without any of you. And though I’ve never met them: Robin Hobb I thank for her wisdom, and Van Morrison and Florence Welch I thank for the best soundtrack any student could wish for. Finally, I’d like to thank my mother, for always having pushed me, for better or for worse. And my dad, for providing the counterweight, telling me he’s proud of me no matter what. Sadly at the time of writing, I remain unconvinced of this. Time will tell. V Abstract The major evolutionary transition from solitary to eusocial living is hallmarked by the reproductive division of labour. I investigated mechanisms underlying reproductive control in a solitary bee (Osmia bicornis), with the aim of informing how and why such mechanisms were co-opted into reproductively constraining workers in a eusocial species (Apis mellifera, mrca 95 mya; Peters et al., 2017). I start out by introducing the problems of reproductive constraint and the evolution of eusociality (Chapter 1). In order to test functional links and perform manipulation, it is imperative to establish a reference species within the laboratory. To address the lack of a temperate European solitary model species, I attempted to establish O. bicornis in a laboratory environment (Chapter 3). Preliminary erratic successes of nesting and egg-laying behaviour were achieved, and future recommendations were laid out. To further facilitate O. bicornis as a model species; microsatellite markers were mined, designed, tested and validated in collaboration with the NERC Biomolecular analysis facility in Sheffield (Chapter 4). The broad applicability of these markers is discussed. The capricious nature of laboratory egg-laying necessitated appraising reproductive control directly. To enable assaying oogenesis, I performed the first microstructural study of the O. bicornis ovary (Chapter 5). Since mating plays an important role in the ovary activation of eusocial queens and other insect species, I concurrently examined the effect of mating status on the ovary of O. bicornis (Chapter 5) — with special reference to the potential role of mating status in reproductive constraint. Finally, I investigated how a known mechanism of reproductive constraint (Duncan et al., 2016) operates in the related solitary bee, to ascertain its ancestral role (Chapter 6).The mechanism was found to be reversed in O. bicornis. Chapter 7 places the overall findings within their wider context, and outlines future avenues of research. VI Table of Contents Acknowledgements .................................................................................... III Abstract ....................................................................................................... V Table of Contents....................................................................................... VI List of Figures ............................................................................................. X List of Tables ............................................................................................. XII Abbreviations ..........................................................................................