Advancing molecular crustacean chronobiology through the characterisation of the circadian clock in two malacostracan species, Euphausia superba and Parhyale hawaiensis Thesis submitted for the degree of Doctor of Philosophy at the University of Leicester by Benjamin James Hunt Department of Genetics University of Leicester 2016 Advancing molecular crustacean chronobiology through the characterisation of the circadian clock in two malacostracan species, Euphausia superba and Parhyale hawaiensis Benjamin James Hunt Abstract The ability to entrain to environmental cycles and therefore anticipate and prepare for the changes they predictably bring is the preserve of the endogenous biological clock, most widely studied at the circadian level. Despite a rich history of research into the behavioural and physiological rhythms shown by many crustacean species, the underlying molecular system driving such traits is not well understood. The aim of this research was to develop our understanding of crustacean clocks through the study of two species, one of major ecological importance and the other a powerful model organism. The Antarctic krill Euphausia superba is a keystone species in the Southern Ocean ecosystem, and evidence suggests that the clock may influence both daily and seasonal rhythms. Using a variety of approaches, including the creation of a de novo assembled head transcriptome, a full suite of clock-related genes have now been cloned and characterised. Unlike many species Euphausia superba possesses orthologs of every canonical core clock gene, and cell culture assays indicate that the central feedback loop has the capacity for complete transcriptional inhibition via two separate pathways, raising the possibility that the krill clock may be an ancestral type or employ multiple oscillators to control rhythms of differing periods. In contrast to the relatively intractable krill, the amphipod Parhyale hawaiensis has simple maintenance requirements and an extensive genetic toolkit with the potential to enable sophisticated dissection of the molecular clock. With the aim of laying the groundwork for future research the clock genes of this species have also been identified, along with the development of a locomotor activity assay. Parhyale hawaiensis shows evidence of bimodal patterns of activity under the control of a molecular clock that combines mammalian-like characteristics with some unique features worthy of further investigation. i Acknowledgements First and foremost my thanks to my supervisor Ezio Rosato, a man of endless and infectious enthusiasm for research who provided me with the perfect level of support and guidance – it has been a pleasure and a privilege to work with him. I am indebted as well to my retired second supervisor Ted Gaten, whose generosity on my return to the fold enabled me to hit the ground, if not running, then at least hobbling slightly faster. Three other people require special thanks. Firstly to my unofficial supervisor Özge Özkaya, who not only laid much of the groundwork for this research but who shouldered the unenviable burden of teaching a ham-fisted zoology graduate the art of molecular benchwork without (visibly) flinching. Secondly to computer wizard Nathaniel Davies, creator of the Euphausia superba Transcriptome Database: Online Edition. Thirdly to Lin Zhang, the other member of lab 124’s tiny crustacean enclave and an ever-reliable source of insight and S2 plasmids. Thank you to every member of our lab past and present, with particular nods to Giorgio Fedele, Celia Hansen, Kam Chana, Jon Shand, Marie Nugent, Marcela Buricova, Ed Green, Laura Delfino, Laura Flavell, Mirko Pegoraro, Elise Facer-Childs and Wilhelmina Assam, all of whom were generous with time, assistance and opinions. Thanks too to the technical and administrative staff, particularly Helen Roe and Liam Gretton, and to Paul Seear for sharing his extensive knowledge. Outside of Leicester, Ana Patricia Ramos and Michalis Averof were generous with their advice and assistance on amphipod wrangling and in their willingness to share data and animals, and am I deeply grateful to Aziz Aboobaker, nastasios Pavlopoulos and Damian Kao for granting me early access to the Parhyale hawaiensis genome. If I tried to fully express my gratitude to family and friends, the acknowledgements section alone would break the 50,000 word limit this thesis is subject to. Mum, dad, granddad, Paula, Chris, Nathan, Manu, Sophie, Isabel and the rest of my family; and everyone who joins me for dinner or drinks from time to time - you have provided support, love and laughs in abundance and I thank you all. Finally, a tribute to two surpassingly wonderful people. Firstly to my fiancée Anna, whose unstinting support has sustained me through this challenge, and others. Even if there was no word limit I could not do you justice - I love you and need you, always. And to my grandma Betty, source of happy childhood memories beyond counting. Gron, I love you too, and I miss you so much. ii Contributors This thesis could not have been produced without the valuable contributions of the following colleagues. Dr. Ted Gaten: Collection and storage of Euphausia superba samples, as described in section 2.2.1.1. Entrainment of Euphausia superba in LD conditions, subsequent collection and storage of samples in LD and DD for use in RT-PCR, as discussed in section 2.4.3.3. Dr. Özge Özkaya: Euphausia superba RNA extraction and storage as described in section 2.2.1.2. Cloning and sequencing of the initial Esbmal1 340 bp fragment through degenerate and semi-degenerate PCR, as detailed in 2.3.1.1. Tissue-specific PCR of Esbmal1, Escry1 and Escry2 (Figure 2.7). Full cloning of Escry1 and Escry2 and characterisation of the former as light-sensitive (Figure 2.13). RT-PCR for Esbmal1, Esclk, Escry1 and Escry2 (2.4.3.3; Figure 2.15). Nathaniel Davies: Production of the Euphausia superba Transcriptome Database, covering the formatting of transcript and annotation data and the production of the website itself. Section 3.2.2.4 details this process in his words. This research used the ALICE High Performance Computing Facility at the University of Leicester and was funded by the National Environmental Research Council (NERC). iii Table of Contents Abstract i Acknowledgements ii Contributors iii Table of Contents iv List of Tables xiii List of Figures xv Common abbreviations and author’s note xix CHAPTER 1 GENERAL INTRODUCTION 1 1.1 The biological clock 1 1.1.1 Ancient rhythms 1 1.1.2 The benefits of a biological clock 1 1.2 The molecular clock 2 1.2.1 The circadian clock in Drosophila melanogaster 2 1.2.2 The core 3 1.2.3 Supporting roles 4 1.2.4 Variations on a theme 5 1.3 Crustacean clocks 5 1.3.1 A history of crustacean chronobiology 5 1.3.2 Molecular data: a long deficit, then a sudden deluge 9 1.4 Euphausia superba 10 1.4.1 Distribution and biomass 10 1.4.2 Life history 11 1.4.3 Ecological and economic importance 11 iv 1.5 The rhythms of Euphausia superba 12 1.5.1 Timekeeping in the Southern Ocean 12 1.5.2 Circadian behaviour 13 1.5.3 Seasonal variations 16 1.6 On the productivity of model and non-model organisms 17 1.6.1 Euphausia superba: high importance, high maintenance 17 1.6.2 The very model of a modern research animal 17 1.6.3 Seeking a pacemaker 19 1.6.4 Parhyale hawaiensis: a model organism par excellence? 22 1.7 Research aims and outline 22 CHAPTER 2 THE CLONING AND CHARACTERISATION OF THE CANONICAL CLOCK GENES OF EUPHAUSIA SUPERBA 24 2.1 Introduction 24 2.1.1 The core genes of the circadian clock 24 2.1.1.1 The central heterodimer 24 2.1.1.2 Repressive elements 26 2.1.1.3 The cryptochromes 27 2.1.1.4 Tying it all together 28 2.1.2 Other organisms 29 2.1.2.1 Insecta 29 2.1.2.2 Crustacea 30 2.1.3 Research aims 30 2.2 Methods 31 2.2.1 Sampling 31 2.2.1.1 Euphausia superba capture and storage 31 2.2.1.2 RNA extraction and cDNA synthesis 31 2.2.2 Gene cloning 32 2.2.2.1 Degenerate PCR 32 2.2.2.2 Searching the SRA 33 2.2.2.3 RACE extension 34 2.2.2.4 Transcriptome mining 34 2.2.2.5 Confirmation 34 2.2.3 Gene characterisation 36 2.2.3.1 Tissue expression 36 v 2.2.3.2 Protein sequence analysis 36 2.2.3.3 Phylogeny 36 2.2.3.4 Transcriptional activity assay 36 2.3 Results 39 2.3.1 Gene cloning 39 2.3.1.1 Esbmal1 39 2.3.1.2 Esclock 39 2.3.1.3 Esperiod 39 2.3.1.4 Estimeless 40 2.3.2 Protein sequence analysis 40 2.3.2.1 EsBMAL1 40 2.3.2.2 EsCLOCK 41 2.3.2.3 EsPERIOD 42 2.3.2.4 EsTIMELESS 42 2.3.2.5 Comparative analysis 43 2.3.3 Tissue expression 44 2.3.4 Phylogenetic trees 48 2.3.4.1 EsBMAL1 48 2.3.4.2 EsCLOCK 48 2.3.4.3 EsPERIOD 48 2.3.4.4 EsTIMELESS 48 2.3.5 Transcriptional activity 53 2.4 Discussion 54 2.4.1 Summary of results 54 2.4.1.1 Esbmal1 54 2.4.1.2 Esclock 54 2.4.1.3 Esperiod 54 2.4.1.4 Estimeless 55 2.4.1.5 The cryptochromes 55 2.4.2 Peptide features and comparisons 56 2.4.2.1 The core heterodimer 56 2.4.2.2 The domains of EsPERIOD and EsTIMELESS 59 2.4.2.3 Evolutionary relationships 60 2.4.3 The molecular clockwork of Euphausia superba 61 2.4.3.1 Transcription, repression and tissue expression 61 2.4.3.2 An ancient clock or a crustacean specialisation? 63 2.4.3.3 Regarding transcript cycling 65 vi 2.4.4 Future work 68 2.4.4.1 Immediate issues 68 2.4.4.2 Following up on a theory 69 2.4.5 Conclusions 70 CHAPTER 3 THE EUPHAUSIA SUPERBA TRANSCRIPTOME DATABASE: DE NOVO ASSEMBLY, ANNOTATION AND OUTPUT.