Microbiome exploration of deep-sea carnivorous Cladorhizidae sponges by Joost Theo Petra Verhoeven A Thesis submitted to the School of Graduate Studies in partial fulfillment of the requirements for the degree of Doctor of Philosophy Department of Biology Memorial University of Newfoundland March 2019 St. John’s, Newfoundland and Labrador ABSTRACT Members of the sponge family Cladorhizidae are unique in having replaced the typical filter-feeding strategy of sponges by a predatory lifestyle, capturing and digesting small prey. These carnivorous sponges are found in many different environments, but are particularly abundant in deep waters, where they constitute a substantial component of the benthos. Sponges are known to host a wide range of microbial associates (microbiome) important for host health, but the extent of the microbiome in carnivorous sponges has never been extensively investigated and their importance is poorly understood. In this thesis, the microbiome of two deep-sea carnivorous sponge species (Chondrocladia grandis and Cladorhiza oxeata) is investigated for the first time, leveraging recent advances in high-throughput sequencing and through custom developed bioinformatic and molecular methods. Microbiome analyses showed that the carnivorous sponges co-occur with microorganisms and large differences in the composition and type of associations were observed between sponge species. Tissues of C. grandis hosted diverse bacterial communities, similar in composition between individuals, in stark contrast to C. oxeata where low microbial diversity was found with a high host-to-host variability. In C. grandis the microbiome was not homogeneous throughout the host tissue, and significant shifts occured within community members across anatomical regions, with the enrichment of specific bacterial taxa in particular anatomical niches, indicating a potential symbiotic role of such taxa within processes like prey digestion and chemolithoautotrophy. The potential for bacteria-mediated chemolithoautotrophy was further supported by stable-isotope analysis in C. grandis but not in C. oxeata. Metagenome analysis further showed a potential ii role for archaea in nitrogenous-waste detoxification, as well as an assemblage of bacteriophages potentially influencing the bacterial community composition and functionality. Lastly, metabolic pathways of associated microbes were diverse and included metabolism of elemental substrates (nitrogen, sulfur) and methane. This thesis provides novel insights into the role of microorganisms in carnivorous sponges and their relation to carnivory; the microbiome of C. grandis is linked to many metabolic functions, including the facilitation of carnivory, whereas heterogeneity and absence of apparent symbiotic potential in C. oxeata suggest that in some sponges, other processes govern carnivory. iii ACKNOWLEDGMENTS Looking back, my journey towards this thesis has been quite an extensive one, starting as a student to become a laboratory technician all the way back in 2003 in The Netherlands. Back then, I would have never thought that one day I would be pursuing a PhD degree abroad, and many people along the way have been so supportive and helped me greatly along the path to get there. I would like to take this opportunity to thank all my previous supervisors and places that offered me internships over the years, including Johnny and Gonda Rottier and all the fine folk at Micro-Analyse Zeeland for putting up with me during my very first internship when I had no idea how a lab worked, Lucy Buyck at the Medical Microbiology Lab Terneuzen, as well as Douglass Turnbull, Robert Taylor and Laura Greaves at the Wellcome Trust Centre for Mitochondrial Research in Newcastle, and Kimberly Benschop at the National Institute for Public Health and the Environment in Bilthoven. A special mention goes out to Lia van der Hoek at the Academic Medical Center for a very formative internship in a great environment (which also included many pubs in Amsterdam!) in which your support and trust in me to manage my own little research project most certainly kindled my confidence to keep learning. Of course, this thesis could not have come to be if not for Suzanne Dufour, who graciously agreed to be my supervisor. Thank you for giving me a chance to pursue an incredibly interesting topic and learn so many new things, I greatly appreciate all the guidance and opportunities you have given me over the course of my PhD program. This iv gratitude also extends to my committee members: Annie Mercier, Lourdes Peña-Castillo and Kapil Tahlan, thank you for all your help and patience over the last few years. I would also like to thank my lab mates for all the nice times in and out of the lab! A source of never-failing help that needs to be acknowledged is my parents, brother and sister. Moving away from home to Canada has not always been easy, and this PhD would not have been possible if it weren’t for your support, continued interest in my work, your visits to Canada, and kindness whenever I had to chance to visit home, thank you so much. Finally, I would like to thank Marta, you have been at the center of this thesis adventure together with me, and I could not have done it without you. Your continued patience, eagerness to teach me things, discuss science (usually over a pint of beer) and all- around encouragement has been an enormous help, and I am looking forward to many more adventures together. v CO-AUTHORSHIP STATEMENT I am the primary author of all chapters in this thesis, for which I performed the associated laboratory work, development of bioinformatic scripts and data analysis. With the exception of Chapter 5 (see below) I authored, in completeness, all the text for the presented manuscripts, which then received editorial inputs from my co-authors. Chapter 2: I wrote the SPONS pipeline software, performed the validation experiments, wrote the thesis chapter and created the supporting material such as figures and tables. Dr. Suzanne Dufour and Dr. Lourdes Peña-Castillo provided editorial inputs which were incorporated into the thesis chapter. Chapter 3: The samples used in this study were collected by Dr. Dufour. All laboratory experiments were performed by me, with the exception of spicule analysis through electron microscopy, which was performed by Dr. Dufour and Alana Kavanagh. I performed the bioinformatic analyses, wrote the manuscript and created the supporting material such as figures and tables. Editorial inputs from my co-authors, and peer reviewers upon publishing in FEMS 1 were incorporated into the chapter. Chapter 4: I collected the samples, performed laboratory experiments and conducted the bioinformatic analyses. Spicule analysis through electron microscopy was performed by Dr. Dufour and Alana Kavanagh. The manuscript, including supporting materials, was written by me and received editorial inputs, incorporated into the thesis 1 Verhoeven JTP, Kavanagh AN, Dufour SC. Microbiome analysis shows enrichment for specific bacteria in separate anatomical regions of the deep-sea carnivorous sponge Chondrocladia grandis. FEMS Microbiol Ecol (2017) 93: doi:10.1093/femsec/fiw214 vi chapter, from co-authors as well as peer reviewers upon submitting the manuscript to Arctic Science2. Chapter 5: This manuscript was the result of a combined effort with Dr. Marta Canuti. Dr. Canuti contributed to the conceptualization and design of the molecular methods, which were validated in a series of experiments performed by both Dr. Canuti and myself. I developed the bioinformatics pipeline. The manuscript was co-written; Dr. Canuti wrote the sections related to virus discovery and influenza virus genome sequencing, as well as the molecular methods, while I wrote the introduction, discussion, bioinformatic methods and the sponge microbiome results. The manuscript further received minor editorial inputs from other co-authors, and peer reviewers after submission to the Canadian Journal of Microbiology3. Chapter 6: All experiments, analysis and manuscript preparation were performed by me. Dr. Dufour provided editorial inputs which were incorporated into the thesis chapter. 2 Verhoeven JTP, Dufour SC. Microbiomes of the Arctic carnivorous sponges Chondrocladia grandis and Cladorhiza oxeata suggest a specific, but differential involvement of bacterial associates. Arct Sci (2017) doi:10.1139/AS-2017-0015. 3 Verhoeven JTP, Canuti M, Munro HJ, Dufour SC, Lang AS. ViDiT-CACTUS: an inexpensive and versatile library preparation and sequence analysis method for virus discovery and other microbiology applications. Can J Microbiol (2018)1–13. doi:10.1139/cjm-2018-0097 vii TABLE OF CONTENTS List of Tables ................................................................................................................... xiii List of Figures .................................................................................................................. xiv List of Abbreviations ....................................................................................................... xvi Chapter 1: Introduction and overview ................................................................................ 1 1.1 Introduction .......................................................................................................... 1 1.1.1 Overview of the phylum Porifera ................................................................. 1 1.1.2 Sponge-associated microbial communities ................................................... 2 1.1.3 The carnivorous