Investigating host-symbiont crosstalk in the coral reef demosponge Amphimedon queenslandica Xueyan Xiang BSc (Biotechnology) 0000-0003-1843-0474 A thesis submitted for the degree of Doctor of Philosophy at The University of Queensland in 2021 School of Biological Sciences Abstract In the last two decades, the widespread application of -omic approaches has revealed the astonishing diversity and ubiquity of microbial communities that inhabit animals. This growing understanding of host-symbiont interactions has revolutionised our view about how symbiotic microbes influence their host animal. Studies in humans and many other animals have revealed that symbiotic microbes can regulate and contribute to the metabolism of different nutrients. These metabolic activities can produce metabolites that affect the host's physiology, development, growth and immunity. In this thesis, I use genomic and transcriptomic approaches to explore host-symbiont crosstalk in the coral reef demosponge Amphimedon queenslandica. This sponge provides a tractable system because it houses a low complexity and low abundance microbiota dominated by just three proteobacterial symbiont types. Complete and reliable holobiont genomic and transcriptomic data can help to investigate interactions between the sponge and its symbionts. To better understand interactions between A. queenslandica and its symbionts, I first resequenced, reassembled and reannotated the existing draft genomes of A. queenslandica and its three primary proteobacterial symbionts – AqS1, AqS2, and AqS3 – using additional in vitro reconstituted chromatin (Chicago) and short paired-end Illumina data. This markedly improved the genome assemblies and gene functional annotations of all four of these symbiotic partners (Chapter 2). The N50 length of A. queenslandica genomic scaffolds increased almost eight-fold, from 120 to 950 kb. The scaffolds N50 of AqS1, AqS2 and AqS3 also substantially increased to 103, 148 and 90 kb, respectively. This reanalysis revealed the relative contribution of these genomes to holobiont functioning, with the three symbionts disproportionately contributing to a majority of the holobiont's metabolic pathways. I then turned to assess the gene activity in this hologenome by developing an approach to accurately analyse holobiont transcriptomes. The efficacy of the various approaches used previously to capture host (eukaryote) and bacterial symbiont transcriptomes indicated biases in capturing both pools of mRNA. In this context, I sought to identify a dual RNA-Seq approach that sufficiently captures both host and symbiont transcriptomes. I compared the transcriptomic data generated from poly(A) captured mRNA-Seq (Poly(A)-mRNA-Seq) and ribosomal RNA depleted RNA-Seq (rRNA- depleted-RNA-Seq) using bacterial-enriched adult tissues of A. queenslandica holobiont, and from previously generated bacterial-unenriched A. queenslandica PolyA-mRNA-Seq data, focusing on the transcriptome depth and coverage (Chapter 3). For the host sponge, no significant difference was found in transcriptomes generated from Poly(A)-mRNA-Seq and rRNA-depleted-RNA-Seq. However, the rRNA-depleted-RNA-Seq performed better than the Poly(A)-mRNA-Seq in capturing i representative symbiont transcriptomes. I also found that bacterial cell enrichment enabled adequate capture of the symbiont transcriptomes, although it reduced the A. queenslandica host transcriptome. This comparison demonstrated that RNA-Seq by ribosomal RNA depletion is an effective and reliable method to obtain an accurate and representative holobiont transcriptome. This dual RNA-Seq approach – rRNA-depleted-RNA-Seq – allows for the accurate generation of A.queenslandica holobiont transcriptome data. I used these new A. queenslandica holobiont genomes and transcriptomes to identify potential host- symbiont metabolic interactions (Chapter 4). For example, genomic and transcriptomic analyses of the sponge A. queenslandica and its three primary symbiotic bacteria, AqS1, AqS2, and AqS3, confirmed host and symbionts are all involved in active carbohydrate metabolism. The host and symbionts also collaborate in assimilating dissolved inorganic nitrogen, sulfur, and phosphate. The symbiotic bacteria can uniquely de novo synthesise – and thus potentially provide the host sponge with – essential amino acids, some vitamins (B1, B2, and B5) and cofactors (folate and siroheme). This analysis of metabolic pathway enzymes suggests the sponge holobiont works cooperatively to assimilate the major marine nutrients, and cycle nutrients in the aquatic ecosystems. A. queenslandica hologenomes and transcriptomes also revealed potential animal-bacterial interkingdom signalling (Chapter 5). For instance, the capacity of the symbiont AqS1 to uptake and degrade sponge-derived γ-aminobutanoate (GABA) suggests that GABA may act as a quorum- sensing (QS) signal between the host and symbionts, which has the potential to influence the holobiont behaviour. The sponge symbionts appear to generate some metabolites that are not produced by the sponge, yet can act as ligands for G protein-coupled receptors (GPCRs) of the sponge. Notably, some of these are neurotransmitters in neural animals, including dopamine, tyramine, tryptamine, acetate, and propionate. Given this may provide a mechanism for the symbionts to regulate sponge signalling pathways and influence sponge physiology, I tested one of these – dopamine – experimentally. I show that the experimental introduction of dopamine can influence larval phototactic swimming behaviour, potentially via a dopamine-related GPCR. Together these results reveal the potential for sponge-bacterial interkingdom signalling communication. In summary, the A. queenslandica holobiont -omics analyses have revealed the molecular-level mechanisms of sponge-microbe cooperation in nutrient assimilation, potential metabolic interdependence amongst the partners of the holobiont, and signalling communication between the host and symbionts. These results reveal potential crosstalk within the A. queenslandica holobiont that establishes and maintains the sponge-bacteria symbiosis. ii Declaration by author This thesis is composed of my original work, and contains no material previously published or written by another person except where due reference has been made in the text. I have clearly stated the contribution by others to jointly-authored works that I have included in my thesis. I have clearly stated the contribution of others to my thesis as a whole, including statistical assistance, survey design, data analysis, significant technical procedures, professional editorial advice, financial support and any other original research work used or reported in my thesis. The content of my thesis is the result of work I have carried out since the commencement of my higher degree by research candidature and does not include a substantial part of work that has been submitted to qualify for the award of any other degree or diploma in any university or other tertiary institution. I have clearly stated which parts of my thesis, if any, have been submitted to qualify for another award. I acknowledge that an electronic copy of my thesis must be lodged with the University Library and, subject to the policy and procedures of The University of Queensland, the thesis be made available for research and study in accordance with the Copyright Act 1968 unless a period of embargo has been approved by the Dean of the Graduate School. I acknowledge that copyright of all material contained in my thesis resides with the copyright holder(s) of that material. Where appropriate I have obtained copyright permission from the copyright holder to reproduce material in this thesis and have sought permission from co-authors for any jointly authored works included in the thesis. iii Publications included in this thesis No publications included iv Submitted manuscripts included in this thesis No manuscripts submitted for publication Other publications during candidature Peer-reviewed papers 1. Hall, M. R., K. M. Kocot, K. W. Baughman, S. L. Fernandez-Valverde, M. E. A. Gauthier, W. L. Hatleberg, A. Krishnan, C. McDougall, C. A. Motti, E. Shoguchi, T. Wang, X. Y. Xiang, M. Zhao, U. Bose, C. Shinzato, K. Hisata, M. Fujie, M. Kanda, S. F. Cummins, N. Satoh, S. M. Degnan and B. M. Degnan (2017). The crown-of-thorns starfish genome as a guide for biocontrol of this coral reef pest. Nature 544(7649): 231-234. 2. Kukekova, A. V., J. L. Johnson, X. Y. Xiang, S. H. Feng, S. P. Liu, H. M. Rando, A. V. Kharlamova, Y. Herbeck, N. A. Serdyukova, Z. J. Xiong, V. Beklemischeva, K. P. Koepfli, R. G. Gulevich, A. V. Vladimirova, J. P. Hekman, P. L. Perelman, A. S. Graphodatsky, S. J. O'Brien, X. Wang, A. G. Clark, G. M. Acland, L. N. Trut and G. J. Zhang (2018). Red fox genome assembly identifies genomic regions associated with tame and aggressive behaviours. Nature Ecology & Evolution 2(9): 1514-1514. 3. Rando, H. M., M. Farre, M. P. Robson, N. B. Won, J. L. Johnson, R. Buch, E. R. Bastounes, X. Y. Xiang, S. H. Feng, S. P. Liu, Z. J. Xiong, J. Kim, G. J. Zhang, L. N. Trut, D. M. Larkin and A. V. Kukekova (2018). Construction of Red Fox Chromosomal Fragments from the Short-Read Genome Assembly. Genes 9(6). 4. Gao, W., Y. B. Sun, W. W. Zhou, Z. J. Xiong, L. N. Chen, H. Li, T. T. Fu, K. Xu, W. Xu, L. Ma, Y. J. Chen, X. Y. Xiang, L. Zhou, T. Zeng, S. Zhang, J. Q. Jin, H. M. Chen, G. J. Zhang, D. M. Hillis, X. Ji, Y. P. Zhang and J. Che (2019). Genomic and transcriptomic investigations of the evolutionary