......................................................................................................................................... ii Preface........................................................................................................................................... iv Table of Contents ...........................................................................................................................v List of Tables ..................................................................................................................................x List of Figures............................................................................................................................... xi List of Abbreviations ................................................................................................................. xiii Acknowledgements ......................................................................................................................xv Dedication ................................................................................................................................... xvi Chapter 1: Introduction ................................................................................................................1 1.1
Co-evolution of animals and their microbiota ................................................................... 1 1.2
Sponge-microbial associations .......................................................................................... 2 1.2.1
Sponge biology ........................................................................................................... 2 1.2.2
Sponge microbiota ...................................................................................................... 4 1.3
Host-symbiont recognition ................................................................................................ 6 1.4
Sponge innate immunity .................................................................................................... 8 1.5
A specific symbiosis: Dragmacidon mexicanum and Cenarchaeum symbiosum............ 10 1.6
Research question and thesis objectives .......................................................................... 12 Chapter 2: Microbial community structure in Dragmacidon mexicanum and other sponge species from northeast Pacific coastal waters ...........................................................................14 2.1
Introduction...................................................................................................................... 14 2.2
Materials and methods ..................................................................................................... 16 vi 2.2.1
Sample collection and processing............................................................................. 16 2.2.2
DNA isolation and purification................................................................................. 17 2.2.3
Quantification of archaeal and bacterial taxonomic marker genes........................... 18 2.2.4
Preparation of PCR amplicons for pyrosequencing.................................................. 19 2.2.5
Three-domain variable region sequence data analysis ............................................. 19 2.2.6
Archaeal SSU rRNA gene amplification, library production and screening ............ 21 2.2.7
Eukaryotic SSU rRNA gene PCR amplification ...................................................... 21 2.2.8
Phylogenetic analysis of SSU rRNA sequences ....................................................... 22 2.3
Results.............................................................................................................................. 24 2.3.1
Composition of sponge sample set ........................................................................... 24 2.3.2
Quantification of archaea and bacteria in sponges ................................................... 26 2.3.3
Community structure relationships between sponges............................................... 27 2.3.4
Taxonomic composition of sponge microbiota ........................................................ 31 2.3.5
Archaeal phylogeny in SB sponges .......................................................................... 34 2.3.6
Indicator species analysis.......................................................................................... 35 2.3.7
Dragmacidon mexicanum core microbiota............................................................... 36 2.4
Discussion........................................................................................................................ 40 2.4.1
Core and indicator sponge microbiota ...................................................................... 40 2.4.2
Taxonomic composition of sponge microbiota ........................................................ 41 2.4.3
Intraspecific variation between Dragmacidon mexicanum microbiota .................... 43 Chapter 3: Genomic and functional characterization of C. symbiosum-encoded genes........46 3.1
Introduction...................................................................................................................... 46 3.2
Materials and methods ..................................................................................................... 48 vii 3.2.1
Comparative genomics of C. symbiosum and other Thaumarchaeota ...................... 48 3.2.2
Protein extraction and peptide mapping ................................................................... 50 3.2.3
Sequence homology and structure prediction ........................................................... 51 3.2.4
Biological activity of C. symbiosum serpins............................................................. 52 3.2.4.1
Cloning, expression and purification of C. symbiosum serpins ......................... 52 3.2.4.2
Protease inhibition assay.................................................................................... 53 3.2.4.3
Serpin-sponge lysate pull-down......................................................................... 54 3.2.4.4
Serpin protein and C-terminal peptide activity in NFκB reporter cell line ....... 55 3.2.4.5
C-terminal peptide activity in human peripheral blood mononuclear cells....... 57 3.2.4.6
C-terminal peptide pull-down in sponge and mammalian cells ........................ 58 3.3
Results.............................................................................................................................. 59 3.3.1
Comparative genomics between C. symbiosum and free-living Thaumarchaeota ... 59 3.3.2
C. symbiosum proteins implicated in host-microbe interactions .............................. 63 3.3.3
Biological activity of C. symbiosum serpins............................................................. 66 3.4
Discussion........................................................................................................................ 73 3.4.1
Putative C. symbiosum symbiosis factors ................................................................. 73 3.4.2
Biological activity of C. symbiosum serpins............................................................. 76 Chapter 4: Microbial recognition and host defense systems in marine sponges ...................80 4.1
Introduction...................................................................................................................... 80 4.2
Materials and methods ..................................................................................................... 83 4.2.1
RNA isolation and purification................................................................................. 83 4.2.2
Sponge housekeeping genes PCR protocol .............................................................. 85 4.2.3
cDNA library production, sequencing and assembly ............................................... 86 viii 4.2.4
Transcriptome annotation ......................................................................................... 86 4.2.5
Identification of putative pathways and interactions between expressed genes ....... 88 4.2.6
Identification of protein-coding genes from existing Oscarella carmela EST data. 89 4.3
Results.............................................................................................................................. 89 4.3.1
Composition of sponge transcriptome datasets ........................................................ 89 4.3.1.1
Taxonomic composition of transcriptomes........................................................ 90 4.3.1.2
Functional annotation of transcriptomes............................................................ 93 4.3.2
Diversity and distribution of pathways and predicted ORFs in sponge transcriptomes 93 4.3.3
Innate immunity genes and pathways in D. mexicanum and T. californiana ........... 98 4.3.3.1
Toll-like receptor signalling............................................................................... 98 4.3.3.2
Nod-like receptor signalling ............................................................................ 100 4.3.3.3
Phagocytosis and autophagy............................................................................ 102 4.3.3.4
Lectins, complement and coagulation ............................................................. 104 4.3.3.5
Viral recognition mechanisms ......................................................................... 107 4.3.3.6
Apoptosis, transendothelial migration and adaptive immunity pathways ....... 108 4.4
Discussion...................................................................................................................... 110 4.4.1
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