Marine animals live and thrive in a literal sea of microorganisms, yet are often able to maintain specific associations that are largely dictated by the environment, host immunity and microbial interactions. Animal-associated microbiomes include bacteria and viruses that vastly outnumber host cells, especially in the gut environment, and are considered to be integral parts of healthy, functioning animals that act as a metaorganism. However, the processes underlying the initial establishment of these microbial communities are not very well understood. This dissertation focuses on the establishment of a well-known developmental animal model, Ciona intestinalis (sea squirt), to study the establishment and maintenance of a stable gut microbiome. Generation of a new model for studying microbial colonization of the gut requires the ability to rear Ciona in the absence of microbes (i.e., germ-free). This dissertation describes the establishment of a germ-free technique for rearing Ciona and the methods utilized for bacterial exposure and colonization. Additionally, to determine the spatial structure of the gut microbiome, viral and bacterial communities within the three main gut compartments (stomach, midgut, hindgut) of Ciona from San Diego, CA, were assessed. The viral community was dominated by phages (viruses infecting bacteria), and numerous prophages (phages integrated into bacterial genomes) matching sequences found in bacteria belonging to the Ciona microbiome were detected within the active viral fraction. To determine the prevalence of prophages within the Ciona microbiome, a total of 70 bacteria cultured from the gut were tested, and 22 isolates were found to possess inducible prophages. When co-cultured with other bacteria, these induced prophages were capable of lytic infection of other members of the microbiome, often exhibiting broad host ranges. The dynamic interactions of gut bacteria and phages were explored further with the isolation and characterization of a novel Shewanella phage-host system from the adult Ciona gut. Lytic phage infection resulted in an increase in biofilm formation correlating with the release of extracellular DNA, a process that was also observed to a lesser degree in control cultures as a result of spontaneous prophage induction. Furthermore, addition of the Ciona immune protein VCBP-C to static cultures of this Shewanella sp. 3313 also enhanced biofilm formation; a similar phenomenon was noted in another bacteria, a Pseudoalteromonas sp. 6751. Interestingly, both of these isolates contained inducible prophages and binding of the VCBP-C protein to these lysogenic strains was found to influence prophage induction in vitro. Colonization of the gut in vivo also correlated with differential up-regulation of VCBP-C expression in germ-free animals and a subsequent induction of prophages. This dissertation makes an important contribution to the symbiosis field by developing a new model system in which novel aspects of host-microbe interactions can be investigated. The discovery that an innate immune effector can influence bacterial biofilms and result in the induction of prophages capable of lytic infection of other co-occurring bacteria reveals a previously unrecognized intersection between secretory immune molecules and phages in shaping the microbiome. These findings establish Ciona as a relevant and tractable model for studying trans-kingdom interactions during colonization of the gut epithelium.

Les activités humaines sont à l’origine de profondes modifications de la distribution naturelle des espèces. Les introductions d’espèces sont en particulier à l’origine de contacts secondaires entre espèces non isolées reproductivement et ayant divergé en allopatrie. Cette situation est un cas d’étude particulièrement intéressant pour étudier des processus éco-évolutifs, tels que la coexistence d’espèces occupant de même niches écologiques ou les flux de gènes contemporains (i.e. hybridation et introgression) entre espèces indigènes et non-indigènes. Cette thèse s’est ainsi intéressée aux interactions écologiques et génomiques entre deux ascidies congénériques et interfertiles, Ciona robusta et Ciona intestinalis. Ces deux ascidies, abondantes dans les habitats portuaires, sont en sympatrie en Manche Occidentale suite à l’introduction récente de C. robusta (supposée originaire du Pacifique Nord-Ouest) dans l’aire de distribution naturelle de C. intestinalis. Par une étude de la distribution spatiale et temporelle (inter-saisonnière et inter- annuelle) des adultes des deux espèces et de la sédentarisation de leurs juvéniles, nous avons montré que les deux espèces coexistent de façon durable, au sein de communautés similaires, à l’échelle régionale. Elles vivent en syntopie dans la majorité des habitats portuaires étudiés, bien qu’il existe une importante dynamique saisonnière de leur abondance relative : C. robusta est surtout présente en fin d’été et en automne, et toujours en moindre abondance par rapport à sa congénère indigène. Cette syntopie, l’interfertilité des deux espèces et le synchronisme de leur maturité sexuelle indiquent un potentiel d’hybridation important entre les deux espèces. L’hybridation réalisée est pourtant faible, comme montré avec quatre marqueurs moléculaires diagnostiques des deux espèces, analysés sur plus de 3000 individus : seulement 4% des individus présentent des génotypes compatibles avec de l’hybridation ou de l’introgression. Ces résultats ont été complétés par une étude de génomique des populations (310 marqueurs SNPs et 450 individus collectés dans différents océans) : ils confirment que les flux de gènes interspécifiques sont très rares. Par ailleurs, les rares locus présentant du polymorphisme partagé montrent un taux d’introgression hétérogène, et l’introgression est également observée dans des localités où seule l’une des espèces existe (ex. côtes américaines et chiliennes). L’ensemble des résultats génétiques et génomiques indiquent que le flux de gènes interspécifique observé résulte d’introgressions anciennes (probablement au Pléistocène) et non contemporaines. Une dernière étude menée à l’échelle mondiale avec des marqueurs mitochondriaux a par ailleurs permis de montrer que les processus d’introduction de C. robusta diffèrent selon les régions d’introduction et pose la question du statut réellement non-indigène des deux espèces dans certaines régions. Ainsi, cette thèse a montré la (quasi)-absence d’hybridation actuelle entre C. robusta et C. intestinalis, en situation de syntopie. Elle ouvre des perspectives quant à l’étude des mécanismes d’isolement reproductif entre ces deux espèces. La question du devenir à long terme des deux espèces, qui par leur coexistence étroite sont en compétition, est posée. Cette compétition pourrait être modulée par leurs préférences environnementales et les changements climatiques en cours dans l’Atlantique Nord. Enfin, cette thèse illustre comment des processus contemporains et anciens interagissent pour façonner la distribution et l’évolution des espèces, la structure génétique de leurs populations et l’architecture de leur génome. / Human activities severely alter species ranges that have been built on evolutionary time scales, and biological introductions promote secondary contacts between non-reproductively isolated species that were in allopatry. Such a situation is a very interesting case-study to examine eco-evolutionary processes, for instance coexistence between species sharing the same ecological niche or inter-specific gene flow (i.e. hybridization and introgression) between native and non-native species. This PhD thesis studied ecological and genomic interactions between two biologically similar, interfertile and congeneric tunicates, namely Ciona robusta and Ciona intestinalis. They are both abundant in harbours and marinas. They are sympatric in the Western English Channel because of the recent introduction of C. robusta (putatively native to Asia) in the natural range of C. intestinalis. Based on spatial and temporal (seasons and years) adult abundance data and examination of recruitment patterns of the two species, we showed that the two species are sustainably coexisting at a regional scale in Brittany, within similar communities. They live in syntopy in most of the study marinas. However, there are significant changes in their relative abundance through time: C. robusta is mostly present at the end of the summer and in the autumn, although always being less abundant than its native congener. Syntopy, interfertility between the two species and synchronous sexual maturity all indicated a high likelihood of hybridization between the two species. The realized hybridization is however very low, as shown by 4 species-diagnostic molecular markers genotyped over more than 3000 individuals: only 4% of the individuals displayed a genotype compatible with hybridization or introgression. This result has been confirmed with a population genomics study (310 SNPs; 450 individuals collected worldwide): inter-specific gene flow is very low. In addition, the few loci showing shared polymorphism displayed variable introgression rates and introgression was observed even in allopatric localities (where only one of the two species exists nowadays). Overall, the genetic and genomic results indicate that interspecific gene flow is most likely due to past introgression events (that may have occurred during the Pleistocene). Finally, a study carried out at a worldwide scale with two mitochondrial loci, showed that the introduction processes of C. robusta are different among the introduced regions. This study also questions the non-native status of the two Ciona species in some regions. This work showed the near absence of contemporary hybridization between C. robusta and C. intestinalis, even in syntopy, opening new research perspectives about the mechanisms preventing their reproduction in the wild. It also questions the fate of these two competing species; this competition may depend on the interaction between their specific environmental preferences and on on-going climatic changes in the North Atlantic. Altogether, this work illustrates the interplay between contemporary and past processes on species distribution and evolution, population genetic structure and genomic architecture.

京都大学 / 0048 / 新制・課程博士 / 博士(理学) / 甲第21946号 / 理博第4524号 / 新制||理||1650(附属図書館) / 京都大学大学院理学研究科生物科学専攻 / (主査)准教授 佐藤 ゆたか, 教授 高橋 淑子, 教授 中務 真人 / 学位規則第4条第1項該当 / Doctor of Science / Kyoto University / DGAM

Marine ecosystems can be a promising reservoir of various kinds of chemical components, applicable as pharmaceutical materials, food, cosmetics, nutraceuticals, and others for different industry. As an example, Tunicates, a group of marine animals, have been attracted a lot of attention in medical application, food market, water pollution issues, and Cellulose nanomaterial production due to their consisting of chemical compounds such as cellulose, amino-sugars, and proteins or protein-polysaccharide complexes e.g. collagen, glycosaminoglycan, chitin, scleroprotein, iodine-binding proteins, and elastin. In this project,  two dominant species of Scandinavian Tunicates, i.e. Ciona intestinalis and Clavelina lepadiformis, harvested from Norwegian ocean have been classified according to body sizes, depths from the ocean surface, ages and species, and separated physically into outer layer and internal organs, followed by measurements of sugar composition, oil content, and  protein content. Application potentials have been investigated by trials for production of pure crystalline cellulose, bioethanol, and biodiesel, and by analysis of amino acid composition of the samples. The cellulose percentage and cellulose yield for the chemically pure cellulose obtained, is around 96% and 54% respectively, and the protein content is decreased step by step by the acid, alkali, and bleaching process applied. Bioethanol can be obtained by fermentation of tunicate hydrolysate with strains A and C which are derived from Saccharomyces cerevisiae. The biodiesel yield of tunicate samples is around 4-6% as an average. The amino acid compositions in our tunicate samples are similar to egg albumin, implying tunicate being an alternative material for animal feed production. Several processing treatments have been conducted with the aims to fractionate tunicate biomass components or enhance the cellulose accessibility and reactivity. After a single processing step, Ba(OH)2 treated samples seemed to be the best in terms of both cellulose preservation (66.5% cellulose) and protein removal (6% protein in the treated residue). Results from the physical separation plus washing reveal that the highest amount of cellulose and protein presents is found in the outer (Tunic) part and internal organs of Tunicate samples respectively. Data obtained from FTIR(Fourier Transform Infrared Spectroscopy) and SEM(Scanning Electron Microscope) indicate that among all processing trials, H3PO4 is the most effective in decreasing the cellulose crystallinity, which renders a higher accessibility for acidic or enzymatic reaction during bioethanol production due to a higher amount of amorphous structure of cellulose. From the analysis results of component contents and structures, it could be concluded that increase of deepness results in a decrease of sugar content of the Tunicate samples while there are no differences in protein and carbohydrate content in different tunicate species. The body size has a positive influence on the protein content and the sample age alters the contents of both sugar and protein. In addition, Tunicate oil has high phospholipid content instead of glycerol ester, the latter being the common oil from vegetable origins. Moreover, lots of free fatty acid is present, and the composition profile of Tunicate fatty acids seems to be similar to fish oil, as revealed by NMR (Nuclear Magnetic Resonance Spectroscopy), FTIR, and GC-MS (Gas Chromatography-Mass Spectrometry).

Marine ecosystems can be a promising reservoir of various kinds of chemical components, applicable as pharmaceutical materials, food, cosmetics, nutraceuticals, and others for different industry. As an example, Tunicates, a group of marine animals, have been attracted a lot of attention in medical application, food market, water pollution issues, and Cellulose nanomaterial production due to their consisting of chemical compounds such as cellulose, amino-sugars, and proteins or protein-polysaccharide complexes e.g. collagen, glycosaminoglycan, chitin, scleroprotein, iodine-binding proteins, and elastin. In this project,  two dominant species of Scandinavian Tunicates, i.e. Ciona intestinalis and Clavelina lepadiformis, harvested from Norwegian ocean have been classified according to body sizes, depths from the ocean surface, ages and species, and separated physically into outer layer and internal organs, followed by measurements of sugar composition, oil content, and  protein content. Application potentials have been investigated by trials for production of pure crystalline cellulose, bioethanol, and biodiesel, and by analysis of amino acid composition of the samples. The cellulose percentage and cellulose yield for the chemically pure cellulose obtained, is around 96% and 54% respectively, and the protein content is decreased step by step by the acid, alkali, and bleaching process applied. Bioethanol can be obtained by fermentation of tunicate hydrolysate with strains A and C which are derived from Saccharomyces cerevisiae. The biodiesel yield of tunicate samples is around 4-6% as an average. The amino acid compositions in our tunicate samples are similar to egg albumin, implying tunicate being an alternative material for animal feed production. Several processing treatments have been conducted with the aims to fractionate tunicate biomass components or enhance the cellulose accessibility and reactivity. After a single processing step, Ba(OH)2 treated samples seemed to be the best in terms of both cellulose preservation (66.5% cellulose) and protein removal (6% protein in the treated residue). Results from the physical separation plus washing reveal that the highest amount of cellulose and protein presents is found in the outer (Tunic) part and internal organs of Tunicate samples respectively. Data obtained from FTIR(Fourier Transform Infrared Spectroscopy) and SEM(Scanning Electron Microscope) indicate that among all processing trials, H3PO4 is the most effective in decreasing the cellulose crystallinity, which renders a higher accessibility for acidic or enzymatic reaction during bioethanol production due to a higher amount of amorphous structure of cellulose. From the analysis results of component contents and structures, it could be concluded that increase of deepness results in a decrease of sugar content of the Tunicate samples while there are no differences in protein and carbohydrate content in different tunicate species. The body size has a positive influence on the protein content and the sample age alters the contents of both sugar and protein. In addition, Tunicate oil has high phospholipid content instead of glycerol ester, the latter being the common oil from vegetable origins. Moreover, lots of free fatty acid is present, and the composition profile of Tunicate fatty acids seems to be similar to fish oil, as revealed by NMR (Nuclear Magnetic Resonance Spectroscopy), FTIR, and GC-MS (Gas Chromatography-Mass Spectrometry).

The ParaHox cluster is the evolutionary sister of the Hox cluster. Like the Hox cluster, the ParaHox cluster is subject to complex regulatory phenomena such as collinearity. Despite the breakup of the ParaHox cluster within many animals, intact and collinear clusters have now been discovered within the chordate phyla in amphioxus and the vertebrates, and more recently within the hemichordates and echinoderms. The archetypal ParaHox cluster of amphioxus places it in a unique position in which to examine the regulatory mechanisms controlling ParaHox gene expression within the last common ancestor of chordates, and perhaps even the wider Deuterostomia. In this thesis, the genomic and regulatory landscape of the amphioxus ParaHox cluster is characterised in detail. New genomic and transcriptomic resources are used to better characterise the B.floridae ParaHox cluster and surrounding genomic region, and conserved non-coding regions and regulatory motifs are identified across the ParaHox cluster of three species of amphioxus. In conjunction with this, the impact of retrotransposition upon the ParaHox cluster is examined and analyses of transposable elements and the AmphiSCP1 retrogene reveal that the ParaHox cluster may be more insulated from outside influence than previously thought. Finally, the detailed analyses of a regulatory element upstream of AmphiGsx reveals conserved mechanisms regulating Gsx CNS expression within the chordates, and TCF/Lef is likely a direct regulator of AmphiGsx within the CNS. The work in this thesis makes use of new genomic and transcriptomic resources available for amphioxus to better characterise the genomic and regulatory landscape of the amphioxus ParaHox cluster, serving as a basis for the improved identification and characterisation of functional regulatory elements and conserved regulatory mechanisms. This work also highlights the potential of Ciona intestinalis as a ‘living test tube' to allow the detailed characterisation of amphioxus ParaHox regulatory elements.

<p>Paralogs arise by duplications and belong to families. Ten paralogs (insulin; <i>IGF-1</i> and <i>-2</i>; <i>INSL3-6</i> and 3-relaxins) constitute the human insulin-relaxin family. The aim of this study was to outline the duplications that gave rise to the vertebrate insulin-relaxin genes and the chromosomal regions in which they reside. Neurotrophin and Trk-receptor families with more than 300, otherwise unrelated, families had paralogs in the regions hosting insulin/relaxin genes, defining two quadruplicate paralogy-regions, namely: insulin/IGF and INSL/relaxin paralogons. Thereby, the localization of insulin/relaxins in human shows that these regions were formed during two genome duplications at the stem of the vertebrates.</p><p>We characterized insulin-like genes (<i>INS-L1</i>, <i>-L2</i> and <i>-L3</i>) in the <i>Ciona intestinalis</i> genome, a species that split from the chordate lineage before the genome duplications. Conserved synteny between the Ciona region hosting the <i>INS-Ls</i> and two human paralogons as well as linkage of the actual paralogons, suggest that a segmental duplication gave rise to the entire region prior to the genome duplications. Synteny together with gene and protein structures demonstrate that <i>INS-L1</i> is orthologous to the vertebrate <i>INSLs</i>/relaxins, <i>INS-L2</i> to insulins and <i>INS-L3</i> to <i>IGFs</i>. This indicates that pro-orthologs of the insulin-relaxin family were formed before Ciona. Our analysis also implies that the INSL/relaxin ancestor switched receptor from tyrosine kinase- to GPCR-type. This probably occurred after the Ciona-stage, but before the genome duplications.</p><p>Using genes residing within the analyzed human paralogons that were present in a chromosomal region in the Ciona-human ancestor, we identified 37 segments with conserved synteny between the <i>Drosophila melanogaster</i> and human genomes. Orthologs residing in Ciona-, sea urchin- and the fly syntenic segments imply that such segments approximate an ancestral region from which the human paralogons originated.</p><p>To conclude, the human paralogons are remnants of genome duplications that in addition to segmental- and single duplications, shaped the extant vertebrate genomes. Using the quadruplicate paralogy-regions we were able to deduce duplication events of the insulin-relaxin genes and their chromosomal regions.</p>

Paralogs arise by duplications and belong to families. Ten paralogs (insulin; IGF-1 and -2; INSL3-6 and 3-relaxins) constitute the human insulin-relaxin family. The aim of this study was to outline the duplications that gave rise to the vertebrate insulin-relaxin genes and the chromosomal regions in which they reside. Neurotrophin and Trk-receptor families with more than 300, otherwise unrelated, families had paralogs in the regions hosting insulin/relaxin genes, defining two quadruplicate paralogy-regions, namely: insulin/IGF and INSL/relaxin paralogons. Thereby, the localization of insulin/relaxins in human shows that these regions were formed during two genome duplications at the stem of the vertebrates. We characterized insulin-like genes (INS-L1, -L2 and -L3) in the Ciona intestinalis genome, a species that split from the chordate lineage before the genome duplications. Conserved synteny between the Ciona region hosting the INS-Ls and two human paralogons as well as linkage of the actual paralogons, suggest that a segmental duplication gave rise to the entire region prior to the genome duplications. Synteny together with gene and protein structures demonstrate that INS-L1 is orthologous to the vertebrate INSLs/relaxins, INS-L2 to insulins and INS-L3 to IGFs. This indicates that pro-orthologs of the insulin-relaxin family were formed before Ciona. Our analysis also implies that the INSL/relaxin ancestor switched receptor from tyrosine kinase- to GPCR-type. This probably occurred after the Ciona-stage, but before the genome duplications. Using genes residing within the analyzed human paralogons that were present in a chromosomal region in the Ciona-human ancestor, we identified 37 segments with conserved synteny between the Drosophila melanogaster and human genomes. Orthologs residing in Ciona-, sea urchin- and the fly syntenic segments imply that such segments approximate an ancestral region from which the human paralogons originated. To conclude, the human paralogons are remnants of genome duplications that in addition to segmental- and single duplications, shaped the extant vertebrate genomes. Using the quadruplicate paralogy-regions we were able to deduce duplication events of the insulin-relaxin genes and their chromosomal regions.