Dr Gilles Bedoux

Keywords: Halymenia floresii, antibiofilm, antifouling, métabolomique, bactéries associées à la surface, quorum sensing, molecules de défense Halymenia floresii, antibiofilm, antifouling, metabolomics, surface-associated bacteria, quorum sensing, defence molecules H. floresii, metabolic fingerprinting, epibiose, QSI, XCMS

Halymenia floresii, une Rhodophycée présente une surface remarquablement exempte d'épiphytes dans les conditions de l'Aquaculture MultiTrophique Intégrée (AMTI). Ce phénomène la présence en surface de composés actifs allélopathiques. L'objectif de ce travail a été d'explorer les mécanismes de défense développés par H. floresii contre l'épibiose, de détecter et d'identifier les métabolites secondaires produits à la surface de l'algue et d'étudier les relations avec les bactéries épiphytes. Nous avons ainsi pu isoler la communauté épibactérienne de H. floresii cultivée dans des conditions contrôlées (AMTI) et non contrôlées (échantillons collectés in situ). Les épibactéries isolées ont été criblées in vitro pour analyser les signaux de détection de Quorum Sensing (QS). Lesextraits produits en surface ont été analysés pour détecter toute interférence avec le Quorum Sensing. Les épibactéries pathogènes et nonpathogènes ont été différenciées par leur capacité à induire une maladie algale, le blanchiment. Vibrio owensii, ainsi que son signal C4-HSL QS, a été identifié comme pathogène opportuniste induisant un blanchiment. Les métabolites extraits de la surface et de cellules entières de H. floresii ont été analysés par LC-MS. Une base de données a été constituée à partir d'une analyse métabolomique non ciblée. Quarante et un putatives actifs ont été identifiés, parmi lesquels les composés halogénés, des furanones et divers inhibiteurs étaient surreprésentés. Fait intéressant, les deux premières classes sont connues comme de puissants composés interférant avec le QS. La présence relativement élevée de putatives allélopathiques à la surface de H. floresii soutient fortement l'hypothèse selon laquelle ils doivent être impliqués dans la protection de l'hôte. Des recherches supplémentaires seront nécessaires pour explorer l'ensemble des métabolites secondaires produits par H. floresii et leurs rôles chez l'algue.

v supervision. Her immense knowledge always influenced me to think critically and develop my ideas. I always admired her way of valuing others TIME equally as she values her own. I could not have imagined having a better supervisor for my PhD study. I equally thank my Co-director Dr. Daniel Robledo for sharing his knowledge on his all-time favourite, macroalgae. I really appreciate his guidance in writing the publications for his insightful comments which incented me to widen my perspectives of the subject.

Nothing would have been possible without their continuous support to my PhD.

My sincere thanks also go to Dr. Gilles Bedoux for his valuable comments and guidance in the chemistry part of the research and his advice on chromatographic analyses is highly valuable. I greatly appreciate his deep knowledge on the subject which really ignited my interest in the field of chemistry. I extend my gratitude to Dr. José Q. Garcia Maldonado for his guidance and comments in the molecular biology part of the research. His direction really helped me a lot and earned me a confidence in the field of molecular biology. I also gratefully acknowledge Prof. Alain Dufour and Dr. Alexis Bazire for their guidance and views in the quorum sensing related work of the research. I also sincerely thank Dr. Bazire for offering me vi the reporter strains that really helped me carry out the QS related works. I also deeply thank Dr. Yolanda Freile-Pelegrín for her care and concern during my research stay in Mexico and also for the GC-MS instrument in her lab for analysing my extracts. Furthermore, I extend my deepest gratitude to Dr. Mahasweta Saha (Plymouth Marine Laboratory, UK) for her very detailed description and guidance through the disease part of the work. I also thank Mme. Florence Le Sann, Mme. Noluenn Chauvin and Mme. Axelle Guitton for their administrative help accompanying throughout the thesis, regarding the mobility's and conferences. I also thank Yohan Duhautois for his administrative assistance regarding the formations.

Technicians are the real heroes of any lab. As my research includes different laboratories, I had an opportunity to meet many heroes during my thesis. First of all, Christel Marty (LBCM, France), I truly thank her for teaching and explaining me the preventive measures of the laboratory and being very supportive all through my research in LBCM. My heartfelt thanks to Crescencia and Edgar Caamal-Fuentes (Phycology, Mexico) for all their support and help, both professionally and morally throughout my stay in Mexico. I will never forget Cres's help in finding me accommodation for my relaxed stay in Merida.

Edgar's knowledge on chemistry, particularly on GC-MS, always astonished me and I am very grateful for sharing it with me. I also thank V. Avila-Velazquez for his skillful technical assistance for the algal cultivation.

My earnest thanks to Philippe Douzenel (UBS, France) for his great help and support in carrying out different experimentations and especially for his support in HPLC related works. My heartfelt thanks extend to Laure Taupin (LBCM, France) for her tremendous and wonderful help and support in LC-MS related works. I should definitely mention her fastest reply to my doubts and her availability in sending me the results. I wholeheartedly thank Bernard Lassard (UBS, France) for all his help and support and very specially for the Luminometer. Being a very happy person, Bernard always gave me answers for my questions and never judged me for my silly doubts. And Abril Gamboa and Roscita (Parasitology and Molecular Biology, Mexico), truly I thank both of them for their immense support in molecular works and maintaining my bacteria. I deeply appreciate their efforts in understanding my needs though I could able to communicate only in English with them. I also truly thank Ana and Dora (Microscopy, Mexico) for their patience and assistance in travelling with me over the surface of my alga and finally getting good pictures of the microbiome.

And my team….LBCM… I should obviously start with Maya with whom I started my PhD days. Thank you, Maya for teaching and assisting me to learn basic experimentations in my early days. I sincerely thank Anne-Sophie getting me the algal samples from Mexico. I wholeheartedly thank Florence for BEing with vii me during the very first isolation of my bacteria. I am also happy that I had a chance to meet Romain who also helped me learning basic manipulations. I happily thank Nolwenn, Marie and Kevin for having been there for me and I shall definitely mention about my coffee time with Nolwenn and chatting about the whole world. I have only few people in UBS to talk with and Hugo, you are first among them. Thank you very much, my friend for everything! For sharing your experiences and stories and moreover your family with me. I think of Paola, Emiliano, Rebecca at this time and of course, Gael. And IRDL… I sincerely owe my gratitude to the whole team for the beautiful smiling morning wishes.

And my team…. Phycology…. I dearly thank Ana for the beautiful friendship and all those big laughs we shared and of course Adrian too, though he did not have a clue of what we were talking he laughed with us.

Well…now, Parasitology, I thank each and every one of the team for the wonderful everlasting experiences, in and out of the lab! I sincerely extend my gratitude to Dr. Victor and Dr. Leo for their very warm love and affection and especially for the Piñata during Christmas! I also thank them for allowing me to use the Epifluorescence microscope. I am very grateful to the whole team. Thanking the friends is not my way, I extend all my love and care to Karen, Eric, Roman, Fer, Sonia, Regina, Celina, Danilu, Catherine and I promise to stand by them as far as I can. I thank Danilu and Catherine, together with their family, for giving me most memorable days in my life by incorporating me in their life.

I also thank Mr. Jean-Roch Sauvé for the pleasant ride to Lorient for few of my manipulations there. Your help really matters a lot for me and moreover I cannot forget the joke you prepared for your daughter on 1 st of April. And finally, Olivier Sire… Thank you for everything! I deeply extend my gratitude and love for BEing with me through my hard times, Just BE, my friend. Thank you very much, Olivier, particularly for clearing the fog on my way and walking me through it.

And my Family… it is hard to find words to thank them. Thank you Mummy…Machi…Thashu…Sunil ma…Kuttima…Mithu… and my dear Don. Thank you, Mummy for believing in me even at times when even I didn't believe me. I promise you all that my every single action will make you all proud. And Raphael… Thank you da for being such a wonderful companion, for being my soul mate, roommate and my everything. Love you all forever and ever. I should definitely mention few of several invisible people and friends who were with me in this journey. I wholeheartedly thank Ambika, Navashree annae and Sahana who supported me during all my visas processes. And I also thank Vimal annae, Baba annae, Sangeeth annae and all friends in Divonne-les bains xi

List of communications

Oral International

A Abdul Malik S., Gamboa A., Bedoux G., Garcia Maldonado J. Q., Bourgougnon N., & Robledo D. Composition, symbiosis and pathogenesis, an equilateral relationship of the surface-associated microbiome of the cultivated Mexican Rhodophyta, Halymenia floresii, 67 th Annual Meeting of The British Phycological Society (07 January -10 January, 2019) Scottish Association of Marine Sciences, Oban, Scotland.

A Abdul Malik S., Bedoux G., Robledo D., Garcia Maldonado J. Q., Freile-Pelegrín Y., & Bourgougnon N. Chemical defence by allelopathic active metabolites on the surface of cultivated Mexican Rhodophyta Halymenia floresii against biofouling, 23 rd International Seaweed Symposium (28 April -03 May, 2019), International Seaweed Association, Jeju, Korea.

Oral National

A Abdul Malik S., Caamal-Fuentes E., Ávila-Velázquez V., Bedoux G., Bourgougnon N., Robledo D. Involvement of allelopathic active metabolites on the surface of the cultivated Halymenia floresii in the biofouling phenomenon, Société Phycologique de France, Paris.

xix

List of Figures (outside the chapters)

Fig. 1 Worldwide macroalgal production (%) under aquaculture per species and countries-year 2014 (Source: FAO, 2016) [START_REF] Buschmann | Seaweed production: overview of the global state of exploitation, farming and emerging research activity[END_REF]

Chapter II

Fig. 1 Induction of bioluminescence in the reporter E.coli pSB406 by Vibrio owensii (B3IM); Maribacter sp., (B9IM); Pseudoalteromonas arabiensis (B4BC); P. tetraodonis (B6BC) and V. owensii (B7BC) No. of cycles (30 minutes per cycle) plotted against relative light units/optical density at 600 nm .................. 1).164 Fig. 2 Left: Relative class occurrence (%) of the Whole-cell metabolites (WCM); surface metabolites (DIP) and common metabolites (CMN) plotted against Activity/Biochemical classes (symbols as in Fig. 1). Right: Metabolic count (A) and Relative Class occurrence (B) in % of the Whole-cell metabolites (WCM=416) and common metabolites (CMN=41) plotted against Activity/Biochemical classes .......... Approximately 70% of Earth's surface is occupied by a continuous stretch of seawater with an average depth of 5 km [START_REF] Baweja | Biology of Seaweeds[END_REF]) with a rich source of both biological and chemical diversity.

Oceans support floating forests with variable sea plants and animals, where marine vegetation is considered to be more primitive and richer than land-based plants. Among them, macroalgae (seaweeds) are important marine resources playing a major role in supporting the rich biodiversity of the sea. Macroalgae constitute a diverse polyphyletic group of predominantly marine, multicellular, photosynthetic, chlorophyll "a" containing, eukaryotic organisms with simple reproductive structures and found from the intertidal zone to 300 m deep (Levine 2016).

History and Classification

Most algae are photosynthetic organisms that acquired the trait some 1.8 billion years ago after an unknown non-photosynthetic unicellular eukaryote engulfed or was invaded by a photosynthetic cyanobacterium similar to Gloeomargarita. This trait ultimately resulted in a cell containing a photosynthetic plastid surrounded by two membranes with highly reduced cyanobacteria derived genome. This event, known as the primary endosymbiosis, gave rise to the extant Archaeplastida, which includes the Glaucophyta, Rhodophyta (red algae), and Chloroplastida (green algae and land plants) [START_REF] Tirichine | Decoding algal genomes: Tracing back the history of photosynthetic life on Earth[END_REF][START_REF] Leliaert | Phylogeny and molecular evolution of the green algae[END_REF][START_REF] Adl | Revisions to the Classification, Nomenclature, and Diversity of Eukaryotes[END_REF]. Brown algae have a very different evolutionary history, as they have acquired the ability to achieve photosynthesis through a secondary endosymbiosis event. The other lines such as Cryptophytes, Haptophytes, Golden brown algae and Dinophytes, are therefore eukaryotes with no direct relationship with terrestrial plants. The lines resulting from secondary or even tertiary endosymbioses between a primitive non-photosynthetic eukaryote and a unicellular red alga include the Ochrophytes within Stramenopiles, Cryptophytes and Haptophytes lines. Derived from endosymbiosis with a green unicellular alga, the lines are grouped within Euglenophyceae and Rhizaria [START_REF] Niklas | The evolution of the land plant life cycle[END_REF]. Such events involved a second heterotrophic eukaryote that engulfed not a cyanobacterium but a green or red photosynthetic eukaryote and resulted in plastids with three or four membranes around them.

The customary notion of macroalgae actually encompasses a polyphyletic complex within eukaryotes [START_REF] Boudouresque | Taxonomy and phylogeny of unicellular eukaryotes[END_REF]. The evolutionarily diverse macroalgae are found in two kingdoms, Plantae and Chromista, and four phyla, Charophyta (Chara), Chlorophyta (green), Rhodophyta, and Ochrophyta (brown). Most marine macroalgae belong to phylum Ochrophyta, class Phaeophyceae, and to phylum Rhodophyta. A few marine macroalgae occur in the phylum Charophyta and about 600 large green algal taxa occur in the phylum Chlorophyta. Macroalgae in these four clades have more fundamental (e.g. cytological, chemical, life histories) differences between one another than with the vascular plants [START_REF] Dawes | Marine Botany, Second[END_REF]. [START_REF] Dring | The Biology of Marine Plant[END_REF] listed 900 species of green algae, 997 brown algae, and 2540 red algae worldwide.

These advancements in phycology have resulted in major changes in the classification of algae (Schneider andWynne 2007, 2013;[START_REF] Wynne | Addendum to the synoptic review of red algal genera[END_REF]. Thus, the number of macroalgal species has increased. [START_REF] Guiry | [END_REF] listed 4885 species in the Charophyta and 6759 in the Chlorophyta, 2057 species in the class Phaeophyceae, and 7303 species in the Rhodophyta. In W.R. Taylor's 1957 flora Seaweeds of the Northeastern Coast of North America, he listed 170 genera (37 green, 57 brown, 76 red algae) and 401 species (95 green, 142 brown, and 164 red algae), while [START_REF] Mathieson | Seaweeds of the Northwest Atlantic[END_REF] have described 248 genera (57 green, 89 brown, 102 red) and 535 species (146 green, 180 brown, and 209 red taxa) for the same geography [START_REF] Dawes | Macroalgae systematics[END_REF]. As multicellular eukaryotes, the classification of marine macroalgae are made according to their photosynthetic pigments like green (chlorophyll α and β), red (phycoerythrin) and brown (xanthophyll/fucoxanthin) [START_REF] Sudhakar | An overview of marine macroalgae as bioresource[END_REF].

Ecology

Macroalgae are ecologically important primary producers, competitors, and ecosystem engineers.

They play a vital role in ecosystems as foundation species by providing habitat for higher trophic levels [START_REF] Fulton | Form and function of tropical macroalgal reefs in the Anthropocene[END_REF]. They constitute one of the important biotic components and play a central role in the different ecosystem in coastal habitats ranging from kelp forests to coral reefs [START_REF] Gattuso | Light availability in the coastal ocean: impact on the distribution of benthic photosynthetic organisms and their contribution to primary production[END_REF][START_REF] Gachon | Algal diseases: spotlight on a black box[END_REF][START_REF] Keith | Global diversity of marine macroalgae: Environmental conditions explain less variation in the tropics[END_REF][START_REF] Singh | Unraveling the Functions of the Macroalgal Microbiome[END_REF][START_REF] Krause-Jensen | Sequestration of macroalgal carbon: the elephant in the Blue Carbon room[END_REF]. Seaweeds serve as the base of the marine food webs and are a direct food source for sea urchins and fish. In addition, they provide shelter and reproductive grounds for fish, invertebrates, birds, and mammals. Seaweeds along with coral animals are the dominant benthic organisms whose relative abundance is often used as an indicator of ecosystem health [START_REF] Baweja | Biology of Seaweeds[END_REF].

The distribution of seaweeds depends upon many factors such as physical (substrate, temperature, light quality and quantity, dynamic tidal activity, winds, and storms), chemical (salinity, pH, nutrients, gases, and pollution level), and biological (herbivores, microbes, epiphytes, endophytes, symbionts, parasites, and diseases). Indeed, as sessile organisms, macroalgae have developed effective mechanisms to survive many biotic and live in a variable, extreme, and hostile abiotic environmental and stress conditions.

As a result, these beings are able to produce a wide range of compounds called "secondary metabolites", like pigments, vitamins, phenolic compounds, sterols, other bioactive agents and some extremely relevant biochemical mechanisms with biomedical and agricultural applications [START_REF] Ioannou | Natural products from seaweeds[END_REF][START_REF] Stengel | Algal chemodiversity and bioactivity: Sources of natural variability and implications for commercial application[END_REF][START_REF] Lalegerie | The stressful life of red and brown seaweeds on the temperate intertidal zone: effect of abiotic and biotic parameters on the physiology of macroalgae and content variability of particular metabolites[END_REF]).

Valorization of Macroalgae

Marine algae contain significant quantities of vitamins, minerals, dietary fibers, proteins, polysaccharides, and various functional polyphenols while nutrient contents can vary with species and, geographical location, season, and temperature. With such potential, macroalgae are subjected to numerous valorizations, particularly in the fields of human and animal nutrition. As human food, the valorization is direct. More than 600 species of seaweeds are edibles and used as vegetables in the world. Their nutritive value may vary depending on the geographic location, the season of the year, the growth stage, part of the seaweed harvested, etc. [START_REF] Leandro | Diverse applications of marine macroalgae[END_REF]. Thus, to assure the nutritional value of seaweeds, they need to be evaluated before being used as supplements. Seaweeds are industrially processed to extract thickening agents such as alginate, agar, and carrageenan cell wall polysaccharides or used, generally in dried powder form, as an animal-feed additive [START_REF] Delaney | Society and Seaweed: Understanding the Past and Present[END_REF]. According to FAO (2018), some 221 species of seaweed are of commercial value and about ten species, such as brown seaweed (Saccharina japonica, Undaria pinnatifida, and Sargassum fusiforme); red seaweed (Porphyra spp. Eucheuma spp. Kappaphycus alvarezii and Gracilaria spp.); and green seaweed (Enteromorpha clathrata, Monostroma nitidum and Caulerpa spp.). Red macroalgae, such as Gelidium, Gracilaria and Pterocladia, are important for human consumption and other uses, mainly as a binder in food products as well as the bacterial substrate in laboratories. Eucheuma and Kappaphycus are essential for the manufacture of carrageenan, used in cosmetics, food processing and industrial usage (FAO, 2018).

Macroalgae and their uses have a long history. Human and macroalgal interactions seem to date back to the Neolithic period and over the years the industrial use of macroalgae has shifted as the potential of the future industry has been viewed as being larger than its actual scale. From exploiting beach-cast macroalgae as fertilizer to their higher value as a source of polysaccharides and their technical applications in agronomics, cosmeceuticals, nutraceuticals and pharmaceuticals [START_REF] Galland-Irmouli | Nutritional value of proteins from edible seaweed Palmaria palmata (Dulse)[END_REF][START_REF] Smit | Medicinal and pharmaceutical uses of seaweed natural products: A review[END_REF][START_REF] Fleurence | What are the prospects for using seaweed in human nutrition and for marine animals raised through aquaculture?[END_REF][START_REF] Buschmann | Seaweed production: overview of the global state of exploitation, farming and emerging research activity[END_REF], the potential of macroalgae has raised the need of their extensive cultivation. By far the largest producers are China and Indonesia followed by the Philippines, Korea, Japan, Malaysia, and Zanzibar in Asia whereas in the Americas only Chile has appeared in the farming statistics tables (Fig. 1). In contrast, Europe, Canada, and Latin America, the macroalgal industries stilly rely on natural harvest [START_REF] Rebours | Seaweeds: an opportunity for wealth and sustainable livelihood for coastal communities[END_REF].

Fig. 1 Worldwide macroalgal production (%) under aquaculture per species and countries-year 2014 (Source: FAO, 2016) [START_REF] Buschmann | Seaweed production: overview of the global state of exploitation, farming and emerging research activity[END_REF] According to FAO statistics (FAO, 2014(FAO, , 2016)), of the top seven most cultivated seaweed taxa, three were used mainly for hydrocolloid extraction: Eucheuma spp. and Kappaphycus alvarezii for carrageenans, and Gracilaria spp. for agar; Saccharina japonica (formerly Laminaria japonica), Undaria pinnatifida, Porphyra/Pyropia spp. and Sargassum fusiforme were most important in human food usage (Fig. 1). The main producing countries were China, Indonesia, and the Philippines, which were also those that cultivated the greatest diversity of seaweed species. While seaweed consumption in South-east Asia has been common and traditional and has depended on taste and price, seaweed use as food in non-Asian European and USA markets has considered additional parameters such as nutritional value and 'food for health', with a strong consumer preference towards organic, sustainable and fair trade products (representing low impacts both on the environment and biodiversity) [START_REF] Chapman | Food or fad? Challenges and opportunities for including seaweeds in a Nordic diet[END_REF][START_REF] Pinchetti | Algae production and their potential contribution to a nutritional sustainability[END_REF]. However, the global market share of seaweed farming production used for food and 'other uses', i.e. other than for hydrocolloids, is still below 1% of the total biomass production [START_REF] Buschmann | Seaweed production: overview of the global state of exploitation, farming and emerging research activity[END_REF]. In 2014, about 28.5 million tonnes of seaweeds and other algae were harvested for direct consumption or further processing for food (traditionally in Japan, the Republic of Korea, and China) or for use as fertilizer and in pharmaceuticals, cosmetics and other purposes (FAO, 2016). In 2015 the total production of seaweed was 30.4 million tonnes, with the culture and capture sectors responsible for 29.4 million tonnes and 1.1 million tonnes, respectively (FAO, 2018).

The global seaweed industry is worth more than USD 6 billion per annum (approximately 12 million tonnes per annum in volume) of which 85% comprises food products for human consumption. The seaweed-derived extracts make up almost 40% of the world's hydrocolloid market in terms of foods (FAO, 2018). The increasing worldwide demand for raw materials raises the issues of sustainability, access, or availability. Aquaculture presents an alternative to ensure the availability of this resource and improve its homogeneous quality. In addition, advances in the development of seaweed cultivation can compensate the wild production and collection, and thus improving productivity and quality of seaweed biomass. Fulfilling aquaculture's growth potential requires responsible technologies and practices. Sustainable aquaculture should be ecologically efficient, environmentally benign, product-diversified, profitable, and societally beneficial. This ultimately paved a way for the emergence of innovative aquaculture practices and one such method is Integrated Multitrophic Aquaculture (IMTA), which can help address many of the environmental impacts of aquaculture. It also aims at a balanced ecosystem-based management approach to aquaculture [START_REF] Chopin | Aquaculture, Integrated Multi-trophic (IMTA)[END_REF][START_REF] Chopin | Challenges of moving Integrated Multi-Trophic Aquaculture along the R&D and commercialization continuum in the western world[END_REF].

IMTA aims to be an ecologically balanced aquaculture practice that co-cultures species from multiple trophic levels to optimize the recycling of farm waste as a food resource [START_REF] Troell | Ecological engineering in aquaculture -Potential for integrated multi-trophic aquaculture (IMTA) in marine offshore systems[END_REF]. By integrating fed aquaculture (finfish or shrimp) with inorganic and organic extractive aquaculture (macroalgae and invertebrates), IMTA can significantly increase the sustainability of aquaculture. The IMTA system is driven only by sunlight, natural inorganic nutrients, as well as carbon dioxide and provides efficient food provisions, nutrient extraction, and climate regulating services to the marine ecosystem [START_REF] Cottier-Cook | Safeguarding the future of the global seaweed aquaculture industry[END_REF]. The land-based IMTA operations have also been advocated as a way of controlling diseases and their transmission, though some concerns have been expressed that multiple species on the site might increase the risk of disease transmission [START_REF] Chopin | Aquaculture, Integrated Multi-trophic (IMTA)[END_REF]. Still, this raises a question on density rather than the diversity of the species.

Macroalgae in Mexico

The coastline of Mexico extends for 11 769 km of which 72% is located at the Pacific Coast and 28% at the Gulf of Mexico and the Caribbean coast [START_REF] Robledo | The Seaweed Resources of Mexico[END_REF]. They include five geographic regions in temperate to tropical latitudes, Baja California, Gulf of California, Tropical Pacific, Gulf of Mexico, and the Mexican Caribbean with distinctive physiographic, geological, and climatic conditions make possible the existence of a very diverse algal flora. The Gulf of Mexico and the Caribbean Sea (Fig. 2) are two outstanding marine ecosystems joining in the Yucatan channel. Even though the Yucatan Peninsula (known as a biotic province) occupies 17.4% of the total of the Mexican coast, the investigation of marine resources has not been intensively investigated [START_REF] Pech-Puch | Marine Natural Products from the Yucatan Peninsula[END_REF]. This particular geographical situation promotes the existence of high diversity and abundance of different marine algal species, which represent a potential source of bioactive compounds and food [START_REF] Morales | Screening of Antibacterial and Antifungal Activities of Six Marine Macroalgae from Coasts of Yucatán Peninsula[END_REF][START_REF] Vázquez-Delfín | Seaweed resources of Mexico: Current knowledge and future perspectives[END_REF][START_REF] Alemañ | Development of seaweed cultivation in Latin America: current trends and future prospects[END_REF]. show a greater species richness. In Mexico, 2,702 macroalgal species were described including freshwater (1,102 species) and marine (1,600 species) and nearly 800 species were reported in the Yucatan Peninsula.

The diversity of habitats in the Yucatan Peninsula favours the most diverse red algae group of the region, with approximately 350 recorded species, distributed in about 15 taxonomic families [START_REF] Dreckmann | Clasificación y nomenclatura de las algas marinas bentónicas del Atlántico mexicano[END_REF][START_REF] Ortega | Catálogo de las algas bénticas de las costas mexicanas del Golfo de México y Mar Caribe[END_REF][START_REF] Mendoza-González | Yucatán seaweeds from the off shore waters of Isla Mujeres, Quintana Roo, Mexico[END_REF], 2016;[START_REF] Wynne | A checklist of benthic marine algae of the tropical and subtropical western Atlantic: fourth revision[END_REF]. In spite of their high diversity algal drifts, however, in this region of Mexico pose a challenge rather than their economic importance. In Yucatan, the most abundant families that drift for both the number of species and the amount of biomass were Gracilariaceae (10 species), Rhodomelaceae (6 species) and Solieriaceae (3 species) [START_REF] Resendiz | Marine red algae (Rhodophyta) of economic use in the algal drifts from the Yucatan Peninsula, Mexico[END_REF].

In Mexico, for about 50 years, the commercial exploitation of phycocolloid-producing algae has occurred continuously and they exported 439 tonnes of macroalgae in 2009 [START_REF] Robledo | Social and economic dimensions of carrageenan seaweed farming in Mexico. In: Social and economic dimensions of carrageenan seaweed farming[END_REF]. The commercial harvesting of natural populations of macroalgae includes three species of Rhodophyta, Gelidium robustum, Chondracanthus canaliculatus, and Gracilariopsis lemaneiformis [START_REF] Robledo | The Seaweed Resources of Mexico[END_REF]) and one species of Phaeophyta, Macrocystis pyrifera [START_REF] Robledo | Social and economic dimensions of carrageenan seaweed farming in Mexico. In: Social and economic dimensions of carrageenan seaweed farming[END_REF].

The Peninsula of Yucatan has 1,940 km of coastline, reportedly with the rich diversity of macroalgae. Rhodophyta has the greatest diversity on the coasts of Yucatan. A large variation in the chemical composition of the macroalgae from Yucatan was observed, particularly in soluble carbohydrates, ash, and protein [START_REF] Robledo | Chemical and mineral composition of six potentially edible seaweeds from Yucatan[END_REF] which encouraged the experimental cultivation of native and exotic species during the years of 2000-2010. The lower seawater temperature may explain the abundance and diversity of macroalgal species as well the cold and warm water currents influence the distribution patterns of marine macroalgae [START_REF] Robledo | The Seaweed Resources of Mexico[END_REF]. The presence of structurally diverse secondary metabolites is invaluable due to its potential application in drug discovery [START_REF] Stengel | Algal chemodiversity and bioactivity: Sources of natural variability and implications for commercial application[END_REF][START_REF] Goulitquer | Mass Spectrometry-Based Metabolomics to Elucidate Functions in Marine Organisms and Ecosystems[END_REF][START_REF] Altmann | Drugs from the oceans: Marine natural products as leads for drug discovery[END_REF][START_REF] Wells | Algae as nutritional and functional food sources: revisiting our understanding[END_REF][START_REF] Hernández-Bolio | Natural Products from the Yucatecan Flora: Structural Diversity and Biological Activity[END_REF][START_REF] Pech-Puch | Marine Natural Products from the Yucatan Peninsula[END_REF]. Being a most conspicuous component on the Yucatan coasts, red algal species are highly valued in the international market for their secondary metabolites [START_REF] Resendiz | Marine red algae (Rhodophyta) of economic use in the algal drifts from the Yucatan Peninsula, Mexico[END_REF].

Part -II-Red Macroalgae

Introduction

Rhodophyta represents an ancient photosynthetic lineage of eukaryotes that arose through primary endosymbiosis, with a fossil record dating back over 1.2 billion years [START_REF] Butterfield | Bangiomorpha pubescens n. gen., n. sp.: Implications for the evolution of sex, multicellularity, and the Mesoproterozoic/ Neoproterozoic radiation of eukaryotes[END_REF]. Presently the phylum contains about 7303 species (Guiry and Guiry 2020) of which 3% is freshwater taxa [START_REF] Sheath | Systematics of Bangia (Rhodophyta) in North America. I. Biogeographic trends in morphology[END_REF]. Based on molecular and morphological studies, [START_REF] Saunders | Assessing red algal supraordinal diversity and taxonomy in the context of contemporary systematic data[END_REF] identified three subphyla in the Rhodophyta: Metarhodophytina, Rhodellophytina, and Eurhodophytina. With the subphylum Eurhodophytina containing macroalgae divided into two classes: Florideophyceae (6950 species) and Bangiophyceae (182 species). Florideophyceae regroups many orders: Ahnfeltiales (11 species), Ceramiales (2690 species), Corallinales (603 species), Gelidiales (235 species), Gigartinales (951 species), Gracilariales (238 species), Halymeniales (357 species), Nemaliales (281 species), Palmariales (45 species), and Rhodymeniales (410 species). Bangiophyceae is a taxonomic unit less diversified. Among the two orders constitutive of the class, the order Bangiales regroups 182 recognized species with specimens of the genera Bangia and Porphyra/Pyropia [START_REF] Schneider | A synoptic review of the classification of red algal genera a half century after Kylin's "Die Gattungen der Rhodophyceen[END_REF][START_REF] Verbruggen | Data mining approach identifies research priorities and data requirements for resolving the red algal tree of life[END_REF]Yoon et al. 2016;[START_REF] Adl | Revisions to the Classification, Nomenclature, and Diversity of Eukaryotes[END_REF].

Many red algae are found in the intertidal and subtidal zones to depths of up to 40, or occasionally, 250 m, and of course, they have the ability to live at greater depths in the ocean than the members of other algal groups. They probably represent one of the oldest groups of eukaryotes and most diversified groups.

From a morphological point of view, the majority of Rhodophyta is filamentous (Antithamnion), pseudoparenchymatous (Palmaria), or parenchymatous (only in Bangiales, Porphyra/Pyropia). Ceramium and Polysiphonia present a vegetative and siphoned filamentous. Some species are calcified. Corallinales contain a form of calcite (calcium carbonate) and are important contributors to the formation of tropical reefs. Red macroalgae are found in tropical, temperate and arctic regions, but are generally the most abundant in temperate regions and in tropical regions on sandy or rocky substrates [START_REF] Keith | Global diversity of marine macroalgae: Environmental conditions explain less variation in the tropics[END_REF]. The overall distribution of genus richness for Rhodophyta had a hotspot on the west coast of North America and also, they had the lowest genus richness in the tropics compared with higher altitudes (Fig. 3).

Red algae are considered as the most important source of many biologically active metabolites in comparison to the other algal class. The majority of macroalgal secondary metabolites has discovered in red macroalgae accounting for >1500 bioactives [START_REF] Amsler | Potential chemical defenses against diatom fouling in Antarctic macroalgae[END_REF][START_REF] Maschek | The Chemistry of Algal Secondary Metabolism[END_REF][START_REF] Wijesekara | Biological activities and potential health benefits of sulfated polysaccharides derived from marine algae[END_REF][START_REF] Stengel | Algal chemodiversity and bioactivity: Sources of natural variability and implications for commercial application[END_REF]. Meanwhile, the red macroalgae are wild-harvested from 32 countries, indicating their widespread popularity, with a total of 216 456 t harvested worldwide (FAO, 2014).

Biology of Red Macroalgae

One of the oldest groups of eukaryotic algae, red algae are an ancient group of eukaryotic plants and the majority of them are filamentous (Polysiphonia), pseudoparenchymatous (Ceramium) or parenchymatous (Porphyra/Pyropia, Halymenia). Rhodophyceae vary considerably in their response to different environmental factors, having adapted to living in nearly freshwater to brackish and marine conditions. Members of red algae are found in the tropical, temperate, and arctic waters, but are most abundant in the temperate and tropical regions. The common features of red algae include eukaryotic cells, a complete lack of flagellar structure, food reserves of floridean starch, the presence of phycobiliproteins (details in "Pigments and Proteins") chloroplasts without stacked thylakoids and no external endoplasmic reticulum [START_REF] Baweja | Biology of Seaweeds[END_REF]. The members of Rhodophyta can tolerate a wider range of light levels than any other groups of photosynthetic organisms and this is supported by the presence of additional accessory pigments together with chlorophyll a. These accessory pigments generally known as phycobiliproteins allow them to live in deep waters. The major product of photosynthesis in red algae is floridoside (O-α-dgalactopyranosyl-(1,2)-glycerol), isofloridoside digeneaside or floridean starch (details in 'Starch').

Another very common feature of red algae is pit connections providing symplastic communication between cells [START_REF] Lee | Phycology[END_REF]. Marine red algae contain significant quantities of vitamins, dietary fibers, proteins, polysaccharides, and various micro-and macro elements [START_REF] Baweja | Biology of Seaweeds[END_REF].

i. Cell wall

The cell wall organization and composition of the Rhodophyta differ significantly from plants and other macroalgae. The cell wall is a rather well-ordered structure as observed by optical and electronic microscopy [START_REF] Tas | Carrageenans in the cell walls of Chondrus crispus Stack. (Rhodophyceae, Gigartinales)[END_REF][START_REF] Craigie | Cell walls[END_REF]). The cell wall is considered here as including polymeric materials originating from the metabolic activities of the algae. Most investigators recognize polysaccharides, proteoglycans, peptides, proteins, lipids, and associated inorganic constituents as components of the native cell wall of red algae. According to previous studies on biological function, the polysaccharides may be grouped with the more rigid structural (β-linked) glycans such as cellulose, mannans, and xylans, as well as with the more flexible and frequently sulfated glycans that comprise the matrix in which the skeletal fibers are embedded [START_REF] Rees | Polysaccharide shapes and their inter-actions-some recent advances[END_REF][START_REF] Baweja | Biology of Seaweeds[END_REF].

The wall of red algae is classically illustrated by two phases or matrices:

1) neutral lower crystalline form with cellulose that forms a weft-like mat [START_REF] Dawes | A light-and electron-microscopic survey of algal cell walls. I. Phaeophyta and Rhodophyta[END_REF]. Some xylan and mannan have been also characterized in the amorphous embedding matrix.

2) charged phase that contains sulphated galactan polymers, some of which are economically important phycocolloids (carrageenan or agar). The cell wall consists of rigid components such as microfibrils and a mucilaginous matrix. (Fig. 4) [START_REF] Stiger-Pouvreau | Carbohydrates from Seaweeds[END_REF].

2016).

Under the optical microscope and in electron microscopic studies Gracilaria corticata reveals cells having thick cell walls that have microfibrils arranged in three distinct layers: (i) the inner-most electrondense (glycoprotein domain), (ii) middle electron-translucent (amorphous matrix) and (iii) outermost electron-dense regions (fibrillar wall). In all three regions, microfibrils are arranged in parallel. The extracellular matrix consists of a cellulose microfibrillar network and an amorphous matrix of cellulose, sulphated galactans, and mucilage.

ii. Polysaccharides: Structural and Storage

a. Carrageenans

Red seaweeds contain large amounts of cell-wall polysaccharides, most of which are sulphated galactans. These galactans are generally built on repeated alternating l,3-linked α-galactopyranose and l,4linked β-galactopyranose units and differ in the level and pattern of sulphation, in the substitution of methoxyl and/or pyruvate groups and in other sugar residues (galactose, xylose). They also differ in 3,6anhydrogalactose content and the configuration of the 1-3-linked α-galactopyranose residue [START_REF] Percival | Chemistry and enzymology of marine algal polysaccharide[END_REF][START_REF] Craigie | Cell walls[END_REF][START_REF] Usov | Structural analysis of red seaweed galactans of agar and carrageenan groups[END_REF]). Among these galactans, carrageenans and agars are widely used as gelling or thickening additives by the food industry and in biotechnologies.

The main carrageenans on the market belong to kappa (κ-; G4S-DA), iota (ι-; G4S-DA2S), and lambda (λ-; G2S-D2S,6S) (Fig. 5). Minor types are mu (µ-; G4S-D6S) and nu (ʋ-; G4S-D2S6S), which are κand ι-precursors, respectively (Fig. 5). 'G' defines the units linked in α (1)(2)(3) and 'D' for the units linked in β (1)(2)(3)(4) and the substituents linked to the units were named as 'M' for methyls and 'S' for sulphates and they can be linked to the carbons from 1-6. The 3, 6 anhydrogalactose units (3,6 AG) are differentiated by the letters DA [START_REF] Knutsen | A modified system of nomenclature for red algal galactans[END_REF]. Natural carrageenans usually occur as mixtures of different hybrid types, such as κ/ι-hybrids, κ/μ-hybrids or μ/ι-hybrids, which often form cyclized derivatives. Especially, the repeating unit of κ-carrageenan is composed of a D-galactose with a sulphated group at C4 linked to a hydrogalactose, the repeating unit of λ-carrageenan is constructed by a D-galactose with a sulphated group at C2 linked to a D-galactose sulphated at C2 and C6, and the repeating unit of ιcarrageenan consists of galactose with a sulphated group at C4 linked to an anhydrogalactose sulphated at C2 [START_REF] Vera | Seaweed polysaccharides and derived oligosaccharides stimulate defense responses and protection against pathogens in plants[END_REF].

Carrageenans constitute 30%-75% of the red algal cell wall by their dry weight and are extensively used for stabilizing and texturing products in the food industry, which accounts for 70-80% of the total world carrageenan production estimated at about 60,000 tonnes/year with a value of US$629 million [START_REF] Mchugh | A guide to the seaweed industry[END_REF][START_REF] Bixler | A decade of change in the seaweed hydrocolloids industry[END_REF][START_REF] Naseri | Biochemical and nutritional composition of industrial red seaweed used in carrageenan production[END_REF]. Based on this, and on assuming a yield of carrageenan extraction in the industrial scale of 20%, it can be estimated that the total dried macroalgal consumption was at least 300,000 tonnes/year [START_REF] Naseri | Biochemical and nutritional composition of industrial red seaweed used in carrageenan production[END_REF].

b. Agar

Agar consists of a family of compounds mainly constituted by agarose and agaropectin. Agarose is a polymer of repeating residues of 3-β-D-galactopyranose and 3,6-anhydro-α-L-galactopyranose. Agar possesses the property of being insoluble in cold water. The principal distinguishing feature of the less sulfated agars is the presence of D-galactose, L-galactose, or anhydro L-galactose. The seminal concept of the masked repeating structure first reported for the agar-like porphyran and the repeating units may be substituted or modified in a number of ways to mask the underlying pattern. In that case, the agarobiose repeating structure of agars may be masked by replacing 3,6-anhydro-L-galactose with L-galactose, and/or adding methyl ethers, sulfate hemiesters, and, exceptionally, pyruvic acid ketal at specific sites on either glycosyl unit [START_REF] Stiger-Pouvreau | Carbohydrates from Seaweeds[END_REF]. Agar is an important phycocolloid, occurs in members of the orders Gelidiales with over 200 species and Gracilariales with 250 taxa [START_REF] Dawes | Macroalgae systematics[END_REF].

c. Starch

The main characteristic reserve storage polysaccharide of red macroalgae is floridean starch, first described by Kutzing in 1843 [START_REF] Stiger-Pouvreau | Carbohydrates from Seaweeds[END_REF]. Floridean starch represents the major sink for photosynthetically fixed carbon and, under certain growth conditions, floridean starch granules can amount to as much as 80% of the total cell volume [START_REF] Ekman | Effects of altered salinity, darkness and algal nutrient status on floridoside and starch content, α-galactosidase activity and agar yield of cultivated Gracilaria sordida[END_REF]. The starch from red algae also differs from higher plant starches in its apparent lack of amylose (except in some unicellular species) [START_REF] Mccracken | Amylose in floridean starch[END_REF]. The floridean starch a branched α-1,4-glucosidic linked glucose homopolymer with α-1,6branches (Fig. 6) functioning as carbon and energy reserve in the cells [START_REF] Yu | Enzymes of floridean starch and floridoside degradation in red alga. Purification, characterization and physiological studies[END_REF]. They were stored in the cytoplasm as a major reserve food and also has an osmoregulatory function [START_REF] Dawes | Macroalgae systematics[END_REF].

Fig. 6 Floridean starch

Red algae constitute an exception to this rule, unlike chlorophytes, as they synthesize and store starch as granules outside their plastids in the cytosol [START_REF] Pueschel | Cell Structure[END_REF]). Compositionally, floridean starch granules are constructed from a polymer more similar to amylopectin than glycogen [START_REF] Peat | Carbohydrates of the red alga, Porphyra umbilicalis[END_REF][START_REF] Manners | α-1,4-Glucosans. Part XIV. The interaction of concanavalin-A with glycogens[END_REF][START_REF] Craigie | Storage products[END_REF] and with similar structural features to higher plant starches, e. g. a radially arranged fibrillar-like pattern and concentric layers [START_REF] Meeuse | Floridean Starch[END_REF][START_REF] Sheath | Ultrastructure of the floridean starch granule[END_REF].

The soluble organic carbon compounds are fairly diverse: floridoside and related compounds.

Floridoside (Fig. 7) is the principal low-molecular-weight carbohydrate present in all orders of the Rhodophyta except the Ceramiales, while the isofloridoside (Fig. 8) only occurs at significant concentrations in members of the Bangiales where it may exceed the levels of floridoside. Isofloridoside could be considered as resulting from the isomerization of floridoside rather than as a direct product of photosynthesis. In most members of the Ceramiales, digeneaside (O-α-D-mannopyranosyl-(1-2)-glyceric) (Fig. 9) is the dominant low-molecular carbohydrate [START_REF] Kremer | Carbon metabolism[END_REF].

iii. Pigments and Proteins

It is well known that red algae have high protein levels [START_REF] Pons | One-step purification of R-phycoerythrin from the red macroalga Palmaria palmata using preparative polyacrylamide gel electrophoresis[END_REF][START_REF] Wells | Algae as nutritional and functional food sources: revisiting our understanding[END_REF]. Reports have shown that they have almost 47% w/w of dry matter [START_REF] Sánchez-Machado | Fatty acids, total lipid, protein and ash contents of processed edible seaweeds[END_REF].

It is noteworthy that the protein content of macroalgae varies not only between species [START_REF] Fleurence | Seaweed proteins[END_REF] but also among seasonal periods, geographic location, culture conditions, and processes, biomass storage, and analytical methods used [START_REF] Mishra | Lipids of the red alga[END_REF][START_REF] Wells | Algae as nutritional and functional food sources: revisiting our understanding[END_REF]. Although the structure and biological properties of algal proteins are still relatively poorly documented, the amino acid composition among several species of algae is known [START_REF] Harnedy | Bioactive proteins, peptides, and amino acids from macroalgae[END_REF]. Most red algae contain all the essential amino acids and are a rich source of the acidic residues asparatic and glutamic acid [START_REF] Fleurence | Seaweed proteins[END_REF]. The predominance of acidic over basic amino acids is typical of red algae [START_REF] Pons | One-step purification of R-phycoerythrin from the red macroalga Palmaria palmata using preparative polyacrylamide gel electrophoresis[END_REF], their high levels being responsible for the algal flavor and taste [START_REF] Mabeau | New seaweed based ingredients for the food industry[END_REF]. Threonine, lysine, tryptophan, cysteine, methionine, and histidine have been shown to be present at low levels in macroalgal proteins. Phycobiliproteins (PBS), the water-soluble proteins (MacColl 1998) are the main proteins of the red algae, representing up to 50% of the total protein content. They are a family of fluorescent proteins covalently linked to tetrapyrrole groups, known as bilins, a prosthetic group [START_REF] Niu | Large-scale recovery of C-phycocyanin from Spirulina platensis using expanded bed adsorption chromatography[END_REF]). These proteins act as antennae, absorbing energy in the portions of the visible spectrum where chlorophyll barely does [START_REF] Sekar | Phycobiliproteins as a commodity: Trends in applied research, patents and commercialization[END_REF]. Unlike carotenoids and chlorophylls, phycobiliproteins are not part of the photosystems located in the lipid bilayer but constitute a structure attached to the cytoplasmic surface of thylakoid membranes named phycobilisomes.

The red colour of the algae results from the dominance of the pigments R-phycoerythrin and Rphycocyanin and these masks the other pigments, chlorophyll a (no chlorophyll b), beta carotene, and a number of unique xanthophylls. Chlorophyll-b is replaced by the chlorophyll-d. The chief xanthophyll is taraxanthin [START_REF] Bold | Introduction to the algae: structure and reproduction[END_REF]. R-phycoerythrin (phycoerythrin from Rhodophyta) (Fig. 10) is an oligomeric water-soluble chromoprotein characterized by an absorption spectrum with three peaks at 499, 545, and 565 nm [START_REF] Denis | Concentration and pre-purification with ultrafiltration of a R-phycoerythrin solution extracted from macro-algae Grateloupia turuturu: Process definition and up-scaling[END_REF]. The apparent molecular weight of R-phycoerythrin is approximately 240 -260 kDa. It has three protein (αβ)6γ subunits: α, β and γ, whose apparent molecular weights are 18, 20 and 30-33 kDa, respectively. R-phycocyanin (Fig. 11) is a minor pigment with an (αβ)3 trimeric structure and is the sole algal biliprotein possessing both phycoerythrobilin (PEB) and phycocyanobilin (PCB) prosthetic groups [START_REF] Zi-Xuan | Isolation and characterization of R-phycocyanin from Polysiphonia Urceolata[END_REF][START_REF] Dumay | Proteins and Pigments[END_REF]. The maximum absorption is observed at 533 and 544 nm and the molecular weights of R-phycocyanin subunits, α and β are 18 and 20 kDa [START_REF] Glazer | Characterization of R-phycocyanin, chromophore content of R-Phycocyanin and C-Phycoerythrin[END_REF]. Allophycocyanin (Fig. 12) is composed of a trimer (αβ)3 with a molecular weight of 110 kDa with maximum absorption at 650 nm. Each subunit of R-phycoerythrin has two PEB chromophores and the R-phycocyanin has one PEB and one PCB chromophores whereas both the subunits of allophycocyanin possess one PCB chromophore [START_REF] Dumay | Proteins and Pigments[END_REF].

iv. Lipids

Despite the lower amount of lipids and fatty acids in red macroalgae, there is a significantly higher level of polyunsaturated fatty acids that act as strong antioxidants, such as omega-3 (n=3) and omega-6 (n=6) [START_REF] Mendis | Present and future prospects of seaweeds in developing functional foods[END_REF]. They are known to contain around twenty carbon-chain fatty acids: eicosapentaenoic acid (EPA, ω-3 C20:5) and arachidonic acid (AA,. Red algae contain a proportion of terpene compounds, tocopherols, and sterols and they differ from others by the presence of oxylipins, oxygenated fatty acids, known to intervene in cell differentiation, immune responses and homeostasis [START_REF] Gerwick | Biogenesis and biological function of marine algal oxylipins[END_REF]. Glycolipids are glycosylated derivatives of glycerols and ceramides, known as glycoglycerolipids and glycosphingolipids, respectively and they play an important role in energy transfer during photosynthesis. Their localization within the membrane contributes to the structural stability of bilayer, to the protection and regulation of any chemical stress and they also act as markers in cell communication [START_REF] Boudière | Glycerolipids in photosynthesis: Composition, synthesis and trafficking[END_REF]. Similarly, the phospholipids are represented as glycophospholipids and sphingophospholipids and they are formed of two fatty acids and a phosphate group binding to different polar compounds such as glycerol, serine, choline, ethanolamine, and inositol. In glycophospholipids, the fatty acids are linked to glycerol via an ester bond to the phosphate group whereas in sphingophospholipids it is linked by an amino alcohol structure of sphingosine type [START_REF] Terme | Selective extraction of lipid classes from Solieria chordalis and Sargassum muticum using supercritical carbon dioxide and conventional solid-liquid methods[END_REF].

Species under Study -Halymenia floresii

The genus Halymenia (Halymeniaceae, Rhodophyta) currently includes 76 taxonomically accepted species [START_REF] Guiry | [END_REF] out of the 178 species names and 44 intraspecific in the database (www.algaebase.org). Recent studies on phylogenetic analyses revealed that Halymenia is a polyphyletic genus, which requires further taxonomic studies [START_REF] Tan | Foliose Halymenia species (Halymeniaceae, Rhodophyta) from Southeast Asia, including a new species, Halymenia malaysiana sp[END_REF], Araujo et al. 2016). The genus is mainly characterized by gelatinous thalli, the presence of anticlinal filaments and refractive ganglionic cells in the medulla, stellate cells in the inner cortex, and auxiliary cell ampullae with branched secondary filaments [START_REF] Balakrishnan | Studies on Indian Cryptonemiales III. Halymenia C. A. Agardh[END_REF][START_REF] Abbott | Studies in some foliose red algae of the Pacific coast. I. Cryptonemiaceae[END_REF][START_REF] Chiang | Morphological studies of red algae of the family Cryptonemiaceae[END_REF][START_REF] De Smedt | Morphology and systematics of the genus Halymenia C. Agardh (Halymeniales, Rhodophyta) in the Philippines[END_REF][START_REF] De Azevedo | Diversity of branched Halymenia (Halymeniales, Rhodophyta) species on the Brazilian coast: molecular and morphological analyses reveal three new species[END_REF]. ampullae as a primary feature to group species at the generic level in the Halymeniaceae [START_REF] Tan | Foliose Halymenia species (Halymeniaceae, Rhodophyta) from Southeast Asia, including a new species, Halymenia malaysiana sp[END_REF].

Classification

Halymenia is a widespread genus distributed throughout the world's oceans [START_REF] Rao | Algae of India : Vol. 3: A Checklist of Indian Marine Algae : Excluding Diatoms and Dinoflagellates by[END_REF], but mainly in tropical and subtropical regions [START_REF] Gargiulo | Structure and reproduction of Halymenia asymmetrica sp. nov. (Rhodophyta) from the Mediterranean Sea[END_REF][START_REF] Kawaguchi | Morphology and culture study of a red alga, Halymenia dilatata Zanardini, from Vietnam and Japan[END_REF][START_REF] Hernández-Kantun | Morphology and anatomy of Halymenia actinophysa (Halymeniales, Rhodophyta) from the southwestern Gulf of California, Mexico[END_REF]. This genus presents mucilaginous to softly cartilaginous thalli, cylindrical or foliose, narrowed or wide fronds or entire, cleft, or branched. The thallus is described as an erected bladelike, with a discoid holdfast with or without a short stipe [START_REF] Guiry | [END_REF].

The species, H. floresii (Fig. 13), is named to honour D. Francisco Flores and the epithet should accordingly be "floresii" not "floresia" as commonly named in the literature. Halymenia floresii (Clemente) C. Agardh, is widely distributed in warm temperate to tropical seas.

Despite their widespread availability the studies on this species, H. floresii, are sparingly made to date.

Until now Halymenia has not been exploited and cultivated commercially although it is an interesting source of raw material for carrageenan and bioactive studies [START_REF] Robledo | Prospects for the cultivation of economically important carrageenophytes in Southeast Mexico[END_REF][START_REF] Freile-Pelegrín | Prospects for carrageenan production in tropical waters of Yucatan peninsula[END_REF]. The microtopography of H. floresii was observed under the different microscopes is shown in Fig. 14. The epifluorescence images depict the intact cortical cells of the alga (Fig. 14 (d)) and the electron micrographs showed a thick matrix of polysaccharide over the cortex (Fig. 14 (f)). An ecophysiological study on H. floresii from the Mexican coast related to growth and chromatic adaptation evidenced the presence of highly sulphated carrageenan, with minor amounts of 3,6anhydrogalactose of lambda-family carrageenan (Godínez- [START_REF] Godínez-Ortega | Growth and pigment composition in the red alga Halymenia floresii cultured under different light qualities[END_REF][START_REF] Freile-Pelegrín | Preliminary characterization of carrageenan from the red seaweed Halymenia floresii[END_REF].

The crude sulphated polysaccharide of H. floresii has been reported to exhibit anticoagulant and antibacterial activity [START_REF] Rodrigues | Anticoagulant activity of sulfated polysaccharides fractions from an aqueous extract obtained from the red seaweed Halymenia floresia (Clemente) C. Agardh[END_REF][START_REF] Meesala | Isolation and structure elucidation of halymeniaol, a new antimalarial sterol derivative from the red alga Halymenia floresii[END_REF]. Halymeniaol isolated from H. floresii off the west coast of India showed significant antimalarial activity [START_REF] Meesala | Isolation and structure elucidation of halymeniaol, a new antimalarial sterol derivative from the red alga Halymenia floresii[END_REF] In addition, [START_REF] Polat | Biochemical composition of some red and brown macro algae from the Northeastern Mediterranean Sea[END_REF] observed a high protein content in H. floresii of 3.05% of its dry weight and 2.46% lipids.

In one of the study areas of this work, H. floresii is cultivated in a IMTA system, a land-based outdoor culture system located at the Cinvestav Coastal Marine Station at Telchac, Yucatan, Mexico (21°20´28´ N, 89°18´25´ W) [START_REF] Peñuela | Environmentally Friendly Valorization of Solieria filiformis (Gigartinales, Rhodophyta) from IMTA Using a Biorefinery Concept[END_REF]). The IMTA system designed for the present study is described as follows: a circular 2000 L tank was stocked with 19 adult snooks (Centropomus undecimalis).

The effluent from this tank was connected to a circular 700 L tank used as sedimentation tank to trap particulate organic matter. In the sedimentation tank, 12 chocolate chip sea cucumbers (Isostichopus badionotus) were placed to feed with the excess organic matter generated. Finally, the effluent from these tanks were used to cultivate H. floresii at a density of 3 g fresh weight L -1 . H. floresii vegetative fragments were placed into 10 replicate 50 L containers supplied with a continuous flow of wastewater from the sedimentation tank (Figure 3). All tanks and containers were continuously aerated.

Other species of Halymenia have also been studied around the world. The alcoholic extract of Halymenia porphyroides, collected from the Gujarat coast, exhibited moderate hypotensive and diuretic activities [START_REF] Lakshmi | Bioactivity of marine organisms: Part IX -Screening of some marine flora from the Indian coasts[END_REF]. Carrageenan, a sulphated polysaccharide, has been isolated from H. porphyroides, while a large number of saturated and unsaturated fatty acids have been isolated and characterized from it by Shameel. Lai et al. (1994) carried out detailed investigations on sulphated polysaccharides obtained from the genus, Halymenia, and reported that due to high heterogeneity, these polysaccharides did not resolve well. H. durvilliae contains a high level of iodine (0.25% dry weight), magnesium (1.65%) and zinc (25.69 ppm) [START_REF] Lakshmi | Bioactivity of marine organisms: Part IX -Screening of some marine flora from the Indian coasts[END_REF].

To the best of our knowledge, this is the first of this kind on this species, H. floresii, regarding surface-associated metabolites, which ultimately led to metabolite fingerprinting of the species, and its surface-associated microbiome.

Part -III-Abiotic and Biotic parameters 1.3.1 Introduction

The external parameters such as the geographic location, environmental, season, sampling conditions, ocean warming, and acidification have direct physiological effects on habitat-forming species and thus directly affecting their secondary and primary metabolites [START_REF] Stengel | Algal chemodiversity and bioactivity: Sources of natural variability and implications for commercial application[END_REF][START_REF] Lalegerie | The stressful life of red and brown seaweeds on the temperate intertidal zone: effect of abiotic and biotic parameters on the physiology of macroalgae and content variability of particular metabolites[END_REF]. Thus, to protect and adapt themselves against these abiotic (temperature, hydrodynamism, salinity, etc.) and biotic (epiphytism, herbivory, etc.) (Fig. 15) stresses, macroalgae produce a wide range of metabolites in order to ensure effective photosynthesis, respiration, growth, and reproduction (Stengel et al., 2011). Indirectly, these stressors change the species' interactions among themselves as well as between other organisms. They especially alter the microbial communities and disturb the symbiotic relationships [START_REF] Qiu | Future climate change is predicted to affect the microbiome and condition of habitatforming kelp[END_REF][START_REF] Van Der Loos | The Macroalgal Holobiont in a Changing Sea[END_REF]. As a consequence, there is a crucial shift from complex and productive forests to simpler, less productive habitats, with significant impacts on ecosystem services [START_REF] Connell | Recovering a lost baseline: missing kelp forests from a metropolitan coast[END_REF][START_REF] Wahl | The responses of brown macroalgae to environmental change from local to global scales: direct versus ecologically mediated effects[END_REF][START_REF] Qiu | Future climate change is predicted to affect the microbiome and condition of habitatforming kelp[END_REF].

i. Hydrodynamism

Hydrodynamism can be referred to as water motions, due to the tidal cycle, and to waves or ocean currents resulting from winds and differences in the densities of water masses [START_REF] Hurd | Water motion[END_REF][START_REF] Lalegerie | The stressful life of red and brown seaweeds on the temperate intertidal zone: effect of abiotic and biotic parameters on the physiology of macroalgae and content variability of particular metabolites[END_REF]. Hydrodynamism influences the distribution and the vertical zonation of algae on the shore. This is mainly due to currents and waves which affect other environmental factors such as light penetration, temperature, and the availability of nutrients and all elements necessary for algal development; it also influences the distribution of fauna, indirectly affecting algae through spatial competition and potential impact of herbivores. Moreover, to survive on exposed sites, macroalgae must be resistant enough to remain attached to their substrate and withstand wave action [START_REF] Lüning | Temperature, Salinity, and Other Abiotic Factors[END_REF][START_REF] Hurd | Water motion, marine macroalgal physiology, and production[END_REF].

Hydrodynamism can also affect the growth, the morphology or the phenology of macroalgae [START_REF] Kraemer | Biomechanics and alginic acid composition during hydrodynamic adaptation by Egregia menziesii (Phaeophyta) juveniles[END_REF]) and the reproduction by affecting gametes and spore releases, and dispersal [START_REF] Gordon | Effects of water motion on propagule release from algae with complex life histories[END_REF][START_REF] Lalegerie | The stressful life of red and brown seaweeds on the temperate intertidal zone: effect of abiotic and biotic parameters on the physiology of macroalgae and content variability of particular metabolites[END_REF]. Moreover, water motion affects the algal nutrient uptake, impacting growth, and photosynthesis [START_REF] Gerard | In situ water motion and nutrient uptake by the giant kelp Macrocystis pyrifera[END_REF][START_REF] Hurd | Water motion, marine macroalgal physiology, and production[END_REF][START_REF] Lalegerie | The stressful life of red and brown seaweeds on the temperate intertidal zone: effect of abiotic and biotic parameters on the physiology of macroalgae and content variability of particular metabolites[END_REF]. Thus, hydrodynamism often makes the macroalgae acclimatize to a type of wave exposure, either sheltered (low hydrodynamism), exposed (high hydrodynamism), or the intermediate, semi-exposed environment. In addition, macroalgae can adapt their size, shape and direction to thrive/disperse with the flow and increase their resistance to currents [START_REF] Boller | The hydrodynamic effects of shape and size change during reconfiguration of a flexible macroalga[END_REF]. They also cope with this stress by having a strong holdfast, a flexible stipe, and blades. Its flexibility allows for bending toward the substratum as wave energy envelops them.

A stable substratum is found to be most important for algal growth in the intertidal area and attachment to rough substratum enhances settlement [START_REF] Baweja | Biology of Seaweeds[END_REF].

ii. Nutrients

To achieve their photosynthesis and growth, algae need nutrients that are essential to their development [START_REF] Lalegerie | The stressful life of red and brown seaweeds on the temperate intertidal zone: effect of abiotic and biotic parameters on the physiology of macroalgae and content variability of particular metabolites[END_REF]. Nutrient dynamics in the sea are inextricably linked to variations in physical processes. Either enhanced nutrient delivery from turbulent mixing or upwelling or enhanced stratification can lead to shifts in microbial assemblages, with significant consequences for nutrient cycling [START_REF] Cullen | Physical influences on marine ecosystem dynamics[END_REF]. The concentration of the nutrients is highly variable in the marine environment and availability greatly depends on the time of day or season. Moreover, nutrient concentrations depend on several abiotic factors, for instance, on hydrodynamism, since the water motion decreases the size of the water boundary layer of the macroalgae [START_REF] Hurd | Water motion, marine macroalgal physiology, and production[END_REF]. To cope with the spatial and temporal variations of nutrients, different species have adapted different strategies, like increased uptake during optimal seasons [START_REF] Hurd | An in vitro nitrate reductase assay for marine macroalgae: Optimization and Characterization of the enzyme for Fucus gardneri (Phaeophyta)[END_REF] and incorporating and storing more or less important nutrients in their cells for a shorter or longer time [START_REF] Gagné | Seasonal patterns of growth and storage in Laminaria longicruris in relation to differing patterns of availability of nitrogen in the water[END_REF].

iii. Desiccation

Being sessile organisms, macroalgae live attached to the substrate where they are faced with desiccation for a short or longer period depending on the location, tidal action, and the alternation of immersion and emersion [START_REF] Lalegerie | The stressful life of red and brown seaweeds on the temperate intertidal zone: effect of abiotic and biotic parameters on the physiology of macroalgae and content variability of particular metabolites[END_REF]. The loss of water induced by desiccation causes morphological changes and to avoid this different algal species display variable desiccation tolerance, for instance, some algae regularly subjected to emersion present a small specific surface to reduce desiccation, as species with a lower surface/volume ratio lose their water content less quickly [START_REF] Lüning | Temperature, Salinity, and Other Abiotic Factors[END_REF][START_REF] Flores-Molina | Desiccation stress in intertidal seaweeds: Effects on morphology, antioxidant responses and photosynthetic performance[END_REF][START_REF] Lalegerie | The stressful life of red and brown seaweeds on the temperate intertidal zone: effect of abiotic and biotic parameters on the physiology of macroalgae and content variability of particular metabolites[END_REF]. The ability of algae to withstand significant water loss and mostly to rehydrate quickly define their tolerance to drought, and not their ability to retain water i.e.

slower water loss, faster rehydration, and greater resistance to high water loss [START_REF] Contreras-Porcia | Tolerance pathways to desiccation stress in seaweeds[END_REF][START_REF] Lalegerie | The stressful life of red and brown seaweeds on the temperate intertidal zone: effect of abiotic and biotic parameters on the physiology of macroalgae and content variability of particular metabolites[END_REF].

iv. Temperature

Temperature is the major factor controlling survival, growth, reproduction and geographical distribution of macroalgae [START_REF] Yarish | Survival strategies and temperature responses of seaweeds belonging to different biogeographic distribution groups[END_REF]) and the macroalgae respond to it in three different ways:

genetic adaptation to the constant temperature of the living environment; phenotypic acclimatization; or short-term physiological regulation in response to the temporal temperature shocks [START_REF] Eggert | Seaweed Responses to Temperature[END_REF].

Moreover, the temperature tolerance of macroalgae depends on latitude [START_REF] Einav | Ecophysiological adaptation strategies of some intertidal marine macroalgae of the Israeli Mediterranean coast[END_REF]. Elevated seawater temperatures resulting from climate change impact metabolic activity as well as growth rates and reproduction. Apart from this temperature also affects the biochemical composition (e.g., lipids, proteins, and pigments) of the macroalgae in various ways [START_REF] Schram | Ocean warming and acidification alter Antarctic macroalgal biochemical composition but not amphipod grazer feeding preferences[END_REF][START_REF] Van Der Loos | The Macroalgal Holobiont in a Changing Sea[END_REF]. The effect of temperature changes perhaps more importantly concerns changing interactions between species across trophic levels. Alteration of the community structure and regime shifts ultimately result in the "dysbiosis", a microbial imbalance (van der Loos et al. 2019).

v. Light

As macroalgae are photosynthetic organisms, light is a key factor for their growth and distribution [START_REF] Lalegerie | The stressful life of red and brown seaweeds on the temperate intertidal zone: effect of abiotic and biotic parameters on the physiology of macroalgae and content variability of particular metabolites[END_REF] and its intensity plays an important role in the photosynthetic reactions. However, the requirements greatly vary depending on the depth and the algae density. Intense sunlight triggers multiple physiological responses and may also cause variations in their defensive capacity [START_REF] Saha | Defence chemistry modulation by light and temperature shifts and the resulting effects on associated epibacteria of Fucus vesiculosus[END_REF]. For their protection, algae are able to produce photoprotective compounds that will dissipate excessive energy [START_REF] Rastogi | Photoprotective compounds from marine organisms[END_REF][START_REF] Bhatia | Mycosporine and mycosporine-like amino acids: A paramount tool against ultra violet irradiation[END_REF]. Whereas under low light intensity algae,

particularly red algae, possess extra pigments called phycobiliproteins (Dumay et al. 2014). Algal growth and metabolism are profoundly influenced by the light characteristics [START_REF] Schramm | Factors influencing seaweed responses to eutrophication: some results from EU-project EUMAC[END_REF][START_REF] Wu | Effect of different light qualities on growth, pigment content, chlorophyll fluorescence, and antioxidant enzyme activity in the red alga Pyropia haitanensis (Bangiales, Rhodophyta)[END_REF], especially in the UV-A and UV-B range can be harmful, mainly for the early development stages [START_REF] Wiencke | Impact of UV-radiation on viability, photosynthetic characteristics and DNA of brown algal zoospores: implications for depth zonation[END_REF][START_REF] Karentz | Chemical defenses of marine organisms against solar radiation exposure: UV-absorbing mycosporine-like aminoacids and scytonemin[END_REF][START_REF] Lalegerie | The stressful life of red and brown seaweeds on the temperate intertidal zone: effect of abiotic and biotic parameters on the physiology of macroalgae and content variability of particular metabolites[END_REF]. Rather than pigments mycosporine-like amino acids are second group compounds that are reported to be photoprotective [START_REF] Rastogi | Photoprotective compounds from marine organisms[END_REF][START_REF] Sonani | Recent advances in production, purification and applications of phycobiliproteins[END_REF].

vi. Salinity

Salinity is an important abiotic factor since it is responsible for the local and/or regional distribution of algae. Extreme salinities decrease the growth rate and concentrations of secondary metabolites in the red alga, Acanthophora spicifera [START_REF] Pereira | Effects of salinity on the physiology of the red macroalga, Acanthophora spicifera (Rhodophyta, Ceramiales)[END_REF]. It has been identified as a major environmental determinant in structuring the microbial communities, showing a high species-level diversity and belowaverage phylogenetic diversity [START_REF] Lozupone | Global patterns in bacterial diversity[END_REF]. Thus, salinity influences the complex epibacterial community associated with the macroalgal host [START_REF] Lachnit | Specific epibacterial communities on macroalgae: Phylogeny matters more than habitat[END_REF][START_REF] Lachnit | Epibacterial community patterns on marine macroalgae are host-specific but temporally variable[END_REF]. This can be affected directly by environmentally induced physiological changes and/or indirectly, via altered biotic interactions. Such a complex shift in the epibacterial community may change the interactions between the host and the environment [START_REF] Stratil | Salinity affects compositional traits of epibacterial communities on the brown macroalga Fucus vesiculosus[END_REF].

Definition and Types of Biotic parameters

i. Herbivory

Herbivory is a key biotic influence on macroalgae. Its effects may be sustained over long periods, often in the face of physiological stress, because grazed individuals are rarely killed. Herbivores affect algal biomass, distribution, abundance, and deterrent chemistry [START_REF] Dethier | Seaweeds under stress: Manipulated stress and herbivory affect critical life-history functions[END_REF][START_REF] Cardoso | Sea urchin grazing preferences on native and non-native macroalgae[END_REF] and they also exert a differential grazing pressure on the native and non-native species of macroalgae where the generalist herbivores were also observed [START_REF] Cardoso | Sea urchin grazing preferences on native and non-native macroalgae[END_REF]). In the coastal communities, grazing is a structuring element because herbivores are able to significantly affect the amount of seaweed biomass available [START_REF] Lubchenco | A unified approach to marine plant-herbivore interactions. I. Populations and communities[END_REF][START_REF] Lalegerie | The stressful life of red and brown seaweeds on the temperate intertidal zone: effect of abiotic and biotic parameters on the physiology of macroalgae and content variability of particular metabolites[END_REF]. Herbivore pressures fluctuate as a function of available habitats and seasonal patterns. And the different stages of the macroalgal life cycle, make it difficult to escape herbivory. Whereas algae can avoid herbivores by growing in habitats where densities of herbivores are reduced, or attempt to escape in space or time by growing at such times when herbivore densities are low. Many algae may grow in crevices or holes where there is no herbivore accessibility. In addition, the strategies of coexistence of palatable and unpalatable algae and, the production of their most palatable tissues during periods of reduced herbivory are also adapted by the macroalgae [START_REF] Baweja | Biology of Seaweeds[END_REF].

ii. Biofilm

Biofilm formation is an interactive process affected by local hydrodynamics of the fluid environment, physicochemical properties of the surface, and behavioral response of bacterial colonisers [START_REF] Geesey | Bacterial behavior at surfaces[END_REF]. Biofilms are composed of bacteria, diatoms, fungi, and other microorganisms within a selfsynthesized polymer matrix (e.g., amino acids, glycoproteins, humic materials) (Fig. 12) (Schafer et al. 2002;[START_REF] Garg | Chemical characterization of a marine conditioning film[END_REF]. Although biofilms are important for macroalgae biology, they can also be detrimental to the host by competing for nutrients, interfering with the gaseous exchange, and forming a barrier to light, which ultimately reduces the photosynthesis rate. According to [START_REF] Wahl | Marine epibiosis. I. Fouling and antifouling: some basic aspects[END_REF], basibiont can be defined as any substrate organism, which is host to the epibiont. Epibionts are organisms growing attached to a living surface (here, the macroalgal host). The terms basibiont and epibiont describe ecological roles. Numerous sessile organisms may live either as basibiont or as epibiont, or both simultaneously (in an epibiosis of the second or third degree) according to circumstances. Epibiosis can be referred to as a non-symbiotic, facultative association between epibionts and basibionts. Rather there are two terms such as Epiphytes i.e. epibiotic plants (mostly algae) and Epizoans i.e. sessile epibiotic animals [START_REF] Wahl | Marine epibiosis. I. Fouling and antifouling: some basic aspects[END_REF].

Biofouling on the surface of macroalgae (called epibiosis) leads to the reduction of access to light, gases and nutrients, and probably increases grazing and infections by pathogens [START_REF] Wahl | The Second Skin: Ecological Role of Epibiotic Biofilms on Marine Organisms[END_REF]. This can lead to the degradation of macroalgal tissue and cause disease outbreak. The epiphytic microbial interactions and their host responses are discussed in detail in Chapter I.

iii. Epiphytism

In the marine environment, epiphytism is a widespread phenomenon that originates due to limited space availability and subsequently competition for a substrate [START_REF] Kersen | Epiphytes and associated fauna on the brown alga Fucus vesiculosus in the Baltic and the North Seas in relation to different abiotic and biotic variables[END_REF]. Following the establishment of primary biofilm, secondary colonisation occurs with the epiphytes and epizoans [START_REF] Lalegerie | The stressful life of red and brown seaweeds on the temperate intertidal zone: effect of abiotic and biotic parameters on the physiology of macroalgae and content variability of particular metabolites[END_REF]. From an anatomical perspective, the wide variety of algae that grows as epibionts on other algae represent a continuum between epiphytes and endophytes [START_REF] Leonardi | Diversity, phenomenology and epidemiology of epiphytism in farmed Gracilaria chilensis (Rhodophyta) in northern Chile[END_REF][START_REF] Da Gama | The antifouling defence mechanisms of marine macroalgae[END_REF].

The abundance of these epiphytic organisms, either prokaryotes or eukaryotes, is determined by abiotic factors such as wave motion, nutrient availability, and tides. The occurrence of epiphytes is specific to different algal frond segments and range of exposure during tides [START_REF] Kersen | Epiphytes and associated fauna on the brown alga Fucus vesiculosus in the Baltic and the North Seas in relation to different abiotic and biotic variables[END_REF]). Epiphytes play a major role in the growth and development of the host macroalgae as the epiphytes secrete metabolites that are important for their survival but may be harmful to their hosts. Five anatomical relationships between epiphytes and their host have been detected from the weak attachment to the host surface with no tissue damage (Type I), up to the deep penetration of the epiphyte into the host cortex, reaching the medullary tissue, and causing the destruction of the host's cells in the area around the infection (Type V) [START_REF] Goulitquer | Mass Spectrometry-Based Metabolomics to Elucidate Functions in Marine Organisms and Ecosystems[END_REF][START_REF] Lalegerie | The stressful life of red and brown seaweeds on the temperate intertidal zone: effect of abiotic and biotic parameters on the physiology of macroalgae and content variability of particular metabolites[END_REF]. Negative effects also include the shading of light and disruption of nutrient uptake by the host [START_REF] Baweja | Biology of Seaweeds[END_REF].

1.4 Part -IV -Influence of the stressors (abiotic and biotic parameters)

Introduction

Macroalgae have evolved a number of long-and short-term acclimation strategies to survive under constant changing and challenging environmental conditions. These involve changes in the thallus morphology [START_REF] Monro | Light quantity and quality induce shade-avoiding plasticity in a marine macroalga[END_REF], cell wall composition at the individual level [START_REF] Carmona | Light quality affects morphology and polysaccharide yield and composition of Gelidium sesquipedale (Rhodophyceae)[END_REF], differences in chloroplast morphology and thylakoid organization at the cellular level (long-term adaptation), and alterations in pigmentation, photosynthetic membrane composition, and functionality at the molecular level (short-term acclimation) [START_REF] Talarico | Light and adaptive responses in red macroalgae: An overview[END_REF].

Chemical Defence

Chemical defence response plays a critical role in the overall defence mechanism of the macroalgae.

The chemical defences of macroalgae such as natural products and oxidative bursts are among the diverse strategies, they employ to remain devoid of harmful epibionts, herbivores, foulers, etc. From the earliest defence response, the oxidative burst, it includes halogenation of the volatile and non-volatile compounds, wound-activated defences by converting the defensive precursors into toxins, production of endogenous chemical signals, oxylipins, and distance signaling by giving water-borne cues [START_REF] Potin | Biotic interactions of marine algae[END_REF]. The chemical defence of Bonnemaisonia hamifera was proven to be metabolically costly, where the algae with impaired defence metabolite production showed an average growth of 20% more than that of the normal metabolite production [START_REF] Nylund | Costs and Benefits of Chemical Defence in the Red Alga Bonnemaisonia hamifera[END_REF]. These defence metabolites are localized in specific structures of the red algae i.e. gland cells (Bonnemaisoniales) and/or at the surface and within the thallus (Delisea pulchra). These unique compartments pave the way for the metabolites to be delivered to the surface of the macroalgae (Steinberg and de Nys 2002). Since Laurencia (Ceramiales, Rhodomelaceae) is one of the most prolific red algae, it has been widely studied in the production of secondary metabolites derived from the sea [START_REF] Bawakid | Isolaurenidificin and bromlaurenidificin, two new C15-acetogenins from the red alga Laurencia obtusa[END_REF]. Sesquiterpenes, diterpenes, triterpenes, and acetogenins (characterized by the presence of halogen atoms in their chemical structures) have been found present in this alga [START_REF] Davis | Seaweed metabolite database (SWMD): A database of natural compounds from marine algae[END_REF].

Allelopathy, one of the direct competitive defence mechanisms, can be a potent way through which macroalgal communities are structured [START_REF] Xu | Allelopathic interactions between the opportunistic species Ulva prolifera and the native macroalga Gracilaria lichvoides[END_REF]. The pioneering marine experiments on allelopathy concerned competition among sessile invertebrates [START_REF] Goodbody | Inhibition of the development of a marine sessile community[END_REF][START_REF] Jackson | Alleopathy and spatial competition among coral reef invertebrates[END_REF][START_REF] Porter | Allelochemical interactions between sponges and corals[END_REF][START_REF] Gross | Allelopathy of aquatic autotrophs[END_REF][START_REF] Harlin | Allelochemistry in marine algae[END_REF]), but later it was also demonstrated that macroalgae have the ability to suppress natural competitors through allelopathic agents (de Nys, Coll and Price 1991; [START_REF] Paul | Chemically mediated interactions between macroalgae Dictyota spp. and multiple life-history stages of the coral Porites astreoides[END_REF][START_REF] Rasher | Macroalgal terpenes function as allelopathic agents against reef corals[END_REF][START_REF] Svensson | Novel chemical weapon of an exotic macroalga inhibits recruitment of native competitors in the invaded range[END_REF]). Thus, it can also be defined as a biological phenomenon by which an organism produces one or more biomolecules that affect the growth, survival, and/or reproduction of other organisms. These biomolecules, which are mostly secondary metabolites, are known as allelochemicals and are produced by certain plants, algae, bacteria, coral, and fungi. These compounds can have beneficial (positive allelopathy) or detrimental (negative allelopathy) effects on the target organism.

Allelopathic interactions are an important factor in determining species distribution and abundance within plant and plankton communities (Bacellar [START_REF] Mendes | Allelopathy as a potential strategy to improve microalgae cultivation[END_REF]. Allelochemicals produced by the macroalgae are a potential source of defensive strategy. Allelopathy can be stimulated or minimized by a number of biotic and abiotic factors (discussed in the previous section "Abiotic and Biotic parameters').

These factors determine the strength of allelopathic interactions [START_REF] Gross | Allelopathy of aquatic autotrophs[END_REF]. Frequently, the epiphyte growth on macroalgal tissue was prevented by allelopathic mechanisms (Harlin et al.1987).

i. Oxidative bursts

The accumulation of reactive oxygen species, known as an oxidative burst, either kills the attacking agent or signals to induce other defensive responses [START_REF] Potin | Biotic interactions of marine algae[END_REF]. The red alga Gracilaria conferta decreases the agarolytic bacterial pathogens by up-regulating bursts of hydrogen peroxide [START_REF] Weinberger | Response of Gracilaria conferta (Rhodophyta) to oligoagars results in defense agains agar-degrading epiphytes[END_REF]. More details are provided in Chapter I.

ii. Halogenation of volatile and non-volatile compounds

The macroalgal haloperoxidases are responsible for the formation of halogenated (iodinated or brominated) compounds. These haloperoxidases fall into two classes: vanadium-dependent haloperoxidases (VHPOs) and chloro-(CPOs), bromo-(BPOs), and iodoperoxidases (IPOs) [START_REF] Wever | Marine Vanadium-Dependent Haloperoxidases, Their Isolation, Characterization, and Application[END_REF]. Oxidation of halides (X -) into hypohalous acids (HXO) by vanadium-haloperoxidases results in the formation of volatile halogenated organic compounds that are often regarded as defensive compounds. The haloperoxidase system can deactivate the bacterial communication signals, AHLs (Acyl Homoserine Lactones) and thus prevent the biofilm formation especially in Gram-negative bacteria, a predominant bacterial group in the marine environment [START_REF] Potin | Biotic interactions of marine algae[END_REF].

Monoterpenes (two isoprene units), the most representative volatile compounds, are used by the macroalgae to defend against the epiphytes or grazers by releasing them into the surrounding seawater after halogenating them. Red algae are considered to be the main source of halogenated monoterpenes (linear or cyclic) with multiple halogen substitutions. Sesquiterpenes (three isoprenoid units), the primary components of marine biologically active substances, play an important role in reproduction and UV protection rather than defending against grazers and fouling organisms [START_REF] Peng | Chemical composition of seaweeds[END_REF]. Diterpenes, derived from geranylgeranyl pyrophosphate, show various types of bioactivity such as antimicrobial, antifungal, antifouling, cytotoxic, etc., (Gross and Königkönig 2006). Meroterpenoids, the prenylated aromatic compounds, may inhibit both the soft and hard fouling organisms [START_REF] Peng | Chemical composition of seaweeds[END_REF]. Three substituted aryl meroterpenoids isolated from Hypnea musciformis have proven to show potential antioxidative activities [START_REF] Chakraborty | Characterization of substituted aryl meroterpenoids from red seaweed Hypnea musciformis as potential antioxidants[END_REF].

iii. Distance signaling

Since the macroalgae are non-vascular plants (not evolved) they do not possess a mechanism to transfer the signals longitudinally. The red algae Chondrus crispus sporophytes bathed in the elicitor, oligosaccharides, show an induced transient resistance against Acrochaete operculata only in the bathed parts of the thallus [START_REF] Potin | Biotic interactions of marine algae[END_REF]. But still, macroalgae can be warned of aggressors by the conspecific neighbours. Recent advanced technologies (such as proteome, transcriptome, metabolome, and other biochemical analyses) have confirmed the role of various macroalgae constituents such as mannitol, proline, abscisic acid, polyamines, polyunsaturated fatty acids, oxylipins, and fatty acid desaturases among others that defend them from diverse environmental stress [START_REF] Egan | Bacterial pathogens, virulence mechanism and host defence in marine macroalgae[END_REF].

Microbial Defence

This part is well detailed and reviewed in Chapter I.

Part -V -Consequence of Holobiont break-up -Macroalgal Disease

Introduction

As dominant habitat-formers and primary producers in the marine ecosystem [START_REF] Longford | Interactions within the microbiome alter microbial interactions with host chemical defences and affect disease in a marine holobiont[END_REF], there is increasing evidence to indicate that they are highly susceptible to infectious diseases [START_REF] Gardiner | VarR controls colonization and virulence in the marine macroalgal pathogen Nautella italica R11[END_REF]. In their natural habitat, several biotic and abiotic parameters affect the macroalgal health but under cultivation, the overall unfavourable environmental conditions result in disease outbreaks [START_REF] Campbell | Climate change and disease: bleaching of a chemically defended seaweed[END_REF][START_REF] Gardiner | VarR controls colonization and virulence in the marine macroalgal pathogen Nautella italica R11[END_REF]. During the last 10 years, seaweed aquaculture has expanded rapidly due to increasing demand for edible seaweed, nutraceuticals, pharmaceuticals, antimicrobials and other compounds with biotechnological uses [START_REF] Cottier-Cook | Safeguarding the future of the global seaweed aquaculture industry[END_REF][START_REF] Alemañ | Development of seaweed cultivation in Latin America: current trends and future prospects[END_REF][START_REF] Shannon | Seaweeds as nutraceuticals for health and nutrition[END_REF]. Despite its niche market, intensive algal aquaculture might favour disease outbreaks [START_REF] Gachon | Algal diseases: spotlight on a black box[END_REF]. As the macroalgal aquaculture industry grows and diversifies into new species and geographical areas, new diseases are likely to emerge and the risk will intensify introducing non-indigenous pathogens to the new areas [START_REF] Cottier-Cook | Safeguarding the future of the global seaweed aquaculture industry[END_REF].

Bacterial diseases are one of the most crucial problems in aquaculture (Natrah et al. 2011a). These microbial infections are observed to be opportunistic in nature. Opportunistic pathogens are defined here as those that are present on both healthy and diseased hosts but only become harmful following a disturbance of their host [START_REF] Brown | Evolution of virulence in opportunistic pathogens: Generalism, plasticity, and control[END_REF]. Thus, identifying the host-associated bacteria is critical in disease management in aquaculture. While macroalgae are increasingly recognized to suffer from the disease, the causative agents are mostly unknown (Kumar et al. 2016). So, the identified microbial community must necessarily be differentiated for their beneficial and/or pathogenic interactions with the host.

Interactions between the hosts and their microbiomes are fundamental for host functioning and resilience [START_REF] Qiu | Future climate change is predicted to affect the microbiome and condition of habitatforming kelp[END_REF]) but such interactions can either be beneficial or detrimental. The disturbance and disruption of the mutualistic association between host and microbiota can be regarded as Holobiont break-up. This can be caused by environmental stress (van der Loos et al. 2019). For example, high organic matter content and high density of the cultured organisms increase the proliferation of opportunistic bacteria and this further induces stress making them more susceptible to diseases [START_REF] Bachère | Anti-infectious immune effectors in marine invertebrates: Potential tools for disease control in larviculture[END_REF]Natrah et al. 2011b). The holobiont breakup resulting from the dynamic shift from symbiotism to pathogenism is well discussed in Chapter I. Thus, we directly focus on the consequence of the Holobiont break up, that is, macroalgal disease.

Definition

The macroalgal disease was defined as "a continuing disturbance to the plant's normal structure and function such that it is altered in growth rate, appearance, or economic importance" [START_REF] Andrews | The Pathology of Marine Algae[END_REF]).

They are emerging as an important impact of global climate change, due to the effects of environmental change on host organisms and their pathogens [START_REF] Campbell | Climate change and disease: bleaching of a chemically defended seaweed[END_REF]. A rapid shift in environmental parameters, followed by fungal, bacterial, and algal infestations, cause the macroalgal disease outbreak [START_REF] Loureiro | Impacts of AMPEP on Epiphytes and Diseases in Kappaphycus and Eucheuma Cultivation[END_REF]. The recent development of intensive and dense aquaculture practices has worsened the situation [START_REF] Gachon | Algal diseases: spotlight on a black box[END_REF] which ultimately results in an increase in the prevalence of diseases and thus a decline in the yields (Ward et al. 2019).

Algal diseases

The study of macroalgal diseases is generally made on the red algae, Eucheuma, and Kappaphycus, particularly on the 'ice-ice' disease phenomenon [START_REF] Largo | Recent developments in seaweed diseases[END_REF]. Diseases reported so far in red algae include, 'rotten thallus syndrome' in Gracilariopsis heteroclada; 'epiphytic filamentous algae' in K. alvarezii; 'iceice' in K. alvarezii and K. striatus; 'red rot, Olpidiopsis and white spot disease in Pyropia (Prophyra) yezoensis; 'red rot and green-spot diseases' in P. tenera; and 'green-spot disease' in P. dentata, particularly in aquaculture (Ward et al. 2019). A commercially important carrageenophyte, K. alvarezii, is often plagued with 'ice-ice' disease due to pathogenic bacterial infections. Generally, ice-ice leads to whitening of the branches initiated with colour changes of the thalli, which become transparent in the end [START_REF] Riyaz | Characterization and identification of isolated bacteria from ice-ice disease infected seaweed Kappaphycus alvarezii[END_REF]). It is then followed by epiphyte infection which ultimately leads to reduced production in highdensity commercial farms [START_REF] Vairappan | Distribution and symptoms of epiphyte infection in major carrageenophyte-producing farms[END_REF] Pathogenic bacteria and fungi may cause rot symptoms and diseases such as bleaching. However, due to the fact that pathogenicity is often associated with the degradation of the cell wall, it is hard to distinguish true pathogens from saprophytic epiphytes [START_REF] Weinberger | Response of Gracilaria conferta (Rhodophyta) to oligoagars results in defense agains agar-degrading epiphytes[END_REF]. As many bacteria and fungi exhibit the potential to degrade algal cell walls, it should be noted that bacteria at first might be commensal and only later become harmful as a result of changes in the environment. The pathogen causing "chytrid blight" of Pyropia in Japan was recently isolated and named Olpidiopsis porphyrae Sekimoto, Yokoo, Kawamura et Honda [START_REF] Sekimoto | Taxonomy, molecular phylogeny, and ultrastructural morphology of Olpidiopsis porphyrae sp. nov. (Oomycetes, Straminipiles), a unicellular obligate endoparasite of Bangia and Porphyra spp. (Bangiales, Rhodophyta)[END_REF]. As the disease-causing agent was not a chytrid, the name of the disease was changed to Olpidiopsis disease. The causative agent of green-spot disease of Pyropia is still unknown. "Diatom felt" (i.e., large concentration of epiphytic diatoms) is produced by many different species of diatoms, but the symptoms and the extent of the damage to Pyropia have not been evaluated [START_REF] Kim | A revaluation of algal diseases in Korean Pyropia (Porphyra) sea farms and their economic impact[END_REF]. The agarolytic Pseudomonas sp. isolated from Pyropia dentata had carboxymethyl cellulase, xylanase, protease, and agarose activities, and was able to macerate algal tissue one week after inoculation at pH 7 and 30° C [START_REF] Park | Characteristics of a marine agarolytic Pseudomonas sp. from Porphyra dentata (Bangiales, Rhodophyta) and some properties of its extracellular agarase[END_REF]. Kordia algicida was identified as a 'significant pathogen' inducing bleaching in Agarophyton under laboratory conditions (16°C and a photon flux of density of 75 µmol m -2 s -1 ) when their natural microbiome was removed (Saha and Weinberger 2019)

Mechanisms of action of pathogen

Bleaching in G. verrucosa is characterized by whitening of the thalli at the base, middle and end of the young thalli [START_REF] Nasmia | Characterization and identification of bacteria isolated from seaweed Gracillaria verrucosa (Linn., 1758) infected by ice-ice[END_REF]) whereas in D. pulchra bleaching is distinctive to the mid-thallus [START_REF] Fernandes | Community structure and functional gene profile of bacteria on healthy and diseased thalli of the red seaweed Delisea pulchra[END_REF]. Bleaching in Halymenia floresii, the species for this study, is characterized by the localized loss of pigment at the apical ends similar to the red alga, Agarophyton [START_REF] Saha | Microbial "gardening" by a seaweed holobiont: Surface metabolites attract protective and deter pathogenic epibacterial settlement[END_REF]. [START_REF] Largo | Time-dependent attachment mechanism of bacterial pathogen during ice-ice infection in Kappaphycus alvarezii (Gigartinales, Rhodophyta)[END_REF] explained the mechanism of the Vibrio infection in seaweed thallus when the algae are under stress. The Vibrio would colonise and reproduce in the cell wall by using polysaccharide as its carbon source. Furthermore, Lin in [START_REF] Yulianto | Macroalgae cultivation of Kappaphycus alvarezii (Linn., 1758) vertically and symptoms of ice ice disease in the water of Pari Island[END_REF] explained that after 2-3 days, Vibrio entered the tissue up to the medullary seam by pumping the carrageenase enzyme which caused the thallus to become pale/white and its tissue to be soft and easily broken [START_REF] Nasmia | Characterization and identification of bacteria isolated from seaweed Gracillaria verrucosa (Linn., 1758) infected by ice-ice[END_REF].

The red rot disease of Pyropia begins with the appearance of distinct, small, red patches on the blades in areas where the zoospores of Pythium porphyrae germinated. The mycelium grows through the host cells killing them. The dead cells change colours, degenerate, and deteriorate forming small holes. The small holes merge into bigger holes, ultimately disintegrating the entire blade [START_REF] Kim | A revaluation of algal diseases in Korean Pyropia (Porphyra) sea farms and their economic impact[END_REF]. To date, several different species of bacteria are thought to be potential causative agents of green-spot disease in Pyropia. The infection process of green-spot disease in Pyropia starts when seawater temperature is below 10° C. The green-spot disease can degenerate a whole blade within a day or two. The blade does not undergo typical maceration process; rather, infected Pyropia cells burst successively from the initial infection site, forming a row of dead cells [START_REF] Kim | A revaluation of algal diseases in Korean Pyropia (Porphyra) sea farms and their economic impact[END_REF] Shifts in the microbial community in response to high temperatures have also been observed on crustose coralline algae (reef-building algae that fulfill an essential role in coral health). [START_REF] Webster | Elevated seawater temperature causes a microbial shift on crustose coralline algae with implications for the recruitment of coral larvae[END_REF] showed that, when seawater temperatures increased to 32°C, these calcified algae experienced bleaching (loss of pigmentation) and reduced maximum quantum yields (an indicator of the performance of photosystem II), together with a shift in the microbial community structure. Temperatures lower than 32°C also led to bleaching and reduced maximum quantum yields but no shifts in microbial community structure. Interestingly, algae cultured at 32°C recovered when temperatures were reduced to the ambient level (27°C), but this was not the case for algae maintained at 32°C. Controversially, a study on the effect of ocean acidification conditions on rhodoliths showed that the associated microbiota remained stable (whereas the communities in both the seawater and on dead rhodoliths shifted). The algae showed no sign of calcium carbonate biomass loss and even increased photosynthetic activity. This resilience to environmental stress was likely provided by the stability of the microbiome [START_REF] Cavalcanti | Rhodoliths holobionts in a changing ocean: Host-microbes interactions mediate coralline algae resilience under ocean acidification[END_REF]).

Impact of disease on the aquaculture sector

Macroalgal diseases are the biggest constraints experienced in the process of their cultivation [START_REF] Nasmia | Characterization and identification of bacteria isolated from seaweed Gracillaria verrucosa (Linn., 1758) infected by ice-ice[END_REF]. The commercial production of macroalgae has expanded greatly over the past century and much of this production is centered in Asia [START_REF] Kim | Seaweed aquaculture: Cultivation technologies, challenges and its ecosystem services[END_REF]Ward et al. 2019). They have been used

as food for centuries in Asia including China, Japan, and Korea. Though Asian countries remain the largest consumers these products are increasingly exported to Europe, North America, and Africa [START_REF] Mchugh | A guide to the seaweed industry[END_REF][START_REF] Chen | Cultured aquatic species information programme, Porphyra spp[END_REF][START_REF] Yang | A review of the bladed Bangiales (Rhodophyta) in China: history, culture and taxonomy[END_REF]Ward et al. 2019). The occurrence of algal diseases results in dramatic economic losses of 25-30% of harvested volumes of Saccharina japonica in China [START_REF] Wang | Assimilation of inorganic nutrients from salmon (Salmo salar) farming by the macroalgae (Saccharina latissima) in an exposed coastal environment: Implications for integrated multi-trophic aquaculture[END_REF]), 20%

of the output of Porphyra/Pyropia in Korea [START_REF] Kim | A revaluation of algal diseases in Korean Pyropia (Porphyra) sea farms and their economic impact[END_REF], and 15% reduction in eucheumatoid stocks in the Philippines [START_REF] Cottier-Cook | Safeguarding the future of the global seaweed aquaculture industry[END_REF]. In order to attempt to eliminate or mitigate the impact and spread of disease on aquaculture, several treatments or mitigation strategies have been used (Ward et al. 2019). The washing of Porphyra/Pyropia blades in acid solutions is a widespread practice and is often used in an attempt to control all diseases [START_REF] Kim | A revaluation of algal diseases in Korean Pyropia (Porphyra) sea farms and their economic impact[END_REF]. Changing cultivation conditions, particularly the repositioning of cultivation ropes to modify exposure to light and more favourable salinities, are effective measures that can be taken to reduce the severity of diseases caused by exposure of S. japonica to unfavourable abiotic conditions [START_REF] Wang | Assimilation of inorganic nutrients from salmon (Salmo salar) farming by the macroalgae (Saccharina latissima) in an exposed coastal environment: Implications for integrated multi-trophic aquaculture[END_REF]. The development of ice-ice symptoms in Kappaphycus and Eucheuma is thought to be the result of stress to the host from abiotic conditions, such as temperature, salinity [START_REF] Vairappan | Distribution and symptoms of epiphyte infection in major carrageenophyte-producing farms[END_REF], light intensity and water movement [START_REF] Hurtado | Occurrence of Polysiphonia epiphytes in Kappaphycus farms at Calaguas Is., Camarines Norte, Phillippines[END_REF] in combination with the action of opportunistic bacteria [START_REF] Uyengco | The "ice-ice" problem in seaweed farming[END_REF][START_REF] Largo | Occasional pathogenic bacteria promoting ice-ice disease in the carrageenan-producing red algae Kappaphycus alvarezii and Eucheuma denticulatum (Solieriaceae, Gigartinales, Rhodophyta)[END_REF]. The triggers behind disease onset and progression are not well understood, and as a result, no effective management protocols that are cost-effective have been developed to date (Ward et al. 2019).

The improvement of disease mitigation strategies in macroalgal aquaculture is reliant on increasing our understanding of the agents and abiotic factors influencing disease onset and progression and the subsequent development of biosecurity programs, including the use of quarantine procedures and improved farm management practices through capacity building [START_REF] Campbell | The environmental risks associated with the development of seaweed farming in Europe -prioritizing key knowledge gaps[END_REF]Ward et al. 2019).

With increasing knowledge of the macroalgal diseases and the range of micro-organisms associated with them, the paradigm of one-disease-one-pathogen is moving towards the pathobiome concept (Bass et al. 2019). As a result of interactions between the host and environmental factors, multiple host-associated organisms are linked with reduced host health status, which is regarded as a pathobiotic system (Ward et al. 2019). Despite serious issues and losses, macroalgal diseases have not drawn more attention. In their natural habitats, macroalgal diseases directly impact the infected organism and population, but may additionally have cascading effects on the ecosystem if keystone species are infected [START_REF] Gachon | Algal diseases: spotlight on a black box[END_REF][START_REF] Egan | Microbial dysbiosis: Rethinking disease in marine ecosystems[END_REF]. A greater understanding of the genetic diversity of cultured seaweeds, appropriate breeding strategies, and crop selection are required to retain this genetic diversity and safeguard the disease and pest resistant cultivars for future use by the industry (Ward et al. 2019).

Context and Aims of the Thesis

Halymenia floresii has been identified as a carrageenophyte with high potential for a land-based IMTA (Integrated MultiTrophic Aquaculture) system. By recycling inorganic nutrients, the IMTA system produces biomass while reducing the environmental impact on coastal ecosystems through responsible aquaculture practices (Godínez- [START_REF] Godínez-Ortega | Growth and pigment composition in the red alga Halymenia floresii cultured under different light qualities[END_REF][START_REF] Robledo | Prospects for the cultivation of economically important carrageenophytes in Southeast Mexico[END_REF]. During the experimental culture of Halymenia floresii, we observed that the surface of this macroalgae was exceptionally free of any fouling organisms compared to any other algae under the same culture conditions.

Even at high concentrations of Dissolved Inorganic Nitrogen (DIN ~ 150 µM) in IMTA system, the epiphytic macrofoulers (such as opportunistic green algae and/or any sessile invertebrates), which usually disturb the culture, were not observed in Halymenia floresii culture tanks [START_REF] Pliego-Cortés | Growth, biochemical and antioxidant content of Rhodymenia pseudopalmata (Rhodymeniales, Rhodophyta) cultivated under salinity and irradiance treatments[END_REF].

This ecological phenomenon could reveal that (i) the presence of H. floresii may protect the surface by releasing allelopathic active compounds that ultimately interfere with the settlement and growth of competitors ( [START_REF] Gal | Communication chimique[END_REF][START_REF] Harlin | Allelochemistry in marine algae[END_REF][START_REF] Gross | Allelopathy of aquatic autotrophs[END_REF] and (ii) the epibacterial community on the surface of H. floresii may prevent the settlement by their symbiotic relationship with the host. With these proposed hypotheses, we framed the objectives of this thesis as follows:

1. Selective extraction of the surface metabolites from H. floresii Chapter six is a general discussion that provides an overview of the major findings of this thesis.

In this context, I mobilized twice to Centro de Investigación y de Estudios Avanzados del Instituto

Politécnico Nacional (CINVESTAV), Mexcio.

Mobility: I

For a period of '4' months (March, 2018-July, 2018) to CINVESTAV, Mexico. Funds provided by ECOS-Nord CONACYT for the collaboration project M14A03 and PN-CONACYT 2015-01-118.

Mobility: II

For a period of '2' months (November, 2019-January, 2020) to CINVESTAV, Mexico. Financial support provided by Ecole Doctorale Science et Mer Littoral (EDSML).

Introduction

Macroalgae, being important habitat formers and primary producers in the marine ecosystem, provide potential settling substrate and protective environments for the early life and development of many invertebrates. Attached to their substrate, marine seaweeds are linked to their environment and confronted with various abiotic and biotic stresses. They are affected by wave action, light radiation, desiccation, and variations in salinity, temperature, nutrient supply, herbivore predation, epibiosis, microfouling and human activity. In the marine environment, competition for space and nutrients is intense. Each species lives in this complex environment according to its own physiological parameters, and its ability to resist changes in the state of the environment, particularly, during low tides or in reduced space. Due to the extraordinary complexity of the marine environment, seaweeds establish close associations with other species to ensure their survival and competitiveness for space.

Macroalgal surfaces represent a major physiological and highly functional interface with the environment. Such an interface is always threatened by fouling, and thus macroalgae need to develop many defence strategies to protect their surface from a vast number of consequences [START_REF] Wahl | The Second Skin: Ecological Role of Epibiotic Biofilms on Marine Organisms[END_REF]. Biofilms on solid surfaces are omnipresent and therefore macroalgae are of no exception to this. Biofilm formation is an interactive process affected by local hydrodynamics of the fluid environment, physicochemical properties of the surface and behavioural response of bacterial colonizers [START_REF] Geesey | Bacterial behavior at surfaces[END_REF]. In biofilms, the self-produced matrix is mainly composed of polysaccharides, proteins, and eDNA synthesized by the associated bacteria, diatoms, fungi, and other microorganisms [START_REF] Garg | Chemical characterization of a marine conditioning film[END_REF].

Host-microbe interactions play a crucial role in marine ecosystems, in a symbiotic community where the largest partner, based on its size, is considered as a host, e.g. macroalgae [START_REF] Dittami | A community perspective on the concept of marine holobionts: state-of-the-art, challenges, and future directions[END_REF].

The host and all of its associated microbiota, which can be acknowledged as all the microorganisms which are present or associated in a particular environment are now considered as a Holobiont [START_REF] Wahl | The Second Skin: Ecological Role of Epibiotic Biofilms on Marine Organisms[END_REF][START_REF] Hollants | What we can learn from sushi: a review on seaweedbacterial associations[END_REF][START_REF] Egan | The seaweed holobiont: understanding seaweed-bacteria interactions[END_REF][START_REF] Da Gama | The antifouling defence mechanisms of marine macroalgae[END_REF][START_REF] Duarte | The energetic physiology of juvenile mussels, Mytilus chilensis (Hupe): The prevalent role of salinity under current and predicted pCO 2 scenarios[END_REF]. The term 'holobiont' was coined by Lynn Margulis which referred to symbiotic associations that last for a significant part of an organism's lifetime (Cavalier-Smith, 1992). As it includes all the associated microbiota, their interactions may be harmful, beneficial or of no consequence, which may be coevolved or opportunistic, competitive, or cooperative [START_REF] Skillings | Holobionts and the ecology of organisms: Multi-species communities or integrated individuals?[END_REF]). The combined genetic information of the associated microbiota is known as a microbiome [START_REF] Skillings | Holobionts and the ecology of organisms: Multi-species communities or integrated individuals?[END_REF][START_REF] Dittami | A community perspective on the concept of marine holobionts: state-of-the-art, challenges, and future directions[END_REF] These microbiomes are functionally connected with their eukaryotic hosts and they are highly diverse. Specific functional genes of the associated microbiome are now regarded as secondary genomes [START_REF] Singh | Unraveling the Functions of the Macroalgal Microbiome[END_REF]. Any disturbances to the microbiome or even uncontrolled microbial colonization could be associated with stress and disease of the host organism [START_REF] Rohde | Decreased depth distribution of Fucus vesiculosus (Phaeophyceae) in the Western Baltic: effects of light deficiency and epibionts on growth and photosynthesis[END_REF][START_REF] Lachnit | Specific epibacterial communities on macroalgae: Phylogeny matters more than habitat[END_REF][START_REF] Morris | Nearshore Pelagic Microbial Community Abundance Affects Recruitment Success of Giant Kelp, Macrocystis pyrifera[END_REF][START_REF] Beattie | Novel ssDNA Viruses Detected in the Virome of Bleached, Habitat-Forming Kelp Ecklonia radiata[END_REF].

Lacking a cell-based adaptive immune response, macroalgae have two different defence capabilities: constitutive defence under normal circumstances, such as the production of metabolites by macroalgae or its bacterial symbionts and induced defence activated by any tissue damage [START_REF] Weinberger | Pathogen-induced defense and innate immunity in macroalgae[END_REF][START_REF] Egan | Bacterial pathogens, virulence mechanism and host defence in marine macroalgae[END_REF]. Surface-associated microbiome proved a wide range of bioactive compounds with a broad spectrum of activities, such as antibacterial, antifungal, antiviral, antiplasmodial, nematicidal, antiinflammatory, anti-cancer and antiangiogenic activities (Singh et al. 2015). Here we consider the defence provided by the macroalgae themselves through the production of secondary metabolites as 'Chemical

Defence' and the defence provided by the associated microbiota as 'Microbial/Biological Defence', however, it is now known that many of the bioactive compounds previously attributed to seaweeds were in fact produced or metabolized by their associated microbiota, the microbiome [START_REF] Wahl | The Second Skin: Ecological Role of Epibiotic Biofilms on Marine Organisms[END_REF][START_REF] Hollants | What we can learn from sushi: a review on seaweedbacterial associations[END_REF][START_REF] Egan | The seaweed holobiont: understanding seaweed-bacteria interactions[END_REF][START_REF] Da Gama | The antifouling defence mechanisms of marine macroalgae[END_REF]. In this association, a highly and dynamic complex set of chemical, physical and biological interactions regulates the relationships between the basibiont and its epibionts [START_REF] Singh | Seaweed-microbial interactions: key functions of seaweed-associated bacteria[END_REF]).

Seaweed surface-microbe interactionsbiofilm development

Biofilms, a consortia of surface-attached microbial cells dispersed in a self-secreted extracellular polymeric matrix, constitute the principal form of microbial growth and a widespread survival strategy amongst microorganisms [START_REF] Costerton | How Bacteria Stick[END_REF][START_REF] Donlan | Biofilms: Microbial Life on Surfaces[END_REF]). In the marine environment, surfaces are rapidly colonized by marine organisms in a process known as biofouling [START_REF] Callow | Marine biofouling: a sticky problem[END_REF]. The adsorption of dissolved macromolecules by the substratum is a preliminary step before bacterial colonization. The algal cell wall structure is a selective factor in the settlement of macromolecules and microorganisms. Bacteria are typically the primary colonizers of submerged surfaces. Marine macroalgae are known to produce secondary metabolites to defend themselves from the colonization of their exposed surfaces. In fact, these metabolites and pioneering bacterial species contribute to the chemical and physicochemical conditions responsible for a secondary recruitment of other microorganisms, but also sponges, macroalgae and invertebrates [START_REF] Dang | Bacterial primary colonization and early succession on surfaces in marine waters as determined by amplified rRNA gene restriction analysis and sequence analysis of 16S rRNA genes[END_REF]. In addition, multi-layered Extracellular Polymeric Substances (EPS), which include polysaccharides and also proteins, nucleic acids and lipids, form a base of biofilm structural organization (Brian-Jaisson et al. 2016; Lage and Graça 2016).

Seaweed Surface: as a substratum

A highly specific association exists between bacterial communities and marine macroalgae. The physiological and biochemical properties of the macroalgae surface predetermine the composition of the adhering microbial communities [START_REF] Goecke | Chemical interactions between marine macroalgae and bacteria[END_REF]. Macroalgae surfaces provide a rich habitat and release large amounts of organic carbon and oxygen into the surrounding environment, providing nutrients for microorganisms, triggering chemotactic behaviour of bacteria or inducing microbial colonization and reproduction (Steinberg et al. 2002;[START_REF] Goecke | Chemical interactions between marine macroalgae and bacteria[END_REF]).

i. Cell wall structure

The cell wall structure of macroalgae is mainly composed of mannans, xylans, cellulose, hemicellulose and sulfated or/and anionic polysaccharides. The cell wall polysaccharides are specific:

Chlorophyta (green seaweeds) contain sulphated xyloarabinogalactans, glucuronoxylorhamnans, glucuronoxylorhamnogalactans and ulvans. Phaeophyceae (brown seaweeds) produce alginate and fucosecontaining sulfated polysaccharides, whereas Rhodophyta (red seaweeds) contain neutral and sulfated polysaccharides such as agars and carrageenans [START_REF] Percival | The polysaccharides of green, red and brown seaweeds: Their basic structure, biosynthesis and function[END_REF][START_REF] Guedes | Algal spent biomass-A pool of applications[END_REF]. Within the algal lineages, these polysaccharides are highly diversified in terms of their degree of sulfation, esterification, molecular weight and conformation of sugar residue [START_REF] Mabeau | Isolation and Analysis of the Cell Walls of Brown Algae: Fucus spiralis, F. ceranoides, F. vesiculosus, F. serratus, Bifurcaria bifurcata and Laminaria digitata[END_REF][START_REF] Kloareg | Structure of the cell walls of marine algae and ecophysiological functions of the matrix polysaccharides[END_REF][START_REF] Popper | Evolution and Diversity of Plant Cell Walls: From Algae to Flowering Plants[END_REF]. Apart from the above mentioned structural polysaccharides, the red, green and, brown macroalgae also contain storage polysaccharides such as floridean starch, and, laminarin and mannitol, respectively [START_REF] Stiger-Pouvreau | Carbohydrates from Seaweeds[END_REF].

ii.

Surface topographical features

The composition and function of surface consortia are significantly influenced by the chemically mediated interactions and communication between the associated microorganisms and the hosts [START_REF] Egan | Unlocking the diversity and biotechnological potential of marine surface associated microbial communities[END_REF]. The surface topographical features include a mucilage and sheath demonstrating a gelatinous covering of the macroalgae [START_REF] Chapman | Bioinspired synthetic macroalgae: Examples from nature for antifouling applications[END_REF]) and it influences the overall epibiosis [START_REF] Sullivan | The characterization, replication and testing of dermal denticles of Scyliorhinus canicula for physical mechanisms of biofouling prevention[END_REF]. The microtopography of the interface is a selective factor in the settlement of macromolecules and microorganisms. [START_REF] Chapman | Bioinspired synthetic macroalgae: Examples from nature for antifouling applications[END_REF][START_REF] Sullivan | The characterization, replication and testing of dermal denticles of Scyliorhinus canicula for physical mechanisms of biofouling prevention[END_REF]. Therefore, the physical and chemical properties of the substratum play a key role in the attachment and establishment of the initial bacterial colonization. Surface roughness is often speculated to have a deterring role either in attaching or promoting the easy release of fouling organisms [START_REF] Callow | Trends in the development of environmentally friendly fouling-resistant marine coatings[END_REF]. In a study on 'bioinspired synthetic macroalgae' the surface topography of the brown algae, Saccharina latissima and Fucus guiryi, was replicated on synthetic elastomer and evaluated for its antifouling nature. Together with the chemical defence, the surface topography efficiently reduced the fouling by 40%, where it has been proved that the topography and chemistry work synergistically [START_REF] Chapman | Bioinspired synthetic macroalgae: Examples from nature for antifouling applications[END_REF]).

Macroalgae defence

Macroalgae control epiphytic microbial colonization by several processes.

Removal of surface layers

Under pressure from epibionts, some algal thalli can decompose, generating a kind of desquamation of tissues on the surface. This phenomenon has been observed in several species of brown algae Ascophyllum nodosum, Sargassum muticum, some Corallinaceae and Dilsea carnosa. In the summer where light intensity and temperature are at extremes, physical processes involve regular shedding of biofilm surface cell layers. For example, the Gigartinales Chondracanthus chamissoi whitens and decomposes.

During this period, many epiphytes and herbivores were observed. Over time, the alga, which is more vulnerable, produces defence molecules. 2014).)

Production of Reactive Oxygen Species

Many seaweed species are known to produce bioactive compounds through their specialized metabolism. The production of ROS or oxidative burst forms an important component of the stress produced in marine organisms. This production is triggered by the contact of the cells with products resulting from the degradation of the algae wall -oligosaccharides (for example oligoguluronates in Laminaria; oligo-agars in Gracilaria) [START_REF] Da Gama | The antifouling defence mechanisms of marine macroalgae[END_REF]. When a macroalga is colonized by bacteria or herbivores, it receives a signal that will enable it to activate its defence systems. These signals of aggression or Elicitors are defined as chemical compounds that can induce physiological changes in the target organism. In marine macroalgae, they are most often oligosaccharides resulting from the degradation of the algae walls. The presence of Polysaccharides (xyloglucan, laminarin), peptides or proteins or lipid derivatives have also been reported [START_REF] Potin | Biotic interactions of marine algae[END_REF])

Antimicrobial compounds

Exuded algal bioactive compounds on the surface have antimicrobial properties, which protect the seaweed's surface from grazers, fouling organisms and bacterial pathogens. In different macroalgae species, the defence molecules rise to the surface as an antifouling. They can also be stored in different types of specialized structures, for example, gland cells of Delisea pulchra, 'corps en cerise' in Laurencia species.

Physodes in brown seaweeds present in the thallus cortex are known for their antifouling compound delivery systems [START_REF] Dworjanyn | Cost of chemical defence in the red alga Delisea pulchra[END_REF][START_REF] Harizani | The Laurencia Paradox: An Endless Source of Chemodiversity[END_REF][START_REF] Dahms | Antifouling Compounds from Marine Macroalgae[END_REF]. In green algae, the identified compounds mainly belong to di-and sesquiterpenes. They have been relatively little studied (Fig. 1). The diversity and richness of Rhodophyte in terms of secondary metabolites are the most important of all macroalgae with more than 1600 compounds identified. Red macroalgae mainly produce terpenoids and nonterpenoids such as acetogenins. Acetogenins are fatty acid derivatives and are usually halogenated, particularly C-15 acetogenins [START_REF] Bawakid | Isolaurenidificin and bromlaurenidificin, two new C15-acetogenins from the red alga Laurencia obtusa[END_REF]. Red algae are also characterized by the production of halogenated compounds containing bromine and chlorine. For example, the red alga Delisea pulchra produces an analogous molecule of bacterial N-acyl-homoserine lactones (AHLs) known to inhibit the signal pathways in the Gram-positive and Gram-negative bacteria Bacillus subtilis [START_REF] Ren | Inhibition of biofilm formation and swarming of Bacillus subtilis by (5Z)-4-bromo-5-(bromomethylene)-3-butyl-2(5H)-furanone[END_REF],

Escherichia coli [START_REF] Ren | Inhibition of biofilm formation and swarming of Escherichia coli by (5Z)-4-bromo-5-(bromomethylene)-3-butyl-2(5H)-furanone[END_REF] and Pseudomonas aeruginosa [START_REF] Hentzer | Inhibition of quorum sensing in Pseudomonas aeruginosa biofilm bacteria by a halogenated furanone compound[END_REF] these lead to selective colonization of bacteria on thalli (Steinberg and de Nys 2002).

Fig. 1 Bioactive compounds produced by the red, green and brown macroalgae.

Defence on surface of Rhodophyta Halymenia floresii: metabolomic profile and interactions with its surface-associated bacteria Shareen Arockiasamy 2020 50

Another mechanism involved in red algae defence reactions against pathogen attack is the oxidation (enzymatic or non-enzymatic) of fatty acids that results in the production of oxylipins. Sugars (floridoside) from photosynthesis are involved in the defence mechanisms. In brown algae, more than 1200 molecules have been characterized, including terpenes, galactoglycerolipids, fatty acids, phlorotannins and small C11 acetogenins. For example, in Dictyota ciliolata, pachydictyol A, dictyol B acetate, dictyodial, and sterols were shown to be present in higher concentrations in older, less palatable tissues than in apical meristem [START_REF] Cronin | Within-plant variation in seaweed palatability and chemical defenses: optimal defense theory versus the growth-differentiation balance hypothesis[END_REF]. Therefore, the different allocation and concentration of the chemical compounds may lead to different microbial communities at the different parts of macroalgae. There is little data about the authenticity and ecological significance of algal chemical defence compounds having verified their production, presence and/or release at ecologically realistic concentrations [START_REF] Maximilien | Chemical mediation of bacterial surface colonisation by secondary metabolites from the red alga Delisea pulchra[END_REF][START_REF] Kubanek | Seaweed resistance to microbial attack: a targeted chemical defense against marine fungi[END_REF][START_REF] Lane | Desorption electrospray ionization mass spectrometry reveals surface-mediated antifungal chemical defense of a tropical seaweed[END_REF][START_REF] Nyadong | Reactive desorption electrospray ionization mass spectrometry (DESI-MS) of natural products of a marine alga[END_REF][START_REF] Nylund | The red alga Bonnemaisonia asparagoides regulates epiphytic bacterial abundance and community composition by chemical defence[END_REF]. In 2010 Lachnit et al.

demonstrated that available surface chemical compounds on Fucus vesiculosus have different main effects on bacterial colonizers; one group of compounds reduced the overall abundance of epiphytic bacteria nondiscriminatively, whereas the other displayed selective and attractant features for bacteria. Consequently, the community patterns and overall densities of epiphytic bacteria are likely to have resulted from some sort of equilibrium between these opposite effects. These authors propose that the effect size will vary over the course of the year due to environmental changes, thus resulting in algal-specific but temporally variable patterns of epibiosis in accordance with previous observations. The results of this study suggest that unidentified compounds and fucoxanthin may be significant drivers of the interaction between the alga and colonizing bacteria. Dimethylsulphopropionate (DMSP) and proline from the surface of F. vesiculosus are active against the attachment of five different bacterial strains isolated from the algae [START_REF] Saha | Dimethylsulphopropionate (DMSP) and proline from the surface of the brown alga Fucus vesiculosus inhibit bacterial attachment[END_REF].

Further dynamics downstream of the initial colonization by bacteria, such as microorganismmicroorganism interactions are expected to add another effective layer that results in spatial heterogeneity of epiphytic bacterial biofilms.

Microbiome on the surface

Bacteria

Descriptive studies of bacteria isolated from the surface of macroalgae were reported as early as 1875 [START_REF] Johansen | Description of Cellulophaga baltica gen. nov., sp. nov. and Cellulophaga fucicola gen. nov., sp. nov. and reclassification of [Cytophaga] lytica to Cellulophaga lytica gen. nov., comb. nov[END_REF]. The interest in bacterial populations living in association with macroalgae has increased during recent decades [START_REF] Goecke | Chemical interactions between marine macroalgae and bacteria[END_REF]. In 2010, Goecke et al. found 107 studies on bacterial communities associated to a total of 148 macroalgae (36 Chlorophyta, 46 Phaeophyceae, 55 Rhodophyta, 12 undetermined algae) within the last 40 yrs. Bacteria often have a protective role through the production of compounds into the water that prevents the installation of biofilm on the surface. The primary biofilm of bacteria serves as cues for the further settlement of unicellular and multicellular eukaryotes. These cues can be either positive or negative [START_REF] Rao | Low densities of epiphytic bacteria from the marine alga Ulva australis inhibit settlement of fouling organisms[END_REF]). The presence of both attractants and antibacterial molecules will determine the promoter or inhibitory effect on biofilm establishment, predation deterrence, and germination of an epiphyte spore or metamorphosis of an epibiont invertebrate.

Earlier studies have mainly focused on elucidation of the roles of the epiphytic bacterial communities in the ecophysiology of the host macroalga. However, mutualistic interactions have become a topic of current interest. It is evident from recent studies that a fraction of epiphytic bacterial communities can be categorized as "core microbial species", suggesting an obligate association. However, the precise composition of microbiomes and their functional partnership with hosts are not fully understood [START_REF] Singh | Unraveling the Functions of the Macroalgal Microbiome[END_REF]. Bacterial communities associated with marine macroalgae differ strongly from those of their surrounding seawater, sediment or substrate (Bengtsson et al. 2010;Bengtsson and Øvreås 2010;[START_REF] Aires | Host and environmental specificity in bacterial communities associated to two highly invasive marine species (Genus Asparagopsis)[END_REF]. For example, the surface microbiome of Fucus vesiculosus was analysed comparatively by amplicon sequencing, using seawater as reference, and it was found that the majority of microbial OTUs (relative abundances) were assigned to Alphaproteobacteria (64% to 68%) and their share in the seawater bacterial community was even higher (84%) [START_REF] Parrot | Mapping the surface microbiome and metabolome of Brown seaweed Fucus vesiculosus by amplicon sequencing, integrated metabolomics and imaging techniques[END_REF]. Bacterial communities associated to marine macroalgae are not fixed and can change temporally and spatially across seasons, lifespans, life stages and tissue types by biotic and abiotic factors [START_REF] Staufenberger | Phylogenetic analysis of bacteria associated with Laminaria saccharina[END_REF][START_REF] Michelou | The Ecology of Microbial Communities Associated with Macrocystis pyrifera[END_REF][START_REF] Mancuso | Diversity and temporal dynamics of the epiphytic bacterial communities associated with the canopy-forming seaweed Cystoseira compressa (Esper) Gerloff and Nizamuddin[END_REF][START_REF] Aires | Host and environmental specificity in bacterial communities associated to two highly invasive marine species (Genus Asparagopsis)[END_REF]. For example, the epibacterial diversity of Laminaria hyperborea (Phaeophyta) increases with the age/successive colonization of the kelp surface [START_REF] Bengtsson | Bacterial diversity in relation to secondary production and succession on surfaces of the kelp Laminaria hyperborea[END_REF].

Bacterial density and community composition follow the brown seaweed seasonal growth cycle. As most of the biofilm seems to consist of bacteria utilizing carbon produced by the host (Bengtsson and Øvreås 2010), microbiome dynamics are probably strongly linked to seasonal changes in the kelp metabolome and seawater temperature (Bengtsson et al. 2010). Table 1 illustrates the list of bacteria isolated from the major '3' phyla, red, green and brown, of macroalgae. Macroalgal growth and morphogenesis are particularly shown to depend on the associated bacterial communities which produce plant growth-promoting substances, quorum sensing signal molecules and several bioactive compounds [START_REF] Singh | Seaweed-microbial interactions: key functions of seaweed-associated bacteria[END_REF]. Bacterial biofilms can have a repellent effect, for example, nonviable bacteria tended to repel cyprids when compared to the surfaces without a biofilm, which ultimately prevent heavy fouling on the surface [START_REF] Nasrolahi | A protective coat of microorganisms on macroalgae: inhibitory effects of bacterial biofilms and epibiotic microbial assemblages on barnacle attachment[END_REF]. Bacterial communities belonging to the phyla Proteobacteria and Firmicutes are generally the most abundant epiphytic bacteria associated with macroalgal host [START_REF] Singh | Unraveling the Functions of the Macroalgal Microbiome[END_REF]. The '71' heterotrophic bacteria isolated from the green alga Ulva rigida belong to the four phyla Proteobacteria, Bacteroidetes, Firmicutes and Actinobacteria [START_REF] Ismail | Heterotrophic bacteria associated with the green alga Ulva rigida: identification and antimicrobial potential[END_REF]. Marinomonas sp. and Bacillus spp. were observed to have the morphogenesis-inducing ability towards its host macroalgae U. fasciata [START_REF] Singh | Isolation and characterization of exopolysaccharides from seaweed associated bacteria Bacillus licheniformis[END_REF]). An in-depth study on the 16s rRNA gene libraries of the bacterial community on the surface of U. australis and the surrounding water determines the uniqueness of the association. Both the surface and seawater libraries were dominated by Alphaproteobacteria and Bacteroidetes families, but they were observed to be clearly distinct from each other through clustering into operational taxonomical units (OTUs) (Burke et al. 2011). Pseudoalteromonas tunicata and Phaeobacter sp. in association with the green alga Ulva australis inhibits the common fouling organisms by producing extracellular compounds even at low densities, 10 2 to 10 3 cells cm -2 and 10 3 to 10 4 cells cm -2 , respectively [START_REF] Rao | Low densities of epiphytic bacteria from the marine alga Ulva australis inhibit settlement of fouling organisms[END_REF]).

The Roseobacter species from the surface of a green alga Ulva mutabilis exhibits a specific chemotactic affinity to the alga and chemically communicates with the alga as well as with another associated bacterium Cytophaga sp. forming a symbiotic tripartite community [START_REF] Spoerner | Growth and thallus morphogenesis of Ulva mutabilis (Chlorophyta) depends on a combination of two bacterial species excreting regulatory factors[END_REF].

Additionally, some bacterial species produce regulatory compounds resembling cytokinin (from Roseobacter, Sulfitobacter, and Halomonas) and auxin (from Cytophaga) that assist in the differentiation of U. mutabilis [START_REF] Spoerner | Growth and thallus morphogenesis of Ulva mutabilis (Chlorophyta) depends on a combination of two bacterial species excreting regulatory factors[END_REF]. The epibiotic bacteria associated with the brown alga Fucus vesiculosus tended to repel the cyprids benefitting the host by preventing heavy fouling on its surface [START_REF] Nasrolahi | A protective coat of microorganisms on macroalgae: inhibitory effects of bacterial biofilms and epibiotic microbial assemblages on barnacle attachment[END_REF]. Specific functionally active bacterial species (FABS) will serve to improve the health and performance of the associated host by suppressing the growth of pathogens [START_REF] Singh | Unraveling the Functions of the Macroalgal Microbiome[END_REF]. [START_REF] Bondoso | Epiphytic Planctomycetes communities associated with three main groups of macroalgae[END_REF] hypothesized that specific association of Planctomycetes and their macroalgal hosts, such as the brown alga Fucus spiralis, the green alga Ulva sp. and the red alga Chondrus crispus, is likely to be determined by the nutrients provided by the host and the set of sulfatases inherent to the associated Planctomycetes species.

In red alga, Asparagopsis armata-associated bacteria exhibited a potential antitumour and antibacterial activity. The extract of Shewanella sp. ASP 26 showed increased antimicrobial activity against Bacillus subtilis and Staphylococcus aureus [START_REF] Horta | Identification of Asparagopsis armata -associated bacteria and characterization of their bioactive potential[END_REF]. [START_REF] Ma | Inhibition of common fouling organisms in mariculture by epiphytic bacteria from the surfaces of seaweeds and invertebrates[END_REF] isolated 192 bacterial strains from the surfaces of seaweeds, of which '63' isolates were shown to inhibit the settlement of algal spores and '62' isolates inhibited larval settlement under laboratory conditions. About '25' heterotrophic algae-associated bacteria from the surfaces of U. rigida show antibacterial activity whereas the free-living bacteria from the surrounding seawater did not show such activity [START_REF] Ismail | Heterotrophic bacteria associated with the green alga Ulva rigida: identification and antimicrobial potential[END_REF]. Sixty of the '280' strains isolated from different marine macroalgae exhibited antibiotic activity contrary to a series of fouling bacteria and therefore have the potential to control the microbial population on the seaweed's surface either by inhibiting their growth or by influencing the tactic behaviour of potentially competing bacteria [START_REF] Boyd | Antibacterial and repellent activities of marine bacteria associated with algal surfaces[END_REF].

A broad range of antibacterial activity contra important food pathogens such as Vibrio parahaemolyticus, V. vulnificus and Aeromonas hydrophilia was observed in Bacillus subtilis associated with the brown alga Anthophycus longifolius (Chakraborty et al. 2017a). Two polyketides from Bacillus amyloliquefaciens, a heterotrophic bacteria associated with the edible red alga, Laurencia papillosa, 3-

(octahydro-9-isopropyl-2H-benzo[h]chromen-4-yl)-2-methylpropyl benzoate and methyl 8-(2- (benzoyloxy)-ethyl)-hexahydro-4-((E)-pent-2-enyl)-2H-chromene-6
-carboxylate exhibits an activity contra to human opportunistic food pathogens (Chakraborty et al. 2017b). Members of the genus Pseudoalteromonas, Bacillus, Vibrio and Shewanella isolated from the surface of the green alga, Ulva lactuca, showed an anti-diatom activity on the pennate diatom, Cylindrotheca fusiformis [START_REF] Kumar | Antidiatom and antibacterial activity of epiphytic bacteria isolated from Ulva lactuca in tropical waters[END_REF]. Figure 2 illustrates bioactive metabolites isolated from the macroalgal associated bacteria.

Fig. 2 Bioactive metabolites from the macroalgal associated bacteria.

Defence on surface of Rhodophyta Halymenia floresii: metabolomic profile and interactions with its surface-associated bacteria Shareen Arockiasamy 2020 57

Fungi

The fungal population on the surface of seaweed mainly consists of parasites, saprobes or asymptomatic fungal endosymbionts where the ascomycetes and anamorphic fungi were dominant. [START_REF] Suryanarayanan | Internal mycobiota of marine macroalgae from the Tamilnadu coast: distribution, diversity and biotechnological potential[END_REF] observed that among the different red, green and brown macroalgae studied, the brown algae supported a higher diversity of fungal endophytes whereas the green algae had a low diversity with dense colonization. In different seaweeds, members of genera Acremonium, Alternaria, Arthrinium, Aspergillus, Cladosporium, Fusarium, Geomyces, Penicillium and Phoma were the most common fungal endosymbionts (reviewed by Singh, Kumari, & Reddy, 2014). More recently, fungi of the Eurotiomycetes class were visualised for the first time on the surface of the brown alga Fucus vesiculosus, by CARD-FISH imaging [START_REF] Parrot | Mapping the surface microbiome and metabolome of Brown seaweed Fucus vesiculosus by amplicon sequencing, integrated metabolomics and imaging techniques[END_REF].

Aspergillus terrreus isolated from Halimeda macroloba, Gracilaria edulis and Ulva lactuca produced metabolites which were active against bacteria, algae and fungi [START_REF] Suryanarayanan | Internal mycobiota of marine macroalgae from the Tamilnadu coast: distribution, diversity and biotechnological potential[END_REF].

However, in contrast to the well-studied bacteria-algae surface interactions, there is only a little evidence on fungi and their role on macroalgal surfaces [START_REF] Egan | The seaweed holobiont: understanding seaweed-bacteria interactions[END_REF]. Most of the studies on macroalgal associated fungi dealt with the endophytic fungi and they are well detailed by [START_REF] Habbu | Antimicrobial metabolites from marine microorganisms[END_REF] together

with their antimicrobial activities. The filamentous fungal community associated with the healthy and the decaying brown alga, Fucus serratus, is observed to be different. Such a change in the community structure could be related to the release of nutrients resulting from tissue breakdown [START_REF] Zuccaro | Detection and identification of fungi intimately associated with the brown seaweed Fucus serratus[END_REF]. A total of '75' fungi were isolated from marine macroalgae of Antarctica and despite the fact that these fungi were isolated from living surfaces most of the isolates are probably saprobic decomposers, which may have an important role in the ecosystem for organic matter recycling [START_REF] Loque | Fungal community associated with marine macroalgae from Antarctica[END_REF]. Table 2 illustrates the list of fungi isolated from the '3' major phyla of macroalgae, such as red, green and brown. A exhibits antifungal activity against Candida albicans [START_REF] Zhang | New sphingolipids with a previously unreported 9-methyl-C20-sphingosine moiety from a marine algous endophytic fungus Aspergillus niger EN-13[END_REF]) (Fig. 3).

Fig. 3 Bioactive metabolites from the macroalgal associated fungi.

Microalgae

Table 3 illustrates the list of microalgae isolated from different macroalgal species. The epiphytic microalgal community is mostly dominated by benthic diatoms and a few centric species possessing an attached mode of life [START_REF] Lage | Biofilms: An Extra Coat on Macroalgae[END_REF]. As photosynthetic eukaryotes, diatoms can also serve as food for heterotrophic bacteria through specific interactions with growing diatoms as well as colonizing the sinking diatom and

Virus

As ubiquitous components of marine holobionts, viruses are now emerging as pathogens or potential pathogens in various marine organisms including macroalgae [START_REF] Lachnit | Expanding our understanding of the seaweed holobiont: RNA viruses of the red alga Delisea pulchra[END_REF][START_REF] Beattie | Novel ssDNA Viruses Detected in the Virome of Bleached, Habitat-Forming Kelp Ecklonia radiata[END_REF]. Viruses can have both positive and negative impacts on the holobiont which either may spread diseases among hosts or may be a part of the holobiont immune system [START_REF] Ying | The effects of marine farm-scale sequentially integrated multi-trophic aquaculture systems on microbial community composition, prevalence of sulfonamide-resistant bacteria and sulfonamide resistance gene sul1[END_REF]. [START_REF] Lachnit | Expanding our understanding of the seaweed holobiont: RNA viruses of the red alga Delisea pulchra[END_REF] reported the viruses associated with a red macroalga, Delisea pulchra, identifying diverse morphotypes of virus-like particles from icosahedral to bacilliform to coiled pleomorphic as well as bacteriophages. The virome sequencing revealed the presence of a diverse group of dsRNA viruses affiliated to the genus Totivirus, known to infect plant pathogenic fungi. Furthermore, an ssRNA virus belonging to the Picornavirales order with a close phylogenetic relationship to a pathogenic virus infecting marine diatoms was also identified in association with D. pulchra. Viruses have a large potential effect by not only infecting the host but also other microorganisms, like bacteriophages which regulate the bacterial community composition, thus adding a complexity to the holobiont structure [START_REF] Gachon | Algal diseases: spotlight on a black box[END_REF][START_REF] Ying | The effects of marine farm-scale sequentially integrated multi-trophic aquaculture systems on microbial community composition, prevalence of sulfonamide-resistant bacteria and sulfonamide resistance gene sul1[END_REF]. Table 4 includes the viruses isolated from different macroalgae.

Role of Microbiome: Microbial Interactions and their Hosts Response

The microbiome performs a 'protective' role with epibiotic bacteria present on surfaces that release chemicals that prevent biofouling by other organisms [START_REF] Armstrong | The symbiotic role of marine microbes on living surfaces[END_REF]. Similarly, macroalgal growth and development are shown to depend on associated microorganisms, particularly on bacterial communities [START_REF] Singh | Seaweed-microbial interactions: key functions of seaweed-associated bacteria[END_REF][START_REF] Wichard | The green seaweed Ulva: a model system to study morphogenesis[END_REF]. With such an obligate association, a fraction of epiphytic bacterial communities can be regarded as 'core microbial species' [START_REF] Singh | Unraveling the Functions of the Macroalgal Microbiome[END_REF]. The group of a microbial community which are consistently present on the surface of different individuals of the macroalgae is known as a 'core community' (Burke et al. 2011). The interactions between the macroalgae and the microbiota are extremely diverse (van der Loos et al. 2019). As the microorganisms in the diverse marine environment are concentrated on microscale patches, their close proximity suggests many potential cell-cell communications [START_REF] Azam | Microbial structuring of marine ecosystems[END_REF][START_REF] Amin | Interactions between Diatoms and Bacteria[END_REF]. The interactions of macroalgae and bacteria are mainly for nutrient exchange, signal transduction and gene transfer [START_REF] Kouzuma | Exploring the potential of algae/bacteria interactions[END_REF].

Quorum Sensing

Quorum Sensing (QS), an essential language of bacteria, is a cell density-dependent communication system that regulates a diverse array of physiological activities such as symbiosis, virulence, competence, conjugation, antibiotic production, motility, sporulation, and biofilm formation. This phenomenon relies on the accumulation of signal molecules in the surrounding environment up to threshold concentrations, at which the target genes are activated [START_REF] Williams | Look who's talking: communication and quorum sensing in the bacterial world[END_REF][START_REF] Romero | Quorum quenching in cultivable bacteria from dense marine coastal microbial communities[END_REF] Apart from defending the attached basibiont the surface-associated bacteria also assist the host in growth and morphogenesis. AHLs, N-acyl homoserine lactones, produced by the macroalgae-associated bacteria facilitates the settlement of zoospores in Ulva spp. [START_REF] Joint | Cell-to-Cell Communication Across the Prokaryote-Eukaryote Boundary[END_REF][START_REF] Williams | Quorum sensing, communication and cross-kingdom signalling in the bacterial world[END_REF]Singh et al. 2015). The bacterial isolates, Shewanella algae, Pseudomonas aeruginosa, Photobacterium sp., P.

lutimaris, Vibrio gallicus, V. fluvialis, V. parahaemolyticus, associated with green macroalgae Ulva (U.

fasciata and U. lactuca) and red macroalgae Gracilaria (G. corticata and G. dura), induced carpospore liberation from cyctocarps of G. dura by producing different AHLs (Singh et al. 2015). The AHLs produced by Acrochaetium sp. control spore release in the red alga, G. chilensis whereas its production was inhibited in the brown alga, Colpomenia sinuosa [START_REF] Weinberger | Spore release in Acrochaetium sp. (Rhodophyta) is bacterially controlled[END_REF][START_REF] Kanagasabhapathy | Presence of quorum-sensing inhibitor-like compounds from bacteria isolated from the brown alga Colpomenia sinuosa[END_REF]). The motile zoospores of Enteromorpha exploit the diffusible signal molecules produced by bacterial biofilms for temporary settlement on the surface [START_REF] Joint | Cell-to-Cell Communication Across the Prokaryote-Eukaryote Boundary[END_REF].

QS-mediated interactions widely influence algal-bacterial symbiotic relationships, which in turn determine community organization, population structure, and ecosystem functioning [START_REF] Zhou | Quorum Sensing Is a Language of Chemical Signals and Plays an Ecological Role in Algal-Bacterial Interactions[END_REF]). In

U. mutabilis the chemical communication signals between the associated bacteria, Roseobacter and

Cytophaga, up-regulate the growth and thallus morphogenesis [START_REF] Spoerner | Growth and thallus morphogenesis of Ulva mutabilis (Chlorophyta) depends on a combination of two bacterial species excreting regulatory factors[END_REF]. A chemical elicitor protein, probably a quorum signal N-acyl homoserine lactone produced by a gram-positive probiotic

Lysinibacillus xylanilyticus associated with a red alga G. edulis promotes the growth of the host (Suvega and Arunkumar 2019).

Quorum Quenching

Quorum Quenching (QQ), is defined as any interference on the QS communication system either by enzymatic degradation of the AHLs, or by production of inhibitors or antagonists of signal reception.

QQ strategy plays an important role in preventing microbial disease and affecting beneficial bacteria [START_REF] Chen | Quorum quenching enzymes and their application in degrading signal molecules to block quorum sensing-dependent infection[END_REF]. Many algae use defence strategies to protect themselves from the potentially opportunistic pathogens associated with them by altering their QS communication systems i.e. quenching their signal molecules.

The first QS inhibitory activity was reported by the halogenated furanones from the red alga Delisea pulchra which exhibited antibiofilm activity against Bacillus subtilis, Pseudomonas aeruginosa and Escherichia coli, and these compounds were released at the surface at concentrations capable of inhibition (Givskov et al. 1996;[START_REF] Ren | Inhibition of biofilm formation and swarming of Escherichia coli by (5Z)-4-bromo-5-(bromomethylene)-3-butyl-2(5H)-furanone[END_REF][START_REF] Ren | Inhibition of biofilm formation and swarming of Bacillus subtilis by (5Z)-4-bromo-5-(bromomethylene)-3-butyl-2(5H)-furanone[END_REF]Steinberg and de Nys 2002) . In particular the brominated furanones significantly inhibited the settlement of cyprid larvae of Balanus amphitrite [START_REF] De Nys | Broad spectrum effects of secondary metabolites from the red alga Delisea pulchra in antifouling assays[END_REF].

A number of halogenated furanones from this alga antagonize the bacterial AHLs and interfere with their associated biological activity, such as competitively inhibiting swarming motility (Givskov et al. 1996). A mixture of relatively less lipophilic compounds, floridoside, betonicine and isethionic acid, isolated from the red alga Ahnfeltiopsis flabelliformis was observed to inhibit the exogenous QS signalling molecule, Noctanoyl-DL-homoserine lactone (OHL), in the quorum-sensing inhibition assay [START_REF] Kim | Quorum sensing inhibitors from the red alga, Ahnfeltiopsis flabelliformis[END_REF]). The methanolic extract of the red alga, Asparagopsis taxiformis, showed quorum sensing inhibitory activity against the reporter strain, Chromobacterium violaceum CV026 [START_REF] Jha | Quorum sensing inhibition by Asparagopsis taxiformis, a marine macro alga: separation of the compound that interrupts bacterial communication[END_REF] whereas the polar extracts of the green alga Ulva fasciata and Codium sp. exhibited QS inhibitory activity on C. violaceum CV017 [START_REF] Batista | Extracts of macroalgae from the Brazilian coast inhibit bacterial quorum sensing[END_REF]). In the brown alga Laminaria digitata, QQ activity was demonstrated by the inactivation of AHLs based on the enzymatic production of the oxidized halogen HOBr which reacts specifically with 3-oxo-acyl HSLs [START_REF] Borchardt | Reaction of acylated homoserine lactone bacterial signaling molecules with oxidized halogen antimicrobials[END_REF][START_REF] Romero | Quorum quenching in cultivable bacteria from dense marine coastal microbial communities[END_REF]. The polar and non-polar extracts of the different brown algae, Sargassum spp. S. vulgare, S. furcatum, showed anti-quorum sensing activity on the reporter strain, C. violaceum CV017 [START_REF] Batista | Extracts of macroalgae from the Brazilian coast inhibit bacterial quorum sensing[END_REF][START_REF] Schwartz | Comparison of antifouling properties of native and invasive Sargassum (Fucales, Phaeophyceae) species[END_REF][START_REF] Carvalho | Extracts of seaweeds as potential inhibitors of quorum sensing and bacterial growth[END_REF].

By interfering with the AHL signalling of Vibrio anguillarum, the settlement of Enteromorpha, an important fouling organism, can be prevented [START_REF] Joint | Cell-to-Cell Communication Across the Prokaryote-Eukaryote Boundary[END_REF]. In aquaculture systems, several algal species were used for controlling pathogenic bacteria by disrupting the quorum sensing communication between them, for example, Vibrio harveyi [START_REF] Natrah | Significance of microalgal-bacterial interactions for aquaculture[END_REF][START_REF] Ramanan | Algae-bacteria interactions: Evolution, ecology and emerging applications[END_REF]. The halogenated furanones of D. pulchra protect both shrimp and fish from vibriosis by interfering with their AHL signalling system [START_REF] Rasch | An Inhibitor of bacterial quorum sensing reduces mortalities caused by vibriosis in rainbow trout (Oncorhynchus mykiss, Walbaum)[END_REF][START_REF] Torres | Saline environments as a source of potential quorum sensing disruptors to control bacterial infections: A Review[END_REF]. A QS antagonist, (5Z)-4-bromo-5-(bromomethylene)-3butyl-2(5H)-furanone (furanone) from the D. pulchra was found to inhibit the formation of the siderophore produced by Pseudomonas putida and P. aeruginosa [START_REF] Ren | Quorum-sensing antagonist (5Z)-4-bromo-5-(bromomethylene)-3-butyl-2(5H)-furanone influences siderophore biosynthesis in Pseudomonas putida and Pseudomonas aeruginosa[END_REF]. QQ is a promising approach for very effective next-generation antibacterial drugs which block the QS-mediated pathogenic infection by interfering with the bacterial communication [START_REF] Chen | Quorum quenching enzymes and their application in degrading signal molecules to block quorum sensing-dependent infection[END_REF]).

Positive Effects: Symbiotism

There are many beneficial functions for the macroalgal host, for example, normal growth, morphological development, spore germination and nitrogen fixation activity. This is despite the low average microbial density (10 5 to 10 6 cells per gram of seawater), the close proximity and the heterogeneous distribution that suggest their interaction is across multiple spatial scales [START_REF] Amin | Interactions between Diatoms and Bacteria[END_REF]. Generally, the associated microorganisms were assumed to use the readily available range of organic carbon sources produced by the macroalgae in the form of complex diversified polysaccharides [START_REF] Martin | Microorganisms living on macroalgae: diversity, interactions, and biotechnological applications[END_REF]). The microbial metabolites produced by these symbiotic microorganisms help in chemical defences of the macroalgae and its biological interactions with foulers and pathogens (Wahl et al. 2012a;Chakraborty et al. 2017a). In such a microhabitat, the macroalgae supply bacteria with oxygen and fixed carbon, and the bacteria supply CO2, minerals, vitamins and growth factors. The microbial community on the surface of the macroalgae was mainly formed by bacteria but also included different eukaryotes in a ratio of 640:4:1 of bacteria:diatoms: flagellates (Railkin 2004;[START_REF] Lage | Biofilms: An Extra Coat on Macroalgae[END_REF].

Epiphytic bacteria are known to maintain the health of the host. Several algal species need specific vitamins for their growth and bacteria may be partly responsible for the production of these substances [START_REF] Villarreal-Gómez | Antibacterial and anticancer activity of seaweeds and bacteria associated with their surface[END_REF]. Conversely, bacterial colonization could be especially rapid if the surface is a potential source of nutrients, such as polysaccharides in exudates of seaweeds that may serve as a source of carbon for heterotrophic bacteria living on the surface. For example, Kelp exudates could shape bacterial community composition, and create communities that are kelp-specific rather than randomly assembled from the surrounding seawater [START_REF] Collen | Seasonality and thermal acclimation of reactive oxygen metabolism in Fucus vesiculosus (Phaeophyceae)[END_REF][START_REF] Michelou | The Ecology of Microbial Communities Associated with Macrocystis pyrifera[END_REF]. Bacteria may degrade algal polysaccharides such as for fucoidan [START_REF] Nedashkovskaia | Characterization of marine bacteria Pseudoalteromonas citrea degradating fucoidan[END_REF] or alginates [START_REF] Preston | Selective alginate degradation by marine bacteria associated with the algal genus Sargassum[END_REF][START_REF] Brown | Cloning of alginate lyase gene (alxM) and expression in Escherichia coli[END_REF]).

An overarching clade of bacteria such as Proteobacteria and Bacteroidetes are more likely to be associated with the green alga and furthermore, they are functionally equipped to be associated with them [START_REF] Ramanan | Phycosphere bacterial diversity in green algae reveals an apparent similarity across habitats[END_REF][START_REF] Ramanan | Algae-bacteria interactions: Evolution, ecology and emerging applications[END_REF]. The temperate green alga Ulva lactuca relies on the defence provided by the microbial community on its surface to regulate the fouling by colonizers [START_REF] Kumar | Antidiatom and antibacterial activity of epiphytic bacteria isolated from Ulva lactuca in tropical waters[END_REF] and '73' different bacterial strains were isolated from the surfaces of U. lactuca, U. pertusa and Enteromorpha sp. [START_REF] Ma | Inhibition of common fouling organisms in mariculture by epiphytic bacteria from the surfaces of seaweeds and invertebrates[END_REF]). In the green alga, Ulva rigida '71' bacterial phylotypes were isolated from its surface which belongs to four phyla: Proteobacteria, Bacteroidetes, Firmicutes and Actinobacteria [START_REF] Ismail | Heterotrophic bacteria associated with the green alga Ulva rigida: identification and antimicrobial potential[END_REF]. Different studies showed that induction of cell divisions, differentiation, wall formation, and normal A study by [START_REF] Ji | MetaSort untangles metagenome assembly by reducing microbial community complexity[END_REF] suggested a complementary and mutualistic relationship between the female gametophyte of the Brown seaweed Saccharina japonica and its microbiome, in which bacteria seem to benefit from seaweed polysaccharides, and the kelp profits from enhanced growth and nutrient uptake by bacterial bioactive compounds such as vitamins and hormones. Analysis of gene functions within this kelp epimicrobiome was mainly symbiosis-associated, indicating that selective pressures shape these microbiomes to sustain a mutual benefit for both kelp and bacteria [START_REF] Ji | MetaSort untangles metagenome assembly by reducing microbial community complexity[END_REF]. Fucus vesiculosus L. is recognized as an important habitat-forming macroalga and could be able to produce secondary metabolites that directly act on the invertebrate larvae to prevent fouling [START_REF] Lachnit | Isolated thallus-associated compounds from the macroalga Fucus vesiculosus mediate bacterial surface colonization in the field similar to that on the natural alga[END_REF][START_REF] Kersen | Epiphytes and associated fauna on the brown alga Fucus vesiculosus in the Baltic and the North Seas in relation to different abiotic and biotic variables[END_REF].

Several species of marine bacteria have been isolated from brown algae such as the spot-wounded fronds of Laminaria japonica [START_REF] Sawabe | Assignment of Alteromonas elyakovii KMM 162(T) and five strains isolated from spot-wounded fronds of Laminaria japonica to Pseudoalteromonas elyakovii comb. nov. and the extended description of the species[END_REF], the surface of Fucus serratus [START_REF] Johansen | Description of Cellulophaga baltica gen. nov., sp. nov. and Cellulophaga fucicola gen. nov., sp. nov. and reclassification of [Cytophaga] lytica to Cellulophaga lytica gen. nov., comb. nov[END_REF][START_REF] Lachnit | Specific epibacterial communities on macroalgae: Phylogeny matters more than habitat[END_REF]) and from the rotten thallus of Fucus evanescens [START_REF] Ivanova | Bacillus algicola sp. nov., a novel filamentous organism isolated from brown alga Fucus evanescens[END_REF]), Undaria pinnatifida [START_REF] Lee | Marine bacteria associated with the Korean brown alga, Undaria pinnatifida[END_REF], Padina arborescens, P. pavonica, P.tetrastromatica [START_REF] Ismail | Antimicrobial activities of bacteria associated with the brown alga Padina pavonica[END_REF], Macrocystis pyrifera [START_REF] Michelou | The Ecology of Microbial Communities Associated with Macrocystis pyrifera[END_REF], Ecklonia radiata [START_REF] Qiu | Future climate change is predicted to affect the microbiome and condition of habitatforming kelp[END_REF], Taonia otamaria [START_REF] Othmani | Surface metabolites of the brown alga Taonia atomaria have the ability to regulate epibiosis[END_REF]. Around '18' different bacteria were isolated from the surfaces of two different brown algae, Sargassum thunbergii and Sargassum sp. [START_REF] Ma | Inhibition of common fouling organisms in mariculture by epiphytic bacteria from the surfaces of seaweeds and invertebrates[END_REF]. Such a complex microbiome on the surface of macroalgae is regarded as an indirect defence mechanism which may strengthen the direct antifouling defence mechanism of the host against any macrofoulers [START_REF] Nasrolahi | A protective coat of microorganisms on macroalgae: inhibitory effects of bacterial biofilms and epibiotic microbial assemblages on barnacle attachment[END_REF]).

Negative Effects: Pathogenism

Any disturbance and disruption of the mutualistic association between host and microbiota are regarded as an 'Holobiont break-up' which can be caused by environmental stress [START_REF] Ying | The effects of marine farm-scale sequentially integrated multi-trophic aquaculture systems on microbial community composition, prevalence of sulfonamide-resistant bacteria and sulfonamide resistance gene sul1[END_REF]. Such disturbances are widely caused by spatial and temporal effects. The spatial and temporal shifts of seaweed associated microbiota are induced by the changing environmental conditions and seaweed physiology. Such shifts may result in potential consequences provoked by the structural, functional and behavioural changes in the host posing a challenge for its fitness [START_REF] Serebryakova | Summer shifts of bacterial communities associated with the invasive brown seaweed Sargassum muticum are location and tissue dependent[END_REF]. This shifts the symbiotic relationship of the microbiota to a potential pathogenic relationship with its associated hosts. The interplay between temperature, the algal chemical defence mechanism and bacterial virulence demonstrates a complex environmental change impact on an ecosystem [START_REF] Case | Temperature induced bacterial virulence and bleaching disease in a chemically defended marine macroalga[END_REF]. Though the epibacterial community structures on the surface of macroalgae are host-specific, they are highly prone to spatial and temporal variation [START_REF] Wu | Phylogenetic analysis of epibacterial communities on the surfaces of four red macroalgae[END_REF]. A shift in the microbiome from a stable state to a disturbed state where the colonizers may become opportunist pathogens when the host system is stressed by abiotic or biotic challenges is referred to as 'dysbiosis' [START_REF] Minich | Elevated temperature drives kelp microbiome dysbiosis, while elevated carbon dioxide induces water microbiome disruption[END_REF].

Pathogenic bacteria can cause severe degradation of algal host cells or even lead to seaweed mortality, causing major financial losses for seaweed mariculture every year [START_REF] Correa | Deformative Disease in Iridaea laminarioides (Rhodophyta): Gall development associated with an endophytic cyanobacterium[END_REF][START_REF] Vairappan | Distribution and symptoms of epiphyte infection in major carrageenophyte-producing farms[END_REF][START_REF] Goecke | Chemical interactions between marine macroalgae and bacteria[END_REF]. Also, biofouling forms a permanent threat to macroalgae as bacterial biofilms increase the hydrodynamic drag on their host and enhance the attachment of other fouling organisms and grazers. Biofilms may also compete for nutrients, inhibit gaseous exchange, or block light essential for photosynthesis.

Microbial colonization can lead to the degradation of algal tissue, decreased photosynthesis and gas and nutrient exchange and ultimately disease. Marine macroalgal diseases have been recognised for a long time, but identifying the causative agents and distinguishing them from other opportunistic bacteria is still an obstacle for describing the virulence mechanism [START_REF] Egan | The seaweed holobiont: understanding seaweed-bacteria interactions[END_REF]). Macroalgae are under constant colonization pressure from the millions of microorganisms within the surrounding seawater, some of which are potential pathogens. The primary biofilm undergoes a transition to a much more complex community which eventually ends in fouling on the surface [START_REF] Bhadury | Exploitation of marine algae: biogenic compounds for potential antifouling applications[END_REF]). All such biofouling communities vary spatially and temporally [START_REF] Fitridge | The impact and control of biofouling in marine aquaculture: a review[END_REF]. Due to the effects of environmental change on host organisms and their pathogens, temporal-mediated diseases can have severe consequences for marine macroalgal ecosystems [START_REF] Campbell | Climate change and disease: bleaching of a chemically defended seaweed[END_REF].

i. Spatial effects

The close spatial proximity of surface-associated microorganisms on macroalgae operates and controls specific intercellular interactions [START_REF] Egan | Unlocking the diversity and biotechnological potential of marine surface associated microbial communities[END_REF]. The spatial distribution and the abundance of the epiphytes are largely determined by several abiotic and biotic factors which ultimately leads to uncontrolled growth (Nylund and Pavia 2005;[START_REF] Kotta | Contribution of scale-dependent environmental variability on the biomass patterns of drift algae and associated invertebrates in the Gulf of Riga, northern Baltic Sea[END_REF][START_REF] Kotta | Regional-Scale Patterns[END_REF][START_REF] Kersen | Epiphytes and associated fauna on the brown alga Fucus vesiculosus in the Baltic and the North Seas in relation to different abiotic and biotic variables[END_REF]. Spatial variability of the epiphytic communities varies both on the large and small scale [START_REF] Fitridge | The impact and control of biofouling in marine aquaculture: a review[END_REF]. The protective effects of the microbiome on the surface were affected by severe settlement pressure and stressful temperatures as observed in Fucus vesiculosus [START_REF] Nasrolahi | A protective coat of microorganisms on macroalgae: inhibitory effects of bacterial biofilms and epibiotic microbial assemblages on barnacle attachment[END_REF], in which the composition and dominance were significantly influenced by wave exposure and frond segment [START_REF] Kersen | Epiphytes and associated fauna on the brown alga Fucus vesiculosus in the Baltic and the North Seas in relation to different abiotic and biotic variables[END_REF]. Also, reduced light conditions due to the uncontrolled colonization of the surface have detrimental effects on the growth of the macroalgae [START_REF] Rohde | Decreased depth distribution of Fucus vesiculosus (Phaeophyceae) in the Western Baltic: effects of light deficiency and epibionts on growth and photosynthesis[END_REF]). The occurrence and richness of the epiphytes were observed to be specific and different, according to the different parts of the host macroalgae [START_REF] Kersen | Epiphytes and associated fauna on the brown alga Fucus vesiculosus in the Baltic and the North Seas in relation to different abiotic and biotic variables[END_REF]. The spatial distribution of epibiotic organisms is determined by their ability to tolerate desiccation during low tides [START_REF] Molina-Montenegro | Positive associations between macroalgal species in a rocky intertidal zone and their effects on the physiological performance of Ulva lactuca[END_REF]. Multiple opportunistic pathogens, Alteromonas sp. BL110, Aquimarina sp. AD1 and BL5 and Agarivorans sp BL7 rather than the previously identified Nautella R11, cause bleaching disease in a temperate red alga Delisea pulchra with loss of its photosynthetic pigments followed by tissue necrosis and death. Multiple opportunistic pathogens are involved in bleaching disease in D. pulchra (Gonzalez and Keshavan 2006;[START_REF] Case | Temperature induced bacterial virulence and bleaching disease in a chemically defended marine macroalga[END_REF]Campbell et al. 2011;[START_REF] Fernandes | Genomes and virulence factors of novel bacterial pathogens causing bleaching disease in the marine red alga Delisea pulchra[END_REF]Kumar et al. 2016).

ii. Temporal Effects

The temporal variation of the macroalgal associated microbiome may be affected by biotic factors such as algal growth, exudation of macroalgal metabolites, the composition of the microbial community in the surrounding seawater and abiotic factors like temperature, wave action, light conditions and nutrient levels in seawater (Bengtsson et al. 2010). Different seasons affect the temporal variations of macroalgal surface-associated bacterial communities (Singh et al. 2015). As a result of climate change, environmental stress can render macroalgae susceptible to the presence of opportunistic pathogens which are otherwise commensal to them, but can still take advantage of the weakened host and this results in disease outbreak [START_REF] Egan | Bacterial pathogens, virulence mechanism and host defence in marine macroalgae[END_REF]. As most of the biofilm seems to consist of bacteria utilizing carbon produced by the host (Bengtsson and Øvreås 2010), microbiome dynamics are probably strongly linked to seasonal changes in the kelp metabolome and seawater temperature (Bengtsson et al. 2010).

The temporal shifts of bacterial communities associated with the brown alga Sargassum muticum were reflected in a significant abundance of unidentified Rhodobacterales and Laktonella in September-March and the substantial prevalence of unidentified Pirellulales in the summer. Such changes were related to the temporally changing algal exudates [START_REF] Serebryakova | Summer shifts of bacterial communities associated with the invasive brown seaweed Sargassum muticum are location and tissue dependent[END_REF]. In another brown alga Laminaria hyperborea, the substratum and seawater temperature manifested a variation in their associated microbiome

where Planctomycetes and Alphaproteobacteria were frequently detected throughout the year and Verrucomicrobia, Cyanobacteria, Gammaproteobacteria, Betaproteobacteria, and Bacteroidetes were more sporadically detected (Bengtsson et al. 2010). The kelp microbiome was most influenced by an elevated temperature exhibiting a reduction in the dominant kelp-associated Alteromondales, which negatively affects its growth [START_REF] Minich | Elevated temperature drives kelp microbiome dysbiosis, while elevated carbon dioxide induces water microbiome disruption[END_REF]. Any temporal variations in the primary biofilm will cause major temporal changes in the marine invertebrate populations, particularly, seasonality [START_REF] Fitridge | The impact and control of biofouling in marine aquaculture: a review[END_REF]).

Future Perspectives

Increased physical disturbances and stress, resulting from changing environmental conditions, are known to affect the macroalgae-associated microbial composition (directly or via its physiological responses) and cause its structural, functional or behavioural changes. In the marine environment, changing climatic conditions such as ocean warming and acidification can have direct physiological effects on the marine-habitat forming macroalgal species, which sequentially, can have negative impacts on the associated microbial communities. The microbiome of the brown alga, Ecklonia radiata, was affected by warming, followed by acidification which resulted in disease-like symptoms in the host [START_REF] Qiu | Future climate change is predicted to affect the microbiome and condition of habitatforming kelp[END_REF]). The characteristic 'core' microbial community was replaced by taxonomically diverse microbial communities following the host's stress [START_REF] Marzinelli | Continental-scale variation in seaweed host-associated bacterial communities is a function of host condition, not geography[END_REF]. [START_REF] Case | Temperature induced bacterial virulence and bleaching disease in a chemically defended marine macroalga[END_REF] showed that temperate marine macroalga, D. pulchra, has become more susceptible to the temperature-enhanced bacterial virulence of Ruegeria sp. R11 as a result of high-water temperatures or increased solar radiation. This also affects the production or delivery of furanones potentially leading to in situ bleaching [START_REF] Case | Temperature induced bacterial virulence and bleaching disease in a chemically defended marine macroalga[END_REF]. Ice-ice disease in the red alga Eucheuma/Kappaphycus is still a major problem in this seaweed cultivation where the changes in extreme environmental conditions make the alga susceptible to the infection of Vibrio sp. [START_REF] Irmawati | IOP Conference Series: Earth and Environmental Science Infection Vibrio sp. bacteria on Kappaphycus Seaweed varieties brown and green[END_REF].

Bioactive metabolites of macroalgae surface-associated bacteria open up a new approach as a novel source of therapeutic molecules. For instance, Asparagopsis armata-associated bacteria revealed to be a potential source of compounds with antitumor and antibacterial activity [START_REF] Horta | Identification of Asparagopsis armata -associated bacteria and characterization of their bioactive potential[END_REF]. Marine bacteria can be a vast source of new antimicrobial molecules and antibiofilm molecules with a wide range of applications in human health and Industry [START_REF] Desriac | Antimicrobial and antibiofilm molecules produced by marine bacteria[END_REF]). In the future, beneficial bacteria identified from healthy seaweed holobionts could be applied to diseased plantlets in order to suppress the growth of detrimental ones and/or to prevent disease outbreaks in aquaculture settings. In addition to bacteria, these macroalgae frequently host endophytic fungi that may have protective functions for the algae [START_REF] Dittami | A community perspective on the concept of marine holobionts: state-of-the-art, challenges, and future directions[END_REF]). Thus, metabolites of these bacteria will provide a new possibility of disease management in aquaculture systems.

The surface-associated microbiome of macroalgae is phylogenetically diverse and ecologically important. Thus, assessing the microbial functions and interactions has emerged as a critical need in macroalgal research. Massive sequencing of the 16S rRNA gene and using a metagenomic approach have been the main tools to explore these aspects. However, the complete understanding of the relationships between host and microorganisms, as well as the dynamics of the symbiotic community, probably requires the generation of comprehensive insights with another novel integrative "omics" approach, such as singlecell sequencing, metagenome binning, GeoChip, RNA-Seq, etc. To characterise the interactions of organisms with their environment, environmental metabolomics shall be further explored in this area [START_REF] Bundy | Environmental metabolomics: A critical review and future perspectives[END_REF]. Bioactive compounds from macroalgae and its associated microbiome may provide candidates with a high-efficiency and have broad implications in disease control. Concentration and quantity are the two main challenges to be addressed here. Different biotechnological tools should be developed for the isolation and identification of these compounds even at lesser concentrations, which again are the biggest challenges in this field.

Isolation and Identification of the surface-associated bacteria of Halymenia floresii and Screening

for their Quorum Sensing activity

Macroalgae host a dense bacterial epibiome forming surface biofilms, and those microbial biofilms act as a biological defense by protecting the surface from macrofoulers. The surface of Halymenia floresii was observed to be free from any fouling organisms when cultivated under Integrated MultiTrophic Aquaculture (IMTA). Moreover, the culture tanks remained clean of any exogenic algae colonization. Thus, we hypothesize that the epibacterial community on the surface of the alga may protect the surface by forming a 'protective coat' [START_REF] Wahl | The Second Skin: Ecological Role of Epibiotic Biofilms on Marine Organisms[END_REF]. In order to explore the ecological phenomenon behind this, we started the study by isolating the epibacterial community of H. floresii from different habitats. Following isolation, they have screened for any production of quorum sensing (QS) signals as these communication signals play a vital role in biofilm formation.

In this chapter, we dealt with the isolation and identification of those epibacterial community and then we further proceeded to detect any QS bacteria among them. Here we outline the work of the following Chapter II.

The objectives of this chapter are as follows:

STEP I

The cultivable epibacterial of H. floresii was isolated swabbing the surface of the algal specimens and streaking on the Marine Agar (MA) after serially diluting. The plates were incubated at 25° C for up to 7 days and the bacterial colonies were selected based on their unique morphological features, such as size, shape, surface, chromogenesis. The selected colonies were carried over for further sub-culturing to obtain axenic cultures (Fig. 18).

Fig. 18 Isolation of the epibacterial strains by swabbing, streaking, and sub-culturing

STEP II

The isolated bacterial strains were characterized morphologically and biochemically. The phenotypic characteristics were determined by using API 20 NE and APIZYM galleries (bioMérieux) (Fig. 19) and the results were listed in the ) for DNA extraction and the 16S rRNA (Fig. 19) was amplified and the bacterial isolates were commercially sequenced by GENEWIZ Laboratories. The protocol is detailed in Section 3.3.3 and the identified isolates were listed in the Table 2.

Fig. 19 Identification of the isolates by biochemical and molecular characterisation

STEP III

The ability of the isolated to produce QS signals was screened using two reporters, Chromobacterium violaceum CV026 by cross-feeding assay and Escherichia coli pSB406 by luminescent assay (Fig. 20). In both the assays Pseudomonas aeruginosa PA01 was used as a positive control. The positively responded isolates were listed in the Table 3. Keywords: Halymenia floresii, quorum sensing, AHLs, 16S rRNA.

Introduction

Macroalgae and their associated microbiota form a functional unit termed 'holobiont', characterized by its complex mutualistic relations. In a holobiont, the microbial communities contain a diverse assembly of organisms that include bacteria, fungi, microalgae, viruses, protozoa, and archaea [START_REF] Ying | The effects of marine farm-scale sequentially integrated multi-trophic aquaculture systems on microbial community composition, prevalence of sulfonamide-resistant bacteria and sulfonamide resistance gene sul1[END_REF]). The Without their associated microbiota, macroalgae cannot function optimally, indicating that the association influences the provision of ecosystem services [START_REF] Barott | Microbial diversity associated with four functional groups of benthic reef algae and the reef-building coral Montastraea annularis[END_REF][START_REF] Busetti | Marine macroalgae and their associated microbiomes as a source of antimicrobial chemical diversity[END_REF][START_REF] Ying | The effects of marine farm-scale sequentially integrated multi-trophic aquaculture systems on microbial community composition, prevalence of sulfonamide-resistant bacteria and sulfonamide resistance gene sul1[END_REF][START_REF] Van Der Loos | The Macroalgal Holobiont in a Changing Sea[END_REF].

A definite core bacterial community, consisting of Gammaproteobacteria, CFB group, Alphaproteobacteria, Firmicutes, and Actinobacteria species, seems to specifically adapt to an algal hostassociated lifestyle [START_REF] Hollants | What we can learn from sushi: a review on seaweedbacterial associations[END_REF]). Among these bacteria, the Gram-negative Proteobacteria and

Bacteroidetes phyla are the most abundant bacterial group associated with the macroalgae. These structured bacterial communities form a protective layer on the host surface acting as a physical and physiological barrier between the host and the environment and thus providing an insulating effect (van der Loos et al.

2019

). On such layers, they functionally interact and communicate through quorum sensing, although the cells are genetically different [START_REF] Thajuddin | Algae -Organisms for Imminent Biotechnology[END_REF].

Discovered in the 1990s, the Quorum Sensing (QS) bacterial communication is a system of stimuli Thus, we hypothesized that the epibacterial community on the surface of Halymenia floresii may prevent the settlement by forming a primary insulating coat possibly by their QS communication system that interfere with the growth of competitors. In this study, we propose to isolate and identify the cultivable epibacterial community from the surface of Halymenia floresii from three different thalli: Beach-Cast (BC),

Halymenia floresii collected from the Yucatan beach, and the collected BC samples cultivated under two different conditions: Cultivar Chambers (CC) and Integrated MultiTrophic Aquaculture (IMTA). After identification of the epibacterial community, the objective is to screen the identified surface-associated bacteria for Quorum Sensing activity using bioreporters. with an intensity of 110 ± 7.2 µmol photons m -2 s -1 inside a cultivation chamber. Every week, the CC samples were pulse-fed with Provasoli (0.3%) media for 24 h. The cultivation was carried for '2' weeks and '4' weeks in IMTA and CC, respectively, after several weeks of acclimatization.

Materials and methods

Algal

Isolation of surface-associated bacterial strains

After collection and/or algae cultivation for a given period of time (see section 3.3.1), the samples were immediately brought to the laboratory under cool conditions. Specimens were washed once with sterile filtered seawater and rinsed twice with Phosphate Buffered Saline (PBS) to remove the loosely attached bacteria or any other debris from the surface. The algal samples were swabbed with sterile cotton swabs and were serially diluted in sterile seawater or in distilled water with sea salts. A 0.1 mL of each dilution (10 0 ; 10 -1 ; 10 -2 ) was spread on marine agar plates and immediately incubated at 25° C for up to 7

days. Bacterial colonies were selected based on their unique morphological features, such as size, shape, surface, chromogenesis. The selected colonies were carried over for further sub-culturing to obtain axenic cultures. The sub-culturing was continued until single pure morphotype colonies were obtained. The isolates were cryopreserved at -80° C in 20% glycerol.

Characterization of selected bacterial strains

i.

Morphological and biochemical characterization

A single drop from the 24h broth culture of the bacterial isolates was observed under a phasecontrast microscope for swimming motility. To determine the twitching motility, a loopful of 24h broth culture was stabbed onto the semisolid agar (0.5 %) and incubated at 25° C for 24h. The motility was determined by observing the growth of the bacteria away from the stabbed line. The Gram type of each strain was determined by both a rapid non-staining procedure, KOH [START_REF] Buck | Nonstaining (KOH) method for determination of gram reactions of marine bacteria[END_REF], and the classical Gram staining. Moreover, phenotypic characteristics were determined by using API 20 NE and APIZYM galleries (bioMérieux) as recommended by the manufacturers. In brief, 65 µL of culture were dispensed into each cupule and inoculated at 30°C for 4 hours. After incubation, one drop of ZYM A and ZYM B was added. After 5 minutes, a value ranging from 0 to5 was assigned, corresponding to the color reaction, according to the table given by the manufacturer.

ii.

Molecular characterization

DNA was extracted from the 24h broth culture of the bacterial isolates using two different kits: iii.

DNeasy

Phylogenetic analyses

To obtain the phylogenetic affiliations from the 16S rRNA amplicons, each sequence was preliminarily aligned with ClustalW Multiple Alignment [START_REF] Susilowati | Isolation and characterization of bacteria associated with brown algae Sargassum spp. from Panjang Island and their antibacterial activities[END_REF], and the phylogenetic analyses were performed using MEGA 7. The sequences were compared against the GenBank database using BLAST (www.ncbi.nlm.nih.gov/BLAST/). The sequence similarity of greater than 97% was considered as the closest neighbor. All the 16S rRNA gene sequences retrieved from this study were deposited to NCBI under accession number MT176133 -MT176163.

Screening of the selected bacterial for QS signals -Reporter assay

Reporter strains

Two reporters, Chromobacterium violaceum CV026 and Escherichia coli pSB406, were used to screen the ability of the isolates to produce QS signals. The reporters are routinely maintained in Luria Bertani (LB) media with 100 µg mL -1 of Ampicillin for E. coli pSB 406.

Cross-feeding assay

The Both the isolates and the reporter were revived from -80°C by culturing on Marine Agar (MA) and LB agar respectively and incubated at 30°C for 24h. After incubation, a loopful of isolate and the reporter were cross-streaked parallel to each other, and the streaked plates were incubated at 30°C. After 24h, the results were observed for any purple colour production by the reporter against each isolate. An overnight culture of Pseudomonas aeruginosa PA01 was used as a positive control, and the reporter strain itself was served as a negative control. The production of violacein by C. violaceum CV026 indicated the presence of QS signals (McClean et al. 1997).

Bioluminescent assay

The bioluminescent assay was performed with the bioreporter E. coli pSB406. pSB406 contains rhlRI'::luxCDABE with EcoRI-flanked luxCDABE cassette from pSB390 [START_REF] Winson | Construction and analysis of luxCDABE -based plasmid sensors for investigating N -acyl homoserine lactone-mediated quorum sensing[END_REF]) with an ampicillin-resistant gene. With this plasmid, E. coli pSB406 can sense and detect the QS signals ranging from short-to long-chain (C-4 to C-12) [START_REF] Winson | Construction and analysis of luxCDABE -based plasmid sensors for investigating N -acyl homoserine lactone-mediated quorum sensing[END_REF]. For the assay, the E. coli pSB406 was grown in the LB broth with the ampicillin (100 mg mL -1 ) at 37°C to reach OD600 = 0.5.

The isolates were cultured in 25 mL of Marine Broth (MB) and incubated at 30°C with stirring, 120 rpm, for a previously determined period of time (16 h -36 h). After incubation, 10 mL of culture was harvested, centrifuged at 5,000 g for 10 min at 4 °C, and the supernatants were collected through a 0.2 µm (Sartorius) sterile filter. The supernatant extracts were stored at -20°C for 1-2 days. The supernatant of P.

aeruginosa PA01 was used as a positive control and that of the reporter strain itself as a negative control.

The luminescent assay was carried out in a 96-well sterile white microplate [START_REF] Class | High-throughput viability assay using an autonomously bioluminescent cell line with a bacterial Lux reporter[END_REF] .200 µL of the reporter strain E.coli pSB406 and 100 µL of the supernatant were added to each well. After incubation, the luminescence was measured by a luminometer [START_REF] Fletcher | Biosensor-based assays for PQS, HHQ and related 2-alkyl-4-quinolone quorum sensing signal molecules[END_REF]) every 30 min for 24 h

-48 h at 37 °C with a 5s shaking period prior to each measurement. All measurements were performed in triplicates. The production of luminescence of E. coli pSB406 is the reporting character of this strain for the presence of QS signals. The RLU of the negative control, i.e. E. coli pSB406, was deducted from the tests as the reporter strain itself produces luminescence.

Statistical analysis

The data were analyzed by one-way analysis of variance (ANOVA) to determine the significance of the results obtained. The normality of the QS data was tested using the Normal Distribution Comparison of SigmaPlot Version 14.0. Out of the total '96' dataset for each bacterial strain, only the normally distributed datasets were taken for the one-way ANOVA. A post-hoc test was performed following the oneway ANOVA to compare the significant differences between the groups.

Results

In this study, for the first time, we identified the cultivable epibacterial community on the surface of H. floresii under different environmental conditions and the presence of QS bacteria among them.

+ + - + - - +/- + /- - - n.d - - - - - Trypsin - - - - - - - - - - n.d - - - - - α-chymotrypsin - + - - - +++ - - - ++++
- - +++++ - - - - - - n.d - - - - - β-galactosidase - - - +++++ - - - - - - n.d - - - - - β-glucuronidase - - - - - - - - - - n.d - - - - - α-glucosidase - - - - - - - - - - n.d - - - ++++ - β-glucosidase - - - - - - - - - - n.d - - - - - N-acetyl-β- glucosaminidase - - - - - - - - - - n.d - - - - - α-mannosidase - - - - - - - - - - n.d - - - - - α-fucosidase - - - - - - - - - - n.d - - - - -

d. Firmicutes

In this study, the Firmicutes phylum was represented by only one phylotype affiliating to Bacillus circulans (B10CC Accession No. MT176154).

Of the '31' isolates from three different samples of H. floresii, Gammaproteobacteria, Alphaproteobacteria, Bacteroidetes and Firmicutes comprise 47%, 37%, 13%, and 3%, respectively.

Detection and screening of QS signal production

In this study, we used '2' different reporter strains to detect the presence of extracellular QS signal molecules and screen all the isolates for the production of QS signals (Table 3). First, all the isolates were screened against the Gram negative AHL-based reporter strain C. violaceum CV026 by cross-feeding each other. This assay did not show a positive response for any of the isolates as none of the isolates induced the purple color formation of the C. violaceum CV026.

Second, all the isolates were screened against E. coli pSB406 in a bioluminescent assay. Out of the '31' isolates, '14' strains did not induce any luminescence in E. coli pSB406, and others induced a light production. The phylotypes that did not induce the light production are as follows: in the class of The luminescent curve of the isolates plotted by number of cycles (30 min interval per cycle) against the relative light units by optical density of the reporter, E. coli pSB406 (Fig. 1234). The light production of the '36h' culture isolates seemed quite similar to the positive control, P. aeruginosa PA01 (1-factor ANOVA: F (1, 68) = 414.45, p < 0.0001). The light production was observed to increase after 10 cycles of interval. Among the three strains of the Bacteroidetes group, Tenacibaculum sp. cycles of interval (Fig. 4). These two isolates were cultured for a period of 24H.

95 units/optical density at 600 nm. Culture period, in hours, of the isolates, were denoted as 'h'.

Discussion

In recent years, marine macroalgae have emerged as a rich source of microbial diversity and biologically active secondary metabolites (Singh et al. 2015;[START_REF] Leiva | Diversity of pigmented Gram-positive bacteria associated with marine macroalgae from Antarctica[END_REF]. A fraction of epiphytic bacterial communities can be categorized as "core microbial species", suggesting an obligate association (Singh and Reddy 2016).

Isolation and identification of selected surface-associated bacteria of H. floresii

Motility is very important for several biofilm-forming bacteria [START_REF] Fernandes | Genomes and virulence factors of novel bacterial pathogens causing bleaching disease in the marine red alga Delisea pulchra[END_REF]. All the isolates in this study were motile in nature; in particular, the Bacteroidetes members were observed to have gliding motility, which is a surface-dependent mode of motility [START_REF] Raina | The role of microbial motility and chemotaxis in symbiosis[END_REF]. A mutant Capnocytophaga ochracea lacking gliding motility showed significantly attenuated biofilm formation, compared with that of the wild-type strain [START_REF] Kita | Involvement of the type IX secretion system in Capnocytophaga ochracea gliding motility and biofilm formation[END_REF]). Thus, we assume that the gliding motility of the Bacteroidetes members may play a significant role in the initial biofilm formation on the surface of H.

floresii. Gram-negative bacteria have previously been reported as the predominant group in the marine and marine macrophyte environment [START_REF] Cottrell | Community composition of marine bacterioplankton determined by 16S rRNA gene clone libraries and fluorescence in situ hybridization[END_REF]Steinberg and de Nys 2002). This study shows no exception to this rule of thumb as all the strains isolated from H. floresii were Gram-negative, except for one phylotype (Table 1). The main groups of bacterial phyla associated with macroalgae include Alphaproteobacteria, Gammaproteobacteria, Bacteroidetes, Firmicutes and Actinobacteria [START_REF] Goecke | Chemical interactions between marine macroalgae and bacteria[END_REF]. Except for Actinobacteria, H. floresii associated bacteria identified in the study essentially also fall under the same groups. [START_REF] Lachnit | Specific epibacterial communities on macroalgae: Phylogeny matters more than habitat[END_REF] demonstrated that bacterial communities derived from macroalgae belonging to the same species but originating from a different habitat were more similar [START_REF] Lachnit | Specific epibacterial communities on macroalgae: Phylogeny matters more than habitat[END_REF][START_REF] Goecke | Chemical interactions between marine macroalgae and bacteria[END_REF]). The same was observed in the red macroalgal species Bonnemaisonia asparagoides, Lomentaria clavellosa and Polysiphonia stricta [START_REF] Nylund | The red alga Bonnemaisonia asparagoides regulates epiphytic bacterial abundance and community composition by chemical defence[END_REF]). However, with H. floresii, the bacterial communities were quite dissimilar with different habitats, such as BC, IMTA, and CC. We assume that this result may be mainly attributed to the macroalgal cultivation system. The abiotic physiological responses such as temperature, light, water turbulence, and nutrient availability in different habitats are believed to play a major role in this dissimilarity. Understanding the temporal dynamics of epiphytic bacteria can help identify the possible modifications in the 'protective layer' due to external factors of stress [START_REF] Mancuso | Diversity and temporal dynamics of the epiphytic bacterial communities associated with the canopy-forming seaweed Cystoseira compressa (Esper) Gerloff and Nizamuddin[END_REF]). In the case of H. floresii, the controlled and uncontrolled culture conditions in CC and IMTA, respectively, significantly contributed to the stress, whereas in BC, it was mainly caused by the overexposure to increased sunlight (i.e. UV radiation).

The macroalgal-associated microbial communities belong to various genera such as the Pseudomonas, Pseudoalteromonas, Stenotrophomonas, Vibrio, Alteromonas, Shewanella, Streptomyces, and Bacillus species, and evolved through a highly competitive environment due to nutrient and space limitation on their host surface, subsequently producing allelochemicals capable of preventing secondary colonization [START_REF] Egan | Pseudoalteromonas ulvae sp. nov., a bacterium with antifouling activities isolated from the surface of a marine alga[END_REF][START_REF] Egan | Unlocking the diversity and biotechnological potential of marine surface associated microbial communities[END_REF]Wiese et al. 2009;[START_REF] Tujula | Variability and abundance of the epiphytic bacterial community associated with a green marine Ulvacean alga[END_REF][START_REF] Uzair | Isolation, purification, structural elucidation and antimicrobial activities of kocumarin, a novel antibiotic isolated from actinobacterium Kocuria marina CMG S2 associated with the brown seaweed Pelvetia canaliculata[END_REF]).

Among the 21, 25, and 27 phylotypes isolated from the red algae, Chondrus crispus, Palmaria palmata and Porphyra umbilicalis, respectively, Gammaproteobacteria was the dominant phyla followed by the Firmicutes (del Olmo et al. 2018). We observed the same result regarding the Gammaproteobacteria (48%), whereas the Firmicutes were the least dominant in H. floresii. After a review of numerous studies, [START_REF] Hollants | What we can learn from sushi: a review on seaweedbacterial associations[END_REF] conclude that macroalgal-bacteria interactions primarily consist of Gammaproteobacteria (37%) followed by Bacteroidetes (20%), Alphaproteobacteria (13%), Firmicutes (10%), and lastly Actinobacteria (9%). We also perceived the same pattern in H. floresii, whereas Alphaproteobacteria (37%) was followed by Bacteroides (13%).

To conclude, among the '31' strains identified in this study, few isolates were described as opportunistic putative pathogens in different marine macroalgae and other organisms. For instance, Nautella italica R11 and Phaeobacter gallaeciensis LSS9, which have been shown to cause bleaching symptoms in vitro, just like Alteromonas, Aquimarina, Agarivorans, are associated with the bleaching disease in a temperate red alga Delisea pulchra [START_REF] Fernandes | Genomes and virulence factors of novel bacterial pathogens causing bleaching disease in the marine red alga Delisea pulchra[END_REF][START_REF] Zozaya-Valdes | A comprehensive analysis of the microbial communities of healthy and diseased marine macroalgae and the detection of known and potential bacterial pathogens[END_REF]Kumar et al. 2016). Several taxa belonging to Vibrio, Alteromonas, Aquimarina, Flavobacteriaceae, and

Rhodobacteriaceae were found to be associated with the bleached brown alga Ecklonia and Delisea and diseased sponges and coral. Members belonging to the same genera mentioned in these studies were also isolated in our study, but from the good healthy surface of H. floresii without any bleaching or other disease symptoms. This may open a new avenue for exploring their opportunistic pathogenicity to the host, and in our future research work, we intend to find out whether there are any opportunistic pathogens under any diseased condition (for example, bleaching) of H. floresii.

Detection and screening of QS signals

Among

i. Cross-feeding assay

In this assay, with the C. violaceum CV026 reporter strain, we did not observe any positive reaction by the formation of purple color, even though for several reasons, it is not possible to conclude from the nonappearance of a signal that the strains do not produce AHLs. They might produce structurally different and novel AHLs that are not easily detected by the biosensors used, so it is rather unlikely to be excluded.

Moreover, the most challenging issue in AHLs detection is that the bacterial strains may produce the signals in very low concentration, so that they are below the threshold of sensitivity of the biosensors used [START_REF] Steindler | Detection of quorum-sensing N -acyl homoserine lactone signal molecules by bacterial biosensors[END_REF].

ii.

Bioluminescence assay

The bioluminescent assay was performed with the reporter strain E. coli pSB406. Using this reporter strain, it was possible to screen the isolates qualitatively and quantitatively for the production of exogenous AHLs by their luminescence induction. QS systems were intensively confirmed to be involved in the regulation of pathogen virulence in aquaculture. Virulence factors of the several opportunistic pathogens in the aquatic environments such as shrimp and freshwater prawn aquaculture were regulated and/or attenuated by their corresponding QS system [START_REF] Zhang | Exploiting quorum sensing interfering strategies in gram-negative bacteria for the enhancement of environmental applications[END_REF]. In our study, '3' isolates were found to be affiliated to Ruegaria sp., but the QS signal production was observed only in Ruegaria lacuscaerulensis (B19CC), isolated from CC H.

floresii. A QS bacterium, Ruegaria sp. R11, isolated from the red alga Delisea pulchra, produces two AHLs, N-octanoyl-DL-homoserine lactone (OHL) and N-hexanoyl-DL-homoserine lactone (HHL), which play a crucial role in causing the bleaching disease.

The pathogenicity of microbes that infect aquaculture systems is closely related to the release of virulence factors, which is regulated by the QS system [START_REF] Zhao | Mechanisms of quorum sensing and strategies for quorum sensing disruption in aquaculture pathogens[END_REF]. There seems to be a link between QS and diseases through the regulation of certain phenotypes and the induction of virulence factors responsible for the pathogen-host association. Macroalgae interfere with bacterial QS systems that regulate bacterial cell-to-cell communication [START_REF] Goecke | Chemical interactions between marine macroalgae and bacteria[END_REF]. Such disruption or interference of the QS mechanism of pathogens is a novel and environment-friendly way to control disease outbreaks in aquaculture (Natrah et al. 2011a). Thus, it is necessary to differentiate the potential pathogens and nonpathogens among the QS bacteria of H. floresii and to identify the QS interfering compounds produced by H. floresii, if any.

Conclusions

Our results provide an important knowledge base as first step to illustrate the epibacterial community of H. floresii under different environmental conditions, such as BC, CC, and IMTA. In this study, we have reported the cultivable epibacterial community isolated from the surfaces of BC, IMTA, and CC Halymenia floresii (see section 3.3.1 for details) of Yucatan, Mexico. This study is the first report about the epibacterial community of H. floresii and their QS activity, particularly in culture conditions.

Thus, the results may open new avenues to explore the H. floresii holobiont and the possible interference in the QS system of its own epibiome. However, the epibacterial community identified in this study by culture-dependent techniques does not provide an entire picture of the bacteria present in H. floresii. Thus, this study must be extended by the metagenomics approach to elucidate a complete bacterial community of H. floresii for greater understanding of the benefits of the associated host and its environment. As this study has revealed the presence of QS signals in different opportunistic pathogens (as reported in several literatures), their pathogenic shift may be provided by their respective communication system. We also assume that any adverse environmental conditions may affect the bacterial-host relationship, and it is very important to identify the potential pathogens or non-pathogens and/or significant protectors in these cultivable bacterial isolates. A further in-depth exploration of the QS communication system and isolation and characterization of the AHLs in these isolates will provide a functional perspective of the signals towards their pathogenicity shift.

Future perspectives

In the Yucatan peninsula, H. floresii represent a potential raw material source for the production of carrageenan [START_REF] Robledo | Prospects for the cultivation of economically important carrageenophytes in Southeast Mexico[END_REF]. As a lamda-carrageenophyte, H. floresii was identified as a good candidate to recycle inorganic nutrients in an IMTA system (Pliego-Cortés et al. 2019), and bacteriadegrading algal polysaccharides are key players in algal biomass recycling [START_REF] Martin | The cultivable surface microbiota of the brown alga Ascophyllum nodosum is enriched in macroalgal-polysaccharidedegrading bacteria[END_REF]. Identifying the bacterial community associated with the carrageenophyte (particularly in aquaculture) is an essential requirement to further explore the production of carrageenan-degrading enzymes, as it is a primary 'carbon' source for the epibiome. Using our results, it is worth conducting experiments to identify the carrageenandegrading bacteria, and particular consideration should be given to Proteobacteria and Bacteroidetes, as they are the main producers of carrageenases [START_REF] Chauhan | Bacterial carrageenases: an overview of production and biotechnological applications[END_REF].

Chemical defense against microfouling by allelopathic active metabolites of Halymenia floresii (Rhodophyta)

Following the identification of the surface-associated bacteria of H. floresii and detecting the Quorum Sensing (QS) strains (Chapter II), we further proceeded to evaluate the chemical defence of the alga on the surface by means of allelopathic active metabolites. The interference of the surface metabolites with the QS activity of the chosen strains was also studied and the metabolite identification was done by untargeted metabolomic analysis. Here we outline the work of the following Chapter III.

Many macroalgae appear to be chemically defended at constantly high levels by producing a rich variety of secondary metabolites [START_REF] Weinberger | Pathogen-induced defense and innate immunity in macroalgae[END_REF][START_REF] Nylund | Metabolomic assessment of induced and activated chemical defence in the invasive red alga Gracilaria vermiculophylla[END_REF]. As the surface of Halymenia floresii was observed to be remarkably free from any fouling organisms when cultivated under Integrated MultiTrophic Aquaculture (IMTA), we also hypothesized that the chemical defence of H. floresii may be responsible for this ecological phenomenon. The allelopathic active compounds may contribute to algal's defence on the surface by preventing biofilm formation that ultimately interferes with the settlement and growth of competitors. In this chapter, we explored the chemical defence of H. floresii by extracting its surface metabolites and evaluating their interference in the quorum sensing (QS) communication of the surface-associated bacteria (more details inside the Chapter III). The objectives of this chapter are as follows:

Methodology

Step I As represented below, the surface metabolites (DIP) of H. floresii were extracted by the method of DIP as proposed by de Nys et al (1998) (Fig. 21). The algal specimens were dipped in different organic solvents (n-hexane, dichloromethane, dichloromethane: methanol (1:1 v/v) and methanol) and assessed for their effect on the lysis of macroalgal cortical cells at different times (10, 20, 30, 40, 50 and 60 seconds) using an epifluorescence microscope. The effect of the solvent on the surface cells was determined by analyzing the microscopic images using Image J software to count the number of lysed versus intact cells in five different fields of view for each replicate. Additionally, the whole-cell metabolites (WCM) were extracted by complete maceration of the algal samples in the chosen solvent (same as determined for the DIP method). This step is well detailed in (Section 4.3.2).

Objectives

1. Extraction of the surface metabolites from H. floresii by DIP method using different solvents with increasing polarity and immersion periods 2. Evaluation of the QQ activity behaviour of the surface extracts on the H. floresii associated bacterial population by bioluminescent assay 3. Identification of compounds by LC-MS metabolomics approach.

Fig. 21 Extraction of surface-associated metabolites by the method of DIP

Step II

The surface-associated bacteria of H. floresii were previously isolated and screened for their QS activity (detailed in Chapter II). In this step, we evaluated the interference of the surface-extracts with the QS activity of the chosen bacterial strains by Bioluminsecent assay using Escherichia coli pSB406 as a reporter. The luminescent assay was carried out in a 96-well sterile white microplate (Fig. 22). To each well, the reporter strain E. coli pSB406, the supernatant of the bacterial culture, and DIP n-hexane extract at '3' different concentrations (250, 500 and 1000 ng cm -2 ) was added. The luminescence (RLU -Relative Light Units) of the reporter was measured by a luminometer at an interval of 30 min for 24 h -48 h at 37 °C. This step is explained in detail in (Section 4.3.3).

Introduction

Marine biofouling is a natural process of colonization involving a wide range of organisms on any surface in aqueous environments. This phenomenon generally takes place over a four-step sequential process (i) initial 'conditioning' of the surfaces by organic compounds, (ii) subsequent development of bacterial biofilm, (iii) secondary colonization by unicellular eukaryotes (maturation I), and (iv) a final step of attachment of invertebrate larvae and algal spores (maturation II) [START_REF] Davis | Epibiosis of marine algae and benthic invertebrates: natural products chemistry and other mechanisms inhibiting settlement and overgrowth[END_REF][START_REF] Wahl | Marine epibiosis. I. Fouling and antifouling: some basic aspects[END_REF][START_REF] Parsek | Sociomicrobiology: the connections between quorum sensing and biofilms[END_REF]. In artificial substrates, cylinders and panels, maturation II is possible without initial conditioning of the surface [START_REF] Rittschof | Fouling in natural flows: Cylinders and panels as collectors of particles and barnacle larvae[END_REF]). The seaweed's surface provides a suitable substratum for the settlement of microorganisms including bacteria, diatoms, protozoa and fungi [START_REF] Longford | Comparisons of diversity of bacterial communities associated with three sessile marine eukaryotes[END_REF][START_REF] Wahl | The Second Skin: Ecological Role of Epibiotic Biofilms on Marine Organisms[END_REF][START_REF] Hollants | What we can learn from sushi: a review on seaweedbacterial associations[END_REF][START_REF] Egan | The seaweed holobiont: understanding seaweed-bacteria interactions[END_REF]. The seaweed's surface secretes various organic substances that function as nutrients for the multiplication of bacteria and the formation of microbial biofilms [START_REF] Singh | Unraveling the Functions of the Macroalgal Microbiome[END_REF]. In this association, known as 'holobiont' (Zilber- Rosenberg and Rosenberg 2008;[START_REF] Vandenkoornhuyse | The importance of the microbiome of the plant holobiont[END_REF] a highly complex set of chemical, physical and biological interactions regulate the relationships between the basibiont and its epibionts. To avoid consequences of epibiosis, some seaweeds have developed a variety of defensive strategies against biofouling settlement.

Biofouling is a great challenge in seaweed aquaculture and in Integrated MultiTrophic Aquaculture (IMTA) as it increases the cost of cultivation [START_REF] Buck | State of the art and challenges for offshore Integrated multi-trophic aquaculture (IMTA)[END_REF]. It also results in the potential loss of biomass by affecting their flexibility, decreasing the photosynthesis and nutrient uptake, and inhibiting reproduction [START_REF] Walls | Successional changes of epibiont fouling communities of the cultivated kelp Alaria esculenta: Predictability and influences[END_REF]). In addition, biofouling poses a health risk to cultured fish as it can facilitate and amplify the presence of pathogens that cause various diseases. This issue is also a concern for IMTA sites, where fish and shellfish are intentionally cultivated in close proximity [START_REF] Bannister | Biofouling in marine aquaculture: a review of recent research and developments[END_REF].

One of the vital steps in biofouling is chemically mediated and regulated by the microbial Quorum Sensing (QS) activity. QS is a cell density-dependent communication system, which regulates a diverse array of physiological activities including symbiosis, virulence, competence, conjugation, antibacterial molecule production, motility, sporulation, and biofilm formation [START_REF] Miller | Quorum Sensing in Bacteria[END_REF][START_REF] Wahl | The Second Skin: Ecological Role of Epibiotic Biofilms on Marine Organisms[END_REF][START_REF] Zhou | Quorum Sensing Is a Language of Chemical Signals and Plays an Ecological Role in Algal-Bacterial Interactions[END_REF]. In biofilms, QS systems are expected to be active and are thought to play a key role in their formation when high cell densities are reached. The process which prevents the QS is known as Quorum Quenching (QQ) [START_REF] Alagarasan | Shoot the message, not the messenger-combating pathogenic virulence in plants by inhibiting quorum sensing mediated signaling molecules[END_REF] and it refers to all processes involved in the disturbance of QS [START_REF] Dong | Quenching quorum-sensingdependent bacterial infection by an N-acyl homoserine lactonase[END_REF]. QQ can be achieved through inhibition, degradation or by producing antagonistic compounds that interfere with the QS signals [START_REF] Chen | Quorum quenching enzymes and their application in degrading signal molecules to block quorum sensing-dependent infection[END_REF]. For example, the red alga Delisea pulchra produces an analogous molecule of bacterial N-Acyl-Homoserine Lactones (AHLs) known to inhibit the signal pathways in Gram-negative bacteria that leads to selective colonization of Grampositive bacteria on this species thalli (Steinberg and de Nys 2002;[START_REF] Dahms | Antifouling Compounds from Marine Macroalgae[END_REF][START_REF] Saha | Minireview: algal natural compounds and extracts as antifoulants[END_REF]. QQ would thus represent an alternative strategy to control biofilm formations [START_REF] Chen | Quorum quenching enzymes and their application in degrading signal molecules to block quorum sensing-dependent infection[END_REF][START_REF] Barre | Blue biotechnology : production and use of marine molecules[END_REF]. Understanding and harnessing the natural antifouling defenses of seaweeds could be a good strategy to control the biofouling by prevention and inhibition [START_REF] Bannister | Biofouling in marine aquaculture: a review of recent research and developments[END_REF].

The genus Halymenia includes several species that are appreciated and consumed in Asian countries (H. discoidea, H. durvillei, H. venusta, H. dilatata, H. formosa). Halymenia spp. has economic importance as a lambda-carrageenophyte and R-phycoerythrin content. Halymenia floresii a native species in the Yucatan peninsula coast of Mexico dominates rocky substrates and has also been identified as a species with a high potential for cultivation. Studies related to growth under different light qualities and the characterization of its carrageenan have also been published, suggesting their use as raw material or for pharmacological utilization [START_REF] Freile-Pelegrín | Preliminary characterization of carrageenan from the red seaweed Halymenia floresii[END_REF][START_REF] Robledo | Prospects for the cultivation of economically important carrageenophytes in Southeast Mexico[END_REF][START_REF] Pliego-Cortés | Growth, biochemical and antioxidant content of Rhodymenia pseudopalmata (Rhodymeniales, Rhodophyta) cultivated under salinity and irradiance treatments[END_REF]. For example, the use of light quality treatments in the cultivation of H. floresii can be used to manipulate lutein, antioxidants and a Mycosporine-like Amino acids composition which may be of particular interest for the market of edible seaweeds and Human Health (Godínez- [START_REF] Godínez-Ortega | Growth and pigment composition in the red alga Halymenia floresii cultured under different light qualities[END_REF]). H. floresii was also observed as a good candidate to recycle inorganic nutrients in the IMTA system, producing biomass while reducing the environmental impact of coastal ecosystems through responsible aquaculture practices [START_REF] Godínez-Ortega | Growth and pigment composition in the red alga Halymenia floresii cultured under different light qualities[END_REF][START_REF] Robledo | Prospects for the cultivation of economically important carrageenophytes in Southeast Mexico[END_REF]. In addition, the establishment of opportunist green algae and the colonization of sessile invertebrates, which usually disturb the cultivation,

were not observed during the experimental cultivation of H. floresii under IMTA. The culture tanks were clean and the surface of the H. floresii was remarkably free from any fouling organisms. In addition, even at high concentrations of Dissolved Inorganic Nitrogen (DIN ~ 150 µM), no noticeable growth of epiphytes was observed over H. floresii [START_REF] Pliego-Cortés | Growth, biochemical and antioxidant content of Rhodymenia pseudopalmata (Rhodymeniales, Rhodophyta) cultivated under salinity and irradiance treatments[END_REF]. This phenomenon could reveal that the presence of H. floresii may prevent biofilm formation by releasing allelopathic active compounds, which ultimately interfere with the settlement and growth of competitors.

To understand this allelopathic phenomenon and how H. floresii could chemically control bacterial epibiosis, we focused mainly on the algal surface metabolites that may affect or control the associated bacteria. For that, H. floresii was cultivated under controlled environmental conditions (CC) after previously being collected from the beach (Beach-Cast, BC). The objectives of our study include (i) the extraction of the surface metabolites from H. floresii carrying out the DIP extraction method using different solvents with increasing polarity and immersion periods; (ii) the isolation and identification of bacteria living on the surface of H. floresii; (iii) the evaluation of the QQ activity behavior of the surface extracts on the H. floresii associated bacterial population by bioluminescent assay and (iv) the identification of compounds using the LC-MS metabolomics approach.

Materials & Methods

Algal material

Beach

Optimization of Selective Extraction of surface-associated metabolites i. Surface-associated metabolites (DIP) extraction -Determination of Immersion solvent and Immersion period

The surface-associated metabolites were extracted using the DIP extraction method proposed by de Nys et al. (1998). In brief, four different organic solvents with increasing polarity were selected: n-hexane, dichloromethane (DCM), dichloromethane: methanol (1:1 v/v) (DCM:MeOH) and methanol (MeOH) and assessed for their effect on the lysis of macroalgal cortical cells at different times using an epifluorescence microscope. Approximately 1.2 g of H. floresii tissue fragments were dipped in 15 mL of each solvent for a period of 10, 20, 30, 40, 50 and 60s under stirring. The macroalgal thalli were previously dried using cotton wipes before dipping. After each immersion, the whole axes of dipped macroalgae were rinsed and small cut sections were finely rinsed with sterile seawater. The finely rinsed algal portions were immediately observed under an epifluorescence microscope (Leica DM2500, Wetzlar, Germany) The cortical cells of H. floresii were observed for their autofluorescence in blue light 2-490 nm-450nm; magnification at 400x and qualitatively assessed for the presence of lysed cells. Intact cells have discrete chloroplasts whereas in lysed cells chloroplasts are disrupted, filling the whole cell with pigments. The effect of the solvent on the surface cells was determined by analyzing the microscopic images using Image J software, to count the number of lysed versus intact cells in five different fields of view for each replicate.

A validation step was performed with the selected solvent with just one cut end of H. floresii in order to eliminate the effect of cut ends while dipping. The extracts of all the immersion periods of each solvent

Quorum Quenching activity by Bioluminescent reporter assay i. Bacterial strains -Isolation from the surface of H. floresii

The algal sample was washed once with sterile filtered seawater and rinsed three times with Phosphate-buffered saline (pH 7.4), to remove the loosely attached bacteria or any other debris from the surface. Samples were swabbed with sterile cotton swabs and were serially diluted using sterile filtered seawater. Care was taken not to damage the algal fronds and the finest branchlets. A 0.1 mL of each dilution (up to 10 -2 ) was spread on the marine agar plates. Plates were incubated at room temperature (25° C) from 24 h to 7 days. The plates were observed intermittently for the growth of the bacteria. Bacterial colonies with distinct morphological features were selected and carried over for further sub-culturing to obtain an axenic culture. The sub-culturing was continued until single pure morphotype colonies were obtained. The The PCR products (4 µL) were analyzed by 1% agarose gel in 1XTAE buffer, electrophoresed at 90V. The results were visualized under a BioDoc-It Imager. The amplified 16S rRNA genes of the bacterial isolates were sent to the GENEWIZ Laboratories, USA for sequencing.

iii. Bioluminescence -Reporter Assay a. Preparation of supernatants

The isolates were cultured in 25 mL of Marine Broth (MB) and incubated at 30°C with stirring, at 120 rpm, for a previously determined period of time (16 h -36 h). After incubation, a 10 mL of culture was harvested, centrifuged at 5,000 g for 10 min at 4 °C and the supernatants were collected by passing it through a sterile 0.2 µm (Sartorius) filter. The supernatant extract was stored at -20°C for 1-2 days.

b. Reporter strain

Escherichia coli pSB406 is the reporter strain used in this study. E. coli pSB406, a construct of rhlRI'::luxCDABE with EcoRI-flanked luxCDABE cassette from pSB390 [START_REF] Winson | Construction and analysis of luxCDABE -based plasmid sensors for investigating N -acyl homoserine lactone-mediated quorum sensing[END_REF]) with an ampicillin-resistant gene. With this plasmid construct, E. coli pSB406 can sense and detect the QS signals ranging from short to long-chain (C-4 to C-12) [START_REF] Winson | Construction and analysis of luxCDABE -based plasmid sensors for investigating N -acyl homoserine lactone-mediated quorum sensing[END_REF]). Thus, the reporter was routinely maintained in Luria Bretani (LB) media containing Ampicillin 100 µg mL -1 . For the assay, the E. coli pSB406 was grown in the LB broth with the ampicillin (100 µg mL -1 ) at 37°C to reach OD600 = 0.5.

c. Bioluminescent assay

The luminescent assay was carried out in a 96-well, sterile white microplate. This assay was performed only with the DIP n-hexane extract, as n-hexane was our eventual choice of immersion solvent.

A 200 µL of the reporter strain E. coli pSB406, 100 µL of the supernatant and 1 µL DIP n-hexane extract at '3' different concentrations (250, 500 and 1000 ng cm -2 ) were added to each well. The plate was covered with a sterile sealing membrane and incubated at 37 °C for 30 minutes. After incubation, the luminescence (RLU -Relative Light Units) was measured by a luminometer at an interval of 30 min for 24 h -48 h at 37 °C, with a 5 seconds agitation period prior to each measurement. The OD600 of the reporter was measured to calculate the RLU/OD. Controls without any extract, and a blank with the solvent, n-hexane, were also included. The RLU of the control was deducted from the tests as the reporter strain itself produces luminescence. All measurements were performed in triplicate.

Untargeted Metabolomic profiling -Identification of the compounds by LC-MS

In this study, a straightforward approach to identify and characterize surface-associated metabolites was not possible due to the low concentration and quantity of the DIP extracts. Thus, the chemical fingerprints of surface metabolites (DIP-n-hexane extract) were compared with the whole-cell extracts (WCM-n-hexane) of H. floresii, for which raw LC-MS data of both the DIP and WCM extracts were preprocessed by the XCMS Online metabolomics platform to search for any possible candidates in a Mass Bank, the Madison Metabolomics Consortium Database.

i. LC-MS conditions

The chemical profiling of the DIP and WCM extracts were analyzed by LC-MS using an LC-ESI-Q-TOF-MS (Dionex, Ultimate 3000, Bruker, micrOTOF-QII) system (Bruker Daltonik GmbH, Bremen, Germany). Separations were performed using an analytical reversed-phase column. A sample of 10 µL of extract was injected for every analysis, at a flow rate of 0.5 mL min -1 with a column temperature of 30 °C and the elution gradient was adopted from [START_REF] Othmani | Surface metabolites of the brown alga Taonia atomaria have the ability to regulate epibiosis[END_REF]. Briefly, a binary programming of methanol (MeOH) and water (H2O) was involved at a linear gradient from MeOH/ H2O (40:60, v/v) to MeOH/ H2O (85:15, v/v) for 3 min and to 100% MeOH for 4 min, followed by a final isocratic step with 100% MeOH for 18 min, finishing by a return to the initial conditions (0.1 min) and equilibration of the column (9.9 min). Regarding the mass spectrometer, operating conditions were set as follows: drying temperature: 350 °C, capillary voltage: 4 kV, nebulizer pressure: 3.45 bars, drying gas: helium at a flow rate of 12 L min -1 [START_REF] Othmani | Surface metabolites of the brown alga Taonia atomaria have the ability to regulate epibiosis[END_REF]. Mass spectra acquisition was set at 0.5 Hz from m/Z 50 to 1000.

ii.

Pre-processing and processing of data: XCMS Online and the Madison Metabolite Consortium

Database LC/MS data were exported directly from the instrument in .mzXML format. The .mzXML data were pre-processed by XCMS Online (https://xcmsonline.scripps.edu/) [START_REF] Gowda | Interactive XCMS Online: Simplifying Advanced Metabolomic Data Processing and Subsequent Statistical Analyses[END_REF]), an automated cloud-based method, for filtering, identifying the peaks, alignment and retention time correction using the centWave method with signal/noise threshold -6 and mz difference as 0.01. Single, Pairwise and Multigroup jobs were assigned based on the sample's requirement. The pre-processed data were uploaded directly as a flat text with a mass/charge ratio, retention time and relative intensity based on mass at a tolerance level of 5 ppm, to the Madison Metabolomics Consortium Database (MMCD http://mmcd.nmrfam.wisc.edu/) [START_REF] Cui | Metabolite identification via the Madison Metabolomics Consortium Database[END_REF] to search for possible candidates present in our extracts.

Statistical Analysis

Results are expressed as mean ± standard deviation (SD). The data were analyzed by one-way analysis of variance to determine the significance of the results obtained. The normality of the QQ data was tested using Normal Distribution Comparison of SigmaPlot Version 14.0. Out of the total '96' datasets for each bacterial strain, only the normally distributed datasets were taken for one-way ANOVA. A post-hoc test was performed following one-way ANOVA to compare the significant differences between the groups.

Results

Algal material -Culture of H. floresii

H. floresii growth was low under pulsed nutrient supply. The macroalgae growth rates ranged from 0.6 to 2.8 % d -1 after 8 weeks of cultivation without significant differences (p > 0.05).

Optimization of Selective Extraction of surface-associated metabolites i. DIP Extraction

The main objective was to define the highest possible exposure time in a specific solvent (or a mixture of solvents) in order to maximize the extraction efficiency of surface-associated metabolites without disturbing the cortical cells and extracting whole-cell metabolites. The effect of four organic solvents (n-hexane, dichloromethane, dichloromethane: methanol (1:1 v/v) and methanol) on the surface cells of Halymenia floresii was tested.

Epifluorescence microscopy was used to estimate the cell lysis, as chloroplasts fill the whole cell with auto-fluorescent pigments when disrupted, in comparison with the control (treated only with sterile seawater). All these solvents showed a destructive effect, except n-hexane, which had the advantage of maintaining the integrity of cell membranes up to 60s. with the other solvents the cells were lysed from the initial period of immersion onwards (data not shown).

After determining the particular immersion solvent and immersion period, the same method was applied to the rest of the samples. In comparison to other solvents preliminarily tested for DIP, the n-hexane extract was the least concentrated (Table 1). ii.

WCM Extraction

Table 1 illustrates the mean levels of secondary metabolites extracted by WCM protocol after DIP extraction with n-hexane. The increase in the immersion period during DIP extraction affected the wholecell metabolite extraction. The concentration of WCM extracts was observed at 29.3 µg mL -1 after 10s of dipping whereas it was 4.5 µg mL -1 after 60s of dipping.

Quorum Quenching activity of DIP extracts by Bioluminescence -Reporter Assay i. Identification -Taxonomic affiliation of bacterial strains

The sequences were compared against GenBank database using BLAST. The taxonomic affiliations of the isolated phylotypes (> 97% similarity) are shown in Based on the 16S rRNA gene sequencing compared against GenBank using NCBI. The assigned reference name of the phylotypes. Bacterial group to which the phylotypes belong, closest neighbor and their similarity in percentage.

ii. Bioluminescence -Reporter Assay

The quorum quenching activity of crude DIP-n-hexane extract was analyzed using the abovementioned phylotypes at three different concentrations. Thus, 1,000 ng cm -2 was the original concentration observed on the surface of extracted H. floresii and it was further diluted to obtain 500 ng cm -2 and 250 ng cm -2 , in order to validate their activity more precisely. plotted against relative light units/optical density at 600 nm.

Untargeted metabolomic profiling

The raw DIP LC-MS data in duplicates, were uploaded to the XCMS platform assigning a 'Single' job treatment whereas the raw WCM LC-MS data were assigned a 'Multi' job as the WCM data includes 10s, the 20s, 30s, 40s, 50s and 60s individually in duplicate. Both sets of data were treated with the same parameters. Initially, the Total Ion Chromatograms (TIC) of DIP and WCM were overlaid and compared for similar peaks based on their retention time (Fig 5). The WCM chromatogram was quite complex with a large diversity of compounds with high intensity than that of the DIP chromatogram. Most of the WCM extract's metabolites were detected between 10 to 17 min. The peaks of DIP chromatogram were observed to be less intensified which may be due to the lower concentration of the surface extract. But interestingly we observed '2' peaks of DIP at high intensity at 14.0 -14.5 min and 17.0 -17.5 min.

The pre-processed data of DIP and WCM were uploaded separately to the MMCD Mass Bank. For DIP LC-MS data, the Mass Bank proposed '83' metabolites with a one-to-one match of '57' metabolites.

For WCM LC-MS data totaled '335' metabolites, with a one-to-one match of '219' metabolites. The proposed list of metabolites for DIP and WCM was quite different from each other where only '8' proposed metabolites were observed to be common among them. The common metabolites of DIP and WCM are shown in Table 3. These proposed metabolites may possibly be smaller compounds as their m/z ratio was observed to be from 200 to 450. 123 458.12 ND 1-deoxy-1- (7,8-dimethyl-2,4-dioxo-3,4-dihydro-2H-benzo[g] We grouped those common metabolites into three groups. Group 1, that includes Compounds 1 and 2, 2-furaldehyde and 2-(2,4-dichloro-phenoxy)-n-(2-mercapto-ethyl)-acetamide, which were not quite relevant to macroalgal secondary metabolites. Group 2 consisted of flavonoid family members such as, Quercetin 3-sulphate, Quercetin 7-O-glucoside, Reduced Flavin Mononucleotide and 1-deoxy-1- (7,8dimethyl-2,4-dioxo-3,4-dihydro-2h-benzo[g]

Discussion

H. floresii is a good candidate for recycling inorganic nutrients in a land-based IMTA system, producing biomass whilst reducing the environmental impact of coastal ecosystems through responsible aquaculture practices. Epiphytes and fouling organisms usually induce problems in aquaculture, reducing the yield. The presence of H. floresii in the culture ponds significantly limits the establishment of opportunist green algae usually disturbing the cultures. From these ecological observations, it was interesting to understand these phenomena. So, at first, H. floresii was cultivated under controlled environmental conditions (CC). The growth rate of the CC H. floresii sample was observed to be significantly lesser when compared to the red alga Rhodymenia pseudopalmata, under stress conditions with higher solar radiation [START_REF] Pliego-Cortés | Stress tolerance and photoadaptation to solar radiation in Rhodymenia pseudopalmata (Rhodophyta) through mycosporine-like amino acids, phenolic compounds, and pigments in an Integrated Multi-Trophic Aquaculture system[END_REF]. We found that this might be an ideal environment to study the defense mechanism of H. floresii, as growth and defense were inversely related to each other [START_REF] Nylund | Costs and Benefits of Chemical Defence in the Red Alga Bonnemaisonia hamifera[END_REF].

Selective extraction of surface-associated metabolites

In order to selectively extract the surface-associated metabolites, we used epifluorescence microscopy to differentiate between the intact and lysed cortical cells of H. floresii after dipping them in different solvents, such as n-hexane, dichloromethane, methanol and dichloromethane: methanol (1:1), without any staining, contrary to the work on the brown alga Taonia atomaria [START_REF] Othmani | Surface metabolites of the brown alga Taonia atomaria have the ability to regulate epibiosis[END_REF]. In a similar study using Delisea pulchra, surface metabolite extraction by n-hexane did not show any cell lysis up to 30s but exposure at 60s lysed the cells, whereas cells of Laurencia obtusa remained intact even at 60s [START_REF] De Nys | A new method for determining surface concentrations of marine natural products on seaweeds[END_REF]. For Delisea pulchra and Laurencia obtusa, n-hexane was found to be a suitable solvent for surface-metabolites extraction (de Nys et al. 1998) whereas for Taonia atomaria it was ineffective [START_REF] Othmani | Surface metabolites of the brown alga Taonia atomaria have the ability to regulate epibiosis[END_REF].

The non-polar solvents, hexane, and dichloromethane (100%) did not discolor algal tissue in different red and brown algae [START_REF] Kientz | A new method for removing microflora from macroalgal surfaces: an important step for natural product discovery[END_REF]) and the same pattern was also seen during DIP extraction of H. floresii. N-hexane has been proved to extract non-polar lipophilic molecules in previous studies (de Nys et al. 1998). In our study, n-hexane also showed extraction of lipophilic metabolites associated on the surface of H. floresii. [START_REF] Jennings | Phlorotannins versus other factors affecting epiphyte abundance on the kelp Ecklonia radiata[END_REF] suggested that they have a higher chance of accumulating at the surface, though in the absence of strong interionic interactions they may have a higher probability of diffusion [START_REF] Saha | Dimethylsulphopropionate (DMSP) and proline from the surface of the brown alga Fucus vesiculosus inhibit bacterial attachment[END_REF]. As a rule of thumb, the defense molecules associated with the surface are expected to be non-polar in nature, thus they will protect the surface without being easily dissolved in the surrounding seawater. The polar defense molecules are expected to dissolve more easily and this may be metabolically costly for the macroalgae, particularly under culture conditions.

So far, the compounds identified on the surface are polar. Most of the surface-associated molecules identified until now have low to moderate polarity for example, halogenated furanones from Delisea pulchra [START_REF] De Nys | A new method for determining surface concentrations of marine natural products on seaweeds[END_REF] or fucoxanthin [START_REF] Saha | Surface-associated fucoxanthin mediates settlement of bacterial epiphytes on the rockweed Fucus vesiculosus[END_REF]). These compounds are characterized by a degree of hydrophobicity (Steinberg and de Nys 2002;[START_REF] Harder | Waterborne polar macromolecules act as algal antifoulants in the seaweed Ulva reticulata[END_REF]) which may allow them to diffuse less efficiently in seawater and thus remain close to the surface of the producing organism. On the contrary, DiMethylSulphoPropionate (DMSP) and proline [START_REF] Saha | Dimethylsulphopropionate (DMSP) and proline from the surface of the brown alga Fucus vesiculosus inhibit bacterial attachment[END_REF]) described on the surface of Fucus vesiculosus are polar compounds, such molecules may have a high diffusion capacity in the surrounding seawater. Fucoxanthin isolated from the algal surface showed a modulatory effect on epiphytic bacteria at environmentally realistic concentrations [START_REF] Saha | Surface-associated fucoxanthin mediates settlement of bacterial epiphytes on the rockweed Fucus vesiculosus[END_REF]. Proline and DMSP have been shown to play an ecologically relevant role as surface inhibitors against the attachment of bacterial strains isolated from algae.

Solely on the basis of its hydrophilic properties, it is quite difficult to determine whether a single compound may play an ecological role at the algal interface. Physicochemical interactions between such a molecule and the algal surface itself (e.g. ionic interactions or hydrogen bonds) on the one hand, but also with molecules and/or organisms on the surface on the other hand, could enhance or limit its diffusion in seawater. Furthermore, due to the high cell density and the presence of EPS, biofilms are well known for their capacity to limit the diffusion of molecules in water [START_REF] Stewart | Diffusion in biofilms[END_REF]. The laboratory experiment where algal incubation in natural seawater was performed showed that the three major surface-associated compounds identified in this study (Group 1, 2 and 3 in Table 3) were released into seawater. This result reinforces the potential of these molecules to be involved in surface interactions.

Although the concentration of the DIP extract was very low, n-hexane was observed to be an efficient solvent in extracting the surface-metabolites of H. floresii. As far as we understand, the surface metabolites on macroalgae studied so far, such as in the red algae Delisea pulchra, Laurencia obtuse, Bonnemaisonia asparagoides, in the brown algae Taonia atomaria and in the green alga Caulerpa filiformis, have reported to be at a very low concentration [START_REF] De Nys | A new method for determining surface concentrations of marine natural products on seaweeds[END_REF][START_REF] Nylund | Surface chemistry versus whole-cell extracts: antifouling tests with seaweed metabolites[END_REF][START_REF] Othmani | Surface metabolites of the brown alga Taonia atomaria have the ability to regulate epibiosis[END_REF]. The concentration of a DIP n-hexane extract of H. floresii was observed to be in accordance with the lower mean surface concentration of secondary metabolites from D. pulchra [START_REF] De Nys | A new method for determining surface concentrations of marine natural products on seaweeds[END_REF].

The chemical defense in macroalgae is metabolically very costly which outweighs the benefits provided through protection against harmful bacterial colonization. Thus, it is not surprising that a low concentration of DIP-n-hexane extract of H. floresii may be costly to the macroalgae (Nylund et al. 2005). In Bonnemaisonia hamifera the chemical defense was observed to be costly in a naturally defended surface compared to that of the undefended macroalgal surface [START_REF] Nylund | Costs and Benefits of Chemical Defence in the Red Alga Bonnemaisonia hamifera[END_REF].

Influence of DIP n-hexane extract on bacterial communication

The host-specific associations between algae and bacteria suggest that algae may control the associated epibacterial community rather the bacterial biofilm conferring protection on the host alga [START_REF] Okami | Marine microorganisms as a source of bioactive agents[END_REF][START_REF] Kelecom | Secondary metabolites from marine microorganisms[END_REF][START_REF] Zheng | Marine bacteria associated with marine macroorganism: the potential antimicrobial resources[END_REF][START_REF] Lachnit | Specific epibacterial communities on macroalgae: Phylogeny matters more than habitat[END_REF][START_REF] Mayer | Marine pharmacology in 2005-6: Marine compounds with anthelmintic, antibacterial, anticoagulant, antifungal, anti-inflammatory, antimalarial, antiprotozoal, antituberculosis, and antiviral activities; affecting the cardiovascular, immune and nervous systems, and other miscellaneous mechanisms of action[END_REF][START_REF] Kientz | A new method for removing microflora from macroalgal surfaces: an important step for natural product discovery[END_REF]. Consequently, rather than the symbiotic relationships with the host, it was hypothesized that the dominant communities secret QS-disruptive compounds in order to control the maturation of biofilms (La Barre and Bates 2018). Thus, interfering with the QS systems is always considered to be a potential tool in pathogenicity and disease control. Quorum Quenching, a QS impairment phenomenon, represents an alternative strategy to control bacterial biofilms. Quorum Quenching mechanisms in marine algae mainly include the production of inhibitors or antagonists of signal receptors (Givskov et al, 1996;[START_REF] Kim | Quorum sensing inhibitors from the red alga, Ahnfeltiopsis flabelliformis[END_REF][START_REF] Romero | Quorum quenching in cultivable bacteria from dense marine coastal microbial communities[END_REF]. In this work, the promising effect of the crude DIP_n-hexane extract was explored for its quenching behavior of the signal molecules being produced by the bacterial strains. The surfaceassociated metabolites from H. floresii were assumed to interfere with the establishment of its associated bacteria, which was evident from the quenching activity of the DIP_n-hexane extract on the gram-negative isolates being tested.

Bacterial strains isolated and used in this work basically fall into three different groups such as were identified as main epiphytic bacteria in macroalgal surfaces [START_REF] Wang | Phylogenetic analysis of epiphytic marine bacteria on Hole-Rotten diseased sporophytes of Laminaria japonica[END_REF]). Alphaproteobacteria, Ruegeria sp. and Gammaproteobacteria such as Vibrio owensii, Alteromonas sp. and Pseudoalteromonas arabiensis were specifically isolated from the surface of D. pulchra and these phylotypes were observed to be potential opportunistic pathogens in different circumstances in red algae (Kumar et al. 2016).

Presumably, the QQ behavior of surface metabolites in the crude extract may justify our observation that the surface of H. floresii was remarkably free from any settling organisms which primarily depends on the well-established biofilm. The four isolates that responded positively, included Vibrio owensii, Tenacibaculum sp., Maribacter sp., Aquimarina sp., are known to be causative agents of various diseases.

Here, we assumed that these isolates may be opportunistic pathogens to the associated host, H. floresii.

Particularly, in D. pulchra, these strains were observed either in bleached or in adjacent bleached areas (Kumar et al. 2016). Vibrio owensii was identified as a causative agent of AHPND (Acute Hepato Pancreatic Necrosis Disease) in Litopenaeus vannamei ponds [START_REF] Liu | A Vibrio owensii strain as the causative agent of AHPND in cultured shrimp, Litopenaeus vannamei[END_REF]. Tenacibaculum maritimum, a worldwide known fish pathogen, is a filamentous biofilm-forming bacteria causing 'Tenacibaculosis' in the cultured Cyclopterus lumpus [START_REF] Småge | First isolation, identification and characterisation of Tenacibaculum maritimum in Norway, isolated from diseased farmed sea lice cleaner fish Cyclopterus lumpus L[END_REF]. Different species of Maribacter were isolated from the bleached surface of D. pulchra (Kumar et al. 2016). Aquimarina latercula had the strongest bleaching pathogenicity on healthy Gracilaria lemaneiformis [START_REF] Liu | Isolation and pathogenicity identification of bacterial pathogens in bleached disease and their physiological effects on the red macroalga Gracilaria lemaneiformis[END_REF]. The quenching of the communication signals produced by these bacteria may possibly limit the population from reaching a threshold for activating their respective virulence system. In that case, the surface-associated metabolites from H. floresii may be an alternative strategy in disease control.

Untargeted Metabolomic Profiling

Macroalgae are known to produce a wide variety of chemical compounds, with a range of bioactivity, including sulphated polysaccharides, proteins or peptides or amino acids, lipids, polyphenols and fucoxanthin [START_REF] Mohamed | Seaweeds: A sustainable functional food for complementary and alternative therapy[END_REF][START_REF] Saha | Dimethylsulphopropionate (DMSP) and proline from the surface of the brown alga Fucus vesiculosus inhibit bacterial attachment[END_REF]. Red algae primarily synthesize isoprenoid and acetogenin derivatives, as well as amino acid, shikimate and nucleic acid derivatives (Amsler 2008).

Volatile-halogenated organic compounds such as monoterpenes, sesquiterpenes, diterpenes and meroterpenes, play a significant defensive role [START_REF] Tiwari | Seaweed sustainability : food and non-food applications Tohge T, Fernie AR (2009) Web-based resources for mass-spectrometry-based metabolomics: A user's guide[END_REF]. In order to understand and hypothesize about the metabolites involved in the chemical defense on the surface of H. floresii, the extracts were analyzed by LCMS and the data obtained were processed on the metabolomic platform. The brominated compound in H. floresii may contribute to its antifouling activity by quenching the communication signals of the associated bacteria. Mostly, the QQ behavior of macroalgae was observed to be related to halogenated compounds on the surface. For instance, different polyhalogenated and nonhalogenated surface metabolites of B. asparagoides seemed to regulate the epiphytic bacterial community [START_REF] Nylund | The red alga Bonnemaisonia asparagoides regulates epiphytic bacterial abundance and community composition by chemical defence[END_REF]. A synthetic halogenated furanone derived from the secondary metabolite of D. pulchra was found to inhibit the AHL-mediated quorum sensing in Pseudomonas aeruginosa [START_REF] Hentzer | Inhibition of quorum sensing in Pseudomonas aeruginosa biofilm bacteria by a halogenated furanone compound[END_REF].

The halogenated furanones from D. pulchra use the same specific receptor binding sites of AHLs, thus interfering with important bacterial communication-regulated processes, such as biofilm formation [START_REF] Harder | Chemical mediation of ternary interactions between marine holobionts and their environment as exemplified by the red alga Delisea pulchra[END_REF]). α-D-galactopyranosyl-glycerol, betonicine and isoethionic acid produced by the red algae Ahnfeltiopssis flabelliformis, compete with the synthetic AHL signals in a laboratory assay with Agrobacterium tumefaciens [START_REF] Kim | Quorum sensing inhibitors from the red alga, Ahnfeltiopsis flabelliformis[END_REF]). The unidentified crude extract of the members of Galaxauraceae and Laurencia sp. inhibits the AHL signals [START_REF] Skindersoe | Quorum Sensing Antagonism from Marine Organisms[END_REF]. Bromoperoxidases produced by Laurencia digitata deactivates the AHLs by an oxidation process, where this enzyme is responsible for the formation of brominated compounds in red algae [START_REF] Wever | Marine Vanadium-Dependent Haloperoxidases, Their Isolation, Characterization, and Application[END_REF].

To conclude, we hypothesized different groups of allelopathic secondary metabolites such as brominated compounds, Volatile Halogenated Organic Compounds (VHOCs) like diterpenes and flavonoids assuming that they may contribute to the macroalgal chemical defense on the surface of the red algae Halymenia floresii. The XCMS Online metabolomics platform and the consequent Mass Bank (MMCD) search may be considered as a potential tool for a metabolomics study. The Quorum Quenching activity of the crude extract may justify our proposed hypothesis that it may interfere with the communication of the bacterial community on the surface and thus may prevent biofouling under culture conditions. Rather than the secondary metabolites with antimicrobial activity, the interfering activity of macroalgae on quorum sensing of any opportunist pathogens may help reduce the emergence of resistant strains in aquaculture systems. In that way, H. floresii will be an excellent model in a multi-trophic culture environment.

In the future we aim to work on the defensive role of surface extracts on chemically undefended H.

floresii, for example on the bleached surface, to further explore the activity of surface-associated metabolites.

Acknowledgements

The authors would like to thank E. Caamal Fuentes, L. Taupin for their skilful technical assistance for the chromatographic analyses and V. Avila-Velazquez for the H. floresii cultivation. Financial support from ECOS-Nord CONACYT for the collaboration project M14A03 and PN-CONACYT 2015-01-118 is also acknowledged.

The references of this article are available at the end of the manuscript ('References', page no 206)

Chapter IV

Identification and characterisation of the quorum sensing signal of the opportunistic pathogen causing bleaching disease in Halymenia floresii by HPLC/MS method

Will be submitted as a 'Research Article' to Marine Drugs_Special Issue "Health Promoting Bioactives from Marine Algae and Their Holobionts" Shareen et al.

Identification and characterisation of the quorum sensing signal of the opportunistic pathogen causing bleaching disease in Halymenia floresii by HPLC/MS method

In this part (Chapter IV), we identified the opportunistic pathogen inducing bleaching disease in H. floresii and also characterised its Quorum Sensing (QS) signal (here, homoserine lactone) by HPLC/MS method.

This chapter is still under preparation to be submitted as a research article.

Following the identification of the H. floresii epibacteria and differentiating the QS isolates among them (Chapter II), we observed and evaluated the H. floresii surface extract's interference with their communication signals (Chapter III). In Chapter II we also discussed that different isolates from H. floresii were found to be associated with the strains causing bleaching in other algal species. With this idea, we hypothesized that one or more of the epibacterial strains of H. floresii shall be opportunistic in nature. In order to evaluate our proposed hypothesis, we studied the ability of these strains to induce bleaching in H.

floresii. We also examined the presence of any QS signals in any opportunistic pathogen(s), since quorum sensing plays a significant role in causing diseases in macroalgae.

Materials and Methods

Algal material

H. floresii, beach-cast (BC) was initially collected from the shores near CINVESTAV Coastal Marine Station at Telchac, Yucatán, México (21.3419° N, 89.2636° W). They were washed thoroughly with natural seawater from the site of the collection in order to remove sand and epiphytes. After packing them in polythene bags, they were transported to the lab in an icebox. Rinsed with sterile seawater (30 psu)

and transferred to 25 L acrylic cylinders for acclimatization (15 days) under continuous aeration at a 12:12 light/dark cycle with a light intensity of 110 ± 7.2 µmol photons m -2 s -1 . After acclimatization, healthy tips of H. floresii were selected for the tip bleaching assay.

Bacterial isolates

To evaluate the potential capacity of epibacterial strains for induction of thallus bleaching in Halymenia floresii, '25' of the cryopreserved bacterial strains were revived. These strains were previously isolated from the surface of H. floresii and identified by 16S in November 2018. They were maintained in Marine Agar medium in darkness. All cultures were incubated at room temperature. The isolation of the H.

floresii epibacterial strains has been detailed in Chapter II.

Tip bleaching assay with single epibacterial strains

H. floresii tips excised from healthy material (n=6 in total for each bacterial strain, each tip was c.

2 -3 cm long) were individually placed into separate 6-well plate containing 3 mL of sterile seawater (SSW, 30 psu). In order to eliminate epibacterial from these material, two antibiotics, Vancomycin and Cefotaxim at a concentration of 0.1 mg/mL each were added to each well. Further the plates were incubated at 20 °C at a photon flux density of 38.3 µmol photon m -2 s -1 for two days. After this pre-treatment, plates were carefully emptied and H. floresii tips rinsed by washing with 1.5 mL of SSW in order to remove antibiotics. Finally, 3 mL of SSW were added into each well and previously revived and cultured bacteria were immediately inoculated.

Prior to their inoculation, all bacterial cultures were grown in sterile Marine Broth medium overnight at room temperature in darkness until they had reached an OD600 of 0.2 to 0.3. A volume of 30 µL bacterial cells along with the medium were added into the wells containing H. floresii tips (n = 6), individual plates were used for each strain. Controls consisted of the same volume of sterile bacterial culture medium added into the wells containing H. floresii tips (n=6) and treated as above described. After '5' days of incubation under laboratory conditions all wells were checked under the binocular microscope (magnification factor: 45) and the number of bleached and non-bleached tips in each well were counted using a dark background. The relative risk of thallus tip bleaching in all treatments with the addition of bacteria relative to control treatments was calculated as odds ratios of numbers of bleached and nonbleached tips as follows:

The data have been statistically analysed by using Fisher's exact test for significance. The Mantel-Haenszel extension of Fisher's exact test has been used for replicated test designs, and Bonferroni correction applied to reduce the risk of type I error. Isolates that turned out to be pathogenic after applying Bonferroni correction (i.e. p < 0.00036) were designated as 'significant pathogens' and those which were non-significant after Bonferroni correction (p < 0.05) were called 'potential pathogens'. Isolates that reduced the risk of thallus tip bleaching were designated as 'non-pathogens' or 'protectors'. Nevertheless, after applying Bonferroni correction isolates that significantly reduced the risk of thallus tip bleaching (p < 0.00036) were also called 'significant non-pathogens or protectors' while those that also reduced the tip bleaching (p < 0.05), but were not significant after applying Bonferroni correction are designated as 'potential non-pathogens or protectors' (Saha and Weinberger 2019).

Extraction of HomoSerine Lactones (HSLs) from the 'significant pathogen'

The so called 'significant pathogen' identified by the tip-bleaching assay (see Section 5.1.3) was used to extract HSLs (Fig. 1). The bacterium chosen was previously revived on Marine Agar (MA) plates.

A single colony was inoculated into 100 mL of Marine Broth (MB) medium and incubated for 24 hours under constant agitation (125 rpm) at 25° C. After 24 hours of culture liquid: liquid extraction was performed to extract the HSLs according to previously described methods [START_REF] Fletcher | Biosensor-based assays for PQS, HHQ and related 2-alkyl-4-quinolone quorum sensing signal molecules[END_REF][START_REF] Wang | Extraction, purification and identification of bacterial signal molecules based on N-acyl homoserine lactones[END_REF]. Briefly, the supernatant (100 mL) was extracted by two equal volumes of acidified ethyl acetate (0.5% of acetic acid) for three times. The solvent was dried under vacuum at 35 °C and the residue was resuspended in acetonitrile. The HSL sample was further dried to remove acetonitrile using nitrogen gas and the dried sample was stored at -20° C until further analysis. The methodology was illustrated in the Fig.

1.

Relative risk of bleaching = (Bleached tips in treatments/healthy tips in treatments) / (Bleached tips in control) healthy tips in controls)

Results

The aim of this part was to differentiate the significant opportunistic pathogen(s) out of the epibacterial community of H. floresii and to identify and characterise the corresponding quorum sensing signal of the opportunistic pathogen(s).

Tip bleaching assay with single epibacterial strains

Of the 25 epibacterial strains isolates evaluated in this work, Vibrio owensii was found to significantly increase the risk of tip bleaching when compared to control treatments without bacterial inoculation. Vibrio owensii was identified as a "significant pathogen" after Bonferroni correction (Table I: Alteromonadaceae bacterium) had the same effect, but were not significantly pathogenic after Bonferroni correction and were thus considered as 'potential pathogens' (Table I; Fig. 2 p < 0.05). Ten out of the 16 remaining isolates were found to significantly reduce the risk of tip bleaching and were considered 'significant protectors' (Table I: Fig. 2 p < 0.00036). The last six had the same effect, however after Bonferroni correction they were not significantly protective (Table I; Fig. 2 p < 0.05). Thus, the relative risk of the bacteria analysed that induce or reduce bleaching in H. floresii were compared and followed the pattern of 40% vs 4% for significant non-pathogens or protectors versus pathogens and 24% vs 32% for potential non-pathogens or protectors versus pathogens. Relative risk of bleaching (%)

Table I. Epibacterial strains isolated from Halymenia floresii evaluated for their pathogenicity and/or nonpathogenicity by the tip bleaching assay.

Accession

No. Defence on surface of Rhodophyta Halymenia floresii: metabolomic profile and interactions with its surface-associated bacteria Shareen Arockiasamy 2020

Code

Discussion

The epibacterial communities are organized as highly structured biofilms in many different microniches and habitats within the marine environment, reaching sufficient abundances to induce quorum sensing-based dialogues [START_REF] Lami | Quorum Sensing in Marine Biofilms and Environments[END_REF]. The recognition of the importance of the bacterial microbiota for other organisms has led to the concept of holobiont, which in turn makes possible the occurrence of bacterial interactions such as quorum sensing [START_REF] Teplitski | Microbial Interactions on Coral Surfaces and Within the Coral Holobiont[END_REF][START_REF] Lami | Quorum Sensing in Marine Biofilms and Environments[END_REF]. The elucidation of quorum sensing roles in the holobionts microbial balance can lead to important applications in aquaculture and/or antifouling industries [START_REF] Lami | Quorum Sensing in Marine Biofilms and Environments[END_REF]. From this work, we explored the H. floresii holobiont that allow us to differentiate significant and/or potential pathogens by their ability to induce bleaching. As a result, V.

owensii was found to be a significant pathogen inducing bleaching in H. floresii. Subsequently, the extracellular AHL of V. owensii was also identified from liquid: liquid extracts.

V. owensii, a member of Gammaproteobacteria, isolated from cultivated H. floresii was previously identified as a QS bacterium. Quorum sensing behaviour of V. owensii was detected by a bioluminescent assay using a reporter strain, Escherichia coli pSB406 (see Chapter II and III). Subsequent to the identification of V. owensii as a "significant pathogen" inducing bleaching in H. floresii, we were driven to identify the particular QS signal (here, HSL) responsible for its QS activity. The presence of C4-HSL in V.

owensii emphasized the QS critical role in the induction of bleaching in H. floresii.

Eight epibacterial isolates from H. floresii, besides V. owensii, also revealed some potential to induce bleaching symptoms. Since these detrimental isolates originated from healthy hosts, it has been proved that the epibacterial community of this alga is obviously composed of opportunistic pathogens that can induce similar bleaching symptoms as described in several red algae [START_REF] Weinberger | Bacterial induction and inhibition of a fast-necrotic response in Gracilaria conferta (Rhodophyta)[END_REF][START_REF] Weinberger | Pathogen-induced defense and innate immunity in macroalgae[END_REF][START_REF] Case | Temperature induced bacterial virulence and bleaching disease in a chemically defended marine macroalga[END_REF][START_REF] Saha | Microbial "gardening" by a seaweed holobiont: Surface metabolites attract protective and deter pathogenic epibacterial settlement[END_REF]. The "significant" or "potential" pathogens (9 out of 25 strains) include 36% of the total isolates assayed here, whereas, [START_REF] Saha | Microbial "gardening" by a seaweed holobiont: Surface metabolites attract protective and deter pathogenic epibacterial settlement[END_REF] observed that approximately 5% of the Agarophyton spp. epibiome induced bleaching. However, this calculation is just a representative sample of the whole microbiome as stated by the authors (Saha and Weinberger 2019). homoserine Lactone (ODHL), etc. Moreover, V. owensii has been identified as a causative agent of AHPND (Acute Hepato Pancreatic Necrosis Disease) in Litopenaeus vannamei ponds [START_REF] Liu | A Vibrio owensii strain as the causative agent of AHPND in cultured shrimp, Litopenaeus vannamei[END_REF]. Interestingly, the V. owensii strain which was observed to induce bleaching in H. floresii was originated from the surface of the alga, which was cultivated in a land-based IMTA system whereas V. owensii (B7BC) isolated from the beach-cast H. floresii samples failed to induce bleaching.

Previously we also identified that the surface extracts of H. floresii, at its 1-fold natural surface concentration, significantly interfered with the QS signals of V. owensii by reducing the luminescence of the reporter strain, E. coli pSB406. Whereas it increased the luminescence at a higher concentration of the extract, which may be assumed as an antagonistic approach of the surface-extract (A Abdul Malik et al. Interestingly both these compounds were brominated compounds which are widely observed in red algae such as B. asparagoides, D. pulchra, and Asparagopsis taxiformis and have always contributed for their reported bioactivities [START_REF] De Nys | A new method for determining surface concentrations of marine natural products on seaweeds[END_REF][START_REF] Nylund | The red alga Bonnemaisonia asparagoides regulates epiphytic bacterial abundance and community composition by chemical defence[END_REF][START_REF] Greff | Mahorones, Highly Brominated Cyclopentenones from the Red Alga Asparagopsis taxiformis[END_REF].

Bacterial infectious diseases produced by Vibrio are the main cause of economic losses in aquaculture. The expression of virulence genes in some Vibrio species is controlled by the diffusion of signal molecules such as AHLs. One of the aquaculture-related pathogenic Vibrio strains, V. owensii VibC-Oc-106, isolated from the seawater, was reported to produce 3-OH-C6-HSL, 3-OH-C7-HSL and C13-HSL [START_REF] Torres | AHLlactonase expression in three marine emerging pathogenic Vibrio spp. reduces virulence and mortality in brine shrimp (Artemia salina) and Manila clam (Venerupis philippinarum)[END_REF]. In our work, it was observed that the macroalgae-associated V. owensii produced the extracellular C4-HSL and, previously, we confirmed its QS activity by a biosensor assay. However, [START_REF] Girard | Characterization of N-Acyl homoserine lactones in Vibrio tasmaniensis LGP32 by a biosensorbased UHPLC-HRMS/MS method[END_REF] did not detect any AHLs from strains of V. owensii from natural collections even after using three different biosensors which covered a large spectrum of AHLs (Pseudomonas putida [pKR-C12],

E.coli and Chromobacterium violaceum [CV026]. QS systems mediated by AHLs have been found in many species of marine pathogenic bacteria and AHLs also seem to play an important role in the host-microbe interactions in the marine environment [START_REF] Romero | Acylhomoserine lactone production and degradation by the fish pathogen Tenacibaculum maritimum , a member of the Cytophaga-Flavobacterium-Bacteroides (CFB) group[END_REF].

World production of carrageenophytes its around 160 000 tons of macroalgae (dry weight), with 28 000 tons of carrageenan obtained, valued at USD270 million (Freile-Pelegrín and Robledo 2016).

Several studies have shown that the thallus whitening or bleaching is quite common in carrageenophyte farms in which red algae are more vulnerable to this disease symptom. Under cultivation, affected segments were cut off allowing the unaffected thallus to continue to regenerate and regrow, though with reduced overall productivity [START_REF] Hurtado | Phyconomy: the extensive cultivation of seaweeds, their sustainability and economic value, with particular reference to important lessons to be learned and transferred from the practice of eucheumatoid farming[END_REF]. Preventing the disease outbreak will be a better approach instead of treating or removing the diseased segments after the disease outbreak. Thus, QS interference may be an effective method for preventing and/or inhibiting the virulence of pathogens and hence reduce diseases and increase productivity in aquaculture systems [START_REF] Zhao | Mechanisms of quorum sensing and strategies for quorum sensing disruption in aquaculture pathogens[END_REF]. The surface-extracts of H. floresii interfere with the QS activity of V. owensii, as we previously observed thus H. floresii seems to be a candidate to obtain compounds for disease control in aquaculture. [START_REF] Pliego-Cortés | Stress tolerance and photoadaptation to solar radiation in Rhodymenia pseudopalmata (Rhodophyta) through mycosporine-like amino acids, phenolic compounds, and pigments in an Integrated Multi-Trophic Aquaculture system[END_REF] described H. floresii as a efficient cultivar to recycle inorganic nutrients in a land-based IMTA reducing the environmental impact at coastal ecosystems through responsible aquaculture practices. Besides its potential use as a carrageenophyte, H. floresii could possibly play a significant role among green approaches to improve aquaculture productivity and sustainable environmental practices.

Conclusion and perspectives

The identification of V. owensii as a significant pathogen inducing the risk of bleaching in H. floresii shall be viewed as a shift from their symbiotic to a pathogenic relationship, since the strain used was isolated from healthy algal specimens (microbiont). AHL production in Vibrio owensii is significant as this bacterium is a well-known pathogen in shrimp aquaculture. Besides the AHLs regulation of virulence mechanisms in other Vibrio species described so far [START_REF] Lilley | Regulation of quorum sensing in Vibrio harveyi by LuxO and Sigma-54[END_REF][START_REF] Zhu | Quorum-sensing regulators control virulence gene expression in Vibrio cholerae[END_REF][START_REF] Frans | Vibrio anguillarum as a fish pathogen: Virulence factors, diagnosis and prevention[END_REF][START_REF] Girard | Characterization of N-Acyl homoserine lactones in Vibrio tasmaniensis LGP32 by a biosensorbased UHPLC-HRMS/MS method[END_REF], our results might increase our understandings of the role of AHLs in the physiology and pathogenicity of these microorganisms [START_REF] Girard | Characterization of N-Acyl homoserine lactones in Vibrio tasmaniensis LGP32 by a biosensorbased UHPLC-HRMS/MS method[END_REF]).

As we previously identified that V. owensii is a QS bacterium and that surface-extracts of H. floresii effectively interfere with this QS behaviour, future works shall particularly focus on in vivo experimentation to assess host's interference on the pathogenicity of V. owensii in H. floresii bleaching. This may contribute to enhance disease control and macroalgal health management in aquaculture systems. In addition, the AHL extracts can be studied further to evaluate the release of other small molecules as has been recently

described with Roseobacter clade of macroalgae associated bacteria [START_REF] Ziesche | Homoserine Lactones, Methyl Oligohydroxybutyrates, and Other Extracellular Metabolites of Macroalgae-Associated Bacteria of the Roseobacter Clade: Identification and Functions[END_REF].

Screening Halymenia floresii secondary metabolites which underly its surface defence mechanisms

In the Chapter III, the allelopathic metabolites of Halymenia floresii present on the surface was identified by comparing the surface-associated (DIP) and whole-cell (WCM) metabolites using an untargeted metabolomics approach. By comparing the pooled data of DIP and WCM, we identified an '8' compound through this approach (Section 4.5.4 Table 3) but there was quite a lot of individual set of data resulted from the pre-processing of both the DIP and WCM extracts. In order to widen the knowledge on the metabolic profile of H. floresii, we further proceeded to investigate the potentially active compounds in each set of data. The schematic workflow of this metabolomic work was illustrated in Scheme I and each step involved were detailed in the Chapter V. Here we outline the work of the following chapter. The objectives of this chapter are as follows:

STEP O

Previously, the surface-associated (DIP) and whole-cell metabolites of H. floresii were extracted by the method of DIP and complete maceration, respectively, as detailed in the Chapter III. Both the DIP and WCM extracts were analyzed by LC/MS (analytical tool) and the raw data were retrieved from the LC/MS system as a .mzXML file (Fig. 24).

STEP III

In this step, both the list of metabolites (DIP and WCM) were compared and the common metabolites were taken for further processing. To post-process the results, we used the SMILES system and exhaustive online search ('Google' 'Google Scholar') to obtain the corresponding structural descriptors (chemical structures) and the functional descriptors (biochemical class/activity), respectively (Fig. 27).

Screening Halymenia floresii secondary metabolites which underly its surface defence mechanisms

Shareen A Abdul Malik 1, *; Gilles Bedoux 1 ; Yolanda Freile-Pelegrin 2 ; Nathalie Bourgougnon 1 and Daniel Robledo 21 Université Bretagne Sud, EA 3884, LBCM, IUEM, F-56000 Vannes, France;

gilles.bedoux@univ-ubs.fr (G.B.); nathalie.bourgougnon@univ-ubs.fr (N.B.)

2 Marine Resources Department, CINVESTAV-Unidad Merida, Mexico;

yolanda.freile@cinvestav.mx (Y.F.P.); daniel.robledo@cinvestav.mx (D.R.)

*Correspondence : shareen.a-abdul-malik-arockiasamy@univ-ubs. fr; Tel: +33 7 82 56 34 94

Abstract

The surface of Halymenia floresii Mexican Rhodophyta revealed to be clean and remarkably free of epiphytes under culture conditions. The objectives of the present study were the investigation and identification of H. floresii secondary metabolites which could prevent colonization by microorganisms on its surface. Algal metabolites were selectively extracted from surface-associated (DIP) and whole-cell (WCM) samples by using n-hexane, which was previously identified to selectively extract all surfaceassociated metabolites. In order to propose a global metabolomic fingerprinting of H. floresii, both the DIP and WCM extracts were considered for a four-step metabolomic analysis using mass spectrometry (LC-MS), data pre-processing (XCMS Online), search for putatives in the Madison Metabolomics Consortium Database (MMCD) mass bank, and hierarchical clustering of those putatives. A total of 416 and 110 compounds were detected in the WCM and DIP extracts respectively, among which 41 compounds were present in both extracts. These 41 compounds were characterized for their structures and/or chemical groups (i.e. fatty acids, polysaccharides, peptides, terpenes, sterols, alkaloids, shikimic acid, hydroquinones, aldehydes, ketones, lactones, halogenated or non-halogenated furanones) including their reported effects (i.e. antioxidant, antibacterial, antiviral, antifungal, antitumoral, enzyme inhibitors, cell proliferation inhibitors, insect repellent). As a whole there were 26 compounds that have been identified as secondary metabolites. The structures were encoded into SMILES (for Simplified Molecular Input Line Entry System) strings to build structural descriptors. Further, functional and chemical descriptors were subjected to a Hierarchical Cluster Analysis to investigate the chemical similarities and their possible correlation with bioactivities. By using 'omic' studies such as metabolomic fingerprinting, we provide a functional signature of phenotype with biological relevance to advance our understanding of the biosynthetic pathways which

Introduction

Macroalgae are a large and diverse group of marine photosynthetic organisms that are commonly found in coastal areas and they represent a high source of chemical diversity (Gaubert et al. 2019a).

Macroalgal metabolome includes diverse primary and secondary metabolites with immediate biochemical and physiological consequences likely to play important ecological functions (Gaubert et al. 2019a, b).

They are involved in cell-cell chemical communication with their associated members and play diverse ecological functions in macroalgae (Gaubert et al. 2019b). Some of these compounds exhibit a wide range of therapeutic properties, including anti-cancer, anti-oxidant, anti-inflammatory and anti-diabetic activities [START_REF] Collins | Looking beyond the terrestrial: The potential of seaweed derived bioactives to treat non-communicable diseases[END_REF]) and thus have been described as an excellent source of biologically active metabolites [START_REF] Blunt | Marine natural products[END_REF] whose structural diversity makes them invaluable in drug discovery [START_REF] Pech-Puch | Marine Natural Products from the Yucatan Peninsula[END_REF]. More recently, 'omic' techniques have been used to study a wide array of metabolomes, a complete set of metabolites, through three different metabolomics approach, untargeted metabolite profiling, targeted metabolite analysis and metabolite fingerprinting. Untargeted metabolic profiling is used for the identification and (relative) quantification of as many compounds as possible whereas the targeted metabolite analysis focuses on a limited number of metabolites chosen based on prior knowledge [START_REF] Pandey | Physio-biochemical composition and untargeted metabolomics of Cumin (Cuminum cyminum L.) Make it promising functional food and help in mitigating salinity stress[END_REF][START_REF] Pandey | Physio-biochemical composition and untargeted metabolomics of Cumin (Cuminum cyminum L.) Make it promising functional food and help in mitigating salinity stress[END_REF][START_REF] Patel | Non-targeted metabolite profiling and scavenging activity unveil the nutraceutical potential of psyllium (Plantago ovata Forsk)[END_REF][START_REF] Tanna | Plant Metabolites and Regulation under Environmental Stress[END_REF]. Metabolite fingerprinting is commonly performed to classify samples based on a comprehensive metabolite profiling without identification of individual peaks [START_REF] Steinfath | Metabolite profile analysis: from raw data to regression and classification[END_REF][START_REF] Tanna | Plant Metabolites and Regulation under Environmental Stress[END_REF]. Any metabolomic study shall include one or more of these approaches to identify known or unknown metabolites.

Until now, the chemical profiling of marine algal extracts has been mainly restricted to targeted identification and quantification of selected compound classes with bioactivity [START_REF] Goulitquer | Mass Spectrometry-Based Metabolomics to Elucidate Functions in Marine Organisms and Ecosystems[END_REF]). On the other hand, untargeted metabolomics aims at identifying the extracted metabolites of a sample for revealing novel and unanticipated activities. As a more global metabolomics approach, the fingerprinting can be used as a diagnostic tool to screen the metabolic diversity of any organism [START_REF] Fiehn | Metabolomics -The link between genotypes and phenotypes[END_REF][START_REF] Weckwerth | Metabolomics: From pattern recognition to biological interpretation[END_REF][START_REF] Nobeli | A bioinformatician's view of the metabolome[END_REF][START_REF] Ellis | Metabolic fingerprinting as a diagnostic tool[END_REF][START_REF] Ivanišević | Metabolic fingerprinting as an indicator of biodiversity: Towards understanding inter-specific relationships among Homoscleromorpha sponges[END_REF]. This technique uses signals from hundreds to thousands of metabolites for a rapid sample classification via statistical analysis [START_REF] Hegeman | Plant metabolomics-meeting the analytical challenges of comprehensive metabolite analysis[END_REF][START_REF] Paix | Temporal covariation of epibacterial community and surface metabolome in the Mediterranean seaweed holobiont Taonia atomaria[END_REF][START_REF] Paix | A multi-omics analysis suggests links between the differentiated surface metabolome and epiphytic microbiota along the thallus of a mediterranean seaweed holobiont[END_REF]. As a matter of example, a broad metabolomic study has been performed on the red alga Gracilaria vermiculophylla, in relation to its defence response. Structural elucidation of these metabolites revealed novel eicosanoids as major components, which are part of the innate defence system of G. vermiculophylla against herbivores [START_REF] Nylund | Metabolomic assessment of induced and activated chemical defence in the invasive red alga Gracilaria vermiculophylla[END_REF][START_REF] Goulitquer | Mass Spectrometry-Based Metabolomics to Elucidate Functions in Marine Organisms and Ecosystems[END_REF].

Nowadays, studies on macroalgal metabolomic fingerprinting provide useful information on particular compounds and their responses to environmental and/or culture conditions [START_REF] Gupta | Seaweed metabolomics: A new facet of functional genomics[END_REF]. Recently, Gaubert et al (2019) (Gaubert et al. 2019b) studied the temporal and spatial variabilities of the metabolome in the brown alga Lobophora by using an untargeted UHPLC-QToF metabolomic fingerprinting approach.

LC-MS based metabolomics is more sensitive than other analytical tools [START_REF] Hegeman | Plant metabolomics-meeting the analytical challenges of comprehensive metabolite analysis[END_REF], and has a great potential in relation to the 'drugability' of natural products [START_REF] Zhao | Application of Metabolomics in the Study of Natural Products[END_REF]. In mass spectrometrybased untargeted metabolomic studies, metabolite's identification is a key challenge [START_REF] Zhou | Metabosearch: Tool for mass-based metabolite identification using multiple databases[END_REF].

Recently, several advances in database management and processing tools have greatly simplified the most laborious tasks for metabolite identification [START_REF] Lewis | Novel NMR and MS approaches to metabolomics[END_REF]. For example, Madison-Qingdao Metabolomics Consortium Database (MMCD) a web-based metabolomic tool contains data related to biologically relevant small molecules from a variety of species. This database is primarily designed to facilitate the search of metabolites mainly based on mass. [START_REF] Cui | Metabolite identification via the Madison Metabolomics Consortium Database[END_REF]). Hence, MMCD has been widely used for macroalgal metabolomic studies [START_REF] Goulitquer | Mass Spectrometry-Based Metabolomics to Elucidate Functions in Marine Organisms and Ecosystems[END_REF][START_REF] Gupta | Seaweed metabolomics: A new facet of functional genomics[END_REF]Kumar et al. 2016;[START_REF] Tanna | Plant Metabolites and Regulation under Environmental Stress[END_REF]. The mass-based search of metabolites in the databases will primarily result in a putative identification of the metabolites which can be further addressed either by experimental verification of the metabolites using authentic standards or by several computational approaches [START_REF] Tautenhahn | Annotation of LC/ESI-MS mass signals[END_REF][START_REF] Cui | Metabolite identification via the Madison Metabolomics Consortium Database[END_REF][START_REF] Wolf | In silico fragmentation for computer assisted identification of metabolite mass spectra[END_REF][START_REF] Horai | MassBank: A public repository for sharing mass spectral data for life sciences[END_REF]. MMCD goes with well-accepted computational algorithms that has been developed to ease metabolite identification [START_REF] Cui | Metabolite identification via the Madison Metabolomics Consortium Database[END_REF][START_REF] Zhou | Prioritization of putative metabolite identifications in LC-MS/MS experiments using a computational pipeline[END_REF].

Halymenia floresii, a tropical red macroalga common to the coast of the Yucatan peninsula, Mexico, has been previously identified with economic interest due to its lambda carrageenan content and as an edible seaweed with high potential for cultivation [START_REF] Pliego-Cortés | Growth, biochemical and antioxidant content of Rhodymenia pseudopalmata (Rhodymeniales, Rhodophyta) cultivated under salinity and irradiance treatments[END_REF][START_REF] Alemañ | Development of seaweed cultivation in Latin America: current trends and future prospects[END_REF].

Except for the studies of Godinez- [START_REF] Ortega | Composición estacional de ácidos grasos de Halymenia floresii (Rhodophyta) de Yucatán, México[END_REF] and [START_REF] Meesala | Isolation and structure elucidation of halymeniaol, a new antimalarial sterol derivative from the red alga Halymenia floresii[END_REF], little is known about H. H. floresii thalli were rinsed with sterile seawater (30 psu) and individually freeze-dried. The lyophilized samples were then exhaustively extracted with n-hexane by maceration for 24 h under agitation at room temperature to yield the Whole Cell Metabolites fraction (WCM). For comparison purposes, the same solvent was used. The extracts were then filtered through Whatman filter to remove any particles and the solvent was evaporated under vacuum. Henceforth, the surface metabolites and whole-cell metabolites will be referred to as DIP and WCM, respectively.

LC-MS conditions and data pre-processing

DIP and WCM extracts were analyzed by LC-MS as described in [85]. Briefly, the chemical profiling of H. floresii extracts were analysed by LC/MS using an LC-ESI-Q-TOF-MS (Dionex, Ultimate 3000, Bruker, micrOTOF-QII) system (Bruker Daltonik GmbH, Bremen, Germany). Separations were performed using an analytical reversed-phase column. A sample of 10 µL of the extract was injected for every analysis at a flow rate of 0.5 mL min -1 with a column temperature of 30 °C and the elution gradient was adopted from [START_REF] Othmani | Surface metabolites of the brown alga Taonia atomaria have the ability to regulate epibiosis[END_REF]. Binary programming of methanol (MeOH) and water (H2O) was injected at a linear gradient from MeOH/ H2O (40:60, v/v) to MeOH/ H2O (85:15, v/v) in 3 min and to 100% MeOH in 4 min, followed by a final isocratic step with 100% MeOH for 18 min, finishing by a return to the initial conditions (0.1 min) and equilibration of the column (9.9 min). Regarding the mass spectrometer, operating conditions were set as following: drying temperature: 350 °C, capillary voltage: 4

kV, nebulizer pressure: 3.45 bars, drying gas: helium at a flow rate of 12 L min -1 [START_REF] Othmani | Surface metabolites of the brown alga Taonia atomaria have the ability to regulate epibiosis[END_REF] .

Mass spectra acquisition was set to 0.5 Hz from m/z 50 to 1,000.

The raw data files were exported as ".mzXML" format from the Agilent LC/MS instrument used for the analyses. Initially, a free user profile account was created on the XCMS Online home page. The LC/MS raw data in .mzXML format were uploaded into the platform. The DIP LC/MS data in duplicates were uploaded to the assigning a 'Single' job treatment whereas the WCM LC/MS data were either assigned a 'Multi' job as the WCM data included six extraction periods of 10, 20, 30, 40, 50 and 60 seconds in duplicates or a 'Single' job which included individual sets. Both data sets were treated with the same default parameters, UPLC / Bruker Q-TOF positive, with mzdiff of '0.01' and SNR (signal-noise ratio) threshold of '6'. The method used for the feature detection is 'centWave' with 10 ppm of m/z tolerance and minimum/maximum peak width of 5 seconds/20 seconds. The p-value threshold was set between 0.001 (highly significant) to 0.05 (not significant). After several steps of treatment such as filtration, retention time correction, fill-in missing peak, and annotation of adducts and isotopes, the pre-processed data were downloaded from the platform. For grouping and annotation, the inbuilt CAMERA package was used by the platform. The schematic workflow of this untargeted metabolomics study is displayed in Scheme I.

H. floresii .mzXML data obtained from the LC/MS analysis were fed to the XCMS Online platform that decoded mass spectra based on mass (charge, retention time, and intensity) reported a list of every 'Single Class'. By using XCMS Online to pre-process LC-MS metabolomic data the retention time was corrected resulting from any experimental drifts from run to run, as the features of ions were defined by unique m/z ratio and retention time [START_REF] Forsberg | Data processing, multi-omic pathway mapping, and metabolite activity analysis using XCMS Online[END_REF]. One of the most critical challenges in the preprocessing of metabolomic data is that, the '.mzXML' files of LC/MS analysis took a long time to be uploaded to the XCMS Online Platform, as the job sizes increased. This issue shall be addressed by using a data-streaming application XCMStream, an update of the metabolomic platform, which directly uploads data from the instrument computer as it is generated and automatically initiates the job once complete [START_REF] Smith | XCMS: Processing Mass Spectrometry Data for Metabolite Profiling Using Nonlinear Peak Alignment, Matching, and Identification[END_REF][START_REF] Montenegro-Burke | Data streaming for metabolomics: Accelerating data processing and analysis from days to minutes[END_REF]).

LC-MS data processing -Madison Metabolomics Consortium Database

H. floresii metabolomic study searched Madison Metabolomics Consortium Database (MMCD) for possible candidates based on the large profile of exact mass since this DB was the most appropriate for algae metabolome (Tohge and Fernie 2009;[START_REF] Goulitquer | Mass Spectrometry-Based Metabolomics to Elucidate Functions in Marine Organisms and Ecosystems[END_REF]) . The metabolite search was based on 'mass' in a 'batch mode' for mixture analysis. Thus, the pre-processed XCMS data were downloaded from the platform and 'flattext' was created with the retention time (rtmax), mass/charge ratio (mzmax), and relative intensity (maxint), all at maximum value. The text file was created only with the numerical data, separating each column by a 'tab space', without any text. The flattext was uploaded to the database and the 'Spectral_Type' was chosen as 'LC/MS'. The predicted metabolites from the database were individually coded with unique numbers/letters. All the recovered metabolites were searched for any previously reported bioactivity and they were grouped and coded accordingly (Table 1). Typically, the algorithm used to categorize the various metabolites is based on a first sorting key after reported bioactivity, secondary, if no such activity was identified (i.e. no specific activity was reported), metabolites were sorted after the presence of a specific chemical group usually involved in defence mechanisms (like ketones or halogens)

and eventually from their biochemical group in its conventional sense. In the case were a particular metabolite exhibited various activities like antibiotic and fungicide and pesticide, it was sorted in the antibiotic class to simplify the post-processing.

Data post-processing -SMILES

Simplified Molecular Input Line Entry System, SMILES, is a chemical notation language and the strings encode a molecular structure as a two-dimensional valence-oriented graph [START_REF] Weininger | SMILES, a chemical language and information system. Introduction to methodology and encoding rules[END_REF]). Thus, it is widely used by large chemical databases that can be reverted or derived from chemical structures or CAS (Chemical Abstracts Service) numbers. [START_REF] Cronin | Structure-Based Classification of Antibacterial Activity[END_REF] converted the names of the '661' drugs into SMILES strings to calculate their structural descriptors and these SMILES strings were further entered into QSARis software to examine the 2-D descriptors of the structures. SMILES are used to build the structural descriptors of the metabolites proposed by the mass bank. In this work, we used the SMILES system to post-process our results. First, the structures were encoded into SMILES strings according to [START_REF] Weininger | SMILES, a chemical language and information system. Introduction to methodology and encoding rules[END_REF] to build structural descriptors. Briefly, the SMILES strings were obtained from the 'Chemical Resolver Identifier' website (https://cactus.nci.nih.gov/chemical/structure) by feeding each metabolite's name. Next, functional and chemical descriptors were added to the selected compounds to fill a matrix with zero or one depending on the lacking or presence of such an effect or chemical group of relevance. The converted structural descriptors of the metabolites were used to build a text file including the ID (previously assigned codes, see above) and their corresponding SMILES strings. This text file was used to build the required structural descriptors by a cheminformatic program, Data Warrior (freely available online and downloaded from http://www.openmolecules.org/). The structural descriptors were downloaded as a '.sdf file', which were further imported for statistical analysis. SMILES hence encode structural descriptors of the molecules that can be matched following strings analysis of their respective Euclidean distances, which is not feasible from their original chemical naming. Therefore, the lowest or highest the Euclidean distance, the shorter or greater their chemical similarity.

Data Validation & Interpretation -Statistical Analysis

Robust computational tools are essential to analyse and interpret these metabolomics data. After one or more pre-processing and post-processing of metabolomic data, the raw data including structural and functional descriptors are converted into a numerical format that can be used for downstream statistical analysis. Such downstream processing is quite feasible with open source commercial software, R, as it is freely available to the users. R is a software environment for statistical computing, data analysis and graphics, which has now become an essential tool in all areas of bioinformatics research [START_REF] Grace | Processing and Visualization of Metabolomics Data Using R[END_REF]. The ChemmineR package was used for the data post-processing [START_REF] Cao | ChemmineR: A compound mining framework for R[END_REF]). An HCA multivariate analysis [START_REF] Ivanišević | Metabolic fingerprinting as an indicator of biodiversity: Towards understanding inter-specific relationships among Homoscleromorpha sponges[END_REF][START_REF] Farag | Metabolite profiling and fingerprinting of commercial cultivars of Humulus lupulus L. (hop): A comparison of MS and NMR methods in metabolomics[END_REF]Farag et al. , 2013a, b;, b;[START_REF] Porzel | Metabolite profiling and fingerprinting of Hypericum species: A comparison of MS and NMR metabolomics[END_REF][START_REF] Rácz | Binary similarity measures for fingerprint analysis of qualitative metabolomic profiles[END_REF], was performed to group the treatments (SMILES and chemical groups identified) that are close to each other in clusters i.e. similar structural descriptors. The HCA of metabolomic data was performed by using the packages 'ChemmineR', 'fmcsR', 'dendextend', and 'gplots'. They allowed computing dendrograms of the metabolites based on their structural (dis)similarities. The (dis)similarity between each pair of observations was measured using distance measures i.e. Euclidean distance in R according the Ward method, for which, the data were feed as '.sdf files' by using the DataWarrior software. It included the code of each observations, SMILES strings and the structural descriptors. The functional descriptors (Table 1)

were separately fed as 'text' files. Further analysis was focused on H. floresii metabolites which were identified in both DIP and WCM extracts which feature nearly 10% of all metabolites recovered. Interestingly, antiproliferative, antiviral and biochemical classes such as hormones, ketones and vitamins were not identified in the surfaceextract i.e. DIP. Thus, in Figure 2 Right A, the metabolic count between WCM and CMN was compared and the most represented classes were IME followed by NUC and ATB with 70, 60, and 41 metabolites, respectively. Similarly, the relative occurrences of bioactive molecular classes' in these two extracts were compared and we found that halogenated (HAL), furanones (FUR) and inhibitors (INH) compounds were relatively higher in the CMN (12% vs 3% / 7% vs 3% / 15% vs 8%, respectively) whereas antibiotics (ATB), antioxidant (ATO), anticancerous (ATC) and antifungal (ATF) compounds are present in similar ratios (Figure 2 Right B). On the contrary, ATP, ATV, HOR, KET, PIG, and VIT were under-represented in these extracts.

In Table 1 we present the properties of the '41' CMN putatives with their corresponding codes, bioactivity/chemical class identified and lipophilicity (Log(KOW)) [START_REF] Cheng | Computation of octanol-water partition coefficients by guiding an additive model with knowledge[END_REF]). The putatives names, their actual mass and the chemical formula were also listed (Table 1) as annotated by the MMCD database used. The SMILES strings of the putatives, CMN and WCM were provided in the Supplementary section 166 (Table S1). The confidence level (∆) showed the difference between the MW of the compounds (available in the database) and the m/z features of the putatives in our extracts. The experimental m/z features (Exp m/z features) from the pre-processed DIP and WCM data matching the putatives were also listed. The experimental m/z features also allowed us to find the retention time (RT in minutes) and the maximum intensity.

H. floresii secondary metabolites

The structural and functional diversity of the '41' compounds which correspond to the CMN cluster were further analysed by HCA method to set a structural typology and then, look for to a correspondence with their reported bioactivities, if any. A dendrogram tree was therefore computed by using the Ward clustering method, and tree was cut at a definite height to yield a reasonable number (here 8) of clusters for further analysis. The dendrogram (see Figure . 3) displays the distances (Ward method) calculated from the metabolite's structural descriptors only. Cutting the tree at a distance of 1.0 yielded '8' clusters numbered hereafter as CI to CVIII roman numbers. The leaf labels which correspond to the '26' compounds for which a defence activity has been reported and are consequently proposed as surface secondary metabolites, are green labelled. . The last molecule is a 2-fluoro-2-deoxyglucuronic acid S07. In cluster IV five halogenated polyaromatic molecules are grouped (S132, S140, S20, S332, and S44). Cluster V contains two molecules with a phosphono-propyl (S72) and a phenylmethyl ester of phosphoric acid (S107). A structurally heterogeneous group with five compounds that seem to be based on a chemical motif close to a hydroxyphenyl is found in cluster VI. Cluster VII is constituted of trinitrotoluene (S166) and Dichlorphenamide (S365). Finally, S251 and S451 from cluster VIII are very similar di-phosphorylated structures with aminopyrimidine linked to substituted thiazole ring and two substituted 3-hydroxy-2-methylpyridine S128 and S194.

As a whole, among the '41' metabolites identified, 15 of them were sorted as intermediary metabolites (IME). Among them, S49 (NUC) and S50 (IME) no dissimilarity in their structures were evident as the Euclidean distance between them is '0'. The IMEs, S152 (NUC) and S402 (NUC), S338

(SAC), S22 (SAC), and S291 (SAC), were grouped into Cluster III at a Euclidean distance of 1.0 to 0.5.

The latter distance was then further branched out into two groups. At 0.5, S152 and S402 were closely similar by having 'purine' rings with 'NH2' groups. The predicted metabolites with antibiotic activity (ATB) such as S146, S07, S107 and S365, were found to be linked to biochemically distinct classes S348 (FUR), S291 (SAC), S72 (INH) and S166 (DIV) at a Euclidean distance below 0.5.

Of the four antioxidant (ATO) metabolites, identified as S264, S126, S125, and S251, predicted in this work, all fell into four different clusters: I, III, VI, and VII, respectively. Moreover, they were closely distanced with different classes of metabolites, such as S264 to S146 (ATB), S126 to S106 (ATC), S251 to S451 (INH) at a Euclidean distance lower than 0.5 and S125 to S316 (IME) higher than 0.5. The INH metabolites, S196 and S223, were observed to be in the same cluster (I) and also at the same distance, whereas S140, S72, S158 and S451 fall under different clusters: IV, V, VI and VII, respectively. This shows that there is no significant correlation between the structural properties of the compounds and their reported bioactivities.

Hydrophobicity of the secondary metabolites

As it is well assessed that the lipophilicity of "defence biomolecules" is critical in their mechanism of action, attempts were made to evaluate a possible (Steinberg and de Nys 2002;[START_REF] Harder | Waterborne polar macromolecules act as algal antifoulants in the seaweed Ulva reticulata[END_REF]), or expected, correlation between the compound's chemical structures and this physico-chemical parameter.

Accordingly, a tanglegram was computed which allows comparing two dendrograms calculated from distinct parameters with the same individuals. Here the analysis was restricted to the 26 secondary metabolites identified. As a whole, this indicates that there is no straightforward correlation between the molecular structure and its lipophilicity. This point will be further discussed. Similarly, the lipophilic pattern of these secondary metabolites was plotted (see Fig. 6) as a function of bioactivity to unveil possible tendencies. Again, it is observed that there is no correlation between lipophilicity and a particular class of activity. All these findings evidence that H. floresii secondary metabolites exhibit a large diversity in the structural, physico-chemical and functional domains.

Discussion

Metabolomics, a rapidly evolving discipline, applies advanced separation and detection methods to capture the collection of small molecules that characterize metabolic pathways [START_REF] Kouskoumvekaki | Navigating the human metabolome for biomarker identification and design of pharmaceutical molecules[END_REF]. It can bridge the information gap between the identification of metabolite profile dynamics and their closest phenotypic responses [START_REF] Kaddurah-Daouk | Metabolic profiling of patients with schizophrenia[END_REF][START_REF] Holmes | Metabolic profiling of CSF: Evidence that early intervention may impact on disease progression and outcome in schizophrenia[END_REF][START_REF] Morvan | Metabolomics by proton nuclear magnetic resonance spectroscopy of the response to chloroethylnitrosourea reveals drug efficacy and tumor adaptive metabolic pathways[END_REF][START_REF] Kouskoumvekaki | Navigating the human metabolome for biomarker identification and design of pharmaceutical molecules[END_REF]. In order to contribute to the knowledge of secondary metabolites of H. floresii, we performed an untargeted metabolomic approach to putatively identify and propose active metabolites from this species, as a preliminary step towards its metabolomic comprehension. As it is a very first kind of work on H. floresii metabolites with little a priori knowledge, the putative identification and peak prediction are completely based on the metabolite database, MMCD.

MMCD mainly list the molecular weight of compounds represented in the literature (Tohge and Fernie 2009) and as we made our predictions only on this basis, we regard them as 'putatives'. As any untargeted study, MMCD did not allow us to identify the individual peaks (more than one experimental m/z features and RT, Table 1), but it greatly assisted to predict these putatives even at very low intensity. Thus, the challenge of low concentration of the surface extracts was efficiently managed.

The first informatic task was to identify the multiple peaks resulting from the chromatographic floresii metabolome, which was categorized as ATB (AnTiBiotics), and most of them ('38') were present in the WCM extract only (Figure 1). Besides this large potential, the macroalgae also exhibit a large diversity of biochemical pathways as deduced from the variety of antibiotics chemical classes. To cite a few, quinones groups (kinobscurinone, romycin, bikaverin, quinquangulin), flavonoids group (isosakuranetin, sakuranetin), benzofuran derivatives (moracin A), coumarin derivatives (chlorobiocin), aminoglycosides (spectinomycin, cyclomycin, manumycin, coformycin), nitrogen heterocycles porphyrines (formylphenyl) or derivatives of tetracyclines. It is also noteworthy that some of the antibacterial molecules have yet been identified in plants like brazilin, phaseolic acid (a plant hormone), adonitoxin, malvin or geniposidic acid. Moreover, they often present a bunch of biological activity as concomitant antibacterial, antifungal (quinquangulin, manumycin) and antiviral (formycin) antioxidant (brazilin), antitumoral (manumycin) effects [START_REF] French | Targeted and untargeted metabolic profiling of wild grassland plants identifies antibiotic and anthelmintic compounds targeting pathogen physiology, metabolism and reproduction[END_REF]. One can hypothesize that these potentially concomitant effects may be stimulated or activated depending on the stress (culture conditions or competitors) to which the alga is exposed. This multi-dimensional diversity, both structural, and biological should promote further investigations. Indeed, the putatives of H. floresii identified in this study can now be considered as a baseline knowledge for future targeted metabolomic study. This predicted fingerprint can also be used to compare the spatial and temporal differences in the metabolite distribution of H. floresii from different habitats. Rather, these putatives or their derivatives were also observed by various authors in different macroalgal or plant species with vast bioactivity as discussed below.

In cluster I, Fucofuroeckol B (predicted for H. floresii as metabolite S348) isolated from Eisenia bicylis, an edible perennial brown alga, has been shown as a potent anti-inflammatory agent [START_REF] Lee | Fucofuroeckol-A from Eisenia bicyclis Inhibits Inflammation in Lipopolysaccharide-Induced Mouse Macrophages via Downregulation of the MAPK/NF-B Signaling Pathway[END_REF]). Phlorofucofuroeckol-A derived from the brown alga, Ecklonia kurome showed antimicrobial activity by inhibiting Campylobacter jejuni, Escherichia coli, Salmonella enteritidis, Salmonella typhimurium, Vibrio parahaemolyticus [START_REF] Nagayama | Bactericidal activity of phlorotannins from the brown alga Ecklonia kurome[END_REF][START_REF] Eom | Antimicrobial effect of phlorotannins from marine brown algae[END_REF]. This compound was clustered together with S146, 11-O-demethylpradinone-I, a member of the Pradinone I antibiotic group with a tetracyclic hydrocarbon structure, a basic structure of anthracyclines as a responsible class of compound [START_REF] Lee | Fucofuroeckol-A from Eisenia bicyclis Inhibits Inflammation in Lipopolysaccharide-Induced Mouse Macrophages via Downregulation of the MAPK/NF-B Signaling Pathway[END_REF]). In the red alga, Halopithys incurva and in the brown algae Fucus spiralis and Treptacantha abies-marina, polyphenols (predicted for H. floresii as S196) and pigments (predicted for H.

floresii as S123 and S250) respectively, were reported to exhibit high antioxidant activity by preventing oxidative stress under IMTA [START_REF] Vega | Antioxidant activity of extracts from marine macroalgae, wild-collected and cultivated, in an integrated multi-trophic aquaculture system[END_REF]. Gracilaria gracilis bio-oil samples were composed of the same compounds classes identified in H. floresii, such as alcohols, ketones, aldehydes fatty acids, esters, aromatics, amino acids, nitrogen-containing heterocyclic compounds and chlorinated/fluorinated compounds [START_REF] Parsa | Hydrothermal liquefaction of Gracilaria gracilis and Cladophora glomerata macro-algae for biocrude production[END_REF]. Although rarely detected, fluorinated compounds (S155 and S212) shall not be excluded from the macroalgal compounds.

The presence of furans in cluster II suggests interesting applications as a biofuel additive or as a base chemical from which a large variety of bio-based compounds can be produced [START_REF] Lange | Furfural-A promising platform for lignocellulosic biofuels[END_REF][START_REF] Van Putten | Hydroxymethylfurfural, a versatile platform chemical made from renewable resources[END_REF]). This has been reported for some of the wide diversity of carbohydrates found in macroalgae and their corresponding degradation resulting in furans, such as furfural (degradation of xylose) from P. palmata, laminarin (conversion of glucose) from brown algae, hydroxymethylfurfural (conversion of glucose) and 5-methyl furfural (conversion of rhamnose) from the green alga Ulva spp. [START_REF] Amin | Biodiesel production from macroalgae[END_REF].

In Cluster III, we could able to group, Scopolin (S127), Biflorin (S126), Pancratistatin (S106), Asperuloside tetra-acetate (S338), and galacturonate derivative (S291). Asperuloside tetra-acetate, a terpenoid, found in a terrestrial plant Morinda officinalis (IARC Working Group on the Evaluation of Carcinogenic Risk to Humans. 2002), was clustered together with the galacturonate derivative (S291).

Terpenes and polysaccharides are well-known for their bioactivity in macroalgae which essentially includes to combat bacterial invasion [START_REF] Pérez | Antimicrobial Action of Compounds from Marine Seaweed[END_REF]. Terpenes in algae also act as allelochemical deterrents to herbivores, inhibitors to bacterial biofilm formation, and also act as antioxidants against UV damage [START_REF] Potin | Biotic interactions of marine algae[END_REF][START_REF] Shannon | Antibacterial derivatives of marine algae: An overview of pharmacological mechanisms and applications[END_REF][START_REF] Dahms | Antifouling Compounds from Marine Macroalgae[END_REF]. Recently polysaccharides of the red macroalgae were widely studied for antiviral activity [START_REF] Bouhlal | Antiviral activity of the extracts of Rhodophyceae from Morocco[END_REF][START_REF] Jiao | Chemical structures and bioactivities of sulfated polysaccharides from marine algae[END_REF][START_REF] Gomaa | Antiviral activity of sulfated polysaccharides carrageenan from some marine seaweeds[END_REF][START_REF] Shi | Overview on the antiviral activities and mechanisms of marine polysaccharides from seaweeds[END_REF][START_REF] Peñuela | Environmentally Friendly Valorization of Solieria filiformis (Gigartinales, Rhodophyta) from IMTA Using a Biorefinery Concept[END_REF]) moreover the depolymerization of the algal polysaccharides also induce protection against fungal and bacterial infections [START_REF] Vera | Seaweed polysaccharides and derived oligosaccharides stimulate defense responses and protection against pathogens in plants[END_REF][START_REF] Pérez | Antimicrobial Action of Compounds from Marine Seaweed[END_REF]. Pancratistatin (PST) is a natural compound that was isolated from a terrestrial plant Pancratium littorale and shown to exhibit antineoplastic activity [START_REF] Mclachlan | Pancratistatin: A natural anti-cancer compound that targets mitochondria specifically in cancer cells to induce apoptosis[END_REF] In cluster V, two different compounds, such as Ibandronate (S72) and Cyclohexylmethyl 2formylphenyl hydrogen phosphate (S107) were grouped together where they were shown to have antiresorptive/anti-osteoclastogenesis [START_REF] Oh | Biological activities and biomedical potential of sea cucumber (Stichopus japonicus): A review[END_REF]) and antibiotic activity (Deprez et al. 2002), respectively, but their role in macroalgae or marine environment were not known. In cluster VI, we observed 2- 5-hydroxymethyl-2-furaldehyde was previously derived from a marine bacterium Bacillus subtilis isolated from the Gulf of Mannar (India), which inhibits the biofilm formation and virulence of Candida albicans [START_REF] Subramenium | 5-hydroxymethyl-2furaldehyde from marine bacterium Bacillus subtilis inhibits biofilm and virulence of Candida albicans[END_REF] and it was also identified as a contaminant of the aquatic environment resulting from different industrial processes [START_REF] Cary | Concise International Chemical Assessment Document 21. Geneva Cavalier-Smith T[END_REF]. Thus, interestingly this compound shall be extracted from the associated microbiota. However, it shall be ruled out as our solvent of extraction nhexane was inefficient to lyse the bacterial membrane and extract its metabolites [START_REF] Kientz | A new method for removing microflora from macroalgal surfaces: an important step for natural product discovery[END_REF]. Thus, there is another possibility of the xenobiotic behaviour where the compound is not naturally present in the algae, but being absorbed as a result of contamination.

In cluster VII we observed trinitrotoluene (S166), can be referred to as Porphyra yezoensis and Portieria hornemannii, possessed a metabolic route to remove this compound from the seawater and in such process trace amounts of TNT were found within the biomass. Thus, this cluster of metabolites give evidence for the ability of the alga to resist xenobiotics, which shall be considered as a most important ecological activity as contaminants are posing a serious problem in the marine environment.

Dichlorphenamide (S365), widely known as a diclofenamide (an ophthalmology drug as per the PubChem), is a carbonic anhydrase inhibitor. Carbonic anhydrase (CA) is an enzyme for CO2 uptake by a photosynthetic organism under external high concentration of HCO3 -. CA activity is tightly regulated by CO2 availability to reverse CO2/HCO3 -and during intense photosynthesis, the CO2 equilibrium is decreased at the algal surface and the red algae have adopted a mechanism to inhibit the CA activity by producing inhibitors [START_REF] Beer | Photosynthetic properties of protoplasts, as compared with thalli, of Ulva fasciata (chlorophyta)[END_REF][START_REF] Larsson | Bicarbonate uptake and utilization in marine macroalgae[END_REF][START_REF] Moulin | Different mechanisms of inorganic carbon acquisition in red macroalgae (Rhodophyta) revealed by the use of TRIS buffer[END_REF].

Cluster VIII mainly grouped nucleotides derivatives like di-phosphorylated structures with aminopyrimidine and substituted hydroxymethylpyridine (S124 and S198). Macroalgal originated nucleosides and nucleotides are involved in several key biological processes [START_REF] Huang | Marine nucleosides: Structure, bioactivity, synthesis and biosynthesis[END_REF] and in red alga Hypnea valentiae has furnished 5-iodo-5-deoxytubercidine, which displayed prominent muscle relaxant property and hypothermia in mice [START_REF] Kazlauskas | Halogenated Pyrrolo[2,3d]pyrimidine Nucleosides from Marine Organisms[END_REF][START_REF] De Koning | Purine and pyrimidine transport in pathogenic protozoa: From biology to therapy[END_REF][START_REF] Huang | Marine nucleosides: Structure, bioactivity, synthesis and biosynthesis[END_REF]. Different pyroglutamic and deoxyguanosine derivatives coupled with bromophenols were obtained in the red alga, Rhodomela confervoides [START_REF] Zhao | Bromophenols coupled with derivatives of amino acids and nucleosides from the red alga Rhodomela confervoides[END_REF].

Five halogenated metabolites identified in our extracts essentially falls into two different clusters.

S155 and S212 are observed to be structurally similar with two benzene rings, an amide function, and five fluorines, and they fall into Cluster I whereas S20 and S44 were structurally similar with two phenyls, bromine, and they further linked to S132, instead of 'Br', which has 'Cl' and fall into Cluster IV.

Halogenation often provides these compounds with interesting key features and marine algae hold diverse and unique biosynthetic pathways for the production of halogenated metabolites [START_REF] Cabrita | Halogenated compounds from marine algae[END_REF]).

Brominated compounds are widely observed in red algae such as Bonnemaisonia asparagoides, Delisea pulchra, and Asparagopsis taxiformis and have always contributed to several bioactivities. Four different polyhalogenated compounds, with bromine and chlorine isotopic patterns, were observed in the surface extracts of B. asparagoides [START_REF] Nylund | Seaweed defence against bacteria: a poly-brominated 2-heptanone from the red alga Bonnemaisonia hamifera inhibits bacterial colonisation[END_REF]. Mahorone and 5-bromomahorone were identified in DCM:

MeOH extract of A. taxiformis and they exhibited mild antibacterial activity against Acinetobacter baumannii, a human pathogen [START_REF] Greff | Mahorones, Highly Brominated Cyclopentenones from the Red Alga Asparagopsis taxiformis[END_REF]).

Among the compounds identified at the algal surface, halogenated ones, furanones, and inhibitors are overrepresented. Interestingly, the first two classes (HAL and FUR) have been identified as potent quorum-sensing-interfering (QSI) compounds (Gonzalez and Keshavan 2006;[START_REF] Huigens | The Path to New Halogenated Quinolines With Enhanced Activities Against Staphylococcus epidermidis[END_REF] with bacterial communication with macroalgal-derived QSI compounds arose as a very promising alternative.

Relatively higher occurrences of bioactive molecular classes found in the WCM and DIP extracts strongly supports the hypothesis that they must be involved in the organism protection against herbivores or epiphytes, as the initial damage to the host begins at their surface (adhesion). Investigations suggest that the primary function of allelopathic (secondary) metabolites of macroalgae is to deter herbivory, however, they may have multiple functions [START_REF] Renaud | Interactions of plant stress and herbivory: intraspecific variation in the susceptibility of a palatable versus an unpalatable seaweed to sea urchin grazing[END_REF][START_REF] Paul | Marine chemical ecology[END_REF][START_REF] Fong | Allelopathic effects of macroalgae on Pocillopora acuta coral larvae[END_REF].

Particularly in red algae, these biologically active compounds range in structure from simple aliphatic haloketones and brominated phenols to more complex monoterpenes, sesquiterpenes, and diterpenes exhibiting that the strong biological activity has always been linked to the several (closely) related compounds [START_REF] Hay | Marine Plant-Herbivore Interactions: The Ecology Of Chemical Defense[END_REF][START_REF] Renaud | Interactions of plant stress and herbivory: intraspecific variation in the susceptibility of a palatable versus an unpalatable seaweed to sea urchin grazing[END_REF]. For example, elatol, a sesquiterpene, one of the well-known major allelopathic metabolites of Laurencia plays important role in ecological interactions, such as antiherbivore activity and potential defence against infection by microorganism ( [START_REF] König | Sesquiterpene content of the antibacterial dichloromethane extract of the marine red alga Laurencia obtusa[END_REF][START_REF] Juagdan | Two new chamigranes from an Hawaiian red alga, Laurencia cartilaginea[END_REF][START_REF] Vairappan | Potent antibacterial activity of halogenated metabolites from Malaysian red algae, Laurencia majuscula (Rhodomelaceae, Ceramiales)[END_REF][START_REF] Lhullier | Isolation of elatol from Laurencia microcladia and its palatability to the sea urchin Echinometra lucunter[END_REF][START_REF] Santos | Effect of elatol, isolated from red seaweed Laurencia dendroidea, on Leishmania amazonensis[END_REF]). Previously we also identified that different groups of allelopathic secondary metabolites such as brominated compounds, Volatile Halogenated Organic Compounds (VHOCs) like diterpenes and flavonoids might contribute to the chemical defence on the surface of the H. floresii. And their activity was mainly attributed to the corresponding interference with the surface-associated bacterial communication [START_REF] Abdul Malik | Chemical defense against microfouling by allelopathic active metabolites of Halymenia floresii (Rhodophyta)[END_REF]. Likewise, the large number of potentially bioactive molecules of H. floresii shall obviously be related to several ecological functions that require more study on the 'Systems Biology' of the predictive putatives.

Conclusion

H. floresii research is still at its infancy and with very limited knowledge of its metabolomics. With this study, we propose a metabolomic fingerprint for H. floresii as a preliminary step in its metabolomic study. This untargeted study should be further extended including multiple analytical tools and/or carried to a targeted metabolomic study for exploring its bioactive or drug-like compounds, such as promising QSI for counteracting global bacterial multi-resistance. Additionally, the same approach shall be widened to include H. floresii from different habitats, to understand how the environmental (including seasonal) factors influence the stimulation of these secondary metabolites' biosynthetic pathways.

In our previous study, allelopathic secondary metabolites on the surface of the H. floresii were found to contribute to the macroalgal chemical defence [START_REF] Abdul Malik | Chemical defense against microfouling by allelopathic active metabolites of Halymenia floresii (Rhodophyta)[END_REF]). The metabolites presented in this study were determined for their biological activities in relation to their potential valorization (Table 2) and those bioactivities shall be treated as a primary line of defence of H. floresii as they were observed in the surface extracts of the alga. Irrespective of any predetermined bioactivity the predicted putatives may have the ability to protect the host either by destroying or interfering with certain biotic interactions (for instance QS communication) based on their structure-activity relationship.

A large set of bioactive putatives from H. floresii were screened in this study. The next step is to use targeted analysis for the corresponding metabolic pathways by using the KEGG pathway database [START_REF] Kanehisa | New approach for understanding genome variations in KEGG[END_REF]. This in silico approach could benefit from experimental design to enhance the production of a particular QSI under culture conditions. This could benefit from existing standard tests, which allow identifying compounds exhibiting QSI activities [START_REF] Mclean | A simple screening protocol for the identification of quorum signal antagonists[END_REF][START_REF] Rasmussen | Screening for quorum-sensing inhibitors (QSI) by use of a novel genetic system, the QSI selector[END_REF].

Additionally, the presence of brominated compounds, which are well-known for their defensive activity against bacteria and antimethanogenic properties, in H. floresii could make them good ingredient candidates for cattle feeding (Øverland et al. 2019) since they fundamentally eliminates the enteric methane resulting from a high grain diet providing evidence of improved livestock production performance (Nys et al. 2016).

H. floresii will be an excellent model in a multi-trophic culture environment as algal secondary metabolites with antimicrobial activity, the interfering activity of macroalgae on quorum sensing of any opportunist pathogens may help reduce the emergence of resistant strains in aquaculture systems.

General Discussion

Origin of the Thesis

During the experimental cultivation of Halymenia floresii under Integrated MultiTrophic Aquaculture, the establishment of opportunist green algae and the colonisation of sessile invertebrates, which usually disturb this type of cultivation, were not observed. The culture tanks were clean and the surface of the H. floresii was remarkably free from any fouling organisms. This phenomenon could reveal that the presence of H. floresii may prevent the biofilm formation either by releasing allelopathic active compounds that ultimately interfere with the settlement and growth of competitors or its epibacterial community may act as a protective layer, thus preventing any fouling on the host. To assess these hypotheses, we proceeded to investigate the chemical and biological defence mechanisms of H. floresii by means of active secondary metabolites and the associated microbiota, through allelopathy and 'protective coat' on the surface of the algae.

The chemical defence of H. floresii was studied differentially by evaluating the surface and wholecell metabolites, which allowed us to identify the bioactive metabolites on the surface. These were followed by an untargeted metabolomic study that helped us to identify and propose a global metabolomic profile of H. floresii. In this section, the biological defence is emphasized on the microbial defence on the surface of the algae (as detailed in Chapter I). This was analysed by its epibacterial community and its communication.

Subsequently, the protective/non-pathogenic and pathogenic bacteria were identified by their ability to induce the risk of bleaching, an algal disease. The analysis of the epibacterial community and the identification of significant pathogens led us to consider the forthcoming disease problems in Aquaculture of algae.

Holobiont: Halymenia floresii and the associated bacterial community

Throughout their evolutionary history, multicellular organisms have been engaged in symbiotic relationships with microorganisms (Moran 2006). In particular, an assemblage of different organisms forming an ecological functional unit, Holobiont, often defined as the host organisms with all of its microbial symbionts, Microbiont. Through several recent studies, it has become increasingly clear that the microbiota are essential for host functioning although the current knowledge on the functional consequences of the macroalgal holobiont is limited [START_REF] Van Der Loos | The Macroalgal Holobiont in a Changing Sea[END_REF]. In this work, we began to explore H. floresii and its associated epibacterial community from different environmental conditions.

However, we dealt only with the cultivable strains of H. floresii, a small fraction of all bacteria that were found associated on the surface of the algae, and thus they could be treated as a representative sample of the H. floresii whole microbiome. This is because only 5% of the marine bacterial strains are cultivable and in order to perform any assays there is no alternative technique to date to separate the selected microbial components from natural microbial communities [START_REF] Haglund | Large differences in the fraction of active bacteria in plankton, sediments, and biofilm[END_REF][START_REF] Saha | Microbial "gardening" by a seaweed holobiont: Surface metabolites attract protective and deter pathogenic epibacterial settlement[END_REF].

7.3

The partner: associated bacteria

Characterisation of bacterial community

Thirty-one axenic bacterial strains were isolated from the surfaces of H. floresii, and they belonged to '4' phyla, '20' genera, and '25' species. The main groups of bacterial phyla associated with H. floresii included Gammaproteobacteria (47%) Alphaproteobacteria (37%), Bacteroidetes (13%), Firmicutes (3%) (Fig. 1). Del Olmo et al (2018) observed that Gammaproteobacteria was the dominant phyla in accordance with our results observations. After a review of numerous studies, [START_REF] Hollants | What we can learn from sushi: a review on seaweedbacterial associations[END_REF] conclude that macroalgal-bacteria interactions primarily consist of Gammaproteobacteria (37%) followed by Bacteroidetes (20%), Alphaproteobacteria (13%), and Firmicutes (10%). We found the same pattern for H. floresii except that Alphaproteobacteria (37%) was followed by Bacteroidetes (13%). Several factors, biological, physical and chemical are likely to influence the colonisation of bacteria and their qualitative and quantitative structuring, and to whether or not to maintain a stable association between algae and their biofilms. Many of the algal-associated microbiome profiles studied so far were structured by variation of several factors such as temperature and nutrients (i.e. NH + 4 ) [START_REF] Florez | Structure of the epiphytic bacterial communities of Macrocystis pyrifera in localities with contrasting nitrogen concentrations and temperature[END_REF][START_REF] Califano | Cultivating the Macroalgal Holobiont: Effects of Integrated Multi-Trophic Aquaculture on the Microbiome of Ulva rigida (Chlorophyta)[END_REF]; different geographical and seasonal gradients (Bengtsson et al. 2010;Burke et al. 2011;[START_REF] Michelou | The Ecology of Microbial Communities Associated with Macrocystis pyrifera[END_REF][START_REF] Campbell | Spatial variability of microbial assemblages associated with a dominant habitat-forming seaweed[END_REF]; their physiological and morphological traits during the host life cycle (Bengtsson et al. 2010;[START_REF] Lemay | Alternate life history phases of a common seaweed have distinct microbial surface communities[END_REF]; physiological conditions of the host under optimal or stressing conditions [START_REF] Marzinelli | Continental-scale variation in seaweed host-associated bacterial communities is a function of host condition, not geography[END_REF][START_REF] Marzinelli | Coastal urbanisation affects microbial communities on a dominant marine holobiont[END_REF][START_REF] Califano | Cultivating the Macroalgal Holobiont: Effects of Integrated Multi-Trophic Aquaculture on the Microbiome of Ulva rigida (Chlorophyta)[END_REF]. floresii from different habitats (BC, IMTA, and CC) were quite dissimilar. This could be related to several factors as discussed earlier but maybe mainly attributed to cultivation conditions. The physiological responses to abiotic factors such as temperature, light, water turbulence, and nutrient availability under different conditions are also believed to play a major role in this dissimilarity. Understanding the temporal dynamics of epiphytic bacteria can help identify the possible modifications in the 'protective layer' due to external stress factors [START_REF] Mancuso | Diversity and temporal dynamics of the epiphytic bacterial communities associated with the canopy-forming seaweed Cystoseira compressa (Esper) Gerloff and Nizamuddin[END_REF]). In the case of H. floresii, the controlled and uncontrolled culture conditions in CC and IMTA, respectively, might significantly contribute to the stress, whereas in BC, it was mainly caused by the overexposure to increase sunlight (i.e. UV radiation or desiccation). Nevertheless, we may not disregard differences attributed to specimens collected or cultivated (individuals, part of the plant, unnoticed bleached fragments, etc.).

The occurrence and richness of epiphytic coverage have been observed to be specific and different in relation to different parts of the host macroalgae leading to a spatial dynamic of the microbiome (Kersen This variation may be due to the overall controlled culture condition of H. floresii. Thus, the spatial and temporal dynamics of macroalgal-associated bacteria shall both be determined by the stressors.

Influence of the epibacterial community

The surface-associated microbiome of macroalgae plays a crucial role in the growth, morphogenesis, and defence of the macroalgae under favourable environmental conditions (Symbiotism).

However, under unfavourable conditions, the microbiome reverses its symbiotic nature affecting the growth and causing diseases leading to macroalgae degradation (Pathogenism) (A Abdul Malik et al. 2020a).

As every organism is associated with microbes, it is becoming increasingly obvious that these associations can greatly influence host health by protecting the host from pathogen intrusion and fouling by the production of bioactive compounds [START_REF] Egan | Phylogenetic relationship and antifouling activity of bacterial epiphytes from the marine alga Ulva lactuca . Brief report[END_REF][START_REF] Ley | Evolution of mammals and their gut microbes[END_REF][START_REF] Turnbaugh | A core gut microbiome in obese and lean twins[END_REF][START_REF] Shnit-Orland | Coral mucus-associated bacteria: a possible first line of defense[END_REF][START_REF] Barott | Unseen players shape benthic competition on coral reefs[END_REF][START_REF] Greff | The interaction between the proliferating macroalga Asparagopsis taxiformis and the coral Astroides calycularis induces changes in microbiome and metabolomic fingerprints[END_REF]. [START_REF] Boyd | Antibacterial and repellent activities of marine bacteria associated with algal surfaces[END_REF] found that sixty of the '280' strains isolated from different marine macroalgae exhibited antibiotic activities against a series of fouling bacteria and therefore have the potential to control the microbial population on the seaweed surface either by inhibiting their growth or by influencing the tactic behaviour of potentially competing bacteria. Moreover, it is widely accepted that bacterial epiphytes can inhibit the colonisation of surfaces by common fouling organisms [START_REF] Egan | Phylogenetic relationship and antifouling activity of bacterial epiphytes from the marine alga Ulva lactuca . Brief report[END_REF]. It has also been suggested that the production of antimicrobial compounds by epibiotic bacteria provides a competitive advantage against potential microbial competitors during algal surface colonisation [START_REF] Franks | Inhibition of fungal colonization by Pseudoalteromonas tunicata provides a competitive advantage during surface colonization[END_REF][START_REF] Alvarado | Phylogeny and bioactivity of epiphytic grampositive bacteria isolated from three co-occurring Antarctic macroalgae[END_REF]. Recently, the associated microbiota is functionally regarded as a 'second skin' of the host [START_REF] Wahl | The Second Skin: Ecological Role of Epibiotic Biofilms on Marine Organisms[END_REF] and thus acts as the first line of defence of macroalgae. Based on our results, H. floresii first line of defence it's essentially composed of at least '31' epibacterial strains identified in our work. Interestingly they were originated from the specimens collected at different habitats revealing the dynamic differences in their association, though we actively dealt with the cultivable strains.

The Host -H. floresii

As a preliminary step towards the metabolomic comprehension of H. floresii, the untargeted metabolomic approach helped us to identify and predict a global metabolomic profile for H. floresii. By comparing the surface and whole-cell metabolites, the untargeted metabolomics analysis allowed us to identify '41' bioactive metabolites that were grouped into '8' clusters based on their structural (dis)similarity by Hierarchical Cluster Analysis (HCA). The '41' metabolites were categorized into different classes based on their bioactivity and/or biochemical class (Fig. 2). To better characterize the chemical similarities within each of the eight clusters, molecular structures were also found (Chapter IV, Fig. 5). Distances were calculated from the presence/absence of chemical patterns within the whole molecular structure according to the SMILES encoding. floresii strongly supports the hypothesis that they must be involved in the organism protection against herbivores or epiphytes, as the initial damage to the host begins at their surface (adhesion). Investigations suggest that the primary function of allelopathic (secondary) metabolites of macroalgae is to deter herbivory; however, they may have multiple functions [START_REF] Renaud | Interactions of plant stress and herbivory: intraspecific variation in the susceptibility of a palatable versus an unpalatable seaweed to sea urchin grazing[END_REF][START_REF] Paul | Marine chemical ecology[END_REF][START_REF] Fong | Allelopathic effects of macroalgae on Pocillopora acuta coral larvae[END_REF]). Previously we also identified that different groups of allelopathic secondary metabolites Among the compounds identified at the algal surface, halogenated ones, furanones, and inhibitors are overrepresented. Interestingly, the first two classes (HAL and FUR) have been identified as potent quorum-sensing-interfering (QSI) compounds (Gonzalez and Keshavan 2006;[START_REF] Huigens | The Path to New Halogenated Quinolines With Enhanced Activities Against Staphylococcus epidermidis[END_REF]) since they mimic the chemical structures of bacterial auto Inducers. Indeed, quorum sensing allows bacterial biofilms, the sessile "way of life" of bacteria, to regulate gene expression and toxin production. By interfering with the communication within the bacterial populations, the QSI compounds provide to eukaryotes potent antibacterial defences. When we consider the pathogenic role of biofilms as they recruit higher eukaryotes i.e. macrofoulers, H. floresii appears as a potent source for such compounds.

Of the '8' different clusters (Chapter IV Fig. 5) of H. floresii metabolites, cluster I possesses Fucofuroeckol B, which has been previously identified as an antimicrobial compound [START_REF] Nagayama | Bactericidal activity of phlorotannins from the brown alga Ecklonia kurome[END_REF][START_REF] Eom | Antimicrobial effect of phlorotannins from marine brown algae[END_REF]). Interestingly, cluster III mainly includes terpenes and polysaccharides (Asperuloside tetra-acetate (S338), and galacturonate derivative (S291)) that are well-known for their bioactivity in macroalgae particularly in combating bacterial invasion [START_REF] Pérez | Antimicrobial Action of Compounds from Marine Seaweed[END_REF]. Terpenes in algae also act as allelochemical deterrents to herbivores, inhibitors to bacterial biofilm formation, and also act as antioxidants against UV damage [START_REF] Potin | Biotic interactions of marine algae[END_REF][START_REF] Shannon | Antibacterial derivatives of marine algae: An overview of pharmacological mechanisms and applications[END_REF][START_REF] Dahms | Antifouling Compounds from Marine Macroalgae[END_REF]. Cluster VI includes 2-furaldehyde (S108), where a derivative of it 5-hydroxymethyl-2furaldehyde was previously derived from a marine bacterium Bacillus subtilis isolated from the Gulf of Mannar (India), which inhibits the biofilm formation and virulence of Candida albicans [START_REF] Subramenium | 5-hydroxymethyl-2furaldehyde from marine bacterium Bacillus subtilis inhibits biofilm and virulence of Candida albicans[END_REF]. Thus, interestingly this compound shall be extracted from the associated microbiota. However, it shall be ruled out as our solvent of extraction n-hexane was inefficient to lyse the bacterial membrane and extract its metabolites [START_REF] Kientz | A new method for removing microflora from macroalgal surfaces: an important step for natural product discovery[END_REF]. Thus, there is another possibility of the xenobiotic behaviour where the compound is not naturally present in the algae, but being absorbed as a result of contamination.

Supporting this hypothesis, we observed the compound trinitrotoluene (S166), under cluster VII, that can be referred to the explosive known as TNT, often present in seawater as a pollutant [START_REF] Cruz-Uribe | Comparison of TNT removal from seawater by three marine macroalgae[END_REF].

The same authors found that the red algae, Porphyra yezoensis and Portieria hornemannii, possessed a metabolic route to remove this compound from the seawater and in such process trace amounts of TNT were found within the biomass. Thus, this cluster or metabolites give evidence for the ability of the alga to resist xenobiotics, which shall be considered as a most important ecological activity as contaminants are posing a serious problem in the marine environment. Recently the ability of the green alga Ulva under IMTA system was evaluated for its potential uptake of enrofloxacin, a commonly used antibiotic in aquaculture, and the results showed that the alga could assimilate the antibiotic and reached ~5% of the maximum residue limit [START_REF] Rosa | Uptake of enrofloxacin from seawater to the macroalgae Ulva and its use in IMTA systems[END_REF]. IMTA systems are believed to play an important role to solve many of the environmental impacts of aquaculture by reaching a balanced ecosystem-based management approach to aquaculture [START_REF] Chopin | Aquaculture, Integrated Multi-trophic (IMTA)[END_REF][START_REF] Chopin | Challenges of moving Integrated Multi-Trophic Aquaculture along the R&D and commercialization continuum in the western world[END_REF], since the uptake of xenobiotics can have cumulative effects potentiating the problems associated with exposure to such compounds in the target species. In this sense, the ability of H. floresii to uptake or resist the xenobiotics should be considered for bioremediation in aquaculture system.

Interactions among bacteria and the Host's Interference

Biological interactions between different marine surface-associated organisms play a major role in the development and maintenance of a biofouling community [START_REF] Egan | Phylogenetic relationship and antifouling activity of bacterial epiphytes from the marine alga Ulva lactuca . Brief report[END_REF], particularly on the macroalgal surface. Such a dynamic epibiotic microbiome protects the surface by forming a protective coat, biofilm [START_REF] Armstrong | The symbiotic role of marine microbes on living surfaces[END_REF]) and this itself acts as a biological defence mechanism of the macroalgal host (A Abdul Malik et al. 2020a). A revolutionary discovery of the quorum sensing (QS) system, a mechanism of cooperative behaviour in bacteria [START_REF] Fuqua | Quorum sensing in bacteria: The LuxR-LuxI family of cell density-responsive transcriptional regulators[END_REF] opened a new avenue in the regulation of macroalgal microbiome [START_REF] Singh | Unraveling the Functions of the Macroalgal Microbiome[END_REF] as the vital process of biofilm formation is mainly regulated by QS [START_REF] Paluch | Prevention of biofilm formation by quorum quenching[END_REF].

Of the '31' epibacterial strains of H. floresii, '17' isolates were identified as QS bacteria by the presence of their extracellular QS signals. As the epibacterial components of H. floresii were isolated from healthy specimens, they can be easily inferred to play a protective role on the algal surface. Bacterial interactions with macroalgae are important at two basic levels. It can be beneficial, acting as symbionts (i.e.

symbiotism), and can also be detrimental, acting as pathogens (i.e. pathogenism). Both processes have been shown to involve QS [START_REF] Joint | Bacterial conversations: talking, listening and eavesdropping. A NERC Discussion Meeting held at the Royal Society on 7 December 2005[END_REF]A Abdul Malik et al. 2020a). In our work, the release of virulence factors microorganisms and the prevalence of putative virulence factors in marine bacteria, it may be argued that a specific disease phenotype in the marine environment can be mediated by different bacterial pathogens [START_REF] Persson | High abundance of virulence gene homologues in marine bacteria[END_REF][START_REF] Zinger | Global Patterns of Bacterial Beta-Diversity in Seafloor and Seawater Ecosystems[END_REF][START_REF] Gennari | Virulence genes and pathogenicity islands in environmental Vibrio strains nonpathogenic to humans[END_REF][START_REF] Lynch | Ecology and exploration of the rare biosphere[END_REF]Kumar et al. 2016).

The ecology of opportunistic pathogens in marine systems has been recognized as an emerging field. In this regard, opportunistic marine pathogens only cause disease in immune-compromised or stressed hosts [START_REF] Burge | Special Issue Oceans and Humans Health: The Ecology of Marine Opportunists[END_REF]). Among several of the macroalgae diseases studied so far, bleaching (whitening of macroalgal thalli), may be caused by diverse microbial pathogens together with environmentally mediated stress such as climate change and global warming. This results in the subsequent decline of these important habitat-former organisms [START_REF] Campbell | Climate change and disease: bleaching of a chemically defended seaweed[END_REF].

Aquaculture/Disease/Global Change

Marine algae, important dominant habitat-formers, and primary producers in the marine ecosystem [START_REF] Longford | Interactions within the microbiome alter microbial interactions with host chemical defences and affect disease in a marine holobiont[END_REF] are highly susceptible to infectious diseases according to increasing evidence [START_REF] Gardiner | VarR controls colonization and virulence in the marine macroalgal pathogen Nautella italica R11[END_REF]. In their natural habitat, several biotic and abiotic parameters affect macroalgal health, whereas under cultivation overall unfavourable environmental conditions may result in disease outbreak [START_REF] Campbell | Climate change and disease: bleaching of a chemically defended seaweed[END_REF][START_REF] Gardiner | VarR controls colonization and virulence in the marine macroalgal pathogen Nautella italica R11[END_REF]. Microbiomes of habitat-formers may be impacted by ocean climate change with implications for the health, persistence and resilience of entire marine ecosystems. The impact of climate change is closely linked to the physiological concept of macroalgal health. Relatively little is known about the prevalence and magnitude of diseases in macroalgae [START_REF] Gachon | Algal diseases: spotlight on a black box[END_REF]), but they are predicted to increase under elevated environmental stress [START_REF] Egan | Bacterial pathogens, virulence mechanism and host defence in marine macroalgae[END_REF] or could change geographical distribution and virulence of diseases. If diseases directly impact the infected organism and its population, they may additionally have cascading effects on the ecosystem if keystone species are infected [START_REF] Gachon | Algal diseases: spotlight on a black box[END_REF][START_REF] Egan | Microbial dysbiosis: Rethinking disease in marine ecosystems[END_REF]. Many diseases are now interpreted as the result of a microbial imbalance and the rise of opportunistic or polymicrobial infections upon host stress [START_REF] Egan | Microbial dysbiosis: Rethinking disease in marine ecosystems[END_REF][START_REF] Dittami | A community perspective on the concept of marine holobionts: state-of-the-art, challenges, and future directions[END_REF]. The direct physiological impacts of ocean warming could have led to changes in the microbiomes of important species such as kelps, which may also be a direct response of ocean acidification [START_REF] Qiu | Future climate change is predicted to affect the microbiome and condition of habitatforming kelp[END_REF]. On the other hand, due to the increasing economic demand of macroalgae, its aquaculture has expanded rapidly during the last 10 years [START_REF] Cottier-Cook | Safeguarding the future of the global seaweed aquaculture industry[END_REF][START_REF] Alemañ | Development of seaweed cultivation in Latin America: current trends and future prospects[END_REF][START_REF] Shannon | Seaweeds as nutraceuticals for health and nutrition[END_REF]. Despite its large niche market, intensive algal aquaculture might favour disease outbreaks [START_REF] Gachon | Algal diseases: spotlight on a black box[END_REF]. One of the most crucial problems in aquaculture is bacterial diseases (Natrah et al. 2011a) and these microbial infections are being observed to be opportunistic in nature. There is an increased need to find an alternative strategy to control disease outbreak in aquaculture as it raises the production cost and reduces the profit/outcome. Interactions between the hosts and their microbiomes are fundamental for host functioning and resilience [START_REF] Qiu | Future climate change is predicted to affect the microbiome and condition of habitatforming kelp[END_REF], and such interactions can either be beneficial or detrimental. Any disturbance and disruption of the mutualistic association between host and microbiota can result in diseases by means of holobiont break-up. Through the holobiont approach, the understanding of microbial disease is undergoing a paradigm shift [START_REF] Longford | Interactions within the microbiome alter microbial interactions with host chemical defences and affect disease in a marine holobiont[END_REF].

For instance, the red alga Agarophyton (Gracilariaceae) possesses a protective epimicrobial community which acts as chemical 'gardens' and at the same time deters opportunistic pathogens. Thus, Agaraphyton wisely recruits its microbiota that maintain its overall health and disease management [START_REF] Saha | Microbial "gardening" by a seaweed holobiont: Surface metabolites attract protective and deter pathogenic epibacterial settlement[END_REF]. The recognition of the bacterial microbiota importance for other organisms lead to the concept of holobiont, which in turn allows the occurrence of bacterial interactions, such as quorum sensing [START_REF] Teplitski | Microbial Interactions on Coral Surfaces and Within the Coral Holobiont[END_REF][START_REF] Lami | Quorum Sensing in Marine Biofilms and Environments[END_REF]).

H. floresii epibacteria were assayed in vitro to identify any opportunistic pathogens by their ability to induce bleaching under laboratory condition (more details in Chapter V) and we found that V. owensii has a "significant pathogen" that increased the risk of tip bleaching (

Future perspectives

Marine macroalgae are consistently exposed to both biotic and abiotic pressures in their natural environments and any external pressure may influence algal physiology leading to the production of metabolites, some of which acting as bioactive compounds [START_REF] Collins | Looking beyond the terrestrial: The potential of seaweed derived bioactives to treat non-communicable diseases[END_REF]. This potential can be tapped for developing new functional ingredients and health-promoting treatments. Indeed, several therapeutic properties have already been demonstrated for several species of marine macroalgae, including anti-cancer, anti-oxidant, anti-inflammatory, and anti-diabetic activities [START_REF] Wijesinghe | Enzyme-assistant extraction (EAE) of bioactive components: A useful approach for recovery of industrially important metabolites from seaweeds: A review[END_REF][START_REF] Collins | Looking beyond the terrestrial: The potential of seaweed derived bioactives to treat non-communicable diseases[END_REF]. The range of ailments reported to be treated experimentally by macroalgae or their derivated compounds may become a triggering factor for the development of novel functional ingredients for pharmaceutical purposes [START_REF] Wijesinghe | Enzyme-assistant extraction (EAE) of bioactive components: A useful approach for recovery of industrially important metabolites from seaweeds: A review[END_REF]. H. floresii compounds are not the exception since in this exhaustive study we predicted and proposed a list of bioactive metabolites and clustered them based on their structural similarity. These endogenous metabolites have good chances for being successful drug candidates based on their metabolite -likeness and biological relevance, as the 'chemical similarity principle' states that molecules with similar structures likely exhibit similar biological properties [START_REF] Kouskoumvekaki | Navigating the human metabolome for biomarker identification and design of pharmaceutical molecules[END_REF].

Regarding this point, our untargeted metabolomics study of H. floresii revealed the possible presence of compounds with potent pharmaceutical benefits. Among these compounds, the bioactive potential of H. floresii is illustrated in Fig. 3 and discussed below.

In the cluster, I, 11-O-demethylpradinone-I (S146), a member of the Pradinone I antibiotic group with a tetracyclic hydrocarbon structure, was found which is a basic structure of anthracyclines as a responsible class of compound [START_REF] Lee | Fucofuroeckol-A from Eisenia bicyclis Inhibits Inflammation in Lipopolysaccharide-Induced Mouse Macrophages via Downregulation of the MAPK/NF-B Signaling Pathway[END_REF]. Similarly, in cluster III we found Pancratistatin (PST), a natural compound previously isolated from a terrestrial plant Pancratium littorale that has shown antineoplastic activity [START_REF] Mclachlan | Pancratistatin: A natural anti-cancer compound that targets mitochondria specifically in cancer cells to induce apoptosis[END_REF]. In cluster V, two different compounds, Ibandronate (S72)

and Cyclohexylmethyl 2-formylphenyl hydrogen phosphate (S107) were grouped together and have been described to show anti-resorptive/anti-osteoclastogenesis [START_REF] Oh | Biological activities and biomedical potential of sea cucumber (Stichopus japonicus): A review[END_REF]) and antibiotic activities (Deprez et al. 2002). For cluster VIII, mainly nucleotide derivatives groups like di-phosphorylated structures with amino-pyrimidine and substituted hydroxymethylpyridine were found (S124 and S198). These macroalgal compounds are involved in several key biological processes [START_REF] Huang | Marine nucleosides: Structure, bioactivity, synthesis and biosynthesis[END_REF]) and some have displayed prominent muscle relaxant property and hypothermia in mice such as 5-iodo-5-deoxytubercidine from the red alga Hypnea valentiae [START_REF] Kazlauskas | Halogenated Pyrrolo[2,3d]pyrimidine Nucleosides from Marine Organisms[END_REF][START_REF] De Koning | Purine and pyrimidine transport in pathogenic protozoa: From biology to therapy[END_REF][START_REF] Huang | Marine nucleosides: Structure, bioactivity, synthesis and biosynthesis[END_REF].

On the other hand, bacterial resistance to therapeutic drugs has been a serious issue over the last decades. Thus the quick development of resistance mechanisms by microorganisms due to the overuse of antimicrobials has turned into a major public health issue [START_REF] Corrêa | Bacterial Resistance: antibiotics of last generation used in clinical practice and the arise of natural products as new therapeutic alternatives[END_REF]. Such emergence of antibioticresistant bacterial strains has driven attention towards the discovery of alternative innovative strategies to combat pathogens. Since the druggability of secondary metabolites drove this study, it is worth noting that bacterial multi-resistance become a global public health challenge. Due to gene transfers among species and strong efflux pumps, "conventional" antibiotics become less and less efficient. This promoted the search for alternative pathways and, in such a context, "messing" with bacterial communication with macroalgal-derived Quorum Sensing Interference (QSI) compounds arose as a very promising alternative. The challenge in most metabolomic studies is to relate the identified metabolites to their biological activities [START_REF] Johnson | Metabolomics: Beyond biomarkers and towards mechanisms[END_REF]. However, to evaluate the biological roles of one or several metabolites (a metabolic signature), the first step is to determine their function in metabolic pathways and their interconnectivity. In our metabolomic study, the predicted metabolites of H. floresii were exhaustively searched for any related bioactivity using Google Scholar. The KEGG and "Cyc" databases are just some examples of the most popular metabolic pathway databases and were used as a targeted approach to proceed with our baseline study. Once pathways have been reconstructed and analysed, it will be possible to perform comparative and evolutionary analyses of metabolic processes among different lineages with different phenotypes and metabolic capabilities or help to understand peculiarities in metabolic processes, including those at primary and secondary metabolism. These metabolomic data can be considered for systems biology approach in the framework of chemical ecology, which ultimately helps us understand the underlying biotic interactions between the host and the associated microbiota, or to different types of organisms in a community [START_REF] Goulitquer | Mass Spectrometry-Based Metabolomics to Elucidate Functions in Marine Organisms and Ecosystems[END_REF].

With this untargeted metabolomics approach, we were able to unveil the potential of H. floresii to produce a vast number of bioactive metabolites under culture conditions. Further questions arise about what may happen if H. floresii culture conditions are changed or disturbed. The metabolic profile of H. floresii arising from this work may help as basic data to compare metabolic spatially and temporally differences, not only in cultivation but also in its natural habitat. H. floresii as a potential carrageenophyte for the food floresii metabolic shift will help us to understand its ecological role as a host. In aquaculture, it will provide useful information on culture conditions affecting their physiology and metabolism in order to optimize the benefit of its bioactive compounds and their pharmacological purposes.

Additionally, the presence of brominated compounds in H. floresii, which are well-known for their defensive activity against bacteria and antimethanogenic properties, could make them suitable as feed ingredients to cattle (Øverland et al. 2019). Thus, it fundamentally eliminates the enteric methane resulting from a high grain diet providing evidence of improved livestock production performance (de Nys et al.

2016

). H. floresii will be an excellent model in a multi-trophic culture environment as algal secondary metabolites with antimicrobial activity, the interfering activity of macroalgae on quorum sensing of any opportunist pathogens may help reduce the emergence of resistant strains in aquaculture systems.

Marine macroalgae are one of the most important economically renewable resources of the oceans.

Despite the advancement in macroalgae transcriptomics, genetic regulations controlling various biochemical pathways are still in its inception and largely remain unexplored [START_REF] Gupta | Seaweed metabolomics: A new facet of functional genomics[END_REF]. Further steps, based on the potential of H. floresii, would be to undergo a genetic comparative study of gene activation under biotic and abiotic stresses. This can be done under cultivation in order to track metabolic pathways (and therefore the secondary metabolites) which are actually engaged in counteracting defined stresses as well as the use of 'Proteomics' as a highly useful method to explore molecular changes that may occur following any disease incidence [START_REF] Khan | Identification of proteins responding to pathogen-infection in the red alga Pyropia yezoensis using iTRAQ quantitative proteomics[END_REF].

World production of carrageenophytes is around 160 000 tons (dry weight), with over 28 000 tons of carrageenan obtained, valued at USD270 million (Freile-Pelegrín and Robledo 2016). Recent reports in commercial carrageenophyte farms show that thallus whitening or bleaching is quite common and most cultivars are quite vulnerable to this disease symptom [START_REF] Largo | Occasional pathogenic bacteria promoting ice-ice disease in the carrageenan-producing red algae Kappaphycus alvarezii and Eucheuma denticulatum (Solieriaceae, Gigartinales, Rhodophyta)[END_REF]. The outbreak of disease resulted in diminishing culture stocks and reduced carrageenan quality, which in turn leads to low market value, loss of income, loss of job opportunities and ultimately cause economic loss [START_REF] Hurtado | Phyconomy: the extensive cultivation of seaweeds, their sustainability and economic value, with particular reference to important lessons to be learned and transferred from the practice of eucheumatoid farming[END_REF]Ward et al. 2019). Preventing the disease outbreak will be a better approach instead of treating or removing the diseased segments after the disease outbreak. Thus, QS interference may be an effective method for preventing and/or inhibiting the virulence of pathogens and hence reduce diseases and increase productivity in aquaculture systems [START_REF] Zhao | Mechanisms of quorum sensing and strategies for quorum sensing disruption in aquaculture pathogens[END_REF]

Future possible experimentations

A metagenomic study on H. floresii (beach-cast) collected from the shores of the Yucatan Peninsula is underway. The results of this analysis will complete our understanding of H. floresii holobiont and its 'core microbial community' as described for other macroalgae [START_REF] Singh | Unraveling the Functions of the Macroalgal Microbiome[END_REF]. In this case, the outcome of this work will become part of the fundamental knowledge of H. floresii.

Additionally, this thesis opens several research opportunities that can be addressed in the future, some of which are:

i.

Targeted metabolomic approach to isolate and characterize the allelopathic active metabolites H. floresii metabolic data shall be taken further for an in-depth analysis of each metabolite by selectively isolating them from the extracts or by mapping genes activation. More precisely, the work can be framed to focus on halogenated metabolites, as they are expected to play a significant role in interfering QS system.

This requires a tremendous amount of algal material to extract, which may be possible by cultivation.

ii. Attenuation of the QS system in Vibrio owensii to prevent disease outbreak

The interference with QS pathways provides an opportunity to attenuate bacterial pathogenicity and thereby the QS antagonists represent novel therapeutic agents to combat bacterial infections [START_REF] Meschwitz | Antagonism of quorum sensing phenotypes by analogs of the marine bacterial secondary metabolite 3-methyl-N-(20-phenylethyl)-butyramide[END_REF]. In such a case, attenuation of the V. owensii QS system by H. floresii shall be explored further for its application in aquaculture in order to prevent disease outbreak.

iii. Bacteria degrading algal-polysaccharides from the epibiome of H. floresii 201 Bacteria degrading algal polysaccharides are key players in the global carbon cycle and in algal biomass recycling [START_REF] Martin | The cultivable surface microbiota of the brown alga Ascophyllum nodosum is enriched in macroalgal-polysaccharidedegrading bacteria[END_REF]. The enzymes degrading algal polysaccharides are highly expected from the macroalgal-associated bacteria, particularly, surface-associated bacteria [START_REF] Martin | Microorganisms living on macroalgae: diversity, interactions, and biotechnological applications[END_REF]). These bacteria have essentially been found in two phyla, Proteobacteria and Bacteroidetes. H. floresii was previously reported to produce highly diverse sulphated polysaccharides [START_REF] Robledo | Prospects for the cultivation of economically important carrageenophytes in Southeast Mexico[END_REF] and the fact that we also identified the epibacteria under these phyla increased our attention towards their possible unique enzymatic profile that could degrade polysaccharides.

Experimentation Setup

1 .

 1 General Introduction ................................................................................................................................. 2 1.1 Part I -Introduction to Macroalgae .................................................................................................... 2 1.1.1 Macroalgae ................................................................................................................................... 2 1.1.2 History and Classification ............................................................................................................ 2 1.1.3 Ecology ........................................................................................................................................ 3 1.1.4 Valorization of Macroalgae ......................................................................................................... 4 1.1.5 Macroalgae in Mexico ................................................................................................................. 6 1.2 Part -II-Red Macroalgae .................................................................................................................. 8 1.2.1 Introduction .................................................................................................................................. 8 1.2.2 Biology of Red Macroalgae ....................................................................................................... 10 i. Cell wall .................................................................................................................................. 10 ii. Polysaccharides: Structural and Storage ............................................................................. 11 a. Carrageenans ....................................................................................................................... 11 b. Agar ..................................................................................................................................... 13 c. Starch ................................................................................................................................... 13 iii. Pigments and Proteins ......................................................................................................... 16 iv. Lipids .................................................................................................................................. 18 1.2.3 Species under Study -Halymenia floresii .................................................................................. 18 1.3 Part -III-Abiotic and Biotic parameters .......................................................................................... 23 1.3.1 Introduction ................................................................................................................................ 23 1.3.2 Definition and Types -Abiotic parameters ................................................................................ 24 i. Hydrodynamism ...................................................................................................................... 24 ii. Nutrients .............................................................................................................................. 25 iii. Desiccation .......................................................................................................................... 25 iv. Temperature ........................................................................................................................ 26 v. Light .................................................................................................................................... 26 vi. Salinity ................................................................................................................................ 26 1.3.3 Definition and Types of Biotic parameters ................................................................................ 27 i. Herbivory ................................................................................................................................ 27 ii. Biofilm .................................................................................................................................... 27 iii. Epiphytism .............................................................................................................................. 28 1.4 Part -IV -Influence of the stressors (abiotic and biotic parameters) ............................................... 28 xii 1.4.1 Introduction ................................................................................................................................ 1.4.2 Chemical Defence ...................................................................................................................... i. Oxidative bursts ...................................................................................................................... ii. Halogenation of volatile and non-volatile compounds ........................................................... iii. Distance signaling................................................................................................................... 1.4.3 Microbial Defence...................................................................................................................... 1.5 Part -V -Consequence of Holobiont break-up -Macroalgal Disease ............................................ 1.5.1 Introduction ................................................................................................................................ 1.5.2 Definition ................................................................................................................................... 1.5.3 Algal diseases ............................................................................................................................. xv 4.6 Discussion ...................................................................................................................................... 4.6.1 Selective extraction of surface-associated metabolites ........................................................... 4.6.2 Influence of DIP n-hexane extract on bacterial communication ............................................. 4.6.3 Untargeted Metabolomic Profiling ......................................................................................... 4.7 Acknowledgements ........................................................................................................................ 4.8 References ...................................................................................................................................... 5. Chapter IV -Identification and characterisation of the quorum sensing signal of the opportunistic pathogen causing bleaching disease in Halymenia floresii by HPLC/MS method .................................. 5.1 Materials and Methods ................................................................................................................... 5.1.1 Algal material .......................................................................................................................... 5.1.2 Bacterial isolates ..................................................................................................................... 5.1.3 Tip bleaching assay with single epibacterial strains ............................................................... 5.1.4 Extraction of HomoSerine Lactones (HSLs) from the 'significant pathogen' ........................ 5.1.5 Chromatographic conditions -LC-MS QTOF analysis .......................................................... 5.2 Results ............................................................................................................................................ 5.3.1 Tip bleaching assay with single epibacterial strains ............................................................... 8.3.2 Identification of homoserine lactones (HSLs) from the 'significant pathogen' ...................... 5.3 Discussion ...................................................................................................................................... 5.5 Conclusion and perspectives .......................................................................................................... 6. Chapter V -Screening Halymenia floresii secondary metabolites which underly its surface defence mechanisms .............................................................................................................................................. 6.1 Abstract .......................................................................................................................................... 6.2 Introduction .................................................................................................................................... 6.3 Materials and Methods ................................................................................................................... 6.3.1 Algal material and extractions ................................................................................................ 6.3.2 LC-MS conditions and data pre-processing ............................................................................ 6.3.3 LC-MS data processing -Madison Metabolomics Consortium Database.............................. 6.3.4 Data post-processing -SMILES .............................................................................................. 6.3.5 Data Validation & Interpretation -Statistical Analysis ........................................................... 6.4 Results ............................................................................................................................................ 6.4.1 LC-MS analyses ...................................................................................................................... 6.4.2 Identifying the H. floresii secondary metbolites ..................................................................... 6.4.3 H. floresii secondary metabolites ............................................................................................ 6.4.4 Hydrophobicity of the secondary metabolites ......................................................................... 6.5 Discussion ...................................................................................................................................... Defence on surface of Rhodophyta Halymenia floresii: metabolomic profile and interactions with its surface-associated bacteria Shareen Arockiasamy xvi xvii Defence on surface of Rhodophyta Halymenia floresii: metabolomic profile and interactions with its surface-associated bacteria Shareen Arockiasamy 2020 xviii

5 Fig. 2 7 Fig. 3 Fig. 4 Fig. 5 Fig. 6 Floridean

 5273456 Fig. 1 Worldwide macroalgal production (%) under aquaculture per species and countries-year 2014 (Source: FAO, 2016) (Buschmann et al. 2017)............................................................................................. 5 Fig. 2 Coastline of the Yucatan Peninsula including the two outstanding marine ecosystems, Gulf of Mexico and Caribbean Sea ........................................................................................................................... 7 Fig. 3 Spatial distribution and Genus richness of Rhodophyta clade worldwide as depicted in (Keith et al. 2014). ............................................................................................................................................................ 9 Fig. 4 Schematic cell wall of red algae with characteristic polysaccharides (Stiger-Pouvreau et al. 2016). .................................................................................................................................................................... 11 Fig. 5 Types of carrageenans (Jiao et al. 2011).......................................................................................... 13 Fig. 6 Floridean Starch ................................................................................................................................ 14 Fig. 7 Floridoside ........................................................................................................................................ 15 Fig. 8 Isofloridoside .................................................................................................................................... 15 Fig. 9 Digeneaside ...................................................................................................................................... 15 Fig. 10 R-Phycoerythrin ............................................................................................................................. 17

Fig. 11 R

 11 Fig. 11 R-Phycocyanin ................................................................................................................................ 17

Fig. 12

 12 Fig. 12 Allophycocyanin ............................................................................................................................. 17

Fig. 13

 13 Fig. 13 Halymenia floresii, (Clemente & Rubio) C.A. Agardh (Rhodophyta, Halymeniales), from the Yucatan peninsula, Mexico ......................................................................................................................... 19 Fig. 14 Observation of the surface of H. floresii under different microscopes; (a, b -cross and longitudinal section view of H. floresii under 'Phase Contrast' microscope; c, d -the surface of H. floresii under 'Epifluorescence microscope' (40x and 400x); e, fthe surface of H. floresii under 'Scanning Electron Microscope' (10 µm and 1 µm) .................................................................................................................. 21 Fig. 15 Main abiotic and biotic factors influencing the ecology and physiology of macroalgae (Lalegerie et al. 2020) .................................................................................................................................................. 24 Fig. 16 Defence mechanism of marine macroalgae against epibionts (da Gama et al. 2014) .................... 29 Fig. 17 Macroalgal diseases in different algal species. a -Red-rot in Pyropia (Kim et al. 2014); b -Redrot in Pythium porphyrae mycelium (Kim et al. 2014); cbleached D. pulchra (Zozaya-Valdés et al. 2016); dbleached K. alvarezii green (Arasamuthu and Patterson Edward 2018); ebleached coralline algae (downloaded from http://coralreefdiagnostics.com/); fbleaching in H. floresii ............................ 34 Fig.18 Isolation of the epibacterial strains by swabbing, streaking, and sub-culturing .............................. 76 Fig. 19 Identification of the isolates by biochemical and molecular characterization ................................ 76 Fig. 20 Reporter assays to screen the isolates for QS activity .................................................................... 77 Fig. 21 Extraction of surface-associated metabolites by the method of DIP ............................................ 105 Fig. 22 Evaluation of Quorum Quenching activity by bioluminescent assay using E. coli pSB406 ........ 105 Fig. 23 Identification of the surface-metabolites by untargeted metabolomic approach .......................... 106 Fig. 24 Extraction and analysis of surface (DIP) and whole-cell metabolites of H. floresii ..................... 149

Fig. 27

 27 Fig. 27 Metabolite organization to yield the corresponding structural and functional descriptors of the predicted metabolites ................................................................................................................................

Fig. 28

 28 Fig. 28 Statistical analysis to validate the data performed in 'R Studio' .................................................. Fig. 29 Different classes of bacteria (%) isolated from the surface of H. floresii .....................................

Fig. 30 Fig. 1

 301 Fig. 30 Scanning electron micrographs of the surface of H. floresii; aapical fronds; bmid-thallus; cstalk. .......................................................................................................................................................... Fig. 31 Count of Metabolites ('41') grouped based on their Activity/Biochemical classes (ATB -Antibiotics; ATC -Anti-cancerous; ATF -Antifungal; ATI -Anti-inflammatory; ATO -Anti-oxidant; ATP -Antiproliferative; ATV -Antiviral; INH -Inhibitors; DIV -Various; FUR -Furans; HAL -Halogenated; HOR -Hormones; IME -Intermediary Metabolites; KET -Ketones; LIP -Lipids; NUC -Nucleotides; PIG -Pigments; SAC -(Poly)-Saccharides; VIT -Vitamins) ............................................. Fig. 32 Bioactive potential of H. floresii identified by untargeted metabolomics study ..........................

Fig. 2

 2 Fig. 2 Bioactive metabolites from the macroalgal associated bacteria .......................................................

Fig. 3 Fig. 4

 34 Fig. 3 Bioactive metabolites from the macroalgal associated fungi ........................................................... Fig. 4 Diatoms embedded on the extracellular matrix of H. floresii (left) and associated with the macroalgal surface bacteria (right) ............................................................................................................. Fig. 5 Different homoserine lactones with differing chain length produced by the bacteria ......................

Fig. 2

 2 Fig. 2 Induction of bioluminescence in the reporter E.coli pSB406 by Tenacibaculum sp., (B9BC); Spongiimicrobium sails (B9CC); Aquimarina sp., (B9.1CC); Alteromonas sp., (B12CC) and Pseudomonas aeruginosa PA01 (positive control). No. of cycles (30 minutes per cycle) plotted against relative light units/optical density at 600 nm ..............................................................................................

Fig. 3 Fig. 4 Fig. 5 Fig. 1 Fig. 1 Fig. 2 Fig. 3 Fig. 4 Fig, 5 Fig. 1

 3451123451 Fig. 3 Induction of bioluminescence in the reporter E.coli pSB406 by Pseudoalteromonas sp., (B5BC); North sea bacterium (B6.1BC) and Alteromonas marina (B16CC). No. of cycles (30 minutes per cycle) plotted against relative light units/optical density at 600 nm. Culture period, in hours, of the isolates were denoted as 'h'. .............................................................................................................................................

Fig. 3 Fig. 6 Table 1

 361 Fig. 3 Dendrogram resulting from the hierarchical cluster analysis (HCA) of '41' metabolites ordered into '8' clusters (I -VIII) at a Ward distance of 1.0. The '26' compounds for which a bioactivity has been reported are green labelled ........................................................................................................................ Fig. 4 Clustering of the H. floresii '41' common metabolites (CMN) according to chemical structures (retrieved from Data Warrior) calculated from their SMILES strings. Numbers correspond to Table 2 ordering. .................................................................................................................................................... Fig. 5 Entanglement of chemical (left) and lipophilicity (right) dendrograms. The level of entanglement is 0.535 (intermediate between 0 and 1) ...................................................................................................... Fig. 6 Lipophilicity of the H. floresii secondary metabolites as a function of the compound's bioactivity. Octanol-water partition coefficients are calculated from the XLOGP3 method .......................................

Table 1 1 1

 11 floresii, DIP extracts with different solvents and WCM extracts with nhexane ....................................................................................................................................................... 118 Table 2. Taxonomic affiliation of the isolated phylotypes. ...................................................................... 119 Table 3. List of common metabolites in DIP and WCM n-hexane extracts proposed by the MMCD mass bank ........................................................................................................................................................... 123 Chapter IV Table I Epibacterial strains isolated from Halymenia floresii tested for their pathogenicity and/or nonpathogenicity in tip bleaching assay ......................................................................................................... 139 Chapter V Codes assigned, based on the bioactivity (functional descriptors) and/or chemical class (chemical descriptors), to the metabolites (DIP and WCM) proposed by the massbank .......................................... 159 Table 2 Codes assigned to the '41' CMN metabolites, names of the putatives, raw/chemical formula, confidence level (∆), and actual mass as proposed by the database MMCD, experimental m/z features (Exp m/z), retention time (RT in minutes) and maximum intensity resulted from the XCMS Online platform, the corresponding Bioactivity /Biochemical classes of putatives (BIO) and their LOGP3 octanol water partition coefficients (XLogP3-AA). ND -Not Detected. ............................................................. 166 xxiv Defence on surface of Rhodophyta Halymenia floresii: metabolomic profile and interactions with its surface-associated bacteria Shareen Arockiasamy 2020 xxv In macroalgal cultivation, epiphytism and fouling are the major problems, severely reducing the productivity and cost efficiency of a diverse range of open-water and on-shore farms. Antifouling practices predominantly include the use of copper-based antifoulant coating, in combination with practical fish husbandry and site management practices. With rapid expansion of the aquaculture industry, coupled with the tightening of legislation on the use of antifouling biocides, the problems of aquaculture biofouling are increasing. The development of environmentally safe antifouling substances is urgently needed. In such a circumstance, during the experimental cycle of culture of Mexican Rhodophyta, Halymenia floresii (Clemente) C. Agardh cultivated in experimental Integrated Multi-trophic Aquaculture systems (IMTA) at onshore tanks, it was observed that, contrary to the tank cultures with Eucheuma, the walls and the propellers remained clean of any exogenic algae colonisation. The presence of H. floresii appeared to limit the establishment of opportunist green algae (Cladophora, Chaetomorpha or Ulva) and the colonisation of barnacles usually disturbing the cultures. Furthermore, the surface of the alga was remarkably free from settlement by fouling organisms. This ecological phenomenon could reveal the release of allelopathically active compounds interfering with the settlement and growth of competitors. The ECOS Nord project is an international scientific cooperation project between FRANCE and MEXICO with the aim of upgrading the macroalgae by combining innovative biotechnologies and process for human health. This project brings together the Laboratory of Marine Biotechnology and Biochemistry (LBCM), University of South Brittany (UBS), FRANCE and the research team of the Department of Marine Resources integrated into The Center for Research and Advanced Studies of the National Polytechnic Institute (CINVESTAV), MEXICO. With this perspective of ECOS Nord, the objective of this project was proposed to better understand the ecological phenomenon behind the exceptional observation in onshore tanks of H. floresii culture.

Fig. 2

 2 Fig. 2 Coastline of the Yucatan Peninsula including the two outstanding marine ecosystems, Gulf of Mexico and the Caribbean Sea

Fig. 3

 3 Fig. 3 Spatial distribution and Genus richness of Rhodophyta clade worldwide as depicted in (Keith et al.2014).

Fig. 4 .

 4 Fig. 4. The schematic cell wall of red algae with characteristic polysaccharides (Stiger-Pouvreau et al.

Fig. 5

 5 Fig.5Types of carrageenans[START_REF] Jiao | Chemical structures and bioactivities of sulfated polysaccharides from marine algae[END_REF]

Fig. 7 FloridosideFig

 7 Fig. 7 Floridoside

Fig

  Fig. 10 R-Phycoerythrin

Fig. 13

 13 Fig. 13 Halymenia floresii (Clemente & Rubio) C.A. Agardh (Rhodophyta, Halymeniales), from the Yucatan peninsula, Mexico

Fig. 14

 14 Fig. 14 Observation of the surface of H. floresii under different microscopes; (a, b -cross and longitudinal section view of H. floresii under 'Phase Contrast' microscope; c, d -the surface of H. floresii under 'Epifluorescence microscope' (40x and 400x); e, fthe surface of H. floresii under 'Scanning Electron Microscope' (10 µm and 1 µm)

Fig. 15

 15 Fig. 15 Main abiotic and biotic factors influencing the ecology and physiology of macroalgae (Lalegerieet al. 2020)

Fig. 16

 16 Fig. 16 The defence mechanism of marine macroalgae against epibionts (esepithallus sloughing; epepibionts; oboxidative bursts; qsquorum sensing; phphysodes; cdchemical defence; cccorps en cerise; gcgland cells mtmicrotopography) (da Gama et al. 2014)

Fig. 17 1 . 5 . 4

 17154 Fig. 17 Macroalgal diseases in different algal species. a -Red-rot in Pyropia (Kim et al. 2014); b -Redrot in Pythium porphyrae mycelium (Kim et al. 2014); cbleached D. pulchra (Zozaya-Valdés et al. 2016); dbleached K. alvarezii green (Arasamuthu and Patterson Edward 2018); ebleached coralline algae (downloaded from http://coralreefdiagnostics.com/); fbleaching in H. floresii

2 . 5 .

 25 Identification of the epibacterial community of H. floresii 3. Screening the epibacteria for their quorum sensing activity 4. Evaluation of H. floresii secondary metabolites for their corresponding interference in the QS behaviour Assessment of the H. floresii secondary metabolites by Untargeted Metabolomic approach 6. Differentiating the H. floresii epibacteria for their significant pathogenic and nonpathogenic/protective interactions. These objectives were studied and the outcomes were detailed in the following chapters: Chapter one reviews the biological defence systems of macroalgae, by means of their associatedmicrobionts, as the microbiome confers a protective shield on the surface of the host (Title: Defence on Surface: Macroalgae and their surface-associated microbiome; A Abdul Malik S., Bedoux G., Garcia Maldonado J. Q., Freile-Pelegrín Y., Robledo D., & Bourgougnon N.; Book: Advances in Botanical research (2020)). Chapter two focuses on the isolation and identification of the cultivable epibacterial community of wild and cultivated Halymenia floresii from the Yucatan peninsula, Mexico. Consecutively, the screening and detection of the QS activity were conducted among the total isolates (Title: Screening of surface-associated bacteria from the Mexican red alga Halymenia floresii for Quorum Sensing activity; A Abdul Malik S., Bazire A., Gamboa A., Bedoux G., Robledo D., Garcia Maldonado J. Q., & Bourgougnon N. Submitted to Microbiology on 26 March 2020). Chapter three studies the surface-associated allelopathic metabolites of H. floresii and their interference in the QS activity of the chosen isolates (Title: Chemical defence against microfouling by allelopathic active metabolites of Halymenia floresii (Rhodophyta). A Abdul Malik S., Bedoux G., Robledo D., Garcia Maldonado J. Q., Freile-Pelegrín Y., & Bourgougnon N. Journal of Applied Phycology (2020)) Chapter four concerns identification of defence molecules from H. floresii by untargeted metabolomic analysis (Title: Investigating the Potential of Halymenia floresii to Produce Bioactive Compounds through Untargeted Metabolomic Profiling; A Abdul Malik S., Bedoux G., Freile-Pelegrín Y., Bourgougnon N & Robledo D., Submitted to Marine Drugs on 27 April 2020) Chapter five determines the significant pathogen, from the epibacterial strains previously isolated from H. floresii, inducing Bleaching in H. floresii (under preparation).

  2010;[START_REF] Amin | Interactions between Diatoms and Bacteria[END_REF]. The scanning electron micrographs of the red alga Halymenia floresii depict the association of different bacterial phylotypes with the surface attached diatoms (Figure4). The abundance and taxonomic composition of the diatoms on the macroalgae are highly affected by the architecture of the macroalgal thallus and its surface characteristics[START_REF] Totti | Epiphytic diatom communities on intertidal seaweeds from Iceland[END_REF]).

Fig. 4

 4 Fig. 4 Diatoms embedded on the extracellular matrix of H. floresii (left) and associated with the macroalgal surface bacteria (right) (unpublished work of the author).

Fig 5

 5 Fig 5 Different homoserine lactones with differing chain lengths produced by the bacteria.

  morphogenesis in green algae belonging to Ulvophyceae depend on bacterial compounds[START_REF] Matsuo | Isolation of an Algal Morphogenesis Inducer from a Marine Bacterium[END_REF][START_REF] Wichard | Exploring bacteria-induced growth and morphogenesis in the green macroalga order Ulvales (Chlorophyta)[END_REF][START_REF] Grueneberg | Macroalgal morphogenesis induced by waterborne compounds and bacteria in coastal seawater[END_REF]. Pseudoalteromonas ulvae and Pseudoalteromonas tunicata isolated from the surface of the green seaweed U. lactuca have shown the ability to reduce the adhesion of colonizing organisms and inhibit the germination of spores of this alga. This result was also observed in the red algae Polysiphonia sp. in the presence of P. tunicata. Ulva spores can be attracted by the emission of QS molecules from biofilms dominated by Pseudoalteromonas. In other species, spore germination may be inhibited by the presence of P. tunicata. Ulva australis is colonized by a very poorly diversified microflora, dominated by Roseobacter gallaeciensis, which facilitates the selective establishment of the bacterium Pseudoalteromonas tunicata. The two bacteria use different strategies. R. gallaeciensis invades and disintegrates the biofilms of competing bacteria, with the exception of P. tunicata. P. tunicata takes advantage of its "immunity" to proliferate in the face of competition that it cannot manage alone and then forms spatially separated microcolonies. These bacterial behaviours on the surface of U. australis allow the algae to have an inexpensive metabolic control of the bacterial biofilm.The red alga Agarophyton vermiculophyllum chemically 'gardens' and recruits protective epibacterial strains in order to strengthen its disease resistance against potentially pathogenic microbes[START_REF] Saha | Microbial "gardening" by a seaweed holobiont: Surface metabolites attract protective and deter pathogenic epibacterial settlement[END_REF]. Twenty one phenotypically different bacterial strains were isolated from the red alga Asparagopsis armata which belong to seven different genera: Vibrio, Staphylococcus, Bacillus, Cobetia, Photobacterium, Shewanella and Alteromonadaceae[START_REF] Horta | Identification of Asparagopsis armata -associated bacteria and characterization of their bioactive potential[END_REF].[START_REF] Ma | Inhibition of common fouling organisms in mariculture by epiphytic bacteria from the surfaces of seaweeds and invertebrates[END_REF] isolated'88' bacterial strains from the surfaces of the red algae: Chondrus ocellatus, Dumontia simplex, Grateloupia filicina, Halymenia sinensis and Porphyra sp.

Fig. 20

 20 Fig. 20 Reporter assays to screen the isolates for QS activity

  community is mostly dominated by bacteria (in a ratio of 640:4:1 of bacteria:diatoms:flagellates) as stipulated by metagenomic and transcriptomic research. However, other members of the microbial community may also play a significant role in the functioning of the holobiont[START_REF] Railkin | Marine biofouling: Colonization processes and defenses[END_REF][START_REF] De Oliveira | Transcriptomic analysis of the red seaweed Laurencia dendroidea (Florideophyceae, Rhodophyta) and its microbiome[END_REF][START_REF] Thajuddin | Algae -Organisms for Imminent Biotechnology[END_REF][START_REF] Ying | The effects of marine farm-scale sequentially integrated multi-trophic aquaculture systems on microbial community composition, prevalence of sulfonamide-resistant bacteria and sulfonamide resistance gene sul1[END_REF]. A broad range of beneficial or detrimental interactions between macroalgae and epi-and endosymbiotic bacteria have been found. Microbes often play a crucial part in macroalgal health, functioning, and development through mutualistic interactions during the various life cycle stages of the host. The interplay of macroalgae with their microbial component affectsamong other thingsnutrient exchange, defence chemical mechanisms, morphology, reproduction, and settlement.

  and response correlated to population density[START_REF] Waters | Quorum Sensing: Cell-to-Cell Communication in Bacteria[END_REF][START_REF] Zhou | Quorum Sensing Is a Language of Chemical Signals and Plays an Ecological Role in Algal-Bacterial Interactions[END_REF]. The epiphytic bacterial community of macroalgae communicate and cooperate with each other (within a species or between species)[START_REF] Whitehead | Quorum-sensing in Gramnegative bacteria[END_REF][START_REF] Waters | Quorum Sensing: Cell-to-Cell Communication in Bacteria[END_REF][START_REF] Abisado | Bacterial quorum sensing and microbial community interactions[END_REF][START_REF] Borges | Quorum Sensing Inhibition by Marine Bacteria[END_REF]. They also interact with the host, using chemical signal molecules through the QS (inter-kingdom) phenomenon. During QS, bacteria produce and excrete signals that accumulate to a threshold level within a diffusion-limited environment[START_REF] Dobretsov | Mini-review: quorum sensing in the marine environment and its relationship to biofouling[END_REF]. QS regulates several bacterial activities such as bacterial virulence and resistance, bioluminescence, and biofilm formation. Such coordinated activities are achieved only when the bacterial population reaches a high density[START_REF] Turan | Quorum sensing: Little talks for an effective bacterial coordination[END_REF]. QS operates through the synthesis of small molecules called Autoinducers (AIs) whereas AutoInducing Peptides (AIPs) and AcylHomoserine Lactones (AHLs) have been shown to regulate QS in Gram-positive and Gram-negative bacteria, respectively[START_REF] Schuster | Acyl-Homoserine Lactone Quorum Sensing: From Evolution to Application[END_REF][START_REF] Monnet | Peptide conversations in Gram-positive bacteria[END_REF][START_REF] Papenfort | Quorum sensing signal-response systems in Gram-negative bacteria[END_REF][START_REF] Kalia | Quorum sensing inhibitors as antipathogens: biotechnological applications[END_REF].Halymenia floresii (Clemente & Rubio) C.A. Agardh (Rhodophyta, Halymeniales) dominates in sublittoral rocky substrates from the Yucatan Peninsula (Mexico) between 3m and 40m[START_REF] Godínez-Ortega | Growth and pigment composition in the red alga Halymenia floresii cultured under different light qualities[END_REF]. Halymenia floresii has been also identified as a carrageenophyte with high potential for landbased IMTA (Integrated MultiTrophic Aquaculture) system. By recycling inorganic nutrients, the IMTA system produces biomass while reducing the environmental impact of coastal ecosystems through responsible aquaculture practices (Godínez-[START_REF] Godínez-Ortega | Growth and pigment composition in the red alga Halymenia floresii cultured under different light qualities[END_REF][START_REF] Robledo | Prospects for the cultivation of economically important carrageenophytes in Southeast Mexico[END_REF]. During the experimental culture of Halymenia floresii, we observed that the surface of this macroalgae was exceptionally free of any fouling organisms compared to any other algae under the same culture conditions.Even at high concentrations of Dissolved Inorganic Nitrogen (DIN ~ 150 µM) in IMTA system, the epiphytic macrofoulers (such as opportunistic green algae and/or any sessile invertebrates), which usually disturb the culture, were not observed over Halymenia floresii culture tanks[START_REF] Pliego-Cortés | Growth, biochemical and antioxidant content of Rhodymenia pseudopalmata (Rhodymeniales, Rhodophyta) cultivated under salinity and irradiance treatments[END_REF]).

  material H. floresii, Beach Cast (BC) was initially collected from the shores near the CINVESTAV Coastal Marine Station at Telchac, Yucatan, Mexico (21.3419° N, 89.2636° W) during autumn 2017 (October-November). The BC collected samples were used for cultivation by IMTA and by Cultivar Chambers (CC), under uncontrolled and controlled environmental conditions, respectively. For IMTA cultivation, landbased aquaculture ponds were set to receive an independent flow of water (mix of shrimp pond effluent with clean, filtered seawater) and constant aeration by air diffusers placed in the bottom of the tanks. For CC cultivation, the macroalgae were grown in transparent polyacrylic cultivation cylinders with sterilized natural seawater, which was further filtered (normal filter 0.5 microns) and UV-sterilized (UV-filter 0.4 microns) at 30 psu, 22 ± 2° C and pH around 8.0 ± 0.3 under continuous aeration at a 12:12 light/dark cycle

  Blood and Tissue Kit (Van der Zee et al. 2002) andZymoBIOMICS DNA Miniprep Kit (Al- Hebshi et al. 2019). Extracted DNA was observed on 1% agarose gel electrophoresis in 1X TAE buffer and quantified by Qubit Fluorometer. The 16S rRNA gene was amplified by PCR using the primers 16sF (5'-AGAGTTTGATCCTGGCTC-3') and 16sR (5'-CGGGAACGTATTCACCG-3') with the following protocol: 12.5 µL of Promega GoTaq @ Green Master Mix 2X (2X Green GoTaq reaction buffer_pH 8.5; 400 µM of dNTPs; 3mM MgCl2), 010µM of each primer, 11 µL DNase free H2O and 10 ng of DNA template. The PCR reaction was performed in the Axygene Thermal Cycler. The PCR amplifications were visualized by agarose gel and documented under BioDoc-It Imager. The amplified 16S rRNA gene of all the bacterial isolates were commercially sequenced by GENEWIZ Laboratories (New Jersey, USA).

  cross-feeding assay was performed with the bioreporter C. violaceum CV026. C. violaceum, a Gram-negative bacterium, produces a characteristic purple pigment violacein whereas its double mini-Tn5 mutant C. violaceum CV026 is derived from C. violaceum ATCC31532, Hgr, cviI::Tn5 xylE, Km r , plus spontaneous Sm r (McClean et al. 1997), uses a class of signal molecules, acyl-homoserine lactones, and exhibits violet pigmentation only in the presence of exogenous short-chain, C-4 and/or C-6 HSLs (González et al. 2001).

  (1-factor ANOVA: F (1, 68) = 65.25, p < 0.0001) induced higher light production compared to that of Spongiimicrobium salis (1factor ANOVA: F (1, 66) = 100.61, p < 0.0001) and Aquimarina sp. (1-factor ANOVA: F (1, 66) = 70.81, p < 0.0001). Fig. 1 also shows the bioluminescence induced by Alteromonas sp. (B12CC) (1-factor ANOVA: F (1, 66) = 71.55, p < 0.0001).

Fig. 1

 1 Fig. 1 Induction of bioluminescence in the reporter E. coli pSB406 by Tenacibaculum sp., (B9BC); Spongiimicrobium sails (B9CC); Aquimarina sp., (B9.1CC); Alteromonas sp., (B12CC) and Pseudomonas aeruginosa PA01 (positive control). No. of cycles (30 minutes per cycle) plotted against relative light units/optical density at 600 nm

Fig. 2 Fig. 3

 23 Fig. 2 Induction of bioluminescence in the reporter E.coli pSB406 by Vibrio owensii (B3IM); Maribacter sp., (B9IM); Pseudoalteromonas arabiensis (B4BC); P. mariniglutinosa (B6BC) and V. owensii (B7BC) No. of cycles (30 minutes per cycle) plotted against relative light units/optical density at 600 nm

Fig. 4

 4 Fig. 4 Induction of bioluminescence in the reporter E.coli pSB406 by Ruegeria lacuscaerulensis (B9CC); Uncultured bacterium clone (B9.2CC) and Erythrobacter sp., (B14CC). No. of cycles (30 minutes per cycle) plotted against relative light units/optical density at 600 nm. Culture period, in hours, of the isolates, were denoted as 'h'.

Fig. 5

 5 Fig. 5 Induction of bioluminescence in the reporter E.coli pSB406 by Roseobacter sp., (B13CC) and Epibacterium sp., (B20CC). No. of cycles (30 minutes per cycle) plotted against relative light

  the various means of bacterial communication, cell density-dependent communication, QS, plays a major role in achieving a dynamic and equilibrated biofilm. Such a biofilm is the most evident form of biofouling. A potential approach to inhibit the process of biofouling is to interfere with bacterial QS signals. Hence, the very first step is to detect the presence of QS communication signals in the epibacterial community of the macroalgae. This study has detected and screened the surface bacteria isolated from H. floresii for the production of exogenous QS signals, and the results of the tests revealed that '17' bacterial isolates produce communication signals. As mentioned earlier, '2' different biosensors, Chromobacterium violaceum CV026 and Escherichia coli pSB406, were used in the study. The biomonitor strains enable sensitive, quantitative, and real-time detection of QS signals such as AHLs (Kalia 2013).

Fig. 22

 22 Fig. 22 Evaluation of Quorum Quenching activity by bioluminescent assay using E. coli pSB406

Fig. 23 1

 23 Fig. 23 Identification of the surface-metabolites by untargeted metabolomic approach

  -Cast (BC) H. floresii was initially collected from the shores near Centro de Investigación y de Estudios Avanzados del Instituto Politécnico Nacional (CINVESTAV) Coastal Marine Station at Telchac, Yucatán, México (21.3419° N, 89.2636° W). The collected BC H. floresii samples were cultivated (CC) under controlled environmental conditions in transparent acrylic cultivation cylinders inside a cultivation chamber after several weeks of acclimatization. The cultivation was performed in sterilized natural seawater, which was further filtered (0.5 microns) and UV-sterilized (UV-filter 0.4 microns) at 30 ups, at a temperature of 22 ± 2° C and a pH of around 8.0 ± 0.3 under continuous aeration at a 12:12 light/dark cycle with an intensity of 110 ± 7.2 µmol photons m -2 s -1 . Every week CC H. floresii was pulsefed with Provasoli Enriched Seawater Media (0.3%) for 24 h. After eight weeks of cultivation, approximately 60 g was harvested and taken for extraction (~ 5 g for each time period in duplicate).

  isolates were cryopreserved at -80° C in 20% glycerol. Around thirty different bacterial isolates were isolated from the surface of H. floresii.ii.Identification -Taxonomic affiliation of the surface-associated bacteria Bacterial strains were grown overnight in Marine broth (MB) at room temperature. The DNA was extracted using two different kits, DNeasy Blood and Tissue Kit (Qiagen, USA) and ZymoBIOMICS DNA Miniprep Kit (Zymoresearch, USA) according to the manufacturer's instructions. The extracted DNA was amplified by PCR for their 16S rRNA gene sequences. To which 12.5 µL of Promega GoTaq @ Green Master Mix 2X (2X Green GoTaq reaction buffer_pH -8.5; 400 µM of dATP, dGTP, dTTP & dCTP; 3mM MgCl2), 0.25 µL of each primer, 16s F -5' -AGAGTTTGATCCTGGCTC -3', 10 µM and 16s R -CGGGAACGTATTCACCG -3', 10 µM, 11 µL DNase free H2O and 1 µL of the extracted DNA template was added. The PCR reaction was performed in the Axygene Thermal Cycler. The 25 µL reaction mixture was then heated to 94° C for 3 min, followed by 30 cycles of denaturation at 94° C for 30 sec and annealing at 52° C for 1 min and extension at 72° C for 1 min. A final extension was carried out at 72° C for 10 min.

  Fig 2 illustrates the epifluorescence micrographs of n-hexane dipped cortical cells of H. floresii where only a few cells were lysed for 60s (1-factor ANOVA: F (1,10) = 136.35, p < 0.0001). Fig 3 represents the cell counts for n-hexane dipped H. floresii, whereas

Fig 2

 2 Fig 2 Epifluorescence micrographs showing the cortical cells of H. floresii after being dipped in n-hexane for 10s; 20s; 30s; 40s; 50s and 60s and positive control treated in exactly the same way with sterile seawater for 30s.

  Fig 3 Number of intact and lysed cells vs. immersion period (in seconds) after dipping in n-hexane counted using ImageJ software. The y-axis represents the Means ± SE of epifluorescence micrographs of H. floresii at '5' different angles (n=3).

  Fig 4 (a-d) Quorum quenching activity of the DIP extract on the chosen phylotypes. a -Vibrio owensii; b -Tenacibaculum sp.; c -Maribacter sp. and d -Aquimarina sp. No. of cycles (30 minutes per cycle)

Fig 5

 5 Fig 5 TIC of DIP_n-hexane (red) and WCM_n-hexane (blue) crude extract after retention time correction by XCMS Online platform.

  pteridin-1-id-10(5H)-yl)-5bromophenyl)[4-({(2E)-4-[cyclopropyl(methyl)amino]but-2-enyl}oxy)phenyl]methanone 399.08 14.0-14.5 (2E)-N-allyl-4-{[3-(4-bromophenyl)-5-fluoro-1-methyl-1h-indazol-6-yl]oxy}-N-methyl-2buten-1-amine 443.10 14.0-14.5 m/zmass/charge ratio; RT -Retention Time in minutes; ND -Not Detected Defence on surface of Rhodophyta Halymenia floresii: metabolomic profile and interactions with its surface-associated bacteria Shareen Arockiasamy 2020 124

  pteridin-1-id-10(5h)-yl)-5-o-phosphonato-d-ribitol. Group 3 essentially included halogenated compounds, particularly brominated compounds, (4-bromophenyl)[4-({(2e)-4-[cyclopropyl(methyl)amino]but-2-enyl}oxy)phenyl]methanone and (2e)-n-allyl-4-{[3-(4bromophenyl)-5-fluoro-1-methyl-1h-indazol-6-yl]oxy}-n-methyl-2-buten-1-amine. Additionally, we also observed one compound showing mass spectra with bromine isotopic patterns during LC-MS analysis.

Alphaproteobacteria, Gammaproteobacteria ,

 Gammaproteobacteria and Bacteroides. They are all Gram-negative, a predominant biofilm-forming bacterial group in the marine environment (Brian-Jaisson et al. 2016; A Abdul Malik et al. 2019). Bacteroidetes, which essentially include Tenacibaculum sp., Maribacter sp. and Aquimarina sp.

Compound 1 , 2 -

 12 Fig 6 LC-MS spectrum of surface extract showing bromine isotopic pattern (red arrow marks) a characteristic of halogenated metabolite.

Fig. 1 5 . 1 . 5

 1515 Fig.1 Schematic illustration of the HSLs extraction process.

Fig. 2 p

 2 Fig. 2 p < 0.00036). Eight additional isolates (Pseudoalteromonas arabiensis; P. mariniglutinosa; Tateyamaria omphalii; Ruegeria sp.; Alteromonas sp. (B7CC and B12CC); Epibacterium sp.;

Fig. 2

 2 Fig. 2 Risk of thallus tip bleaching in H. floresii after inoculation of '25' bacterial strains relative to control thalli without bacterial inoculation. No. of replicates (n=6) was assigned for each isolate. Error bars ± in %.

Fig. 3

 3 Fig. 3 Chromatogram of V. owensii HSL extract pre-processed in XCMS Online platform.

Fig. 4

 4 Fig. 4 Chromatogram showing the presences of C4 HSL in the standard (upper panel) and V. owensii extract (lower panel) at a retention time of 6.2 to 7.0 minutes.

Fig 5 .

 5 Fig 5. Mass Spectrum showing the fragments of C4 HSL in the standard (upper panel) and V. owensii extract (lower panel) as 102.05 (lactone ring); 172.09 (M+H) + and 194.08 (M+Na) + .

Fernandes

  et al. (2011) elucidated the ability of Nautella sp. R11 to induce bleaching in D. pulchra and they observed that a combination of virulence factors and QS-dependent regulatory mechanisms enabled a shift from symbiotic to a pathogenic lifestyle of the epibacterial community, especially under environmental conditions unfavorable for the host. As reviewed by Natrah et al. (2011b), several different virulence factors of Vibrio causing diseases in aquaculture systems were strongly related to the AHLs, such as N-hexanoyl-L-homoserine lactone (HHL), N-(3-oxohexanoyl)-L-homoserine lactone (OHHL), Nbutanoyl-L-homoserine lactone (BHL), N-3-octanoyl homoserine lactone (OHL), N-(3-oxodecanolyl)-L-

  2020b). The quorum sensing interference of H. floresii may remarkably contributed by the bromophenyl)[4-({(2E)-4-[cyclopropyl(methyl)amino]but-2-enyl}oxy)phenyl]methanone (Compound 7) and (2E)-N-allyl-4-{[3-(4bromophenyl)-5-fluoro-1-methyl-1H-indazol-6-yl]oxy}-N-methyl-2-buten-1-amine (Compound 8).

Fig. 24

 24 Fig. 24 Extraction and analysis of surface (DIP) and whole-cell metabolites of H. floresii

Fig. 26

 26 Fig. 26 Processing of the data in Madison Metabolomics Consortium Database (MMCD)

Fig. 27

 27 Fig. 27 Metabolite organization to yield the corresponding structural and functional descriptors of the predicted metabolites

  photons m-2 s-1. Every week H. floresii was pulse-fed with Provasoli Enriched Seawater Media (0.3%) for 24 h. After eight weeks of cultivation, approximately 60 g fw were harvested and extracted (~ 5 g for each time period in duplicates). In order to selectively extract the surface-associated metabolites, samples were taken for the DIP extraction as previously reported (A Abdul Malik et al. 2020) since the use of n-hexane allows to extract the surface metabolites of H. floresii without lysis of the cortical cell's wall. Thus, the surface metabolites were gently and selectively extracted using n-hexane, a non-polar solvent, using the dipping method (DIP) previously described by[START_REF] De Nys | A new method for determining surface concentrations of marine natural products on seaweeds[END_REF].

Fig. 1

 1 Fig. 1 Metabolite counts of the Whole-cell metabolites (WCM); surface metabolites (DIP) and common metabolites (CMN) plotted against Activity/Biochemical classes (symbols are from Table1).

Fig. 3

 3 Fig. 3 Dendrogram resulting from the hierarchical cluster analysis (HCA) of '41' metabolites ordered into '8' clusters (I -VIII) at a Ward distance of 1.0. The '26' compounds for which a bioactivity has been reported are green labelled.

Fig. 5 Fig. 5

 55 Fig. 5 Entanglement of chemical (left) and lipophilicity (right) dendrograms. The level of entanglement is 0.535 (intermediate between 0 and 1).

Fig. 6

 6 Fig. 6 Lipophilicity of the H. floresii secondary metabolites as a function of the compound's bioactivity. Octanol-water partition coefficients are calculated from the XLOGP3 method.

  furaldehyde (S108) linked to Trans-5-o-caffeoyl-d-quinate (S125) and further with [(4Z)-2-(aminomethyl)-4-(4-hydroxybenzylidene)-5-oxo-4,5-dihydro-1h-imidazol-1-yl] acetic acid (S316). A derivative of S108,

Fig. 29

 29 Fig. 29 Different classes of bacteria (%) isolated from the surface of H. floresii

  et al. 2011).[START_REF] Parrot | Mapping the surface microbiome and metabolome of Brown seaweed Fucus vesiculosus by amplicon sequencing, integrated metabolomics and imaging techniques[END_REF] used scanning electron microscopy to study the biofilm density on the surface of the brown alga Fucus vesiculosus and showed a lower biofilm thickness at the uppermost tip than at the older thallus regions. We also analysed different parts of the thallus of H. floresii cultivated in controlled conditions (CC), such as apical fronds, mid-thallus and stalk, to understand microbial distribution on the macroalgal surface under the scanning electron microscope. We observed increased microbial load at the mid-thallus than apical fronds showing a spatial variation of the microbiome on the thallus surface.

Fig. 30

 30 Fig. 30 Scanning electron micrographs of the surface of H. floresii; aapical fronds; bmid-thallus; cstalk.

Fig. 31

 31 Fig. 31 Count of Metabolites ('41') grouped based on their Activity/Biochemical classes (ATB -Antibiotics; ATC -Anti-cancerous; ATF -Antifungal; ATI -Anti-inflammatory; ATO -Anti-oxidant;

  such as brominated compounds, Volatile Halogenated Organic Compounds (VHOCs) like diterpenes and flavonoids might contribute to the chemical defence on the surface of H. floresii. Their activity was mainly attributed to the corresponding interference with the surface-associated bacterial communication (A Abdul Malik et al. 2020b).

Fig. 1 p

 1 < 0.05) and thus were considered as 'potential pathogens'. V. owensii, a member of Gammaproteobacteria, isolated from H. floresii cultivated under IMTA was previously identified as a QS bacterium (see Chapter II) and LC/MS confirmed the presence of N-butyryl-Lhomoserine lactone (C4-HSL) in the culture medium of V. owensii grown in Marine Broth (MB -liquid) medium (see Chapter IV). The presence of C4-HSL in V. owensii emphasized the QS critical role in the induction of bleaching in H. floresii.

Fig. 32

 32 Fig. 32 Bioactive potential of H. floresii identified by untargeted metabolomics study

  industry and pharmacological purposes[START_REF] Freile-Pelegrín | Preliminary characterization of carrageenan from the red seaweed Halymenia floresii[END_REF], can be cultured under favourable culture conditions leading to the improvement of their bioactive secondary metabolites. Under IMTA cultivation, environmental stressors such as temperature, sunlight, nutrient availability (eutrophic or oligotrophic), epibiosis and disease outbreak in any of the trophic levels might result in a putative metabolic shift, exhibiting a high and/or low bioactivity ultimately linking to its chemical defence. With the metabolic profile of H. floresii proposed in this study, this metabolic shift can be assessed. In their natural habitat, H.

Table of Contents

 of

Table 1

 1 Motility, Gram stain and Biochemical characteristics of the isolates ............................................ 86

Table 2

 2 Halymenia floresii surface-associated bacteria; * -Strain with <97% similarity. ......................... 89

Table 3

 3 Screening of the isolates based on QS signals production; * -Strain with <97% similarity. ........ 91

	Chapter III

Table 1 .

 1 Concentration of the H.

Table 1

 1 List of bacteria isolated from the '3'major phyla of macroalgae

	Macroalgae	Bacteria Isolated	References
	Red
	Polysiphonia stricta	Pseudoalteromonas arctica	(Nasrolahi et al. 2012)
		Shewanella basaltis
	Gracilariopsis heteroclada	Vibrio parahaemolyticus; V. alginolyticus	(Martinez and Padilla 2016)
	Gracilaria blodgetti	Agarilytica rhodophyticola	(Ling et al. 2017)
	Laurencia papillosa	Bacillus amyloliquefaciens	(Chakraborty et al. 2017b)
	Ahnfeltia tobuchiensis	Polaribacter staleyi	(Nedashkovskaya et al.
			2018a)

Cobetia amphilecti; Shewanella sp.; Sphingomonas sp.; Sulfitobacter sp.; Polaribacter sp.; Bacillus sp.; Staphylococcus saprophyticus. (Albakosh et al. 2016) Padina pavonica Paracoccus sp.; Devosia sp.; Vibrio sp.; Pseudomonas sp.; Pseudomonas putida; Brevibacterium iodinum; Staphylococcus sp.; Bacillus pumilus; Pseudoalteromonas sp.; Acinetobacter sp.; Planomicrobium glaciei; Erwinia bilingiae. (Ismail et al. 2016)

Table 2

 2 List of fungi isolated from the '3'major phyla of macroalgae Aspergillus sp., Cladosporium sp., Curvularia sp., Emericella nidulans, Trichoderma sp., Pestalotiopsis sp. Aspergillus spp. and Penicillium were common colonizers and also serve as a potential source of pharmacologically active compounds(Singh et al. 2015). A. terreus from the tissues of Caulerpa scalpelliformis, C. sertularioides, G. edulis, Sargassum ilicifolium and U. lactuca produced metabolites which have insecticidal activity[START_REF] Suryanarayanan | Internal mycobiota of marine macroalgae from the Tamilnadu coast: distribution, diversity and biotechnological potential[END_REF]. Xylaria sp. isolated

	Macroalgae	Fungi Isolated	References
	Red
	Eucheuma sp.	Aspergillus flavus, A. niger	(Vala et al., 2004)
	Palmaria decipiens	Metschnikowia australis, Cryptococcus	(Loque et al. 2010)
		carnescens
	Grateloupia lithophila	Cladosporium sp., Chaetomium sp.,	(Murali 2011)
		Nigrospora sp.,

from red alga Bostrychia tenella synthesize cytochalasin D, a known antibiotic and antitumour compound (de Felício et al. 2015) whereas A. niger EN-13, isolated from brown alga, Colpomenia sinuosa, produces a sphingolipid and their corresponding glycosphingolipid named Asperamides A and B, where compound

Table 3

 3 List of microalgae isolated from the macroalgae

	Macroalgae	Microalgae Isolated	References
	Chaetomorpha linum	Leptocylindrus danicus; Navicula distans;	(Al-Harbi 2017)
		Nitzschia hungarica; Thalassionema
		frauenfeldi
	Enteromorpha intestinalis	Chaetoceros fragile ; Gyrosigma fasciola ; L.
	Ulva lactuca	Licmophora flabellata ; L. abbreviata ; P.	(Al-Harbi 2017)
		normanii ; T. frauenfeldii
	Laminaria sp.	L. danicus; L. flabellata; Navicula	(Marzoog Al-Harbi 2017)
		ramosissma;
	Padina fraseri	Cylindrotheca closterium; Navicula sp.;	(Marzoog Al-Harbi 2017)
		Nitzschia sp.; Prorocentrum lima; Cocconeis
		lineatus
	Sargassum muticum	N. distans ; N. socialis ; N. vanhoeffeni	(Marzoog Al-Harbi 2017)
	Turbinaria ornata	Leptocylindrus minimus; L. danicus;	(Marzoog Al-Harbi 2017)
		Bacillaria paxillifer ; N. hungarica; T.
		frauenfeldii
	Ecklonia maxima	Amphora; Gomphoseptatum; Navicula,	(Mayombo et al. 2019)
		Rhoicosphenia

danicus ; N. distans ; N. transitans ;

Pleurosigma angulatum ; P. normanii

[START_REF] Sm | Epiphytic microalgal species composition and dynamics on host green seaweeds (Ulvaphyceae) on the northern coast of Jeddah, Saudi Arabia[END_REF]

Table 4

 4 List of viruses isolated from the macroalgae

	Macroalgae	Viruses	References
	Delisea pulchra	Totivirus; Picornavirales	(Lachnit et al. 2016)
	Chondrus crispus	Totivirus-like entities	(Rousvoal et al. 2016)
	Ectocarpus siliculosus	Ectocarpus DNA virus	(Lanka et al. 1993)
	Heterosigma akashiwo	Phycodnaviridae	(Nagasaki et al. 2005)
	Ecklonia radiata	Siphoviridae; Myoviridae; Inoviridae;	(Beattie et al. 2018)
		Podoviridae
	Laminaria sp. and	Phaeovirus	(McKeown et al. 2017)
	Saccharina sp.

  . These QS signals, otherwise known as Auto Inducers (AI), broadly fall into three categories. Acyl Homoserine Lactones (AHLs) are the major group of auto inducer signals in gram-negative bacteria (Di Cagno et al. 2011) (Figure 5 ); Auto

Inducer Peptides (AIPs) are the major auto inducers in gram-positive bacteria

[START_REF] Thoendel | Peptide Signaling in the Staphylococci[END_REF][START_REF] Zhang | Exploiting quorum sensing interfering strategies in gram-negative bacteria for the enhancement of environmental applications[END_REF]

, whereas, regardless of gram-positive or gram-negative bacteria, Auto inducer 2 (AI 2) known as universal language, is used for intra-and inter-species communication

[START_REF] Lowery | Interspecies and interkingdom communication mediated by bacterial quorum sensing[END_REF]

. Besides, there have been cyclic dipeptides, bradyoxetin, AI -3 in Escherichia coli and diffusible signal factors, documented in different QS systems

(Gonzalez and Keshavan 2006)

.

Table 1

 1

(Chapter II). The molecular identification of the isolates was performed using DNeasy Blood and Tissue Kit (Van der Zee et al. 2002) and ZymoBIOMICS DNA Miniprep Kit (Al-Hebshi et al. 2019

Table 1

 1 Motility, Gram stain and Biochemical characteristics of the isolates

	Bacterial isolates/	B1BC	B4BC	B5BC	B6BC	B6.1BC	B7BC	B8BC	B9BC	B2IM*	B3IM	B9IM	B1CC	B2CC	B3NBCC	B4CC	B7CC
	Characteristics
	Motility	Motile a	Motile a	Motile a	Motile a	Motile a	Motile a	Motile a	Motile c	Motile	Motile	Motile c	Motile b	Motile a	Motile a	Motile a	Motile a
	Gram reaction	--	-	-	-
	Alkaline	+++++	+++++	+++++	+++++	+++++	+++++	+++++	+++++	+++++	+++++	n.d	+	-	-	++	+++
	phosphatase
	Esterase (C 4)	++	+++	+	+/-	-	-	-	++++	+	+++	n.d	++++	+++	-	++	++++
	Esterase Lipase	+++	++++	+	++	++	-/+	+/-	+++	+	++	n.d	+++	++	-	++	+++++
	(C8)
	Lipase (C18)	-	-	-	-	-	-	-	-	-	-	n.d	-	-	-	-	-
	Leucine	+++++	+++++	+++++	+++++	+++++	++	+++++	+++++	+	+	n.d	-	++	-	-	+++
	arylamidase
	Valine arylamidase	+	++	++	+	+	-	++	+	-	-	n.d	-	-	-	+	-
	Cystine
	arylamidase

  Alphaproteobacteria, Gammaproteobacteria, Bacteroidetes, and Firmicutes (Table 2). The bacterial isolates from BC, IMTA, and CC H. floresii included the members of Gammaproteobacteria and Bacteroidetes, whereas the members of Alphaproteobacteria and Firmicutes

	Bacterial isolates/ Characteristics Motility Gram reaction corresponded to four phyla: were found only in the CC H. floresii. B7.1CC B8CC B8.1CC B9CC Motile a Motile a Motile a Motile c Molecular characterization BLAST analyses of the 16S rRNA gene sequences revealed that all the '31' bacterial strains B9.1CC B9.2CC B10CC B10.1CC B10.2CC B12CC B13CC Motile c Motile a Motile a Motile a Motile a Motile a Motile a -ii.	B14CC Non-motile	B16CC Motile a	B17CC Motile a	B19CC Motile a	B20CC Motile b
	a. Alphaproteobacteria
	The genera identified within Alphaproteobacteria from CC H. floresii were: Tateyamaria omphalii
	(B1CC_Accession No. MT176143 and B3CC_Accession No. MT176145); Phaeobacter sp.
	(B2CC_Accession No. MT176144); Ruegeria sp. (B4CC_Accession No. MT176146); Ruegeria
	lacuscaerulensis (B8CC_Accession No. MT176149 and B19CC_ Accession No. MT176162); Uncultured
	Kordiimonas sp. (B9.2CC_Accession No. MT176153) Epibacterium sp. (B10.1CC_ Accession No.
	MT176155; B10.2CC_ Accession No. MT176156 and B20CC_ Accession No. MT176163); Roseobacter
	Tenacibaculum sp. (B9BC_Accession No. MT176141) and Maribacter sp. (B9IM_Accession No.
	MT176142) respectively and Spongiimicrobium salis (B9CC_Accession No. MT176151) and Aquimarina
	sp. (B9.1CC_Accession No. MT176152) from CC H. floresii. The members of this class have shown the
	same pattern of chromogenesis by the production of yellow pigments. Another interesting characteristic of
	these members was their gliding motility. Bacteroidetes isolated in this study also showed iridescence; in

sp. (B13CC_ Accession No. MT176158); Erythrobacter sp. (B14CC_ Accession No. MT176159). b. Gammaproteobacteria The Gammaproteobacteria from the BC H. floresii mainly included the Pseudoalteromonas species, whereas the CC samples included the Alteromonas species. Vibrio owensii was observed in BC (B7BC_Accession No. MT176139) and IMTA (B3IM_Accession No. MT176134) samples, but one strain, Vibrio sp. (results not shown) from IMTA, showed <97% of similarity. We could not observe any Pseudoalteromonas sp. in IMTA H. floresii and Vibrio sp. from CC H. floresii.

c. Bacteroidetes

The phylotypes belonging to the class Bacteroidetes were observed in H. floresii regardless of its origins such as BC, IMTA, and CC. Bacteroidetes of BC, IMTA H. floresii were found to be affiliated to particular, Tenacibaculum sp. from BC H. floresii is observed to have higher iridescence (observed macroscopically) compared to the other two members. This strain showed a glitter-like iridescence macroscopically on the culture plate itself.

Table 2

 2 Halymenia floresii surface-associated bacteria. Group, Code, Closest neighbor, similarity and sample origin were listed in the table

	Accession No.	Group	Code	Closest Neighbour	Similarity	Sample Origin
	MT176133 Gammaproteobacteria B1BC	Pseudoalteromonas arctica.	100% Beach Cast
	MT176135 Gammaproteobacteria B4BC	Pseudoalteromonas arabiensis	100% Beach Cast
	MT176136 Gammaproteobacteria B5BC	Pseudoalteromonas sp.	99.83% Beach Cast
	MT176138 Gammaproteobacteria B6BC	Pseudoalteromonas mariniglutinosa	99% Beach Cast
	MT176137 Gammaproteobacteria B6.1BC	Pseudoalteromonas sp.	100% Beach Cast
	MT176139 Gammaproteobacteria B7BC	Vibrio owensii	100% Beach Cast
	MT176134 Gammaproteobacteria B3IM	Vibrio owensii	100% IMTA
	MT176140 Gammaproteobacteria B8BC	Pseudoalteromonas arctica	99.84% Beach Cast
	MT176147 Gammaproteobacteria B7CC	Alteromonas sp.	99%	Cultivars Cylinders
	MT176148 Gammaproteobacteria B7.1CC	Alteromonadaceae bacterium strain	98.68%	Cultivars Cylinders
	MT176150 Gammaproteobacteria B8.1CC	Alteromonas sp.	99.37%	Cultivars Cylinders
	MT176157 Gammaproteobacteria B12CC	Alteromonas sp.	99.53%	Cultivars Cylinders
	MT176160 Gammaproteobacteria B16CC	Alteromonas sp.	99.68%	Cultivars Cylinders
	MT176161 Gammaproteobacteria B17CC	Alteromonadaceae bacterium	98.74%	Cultivars Cylinders
	MT176143 Alphaproteobacteria	B1CC	Tateyamaria omphalii	100%	Cultivars Cylinders
	MT176144 Alphaproteobacteria	B2CC	Phaeobacter sp.	100%	Cultivars Cylinders
	MT176145 Alphaproteobacteria	B3CC	Tateyamaria omphalii	99%	Cultivars Cylinders
	MT176146 Alphaproteobacteria	B4CC	Ruegeria sp.	100%	Cultivars Cylinders
	MT176149 Alphaproteobacteria	B8CC	Ruegeria lacuscaerulensis	99.83%	Cultivars Cylinders

  Pseudoalteromonas sp., and the Bacteroidetes, Maribacter sp.; (ii) the isolates of 36H culture period include the Gammaproteobacteria, Alteromonas sp.(B12CC); the Bacteroidetes, Tenacibaculum sp., Spongiimicrobium salis, Aquimarina sp. Apart from these '2' major culture periods, the following strains, such as Epibacterium sp., Roseobacter sp. and

	30	B19CC	Ruegeria lacuscaerulensis	-	+
	31	B20CC	Epibacterium sp.	-	+
		Alteromonas sp. (B16CC), induce the luminescence after 12h, 24h, and 48h of the culture period,
		respectively.
		Table 3 Screening of the isolates based on QS signals production; * -Strain with <97% similarity.
	S.No	Bacterial	Closest neighbour	Chromobacterium violaceum	Escherichia coli pSB
		isolates		CV026	406
	1	B1BC	Pseudoalteromonas arctica	-	-
	2	B4BC	Pseudoalteromonas arabiensis	-	+
	3	B5BC	Pseudoalteromonas sp.	-	+
	4		Pseudoalteromonas	-	+
		B6BC	mariniglutinosa
	5	B6.1BC	Pseudoalteromonas sp.	-	+
	6	B7BC	Vibrio owensii	-	+
	7	B8BC	Pseudoalteromonas arctica	-	-
	8	B9BC	Tenacibaculum sp.	-	+
	9	B3IM	Vibrio owensii	-	+
		B9IM	Maribacter sp.	-	+
		B1CC	Tateyamaria omphalii	-	-
		B2CC	Phaeobacter sp.	-	-
		B3CC	Tateyamaria omphalii	-	-
		B4CC	Ruegeria sp.	-	-
		B7CC	Alteromonas sp.	-	-
		B7.1CC	strain Alteromonadaceae bacterium	-	-
		B8CC	Ruegeria lacuscaerulensis	-	-
		B8.1CC	Alteromonas sp.	-	-
		B9CC	Spongiimicrobium salis	-	+
		B9.1CC	Aquimarina sp.	-	+
		Gammaproteobacteria, Pseudoalteromonas arctica (B1BC and B8BC); Alteromonas sp. (B7CC and B9.2CC Uncultured Kordiimonas sp. -+
		B8.1CC); Alteromonadaceae bacterium strain (B7.1CC and B17CC); in the class of Alphaproteobacteria, B10CC Bacillus circulans --
		Tateyamaria omphalii (B1CC and B3CC), Phaeobacter sp., Ruegaria sp., Ruegeria lacuscaerulensis B10.1CC Epibacterium sp. --
		B10.2CC (B8CC), and Epibacterium sp. (B10.1CC and B10.2CC); and also in the only Firmicutes, B. circulans. We Epibacterium sp. --Alteromonas sp. -+ B12CC also observed that the induction of bioluminescence in E. coli pSB406 was highly influenced by the culture B13CC Roseobacter sp. -+
		B14CC	Erythrobacter sp.	-	+
		B16CC	Alteromonas sp.	-	+
		B17CC	Alteromonadaceae bacterium	-	-
					92

period of the isolate supernatant. The phylotypes that induced luminescence are grouped according to the culture period, and they are as follows:

(i)

the isolates of 16H culture period include the Gammaproteobacteria, Pseudoalteromonas arabiensis, Pseudoalteromonas sp., Pseudoalteromonas mariniglutinosa, Vibrio owensii (B3BC and B3IM),

  Except for B. circulans (B10CC), all the bacterial strains isolated from the surface of H. floresii, in this study were Gram-negative; hence, the main focus of QS signals is on the AHLs, irrespective of their classes. AHLs are classified based on the N-linked acyl chains, 4-16 carbon long and substitution on the C3 carbon of the N-linked acyl chain, usually with a hydroxy or oxo group, and they are known to modulate biofilm formation, motility, antibiotic production, and the exchange of genetic material[START_REF] Chhabra | Autoregulation of carbapenem biosynthesis in Erwinia carotovora by analogues of N-(3oxohexanoyl)-L-homoserine lactone[END_REF][START_REF] Singh | Seaweed-microbial interactions: key functions of seaweed-associated bacteria[END_REF]. Of the '31' epibacterial strains isolated from H. floresii, more than 50% of the strains were detected as QS bacteria.[START_REF] Girard | Characterization of N-Acyl homoserine lactones in Vibrio tasmaniensis LGP32 by a biosensorbased UHPLC-HRMS/MS method[END_REF] did not detect any QS signals produced by V.

owensii using C. violaceum CV026, Pseudomonas putida F117, and E.coli MT102, but at the same time,

[START_REF] Torres | AHLlactonase expression in three marine emerging pathogenic Vibrio spp. reduces virulence and mortality in brine shrimp (Artemia salina) and Manila clam (Venerupis philippinarum)[END_REF]

found AHLs using C. violaceum CV026. Though we could not detect the QS activity of V. owensii using C. violaceum CV026, we confirmed its presence using E. coli pSB406. The detection of QS activity in V. owensii from the IMTA cultivated H. floresii may open a new way of disease control in the aquaculture system because V. owensii is the most important aquaculture-related pathogen, particularly in shrimp ponds, causing AHPND (Acute HepatoPancreatic Necrosis Disease)

[START_REF] Liu | A Vibrio owensii strain as the causative agent of AHPND in cultured shrimp, Litopenaeus vannamei[END_REF][START_REF] Torres | AHLlactonase expression in three marine emerging pathogenic Vibrio spp. reduces virulence and mortality in brine shrimp (Artemia salina) and Manila clam (Venerupis philippinarum)[END_REF]

).

Table 1

 1 Concentration of the H. floresii, DIP extracts with different solvents and WCM extracts with n-

	hexane
	Extracts	Mean levels of
		secondary metabolites
	DIP Extracts	(ng cm -2 )
	n-hexane	600
	Dichloromethane	ND
	Methanol	76.9
	Dichloromethane:methanol (1:1 v/v)	36.9
	WCM Extracts -n-hexane	(µg mg -1 )
	10s	29.38
	20s	19.00
	30s	10.25
	40s	7.00
	50s	3.25

Table 2

 2

, which include Vibrio owensii (B3IM), Alteromonas sp. (B7CC), Pseudoalteromonas arabiensis (B4BC), Ruegeria sp. (B4CC), Tenacibaculum sp.(B9BC), Maribacter sp. (B9IM) and Aquimarina sp. (B9.1CC). All the strains belonged to Alphaproteobacteria, Gammaproteobacteria, and Bacteroides. Except for the phylotypes Maribacter sp.

(97%) and Alteromonas sp. (99%) all other strains showed 100% similarity to their closest neighbor.

Table 2

 2 Taxonomic affiliation of the isolated phylotypes.

	Name	Group	Closest matching strain in	Similarity	Designation
			Genbank
	B4CC	Alphaproteobacteria Ruegeria sp.	100%	Ruegeria sp.
	B3IM		Vibrio owensii	100%	Vibrio owensii
	B7CC	Gammaproteobacteria	Alteromonas sp	99%	Alteromonas sp.
	B4BC		Pseudoalteromonas	100 %	Pseudoalteromonas arabiensis
			arabiensis
	B9BC		Tenacibaculum sp.	100%	Tenacibaculum sp.
	B9IM	Bacteroidetes	Maribacter sp.	97%	Maribacter sp.
	B9.1CC		Aquimarina sp.	100%	Aquimarina sp.

Table 3

 3 List of common metabolites in DIP and WCM n-hexane extracts proposed by the MMCD mass bank

	Group	S.No

Table 1 .

 1 Codes assigned, based on the bioactivity (functional descriptors) and/or chemical class (chemical descriptors), to the metabolites (DIP and WCM) proposed by the massbank.

	Code	Biological activity/Biochemical class
	ATB	Antibiotics
	ATC	Anti-cancerous
	ATF	Antifungal
	ATI	Anti-inflammatory
	ATO	Anti-oxidant
	ATP	Antiproliferative
	ATV	Antiviral
	INH	Inhibitors

Table 2 .

 2 Codes assigned to the '41' CMN metabolites, names of the putatives, raw/chemical formula,

	confidence level (∆), and actual mass as proposed by the database MMCD, experimental m/z features
	(Exp m/z), retention time (RT in minutes) and maximum intensity resulted from the XCMS Online
	platform, the corresponding Bioactivity/Biochemical classes of putatives (BIO) and their LOGP3 octanol
	water partition coefficients (XLogP3-AA). ND -Not Detected.

Defence on surface of Rhodophyta Halymenia floresii: metabolomic profile and interactions with its surface-associated bacteria Shareen Arockiasamy 2020 Defence on surface of Rhodophyta Halymenia floresii: metabolomic profile and interactions with its surface-associated bacteria Shareen Arockiasamy 2020 Defence on surface of Rhodophyta Halymenia floresii: metabolomic profile and interactions with its surface-associated bacteria Shareen Arockiasamy 2020

Table 1

 1

	ordering.

  separation afforded by LC-MS(Tohge and Fernie 2009) and for which we used two different extracts. The untargeted metabolic protocol for H. floresii is described in Scheme I. Initially we used n-hexane, a nonpolar lipophilic solvent, to extract the surface-associated (DIP extract) and whole-cell metabolites (WCM extract). Interestingly, n-hexane was observed to extract both the hydrophilic and lipophilic compounds in accordance with[START_REF] Paix | A multi-omics analysis suggests links between the differentiated surface metabolome and epiphytic microbiota along the thallus of a mediterranean seaweed holobiont[END_REF] [START_REF] Paix | A multi-omics analysis suggests links between the differentiated surface metabolome and epiphytic microbiota along the thallus of a mediterranean seaweed holobiont[END_REF]). These authors also observed that a polar solvent, methanol, was useful to extract the lipophilic compounds rather than hydrophilic ones. Anyway, only 10% of the total classes in the surface-extracts (DIP) of H. floresii were reported. Indeed, surface molecules, any environmental detriments. As the putatives predicted in this study were based on MS1 future studies should be done to confirm them by MS-MS fragmentation (MS 2 ) using any authentic standards available.The H. floresii putatives predicted in this study essentially fell under different biochemical classes with characteristic bioactivity based on their chemical structures. Up to '41' putatives are present in the H.

	A challenging issue for marine organisms metabolomic analysis is the high number of compounds
	with unknown structures (Goulitquer et al. 2012). By means of HCA, '41' putatives of H. floresii were
	clustered into '8' clusters (I-VIII) based on their corresponding structural descriptors i.e. structural
	(dis)similarity (Figure 4). H. floresii untargeted metabolomic approach revealed a small number of putatives
	that were structurally similar to different bioactive compounds proposed by the mass bank. The 'chemical
	similarity principle' states that molecules with similar structures may likely exhibit similar biological
	properties (Kouskoumvekaki and Panagiotou 2010). Based on the metabolite-likeness and biological
	relevance filters, chemical compounds from virtual screens of large pharmaceutical libraries that are similar

when identified as bioactive molecules may play a significant role in the defence mechanisms of H. floresii, since some of these microorganisms first damage multicellular organisms through primary surface adhesion.

With this approach, a fingerprint of '41' H. floresii putatives was proposed from the comparison of DIP and WCM extracts. Confidence in the bioactive putatives identified relies on a robust experimental protocol based on duplicate experiments performed at various extraction durations (10s -60s). Although to analyse H. floresii crude metabolic extracts we used LC-MS as the sole analytical tool, the increased number of samples ('26' samples) resulted in a large data set to process and analyse.

to endogenous metabolites stand more chances for being successful drug candidates

[START_REF] Gupta | Comparing the chemical spaces of metabolites and available chemicals: Models of metabolite-likeness[END_REF][START_REF] Kong | Do Biologically Relevant Compounds Have More Chance To Be Drugs?[END_REF][START_REF] Kouskoumvekaki | Navigating the human metabolome for biomarker identification and design of pharmaceutical molecules[END_REF]

. Thus, H. floresii may become a potential candidate with several bioactive compounds which may be inducible or controlled culture conditions without

  TNT, an explosive, often present in seawater as a pollutant; Cruz-Uribe et al. (2007) (Cruz-Uribe et al. 2007) found that the red algae,

  since they mimic the chemical structures of bacterial auto Inducers. Indeed, quorum-sensing allows bacterial biofilms, the sessile "way of life" of bacteria, to regulate gene expression and toxin production. By interfering with the communication within the bacterial populations, the QSI compounds provide to eukaryotes potent antibacterial defences. If one reminds us that biofilms are mainly responsible for fouling on marine organisms, H. floresii appears as a potent source for such compounds. Since the drugability of secondary metabolites drove this study, it is worth noting that bacterial multi-resistance became a global public health challenge. Due to gene transfers among species and strong efflux pumps, "conventional" antibiotics become less and less efficient. This promoted the search for alternative pathways and, in such a context, "messing"

Table 1 :

 1 Fig. 1 p < 0.00036) when compared to control treatments without bacterial inoculation. Eight additional isolates (Pseudoalteromonas

arabiensis; P. mariniglutinosa; Tateyamaria omphalii; Ruegeria sp.; Alteromonas sp. (B7CC and B12CC); Epibacterium sp.; Alteromonadaceae bacterium) had the same effect, but were not pathogenic (according to Bonferroni test; Table

1;

  . As we previously observed how the surface-extracts of H. floresii interfere with the QS activity of V. owensii (see Chapter I), we believe that H. floresii can be a good candidate for disease control in aquaculture. Pliego-Cortés et al. (2017) considered H. floresii as a good candidate to recycle inorganic nutrients in a land-based IMTA by reducing the environmental impact of coastal ecosystems through responsible aquaculture practices. As a potential carrageenophyte, H. floresii could possibly play a significant role among green approaches to improve aquaculture productivity by means of an effective disease mitigation strategy.

Defence on surface of Rhodophyta Halymenia floresii: metabolomic profile and interactions with its surface-associated bacteria Shareen Arockiasamy 2020

2.8 ReferencesThe references of this article are available at the end of the manuscript('References', page no. 206)

-----------

-----+ -----

General DiscussionDefence on surface of Rhodophyta Halymenia floresii: metabolomic profile and interactions with its surface-associated bacteria Shareen Arockiasamy 2020

8.5 PCR Amplification -16SDefence on surface of Rhodophyta Halymenia floresii: metabolomic profile and interactions with its surface-associated bacteria Shareen Arockiasamy 2020

ReferencesDefence on surface of Rhodophyta Halymenia floresii: metabolomic profile and interactions with its surface-associated bacteria Shareen Arockiasamy 2020

Acknowledgements

Wow... Expressing gratitude is such a greatest human emotion and it's very exciting to start acknowledging the BEings who have been and/or being with me throughout my PhD. This period of '3' years in my life gave me a lot of people and lots and lots of wonderful experiences. And I am very grateful for it. Firstly, I sincerely thank Prof. Gérald Culioli, Associate Professor of the Laboratory of Matériaux Polymères Interfaces Environnement Marin (MAPIEM), Université de Toulon (La Garde) and Dr. Leila Tirichine, Research Director of the Epigénomique des microalgues et interactions avec l'environment, Université de Nantes, for being rapporteurs and examining the work. I also thank other members of jury Prof. Claire Gachon, Professor, Museum National d'Histoire Naturelle and Prof. Gwenaëlle Le Blay, Professor, Université Bretagne Occidentale (UBO) for equally participating in examining my work. I profoundly thank my doctoral school, Ecole Doctorale Sciences de la Mer et du Littoral (EDSML) for constantly supporting throughout my thesis and granting me scholarship for my international mobility. I also thank ECOS-Nord CONACYT for their financial support towards my research stay in Mexico. I sincerely thank GlobalSeaweedSTAR for their financial help towards attending the conference of ISS, 2019, Korea. Well, my Directors of the thesis... Words are not enough to express my deep gratitude to Prof. Nathalie Bourgougnon, firstly for the offering me this wonderful opportunity to pursue my PhD under her wonderful

Acknowledgements

Financial support from ECOS-Nord CONACYT for the collaboration project M14A03 and PN-CONACYT 2015-01-118 is gratefully acknowledged.

The references of this article are available at the end of the manuscript ('References', page no. 206)

Acknowledgements

The authors thank L. Taupin for her skillful technical assistance for chromatographic analyses. IBT. V.

Avila for culturing the Halymenia floresii material used in this experiment. Financial support from ECOS-Nord CONACYT for the collaboration project M14A03 and PN-CONACYT 2015-01-118 are also acknowledged.

The references of this article are available at the end of the manuscript ('References', page no. 206)

Chapter I

Defence on Surface: Macroalgae and their associated-microbiome

Shareen A Abdul Malik, Gilles Bedoux, Jose Q. Garcia Maldonado, Yolanda Freile-Pelegrín, Daniel Robledo, Nathalie Bourgougnon Published as a Book Chapter to "Seaweeds Around the World: State of Art and Perspectives. Advances Botanical Research.

Shareen at al.

January, 2020

Defence on Surface: Macroalgae and their surface-associated microbiome Shareen A Abdul Malik 1 , Gilles Bedoux 1 , Jose Q. Garcia Maldonado 2 , Yolanda Freile-Pelegrín 2 , Daniel Robledo 2 , Nathalie Bourgougnon 1 1 Laboratoire de Biotechnologie et Chimie Marines, Université Bretagne Sud, France 2 CINVESTAV Marine Resources Department, Mexico

Abstract

Macroalgae surfaces are considered as a potentially suitable substratum present underwater for the dynamic epibiotic microbiome. They secrete various organic substances as nutrients for the multiplication of bacteria and the formation of microbial biofilms. The microbial biofilm itself acts as a biological defence mechanism to prevent the establishment of pathogenic microbes as well as to protect the surface of the seaweed from the macrofoulers. In most cases, the presence and diversity of these organisms are host-specific. The macroalgal host and their associated microbiont are often referred to as a 'Holobiont'.

The surface-associated microbiome of macroalgae plays a crucial role in the growth, morphogenesis and defence in the normal favourable environmental conditions of the macroalgae (Symbiotism). Whereas, when there is a change i.e. reversed, unfavourable conditions, the microbiome reverses its symbiotic nature and affects growth and degrades the macroalgae by causing different diseases in different species (Pathogenism). In parallel, marine macroalgae employ different defence mechanisms, a physical defence such as continuous shedding of the outer layer of cells, and the mucilaginous covering and chemical defence such as the production of secondary metabolites.

The following chapter focuses on the macroalgal surface-associated microbiome and its defence system with a certain number of relevant examples from related literature on this subject.

Keywords: Surface-associated microbiome; microbionts; holobionts; symbiotism; secondary metabolites; microbial colonization; biofilm;

Chapter II

Screening of surface-associated bacteria from the Mexican red alga

Halymenia floresii for Quorum Sensing activity

Isolation and identification of surface-associated bacteria

Before molecular identification of the isolated strains, these were preliminarily identified and grouped based on their motility, Gram staining, and the biochemical characteristics determined by APIZYM (Table 1).

i. Morphological and biochemical characterization

Except for the strain B14CC, all strains were observed to be motile in nature (either swimming or twitching, see Table 1). The strains B9BC, B9IM, B9CC, and B9.1CC did not show any motility in agarstabbing, and it is worth noting that they glide when observed by hanging-drop. Using our methodology, all the isolated bacteria were identified as Gram negative, and the B10CC strain was the only Gram-positive bacteria.

Biochemical characterization of the isolates performed using APIZYM (Biomerieux) consistently tested '20' different characteristics. Among the '31' isolated strains, almost all of the strains reacted negatively to the following enzymatic evaluations: β-glucuronidase; α-mannosidase; α-fucosidase.

Similarly, all the isolates showed a negative reaction to α-glucosidase, except for B4CC. An intermediate reaction was observed in B9.1CC towards the β-glucosidase test and in B10CC towards the N-acetyl-βglucosaminidase test, whereas the rest of the isolates showed a negative reaction. Only B6BC reacted positively for α-galactosidase and β-galactosidase, whereas B9.1CC and B10CC were the only strains to react positively to β-galactosidase. Only B16CC showed a positive reaction towards the Lipase (C18) test.

All the isolates showed a positive reaction to Naphthol-AS-BI-phosphohydrolase, though their intensity varies depending on the phylotypes. ---- were pooled together, evaporated under pressure and quantitatively assessed for the presence of surfaceassociated metabolites using LC-MS. Henceforth, the surface metabolites will be referred to as DIP.

ii. Whole-Cell Metabolites (WCM) extraction

After DIP extraction protocols described above, the H. floresii samples were rinsed with sterile seawater and individually freeze-dried. The lyophilized samples were homogenized and extracted with nhexane. Briefly, 400 mg of the lyophilized sample was exhaustively extracted by 5sequential extractions (i.e. initially with 8 mL of solvent for two hours, followed by 4 mL x 3 for every two hours and finally again with 8 mL up to 24 h under agitation) (de Nys et al. 1996). The extract was dried under vacuum and suspended again in the respective solvents, filtered through a 0.45 µm PTFE filter and analyzed by LC-MS.

Henceforth, the whole-cell metabolites will be referred to as WCM. The workflow of both the extractions is illustrated in Fig 1.

Identification of homoserine lactones (HSLs) from the 'significant pathogen'

The presence of AHLs was investigated in the acidified ethyl acetate extracts of the significant pathogen, identified here as V. owensii (Accession No. MT176134) after 24h of culture. The QS activity of V. owensii was previously detected by the bioluminescent reporter strain, Escherichia coli pSB406 (for more details, see Chapter II and III). E. coli pSB406 is a lux-based biosensor containing a fusion of rhlRIP:

:luxCDABE on a pUC18 plasmid backbone, which enables the detection of a wide range of AHLs differing in the length of their acyl chain [START_REF] Winson | Construction and analysis of luxCDABE -based plasmid sensors for investigating N -acyl homoserine lactone-mediated quorum sensing[END_REF]. LC/MS confirmed the presence of N-butyryl-Lhomoserine lactone (C4-HSL) in the culture medium of V. owensii grown in MB liquid media. The presence of AHL in the sample was identified according to the retention times of their corresponding standards. The chromatograms generated by the analytical instrument illustrates the separation of the standard and the sample of C4-HSL at a retention time of 6.2 -7.0 minutes (Fig. 3). This AHL was unambiguously identified by a comparison of its mass spectra with those of pure standards. In Fig. 4, the MS spectrum of the C4-HSL in the standard and the sample are shown as fragments at 102.05 (lactone ring); 172.09 (M+H) + and at 194.08 (M+Na) + .

The Extracted Ion Chromatogram (EIC) of the AHL extract of V. owensii (significant pathogen), shown in Fig. 2 was previously treated in the XCMS Online Platform (for details see Chapter V). Besides the chemical structure of C4-HSL was depicted in Fig. 5 for a better understanding of its fragments fundamentally based on the lactone moiety. Shareen at al.

Chapter V

Submitted on 21 September 2020

Defence on surface of Rhodophyta Halymenia floresii: metabolomic profile and interactions with its surface-associated bacteria Shareen Arockiasamy 2020

STEP I

The raw data files were exported as ".mzXML" format from the Agilent LC/MS instrument used for the analyses. Initially, a free user profile account was created on the XCMS Online home page. The LC/MS raw data in .mzXML format were uploaded into the platform. The parameters to pre-process were set in accordance with the analytical tool used. After the (pre) processing of the data, the results were viewed and downloaded as an 'excel file' (Fig. 25).

Fig. 25 Pre-processing of the raw data (.mzXML) in XCMS Online platform

STEP II

The processing of the data was performed in Madison Metabolomics Database Consortium (MMCD). The pre-processed XCMS data were downloaded from the platform and 'flattext' was created with the retention time (rtmax), mass/charge ratio (mzmax), and relative intensity (maxint). The metabolite search was based on 'mass' in a 'batch mode' for mixture analysis. The results were downloaded as an 'excel file' (Fig. 26).

STEP IV

The downstream statistical analysis was performed to validate and interpret the metabolomic data. This was performed by using the command-line program in 'R Studio' and multivariate analysis, Hierarchical Clustering Analysis (HCA), was performed to group the treatments (SMILES and chemical groups identified) that are close to each other in clusters i.e. similar structural descriptors (Fig. 28).

LC-MS analyses

Once the LC/MS data were pre-processed by using the XCMS Online platform, results were downloaded from the website. The downloaded files were Extracted Ion Chromatogram (EIC) and Spectrum (spec), which contain EICs of every single peak detected and their spectrum (m/z versus intensity), respectively. Moreover, the 'RTcor' file showed the corrected retention time between the samples analysed, from both DIP and WCM as well as among the replicates and 'Total Ion Chromatogram (TIC)' file showed the chromatogram of the samples after correcting the retention time. Apart from the above-mentioned files, the platform also produces an excel file 'XCMS-Report-Annotated-SingleClass', which includes the following details: m/z (median, minimum and maximum) of the group; retention time (rt) (median; minimum and maximum) in the group; the number of peaks (npeaks); maximum intensity (maxint); isotopes and adducts.

Identifying the H. floresii metabolic diversity

The 'Mass Batch Search Results' were downloaded from the MMCD, after being processed. This resulted in '110' and '416' compounds for the surface and whole-cell extracts, respectively. After carefully analysing data by overlaying the compounds which were observed in both extracts and removing duplicated compounds, a set of '41' compounds was retained hereafter labelled as the CMN pool. Most of these compounds exhibit various biological activities as compared with previous studies. Nevertheless, the identification of these macroalgal metabolites is a bottleneck in metabolomic studies, because of the vast metabolite diversity (i.e. 100,000 known and 200,000 unknown metabolites) reported so far [START_REF] Goulitquer | Mass Spectrometry-Based Metabolomics to Elucidate Functions in Marine Organisms and Ecosystems[END_REF][START_REF] Gupta | Seaweed metabolomics: A new facet of functional genomics[END_REF]. For instance, [START_REF] Paix | Temporal covariation of epibacterial community and surface metabolome in the Mediterranean seaweed holobiont Taonia atomaria[END_REF] [START_REF] Paix | Temporal covariation of epibacterial community and surface metabolome in the Mediterranean seaweed holobiont Taonia atomaria[END_REF]) identified 3065 m/z features composed of 429 variables in the surface extracts of the brown alga, Taonia atomaria however, its raw data were treated with the XCMS package under R environment whereas we used XCMS Online. Such a metabolomic study on marine macroalgae is highly dependent on the availability and quality data and the use of a wide variety of electronic resources.

is also being closely related to the QS and thus there is a possibility that the putative opportunistic pathogenicity of these strains may also be linked to it.

Analysis of positive relation-symbiotism

The macroalgal-associated microbial communities have evolved through a highly competitive environment due to nutrient and space limitation on their host surface, subsequently producing allelochemicals capable of preventing secondary colonisation [START_REF] Egan | Pseudoalteromonas ulvae sp. nov., a bacterium with antifouling activities isolated from the surface of a marine alga[END_REF][START_REF] Egan | Unlocking the diversity and biotechnological potential of marine surface associated microbial communities[END_REF]Wiese et al. 2009;[START_REF] Tujula | Variability and abundance of the epiphytic bacterial community associated with a green marine Ulvacean alga[END_REF][START_REF] Uzair | Isolation, purification, structural elucidation and antimicrobial activities of kocumarin, a novel antibiotic isolated from actinobacterium Kocuria marina CMG S2 associated with the brown seaweed Pelvetia canaliculata[END_REF]. Thus, as we hypothesized, the microbial biofilms act as a biological defence by protecting the surface from macrofoulers. The epibacterial community of H. floresii identified in this study was obviously from the healthy surfaces of the algae, rather than those collected from stressful environments (Chapter II). Hence, the epibacteria identified were symbiotically associated with the host, H. floresii. According to the symbiosis theories by Lynn Margulis (Cavalier-Smith 1992), we can consider H. floresii and its epibacterial community as a functional entity that referred to symbiotic associations that last for a significant part of an organism's lifetime (Cavalier-Smith 1992). Moreover, when we trace back to the four-step sequential process of biofouling, after an initial 'conditioning' (step I) of the surfaces by organic compounds, this resulted in the subsequent development of bacterial biofilm (step II) [START_REF] Davis | Epibiosis of marine algae and benthic invertebrates: natural products chemistry and other mechanisms inhibiting settlement and overgrowth[END_REF][START_REF] Wahl | Marine epibiosis. I. Fouling and antifouling: some basic aspects[END_REF][START_REF] Parsek | Sociomicrobiology: the connections between quorum sensing and biofilms[END_REF]. With our study, we shall prove that the bacterial biofilm, though regarded as one of the steps towards biofouling, may also acts as a defence mechanism itself by protecting the host surface by symbiotic association.

Putative opportunistic pathogenicity

The pathogenic shift of the macroalgal associated microbiota can be caused by environmental stresses resulting in a 'Holobiont break-up' [START_REF] Ying | The effects of marine farm-scale sequentially integrated multi-trophic aquaculture systems on microbial community composition, prevalence of sulfonamide-resistant bacteria and sulfonamide resistance gene sul1[END_REF]. In order to survive under such circumstances, marine macroalgae are able to stimulate, inhibit, or compromise QS signals in bacteria [START_REF] Saurav | Quorum Sensing Inhibitors from the Sea Discovered Using Bacterial N-acyl-homoserine Lactone-Based Biosensors[END_REF].

The first QS inhibitory compound, Furanones which mimic the bacterial AHL signals, was isolated from the red macroalga Delisea pulchra [START_REF] Dahms | Antifouling Compounds from Marine Macroalgae[END_REF][START_REF] Saurav | Quorum Sensing Inhibitors from the Sea Discovered Using Bacterial N-acyl-homoserine Lactone-Based Biosensors[END_REF]. Thus by 'eavesdropping' the bacterial talk [START_REF] Joint | Bacterial conversations: talking, listening and eavesdropping. A NERC Discussion Meeting held at the Royal Society on 7 December 2005[END_REF], the macroalgae produce the antagonistic signals which

interfere the bacterial communication i.e. Quorum Sensing Interference and control the pathogenic disease outbreak. QSI may be an effective method for inhibiting the virulence of pathogens, further reducing disease in aquaculture systems, and increasing productivity [START_REF] Zhao | Mechanisms of quorum sensing and strategies for quorum sensing disruption in aquaculture pathogens[END_REF].

Mass mortalities of a diverse range of habitat-forming organisms like seagrasses, corals, and macroalgae have been associated with pathogen-induced disease events [START_REF] Littler | Impact of CLOD pathogen on pacific coral reefs[END_REF][START_REF] Rosenberg | Microbial diseases of corals and global warming[END_REF][START_REF] Case | Temperature induced bacterial virulence and bleaching disease in a chemically defended marine macroalga[END_REF]. Obviously, these pathogens are opportunistic in nature by forming an essential part of a 'holobiont' [START_REF] Case | Temperature induced bacterial virulence and bleaching disease in a chemically defended marine macroalga[END_REF][START_REF] Saha | Microbial "gardening" by a seaweed holobiont: Surface metabolites attract protective and deter pathogenic epibacterial settlement[END_REF]. With a large diversity of