Juliana Ivanisevic

Pierre Sasal

Isabelle Bonnard

Cédric Bertrand

Nicolas Inguimbert

Valeriano Parravicini

Suzanne Mills

Ricardo Beldade

Joaquim Claudet

Bruno Delesalle

Maggie Nugues

René Galzin

Fabien Modat

Ophélie Ladrière

Guillaume Mitta

Rodney Bray

Scott C Cutmore

Thomas H Cribb

Véronique Special

Berteaux

Nathalie Toulou

Elina Burns

Yannick Chancerelle

Gilles Siu

Lauric Thiault

Isis Guibert

Catherine Dubois

Noémie Aubin

Antoine Puisay

Charlotte Exposito

Catherine Ninane

Christina Skinner

Fanny Musson

Laura Gajdzik

Flora Artzner

Jérémie Carlot

Vanessa Andreu

Anaïs Amiot

Sana Romhdan

Emilie Boissin

Carole Blay

Gaëlle Queré

Ornella Weideli

Valentina Neglia

Erwan Delrieu-Trottin

Jeanine Almany

Johann Mourier

Océane Salles

Caroline Dubé

Mélodie Dubois

Kevin Magré

Agnès Bardon-Bardet

Alexandre Tayale

Emmanuelle Aliotti

Vincent Mehn

Antoine Herné Special

Sophie Escaich

Maururu

Odette Tani

Miriam Reverter

Reverter Miriam

Bontemps-Tapissier Nathalie

Lecchini David

Banaigs Bernard

Sasal Pierre

Gill monogenean communities (Platyhelminthes, Monogenea, Dactylogyridae) of butterflyfishes from tropical Indo-west Pacific Islands

Keywords: monogenean, dactylogyridae, butterflyfish, parasite community, specificity, hostparasite interactions biogeography, dactylogyrids, butterflyfish, host specificity, turnover, nestednees chemical mediation, fish mucus, interspecific communication, mucus microbiome, mucus molecules, mucus bioactivities fish gill mucus, microbiome, butterflyfishes, coral reef fishes. Root.p__Spirochaetes.c__Spirochae Root.p__Fusobacteria.c__Fusobacte Root.p__Proteobacteria.c__Gamma Root.p__Spirochaetes.c__Spirochae

Perpignan and Moorea (French Polynesia

General introduction

Ecosystems are dynamic systems composed by the biological community and the abiotic environment. Organisms from the same ecological communities interact directly and indirectly forming the basis for many ecosystem properties and processes such as nutrient cycling and food webs [START_REF] Agrawal | Filling key gaps in population and community ecology[END_REF]). Organisms' interactions play an important role in the maintenance of biodiversity, mediation of ecosystem responses to global changes and ecosystem stability and resilience [START_REF] Bascompte | Asymmetric coevolutionary networks facilitate biodiversity maintenance[END_REF][START_REF] Ives | Stability and diversity of ecosystems[END_REF]Suttle et al. 2007;[START_REF] Tylianakis | Global change and species interactions in terrestrial ecosystems[END_REF].

At the coarsest level, ecological interactions can be defined as intra-specific interactions, when they occur between individuals of the same species, or inter-specific interactions, occurring between different species (Fig. 1). However, since these interactions occur within ecological communities they can be affected and indirectly influenced by other species and their interactions (Land et al. 2015). Some of the most studied ecological interactions include competition, predation, symbiosis, commensalism and parasitism.

Parasitism

Parasitism is a non-mutual relationship between species, where one species, the parasite, lives at the expense of the other, the host, causing deleterious effects [START_REF] Combes | Parasitism: the ecology and evolution of intimate interactions[END_REF].

Parasites are ubiquitous, occurring in all food webs at all trophic levels and parasitism is considered to be the most common lifestyle on earth [START_REF] Price | Evolutionary biology of parasites[END_REF]Lafferty et al. 2008a).

Parasites are often classified depending their life cycle and their habitat within the hosts. A direct parasite has only one host, while an indirect parasite has multiple hosts, with one or more intermediate hosts and a definitive host. Parasites that live on the outer surfaces of the host like skin or gills are called ectoparasites, while those living inside the host are called endoparasites. There are two types of endoparasites, intercellular parasites like some worms, which inhabit spaces in the host's body and intracellular parasites such as protozoa, bacteria or viruses, that inhabit host cells. Transmission of ectoparasites, such as in monogenean worms, happens through direct contact between hosts. Endoparasites can infect their hosts either by penetrating its external surface or through parasite ingestion.

In host-parasite interactions, host populations develop defence mechanisms to avoid parasite infections, and parasites evolve counter-adaptations to overcome host defence mechanisms, which results in a tight host-parasite co-evolution [START_REF] Klein | Seoul virus infection increases aggressive behaviour in male Norway rats[END_REF]). According to the Red Queen Hypothesis, hosts are at disadvantage during the course of co-evolution with their parasites because they tend to have longer generation times and smaller population sizes than their parasites [START_REF] Kaltz | Local adaptation in host-parasite systems[END_REF].

If the host population is homogeneous and remains constant over time, evolution is expected to favor parasites that are best adapted for this host, what is known as parasite specialisation 1 . In more rapidly changing or spatially heterogeneous host populations, parasites might find themselves exposed to a wider range of hosts, and therefore parasites tend to be generalists 2 , favouring the highest geometric mean fitness across different hosts [START_REF] Futuyma | The Evolution of Ecological Specialization[END_REF][START_REF] Rohstein | Phylogeny, specialization, and brood parasite-host coevolution: some possible pitfalls of parsimony[END_REF][START_REF] Legros | Experimental evolution of specialization by a microsporidian parasite[END_REF]. A high parasite specialisation increases host exploitation efficiency, however they are at the same time more susceptible to co-extinction since they rely strictly on one or few host species. On the other hand, generalist parasites have the advantage of lower extinction probability, but their decreased fitness in their hosts can reduce parasite establishment, adult survivorship and fecundity in novel hosts [START_REF] Gemmill | The evolutionary ecology of host-specificity: experimental studies with Strongyloides ratti[END_REF]Poulin, 2005;[START_REF] Koprivnikar | Benefits of fidelity: does host specialization impact nematode parasite life history and fecundity?[END_REF]. A recent study, has estimated the probability of fish parasites to go co-extinct on the basis of its specificity and of the host extinction vulnerability and they found that specialized parasites use few, but less vulnerable hosts, reducing their co-extinction risk [START_REF] Strona | (Temminck and Schlegel) larvae against Vibrio anguillarum: Effect of medicinal herb on larval red sea bream[END_REF].

In many cases, the parasite counter-adaptations involve direct manipulation of host behaviour to increase probability of transmission and reproductive success of the parasite [START_REF] Moore | An overview of parasite-induced behavioural alterations-and some lessons from bats[END_REF]. If a parasite has a direct life cycle, survival and reproduction can be increased through social contact among conspecifics [START_REF] Klein | Parasite manipulation of host behavior: mechanisms, ecology, and future directions[END_REF]. For example, viruses that have direct life cycles typically cause an increased aggression and physical contact in the host population [START_REF] Klein | Seoul virus infection increases aggressive behaviour in male Norway rats[END_REF]). Parasites relying on trophic transmission to complete their life cycles often induce modifications in their intermediate host's behaviour to increase their susceptibility to predation by final hosts [START_REF] Lagrue | Modification of hosts' behaviour by a parasite: field evidence for adaptative manipulation[END_REF][START_REF] Poulin | Parasite manipulation of host personality and behavioural syndromes[END_REF]. For example, the acanthocephalan Pomphorhynchus laevis inhibits attraction to conspecifics in the amphipod intermediate host Gammarus pulex in order to increase predation by fish definitive hosts [START_REF] Durieux | Parasite-induced suppression of aggregation under predation risk in a freshwater amphipod: Sociality of infected amphipods[END_REF]) (Fig. 2).

Importance of parasitism in ecosystems

Parasites play an important role in population dynamics and community structure. Their impact reducing host fitness and therefore modifying competitive and trophic interactions among species have profound effects on the abundance of different species and the interactions between them [START_REF] Hudson | Is a healthy ecosystem one that is rich in parasites?[END_REF]. Parasite species affect their hosts differently, thus they can mediate the co-existence of host species by harming some species more than others, which can contribute to the maintenance of host diversity [START_REF] Poulin | Geographical distances and the similarity among parasite communities of conspecific host populations[END_REF].

For example, the amphipods Corophium volutator and Corophium arenanium coexist in nature and share a trematode parasite (Microphallus claviformis). C. volutator is the superior competitor, but it is also more sensitive to M. claviformis, presenting higher mortalities than C. aeranium. In this case, the parasite cancels the advantage of the superior competitor, which could lead to C. aeranium extinction and promotes the coexistence of the two congeners [START_REF] Jensen | The influence of the trematode Microphallus claviformis on two congeneric intermediate host species (Corophium): infection characteristics and host survival[END_REF].

Another way in which parasites can influence community structure is by indirectly modifying the functional importance of their host species in the community. Modifications of host behaviour or phenotype can lead to modifications of the host interactions with other species [START_REF] Poulin | Geographical distances and the similarity among parasite communities of conspecific host populations[END_REF]. For example, rhizocephalan parasites modify the physiology of their crab host and infected crabs do not moult, which causes an increase of epibiotic invertebrates. Parasitized crabs become then functionally more important for the rest of the invertebrate community [START_REF] Thomas | Parasites and ecosystem engineering: what roles could they play?[END_REF].

Host modifications can also alter the host availability to other parasite species. Many parasite species interfere with the host immune system and immunosuppressed hosts can become more available to other parasite species [START_REF] Wakelin | Immunity to parasites: how animals control parasitic infections[END_REF].

Further evidence of parasite importance in shaping communities comes from biological invasion studies. Invader species tend to loose their co-evolved parasites in the process of invasion (enemy release hypothesis), increasing invaders performance over the native species [START_REF] Torchin | Introduced species and Bibliography -324 -their missing parasites[END_REF]. Parasites that survive the host translocation can in turn infect naïve native host populations that have not developped defence mechanisms against them, which can eventually lead to local host extinction [START_REF] Prenter | Roles of parasites in animal invasions[END_REF][START_REF] Hudson | Is a healthy ecosystem one that is rich in parasites?[END_REF]. For example, the native European crayfish Austropotamobius pallipes has been driven locally extinct as a result of transmission of the parasitic fungi Aphanomyces astaci from the introduced North American crayfish Pascifastacus leniusculus [START_REF] Holdich | Distribution of freshwater crayfish in the British Isles, with particular reference to crayfish plague, alien introductions and water quality[END_REF]. Invader populations harbour significantly fewer parasites than native populations, since parasites from native hosts are often host specific and unable to spread to invaders [START_REF] Dunn | Parasitism and epibiosis in native and non-native gammarids in freshwater in Ireland[END_REF].

Therefore, parasites play a major role mediating the interaction of invaders and native hosts and determining invaders success or failure [START_REF] Prenter | Roles of parasites in animal invasions[END_REF].

Finally, parasites are a valuable indicator of ecosystem health. Healthy ecosystems, that is, ecosystems that persist, maintain vigour, organisation and resilience to change, are reported to harbour a high diversity of parasites [START_REF] Hudson | Is a healthy ecosystem one that is rich in parasites?[END_REF]. Given that pollution, climate change and other stressors may have impacts on populations of organisms, examination of parasite assemblages may reflect alterations in the food web structure and function, resulting from ecological disturbances to host distributions [START_REF] Marcogliese | Parasites: Small Players with Crucial Roles in the Ecological Theater[END_REF][START_REF] Marcogliese | Parasites of the superorganism: are they indicators of ecosystem health?[END_REF]. Some studies have reported that pollution and stress are often associated with a reduction in parasite richness due to the reduction of host species. However, some ectoparasite populations like protozoans or monogeneans may increase linked to a host's compromised immune response (McKenzie et al. 1995;McKenzie, 1999). Since parasites depend on host density for their transmission and some might depend on several host species, some studies have concluded that parasites are more sensitive to environmental degradation than their hosts [START_REF] Marcogliese | Parasites of the superorganism: are they indicators of ecosystem health?[END_REF]. Recently, [START_REF] Johnson | Host and parasite diversity jointly control disease risk in complex communities[END_REF] found that host and parasite richness interacted to negatively affect the transmission of the deadliest parasite Ribeiroia ondatrae in amphibians, emphasising the role of parasite communities regulating disease risk.

Despite increasing interest in host-parasite interactions, they remain poorly understood in reef fishes [START_REF] Finley | Impact of ectoparasites on the demography of a small reef fish[END_REF]. Understanding the relationship between environmental variables, parasitism and disease in reef fishes is highly important to understand the complexity and resilience of coral reef ecosystems [START_REF] Sikkel | Habitat and species differences in prevalence and intensity of Neobenedenia melleni (Monogenea: Capsalidae) on sympatric Caribbean surgeonfishes (Acanthuridae)[END_REF]. Lafferty et al. (2008b) showed that fish parasite richness was higher in a well-conserved atoll than in a heavily fished island, indicating the effect of some ecosystem disturbances on the parasite communities of coral reef fish. A recent study found a dramatically higher rate of infection of dermal parasites among coral reef fishes in Curaçao compared to other Carbibbean sites like Belize and Mexico. Although responsible factors of this phenomenon remain unknown it is likely that the lack of predators in Curaçao contributes to a higher coral reef fish population and thus parasite abundance (Bernal et al. 2016). The study of host-General introduction -25 -parasite interactions in cultured fish is also of crucial importance, since they are the often more sensible to parasite outbreaks [START_REF] Leung | More rapid and severe disease outbreaks for aquaculture at the tropics: implications for food security[END_REF].

Parasitism in aquaculture

The emergence of infectious diseases in aquaculture is a major problem that causes severe financial losses through decreased production of farmed species or increased production costs, and important environmental consequences. For example, sea lice infections cause £15-30 million annual losses to Scottish salmon farms (Pike and Wadsworth, 2000).

Owing to the cost effective nature of aquaculture, fish may be cultured in densities more than 1000 times higher than under natural conditions. High fish densities increase the probability of contact between infected fish and susceptible hosts, facilitating the spread of pathogens (Pulkkinen et al. 2009). Besides, aquaculture is often linked to high stress levels in cultured animals and environment degradation, such as low water quality, low oxygen and high nitrogen levels, which reduce fish fitness and enhances infection by opportunistic pathogens [START_REF] Pickering | Stress responses of farmed fish[END_REF][START_REF] Martin | The effects of anthropogenic global changes on immune functions and disease resistance[END_REF].

Another major problem of parasitic infections in aquaculture is the increase of virulence [START_REF] Kennedy | Potential drivers of virulence evolution in aquaculture[END_REF]. Virulence is defined as the deleterious health effects of a pathogen infection in a host. Several studies have shown that aquaculture can create conditions that favour the development of highly virulent pathogens [START_REF] Murray | A framework for understanding the potential for emerging diseases in aquaculture[END_REF]Pulkkinen et al. 2009). Parasites normally favour to exploit their hosts prudently to prolong infection and avoid killing the host, but they also need to exploit host resources to optimise reproduction and transmission. Therefore, there is a trade off between prudent host exploitation and rapid parasite reproduction (Figure 3). In aquaculture systems, the high number of hosts does not limit the fitness gain of increased infectiousness, and thus can lead to evolutionary increases in parasite virulence [START_REF] Kennedy | Potential drivers of virulence evolution in aquaculture[END_REF]. Evolution of virulence theory predicts that virulence levels depend on host lifespans, so shortening the effective host lifespan, for example by compressing the rearing cycle duration, can also favour evolution of increased pathogen virulence [START_REF] Nidelet | Effects of shortened host life span on the evolution of parasite life history and virulence in a microbial host-parasite system[END_REF]. Selective breeding and inbreeding in broodstock populations are common in aquaculture leading to populations with limited genetic diversity.

Host diversity can prevent parasite specialisation, however pathogen strains that specialise on low diversity populations can develop high virulence in those populations (Garamszegi, 2004; General introduction -26 - [START_REF] Kennedy | Potential drivers of virulence evolution in aquaculture[END_REF]. Finally, continuous treatment against disease can also contribute to virulent form selection [START_REF] Porco | The effect of treatment on pathogen virulence[END_REF]. Aquaculture pathogens can come from wild populations, however most of the agents of emerging diseases in aquaculture seem to come from other geographical areas or other host species via the artificial movement of live animals (Nylund and Jackobsen, 1995;[START_REF] Hedrick | Movements of pathogens with the international trade of live fish: problems and solutions[END_REF].

Warmer climate promotes higher pathogen proliferation and transmission rate, therefore mortality due to infectious diseases is likely to be higher at lower latitudes [START_REF] Robar | Tropics, trophics and taxonomy: the determinants of parasiteassociated host mortality[END_REF].

The combination of overcrowding with warm temperatures provide the perfect conditions for disease outbreaks, thus aquaculture at low latitudes is exposed to more severe mortality and rapid progression of diseases [START_REF] Mennerat | Intensive farm-ing: evolutionary implications for parasites and pathogens[END_REF][START_REF] Leung | More rapid and severe disease outbreaks for aquaculture at the tropics: implications for food security[END_REF]. This is of particular significance, since lower latitudes are also the most vulnerable towards climate change.

Understanding patterns of parasite distribution along geographical gradients might provide insights in the parasite responses to different climatic conditions and the risk of parasite outbreaks.

Distribution of parasite communities

Parasite community analysis often uses several metric indices such as prevalence, mean intensity, and richness that allow quantification of the infections. Prevalence is the proportion of infected hosts among all the hosts examined. Intensity is the mean number of parasites found within infected hosts. Parasite richness is the number of parasite species that are found in a given host species [START_REF] Morand | macro-)Evolutionary ecology of parasite diversity: From determinants of parasites species richness to host diversification[END_REF].

Animal parasite communities are highly dynamic and complex and they are generally influenced by host physiology, ecology and phylogeny [START_REF] Muñoz | Endoparasite communities of five fish species (Labridae: Cheilininae) from Lizard Island: how important is the ecology and phylogeny of the hosts?[END_REF]. Several studies have shown that larger hosts offer a larger number of habitats for parasite colonisation and thus, they may be expected to harbour richer parasite communities [START_REF] Sasal | Determinants of parasite species richness in mediterranean marine fishes[END_REF][START_REF] Sasal | Comparative analysis: a tool for studying monogenean ecology and evolution[END_REF]. Host diet seems to play an important role in parasite richness, especially in parasite with complex life cycle since hosts with broader diet ranges could acquire more trophically transmitted parasites 3 than those with narrow, specialised diets (Simkova et al. 2001;[START_REF] Cirtwill | Are parasite richness and abundance linked to prey species richness and individual feeding preferences in fish hosts?[END_REF]. A recent study has shown that different foraging habits of animals can also expose them to encounter different ectoparasite species and thus be related to parasite richness and abundance [START_REF] Luguterah | Effect of dietary guild (frugivory and insectivory) and other host characteristics on ectoparasite abundance (mite and nycteribiid) of chiropterans[END_REF]. Similarly, hosts with larger depth ranges or geographic ranges could have greater chances of encountering more parasite species, and therefore, present richer parasite faunas (Simkova et al. 2001;[START_REF] Luque | Parasite biodiversity and its determinants in coastal marine teleost fishes of Brazil[END_REF][START_REF] Garrido-Olivera | The influence of host ecology and biogeography on the helminth species richness of freshwater fishes in Mexico[END_REF]). Although results from different studies do not fully agree, host phylogeny could play a role in parasite diversity and richness [START_REF] Morand | Endoparasite species richness of New Caledonian butterfly fishes: host density and diet matter[END_REF]Poulin et al. 2011;Alarcos and Timi, 2011). Inter-specific relationships within a host species (e.g. competition between parasite species) have been less studied, but are also potentially important in host-parasite relationships and structure of parasite communities [START_REF] Hughes | Let your enemy do the work: within-host interactions between two fungal parasites of leaf-cutting ants[END_REF]. [START_REF] Rohde | Latitudinal gradients in species diversity: the search for the primary cause[END_REF] and [START_REF] Rohde | Aspects of the ecology of metazoan ectoparasites of marine fishes[END_REF] observed that marine fish from tropical latitudes typically harbour richer ectoparasite communities than fish from temperate latitudes, following the latitudinal diversity gradient. Although there is still some controversy, temperature seems a major force driving parasite richness and increasing their taxonomic complexity over evolutionary time, mediated by greater rates of speciation and host colonisation [START_REF] Luque | Parasite biodiversity and its determinants in coastal marine teleost fishes of Brazil[END_REF][START_REF] Garrido-Olivera | The influence of host ecology and biogeography on the helminth species richness of freshwater fishes in Mexico[END_REF]. Although parasite biodiversity in coral reefs has not been evaluated, recent studies show high parasite diversity in coral reef fishes, suggesting an estimate of 10 parasite species per fish species [START_REF] Justine | Parasite biodiversity in a coral reef fish: twelve species of monogeneans on the gills of the grouper Epinephelus maculatus (Perciformes: Serranidae) off New Caledonia, with a description of eight new species of Pseudorhabdosynochus (Monogenea: Diplectanidae)[END_REF][START_REF] Justine | An annotated list of fish parasites (Isopoda, Copepoda, Monogenea, Digenea, Cestoda, Nematoda) collected from Snappers and Bream (Lutjanidae, Nemipteridae, Caesionidae) in New Caledonia confirms high parasite biodiversity on coral reef fish[END_REF] Abundance of parasites may differ geographically coupled to environmental gradients following the "favourable centre" hypothesis, which states that a species reaches its highest abundance at the centre of its range, where optimal conditions for survival and reproduction exist. With increasing distance from this optimal site the conditions become less favourable and population size declines (Sagaring and Gaines, 2002). Similarity of parasite communities of the same host species tends to decrease exponentially with increasing distance between them, or increasing habitat differences [START_REF] Poulin | Geographical distances and the similarity among parasite communities of conspecific host populations[END_REF]Poulin et al. 2011).

Distance decay in similarity results from three processes: species-sorting along environmental gradients, dispersal constraints imposed by topography, and intrinsic species differences in dispersal abilities [START_REF] Soininen | The distance decay of similarity in ecological communities[END_REF]).

Chemical ecology in host-parasite interactions and host protection strategies from parasites

Chemical ecology is the study of chemicals involved in the interactions of living organisms and provides significant insights into the ecology and evolution of populations, from the organisation of communities and the function of ecosystems (Hay, 2009).

Parasites have evolved numerous strategies to evade host defences and succeed in infection, some of which involve chemical mediation. Social insects and their parasites are the best-studied group for chemical mediated host-parasite interactions. Social insects are known to produce semiochemicals (chemical substances involved in communication) in order to recognise their nest-mates and detect intruders. Many social parasites have evolved chemical mimicry in order to take advantage of their hosts without being noticed [START_REF] Lenoir | Chemical ecology and social parasitism in ants[END_REF].

For example, the larvae of the parasitic butterfly Maculinea rebeli produce mimetic hydrocarbons in the surface of their body that make the foraging Mirmica schencki workers recognize them as their own ant larvae and bring them to their nest brood chambers [START_REF] Akino | Chemical mimicry and host specificity in the butterflyfish Maculinea rebeli, a social parasite of Myrmica ant colonies[END_REF].

Chemical mediation in host-parasite interactions in the marine ecosystem is still poorly understood. However, some studies have found that parasites use chemical cues to identify their hosts. For example, the pathogenic bacteria Vibrio coralliilyticus, uses chemotaxis4 and chemokinesis5 to target the mucus of its stressed coral host, Pocillopora damicornis [START_REF] Garren | A bacterial pathogen uses dimethylsulfoniopropionate as a cue to target heat-stressed corals[END_REF]. Similarly, the pathogen Flavobacterium columnare is attracted by mucus from channel catfish (Ictalurus punctatus) [START_REF] Klesius | Flavobacterium columnare chemotaxis to channel catfish mucus[END_REF]. [START_REF] Kallert | No shot in the dark: myxozoans chemically detect fresh fish[END_REF] found that the nucleosides inosine and its 2'-deoxyinosine and guanosine from fish mucus attracted actinospores from 3 species of myxozoan and induced polar filament discharge and sporoplasm emission (myxozoan way of host invasion). Other studies have investigated the specificity of monogenean parasites and found that glycoproteins present in fish mucus induced the attachment of oncoramicidia [START_REF] Yoshinaga | Attachment-inducing capacities of fish skin epithelial extracts on oncomiracidia of Benedenia seriolae (Monogenea: Capsalidae)[END_REF][START_REF] Ohashi | Purification and identification of a glycoprotein that induces the attachment of oncomiracidia of Neobenedenia girellae (Monogenea, Capsalidae)[END_REF].

Host defences against parasites usually involve the activation of the immune cascade involving the innate and adaptative immune systems. Innate immune initiation relies on the recognition of pathogen-associated molecular patterns (PAMPs) by pathogen recognizing receptors (PRRs), such as Toll-like receptors (TLRs) and C-type lectin receptors [START_REF] Buchmann | Lectins in fish skin: do they play a role in host-monogenean infections?[END_REF][START_REF] Alvarez-Pellitero | Fish immunity and parasite infections: from innate immunity to immunoprophilactic prospects[END_REF]. Both TLR receptors and physical attachment of parasites (e.g. ectoparasites) initiate inflammatory responses, activating macrophages to produce cytokines, including tumour-necrosis factor (TNF), interleukins (IL-1, IL-6, IL-8), complement factor, lysozyme, antimicrobial proteins and peptides, the enzyme inducible nitric-oxide synthase (iNOS) and other oxidative factors [START_REF] Jones | The occurrence and mechanisms of innate immunity against parasites in fish[END_REF][START_REF] Alvarez-Pellitero | Fish immunity and parasite infections: from innate immunity to immunoprophilactic prospects[END_REF]. In ectoparasite infections, hypersecretion of mucus is the first response to infection. Excess of mucus interferes with parasite infection, making difficult parasite access to epithelial layers and removing parasites via mucus sloughing [START_REF] Jones | The occurrence and mechanisms of innate immunity against parasites in fish[END_REF]. Fish mucus contains several innate and humoral substances with biostatic or bioacidal activity such as immunoglobulin, complement, C-reactive proteins, lectins, lysozymes and antimicrobial peptides [START_REF] Jones | The occurrence and mechanisms of innate immunity against parasites in fish[END_REF].

Mucosal immune system also possesses lymphocytes with high specificity and memory capacities, which are able to differentiate commensals from pathogens (Gomez et al., 2013b).

The development of new techniques such as metabolomics and proteomics allows a more comprehensive study of chemical molecules from organisms, providing new opportunities to study differential expression of signalling molecules in organism's interactions [START_REF] Kuhlisch | Metabolomics in chemical ecology[END_REF][START_REF] Brinchmann | Immune relevant molecules identified in the skin mucus of fish using -omics technologies[END_REF].

Metabolomics

Metabolomics is one of the most recent "omics" sciences, joining genomics, transcriptomics and proteomics as tools in global systems biology (Sumner et al. 2003).

"omics" sciences use high-throughput technologies to study comprehensively biochemical and molecular processes within an organism, tissue or cell type [START_REF] Weckwerth | Metabolomics in systems biology[END_REF].

Metabolomics is the comprehensive, qualitative, and quantitative study of the small molecules in an organism, the metabolome [START_REF] Dunn | Measuring the metabolome: current analytical technologies[END_REF]. Metabolites are the end products of cellular regulatory processes, and their levels can be regarded as the ultimate response of biological systems to genetic or environmental changes [START_REF] Fiehn | Metabolomics--the link between genotypes and phenotypes[END_REF]. Combined with proteomics and genomics, metabolomics can help gain insight into systems biology by studying the metabolite alterations and their relationships to changes in gene expression, protein expression and enzyme activity [START_REF] Want | Global metabolic profiling procedures for urine using UPLC-MS[END_REF]) (Figure 4). The metabolome of a sample provides a snapshot of the state of the organisms at that time under specific environmental conditions [START_REF] Kosmides | Metabolomic fingerprinting: challenges and opportunities[END_REF]. Metabolomics approaches prove useful to distinguish among individual signals and thus might serve in biomarker discovery in a wide range of topics, from disease biomarkers to indicators of short-term environmental changes in ecology [START_REF] Wolfender | MS-based plant metabolomic approaches for biomarker discovery[END_REF]. Metabolomics methodologies fall into two distinct groups, depending on the goal of the experiment. Targeted metabolomics is the measurement of defined groups of chemically characterised and biochemically annotated metabolites. Since targeted metabolomics analyses

General introduction -31 -known metabolites, sample preparation and analytical techniques can be optimised depending the chemical properties of these compounds. Untargeted metabolomics intends to study all the measurable analytes in a sample including chemical unknowns, therefore, special attention to sample preparation procedures and analytical techniques must be taken to avoid exclusion of metabolites [START_REF] Fiehn | Metabolomics--the link between genotypes and phenotypes[END_REF][START_REF] Roberts | Targeted metabolomics[END_REF].

Most used analytical approaches in metabolomics include nuclear magnetic resonance (NMR) spectroscopy and mass spectrometry (MS) coupled techniques, including liquid chromatography (LC-MS n ), gas chromatography (GC-MS). NMR spectroscopy is the most common technique used to generate metabolomic data from biofluids and the main advantages is that it is a non-destructive, highly reproducible and fully quantitative analytic technique, taking only a few minutes per sample. MS methods, on the other hand, provide much higher sensitivity, enabling the quantitative measurement of a broader spectrum of metabolites at lower cost, however the injected part of the sample is destroyed during ionization [START_REF] Reo | NMR-based metabolomics[END_REF][START_REF] Kanani | Standardizing GC-MS metabolomics[END_REF]Zhou et al. 2012;[START_REF] Kosmides | Metabolomic fingerprinting: challenges and opportunities[END_REF].

Metabolomic analysis produces large quantities of data that have to be pre-processed before being exploited by statistical analysis and chemometrics. Experiment design is of vital importance to have biologically significant data and that includes careful sampling design, extraction procedure, analytical analysis and data pre-processing and treatment. Due to the high number of samples analysed simultaneously, there is a high likeliness of bias introduction during sample analysis related to technical drift. Randomisation of samples or the use of a Latin square order in the analytical sequence can partly solve the problem, however, several algorithms have also been developed to correct technical drift in retention times between samples and deconvolute and align peaks, such as the free software XCMS.

Once the data has been pre-processed (for example with XCMS), several univariate and multivariate statistics are often applied in order to extract the biological information of interest (Figure 5).

Environmental metabolomics

Environmental metabolomics studies the organism's metabolic response to natural or anthropogenic stressors in its environment. Environmental metabolomics can be a powerful tool in 1) ecophysiology studies to unveil organism's biochemical responses to biotic or abiotic conditions such as effects of feeding behaviours on metabolism, 2) ecotoxicology to characterise metabolic responses of organisms to anthropogenic stressors, such as pollution and climate change and 3) to characterize the metabolites involved in organisms interactions [START_REF] Viant | Recent developments in environmental metabolomics[END_REF].

One of the main advantages of metabolomics is the possible application to non-model species, since differences in metabolite fingerprints can be detected and analysed without knowing the metabolite identifications. However, a posteriori metabolite identification is an important step to biomarker discovery and identification of metabolic pathways that are altered in response to a biological perturbation, or discovering novel signalling compounds [START_REF] Bundy | Environmental metabolomics: a critical review and future perspectives[END_REF].

In comparison to the large number of metabolomics studies in plants, human and model organisms (e.g. yeast or Escherichia coli), fewer metabolic profiles have been collected from aquatic organisms [START_REF] Viant | Metabolomics of aquatic organisms: the new "omics" on the block[END_REF][START_REF] Yanes | Expanding Coverage of the Metabolome for Global Metabolite Profiling[END_REF][START_REF] Quanbeck | Metabolomics as a Hypothesis-Generating Functional Genomics Tool for the Annotation of Arabidopsis thaliana Genes of "Unknown Function[END_REF]. Marine metabolomics, has however seen a rapid development in the last years, with increasing studies on wide-range of organisms including marine bacteria, micro and macroalgae, shellfish, sponges, corals and some fish species [START_REF] Motti | Environmental marine metabolomics: from whole organism system biology to ecosystem management[END_REF][START_REF] Goulitquer | Mass spectrometry-based metabolomics to elucidate functions in marine organisms and ecosystems[END_REF]).

Several studies have been performed to identify marine animal biochemical responses to stress caused by changing environmental conditions. For example, the metabolic response of mussels (Mytilus edulis) and marine copepods to acidification and increased temperature were studied by 1 H-NMR and LC-MS metabolomics [START_REF] Ellis | 1)H NMR metabolomics reveals contrasting response by male and female mussels exposed to reduced seawater pH, increased temperature, and a pathogen[END_REF][START_REF] Mayor | The metabolic response of marine copepods to environmental warming and ocean acidification in the absence of food[END_REF].

Metabolomics was also used to study metabolic response of Atlantic salmon (Salmo salar)

and shrimp (Litopenaeus vannamei) to culture stressors (Karakach et al. 2009;Shock et al. 2013).

Marine metabolomics has also proven useful for chemotaxonomy purposes, to identify between different Homoscleromorpha sponges and Symbiodinium dinoflagellates species (Ivanisevic et al. 2010;[START_REF] Klueter | Taxonomic and environmental variation of metabolite profiles in marine dinoflagellates of the genus Symbiodinium[END_REF]. A recent study used metabolomics and proteomics to study chemical mediated competition in marine plankton [START_REF] Poulson-Ellestad | Metabolomics and proteomics reveal impacts of chemically mediated competition on marine plankton[END_REF].

The use of metabolomic techniques to understand coral reef ecosystems has also exploded in the recent years. However most of the studies concentrate mainly on coral species or coral symbionts (He et al. 2014;[START_REF] Sogin | Application of 1H-NMR metabolomic profiling for reef-building corals[END_REF][START_REF] Klueter | Taxonomic and environmental variation of metabolite profiles in marine dinoflagellates of the genus Symbiodinium[END_REF]. He et al. (2014) studied the metabolic spatial variation of soft corals at two geographic locations in the South China Sea with NMR-bases metabolomic analysis. [START_REF] Sogin | Application of 1H-NMR metabolomic profiling for reef-building corals[END_REF][START_REF] Sogin | Metabolomic signatures of increases in temperature and ocean acidification from the reef-building coral, Pocillopora damicornis[END_REF] showed that 1 H-NMR metabolomic profiles were species-specific among four species of reef-building corals and they studied the metabolic changes of Pocillopora damicornis under thermal and

General introduction -34 -acidification stress. A recent article [START_REF] Quinn | Metabolomics of reef benthic interactions reveals a bioactive lipid involved in coral defence[END_REF]) used metabolomics to study reef benthic interactions between corals and algae and found that a bioactive lipid was involved in coral defence against algae. However, to date no study has yet assessed metabolic profiles and metabolic variability of coral reef fishes.

Objectives

Fish parasites are an important part of ecosystems, however, in certain cases such as in aquaculture they cause severe disease outbreaks and host mortality. The objective of this Ph.D. is to gain knowledge of host-parasite interactions in coral reef fishes, through their study in both the natural and culture environments.

Parasite prevalence, intensity and virulence depend highly on parasite specificity.

Therefore, the first objective of this thesis is to investigate parasite specificity and understand which factors shape it. To accomplish this goal we have analysed parasite specificity of monogenean parasites in the highly diversified Chaetodontidae family. We have surveyed the distribution of 13 species of dactylogyrids (Monogenea) among 34 butterflyfishes in the tropical Indo-Pacific islands (Chapter I), and we have studied the parasite biogeographical patterns in order to better understand evolution of host specificity among a large spatial scale (Chapter II).

Control of parasitic infections is crucial to host well-being. In aquaculture, spread of parasitic infections leads to high fish mortalities, and consequent important economic losses.

Parasite control in aquaculture is mainly done by the use of synthetic persistent drugs, which have numerous side effects on fish and local biodiversity. The second and largest objective of the Ph.D. is to examine sustainable parasite control methods. This objective has been approached under two different directions. On one side, I have investigated parasite evasion methods in natural fish populations, which might provide new opportunities for parasite control in aquaculture. I observed that among the 34 butterflyfishes studied, only Chaetodon lunulatus was never parasitized by gill monogeneans. Therefore, I researched the speciesspecific factors in this species that might be related to this marked parasite absence. I have analysed the fish mucus, since it is the first surface of exchange between the fish and the environment and is known to be involved both in pathogen defence and parasite recognition (Chapter III). I then studied the gill mucus microbiome and metabolome of eight butterflyfish species (Chapter IV and V) and integrated the results in order to find specific microbial taxons and metabolites overexpressed in C. lunulatus (Chapter VI). On the other side, I assessed the use of medicinal plants as an alternative to chemotherapy in fish aquaculture (Chapter VII). I investigated the potential of several local Polynesian plants and algae against the two major pathogenic agents (Vibrio harveyi and Tenacibaculum maritimum), by

Scientific context and model species

Butterflyfishes (Order: Perciformes, family: Chaetodontidae) are a diverse and conspicuous coral reef fish family widely distributed in all tropical seas. They are almost exclusively associated with coral reefs, but a few species are found in estuarine areas and over soft bottom habitats [START_REF] Roberts | Butterflyfish social behaviour, with special reference to the incidence of territoriality: a review[END_REF]. There are 130 species of butterflyfish described to date, belonging to 10 genera (Tricar and Boyle, 2015). Although, most butterflyfish species are easily distinguished due to their colorful patterns, several hybrids (34% of the species) are known to occur [START_REF] Montanari | Does genetic distance between parental species influence outcomes of hybridization among coral reef butterflyfishes?[END_REF]. Butterflyfish possess a highly compressed body, a small protractile mouth with brush-like teeth and their maximum lengths vary from 10 to 30 cm depending on the species. Most are highly social species, which form long-term monogamous pairs with territorial and "home-ranging" behaviors. The few planktivorous butterflyfish species tend to form larger associations or schools [START_REF] Roberts | Butterflyfish social behaviour, with special reference to the incidence of territoriality: a review[END_REF][START_REF] Pratchett | Biology of Butterflyfishes[END_REF]. Recent studies have showed that a high number of butterflyfish species produce sounds during social interactions (Tricar and Boyle, 2015). Chaetodon. The genus Chaetodon itself is distributed within 4 clades [START_REF] Littlewood | Molecular phylogenetics of Chaetodon and the Chaetodontidae (Teleostei: Perciformes) with reference to morphology[END_REF][START_REF] Fessler | Molecular phylogenetics of the butterflyfishes (Chaetodontidae): Taxonomy and biogeography of a global coral reef fish family[END_REF] (Figure 2). Butterflyfish consume a wide variety of prey including scleractinian corals, alcyonacean corals, algae, polychaetes, crustaceans and plankton. However, most species feed primarily, if not exclusively, on scleractinian corals. Butterflyfishes can be categorized upon their diet as obligate corallivores, facultative corallivores, omnivorous and planktivorous [START_REF] Pratchett | Dietary overlap among coral-feeding butterflyfishes (Chaetodontidae) at Lizard Island, northern Great Barrier Reef[END_REF], Pratchett et al. 2014a).

Different butterflyfishes also exhibit different levels of diet specialization. For example,

Chaetodon trifascialis is among the most specialized of all coral reef fish, feeding almost exclusively on one single coral species, Acropora hyacinthus. On the other side, Chaetodon lunulatus is among the most generalist obligate corallivore [START_REF] Pratchett | Dietary overlap among coral-feeding butterflyfishes (Chaetodontidae) at Lizard Island, northern Great Barrier Reef[END_REF]. A recent study showed that specificity of a corallivorous butterflyfish (Chaetodon triangulum) varied depending level of coral coverage, with a lower feeding specificity and a higher territory size in low coral cover sites [START_REF] Chandler | Influence of resource availability on the foraging strategies of the triangle butterflyfish Chaetodon triangulum in the Maldives[END_REF]. Several butterflyfish species (e.g. corallivorous species or with diets depending on coral-rich habitats) are sensible to coral loss and readily respond to changes in reef environments, with marked declines of some butterflyfish species upon extensive coral depletion (Pratchett et al. 2006b[START_REF] Pratchett | Changes in biodiversity and functioning of reef fish assemblages following coral bleaching and coral loss[END_REF](Pratchett et al. , 2014b)).

Although butterflyfish are one of the best-studied coral reef fish, study of their parasite communities remains limited. Parasites, are an intrinsic part of organisms, and therefore play a major role in the evolution, adaptation and success or extinction of host species. A better comprehension on butterflyfish parasite communities is therefore important to better understand their stressors and possible adaptations. Also, since butterflyfish ecology and phylogeny has been extensively studied, butterflyfishes provide a good model to study parasite communities in large geographic areas.

Butterflyfishes are parasitized by several endo-and ectoparasite species. [START_REF] Morand | Endoparasite species richness of New Caledonian butterfly fishes: host density and diet matter[END_REF] analyzed butterflyfishes endoparasite richness and identified 10 species of digeneans, 2 nematode species and 1 Acanthocephalean species. Some ectoparasites that have been found in butterflyfish include isopods (family Gnathiidae and Cymothoidae) [START_REF] Meadows | Behavioral and ecological correlates of foureye butterflyfish, Chaetodon capistratus, (Perciformes: Chaetodontidae) infected with Anilocra chaetodontis (Isopoda: Cymothoidae)[END_REF]Coile and Sikel, 2013), transversotrematid trematodes [START_REF] Cribb | Biogeography of tropical Indo-West Bibliography -293 -Pacific parasites: a cryptic species of Transversotrema and evidence for rarity of Transversotrematidae (Trematoda) in French Polynesia[END_REF], faustulids [START_REF] Diaz | Paradiscogaster flindersi and P. oxleyi n. sp. (Digenea: Faustulidae): overlapping host and geographical distributions in corallivore chaetodontid fishes in the tropical Indo-west Pacific[END_REF][START_REF] Diaz | A complex of species related to Paradiscogaster glebulae (Digenea: Faustulidae) in chaetodontid fishes (Teleostei: Perciformes) of the Great Barrier Reef[END_REF], and monogeneans (Plaisance and[START_REF] Plaisance | Dactylogyrids (Platyhelminthes: Monogenoidea) parasitizing butterfly fishes (Teleostei: Chaetodontidae) from the coral reefs of Palau, Moorea, Wallis, New Caledonia, and Australia: species of Euryhaliotrematoides n. gen. and Aliatrema n[END_REF]).

Monogenean parasites (Plathelminthes, Bychowsky 1937) are direct cycle ectoparasites mainly found on fish skin or gills. Monogeneans are a morphologically and ecologically diverse group, with several thousand species described [START_REF] Poulin | The evolution of monogenean diversity[END_REF]. They can be divided into two major groups based on the complexity of their attachment structures (haptor): the monopisthocotyleans, which have hook-like organs on their haptors, and the polyopisthocotyleans, which use clamp-like structures for attachment (Reed et al. 2009, Figure 3). Most monogeneans are oviparous, releasing heavy ciliated free-swimming larvae (oncoramicidium) that are able to seek their hosts actively over short distances. Monogeneans tend to be host specific, one monogenean species infecting only one or few host species (Sasal et al. 1999). Buchmann and Linderstrøm (2002) proposed that monogenean specificity must be governed by factors on the host surface. It has been suggested that chemical stimuli emitted from the host could attract the parasites and initiate certain behavioural and physiological changes in the parasite. [START_REF] Hirazawa | Mucus pH of the tiger puffer Takifugu rubripes is an important factor for host identification by the monogenean Heterobothrium okamotoi[END_REF] found that Heterobothrium okamotoi oncomiracidia attached preferentially to skin mucus of its host species (tiger puffer, Takifugu rubripes) when several fish skin mucus were offered, and proposed that pH of the host mucus could be used by the parasite to identify the host. Two other studies found that capsalid oncoramicidia attachment to the hosts was induced by skin and mucus extracts, and they identified a host glycoprotein as the possible responsible of the recognition [START_REF] Yoshinaga | Attachment-inducing capacities of fish skin epithelial extracts on oncomiracidia of Benedenia seriolae (Monogenea: Capsalidae)[END_REF][START_REF] Ohashi | Purification and identification of a glycoprotein that induces the attachment of oncomiracidia of Neobenedenia girellae (Monogenea, Capsalidae)[END_REF]).

Butterflyfishes are parasitized by gill monogeneans belonging to the family Dactylogyridae (Yamaguti, 1963). A total of 15 dactylogyrid species belonging to the genus Haliotrema and Euryhaliotrematoides have been described to date in the Chaetondontidae family [START_REF] Plaisance | Dactylogyrids (Platyhelminthes: Monogenoidea) parasitizing butterfly fishes (Teleostei: Chaetodontidae) from the coral reefs of Palau, Moorea, Wallis, New Caledonia, and Australia: species of Euryhaliotrematoides n. gen. and Aliatrema n[END_REF]Plaisance et al. 2004a). Haliotrema is a highly diversified genus that contains more than 100 species and is found in 6 orders of teleost fish.

Six Haliatrema species are known to paratisize butterflyfishes. Euryhaliotrematoides is only found on fish from the Chaetodontidae family (Figure 3). To date, 9 species have been described in the genus Chaetodon, Forcipiger and Heniochus [START_REF] Plaisance | Dactylogyrids (Platyhelminthes: Monogenoidea) parasitizing butterfly fishes (Teleostei: Chaetodontidae) from the coral reefs of Palau, Moorea, Wallis, New Caledonia, and Australia: species of Euryhaliotrematoides n. gen. and Aliatrema n[END_REF]Plaisance et al. 2004[START_REF] Plaisance | Molecular phylogeny of gill monogeneans (Platyhelminthes, Monogenea, Dactylogyridae) and colonization of Indo-West Pacific butterflyfish hosts (Perciformes, Chaetodontidae)[END_REF]. [START_REF] Plaisance | Dactylogyrids (Platyhelminthes: Monogenoidea) parasitizing butterfly fishes (Teleostei: Chaetodontidae) from the coral reefs of Palau, Moorea, Wallis, New Caledonia, and Australia: species of Euryhaliotrematoides n. gen. and Aliatrema n[END_REF].

In this first part of the thesis I have studied parasite specificity of butterflyfish monogenean parasites and I have investigated which factors shape it. I have studied the monogenean communities of 34 butterflyfish species from the Indo West-Pacific and I have identified 13 dactylogyrid species. I have studied the dactylogyrid intensity, prevalence, richness and diversity in each butterflyfish species as well as the taxonomic composition of their dactylogyrid assemblages. I then investigated the host factors that could be involved in the observed patterns (phylogenetic signal, size, diet, geographic range and depth range). I have investigated the biogeography of dactylogyrid parasites in the West-Indo Pacific and I have assessed the effect of locations geographic distance, similarity in host communities and environmental similarities on the differences of parasite communities. Chaetodon lunulatus was the only butterflyfish species never found parasitized by gill monogeneans. Therefore, I studied which are the species-specific factors in this species that might be related to this marked parasite absence. I have studied the fish mucus, since it is the first surface of exchange between the fish and the environment and is known to be involved both in pathogen defence and parasite recognition. I then studied the gill mucus microbiome and metabolome of several butterflyfish species and integrated the results in order to find specific microbial taxons and metabolites overexpressed in C. lunulatus. [START_REF] Gómez | Neglected wild life: Parasitic biodiversity as a conservation target[END_REF]. Given that parasites rely on their hosts for resources, changes in the host population also affect parasite communities, these interactions leading to on-going hostparasite coevolution [START_REF] Quigley | The mode of host-parasite interaction shapes coevolutionary dynamics and the fate of host cooperation[END_REF]).

Gill monogenean communities (Platyhelminthes

Parasite assemblages are highly complex and dynamic, resulting from the combination of parasite physiological traits and ecology, and multiple host ecological parameters and phylogenetic histories [START_REF] Muñoz | Endoparasite communities of five fish species (Labridae: Cheilininae) from Lizard Island: how important is the ecology and phylogeny of the hosts?[END_REF]. For fishes, several studies have shown that parasite communities are influenced by host size, diet, geographic range and phylogenetic affiliation [START_REF] Sasal | Determinants of parasite species richness in mediterranean marine fishes[END_REF][START_REF] Morand | Endoparasite species richness of New Caledonian butterfly fishes: host density and diet matter[END_REF]Simkova et al. 2001). Inter-specific relationships within a host species (e.g. competition between parasite species) have been less well studied, but are also potentially important in host-parasite relationships and structure of parasite communities [START_REF] Hughes | Let your enemy do the work: within-host interactions between two fungal parasites of leaf-cutting ants[END_REF]. Parasite specificity can vary dramatically between species, and specificity patterns and drivers are still being explored (Simkova et al. 2006). However, factors associated with recognition and selection of the host certainly play a major role in the structure of parasite assemblages (Buchmann and Linderstrøm, 2002).

Therefore, studies of parasite communities should also take into account the possibility of chemical cues or other factors playing major roles in parasite specificity.

CHAPTER I

-60 -Butterflyfishes (family Chaetodontidae) are a diverse and emblematic family of coral reef fishes distributed widely in all tropical seas. It has been proposed that butterflyfish might be used to monitor health status of coral reefs or as a proxy of total reef fish assemblage [START_REF] Kulbicki | The use of butterflyfish (Chaetodontidae) species richness as a proxy of total species richness of reef fish assemblages in the Western and Central Pacific[END_REF]. Butterflyfish ecology and behaviour has been extensively studied, and they can consume a variety of prey, including algae, polychaetes, crustaceans and coral [START_REF] Pratchett | Dietary overlap among coral-feeding butterflyfishes (Chaetodontidae) at Lizard Island, northern Great Barrier Reef[END_REF]. Most species of butterflyfish are associated with coral cover, and some studies have shown butterflyfish vulnerability to coral loss [START_REF] Pratchett | Changes in biodiversity and functioning of reef fish assemblages following coral bleaching and coral loss[END_REF].

Although some studies have investigated parasite communities on butterflyfishes in the tropical Indo-west Pacific (TIWP), little is known about the ectoparasite assemblages among different species of butterflyfish [START_REF] Morand | Endoparasite species richness of New Caledonian butterfly fishes: host density and diet matter[END_REF][START_REF] Yong | The ghost of parasites past: eggs of the blood fluke Cardicola chaetodontis (Aporocotylidae) trapped in the heart and gills of butterflyfishes (Perciformes: Chaetodontidae) of the Great Barrier Reef[END_REF][START_REF] Cribb | Biogeography of tropical Indo-West Bibliography -293 -Pacific parasites: a cryptic species of Transversotrema and evidence for rarity of Transversotrematidae (Trematoda) in French Polynesia[END_REF][START_REF] Mcnamara | Evidence for extensive cryptic speciation in trematodes of butterflyfishes (Chaetodontidae) of the tropical Indo-West Pacific[END_REF]. Because of butterflyfishes high diversity and abundance in coral reefs, they provide a good model to study the arrangement of parasite communities between phylogenetically and ecologically similar host species along large geographical scales. Also, the knowledge on butterflyfishes phylogeny and ecology can help in the study of the underlying factors shaping parasites communities.

Platyhelminth monogeneans have direct life cycles, living predominantly on the gills and skin of fishes [START_REF] Kearn | Evolutionary expansion of the Monogenea[END_REF]. Butterflyfishes are parasitised by gill monogeneans belonging to the family Dactylogyridae, with a total of 15 species reported from chaetodontids to date [START_REF] Plaisance | Dactylogyrids (Platyhelminthes: Monogenoidea) parasitizing butterfly fishes (Teleostei: Chaetodontidae) from the coral reefs of Palau, Moorea, Wallis, New Caledonia, and Australia: species of Euryhaliotrematoides n. gen. and Aliatrema n[END_REF]Plaisance et al. 2004). Dactylogyrids are the primary monogenean group found on this host family; a few capsalids are the only other known monogeneans known to infect chaetodontids [START_REF] Bullard | Six new host records and an updated list of wild hosts for Neobenedenia melleni (MacCallum) (Monogenea: Capsalidae)[END_REF]. Although some recent studies have revised the classification of the monogenean fauna of butterflyfish and their molecular phylogeny [START_REF] Plaisance | Dactylogyrids (Platyhelminthes: Monogenoidea) parasitizing butterfly fishes (Teleostei: Chaetodontidae) from the coral reefs of Palau, Moorea, Wallis, New Caledonia, and Australia: species of Euryhaliotrematoides n. gen. and Aliatrema n[END_REF]Plaisance et al. 2004;[START_REF] Plaisance | Molecular phylogeny of gill monogeneans (Platyhelminthes, Monogenea, Dactylogyridae) and colonization of Indo-West Pacific butterflyfish hosts (Perciformes, Chaetodontidae)[END_REF][START_REF] Kritsky | Dactylogyrids (Monogenoidea: Polyonchoinea) parasitizing the gills of snappers (Perciformes: Lutjanidae): Revision of Euryhaliotrema with new and previously described species from the Red Sea, Persian Gulf, the eastern and Indo-west Pacific Ocean, and the Gulf of Mexico[END_REF], little is known about monogenean community structure.

In this study we aim to better understand the structure of dactylogyrid communities (intensity, prevalence, diversity and taxonomic composition) infecting multiple butterflyfish species in the TIWP and the factors shaping them. Since dactylogyrids are direct cycle parasites, we could expect that sympatric butterflyfishes with close ecologies and phylogeny would present similar parasitism levels and taxonomic compositions [START_REF] Tavares | Similarity between metazoan parasite communities of two sympatric brackish fish species from Brazil[END_REF], unless strong species-specific factors between different butterflyfishes play a major role in structuring dactylogyrid communities. Firstly, we studied variation of dactylogyrid intensities, prevalence, richness and diversity among 34 butterflyfish species and we analysed

CHAPTER I -61 -
the influence of host factors (phylogenetic signal, size, diet, geographic range and depth range) on the parasitism differences between host species. Secondly, we studied the taxonomic composition of dactylogyrid communities among the 34 butterflyfishes, and we identified different groups of butterflyfish species with similar dactylogyrid assemblages. We studied the relative effects of host phylogenetic, ecological (body size and feeding behavior)

and geographic distribution (geographic range and depth range) similarities on the taxonomic structuring of dactylogyrid assemblages between butterflyfish species using multivariate regression on distance matrices. Correlations between parasites species were used to see if there were strong antagonisms or synergisms between dactylogyrid species that could influence different taxonomic composition between hosts.

Materials and methods

Sampling

Thirty-four species of butterflyfish (n = 560) were collected at 8 localities in the Pacific:

Palau, Wallis and Futuna, Lizard Island (Australia), and the 5 archipelagos in French Polynesia (Society Islands, Tuamotu Islands, Austral Islands, Gambiers Islands and Marquesas Islands). Butterflyfish were also sampled from a single locality in the Indian

Ocean, from Ningaloo Reef on the west Australian coast (Fig. 1).

Adult butterflyfish were captured, put in individual plastic bags with seawater and brought immediately to a laboratory for dissection. Fish total and standard lengths were measured and gills were removed and fixed in 70% ethanol. Dactylogyrids were removed from the gills using a stereo microscope and were mounted on glass slides in Malmberg's ammonium picrate-glycerine.

All dactylogyrids from each individual fish were observed under a compound microscope, and parasite species and number were recorded. Dactylogyrid species determination was according to published literature (Plaisance and[START_REF] Plaisance | Dactylogyrids (Platyhelminthes: Monogenoidea) parasitizing butterfly fishes (Teleostei: Chaetodontidae) from the coral reefs of Palau, Moorea, Wallis, New Caledonia, and Australia: species of Euryhaliotrematoides n. gen. and Aliatrema n[END_REF][START_REF] Kritsky | Dactylogyrids (Monogenoidea: Polyonchoinea) parasitizing the gills of snappers (Perciformes: Lutjanidae): Revision of Euryhaliotrema with new and previously described species from the Red Sea, Persian Gulf, the eastern and Indo-west Pacific Ocean, and the Gulf of Mexico[END_REF] and was based on the morphological characters of the sclerotised structures (the haptor and the reproductive organs).

Data analysis

Dactylogyrid intensity, prevalence, richness and diversity

Counts of total monogeneans per fish were used to calculate intensity and prevalence for each host species. Normality of parasite intensities and prevalence (Saphiro-Wilk test) and homogeneity of variances (Levene tests) were tested and not satisfied, thus non-parametric tests were used in this data analysis sub-section. Non-parametric Kruskal-Wallis test and Kruskal post-hoc test were performed in order to detect parasite intensity differences between chaetodontid species. Host species intensity and prevalence were displayed using boxplots, where the median, first and third quartile were plotted.

Detailed counts of each dactylogyrid species per fish were used to calculate parasite richness, evenness and diversity [Shannon (H)] for each species of butterflyfish. Species CHAPTER I -63 -accumulation curves for each butterflyfish species were generated with the "vegan" package for R, and expected richness were calculated using the "exact" method.

The Chaetodontidae phylogenetic tree was reconstructed under the newick format using the phylogenetic tree from [START_REF] Fessler | Molecular phylogenetics of the butterflyfishes (Chaetodontidae): Taxonomy and biogeography of a global coral reef fish family[END_REF], and the package "phytotools" for R was used to read the tree. Since phylogenetic tree branch lengths were not available, phylogenetic signal on the parasite intensity and richness was measured using the Abouheif-Moran test, which has been recently defined as a powerful alternative to the K statistic when branch lengths are not accurate (package "adephylo" for R) [START_REF] Abouheif | A method for testing the assumption of phylogenetic independence in comparative data[END_REF][START_REF] Pavoine | Testing for phylogenetic signal in phenotypic traits: New matrices of phylogenetic proximities[END_REF][START_REF] Pavoine | Testing for phylogenetic signal in biological traits: the ubiquity of crossproduct statistics[END_REF].

Linear regression and Spearman correlation analyses were performed to analyse the influence of host factors (size, host geographical range, depth range and feeding behaviour)

on parasite intensity and parasite richness. Host geographical range (km 2 ), depth range and feeding behaviour data were obtained from FishBase [START_REF] Froese | FishBase. World wide web electronic publication[END_REF] and the IUCN Red List (IUCN, 2016). Feeding behaviour was categorized into the following groups depending on the level of specificity: 1 = specialist (feeding on restricted species), 2 = corallivore (feeding mostly on scleractinians), 3 = omnivore (feeding on a wide variety of organisms including corals, algae and invertebrates).

Taxonomic composition of dactylogyrid assemblages

Analyses of the dactylogyrid taxonomic composition were done using detailed counts of each dactylogyrid species per fish. A hierarchical cluster analysis («hclust » package for R, Euclidian distance) was used to identify groups of butterflyfish species with similar taxonomic composition of dactylogyrids. The "Average" algorithm was chosen after analysis of the cophenetic correlation coefficients (Pearson correlation between the cophenetic distances calculated on cluster branches and the parasite dissimilarity matrix) [START_REF] Kulbicki | Global biogeography of reef fishes: A hierarchical quantitative delineation of regions[END_REF]. The Kelley-Gardner-Sutcliffe (KGS) penalty function was used to prune the dendrogram [START_REF] Bottegoni | AClAP, Autonomous hierarchical agglomerative Cluster Analysis based protocol to partition conformational datasets[END_REF]. A Principal Component Analysis (PCA) was employed to analyse the correlations between the dactylogyrid species with the use of a correlation circle.

Multivariate regression on distance matrices (function MRM, package "ecodist" for R)

was used to evaluate the relative effects of host phylogeny, host ecology and host geographical distribution on the taxonomic composition of dactylogyrid assemblages among CHAPTER I -64 -different butterflyfish species. A matrix of phylogenetic distances between pairs of Chaetodontidae species was obtained using the distTips function ("adephylo" R package, method = nNodes) from the reconstructed phylogenetic tree (see the 'Dactylogyrid intensity, prevalence, richness and diversity' section). The chaetodontid ecology (host maximum size and feeding behavior) and geographical distribution (geographic range and depth range) matrices of dissimilarity were obtained using the vegdist function ("vegan" R package, Euclidean distance).

Results

Species identification

Thirteen dactylogyrid species were identified based on the morphology of the sclerotised structures, two of which are undescribed (Euryhaliotrema grandis (Mizelle & Kritsky, 1969),

Euryhaliotrema annulocirrus [START_REF] Yamaguti | Monogenetic trematodes of Hawaiian fishes[END_REF], Euryhaliotrema triangulovagina [START_REF] Yamaguti | Monogenetic trematodes of Hawaiian fishes[END_REF], Euryhaliotrema pirulum [START_REF] Plaisance | Dactylogyrids (Platyhelminthes: Monogenoidea) parasitizing butterfly fishes (Teleostei: Chaetodontidae) from the coral reefs of Palau, Moorea, Wallis, New Caledonia, and Australia: species of Euryhaliotrematoides n. gen. and Aliatrema n[END_REF], Euryhaliotrema microphallus [START_REF] Yamaguti | Monogenetic trematodes of Hawaiian fishes[END_REF], Euryhaliotrema berenguelae [START_REF] Plaisance | Dactylogyrids (Platyhelminthes: Monogenoidea) parasitizing butterfly fishes (Teleostei: Chaetodontidae) from the coral reefs of Palau, Moorea, Wallis, New Caledonia, and Australia: species of Euryhaliotrematoides n. gen. and Aliatrema n[END_REF], Euryhaliotrema aspistis [START_REF] Plaisance | Dactylogyrids (Platyhelminthes: Monogenoidea) parasitizing butterfly fishes (Teleostei: Chaetodontidae) from the coral reefs of Palau, Moorea, Wallis, New Caledonia, and Australia: species of Euryhaliotrematoides n. gen. and Aliatrema n[END_REF], Euryhaliotrema cribbi [START_REF] Plaisance | Dactylogyrids (Platyhelminthes: Monogenoidea) parasitizing butterfly fishes (Teleostei: Chaetodontidae) from the coral reefs of Palau, Moorea, Wallis, New Caledonia, and Australia: species of Euryhaliotrematoides n. gen. and Aliatrema n[END_REF],

Haliotrema angelopterum [START_REF] Plaisance | Description and redescription of Haliotrema species (Monogenoidea: Poloyonchoinea: Dactylogyridae) parasitizing butterfly fishes (Teleostei: Chaetodontidae) in the Indo-West Pacific Ocean[END_REF], Haliotrema aurigae [START_REF] Yamaguti | Monogenetic trematodes of Hawaiian fishes[END_REF], Haliotrema scyphovagina [START_REF] Yamaguti | Monogenetic trematodes of Hawaiian fishes[END_REF]

Dactylogyrid intensity, prevalence, richness and diversity

Monogenean prevalence and intensity was found to be highly variable between sympatric butterflyfish species. Fifteen chaetodontid species were found to always be parasitised by dactylogyrids, whereas Chaetodon lunulatus (n = 30) was the only species that was never found to be parasitised (Figure 2). Kruskal- 3). Species accumulation curves for dactylogyrid species for each chaetodontid species showed that despite differences in sampling effort between fish species, parasite richness seems well described for nearly all fish species (Supp. material semeion, H = 0) was found on C. quadrimaculatus (H = 0.17). Evenness (E) followed the same trend as diversity, with F. flavissimus having the highest evenness (E = 0.87) and C.

quadrimaculatus the lowest (E = 0.10) (Table 1). Host phylogenetic signal and ecological factors (size, geographical range, depth range and feeding behaviour) were studied in order to estimate their influence on the differences of parasitism intensities and richness between different butterflyfish species. Host phylogenetic signal (Abouheif-Moran test) was non significant for neither parasite intensity (P = 0.468) nor

Taxonomic composition of dactylogyrid assemblages

Thirteen dactylogyrid species were identified from the gills of the butterflyfish examined from the TIWP. Although dactylogyrid specificity varied between species, all species were found to be generalists among the Chaetodontidae. Euhaliotrematoides triangulovagina had the lowest specificity, being found on 29 host species, followed by H. aurigae and E. grandis (28 host species each). Haliotrema scyphovagina had the highest specificity, being found only on C. unimaculatus and C. vagabundus (Figure 5). Structure of the dactylogyrid communities was analyzed for each species of butterflyfish (Figure 6), and significant differences in the structure of communities were found between host species. Most fish species were parasitised by several dactylogyrid species, but relative abundances differed dramatically between host species. Chaetodon semeion was the only fish species parasitised by just one monogenean species (H. aurigae). Chaetodon baronessa also showed little parasite diversity, harbouring just two monogenean species (H. aurigae and E. triangulovagina) (Figure 6).

In order to analyse differences between the dactylogyrid communities for different fish species, a hierarchical cluster analysis was performed. Six groups based on monogenean community similarity were recognised (Figure 7). While the fifth group was constituted by fish species with dactylogyrid communities highly dominated by H. aurigae, the sixth group communities were more diversified (Figure 6 and [START_REF] Conceição | Orpotrin: a novel vasoconstrictor peptide from the venom of the Brazilian stingray Potamotrygon gr. orbignyi[END_REF]. The parasite correlation circle showed that there were no marked negative correlations between parasite species, however some parasite species had positive correlations such as H.

aurigae, E. pirulum and E. grandis and H. angelopterum and E. microphallus (Fig. 8).

Results from multivariate regression on distance matrices (MRM) showed that host phylogenetic similarities was the only significant factor (P = 0.003) explaining different taxonomic composition of dactylogyrid assemblages in butterflyfishes, while host ecology and geographic distributions were non significant (P > 0.05). However, the regression coefficient obtained for the model was extremely low (R 2 = 0.018).

CHAPTER I -71 -

Discussion

Dactylogyrid intensity, prevalence, richness and diversity

Since parasite communities are influenced by host physiology, ecology and phylogeny, phylogenetically close host species with similar ecologies can be expected to be infected by similar parasite communities [START_REF] Muñoz | Endoparasite communities of five fish species (Labridae: Cheilininae) from Lizard Island: how important is the ecology and phylogeny of the hosts?[END_REF][START_REF] Tavares | Similarity between metazoan parasite communities of two sympatric brackish fish species from Brazil[END_REF]. In this study, the dactylogyrid communities of 34 butterflyfish species from tropical Indo-west Pacific were studied and results indicated that parasitism intensities between closely related sympatric fish species varied significantly. Dactylogyrid intensity differences were broadly independent of the host ecological factors studied (size, depth range, geographical range and feeding behaviour) as well as host phylogeny. [START_REF] Sikkel | Habitat and species differences in prevalence and intensity of Neobenedenia melleni (Monogenea: Capsalidae) on sympatric Caribbean surgeonfishes (Acanthuridae)[END_REF] also found significant differences in intensity and prevalence of Neobenedenia melleni (McCallum, 1927) (Monogenea: Capsalidae) on three species of sympatric surgeonfishes and hypothesised that those can arise from microhabitat differences or from species-specific chemical cues. Our results revealed that fifteen butterflyfish species were always parasitised by dactylogyrids whereas C. lunulatus was never parasitised.

The ecology and phylogenetic position of C. lunulatus has been extensively studied [START_REF] Pratchett | Dietary overlap among coral-feeding butterflyfishes (Chaetodontidae) at Lizard Island, northern Great Barrier Reef[END_REF]Pratchett et al. 2006a;2014[START_REF] Fessler | Molecular phylogenetics of the butterflyfishes (Chaetodontidae): Taxonomy and biogeography of a global coral reef fish family[END_REF][START_REF] Hsu | Molecular phylogeny of Chaetodon (Teleostei: Chaetodontidae) in the Indo-west Pacific: evolution in geminate species pairs and groups[END_REF] and there is no obvious peculiarity that could explain this marked difference in parasitism. For example, C. lunulatus and C. ornatissimus are ecologically and phylogenetically close, both species being obligate corallivores and occurring in coral-rich areas of lagoons [START_REF] Hsu | Molecular phylogeny of Chaetodon (Teleostei: Chaetodontidae) in the Indo-west Pacific: evolution in geminate species pairs and groups[END_REF][START_REF] Berumen | Relative gut lengths of coral reef butterflyfishes (Pisces: Chaetodontidae)[END_REF] [START_REF] Bray | A new species of Aponurus Looss, 1907 (Digenea: Lecithasteridae) in butterflyfishes (Perciformes: Chaetodontidae) from New Caledonia[END_REF] and the blood fluke Cardicola chaetodontis Yamaguti, 1970, reported in previous studies [START_REF] Bray | A new species of Aponurus Looss, 1907 (Digenea: Lecithasteridae) in butterflyfishes (Perciformes: Chaetodontidae) from New Caledonia[END_REF]McNamara and Cribb, 2011;[START_REF] Mcnamara | Monorchiids (Platyhelminthes: Digenea) of chaetodontid fishes (Perciformes): Biogeographical patterns in the tropical Indo-West Pacific[END_REF]Díaz et al. 2013;[START_REF] Yong | The ghost of parasites past: eggs of the blood fluke Cardicola chaetodontis (Aporocotylidae) trapped in the heart and gills of butterflyfishes (Perciformes: Chaetodontidae) of the Great Barrier Reef[END_REF]. It seems that the differences in monogenean intensities between closely related species such as C. lunulatus and C. ornatissimus must therefore arise from gill or gill mucus species-specific CHAPTER I -74 -characteristics. Some studies [START_REF] Buchmann | Immune mechanisms in fish skin against monogeneans -a model[END_REF][START_REF] Buchmann | Interactions between monogenean parasites and their fish hosts[END_REF] have analysed monogenean-host interactions and have found that immune mechanisms in fish skin and mucus play a role in monogenean specificity. Chemical composition of fish skin and mucus seems to be species-specific and studies, like those of [START_REF] Yoshinaga | Attachment-inducing capacities of fish skin epithelial extracts on oncomiracidia of Benedenia seriolae (Monogenea: Capsalidae)[END_REF] and [START_REF] Ohashi | Purification and identification of a glycoprotein that induces the attachment of oncomiracidia of Neobenedenia girellae (Monogenea, Capsalidae)[END_REF], have identified some glycoproteins in fish mucus that induce oncomiracidia attachment in two species of capsalids. Recently, numerous studies have focused on fish mucus molecules and bioactivities and an antimicrobial peptide derived from the catfish gill hemoglobin was found to display potent antiparasitic activity against the ciliate parasites Ichthyophthirius multifiliis (Fouquet) (Ullal et al;2008;[START_REF] Gómez | Neglected wild life: Parasitic biodiversity as a conservation target[END_REF][START_REF] Rakers | Antimicrobial peptides (AMPs) from fish epidermis: perspectives for investigative dermatology[END_REF].

2012), one lecithasterid (Aponurus chelebesoi

Skin and mucus microbiome are also species-specific and some recent studies showed that mucus commensal microbiota play a role in providing protection against opportunistic infections [START_REF] Larsen | Diversity of the skin microbiota of fishes: evidence for host species specificity[END_REF][START_REF] Boutin | Inter individual variations of the fish skin microbiota: host genetics basis of mutualism?[END_REF]. [START_REF] Lowrey | Topographical Mapping of the Rainbow Trout (Oncorhynchus mykiss) Microbiome Reveals a Diverse Bacterial Community with Antifungal Properties in the Skin[END_REF] found antifungal activity in bacteria isolates from skin of rainbow trout (Oncorhynchus mykiss Walbaum, 1792) and [START_REF] Zbakh | Antibacterial activity of benthic marine algae extracts from the mediterranean coast of Morocco[END_REF] found that bacteria from fish intestinal mucus produced a novel bioactive lipid sebastanoic acid with antibacterial activities. We thus suggest that studying C. lunulatus gill mucus molecules and microbiome could be highly interesting in order to investigate whether this species possesses some chemical or microbiota particularities that could explain the absence of dactylogyrid parasites. C. lunulatus and their sister species C. trifasciatus Park 1797 (Indian Ocean) and C. austriacus Rüpell, 1836 (Red Sea) are reported to be among the most dominant species in coral reefs worldwide, highlighting the functional importance of these butterflyfish species in coral reefs [START_REF] Findley | Global, regional and local patterns in species richness and abundance of butterflyfishes[END_REF].

Analysis of the dactylogyrid community infecting the sister species of C. lunulatus, might also give some insights into the mechanisms of parasite evasion in C. lunulatus and maybe clarify whether the absence of some parasitic groups such as dactylogyrids in C. lunulatus can be related to its demographic success over its congeners. [START_REF] Kearn | Evolutionary expansion of the Monogenea[END_REF] proposed that direct life cycles and the absence of specialised transmission stages in monogenean parasites could favour host switching. This hypothesis would predict that closely related sympatric host species would have similar parasite richness and diversity.

However, dactylogyrid richness and diversity in sympatric butterflyfish species was highly variable in this study. For instance, C. auriga (host to 11 dactylogyrid spp.) and C. ephippium (host to 4 dacytlogyrid spp.) inhabit similar ecological niches but exhibit very different CHAPTER I -75 -parasite richness. C. quadrimaculatus (host to 6 dactylogyrid spp., H = 0.17) and F. flavissimus (host to 8 dactylogyrid spp., H = 1.82) are also frequently found in the same habitats but have dramatically different monogenean diversity, indicating the possible presence of species specificities.

Several studies have shown that larger hosts can sustain greater number of parasites and may be expected to harbor richer parasite communities [START_REF] Sasal | Determinants of parasite species richness in mediterranean marine fishes[END_REF][START_REF] Sasal | Comparative analysis: a tool for studying monogenean ecology and evolution[END_REF]. In our study, no correlation was found between dactylogyrid richness and host size, and one of the smallest butterflyfish species (C. citrinellus) possessed the highest parasite richness. [START_REF] Morand | Endoparasite species richness of New Caledonian butterfly fishes: host density and diet matter[END_REF] also failed to find a relationship between endoparasite communities and host size of butterflyfish species in New Caledonia. Host diet is also known to be an important parameter in parasite richness, especially in parasites with complex life cycles where hosts with broader diet ranges could acquire more trophically transmitted parasites than those with narrow, specialised diets (Simkova et al. 2001;[START_REF] Cirtwill | Are parasite richness and abundance linked to prey species richness and individual feeding preferences in fish hosts?[END_REF].

Such is the case for endoparasites in butterflyfish where host diet was a good predictor of parasite richness [START_REF] Morand | Endoparasite species richness of New Caledonian butterfly fishes: host density and diet matter[END_REF]. A recent study has showed that host diet affected ectoparasite communities in bats due to different host fitness directly related to host diet [START_REF] Luguterah | Effect of dietary guild (frugivory and insectivory) and other host characteristics on ectoparasite abundance (mite and nycteribiid) of chiropterans[END_REF]. In our study we did not find any correlation between parasite intensity or richness and feeding specificity, since one of the species with the most diverse monogenean communities, C. trifascialis, is among the most specialised, feeding almost exclusively on Acropora hyacinthus [START_REF] Pratchett | Dietary overlap among coral-feeding butterflyfishes (Chaetodontidae) at Lizard Island, northern Great Barrier Reef[END_REF]. Some authors have proposed that hosts with a larger depth range would encounter more parasite species and therefore have richer parasite faunas [START_REF] Luque | Parasite biodiversity and its determinants in coastal marine teleost fishes of Brazil[END_REF], however this was not the case for the dactylogyrids communities. Although some authors disagree, host species with large geographical ranges could have greater chances of acquiring new parasite species via colonisation (Simkova et al. 2001;Poulin, 2005a). In this study, the positive relationship between host geographic range and parasite species richness seemed to explain some of the observed parasite richness variability on different butterflyfish species, but the effect was moderate. Finally, the role of host phylogeny in the diversity and structure of parasites communities has been discussed extensively. While some studies like those of [START_REF] Morand | Endoparasite species richness of New Caledonian butterfly fishes: host density and diet matter[END_REF] and Poulin et al. (2011) confirmed that host phylogenetic relationships have a strong influence on patterns of parasite richness, other provide evidence that host phylogeny might have only a weak influence on parasite richness and diversity [START_REF] Muñoz | Endoparasite communities of five fish species (Labridae: Cheilininae) from Lizard Island: how important is the ecology and phylogeny of the hosts?[END_REF][START_REF] Alarcos | Parasite communities in three sympatric flounder species (Pleuronectiformes: Paralichthyidae): similar ecological filters driving toward repeatable assemblages[END_REF]. In our study, CHAPTER I -76 -host phylogeny was not related to parasite species richness. However, we should highlight that this is one of the first studies where quite homogeneous parasite communities (13 species of parasites belonging to the same family) were assessed in a highly diversified host family. [START_REF] Mcnamara | Monorchiids (Platyhelminthes: Digenea) of chaetodontid fishes (Perciformes): Biogeographical patterns in the tropical Indo-West Pacific[END_REF] studied the monorchiid communities from chaetodontids and their results also show a weak influence of chaetodontids phylogeny on prevalence and richness of monorchiids. [START_REF] Yong | The ghost of parasites past: eggs of the blood fluke Cardicola chaetodontis (Aporocotylidae) trapped in the heart and gills of butterflyfishes (Perciformes: Chaetodontidae) of the Great Barrier Reef[END_REF] found that prevalence of the blood fluke C. chaetodontis varied importantly among closely related chaetodontids.

Taxonomic composition of dactylogyrid assemblages

Monogeneans are generally considered to be highly host-specific [START_REF] Whittington | Host-specificity of monogenean (platyhelminth) parasites: a role for anterior adhesive areas?[END_REF]. In this study we found just one species of dactylogyrid (H. scyphovagina) to be relatively host-specific and that most species parasitise a high number of chaetodontid species. Significant differences between taxonomic composition of dactylogyrid assemblages of sympatric host species were found, but no relationship was found between dactylogyrid community structure and host ecological factors and host geographic distribution. Only a very week relationship was found between dactylogyrid taxonomic composition and host phylogeny, indicating the presence of other species-specific factors driving structure of dactylogyrid communities. C. semeion was uniquely parasitised only by H. aurigae, despite this species being found living among other fish species that harboured more dactylogyrid species. Although six dactylogyrid species were found infecting C. quadrimaculatus, over 95% of the dactylogyrid assemblage was constituted of H. angelopterum. Relative abundances of parasite species varied among chaetodontid species and we identified 6 groups of species that were dominated by different dactylogyrid species. The majority of the species (cluster 5 and most of cluster 6) were characterised by a dominance of H. aurigae, with cluster 5 having very high abundances of H. aurigae. The second most abundant dactylogyrid on butterflyfish was H. angelopterum, which clearly dominated monogenean assemblages of six fish species. H. aurigae and H. angelopterum are two of the largest dactylogyrid species studied and thus we suspect that, when in competition, they may appropriate most of the available space. H. varius, which constituted a group by itself, had the most distinct dactylogyrid community, dominated by E. microphallus; however, neither host ecology or phylogeny explains this marked divergence. H. polylepis had the second most different CHAPTER I -77 -parasite assemblage. This species possesses a very particular ecology, being the only species in the analysis always found on outer-shelf reefs and feeding on plankton which could explain different dactylogyrid assemblages [START_REF] Emslie | Great Barrier Reef butterflyfish community structure: the role of shelf position and benthic community type[END_REF]. H. acuminatus is also a planktivorous fish that inhabits unusually deep waters, which might partly explain its distinct parasite community, but little is known about the ecology of the Australian C. marginalis that could explain the similarity of its dactylogyrid community to that of H. acuminatus.

Since little is known about the ecology of butterflyfish dactylogyrids, their community assemblages could be partly determined by inter-parasite interactions on their hosts as shown in some other systems (e.g. [START_REF] Hughes | Let your enemy do the work: within-host interactions between two fungal parasites of leaf-cutting ants[END_REF]. We did not observe strong negative correlations between dactylogyrid species, but however positively related species could occupy different gill microhabitats in order to avoid direct competition as proposed by Soler-Jiménez and Fajer-Ávila (2012) in dactylogyrids from wild spotted snapper (Lutjanus guttatus, Steindachner, 1868). However, to better understand dactylogyrid interaction with and within the host, more studies should be conducted on parasite microhabitats.

In summary, the gill monogenean communities of 34 sympatric butterflyfish species differ considerably in terms of intensities and in the structure of the dactylogyrid assemblages.

One otherwise unremarkable species, C. lunulatus, was never parasitised, suggesting the involvement of gill species-specific parameters such as chemical cues. Dactylogyrid richness among butterflyfish species is not detectably related to host size, diet, depth range or phylogeny. However, there was a positive (although not strong) correlation between monogenean richness and host geographical range. Finally, we were able to identify six distinct groups of butterflyfishes based on the similarity of their dactylogyrid communities.

Dactylogyrid communities in chaetodontids are evidently extremely complex, and although some differences between fish species could be explained by host geographical range, most of the drivers remain unclear. This suggests that other species-specific parameters or within-host interactions have major roles in shaping the dactylogyrid communities of butterflyfish.

CHAPTER II

Biogeography of gill monogenean communities (Platyhelminthes, Monogenea, Dactylogyridae) of butterflyfish from tropical Indowest Pacific Islands Reverter, M., Cribb, T.H., Cutmore, S.C., Bray, R., Parravicini, V., Sasal, P. Gill monogenean communities (Platyhelminthes, Monogenea, Dactylogyridae) of butterflyfishes from tropical Indo-West Pacific Islands. Parasitology. To be submitted to Journal of Biogeography (IF = 3.9)

Introduction

Parasitism is considered to be the most common lifestyle on earth [START_REF] Lafferty | Parasites in food webs: the ultimate missing links[END_REF].

Parasite impact on host fitness and consequent modification of competitive and trophic interactions among species have profound effects on the community structure and population dynamics [START_REF] Hudson | Is a healthy ecosystem one that is rich in parasites?[END_REF]. Healthy ecosystems are reported to harbor a high diversity of parasites, however, current scenarios of environmental change, biological invasions and emerging diseases are likely to have numerous impacts on parasitic populations [START_REF] Perkins | Invasion biology and parasitic infections[END_REF]McLeod and Poulin, 2012;[START_REF] Altizer | Climate change and infectious diseases: from evidence to a predictive framework[END_REF]. [START_REF] Lafferty | Parasites in food webs: the ultimate missing links[END_REF] showed that fish parasite richness was higher in a well-conserved atoll than in a heavily fished island, indicating the effect of some ecosystem disturbances on the parasite communities of coral reef fish. Therefore, understanding the parasite distribution, biogeography and the relationship between environmental variables and the parasitic communities is highly important in studies on coral reef resilience [START_REF] Sikkel | Habitat and species differences in prevalence and intensity of Neobenedenia melleni (Monogenea: Capsalidae) on sympatric Caribbean surgeonfishes (Acanthuridae)[END_REF]).

Biogeography of free-living organisms such as fish, has been studied consistently and nowadays there are several universal biogeographical theories that explain their patterns of biodiversity at large spatial and temporal scales (Floeter et al. 2007;[START_REF] Briggs | Marine shelf habitat: biogeography and evolution[END_REF][START_REF] Kulbicki | Global biogeography of reef fishes: A hierarchical quantitative delineation of regions[END_REF]. For example, the latitudinal diversity gradient (LDG) which states that diversity increases with latitude decrease or the distance decay in similarity that proposes that similarity in biological communities decreases with increasing biogeographical distance between them [START_REF] Gaston | Global patterns in biodiversity[END_REF][START_REF] Soininen | The distance decay of similarity in ecological communities[END_REF]). However, biogeography of parasites, especially of coral reef fish parasites remains poorly studied and elusive. Since parasites are strictly dependent on their hosts for essential resources (nutrients and physical attachment), their geographical distribution, biodiversity and evolutionary history is closely related to host communities (Poulin et al. 2011b). Besides, in host-parasite interactions, the evolution of parasite-avoidance strategies by hosts and parasite counter-adaptations to overcome host defence mechanisms leads to a tight host-parasite coevolution and parasite specificity [START_REF] Klein | Seoul virus infection increases aggressive behaviour in male Norway rats[END_REF]. Past geological episodes such as geographic isolation between host communities could have been a major determinant in the host-parasite coevolution and current parasite specificity (Poulin et al. 2011a;Martinez-Aquino et al. 2014). Therefore, the study of the biogeography of parasite communities coupled to the study of parasite specificities among multiple hosts can provide insights into the differential evolution of parasitic communities over spatial scales and help us better understand parasite-host interactions.

Beta-diversity was first defined by [START_REF] Whittaker | Vegetation of the Siskiyou Mountains, Oregon and California[END_REF] as the extent of change in community composition among sites. Two different phenomena can produce differences in species compositions between two sites: spatial turnover and nestedness [START_REF] Baselga | Partitioning the turnover and nestedness components of beta diversity[END_REF]. Spatial turnover is the replacement of some species by others from site to site, while nestedness of species assemblages occurs when the community of a poorer site is a subset of a richer site [START_REF] Baselga | Partitioning the turnover and nestedness components of beta diversity[END_REF]2012). The differentiation of the spatial turnover and nestedness components of community dissimilarity is crucial to understand the biogeographic patterns of species, since they can be generated from different underlying processes [START_REF] Williams | Interpreting biogeographical boundaries among Afrotropical birds: spatial patterns in richness gradients and species replacement[END_REF]. For example, spatial turnover patterns suggest the existence of spatial and historical constraints, whereas nestedness reflects a non-random process of species loss [START_REF] Baselga | Partitioning the turnover and nestedness components of beta diversity[END_REF].

Butterflyfishes (Chaetodontidae) are a diverse and emblematic family of coral reef fishes.

They are among the most broadly distributed reef fishes, and their ecologies and distribution patterns have been studied consistently [START_REF] Pratchett | Biology of Butterflyfishes[END_REF][START_REF] Dibattista | Blinded by the bright: a lack of congruence between colour morphs, phylogeography and taxonomy for a cosmopolitan Indo-Pacific butterflyfish, Chaetodon auriga[END_REF]. They, thus, present an outstanding system to study spatial distribution and biogeography of parasites. Butterflyfishes are parasitized by gill monogeneans belonging to the family Dactylogyridae, with a total of 15 species reported from chaetodontids to date [START_REF] Plaisance | Dactylogyrids (Platyhelminthes: Monogenoidea) parasitizing butterfly fishes (Teleostei: Chaetodontidae) from the coral reefs of Palau, Moorea, Wallis, New Caledonia, and Australia: species of Euryhaliotrematoides n. gen. and Aliatrema n[END_REF]Plaisance et al. 2004). Dactylogyrids are the primary monogenean group found on this host family; a few capsalids are the only other monogeneans known to infect chaetodontids [START_REF] Bullard | Six new host records and an updated list of wild hosts for Neobenedenia melleni (MacCallum) (Monogenea: Capsalidae)[END_REF]. Dactylogyrids are small flatworms (ranging from 160 to 490 μm in length) that attach to the gills where they feed and reproduce sexually (Plaisance et al. 2004;[START_REF] Plaisance | Dactylogyrids (Platyhelminthes: Monogenoidea) parasitizing butterfly fishes (Teleostei: Chaetodontidae) from the coral reefs of Palau, Moorea, Wallis, New Caledonia, and Australia: species of Euryhaliotrematoides n. gen. and Aliatrema n[END_REF]. Their eggs leave the host and hatch in the water to release a swimming larva (oncomiracidia) that must find a new host within 12-24 hours [START_REF] Gannicott | Larval survival characteristics and behaviour of the gill monogenean Discocotyle sagittata[END_REF].

The aim of this study is to investigate the biogeography of dactylogyrid parasites from butterflyfishes in order to better understand the spatial structure of coral reef fish ectoparasites in the West Indo-Pacific and to investigate if biogeography is related to the observed dactylogyrid specificity among butterflyfishes [START_REF] Reverter | Gill monogenean communitiyes (Platyhelminthes, Monogenea, Dactylogyridae) of butterflyfishes from tropical Indo-West Pacific Islands[END_REF]. Since the biogeography of dactylogyrid parasites from butterflyfish has been little studied, we first determined the area of distribution, local prevalences and abundances for thirteen dactylogyrid species. We then studied the structure of the dactylogyrid communities at nine geographical locations, and assessed the effect of geographic distance, similarity in host communities and environmental similarities on both components of parasite community dissimilarity: species turnover and community nestedness.

Materials and methods

Sampling

Thirty-four species of butterflyfish (n = 560) were collected at 8 localities in the Pacific: Palau, Wallis and Futuna, Lizard Island (Australia), and the 5 archipelagos in French Polynesia (Society Islands, Tuamotu Islands, Austral Islands, Gambiers Islands and Marquesas Islands). Butterflyfish were also sampled from a single locality in the Indian Ocean, from Ningaloo Reef on the west Australian coast (Figure 1). Adult butterflyfish were captured, put in individual plastic bags with seawater and brought immediately to a laboratory, where gills were dissected and fixed in 70% ethanol.

Dactylogyrids were removed from the gills using a stereo microscope and were mounted on glass slides in Malmberg's ammonium picrate-glycerine.

All dactylogyrids from each individual fish were observed under a compound microscope, and parasite species and number were recorded. Dactylogyrid species determination was according to published literature (Plaisance and[START_REF] Plaisance | Dactylogyrids (Platyhelminthes: Monogenoidea) parasitizing butterfly fishes (Teleostei: Chaetodontidae) from the coral reefs of Palau, Moorea, Wallis, New Caledonia, and Australia: species of Euryhaliotrematoides n. gen. and Aliatrema n[END_REF][START_REF] Kritsky | Dactylogyrids (Monogenoidea: Polyonchoinea) parasitizing the gills of snappers (Perciformes: Lutjanidae): Revision of Euryhaliotrema with new and previously described species from the Red Sea, Persian Gulf, the eastern and Indo-west Pacific Ocean, and the Gulf of Mexico[END_REF] and was based on the morphological characters of the sclerotised structures (the haptor and the reproductive organs).

Sampling was performed in order to be as representative as possible of the butterflyfish community for each of the localities, so differential sampling of butterflyfish species reflects changes in butterflyfish communities between localities.

2 Data analysis

Dactylogyrid prevalence (% of parasitized fish) and mean intensity (number of parasites in parasitized fish) were determined for each dactylogyrid species at each geographic site.

Dactylogyrid areas of distribution were determined using presence-absence of the dactylogyrid species at the 9 studied geographic locations, combined with previous records found in the literature [START_REF] Yamaguti | Monogenetic trematodes of Hawaiian fishes[END_REF][START_REF] Dyer | Monogeneans from marine fishes of Okinawa, Japan[END_REF][START_REF] Zhang | Monogeneans of Chinese marine fishes[END_REF]Plaisance and[START_REF] Plaisance | Dactylogyrids (Platyhelminthes: Monogenoidea) parasitizing butterfly fishes (Teleostei: Chaetodontidae) from the coral reefs of Palau, Moorea, Wallis, New Caledonia, and Australia: species of Euryhaliotrematoides n. gen. and Aliatrema n[END_REF]. Pearson correlation was used to test correlations between dactylogyrid species prevalences.

Parasite communities were studied at each geographic site, and the relative abundance of dactylogyrid species (contribution of a dactylogyrid species to the overall dactylogyrid community expressed in %) and the weighted parasitism intensity were calculated. The weighted parasitism intensity at each geographic site (Iw site ) was computed in order to correct the effect of differential parasitism intensities from different butterflyfish species sampled at different geographic locations [START_REF] Reverter | Gill monogenean communitiyes (Platyhelminthes, Monogenea, Dactylogyridae) of butterflyfishes from tropical Indo-West Pacific Islands[END_REF]. Weighted parasitism (Iw) intensity was calculated according to the following equation:

Iw site = ∑ (Isp site Isp) ⁄ spx sp1 n° sp site
where Isp site is the mean parasitism intensity of a fish species (sp 1,2,x ) at one geographic site, Isp is the global mean parasitism intensity of that fish species and n° sp site is the number of fish species sampled at that geographic site.

Parasite diversity at each geographic site was computed using the Shannon diversity index (H) and parasite richness was also calculated. A hierarchical cluster analysis («hclust » package for R) using the Bray-Curtis distance was used to identify geographic regions with similar monogenean assemblages. The "Average" algorithm was chosen after analysis of the cophenetic correlation coefficients (Pearson correlation between the cophenetic distances calculated on cluster branches and the parasite dissimilarity matrix) [START_REF] Kulbicki | Global biogeography of reef fishes: A hierarchical quantitative delineation of regions[END_REF].

The Kelley-Gardner-Sutcliffe (KGS) penalty function was used to prune the dendogram [START_REF] Bottegoni | AClAP, Autonomous hierarchical agglomerative Cluster Analysis based protocol to partition conformational datasets[END_REF]).

The function "bray.part" from the package betapart (R Software) was used to obtain 3 distance matrices of the parasite communities at different locations, accounting for the balanced variation (species turnover, dBC-bal) and the abundance gradient (species nestedness, dBC-gra) components of Bray-Curtis dissimilarity and the sum of both values (Bray-Curtis dissimilarity, dBC) [START_REF] Baselga | Separating the two components of abundance-based dissimilarity: balanced changes in abundance vs. abundance gradients[END_REF]. Multiple regression on distance matrices (MRM, package ecodist for R, 1000 permutations) was used to study the contribution of sampled fish species, geographic distance and environmental differences between localities on dissimilarity in parasite communities (turnover and nestednees and Bray-Curtis dissimilarity) [START_REF] Lichstein | Multiple regression on distance matrices: a multivariate spatial analysis tool[END_REF]. Linear geographic distances between pairs of geographic locations were obtained. The environmental parameters considered in the MRM analyses were chlorophyll concentration, cloud cover, par radiation, dissolved oxygen, nitrate concentration, phosphate concentration, silicate concentration, salinity and sea surface temperature. Distance-decay curves were generated for factors that had a significant effect in the dissimilarity of parasite communities (Chaetodon species and environmental conditions).

Results

Dactylogyrid area of distribution

Dactylogyrid parasites from butterflyfishes are widely distributed throughout the Indo-West Pacific region, from Ningaloo Reef in the Indian Ocean and the South China Sea to the most western Polynesian archipelago, the Gambiers (Figure 1). Most dactylogyrid species were found at all the sampled locations, and the absence of some rare dactylogyrid species in some locations (prevalence lower than 20% in all the locations) are likely explained by their low prevalence and sampling difficulty (Supp. Material, Table 1). For example, the absence of Euryhaliotrema annulocirrus and E. aspistis in samples from Wallis could be explained by their low prevalence rather than an actual absence and consequent fragmentation in their range between Australia and French Polynesia. Although the different dacytlogyrid species share their habitat, their prevalences and intensities vary at different locations. Thus, 2).

Haliotrema
H. sp. B - 12 - - - 41 - - - - - - - H. scyphovagina - - - - - 23 - - - - - X -

Biogeographic structure of dactylogyrid communities

Weighted intensity, that is, dactylogyrid intensity where the effect of differential host species intensities has been corrected, showed striking differences between geographic locations. Fish from the Marquesas archipelago presented the lowest parasitism intensity (Iw Marquises = 0.57), followed by Palau (Iw Palau = 0.65) and the Gambiers archipelago (Iw Gambiers = 0.67). Fish from the Tuamotu archipelago had the highest number of dactylogyrid parasites (Iw Tuamotu = 1.49) followed by the Society archipelago (Iw Society = 1.12) and Ningaloo Reef (Iw NR = 1.09) (Table 2, Figure 3).

Twelve out of the thirteen identified dactylogyrid species were found at the Tuamotu archipelago and Lizard Island (Table 1). Wallis and the Marquesas islands were the two locations where the lowest number of dactylogyrid species were found (6) (Table 2).

Dactylogyrid diversity (H) was variable between geographic locations. Highest dactylogyrid diversity was found at Lizard Island (1.96) on the Great Barrier Reef, followed by the were the most abundant parasites in the Marquesas (Table 2, Figure 3).

Factors shaping biogeographic differences in dactylogyrid communities

Multiple regression models showed that most of the differences between dactylogyrid communities from geographic sites is due to differences in the butterflyfish communities (Table 2). Distance decay curve showed that similarity of dactylogyrid communities decreases with increasing butterflyfish dissimilarity (R 2 = 0.64) (Figure 5a). The Chaetodon species sampled at different locations (both balanced variation and abundance gradient) had a significant effect on the balanced variation (dBC-bal) of the dactylogyrid communities (Table 2). Distance decay curves showed that balanced variation similarity of dactylogyrid communities (dBC-bal) decreases with an increase in the dissimilarity of the butterflyfish communities (dBC-gra, R 2 = 0.39) (Figure 5b). Environmental parameters had a significant effect on the gradient variation (dBC-gra) of dactylogyrid communities and distance decay curve showed that the gradient variation similarity of dactylogyrid communities decreased with increasing environmental dissimilarity (R 2 = 0.32) (Table 2, Figure 5c). Linear distances between geographic locations did not have a significant effect on the dissimilarity (dBC-bal, dBC-gra and dBC) of the dactylogyrid communities between sites (Table 2). Most dactylogyrid species from butterflyfishes were broadly distributed throughout the Pacific Ocean with the exception of some rare species that were absent at some locations.

However, since prevalences of rare dactylogyrid species were, by definition, low, they could have been missed from our sampling. Polynesia and the western Indo-Pacific region.

The Coral Triangle that encompasses the Philippines, Indonesia and New Guinea is the area of highest marine biodiversity in the world, with marine richness radially decreasing from it [START_REF] Bellwood | Regional-scale assembly rules and biodiversity of coral reefs[END_REF][START_REF] Carpenter | The center of the center of marine shore fish biodiversity: the Philippine Islands[END_REF]. For example, richness of shore fishes declines from 1693 at the center of the Coral Triangle in the Philippines to 1579 in the main island of New Caledonia, 702 fish species in the Society Islands and just 245 fish species in the Gambier Archipelago (Kulbicki, 2007). Biodiversity decrease of parasitic organisms with increasing distance from the Coral Triangle has been much less studied.

However, a few works have shown a dramatic reduction of parasite richness from west to east Pacific [START_REF] Rigby | Patterns of species diversity in the gastrointestinal helminths of a coral reef fish, Epinephelus merra (Serranidae), from French Polynesia and the South Pacific Ocean[END_REF][START_REF] Mcnamara | Monorchiids (Platyhelminthes: Digenea) of chaetodontid fishes (Perciformes): Biogeographical patterns in the tropical Indo-West Pacific[END_REF][START_REF] Cribb | Biogeography of tropical Indo-West Bibliography -293 -Pacific parasites: a cryptic species of Transversotrema and evidence for rarity of Transversotrematidae (Trematoda) in French Polynesia[END_REF]. In contrast, the few studies on butterflyfish dactylogyrids seemed to indicate greater parasite richness in French Polynesia (Plaisance et al. 2004, Plaisance and[START_REF] Plaisance | Dactylogyrids (Platyhelminthes: Monogenoidea) parasitizing butterfly fishes (Teleostei: Chaetodontidae) from the coral reefs of Palau, Moorea, Wallis, New Caledonia, and Australia: species of Euryhaliotrematoides n. gen. and Aliatrema n[END_REF]. Results from our larger study including 34 butterflyfishes and 13 monogenean parasites do not support a decrease in parasite richness or diversity from the Coral Triangle. Instead, we have discovered high parasite richness and diversity both in French Polynesia and the Great Barrier Reef. The lowest parasite richness in the Marquesas Islands is potentially explained by the isolation of this archipelago together with the absence of lagoons and very low coral cover and therefore a lower number of susceptible host species and number of individuals (Kulbicki, 2007). [START_REF] Bellwood | Regional-scale assembly rules and biodiversity of coral reefs[END_REF] proposed that island size influenced local diversity, with smaller island supporting fewer species. Sampling in the Society archipelago was done in Scilly and Mopelia, two young small islands situated at the most western part of French Polynesia. Lower fish richness has been observed in those islands than in other Society Islands like Moorea, which is consistent with the lower dactylogyrid diversity observed there.

The cluster analysis showed that three main biogeographic regions can be recognised based on differences in the dactylogyrid communities of butterflyfishes: French Polynesia, the Distance decay in similarity, which states that similarity in species composition decreases with increasing distance between localities, is considered a universal pattern in ecology [START_REF] Soininen | The distance decay of similarity in ecological communities[END_REF][START_REF] Morlon | A general framework for the distance-decay of similarity in ecological communities[END_REF]. Numerous studies have shown that similarity of parasite communities decreases with increasing distances (Oliva and Gonzalez, 2005;[START_REF] Pérez-Del-Olmo | Not everything is everywhere: the distance decay of similarity in a marine host-parasite system[END_REF][START_REF] Thieltges | Distance decay of similarity among parasite communities of three marine invertebrate hosts[END_REF][START_REF] Timi | Host ontogeny and the temporal decay of similarity in parasite communities of marine fish[END_REF]. However, some other studies have shown that other parameters such as host phylogenetic distance or environmental conditions might play a more important role in similarity of parasite communities than geographical distance [START_REF] Krasnov | Spatial variation in species diversity and composition of flea assemblages in small mammalian hosts: geographical distance or faunal similarity?[END_REF][START_REF] Vinarski | Decay of similarity of gamasid mite assemblages parasitic on Palaearctic small mammals: geographic distance, host-species composition or environment[END_REF]Seifertova et al. 2008). To date, all the studies on parasite community similarities have been either on qualitative (i.e.

species presence or absence) or quantitative data (i.e. species abundances). However, recent studies have shown that incidence-based measures of dissimilarities reflect two different phenomena, spatial species turnover, which is the species replacement, and nestedness of assemblages, which accounts for abundance gradients [START_REF] Baselga | Partitioning the turnover and nestedness components of beta diversity[END_REF]2012). To the best of our knowledge, this is the first study on the biogeography of parasite communities that distinguished between the balanced variation (turnover) and abundance gradient (nestedness) components of dissimilarity in order to identify which parameters are driving each of the processes. Our results show that geographic distance between host populations was not a significant factor on either the turnover or nestedness of the parasite community. However, similarity of the host populations between sites was identified as the main driver responsible for the turnover of the parasite community and for the overall similarity in parasite communities. In a previous study, we showed that different butterflyfish species, even sympatric species, presented well delineated communities of dactylogyrids, and we identified ornatissimus) [START_REF] Reverter | Gill monogenean communitiyes (Platyhelminthes, Monogenea, Dactylogyridae) of butterflyfishes from tropical Indo-West Pacific Islands[END_REF]). Prevalence differences (and a possible turnover) of these two dactylogyrid species, as discussed above, are among the main differences between the two geographic regions identified (French Polynesia and the western Indo-Pacific). H. aurigae and H. angelopterum are phylogenetically close, and they are among the biggest species of dactylogyrid parasites found in butterflyfishes [START_REF] Plaisance | Molecular phylogeny of gill monogeneans (Platyhelminthes, Monogenea, Dactylogyridae) and colonization of Indo-West Pacific butterflyfish hosts (Perciformes, Chaetodontidae)[END_REF]. We could therefore imagine that the evolutionary history of these two parasite species, coupled to geological processes that determine their biogeography, have shaped parasite specificity over time through tight parasite-host coevolution. We suggest that H. aurigae and H. angelopterum might have diverged from a common ancestor for example during French Polynesia Pleistocene isolation.

Recent studies have shown that environmental factors such as salinity or temperature are important factors in in driving the composition of marine parasite communities [START_REF] Thieltges | Salinity gradient shapes distance decay of similarity among parasite communities in three marine fishes[END_REF]Poulin et al. 2011b;[START_REF] Blasco-Costa | Biogeography of parasitism in freshwater fish: spatial patterns in hot spots of infection[END_REF]. In our study, environmental dissimilarity was the most important factor explaining nestedness of parasite communities. [START_REF] Pietrock | Free-living endohelminth stages: at the mercy of environmental conditions[END_REF] reported that free-living infective stages of helminth parasites can only survive and successfully infect a host within narrow and species-specific set of environmental conditions. Since chaetodontid larvae are not parasitized by monogenean parasites and dactylogyrid oncoramicidia can only live a few hours without a host, it has been proposed that dactylogyrid dispersal takes places through the passive dispersion of their eggs by oceanic currents (Ganicott and Tinsley, 1998;[START_REF] Plaisance | Colonization of Pacific islands by parasites of low dispersal ability: phylogeography of two monogenean species parasitizing butterflyfishes in the South Pacific Ocean[END_REF]. Several studies have shown that monogenean egg hatching is highly dependent on environmental parameters such as water temperature [START_REF] Cecchini | Influence of temperature on the life cycle of Diplectanum aequans (Monogenea, Diplectanidae), parasitic on sea bass, Dicentrarchus labrax (L.)[END_REF][START_REF] Tubbs | Effects of temperature on fecundity in vitro, egg hatching and reproductive development of Benedenia seriolae and Zeuxapta seriolae (Monogenea) parasitic on yellowtail kingfish Seriola lalandi[END_REF]. Our results show that environmental parameters affect the gradient abundance of parasite species but not the parasite turnover, indicating the effect of environmental conditions on the suitability for egg hatching, larval development and successful infection of their hosts.

This study dramatically increases knowledge of dactylogyrid communities of butterflyfishes and their distribution in the West Indo-Pacific region. However, more studies should be conducted to determine the full distribution of these species, which might include other oceans (e.g. E. annulocirrus has been recorded in the Red Sea) [START_REF] Paperna | Monogenea of Red Sea Fishes: III. Dactylogyridae from littoral and reef fishes[END_REF]. We have observed two main biogeographic regions that are mainly differentiated by a turnover in the two major parasite species belonging to the genus Haliotrema. Differences between Chaetodon assemblages are the main factor influencing parasite community turnover, and coupled with the results from a previous work [START_REF] Reverter | Gill monogenean communitiyes (Platyhelminthes, Monogenea, Dactylogyridae) of butterflyfishes from tropical Indo-West Pacific Islands[END_REF], this suggests that biogeographical processes shaped parasite specificity probably through tight parasite-host coevolution. Environmental dissimilarity arose as the main factor in the nestedness of parasite communities, indicating the importance of favorable environmental condition for dactylogyrid egg hatching and effective host infection.

Finally, butterflyfishes are well known for their propensity to hybridize [START_REF] Montanari | Does genetic distance between parental species influence outcomes of hybridization among coral reef butterflyfishes?[END_REF], thus the study of dactylogyrid communities between hybrids and closely related species from different oceans might provide helpful insights in their biogeography and evolutionary history. Similarly, the integrated study of the biogeography of direct cycle parasites (monogeneans) and complex cycle parasites (digeneans) might also provide new insights in the biogeography of coral reef parasites.

Introduction

To better understand ecosystem dynamics, it is necessary to understand the complex network of interactions between organisms [START_REF] Rossberg | A complete analytic theory for structure and dynamics of populations and communities spanning wide ranges in body size[END_REF]. Fish are among the most biologically complex organisms inhabiting marine ecosystems, presenting specialised organs and a central nervous system like higher vertebrates with well-developed sensory systems [START_REF] Bone | Biology of fishes. 3rd Ed[END_REF]. Fish biology and ecology have been studied for a long time, and there is extensive information of intraspecific communication between fish [START_REF] Timms | Intraspecific communication in goldfish[END_REF].

Studies have shown that fish interact through visual, acoustic, electric or chemical signals (e.g., [START_REF] Sorensen | Brief review of fish pheromones and discussion of their possible uses in the control of non-indigenous teleost fishes[END_REF][START_REF] Moller | Electrocommunication: history, insights, and new questions[END_REF]). However, there is still little knowledge on how interspecific interactions occur and what are the signals involved. Some studies have shown that interspecific communication mainly occurs through chemical signaling [START_REF] Wisenden | Anti-predator behaviour in response to conspecific chemical alarm cues in an esociform fish, Umbra limi (Kirtland 1840)[END_REF]Hay 2009). For example, [START_REF] Kidawa | Fish blood as a chemical signal for Antarctic marine invertebrates[END_REF] showed that blood from Black rockcod (Notothenia corriceps, Nototheniidae) acted as a chemical attracting signal to several necrophagous invertebrate species. However, there are very little studies where the chemical molecules involved in interspecific communication in fish have been identified and characterised, most probably because of the biological complexity of fish, rendering chemical isolation especially difficult [START_REF] Ito | Detection of tetrodotoxin (TTX) from two copepods infecting the grass puffer Takifugu niphobles: TTX attracting the parasites?[END_REF][START_REF] Ohashi | Purification and identification of a glycoprotein that induces the attachment of oncomiracidia of Neobenedenia girellae (Monogenea, Capsalidae)[END_REF]).

In fish, the mucosal layers recovering the epidermis and gills is the main surface of exchange between them and their surrounding environment. Several studies have shown fish mucus involvement in intra and interspecific chemical communication [START_REF] Todd | Chemical communication in social behavior of a fish, the yellow bullhead (Ictalurus natalis)[END_REF]Bekliougu et al. 2006). Mucus acts as a thin dynamic physical and biochemical barrier that enables nutrient, water, gas and molecule diffusion (Shepard 1993). Mucus displays numerous biological and ecological roles such as osmoregulation [START_REF] Shephard | Mucus on the epidermis of fish and its influence on drug delivery[END_REF], protection against abrasion [START_REF] Oosten | Skin and scales[END_REF], protection against environmental toxins and heavy metal toxicity [START_REF] Coello | Protection against heavy metal toxicity by mucus and scales in fish[END_REF], parental feeding [START_REF] Chong | Proteomics profiling of epidermal mucus secretion of a cichlid (Symphysodon aequifasciata) demonstrating parental care behavior[END_REF], protection against pathogens [START_REF] Gómez | Neglected wild life: Parasitic biodiversity as a conservation target[END_REF]) and chemical mediation [START_REF] Leonard | Finding paradise: cues directing the migration of the waterfall climbing Hawaiian gobioid Sicyopterus stimpsoni[END_REF]).

Teleost mucus is very similar to mammalian mucus and is mainly composed of highly glycosylated glycoproteins called mucins [START_REF] Shephard | Mucus on the epidermis of fish and its influence on drug delivery[END_REF]. Fish mucus contains numerous immune molecules such as lysozymes, immunoglobulins, complement and lectins, antimicrobial peptides (AMPs) and antimicrobial proteins [START_REF] Esteban | An overview of the immunological defenses in fish skin[END_REF]. Other molecules present in fish mucus include mycosporine-like amino acids (MAAs), toxins or kairomones (uncharacterized molecules that serve as infochemicals to other species) [START_REF] Shephard | Mucus on the epidermis of fish and its influence on drug delivery[END_REF][START_REF] Purcell | Electrical responses to water-soluble components of fish mucus recorded from the cnidocytes of a fish predator, Physalia physalis[END_REF].

Interest in fish mucus has been growing in the last decade due to the discovery of numerous bioactive molecules (antibacterial, antiviral, antifungal and antiparasitic) and their potential applications to human medicine and aquaculture [START_REF] Rakers | Antimicrobial peptides (AMPs) from fish epidermis: perspectives for investigative dermatology[END_REF][START_REF] Beck | Mucosal Health in Aquaculture[END_REF]. Most studies have focused on immune-related molecules and AMPs, however, few studies have analysed other mucus molecules and their ecological roles in the environment. Secondary metabolites, for example, even though they are known to play key roles in the communication of a wide range of species including plants, invertebrates and microorganisms have been rarely studied in fish tissues or mucus (Hay et al. 2009).

There are numerous research and review articles studying specific topics on fish mucus (e.g. [START_REF] Rakers | Antimicrobial peptides (AMPs) from fish epidermis: perspectives for investigative dermatology[END_REF][START_REF] Lazado | Mucosal immunity and probiotics in fish[END_REF][START_REF] Larsen | Community Structure of Skin Microbiome of Gulf Killifish, Fundulus grandis, Is Driven by Seasonality and Not Exposure to Oiled Sediments in a Louisiana Salt Marsh[END_REF]. However, none of them have studied fish mucus under a holistic approach, assessing its importance both for the fish and the ecosystem. This original critical synthesis aims to gather and analyze most of the current knowledge on skin and gill fish mucus, in order to highlight fish mucus importance in the marine ecosystem as well as identify the gaps in knowledge and provide future directions. We discuss the mucus composition, including the hardly studied mucus microbiome and mycobiome, and we discuss for the first time the fish mucus biological and ecological roles in species interactions. We also examine the state of fish mucus research and identify novel research priorities, focusing on the use of "omics" technologies to discover novel mucus molecules and study the mucus microbiome.

Mucus composition and biological activities

Mucus is produced by goblet and club cells found in the epithelium and it is constantly being secreted and shed, rendering pathogen infections difficult [START_REF] Shephard | Mucus on the epidermis of fish and its influence on drug delivery[END_REF][START_REF] Cone | Barrier properties of mucus[END_REF].

Mucus composition and its rheological properties are vital for the maintenance of mucus functions [START_REF] Lai | Micro-and macrorheology of mucus[END_REF]. For example, mucus viscoelasticity determines the ability to block many types of motile bacteria [START_REF] Cone | Barrier properties of mucus[END_REF], and decreased secretion rates have been associated with increased infection rates (Tabak 1995).

Numerous biological activities have been found in fish mucus, contributing greatly to recent development of fish mucus research and rendering fish mucus molecules (e.g. AMPs)

interesting to veterinary and human medicine research. Antibacterial, antifungal and antiviral activities against human and fish pathogens have been found in many species of fish as well as proteolytic, cytotoxic, pro-apoptotic and antitumor activities (e.g. [START_REF] Hellio | Antibacterial, antifungal and cytotoxic activities of extracts from fish epidermis and epidermal mucus[END_REF][START_REF] Kwak | Induction of Apoptosis and Antitumor Activity of Eel Skin Mucus, Containing Lactose-Binding Molecules, on Human Leukemic K562 Cells[END_REF]. Even if mucus composition has been studied intensively, especially its antimicrobial compounds, there is still limited knowledge on most of the fish mucus components. Here, we summarise the different identified fish mucus components to date, and discuss their biological roles and the gaps in knowledge (Figure 1 -mucus composition and ecological roles). CHAPTER III -111 -

Glycoproteins and other carbohydrate derivatives

Fish mucus is mainly constituted by high molecular weight, filamentous, O-glycosylated proteins (GPs) also called mucins. GPs are responsible for the viscoelastic and adhesive properties of mucus. Since glycoprotein characteristics depend on their carbohydrate chains, characterisation of the carbohydrate chains is important to better understand mechanisms of fish disease resistance (Estensoro et al. 2012;[START_REF] Pérez-Sánchez | Mucins as Diagnostic and Prognostic Biomarkers in a Fish-Parasite Model: Transcriptional and Functional Analysis[END_REF].

Several studies showed differences in glycoprotein composition depending on the species habits and habitat characteristics [START_REF] Gona | Mucous glycoproteins of teleostean fish: a comparative histochemical study[END_REF]), but also between different fish tissues (epithelium, gills and intestines) [START_REF] Jin | Atlantic Salmon carries a range of novel O-glycan structures differentially localized on skin and intestinal Mmucins[END_REF]. For example, [START_REF] Kumari | Histochemical analysis of glycoproteins in the secretory cells in the gill epithelium of a catfish, Rita rita (Siluriformes, Bagridae)[END_REF] found differences in the composition and concentration of GPs between the epithelium of the gill arches and the gill rakers (abundance of GPs with O-sulphate esters) and the gill fillaments and the secondary lamellae (abundance of GPs with sialic acid) in the catfish Rita rita (Bagridae), indicating an adaptation of mucus viscosity to optimise physiological functions.

Based on their structural and functional features, mucins are grouped in two different families: secreted mucin glycoproteins, which are highly O-glycosylated proteins assembled in oligomers giving mucus its viscosity [START_REF] Thornton | Structure and function of the polymeric mucins in airways mucus[END_REF], and membrane-bound forms, which are cell-surface mucins containing an extracellular O-glycosylated domain and can form long filaments carrying oligosaccharides, which can act as ligands in microbial adhesions [START_REF] Mcguckin | Mucin dynamics and enteric pathogens[END_REF][START_REF] Beck | Mucosal Health in Aquaculture[END_REF]. Histochemical studies have shown presence of neutral and acidic mucins, the latter including both sialomucins and sulfomucins [START_REF] Mittal | Histochemical analysis of glycoproteins in the unicellar glands in the epidermis of an Indian freshwater fish Mastacembelus pancalus (Hamilton)[END_REF]. [START_REF] Ebran | Isolation and characterization of novel glycoproteins from fish epidermal mucus: correlation between their pore-forming properties and their antibacterial activities[END_REF] and [START_REF] Molle | First evidence of the poreforming properties of a keratin from skin mucus of rainbow trout (Oncorhynchus mykiss, formerly Salmo gairdneri)[END_REF] isolated and characterised novel glycoproteins from different fish skin mucus with strong pore-forming and antibacterial activities. Transferrin, a globular glycoprotein with a high iron chelator power, was also identified in the mucus of Atlantic cod (Gadus morhua, Gadidae) and Atlantic salmon (Salmo salar, Salmonidae), suggesting an involvement in innate immunity [START_REF] Raeder | Effect of fish skin mucus on the soluble proteome of Vibrio salmonicida analysed by 2-D gel electrophoresis and tandem mass spectrometry[END_REF][START_REF] Easy | Identification of transferrin in Atlantic cod Gadus morhua epidermal mucus[END_REF]). However, even if there is extensive literature on the subject, the chemical composition and properties of most GPs in fish mucus remain poorly understood (Sumi et al.

2004).

Besides GPs, other types of carbohydrate molecules exist in fish mucus. Qin et al. (2002a) isolated a novel polysaccharide from Pond loach (Misgurnus anguillicaudatus, Cobitidae), known as misgurnan or MAP. MAP proved to have multiple bioactivities, such as, antioxidant, apoptotic and immunomodulating activities (Qin et al. 2002b;Zhang andHuang 2005a, 2005b).

Proteins and peptides

Mucosal tissues in fish contain a very diverse array of proteins, including some structural proteins, immune-related proteins and antimicrobial peptides, which display varied biological roles within the fish biology.

Although, reports of structural proteins in mucus are scarce, some studies have shown the presence of high levels of actin in fish epidermal mucus, suggesting that actin may have a discrete role in mucus structure [START_REF] Easy | Changes in Atlantic salmon Salmo salar mucus components following shortand long-term handling stress[END_REF]. Keratin and calmodulin were also identified in salmon epidermal mucus [START_REF] Easy | Changes in Atlantic salmon Salmo salar mucus components following shortand long-term handling stress[END_REF]. Presence of calmodulin in fish mucus suggests an enzymatic involvement in the control of integumental permeability [START_REF] Flik | Evidence for the presence of calmodulin in fish mucus[END_REF]. Cholinesterases have also been identified in skin mucus of carps, and it has been suggested that they can interact with organophosphate pesticide molecules and thereby neutralise them and decrease skin penetration [START_REF] Nigam | The first evidence of cholinesterases in skin mucus of carps and its applicability as biomarker of organophosphate exposure[END_REF].

Recently, the use of new techniques such as proteomics has enabled the identification of many proteins unreported to date in fish mucus, including some immune-related proteins (galectins, caspases, concanavalin A, FK-506 binding protein, cyclophilin A and cystitis B), proteins involved in cellular metabolism (ubiquitin, glutathione hydrolase and transferase and calreticulin), carbohydrate metabolism (citrate synthase and enolase andglyceraldehyde-3phosphate dehydrogenase) and proteins involved in lipid metabolism (preapolipoprotein Aa and fatty acid-binding protein) [START_REF] Rajan | Proteome reference map of the skin mucus of Atlantic cod (Gadus morhua) revealing immune competent molecules[END_REF][START_REF] Rajan | Differentially expressed proteins in the skin mucus of Atlantic cod (Gadus morhua) upon natural infection with Vibrio anguillarum[END_REF][START_REF] Easy | Identification of transferrin in Atlantic cod Gadus morhua epidermal mucus[END_REF][START_REF] Cordero | Skin mucus proteome map of European sea bass (Dicentrarchus labrax)[END_REF].

Immune related proteins

Fish mucus is enriched with a multitude of immune related proteins such as lysozymes, phosphatases, esterases, proteolytic enzymes, complement factors, lectins, immunoglobulins, C-reactive proteins and lymphocytes that attempt to eliminate pathogens and launch the immune cascade when an infection occurs [START_REF] Guardiola | Comparative skin mucus and serum humoral defence mechanisms in the teleost gilthead seabream (Sparus aurata)[END_REF].

Lysozyme, is a well-studied bacteriolytic enzyme that acts disrupting mucopolysaccharides in the bacterial cell walls and thus, cause bacteria death [START_REF] Saurabh | Lysozyme: an important defence molecule of fish innate immune system[END_REF]. Two isoforms of lysozyme that are similar to goose (g) type and chicken (c) type in vertebrates have been detected in fish mucus [START_REF] Nigam | Comparative analysis of innate immune parameters of the skin mucous secretions from certain freshwater teleosts, inhabiting different ecological niches[END_REF].

Acid and alkaline phosphatases and esterases are also important enzymes found in fish mucus; however their function in fish mucosal immunity is not completely understood [START_REF] Nigam | Comparative analysis of innate immune parameters of the skin mucous secretions from certain freshwater teleosts, inhabiting different ecological niches[END_REF]. Alkaline phosphatases in mucus have shown to act as antibacterial agent, to play a protective role in the initial stages of wound healing and to be potential stress indicators in skin mucus of fish [START_REF] Rai | Histochemical response of alkaline phosphatase activity during the healing of cutaneous wounds in a cat-fish[END_REF][START_REF] Ross | Changes in hydrolytic enzyme activities of naïve Atlantic salmon Salmo salar skin mucus due to infection with the salmon louse Lepeophtheirus salmonis and cortisol implantation[END_REF].

Proteases provide protection against fish infections, either directly, by cleaving pathogen proteins, or indirectly, by modifying mucus consistency and activating the production of other immune molecules such as complement, immunoglobulins or AMPs [START_REF] Esteban | An overview of the immunological defenses in fish skin[END_REF].

Different types of proteases (trypsins, metalloproteases, cathepsins and aminopeptidases)

have been identified in fish skin mucus [START_REF] Esteban | An overview of the immunological defenses in fish skin[END_REF]. Serine-and metalloproteases have been reported as the predominant proteases in several fish species [START_REF] Salles | Identification and characterization of proteases from skin mucus of tambacu, a Neotropical hybrid fish[END_REF][START_REF] Tvete | Purification and characterization of a 630 kDa bacterial killing metalloprotease (KilC) isolated from plaice Pleuronectes platessa (L.), epidermal mucus[END_REF]. Metalloproteases are associated with the immune response, tissue repair and in the activation of pro-cathepsin D in cathpesin D, a protease that regulates secretion of AMPs [START_REF] Cho | Matrix metalloproteinase 2 is involved in the regulation of the antimicrobial peptide parasin I production in catfish skin mucosa[END_REF][START_REF] Pedersen | Matrix metalloproteinases in fish biology and matrix turnover[END_REF]).

The complement system plays an important role in the innate immune system and elimination of pathogens in fish [START_REF] Boshra | Recent advances on the complement system of teleost fish[END_REF]. And some studies have identified complement components present in fish mucus, such as C7 in skin and intestine mucus of grass carp, C3 in skin mucus of Atlantic halibut (Hippoglossus hippoglossus, Pleuronectidae) and C1q in skin mucus of Siberian sturgeon (Acipenser baerii, Acipenseridae) [START_REF] Shen | Expression of complement component C7 and involvement in innate immune responses to bacteria in grass carp[END_REF][START_REF] Fan | Functional C1q is present in the skin mucus of Siberian sturgeon (Acipenser baerii)[END_REF].

Fish mucus also contains a wide variety of lectins (e.g. Suzuki et al. 2003;[START_REF] Tsutsui | A unique epidermal mucus lectin identified from catfish (Silurus asotus): first evidence of intelectin in fish skin slime[END_REF]. Several studies have shown that lectins in fish mucus are able to agglutinate several bacterial strains or bind to other parasites such as the parasitic nematode Heterobothrium okamotoi, suggesting the important role of mucus lectins in host defences [START_REF] Tsutsui | Possible role of a skin mucus lectin from fugu Takifugu rubripes in excluding marine bacteria from the body surface[END_REF].

Natural antibodies such as immonuglobulins are also present in fish mucus and they play a key role both in innate and adaptive immunity, producing specific antibody responses against various antigens [START_REF] Coscia | Investigation of immunoglobulins in skin of the Antarctic teleost Trematomus bernacchii[END_REF]. Although three types of immunoglobulins have been identified in teleost fish, (IgM, IgT and IgD), only IgM and IgT have been found in fish mucus (Sunyer 2013). IgT seems to play a major role in fish mucosal immunity, while IgM is present at low levels in mucosal surfaces [START_REF] Zhang | IgT, a primitive immunoglobulin class specialized in mucosal immunity[END_REF][START_REF] Xu | Teleost skin, an ancient mucosal surface that elicits gut-like immune responses[END_REF].

Although most immune-related proteins in fish mucus have been discovered by classic biochemical approaches, the use of new techniques such as genomics, transcriptomics and proteomics is allowing researchers a more rapid and efficient identification of fish mucus immune-related molecules, as well as elucidating their sources of variability and biological roles [START_REF] Cordero | Skin mucus proteome map of European sea bass (Dicentrarchus labrax)[END_REF].

AMPs and antimicrobial proteins

An important component of fish mucus with multiple biological activities are antimicrobial peptides (AMPs). AMPs are nearly found in all life forms and are normally small cationic peptides (less than 50 aminoacids, < 10 kDa), amphipathic and encoded by single genes [START_REF] Zasloff | Antimicrobial peptides of multicellular organisms[END_REF]. AMPs typically exhibit activities against multiple pathogens including Gram-positive and Gram-negative bacteria, fungi, viruses and parasites. Certain AMPs can also function as immunomodulators [START_REF] Bowdish | Immunomodulatory activities of small host defense peptides[END_REF]. Interest in fish AMPs, and especially in fish mucus AMPs has risen recently, for their potential use both in aquaculture and human medicine as antimicrobial agents, vaccine adjuvants, inactivated vaccines, antitumor agents and even in dermatology [START_REF] Noga | Application of antimicrobial polypeptide host defenses to aquaculture: Exploitation of downregulation and upregulation responses[END_REF]Rajanabu and Chen 2011;[START_REF] Rakers | Antimicrobial peptides (AMPs) from fish epidermis: perspectives for investigative dermatology[END_REF].

Although AMPs are highly heterogeneous in their primary structure, three main classes of AMPs have been described in teleost fish (α-helical peptides, cysteine-rich AMPs and AMPs derived from functional proteins). α-helical peptides, comprise the most common AMP family in fish, the piscidins, peptides with a short, linear structure, which adopt α-helical conformation when interacting with bacterial membranes [START_REF] Smith | Conventional and Unconventional Antimicrobials from Fish, Marine Invertebrates and Micro-algae[END_REF]. The piscidins family includes the moronecidins (piscidins), pleurocidins, dicentracins and chrysopsins (Masso- [START_REF] Masso-Silva | Antimicrobial peptides from fish[END_REF]. Several immunohistochemistry and genomic studies have reported piscidins to be widespread among teleost fish [START_REF] Silphaduang | Evidence for widespread distribution of piscidin antimicrobial peptides in teleost fish[END_REF][START_REF] Salerno | cDNA sequence and tissue expression of an antimicrobial peptide, dicentracin; a new component of the moronecidin family isolated from head kidney leukocytes of sea bass, Dicentrarchus labrax[END_REF]. Piscidins 1 and 2 (Pis1 and Pis2 genes) have been found in Atlantic cod epidermal mucus and whereas Pis2 was found expressed similarly in all tissues, the strong expression of Pis1 in mucus suggests its involvement with systemic and mucosal immunity [START_REF] Ruangsri | Differential expression and biological activity of two piscidin paralogues and a novel splice variant in Atlantic cod (Gadus morhua L.)[END_REF]. Piscidins present broad antibacterial, antifungal, antiparasitic and antitumoral activities (Masso- [START_REF] Masso-Silva | Antimicrobial peptides from fish[END_REF]. Pleurocidins, first isolated from the skin mucus of winter flounder (Pleuronectes americanus, Pleuronectidae), have broad-spectrum antimicrobial activities and inhibit DNA, RNA and protein synthesis [START_REF] Cole | Isolation and characterization of pleurocidin, an antimicrobial peptide in the skin secretions of winter flounder[END_REF][START_REF] Cole | Characterization of a fish antimicrobial peptide: gene expression, subcellular localization, and spectrum of activity[END_REF]. Dicentracin is a peptide isolated from several organs of european sea bass (Dicentrarchus labrax, Moronidae), including skin and gills and seems to have an important defensive role in the fish mucosal tissues [START_REF] Salerno | cDNA sequence and tissue expression of an antimicrobial peptide, dicentracin; a new component of the moronecidin family isolated from head kidney leukocytes of sea bass, Dicentrarchus labrax[END_REF]. Chrysopsin is also an AMP from the piscidin family found in mucus of European sea bass [START_REF] Fekih-Zaghbib | A complementary LC-ESI-MS and MALDI-TOF approach for screening antibacterial proteomic signature of farmed European Sea bass mucus[END_REF]. Other linear AMPs described in fish mucus include pardaxin from the skin mucus of the Moses sole (Pardachirus marmoratus, Soleidae) and pelteobagrin from the skin mucus of the yellow catfish (Pelteobagrus fulvidraco, Bagridae) [START_REF] Oren | A class of highly potent antibacterial peptides derived from pardaxin, a poreforming peptide isolated from Moses sole fish Pardachirus marmoratus[END_REF]Su, 2011).

Amongst the cysteine-rich AMPs in teleost fish are three families: defensins, cathelicidins and hepcidins [START_REF] Smith | Conventional and Unconventional Antimicrobials from Fish, Marine Invertebrates and Micro-algae[END_REF]. The most prominent group of cysteine-rich AMPs are the defensins. Several β-defensin genes have been identified in mucosal tissues of several fish species and besides their broad antibacterial and antiviral activity they seem to be an important component of the innate defences of fish [START_REF] Casadei | Bibliography -291 -Characterization of three novel beta-defensin antimicrobial peptides in rainbow trout (Oncorhynchus mykiss)[END_REF][START_REF] Marel | Molecular cloning and expression of two β-defensin and two mucin genes in common carp (Cyprinus carpio L.) and their up-regulation after β-glucan feeding[END_REF][START_REF] Ruangsri | A Novel Beta-Defensin Antimicrobial Peptide in Atlantic Cod with Stimulatory Effect on Phagocytic Activity[END_REF]. Although some cathelicidins have been reported in several fish species, little is known about their function and importance in fish [START_REF] Scocchi | The salmonid cathelicidins: a gene family with highly varied C-terminal antimicrobial domains[END_REF]). To date, cathelicidins have not been reported in fish mucus, however, [START_REF] Chang | Identification of a novel cathelicidin gene in the rainbow trout, Oncorhynchus mykiss[END_REF] found expression of a cathelicidin gene (rtCATH_1) on the skin of the rainbow trout. Hepcidins are highly disulphide bonded β-sheet peptides, and have been identified in various tissues of fish, including skin; although, higher expression levels have been found in liver and head kidneys [START_REF] Huang | Three different hepcidins from tilapia, Oreochromis mossambicus: analysis of their expressions and biological functions[END_REF][START_REF] Yang | Genomic organization and tissue-specific expression analysis of hepcidin-like genes from black porgy (Acanthopagrus schlegelii B)[END_REF]. AJN-10 is a novel cysteine-rich AMP isolated from the skin mucus of Japanese eel (Anguilla japonica, Anguillidae) [START_REF] Liang | Isolation and identification of a novel inducible antibacterial peptide from the skin mucus of Japanese eel, Anguilla japonica[END_REF].

While most AMPs are derived from a biologically inactive proprotein that is processed to the active form, some AMPs are derived from larger, functional proteins that have primary functions other than immune defense [START_REF] Ullal | Antimicrobial peptides derived from hemoglobin are expressed in epithelium of channel catfish (Ictalurus punctatus, Rafinesque)[END_REF]. The most common of these are histone-like proteins. Histones are mainly known for their involvement in nuclear regulation and as an integral part of chromatin structure, however, more recent studies have shown they present strong antibacterial activities suggesting they may play a role in innate defence in many animals [START_REF] Parseghian | Beyond the walls of the nucleus: the role of histones in cellular signaling and innate immunity[END_REF][START_REF] Noga | Application of antimicrobial polypeptide host defenses to aquaculture: Exploitation of downregulation and upregulation responses[END_REF]. Several histones (H1, H2A, H2B) with antibacterial, antifungal and antiparasitic activities have been identified in skin mucus of several fish families including Salmonidae, Gadidae and Ictaluridae [START_REF] Robinette | Antimicrobial activity in the skin of the channel catfish Ictalurus punctatus: characterization of broad-spectrum histone-like antimicrobial proteins[END_REF][START_REF] Fernandes | Anti-microbial properties of histone H2A from skin secretions of rainbow trout, Oncorhynchus mykiss[END_REF][START_REF] Bergsson | Isolation and identification of antimicrobial components from the epidermal mucus of Atlantic cod (Gadus morhua)[END_REF]. Also, an increasing number of AMPs derived from terminal parts of histones (N-terminal and C-terminal regions) and other proteins have been described including parasin-I from the skin mucus of catfish (Parasilurus asotus, Siluridae), hipposin from the Atlantic halibut, SAMP H1 from Atlantic salmon and onchorrycin II from the rainbow trout. [START_REF] Park | Parasin I, an antimicrobial peptide derived from histone H2A in the catfish, Parasilurus asotus[END_REF][START_REF] Birkemo | Hipposin, a histone-derived antimicrobial peptide in Atlantic halibut (Hippoglossus hippoglossus L.)[END_REF][START_REF] Fernandes | Isolation and characterisation of oncorhyncin II, a histone H1-derived antimicrobial peptide from skin secretions of rainbow trout, Oncorhynchus mykiss[END_REF][START_REF] Lüders | Proline conformation-dependent antimicrobial activity of a proline-rich histone h1 N-terminal Peptide fragment isolated from the skin mucus of Atlantic salmon[END_REF].

Finally, other proteins displaying antibacterial properties in the skin mucus of several fish species include L-amino acid oxidases (LAOs), like Sebastes schlegeli antibacterial protein (SSAP) from rockfish (Sebastes schlegeli, Sebastidae), ribosomal proteins like L40, L36A, L35 and S30 from Atlantic cod and rainbow trout and hemoglobin-like proteins (Hb-β) from channel catfish [START_REF] Fernandes | Anti-microbial properties of histone H2A from skin secretions of rainbow trout, Oncorhynchus mykiss[END_REF][START_REF] Bergsson | Isolation and identification of antimicrobial components from the epidermal mucus of Atlantic cod (Gadus morhua)[END_REF][START_REF] Kitani | Identification of an antibacterial protein as L-amino acid oxidase in the skin mucus of rockfish Sebastes schlegeli[END_REF][START_REF] Ullal | Antimicrobial peptides derived from hemoglobin are expressed in epithelium of channel catfish (Ictalurus punctatus, Rafinesque)[END_REF].

Although, research on fish mucus AMPs is rapidly progressing, most studies are essentially descriptive and focused on characterisation of new AMPs in single fish species.

Despite the vast diversity in fish AMPs, studies show that AMPs are evolutionary well conserved, and many fish AMPs are preserved in higher vertebrates [START_REF] Rakers | Antimicrobial peptides (AMPs) from fish epidermis: perspectives for investigative dermatology[END_REF].

However, besides piscidins, most fish AMPs have been described in very few species, and there is still little knowledge on their distribution among fish families. In the future, fish mucus AMP research should focus on better understanding AMP evolution in fish, and their spread among fish families. Fish mucus AMPs show great potential as antimicrobial agents for therapeutic applications, however, to become exploitable, more research has to be conducted to understand their origin and mode of action in order to identify the conserved or most active parts.

Lipids

Lipids in fish mucus have been very rarely studied, however those studies indicate that they are also major constituents of fish mucus. [START_REF] Lewis | Fish cutaneous mucus: A new source of skin surface lipid[END_REF] showed that lipid quantity in fish mucus is surprisingly high when compared to human sebum, being phospholipids (lecithin and phosphatidyl ethanolamine) the most abundant lipids. Other lipids present are squalene, sterols, triglycerides, free fatty acids and carotenoids like β-carotene [START_REF] Lewis | Fish cutaneous mucus: A new source of skin surface lipid[END_REF][START_REF] Jais | Fatty acid compositions in mucus and roe of Haruan, Channa striatus, for wound healing[END_REF]. It seems that free fatty acids might have protective functions and the interaction between the phospholipids and glycoproteins may determine the relative viscosity of mucus [START_REF] Lewis | Fish cutaneous mucus: A new source of skin surface lipid[END_REF]).

Toxins and other specific molecules

To date, fish mucus research has focused mainly on the study of macromolecules.

Nevertheless, fish mucus is a rich and yet unexplored source of novel bioactive molecules.

For example, fish mucus toxins or mycosporine like amino acids (MAAs) have been described in fish mucus before, but they have rarely been integrated in studies of fish mucus composition and roles (Table 1).

Fish toxins are bioactive secretions normally stored in specialized glands but frequently found in fish epidermal mucus, which possess a variety of biological functions such as defence and competitor and predator deterrence [START_REF] Baumann | A ray of venom: Combined proteomic and transcriptomic investigation of fish venom composition using barb tissue from the blue-spotted stingray (Neotrygon kuhlii)[END_REF]. Ichtyocrinotoxins (fish epidermal toxins not associated with any venom apparatus) with very diverse structures have been reported in over 50 species of teleost fish from 14 families [START_REF] Halstead | Poisonous and Venomous Marline Animals of the World[END_REF][START_REF] Munday | Skin toxins and external parasitism of coral-dwelling gobies[END_REF]) (Table 1). Most crinotoxins are released into mucus layers when fish are stressed or injured, and they normally have hemolytic activity and can have antibacterial properties [START_REF] Nair | Fish skin toxins[END_REF]Sugiyama et al. 2005).

Tetrodotoxin (TTX), secreted by numerous pufferfish species (Tetrodontidae and Diodontidae), is one of the best studied marine fish crinotoxins [START_REF] Williams | Behavioral and Chemical Ecology of Marine Organisms with Respect to Tetrodotoxin[END_REF]; Figure 2).

TTX is a neurotoxin that inhibits nerve and muscle conduction by selectively blocking sodium channels, resulting in respiratory paralysis that causes death [START_REF] Narahashi | Tetrodotoxin: a brief history[END_REF]. TTX has been identified in multiple organisms such as starfish and mollusks, but also in terrestrial invertebrates and vertebrates [START_REF] Noguchi | Tetrodotoxin -distribution and accumulation in aquatic organisms, and cases of human intoxication[END_REF][START_REF] Stokes | Presence and function of tetrodotoxin in terrestrial vertebrates and invertebrates[END_REF]. Studies have suggested bacteria as the primary source of TTX, and have found that TTX accumulation in pufferfish occurs via the food chain [START_REF] Noguchi | Tetrodotoxin -distribution and accumulation in aquatic organisms, and cases of human intoxication[END_REF]. Although, the origin of most ichtyocrinotoxins remains unstudied, it is likely that like TTX, most fish mucus toxins are either produced by symbiotic microorganisms or accumulated by other means [START_REF] Daly | Marine toxins and nonmarine toxins: convergence or symbiotic organisms?[END_REF]). Recent studies have shown that fish mucus contains UV absorbing molecules, which have been identified as micosporine-like amino acids (MAAs), which are water-soluble nitrogenous compounds that perform a photo protective function against solar radiation [START_REF] Zamzow | Ultraviolet-absorbing compounds in the mucus of shallow-dwelling tropical reef fishes correlate with environmental water clarity[END_REF]. MAAs are selectively sequestered and accumulated via trophic transfer from corals and producer species [START_REF] Mason | Ultraviolet radiation-absorbing mycosporine-like amino acids (MAAs) are acquired from their diet by medaka fish (Oryzias latipes) but not by SKH-1 hairless mice[END_REF]. Three different MAAs have been identified to date in fish mucus: palythene, asterina-33 and mycosporine-N-methylamine serine. Different fish species present different combinations of MAAs [START_REF] Eckes | Ultraviolet sunscreens in reef fish mucus[END_REF]; Figure 2). Finally, fish mucus it is highly likely to contain other specialised and yet unknown metabolites, that could be 1) produced by the fish itself, 2) produced by associated microbiota or 3) accumulated in the mucus by other means such as diet.

Fish family

Mucus microbiome

Living organisms harbor a diverse community of microorganisms that have important functions in maintaining the hosts' health and homeostasis [START_REF] Maranduba | Intestinal Microbiota as Modulators of the Immune System and Neuroimmune System: Impact on the Host Health and Homeostasis[END_REF]. In fish, native microbiota influence the innate immune system and thus is involved in disease resistance (Gomez and Balcazar, 2008). Although interest in organisms' microbiota and microbiome has exploded in the last decade, studies exploring fish mucosal microbiota remain limited [START_REF] Llewellyn | Teleost microbiomes: the state of the art in their characterization, manipulation and importance in aquaculture and fisheries[END_REF]; Table 2).

Several studies have shown that the composition of the fish mucosal microbiota is highly variable. Higher bacterial diversity has been found in external organs such as skin or gills than the gut [START_REF] Smith | Characterization of the bacterial community associated with the surface and mucus layer of whiting (Merlangius merlangus)[END_REF][START_REF] Lowrey | Topographical Mapping of the Rainbow Trout (Oncorhynchus mykiss) Microbiome Reveals a Diverse Bacterial Community with Antifungal Properties in the Skin[END_REF]. Other studies showed that bacterial population associated with epidermal fish mucus was species specific and did not reflect the bacterial communities in the surrounding water [START_REF] Smith | Characterization of the bacterial community associated with the surface and mucus layer of whiting (Merlangius merlangus)[END_REF][START_REF] Larsen | Diversity of the skin microbiota of fishes: evidence for host species specificity[END_REF]. Besides, some studies point out mucus microbiome as highly dynamic, dominant bacterial taxa changing upon environmental conditions such as seasonality, suggesting the most adapted bacteria to a certain environment would appropriate the most niche space [START_REF] Wilson | The diversity of bacterial communities associated with Atlantic cod Gadus morhua[END_REF][START_REF] Larsen | Community Structure of Skin Microbiome of Gulf Killifish, Fundulus grandis, Is Driven by Seasonality and Not Exposure to Oiled Sediments in a Louisiana Salt Marsh[END_REF][START_REF] Schmidt | Community assembly of a euryhaline fish microbiome during salinity acclimation[END_REF] Commensal microbiota in mucosal surfaces are suggested to provide protection against opportunistic infection. Some studies showed that when microbiota homeostasis was disrupted (dysbiasis) by stressful conditions, the abundance of probiotic-like bacteria decreased while pathogenic bacteria increased [START_REF] Boutin | Probiotic treatment by indigenous bacteria decreases mortality without disturbing the natural microbiota of Salvelinus fontinalis[END_REF]. [START_REF] Boutin | Inter individual variations of the fish skin microbiota: host genetics basis of mutualism?[END_REF] showed a strong negative correlation between the indigenous bacteria Methylobacterium and the pathogenic Flavobacterium suggesting a mutually competitive relationship.

Methylobacterium rhodesianum is known to produce a poly--hydroxybutyrate, a polymer of short-chain fatty acid that inhibits growth of bacterial pathogens like enterobacteria and Vibrio sp. [START_REF] Halet | Poly--hydroxybutyrateaccumulating bacteria protect gnotobiotic Artemia franciscana from pathogenic Vibrio campbellii[END_REF]).

To date, very few studies have analysed the potential of vertebrate-associated microorganisms to produce bioactive compounds. Recent studies are revealing that microorganisms are the main producers of most of the natural products found in marine invertebrates [START_REF] Balskus | Natural products: Sponge symbionts play defense[END_REF]. Since vertebrate organisms such as fish possess diverse microbiomes, it is likely to think that microorganisms could also synthesize bioactive secondary metabolites. [START_REF] Sanchez | Examining the fish microbiome: vertebrate-derived bacteria as an environmental niche for the discovery of unique marine natural products[END_REF] showed that some isolated bacterial strains from fish intestines possessed antibacterial activity and they identified a novel bioactive lipid, sebastenoic acid, as antimicrobial agent (Figure 2). Another study analysed the rainbow trout microbiome and found antifungal activity in two Arthrobacter sp. skin isolates, a

Psychrobacter sp. strain and a combined skin aerobic bacterial sample [START_REF] Lowrey | Topographical Mapping of the Rainbow Trout (Oncorhynchus mykiss) Microbiome Reveals a Diverse Bacterial Community with Antifungal Properties in the Skin[END_REF].

Most studies on the associated microorganisms from animals have focused on bacteria, however, very recent studies are showing the key role of commensal fungi (mycobiota) and their effect on host immune system and health [START_REF] Underhill | The mycobiota: interactions between commensal fungi and the host immune system[END_REF]. Most of those studies have focused on human organs and human mucosal surfaces, but some studies are starting to investigate the mycobiome of marine species [START_REF] Borzykh | Mycobiota of the bivalve mollusk Anadara broughtoni (Schrenck, 1867) from various parts of peter the great Bay, Sea of Japan[END_REF].

Moreover, symbiotic fungi might also be a potential source for bioactive natural products that might benefit their hosts [START_REF] Debbab | Endophytes and associated marine derived fungi-ecological and chemical perspectives[END_REF]. Therefore, it will be of great importance to study the fish mucus mycobiota to gain a better understanding of the whole mucosal system, its interactions with other microorganisms (commensal and pathogenic) as well as their involvement in the fish immunity and health.

Mucus roles in ecological interactions

Molecules found in skin and gill fish mucus display key roles in the fish physiology and especially in fish protection against infections. However, some of those molecules also possess very important ecological roles in the ecosystem as a tool for communication. Studies have shown that epideraml and gill mucus substances can act as infochemicals in different interspecific interactions, such as in prey-predator relationship or parasite-host interaction (Figure 1). Fish mucus molecules can be perceived by a wide range of organisms and generate different type of responses. Several studies have shown that different pathogenic bacteria exhibit positive chemotaxis towards their hosts' mucus [START_REF] Bordas | Chemotaxis of pathogenic Vibrio strains towards mucus Surfaces of gilt-head sea bream (Sparus aurata L.)[END_REF][START_REF] Larsen | Chemotaxis of Vibrio anguillarum to fish mucus: role of the origin of the fish mucus, the fish species and the serogroup of the pathogen[END_REF]. Although specific molecules responsible for this activity have not yet been identified, O'toole et al. (1999) suggested free amino acids and carbohydrates could act as chemoattractant molecules in fish mucus while [START_REF] Klesius | Flavobacterium columnare chemotaxis to channel catfish mucus[END_REF] suggested that a lectinlike substance may be responsible for the chemotaxis.

Chemodetection of fish mucus and behavior modification has also been reported in other parasites such as the actinospores from oligochaetes, myxozoans and ectoparasites like copepods and monogeneans (Xiao and Dresser 2000;[START_REF] Ohashi | Purification and identification of a glycoprotein that induces the attachment of oncomiracidia of Neobenedenia girellae (Monogenea, Capsalidae)[END_REF][START_REF] Kallert | No shot in the dark: myxozoans chemically detect fresh fish[END_REF][START_REF] Brooker | Role of kairomones in host location of the pennellid copepod parasite, Lernaeocera branchialis (L. 1767)[END_REF]. [START_REF] Kallert | No shot in the dark: myxozoans chemically detect fresh fish[END_REF] found that free nucleosides present in trout mucus (inosine, 2'-deoxyinosine and guanosine) stimulated myxozoan attachment. Another study showed the glycoprotein WAP 65-2 in tigger puffer fish (Takifugu rubripes, Tetradontidae) skin mucus induced attachment of Neobenedenia girellae oncoramicidia (Monogenea, capsalidae) [START_REF] Ohashi | Purification and identification of a glycoprotein that induces the attachment of oncomiracidia of Neobenedenia girellae (Monogenea, Capsalidae)[END_REF]). It has also been proposed that TTX from pufferfish may also act as an attractant of the infective copepodids stages of Pseudocaligus fugu [START_REF] Ito | Detection of tetrodotoxin (TTX) from two copepods infecting the grass puffer Takifugu niphobles: TTX attracting the parasites?[END_REF].

Fish mucus substances act often as semiochemicals in predator-prey relationships, either as predator deterrents or predator and prey signaling. For example it is known that pavoninins, pardaxin and mosesins secreted by several species of soles repell sharks, by acting on their olfactory sense (Tachibana et al. 1984) (Table 1, Figure 2). TTX from puffer fish has also been reported to repel predation by groupers and act as a sex pheromone attracting males towards fertile females [START_REF] Matsumura | Tetrodotoxin as a pheromone[END_REF][START_REF] Williams | Behavioral and Chemical Ecology of Marine Organisms with Respect to Tetrodotoxin[END_REF]. [START_REF] Itoi | Larval pufferfish protected by maternal tetrodotoxin[END_REF] suggested that the female parent transferred vertically TTX to the larvae (through TTX accumulation in the ovaries) to increase egg and larvae survival.

Purcell and [START_REF] Purcell | Electrical responses to water-soluble components of fish mucus recorded from the cnidocytes of a fish predator, Physalia physalis[END_REF] found that Physalia physalis could identify its preys by chemical cues from fish epidermal mucus, although the chemical nature of these molecules remains unknown. Several organisms are also able to identify fish mucus chemicals to detect predators and avoid predation. Several studies indicate that nocturnal dial vertical migration (DVM) in zooplankton is affected by uncharacterised kairomones present in mucus of planktivorous fish [START_REF] Forward | Alteration of photoresponses involved in diel vertical migration of a crab larva by fish mucus and degradation products of mucopolysaccharides[END_REF][START_REF] Beklioglu | Fish and mucus-dwelling bacteria interact to produce a kairomone that induces diel vertical migration in Daphnia[END_REF]). [START_REF] Forward | Alteration of photoresponses involved in diel vertical migration of a crab larva by fish mucus and degradation products of mucopolysaccharides[END_REF] proposed that disaccharide degradation products of predator mucus containing sulfated and acetylated amines can serve as kairomones, whereas [START_REF] Beklioglu | Fish and mucus-dwelling bacteria interact to produce a kairomone that induces diel vertical migration in Daphnia[END_REF] suggested that both fish and mucus-dwelling bacteria interact in the release of kairomones. Recent research showed that the polychaete Nereis developed chemosensory mechanisms for predator detection (chemical cues from fish mucus) to minimise predation risks [START_REF] Schaum | Smelling Danger -Alarm Cue Responses in the Polychaete Nereis (Hediste) diversicolor (Mvller, 1776) to Potential Fish Predation[END_REF].

Goby epidermal toxins and their ecological implications are well studied and provide a good example of the importance and complexity of fish mucus molecules in ecosystems dynamics. Coral gobies are known to possess different epidermal mucus toxins that exhibit predator deterrence and parasite avoidance [START_REF] Munday | Skin toxins and external parasitism of coral-dwelling gobies[END_REF][START_REF] Gratzer | Skin toxins in coral-associated Gobiodon species (Teleostei: Gobiidae) affect predator preference and prey survival[END_REF]. Recent studies have shown how goby mucus substances play a key role in the mutualistic association between gobies and corals. [START_REF] Dixson | Corals Chemically Cue Mutualistic Fishes to Remove Competing Seaweeds[END_REF] showed that Acropora, chemically attracted gobies when attacked by toxic seaweeds. In turn, gobies would trim the seaweed and increase their own mucus toxicity, protecting them against predators and parasites.

Dirnwoeber and Herler (2013) also showed that goby skin mucus functioned as a corallivore repellent, protecting corals from predation. Although, role of goby epidermal mucus toxins has been ecologically well studied, their chemical structures are still unknown.

The study of chemical mediation in marine ecosystems is vital to understand ecosystem dynamics and thus understand how ecoystems can respond and adapt to changing conditions.

Mucus dynamics

Mucus is a dynamic matrix and its composition varies among fish species, being influenced by endogenous factors such as sex and developmental stage and exogenous factors such as stress, water temperature, pH and infections [START_REF] Esteban | An overview of the immunological defenses in fish skin[END_REF]. [START_REF] Chong | Characterisation of proteins in epidermal mucus of discus fish (Symphysodon spp.) during parental phase[END_REF] found that adult fish presented higher amounts of protein in epidermal mucus than juvenile fish. Higher salinities have been associated with a decrease in density of mucus cells and an increase in mucus viscosity [START_REF] Shephard | Mucus on the epidermis of fish and its influence on drug delivery[END_REF][START_REF] Roberts | The viscosity and glycoprotein biochemistry of salmonid mucus varies with species, salinity and the presence of amoebic gill disease[END_REF]. Changes in protein levels and immune-related molecules have been observed

upon different temperatures, with increased levels of lysozyme, hepcidin, IgM, lectin and mucosal antibodies at higher temperatures, which might be related to the increasing pathogen pressure in warmer temperatures [START_REF] Huang | The immune response of turbot, Scophthalmus maximus (L.), skin to high water temperature[END_REF][START_REF] Jung | Seasonal variation and comparative analysis of nonspecific humoral immune substances in the skin mucus of olive flounder (Paralichthys olivaceus)[END_REF][START_REF] Ma | Changes in protein composition of epidermal mucus in turbot Scophthalmus maximus (L.) under high water temperature[END_REF]. Toxic and irritating substances such as environmental contaminants produce irregular and excessive mucous secretions, which results in a decrease of mucus cell density after the chemical exposure, compromising fish health (Al-Zaidan et al. 2013).

Acute stress seems to cause changes in mucus proteins. [START_REF] Terova | Impact of acute stress on antimicrobial polypeptides mRNA copy number in several tissues of marine sea bass (Dicentrarchus labrax)[END_REF] showed an increase in the expression levels of histone-like proteins due to confinement, whereas, Easy and Ross (2010) observed changes in mucus proteases, in response to long-term handling stress in Atlantic salmon.

Several studies have shown that fish increase their mucus secretion as well as change their composition upon biological stressors such as pathogen presence and infection in order to better protect themselves [START_REF] Gustafsson | Dynamic Changes in Mucus Thickness and Ion Secretion during Citrobacter rodentium Infection and Clearance: Infection PLoS One[END_REF][START_REF] Rajan | Differentially expressed proteins in the skin mucus of Atlantic cod (Gadus morhua) upon natural infection with Vibrio anguillarum[END_REF]. Van der Marel et al.

(2010) showed that fish secreted higher quantities of mucins with higher glycosilation as an adaptation to higher bacterial loads in the water. Several studies have also showed a higher mucus production following parasites infection [START_REF] Maki | Systemic and Cutaneous Mucus Antibody Responses of Channel Catfish Immunized against the Protozoan Parasite Ichthyophthirius multifiliis[END_REF][START_REF] Valdenegro-Vega | Differentially expressed proteins in gill and skin mucus of Atlantic salmon (Salmo salar) affected by amoebic gill disease[END_REF]. Higher quantities of immune-related molecules (lectins, lyzoyme, immunoglobulins and cleaved transferrin) have been detected following different types of pathogenic infections [START_REF] Easy | Changes in Atlantic salmon Salmo salar mucus components following shortand long-term handling stress[END_REF][START_REF] Kar | Transcriptional changes in three immunoglobulin isotypes of rohu, Labeo rohita in response to Argulus siamensis infection[END_REF][START_REF] Makesh | Systemic and mucosal immune response of rainbow trout to immunization with an attenuated Flavobacterium psychrophilum vaccine strain by different routes[END_REF]. Finally, some mucus changes can be directly triggered by parasites, for example, monogenean and crustacean ectoparasites seem to modulate mucus production during attachment by reducing the density of fish mucus cells through disruption of cell dynamics within the epidermis in an attempt to minimise inhibitory effects of the mucus [START_REF] Jones | The occurrence and mechanisms of innate immunity against parasites in fish[END_REF][START_REF] Wells | Experimental studies on the effect of Gyrodactylus colemanensis and G. salmonis (Monogenea) on density of mucous cells in the epidermis of fry of Oncorhynchus mykiss[END_REF].

Considering the mucus dynamics when studying fish mucus is important since they give insights on fish responses to environmental conditions. Modifications in fish mucus can compromise fish health but it could also affect or disrupt fish chemical mediations.

Understanding mucus composition changes can be extremely useful in disease control

CHAPTER III -126 -strategies in aquaculture, but also in conservation studies.

Research advances and priorities

Fish mucus research is rapidly growing [START_REF] Makesh | Systemic and mucosal immune response of rainbow trout to immunization with an attenuated Flavobacterium psychrophilum vaccine strain by different routes[END_REF][START_REF] Larsen | Community Structure of Skin Microbiome of Gulf Killifish, Fundulus grandis, Is Driven by Seasonality and Not Exposure to Oiled Sediments in a Louisiana Salt Marsh[END_REF].

Technological advances in the "omics" sciences such as proteomics has recently allowed the discovery of many unreported proteins in fish mucus [START_REF] Rajan | Proteome reference map of the skin mucus of Atlantic cod (Gadus morhua) revealing immune competent molecules[END_REF]. Developments in the field of genomics facilitate the study of genes present in fish mucus, and it is allowing the rapid expansion of microbiomics (microbiome characterisation). Studying fish mucus microbiome and mycobiome together with their roles and their associated secondary metabolites are some of the novel priorities in fish mucus research. The use of metabolomics could also be highly useful in fish mucus research. Firstly, on the discovery of small metabolites, which have been poorly studied, and secondly, metabolomics provide metabolic signatures at specific moments of time so it could cast light on mucus dynamics (Figure 3).

Finally, fish mucus research needs to be approached from an integrative perspective in order to study the whole mucosal system. Fish mucus is a very complex biological material, involved in numerous biological functions within the fish and the ecosystem, which needs to be more studied and understood in order to evaluate its full potential. Numerous fish mucus molecules have drawn attention as potential candidates for drug and vaccine development.

Fish mucus plays an important role in intra and interspecific relationships, and therefore a better comprehension on those relationships could clarify methods of co-evolution between fish and pathogens as well as provide important information to understand species adaptation in changing environments (Figure 3). This article is the first attempt to study fish mucus under a holistic approach, combining current knowledge in different disciplines to critically discuss the biological and ecological importance of mucus, and identifying new opportunities in the research of fish mucus.

Summary and conclusions

Fish mucus surfaces are dynamic layers that display very important functions in fish, playing major roles in physiological functions such as osmoregulation and protection against infections, but also as a tool of interspecific communication (Bekliougu et al. 2006). Mucus contains a wide variety of biologically active molecules that take part in numerous roles and biological interactions. Study of fish mucus components has exploded these last years for its potential use both in aquaculture and human medicine. However, as we stated in this article, there are numerous mucus components that need further research in order to better characterise the molecules present, but specially in order to elucidate their roles. We have discussed the multiple roles of mucus in organism interactions of several nature (predatorprey, parasite-host), highlighting mucus ecological importance and the need to identify the molecules responsible for these interactions. Elucidating the molecules responsible for the

Fish mucus research

Microbiome and mycobiome characterization

Fish mucus molecules discovery

Antibacterial, antiviral, cytotoxic and antiparastic activities.

Kairomones, infochemicals

Improve understanding of fish biology, ecology and ecosystem dynamics

Marine conservation and management

• Adaptation to changing environments

• Co-evolution of species

Aquaculture

• Disease management

• Vaccine development

Human medecine

• Drug development

Genomics

Proteomics Metabolomics chemical mediation between fish mucus and other organisms will allow a deeper understanding of marine ecosystem dynamics and thus will give new insights in both the aquaculture management of parasites but also in marine conservation studies. In addition, better understanding of mucus shifts and changes due to biological and physical stressors could help to prevent fish disease outbreaks but also monitor the health of the marine environment. Finally, nearly all studies have focused on the study of macromolecules, via multiple techniques such as proteomics, but little studies have tried to elucidate smaller specialised metabolites. We would like to draw attention to the importance of secondary metabolites and their varied bioactivities in better-studied species such as marine invertebrates and their potential presence and importance in fish mucus. Furthermore, the development of new disciplines such as metabolomics will allow studying fish secondary metabolites more efficiently. Recent studies have shown the importance of microbiome, and some bacterial synthesised molecules have been described [START_REF] Zbakh | Antibacterial activity of benthic marine algae extracts from the mediterranean coast of Morocco[END_REF]. Therefore, the study of fish mucus microbiome and mycobiome is another promising field to both the discovery of new metabolites and better comprehension of the mucus entity and biological and ecological roles. To summarize, fish mucus is a key tissue in fish fitness and communication. Fish mucus contains a wide range of bioactive molecules that display numerous roles within the fish and other organisms and are of interest for medicine research.

We propose here a new perspective on fish mucus research, using an integrative approach to elucidate new bioactive mucus molecules and microorganisms and study their biological and ecological roles using the "omics" technologies.

Introduction

The associations between metazoans and commensal microorganisms are among the most ancient and successful associations in nature [START_REF] Mcfall-Ngai | Animals in a bacterial world, a new imperative for the life sciences[END_REF][START_REF] Lowrey | Topographical Mapping of the Rainbow Trout (Oncorhynchus mykiss) Microbiome Reveals a Diverse Bacterial Community with Antifungal Properties in the Skin[END_REF]. Animals and their associated microorganisms share a common evolutionary history, since they have coevolved in response to environmental selective pressures over hundreds of millions of years (Zilber-Rosenberg and Rosenberg, 2008). Since host-microbiota interactions are linked to host physiology, maintaining microbiota homeostasis is a key factor to avoid pathogen proliferation and diseases [START_REF] Kamada | Control of Pathogens and Pathobionts by the Gut Microbiota[END_REF][START_REF] Sommer | The gut microbiota -masters of host development and physiology[END_REF].

Studies on human gut microbiota show that microbiota provide essential health benefits to its hosts, particularly by regulating immune homeostasis [START_REF] Wu | The role of gut microbiota in immune homeostasis and autoimmunity[END_REF]. In fish, disruption of microbiota homeostasis (dysbiasis) resulted in a decrease of probiotic-like bacteria and an increase in pathogenic bacteria [START_REF] Boutin | Probiotic treatment by indigenous bacteria decreases mortality without disturbing the natural microbiota of Salvelinus fontinalis[END_REF][START_REF] Boutin | Inter individual variations of the fish skin microbiota: host genetics basis of mutualism?[END_REF]. Recent studies also show that bacterial isolates from fish gut and skin displayed antibacterial and antifungal activities against human and fish pathogens, suggesting a protective role of fish native microbiota against pathogens [START_REF] Sanchez | Examining the fish microbiome: vertebrate-derived bacteria as an environmental niche for the discovery of unique marine natural products[END_REF][START_REF] Lowrey | Topographical Mapping of the Rainbow Trout (Oncorhynchus mykiss) Microbiome Reveals a Diverse Bacterial Community with Antifungal Properties in the Skin[END_REF].

Fish external microbiota diversity (skin and gills) has been reported to be higher than internal (gut) microbiota [START_REF] Smith | Characterization of the bacterial community associated with the surface and mucus layer of whiting (Merlangius merlangus)[END_REF][START_REF] Lowrey | Topographical Mapping of the Rainbow Trout (Oncorhynchus mykiss) Microbiome Reveals a Diverse Bacterial Community with Antifungal Properties in the Skin[END_REF]. Fish microbiota seems to be species-specific and fish skin and gut microbiome is in most cases, significantly different than the surrounding water microbiome [START_REF] Smith | Characterization of the bacterial community associated with the surface and mucus layer of whiting (Merlangius merlangus)[END_REF][START_REF] Larsen | Diversity of the skin microbiota of fishes: evidence for host species specificity[END_REF][START_REF] Larsen | Community Structure of Skin Microbiome of Gulf Killifish, Fundulus grandis, Is Driven by Seasonality and Not Exposure to Oiled Sediments in a Louisiana Salt Marsh[END_REF]. However, [START_REF] Giatsis | The impact of rearing environment on the development of gut microbiota in tilapia larvae[END_REF] found that changes in water bacterial communities were highly correlated with changes in the gut of tilapia larvae. Similarly, And Mouchet et al. (2011) found that gut bacterial genetic diversity was significantly influenced by sampling site, both studies indicating that microorganisms in surrounding water might be a significant contributor to fish gut microbiomes.

To date, most studies on fish microbiota have investigated fish gut and skin microbiome, but gill microbiome diversity and their functional roles remain poorly understood (Larsen et al. 2013[START_REF] Larsen | Characterization of the gut microbiota of three commercially valuable warmwater fish species[END_REF][START_REF] Giatsis | The impact of rearing environment on the development of gut microbiota in tilapia larvae[END_REF]. Gills are the main respiratory organ in fish, and are composed of four pairs of vascularised gill arches with hundreds of gill filaments, which increase their contact surface for oxygen diffusion by folding into secondary lamella. In addition to respiration, gills perform other functions including osmoregulation, pH balance, ammonia excretion, hormone regulation, detoxification and immune defence [START_REF] Maina | Structure, function and evolution of the gas exchangers: comparative perspectives[END_REF].

Gills are constantly in contact with water, and therefore they are continuously exposed to pathogens [START_REF] Xu | Mucosal immunoglobulins at respiratory surfaces mark an ancient association that predates the emergence of tetrapods[END_REF]. They are covered by a mucus layer that acts as the first physical and biochemical barrier against pathogens [START_REF] Roberts | Comparative ionic flux and gill mucous cell histochemistry: effects of salinity and disease status in Atlantic salmon (Salmo salar L.)[END_REF]. Gill mucus production increases following gill infection, and a recent study reported decreased oxygen diffusion and an increase of pathogenic bacteria in fish exposed to high sediment concentrations, impairing normal gill function [START_REF] Ferguson | Responses of mucus-producing cells in gill disease of rainbow trout (Oncorhynchus mykiss)[END_REF][START_REF] Hess | Exposure of clownfish larvae to suspended sediment levels found on the Great Barrier Reef: Impacts on gill structure and microbiome[END_REF]. However, despite their importances in conserving gill homeostasis and preventing gill infection, native gill microbiota remain poorly studied.

In this study, we aimed to cast light on the gill microbiome of four sympatric butterflyfish species (Chaetodon lunulatus, C. ornatissimus, C. reticulatus and C. vagabundus) from Moorea (French Polynesia). Butterflyfishes (family Chaetodontidae) are emblematic and widely distributed coral reef fish species, which feed mostly but not exclusively on coral [START_REF] Pratchett | Dietary overlap among coral-feeding butterflyfishes (Chaetodontidae) at Lizard Island, northern Great Barrier Reef[END_REF]. Coral reefs are among the most productive and biologically diverse ecosystems on Earth, and despite the fact that coral microbiome has attracted increasing attention in recent years, most microbial communities of reef-associated organisms remain poorly studied [START_REF] Ainsworth | The coral core microbiome identifies rare bacterial taxa as ubiquitous endosymbionts[END_REF]. The main objectives of this study were to characterise the bacterial diversity and taxonomic composition of gill mucus of four butterflyfishes and compare the microbiome variability between four sympatric species. This is one of the first studies evaluating the gill mucus microbiome and to the best of our knowledge it is the first comprehensive study of gill microbiome in coral reef fishes using high-throughput sequencing.

Materials and methods

Data treatment

Multiplex raw sff files were analysed using a hybrid analysis pipeline as previously described [START_REF] Croué | A single betaproteobacterium dominates the microbial community of the crambescidine-containing sponge Crambe crambe[END_REF]) with some modifications. In brief, denoising was done by AmpliconNoise V1.25 [START_REF] Quince | Removing noise from pyrosequenced amplicons[END_REF]) implemented in Qiime V1.5 [START_REF] Caporaso | QIIME allows analysis of high-throughput community sequencing data[END_REF]) with a small modification to allow it to be ran in an iDataplex (IBM) cluster. De novo chimera detection and removal was performed with the uchime module [START_REF] Edgar | Search and clustering orders of magnitude faster than BLAST[END_REF][START_REF] Cipriano | Far from superficial: microbial diversity associated with the dermal mucus of fish[END_REF] [START_REF] Lane | 16S/23S rRNA sequencing[END_REF] using mothur v.1.38.0 and a of neighbour joining tree constructed using phylip v3.6a3 . Alpha-diversity indices (observed richness, Chao 1, Shannon and evenness)

were calculated using an OTU table rarefied to 1499 sequences using the single_rarefaction.py function of Qiime v1.5 summarised per fish species (mean and standard deviation). ANOVA and Tukey post-hoc test were used to detect significant differences in Shannon's diversity (H) between fish species. OTUs expected richness of fish species was computed using the specaccum function of the vegan package for R. Principal coordinates analysis (PCoA) using weighted unifrac distance, which takes in account both the OTUs abundance and the phylogenetic distance between the OTUs, was used to assess the differences between the microbiomes of the different fish species [START_REF] Lozupone | UniFrac: an effective distance metric for microbial community comparison[END_REF] using a relativized (percentage of reads per sample) OTU table. Permutational multivariate analysis of variance (PERMANOVA, function adonis of the vegan package for R) and pairwise comparisons between group levels with corrections for multiple testing (function pairwise.perm.manova of the RVAideMemoire package for R) were used to evaluate statistically significant differences of PCoA groups between fish species. The number of shared OTUs among all fish species combinations were calculated and represented using a Venn diagram (using the rarefied OTU table ). Microbiome composition of the four butterflyfish species was characterised and results are displayed by bacterial phylum abundances. The bacterial families and genera with abundances higher than 5% at least in one of the samples were also identified.

Results

Microbiome diversity

A total of 1940 OTUs were obtained from the gill mucus of four butterflyfish species (C. 1).

Table 1. Alpha-diversity estimates for the four butterflyfish species (mean ± standard deviation). * indicates significant difference (p-value < 0.05).

Microbiota differences between butterflyfishes

Among the 1041 OTUs, only 27 OTUs were found in all four butterflyfishes. This "core microbiota" (27 OTUs) represented between 18 and 35% of all bacterial sequences in each fish species. The core microbiota (at the OTU level) consisted mainly of Proteobacteria, Actinobacteria and Firmicutes (class Clostridia) and one cyanobacteria OTU ( 2).

Microbiome composition

We are fully aware that percentage of OTUs does not represent exactly the percentage of cellular abundances, and thus hereafter abundances refer to the percentage of total sequence reads in the final OTU 11% and 17% respectively). C. lunulatus also presented a notably higher abundance of Fusobacteria (4%) which was nearly absent in the other fish species (Figure 3). At the family level, we saw some consistent associations between some bacterial families and Chaetodon species, but also high inter-individual variation (Figure 4).

Discussion

Gills are an essential organ to fish well-being, thus studying their normal microbiota is of great importance to understand their role in gill homeostasis and prevention of infections.

Fish gill mucus is in constant contact with the environment, proportioning a rich nutrient habitat for heterotrophic bacteria, compared to the surrounding oligotrophic coral reef waters.

In this study we characterized and compared the gill mucus microbiome of four sympatric coral reef fish species of Moorea (French Polynesia) using tag 454-pyrosequencing. Although the microbiome diversity of gill mucus of butterflyfishes varied significantly between species, Shannon diversities were surprisingly and consistently high (H = 4.8 -5.7, 26 phyla, with the exception of C. reticulatus that displayed a significant lower diversity H = 3.7) when compared to gill microbiome of cultured rainbow trout (Oncorhynchus mykiss) (H < 4.5, 11 phylum), gut microbiota of surgeonfishes (H = 0.5 -3.9), tropical sponges (H = 1.6 -4.3), some coral species (H = 0.8 -2.0), coral reef water (H = 0.6) or coral reef sediment (H = 0.8) [START_REF] Bayer | The microbiome of the Red Sea coral Stylophora pistillata is dominated by tissue-associated Endozoicomonas bacteria[END_REF][START_REF] Moitinho-Silva | Specificity and transcriptional activity of microbiota associated with low and high microbial abundance sponges from the Red Sea[END_REF][START_REF] Lowrey | Topographical Mapping of the Rainbow Trout (Oncorhynchus mykiss) Microbiome Reveals a Diverse Bacterial Community with Antifungal Properties in the Skin[END_REF][START_REF] Miyake | Diet strongly influences the gut microbiota of surgeonfishes[END_REF][START_REF] Glasl | The microbiome of coral surface mucus has a key role in mediating holobiont health and survival upon disturbance[END_REF]. Although these higher levels could be partly attributed to the higher OTU cutoff (98%) used in our study as well as the primers used for amplification. Nonetheless regarding the difference to rainbow trout, it is to be expected that the microbiome of cultured fish would be less diverse than wild fish, which have higher genetic variability as well as higher dietary plasticity [START_REF] Givens | Comparison of the gut microbiomes of 12 bony fish and 3 shark species[END_REF]. We would also like to note that higher Shannon diversity from other coral species (Montastrea faevolata) has also been reported (H = 6.5) (Kimes et vagabundus has a more even bacterial community.

Although all fish species were collected at the same sampling site, they presented significant microbiome differences. While we saw high inter-individual variation, by comparing at least 4 individual microbiomes per species, we were able to highlight some general trends regarding the Chaetodon microbiomes. C. lunulatus and C. ornatissimus were the two species clearly with the most similar microbiomes. These two species are the closest phylogenetic relatives and they are both obligate corallivores [START_REF] Pratchett | Dietary overlap among coral-feeding butterflyfishes (Chaetodontidae) at Lizard Island, northern Great Barrier Reef[END_REF]Fessler and Wesneat, 2007). In contrast, C. vagabundus is an omnivorous species that belong to a different phylogenetic clade [START_REF] Pratchett | Dietary overlap among coral-feeding butterflyfishes (Chaetodontidae) at Lizard Island, northern Great Barrier Reef[END_REF]Fessler and Wesneat, 2007). Therefore, a similarity between their gill microbiomes is not unexpected and these results suggest that gill mucus microbiome of fishes might be influenced together by phylogeny and/or ecological characteristics of the host species. On the other hand, we cannot explain why C. reticulatus, High abundances of Vibrio and Photobacterium are reported in normal microbiota of fish skin, [START_REF] Smith | Characterization of the bacterial community associated with the surface and mucus layer of whiting (Merlangius merlangus)[END_REF][START_REF] Yücel | Prevalence of listeria, Aeromonas, and Vibrio species in fish used for human consumption in Turkey[END_REF][START_REF] Larsen | Community Structure of Skin Microbiome of Gulf Killifish, Fundulus grandis, Is Driven by Seasonality and Not Exposure to Oiled Sediments in a Louisiana Salt Marsh[END_REF] but to date only one study has reported a high incidence of Vibrio spp. in the gills of marine fish used for human consumption [START_REF] Yücel | Prevalence of listeria, Aeromonas, and Vibrio species in fish used for human consumption in Turkey[END_REF]. Vibrio species are widespread in the marine environment and are also known to be associated to coral mucus and tissues and in some cases responsible for coral disease (reviewed in Rosemberg et al 2007). Unfortunately, it is very difficult if not impossible to discriminate between Vibrio species based on 16S rRNA sequences alone, so we prefer not to speculate the origin or the role of the OTUs associated with, C. lunulatus and C. ornatissimus gill mucus [START_REF] Sawabe | Updating the Vibrio clades defined by multilocus sequence phylogeny: proposal of eight new clades, and the description of Vibrio tritonius sp[END_REF]). However, the fact that Vibrionaceae are less abundant in C. reticulatus and C. vagabundus, suggest that these bacteria are not just passively transferred from the corals or surrounding seawater, but can be in fact have a stronger association to these species. Endozoicomonas species are often found as part of the corals holobiont or in close association to other marine invertebrates (e.g. bivalves, sea slugs; [START_REF] Kurahashi | Endozoicomonas elysicola gen. nov., sp. nov., a gamma-proteobacterium isolated from the sea slug Elysia ornata[END_REF][START_REF] Bayer | The microbiome of the Red Sea coral Stylophora pistillata is dominated by tissue-associated Endozoicomonas bacteria[END_REF][START_REF] Hyun | Endozoicomonas atrinae sp. nov., isolated from the intestine of a comb pen shell Atrina pectinata[END_REF], and interestingly a recent study also identified Endozoicomonas strain [START_REF] Katharios | Environmental marine pathogen isolation using mesocosm culture of sharpsnout seabream: striking genomic and morphological features of novel Endozoicomonas sp[END_REF] pathogenic to fish, but with a sequence quite distant to the Endozoicomonas found in our samples.

which
Verrucomicrobia species, which were abundant in C. lunulatus and C. ornatissimus (1 -15%, mainly an Akkermansia unidentified species) have been found in low abundances in fish guts (0.3 -1%), fish skin and coral tissues (1 -3%) [START_REF] Kooperman | Coral mucusassociated bacterial communities from natural and aquarium environments[END_REF][START_REF] Chiarello | High diversity of skin-associated bacterial communities of marine fishes is promoted by their high variability among body parts, individuals and species[END_REF][START_REF] Miyake | Diet strongly influences the gut microbiota of surgeonfishes[END_REF][START_REF] Lawler | Coral-associated bacterial diversity is conserved across two deep-sea Anthothela species[END_REF]. Verrucomicrobia are ubiquitous (although rarely in very high proportions) in the marine environment, both in the water column and sediment, but little is known of the functional role they might play in association with organisms such as fish [START_REF] Freitas | Global distribution and diversity of marine Verrucomicrobia[END_REF]).

The nearly specific presence of Fusobacteria in C. lunulatus was also remarkable, especially considering that this fish species has the particular trait of not being infected by gill monogenean parasites. Fusobacteria are anaerobic bacteria found in large quantities in fish gut [START_REF] Clements | Intestinal microbiota in fishes: what's known and what's not[END_REF], however to the best of our knowledge this is the first record of Fusobacteria in external surfaces of fish. Fusobacteria are known to produce a short-chain fatty acid, butyrate, which is the end product of fermentation of carbohydrates including those found in mucins [START_REF] Bennett | Fusobacteria: new taxonomy and related diseases[END_REF]. In mammals, butyrate provides many benefits to the host, enhancing mucus production and acting as an anti-carcinogen and anti-inflammatory (von [START_REF] Engelhardt | Role of short-chain fatty acids in the hind gut[END_REF][START_REF] Andoh | Physiological and anti-inflammatory roles of dietary fiber and butyrate in intestinal functions[END_REF]. Some Fusobacteria species such as Cetobacterium somerae, which has an identical sequence to an OTU present in the gill mucus of C. lunulatus, is also known to produce abundant amounts of vitamin B12 [START_REF] Merrifield | The fish microbiome and its interactions with mucosal tissues[END_REF].

C. reticulatus and C. vagabundus had a much higher proportion of Alphaproteobacteria, that corresponded mostly to higher abundances in Rhodobacteraceae (mainly Paracoccus)

and remarkable abundances of Neorickettsia in two samples of C. reticulatus. Paracoccus has been previously identified in fish skin, fish gut and corals [START_REF] Sheu | Paracoccus stylophorae sp. nov., isolated from the reef-building coral Stylophora pistillata[END_REF][START_REF] Liu | Paracoccus siganidrum sp. nov., isolated from fish gastrointestinal tract[END_REF][START_REF] Larsen | Community Structure of Skin Microbiome of Gulf Killifish, Fundulus grandis, Is Driven by Seasonality and Not Exposure to Oiled Sediments in a Louisiana Salt Marsh[END_REF]. Neorickettsia species are normally intracellular pathogens that cause severe illnesses in mammals and are transmitted by flukes (Platyhelminthes: Digenea) that infect fishes [START_REF] Vaughan | Neorickettsial endosymbionts of the digenea: diversity, transmission and distribution[END_REF]. A potential new species of Neorickettsia has been identified as species specific from skin microbiota of striped mullet (Mugil cephalus),

however their role and importance in fish microbiota remains unknown [START_REF] Larsen | Community Structure of Skin Microbiome of Gulf Killifish, Fundulus grandis, Is Driven by Seasonality and Not Exposure to Oiled Sediments in a Louisiana Salt Marsh[END_REF].

Actinobacteria and Firmicutes, which seem to constitute the fish core microbiota along with Proteobacteria, were abundant in gill mucus of all fish species, especially in C. reticulatus and C. vagabundus. Interestingly, 10% of the microbial communities of C. vagabundus and C. reticulatus were composed of Corynebacterium, a pathogenic bacteria which was found to increase in gill mucus of clownfish exposed to high concentrations of sediment [START_REF] Hess | Exposure of clownfish larvae to suspended sediment levels found on the Great Barrier Reef: Impacts on gill structure and microbiome[END_REF]. Finally, we found remarkable high abundances of Propionibacterium and Ralstonia (2 -8% and 1 -25% respectively), which have been described as widespread coral rare associate species, which are closely associated to coral dinoflagellate symbionts, but undetectable on water surrounding corals [START_REF] Ainsworth | The coral core microbiome identifies rare bacterial taxa as ubiquitous endosymbionts[END_REF].

In conclusion, we found a high bacterial diversity in gill mucus of butterflyfishes, many of which are also found in the coral holobiont. Mucus layers are nutrient hotspots for marine heterotrophic bacteria living in oligotrophic environments such as coral reefs. Therefore, we hypothesise that external fish mucus surfaces can act as a reservoir for coral reef bacterial diversity. Our study shows that different fish species possess different microbiomes, indicating the presence of species specificities in some bacterial OTUs that might arise from bacterial-host coevolution and ecological parameters. However, there is a large part of the bacterial community (50%) that is common in the four fish species studied. We propose that there might be a bacterial transfer between sympatric organisms that could include other organisms such as corals. Bacterial chemotaxis has been shown between coral associate bacteria (e.g. Endozoicomonaceae, Rhodobacteraceae and Vibrionaceae) and coral mucus amino acids [START_REF] Tout | Chemotaxis by natural populations of coral reef bacteria[END_REF]. However, fish mucus is known to contain a wide array of proteins, including the same amino acids found in coral mucus, therefore there might exist also a bacterial chemotaxis between natural occurring coral reef bacteria and coral reef fish mucus [START_REF] Valdenegro-Vega | Differentially expressed proteins in gill and skin mucus of Atlantic salmon (Salmo salar) affected by amoebic gill disease[END_REF]. Finally, we have observed the presence of several potentially pathogenic bacteria in the gill mucus of healthy butterflyfishes. We suggest that a high bacterial diversity could control proliferation of pathogenic agents by direct competition as it has been proposed for the gut microbiota [START_REF] Kamada | Control of Pathogens and Pathobionts by the Gut Microbiota[END_REF]). However, in stress conditions, a microbiota dysbiasis could lead to proliferation of pathogens and disease outbreaks such as shown by [START_REF] Hess | Exposure of clownfish larvae to suspended sediment levels found on the Great Barrier Reef: Impacts on gill structure and microbiome[END_REF].

Fish mucus metabolomics reveals fish life history traits.

Reverter, M. 1,2,3 , Sasal, P. 2,3 , Banaigs, B. 1,3 , Lecchini, D. 2,3 , Lecellier, G. 2,4,5 . Tapissier-Bontemps, N.

Abstract

Fish mucus has important biological and ecological roles such as defense against fish pathogens and chemical mediation between several species. A non-targeted LC-MS metabolomic approach was developed to study gill mucus of eight butterflyfish species in Moorea (French Polynesia), and the influence of several fish traits (site of collection i.e.

geographic site and reef habitat, species taxonomy, phylogeny, diet and parasitism levels) on the metabolic variability was investigated. A biphasic extraction yielding two fractions (polar and apolar) was used. Fish diet (obligate corallivorous, facultative corallivorous or omnivorous) arose as the main driver of the metabolic differences in the gill mucus in both fractions, accounting for 23% of the observed metabolic variability in the apolar fraction and 13% in the polar fraction. Level of monogenean gill parasites was the second most important factor shaping the gill mucus metabolome, and it explained 10% of the metabolic variability in the polar fraction and 5% in the apolar fraction. A multiple regression tree revealed that the metabolic variability due to parasitism in the polar fraction was mainly due to differences between non-parasitized and parasitized fish. Phylogeny and butterflyfish species were factors contributing significantly to the metabolic variability of the apolar fraction (10% and 3% respectively) but had a less pronounced effect in the polar fraction. Finally, geographic site and reef habitat of butterflyfish species did not influence the gill mucus metabolome of butterflyfishes.

Keywords: fish mucus, metabolomics, coral reefs, butterflyfishes.

Introduction

Ecosystems are dynamic assemblies of organisms that interact with each other in multiple ways, forming complex networks of ecological interactions [START_REF] Olff | Parallel ecological networks in ecosystems[END_REF].

Chemically mediated interactions either within one species (intraspecific) or between different species (interspecific) are by far, the most widespread interactions in the living world. Chemical cues determine species foraging behavior, competitive interactions, commensal associations, pathogenic invasions and selection of mates and habitats (Hay, 2009). Metabolic diversity is associated to ecosystem complexity, where a high biodiversity and the associated multiple ecological interactions would maintain and promote diversity of metabolites [START_REF] Moore | Explaining intraspecific diversity in plant secondary metabolites in an ecological context[END_REF]). Metabolite production is highly dependent on environmental factors such as temperature, light and nutrients, being very susceptible to biotic and abiotic stresses such as nearby competitor's pressure [START_REF] Kooke | Multi-dimensional regulation of metabolic networks shaping plant development and performance[END_REF][START_REF] Lages | Proximity to competitors changes secondary metabolites of non-indigenous cup corals, Tubastraea spp., in the southwest Atlantic[END_REF][START_REF] De Caralt | Sources of Secondary Metabolite Variation in Dysidea avara (Porifera: Demospongiae): The Importance of Having Good Neighbors[END_REF].

The mucosal layers covering fish epidermis and gills are the main surface of exchange between fish and the surrounding environment (Shepard 1993). Fish mucus presents numerous biological and ecological roles including respiration, osmotic regulation, excretion, defense against pathogens and chemical mediation between species [START_REF] Purcell | Electrical responses to water-soluble components of fish mucus recorded from the cnidocytes of a fish predator, Physalia physalis[END_REF][START_REF] Brinchmann | Immune relevant molecules identified in the skin mucus of fish using -omics technologies[END_REF]. Therefore, mucosal surfaces have a high functional importance reflecting both fish physiology and responses to environment. Mucus is mainly constituted of glycoproteins, called mucins, but it also contains immune-related molecules such as lysozyme, immunoglobulins, lectins and antimicrobial peptides, lipids and carbohydrates [START_REF] Brinchmann | Immune relevant molecules identified in the skin mucus of fish using -omics technologies[END_REF]. Smaller molecules composition of fish mucus has been much less studied, but some toxins (e.g. tetrodotoxin) or mycosporine-like aminoacids, which are acquired through diet, have been found in fish skin mucus [START_REF] Eckes | Ultraviolet sunscreens in reef fish mucus[END_REF][START_REF] Noguchi | Tetrodotoxin -distribution and accumulation in aquatic organisms, and cases of human intoxication[END_REF]. Most studies on fish mucus have focused on one or few molecules at a time, giving a fragmented idea on fish mucus composition. Recent advances in omics technologies, however, have allowed studying fish mucosal surfaces using more global approaches [START_REF] Brinchmann | Immune relevant molecules identified in the skin mucus of fish using -omics technologies[END_REF]. For example, the use of proteomics, which gives a detailed protein composition, revealed a high diversity of immune competent molecules previously unreported in fish mucus [START_REF] Rajan | Proteome reference map of the skin mucus of Atlantic cod (Gadus morhua) revealing immune competent molecules[END_REF][START_REF] Cordero | Skin mucus proteome map of European sea bass (Dicentrarchus labrax)[END_REF]. Metabolomics, which is the comprehensive study of metabolites, is one of the most recent "omics" sciences (Sumner et , 2003). Metabolites are the end products of cellular regulatory processes, and their levels can be regarded as the ultimate response of biological systems to genetic or environmental changes [START_REF] Fiehn | Metabolomics--the link between genotypes and phenotypes[END_REF]. A major benefit of metabolomics is the simultaneous analysis of multiple metabolites that do not necessarily need to be known, providing a powerful tool to analyze chemical diversity and changes in the natural environment [START_REF] Kusano | Assessing metabolomic and chemical diversity of soybean lineage representing 35 years of breeding[END_REF].

CHAPTER V -152 - al.
Metabolomic approaches have proven useful to distinguish among individual signals and thus might serve in biomarker discovery in a wide range of topics, from disease biomarkers to indicators of short-term environmental changes in ecology or even in chemotaxonomy studies [START_REF] Wolfender | MS-based plant metabolomic approaches for biomarker discovery[END_REF]. A recent study investigated for the first time the fish mucus metabolome and found over 200 distinct metabolites in fish skin mucus, some of which showed altered levels in response to bisphenol A exposure [START_REF] Ekman | Metabolite Profiling of Fish Skin Mucus: A novel approach for minimally-invasive environmental exposure monitoring and surveillance[END_REF].

Coral reefs are among the most productive and biologically diverse ecosystems on Earth.

However, anthropogenic activities such as overfishing and pollution and rapid climate change compromise their persistence [START_REF] Pandolfi | Projecting coral reef futures under Global warming and ocean acidification[END_REF]. Continued loss of coral cover and the erosion of the complex habitat structure associated with healthy coral reefs are expected to have significant consequences for a vast number of reef-associated species such as butterflyfishes [START_REF] Munday | Climate change and the future for coral reef fishes[END_REF]. Butterflyfishes (family Chaetodontidae) are a diverse and conspicuous family of coral reef fishes distributed widely in all tropical seas.

Butterflyfish ecology and behavior have been extensively studied, and although they can consume a variety of prey, including algae, polychaetes, crustaceans and coral, most species feed primarily if not exclusively on scleractinian corals [START_REF] Pratchett | Dietary overlap among coral-feeding butterflyfishes (Chaetodontidae) at Lizard Island, northern Great Barrier Reef[END_REF].

The present work aimed to study butterflyfishes gill mucus metabolome and explore the relationship between fish mucus metabolome, fish life history and the environment in which butterflyfish live. Understanding which fish traits affect the mucus metabolome is the first step towards the comprehension of the fish mucus roles and its dynamics. Besides, since fish mucus is in direct contact with the environment, it can provide useful information about both the surrounding environment and the fish ecological interactions. A non-targeted LC-MS metabolomic approach was developed to study fish gill mucus. The influence of several fish traits (site of collection, species taxonomy, diet, parasitism level, and phylogeny) on the metabolic variability was investigated. Metabolic profiles of gill mucus of eight Chaetodon species, belonging to three phylogenetic clades and displaying contrasting diet preferences

CHAPTER V -153 -
and levels of parasitism were studied at three geographic sites in Moorea Island (French Polynesia) and at different reef habitats (fringing reef, barrier reef, outer slope).

Material and methods

Sampling and chemical extraction

Eight and acetonitrile (B) as carriers, both containing 0.1 % v/v formic acid. The flow rate was 500 µL/min, column temperature was maintained at 30 °C and injection volume was 10 μL.

For the polar fraction a linear biphasic gradient from 2 to 100 % acetonitrile in water was applied over a period of 30 min, followed by 10 min washing and 15 min re-equilibration with 2% of B before the next injection. For the apolar fraction a linear gradient was conducted from 30% to 70% of B in 5 min, then from 70% to 80% of B in 20 min and from 80% to the sample sequences (polar and apolar). QC samples were analyzed at the beginning, the end and equidistantly throughout the sequence. In order to detect column contamination throughout the sequence methanol blank samples were analyzed just before each QC sample. Metabolomic diversity of each sample was determined by the Shannon's diversity index (H), and differences across fish traits were tested using the analysis of variance (ANOVA)

and the Tukey's HSD test of an ANOVA. Permutational multivariate analysis of variance using distance matrices (PERMANOVA) (adonis function, package vegan for the R software)

was used to identify the significant fish traits linked to the metabolic variability in gill mucus of butterflyfishes in polar and apolar fractions. Principal component analysis (PCA) was then used to visualize the gill metabolic variation on the significant fish traits (determined with the PERMANOVA analysis) and the 10 most important contributors (features) were represented.

Multiple regression trees (MRT, package mvpart for the R software) were used to obtain further details of the fish traits explaining the chemical variability in both fractions. MRT allow to rank fish traits according to their contribution, by producing a tree where at each split data is partitioned into two mutually exclusive groups, each of which is as homogeneous as possible, displaying this way the major factors determining the variability (De'ath, 2002; [START_REF] Vignon | Multiscale determinants of parasite abundance: A quantitative hierarchical approach for coral reef fishes[END_REF]. Only the fish traits with a contribution higher than 0.1% were plotted.

Technical vs. biological variance

Two types of variance should be considered in metabolic studies: the technical variance due to variation of sample preparation and instrumental analysis and the biological variance, existing between the analyzed samples. To study the biological variance, the technical variance must be lower. Biological variance was monitored by extracting and analyzing 5 different mucus samples from the fish species C. lunula. Technical variance (repeatability)

was assessed by analyzing 5 independently prepared extracts (technical replicates) from a pool constituted of the same 5 samples of C. lunula. Samples were extracted and analyzed as described before (including the randomized analytical sequences and the QC). The two datasets (polar and apolar) were then pre-treated and statistical analyses were used to determine technical and biological variance.

Results

Technical vs. biological variance

Technical variability due to extraction procedure, analytical analysis and data processing was significantly smaller than biological variability. PCA revealed close clustering between technical replicates, while biological replicates were significantly more dispersed in both fractions (Figure 2). These results show the suitability of the metabolomic approach used for CHAPTER V

-157 -

the study of metabolic differences between different biological samples of butterflyfish gill mucus.

Metabolomic diversity

Metabolomic diversity (Shannon's index, H) calculated according to the analyzed fish traits (fish species, reef habitat, geographic site, phylogeny, diet and intensity of parasitism)

showed no significant differences (P > 0.05) in the polar fraction. However, significant differences in the metabolomic diversity were found in the apolar fraction depending on the fish species, phylogeny, diet and level of parasitism (Figure 3). C. ulietensis, C. lunula and C.

vagabundus had significant higher metabolomic diversity (P < 0.001) than C. lunulatus and C. ornatissimus. C. ulietensis and C. lunula also presented a higher metabolomic diversity (P < 0.001) than C. reticulatus. Butterflyfish from the clade 4 had higher metabolomic diversity than clade 3. Obligate corallivores presented significantly (P < 0.001) lower metabolomic diversity than facultative corallivores and omnivores. Fish never parasitized presented also lower metabolomic diversity than fish with low levels of parasites (P < 0.001) (Figure 3).

Metabolic variability of gill mucus

We conducted a PERMANOVA analysis on distance matrices to identify the fish traits that contributed significantly to the metabolic variability of the two fractions of gill mucus extract. Geographic site (Afareiatu, Ta'haiamanu and Haapiti) and reef habitat (fringing reef, barrier reef and outer slope) did not affect significantly the metabolic profiles in any of the gill mucus fractions (polar and apolar). Butterflyfish species played a significant role in the metabolic variability of the apolar fraction, explaining 10% of the variability observed.

Phylogeny, diet and parasitism were significant factors in both fractions. Diet explained most of the metabolic variability observed in both fractions (23% of the apolar fraction and 13% of the polar fraction), followed by parasitism (5% of the apolar fraction and 10% of the polar fraction). Phylogeny explained 3% of the metabolic variability in both fractions (Table 1).

PERMANOVA analysis shows that studied variables (species, geographic site, reef To visualize the significant sources of gill mucus metabolite variation and their relative distribution, we conducted PCA analysis for both polar (Figure 3) and apolar (Figure 4)

fractions. The ten most important contributors of the polar fraction discriminate diagonally (bottom left -top right) the fish traits (Figure 3), whereas the ten most important contributors of the apolar fraction discriminate the fish traits on the top leftbottom right diagonal (Figure 4). Multiple regression tree analysis also shows that diet, parasitism and less importantly phylogeny are the main factors shaping the metabolomic differences in the butterflyfish gill mucus. In the polar fraction the main metabolomic differences are due to differences between obligate corallivores/omnivores and facultative corallivores, followed by differences between parasitized fish (high and low intensities) and non-parasitized fish (7.4% out of the 10% of metabolic variability explained by parasitism). Metabolomic diversity in parasitized fish is explained by differences between obligate corallivore fish and omnivores (Figure 5a).

The main factor driving metabolomic variability in the apolar fraction is the difference between facultative corallivores/omnivores and obligate corallivores. Differences in phylogeny (clade 2/4 and clade 3) explain the metabolic variability within the facultative corallivore and omnivorous fish. While, parasitism (non-parasitized vs. highly parasitized)

explains the metabolic differences of obligate corallivorous fish (Figure 5b).

Discussion

Mucus is involved in many regulatory processes in fish, playing a major role in both fish physiology and fish environmental interactions [START_REF] Brinchmann | Immune relevant molecules identified in the skin mucus of fish using -omics technologies[END_REF]. This study at Moorea, is the first attempt to study the gill mucus metabolome and the factors that might explain some of its variability. Improving the knowledge on fish mucus constituents and dynamics will cast light on some biological and ecological roles of fish mucus, as well as help future research dealing with fish mucus variations.

Distinct metabolite profiles are often observed between different species of similar organisms, and sometimes these metabolic species-specific signatures have been proposed as chemotaxonomic markers (Ivanisevic et al. 2010;[START_REF] Sogin | Application of 1H-NMR metabolomic profiling for reef-building corals[END_REF]). In our study, different metabolic fingerprints between species of butterflyfish were observed in the apolar fraction, but no significant differences were observed in the polar fraction (Figure 4), which indicates that apolar metabolites are species-specific. However, other factors such as diet seemed to play a bigger role in the metabolite fingerprints than butterflyfish species (Figure 7).

Butterflyfish species are phylogenetically close, so our results could indicate that in historically close complex organisms (such as fish) other factors than species could have a major impact in the metabolic profiles.

Spatial variations in metabolic fingerprints have been observed for a number of species, including soft corals [START_REF] Matlock | Spatial variability in secondary metabolite production by the tropical red alga Portieria hornemannii[END_REF][START_REF] Rohde | Spatial variability in secondary metabolites of the indo-pacific sponge Stylissa massa[END_REF]He et al. 2014). Our results

show that metabolic fingerprints of butterflyfish mucus did not vary significantly over the three geographic sites studied or between different reef habitats (Figure 5). Although there are certain spatial differences in the reefs of Moorea, most of the environmental properties (temperature, salinity and pH) are quite homogeneous [START_REF] Lamy | Understanding the spatio-temporal response of coral reef fish communities to natural disturbances: insights from beta-diversity decomposition[END_REF]. We thus hypothesize that since butterflyfish gill mucus metabolome responds to ecological drivers, spatial variations might be observed in environmentally different geographic locations, but they could be masked in similar environments.

Differences in metabolic profiles in fish internal organs like liver have been observed in cultured fish after the administration of different diets [START_REF] Abro | Metabolic insights in Arctic charr (Salvelinus alpinus) fed with zygomycetes and fish meal diets as assessed in liver using nuclear magnetic resonance (NMR) spectroscopy[END_REF][START_REF] Silva | Metabolic fingerprinting of gilthead seabream (Sparus aurata) liver to track interactions between dietary factors and seasonal temperature variations[END_REF].

Different food intake can stimulate contrasted physiological reactions in fish, which result in the differential production of metabolites in fish internal and external organs or tissues CHAPTER V -164 - [START_REF] Roosta | The effects of dietary vitamin C on mucosal immune responses and growth performance in Caspian roach (Rutilus rutilus caspicus) fry[END_REF], Silva et al. 2014). For example, [START_REF] Ghehdarijani | The effects of garlic-supplemented diets on skin mucosal immune responses, stress resistance and growth performance of the Caspian roach (Rutilus rutilius) fry[END_REF] found that fish fed with a supplemented diet in garlic displayed higher protein levels and alkaline phosphatase in fish skin mucus. Besides, feeding on chemically defended organisms (such as algae, corals or sponges) can result in dietary sequestration of secondary metabolites [START_REF] Pennings | Sequestration of dietary secondary metabolites by three species of sea hares: location, specificity and dynamics[END_REF][START_REF] Thoms | Sequestration of Dietary Alkaloids by the Spongivorous Marine Mollusc Tylodina perversa[END_REF]. Our results show for the first time the importance of fish diet in the chemical composition of gill mucus. Butterflyfish feed on a wide variety of invertebrates, thus we can imagine that different diet preferences such as scleractinian corals, algae or polychaetes will expose butterflyfish to different secondary metabolites, that could significantly alter fish mucus chemical composition by 1) affecting fish physiology differently, for example depending on the toxicity of the secondary metabolites ingested and the detoxifying pathways developed [START_REF] Mclean | Pharmacological perspectives on the detoxification of plant secondary metabolites: implications for ingestive behavior of herbivores[END_REF] or

2) assimilating some of the secondary metabolites for defense purposes [START_REF] Thoms | Sequestration of Dietary Alkaloids by the Spongivorous Marine Mollusc Tylodina perversa[END_REF].

Butterflyfish with broader diets (omnivorous and facultative corallivores) had a significant higher metabolic diversity than obligate corallivorous fish, indicating that gill metabolome reflects the diversity of food intake. Trophic transfer of metabolites and their accumulation in external fish mucus has been observed before, such as the UV-absorbing molecules (MAAs), which are selectively sequestered via trophic transfer from corals, or tetrodotoxin, which is also accumulated through the food chain [START_REF] Eckes | Ultraviolet sunscreens in reef fish mucus[END_REF][START_REF] Noguchi | Tetrodotoxin -distribution and accumulation in aquatic organisms, and cases of human intoxication[END_REF].

Fish mucus also possesses a complex community of commensal microorganisms that play an important role in maintaining mucus homeostasis and protection against pathogens [START_REF] Boutin | Probiotic treatment by indigenous bacteria decreases mortality without disturbing the natural microbiota of Salvelinus fontinalis[END_REF][START_REF] Llewellyn | Teleost microbiomes: the state of the art in their characterization, manipulation and importance in aquaculture and fisheries[END_REF]. A recent study showed that some mucus bacteria are able to produce bioactive secondary metabolites, which could contribute to the overall mucus metabolome [START_REF] Sanchez | Examining the fish microbiome: vertebrate-derived bacteria as an environmental niche for the discovery of unique marine natural products[END_REF]. Different fish foraging behaviors could affect the mucus microbiome by encountering different bacterial communities or by benefiting some bacterial populations more than others, and this could be reflected in the mucus metabolome [START_REF] Antwis | Ex situ diet influences the bacterial community associated with the skin of red-eyed tree frogs (Agalychnis callidryas)[END_REF].

The loss of coral reef resilience is often cryptic, and it has been hard to characterize the instability of coral reef ecosystems before their collapse [START_REF] Bellwood | Confronting the coral reef crisis[END_REF]). When coral reefs fail to regenerate after natural and human impacts they undergo shifts to alternate states where coral dominance is often replaced by algae dominance [START_REF] Hughes | Catastrophes, phase shifts, and large-scale degradation of a Caribbean coral reef[END_REF][START_REF] Bellwood | Confronting the coral reef crisis[END_REF]). Butterflyfishes are closely related to coral cover, and since their mucus reflects directly their feeding habits we could suppose that butterflyfish mucus metabolome might prove CHAPTER V -165 -useful detecting changes in coral reefs. Decreases in coral cover might induce some facultative corallivorous feeders to adapt to the available food resources and such changes might be reflected in the mucus metabolome [START_REF] Wen | Dietary shift in juvenile coral trout (Plectropomus maculatus) following coral reef degradation from a flood plume disturbance[END_REF]. Selective feeding of butterflyfish on coral lesions associated to infective diseases where there is a high density of pathogenic organisms absent in healthy coral tissues could also produce remarkable differences in the fish mucus metabolome [START_REF] Frias-Lopez | Cyanobacteria associated with coral black band disease in Caribbean and Indo-Pacific Reefs[END_REF]Chong-Seng et al. 2011).

Some recent studies have shown that metabolic profiles of corals and their symbiotic dinoflagellates change under temperature stress [START_REF] Klueter | Taxonomic and environmental variation of metabolite profiles in marine dinoflagellates of the genus Symbiodinium[END_REF][START_REF] Sogin | Metabolomic signatures of increases in temperature and ocean acidification from the reef-building coral, Pocillopora damicornis[END_REF].

Therefore, changes in the metabolic profiles of butterflyfish preys are also likely to infer variations in butterflyfish mucus metabolome.

Parasitism also influences the gill mucus metabolome of butterflyfishes, especially in the polar fraction. There are several possible explanations for the relationship between the mucus metabolome and gill parasitism 1) fish mucus molecules could be responsible for the presence or absence of parasites, either by the presence of antiparasitic molecules or by the presence of recognition molecules that would enhance parasitism [START_REF] Kallert | No shot in the dark: myxozoans chemically detect fresh fish[END_REF]), 2) parasites can modulate the production of certain fish mucus molecules and 3) metabolic differences arise from fish physiological answers to parasitism.

Parasites are often more sensible to environmental degradation than their hosts [START_REF] Marcogliese | Parasites of the superorganism: are they indicators of ecosystem health?[END_REF]. Thus habitat modification and environmental degradation would have major impacts in the parasite communities, with a decrease in parasite richness and an increase in the abundance of some virulent parasites [START_REF] Tylianakis | Habitat modification alters the structure of tropical host-parasitoid food webs[END_REF][START_REF] Johnson | Host and parasite diversity jointly control disease risk in complex communities[END_REF]. Dactylogyrids are the primarily parasites of butterflyfish gills and we have observed they significantly influenceour results revealed that their presence is related with the gill metabolome of butterflyfishes. Therefore, changes in dactylogyrid communities and intensities due to coral reef degradation and perturbations are likely to be observed in the mucus metabolome.

To the best of our knowledge, this is the first study investigating the gill mucus metabolome. We have developed a non-targeted LC/MS metabolomic approach to study gill mucus metabolome of butterflyfishes and have studied how several butterflyfish life traits influence metabolic variability. Diet appeared as the major determinant in the differences observed among butterflyfish mucus samples followed by level of dactylogyrid parasites.

CHAPTER V -166 -Butterflyfish phylogeny and species were also responsible for some of the metabolic variability observed. Our results show that fish mucus reflects simultaneously several life traits of their hosts and could potentially be used as indicator of the environmental status of their host habitat. Metabolomics is a powerful tool to compare the biological response of organisms to several conditions and perturbations. Mucus is the first surface of exchange between fish and its environment, and therefore the analysis of the mucus metabolome offers a unique opportunity to study organism responses to environmental disturbances which might allow an early detection of environment degradation, before the known macroecology indicators take place (e.g. lost of fish richness -abundance, increase in seaweed cover) [START_REF] Heenan | Monitoring herbivorous fishes as indicators of coral reef resilience in American Samoa[END_REF]. This is the first study exploring coral reef fish mucus metabolome and some of the drivers of its variability, but an important part of the variability remains unexplained. We think that maybe other factors not considered in this study like fish age, gender or exact health status of the fish could also play a role in the metabolic variability of fish mucus.

Do mucus microbiome and metabolome interact to prevent ectoparasitic infection in a coral reef fish?

Reverter, M. 1,2 , Suzuki, M. 3 , Sasal, P. 1,2 , Fagervold, S. 3 , Lecchini, D.

Abstract

Chaetodon lunulatus is a butterflyfish that unlike other sympatric and phylogenetically close butterflyfishes with similar ecologies, has never been found parasitized in the gills by monogenean Platyhelminthes. In order to find a possible explanation for this intriguing phenomenon we first tested whether C. lunulatus mucus would be deterrent to these parasites in an in vivo study. C. lunulatus gill mucus induced a significant decrease in the number of parasites in three highly parasitized Chaetodon species. We followed this in vivo study by characterizing the chemical composition of the mucus in conjunction to a microbiome analysis in order to better understand the factors leading to parasite deterrence in this butterflyfish species. The microbiome of C. lunulatus contained higher relative abundances of Fusobacteria, Spirochaetes and Vibrionaceae when compared to other Chaetodon species.

These bacteria were strongly positively correlated with an overexpression of β-hemoglobin peptides in C. lunulatus, suggesting that might exist a relation between them. As hemoglobin peptides are known to present a wide array of bioactivities, we hypothesize that they might play a role in preventing monogenean infection of C. lunulatus. However, further evaluation of the biological activities of purified peptides will be performed in the laboratory to confirm the previous hypotheses. This is one of the first studies where fish metabolome and microbiome has been related to fish parasitism. However, more research is needed to clarify the exact role of these bacteria and peptides to the absence of parasites in C. lunulatus.

Keywords: fish mucus, metabolomics, microbiome, coral reefs, butterflyfishes, parasitism.

Introduction

Parasites modify competitive and trophic interactions between organisms, playing a major role in community and ecosystem organisation [START_REF] Hudson | Is a healthy ecosystem one that is rich in parasites?[END_REF]). However, environmental degradation due to pollution, and climate change or culture conditions such as overcrowding can lead to parasite outbreaks and high host mortalities [START_REF] Leung | More rapid and severe disease outbreaks for aquaculture at the tropics: implications for food security[END_REF][START_REF] Fox | Climate-driven tipping-points could lead to sudden, high-intensity parasite outbreaks[END_REF].

Host-parasite interactions, which result from a tight host-parasite co-evolution, are extremely complex [START_REF] Quigley | The mode of host-parasite interaction shapes coevolutionary dynamics and the fate of host cooperation[END_REF][START_REF] Song | Host-parasite coevolution in populations of constant and variable size[END_REF]. Hosts develop defence mechanisms to avoid parasite infections, and parasites develop counter-adaptations to overcome these defence mechanisms (Schmid-Hempel, 2008). Host defence mechanisms include physical barriers such as epithelial surfaces or mucous membranes and innate and adaptive immune responses [START_REF] Janeway | The front line of host defense[END_REF], Magnadottir, 2006).

The mucosal layers are the first protective barrier against pathogens, avoiding parasite contact with epithelial cells and promoting their clearance [START_REF] Hansson | Role of mucus layers in gut infection and inflammation[END_REF]. Mucus is mainly constituted of glycoproteins but it also contains a wide array of innate immune molecules such as lysozyme, immunoglobulins, lectins and numerous antimicrobial peptides [START_REF] Gómez | Neglected wild life: Parasitic biodiversity as a conservation target[END_REF]. Antimicrobial peptides (AMPs), which can either be derived from a biologically inactive proprotein that is processed to the active form or derived from larger, functional proteins, display biological activities against numerous pathogens including bacteria, fungi and metazoan parasites [START_REF] Vizioli | Antimicrobial peptides versus parasitic infections?[END_REF][START_REF] Radek | Antimicrobial peptides: natural effectors of the innate immune system[END_REF].

Several fish AMPs with antiparasitic activities (e.g. piscidins and HbβP-1) have been isolated

from different tissues of cultured fish including gill epithelium and epidermal mucus [START_REF] Colorni | Activity of the antimicrobial polypeptide piscidin 2 against fish ectoparasites[END_REF][START_REF] Ullal | Antimicrobial peptides derived from hemoglobin are expressed in epithelium of channel catfish (Ictalurus punctatus, Rafinesque)[END_REF][START_REF] Ullal | Antiparasitic activity of the antimicrobial peptide HbβP-1, a member of the βhaemoglobin peptide family[END_REF].

Native microbiota have multiple effects on hosts, such as modulation of the host innate immune system, which can influence disease resistance and parasitic infections (Gomez and Balcazar, 2008;Cirimotich et al. 2011aCirimotich et al. , 2011b)). For example, Cirimotich et al. (2011a) found that production of reactive oxygen species (ROS) by mosquito endogenous bacteria inhibited the development of malaria Plasmodium parasites. However, relationships between vertebrate microbiota and parasite co-ocurrence are poorly understood [START_REF] Bär | The Interplay of Host Microbiota and Parasitic Protozoans at Mucosal Interfaces: Implications for the Outcomes of Infections and Diseases[END_REF][START_REF] Kreisinger | Interactions between multiple helminths and the gut microbiota in wild rodents[END_REF] CHAPTER VI -172 -2015; [START_REF] Reynolds | Worming their way into the picture: microbiota help helminths modulate host immunity[END_REF]. In fish, some studies have highlighted the importance of fish mucus microbiota as protection against opportunistic bacteria [START_REF] Boutin | Probiotic treatment by indigenous bacteria decreases mortality without disturbing the natural microbiota of Salvelinus fontinalis[END_REF]), but to our knowledge no study has yet studied the interaction between fish microbiome and parasite infection.

Butterflyfishes (family Chaetodontidae) are a diverse and emblematic family of coral reef fishes distributed widely in all tropical seas. Butterflyfish phylogeny, ecology and behaviour have been extensively studied, and they are mostly territorial monogamous species that can consume a variety of prey including algae, polychaetes, crustaceans and coral [START_REF] Pratchett | Dietary overlap among coral-feeding butterflyfishes (Chaetodontidae) at Lizard Island, northern Great Barrier Reef[END_REF]. Butterflyfishes are naturally parasitized by monogeneans belonging to the family Dactylogyridae, which are direct cycle flatworms that attach to fish gills by two pair of hooks [START_REF] Kearn | Evolutionary expansion of the Monogenea[END_REF]Plaisance et al. 2004). In a recent study we found that out of 34 butterflyfish species, only Chaetodon lunulatus was never parasitized by gill monogeneans [START_REF] Reverter | Gill monogenean communitiyes (Platyhelminthes, Monogenea, Dactylogyridae) of butterflyfishes from tropical Indo-West Pacific Islands[END_REF]). This species does not present any remarkable different ecological or behavioural traits, therefore we hypothesised that specific parasitism differences between closely related butterflyfishes might arise from gill or gill mucus characteristics.

This study aims to investigate the role of C. lunulatus gill mucus in the absence of gill monogenean parasites in this fish species. We have performed an in vivo essay to test whether C. lunulatus gill mucus decreased monogenean rates on three highly parasitized butterflyfishes. Then, we studied the gill mucus microbiome and metabolome of several parasitized butterflyfishes versus the non-parasitised C. lunulatus. We found a higher abundance of strictly anaerobic bacteria (mostly Fusobacteria and sulfate-reducing bacteria)

and Vibrio bacteria in C. lunulatus, which are positively correlated with overexpressed haemoglobin-derived peptides in this latter species. We suggest that C. lunulatus haemoglobin-derived peptides might display biological activities and might prevent ectoparasitic infection in C. lunulatus. This is the first study, to the best of our knowledge, where a systems biology approach combining metabolomics and microbiomics was used to study host-parasite interactions in fish.

Material and methods

In vivo test

Butterflyfishes (C. ornatissimus, C. reticulatus and C. vagabundus) were collected alive at night using a hand-net. They were immediately placed and transported to the laboratory in a 10 L aerated bucket and placed in flow through 200 L tanks with a water renewal of 20 L/h and aeration via an airstone. After 36 h of acclimatisation, fish were anesthetized for 1 min with 0.1% of clove oil (Syzygium aromaticum). Five fish of each species were carefully injected 1 mL of freshly collected C. lunulatus mucus on the gill arcs. Control fish (n = 5 for each species) were anesthetized and handled as the treated fish, but without actually injecting mucus on the gill arcs. Treated fish and control fish were then placed into two different 200 L tanks. 24 h after the treatment, all fish were euthanised, gills were dissected and monogenean parasites were counted under a binocular microscope.

Fish mucus sampling

Eight butterflyfish species (C. further treated to remove (using custom bash and awk scripts) OTUs for which the representative sequence was shorter than 372 base pairs (400 bp minus the length of the primer), OTUs which were represented by a single sequence in the ensemble of samples and a "Root" taxonomy status by the rdp_classifier analysis, as well as sequences not alignable by pynast.

Data statistical analysis

Microbiome differences between parasitized and non-parasitized fish were assessed on the classified OTUs at family level, since it there was a considerable amount of OTUs not identified to genus and species level. Principal component analysis (PCA) was used to assess the microbiome differences between the sample groups (parasitized vs. non-parasitized) in an unbiased manner and detect patterns (package ade4 for the R software). Permutational Multivariate Analysis of Variance (PERMANOVA, function adonis, package vegan for R) was used to test significance of PCA group separations. Partial least squares discriminant analysis (PLS-DA), a form of supervised multivariate analysis that aims to find the maximum separation between a priori groups was performed (package mixOmics for R). PLS-DA model accuracy was calculated using a two-fold cross validation (2CV) and the resulting number of misclassifications (NMC) was compared to its permutated null distribution (999 permutations) to test for model significance (p-value < 0.05). To identify OTUs driving separation in the PLS-DA model, Variables Importance in Projection (VIP) were obtained (package RVAideMemoire for R). OTUs with a VIP score higher than 1.5 were retained and Kruskal-Wallis test was used to identify significant abundance differences of OTUs between the groups (parasitized and non-parasitized). filter (Phenomenex, CA, USA). The quality control (QC) samples were prepared by pooling small aliquots from the polar and apolar extracts, respectively.

Metabolomics analyses

Chemical extraction of antimicrobial peptides

An acidic extraction was performed to enrich the extract in peptides following the method described in Vidal-Dupiol et al (2011). Briefly, 10 mg of freeze-dried mucus were re-suspended in 1 mL of 2M acetic acid. The suspension was placed in a 4°C water bath, sonicated (Vibra-cell TM 75185, 50% power, three pulses of 30 s) and centrifuged at 10,000 x g for 20 min at 4°C, to remove the cellular fragments. The supernatant was immediately collected and prefractionated using a Strata C18 cartridge (500 mg/ 6 mL; Phenomenex, CA, USA). Briefly, the C18 column was washed using acidified water with TFA (0.05%), and three successive elutions were performed with 10, 60, and 80% acetonitrile in acidified water.

The fractions obtained were lyophilised and reconstituted with 1 mL of a H 2 O/acetonitrile

(1:1) mixture. To randomise analytical sequences and reduce systematic error associated with instrumental drift, a Latin square was carried out for each of the sample sequences (polar and apolar). QC samples were analyzed at the beginning, the end and equidistantly throughout the sequence. In order to detect column contamination throughout the sequence methanol blank samples were analysed just each the QC sample.

LC-MS analysis

UHPLC-HRMS/MS

Ultra high performance liquid chromatography tandem mass spectrometry (UHPLC-HRMS/MS) analyses were performed on selected samples of non-parasitized fish (aqueous fractions and acidic extractions) in order to obtain a higher mass accuracy of the discriminant compounds obtained by the metabolomic analyses. Analyses were performed on a Dionex

Data pre-treatment and statistical analysis

LC-MS raw data files were converted to netcdf files with the Excalibur Software. Netcdf files were processed using the R package XCMS to detect, deconvolute and align features (molecular entities with a unique m/z and a specific retention time). XCMS analysis of these data provided a matrix containing the retention time, m/z value and integrated peak area of the identified features. Data were normalised by log transformation prior to statistical analysis.

The gill mucus metabolome data was analysed using the same pipeline than the microbiomics data (section 2.3.2). Briefly, a PCA was performed to observe metabolite differences between the sample groups (parasitized and non-parasitized), and a PERMANOVA test was used to check for PCA group significant separation. Then a PLS-DA (2CV validation and permutation test) was performed and the VIP (metabolites responsible for the separation between the two groups) were obtained. Initially, features with VIP scores higher than 1.5 were retained. Kruskal-Wallis test was applied to identify significant differences of the selected feature (VIP) expression between the groups (parasitized and non parasitized). Pertinence of selected features was also checked in the LC-MS chromatograms, and finally features with VIP scores higher than 2 were retained.

Peptide de novo sequencing

Peptide de novo sequencing, which consists in the determination of peptide sequences directly from tandem mass spectrometry (MS/MS) data, was carried out with the PEAKS Studio 7.5 (Bioinformatics Solutions, Waterloo, Ontario, Canada) software. PEAKS outputs a peptide significance score (-10lgP) [START_REF] Ma | PEAKS: powerful software for peptide de novo sequencing by tandem mass spectrometry[END_REF]2005). Error tolerances for de novo sequencing were set as follows: precursor mass 15 ppm and fragment ion 0.5 Da. PEAKS software can also identify amino-acid post-translational modifications, and modified peptides are evaluated with the same score (-10lgP) [START_REF] Zhang | PEAKS DB: de novo sequencing assisted database search for sensitive and accurate peptide identification[END_REF]. After obtention of the peptide sequences they were matched to the Swissprot database without taxonomic restriction and coverage percentage (%) of the matched proteins were obtained.

Comparison of gill mucus metabolome and microbiome (Relationship between the gill mucus metabolome and microbiome)

In order to study whether there was a significant relationship between the metabolites' VIP (> 1.5) and the OTUs' VIP (> 1.5), a Mantel test using Euclidean distances was performed. Relationship between the three haemoglobin metabolites overexpressed in C.

lunulatus and the bacteria overexpressed in C. lunulatus (strictly anaerobic bacteria, Fusobacteria, sulphate-reducing bacteria and Vibrio) were analysed by Pearson correlation.

Results

In vivo test

Microbiome results

Microbiome of one non-parasitized butterflyfish species (C. lunulatus) and three highly parasitized butterflyfish species (C. ornatissimus, C. reticulatus and C. vagabundus) were studied. PCA analysis was used to assess the differences on the gill mucus microbiome between the parasitized and non-parasitized fish. The PERMANOVA test on the three principal components (PC1, PC2 and PC3) indicated that the groups were significantly different (p-value = 0.028) (Figure 2a). The PLS-DA model was accurate to predict bacterial differences (family level) between parasitized and non-parasitized fish (NMC = 0.16, p-value = 0.023) (Figure 2b). Twenty-six bacterial families presented a VIP score higher than 1.5. However, only 14 of these families were significantly more abundant in C. lunulatus (Kruskal-Wallis, p < 0.05) (Figure 2c). Fusobacteriaceae was the bacterial family with the second highest VIP score, but presented the most significant difference between parasitized and non-parasitized fish (Figure 4). Spirochaetes (Spirochaetaceae and Sphaerochaetaceae) and Vibrionaceae were the other bacterial families that were significantly more abundant in C. lunulatus, representing more than 1% of total bacterial sequences (Figure 4).

Metabolomic results

Multivariate analysis and VIP obtention

Metabolomics analyses were performed on both polar and apolar fractions. None of the VIP features obtained from the apolar fraction was significantly overexpressed in the nonparasitized fish (Supplementary material). Therefore, all the following results, including VIP obtention and identification are based on the analysis of the polar fraction.

Principal component analysis (PCA) was used to assess the metabolic differences between the sample groups (parasitized vs. non-parasitized). The PERMANOVA test on the three principal components (PC1, PC2 and PC3) indicated that the groups in the polar fraction were significantly different (p-value < 0.001) (Figure 4a).The PLS-DA model was accurate to predict metabolite differences between parasitized and not parasitized fish (NMC = 0.077, pvalue = 0.001) (Figure 4b). 190 over 1835 identified features after feature alignment presented a VIP score higher than 1.5 and they were all significantly overexpressed in the non-parasitized fish (C. lunulatus) (Figure 5). Features with VIP scores > 1.5 were checked in the LC-MS chromatograms and due to signal intensity only VIP > 2 (43 features) were retained for further VIP characterisation from HR-LCMS analysis (Figure 4c). All the 43 *** * * * metabolites with VIP scores higher than 2 presented concentrations significantly higher in the non-parasitized fish than parasitized fish (Kruskal-Wallis p-value > 0.001).

Non-parasitised Parasitised

A B M531T544 M335T598 M336T511 M345T554 M360T581 M363T544 M370T530 M370T650 M371T541 M373T1000 M394T521 M397T487 M426T602 M440T480 M443T570 M448T779 M452T606 M458T551 M473T645 M495T542 M528T627 M529T573 M532T681 M534T744 M547T693 M582T522 M589T722 M645T780 M658T837 M668T837 M756T999 M757T861 M782T855 M787T848 M806T656 M829T797 M846T802 M891T798 M895T918 M912T942

VIP characterisation

Four different C. lunulatus extracts (polar fractions) were analysed by UHPLC-HRMS/MS in order to obtain a higher mass accuracy of the 43 features with VIP scores higher than 2. Forty-two of the features were multicharged ions corresponding to metabolites with masses ranging from 720 Da to 4556 Da. 17% of the metabolites presented a mass <1000 Da, 48% of the metabolites presented a mass between 1000-2000 Da and 33% of the metabolites presented a mass > 2000 Da (Figure 3a). LC-MS/MS profiles of some of the most important VIPs were characteristic of peptides, with several multi-charged ions and typical fragmentations (Figure 3b). Therefore, we decided to optimise the extraction procedure using an acidic extraction to selectively precipitate large proteins while enhancing the solubility of peptides. The UHPLC-HRMS/MS analysis of the acidic extract showed enrichment in three VIPs > 2 (M644T680, M657T837 and M782T855) (Figure 4). An acidic extraction of one heavily parasitized species (C. reticulatus) was also performed and we did not found any of the VIPs previously identified. The de novo sequencing done with the PEAKS software and the Swissprot database was achieved for the two most abundant VIP peptides at m/z 644.8725 and m/z 657.3557, which seem to match to amino acids sequences (respectively 10 and 30 residues) from the β-chain of the haemoglobin of the Japanese amberjack Seriola quinqueradiata (Table 1).

Relationship between metabolome and microbiome

The Mantel test revealed a strong correlation (R = 0.62, p-value = 0.001) between the metabolites and OTUs responsible for the differences between parasitized and non-parasitized fish (VIP > 1.5). Pearson correlation between the overexpressed bacteria in C. lunulatus and the three haemoglobin-derived peptides (M644T780, M657T837 and M782T855), showed that peptide M657T837 displayed the strongest correlation with Fusobacteriaceae (ρ = 0.85), while M644T780 and M782T855 displayed strong correlations with Spirochaetaceae (ρ M644T780 = 0.79 and ρ M782T855 = 0.81 respectively) and Sphaerochaetaceae (ρ M644T780 = 0.91 and ρ M82T855 = 0.91 respectively). The three hemoglobin-derived peptides had the weakest correlations with Vibrionoaceae (Figure 7).

Discussion

Most butterflyfishes are parasitized by gill monogenean parasites. To date, only one butterflyfish species, C. lunulatus, has never been found parasitized by gill monogeneans [START_REF] Reverter | Gill monogenean communitiyes (Platyhelminthes, Monogenea, Dactylogyridae) of butterflyfishes from tropical Indo-West Pacific Islands[END_REF]. C. lunulatus lives among parasitized butterflyfishes, and its similar ecology, behaviour and phylogenetic position cannot explain this marked absence of gill monogeneans. In this study, we have analysed the role of C. lunulatus gill mucus in the lack

M644T780 M657T7837 M782T855
of gill monogeneans in this species. We found that C. lunulatus mucus reduced significantly the number of parasites in three highly parasitized species, indicating a possible antiparasitic activity of C. lunulatus gill mucus. In our experiment, control fish followed the exact same stress as treated fish, but no mucus or other substance was injected on their gills. Therefore, we cannot exclude that the decrease in the number of parasites could be related to fish innate immune response to exogenous substances (gill mucus of another fish species), or even related to a decrease in oxygen diffusion due to an increase of mucus quantity.

Host commensal microbiota influences host physiology, playing an active role regulating host immune homeostasis [START_REF] Wu | The role of gut microbiota in immune homeostasis and autoimmunity[END_REF]. Fish microbiota homeostasis is important to avoid proliferation of pathogenic bacteria, and recent studies have shown that fish skin and gut isolates displayed antibacterial and antifungal properties against fish and human pathogens [START_REF] Sanchez | Examining the fish microbiome: vertebrate-derived bacteria as an environmental niche for the discovery of unique marine natural products[END_REF][START_REF] Boutin | Probiotic treatment by indigenous bacteria decreases mortality without disturbing the natural microbiota of Salvelinus fontinalis[END_REF][START_REF] Boutin | Inter individual variations of the fish skin microbiota: host genetics basis of mutualism?[END_REF][START_REF] Lowrey | Topographical Mapping of the Rainbow Trout (Oncorhynchus mykiss) Microbiome Reveals a Diverse Bacterial Community with Antifungal Properties in the Skin[END_REF]. In a previous study we characterised the core microbiota of three highly parasitized butterflyfishes and the non-parasitized C. lunulatus (Chapter 5). In this study we found that the strictly anaerobic bacteria Fusobacteria and Spirochaetes and Vibrionaceae (facultative anaerobic)

were significantly more abundant in C. lunulatus. These results indicate that C. lunulatus gill mucus might have less oxygen concentrations than the three highly parasitized butterflyfishes.

Fusobacteria produce a short-chain fatty acid, butyrate, that provides benefits to mammal hosts, enhancing mucus production and acting as an anti-carcinogen and anti-inflammatory (von [START_REF] Engelhardt | Role of short-chain fatty acids in the hind gut[END_REF][START_REF] Andoh | Physiological and anti-inflammatory roles of dietary fiber and butyrate in intestinal functions[END_REF]. Although, Fusobacteria role in fish is not well established, we can hypothesise that if they do enhance mucus production, this would increase gill mucus layer thickness, increasing oxygen diffusion distance, and eventually promoting higher levels of hypoxia [START_REF] Hess | Exposure of clownfish larvae to suspended sediment levels found on the Great Barrier Reef: Impacts on gill structure and microbiome[END_REF]. Spirochaetes bacteria are associated with a wide range of human and animal pathologies such as Lyme disease, syphilis, and dermatitis [START_REF] Radolf | Characterization of outer membranes isolated from Treponema pallidum the syphilis spirochaete[END_REF]2012;Fraser et al. 1998). Spirochaetes have also been found associated to marine invertebrates such as sponges and molluscs, some of them being infectious, however to date, we know little of their presence and role in fihs [START_REF] Neulinger | New Phylogenetic Lineages of the Spirochaetes Phylum Associated with Clathrina Species (Porifera)[END_REF]. Vibrionaceae bacteria are often reported as opportunistic pathogens causing numerous diseases on a wide range of marine organisms including fish, shellfish and corals [START_REF] Diggles | Vibrio species associated with mortalities in hatchery-reared turbot (Colistium nudipinnis) and brill (C. guntheri) in New Zealand[END_REF]Sung et al. 2001;[START_REF] Cervino | The Vibrio core group induces yellow band disease in Caribbean and Indo-Pacific reef-building corals[END_REF]. Increased mucus production on stressed or infected fish is well reported, therefore a higher abundance of Vibrionaceae bacteria and eventually Spirochaetes could also be related to an increase in mucus production and decrease of oxygen [START_REF] Ferguson | Responses of mucus-producing cells in gill disease of rainbow trout (Oncorhynchus mykiss)[END_REF][START_REF] Hess | Exposure of clownfish larvae to suspended sediment levels found on the Great Barrier Reef: Impacts on gill structure and microbiome[END_REF].

We have also studied the overexpressed metabolites in C. Lunulatus and we identified several peptides that could be derived the β-subunit of haemoglobin that were absent in other fish species, which were especially well correlated to Fusobacteria and Spirochaetes.

Haemoglobin (Hb) is an iron-containing oxygen-transport metalloprotein with a quaternary structure composed of two α-globin and two β-globin chains [START_REF] Anthea | Human Biology and Health[END_REF]. Hb is mainly found in the red blood cells of vertebrates, however, it is also expressed in nonerythroid cells such as alveolar epithelial cells, macrophages, retinal pigment epithelial cells and hepatocytes [START_REF] Saha | Hemoglobin expression in nonerythroid cells: novel or ubiquitous?[END_REF]. Haemoglobin play a crucial role in the transport of oxygen to the cells, and several studies reported an increased expression of haemoglobin under low oxygen levels in organisms such as fish, plants or humans [START_REF] Hunt | Increased level of hemoglobin 1 enhances survival of hypoxic stress and promotes early growth in Arabidopsis thaliana[END_REF][START_REF] Heinicke | Long-term exposure to intermittent hypoxia results in increased hemoglobin mass, reduced plasma volume, and elevated erythropoietin plasma levels in man[END_REF]. Haemoglobin up-regulation has also been observed in several fish and shellfish tissues such as skin, gill or internal organs following Vibrio infections [START_REF] Bao | Structure and immune expression analysis of hemoglobin genes from the blood clam Tegillarca granosa[END_REF][START_REF] Meloni | Preliminary study on expression of antimicrobial peptides in European sea bass (Dicentrarchus labrax) following in vivo infection with Vibrio anguillarum. A time course experiment[END_REF]. In addition to its primordial function as an oxygen carrier, haemoglobin is a known source of biologically active peptides that exhibit varied functions such as analgesic, antimicrobial and antiparasitic (Takagi et al. 1979;[START_REF] Liepke | Human hemoglobin-derived peptides exhibit antimicrobial activity: a class of host defense peptides[END_REF][START_REF] Ullal | Antiparasitic activity of the antimicrobial peptide HbβP-1, a member of the βhaemoglobin peptide family[END_REF]. [START_REF] Ullal | Antimicrobial peptides derived from hemoglobin are expressed in epithelium of channel catfish (Ictalurus punctatus, Rafinesque)[END_REF] found that expression of three β-chain haemoglobin peptides (HbβP-1, HbβP-2 and HbβP-3) increased after ich (Ichthyophthirius multifiliis)

infection in gill epithelium of channel catfish (Ictalurus punctatus). HbβP-1 displayed a moderate activity against bacterial pathogens, but had a high specific activity against different ectoparasite species ( [START_REF] Ullal | Antiparasitic activity of the antimicrobial peptide HbβP-1, a member of the βhaemoglobin peptide family[END_REF]. Cell-free Hb is toxic to the organism because it generates reactive oxygen species (ROS) that perturb the innate immune homeostasis [START_REF] Lee | A Perspective on the role of extracellular hemoglobin on the innate immune system[END_REF]. However, [START_REF] Jiang | Respiratory protein-generated reactive oxygen species as an antimicrobial strategy[END_REF] found that the Hb redox activity is specifically activated by microbial virulence factors such as lipopolysaccharides (LPS) and proteases. A recent study showed that Hb is specifically and progressively cleaved by the extracellular proteases of the invading microbe [START_REF] Du | Rapid reprogramming of haemoglobin structure-function exposes multiple dual-antimicrobial potencies[END_REF]. Some active Hb peptides then display a dual function, they bind to the pathogen-associated molecular patterns (PAMPs) and they produce cytotoxic free radicals, killing the pathogen in proximity and limiting at the same time diffusion of free radicals in the host [START_REF] Du | Rapid reprogramming of haemoglobin structure-function exposes multiple dual-antimicrobial potencies[END_REF]. Altogether, these results indicate that the hemoglobin peptides found in C. lunulatus might have biological activities such as antiparasitic and thus be responsible for the absence of monogeneans in this fish species. We suggest that expression of hemoglobin peptides in C. lunulatus might be enhanced either from a low oxygen environment, or after microbial cleaving of the hemoglobin protein. In turn, some of these peptides, such as peptide M657T837, which possess a high homology to HbβP-3, might display antimicrobial and anti-parasitic activities. Peptide purification, further peptide sequence confirmation of purified peptides, peptide synthesis and evaluation of the biological activities are being currently performed.

It is noteworthy to raise the question whether a durable association between potentially pathogenic Vibrio bacteria and C. Lunulatus could exist, or the observed higher Vibrio lunulatus. Further evaluation of purified peptides biological activities will be performed in the laboratory.

M531T544 M335T598 M336T511 M345T554 M360T581 M363T544 M370T530 M370T650 M371T541 M373T1000 M394T521 M397T487 M426T602 M440T480 M443T570 M448T779 M452T606 M458T551 M473T645 M495T542 M528T627 M529T573 M532T681 M534T744 M547T693 M582T522 M589T722 M645T780 M658T837 M668T837 M756T999 M757T861 M782T855 M787T848 M806T656 M829T797 M846T802 M891T798 M895T918 M912T942 M952T878
Non-parasitised Parasitised

Scientific context and model species

Scientific context

Fish and fishery products are an important source of proteins with crucial nutritional value, especially in developing and densely populated countries (FAO, 2014). Global fish production has been growing steadily with an average annual rate of 3.2% in the last five decades. Since fish captures have remained stable at about 90 million tones since the 1990s, aquaculture has been the main source for increasing fish supply. Global aquaculture production attained 90.4 million tones in 2012, and according to estimates, world food fish aquaculture production rose by 5.8% in 2013. World aquaculture production is imbalanced, with Asia accounting for about 88% of aquaculture production by volume, and China itself for nearly 62%. Oceania on the other side produced only 0.28% (184 191 tones) of world aquaculture production in 2012 (FAO, 2014).

French Polynesians, as other remote maritime areas, rely on sea resources as an important part of their daily diet, and are among the highest fish consuming nations in the world, with a fish consumption per capita of 54 kg/year (Dewailly et al. 2008a(Dewailly et al. , 2008b)).

Although, local fishing is still the main source of fish and seafood in French Polynesia, local aquaculture has emerged during the last years.

Aquaculture in French Polynesia is dominated by the black pearl aquaculture (Pinctada margaritifera cumingii) that takes places mainly in the Tuamotu and Gambiers archipelagos. French Polynesia, due to its culture performance and its economic value [START_REF] David | Molecular detection of betanodavirus from the farmed fish, Platax orbicularis (Forsskal) (Ephippidae), in French Polynesia[END_REF].

The orbicular batfish is indeed a highly appreciated fish by the local Polynesian and Chinese communities for its meat quality and taste, that has become uncommon in French Polynesian waters due to overexploitation. In 2010, a national hatchery was built, which has nowadays the total control of the orbicular batfish biological cycle, and in 2011 the first private orbicular batfish farm was established. In 2014, 13 tonnes of P. orbicularis were produced in Tahiti accounting for nearly 200.000 USD (FAO Global Aquaculture Production Database).

Platax orbicularis

Platax orbicularis (Forsskal, 1775; Ephippidae) is a tropical batfish found throughout the Indo-Pacific, which is normally associated to coastal ecosystems such as coral reefs and mangroves [START_REF] Barros | Comparative Allometric Growth of the Mimetic Ephippid Reef Fishes Chaetodipterus faber and Platax orbicularis[END_REF]. It reaches a maximum size of 40 cm and is usually found at depths of 30 m. Platax juveniles present extremely tall and deep dorsal and anal fins, which proportionally decrease in size with growth, as the adults become more "bat-like" with round and deeply compressed bodies [START_REF] Cavalluzzi | Ephippidae (Batfishes, Spadefishes)[END_REF]. The juveniles are usually found in shallow water, drifting around floating materials and mimicking dead leaves on the surface of the water through their coloration, body shape and swimming behavior (Barros et al. 2008).

Platax fingerlings feed on algae during the day and zooplankton during the night (Barros et al. 2008). Although ecology of adult orbicular batfish is not well studied, they are described as omnivorous fish [START_REF] Leis | Pelagic to demersal transition in a coral-reef fish, the orbicular batfish Platax orbicularis[END_REF]. Orbicular batfish are also produced in Taiwan for food purposes and in Thailand as ornamental fish for the aquarium market. In Tahiti, P. orbicularis fingerlings are produced in the indoor bio-secured Vaia hatchery and then transferred and reared in floating offshore netcages in the lagoon of Tahiti Iti. However, P. orbicularis fingerlings have been suffering

severe mortality episodes shortly after the transfer due to bacterial disease caused jointly by Tenacibaculum maritimum and Vibrio harveyi. Around five days after the transfer, fish show signs of disorientation, lost of appetite and reduced swimming ability. Shortly after, fish start showing symptoms of disease with white patches on tegument that evolve in necrosis, damaged pectoral and caudal fins and in some cases ulcers in the mouth too (Saulnier et al.

pers. comm).

Tenacibaculum maritimum (Flavobacteriaceae) is a Gram-negative, filamentous bacteria formerly known as Flexibacter maritimus that causes the marine disease tenacibaculosis, which affects a large number of fish species and causes heavy aquaculture losses worldwide (Avendano-Herrera et al. 2006b). Several authors studied the infection pathway of T.

maritimum, and although it isn't completely clear, it seems that T. maritimum would attack the body surfaces of immunosuppressed or already abraded fish, causing external lesions (ulcers, necrosis, eroded mouths, frayed fins and tail-rot), and once it penetrates the body, it would easily spread along internal organs causing septicemia (Avendano-Herrera et al. 2004, 2006b;Failde et al. 2013). An increased prevalence and severity of the disease has been reported at higher temperatures (above 15°C) and salinities (30 to 35‰) as well as with low water quality (Avendano-Herrera et al. 2006b).

Besides, external lesions caused by tenacibaculosis could then favor the entrance of other pathogenic bacteria such as Vibrio sp. (Avendano-Herrera et al. 2004). Vibrio harveyi (Vibrionaceae) is a Gram-negative, bioluminescent bacteria that is found normally in the intestinal microbiota of aquatic vertebrates and invertebrates. Although, it is often considered to be an opportunistic pathogen, V. harveyi has also been reported to be a primary diseasecausing agent with highly virulent strains [START_REF] Austin | Vibrio harveyi: a significant pathogen of marine vertebrates and invertebrates[END_REF]. Several diseases such as gastro-enteritis, skin ulcers and dermal and eye lesions as well as mortality episodes in different fish species have been attributed to V. harveyi infections [START_REF] Bertone | Vibrio carchariae associated with a chronic skin ulcer on a shark, Carcharhinus plumbeus (Nardo)[END_REF][START_REF] Ishimaru | Taxonomical re-examination of two pathogenic Vibrio species isolated from milkfish and swimming crab[END_REF][START_REF] Lee | Pathogenesis of gastroenteritis caused by Vibrio carchariae in cultured marine fish[END_REF]. The current treatment for tenacibaculosis and vibriosis is done by chemotherapy and antibiotic administration. Several studies have shown that bath treatments might be more effective against tenacibaculosis than oral treatments, since infected fish become anorexic immediately post-infection (Soltani et al. 1995). An alternative treatment to antibiotics is the use of immersion baths with surface acting disinfectants such as formalin or hydrogen peroxide. However, these baths are often agressive to fish and some physiology problems have been noted (McVitar and White, 1979;Avendano-Herrera et al. 2006a, 2006b).

This chapter aims to study alternative more sustainable solutions for the prevention and  There is an increasing use of natural products in aquaculture for disease control due to their multiple bioactivities.

 There is a great need to normalize the use of natural products in aquaculture in order to assure their efficacy.

 Algae natural products should be favoured to avoid any exogenous molecule to be introduced in the sea.

Introduction

Fish and fishery products represent a very valuable source of protein and essential micronutrients for balanced nutrition and good health. In 2009, fish accounted for 17% of the world population intake of animal protein and 6.5% of all protein consumed. World fish food supply has grown considerably in the last five decades, with an average growth rate of 3% per year in the period 1961-2009. Since fish capture has remained stable at about 90 million tonnes since 1990, aquaculture has been revealed as the main source for increasing fish supply. World aquaculture production reached 62 million tonnes in 2011 (excluding seaweeds and non-food products), with an estimated value of US$ 130 billion. Aquaculture is the fastest-growing animal food producing sector with an average annual increase of 6% per year in the period [START_REF] Kajita | The immunomodulatory effects of levamisole on rainbow trout, Oncorhynchus mykiss[END_REF]-2010(FAO, 2012)).

However, world aquaculture production is vulnerable and an increase of diseases outbreaks has been reported due to culture intensification, resulting in partial or total loss of production (Bondad-Reantaso et al., 2005). Factors such as overcrowding, periodic handling, high or sudden changes in temperature, poor water quality and poor nutritional status contribute to physiological changes in fish such as stress or immunosuppression and thus, heighten susceptibility to infection. Moreover, high concentrations of fish and lack of sanitary barriers facilitate the spread of pathogens, producing high mortality levels [START_REF] Naylor | Effect of aquaculture on world fish supplies[END_REF]Cabello, 2006;[START_REF] Quesada | Considerations on the aquaculture development and on the use of veterinary drugs: special issue for fluoroquinolones-A review[END_REF].

CHAPTER VII -210 -

In order to avoid economic losses related to sanitary shortcomings, several veterinary drugs are commonly used in aquaculture to prevent or treat diseases outbreaks.

Antimicrobials and other veterinary drugs are administered regularly as additives in fish food or sometimes in baths and injections and are used as prophylactics (prevent diseases before they occur), therapeutics (treat sick animals) or growth promoters (Rico et al., 2013).

Nevertheless, the use of veterinary drugs is becoming more restricted since they present numerous side-effects for the environment and health safety. For example, massive use of antibiotics have resulted in the development of resistant bacteria strains (e.g. [START_REF] Mirand | Antimicrobial multiresistance in bacteria isolated from freshwater Chilean salmon farms[END_REF]Seyfried et al., 2010) or the presence of residual antibiotics in the muscle of commercialized fish and thus has potential consequences on human health (e.g. Cabello et al., 2006;Romero Ormázabal et al., 2012). The use of drugs like trichlorfon or praziquantel in bath treatments for ectoparasites has also numerous disadvantages like development of resistance (Umeda et al., 2006), hazardous for animal health [START_REF] Kiemer | The effects of hydrogen peroxide on the gill tissues of Atlantic salmon, Salmo salar[END_REF]Forwood et al., 2013) and environmental disadvantages. Vaccination has also been regarded as a potential treatment against diseases outbreaks in aquaculture. However, commercial vaccines are too expensive for a widespread use by fish producers and they have the downside that a single vaccine is effective against only one type of pathogens (Sakai, 1999;Pasnik et al., 2005;Harikrishnan et al., 2011a).

Considering the potential harm of veterinary drug treatments on environment and human health and in some cases their limited efficacy, disease management should concentrate on harmless, preventive and lasting methods. Moreover, disease outbreaks are frequently associated with fish fitness and health, most of pathogens being opportunistic and taking advantage of immunocompromised or stressed fish, thus alternate solutions should maximize fish immunity and fitness to avoid and face pathogen infections (e.g., [START_REF] Ruane | Modulation of the response of rainbow trout (Oncorhynchus mykiss Walbaum) to confinement, by an ectoparasitic (Argulus foliaceus L.) infestation and cortisol feeding[END_REF]Davis et al., 2002;Iguchi et al., 2003;Ashley 2007). Some of the proposed solutions are the use of natural products (plant extracts) or probiotics (beneficial microbial strains) in the culture of fish and shrimps (e.g., [START_REF] Panigrahi | Microbial intervention for better fish health in aquaculture: the Indian scenario[END_REF][START_REF] Makkar | Bioactivity of phytochemicals in some lesser-known plants and their effects and potential applications in livestock and aquaculture production systems[END_REF][START_REF] Citarasu | Herbal biomedicines: a new opportunity for aquaculture industry[END_REF]Lee and Gao, 2012 ;[START_REF] Mohapatra | Aquaculture and stress management: a review of probiotic intervention: Stress management by probiotics administration[END_REF]. Finally, there is an increasing interest in consuming organic and environmentally friendly food. Thereafter, the limitation of chemical products in aquaculture and the use of natural treatments could enhance the consumption of aquaculture products.

In this article we aim to review the potential of plant extracts as a sustainable and effective substitute of chemical treatments in fish aquaculture. In order to be as exhaustive as possible we performed several databases crossed searches with the keywords « plant extracts » and « fish aquaculture » (Web of Knowledge, PubMed, Scopus, ScienceDirect, SpringerLink Journals and Wiley Online Library) and we found 115 results that matched our criteria. The results clearly show that there is an increasing number of published studies highlighting the potential application of natural products and plant extracts in aquaculture either as immunostimulant or to fight parasite diseases (Figure 1).

Potential of plant extracts in aquaculture

Plant extracts have been reported to favor various activities like anti-stress, growth promotion, appetite stimulation, enhancement of tonicity and immunostimulation, maturation of culture species, aphrodisiac and anti pathogen properties in fish and shrimps aquaculture due to the active principles such as alkaloids, terpenoids, tannins, saponins, glycosides, flavonoids, phenolics, steroids or essential oils [START_REF] Citarasu | Herbal biomedicines: a new opportunity for aquaculture industry[END_REF]Chakraborty and Hancz, 2011). Besides, their use could reduce costs of treatment and be more environmentally friendly as they tend to be more biodegradable than synthetic molecules and they are less likely to produce drug resistance on parasites due to the high diversity of plant extract molecules [START_REF] Logambal | Immunostimulatory effect of leaf extract of Ocimum sanctum Linn. in Oreochromis mossambicus (Peters)[END_REF][START_REF] Blumenthal | Integrative Medicine Communications[END_REF][START_REF] Olusola | The potentials of medicinal plants extracts as bioantimicrobial in aquaculture[END_REF].

CHAPTER VII -212 -

Plant extracts as appetite stimulators and growth promoters

Several plant extracts are reported to stimulate appetite and promote weight gain when they are administered to cultured fish (Pavaraj et al., 2011;Harikrishnan et al., 2012a[START_REF] Takaoka | Effect of rotifer enrichment with herbal extracts on growth and resistance of red sea bream, Pagrus major (Temminck & Schlegel) larvae against Vibrio anguillarum[END_REF] (Table 1,2). [START_REF] Shalaby | Effects of Garlic (Allium sativum) and chloramphenicol on growth performance, physiological parameters and survival of Nile tilapia (Oreochromis niloticus)[END_REF] showed that food intake, specific growth rate and final weight of Nile tilapia (Oreochromis niloticus) increased when garlic was incorporated in the diet. In another study, grouper Ephinephelus tauvina fed with a diet supplemented with a mixture of methanolic herb extracts (Bermuda grass (Cynodon dactylon), Long pepper (Piper longum), stonebreaker (Phyllanthus niruri), coat buttons (Tridax procumbens) and ginger (Zingiber officinalis)) displayed 41% higher weight than fish fed with the control (Punitha et al., 2008). Ji et al. (2007a) showed that olive flounder (Paralichthys olivaceus) fed with a herbal mixture of medicated leaven (Massa medicata fermentata), hawthorne (Crataegi fructus), virgate wormwood (Artemisia capillaries) and Cnidium officiale (2:2:1:1) had higher weight gain than the control fish and showed higher total carcass unsaturated fatty acid content and lower carcass saturated fatty acid content, indicating that feeding with the herbal mixture improves fatty acid utilization. The authors suggested that this could be caused by a lower plasma triglyceride and high plasma HDL-CHO (high-density lipoprotein cholesterol) levels in the herbal mixture diets.

Besides, plant extracts have been shown to improve digestibility and availability of nutrients resulting in an increase in feed conversion and leading to a higher protein synthesis (Nya and Austin, 2009b;[START_REF] Citarasu | Herbal biomedicines: a new opportunity for aquaculture industry[END_REF]Talpur et al. 2013b). For example, Putra et al. (2013) showed that supplemented diet with 1% of ethanolic katuk extract (Sauropus androgynous) stimulated appetite, growth and improved food utilization (lower feed conversion ratio) in grouper Ephinephelus coioides. However, E. coioides fed with enriched diets with higher percentages of katuk extract (2.5 and 5%) presented lower growth. These results also indicate the importance of suitable dosing to obtain the desired effects and thus the need of further studies to chemically characterize extracts in order to be able to quantify active molecules and establish adequate doses.

Plant extracts as immunostimulants

The immune system is classified into innate (non-specific) and adaptive (specific) immune systems. The innate immune system is the first line of defense against invading pathogens and their major components are macrophages, monocytes, granulocytes, and humoral elements, including lysozyme or complement system (Harikrishnan et al., 2009b).

An immunostimulant is a substance that enhances the defense mechanisms or immune response (both specific and non-specific), thus rendering the animal more resistant to diseases and external agressions (Anderson, 1992).

An increasing interest in the use of plant extracts as fish immunostimulants has arisen in the last decade [START_REF] Galina | The use of immunostimulating herbs in fish. An overview of research[END_REF][START_REF] Vaseeharan | Medicinal plant derivatives as immunostimulants: an alternative to chemotherapeutics and antibiotics in aquaculture[END_REF]. Several studies have monitored the immunological parameters after intraperitoneal injection or orally administered plant extracts on distinct fish species and they found that treated fish showed increased lysozyme activity, phagocytic activity, complement activity, increased respiratory burst activity and increased plasma protein (globulin and albumin) (e.g. Dügenci et al., 2003;Yuan et al., 2007;[START_REF] Wu | Effects of hot-water extract of Toona sinensis on immune response and resistance to Aeromonas hydrophila in Oreochromis mossambicus[END_REF] (Table 1,2). Lysozyme plays an important role in the defense of fish by inducing antibacterial activity in the presence of complement (Harikrishnan et al., 2012b). Phagocytosis is one of the main mediators of innate immunity to pathogens, while respiratory burst is also a crucial effector mechanism for limiting the growth of fish pathogens [START_REF] Divyagnaneswari | Enhancement of nonspecific immunity and disease resistance in Oreochromis mossambicus by Solanum trilobatum leaf fractions[END_REF]. Increase in plasma protein, albumin and globulin, is considered as a strong innate response in fishes.

Other studies analyzed the hematological parameters which provides a clue of the fish health status and they found that erythrocytes, lymphocytes, monocytes, haemoglobin and haemocrit levels significantly increased in fish treated with plant extracts compared to control fish (e.g. [START_REF] Shalaby | Effects of Garlic (Allium sativum) and chloramphenicol on growth performance, physiological parameters and survival of Nile tilapia (Oreochromis niloticus)[END_REF][START_REF] Innocent | Studies on the immunostimulant activity of Coriandrum sativum and resistance to Aeromonas hydrophila in Catla catla[END_REF]Harikrishnan et al., 2012a).

For example, Park and Choi (2012) showed that Nile tilapia (Oreochromis niloticus) fed with diets containing different doses of mistletoe (Viscum album coloratum) for a period of 80 days in which immunological parameters were monitored (lysozyme activity, respiratory burst activity, alternative complement activity and phagocytic activity), displayed an increase in activity. After that period, fish were challenged against Aeromonas hydrophila and treated fish had 42% more survivability than control group.

Another study showed that olive flounder (Paralichthys olivaceus) infected with the scutociliate Philasterides dicentrarchi presented less mortality (30 -45%) when was fed with monkey head mushroom (Hericium erinaceum) enriched diet (0.1 and 1% respectively) compared to control group which had a mortality of 90%. Lysozyme activity and burst respiratory activity of olive flounder increased more in the treated groups, indicating that enhancement of immunological system leads to a better protection of olive flounder against P. dicentrarchi (Harikrishnan et al., 2011b). [START_REF] Kim | Effects of dietary kelp (Ecklonia cava) on growth and innate immunity in juvenile olive flounder Paralichthys olivaceus (Temminck and Schlegel)[END_REF] showed that non-specific immune response in juvenile olive flounder (P. olivaceus) fish was enhanced (increased phagocytes' respiratory burst, serum lysozyme and myeloperoxidase activities) with dietary supplementation of kelp (Ecklonia cava), most probably due to the high antioxidant and polyphenolic content in E. cava.

These results indicate that enriched diets with plant extracts have beneficial effects on fish health and enhance immune system and hence they could play an important role avoiding diseases outbreaks in aquaculture systems. However, in most cases mechanisms responsible

for the physiological answer in fish are still unknown.

Table 1. Growth-promoter and immunostimulant effect of plant extracts on fish.

Plant extracts as fish anti-pathogenic

Numerous studies have reported a wide range of bioactivities displayed by natural products from plants, fungus and algae, which revealed to be of great interest in the prevention or treatment of pathogens (e.g. Tagboto and Townson, 2001;[START_REF] Zheng | Natural products in clinical trials: antiparasitic, antiviral and neurological drugs[END_REF][START_REF] Zahir | Laboratory determination of efficacy of indigenous plant extracts for parasites control[END_REF]. Several current studies showed that plant extracts presented anti parasitic properties both in vitro and in vivo against fish pathogens (bacteria, virus, fungus, helminths and other parasites) (Table 2).

Antibacterial

Tests in vitro

Antibacterial properties of plant products have been by far the most studied bioactivity with potential application in aquaculture systems (Table 2; Table 3). Castro et al. (2008) found found that all garlic extracts were effective against the tested pathogenic bacteria.

Some recent studies reveal seaweed and algae as a potential source of antimicrobial products (e.g. [START_REF] Alghazeer | In vitro antibacterial activity of alkaloid extracts from green, red and brown macroalgae from western coast of Lybia[END_REF][START_REF] Mendes | Antimicrobial activity and lipid profile of seaweed extracts from the North Portuguese Coast[END_REF][START_REF] Al-Saif | Antibacterial substances from marine algae isolated from Jeddah coast of Red sea, Saudi Arabia[END_REF]. [START_REF] Dubber | Extracts of Ceramium rubrum, Mastocarpus stellatus and Laminaria digitata inhibit growth of marine fish pathogenic bacteria at ecologically realistic concentrations[END_REF] showed that methanol extracts of red hornweed (Ceramium rubrum) (10 mg dry weight/mL) and hexane extracts of oearweed (Laminaria digitata) (31 mg dry weight/mL) evoked strong antibacterial activities against 16 different bacteria tested (marine bacteria and fish pathogenic bacteria). They also showed that Gram-positive marine Bacillaceae were generally more susceptible than Gram-negative marine Vibrionaceae. Another study showed the broad antibacterial activity (agar diffusion assay) of ethanol extract of algae limu kohu (Asparagopsis taxiformis) (100 mg/mL) against 9 fish pathogenic bacteria, which was specially strong against Vibrio alginolyticus (17.0 ± 1.4 mm), Vibrio vulnificus (16.8 ± 1.0 mm) and Aeromonas salmonicida subsp. salmonicida (15.0 ± 0.9 mm) [START_REF] Genovese | In vitro evaluation of antibacterial activity of Asparagopsis taxiformis from the Straits of Messina against pathogens relevant in aquaculture[END_REF].

Tests in vivo

Indian major carp (Labeo rohita) fed with enriched diets in indian ginseng (Achyrantes aspera) (0.2%) and prickly chaff flower (Whitania somnifera) (0.5%) showed 41% and 49% respectively of reduced mortality when it was challenged against A. hydrophila compared to control groups (Vasudeva-Rao et al., 2006;Sharma et al., 2010)

Anthelminthic (monogeneans)

Monogenean parasites are flatworms which inhabit skin, gills and eventually eyes of fish. Monogeneans from the genus Dactylogyrus, Gyrodactylus and Neobenedenia are widespread parasites affecting a large number of cultured fish and inducing significant economic losses worldwide [START_REF] Deveney | First published record of the pathogenic monogenean parasite Neobenedenia melleni (Capsalidae) from Australia[END_REF][START_REF] Woo | Diseases and disorders of finfish in cage culture[END_REF]. Up to now, there are no effective methods to prevent monogenean infections in open aquaculture systems and the only available technique is to remove attached parasite stages through different bath treatments.

Moreover, monogenean eggs are highly resistant to physical and chemical treatments due to their sclerotised protein shell protecting the developing embryo [START_REF] Ernst | Effects of temperature, salinity, desiccation and chemical treatments on egg embryonation and hatching success of Benedenia seriolae (Monogenea: Capsalidae), a parasite of farmed Seriola spp[END_REF]Whittington, 2012).

In consequence, several studies have been performed recently to assess the anthelminthic activity of plant extracts in order to treat monogenean infections (Table 4).

Plant extracts like methanolic extract of bupleurum root (Radix bupleuri chinensis), aqueous and methanolic extracts of cinnamon (Cinnamomum cassia), methanolic extract of Chinese spice bush (Lindera agreggata) and methanolic and ethyl acetate extracts of golden larch (Pseudolarix kaempferi) have shown to possess 100% in vivo efficacy against monogenean Dactylogyrus intermedius when added to water of infected goldfish (Carassius auratus) (Wu et al., 2011;Ji et al., 2012). [START_REF] Tu | Anthelmintic efficacy of Santalum album (Santalaceae) against monogenean infections in goldfish[END_REF] studied the effect of several extracts of indian sandalwood (Santalum album) against Dactylogyrus intermedius and Gyrodactylus elegans on goldfish and found that chloroform extract was the most effective and safest for the fishes.

They also observed that bath treatment with long duration and multiple administrations could eliminate a greater proportion of monogenean infections.

Other parasites

Until now only few studies have reported plant antiviral, antifungal or antiprotozoal activities on fish pathogens (Table 5). Yellow-Head Virus (YHV) in vitro at concentrations as low as 1 μg/mL, and when black tiger shrimp (Penaeus monodon) were fed with the extract, the cumulative mortality decreased from 75% in control group to 33% in 1% enriched diet. In other studies P. monodon challenged with White Spot Syndrome Virus (WSSV) and intramuscular injected or orally administered aqueous extract of Bermuda grass displayed no mortality and no signs of white spot disease compared to 100% mortality observed in control group (Balasubramanian 2007(Balasubramanian , 2008a(Balasubramanian and 2008b)). All these studies point out that virus inactivation may occur by the interaction between the extract and the envelope of the virus.

Xue-Gang et al. ( 2013) showed that 10 plant species used in traditional Chinese medicine displayed strong inhibition against Saprolegnia and Achlya klebsiana, being the petroleum ether extracts of conidium fruit (Cnidium monnieri), magnolia bark (Magnolia officinalis) and aucklandia root (Aucklandia lappa) the ones with the best antifungal activity. Hashemi-Karouei et al. (2011) found that ethanol extracts of common rue (Ruta graveolens) had anti fungal effects and prevented growth of Saprolegnia sp., while [START_REF] Genovese | The Mediterranean red alga Asparagopsis taxiformis has antifungal activity against Aspergillus species[END_REF] found that the red algae Asparagopsis taxiformis presented antifungal activity against Aspergillus species. In an in vivo study, Chitmanat et al. (2005) showed that indian almond leaves (Terminalia catappa) extract reduced the fungal infection in tilapia eggs. [START_REF] Buchmann | Effects of sodium percarbonate and garlic extract on Ichthyophthirius multifiliis theronts and tomocysts: In Vitro experiments[END_REF] found that garlic extract killed theronts of the ciliate Ichthyophthirius multifilis responsible for the white spot disease at 62.5 mg/L and tomocysts of the same parasite at 570 mg/L. Harikrishnan et al. (2012c) showed that mortality of infected olive flounder by the ciliate Miamiensis avidus decreased from 80% in the control diet to 40% when fish were fed with a supplemented diet in Suaeda maxima extract.

These studies highlight the antiparasitic activities of numerous plant extracts and their potential to be used as an alternative to chemical treatments. However, the use of natural products in aquaculture is a very recent trend so even if most of the studies show promising results there is still little studies who have analyzed the long-term effects on fish physiology.

Several studies have shown that plant extracts can have a toxic effect on fish if applied at inadequate doses (e.g. Sambasivam et al., 2003;Ekanem et al., 2007 ;Kavitha et al. 2012).

Consequently, further regulation and standardization is required in order to use plant products in the aquaculture industry. Normalized extraction procedure depending on the bioactive CHAPTER VII -222 -molecules identified, application procedure, doses and duration of treatment depending on the type of parasite should be established.

Isolated natural products with anti parasitic properties

As the interest in the use of plant products in aquaculture is relatively recent, most of the efforts have been made in identifying potential activities on different plant species. However, identifying the active molecules responsible for the observed bioactivities is the next step and would allow 1) to optimize the extraction procedure 2) enable appropriate dosage and 3) study mechanisms of action.

A few isolated and characterized natural products have been evaluated for their activity in preventing and treating disease outbreaks in aquaculture. Kumar et al. (2013) administered oral azadirachtin (1) to goldfish and observed that all immunologic parameters were enhanced (p < 0.05) as well as hematologic parameters and fish presented a higher relative rate survival (42.6%) compared with the control against A. hydrophila infection. The same molecule also displayed potential to treat Argulus sp. infection in goldfish, presenting a therapeutic index (difference in toxicity between the parasite and the fish) of 4.10 (Kumar et al., 2012b).

Azadiracthin is a highly oxygenated triterpenoid isolated from neem (Azadirachta indica)

known to have antimicrobial and pesticidal properties while being non-toxic to vertebrates [START_REF] Govindachari | Insect antifeedant and growth regulating activities of neem seed oil -the role of major tetranortriterpenoids[END_REF]. Some works indicated that azadirachtin block microtubule formation and thus inhibiting cell division, and other studies indicated that azadirachtin antiparasitic effect can be explained by its effect on molting and juvenile hormones [START_REF] Mitchell | Effects of the neem tree compounds azadirachtin, salannin, nimbin, and 6-desacetylnimbin on ecdysone 20-monooxygenase activity[END_REF][START_REF] Mordue (luntz | Azadirachtin: an update[END_REF]) (Figure 2). Kumar et al. (2012a) evaluated the effect of piperine (2) on the ectoparasite Argulus sp.

parasitizing C. auratus and found that Argulus mortalities were positively correlated with concentration of piperine, whose therapeutic index was 5.8. This study confirmed that increase in time of exposure of parasitized fish to compound led to drastic reduction in the parasitic load to a minimum level, confirming that low dosage of piperine can effectively control ectoparasites from fish as long as the duration of exposure to the treatment is increased. Piperine is a bioactive amide isolated from long pepper (Piper longum) known to have insect repellent activity [START_REF] Traxler | Piperine, a pungent component of blackpepper[END_REF]) (Figure 2).

Several natural products such as sanguinarine (3), chelerythrine (4), chelidonine (5), dioscin

and isopimpinellin (13) have been isolated from different plants through bioassayguided fractionations and proved to have better anthelminthic activity against D. intermedius than positive controls of mebendazole (a commercial anthelminthic drug) [START_REF] Wang | Osthol and isopimpinellin from Fructus cnidii for the control of Dactylogyrus intermedius in Carassius auratus[END_REF](Wang et al., , 2010a(Wang et al., , 2010b[START_REF] Wang | In vivo anthelmintic activity of bruceine A and bruceine D from Brucea javanica against Dactylogyrus intermedius (Monogenea) in goldfish (Carassius auratus)[END_REF][START_REF] Li | Activity of the chelerythrine, a quaternary benzo[c]phenanthridine alkaloid from Chelidonium majus L. on Dactylogyrus intermedius[END_REF][START_REF] Yao | In vivo anthelmintic activity of chelidonine from Chelidonium majus L. against Dactylogyrus intermedius in Carassius auratus[END_REF][START_REF] Hao | Bioassay-guided isolation and identification of active compounds from Semen pharbitidis against Dactylogyrus intermedius (Monogenea) in goldfish (Carassius auratus)[END_REF], Liu et al., 2012) (Figure 2). Sanguinarine (3) is a benzo[c]phenanthridine alkaloid isolated from Kelway's plume poppy (Macleaya microcarpa) that has the ability to cause DNA single and double strand break resulting in DNA damage, which may be responsible for the anthelminthic activity [START_REF] Matkar | Sanguinarine causes DNA damage and p53independent cell death in human colon cancer cell lines[END_REF]. Chelerythrine (4) and chelidonine (5) are also benzo[c]phenanthridines with antimicrobial, antifungal and anti-inflammatory activity isolated from greater celandine (Chelidonium majus L.) (Walterova et al., 1995;Malikova et al., 2006). Chelerythrine (4) exhibits high cytotoxic potency against a wide number of cells, through multiple apoptosis-inducing pathways, so a direct action on mitochondria may be involved in the eradication of parasites (Slaninova et al., 2001;Kémeny-Beke et al., 2006;Kaminsky et al., 2008). Some studies like Philchenkov et al. (2008) indicated that the mechanism of action of chelidonine (5) against D. intermedius may be through the mithocondrial cell death pathway activated through the caspase-9.

Dioscin (6) and polyphyllin D (7) are two diosgenyl saponins isolated from Himalayan paris (Paris polyphylla) that display antibacterial [START_REF] Hufford | Antifungal activity of Trillium grandiflorum constituents[END_REF], antiviral (Ikeda et al.,

CHAPTER VII -224 - 2000)
, antifungal [START_REF] Takechi | Structure-activity relationships of the saponins dioscin and dioscinin[END_REF][START_REF] Sautour | Antifungal steroid saponins from Dioscorea cayenensis[END_REF], inflammatory [START_REF] Kim | Inhibition of mouse ear edema by steroidal and triterpenoid saponins[END_REF] and high cytotoxic activities [START_REF] Mimaki | Cytotoxic activities and structure-cytotoxic relationships of steroidal saponins[END_REF][START_REF] Cheung | Polyphyllin D is a potent apoptosis inducer in drug-resistant HepG2 cells[END_REF]. Recent works found that mode of action of saponins is similar to that of conventional anthelminthic drugs such as praziquantel, so they would affect the permeability of the cell membrane of the parasites and cause vacuolization and disintegration of monogenea teguments [START_REF] Schmahl | Treatment of fish parasites. 2. Effects of praziquantel, niclosamide, levamisole-HCl, and metrifonate on monogenea (Gyrodactylus aculeati, Diplozoon paradoxum)[END_REF]Wang et al., 2010a). Brucein A and D (8,9) are two quassinoids isolated from (Brucea javanica) that have displayed activity against several protozoan parasites (O' Neill et al., 1987;[START_REF] Wright | Use of microdilution to assess in vitro antiamoebic activities of Brucea javanica fruits, Simarouba amara stem, and a number of quassinoids[END_REF][START_REF] Wright | Quassinoids exhibit greater selectivity against Plasmodium falciparum than against Entamoeba histolytica, Giardia intestinalis or Toxoplasma gondii in vitro[END_REF][START_REF] Nakao | Evaluation of efficacy of bruceine A, a natural quassinoid compound extracted from a medicinal plant, Brucea javanica, for canine babesiosis[END_REF] as well as anti-inflammatory, antitrypanosomal and antimalarial activities [START_REF] Hall | Anti-inflammatory agents III: Structureactivity relationships of brusatol and related quassinoids[END_REF][START_REF] Saxena | Antimalarial agents from plant sources[END_REF][START_REF] Bawm | In vitro antitrypanosomal activities of quassinoid compounds from the fruits of a medicinal plant, Brucea javanica[END_REF] and it seems that their major cellular target on parasites are proteins [START_REF] Guo | Biologically active quassinoids and their chemistry: potential leads for drug design[END_REF].

Synergistic effect of mixed herbal extracts has been reported before, so it is of great importance to determine whether the bioactivity observed is due to isolated molecules or it is rather a consequence of a synergistic effect between several molecules contained in the extracts (Bhuvaneswari and Balasundaram, 2006;Ji et al., 2007b;Harikrishnan et al., 2009a[START_REF] Harikrishnan | Indian ginseng (Solanales, Solanaceae) has been used for centuries in Ayurvedic medicine to increase longevity and vitality. Scientific research has indeed showed antioxidant, anti-inflammatory, immune-modulating, and anti-stress properties in the whole plant extract. W. somnifera biologically active chemical constituents include alkaloids (isopelletierine, anaferine), steroidal lactones (withanolides, withaferins) and saponins containing an additional acyl group (sitoindoside VII and VIII) (Mishra et al. 2000). W. somnifera showed to have antibacterial, antiviral, immunostimulant and growth promoting activities in aquaculture[END_REF]. For example, [START_REF] Lee | Chemical composition and antimicrobial activity of the essential oil of Syzygium aromaticum flower bud (Clove) against fish systemic bacteria isolated from aquaculture sites[END_REF] showed that the minimum inhibitory concentration (MIC) of clove (Syzygium aromaticum) essential oil against bacteria isolated from aquaculture sites was lower than MIC from the supposed bioactive molecule eugenol (14) (Figure 2). However, Rattanachaikunsopon and Phumkhachorn (2010) and Abd el-Galil and Hashiem (2012a) showed that antibacterial activity of carvacrol (15) against Edwarsiella tarda and Tenacibaculum maritimum was enhanced when administered with cymene (16) (Figure 2).

Conclusion and perspectives

The beneficial properties and efficacy of plant products on the health of cultured fish depends on the plant part, the method of extraction and the extract concentration. Although numerous studies have reported multiple activities and potential application of plant extracts in aquaculture, there has been little effort to homogenize the extraction procedure, the extract concentration and the administration way. Intraperitoneal injection has proved to be the most rapid and efficient way of administration although it is expensive, laborious and stressful for fish, specially for very young specimens; thus oral administration seems the most suitable for aquaculture [START_REF] Blazer | Nutrition and disease resistance in fish[END_REF]Anderson, 1992;Yoshida et al., 1995). Besides, the effect of plant products on fish is dose-dependent and there is a potential for overdosing, so determining the suitable extract concentration is of great importance [START_REF] Kajita | The immunomodulatory effects of levamisole on rainbow trout, Oncorhynchus mykiss[END_REF], Harikrishan et al., 2011a). Therefore, the upcoming necessities are quantifying and characterizing chemically plant extracts in order to identify active molecules responsible for the observed activity and thus facilitate the establishment of a standardized protocol including different methods of extraction depending on the bioactive molecules observed, adequate extract concentration and regularized administration way.

Terrestrial plants have been studied and exploited extensively over centuries for the bioactivities of their extracts. Algae are also considered to be a rich source of bioactive molecules, but only recent researches have started to study their chemical composition and to evaluate their potential bioactivities, mainly against human pathogens (e.g. [START_REF] Lima-Filho | Antibacterial activity of extracts of six macroalgae from the Northeastearn Brazilian Coast[END_REF][START_REF] Sukoso | Study antibacterial and cytotoxic activ-ities of seaweed Porphyra sp. extract[END_REF]Zbakh et al., 2012). Nevertheless, they present an enormous potential to be used in aquaculture as immunostimulant and preventing diseases outbreaks as 1) they present a wide range of original bioactive molecules (e.g. [START_REF] Cannell | Algae as a source of biologically active products[END_REF][START_REF] Holdt | Bioactive compounds in seaweed: Functional food applications and legislation[END_REF][START_REF] Stengel | Algal chemodiversity and bioactivity : sources of natural variability and implications for commercial applications[END_REF] they seem to possess multiple strong bioactivities such as antibacterial and anthelminthic (e.g. [START_REF] Mayer | Marine pharmacology in 1998: Marine Compounds with Antibacterial, Anticoagulant, Antifungal, Antiinflammatory, Anthelmintic, Antiplatelet, Antiprotozoal, and Antiviral Activities; with actions on the Cardiovascular, Endocrine, Immune, and Nervous Systems; and other Miscellaneous Mechanisms of Action[END_REF][START_REF] Al-Saif | Antibacterial substances from marine algae isolated from Jeddah coast of Red sea, Saudi Arabia[END_REF] and 3)

the utilization of local algae can avoid the introduction of exogenous molecules in the marine environment. Therefore, more studies should be conducted in this discipline, following the same directions that plants in order to assure a regulated and appropriate use of either plant or algae extracts in aquaculture.

Regarding treatment of ectoparasites like monogeneans, studies have frequently focused on finding extracts or chemical compounds with anthelminthic properties which can be added

Introduction

Bacterial diseases present a major constraint to aquaculture development (Deifordt et al. 2011). Opportunistic and pathogenic bacteria are responsible for elevated mortality rates and decreased efficiency in aquaculture, causing high economic losses to fish farmers (Subasinghe et al. 2001;Sitja-Bobadilla et al. 2007). Antibiotics (such as oxytetracycline, chloramphenicol and erythromycin) are widely used to prevent bacterial diseases in fish. However, continuous and intensive use of antibiotics can lead to the emergence of drug resistant strains, leading to reduced efficacy of the drugs. Besides, antibiotic resistance can be transferred by horizontal gene transmission to environmental and human pathogenic bacteria, creating serious public health problems [START_REF] Cabello | Antimicrobial use in aquaculture re-examined: its relevance to antimicrobial resistance and to animal and human health[END_REF].

Therefore, novel strategies to control bacterial infections are needed for a further sustainable development of the aquaculture industry. Prevention of bacterial outbreaks is one of the most important health management options. However, since not all infections can be prevented, new techniques controlling pathogenic bacteria are also needed [START_REF] Defoirdt | Alternatives to antibiotics for the control of bacterial disease in aquaculture[END_REF]. Several biocontrol strategies have been proposed such as the use of probiotic bacteria, bacteriophages that kill specifically pathogenic bacteria or inhibition of bacteria virulence by quorum sensing disruption (Verschuere et al., 2001;[START_REF] Nakai | Bacteriophage therapy of infectious diseases in aquaculture[END_REF][START_REF] Natrah | Disruption of bacterial cell-to-cell communication by marine organisms and its relevance to aquaculture[END_REF]). However, most of these biocontrol strategies are still in the research phase and are not yet applicable to commercial aquaculture facilities [START_REF] Qi | Probiotics in aquaculture of China -Current state, problems and prospect[END_REF][START_REF] Defoirdt | Alternatives to antibiotics for the control of bacterial disease in aquaculture[END_REF]). The use of medicinal plants in aquaculture has also been proposed as an alternative to chemotherapy (Reverter et al. 2014).

Rural fish farmers around the world have long been using medicinal plants to improve water quality, reduce fish stress and prevent and treat fish diseases (Caruso et al. 2013). Plants and algae possess a wide variety of metabolites that confer them numerous biological activities such as antibacterial, antiviral, antiparasitic and antifungal properties. Numerous plants have been reported to favour various activities like anti-stress, growth promotion, appetite stimulation, and immunostimulation in fish and shellfish (Reverter et al. 2014). The use of plants in aquaculture could therefore enhance fish fitness and immunity, decreasing risk of bacterial outbreaks but also contribute to bacteria control in infections due to their antibacterial properties. Most investigations have focused on the study of terrestrial plants, however recent works show that algae possess structurally novel and biologically active metabolites with interesting activities on fish pathogens [START_REF] Sukoso | Study antibacterial and cytotoxic activ-ities of seaweed Porphyra sp. extract[END_REF]Zbakh et al. 2012).

Vibrio harveyi (Vibrionaceae) is Gram-negative luminous bacteria recognized as an important cause of disease in fish and marine invertebrates like penaeid shrimps. In fish, diseases include vasculitis, gastro-enteritis and eye lesions, while in shrimp V. harveyi is associated with luminous vibriosis (the shrimp glow in the dark, [START_REF] Prayitno | Experimental infections of crustaceans with luminous bacteria related to Photobacterium and Vibrio -effect of salinity and pH on infectiosity[END_REF], Bolitas negricans (diseased animals contain balled epidermal tissue blocking the digestive tract, [START_REF] Robertson | Experimental Vibrio harveyi infections in Penaeus vannamei larvae[END_REF]) and bacterial white tail disease [START_REF] Austin | Vibrio harveyi: a significant pathogen of marine vertebrates and invertebrates[END_REF]Zhou et al. 2012a). Recent studies have reported that several V. harveyi strains display multiple resistance against antibiotics like cephalothin, vancomycin, ampicillin, cefepime, cefotetan, ciprofloxacin, penicillin, rifampicin and streptomycin, indicating the urgent need for targeted alternative biocontrol strategies in aquaculture [START_REF] Kang | Antibiotic resistance of Vibrio harveyi isolated from seawater in Korea[END_REF][START_REF] Stalin | Molecular characterization of antibiotic resistant Vibrio harveyi isolated from shrimp aquaculture environment in the south east coast of India[END_REF]. Several medicinal plants (e.g. garlic-Allium sativum and ginger -Zingiber officinalis) and algae (e.g. Asparagopsis taxiformis and Sargassum latifolium) have been reported to display antibacterial activity against some V. harveyi strains [START_REF] Dashtiannasab | In vitro effects of Sargassum latifolium (Agardeh, 1948) against selected bacterial pathogens of shrimp[END_REF][START_REF] Genovese | In vitro evaluation of antibacterial activity of Asparagopsis taxiformis from the Straits of Messina against pathogens relevant in aquaculture[END_REF]Talpur and Ikhwanuddin;2012;Talpur et al. 2013).

Tenacibaculum maritimum (Flavobacteriaceae) is also a Gram-negative bacteria with worldwide distribution responsible for the ulcerative disease tenacibaculosis, which causes important losses in fish aquaculture. Tenacibaculosis causes mainly lesions on the body surface of fish such as ulcers, necrosis, eroded mouth, frayed fins and tail rots, and sometimes necrosis on the gills and eyes [START_REF] Ostland | Flexibacter maritimus associated with a bacterial stomatitis in Atlantic salmon smolts reared in net-pens in British Columbia[END_REF]Avendaño-Herrera et al. 2006b). Most tenacibaculosis treatments to date include the use of antibiotics, however, studies indicated that repetitive therapy with the same antimicrobial treatment resulted in T. maritimum resistance [START_REF] Avendaño-Herrera | Evolution of drug resistance and minimum inhibitory concentration to enrofloxacin in Tenacibaculum maritimum strains isolated in fish farms[END_REF]). However, to date only one study has assessed the effect of natural products (carvacrol and cymene, both major compounds in oregano and thyme) on T. maritimum (Abd-el-Galil et al. 2012a).

In this chapter, the antibacterial activity of several local plant or easily found in Frenh Polynesia (garlic -Allium sativum, turmeric -Curcuma longa, ginger -Zingiber officinalis, noni -Morinda citrifolia and tamanu -Calophyllum inophyllum) and algae (Anabaena torulosa, Asparagopsis taxiformis, Dichotomaria marginata, Lyngbya majuscula, Padina CHAPTER VIII -235 -boryana and Turbinaria ornata) were evaluated against the fish pathogenic Tenacibaculum maritimum and Vibrio harveyi. After a first antibacterial evaluation, the most active extract (A. taxiformis) was subjected to fractionation and antibacterial assays coupled to HPLC extract separation were performed in order to identify the bioactive molecules responsible for the observed antibacterial activity.

Material and methods

Extracts preparation for the biological activity screening

Fresh plant material (garlic -A. sativum, ginger -Z. officinalis, and turmeric -C. longa) Absorbance (λ = 340 nm) was monitored with a FLUOStar Optima microplate spectrophotometer during 6 hours (36 cycles, 10 min/cycle).

and

Disk diffusion essays

Disk diffusion assays were used as a first screening of bioactive plants and algae against the fish pathogenic bacteria T. maritimum (Aq9-64 isolated from P. orbicularis from Tahiti, Labofarm Laboratories, Loudeac, France). 20 μL of plant or algae extract diluted in MeOH (25 mg/mL) were imbibed in Whatman filters (6 mm diameter) and placed under an extractor hood for solvent evaporation. Positive controls consisted of disks imbibed with 20 μL of erythromycin solution (2.5 mg/mL) and negative controls consisted of disks imbibed with 20 μL of MeOH. Bacteria was cultured 24 hours prior to in vitro tests in LB Broth (5 g/L yeast extract (Simca), 10 g/L peptone (Simca) and 20 g/L NaCl) at 28°C and diluted at 1/25 (1 mDo). 100 μL of bacterial culture were added on agar plate surfaces and spread homogeneously with sterile glass beads. The impregnated disks (plant or algae extract, positive control and negative control) were placed on the agar plates suitably spaced apart and the plates were incubated at 28°C. Inhibition zones (IZ) were measured after 24 hours of incubation. Triplicates for each extract were performed and the results (inhibition efficiencies)

were expressed as the triplicate's mean of the division between the extract inhibition zone and the erythromycin inhibition zone (IE = IZ extract / IZ erythromycin ). Disk diffusion assays were also performed to evaluate the antibacterial capacity of the three A. taxiformis fractions. Fractions A, B and C and were diluted in MeOH (50 mg/mL) and disk diffusion assays were performed as described above.

Antibacterial assays coupled to HPLC extract separation.

Complementary antibacterial tests of A. taxiformis methanolic extract (fraction B) were done on 96-well plates previously coupled to HPLC fractionation in order to localise the extract bioactivity on a region or regions of the chromatogram. Antibacterial activity was evaluated on T. maritimum (Aq9-64). The experiment was carried out on a system from Waters including Alliance separation module 2695, the 2998 photodiode array detector, the 2424 ELS detector and a Fraction Collector III (WFC III) equipped with a rack supporting 96)

well microtiter plates (Waters Corp. MA, USA). The equipment was controlled, and the data was handled by the Empower Chromatography Data Software. HPLC elution conditions consisted of two eluants, A (water with 1‰ formic acid) and B (acetonitrile with 1‰ formic acid), an elution profile based on a linear gradient from 15 % to 100 % B in from 5 to 25 min, a flow rate of 1 mL.min -1 , and an injection volume of 20 μL of an extract at 50 mg/mL.

Routine detection was set at 254 nm, and the column used was Gemini C6-Phenyl (250 x 3 mm, 5μ, Phenomenex). From the column outlet, the eluate was divided, 0.3 mL/min were analyzed by PDA and ELS detectors, and 0.7 mL/min were collected continuously at uniform volume intervals (262 μL/well) in 96-well microplates.

CHAPTER VIII -238 -T. maritimum was cultured 24 hours prior to in vitro tests in LB Broth at 28°C. Five μl of DMSO (to dissolve the metabolites) and 195 μL of 1/25 diluted bacterial solution (1 mDO)

were added to freeze-dried microplates. Six positive controls were done by microplate (three replicates). Microplates were incubated at 28 °C for 24 h. Bacterial growth was estimated reading optical absorbance at 630 nm with a microplate reader Aviso Sirius HT (Ebersberg, Germany), after inoculation and 24 h later and % of bacteria growth inhibition were calculated.

Results

Antibacterial screening of plant and algae extracts

Broth dilution tests

Broth dilution tests showed that the highest dose ( 2 T. maritimum agglutinated during broth dilution tests forming filaments, and no bacterial exponential growth was observed after 6 hours of incubation. Disk diffusion assays were done to evaluate plant and algae antibacterial activities against T. maritimum.

Disk diffusion assays

Disk diffusion assays revealed that A. taxiformis (2014) was the only active extract against T. maritimum (IE = 0.45 ± 0.22) at the concentration tested (500 μg). None of the other plants or algae (including garlic and turmeric) displayed any antibacterial activity on T. maritimum.

Antibacterial activity of A. taxiformis fractions

Antibacterial activity of A. taxiformis (2014) fractions A (g) and B (g) was tested with disk diffusion assays (20 μL of 50 mg/mL extract) against the least susceptible bacteria T.

maritimum. C fraction (CH 2 Cl 2 ) yielded approximately 1 mg of dried extract, so antibacterial activity could not be evaluated for this fraction. Results show that only the A fraction (MeOH) presents strong antibacterial activities against T. maritimum (3.01 ± 0.53) (Figure 2).

HPLC coupled antibacterial assays

Complementary antibacterial assays coupled to HPLC separation were pursued with the A fraction of A. taxifomis extract (MeOH) (2014 and 2015) to localise bioactive regions of the chromatogram. For each replicate, 100 % of inhibition of T. maritimum growth was observed for the 75 th well (Figure 3). We analysed antibacterial bioactivity and chemical profile of A.

taxiformis extracts from different years and months (2014, 2015 and 2016) and they all displayed strong antibacterial activities and the same chemical profile (Figure 3). Purification and characterisation of the antibacterial compound or compounds corresponding to the 75 th well from the methanolic fraction of A. taxiformis (2015,2016) is being currently performed at the laboratory.

Discussion

V. harveyi and T. maritimum are both serious disease agents in aquaculture, causing heavy losses worldwide (Brian and [START_REF] Austin | Vibrio harveyi: a significant pathogen of marine vertebrates and invertebrates[END_REF]Avendaño-Herrera et al. 2006b). Repetitive antimicrobial treatment has resulted in the development of V. harveyi and T. maritimum resistant strains [START_REF] Avendaño-Herrera | Evolution of drug resistance and minimum inhibitory concentration to enrofloxacin in Tenacibaculum maritimum strains isolated in fish farms[END_REF][START_REF] Stalin | Molecular characterization of antibiotic resistant Vibrio harveyi isolated from shrimp aquaculture environment in the south east coast of India[END_REF]. Therefore, there is an urgent need to develop sustainable biocontrol strategies. In this study we have evaluated the antibacterial capacity of several Polynesian plants and algae against T. maritimum and V.

harveyi strains from Tahiti.

Garlic (Amaryllidaceae) and turmeric (Zingiberaceae) presented strong antibacterial activities against V. harveyi at 2 mg/mL; however, they did not present antibacterial activity against T. maritimum at the concentration tested (500 μg per disk). Garlic antibacterial properties have been widely studied on marine pathogenic bacteria, including on multidrug resistant V. harveyi strains [START_REF] Vaseeharan | Antibacterial activity of Allium sativum against multidrug-resistant Vibrio harveyi isolated from black gill-diseased Fenneropenaeus indicus[END_REF]Lee and Gao, 2012). Garlic multiple biological activities (e.g. antibacterial, antifungal, antiparasitic, antioxidant and immunostimulant) have mostly been associated to organosulfur compounds, particularly cysteine sulphoxides and thiosulphinates (Suleria et al. 2012). Allicin (diallylthiosulphinate), which is enzymatically formed from its percursor alliin when garlic is crushed, is considered the most bioactive compound of garlic, and its antibacterial activities have been confirmed

against a large number of human and fish pathogenic bacteria [START_REF] Ankri | Antimicrobial properties of allicin from garlic[END_REF]Nya et al. 2010). However, allicin is a labile compound easily transformed to a number of stable lipid-soluble allylsul des such as ajoene, which is also reported to have antibacterial activity [START_REF] Naganawa | Inhibition of microbial growth by ajoene, a sulfur-containing compound derived from garlic[END_REF]Viswanathan et al. 2014). Turmeric is also known to display numerous bioactivities, with reported antibacterial activities against a wide range of human and animal pathogenic bacteria, including V. harveyi and methicillin resistant Staphylococcus aureus [START_REF] Kim | Antibacterial activity of Curcuma longa L. against methicillin-resistant Staphylococcus aureus[END_REF]Lawhavinit et al. 2010). Curcuminoids, which include mainly curcumin, demethoxycurcumin and bisdemethodxycurcumin, are the most bioactive compounds of turmeric [START_REF] Balaji | Toxicity prediction of compounds from turmeric (Curcuma longa L.)[END_REF].

A. taxiformis was the only algae to display antibacterial activity, and the only extract to display activity against both of the bacteria tested, V. harveyi and T. maritimum. A. taxiformis extract has been reported to display potent antibacterial, antiparasitic and antifungal activities against human and marine pathogens (Hutson et al. 2012 ;[START_REF] Genovese | In vitro evaluation of antibacterial activity of Asparagopsis taxiformis from the Straits of Messina against pathogens relevant in aquaculture[END_REF][START_REF] Genovese | The Mediterranean red alga Asparagopsis taxiformis has antifungal activity against Aspergillus species[END_REF][START_REF] Vitale | Effectiveness of red alga Asparagopsis taxiformis extracts against Leishmania infantum[END_REF]. A. taxiformis produces numerous low molecular weight halogenated compounds including methanes, ethenes, acetic acids, acetamides, propanols, propanones, propenes, acrylic acids, propylene oxide, propyl and propanyl acetates, butenols, butenones and the recently described cyclopentenones (mahorone and 5-bromomahorone), which exhibit an array of biological activities including antibacterial and cytotoxic activities (Burresson et al. 1976;[START_REF] Greff | Mahorones, highly brominated cyclopentenones from the red alga Asparagopsis taxiformis[END_REF]. In our study, we found only one compound (or a group of similar compounds being eluted simultaneously) responsible for the antibacterial activity observed, and its purification and characterization is being currently perfomed in our laboratory. The bioactive compound/compounds are eluted at the end of the chromatogram, therefore we think they might be more lypophilic compounds than the previously mahorones described by [START_REF] Greff | Mahorones, highly brominated cyclopentenones from the red alga Asparagopsis taxiformis[END_REF].

A. taxiformis can also inhibit quorum sensing and the proposed compound responsible for this activity is a 2-dodecanoyloxyethanesulfonate (C 14 H 27 O 5 S) [START_REF] Jha | Quorum Sensing Inhibition by Asparagopsis taxiformis, a Marine Macro Alga: Separation of the Compound that Interrupts Bacterial Communication[END_REF]). Both V.

harveyi and T. maritimum are known to have quorum sensing (QS) mechanisms, therefore, A. taxiformis antibacterial activity observed against both bacteria could be related to inhibition of QS [START_REF] Defoirdt | Quorum sensing and quorum quenching in Vibrio harveyi: lessons learned from in vivo work[END_REF][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].

CHAPTER VIII -242 -

We observed a potent antibacterial activity of A. taxiformis methanolic extract against the aquaculture pathogenic bacteria V. harveyi and T. maritimum, and the characterisation of the bioactive compounds is currently being performed at the laboratory. Algae display a promising potential to be used in aquaculture against pathogenic infections due to their varied bioactive components. However, in order to evaluate their true potential in fish and shellfish aquaculture, in vivo experiences must be done to determine their effects on animal's physiology. Some studies have reported A. taxiformis immunostimulant activities in shrimp (Penaeus monodon), however, to date no study has yet evaluated the effect of A. taxiformis on fish immune system (Manilal et al. 2012(Manilal et al. , 2013)). A recent study showed A. taxiformis aqueous extract was highly toxic to fish when they were immersed in it [START_REF] Mata | Water-soluble natural products from seaweed have limited potential in controlling bacterial pathogens in fish aquaculture[END_REF]. Therefore, there is an urgent need to evaluate algae antibacterial activities together with in vivo fish treatments in order to evaluate the algae true potential as treatment of bacterial infections in aquaculture.

Introduction

World aquaculture production keeps steadily growing, reaching 70.2 million tons of farmed fish in 2013, an increase of 5.6% from 2012 (FAO, 2014). However, aquaculture growth is often linked to culture intensification, which leads to overcrowding and poor water quality and facilitates the spread of pathogens and disease outbreaks (Bondad-Reantaso et al. 2005). Several drugs, like antibiotic and anthelmintic drugs, are commonly used by fish farmers to prevent and treat disease outbreaks (Rico et al. 2013). However, the intensive use of drugs presents numerous disadvantages and can cause 1) accumulation in the muscle of commercialized animals, 2) development of resistance and 3) undesirable effects on the local environment (e.g. Cabello, 2006;[START_REF] Marshall | Food animals and antimicrobials: Impacts on human health[END_REF]. Besides, the rising cost of prescription drugs also limits their application in many developing countries where aquaculture is one of the main supplies of animal protein (Hoareau and DaSilva, 1999).

Medicinal plants appear to be a more sustainable and accessible alternative to synthetic drugs, and can at the same time boost fish fitness and immunity and help in pathogen prevention and treatment (Reverter et al. 2014). Medicinal plants have been reported to promote weight gain and enhance immunity in fish and shellfish as well as display antibacterial, antiviral, antifungal and antiparasitic activities against numerous aquaculture pathogens (Reverter et al. 2014). For example, oral administration of garlic powder (Allium sativum) promoted weight, enhanced immunity, showed antibacterial effects against Aeromonas hydrophila and Vibrio harveyi and antiparasitic effects against Neobenedenia sp. in several fish species [START_REF] Sahu | Effect of Allium sativum on the immunity and survival of Labeo rohita infected with Aeromonas hydrophila[END_REF][START_REF] Talpur | Dietary effects of garlic (Allium sativum) on haemato-immunological parameters, survival, growth, and disease resistance against Vibrio harveyi infection in Asian sea bass, Lates calcarifer (Bloch)[END_REF]Militz et al. 2013). Turmeric (Curcuma longa) and ginger (Zingiber officinalis) also showed immunostimulant, antibacterial, antifungal, antiviral and antiparasitic effects on fish and shellfish (Dügenci et al. 2003;Nya and Austin, 2009;[START_REF] Sahu | Effect of dietary Curcuma longa on enzymatic and immunological profiles of rohu, Labeo rohita ( Ham .), infected with Aeromonas hydrophila[END_REF]. Some recent studies are also showing the promising potential of some algae in disease treatment and prevention in aquaculture [START_REF] Dubber | Extracts of Ceramium rubrum, Mastocarpus stellatus and Laminaria digitata inhibit growth of marine fish pathogenic bacteria at ecologically realistic concentrations[END_REF]. Algae are considered to be a rich source of bioactive molecules and several in vitro studies have showed antibacterial, antiviral, antifungal and antiparasitic activities in different algae extracts (Choudhury et al. 2005;Hutson et al. 2012;[START_REF] Genovese | The Mediterranean red alga Asparagopsis taxiformis has antifungal activity against Aspergillus species[END_REF]. For example, the red algae Asparagopsis taxiformis displayed antibacterial, antifungal and antiparasitic activities against several fish pathogens and enhanced immune system of Penaeus monodon [START_REF] Genovese | In vitro evaluation of antibacterial activity of Asparagopsis taxiformis from the Straits of Messina against pathogens relevant in aquaculture[END_REF][START_REF] Genovese | The Mediterranean red alga Asparagopsis taxiformis has antifungal activity against Aspergillus species[END_REF]Manilal et al. 2013).

Orbicular batfish (Platax orbicularis -Forsskâl, 1775, Ephippidae) live in brackish and marine waters around coral reefs, and is highly appreciated by the Polynesian and Chinese communities for its high quality meat and taste [START_REF] Gasset | Le paraha peue (Platax orbicularis), biologie, pêche, aquaculture et marché[END_REF]. P.

orbicularis aquaculture is an emerging industry in French Polynesia, but advances in the control of its biological cycle together with the high economic value of this fish species, have contributed in its rapid development, mainly concentrated on Tahiti Island. However, orbicular batfish farming in Tahiti is suffering severe mortality episodes due to bacterial infections caused jointly by V. harveyi and Tenacibaculum maritimum shortly after the transfer of hatchery fingerlings to off-shore cages (D. Saulnier, pers. commun.). Since batfish aquaculture takes place close to coral reefs, synthetic drug utilization should be restricted and alternative treatments are required.

In the present study, we evaluate the capacity of some common medicinal Polynesian plants (garlic -A. sativum, turmeric -C. longa, ginger -Z. marginalis and noni -Morinda taxifolia) and algae (A. taxiformis) to increase expression of immune-related genes (lysozyme g and transforming growth factor) in orbicular batfish fingerlings when administered orally.

Weight gain or loss of treated fish compared to control was also monitored to evaluate the effect of plants on fish appetite. Plants and algae were chosen according to reported bioactivities and local ethnobotanical knowledge [START_REF] Sahu | Effect of Allium sativum on the immunity and survival of Labeo rohita infected with Aeromonas hydrophila[END_REF][START_REF] Sahu | Effect of dietary Curcuma longa on enzymatic and immunological profiles of rohu, Labeo rohita ( Ham .), infected with Aeromonas hydrophila[END_REF][START_REF] Nayak | Immunostimulant activity of the extracts and bioactivities of the fruits of Morinda taxifolia[END_REF]Nya and Austin, 2009).

Materials and methods

P. orbicularis fingerlings and sampling

Healthy fingerlings of P. orbicularis were obtained from the Vaia hatchery located in Vairao (Tahiti, French Polynesia). Fish were placed in flow through 200 L tanks (35 fish per tank) with a water renewal of 100L/h and aeration via an airstone. Temperature, salinity and dissolved oxygen were measured daily and the unfed and fecal materials were removed once a day. Fish were fed 4 times a day with commercial food AL2G (Le Gouessant, Lamballe, France). Administered food quantities were precisely determined according to a feeding ratio based on standard production, which evolves with fish growth (5 to 10% of biomass depending on fish age, [START_REF] Gasset | Le paraha peue (Platax orbicularis), biologie, pêche, aquaculture et marché[END_REF]. Experiments were carried out in triplicate tanks (3 tanks per treatment) and fish were sampled (3 fish/tank) after two or three weeks of treatment. Fish were weighted and spleen and kidney were collected aseptically and stored in RNA later (Ambion, Austin Texas, USA) at -80°C until RNA extraction (less than 2 months from collection time for most of the samples).

Diet

Fresh plant material (garlic -A. sativum, ginger -Z. officinalis, and turmeric -C. longa)

was bought from local farmers, whereas noni (M. citrifolia) was collected in the Moorea rainforest (French Polynesia) and the red algae A. taxiformis was collected on the coral reef outer slope in Moorea. Noni and A. taxiformis as well as pealed garlic, ginger and turmeric bulbs were freeze-dried, powdered and stored at -20°C until used. Enriched diets were prepared adding 3% of sunflower oil, 3% of cod liver oil and the chosen proportion of the plant per kg of commercial fish food (Table 1). The plant enrichment proportion of 3% was chosen as a standard dose to evaluate efficacy differences between plants and then, two doses (3 and 1.5%) were selected for A. taxiformis to evaluate the dose effect on gene expression of immune-related genes. Control diet was also prepared with 3% of sunflower oil and 3% of cod liver oil to maintain the lipid content. One experiment without oil enrichment was also CHAPTER IX

-250 -performed to study the effect of oil on fish physiology and immunostimulant activity in particular.

Table 1. Dietary plant enrichment specifications.

2.3. Immune-related gene expression study

Primer design

Since P. orbicularis is a non-model fish species, no sequences of immune-related or possible housekeeping genes were available on the GenBank database. Therefore, several primer sets were selected on the basis of a multiple alignment of nucleotide sequences of transforming growth factor (TGF-β1), lysozyme G (Lys G) and alpha-actin genes (-actin) from other fish species. Both lysozyme G and transforming growth factor genes were selected due to their key role in fish immune defense, and their potent regulatory activities on other immune molecules such as cytokines and complement [START_REF] Li | Transforming growth factor-beta regulation of immune responses[END_REF][START_REF] Saurabh | Lysozyme: an important defence molecule of fish innate immune system[END_REF]. These primers were designed using conserved regions of each gene and with Primer3 software to allow an optimal annealing temperature of 60°C +/-2°C. Control or invariant internal genes were necessary for the global normalization of the quantification by real-time PCR (qPCR). The targeted candidate control genes were -actin and elongation factor alpha (EF1), which were validated in several other fish species [START_REF] Varsamos | Pituitary hormone mRNA expression in European seabass Dicentrarchus labrax in seawater and following acclimation to fresh water[END_REF][START_REF] Tang | Validation of zebrafish (Danio rerio) reference genes for quantitative real-time RT-PCR normalization[END_REF][START_REF] Mo | Validation of reference genes for RT-qPCR analysis of CYP4T expression in crucian carp[END_REF].

A total of 17 combinations of forward and reverse primers (4 μM) for the 4 genes were analyzed by qPCR on dilutions of a reference cDNA sample obtained from a pool of cDNAs from spleen and kidney P. orbicularis tissues (see next section below). For each primer combination, seven series of dilutions tested in triplicate were used to establish the relationship between threshold cycle (Ct) qPCR values and log10 of the reference cDNA cDNA synthesis and gene expression analysis.

RNA was isolated from P. orbicularis spleen and kidney tissues using the kit «SV Total RNA Isolation System» from Promega (Madison, WI, USA). Disruption of the cells was carried out by agitation with metal beads for 15 min (30 agitations/s) using a bead-beating device. Concentration of the nucleic acids was quantified by measuring the absorbance at 260 nm using a Thermo Scientific Nanodrop 1000 Spectophotometer (Wilmington, Pennsylvania, USA). Purity of the samples was checked by measuring the ratio of OD 260/280 nm and 230/260 nm, and samples with a ratio lower than 1.8 or higher than 2.1 were purified again by isopropanol and ethanol precipitations. cDNA was generated using the «Transcriptor First The relative expression of TGF-β1 and Lys G genes were calculated using the comparative C t method also referred to as the 2 -ΔΔCt method [START_REF] Livak | Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method[END_REF].

Briefly, each immune-related gene expression level in fresh plant material condition was normalized with two housekeeping genes (-actin and EF1) (ΔCt = Ct target gene -C mean housekeeping genes ) and compared to the control diet condition (ΔΔCt = ΔCt target gene -ΔCt control ) to yield relative immune-related gene expression rate (2 -ΔΔCt ).

Statistical analysis

All experimental tests were performed by triplicate and mean ± S.D. was calculated.

Gene expression results were displayed using boxplots (package ggplot2 for R), where the median and the first and third quartile were represented. Normality of data distribution (Shapiro-Wilk test) and homogeneity of variances (Levene test) were tested and not satisfied, thus non-parametric tests were used. Mann-Whitney U test was used to identify differences among treatments. Significance level was considered at P < 0.05.

CHAPTER IX -253 -

Results

3.1.Growth

None of the enriched diets (plants and algae) displayed a negative effect on fish growth.

Only the fish fed with a diet supplemented in A. taxiformis presented a significantly higher growth (P < 0.05) than those fed with the control diet. Fish fed for two weeks on an enriched diet in A. taxiformis (3%) presented a weight gain of 13.8%, while fish fed for 3 weeks in A.

taxiform is (1.5 and 3%) presented respectively 23.8% and 14.8% weight gain (Table 3).

Immunomodulatory effect

Two immune-related genes were identified for the first time in P. orbicularis. The relative expression of genes encoding lysozyme g (Lys G) and transforming growth factor (TGF-β1) were measured in spleen and kidney of P. orbicularis.

CHAPTER IX

-254 -Since we used a control diet enriched in oil to maintain the same lipid content as our treatment diets, a first assay to test the oil effect on the expression of the studied immunerelated genes was performed. Oil did not increase expression level of neither Lys G nor TGF-β1 in P. orbicularis fingerlings. Fish fed with an enriched diet in turmeric had significant higher expression of Lys G in kidney (P < 0.05, Figure 1), while fish fed with garlic presented significant higher expression of Lys G in spleen (P < 0.05, Figure 1). Fish fed during 3 weeks with an enriched diet in A. taxiformis (3%) had significant higher expression of Lys G in both spleen and kidney and significant higher expression of TGF-β1 in kidney (P < 0.05, Figure 1 and 2). Fish fed during 3 weeks with an enriched diet in A. taxiformis (1.5%) presented significant higher expression levels of Lys g and TGF-β1 in kidney (P < 0.05, Figure 1 and2).

However, fish fed with A. taxiformis for 2 weeks did not display an increased expression level of Lys g or TGF-β1 in any of the organs. Ginger treated fish displayed a moderately but significant higher expression level of TGF-β1 in spleen. No immunostimulatory effect was observed with the noni-enriched diet. No immunosuppression effects were observed in any of the treated fish in our experiments.

Discussion

An increasing number of studies report medicinal plant bioactivities in fish and against fish pathogens, making bioactive plants and algae a new alternative to prevent and treat disease outbreaks in aquaculture (Reverter et al. 2014). Our study has revealed the potential of several local Polynesian plants and algae to enhance the expression of two immune-related genes (Lys G and TGF-β1) in orbicular batfish after 2 and/or 3 weeks of oral administration.

Lysozyme is a bacteriolytic enzyme that acts disrupting mucopolysaccharides in the bacterial cell walls, causing bacteria death [START_REF] Chipman | Mechanism of lysozyme action[END_REF]. Lysozyme can also trigger other immune responses such as the complement system and phagocytic cells (Magnadottir, 2006). Therefore, lysozymes play an important role in the defense of fish and an increased level of lysozyme observed after dietary administration of garlic, turmeric and A.

taxiformis, might improve fish performance against pathogenic infections. For example, Park and Choi (2012) showed that Nile tilapia (Oreochromis niloticus) fed with diets containing mistletoe (Viscum album coloratum) displayed increased levels of lysozyme and when challenged with the bacteria Aeromonas hydrophila, survivability of treated fish increased by 42%. Several studies have shown an increase of lysozyme and immunostimulant activity in other fish species after ginger, garlic and turmeric dietary administration [START_REF] Sahu | Effect of dietary Curcuma longa on enzymatic and immunological profiles of rohu, Labeo rohita ( Ham .), infected with Aeromonas hydrophila[END_REF]Nya and Austin, 2009;[START_REF] Talpur | Dietary effects of garlic (Allium sativum) on haemato-immunological parameters, survival, growth, and disease resistance against Vibrio harveyi infection in Asian sea bass, Lates calcarifer (Bloch)[END_REF]. We did not observe an increased expression of lysozyme G after 3 weeks of ginger enriched diet administration. Plant chemodiversity varies between individuals of the same species depending on plant ontogeny, environmental and genetic factors [START_REF] Moore | Explaining intraspecific diversity in plant secondary metabolites in an ecological context[END_REF]. Variability in secondary metabolites and biological activities have been reported in ginger species, therefore we could think that the absence of immunostimulatory activity of ginger on P. orbicularis fingerlings could be related to the specific ginger chemotype used in this experiment [START_REF] Ghasemzadeh | Variation in secondary metabolite production as well as antioxidant and antibacterial activities of Zingiber zerumbet (L.) at different stages of growth[END_REF]).

However, we can not exclude that maybe ginger dose used (3 %) or treatment length (3 weeks) could also not be the optimal to observe ginger immunostimulant effect on lysozyme g gene.

Transforming growth factor β is an immune regulator cytokine involved in wound repair processes, pro-inflammatory reactions and haematopoiesis [START_REF] Mccartney-Francis | Transforming growth factor beta: a matter of life and death[END_REF][START_REF] Lawrence | Transforming growth factor-beta: a general review[END_REF]. [START_REF] Atiba | Aloe vera oral administration accelerates acute radiation-delayed wound healing by stimulating transforming growth factor-β and fibroblast growth factor production[END_REF] showed that higher levels of TGF-β1 due to Aloe CHAPTER IX -258 -vera administration accelerated wound healing in rats. Other studies have shown that some immunostimulants and probiotics can increase levels of TGF-β1 expression in fish (Panigrahi et al. 2007;[START_REF] Awad | Effect of dietary supplements on cytokine gene expression in rainbow trout, Oncorhynchus mykiss (Walbaum)[END_REF]). In our study, TGF-β1 expression increased significantly in the kidney of fish fed with an enriched diet in A. taxiformis for three weeks, and a moderate increase was also observed in the spleen and kidney of ginger treated fish.

Length of treatment and dose are also important parameters when assessing plant effects on fish physiology, since inappropriate doses can either be not effective or display toxic effects on fish (Kavitha et al. 2012;Militz et al. 2013). In our study, length played a notable effect on the immunostimulant activity of A. taxiformis in orbicular batfish, and a weeklonger treatment caused a significant higher expression of two immune-related genes.

However, we did not observe very different responses between diets with two A. taxiformis doses (1.5 and 3%).

The widely distributed and rapidly spreading red algae A. taxiformis, is known to produce a high diversity of halogenated metabolites with multiple bioactivities [START_REF] Mcconnell | Halogen chemistry of the red alga Asparagopsis[END_REF][START_REF] Greff | Mahorones, highly brominated cyclopentenones from the red alga Asparagopsis taxiformis[END_REF][START_REF] Dijoux | The More We Search, the More We Find: Discovery of a New Lineage and a New Species Complex in the Genus Asparagopsis[END_REF][START_REF] Andreakis | Endemic or introduced? Phylogeography of Asparagopsis (Florideophyceae) in Australia reveals multiple introductions and a new mitochondrial lineage[END_REF]. Some chemical ecology studies have shown that Asparagopsis brominated metabolites are involved in the control of epiphytic bacterial communities and quorum sensing inhibition activities from MeOH extracts of A. taxiformis have been found (Paul et al. 2006a;[START_REF] Jha | Quorum Sensing Inhibition by Asparagopsis taxiformis, a Marine Macro Alga: Separation of the Compound that Interrupts Bacterial Communication[END_REF]. Paul et al. (2006b) showed that halogenated natural products from the sister species Asparagopsis armata deterred herbivorous feeding. In aquaculture, several studies have shown antibacterial, antifungal and antiparasitic properties of A. taxiformis extracts against fish pathogens (Hutson et al. 2012;[START_REF] Genovese | In vitro evaluation of antibacterial activity of Asparagopsis taxiformis from the Straits of Messina against pathogens relevant in aquaculture[END_REF][START_REF] Genovese | The Mediterranean red alga Asparagopsis taxiformis has antifungal activity against Aspergillus species[END_REF]. However, an in vivo study showed high toxicity of A. taxiformis aqueous extract in barramundi (Lates calcarifer) [START_REF] Mata | Water-soluble natural products from seaweed have limited potential in controlling bacterial pathogens in fish aquaculture[END_REF]. This is the first in vivo study to show that A. taxiformis increased growth and expression level of immune-related genes in fish, when administered orally. In this study we fed P.

orbicularis fingerlings during 2 and 3 weeks with a diet enriched in A. taxiformis and fish did not show any sign of deterrence due to the bioactive metabolites from the algae but rather an increased appetite and weight gain. Although we did not find any sign of algae toxicity in fish at any of the doses tested, longer in vivo studies should be done in order to evaluate algae toxicity on fish physiology after long exposures to the algae metabolites. Besides, studies on CHAPTER IX -259 -several fish species would be beneficial to understand whether some fish species are more susceptible than others to A. taxiformis metabolites, or it is rather the exposure or administration procedure which affects fish susceptibility to the algae. Finally, since A.

taxiformis proliferation is increasing in tropical areas like French Polynesia, its commercial use as fish food complement would not involve the introduction of exogenous molecules in the environment (and facility of culturing the algae) [START_REF] Mantelatto | Cost-benefit of different methods for monitoring invasive corals on tropical rocky reefs in the southwest Atlantic[END_REF].

Conclusions

This study showed the potential of some plants like garlic, turmeric and A. taxiformis to be integrated in fish diets to increase expression of immune-related genes. This is the first study, where A. taxiformis was orally adminisitered to fish, and results show its capacity to induce weight gain and increase level of two immune-related genes in the new cultured fish species P. orbicularis. Fish fed with garlic, turmeric and A. taxiformis increased the level of Lys G in the spleen and/or kidney of P. orbicularis fingerlings. Fish fed with and enriched diet in A. taxiformis for 3 weeks also increased the level of TGF-β1 and promoted weight gain in P. orbicularis fingerlings. These data provide interesting information on the effect of A.

taxiformis on orbicular batfish immunity, and it shows the promising potential of this algae to be used as a fish complement to promote weight gain and enhance immunity, without the introduction of exogenous molecules into the environment. Further studies involving in vivo challenges with pathogens in fish fed with enriched diets in A. taxiformis will be needed in order to assess A. taxiformis true potential to prevent or treat diseases outbreaks in aquaculture.

Introduction

The emerging Platax orbicularis aquaculture in French Polynesia is threatened by severe mortality episodes of fingerlings due to tenacibaculosis outbreaks (Saulnier, pers. comm.).

Tenacibaculosis is an ulcerative disease caused by the Gram-negative bacteria Tenacibaculum maritimum (Flavobacteriaceae) that affects a large number of fish species and causes significant economic losses worldwide (Avendaño-Herrera et al. 2006b). Tenacibaculosis causes mainly lesions on the body surface of fish, such as ulcers, necrosis, eroded mouth, frayed fins and tail rots, and sometimes necrosis on the gills and eyes [START_REF] Ostland | Flexibacter maritimus associated with a bacterial stomatitis in Atlantic salmon smolts reared in net-pens in British Columbia[END_REF]Avendaño-Herrera et al. 2006b). Tenacibaculosis outbreaks are mainly reported in cultured fish, following environmental or mechanical stress of fish, but a recent study reported low prevalences of tenacibaculosis in wild coral reef fish (Abd el-Galil and Hashem, 2012b). To the best of our knowledge, no published study has yet reported tenacibaculosis outbreaks in orbicular batfish.

Chemotherapy either by oral administration (e.g. antibiotics like enrofloxacin, florfenicol and oxytetracycline) or bath treatment (e.g. hydrogen peroxide) has been widely used to control tenacibaculosis, but variable results have been obtained [START_REF] Mcvicar | Fish and skin necrosis of cultivated Dover sole, Solea solea[END_REF][START_REF] Cepeda | First isolation of Flexibacter maritimus from farmed Senegalese sole (Solea senegalensis, Kaup) in Spain[END_REF]Santos, 2002, Avendaño-Herrera et al. 2005;2006b). Fish become anorexic immediately post-infection, and thus bath treatment seems to be more effective than oral administration of antibiotics [START_REF] Soltani | The relative susceptibility of fish to infections by Flexibacter columnaris and Flexibacter maritimus[END_REF]. However, bath treatments are expensive, have environmental impacts and induce considerable amounts of stress to fish, especially when applied to young fingerlings [START_REF] Kiemer | The effects of hydrogen peroxide on the gill tissues of Atlantic salmon, Salmo salar[END_REF]. Vaccines are also considered to control tenacibaculosis but there is currently only one vaccine commercially available to prevent turbot (Scophthalmus maximus) tenacibaculosis. However, isolated T. maritimum from different fish species is genetically variable, indicating that the vaccine developed for turbot may not be effective in preventing tenacibaculosis in other fish species [START_REF] Avendaño-Herrera | Intraspecific diversity of the marine fish pathogen Tenacibaculum maritimum as determined by randomly amplified polymorphic DNA-PCR[END_REF][START_REF] Toranzo | Disease trends in the primary marine fish species cultured in Spain: A 20-year study[END_REF]). Since aquaculture of orbicular batfish is still emerging, no studies evaluating the effectiveness of chemotherapy in orbicular batfish against bacterial infections such as tenacibaculosis are available.

Tenacibaculosis outbreaks occur often after the transfer of fish from hatchery to both inshore and offshore net cages, taking advantage of immunosuppressed or already abraded CHAPTER X -266 -fish [START_REF] Wakabayashi | Flexibacter maririmus sp. nov., a pathogen of marine fishes[END_REF] 1).

Material and methods

P. orbicularis fingerlings

Healthy fingerlings of P. orbicularis were obtained from the Vaia hatchery located in Vairao (Tahiti, French Polynesia). Fish were stocked in flow through 200 L tanks (35 fish per tank) with a water renewal of 20L/h and aeration via an airstone. Temperature, salinity and dissolved oxygen were measured daily and the unfed and fecal materials were removed once a day. Fish were fed 4 times a day with commercial food AL2G (LeGouessant, Lamballe, France), according to a feeding ratio based on standard production (5 to 10% of biomass, [START_REF] Gasset | Le paraha peue (Platax orbicularis), biologie, pêche, aquaculture et marché[END_REF]. Experiments were carried out in triplicate (3 tanks per treatment).

CHAPTER X -267 -

Diet

Fresh plant material (garlic -Allium sativum, ginger -Zingiber officinalis, curcuma -Curcuma longa and tamanu oil -Calophyllum inophyllum) was bought from the local market, whereas noni (Morinda citrifolia) was collected in the rainforest and the red algae (Asparagopsis taxiformis) was collected in the outer slope of Moorea (French Polynesia).

Noni, Asparagopsis as well as peeled garlic, ginger and curcuma bulbs were freezedried, powdered and stored at -20°C until used. Enriched diets were prepared adding 3% of sunflower oil, 3% of cod liver oil and the chosen proportion of the plant per kg of commercial fish food. The enriched diet with tamanu was prepared substituting the sunflower oil by tamanu oil (Table 1). The plant enrichment proportion of 3% was chosen as a standard dose to evaluate efficacy differences between plants and then, two doses (3 and 1.5%) were selected for A. taxiformis to evaluate the dose effect. Control diet was also prepared with 3%

of sunflower oil and 3% of cod liver oil to maintain the lipid content. One experiment without oil enrichment was also performed to study the effect of oil on fish physiology and disease resistance.

Tenacibaculum maritimum bath challenge

The fish were initially divided in two groups (6 tanks per group): enriched diet and control diet. On the 15th or 21st day of the experiment, fish were not fed for 24 hours, and then 3 tanks of each group were bath challenged in seawater with the bacteria T. maritimum.

Fish were transferred to 40 L aerated tanks, where 10 mL of T. Maritimum sowed liquid culture were added. T. maritimum (TFA4) was cultured in both solid and liquid cultures. Solid cultures were grown using Zobell Marine Agar (Difco™) and incubatedat 24h at 27°C, while liquid culture of T. maritimum was grown in Zobell Marine Broth (Difco™) for 24-48 hours at 27°C under agitation. T. Maritimum sowed solution was prepared immediately before the challenge and wasobtained using three solid T. maritimum cultures to sow the liquide culture.

After 2 hours of exposure, fish were rinsed with seawater and returned to the 200 L tanks.

Control fish (non challenged) were also placed in 40 L tanks for two hours with seawater in order to have the same stress level than challenged fish. Fish were then fed with their corresponding diets until the end of the experiment (7 days after infection). Dead fish were removed every two hours, and mortality was monitored.

Data analysis

Cumulative fish mortality (%) after 7 days post-infection was calculated for each treatment and control experiments and was expressed as the mean ± standard deviation of the three replicate tanks. T-tests were performed to identify significant different mortalities (pvalue < 0.5) between treated and control fish.

Results and discussion

Experimental infection of P. orbicularis with T. maritimum through bath challenge proved to be poorly reproducible. Mortality rates varied from 0% to 64% in control fish depending on the experiments, which greatly complicated the interpretation of the results concerning the plant treatments. Besides, in some experiments, highly different infection rates were observed among replicates of a same treatment. During the tamanu experiment, bad meteorological conditions contributed to specific culture conditions, with mean salinities of 18 ‰ and a high turbidity in the seawater.

Although, the fish did not show any sign of stress and their growth was normal, the infection was not successful. T. Maritimum is a virulent bacteria, that causes big economic losses in the aquaculture industry, however it appears to be sensible to salinity, and some studies have shown that increased prevalences and severity of tenacibaculosis were observed at higher salinities (30 to 35 ‰) (Avendaño-Herrera et al. 2006b). To the best of our knowledge, no study has yet assessed turbidity effect on T. maritimum virulence and pathogenicity. T. maritimum is a highly filamentous bacteria, so it could be possible that high levels of suspended materials in the water could aggregate the bacteria, diminish available pathogenic bacteria to infect the fish, or decrease T. maritimum fitness, and thus decrease bacterial virulence. In fact, P. orbicularis fingerlings in the wild are found to inhabit shallow turbid waters where they drift around floating materials such as logs and ropes and mimic dead leaves. This habitat selection could result from a natural adaptation of the fish juveniles to protect themselves against pathogens.

CHAPTER X

-270 -

The experimental infection in fish from the noni experiment was also unsuccessful. Fish did not show any behavioural change post-infection such as disorientation or appetite loss, and no signs of tenacibaculosis could be observed on their bodies or fins. Fish from the noni experiment weighted around 30 g, whereas fish in the other experiments weighted between 5 and 10 g. Different susceptibilities to T. maritimum associated with fish body weight have been observed before, however studies showed that T. maritimum resistance was observed in fish above 100 g (Avendaño-Herrera et al. 2005;2006b). Therefore, higher fish weight could be a plausible hypothesis for the non-infection of P. orbicularis fingerlings during the noni experiment, but we cannot reject other explanations.

The third and last experiment in Moorea was on fish fed with an enriched diet of ginger and an enriched diet of garlic. Fish from these experiments were successfully infected, although infection was not even among replicates. Only two out of three control replicates were infected. The control mortality of the infected tanks was 12.86 ± 1.24 %, the ginger treated fish mortality was 27.61 ± 4.36 % and the garlic fed fish mortality was 6.67 ± 4.36 % (Figure 2A). Although, there was a poor repeatability among the control replicates, garlic treated fish had a significant higher survivability than the control fish and the ginger treated fish.

A second experiment with an enriched diet of garlic was performed, along with an enriched diet in curcuma. Despite low cumulative mortalities, (control = 5.37 ± 1.65 %, garlic = 1.07 ± 1.65 % and curcuma = 2.15 ± 1.65 %), garlic and curcuma treated fish displayed significantly (p-value < 0.05) lower mortalities than control fish (Figure 2B).

Finally, two experiments were performed on fish fed with an enriched diet in the red algae A. taxiformis, with different treatment lengths (2 and 3 weeks) and two doses (1,5 and 3 % of A. taxiformis). In the first experiment (2 weeks), a control diet without oil enrichment was also performed in order to test if the presence of high oil content in food played a role against T. maritimum. Results showed no significant differences in the fish mortality between the three treatments (control = 21.66 ± 6.24, control without oil = 13.33 ± In summary, mortality levels varied a lot between the experiments, and different levels of infection could be observed between tanks of the same condition. Garlic seems to be the Mortality was reduced by 80% and 61% in fish fed with an enriched diet of garlic during 2 weeks and 3 weeks correspondingly. Fish fed with an enriched diet in curcuma also presented a reduction of mortality of 60%.

Conclusions and perspectives

Parasites are an ubiquitous component of ecosystems, displaying important functional roles such as agents of natural selection and contributing to ecosystem organisation [START_REF] Marcogliese | Parasites: Small Players with Crucial Roles in the Ecological Theater[END_REF][START_REF] Poulin | Parasite biodiversity revisited: frontiers and constraints[END_REF]. Healthy natural ecosystems support a high parasite diversity, which contributes to the host-parasite balance and control of disease outbreaks [START_REF] Johnson | Host and parasite diversity jointly control disease risk in complex communities[END_REF]). However, under certain circumstances, such as climate perturbations or in high-density cultures, host-parasite interactions are modified, which can lead to outbreaks of infectious diseases [START_REF] Hudson | Is a healthy ecosystem one that is rich in parasites?[END_REF][START_REF] Brooks | How will global climate change affect parasite-host assemblages?[END_REF]Pulkkinen et al. 2009).

Until now, chemotherapy has been widely used to prevent and treat disease outbreaks in culture systems such as aquaculture. Use of chemical drugs, however, presents multiple negative impacts on the environment and human health (e.g. development of bacterial strains and residual accumulation in animal tissue) (Reverter et al. 2014, Chapter VII). Besides, use of drugs such as antibiotics, has been shown to have immunosuppressive effects and alter the balance of the microbial community that provide protection against several infections, predisposing animals to repeated disease outbreaks (Rijkers et al. 1998;[START_REF] Sekirov | Antibioticinduced perturbations of the intestinal microbiota alter host susceptibility to enteric infection[END_REF].

Therefore, disease management should concentrate on sustainable methods that increase fish fitness.

Medicinal plants have attracted increasing attention during the last few years as a potential alternative to chemotherapy in aquaculture. Plant products have been reported to stimulate appetite and promote weight gain, to act as immunostimulants and to have antibacterial and anti-parasitic (virus, protozoans, monogeneans) properties in fish and shellfish due to active molecules such as alkaloids, terpenoids, saponins and flavonoids taxiformis extracts against fish pathogens, however, the only previous in vivo fish study showed high toxicity of A. taxiformis aqueous extract in farmed barramundi (Lates calcarifer) [START_REF] Genovese | In vitro evaluation of antibacterial activity of Asparagopsis taxiformis from the Straits of Messina against pathogens relevant in aquaculture[END_REF][START_REF] Genovese | The Mediterranean red alga Asparagopsis taxiformis has antifungal activity against Aspergillus species[END_REF][START_REF] Mata | Water-soluble natural products from seaweed have limited potential in controlling bacterial pathogens in fish aquaculture[END_REF]. This is therefore, the first study in which oral administration of A. taxiformis in fish has been performed (Chapter IX). Results show a promising potential of A. taxiformis to be included in fish diets to increase fish fitness and enhance immune responses. However, further studies investigating the effectiveness of A.

taxiformis enriched diet on disease protection in fish against bacterial pathogens need to be performed through in vivo challenge trials (Chapter X).

Better understanding of fish-parasite interactions and natural parasite evasion mechanisms can also provide new opportunities for sustainable parasite control in aquaculture. I have comprehensively studied the butterflyfish-monogenean interactions of 34 butterflyfish species from the tropical Indo-West Pacific Islands (Chapter I, II). Dactylogyrid parasites displayed high specificity, sympatric butterflyfish species presenting significantly different dactylogyrid communities (Chapter I). Results suggest that past biogeographical processes might have shaped parasite specificity probably through tight host-parasite coevolution. Spatial differences in the parasite nestedness (abundance gradient) were related to different environmental parameters, indicating the importance of favourable environmental conditions for dactylogyrid egg hatching and effective host infection (Chapter II). Recent studies have shown that environmental factors such as salinity or temperature are important factors in the arrangement of marine parasite communities [START_REF] Thieltges | Salinity gradient shapes distance decay of similarity among parasite communities in three marine fishes[END_REF]Poulin et al. 2011;[START_REF] Blasco-Costa | Biogeography of parasitism in freshwater fish: spatial patterns in hot spots of infection[END_REF]. Higher temperature and salinity improved monogeneans egg hatching success, reduced time to sexual maturity and resulted in parasites reaching sexual maturity at a larger size [START_REF] Tubbs | Effects of temperature on fecundity in vitro, egg hatching and reproductive development of Benedenia seriolae and Zeuxapta seriolae (Monogenea) parasitic on yellowtail kingfish Seriola lalandi[END_REF][START_REF] Lackenby | Effect of water temperature on reproductive development of Benedenia seriolae (Monogenea: Capsalidae) from Seriola lalandi in Australia[END_REF][START_REF] Brazenor | Effects of temperature and salinity on the life cycle of Neobenedenia sp. (Monogenea: Capsalidae) infecting farmed barramundi (Lates calcarifer)[END_REF]. The knowledge of environmental effects on parasite physiology are of high importance for an effective parasite management, and for example, Brazenor and Hutson recommended a more frequent treatment of fish stocks during warm temperature periods when parasites can complete their life cycle more quickly.

Out of the 34 butterflyfish species studied, I found only one species, C. lunulatus, that was never found parasitised by gill monogeneans (Chapter I). The ecology and phylogenetic position of C. lunulatus has been extensively studied [START_REF] Pratchett | Dietary overlap among coral-feeding butterflyfishes (Chaetodontidae) at Lizard Island, northern Great Barrier Reef[END_REF][START_REF] Pratchett | Declines in the abundance of Chaetodon butterflyfishes following extensive coral depletion[END_REF]2014, Fessler and Wesneat, 2007[START_REF] Hsu | Molecular phylogeny of Chaetodon (Teleostei: Chaetodontidae) in the Indo-west Pacific: evolution in geminate species pairs and groups[END_REF]) and there is no obvious peculiarity that IV). Microbiome of outer surfaces such as skin and gills has been reported to display a higher diversity than inner organs such as gut [START_REF] Lowrey | Topographical Mapping of the Rainbow Trout (Oncorhynchus mykiss) Microbiome Reveals a Diverse Bacterial Community with Antifungal Properties in the Skin[END_REF]. Cultured fish microbiome is also expected to be less diverse than wild fish microbiome, which have higher genetic variability as well as higher diet plasticity [START_REF] Givens | Comparison of the gut microbiomes of 12 bony fish and 3 shark species[END_REF]. This study was the first to investigate wild fish gill mucus, therefore, more studies should be conducted to improve the knowledge on gill mucus microbiota. I found that a considerable proportion of butterflyfish gill mucus microbiota consisted of bacteria commonly found in the coral holobiont (Chapter IV). Since mucus layers are nutrient hotspots for marine heterotrophic bacteria living in oligotrophic environments such as coral reefs, I hypothesised that fish external mucus layers might act as a reservoir for coral reef bacterial diversity.

Metabolome study of eight butterflyfish species (four highly parasitized species, three low parasitized species and C. lunulatus) also revealed a high metabolic diversity, which differed significantly between fish species, fish with different diets, phylogenetic clade and parasitism level (Chapter V). Results showed that fish diet (obligate corallivore, facultative corallivore and omnivore) played a major role in the metabolite composition of gill mucus.

Different food intakes can stimulate different physiological reactions in fish, which might

Conclusion and perspectives

-279 -result in the differential production of metabolites in fish organs or tissues [START_REF] Silva | Metabolic fingerprinting of gilthead seabream (Sparus aurata) liver to track interactions between dietary factors and seasonal temperature variations[END_REF].

Diet effect on metabolic profiles of fish internal organs such as liver are well reported, however, some studies have also reported expression of diet acquired metabolites such as mycosporine-like aminoacids (MAAs) or toxins in fish outer surfaces like skin mucus [START_REF] Zamzow | Ultraviolet-absorbing compounds in the mucus of shallow-dwelling tropical reef fishes correlate with environmental water clarity[END_REF][START_REF] Noguchi | Tetrodotoxin -distribution and accumulation in aquatic organisms, and cases of human intoxication[END_REF][START_REF] Abro | Metabolic insights in Arctic charr (Salvelinus alpinus) fed with zygomycetes and fish meal diets as assessed in liver using nuclear magnetic resonance (NMR) spectroscopy[END_REF]. Gill mucus metabolome of butterflyfishes was also influenced significantly by gill monogenean parasitism rates.

Differences between parasitized and non-parasitized fish were the second most important factor explaining metabolic variability in the gill mucus polar fraction.

C. lunulatus gill mucus sustained a higher abundance of the anaerobic bacteria Fusobacteria and Spirochaetes that were positively correlated with overexpressed haemoglobin derived peptides in C. lunulatus gill mucus (Chapter IV). Fusobacteria is known to produce a short-chain fatty acid, butyrate, which enhances mucus production in mammals [START_REF] Andoh | Physiological and anti-inflammatory roles of dietary fiber and butyrate in intestinal functions[END_REF]. If Fusobacteria also enhances mucus production in fish, this would increase the gill mucus layer thickness, increasing oxygen diffusion distance and eventually promoting lower oxygen levels in gills of C. lunulatus. Haemoglobin expression has been reported to increase in several organisms under low oxygen levels, therefore, results suggest that C. lunulatus specific bacteria might play a role enhancing production of haemoglobin derived peptides (Chapter VI). In addition to its primordial function as oxygen carrier, haemoglobin is a known source of biologically active peptides that exhibit varied functions such as antimicrobial and anti-parasitic [START_REF] Liepke | Human hemoglobin-derived peptides exhibit antimicrobial activity: a class of host defense peptides[END_REF][START_REF] Ullal | Antiparasitic activity of the antimicrobial peptide HbβP-1, a member of the βhaemoglobin peptide family[END_REF]. [START_REF] Ullal | Antimicrobial peptides derived from hemoglobin are expressed in epithelium of channel catfish (Ictalurus punctatus, Rafinesque)[END_REF] found that the expression of three β-chain haemoblogin peptides, one of which presented a high similarity with one of the C. lunulatus peptides, increased after ich (Ichthyophthiriums multifiliis) infection in gill epithelium of channel catfish (Ictalurus punctatus). They found antimicrobial activities and strong anti-parasitic activities in one of the peptides, revealing their involvement in fish innate immunity against ectoparasitic infections [START_REF] Ullal | Antiparasitic activity of the antimicrobial peptide HbβP-1, a member of the βhaemoglobin peptide family[END_REF]. Altogether, these results suggest that C. lunulatus native microbiota might play a role enhancing the production of haemoglobin derived peptides, most likely through the decrease of mucus oxygen levels, and therefore might protect this species from gill ectoparasites (Chapter VI). However, purification, synthesis and further evaluation of purified peptides biological activities will be performed in the laboratory, in order to confirm this hypothesis. It would be highly interesting to test whether an increased level of haemoglobin antimicrobial peptides would also occur in other Chaetodon species under lower oxygen levels, or whether exclusive C. lunulatus bacteria (mainly Fusobacteria and Spirochaetes) play a more specific role in the production of haemoglobin peptides, for example by cleaving the haemoglobin protein as shown by [START_REF] Du | Rapid reprogramming of haemoglobin structure-function exposes multiple dual-antimicrobial potencies[END_REF]. Experiments involving bacterial challenges of butterflyfishes and study of the haemoglobin peptides expression could cast light on the effect of bacteria in the production of butterflyfish haemoglobin peptides.

Antimicrobial peptides (AMPs) are normally small cationic peptides (usually less than 50 aa), which can either be derived from a biologically inactive proprotein that is processed to the active form or derived from larger, functional proteins such as histones or haemoglobins [START_REF] Radek | Antimicrobial peptides: natural effectors of the innate immune system[END_REF]. AMPs are one of the components of the non-specific immune system that offer a first line of protection in many animal species including fish [START_REF] Falco | Antimicrobial peptides as model molecules for the development of novel antiviral agents in aquaculture[END_REF]. Besides having direct activity on a wide-range of microorganisms, AMPs modulate host innate and adaptive immunity by induction or modulation of pro-inflammatory cytokines and chemokines, apoptosis, gene transcription activities, inhibition of the inflammatory response, recruitment and stimulation of proliferation of macrophages, neutrophils, eosinophils and T lymphocytes, and the activation and differentiation of dendritic cells [START_REF] Nijnik | Human cathelicidin peptide LL-37 modulates the effects of IFN-γ on APCs[END_REF][START_REF] Katzenback | Antimicrobial peptides as mediators of innate immunity in teleosts[END_REF]. AMPs can be produced at a constitutive level, however, the major pathway of AMP production is through the recognition of pathogen associated molecular patterns (PAMPs) via Toll-like receptors (TLRs) [START_REF] Zasloff | Antimicrobial peptides of multicellular organisms[END_REF][START_REF] Katzenback | Antimicrobial peptides as mediators of innate immunity in teleosts[END_REF]. AMPs provide promising applications in aquaculture and human health as an alternative to antibiotics, vaccine adjuvants and antitumor agents [START_REF] Noga | Application of antimicrobial polypeptide host defenses to aquaculture: Exploitation of downregulation and upregulation responses[END_REF][START_REF] Gautam | Development of antimicrobial peptide prediction tool for aquaculture industries[END_REF]. [START_REF] Jia | Antimicrobial peptides protect Coho Salmon from Vibrio anguillarum Infections[END_REF] showed that continuous peritoneal deliverance of the antimicrobial peptides CEME (a cecropin-melittin hybrid peptide) and pleurocidin amide (a C-terminally amidated form of the natural flounder peptide) reduced mortality in Vibrio anguillarum infected coho salmon (Onchorhynchus kisutch). It has been claimed that bacteria are less likely to evolve resistance towards cationic antimicrobial peptides than conventional antibiotics [START_REF] Zasloff | Antimicrobial peptides of multicellular organisms[END_REF]. However, several studies showed that bacteria could evolve heritable mechanisms of resistance against cationic antimicrobial peptides, suggesting that, like conventional anti-infective agents, the therapeutic use of AMPs could result in the spread of resistant organisms [START_REF] Perron | Experimental evolution of resistance to an antimicrobial peptide[END_REF][START_REF] Sallum | Inducible resistance of fish bacterial pathogens to the antimicrobial Peptide cecropin B[END_REF]. Fish display a widevariety of AMPs, therefore, instead of therapeutical use of targeted AMPs in aquaculture disease management, it would be preferential to trigger fish species-specific expression of

2014).

In the second part of this thesis, as discussed before, I have investigated the use of medicinal plants to fight against bacterial infections in orbicular batfish. It would be interesting to study the AMP composition of P. orbicularis external surfaces, which are the first surfaces to be attacked by T. maritimum, and their bioactivities. Once known which AMPs are naturally found in P. orbicularis, a sustainable method to control bacterial infections could focus on enhancing AMPs expression. A. taxiformis oral administration to orbicular batfish (P. orbicularis) increased the level of two immune-related genes (Lys G and TGF-β1) (Chapter IX). However, the next step would be to test whether A. taxiformis can also induce a higher expression of antimicrobial peptides, and their effectivity against bacterial infections such as tenacibaculosis.

The comprehensive study of fish-parasite interactions can provide helpful insights in parasite evasion mechanisms of potential interest for aquaculture. Fish and their parasites have coevolved together for millions of years; therefore, natural systems can provide the best sustainable strategies against parasite outbreaks. Recent studies in humans and corals are showing that the complex interactions within one organism (the holobiont) play an important role in the organism's health, and disturbances within the complex community have drastic consequences for the well-being of the members [START_REF] Clemente | The Impact of the gut microbiota on human health: an integrative view[END_REF][START_REF] Krediet | Coral-associated micro-organisms and their roles in promoting coral health and thwarting diseases[END_REF].

Aquaculture disease management is mostly focused on the use of chemical drugs that display direct effects against the targeted infectious organisms [START_REF] Cabello | Antimicrobial use in aquaculture re-examined: its relevance to antimicrobial resistance and to animal and human health[END_REF]. Cultured fish are subjected to regular stresses that deeply affect their fitness as well as their interactions with their commensal microbiota, which increase their susceptibility to pathogenic infections [START_REF] Boutin | Probiotic treatment by indigenous bacteria decreases mortality without disturbing the natural microbiota of Salvelinus fontinalis[END_REF]. Therefore, disease management should concentrate on sustainable methods that increase fish fitness and re-establish their natural balance. In this thesis I have studied the potential effect of some plants and algae to enhance fish immunity and I have investigated the natural parasite evasion mechanisms of a species of butterflyfish, which

Conclusion and perspectives

-282 -revealed the importance of the association between the commensal microbiota and the fish innate immune system. This study shows the importance to investigate host-parasite interactions under a global approach. The integration of epidemiology, biogeography, metabolomics and microbiomics show the complexity of fish-parasite relationships and the relevance of integrating different disciplines.

Introduction

For thousands of years, civilisations throughout the world have used medicinal plants to treat a variety of diseases. The oldest written evidence of medicinal plants' usage was found on a Sumerian clay slab, approximately 5000 years old [START_REF] Petrovska | Historical review of medicinal plants' usage[END_REF]. Nowadays, traditional medicinal plants continue to be the primary source of healthcare in many developing countries and rural regions [START_REF] Calixto | Twenty-five years of research on medicinal plants in Latin America: a personal view[END_REF].

Ethnobotanical studies have proved to be highly useful in identifying bioactive plants, and numerous research works have been conducted on biological activities and chemical composition of ethnomedicinal plants (Banksota et al. 2003;[START_REF] Ayyanar | Ethnobotanical survey of medicinal plants commonly used by Kani tribals in Tirunelveli hills of Western Ghats, India[END_REF]. Interest in medicinal plants for human and veterinary health has been fuelled by the countless side effects, and rising costs of prescription drugs (Hoareau and Da-Silva, 1999). Plants also provide the advantage to possess a complex chemical composition which, 1) display varied biological activities, making plants suitable for the treatment of multifactorial diseases and

2) makes plants a suitable alternative to antibiotics with little risk for development of resistance (Srivastava et al. 2014;Gostner et al. 2012).

Aquaculture, is the fastest growing animal food producing sector, with an average annual increase of 6.2% per year in the period [START_REF] Gaston | Global patterns in biodiversity[END_REF]-2012(FAO, 2014)). However, aquaculture growth is often linked to culture intensification, leading to overcrowding and poor water quality, facilitating the spread of pathogens and increasing disease outbreaks and mortality (Bondad-Reantaso et al. 2005). In order to avoid economic losses related to sanitary shortcomings, veterinary drugs are commonly used in aquaculture to prevent and treat disease outbreaks (Rico et al. 2013).

The intensive use of synthetic drugs, however presents numerous disadvantages, both for the environment and health safety. Intensive use of antibiotics have resulted in accumulation in muscle of commercialised animals (e.g. Cabello et al. 2006;Romero-Ormazabal et al. 2012) and the development of resistant bacteria strains (e.g. [START_REF] Miranda | Antimicrobial multiresistance in bacteria isolated from freshwater Chilean salmon farms[END_REF]Seyfried et al. 2010). Also, the use of antiparasitic drugs like trichlorfon or praziquantel in bath treatments is hazardous for animals and the environment and it can also result in the development of resistance (Umeda et al. 2006;Forwood et al. 2013). Vaccines, which are considered to be the most effective method to prevent disease outbreaks in aquaculture, are too expensive for widespread use by fish producers and, since it is extremely difficult to develop multiple strain vaccines, most vaccines are only effective against one type of pathogen (Sakai, 1999;Pasnik et al. 2005). Considering the numerous disadvantages of synthetic drugs, there is an increasing need for the development of alternative strategies in aquaculture disease management.

Moreover, disease outbreaks in aquaculture are often associated with animal fitness and health, most pathogens being opportunistic and taking advantage of immunocompromised or stressed fish, thus alternative solutions should maximise fish immunity and fitness as a strategy to face pathogen infections

In vitro and in vivo studies have showed the potential of medicinal plants against a wide range of marine pathogens (bacteria, virus, fungus and ectoparasites) (e.g. Direkbusarakom et al. 1996 ;Chitmanat et al. 2005;Ji et al. 2012). Antibacterial properties of medicinal plants have been by far the most studied biological activities, with abundant in vitro studies reporting antibacterial activity in numerous plants, against both gram-positive and gram-negative marine bacteria (e.g. Castro et al. 2008;Roomiani et al. 2013). In vivo studies in Indian major carp (Labeo rohita) fed with enriched diets in prickly chaff flower (Achyrantes aspera, 0,2%) and Indian ginseng (Withania somnifera, 0,5%) showed a reduction in mortality of 41% and 49% respectively when fish were challenged with Aeromonas hydrophila (Vasudeva-Rao et al. 2006;Sharma et al. 2010). Antiviral and antifungal activities of medicinal plants are equally interesting to prevent high mortality rates in aquaculture. Balasubramanian et al. (2008aBalasubramanian et al. ( , 2008b) ) showed

Application of medicinal plants in aquaculture

Plants can be administered as a whole plant or parts (leaf, root, seed, fruit) and can either be used fresh or as prepared herbal extracts with different solvents (e.g. water, methanol, chloroform, ethyl acetate) [START_REF] Van Hai | The use of medicinal plants as immunostimulants in aquaculture: A review[END_REF]. Biological activities and chemical composition of plants and extracts can vary greatly depending on the part used and type of extract, and thus previous knowledge of the plants'

bioactive compounds is required. Besides, seasonal variations in production of bioactive metabolites has also been observed in different plants and algae, so a deep study on the plant production of the bioactive molecules is desirable [START_REF] Manilal | Antimicrobial potential and seasonality of red algae collected from the southwest coast of India tested against shrimp, human and phytopathogens[END_REF][START_REF] Chaves | Seasonal variation in the production of secondary metabolites and antimicrobial activity of two plant species used in Brazilian traditional medicine[END_REF].

To date, most scientific studies on the use of medicinal plants in aquaculture have focused on identification of biological activities rather than natural product determination (Balasubramanian et al. 2007;[START_REF] Cox | An assessment of the antioxidant and an-timicrobial activity of six species of edible Irish seaweeds[END_REF]Ji et al. 2012). Exploratory studies on the potential of medicinal plants in aquaculture can therefore be misleading if the wrong part or wrong solvent is used. Also, suitable dosing is crucial to obtain the desired effects, since inappropriate doses can display toxic effects on fish (Sambasivam et al. 2003;Ekanem et al. 2007;Kavitha et al. 2012). For example, grouper Epinephelus coioides fed with a supplemented diet of 1% of ethanol katuk extract (Sauropus androgynous) showed enhanced growth and improved food utilisation, whereas at 2.5 and 5% of katuk extract they presented lower growths (Putra et al. 2013). Treatment length is another important parameter in medicinal plant applications in aquaculture, since it influences directly the effectiveness of the treatment. For example, Militz et al. (2013) showed that farmed barramundii (Lates calcarifer) fed with an enriched diet of garlic (Allium sativum) for 30 days displayed 70% decreased Neobenedenia sp. infection success compared to control and short-term treatment (10 days).

Medicinal plants can be administered to fish and shellfish by injection (intramuscular and intraperitoneal), oral administration and through immersion or baths [START_REF] Wu | Effects of hot-water extract of Toona sinensis on immune response and resistance to Aeromonas hydrophila in Oreochromis mossambicus[END_REF]Ji et al. 2012;Putra et al. 2013). Although intraperitoneal injection has proved to be the most rapid and efficient way of administration, it is expensive, laborious and stressful for fish, special for very young specimens (Anderson, 1992;Yoshida et al. 1995). Bath treatment is an extensively used technique for the treatment of ectoparasites (Whittington, 2012;Forwood et al. 2013). However, this method is also expensive and laborious and involves the release of exogenous molecules in the marine environment, and thus can present some undesired environmental side effects (Umeda et al. 2006). Hence, oral administration seems to be the most suitable for aquaculture, since medicinal plants can induce physiological changes in fish to improve their fitness and reinforce their resistance to pathogens.

Finally, determining plant natural products helps to better understand their mode of action. For example, chelerythrine and chelidonine are two benzo[c]phenanthridines with antimicrobial, antifungal and anti-inflammatory activities isolated from greater celandine (Chelidonium majus L.) (Waltérova et al. 1995;Malikova et al. 2006). Several studies have shown that chelerythrine exhibits high cytotoxic potency against a large number of cells, through multiple apoptosis-inducing pathways, indicating that a direct action on mitochondria may be involved in the eradication of parasites (Slaninová et al. 2001;Kemeny-Beke et al. 2006;Kaminskyy et al. 2008). Other studies showed the mechanisms of action of chelidonine against the monogenean ectoparasite Dactylogyrus intermedium may be through mitochondrial cell death pathway activated through caspase-9 (Philchenkov et al. 2008). Therefore, it is of high importance to identify the bioactive molecules for the observed bioactivities and standardize extraction procedures depending on the bioactive molecules identified, application procedures as well as doses and duration of treatments depending on the type of pathogen (Reverter et al. 2014).

It is important to state that modes of action and toxicity of many plants are still poorly understood, so more research is needed before they can be safely used in aquaculture. Many of the plant bioactive molecules can also be toxic or antinutritional in fish, decreasing efficient utilisation of feed nutrients or causing intestinal dysfunction, so their effect on fish physiology and adequate dosing needs to be studied [START_REF] Krogdahl | Important antinutrients in plant feedstuffs for aquaculture: an update on recent findings regarding responses in salmonids[END_REF]. In order to fill in the gaps in current knowledge, a structured research plan should be followed, focused on bringing light firstly on bioactivities, compounds and modes of actions of the plants, and secondly on the plant effect on organism physiology and its appropriate use. In figure 1 we propose some directions for research on medicinal plants utilisation in aquaculture. In vitro tests should be performed in order to determine plant bioactivities, together with bio-assay guided fractionation to identify and characterise the bioactive natural products. In vitro tests should also be carried to test cytotoxicity of plant extracts on fish cells, in order to have a first idea of the plant toxicity on the animals.

Then in vivo tests on model species should be performed, firstly to understand the plant effects on animal physiology and determine the best conditions for plant application (mode of application, plant part and preparation, dose and length of treatment), and secondly to estimate plant efficiency in organism survival against different pathogenic infections. Hematology indicators such as hematocrit, hemoglobin, erythrocyte and lymphocyte levels can be used to determine fish fitness [START_REF] Haghighi | The effects of powdered ginger (Zingiber officinale) on the haematological and immunological parameters of rainbow trout Oncorhynchus mykiss[END_REF]Kanani et al. 2014). Fish immunity is often analysed to see immunomodulatory power of plants and it can be either studied by classical biochemical approaches (e.g. lysozyme activity, phagocytic activity, respiratory burst activity) or by the study of immune gene expression (e.g. Lys, TNF-α, IL-1, IL-10 genes) (Harikrishnan et al. 2011e;Kumar et al. 2013;[START_REF] Chakrabarti | Effect of seeds of Achyranthes aspera on the immune responses and expression of some immune-related genes in carp Catla catla[END_REF].

RESEARCH STEPS OF MEDICINAL PLANT USE IN AQUACULTURE

In vivo studies

In vitro studies

Analysis of plants used in aquaculture

An extensive literature review was performed on the published studies on plant use in aquaculture in order to identify the most common studied plants and their activities (Table 1). Most of the plants used both by fish farmers and in scientific studies for the potential use in aquaculture correspond to medicinal plants widely used in traditional medicine. We identified over 250 plant species from 75 families and 32 orders, which have been used or reported as potentially interesting in aquaculture.

Plant ordrers most frequently used in aquaculture

Plants from the order Lamiales (family Lamiaceae) were the most often studied plants (12%), followed by the Fabales (family Fabaceae, 11%), Asterales (family Asteraceae, 10%) and Malpighiales (family Euphorbiaceae et Phyllanthaceae, 10%) (Figure 2). Several essential reported to display antimicrobial, antispasmodic, carminative and antiviral activities (Mimika-Duzic and Boizin, 2008). Lamiaceae plants contain a wide range of bioactive metabolites which include numerous monoterpenes and sesquiterpenes such as linalool, geraniol, eugenol, ocimene, carvacrol, p-cymene, thymol and myrcene and flavonoids like luteolin first isolated from Salvia tomentosa (Mimica-Duzic and Boizin, 2008;[START_REF] Bower | Bioactive Compounds from Culinary Herbs Inhibit a Molecular Target for Type 2 Diabetes Management, Dipeptidyl Peptidase IV[END_REF]Malmir et al. 2015) (Figure 3). Plants from the family Lamiaceae also contain several bioactive fatty acids such as palmitic acid and myristic acid and phenolic actives like rosmarinic acid (Pattayanak et al. 2010). Rattanachaikunsopon and Phumkhachorn (2010) and Abd-El-Galil (2012) showed that antibacterial activity of carvacrol against Edwarsiella tarda and Tenacibaculum maritimum was enhanced when administered with cymene (Figure 3). Gormez and Diler (2014) showed that essential oils of Origanum onites and Thymbra spicata were more bioactive against the fungus Saprolegnia parasitica than thymol or carvacrol alone. The latter studies indicate that synergistic effects between different bioactive compounds may exist and enhance the plant bioactivities. [START_REF] Hao | Bioassay-guided isolation and identification of active compounds from Semen pharbitidis against Dactylogyrus intermedius (Monogenea) in goldfish (Carassius auratus)[END_REF] found that palmitic acid was effective against the monogenean parasite D. intermedium in goldfish (Carassius auratus).

into account, that those proportions are biased by the number of studies performed to date on each targeted pathology; for example many more studies have been focused on antibacterial activities than antifungal activities.

Plant bioactivies were also analysed by plant order and we found that Asterales was the order with the highest number of bioactive plants (34), with nearly half of them displaying antibacterial activity (Figure 5). Lamiales and Malpighiales were the two following orders with the highest number of bioactive plants (33 and 32). The Lamiales order had the highest number of plants displaying immunostimulant activity [START_REF] Conceição | Orpotrin: a novel vasoconstrictor peptide from the venom of the Brazilian stingray Potamotrygon gr. orbignyi[END_REF] and antifungal activity (5), while the Malpighiales presented the highest antiviral activities (11) (Figure 5).

Analysis on the plant parts used in aquaculture

Most of the studies on the use of medicinal plants in aquaculture used plant leaves (37%), while 22% of them used the whole plant either as powder, plant essential oil or extract. Root was also often used with (18%), followed by seeds (8%), barks (6%), fruits (6%) and finally flowers (4%) (Figure 6).

Other plants and perspectives

Some algae and some mushrooms have also been studied for their potential application in aquaculture. Algae are considered to be a rich source of bioactive molecules, and most of the studied algae presented high antibacterial activities, and some also presented immunostimulant, antifungal, antiparasitic and antiviral activities (Choudhury et al. 2005;Hutson et al. 2012;[START_REF] Sukoso | Study antibacterial and cytotoxic activ-ities of seaweed Porphyra sp. extract[END_REF]Zbakh et al. 2012;[START_REF] Genovese | The Mediterranean red alga Asparagopsis taxiformis has antifungal activity against Aspergillus species[END_REF]. For example, red algae Asparagopsis taxiformis, which is known to produce a high diversity of halogenated metabolites, displayed antibacterial, antifungal and antiparasitic activities against several fish pathogens [START_REF] Genovese | In vitro evaluation of antibacterial activity of Asparagopsis taxiformis from the Straits of Messina against pathogens relevant in aquaculture[END_REF][START_REF] Genovese | The Mediterranean red alga Asparagopsis taxiformis has antifungal activity against Aspergillus species[END_REF]. Also, A. taxiformis enhanced immune system of Penaeus monodon and was highly efficient in the treatment of Vibriosis in P.

monodon (Manilal et al. 2012 ;2013). Also, some studies like [START_REF] Mai | Quorum sensing inhibitors from Leucette chagonensis Dendy, 1863[END_REF] are starting to show interesting properties of other marine organisms like sponges in the quorum sensing inhibition of marine pathogenic bacteria such as Vibrio harveyi. Nevertheless, more research needs to be performed in this field, in order to stablish the possibilities of application in aquaculture disease management.

Finally, there are numerous medicinal plants used in human medicine that have not yet been tested on aquaculture. For example, the gum rockrose (Cistus ladanifer, Cistaceae, Malvales) is a widely used plant in several Mediterranean countries which possesses anti-inflamatory, antibacterial and antioxidant activities [START_REF] Neves | Ethnopharmacological notes about ancient uses of medicinal plants in Trás-os-Montes (northern of Portugal)[END_REF][START_REF] Ferreira | Screening of antimicrobial activity of Cistus ladanifer and Arbutus unedo extracts[END_REF]. Dandelions (Taraxacum officinale, Asteraceae, Asterales) have long been used in the Northern hemisphere for its diuretic, choleretic, anti-inflammatory, anti-oxidative, anti-carcinogenic, analgesic, anti-hyperglycemic, anti-coagulatory and prebiotic effects [START_REF] Schütz | Taraxacum-A review on its phytochemical and pharmacological profile[END_REF]. Therefore, studies on the medicinal plants potentially used in aquaculture, should also explore the ethnobotanic studies in order to identify other interesting plants.

Conclusion

Medicinal plants (including algae and mushrooms) present promising potential to be used in aquaculture as a substitute of chemotherapy in the treatment of diseases outbreaks. Ethnobotanical studies have shown to be highly useful in the discovery of bioactive plants, and bioactive natural products with interesting applications in aquaculture. However, there is still scarce knowledge on the mode of action of most bioactive plants, as well as the most suitable form for an effective and safe administration. More research is needed to elucidate the plant natural products and their modes of action (establish the bioactive parts of the plant and the most suitable preparations), and to test the plant effects on the organisms physiology in order to establish an appropriate treatment (way of administration, dose and length). Besides, research works could also highly benefit from traditional knowledge of fish farmers who use regularly plants.

Finally, most studies have focused on the potential of terrestrial plants in aquaculture. Although, plants indeed offer interesting biological activities, they are not a natural component of neither the fish alimentation or the marine environment. Thus, continuous introduction of plant extracts and their bioactive compounds (through bath treatment or food diffusion) in the marine or freshwater environment, could eventually cause environmental problems. Therefore, the use of bioactive algae and seaweeds from the local natural environments where the aquaculture is performed, could represent a better alternative to avoid introduction of exogenous molecules into the environment. Algae are considered to be a rich source of original bioactive molecules which display multiple bioactivities. In aquaculture, several recent studies have showed the algae potential for the treatment of pathogens or to improve fish fitness, however they can also present indesirable effects and toxicity on fish, therefore more research needs to be performed on the physiological effects of algae on fish and the suitable dosing [START_REF] Manilal | Bioactivity of the red algae Asparagopsis taxiformis collected from the southwestern coast of India[END_REF].

Bibliography

Figure 1 .

 1 Figure 1. Organisms' interactions within a biological community.

Figure 2 .

 2 Figure 2. Behaviour modification of the amphipod G. pulex by the parasite P. laevis to facilitate transmission to the definitive host Salmo trutta.

Figure 3 .

 3 Figure 3. Illustration of a trade-off between parasite virulence and transmission adapted from Kennedy et al. (2015). Virulence induced host mortality shortens the duration of an infection (A), virulence increases the instantaneous transmission of infection (B). Result of the trade-off between virulence, transmission and infection duration can lead to a maximised pathogen fitness at intermediate levels of virulence in natural conditions (C).

Figure 4 .

 4 Figure 4. Overview from the 4 major "omics" sciences (adapted from www.ebi.ac.uk).

Figure 5 .

 5 Figure 5. Metabolomics workflow for LC-MS.

Figure 1 .

 1 Figure 1. Photographs of two butterflyfish species. (A) Forcipiger longirostris (B) Chaetodon lunulatus

Figure 2 .

 2 Figure 2. Phylogenetic tree of the Chaetodontidae family (adapted from Fessler and Westneat, 2007).

Figure 3 .

 3 Figure 3. Drawing of Euryhaliotrematoides grandis, monopisthocotylean monogenean (adapted from Plaisance

  ., Cutmore, S.C., Bray, R., Cribb, T.H., Sasal, P. Gill monogenean communities (Platyhelminthes, Monogenea, Dactylogyridae) of butterflyfishes from tropical Indo-West Pacific Islands. Parasitology. In press. (IF = 3.0) 1. Introduction Parasites are an essential part of every ecosystem, acting as agents of natural selection and contributing to community and ecosystem organisation. Parasites modify host behaviour and generate pressure for selection and adaptation by their hosts (Hatcher et al. 2012; Gómez

Figure 1 .

 1 Figure 1. Map of sampling sites in the tropical Indo-west Pacific, with the number of species and total fish sampled per site.

Figure 2 .

 2 Figure 2. Dactylogyrid prevalence for the butterflyfish species analyzed. Median, first and third quartile are plotted. White dots represent outlier samples.

Figure 3 .

 3 Figure 3. Dactylogyrid intensities for the butterflyfish species analyzed. Median, first and third quartile are plotted. White dots represent outlier samples. The grey areas have been determined as statistically different by kruskal post-hoc test.

  276). Size, depth range, feeding behaviour (specificity level) did not correlate with either parasite intensity or parasite richness among different chaetodontid species. Host geographical range displayed a moderate positive Spearman correlation (ρ = 0.46; P = 0.01) with parasite richness. Linear regression showed also a positive correlation (r 2 = 0.16) (Figure4).

Figure 4 .

 4 Figure 4. Linear regression between parasite diversity of butterflyfish species and host geographic range. Dark grey zone represents the 95% confidence interval.

Figure 5 .

 5 Figure 5. Host specificity for the 13 dactylogyrid species identified in this study.

Figure 6 .

 6 Figure 6. Dactylogyrid community structure for the butterflyfish species studied in the TIWP.

Figure 7 .

 7 Figure 7. Cluster dendogram of butterflyfish species based on their dactylogyrid community similarities. Colors show the sub-cluster identified with the KGS penalty function and the cutree function for R.

Figure 8 .

 8 Figure 8. Correlation circle between the dactylogyrid species identified in this study.

  . However, unlike C. lunulatus, C. ornatissimus is always parasitised by dactylogyrids. In further contrast, C. lunulatus has a relatively rich fauna of skin and gut trematodes most of which are also found in C. ornatissimus, with two species of faustulids (Paradiscogaster oxleyi Díaz, Bray & Cribb, 2013 and Paradiscogaster flindersi Bray, Cribb & Barker, 1994), one monorchiid (Hurleytrematoides dollfusi McNamara et al.

Figure 1 .

 1 Figure 1. Areas of distribution (blue shadow), prevalence (circle colour) and abundance (circle size) of dactylogyrid parasites from butterflyfishes in the Indo-West Pacific Region. Only parasites with prevalences > 20% for at least one locality were plotted (other dactylogyrid species area of distribution are found in supplementary material).

Figure 2 .

 2 Figure 2. Correlation matrix between dactylogyrid species (Pearson correlation). Only dactylogyrid species found at least at 5 locations were plotted. * indicates p-value < 0.05.

Figure 3 .

 3 Figure 3. Structure of dactylogyrid communities at 9 locations in the West Indo-Pacific. Size of the circles is relative to the weighted intensity (Iw) for each location.

Figure 4 .

 4 Figure 4. Cluster analysis of the parasite communities dissimilarities (Bray-Curtis distance, « average » algorithm).

Figure 5 .

 5 Figure 5. Distance decay curves for significant factors in parasite community dissimilarity.

western

  Indo-Pacific (Palau, Lizard Island and Wallis) and Ningaloo Reef. Altogether our results indicate the possible presence of two different biodiversity hotspots of dactylogyrids in the Indo-West Pacific: the southwest Pacific and French Polynesia, supporting qualitatively and quantitatively different monogenean assemblages. Plaisance et al. (2008) studied the phylogeography of two dactylogyrid parasites from butterflyfishes and found clear genetic differentiation of E. grandis between the population in Moorea (French Polynesia) and all the other populations (West Pacific), suggesting a possible isolation of French Polynesia during Pleistocene sea-level fluctuations. These fluctuations might have been at the origin of the two biodiversity hotspots. Macpherson et al. (2010) also found different communities of galatheid squat lobsters between French Polynesia and the rest of the West Indo-Pacific region with a high proportion of endemism, also suggesting French Polynesia as a diversity center.

6

  clusters containing species with similar dactylogyrid assemblages (Reverter et al. 2016) The present results reinforce the hypothesis of a strong specificity of dactylogyrid parasites, and the non-random organization of parasite communities within butterflyfish populations in the West Indo-Pacific. The two main clusters of butterflyfish species based on similarity of their dactylogyrid parasites were characterized by the dominance of H. aurigae and H. angelopterum respectively, with most species from cluster 1 primarily found in the western Indo-Pacific (e.g. C. baronessa, C. speculum, C. semeion, C. lineolatus) and species from cluster 2 most often found in French Polynesia (e.g. C. reticulatus, C. trichrous, C.

Figure 1 .

 1 Figure 1. Chemical and biological compositions of epidermal fish mucus and its ecological interactions.

Fish

  chemical mediation is still poorly understood, with very few examples where fish mucus molecules have been elucidated and their ecological roles identified. As previously shown for TTX, a single molecule can have multifunctional properties. Species, sex, and tissue specific differences in the distribution of TTX render unclear the exact function of this molecule in pufferfish. Depending on the concentration, TTX may function as a chemical defense against predators, as attractant for parasites or as a chemical pheromone during spawning. These molecules of keystone significance are vital in structuring ecological communities, and therefore a deeper understanding is needed.[START_REF] Ferrer | Molecules of Keystone Significance: Crucial Agents in Ecology and Resource Management[END_REF]. Research on fish mucus, coupled to the development of new technologies will allow to clarify how fish interact and respond to their environment.

Figure 3 .

 3 Figure 3. Diagram on the priorities of fish mucus research and applications.

  lunulatus and C. reticulatus shared the lowest (40 OTUs) (Figure1).

Figure 1 .

 1 Figure 1. Venn diagram representing shared operational taxonomic units (OTUs) between fish species. CL = C. lunulatus, CO = C. ornatissimus, CR = C. reticulatus and CV = C. vagabundus.

Figure 2 .

 2 Figure 2. PCoA analyses of the gill mucus microbiota of four species of butterflyfish (CL = C. lunulatus, CO = C. ornatissimus, CR = C. reticulatus and CV = C. vagabundus) based on Weighted Unifrac distances. The Diagonal black line shows significant difference (p-value < 0.05) between C. reticulatus/ C. vagabundus and C. lunulatus/ C. ornatissimus tested by a PERMANOVA analysis and pairwise comparisons corrected for multiple testing.

Figure 3 .

 3 Figure 3. Bacterial diversity at the phylum level (pie chart) and class level (bars) determined by 454 pyrosequencing. Pie diagrams show the percentage of sequenced clones belonging to different bacterial phylum for each fish species analyzed. Bar graphs represent the percentage of Proteobacteria classes detected in each fish species. CL = C. lunulatus, CO = C. ornatissimus, CR = C. reticulatus and CV = C. vagabundus.

Figure 4 .

 4 Figure 4. Abundant taxa a) family and b) genus of the gill mucus microbiome. Only taxa with abundance ≥ 5% at least in two of the samples is plotted. CL = C. lunulatus, CO = C. ornatissimus, CR = C. reticulatus and CV = C. vagabundus.

  butterflyfish species (C. auriga, C. lunulatus, C. lunula, C. ornatissimus, C. quadrimaculatus, C. reticulatus, C. vagabundus and C. ulietensis) (n = 110) were sampled at three geographic locations in Moorea (French Polynesia). In one of the locations (Ta'ahiamanu), fish were sampled in the different reef habitats: fringing reef, barrier reef and outer reef slope (Figure 1).

Figure 1 .

 1 Figure 1. Map of the Moorea island (French Polynesia) with the sampling sites at three geographic locations (Ta'ahiamanu, Haapiti and Afareiatu) and three reef environments (fringing reef, barrier reef and outer slope) in the Ta'ahiamanu site.

  100% of B in 5 min, followed by a 10 min wash with 100% of B and 15 min re-equilibration with 30 % of B before the next injection. Full scan data were acquired in positive ionization mode for both fractions. The mass spectrometer analyzer parameters were set as follows:sheath gas flow rate 25 a.u., aux gas flow rate 5 a.u., sweep gas flow rate 1 a.u., capillary temperature 275 °C, capillary voltage +2 V, tube lens voltage 120 V and spray voltage 4 kV and a full scan mass window of 130-2000 m/z. To randomize analytical sequences and reduce systematic error associated with instrumental drift, a Latin square was carried out for each of

2. 3 .

 3 Data treatment and statistical analysis LC-MS raw data files were converted to netcdf files with the Excalibur Software. Netcdf files were processed using the R package Xcms to detect, deconvolute and align features (molecular entities with a unique m/z and a specific retention time). Xcms analysis of these data provided a matrix containing the retention time, m/z value and integrated peak area of the identified features. Data were normalized by log transformation prior to statistical analysis.Data were analyzed considering the following fish traits: species taxonomy, collection site (reef habitat and geographic site), diet, phylogeny and level of parasitism. Butterflyfish were classified upon their diets as obligate corallivores (C. lunulatus, C. ornatissimus and C. reticulatus), facultative corallivores (C. quadrimaculatus and C. lunula) and ,omnivorous (C. auriga and C. vagabundus and C. ulietensis) following in situ observations in Moorea and existing bibliography[START_REF] Pratchett | Dietary overlap among coral-feeding butterflyfishes (Chaetodontidae) at Lizard Island, northern Great Barrier Reef[END_REF]. Phylogenetic groups were determined from the phylogenetic tree inFessler and Wesneat (2007) and described as follows: 2 = Chaetodon clade 2 (C. quadrimaculatus); 3 = Chaetodon clade 3 (C. lunulatus, C. ornatissimus, C. reticulatus); 4 = Chaetodon clade 4 (C. auriga, C. vagabundus, C. lunula and C. ulietensis). Butterflyfishes were classified in three groups depending on the gill parasitism intensities of dactylogyrids (Platyhelminthes, Monogenea) described in Reverter et al. (2016): 1 = non parasitized (C. lunulatus), 2 = low parasitism intensity (C. auriga, C. lunula and C. ulietensis), 3 = high parasitism intensity (C. ornatissimus, C. quadrimaculatus, C. reticulatus and C. vagabundus).

Figure 2 .

 2 Figure 2. PCA plots of the technical (M) and biological variability (CL) for the polar (a) and apolar (b) fractions.

Figure 3 .

 3 Figure 3. Shannon's diversity indices of the fish mucus metabolome according to the different factors analyzed. a) butterflyfish species, b) phylogenetic clades, c) diet, O corallivore = obligate corallivore, F corallivore = facultative corallivore and d) intensity of parasitism, No = never parasitized, High = high parasitism intensities, Low = low parasitism intensities. Statistical significance was determined using the Tukey's test of one-way analysis of variance, ***p = 0.001. Light grey and dark grey regions indicate statistically significant differences between categories.

  and parasitism) only partly explain the metabolic variability (42% of the apolar fraction and 30 % of the polar fraction).

Figure 3 .

 3 Figure 3. PCA of gill mucus metabolome of the polar fraction with distribution of the samples on the first two components identified to the significant fish traits (fish phylogenetic clade, fish diet and fish level of parasitism). The ten most important contributors are represented on the circle of correlation in the right graph.

Figure 4 .

 4 Figure 4. PCA of gill mucus metabolome of the apolar fraction with distribution of the sample on the first two components identified to significant fish traits (fish species, fish phylogenetic clade, fish diet and fish level of parasitism). The ten most important contributors to the observed PCA analysis are represented on the circle of correlation in the right graph.

Figure 5 .

 5 Figure 5. MRT of the gill mucus metabolome from the polar (a) and apolar (b) fractions with the % of variance explained by each split. O corallivore = obligate corallivore, F corallivore = facultative corallivore, High = high parasite intensities, Low = low parasite intensities, No = never parasitized.

  extraction for untargeted metabolomics analysis 10 mg of the freeze-dried samples were extracted using a two-step biphasic extraction, yielding two separate fractions, where polar and apolar metabolites could be extracted simultaneously[START_REF] Wu | High-throughput tissue extraction protocol for NMRand MS-based metabolomics[END_REF]. Samples were suspended in 1.5 mL of H 2 O/MeOH (2:0.5), and then 1.5 mL of MeOH/CH 2 Cl 2 (2.5:2) was added. The mixture was extracted 15 min in an ultrasonic bath and then centrifuged for 20 min at 3500 G (4°C). The two fractions were carefully collected with glass pipettes into separate tubes. The apolar fraction was then dried under nitrogen flux, and the polar fraction was freeze-dried. Polar and apolar extracts were resuspended in 0.3 mL MeOH and filtered through a 20 μm polytetrafluoroethylene (PTFE)

  2.4.2.1. LC-MS analysis of the metabolomic sequence LC-MS analyses were performed with a LC-DAD-ESI-MS system from ThermoScientific (MA, USA) equipped with an Accela PDA detector and a LCQ Fleet 2300 mass spectrometer. The equipment was controlled, and the data was handled by the Excalibur Data Software. LC elution conditions consisted of two eluants A (water with 1‰ formic acid) and B (acetonitrile with 1‰ formic acid) with a flow rate of 0.5 mL.min -1 , and an injection volume of 10 μL. The elution profile of the polar extract consisted of a linear gradient from 2% to 100% of B in 30 min followed by 10 min washing and 15 min re-equilibration with 2% of B before the next injection. For the apolar fraction a linear gradient was conducted from 30% to 70% of B in 5 min, then from 70% to 80% of B in 20 min and from 80% to 100% of B in 5 min, followed by a 10 min wash with 100% of B and 15 min re-equilibration with 30 % of B before the next injection. Full scan data were acquired in positive ionization mode for both fractions. The column used for both extracts was a Kinetex C6-Phenyl analytical column (100 × 2.10 mm, 2.6 μm particular size; Phenomenex, CA, USA) equipped with the corresponding pre-column with a fixed temperature of 30 °C. The mass spectrometer analyzer parameters were set as follows: sheath gas flow rate 25 a.u., aux gas flow rate 5 a.u., sweep gas flow rate 1 a.u., capillary temperature 275 °C, capillary voltage +2 V, tube lens voltage 120 V and spray voltage 4 kV and a full scan mass window of 130-2000 m/z.

  UltiMate 3000 UHPLC system (Thermo Scientific) equipped with a Corona® Veo detector and interfaced with a hybrid quadrupole Orbitrap mass spectrometer (Q Exactive, Thermo Scientific). The equipment was controlled by the Chromeleon 7.2 Software, and the data were handled using the Excalibur Data Software. Elution conditions, elution profile and column were the same as described for the polar extract on the LC-DAD-ESI-MS analyses. Mass spectrometric data were collected in continuum full-scan mode from m/z 150 -2000 in positive mode with a resolution of 70,000 and a normalised collision energy (NCE) of 30. CID (Low-energy collisional dissociation) fragmentation was used to obtain MS/MS spectra for the selected ions (RT 1 = 14.10-14.40 min/M 1 = 644.8725 Da ; RT 2 = 15.00-15.50 min/M 2 = 657.3557 Da; RT 3 = 15.40-15.70 min/M 3 = 782.4199 Da) with a resolution of 35,000 and a NCE of 35.

  Number of parasites per fish decreased significantly after an injection of C. lunulatus gill mucus to the three butterflyfish species the experiment was conducted on (C. ornatissimus, C. reticulatus and C. vagabundus) (Figure 1). Parasite intensity decreased 62 ± 17% in treated C. ornatissimus, 70 ± 6% in treated C. reticulatus, and 55 ± 7% in treated C. vagabundus.

Figure 1 .

 1 Figure 1. In vivo test results on C. ornatissimus, C. reticulatus and C. vagabundus. p-value < 0.05, p-value < 0.001.

Figure 2 .

 2 Figure 2. PCA (A) and PLS-DA (B) of the parasitized vs. non-parasitized fish and the heatmap (C) of the OTUs with VIP scores ordered from 1.5 to 2.5 (bottom to top). * indicates p-value < 0.05 (Kruskal-Wallis).

-

Figure 3 .

 3 Figure 3. Relative bacterial abundances of VIP bacterial families found at > 1% in C. lunulatus. * p-value < 0.05; *** p-value < 0.001.

Figure 4 .

 4 Figure 4. PCA (A) and PLS-DA (B) of the parasitized vs. non-parasitized fish and the heatmap (C) of the metabolites with VIP scores higher than 2 (polar fraction). * indicate peptides that were enriched in the acidic extraction. * indicate peptides whose approximate sequence was obtained by the PEAKS software.

Figure 5 .

 5 Figure 5. Boxplot of three peptides over-expressed in C. lunulatus.

Figure 5

 5 Figure 5. a) UHPLC/MS chromatogram of a C. lunulatus polar extract. Peaks detailed with mass (M in Da) and retention time (RT in min.) are three VIP peptides. b) Multi-charged isotopic cluster of the peptide with RT = 15.19 min.

Figure 6 .

 6 Figure 6.UHPLC (A) chromatogram of a C. lunulatus acidic extract. Peaks with detailed mass (M, Da) and retention time (RT, min) are three VIP peptides enriched in the acidic extraction. (B) Mass spectra of the three VIP peptides showing several multi-charged ions.

Figure 7 .

 7 Figure 7.Pearson correlation between the three haemoglobin-derived peptides overexpressed in C. Lunulatus and bacteria overexpressed in C. lunulatus. Relative circle size indicates magnitude of correlation.

  abundance in C. lunulatus was punctual. C. lunulatus has been observed to feed preferentially on bacterial mats of coral disease lesions, harbouring high densities of Vibrio bacteria[START_REF] Arotsker | Vibrio sp. as a potentially important member of the Black Band Disease (BBD) consortium in Favia sp. corals[END_REF] Chong-Seung et al. 2011). Such foraging habitats could promote Vibrio transfer from diseased corals to C. lunulatus external organs such as gills. However, durability and physiological consequences of associations between Vibrio and C. Lunulatus have yet to be properly studied.In summary, we have studied the gill mucus of C. lunulatus in comparison to other parasitized butterflyfish species to investigate the potential role of C. lunulatus mucus in the absence of monogeneans. C. lunulatus gill mucus induced a decrease of the number of parasites in three highly parasitized species. We further studied the microbiome and metabolome of C. lunulatus gill mucus and found a higher presence of Fusobacteria, Spirochaetes and Vibrionaceae bacteria that was positively correlated with an overexpression of hemoglobin peptides. These results suggest that bacteria might play a role enhancing production of hemoglobin peptides, either through the decrease of the oxygen levels or by cleaving the hemoglobin protein. Hemoglobin peptides present a wide array of bioactivities, therefore we hypothesise that they might play a role preventing monogenean infection in C.

  However, since the 1970s, there has been a local effort to develop other aquaculture sectors like the shrimp (Litopenaeus stylirostris) and the giant clam (Tridachna maxima) aquaculture.Fish lagoon aquaculture in Tahiti has been developed very recently with the main objective of promoting local sustainable farming to obtain high quality fish meat free of ciguatoxins and other ciguatera associated toxins[START_REF] Spc | Regional strategy for developing marine finfish aquaculture in the Pacific islands[END_REF]. In 2001, the Fisheries Department of French Polynesia (DRMM) began aquaculture trials on three different marine finfish species: moi (sixfinger threadfin, Polydactylus sexfilis), pa'aihere manini (golden trevally, Gnathanodon speciosus), and paraha peue (orbicular batfish, Platax orbicularis). In 2006, the DRMM and the French Research Institute for the Exploitation of the Sea (Ifremer) targeted the orbicular batfish (Platax orbicularis) as the priority candidate in lagoon aquaculture development in

Figure 1 .

 1 Figure 1. P. orbicularis aquaculture development in Tahiti.

Figure 2 .

 2 Figure 2. Adult (A) and fingerling (B) of orbicular batfish (P. orbicularis).

Figure 3 .

 3 Figure 3. P. orbicularis fingerlings with tenacibaculosis at different infectious stages.



  treatment of the marine tenacibaculosis in P. orbicularis, in order to decrease the severe mortality episodes currently encountered in Tahiti. Since P. orbicularis aquaculture in Tahiti takes place close to the coral reef, synthetic persistent molecules should be restricted. I investigated the potential of several local Polynesian plants plants(garlic, turmeric, noni, ginger and tamanu) and algae (Asparagopsis taxiformis, Padina boryana, Turbinaria ornata, Dichotoma marginata, Lingbya majuscula and Anabaena torulosa) against the two major pathogenic agents (Vibrio harveyi and Tenacibaculum maritimum), by evaluating plant and algae antibacterial activities, studying the capacity of plants and algae to increase level of expression of two immune-related genes in orbicular batfish (Platax orbicularis) and finally testing in vivo efficacy of orally administered plants and algae in reducing infected (Tenacibaculum maritimum) orbicular batfish mortality.Reverter, M., Bontemps, N.,Lecchini, D., Banaigs, B., Sasal, P. (2014). Use of plant extracts as an alternative to chemotherapy: Current status and future perspectives. Aquaculture 433, 50-61. (IF = 1.8) Diseases outbreaks are becoming more recurrent and conventional treatment methods have multiple environmental and health disadvantages.

Figure 1 .

 1 Figure 1. Number of published articles about the use of plant, algae or natural products in aquaculture.

  that 31 methanolic extracts of Brazilian plants presented antibacterial activity (agar diffusion assay) against the fish pathogenic bacteria Streptococcus agalactiae, Flavobacterium columnare and Aeromonas hydrophila, being F. columnare the most susceptible microorganism to most of the tested extracts. Wei and Musa (2008) studied the susceptibility (minimum inhibitory concentration, MIC) of two Gram positive bacteria (Staphylococcus aureus and Streptococcus agalactiae), four Gram negative bacteria (Citrobacter freundii, Escherichia coli, Vibrio parahaemolyticus and Vibrio vulnificus) and 18 isolates of Edwarsiella tarda to aqueous extract of garlic (500, 250, 125, 62.5 mg/mL), and

  Hutson et al. (2012) assessed the effect of aqueous extracts from different algae on the lifecycle of the parasite Neobenedenia sp. They observed that infection success on Lates calcarifer was lower in the presence of Asparagopsis taxiformis (51%) and Ulva sp. (54%) extracts in seawater compared with the control (71%). Moreover, A. taxiformis extract inhibited embryonic development of Neobenedenia sp. and reduced hatching success to 3% compared with 99% for the seawater control. Militz et al. (2013 a,b) fed farmed barramundi (L. calcarifer) with two enriched garlic diets and observed that when fish were challenged with Neobenedenia sp. treated groups during long-term supplementation (30 days) displayed CHAPTER VII -220 -70% reduced infection success compared to control and short-term supplementation (10 days).

  Dierebusarakom et al. (1996) found that 18 Thai traditional herbs presented antiviral activity against Oncorhyncus Mason Virus (OMV) and Infectious Haemotopoietic Necrosis Virus (IHNV), while Orchocarpus siamensis and star goosebarry (Phyllanthus acidus) inhibited the replication of OMV and IPNV (Infectious Pancreatic Necrosis Virus) in cells. Dierebusarakom et al. (1998) showed that Clinacanthus nutans ethanolic extract inactivated

  , polyphyllin D (7), brucein A and D (8,9), palmitic acid (10), pharnilatin (11), osthol

Figure 2 .

 2 Figure 2. Structures of natural products with anti-parasitic activity with their stereochemistry.

  tamanu essential oil (C. inophyllum) were bought from the local market in Moorea in April 2014 (French Polynesia). Noni (M. citrifolia) was collected in the Moorea rainforest in April 2014, whereas red algae (A. taxiformis and D. marginata) and cyanobacteria (A. torulosa and L. majuscula) were collected on the reef outer slope and the brown algae (T. ornata and P. boryana) in the fringing reef in Moorea in April 2014. A. taxiformis was also collected in Febrary 2015; June 2015 and May 2016. Noni and the algae (A. taxiformis, D. marginata, A. torulosa, L. majuscula, T. ornata and P. boryana) as well as pealed garlic, ginger and turmeric bulbs were freeze-dried, powdered and stored at -20°C until used. Plant extracts were prepared extracting 3 times approximately 1g of dried mass with 20 mL of CH 2 Cl 2 -MeOH (1:1) in an ultrasonic bath for 10 min. Extracts were evaporated by vacuum rotatory evaporation (40°C), freeze-dried and stored at -20°C until used. Algae samples (approximately 1g) were extracted the same way (1:1 CH 2 Cl 2 -MeOH) but prior to evaporation 500 mg of octadecyl silica powder were added. Extracts were evaporated to dryness by vacuum rotatory evaporation, and loaded onto RP18 Solid Phase Extraction (SPE) cartridges (Phenomenex, CA, USA). The conditioned cartridges were first washed with 10 mL of H 2 O in order to remove the salts and then with 5 mL of CH 2 Cl 2 -MeOH (1:1). Water soluble fractions were discarded and CH 2 Cl 2 -MeOH fractions were dried under nitrogen and stored at -20°C until used. Fractionation of the most bioactive extract (A. taxiformis) was performed. The algae A. taxiformis (2 g) was extracted as described above and the conditioned cartridges were washed with 20 mL of H 2 O and then with 20 mL of MeOH (A), MeOH-CH 2 Cl 2 (B), and CH 2 Cl 2 (C) to obtain three fractions (A, B and C).

2. 2 .

 2 Antibacterial tests 2.2.1. Broth dilution tests A first evaluation of the antibacterial activities of plants and algae extracts was performed with broth dilution tests and disk diffusion assays. Broth dilution tests of plant and algae extracts were performed against the pathogenic bacteria Tenacibaculum maritimum and Vibrio harveyi. Bacterial strains T. maritimum (TFA4 isolated from P. orbicularis from Tahiti, French Polynesia, Centre IFREMER du Pacifique, Tahiti) and V. harveyi (LMG4044, Centre IFREMER du Pacifique, Tahiti) were cultured 24 hours prior to in vitro tests in Zobell Marine Broth 2216 at 28°C. 20 μL of plant or algae extract diluted in DMSO and 180 μL of 1/25 diluted bacterial solution (T. maritimum or V. harveyi) were added to 96-well microplates. Plant and algae extracts were tested at 3 final concentrations: 2 mg/mL, 0.2 mg/mL and 0.02 mg/mL. Negative controls were done replacing the bacterial solution by Zobell Marine broth and positive controls were done with 20 μL of DMSO (1, 0.1, 0.01%) and 180 μL of bacterial solution. Triplicates were performed for all samples and controls.

  mg/mL) of A. sativum, C. longa and A. taxiformis (2014) inhibited growth of V. harveyi. Partial growth inhibition of V. harveyi was observed at weaker concentrations of C. longa extract (0.2, 0.002 mg/mL), revealing a dose-effect relationship. No bacterial growth inhibition was observed with any of the other plants (M. taxifolia, C. inophyllum and Z. officinalis) and algae (A. torulosa, D. marginata, L. majuscula, P. boryana and T. ornata) at any of the three concentrations studied (Figure 1).

Figure 1 .

 1 Figure 1. V. harveyi growth curves (λ = 340 nm) with plant (A) and algae (B) extracts at three concentrations (2, 0.2 and 0.02 mg/mL).

Figure 2 .

 2 Figure 2. Disk assays tests of the A. taxiformis (At) fraction A. Inhibition zones are delimitated with blue circles.

  Synthesis kit» from Roche (Roche Applied Science, Penzberg, Germany) and 500 ng of total RNA. Each cDNA sample amplification was performed in duplicate using a Mx3000P thermal cycler (Agilent Technologies, Santa Clara, California, USA). Each reaction contained 12.5 μL of Brilliant II SYBR Green qPCR Master Mix (Agilent Tech.), 4 μM forward and reverse primer and 10 μL of template formerly diluted at 1:100 in pure water, in a final reaction volume of 25 µl. The cycling conditions were 10 min at 95°C to allow the enzyme activation followed by 40 cycles (denaturation 30 s at 95°C, annealing 1 min at 60°C and 30 s extension at 72°C) and 1 min at 95°C and finally increasing temperature from 45°C to 95°C to obtain the melting curves. Threshold Cycle (C t ) value corresponded to the PCR cycle number at which an increase in reported fluorescence above the baseline signals was first detected. The threshold was set using an amplification-based algorithm from the Mx3000 software (Agilent Technologies) for the initial plate. For the other plates an interplate calibrator was used to set the threshold manually and ensure the repeatability of measures.

Figure 1 .Figure 2 .

 12 Figure 1. Lys G relative gene expression on kidney and spleen of P. orbicularis following 2 week treatment (a) and 3 week treatment (b) with diets enriched in several plants or algae. Oil enrichment control test is displayed in the grey rectangle. No-oil stands for commercial food alone, and W. oil represents commercial food enriched in oil (control used in the rest of the experiments). Upper hinge and lower hinge represent the first and third quartile, while upper whisker and lower whisker represent maximum and minimum values excluding the outliers (represented as black dots). * indicate P-value < 0.05 (Mann-Whitney U tests).

  If infection was successful, fish condition degraded very fast, and within few hours fish started to present visible signs of infection like damaged fins, tail-rot and white spots and ulcers on the body. Mortality started approximately 24 hours post-infection and stabilized between the 3rd and 5th day. Fish heavily infected presented a considerable loss of colour in spleen and other organs (Figure1).

Figure 1 .

 1 Figure 1. (A) and (B) Heavily infected P. orbicularis fingerlings with T. maritimum and their spleens. (C) Lightly infected P. orbicularis fingerlings and the normal spleen.

Figure 2 .

 2 Figure 2. Mortalities (%) of P. orbicularis fingerlings infected with T. maritimum following different diet enrichments.

  reducing mortality of T. maritimum infected P. orbicularis fingerlings.

(

  Reverter et al. 2014; Chapter VII). The emerging orbicular batfish (P. orbicularis) aquaculture in Tahiti is suffering severe mortality episodes due to bacterial infections (T. maritimum and V. harveyi) that compromise its development. In order to propose a local, cheap and sustainable treatment I have evaluated the potential of several local Polynesian plants and algae against P. orbicularis bacterial infections (Chapters VIII-X). The red opportunistic algae A. taxiformis displayed strong antibacterial activity against V. harveyi and T. maritimum, promoted weight gain and increased the expression level of two immunerelated genes (Lys G and TGF-β1) in orbicular batfish after three weeks of oral administration Conclusion and perspectives -277 -(Chapters VIII, IX). Several studies have shown numerous biological activities of A.

Figure 1 .

 1 Figure 1. Research steps of medicinal plant utilisation in aquaculture.

Figure 2 .

 2 Figure 2. Plant orders used in aquaculture.

Figure 4 .

 4 Figure 4. Plant bioactivities in aquaculture.

Figure 5 .

 5 Figure 5. Plant order bioactivities in aquaculture.

Figure 6 .

 6 Figure 6. Parts of plants used in aquaculture.

  Abdelhadi, Y., A. Saleh, O., F. Sakr, S., 2010. Study on the Effect of Wormseed Plants; Artemisia cina L. and Chamomile; Matricaria chamomilla L. on Growth Parameters and Immune Response of African Catfish, Clarias gariepinus. J Fish Inter 5, 1-7.

  espèces de poisson papillon. Les autres taxons présents chez les quatre espèces de Chaetodon incluent les Actinobactéries et les Firmicutes.J'ai étudié le métabolome du mucus branchial de huit espèces de Chaetodon(C. auriga, C. lunula, C. lunulatus, C. quadrimaculatus, C. ornatissimus, C. reticulatus, C. ulietensis et C. vagabundus) avec des régimes alimentaires différents (corallivores facultatifs, corallivores obligactoires et omnivores), différentes positions phylogénétiques et différents taux de parasitisme branchial. Une extraction biphasique du mucus branchial des Chaetodons a permis d'obtenir un maximum de composés ; les deux fractions obtenues (polaire and apolaire) on été étudiées en utilisant une approche métabolomique non-ciblée en chromatographie liquide couplée à la spectrométrie de masse (LC-MS). J'ai étudié la possible relation entre les différences métaboliques observées et les caractéristiques écologiques et phylogénétiques de chaque espèce. Le régime alimentaire est le paramètre qui explique la plus grand partie de la variabilité métabolique dans les deux fractions, tandis que le parasitisme est le deuxième facteur qui explique la variabilité métabolique de la fraction polaire. J'ai ensuite intégré les résultats obtenus à partir du microbiome et du métabolome du mucus branchial des Chaetodon pour chercher s'il y a des taxons bactériens spécifiques et des métabolites surexprimés chez les poissons non parasités (C. lunulatus) qui pourraient être reliés à l'absence des parasites chez cette espèce. C. lunulatus présente une plus grande abondance des Fusobactéries et Spirochaetes qui sont fortement corrélées avec une surexpression de peptides qui pourraient provenir de la β-hémoglobine. Leur purification et leur séquençage sont en cours au laboratoire et leur synthèse sera réalisée pour pouvoir déterminer leur activité antiparasitaire. Dans une deuxième partie de ma thèse, menée sur des poissons en élevage, j'ai étudié l'utilisation des plantes Des études supplémentaires sont en cours au laboratoire pour identifier et caractériser les molécules antibactériennes d'A. taxiformis. De plus, d'autres études in vivo sont nécessaires pour évaluer la capacité réelle d'A. taxiformis contre des infections bactériennes en aquaculture.médicinales comme alternative à des traitements chimiques en l'aquaculture. En effet, les plantes et les algues ont été utilisées dans l'aquaculture traditionnelle pendant longtemps. Plusieurs études ont montré leur capacité immunostimulante et leur activité contre nombreux pathogènes marins comme des bactéries, champignons, parasites et virus. De plus, elles présentent une alternative plus durable à l'utilisation des médicaments persistants qui sont aujourd'hui utilisés en grande quantité dans le traitement des infections aquacoles et qui présentent de nombreux inconvénients tels que le développement des bactéries résistantes, leur accumulation dans le tissu des animaux ou leur effet sur l'environnement. J'ai testé l'activité antibactérienne et immunostimulante de plusieurs plantes et algues polynésiennes. L'algue rouge Asparagopsis taxiformis montre de fortes activités antibactériennes sur Vibrio harveyi et Tenacibaculum maritmum, deux bactéries responsables de hautes mortalités chez de nombreuses

Table of contents

 of

	GENERAL INTRODUCTION
	1. PARASITISM
	1.1. IMPORTANCE OF PARASITISM IN ECOSYSTEMS
	1.2. PARASITISM IN AQUACULTURE
	1.3. DISTRIBUTION OF PARASITE COMMUNITIES
	1.4. CHEMICAL ECOLOGY IN HOST-PARASITE INTERACTIONS AND HOST PROTECTION STRATEGIES FROM

  Wallis test and Kruskal post-hoc test allowed the identification of a group of 10 butterflyfish species (C. lunulatus Quoy and Gaimard, 1825, C. ephippium Cuvier, 1831, C. baronessa Cuvier, 1829, C. pelewensis Kner, 1868, Hemitaurichthys polylepis Bleeker, 1857, C. semeion Bleeker, 1855, C. speculum Cuvier, 1831, C. ulietensis Cuvier, 1831, Heniochus chrysostomus Cuvier, 1831 and C.

unimaculatus Bloch, 1787) with low dactylogyrid intensities that differed significantly (P < 0.05) from eight fish species that had high intensities and were always parasitised (C. vagabundus Linnaeus, 1758, Chelmon marginalis Richardson, 1846, C. ornatissimus Cuvier, 1831, C. citrinellus Cuvier, 1831, C. reticulatus Cuvier, 1831, C. quadrimaculatus Gray, 1831, C. declivis Randall, 1975, and H. acuminatus Linnaeus, 1758) (Figure

Table 1 .

 1 Parasite richness, diversity (H) and eveness (E) of the 33 butterflyfish species parasitized by dactylogyrids.

	Fish species	Parasite richness	Evenness (E)	Diversity index (H)
	Chaetodon aureofasciatus	5	0.68	1.33
	Chaetodon auriga	10	0.57	1.38
	Chaetodon baronessa	2	0.59	0.41
	Chaetodon citrinellus	11	0.67	1.6
	Chaetodon declivis	5	0.68	1.1
	Chaetodon ephippium	4	0.76	1.05
	Chaetodon flavirostris	9	0.68	1.49
	Chaetodon kleinii	7	0.68	1.33
	Chaetodon lineolatus	5	0.59	0.95
	Chaetodon lunula	10	0.48	1.15
	Chelmon marginalis	5	0.45	0.72
	Chaetodon mertensii	6	0.28	0.5
	Chaetodon ornatissimus	8	0.46	0.95
	Chaetodon pelewensis	8	0.85	1.76
	Chaetodon plebeius	9	0.48	1.06
	Chaetodon quadrimaculatus	6	0.1	0.17
	Chaetodon rafflesii	7	0.74	1.44
	Chaetodon rainfordi	8	0.76	1.59
	Chaetodon reticulatus	8	0.39	0.81
	Chaetodon semeion	1	NA	0
	Chaetodon speculum	3	0.2	0.22
	Chaetodon trichrous	3	0.7	0.77
	Chaetodon trifascialis	10	0.79	1.81
	Chaetodon ulietensis	10	0.68	1.56
	Chaetodon unimaculatus	7	0.5	0.97
	Chaetodon vagabundus	7	0.78	1.52
	Forcipiger flavissimus	8	0.87	1.82
	Forcipiger longirostris	7	0.73	1.42
	Heniochus acuminatus	3	0.53	0.58
	Heniochus chrysostomus	8	0.64	1.34
	Heniochus monoceros	7	0.66	1.28
	Heniochus varius	3	0.18	0.51
	Hemitaurichthys polylepis	3	0.46	0.2

Table 1 .

 1 Abundance of dactylogyrid species at the nine sampled geographic sites. Presence (X) of dactylogyrid species obtained from literature. NR = Ningaloo Reef, LI = Lizard Island, HI = Heron Island, SCS = South China Sea.

	Dactylogyrid species	NR	LI	Palau	Wallis	Society	otu Tuam	Australs	Gambiers	Marquesas	HI	NC	Hawaii	SCS
	H. angelopterum	56	117	5	1	854	764	1970	182	526	-	-	X	-
	H. aurigae	487	469	81	53	134	593	459	127	46	X	X	X	-
	E. triangulovagina	102	194	14	35	16	114	92	12	407	-	-	X	X
	E. grandis	629	190	9	8	205	621	64	49	43	X	X	X	-
	E. berenguelae	1173	94	2	11	10	21	77	37	-	-	-	-	-
	E. microphallus	2	273	1	X	63	87	189	132	120	X	X	X	-
	E. annulocirrus	-	20	X	-	17	4	48	27	-	X	X	X	X
	E. pirulum	1	1	1	X	X	2	5	14	1	-	X	-	-
	E. cribbi	-	79	5	30	4	3	6	1	-	-	-	-	-
	E. aspistis	-	39	X	X	2	29	13	-	-	-	X	-	-
	H. sp. A	-	11	-	-	-	-	92	-	-	-	-	-	-

Table 2 .

 2 Dactylogyrid

	Site	Richness	Shannon index (H)	Intensity	Weighted intensity
	Society	9	1.12	111.3	1.12
	Palau	8	1.22	24.4	0.65
	Marquesas	6	1.22	60.7	0.57
	Australs	11	1.24	79.8	0.99
	Ningaloo Reef	7	1.25	132.4	1.09
	Wallis	6	1.45	19.7	0.90
	Tuamotu	12	1.65	126.5	1.49
	Gambiers	9	1.74	26.5	0.67
	Lizard Island	12	1.96	56.5	0.86
		diversity index (Shannon), richness, intensity and weighted intensity at the
	different geographic locations.
	A hierarchical cluster analysis allowed identification of three main different biogeographic
	regions based on dissimilarities of their dactylogyrid communities, the eastern Pacific region
	containing French Polynesia, the western Indo-Pacific region containing Lizard Island, Palau
	and Wallis and Ningaloo Reef (Figure 4). French Polynesia was characterized by a high
	abundance of H. angelopterum, while localities in the western Indo-Pacific region exhibited
	higher abundances of H. aurigae. Ningaloo reef was highly dissimilar with a high abundance
	of H. berenguelae (48%) (Figure 3, 4).

F. longirostris C. declivis C. trichrous C. rainfordi C. reticulatus F. flavissimus C. unimaculatus C. pelewensis H. chrysostomus C. vagabundus C. plebeius C. rafflesii H. varius

	H. angelopterum	H. aurigae
	E. triangulovagina	E. berenguelae
	E. microphallus	E. grandis
	E. pirulum	E. annulocirrus
	E. cribii	E. aspistis
	H. scyphovagina	H. sp. A
	H. sp. B

C. quadrimaculatus C. mertensii F. longirostris C. declivis C. trichrous C. rainfordi C. reticulatus C. ornatissimus F. flavissimus C. unimaculatus C. pelewensis C. citrinellus C. trifascialis chrysostomus vagabundus C. plebeius rafflesii C. aureofasciatus auriga flavirostris H. polylepis H. acuminatus H. varius

	H. angelopterum	H. aurigae
	E. triangulovagina	E. berenguelae
	E. microphallus	E. grandis
	E. pirulum	E. annulocirrus
	E. cribii	E. aspistis
	H. scyphovagina	H. sp. A
	H. sp. B

C. quadrimaculatus C. mertensii F. longirostris C. declivis C. trichrous C. rainfordi C. reticulatus C. ornatissimus F. flavissimus C. unimaculatus C. pelewensis C. citrinellus C. trifascialis C. plebeius C. aureofasciatus H. polylepis H. acuminatus H. varius

	H. angelopterum	H. aurigae
	E. triangulovagina	E. berenguela
	E. microphallus	E. grandis
	E. pirulum	E. annulocirru
	E. cribii	E. aspistis
	H. scyphovagina	H. sp. A
	H. sp. B

F. longirostris C. declivis C. trichrous C. rainfordi C. reticulatus F. flavissimus C. unimaculatus C. pelewensis H. chrysostomus C. vagabundus C. plebeius C. rafflesii H. varius

	H. angelopterum	H. aurigae
	E. triangulovagina	E. berenguelae
	E. microphallus	E. grandis
	E. pirulum	E. annulocirrus
	E. cribii	E. aspistis
	H. scyphovagina	H. sp. A
	H. sp. B

C. quadrimaculatus C. mertensii F. longirostris C. declivis C. trichrous C. rainfordi C. reticulatus C. ornatissimus F. flavissimus C. unimaculatus C. pelewensis C. itrinellus C. trifascialis chrysostomus vagabundus C. plebeius rafflesii C. reofasciatus auriga flavirostris H. polylepis H. acuminatus H. varius

	H. angelopterum	H. aurigae
	E. triangulovagina	E. berenguelae
	E. microphallus	E. grandis
	E. pirulum	E. annulocirrus
	E. cribii	E. aspistis
	H. scyphovagina	H. sp. A
	H. sp. B

C. quadrimaculatus C. mertensii F. longirostris C. declivis C. trichrous C. rainfordi C. reticulatus C. ornatissimus F. flavissimus C. unimaculatus C. pelewensis C. citrinellus C. trifascialis C. plebeius C. aureofasciatus H. polylepis H. acuminatus H. varius

	H. angelopterum	H. aurigae
	E. triangulovagina	E. berenguelae
	E. microphallus	E. grandis
	E. pirulum	E. annulocirrus
	E. cribii	E. aspistis
	H. scyphovagina	H. sp. A
	H. sp. B

Table 2 .

 2 Results from the multiple regression models (MRM) between the parasite dissimilarity matrices (balanced variation -dBCbal and gradient abundance component dBC-gra and Bray-Curtis dissimilarity -dBC) and the environmental dissimilarity, geographic distance and dissimilarity in Chaetodon species.

	Response or dependent variable	P-value R2 model p-values	F
	Parasite community balanced variation (dBC-
	bal)
	Model 1	0.39	0.005	6.88
	Environmental parameters	0.066
	Linear distance	0.277
	Chaetodon species (dBC-gra)	0.005
	Model 2	0.35	0.004	5.74
	Environmental parameters	0.254
	Linear distance	0.065
	Chaetodon species (dBC-bal)	0.012
	Model 3	0.23	0.06	3.25
	Environmental parameters	0.196
	Linear distance	0.090
	Chaetodon species (dBC)	0.131
	Parasite community gradient abundace (dBC-
	gra)
	Model 1	0.31	0.028	4.89
	Environmental parameters	0.008
	Linear distance	0.486
	Chaetodon species (dBC-gra)	0.418
	Model 2	0.49	0.003	10.37
	Environmental parameters	0.001
	Linear distance	0.335
	Chaetodon species (dBC-bal)	0.014
	Model 3	0.32	0.037	4.93
	Environmental parameters	0.025
	Linear distance	0.089
	Chaetodon species (dBC)	0.322
	Parasite community dissimilarity (dBC)
	Model 1	0.58	0.001	14.77
	Environmental parameters	0.096
	Linear distance	0.615
	Chaetodon species (dBC-gra)	0.038
	Model 2	0.46	0.005	9.23
	Environmental parameters	0.032
	Linear distance	0.087
	Chaetodon species (dBC-bal)	0.881
	Model 3	0.64	0.003	19.30
	Environmental parameters	0.126
	Linear distance	0.765
	Chaetodon species (dBC)	0.003

Table 1 .

 1 Toxins described in fish mucus.

		Fish species	Toxin	Molecule type	Reference
	Tetraodontidae	Takifugu niphobles, T.	Tetrodotoxin	Amino	Kodama et al. 1986;
		pardalis, T. vermiculare		perhydroquinazoline	Ito et al. 2006
		porphyreum, T.
		poecilonotus, T.
		vermiculare radiatum, T.
		xanthopterum
		Teatrodon sp.	Saxitoxin,	Purine base with 3	Ngy et al. 2008
			decarbamolysaxitoxin	carbon bridge linking
				positions 3 and 9 and
				a methyl carbamate at
				position 6
	Ostraciidae	Ostragon lentiginosus,	Ostracitoxin (also	Choline chloride ester	Thompson, 1964;
		Lactophyrs triqueter	called Pahutoxin)	of a fatty acid	Boylan and Scheuer,
					1967
		O. immaculatus, O.	Pahutoxin and	Fatty acid derivative	Fusetani and
		lentiginosus	homopahutoxin		Hashimoto, 1987
		O. cubicus	Boxin	Protein	Kalmanzon and
					Zlotkin, 2000; 2001
	Aracanidae,	Anoplacapros		β-substituted choline	Goldberg et al. 1988
	Ostraciidae	lenticularis, Aracana		chloride esters of
		aurita, Aracana ornata,		palmitic acid
		Lactoria fornasini, O.
		cubicus, Rhinesomas
		reipublicae,
		Strophiurichthys inermis,
		S. robustus
	Soleidae	Pardachirus marmoratus	Pardaxins (I-II	Polypeptide (AMP) Lazarovici et al. 1986
		P. pavonicus	Pavoninins 1-6	Steroidal saponins	Tachibana, 1984;
					Williams and Gong,
					2007
	Plotosidae	Plotosus lineatus	Toxin I, II	Natterine-like	Tamura et al. 2011
				(proteinaceous)
	Ariidae	Cathorops spixii	WAP65	Protein	Ramos et al. 2012
	Potamotrygonidae	Potamotrygon gr.	Orpotrin, porflan	Peptides	Conceiçao et al. 2006
		orbignyi
	Serranidae	Grammistes sexlineatus,	Grammnistins (A-E,	Polypeptides	Hashimoto and
		Pogonoperca punctata	1, 2, Pp1, Pp2, Pp3,		Oshima, 1972;
			Pp4a, Pp4b		Shiomi et al. 2000,
					Sugiyama et al. 2005

Table 2 .

 2 Bacteria found in external fish mucus.

	Fish species	Predominant bacteria phyla	Tissue	Reference
		(genus)
	Lutjanus	Proteobacteria (Vibrio,	Skin mucus	Arias et al .2013
	campechanus	Photobacterium)
	Merlangius	y-Proteobacteria	Skin mucus	Smith et al. 2007
	merlangus	(Photobacterium )
	Mugil cephalus	Proteobacteria	Skin mucus	Larsen et al.2013
	Fundulus grandis	y-proteobacteria (Pseudomonas),	Skin mucus	Larsen et al. 2015
		cyanobacteria
		(Prochlorococchus)
	Megalobrama	y-Proteobacteria (Acinetobacter)	Gills	Wang et al. 2010c
	amblycephala	and firmicutes (Anoxybacillus
		flavithermus)
	Megalobrama	y-Proteobacteria (Acinetobacter) Skin	Wang et al. 2010c
	amblycephala
	Carassius auratus	y-Proteobacteria (Acinetobacter)	Gills	Wang et al. 2010c
		and firmicutes (Anoxybacillus
		flavithermus)
	Carassius auratus	Proteobacteria (Enterobacter)	Skin	Wang et al. 2010
		and firmucutes (Staphylococcus)
	Salvelunus fontinalis α-proteobacteria	Skin mucus	Boutin et al. 2013;
				2014
	Salmo salar	α-proteobacteria (Pseudomonas) Skin mucus	Cipriano, 2011
	Gadus morhua	y-proteobacteria	Skin mucus	Wilson et al. 2008
		(Photobacterium)

Figure 2. Small molecules found in epidermal fish mucus.

  Mucus DNA was extracted right after collection using the DNeasy Blood & Tissue kit(QIAGEN, Courtaboeuf, France). Concentration of the nucleic acids was quantified by measuring the absorbance at 260 nm and their purity was checked by measuring the ratio of OD 260/280 nm and 230/260 nm. DNA samples were sent for 454 pyrosequencing by Mr.

	DNA (Shallowater, TX, USA) as previously described (Croué et al 2013). Briefly a PCR step
	(30 cycles) with a HotStart HiFidelity Polymerase and primers 28F (59
	TTTGATCNTGGCTCAG 39) and 519R (59 GTNTTACNGCGGCKGCTG 39) was
	performed in order to amplify the hypervariable V1-V3 region of the 16S rRNA gene. Tag-
	encoded FLX amplicons were sequenced on the Roche 454 FLX sequencer using Titanium
	(Roche) reagents. Samples were identified by the fish initials and an order number (Ex. CL1-
	CL5 for C. lunulatus replicates)
	2.1. Mucus sampling and DNA extraction
	Four butterflyfish species (C. lunulatus, C. ornatissimus, C. reticulatus and C.
	vagabundus) were sampled (n = 20) on the island of Moorea (French Polynesia). Fish were

spear-fished, placed in individual plastic bags with seawater and brought immediately to the laboratory for gill dissection. Gill mucus was carefully scraped with a sterile spatula into sterile tubes.

  Finally, as one of the samples from C. vagabundus was clearly an outlier in preliminary analyses, the sample was removed from the OTU table along with all OTUs present exclusively in this sample, as was one OTU abundant is sample CV1 corresponding to a chloroplast, but not identified as such by the RDP classifier. Sequences were aligned and filtered

of usearch 5.2 (http://drive5.com/usearch/). Non-chimeric sequences were unweighted and grouped into operational taxonomic units (OTUs) with 98% sequence identity using the usearch V5.2 method implemented in Qiime V1.5. The longest sequences in the OTU were selected as representatives. OTUs were classified using the rdp_classifier software implemented in Qiime V1.5 and a database based on the Green genes August 2013 taxonomy (http://greengenes.secondgenome.com) modified to exclude orders, and to correct to comply to current official nomenclature [(list of prokaryotic names with standing in nomenclature (http://www.bacterio.net)]. Based on this classification, OTUs representing chloroplasts and mitochondria were removed. The resulting OTU table was further treated to remove (using custom bash and awk scripts) OTUs for which the representative sequence was shorter than 372 base pairs (400 bp minus the length of the primer), OTUs which were represented by a single sequence in the ensemble of samples and a "Root" taxonomy status by the rdp_classifier analysis, as well as sequences failing aligning by pynast aligner implemented in Qiime.

  Alpha-rarefaction indicesshowed that one of the C. vagabundus species possessed a remarkably different microbiome (45% of its OTUs were exclusive of this sample, including 5 different phyla). Therefore, we considered it as an outlier and the following analyses included only four C. vagabundus samples (Figure1. Supplementary information). After removal of the outlier sample, 1450 OTUs were obtained in the full OTU table and 1041 in the rarefied table.

	Shannon diversity varied significantly between fish species, with the highest diversity
	found in C. vagabundus (H = 5.7 ± 0.5) and the lowest in C. reticulatus (H = 3.7 ± 0.7). C.
	lunulatus and C. ornatissimus presented similar diversities (H = 4.7 ± 1.2 and H = 4.8 ± 0.5
	respectively). Microbial community evenness (E) was slightly higher in C. vagabundus (E =
	0.8 ± 0.1). Observed and expected richness were both the highest in C. lunulatus (142.8 ±
	62.3 and 473 OTUs respectively) followed by C. ornatissimus (114.4 ± 38.9 and 370 OTUs

lunulatus, C. ornatissimus, C. reticulatus and C. vagabundus). respectively), C. vagabundus (137.0 ± 53.7 426 OTUs respectively) and C. reticulatus (45.6 ± 20.4 and 179 OTUs respectively) (Table

Table 2

 2

	). C.

highest number of specific OTUs (249 OTUs, 26-66% of all OTUs), followed by C. ornatissimus (143 OTUS, 6-15% of all OTUs), and finally C. reticulatus, with only 80 specific OTUs that represented between 16 and 62% of its bacterial community. C. lunulatus and C. ornatissimus shared the highest number of OTUs (total of 167 shared OTUs), while C.

Table 2 .

 2 Core

	OTUs	Phylum	Class	Family	Genus	Species
	44, 68			Corynebacteriaceae	Corynebacterium	-
	131	Actinobacteria	Actinobacteria	Micrococcaceae	Micrococcus	luteus
	3			Propionibacteriaceae	Propionibacterium	acnes
	114	Cyanobacteria	Synechococcophycideae	Synechococcaceae	Synechococcus
	1, 25, 36, 38, 47, 219, 4467	Firmicutes	Clostridia	-	-	-
	58, 208			[Mogibacteriaceae]	-	-
	280 71, 269		Alphaproteobacteria	Rhodobacteraceae	Nautella Ruegeria	--
	854		Betaproteobacteria	Burkholderiaceae	Ralstonia	-
	696			-	-	-
	79				Acinetobacter	-
	42	Proteobacteria		Moraxellaceae	Acinetobacter	venetianus
	142 67		Gammaproteobacteria	Salinisphaeraceae	Enhydrobacter -	--
	5				Photobacterium	-
	0			Vibrionaceae	Vibrio	-
	426				Vibrio	shilonii

microbiota (OTUs and their classification) of the four butterflyfish species (C. lunulatus, C. ornatissimus, C. reticulatus and C. vagabundus). The Principal Coordinate Analysis (PCoA) based on weighted Unifrac distances showed significant microbiota differences between the pairs C. lunulatus/ C. ornatissimus and C. reticulatus/ C. vagabundus (p-value < 0.05, Adonis test and pairwise comparisons) (Figure

  table. Unidentified OTUs (Unassigned) represented less than 10% in all four butterflyfish species. Proteobacteria was the most abundant phylum in the four butterflyfish species. Gammaproteobacteria dominated the microbial assemblage of C.

	lunulatus and C. ornatissimus (90% and 80% of the total Proteobacteria), while
	Alphaproteobacteria was more abundant in C. vagabundus (59% of total Proteobacteria). C.
	reticulatus, whose community presented the highest abundance of Proteobacteria (67% of

total OTUs), presented 41% of Alphaproteobacteria, 19% of Betaproteobacteria and 39% of Gammaproteobacteria. In addition to Proteobacteria, only Actinobacteria and Firmicutes were present in all samples of butterflyfish gill mucus, being particularly well represented in C. vagabundus, C. reticulatus and C. ornatissimus. C. lunulatus and C. ornatissimus presented a higher abundance of Bacteroidetes (

  Endozoicomonas spp.), which were frequent in C. lunulatus and C. ornatissimus, reaching an abundance of 25% in one C. lunulatus individual, while absent in

		CHAPTER IV
	3) Hahellaceae (mostly C. reticulatus samples, 4) Spirochaetaceae (Spirochaeta spp.) with abundances ranging from
	1 to 10% in C. lunulatus and C. ornatissimus, and rarely present in C. vagabundus and totally
	absent in C. reticulatus and 5) Ruminococcaceae (genus undetermined), which represented
	around 5% of the total bacterial assemblage of C. lunulatus and C. ornatissimus, but they
	CL were absent in C. reticulatus.	Unassigned Acidobacteria Actinobacteria	CR	Unassigned Acidobacteria Unassigned
	Bacteroidetes Groups displaying interesting associations with C. reticulatus and C. vagabundus Actinobacteria Acidobacteria Cyanobacteria Firmicutes Bacteroidetes Actinobacteria include 1) Moraxellaceae (mainly Acinetobacter spp.), which were more abundant in C.
		Fusobacteria		Cyanobacteria Bacteroidetes
		Planctomycetes Spirochaetes		Firmicutes Cyanobacteria
		Verrucomicrobia	Fusobacteria Firmicutes
	CO	Alphaproteobacteria Betaproteobacteria Deltaproteobacteria Gammaproteobacteria CV	Fusobacteria Planctomycetes Spirochaetes Verrucomicrobia Planctomycetes Spirochaetes
				Alphaproteobacteria Verrucomicrobia Proteobacteria
				Betaproteobacteria Alphaproteobacteria
				Deltaproteobacteria Betaproteobacteria
				Gammaproteobacteria Deltaproteobacteria
				Gammaproteobacteria
		-141 -

As expected from the PCoA analysis, dominant families were most similar between C. lunulatus and C. ornatissimus, and C. reticulatus and C. vagabundus. Groups with interesting associations with C. lunulatus and C. ornatissimus include 1) Vibrionaceae (mostly Vibrio spp.), generally the most abundant microbial taxa in C. lunulatus (between 3 and 90 %) and C. ornatissimus (between 1 and 41%) 2) Verrucomicrobiaceae (mostly Akkermansia spp.) with abundances up to 15% in C. lunulatus and C. ornatissimus, reticulatus and C. vagabundus than C. lunulatus and C. ornatissimus 2) Propionibacteriaceae (mostly Propionibacterium spp.) with abundances of 2 -8% in C. reticulatus and 0.5 -12% in C. vagabundus and 3) Corynebacteriaceae (genus Corynebacterium), which represented around 10% of the microbiota community in most C. reticulatus and C. vagabundus samples while were nearly absent in all C. lunulatus and C. ornatissimus.

A number of bacterial families appeared to have been particularly associated to a single Chaetoron species. Anaplasmataceae were surprisingly abundant (70 and 33%) in two samples of C. reticulatus, and were mainly constituted by OTUs belonging to the Neorickettsia genus. Burkholderiaceae (genus Ralstonia) were also remarkably more frequent in C. reticulatus (up to 25%) than in the other fish species. Rhodobacteraceae were present in all fish species, but were particularly abundant in C. vagabundus, where they reached abundances up to 32%. As were "Exiguobacteraceae", mainly the Exiguobacterium genus, nearly specific to C. vagabundus, displaying abundances lower than 1% in the other fish species. Finally, Fusobacteriaceae (genus undetermined) had abundances in C. lunulatus ranging from 0.4 to 7.1%, while they were nearly absent in all other three fish species.

  is ecologically and phylogenetically closer to C. lunulatus and C. ornatissimus, presents a microbiome that is more similar to that of C. vagabundus. Overall, our results show that gill mucus microbiota is somewhat host-specific, however more studies should assess variability of gill mucus microbiome over space and time to confirm the associations presented here.The microbiome associated to the gill mucus of the four butterflyfish species were dominated by Proteobacteria, which is in accordance with previous studies on the gill microbiome of rainbow trout and the skin and gut microbiome of several fish species (Sullam

et al. 2012;[START_REF] Larsen | Community Structure of Skin Microbiome of Gulf Killifish, Fundulus grandis, Is Driven by Seasonality and Not Exposure to Oiled Sediments in a Louisiana Salt Marsh[END_REF][START_REF] Lowrey | Topographical Mapping of the Rainbow Trout (Oncorhynchus mykiss) Microbiome Reveals a Diverse Bacterial Community with Antifungal Properties in the Skin[END_REF]

. Gammaprotebacteria represented over 80% of the total Proteobacteria in C. lunulatus and C. ornatissimus gill mucus, which was mainly represented by families Vibrionaceae, Pseudomonaceae and Hahellaceae bacteria.

  1,3 1. CRIOBE, USR3278 -CNRS/EPHE/UPVD, Paris Sciences et Lettres (PSL), University of Perpignan Via Domitia, 58 Avenue Paul Alduy, 66860 Perpignan Cedex, France 2. Laboratoire d'Excellence "CORAIL", 98729 Moorea, French Polynesia 3. University Paris Saclay / Versailles-Saint Quentin en Yvelines, 55 Avenue de Paris 78035 Versailles Cedex, France 4. ENTROPIE, UMR250/9220-CNRS/IRD/UR, 101, promenade Roger-Laroque, BP A5 98848 Nouméa cedex New-Caledonia.

Table 1 .

 1 Results of the PERMANOVA test explaining the metabolic variability of gill mucus of butterflyfishes.

Table 3 .

 3 In

	CHAPTER VII

vivo anti-bacterial studies of several plant extracts and herbal mixture extracts. *Plants are from marine origin (seaweeds or macroalgae).

Table 4 .

 4 In vivo anti-helminth studies of several plant extracts and herbal mixture extracts.

* plants are form marine origin (seaweeds or macroalgae). PE: Petroleum ether, CHL: chloroform, EA: Ethyl acetate, ME: Methanol. Underlined solvents are the solvents that displayed better results, while bold letter solvents didn't show any activity.

Table 5 .

 5 In vitro anti-parasitic studies of several plant extracts and herbal mixture extracts. * plants are from marine origin (seaweed or macroalgae).

  template. Couple of primers yielding both a qPCR efficiency ratio of almost 100% (restricted to a range between 90 and 110%), and the higher qPCR sensitivity, as expressed as the lowest cycle threshold (Ct) values for a given dilution, were selected. The specificity of the retained couple of primers was firstly checked by electrophoresis on a 1% agarose gel of qPCR products, using a DNA fluorescent dye and a DNA molecular weight marker and visualizing a single amplicon of the attempted size. Finally four amplicons of each targeted gene were purified on a QIAEX II gel extraction kit (Qiagen, Courtaboeuf, France), cloned on TOPO® TA cloning® kit (Invitrogen, CergyPontoise, France) and sequenced. For each targeted gene, one single sequence was obtained and deposited in GenBank database (Table2) after verifying edited sequence by Basic Local Alignment Search Tool (BLAST).

			CHAPTER IX
		Plant	2 weeks treatment	3 weeks treatment
			% in fish food (w/w)	% in fish food (w/w)
		Morinda taxifolia	-			3
		Zingiber officinalis	-			3
		Allium sativum	3			-
		Curcuma longa	3			-
		Asparagopsis
		taxiformis	3			3, 1.5
	Gene	Oligonucleotide sequences (5'-3')	qPCR efficiency (%)	Amplicon Length (bp)	Accession n°
	EF1	GGCTGGTATCTCCAAGAACG GTCTCCAGCATGTTGTCTCC	106		239	KU950348
	-actin	GACTACCTCATGAAGATCCTGAC AGCTTCTCCTTGATGTCACG	102		89	KU976283
	Lys G	GCTCTCATTGCTGCCATCAT TCAACCTGCATCAGTCCCA	98		100	KU976284
	TGF-β1	TCCCTCTACAACAGCACCAAG CAGGACCCCATGCAGTAGTT	93		758	KU950349
			-251 -

Table 2 .

 2 Characteristics of the primers used to amplify the different genes.

Table 3 .

 3 Weight results after the different diet treatments of P. orbicularis fingerlings.

	Plant	Concentratio n (%)	Lenght of treatment	Control weight (mean ± S.D. )	Treatment (mean ± S.D. )	Weight gain (%)	P-value
	Oil control	3 (vegetal) +3 (cod)	2	3.43 ± 0.65 3.64 ± 0.74	5.78	> 0.1
	A. sativum	3	2	12.25 ±1.06 12.68 ±1.24	3.39	> 0.1
	C. longa	3	2	12.25 ±1.06 12.28±1.05	0.24	> 0.1
	A. taxiformis	3	2	3.63 ± 0.73 4.13 ± 0.57	13.77	< 0.05
	A. taxiformis	3	3	4.95 ± 0.8	6.13 ± 1.17	23.84	< 0.05
	A. taxiformis	1.5	3	4.95 ± 0.8	5.68 ± 1.1	14.75	0.1
	M. taxifolia	3	3	12.45 ±1.98 12.94 ±2.76	3.78	> 0.1
	Z. officinale	3	3	9.70 ± 1.22 9.64 ± 0.95	-0.62	>0.1

  . Therefore, tenacibaculosis management should focus on the use of preventive sustainable techniques that allow maximization of fish fitness to avoid opportunistic outbreaks. Medicinal plants have been reported to promote weight gain, reduce stress and enhance immunity in fish and shellfish as well as display antibacterial, antiviral, antifungal and antiparasitic activities against numerous aquaculture pathogens(Reverter et al. 2014). Therefore, the administration of medicinal plants as a complement of fish diet might boost fish immunity and decrease susceptibility to T. maritimum infections.Six experimental infections were performed to P. orbicularis fingerlings fed with different enriched diets in plants (Allium sativum, Calophyllum Inophyllum, Curcuma longa, Morinda taxifolia and Zingiber officinalis) and algae (Asparagopsis taxiformis) in order to test efficacy of diet enrichment against T. maritimum infection. Three experiments were performed at the CRIOBE laboratory facilities in Moorea (French Polynesia) and the other three were performed at the IFREMER Pacific Center in Vairao, Tahiti(French Polynesia)

(Table

Table 1 .

 1 Dietary plant enrichment specifications.

	Plant	Site of experiment	2 weeks treatment % in fish food (w/w) % in fish food (w/w) 3 weeks treatment
	Calophyllum inophyllum	Moorea	-	3
	Morinda taxifolia	Moorea	-	3
	Zingiber officinalis	Moorea	-	3
	Allium sativum	Moorea / Tahiti	3	3
	Curcuma longa	Tahiti	3	-
	Asparagopsis taxiformis	Tahiti	3	3, 1.5

  Differences of P. orbicularis susceptibility between different experimental infections could be due to several factors. First of all, although all experimental infections are done following the same procedure, total T. maritimum concentration added to fish tanks during challenge might vary. A recent study has shown quorum sensing mechanisms (bacterial communication correlated to population density) in T. maritimum cultures[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]. Bacterial cell communication and group behaviour provide significant benefits to bacteria in host colonization and biofilm formation, and most importantly, quorum sensing activities control expression of virulence factors of pathogenic bacteria[START_REF] Li | Quorum sensing and bacterial social interactions in biofilms[END_REF][START_REF] Rutherford | Bacterial quorum sensing: its role in virulence and possibilities for its control[END_REF]. Therefore, different concentrations of T. maritimum in experimental infections could play a major role in the virulence of infection. Different P. orbicularis fingerling susceptibilities to tenacibaculosis were also observed in the aquaculture farms, with different larvae productions, regardless of the period of the year. Our experiments, were separated in time, and were therefore performed on different larvae

	CHAPTER X
	6.24, A. taxiformis
	= 18.33 ± 4.71 %). No significant differences in mortality were neither observed in the
	experiment where fish were fed for 3 weeks with an enriched diet of A. taxiformis 3% nor in
	-271 -

fish fed with A. taxiformis 1.5 % (control = 64.70 ± 21.20%, A. taxiformis 3% = 94.74 ± 9.12%, A.taxiformis 1.5% =82.35 ± 8.31%) (Figure

2C and 2D

). production, issued from different reproduction episodes. Since, P. orbicularis larvae production uses different breeders, we could think that some breeders could produce genetically more resistant P. orbicularis fingerlings to tenacibaculosis.

  could explain this marked difference in parasitism. C. lunulatus is ecologically and phylogenetically close to species that are always heavily parasitized by gill monogeneans like C. ornatissimus and C. reticulatus[START_REF] Hsu | Molecular phylogeny of Chaetodon (Teleostei: Chaetodontidae) in the Indo-west Pacific: evolution in geminate species pairs and groups[END_REF][START_REF] Berumen | Relative gut lengths of coral reef butterflyfishes (Pisces: Chaetodontidae)[END_REF]. Besides, C. lunulatus is known to be parasitized by other gut and skin trematodes, indicating that gill monogenean absence must arise from gill or gill mucus species-specific characteristics.I studied fish mucus because it is the first physical and biochemical barrier against pathogens (Chapter III). When C. lunulatus gill mucus was injected on the gills of three highly parasitized butterflyfishes, it induced a decrease in the number of gill monogenean parasites, indicating a possible antiparasitic activity of C. lunulatus gill mucus (Chapter VI). I have studied the gill mucus microbiome and metabolome of several butterflyfish species in order to investigate whether C. lunulatus had a specific microbiota and/or specific metabolites that could be related to the absence of gill monogenean parasites (Chapters IV, V). A microbiome study of four butterflyfish species (three highly parasitized species and C.

	Conclusion and perspectives
	-278 -

lunulatus) revealed a high bacterial diversity in gill mucus of butterflyfishes, compared to previous studies on gill microbiome of cultured rainbow trout (Oncorhynchus mykiss) or gut microbiota of the coral reef surgeonfishes

[START_REF] Lowrey | Topographical Mapping of the Rainbow Trout (Oncorhynchus mykiss) Microbiome Reveals a Diverse Bacterial Community with Antifungal Properties in the Skin[END_REF][START_REF] Miyake | Diet strongly influences the gut microbiota of surgeonfishes[END_REF]

(Chapter

  antimicrobial peptides (Masso-[START_REF] Silva | Metabolic fingerprinting of gilthead seabream (Sparus aurata) liver to track interactions between dietary factors and seasonal temperature variations[END_REF].Casadei et al. (2013) found that several AMPs such as defensins, cathelicidins and liver AMPs were overexpressed after peptidoglycan administration in rainbow trout (O. mykiss). However, to date, little is still known on how induced AMP expression can be performed in cultured fish in order to prevent disease outbreaks. A recent study highlighted the use of probiotics to produce and secrete heterologous endogenous AMPs of interest in the human gastrointestinal tract(Mandal et al.

	Conclusion and perspectives
	-281 -

  that black tiger shrimp (Penaeus monodon) challenged with White Spot Syndrome Virus (WSSV) whilst treated with Bermuda grass (Cynodon dactylon) displayed no mortality and no signs of disease compared to 100% mortality observed in control groups. Several studies have shown in vitro antifungal

activities of several plants like conidinium fruit (Cnidium monnieri), magnolia bark (Magnolia officinalis), aucklandia root (Aucklandia lappa) and common rue (Ruta graveolens)

(Hashemi-Karouei et al. 2011; Xue-Gang et al. 2013)

. Medicinal plants seem to be also an effective alternative for treating ectoparasites. Several studies have shown antiparasitic activities of medicinal plants when added to water or administered orally

(Yi et al. 2012; Huang et al. 2013; Fu et al. 2014)

. Methanol extract of bupleurum root (Radix bupleuri chinensis), aqueous and methanol extracts of cinnamon (Cinnamomum cassia), methanol extract of Chinese spic brush (Lindera agreggata) and methanol and ethyl acetate extracts of golden larch (Pseudolarix kaempferi) had 100% in vivo efficacy against monogenean Dactylogyrus intermedium in infected goldfish (Carassius auratus)

(Wu et al. 2011; Ji et al. 2012)

.

Harikrishnan et al. (2012c)

showed 40% mortality decrease and enhanced immunity in olive flounder (Paralichthys olivaceus) infected by the protozoan ciliate Miamiensis avidus when fish were fed with a supplemented diet on Suaeda maxima.

Table 1 .

 1 Plants, algae and mushrooms used or studied for potential application in aquaculture. AV : antiviral, NA : not available, AB : antibacterial, IM : immunostimulant, AF :antifungal, AP : antiparasitic,

	Order	Family	Species	Activities	Used part	Bigliography
	Acorales	Acoraceae	Acorus calamus	AV	R	Sivasankar et al. 2015
	Alismatales	Araceae	Alocasia macrorrhizos	NA	L	Caruso et al. 2013
	Alismatales	Araceae	Colocasia esculenta	AB	R	Wet at al. 2010
	Apiales	Apiaceae	Angelica membranaceus	IM	R	Jian and Wu, 2004
	Apiales	Apiaceae	Angelica sinensis	IM	R	Wang et al. 2011
	Apiales	Apiaceae	Angelica pubsecens	AF	R	Xue-Gang et al. 2013
	Apiales	Apiaceae	Bupleurum chinense	AP	W	Wu et al. 2011
	Apiales	Apiaceae	Centella asiatica	AB	R	Rattanachaikunsopon and
						Phumkhachorn, 2010b
	Apiales	Apiaceae	Cnidium officinale	AB,GP	L, Fr	Takaoka et al. 2007b
	Apiales	Apiaceae	Corandrium sativum	AB	NA	Innocent et al. 2011
	Apiales	Apiaceae	Heracleum lasiopetalum	AB	R	Pirbalouti et al. 2011
	Apiales	Apiaceae	Radix peucedani	AP	W	Wu et al. 2011
	Apiales	Araliaceae	Kalopanax pictus	AP, IM	S	Harikrishnan et al. 2011e
	Apiales	Araliaceae	Panax ginseng	IM	R	Gooda, 2008
	Apiales	Araliaceae	Eleutherococcus senticosus	AB, IM	R	Won et al. 2008
	Apiales	Araliaceae	Panax quinquefolium	GP	W	Abdel-Tawwab, 2012
	Apiales	Umberlliferae	Carum carvi	GP	L	Abdel-Tawwab, 2011
	Arecales	Arecaceae	Area catechu	AP	L	Caruso et al. 2013
	Arecales	Arecaceae	Phoenix dactylifera	NA	L	hoseinfar 2015
	Asparagales	Amaryllidaceae	Allium sativum	AB, AP,	W	Sahu et al. 2007; Aly et al. 2008;
				IM, GP		Nya and Austin, 2009; Militz et al.
						2013a,b
	Asparagales	Amaryllidaceae	Allium tuberosum	AB, GP	L	Rattanachaikunsopon and
						Phumkhachorn, 2009a
	Asparagales	Xanthorrhoeacea	Aloe vera	AB	W	Kim et al. 1999; Alishani et al. 2010
	Asterales	Asteraceae	Ageratum conzyoides	NA	L	Caruso et al. 2013
	Asterales	Asteraceae	Artemisia annua	AP	L	Martins et al. 2002
	Asterales	Asteraceae	Artemisia argyi	AB, AF,	NA	Ekanem and Brisibe (2010)
				AP
	Asterales	Asteraceae	Artemisia capillaries	AB, GP	W	Ji et al. 2007b
	Asterales	Asteraceae	Artemisia cina	AB, GP	W	Abdelhadi et al. 2010
	Asterales	Asteraceae	Artemisia vulgaris	AB, AV,	NA	El-Deen and Mohamed, 2009
				AP
	Asterales	Asteraceae	Aucklandia lappa	AV	NA	Xue-Gang et al. 2013
	Asterales	Asteraceae	Austroeupatorium inulifolium	NA	NA	Caruso et al. 2013
	Asterales	Asteraceae	Chamaemelum nobile	AB	NA	Syahidah et al; 2015
	Asterales	Asteraceae	Chrysanthemum	AP, IM,	NA	Harikrishnan et al. 2010d
			cinerariaefolium	GP
	Asterales	Asteraceae	Cosmos caudatus	AB	NA	Caruso et al. 2013, Syahidah et al.
						2015
	Asterales	Asteraceae	Echinacea purpura	AB, AP	L, F	Aly and Mohamed, 2010; Caruana
						et al. 2012
	Asterales	Asteraceae	Eclipta alba	AB, GP	L,S	Christytbapita et al. 2007
	Asterales	Asteraceae	Erechtites valerianifolia	NA	NA	Caruso et al. 2013
	Asterales	Asteraceae	Eupatorium fortunei	AP	S	Huang et al. 2013
	Asterales	Asteraceae	Gynura procumbens	NA	NA	Caruso et al. 2013
	Asterales	Asteraceae	Lactuca indica	AB, IM	NA	Harikrishnan et al. 2011c
	Asterales	Asteraceae	Matricaria chamomilla	AB, GP	NA	Abdelhadi et al. 2010
	Asterales	Asteraceae	Mikania scandens	NA	NA	Caruso et al. 2013
	Asterales	Asteraceae	Siegesbeckia glabrescens	AB, IM	W	Harikrishnan et al. 2012b
	Asterales	Asteraceae	Spagneticola calendulacea	AB	NA	Direkbysarakom et al. 2004
	Asterales	Asteraceae	Tagetes erecta	AB, AV	L	Caruso et al. 2013
	Asterales	Asteraceae	Tithonia diversifolia	NA	NA	Caruso et al. 2013
	Asterales	Asteraceae	Tridax procumbens	AV, GP	W	Sivasankar et al. 2015
	Asterales	Asteraceae	Vernonia amygdaluna	AF	NA	Syahidah et al; 2015
	Brassicales	Caricaceae	Carica papaya	AP, GP	NA	Ekanem et al. 2004
	Brassicales	Moringaceae	Moringa oleifera	NA	W	Caruso et al. 2013

GP : growth promoter. R : root, L : leaves, W : whole plant, Fr : fruit, S : seeds, F : flowers.

Parasite specialisation: Parasites adapt themselves to maximise their fitness in one or few closely related host species.

Generalist parasites: They can parasitize a wide range of hosts.

Chemotaxis: movement of an organism in response to a chemical stimulus.

Chemokinesis: Motile response of an organism to chemicals that induce a change in their migratory/swimming behaviour.

Scientific context and model species

Scientific context and objectives Part II

Table 2. In vitro anti-bacterial studies of several plant extracts and herbal mixture extracts. * plants are from marine origin (seaweeds or macroalgae).

This work was supported by the Labex CORAIL and the French Ministry of Overseas. I would like to thank Tim Littlewood (Head of the Life Sciences Department from the Natural History Museum of London), Yves Desdevises solve any material shortcut, Franck Lerouvreur for his field assistance and Christine Sidobre for her laboratory assistance. I would like to thank all the partners working on the Platax orbicularis project in French Polynesia, the Department of Mining and Marine Ressources (DRRM) of French Polynesia including Georges Remoissenet, Rarahu David and Moana Maamaatuaiahutapu.

CHAPTER IV Characterisation of the gill mucus microbiome of four species of butterflyfish: a bacterial diversity reservoir in coral reefs

Reverter, M., Sasal, P., Tapissier-Bontemps, N., Fagervold, S., Lecchini, D., Suzuki, M. Characterisation of the gill mucus microbiome of four species of butterflyfish: a bacterial diversity reservoir in coral reefs. To be submitted to FEMS Microbial Ecology (IF = 3.5).

Characterisation of the gill mucus microbiome of four species of butterflyfish: a bacterial diversity reservoir in coral reefs.

Reverter, M. 1,2 , Sasal, P. 1,2 , Tapissier-Bontemps, N. 1,2 , Fagervold, S. 3 , Lecchini, D. 1,2 , Suzuki, M. 3

CHAPTER V

Metabolomics reveals coral reef fish history traits Reverter, M., Sasal, P., Banaigs, B., Lecchini, D., Lecellier, G.

Part 2

Fish-parasite interactions in aquaculture: Orbicular batfish -bacterial interactions on water to treat infectious stages. However, natural products are not necessarily present in the marine environment and thus their release in the environment (through bath treatment) can have the same side effects than current synthetic drugs (resistance development or side-effects on surrounding wildlife). Hence, a better approach would be to focus on the immunostimulant and anthelminthic properties of these compounds when administered orally (with food) to fish in order to avoid monogenean infection through a reenforced protection of fish against pathogens like the study conducted by Militz et al. (2013 a,b).

Based on this review, it is clear that the use of plant or algae extract can open promising perspectives in terms of parasite treatment in aquaculture. However, it is necessary to carry out tests under homogeneous conditions with controlled amount of identified active molecules along with negative controls for a real estimation of the effectiveness of the tested product.

Finally, it seems important not only to determinate the molecules involved in the activity against pathogens but also to establish the induced response on the fish physiology.

Quantifying the response in fish can be achieved by measuring hematological indices, but also by analyzing the expression of genes directly or indirectly involved in the physiological response like innate immunity genes.

CHAPTER VIII

Antibacterial activity of local Polynesian plants and algae against fish pathogenic bacteria

CHAPTER IX

Effects of local Polynesian plants and algae on growth and expression of two immune-related genes in orbicular batfish (Platax orbicularis)

Reverter, M., Saulnier D, David R, Bardon-Albaret A, Belliard C, Bontemps N, Lecchini D, Sasal P. Effects of local Polynesian plants and algae on growth and expression of two immunerelated genes in orbicular batfish (Platax orbicularis). Fish and Shellfish immunology, in review.

CHAPTER X

Effects of local Polynesian plants and algae against Tenacibaculum maritimum infections in orbicular batfish (Platax orbicularis)

Effects of local Polynesian plants and algae against Tenacibaculum maritimum infections in orbicular batfish (Platax orbicularis)

Abstract

The emerging Platax orbicularis aquaculture in French Polynesia is threatened by severe mortality episodes of fingerlings due to tenacibaculosis outbreaks. Tenacibaculosis is often treated by antibiotic bath treatments, which are expensive and induce considerable amounts of stress to fish, heightening the disease severity. Since tenacibaculosis often appears after a stress shock, its management should be focused on techniques that maximize fish fitness.

Medicinal plants are reported to increase fish fitness by stimulating their growth, enhancing immunity and reducing stress. In this study we have investigated the potential of local Polynesian plants (Allium sativum, Calophyllum Inophyllum, Curcuma longa, Morinda taxifolia and Zingiber officinalis) and algae (Asparagopsis taxiformis) against T. maritimum infections in P. orbicularis fingerlings. We have fed P. orbicularis fingerlings in enriched diets with the plants or algae for a period of two or three weeks. After the feeding period the fish were bath challenged with T. maritimum and cumulative mortality was recorded for a week. Although we encountered several technical problems, mainly on the repeatability of our experiments, the fish fed with an enriched diet in garlic had a significant higher survivability than control fish.

Keywords:

Tenacibaculum maritimum challenge, medicinal plants, Platax orbicularis.

Conclusions and perspectives

ANNEXES

-330 - (Davis et al. 2002;Iguchi et al. 2003;Ashley et al. 2007). Medicinal plants, can therefore provide a cheaper and more sustainable alternative to chemotherapy in aquaculture, since they have been reported

to display numerous bioactivities such as anti-stress, immunostimulant and anti-parasitic (bacterial, fungus, virus and ectoparasites) (Reverter et al. 2014).

Use of medicinal plants in aquaculture

Although interest in the use of medicinal plants and plant extracts in aquaculture has exploded recently, medicinal plants have long been used by some rural fish farmers. Caruso et al. (2013) found that 46% of the fish farmers surveyed in West Java (Indonesia) used plants in their farms, most of which were also traditionally used in human pharmacopeia. In most cases, fresh plants were directly introduced into the rearing water and used to 1) improve water quality, 2) reduce fish stress, 3) increase fish resistance to pathogens and 4) treat fish diseases. Their study showed that fish farmers used plants and doses depending on their personal experience and their ethnic origin.

Numerous scientific studies endorse the use of medicinal plants in aquaculture (e.g. Bhuvansewari and Balasundaram, 2006;Harikrishnan et al. 2011a). Plants have been reported to promote various activities like anti-stress, growth promotion, appetite stimulation, immunostimulation, aphrodisiac and to have anti-pathogen properties in fish and shrimp aquaculture due to highly varied active principles such as alkaloids, terpenoids, tannins, saponins or flavonoids [START_REF] Citarasu | Herbal biomedicines: a new opportunity for aquaculture industry[END_REF]Chakraborty and Hancz, 2011).

Medicinal plants biological activities in aquaculture

Several plant extracts are reported to stimulate appetite and promote weight gain when they are administered to cultured fish (Pavaraj et al. 2011;[START_REF] Takaoka | Effect of rotifer enrichment with herbal extracts on growth and resistance of red sea bream, Pagrus major (Temminck & Schlegel) larvae against Vibrio anguillarum[END_REF]. For example, grouper

Epinephelus tauvina fed with a supplemented diet of a mixture of methanolic herb extracts (Cynodon dactylon, Piper long, Phyllanthus niruri, Tridax procumbens and Zingiber officinalis) displayed 41%

higher weight than fish fed with the control (Punitha et al. 2008). Some other studies have reported that administration of plant extracts improves digestibility and availability of nutrients, resulting in an increase in feed conversion and leading to higher protein synthesis (Putra et al. 2013;Talpur et al. 2013).

Numerous studies showed that fish treated with medicinal plants presented enhanced immune parameters (Dügenci et al. 2003;Yuan et al. 2007). For example, Nile tilapia (Oreochromis niloticus) fed with a diet containing mistletoe (Viscum album coloratum) for a period of 80 days displayed higher lysozyme activity, respiratory burst activity, alternative complement activity and phagocytic activity, which resulted in 42% of increased survivability when they were challenged with the bacterial pathogen Aeromonas hydrophila (Park and Choi, 2012). Studies on fish hematological parameters showed that fish treated with plants displayed higher levels of erythrocytes, lymphocytes, monocytes and hemoglobin than control fish, indicating better fish fitness (Harikrishnan et al. 2012a;[START_REF] Haghighi | The effects of powdered ginger (Zingiber officinale) on the haematological and immunological parameters of rainbow trout Oncorhynchus mykiss[END_REF].

Fabales

The Fabales include the family Fabaceae, commonly known as pea or bean family. The Fabaceae is the third-largest plant family as it includes more than 12.000 plants (Allaby, 2006). Several plants from the family Fabaceae are used in traditional medicine for treatments against diarrhea, parasite infections, inflammations, rheumatism and ulcers [START_REF] Molares | The Usefulness of Edible and Medicinal Fabaceae in Argentine and Chilean Patagonia: Environmental Availability and Other Sources of Supply, The Usefulness of Edible and Medicinal Fabaceae in Argentine and Chilean Patagonia: Environmental Availability and Other Sources of Supply. Evidence-Based Complementary and Alternative Medicine, Evidence-Based Complementary and Alternative Medicine[END_REF]

Asterales

Asterales is an order of dicotyledonous flowering plants that includes the large family Asteraceae, commonly known as the daisy family. The Asteraceae family contains more than 23.000 species and most members are herbaceous, but a significant number are also shrubs, vines, or trees (Allaby, 2006).

Many artemisia species are present in the Chinese pharmacopeia, and they have been used as scholeretic, anti-inflammatory and diuretic agents in the treatment of diseases like epidemic hepatitis and fevers [START_REF] Tang | Chinese drugs of plant origin[END_REF]). The use of Artemisia annua in Chinese traditional medicine was recorded before 168 BC, and in 1971 artemisinin, a sesquiterpene lactone with antimalarial properties was isolated [START_REF] Klayman | Qinghaosu (artemisinin): an antimalarial drug from China[END_REF]Cávar et al. 2012). Artemisinin and its semi-synthetic derivatives are currently used worldwide for the treatment against the malaria parasite Plasmodium falciparum (WHO, 2006) (Figure 3). Artemisia plants also contain abundant monoterpenes (eucalyptol, germacrenes, camphor), coumarins (scopoletin) chlorogenic acids (quinic acid, caffeic acid) and flavonoids (luteolin, isovitexin) [START_REF] Juteau | Antibacterial and antioxidant activities of Artemisia annua essential oil[END_REF][START_REF] Efferth | Cytotoxic activity of secondary metabolites derived from Artemisia annua L. towards cancer cells in comparison to its designated active constituent artemisinin[END_REF][START_REF] Carbonara | Phytochemical analysis of a herbal tea from Artemisia annua L[END_REF], Cávar et al. 2012) (Figure 3). Aucklandia lappa is another plant from the Asteraceae family with numerous bioactivities and widely used in traditional medicines for the treatment of asthma, rheumatism, coughs, tuberculosis, and many other diseases. [START_REF] Zhang | Pharmacokinetic study on costunolide and dehydrocostuslactone after oral administration of traditional medicine Aucklandia lappa Decne. by LC/MS/MS[END_REF][START_REF] Seo | Simultaneous determination of three sesquiterpene lactones in Aucklandia lappa Decne by high-performance liquid chromatography[END_REF]. A. lappa contains the sesquiterpenes lactones costunolide, dehydrocostus lactone, and alantolactone which are known to display multiple bioactivities [START_REF] Li | Preparative isolation and purification of costunolide and dehydrocostuslactone from Aucklandia lappa Decne by high-speed counter-current chromatography[END_REF][START_REF] Seo | Simultaneous determination of three sesquiterpene lactones in Aucklandia lappa Decne by high-performance liquid chromatography[END_REF]. Xue-Gang et al. (2013) showed that A. lappa displayed antifungal activity against Saprolegnia sp. and Achlya klebsiana. Other Asteraceae plants are shown to display interesting bioactivities for their application in aquaculture. Oral administration of Eclipta alba in tilapia Oreochromis mossambicus enhanced non-specific immune parameters and reduced fish mortality when challenged with A. hydrophila [START_REF] Christybapita | Oral administration of Eclipta alba leaf aqueous extract enhances the non-specific immune responses and disease resistance of Oreochromis mossambicus[END_REF]. Siegesbeckia glabrescens enriched diet also displayed an immunomodulatory effect and increase disease resistance in kelp grouper (Epinephelus bruneus) (Harikrishnan et al. 2012b). Artemisia cina, Matricaria chamomilla and Tridax procumbens promoted weight gain and enchanced non-specific immune responses in fish (Abdelhadi et al. 2010;[START_REF] Sivasankar | A review on plants and herbal extracts against viral diseases in aquaculture[END_REF]. Echinacea purpura appeared to have immunostimulant, antibacterial and antifungal properties in fish (Aly and Mohamed, 2010;Caruana et al. 2012).

Malpighiales

Malpighiales is a very diverse order, containing the families of flowering plants Euphorbiaceae and Phyllanthaceae. Euphorbiaceae, the spurge family, is constituted mainly of herbs but also includes some shrubs and trees. The spurge family contains numerous species of medicinal plants such as different species from the genus Euphorbia or the irritan mangrove Exoecaria agallocha [START_REF] Ernst | Global medicinal uses of Euphorbia L. (Euphorbiaceae)[END_REF]. The study of the natural products of the Euphorbia species started in 1968 with the isolation of a tumourpromoting phorbol-12,13-diester from Croton tiglium L., and the description of the anti-tumour activity of a diterpene isolated from Euphorbia esula L. in 1976 [START_REF] Hecker | Cocarcinogenic Principles from the Seed Oil of Croton tiglium and from Other Euphorbiaceae[END_REF][START_REF] Kupchan | Antileukemic principles isolated from euphorbiaceae plants[END_REF]. More recently, a diterpene drug (ingenol mebutate, Picato®) isolated from Euphorbia peplus was released for the treatment of actinic keratosis [START_REF] Berman | New developments in the treatment of actinic keratosis: focus on ingenol mebutate gel[END_REF]. Euphorbia sp. contains several terpenes, such as the ingoltype diterpens euphorantins, flavonoids like quercitrin and tannins (Qi et al. 2014, Trinh andLe, 2014) (Figure 3). Different species of Euphorbia have displayed antibacterial, antiviral, antifungal and antiparastic activities against fish and shellfish pathogens (Direkbusarakom, 2004;[START_REF] Zhang | Pharmacokinetic study on costunolide and dehydrocostuslactone after oral administration of traditional medicine Aucklandia lappa Decne. by LC/MS/MS[END_REF]Huang et al. 2015). The Phyllanthaceae family is a small (about 2000 species) but diverse family, which includes the genus Phyllanthus. Phyllanthus plants are well known by their potent medicinal activities and are used worldwide [START_REF] Kumar | Rapid qualitative and quantitative analysis of bioactive compounds from Phyllanthus amarus using LC/MS/MS techniques[END_REF]. Numerous bioactive phytochemicals have been isolated from Phyllanthus plants including phyllanthin, phyltetralin, phyllangin, corilangin, gallic acid, methylgallate, and rhamnocitrin, protocatechuic acid, kaempferol 3-O-rutinoside, quercitrin, and rutin [START_REF] Wei | Two new compounds from Phyllanthus niruri[END_REF][START_REF] Fang | Anti-oxidant and inflammatory mediator's growth inhibitory effects of compounds isolated from Phyllanthus urinaria[END_REF][START_REF] Kumar | Rapid qualitative and quantitative analysis of bioactive compounds from Phyllanthus amarus using LC/MS/MS techniques[END_REF]. Phyllanthus have proved to possess a high antibacterial and antiviral potential against pathogens in aquaculture ( Balasubramanian et al. 2007;Punitha et al. 2008;[START_REF] Sivasankar | A review on plants and herbal extracts against viral diseases in aquaculture[END_REF]. Mots-clés : interactions hôte-parasite, poissons coralliens, spécificité parasitaire, mucus de poisson, contrôle parasitaire, plantes médicinales.

Title: Host-parasite interactions in coral reef fish. Abstract : Fish parasites are an important part of ecosystems, however, in certain cases such as in aquaculture they can cause severe disease outbreaks. In this thesis I have studied host-parasite interactions in coral reef fishes, both in the natural and culture environments. I have studied the distribution of 13 dactylogyrid species from 34 butterflyfishes in the Indo West-Pacific. Composition of dactylogyrid communities was host specific and together with the biogeography results, where a turnover in the main Haliotrema species was observed, suggest that parasite specificity might result from host-parasite coevolution derived from past biogeographical episodes. Only one butterflyfish species, Chaetodon lunulatus, was never found parasitized by gill monogeneans. I have studied the butterflyfish mucus, which is the first barrier against pathogens, to investigate the C. lunulatus factors that might be related to the monogenean absence. Butterflyfish gill microbiome and metabolome revealed a high diversity. C. lunulatus presented a significantly higher abundance of Fusobacteria which was correlated to a higher expression of potentially derived β-hemoglobin peptides. Synthesis and evaluation of the peptide antiparasitic activities are being performed in the laboratory. I have also studied the use of medicinal plants as an alternative to chemotherapy in fish aquaculture. I investigated the antibacterial and immunostimulant activities of several local Polynesian plants and algae, and I found that Asparagopsis taxiformis displayed a potent antibacterial activity against Vibrio harveyi and Tenacibaculum maritmum and increased expression of two immune-related genes in Platax orbicularis.

Keywords : host-parasite interactions, coral reef fish, parasite specificity, parasite evasion, fish mucus, parasite control, medicinal plants.