Keywords: Ecological importance of brain lateralization in larvae facing predation, Effect of chemical pollution on the visual lateralization of A, triostegus larvae

The persistence and sustainability of coral-reef fish populations depends on the continued larval recruitment, i.e. successful settlement by pelagic larvae into adult reef habitats and post-settlement survival through metamorphosis to a juvenile stage. There is growing evidence that changes to water conditions due to global change and waterborne pollution can impair coral-reef fish sensory abilities to locate settlement habitats that maximize growth while minimizing mortality risk. However, the inner mechanisms of such impairments remain less well studied. In this thesis, I have examined the recruitment phase of the convict surgeonfish Acanthurus triostegus, and determined that the ecological, morphological, physiological, and behavioral changes occurring at recruitment correspond to a metamorphosis mediated by thyroid hormones (TH). Then, I investigated whether this metamorphosis is prone to endocrine disruption under anthropogenic disturbances such as elevated sea water temperature and agricultural pesticide pollution. I demonstrated that such pressures can reduce TH levels at a critical developmental stage in coral-reef fishes, impairing their metamorphic processes such as intestine remodeling, sensory organ maturation, and sensory abilities acquisition, further increasing their mortality rates. Overall, this thesis is a holistic analysis that addresses molecular, histological, anatomical, and behavioral assays of multiple stressors affecting coral-reef fish recruitment. It indicates the importance of a proper endocrine function during coral-reef fish recruitment, highlighting the need for a better understanding of these processes for coral-reef conservation.

Exp. 1: Do A. triostegus larvae exhibit a left-right visual preference when inspecting either negative (predator or positive (mirror self image, conspecific) stimuli? ........

Résumé (FR)

Le maintien et le renouvellement des populations de poissons coralliens dépendent en grande partie du recrutement larvaire, c'est-à-dire de l'installation des larves pélagiques dans les habitats récifaux adultes, et de leur survie après s'être métamorphosées en juvéniles. De plus en plus d'études révèlent que les changements de composition de l'eau, causés par le changement climatique et la pollution, peuvent altérer les capacités sensorielles des poissons coralliens, diminuant leurs aptitudes à localiser des habitats propices (maximisant leur croissance et diminuant leur mortalité) lors de l'installation. Cependant, les mécanismes internes à l'origine de ces phénomènes sont méconnus. Lors de cette thèse, j'ai examiné le recrutement larvaire du poisson chirurgien bagnard Acanthurus triostegus et mis en évidence que les changements écologiques, morphologiques, physiologiques et comportementaux qui s'y déroulent correspondent à une métamorphose contrôlée par les hormones thyroïdiennes (HT). J'ai ensuite analysé comment des stress d'origine anthropique, tels que l'élévation des températures de surface et la pollution par un pesticide d'origine agricole, peuvent perturber sa métamorphose. Lors de cette étape clé de leur cycle de vie, ces perturbations diminuent les taux d'HT, altérant la maturation de leurs organes sensoriels, leurs capacités sensorielles, et augmentant leur mortalité. Cette thèse est donc une analyse holistique de l'impact des perturbations anthropiques sur les processus moléculaires, et les changements histologiques, anatomiques et comportementaux du recrutement larvaire des poissons coralliens. Elle souligne l'importance du système thyroïdien, et invite à une meilleure compréhension des processus endocriniens du recrutement larvaire, dans l'optique d'une amélioration de la conservation des récifs coralliens.

I would like to thank Pr. Stuart Sandin (Scripps Institution of Oceanography, University of California San Diego, USA) and Pr. Stephen Swearer (School of Biosciences, Faculty of Science, University of Melbourne, Australia) for accepting to review my PhD manuscript and to be part of my thesis committee jury. I would also like to thank Pr. Jennifer Caselle (Marine Science Institute, University of California Santa Barbara, USA), Pr. Jodie Rummer (ARC Centre of Excellence for Coral reef Studies, James Cook University, Australia), and Pr. Serge Planes (CRIOBE, USR3278 EPHE-CNRS-UPVD, PSL Research University, Moorea, French Polynesia) for accepting to be also part of my thesis committee jury.

Je remercie mes deux directeurs de thèse, David Lecchini et Vincent Laudet, de m'avoir accueilli dans leurs équipes respectives, de m'avoir guidé, appris, et de m'avoir fait confiance tout au long de ces trois ans. Merci David pour ton encadrement, pour ton soutien infaillible dans tous les moments de cette thèse, pour les nombreuses collaborations dans lesquelles tu m'as inclus, mais surtout pour ton aide sur le terrain à toute heure de la nuit, avec les arrivées mémorables de larves sur le beach rock, les 'petits dejs' sur la route du retour, avec les glacières pleines de larves de poissons dans la benne du pick-up, et les moments moins glorieux en plein océan, de nuit, dans l'eau, avec 2000 mètres de profondeur sous les pieds, à chercher des larves transparentes dans ce noir infini, avec pour seul outil une lampe torche, et un couteau à la cheville, sans doute pour se donner du courage et une illusion de sécurité… Ces bons souvenirs resteront gravés très longtemps. Merci également Vincent pour ton encadrement, ton accueil chaleureux que ce soit à Lyon ou à Banyuls, pour ton soutien dans certains moments difficiles, qu'ils soient professionnels ou plus personnels. Merci pour ce brillant projet sur la métamorphose des poissons coralliens auquel j'ai eu la chance d'apporter ma contribution. Je me souviens encore de notre discussion, alors que je n'étais encore qu'en Master 1 en stage dans ton laboratoire, et où tu dessinais déjà les premières hypothèses sur cette étape relativement méconnue du cycle de vie des poissons coralliens. Je n'imaginais pas alors que je ferai partie de ce projet quelques années plus tard.

Je remercie également Frédéric Flamant et Jean-Olivier Irisson pour leur grande disponibilité et leurs précieux conseils, notamment lors de mes deux comités de thèse.

Je remercie très chaleureusement toutes les différentes personnes que j'ai eu la chance de côtoyer tout au long de ma thèse, et dans les différents laboratoires où j'ai travaillé, de Moorea à Perpignan et de Lyon à Banyuls-sur-mer. Merci Serge pour ton accueil au sein du CRIOBE. Un énorme merci à toi Nathalie, toujours là pour répondre à mes incessantes demandes, commandes, questions… je ne suis pas certain qu'une thèse soit réalisable en 3 ans s'il n'y a pas des gens comme toi pour faire avancer les choses pour tout ce qui accompagne le travail de recherche. Je tiens également à remercier Aurélie, Peter, Océane, Fabien, Morgan et Guillaume pour l'aide apportée durant ces trois ans. A Perpignan je remercie également Frédéric, pour le squat du bureau, l'aide pour les enseignements et pour nos papiers en commun, mais aussi Louis (M. Loulou plus exactement, à prononcer avec l'accent belge), Bruno, Carole, Mélodie, Miriam, Jean-Pierre, et Jeanine. Je me tourne maintenant vers Moorea, car bien qu'inscrit à Perpignan c'est bien sur ce petit bout de terre au milieu du Pacifique que j'ai passé le plus de temps durant cette thèse… et je ne peux du coup pas commencer sans remercier Elina pour son accueil chaleureux à chacune de mes arrivées, à chacun de mes retours. C'est toujours un grand plaisir de revenir, et j'ai de la peine à penser que le prochain sejour sera peut-être un des derniers. Merci également à Benoit pour son aide sur mes nombreux protocoles, merci à Pascal pour les innombrables coups de mains et réparations qui sauvent certaines manips, et merci à Franck pour les formidables anecdotes et son aide sur le terrain. Ce fut un plaisir et une chance de pouvoir compter sur vous pour la mise en place du petit laboratoire où j'ai passé de longues heures durant ma thèse. Merci Gilles également pour ton aide sur de nombreuses manips (acidification notamment) et ton soutien technique et amical (les bons plans plongée et voyage !) tout au long de mes séjours à Moorea. Merci Cécile pour ton aide sur le terrain, au large, au filet de crête, à Temae, sur le thonier et aux anémones ! Merci René pour les bons barbecues, les fruits de la passion, et les discussions du milieu de la nuit, quand moi je rentre du terrain et que toi tu commences ta journée. Mais je tiens également à remercier Simon, Yann, Laurent, Jérémie, Xavier, Antoine, Lauric, Pierpaolo, Yannick, Ricardo, Suzie, Pierre et Françoise (merci pour le bon maa'a) pour le CRIOBE, et les deux Benoits, Moana et Heifara de Vairao. Et que dire des nombreux stagiaires qui m'ont épaulé durant ces fastidieuses sorties de nuit à la pêche aux larves, je crois que je ne vous remercierai jamais assez… Un GRAND merci donc à Camille (le meilleur pêcheur, après moi évidemment…), à Isadora (our record of 296 maninis caught in one night is still unbeaten!), à Loïc (présent jusqu'au bout !), à Anastasha (thank you for the long time that you've spent picturing maninis for me, revealing parts of their mysteries !), à Daan et Peter (fieldwork guys! Thank you for the fishing and surf lessons!) et enfin à Adam (le plus dévoué ! bien que pas mon stagiaire je lui dois beaucoup). A Moorea je tiens également à remercier Francis, pour son accueil, sa sympathie, sa gentillesse, le charme de sa pension et les matchs de foot sur grand écran, à des horaires souvent improbables, mais aussi merci à Damien, Alexis, Céline, Bruno et François pour ces bons moments sur Moorea. Je remercie également Matthieu et Alexia pour leur aide au MEB de Tahiti. Je tiens également à remercier tout particulièrement l'ensemble de l'équipe de Te mana o te moana, vous êtes pour moi comme une petite seconde famille, et j'ai un attachement profond pour vous depuis que je vous ai rencontrés lors de ma césure. Merci donc à Vie, Matthieu, Magalie et Cécile, sans vous il est certain que ma thèse n'aurait pas été la même. Et enfin sache que non je ne t'ai pas oublié, je t'ai juste gardé pour la fin (on garde souvent le meilleur pour la fin !), merci à toi Vetea ! J'ai de la chance de te compter parmi mes amis, et tu me manques beaucoup depuis mon départ de Polynésie. Je pense que ces années là-bas n'auraient pas eu la même saveur sans ces supers moments avec toi, à la pêche, à la découpe, à la cuisine, à la cuisson, toujours dans le thème du poisson. Prépare le kayak, la pagaie (la noire !), et ton souffle, j'arrive bientôt. Du côté de la France et plus particulièrement de Lyon et de l'IGFL, je tiens à remercier tout particulièrement Guillaume, ce fut un plaisir de travailler avec toi sur ce beau projet de la métamorphose des poissons coralliens, à prendre ton relai en quelque sorte. J'espère que notre travail acharné portera bientôt ses premiers fruits. Mais je remercie également Anne pour tout ce qu'elle m'a appris en matière d'histologie, tu as su me donner et m'apprendre sans compter, et je te dois beaucoup. Merci aussi à Alain pour la bonne humeur qui motive à toute heure au labo, et à Marie, Sophie, Luke, Carine et Coralie, mais aussi à tous les autres membres de l'IGFL, notamment pour les afterworks football ! A Dinard je tiens à remercier Samuel, Antoine, Helène et Dorothée de m'avoir accueilli dans leur laboratoire lors de mes séances rédactionnelles de dernière année. Merci également à Eric et à toute l'équipe du Cresco pour les bons moments passés dans ce bel endroit de Bretagne. Du côté de Banyuls enfin je remercie très chaleureusement toute l'équipe pour son accueil lors de ma dernière année de thèse. Merci à Hector pour l'invitation à tenir un talk à la JAP. Merci à Natacha et Laurence pour leur fous-rires et l'aide pour les protocoles. Merci à Laura et Charles-Hubert pour l'accueil et le squat de leur labo, merci à Valentin (l'homme tronc) et Arthur pour les sessions city-stade après le boulot, merci aussi à toi Pauline pour les discussions manips, science, avenir, et pour ta participation à ma fin de thèse, j'espère que cette collaboration portera bientôt ses fruits. Merci à Hugo pour cette colocation maison/bureau, les calamars frais, les bons repas, les moments télés, et pour ton aide (avec Arthur) sur mes dernières manips. Enfin merci Kévin d'avoir été mon deuxième compère de cette dernière année. Avec le recul je réalise que je n'ai su apprécier pleinement la chance de t'avoir eu à mes côtés durant cette dernière année, la faute sans doute aux rushs des manips et aux weekends incompatibles. En tout cas, que de bons moments sur et en dehors de l'eau avec toi, d'innombrables droites, la découverte d'une gauche encore à tester, la bouteille d'eau chaude avec bouchon sport pour rincer les pieds, les arrivées du Tour, et les pintes lors des soirées matchs. Enfin, merci à Marine et Nanuk pour être des BGs, et pour avoir partagé de nombreux moments avec moi, ici et là-bas ! Tous ces remerciements me font me rendre compte que le sport a pris une part importante de ma thèse, et puisque essentiellement rendu en position assise comme tout bon étudiant en thèse, j'ai ma foi plutôt raccroché les baskets et pratiqué ce que d'aucuns appellent le sport de salon. Merci donc à L'Equipe d'avoir su me faire prendre des pauses et me changer les idées lorsqu'arrivé à saturation d'une rédaction ou d'une manip. Merci également à Martin Fourcade, Michel Bourez, et surtout Alberto Contador, de m'avoir fait rêver par procuration.

Plus sérieusement, je tiens également à remercier ceux avec qui j'ai eu la chance de collaborer, et pour qui la langue anglaise n'a aucun secret. Thank you very much Zoe, Rohan, Jack and Will for your help with science and English language. I'm very lucky to know you, and working with you has been a great opportunity for me. I hope we will continue to correspond and work together in a near future.

Enfin je tiens à remercier mes proches, ceux qui n'ont pas forcément un lien direct avec cette thèse, quoique… Merci à mes amis de longue date, qu'ils soient Calaisiens, Parisiens, Mayennais, ou Lyonnais, tous Stylés et désormais éparpillés aux quatre coins de la France et du monde. Merci à toi Mateusz d'avoir fait une fois le voyage avec moi, merci pour ton soutien, ta sincérité et ces nombreuses discussions auxquelles je réponds malheureusement avec trop peu de constance, j'ai hâte de reprendre une vie « normale » et d'en profiter avec toi. Merci à Bastien et Guillaume, mes deux amis du grand nord, d'être toujours à mes côtés. Je pense aussi très fortement à François, Grégoire, Pierre, Clément, bien que je regrette de ne pouvoir vous voir plus souvent. Merci à Nina, Camille, Marie, Mathilde, Félix, Théophile et Michel. Merci également à Vincent (Raafaa Nadaal) pour ses Intérieur Sport, et sa découverte de nouvelles disciplines. Merci à Hugo et Thibault, d'être là dans tous les moments, dans tous les endroits, vous êtes des amis en or, vous faites partie intégrante de cette thèse et de ma vie. Je ne saurai jamais comment vous remercier pour tout ce vous avez fait pour moi. Je remercie très chaleureusement et avec beaucoup d'amour, l'ensemble de ma famille, présente dans toutes les circonstances, toutes les situations, allant jusqu'à faire de nombreux sacrifices pour me suivre dans mes péripéties de recherche au bout du monde, et m'accueillant toujours à bras ouverts durant les (quelques) weekends et (rares) vacances. Plus particulièrement, merci Marie, Paul, Maman, Papa, pour votre amour, pour votre soutien, pour votre présence. Je vous aime. Enfin je tiens à terminer ces remerciements en dédiant ce manuscrit à celle qui m'accompagne et que j'aime, merci Lise d'être à mes côtés et de m'avoir supporté durant ces trois longues années de thèse. Merci d'avoir été là, dans tous les bons et moins bons moments. Merci pour ta patience, ta force, ton amour, et pour l'incroyable aide que tu as su m'apporter tout au long de ce parcours, que ce soit sur le terrain, en laboratoire, dans la mise en place de ce manuscrit ou tout simplement dans la vie de tous les jours (avec Tiama, que je remercie également). Cet aboutissement est en quelque sorte également le tien.

Table of contents

Figure I.2 | Fishes in coral reefs

A: Blacktail snappers (Lutjanus fulvus) on the foreslope reef of Fakarava Atoll (Tuamotu Archipelago, French Polynesia). B: Hawaiian bigeyes (Priacanthus meeki) on the foreslope reef of Rangiroa Atoll (Tuamotu Archipelago, French Polynesia). C: Yellowfin surgeonfishes (Acanthurus xanthopterus) in the channel pass of Rangiroa Atoll (Tuamotu Archipelago, French Polynesia). D: Convict tangs (Acanthurus triostegus) on patch reefs near the barrier reef of Tetiaroa Atoll (Society Archipelago, French Polynesia). E: Blue-green damselfishes (Chromis viridis) and Whitetail dascyllus (Dascyllus aruanus) in the lagoon of Moorea (Society Archipelago, French Polynesia). F: Blue-green damselfishes (C. viridis) around their Pocillopora shelter in a fringing reef of Moorea Island (Society Archipelago, French Polynesia). Pictures: L. Thiault.

Figure I.3 | Herbivorous fishes grazing on algae in a bleached coral reef

A: Two herbivorous fishes: Chaetodon ephippium (front) and Acanthurus nigricans (back) feeding on turf algae that have recently grown on dead Pocillopora following a bleaching event in Moorea Island (French Polynesia). B: same observation with the line surgeonfish (Acanthurus lineatus). Pictures: L. Hedouin.

Figure I.4 | Ten coral reef fishes from iconic coral reef fish families

A: Acanthurus triostegus (Acanthuridae). B: Myripristis berndti (Holocentridae). C: Chaetodon humeralis (Chaetodontidae). D: Dascyllus trimaculats (Pomacentridae). E: Salarias fasciatus (Blenniidae). F: Apogonichthyoides pseudotaeniatus (Apogonidae). G: Stethojulis bandanensis (Labridae). H: Scarus spinus (Scaridae). I: Mulloidichthys flavolineatus (Mullidae). J: Carangoides ferdau (Carangidae). Adapted from FAO species identification guides (FAO 2001).

Figure I.5 | The convict tang Acanthurus triostegus as a model for teleost coral reef fish characteristics

n.: nostril, e.: eye, s.: scales, l.l.: lateral line, a.: anus, op.: operculum, p.op.: pre-operculum, m.: mouth. d.f.: dorsal fin, c.f.: caudal fin, a.f. : anal fin, pel.f. : pelvic fins, pec.f. : pectoral fin. SL: standard length, BH: body height, SnL: snout length. Picture: M. Besson.

Figure I.6 | Life cycle of the convict tang Acanthurus triostegus

A. triostegus is a pelagic egg spawning species. This life cycle is representative of all coral reef fish species with this spawning mode (around 2/3 of all coral reef fishes). In benthic egg laying species, pelagic dispersal only concerns larvae, as hatching occurs within the reefs. Larval recruitment is different from the 'Recruitment' presented in this figure, and encompasses all the steps from the end of larval dispersal to the survival of juveniles in nursery areas. Pictures: M. Besson.

Figure I.8 | Thyroid hormone levels and development in anurans

Dph: days post hatching. Circular structure on the 'guts remodeling' axis correspond to schematic representation of the intestine cross-sections. Adapted from Brown and Cai (2007) and [START_REF] Hasebe | Thyroid hormone-induced cell-cell interactions are required for the development of adult intestinal stem cells[END_REF].

Figure I.9 | Thyroid hormones and deiodinase enzymes

Schematic representation of the biological role of deiodinase enzymes. T4 and T3 are the two active forms of thyroid hormones (TH) that can bind thyroid hormone receptors (TR). Adapted from Bianco and Kim (2006) and [START_REF] Holzer | Origin of thyroid hormone signalling in metazoans and implications in their metamorphosis[END_REF].

Figure I.10 | Thyroid hormone signaling

Environmental and metabolic cues can either induce or repress the release of TRH in the hypothalamus. TRH binds its receptors on the anterior pituitary, which then releases TSH. TSH binds its receptors on the thyroid, which then secretes TH. TH are transported to target cells where the deiodinase metabolism determines their faith. When T3 reaches TR in the nucleus, this activates the expression of metamorphosis genes (e.g., Klf9 gene, involved in brain development).

Figure I.11 | Changes in TH levels (A) and TR expressions (B) during the development of the Pacific Bluefin tuna (Thunnus orientalis)

Peaks of TH levels and TR expressions are observed around the N and O developmental stages, which correspond to the stage where most changes are observed in the metamorphosing larvae. Adapted from Kawakami (2008a)

Figure I.12 | Changes in TH levels (A) and TR expressions (B) during the development of the African clawed frog (Xenopus laevis) and the Japanese flounder (Paralichtys olivaceus)

TH (dotted lines) levels and TR expressions (solid and dashed lines) peak during metamorphosis of X. laevis and P. olivaceus. Contrarily, TRα-B in P. olivaceus (alternating dashdot line) does not peak. Adapted from [START_REF] Shi | Amphibian Metamorphosis: From Morphology to Molecular Biology[END_REF], [START_REF] Yamano | Differential gene expression of thyroid hormone receptor alpha and beta in fish development[END_REF] and figure originating from Schreiber (2013). normalized expression X-axis: gene and sampling point. Far ocean larvae are split in two groups: Far a (black stripes) and Far b (white). For each panel, the color shading is the same one as in Figure II.1A. For each sampling point 3 biological replicates and 3 technical replicates were performed. Statistics: comparisons of each mean to the Crest condition mean (Student's t cumulative distribution function, which was automatically computed by qPCR software CFX Biorad Manager, CFX Manager 2017). Error bars represent standard errors. *: p-value < 0.05; **: p-value < 0.01; ***: p-value < 0.001. (A) Upper panel: schematization of the external slope experiment with control fish, in the lagoon (grey circle), and relocated fish, on the external slope (orange circle). Lower panel: comparison of the pigmentation between the relocated fish (left) and the control lagoon fish (right) at day 2. (B) T4 and (C) T3 dosage of crest captured larvae and day 2, 5 and 8 juveniles, in control (grey shade) or external slope (orange shade) conditions. n = 4 fish for each experimental condition. X-axis: sampling point, Y-axis: T4 or T3 level in pg.g -1 . Statistics: mean comparisons between control and external slope (Wilcoxon-Mann-Whitney U-test). (D) Expression level of TRα-A, TRα-B, TRβ and Klf9 by qPCR, normalized with pold2 and rpl7 housekeeping genes, in control (grey shade) or external slope (orange shade) conditions. Yaxis: Relative normalized expression. X-axis: gene and stages. For each point 3 biological replicates and 3 technical replicates were performed. Statistics: comparisons of "external slope" means to their "control" counterparts (Student's t cumulative distribution function). (E) Intestine length in cm of fish at crest (C), day 2 (d2) or day 5 (d5) under different treatment conditions: solvent-control (grey shade), T3+IOP treated at 10 -6 M (blue shade), NH3 treated at 10 -6 M (green shade), T3+IOP+NH3 treated at 10 -6 M (blue and green dashed shade) or located to the external slope (orange shade). n > 10 fish for each experimental condition. Statistics: ANOVA followed by Tukey posthoc tests, only significant differences with the respective solvent-control condition are indicated. (F) Histological cross section (5 µm width) of intestine stained by haematoxylin/eosin. Scale bars indicate 200 µm. These pictures are representative of n = 2 microtome section and staining technical replicates, performed on n = 3 fish (biological replicate) per treatment and per developmental stage. Two technical replicates of this experiment were performed. (G) Mean number of bites (on turf algae) per fish in a batch of 18 fish at day 2 (d2) and day 5 (d5) under different treatment conditions: solvent control (DMSO, grey shade), T3+IOP at 10 -6 µM (blue shade), NH3 at 10 -6 µM (green shade) or relocated to the external slope (orange shade). n = 3 to 6 independent bite counts (technical replicates) for each condition. Bite counting was not performed on crest captured individuals as A. triostegus larvae do not feed for at least 12 hours after colonizing the reef at night (MB pers. obs.). Statistics: same as in (E). ). Circles bordered in black represent means while alpha transparent circles in the background represent each data. d2 and d5 = day 2 and day 5 post-settlement, respectively. a, T3 level variations under different temperatures (d2 fish at 28.5°C as standards). A<B (p=0.021) at d2 (n=42, Kruskal-Wallis+Nemenyi), no difference at d5 (p=0.105, n=30, Kruskal-Wallis). b, T4 levels variations under different temperatures (d2 fish at 28.5°C as standards). A<B (p=0.001) at d2 (n=29, Kruskal-Wallis+Nemenyi), no difference at d5 (p=0.074, n=27, Kruskal-Wallis c, bpc density variations under different temperatures. A<B (p=0.034) at d2 (n=20, ANOVA+TukeyHSD) and no difference at d5 (p=0.375, n=20, ANOVA). d, prn density variations under different temperatures. No difference at d2 (p=0.637, n=20, ANOVA) and d5 (p=0.983, n=20, ANOVA). e, pes density variations under different temperatures. No difference at d2 (p=0.291, n=19, ANOVA) and d5 (p=0.177, n=20, Kruskal-Wallis). f, nostril lamellae number variations under different temperatures. No difference at d2 (p=0.609, n=29, Kruskal-Wallis) and d5 (p=0.626, n=20, Kruskal-Wallis). g, lateral line canal number variations under different temperatures. A<B (p=0.025) at d2 (n=29, ANOVA+TukeyHSD) and A<B (p=0.002) at d5 (n=20, ANOVA+TukeyHSD). h, T3 level variations under CPF exposure (d2 fish at 28.5°C as standards). A<B (p=0.039) at d2 (n=47, ANOVA+TukeyHSD), no difference at d5 (p=0.698, n=50, ANOVA). i, T4 levels variations under CPF exposure (d2 fish at 28.5°C as standards). No difference at d2 (p=0.255, n=42, ANOVA) d5 (p=0.578, n=50, ANOVA). j, bpc density variations under CPF exposure. A<B (p=0.049) at d2 (n=50, ANOVA+TukeyHSD) and A<B (p=0.004) at d5 (n=50, ANOVA+TukeyHSD). k, prn density variations under CPF exposure. No difference at d2 (p=0.993, n=50, ANOVA) and d5 (p=0.436, n=50, Kruskal-Wallis). l, pes density variations under CPF exposure. No difference at d2 (p=0.978, n=50, ANOVA) and A<B (p=0.022) at d5 (n=50, Kruskal-Wallis+Nemenyi). m, nostril lamellae number variations under CPF exposure. No difference at d2 (p=0.669, n=50, Kruskal-Wallis) and A<B (p=0.018) at d5 (n=49, Kruskal-Wallis+Nemenyi). n, lateral line canal number variations under CPF exposure. a, Design of the two-channel Atema choice flume apparatus used for olfactory preferences assessment in this study. Total length: 25 cm (laminating channel area = 15 cm, choice arena = 5 cm and drained exit area = 5 cm). Width: 5 cm. b, Olfactory preferences and TH signaling when facing olfactory cues from control seawater (Ø) vs seawater with predator Lutjanus fulvus odor (P). d2 and d5 = day 2 and day 5 post-settlement, respectively. *** indicates p<0.001 (n = 8 to 13 per treatment and per cue, Wilcoxon-Mann-Whitney). c, Design of the choice tank apparatus used for visual preference assessment in this study. Total length: 60 cm (release area = 30 cm, each choice area = 15 cm). Width: 15 cm. The tank is equipped with opaque walls expect for the two opposite walls where visual stimuli are presented. d, Impact of TH signaling, water temperature and CPF exposure (results in september) on visual preferences when facing visual cues from a 'fish-empty' tank (Ø) vs tank containing a L. fulvus live predator (P). ** indicates p<0.01 and *** indicates p<0.001 (n = 10 to 20 per treatment and per cue, Wilcoxon-Mann-Whitney). e, Design of the survival arena apparatus for measuring the impact of TH signaling on A. triostegus survival rates at recruitment when facing direct predation by L. fulvus. Size: 1mx1mx1m. f, Relative survival and TH signaling. A<B (p<0.001), B<C (p<0.001), C<D (p<0.001) at d2 (n=20, ANOVA+TukeyHSD) and A<B (p<0.001), B<C (p=0.006) at day 5 (n=20, ANOVA+TukeyHSD). (Methods). g, Similar experiment as in f, has yet to be performed. b,d,f, Error bars indicate standard errors (error bars are not visible when standard errors are too small). Circles bordered in black represent means while alpha transparent circles in the background represent each data. (Methods)

III. Extended Data Fig 1 | Body depth variation along recruitment in Acanthurus triostegus

Body depth was assessed from day 0 (d0) to day 30 (d30) post-settlement. Body depth was calculated as follows: Body Height / Standard length. Error bars indicate standard errors (error bars are not visible when standard errors are too small). Circles bordered in black represent means while alpha transparent circles in the background represent each data. A<B (p<0.001) and B<C (p<0.001) (n=320, Kruskal-Wallis+Nemenyi).

III. Extended Data Fig 2 | Fulton's K condition factor variation along recruitment in Acanthurus triostegus

Fulton's K condition factor was assessed from day 0 (d0) to day 30 (d30) post-settlement. Fulton's K condition factor was calculated as follows: K = 100*(W/SL)^3 (with SL, in cm, and W, in g) (Fulton 1904) Fulton's K condition factor was calculated as follows: K = 100*(W/SL)^3 (with SL, in cm, and W, in g) (Fulton 1904). Error bars indicate standard errors (error bars are not visible when standard errors are too small). Circles bordered in black represent means while alpha transparent circles in the background represent each data. At day 2 (d2) post-settlement, neither TH (p=0.976, n=62, ANOVA), temperature (p=0.849, n=129, Kruskal-Wallis), nor CPF (p=0.529, n=75, Kruskal-Wallis) affect Fulton's K condition factor in A. triostegus. Same observations were made at day 5 (d5) post-settlement: TH (p=0.715, n=92, Kruskal-Wallis); Temp (p=0.733,n=135,and CPF (p=1,n=76,ANOVA). The exact same patterns have been observed for standard length, weight, height, and body depth. (Methods).

Fig. IV.1 | Schematic representation of the test apparatus for eye lateralization determination

The three dark gray walls represent the opaque wall of the aquarium, while the light blue wall corresponds to the transparent wall where the visual stimuli are presented. No eye preference was recorded when the fish was perpendicular to the transparent wall (i.e. binocular stimulation) or when it formed an angle larger than 180° with respect to the transparent wall (i.e. fish looking in the opposite direction).

Fig. IV.2 | Left-right eye preference of A. triostegus larvae during the inspection of visual stimuli in an adjacent aquarium

Fig. IV.3 | Ecological importance of brain lateralization in larval survival facing direct predation

Survival index (SI) was calculated as follow: survival rate of the group minus the overall survival rate in the in situ cage. ). The reproductive strategies, formation and evolution processes, economical benefits and values of coral reef ecosystems have intrigued and enthralled geologists, biologists, and even economists, and their mysteries will probably never be completely elucidated (Duce et al. 2016;Rapuano et al. 2016;[START_REF] Brumbaugh | Protecting Million Dollar Reefs is Key to Sustaining Global Tourism[END_REF]). These iconic tropical reef systems have been present since the Ordovician (around 485 to 444 million years ago) (Adachi et al. 2011) and today they constitute one of the most biologically diverse, productive, and economically important ecosystems worldwide (Odum and Odum 1955;Connell et al. 1978;Moberg and Folke 1999;[START_REF] Brumbaugh | Protecting Million Dollar Reefs is Key to Sustaining Global Tourism[END_REF]. Tropical coral reef edifices dominate many shallowwater environments and harbor more than a third of the ocean's biodiversity while only covering 0.1 to 0.5 % of the ocean floor (Copper 1994;[START_REF] Spalding | New estimates of global and regional coral reef areas[END_REF]McCulloch et al. 2017). With more than 100 countries having coastlines with coral reefs, these ecosystems supply half a billion of people with goods and services such as food, coastal protection from storm and erosion, new medicines, tourism and cultural benefits (Salvat 1992;Jameson et al. 1995). The ecosystem services driven by coral reefs have been estimated at $36 billion per year [START_REF] Brumbaugh | Protecting Million Dollar Reefs is Key to Sustaining Global Tourism[END_REF].

Most tropical coral reefs are dominated by scleractinian corals, which generally only grow at depths shallower than 50 meters, and in a zone extending from approximately 30° N to 30° S of the equator (Dubinsky 1990). Scleractinian corals accumulate and deposit calcium carbonate (CaCO3) to build their limestone exoskeleton [START_REF] Zoccola | Bicarbonate transporters in corals point towards a key step in the evolution of cnidarian calcification[END_REF]. This bio-accumulation of calcium carbonate is often referred to as calcification, and is promoted by the mutualist partnership between scleractinian corals and their endosymbiotic and photosynthetic dinoflagellates: Symbiodinium (zooxanthellae) (Pearse and Muscatine 1971;[START_REF] Furla | Sources and mechanisms of inorganic carbon transport for coral calcification and photosynthesis[END_REF][START_REF] Zoccola | Bicarbonate transporters in corals point towards a key step in the evolution of cnidarian calcification[END_REF]. There are three main types of coral reefs: fringing reefs, barrier reefs and atolls, which can all be encountered in French Polynesia (Figure I.1). Scleractinian corals are the foundation species of all these tropical reef types, as they produce the biogenic matrix on which these ecosystems depend for structure, function, and biodiversity [START_REF] Spalding | Early postsettlement predation on three reef fishes: effects on spatial patterns of recruitment[END_REF][START_REF] Alvarez-Filip | Flattening of Caribbean coral reefs: region-wide declines in architectural complexity[END_REF]. The physical structure of coral reefs provides a large diversity of habitats that facilitate species coexistence (e.g., corals, fish, algae, and invertebrates, Figure I.2) through niche partitioning and by providing, for instance, refuges from predators and environmental stressors (Roberts and Ormond 1987;[START_REF] Almany | Does Increased Habitat Complexity Reduce Predation and Competition in Coral Reef Fish Assemblages?[END_REF][START_REF] Gratwicke | The relationship between fish species richness, abundance and habitat complexity in a range of shallow tropical marine habitats[END_REF]. The great diversity of ecological niches provided by coral reefs has been acknowledged as one of the drivers of the teleost fish diversification [START_REF] Alfaro | Do reefs drive diversification in marine teleosts? Evidence from the pufferfish and their allies (Order Tetraodontiformes)[END_REF][START_REF] Frédérich | Iterative ecological radiation and convergence during the evolutionary history of damselfishes (Pomacentridae)[END_REF]).

However, coral reefs are also among the most rapidly changing environments globally. Over the past three decades, they have faced increasingly frequent and severe natural (e.g., cyclones and Acanthaster planci outbreaks) and anthropogenic perturbations (e.g., pollution from agricultural runoff, rising sea-water temperatures, overfishing), leading to global declines in habitat quality, biodiversity and associated ecosystem services (Moberg and Folke 1999, Hughes et al., 2003, Hoegh-Guldberg et al., 2007, Hughes et al., 2014, Belwood et al., 2004, Hughes et al., 2017). Many tropical reefs phase shift from coral-dominated to macroalgal-dominated habitats (Hoegh-Guldberg et al. 2007;Hughes et al. 2017). In this context, algal-herbivore fish are considered keystone species for their ability to maintain or rescue coral-dominated habitats, in particular facing algal invasions that follow bleaching events (Figure I.3) (Hughes et al. 2007;Ledlie et al. 2007). Along with other reef-fish functional groups, they play important roles in maintaining reef habitats, preserving their associated biodiversity, and increasing their resilience (e.g., through the regulation of coral-algal interactions or by maintaining trophic sustainability) (Russ 1984, Hughes et al., 2007). Furthermore, while fisheries yield from coral reefs are estimated to be worth at least US$ 5 billion annually, with dozens of millions of people directly depending on tropical reefs for food and livelihood, most of them are already over-exploited and may have little resilience to additional impacts from global change (Moberg and Folke 1999;Hoegh-Guldberg et al. 2007;Munday et al. 2008) I.1.2. Coral reef fishes Among vertebrates (around 45.000 species), teleost fishes constitute the most biodiverse group (almost 25.000 species). There are at least 4.000 species of fishes inhabiting coral reefs, and, apart from some species of sharks and rays associated with coral reefs, nearly all fishes found in coral reefs are teleost fishes. Coral reef fish hotspot of biodiversity is located in Indonesian coral reefs, with biodiversity decreasing eastward in the Pacific (Sale 2002). [START_REF] Bellwood | The eocene fishes of Monte Bolca: The earliest coral reef fish assemblage[END_REF] established a list of coral reef fish families regarded as "characteristic reef fish families, the essence of a reef fish fauna; all abundant and speciose on coral reefs" [START_REF] Bellwood | The eocene fishes of Monte Bolca: The earliest coral reef fish assemblage[END_REF][START_REF] Bellwood | The history and biogeography of fishes on coral reefs[END_REF]. This family list therefore provides no diagnostic, but covers most species that one would always find on a coral reef irrespective of its biogeographic location: Acanthuridae, Apogonidae, Blennidae, Carangidae, Chaetodontidae, Holocentridae, Labridae, Mullidae, Pomacentridae and Scaridae (Figure I.4).

All teleost coral reef fishes share anatomical similarities, for example, a dorsal fin, a symmetrical caudal fin, an anal fin and two pairs of bilateral fins: pelvic and pectoral fins (Figure I.5). Breathing functions via the circulation of water from the mouth to the gills, which are located in a cavity protected by opercular bones (e.g., preoperculum and operculum, Figure I.5). Some classical morphological measurements can be conducted for studies at the individual or population levels (e.g., standard length -distance between the extremity of mouth and the end of the notochord, body height -maximum height between the bases of the dorsal and anal fins, perpendicular to the standard length, and snout length -distance between the extremity of the mouth and the base of the eye, Figure I.5). These fish also possess several sensory organs such as the eyes for vision, the nostrils for olfaction, the lateral lines for the mechanical perception of water movements (e.g., predator surging or sounds), and otoliths for hearing and orientation (Myrberg and Fuiman 2002). Some fishes also exhibit some external gustatory (chemosensory) organs such as barbels, or have the ability to detect magnetic fields [START_REF] Mccormick | Development and changes at settlement in the barbel structure of the reef fish, Upeneus tragula (Mullidae)[END_REF]O'Connor and Muheim 2017).

In the wide variety of habitats provided by coral reefs, the vertical and horizontal distributions of coral reef fishes depend on several habitat factors such as the amount of algae, plankton, invertebrates and prey available, but also exposure to wave and currents, light levels, and lastly shape and assemblages of coral species [START_REF] Lieske | Coral Reef Fishes -Caribbean, Indian Ocean and Pacific Ocean, including Red Sea[END_REF]. Each coral reef niche exhibits its own set of characteristic species. For example, small and vulnerable planktivores like the blue-green damselfish (Chromis viridis) and the whitetail dascyllus (Dascyllus aruanus) only feed above their coral shelter . Larger planktivores such as the yellowfin surgeonfish (Acanthurus xanthopterus) and fusiliers school in large groups in deeper and nutrientrich waters (e.g., channel passes) or above fore slope reefs, where they can roam large areas (Figure I.2C) [START_REF] Lieske | Coral Reef Fishes -Caribbean, Indian Ocean and Pacific Ocean, including Red Sea[END_REF]. Large grazers, such as the majority of surgeonfishes and parrotfishes, also occur in groups, swimming at a short distance above reefs (Figure I.2D). Higher in the trophic chain, some piscivores can be bottom- dwellers and school, such as blacktail snappers (Lutjanus fulvus) and Hawaiian bigeyes (Priacanthus meeki) (Figure I.2A-B), but others also live solitary, in pairs, or in small groups occurring in mid-waters such as jacks [START_REF] Lieske | Coral Reef Fishes -Caribbean, Indian Ocean and Pacific Ocean, including Red Sea[END_REF]. However, these examples are not universal, as there are almost as many ecological and social interactions in coral reef fishes as there are species. While most coral reef fishes are diurnal, some of them (e.g., soldier-, squirrel-and cardinal-fishes) only emerge from caves and crevices at night to feed outside from their shelter [START_REF] Lieske | Coral Reef Fishes -Caribbean, Indian Ocean and Pacific Ocean, including Red Sea[END_REF]. Furthermore, most coral reefs fishes do not change sex after reaching sexual maturity, with only a few reef fish species changing sex along their adult growth. For example, some Scaridae, Labridae, and Serranidae first reach sexual maturity as a female, and turn male later in life, while some Pomacentridae, such as clownfish, are first immature juveniles and then males before turning into females when reaching the highest hierarchical position in the anemone) [START_REF] Lieske | Coral Reef Fishes -Caribbean, Indian Ocean and Pacific Ocean, including Red Sea[END_REF].

Coral reef fish reproduction events can be particularly remarkable, such as the massive spawning aggregations encountered in Acanthuridae and Serranidae (Y. Sadovy de Mitcheson and P.L. Colin 2012). The majority of coral reef fishes emit their gametes (eggs and sperms) directly into the water column (pelagic eggs species), while a minority of species deposit and fertilize their eggs on a reef substratum (benthic eggs species, e.g., Pomacentridae and Balistidae) [START_REF] Lieske | Coral Reef Fishes -Caribbean, Indian Ocean and Pacific Ocean, including Red Sea[END_REF]. Eggs generally hatch in a couple of days (pelagic eggs) to a few weeks (benthic eggs) following fertilization, and the emerging larvae are initially swept away from the reefs to the open ocean by oceanic currents (Leis and McCormick 2002). Larvae can drift for as little as a week (clownfish) [START_REF] Thresher | Planktonic duration, distribution and population structure of western and central Pacific damselfishes (Pomacentridae)[END_REF]) to more than 64 weeks (porcupine fish) (Ogden and Quinn 1984) before migrating towards the reefs.

The majority of reef fishes have such a bipartite life cycle, consisting of a pelagic larval stage followed by a largely sedentary reef-associated juvenile and adult stage (Figure I.6) (Leis and McCormick 2002;Cowen and Sponaugle 2009;[START_REF] Clobert | Xenopus Bcl-XL selectively protects Rohon-Beard neurons from metamorphic degeneration[END_REF]. In these species, the pelagic stage (i.e. larvae) primarily facilitates survival (as predation pressure on early life stages is lower in the open ocean) but also dispersal, driven by both physical (e.g. oceanic currents) and biological (e.g. directional swimming and sensory perception) processes (Cowen and Sponaugle 2009;[START_REF] Green | Larval dispersal and movement patterns of coral reef fishes, and implications for marine reserve network design[END_REF]Brooker and Dixson 2016). These processes act at a large spectrum of spatial and temporal scales, with their relevance to dispersal varying depending on larval development and pelagic larval duration. For example, young and poorly developed larvae are usually ineffective swimmers, with initial dispersal largely a result of hydrodynamic processes (Leis and McCormick 2002). However, larvae rapidly develop the ability to orientate themselves within the water column, to swim against the current, and to control their trajectories [START_REF] Fisher | Development of swimming abilities in reef fish larvae[END_REF][START_REF] Fisher | Swimming speeds of larval coral reef fishes: Impacts on selfrecruitment and dispersal[END_REF]). Thus, while larvae often have the potential to travel over vast distances during the pelagic phase, many also have the potential to remain close to their native reefs (Leis and McCormick 2002;Cowen et al. 2005;[START_REF] Irisson | In situ observation of settlement behaviour in larvae of coral reef fishes at night[END_REF], leading to well-connected populations on scales of 0-30 km (Almany et al. 2007;[START_REF] Leahy | Pelagic larval duration and settlement size of a reef fish are spatially consistent, but post-settlement growth varies at the reef scale[END_REF]. While in the ocean, coral reef fish larvae experience rapid development and growth before reaching a developmental stage called 'competence', after which they usually exhibit reduced growth and body maintenance (Cowen and Sponaugle 1997). In the pre-competent stage, larval growth is usually genetically and environmentally mediated (e.g. temperature and food availability) [START_REF] Victor | Delayed metamorphosis with reduced larval growth in a coral reef fish (Thalassoma bifasciatum)[END_REF]Sale 1991;Paris and Cowen 2004). These factors therefore mediate the duration of the pre-competent stage. Once competent, usually near the end of the pelagic phase, larvae are ready to leave the open ocean and rejoin the demersal reef populations (Doherty 2002). The transition between the pelagic and the demersal stages corresponds to settlement (Figure I.6) (Kaufman et al. 1992). This step will be introduced in more details in the next sub-chapter.

In the few days following their colonization of the reef, larval fishes turn into juveniles and select a juvenile reef habitat facilitating their growth and survival (i.e., larval recruitment). Larval recruitment is a generic term used to describe a variety of processes in the reef fish life cycle, usually encompassing all the steps from larval dispersal in the pelagic environment, to the arrival, settlement, and survival in the reef ecosystem (Figure I.6) (Richards and Lindeman 1987). Following larval recruitment, individuals will grow before joining the adult population when sexually mature (i.e., recruitment, in its broad sense, but one should note that larval recruitment is also very often simply called recruitment by scientists when only focusing on the period of larval return to the reefs) (Figure I.6).

Figure I.6 | Life cycle of the convict tang Acanthurus triostegus

A. triostegus is a pelagic egg species. This life cycle is representative of all the pelagic egg coral reef fish species (around 2/3 of all coral reef fishes). In benthic egg species, pelagic dispersal only concerns larvae, as hatching occurs within the reefs. Larval recruitment is different from the 'Recruitment' presented in this figure, and encompasses all the steps from the end of larval dispersal to the survival of juveniles in nursery areas. Pictures: M. Besson.

I.1.3. Coral reef fish settlement and recruitment on the reefs How larval fishes manage to locale coral reefs in the vast ocean has always fascinated ecologists and oceanologists. For example, in French Polynesia, the overall emerged island surface has been estimated as approximately equal to half the area of Corsica island (i.e., half the area of the island of Hawaii), while the oceanic waters around those territories are as vast as Europe (L'ORSTOM 1993). Arriving to the reefs and subsequent return to the benthic environment involves species-specific perception of environmental cues such as reef odors (chemical cues) (Paris et al. 2013;Lecchini et al. 2014b), reef noises (acoustic cues) [START_REF] Vermeij | Coral larvae move toward reef sounds[END_REF], sun compass (visual cues) [START_REF] Berenshtein | Polarized light sensitivity and orientation in coral reef fish post-larvae[END_REF], and magnetic fields (O'Connor and Muheim 2017) (for review, see [START_REF] Leis | How Nemo finds home: the neuroecology of dispersal and of population connectivity in larvae of marine fishes[END_REF] and Barth et al. (2015)). I participated to the latter study, which has been written and published during this PhD, contributing as third author (Barth et al. 2015). This study is available in this thesis (Appendix 1).

Coral larval fish settlement patterns are often species-specific, dependent on nocturnal, lunar, seasonal and inter-annual factors. Some factors appear to be particularly important, with the abundance of larvae settling to reefs often peaking during the night, new moon, summer, and La Niña periods (Dufour and Galzin 1993;Leis and McCormick 2002;Lecchini et al. 2004;Lecchini 2005;[START_REF] Irisson | In situ observation of settlement behaviour in larvae of coral reef fishes at night[END_REF]Lo-Yat et al. 2011;Besson et al. 2017b). I participated to the latter study, which has been written and published during this PhD, contributing as first author (Besson et al. 2017b). This study is available in this thesis (Appendix 2). While experiencing this ecological transition, coral reef fishes often lose many of their planktonic characteristics and develop other features adapted to their new demersal or benthic lifestyle (see fish pictures in Figure I.6). Indeed, after reaching competency, coral reef fish larvae usually encounter a key species-specific period of adaptive changes, which was first described in lizard-fishes (Breder 1944;[START_REF] Gibbs | A synopsis of the postlarvae of western Atlantic lizard-fishes (Synodontidae)[END_REF] and in the convict surgeonfish Acanthurus triostegus (Randall 1961). In this context, nearly all studies have focused on morphological transformations (e.g., morphometrical remodeling, fin spine shortening, pigmentation and skeletal development) (McCormick and Makey 1997;McCormick 1999;McCormick et al. 2002;Parmentier et al. 2004a, 2004b[START_REF] Frédérich | Ontogenetic shape changes in Pomacentridae (Teleostei, Perciformes) and their relationships with feeding strategies: a geometric morphometric approach[END_REF], 2010a[START_REF] Fabricius | Effects of terrestrial runoff on the ecology of corals and coral reefs: Review and synthesis[END_REF], sensory organ development (e.g., evolution of the retina and the barbel structures) (McCormick and Shand 1992;[START_REF] Mccormick | Development and changes at settlement in the barbel structure of the reef fish, Upeneus tragula (Mullidae)[END_REF][START_REF] Shand | Changes in the spectral absorption of cone visual pigments during the settlement of the goatfish Upeneus tragula: the loss of red sensitivity as a benthic existence[END_REF][START_REF] Shand | Changes in the visual system of teleost fishes during growth and settlement: an ecological perspective[END_REF][START_REF] Shand | Ontogenetic changes in retinal structure and visual acuity: a comparative study of coral-reef teleosts with differing post-settlement lifestyles[END_REF], and intestine remodeling (e.g., elongation and coiling) (Randall 1961;[START_REF] Labelle | Some aspects of the early life history of the redlip blenny, Ophioblennius atlanticus (Teleostei: Blenniidae)[END_REF]Nursall and Turner 1985;[START_REF] Green | Ontogeny of the digestive and feeding systems in the anemonefish Amphiprion melanopus[END_REF]. All these changes can occur either (i) in the open ocean (e.g. in some pomacentrids, which undergo most of their developmental changes after hatching, before entering the reef and settling) (McCormick et al. 2002), (ii) along with settlement (e.g. acanthurids, with morphological changes starting a few days before and continuing a few days after settlement in A. triostegus) (Randall 1961;McCormick 1999), or (iii) later on: up to three weeks after larvae settled in the reef habitat (e.g. in some labrids, dartfishes, carapids and scarids) (McCormick and Makey 1997;Parmentier et al. 2004aParmentier et al. , 2004b)). This diversity of time scales among species is also observed at the species level, as this massive period of change can, in some cases, be delayed until favorable environmental cues are encountered (Randall 1961;[START_REF] Victor | Delayed metamorphosis with reduced larval growth in a coral reef fish (Thalassoma bifasciatum)[END_REF]McCormick 1994McCormick , 1999;;Cowen and Sponaugle 1997). Regardless of whether these changes at the end of the larval stage coincide precisely with settlement or not, they occur very rapidly in most species [START_REF] Leis | The larvae of Indo-Pacific coastal fishes: an identification guide to marine fish larvae[END_REF]. For instance, the pigmentation of the transparent A. triostegus larva can occur within 2 hours after entering the reef (McCormick 1999).

Figure I.7 | Settlement patterns or larval coral reef fishes in French Polynesia

Overall, the development of a coral reef fish larva into a juvenile, which occurs at or around settlement (i.e., along larval recruitment), after reaching developmental competency under (currently unknown) environmental signals, involves rapid post-embryonic morphological, behavioral, physiological and ecological changes. This is why this transition is called a 'metamorphosis', as classically defined in the literature [START_REF] Shi | Amphibian Metamorphosis: From Morphology to Molecular Biology[END_REF]Bishop et al. 2006;[START_REF] Heyland | Signaling mechanisms underlying metamorphic transitions in animals[END_REF]Laudet 2011).

I.2. Metamorphosis and thyroid hormone signaling

Metamorphosis has been fascinating scientists, philosophers and poets for centuries, with many novels relating humans and gods transforming into something else, inspirations probably originating from the observation of the well-known and spectacular tadpole-to-frog and caterpillar-to-butterfly metamorphoses (Ovide 1925;[START_REF] George | The Epic of Gilgamesh: The Babylonian Epic Poem and Other Texts in Akkadian and Sumerian[END_REF]. Metamorphosis corresponds to a post-embryonic life transition event that is commonly observed in many animal species. Indeed, 80 % of all animal species associate metamorphosis (not always as spectacular as in frogs and butterflies) and niche shift during their life cycle [START_REF] Werner | Size, scaling, and the evolution of complex life cycles[END_REF], including vertebrates (Paris and Laudet 2008;Laudet 2011). Ecological, physiological, molecular and hormonal proceedings and triggers of metamorphosis have been well described in insects, amphibians and teleost fishes, but remain largely unknown in other arthropods and vertebrates species, as well as in other phyla [START_REF] Gilbert L Irwin | Metamorphosis: Postembryonic Reprogramming of Gene Expression in Amphibians and Insect Cells[END_REF][START_REF] Hall | The Origin and Evolution of larval Forms[END_REF]Nielsen 2000;[START_REF] Shi | Amphibian Metamorphosis: From Morphology to Molecular Biology[END_REF]Bishop et al. 2006;[START_REF] Heyland | Signaling mechanisms underlying metamorphic transitions in animals[END_REF]. Most of our knowledge, from which we build our definition of vertebrate metamorphosis, comes from the study of amphibians.

I.2.1. Metamorphosis in amphibians

Amphibians, particularly the anurans, are the best known models of vertebrate metamorphosis. Their metamorphosis corresponds to the transition of a tadpole, which is herbivorous, and living and breathing underwater, into a carnivorous land-living frog that have essentially a lung-based air respiration. Some environmental parameters such as food availability, temperature, and water volume, trigger metamorphosis by affecting cortisol levels and thyroid hormones (TH) production and release (Denver 1998;Newman 1998;[START_REF] Tata | Amphibian metamorphosis as a model for the developmental actions of thyroid hormone[END_REF]. Thyroxine (T4) and triiodothyronine (T3) are the two TH that endocrinologically trigger metamorphosis [START_REF] Gudernatsch | Feeding experiments on tadpoles[END_REF][START_REF] Buchholz | Molecular and developmental analyses of thyroid hormone receptor function in Xenopus laevis, the African clawed frog[END_REF]. At the climax, when the major metamorphic changes occur, a peak of TH levels is observed in the organism [START_REF] Leloup | La triiodothyronine, hormone de la métamorphose des amphibiens[END_REF][START_REF] Tata | Amphibian metamorphosis as a model for the developmental actions of thyroid hormone[END_REF]. Also, early treatments with TH can induce early metamorphosis [START_REF] Gudernatsch | Feeding experiments on tadpoles[END_REF]. For example, in Xenopus, many organs and tissues undergo an extensive remodeling under TH control. Certain structures like the tail and gills undergo a controlled apoptosis whereas other are protected [START_REF] Brown | The thyroid hormone-induced tail resorption program during Xenopus laevis metamorphosis[END_REF][START_REF] Clobert | Xenopus Bcl-XL selectively protects Rohon-Beard neurons from metamorphic degeneration[END_REF]. The intestine shifts from a long coiled and villi free structure (typical of an herbivorous diet) towards a short, uncoiled and structurally complex carnivorous-like intestine (Figure I.8) [START_REF] Su | Thyroid hormone induces apoptosis in primary cell cultures of tadpole intestine: cell type specificity and effects of extracellular matrix[END_REF]Schreiber et al. 2005). Other organs such as the eyes also undergo such remodeling processes [START_REF] Mann | Control of retinal growth and axon divergence at the chiasm: Lessons from xenopus[END_REF]. The limbs (initially nonexistent) develop, with all the bones, muscles and neuronal remodeling it implies (Figure I.8) [START_REF] Brown | Thyroid hormone controls multiple independent programs required for limb development in Xenopus laevis metamorphosis[END_REF]. It is particularly spectacular to consider that all these major and complex processes such as cell differentiation, cell proliferation and cell death, implying a variety of organ responses, are under the control of a single hormonal peak: the thyroid hormone signaling (Laudet 2011). I.2.2. Thyroid hormone signaling Thyroid Hormones (TH) have a pleiotropic role in animal organisms. They are associated with the catabolism of fat and sugar (Mullur et al. 2014), thermoregulation in mammals, and birds [START_REF] Lebon | Effect of triiodothyronine on mitochondrial energy coupling in human skeletal muscle[END_REF]Ikegami et al. 2015), and thermal-acclimation in zebrafish (Little et al. 2013). The TH signaling pathway is also involved in the physiological response to day lengthening in a wide range of species from fish (e.g., in salmons) (Lorgen et al. 2015) and birds (e.g., in ducks) (Jacquet 1997) to mammals (e.g., in sheep) [START_REF] Bentley | Biological Timing: Sheep, Dr. Seuss, and Mechanistic Ancestry[END_REF]. Moreover, TH are also known for their involvement in developmental processes and organ maturation. This was shown in the previous sub-chapter with regards to metamorphosis, but is also true for less spectacular, but nonetheless important, developmental processes. For example, in humans, there is a peak of TH at birth, and TH are one of the first hormones dosed in new-born infants as any defect in TH levels at this time definitely impairs brain development later in life (Raiti and Newns 1971;Erenberg et al. 1974;Delange 2001;[START_REF] Lopez-Espinosa | Prenatal exposure to organochlorine compounds and birth size[END_REF]Morvan-Dubois et al. 2013). Other studies in mice have

Figure I.8 | Thyroid hormone levels and development in anurans

Dph: days post hatching. Circular structure on the 'guts remodeling' axis correspond to schematic representation of the intestine cross-sections. Adapted from Brown and Cai (2007) and [START_REF] Hasebe | Thyroid hormone-induced cell-cell interactions are required for the development of adult intestinal stem cells[END_REF].

confirmed this importance of TH in the post-embryonic development of the intestine, the bones and the brain [START_REF] Flamant | Congenital hypothyroid Pax8(-/-) mutant mice can be rescued by inactivating the TRalpha gene[END_REF]Morte et al. 2002;[START_REF] Flamant | Thyroid hormone receptors: Lessons from knockout and knock-in mutant mice[END_REF].

TH are synthetized in the thyroid. This gland mainly synthetizes T4, while the conversion of T4 into T3, which has a higher biological activity (i.e., T3 is often referred to as the main metamorphosis hormone), as well as the degradation of both T4 and T3 in inactive forms, are performed by deiodinase enzymes (Figure I.9) (Bianco and Kim 2006a). These enzymes are essentially expressed in peripheral and target tissues, and their expression is highly controlled. This ensures a fine tuned regulation of the T3 level in each tissue in response to any environmental and physiological conditions (Bianco and Kim 2006a). The brain, and more precisely the Hypothalamus-Pituitary-Thyroid (HPT) axis, is the central regulator of TH signaling. HPT integrates metabolic and environmental variations and consequently adjusts TH synthesis through the secretion of thyrotropin releasing hormone (TRH) and thyroid secretary hormone (TSH) (Figure I.10). This centralization allows interconnection and cross-talk with other signaling pathways (e.g., adrenal and gonadal signaling pathways) (Pierantoni et al. 2002;Denver 2009) and feedback loops (Denver 1997;Nakao et al. 2008). Thus, it is not surprising to find that the regulation of such an important hormonal pathway is operated by one of the oldest and most conserved structure of the vertebrate brain [START_REF] Sower | The origins of the vertebrate hypothalamic-pituitary-gonadal (HPG) and hypothalamic-pituitary-thyroid (HPT) endocrine systems: New insights from lampreys[END_REF].

Following TH production, the TH signaling pathway involves the binding of TH to the thyroid hormone receptors (TR), which are ligand dependent transcription factors belonging to the superfamily of nuclear receptors. In the absence of TH, TR repress their targeted gene expression by binding on their regulatory sequences and actively recruiting co-repressors (Figure I.10) [START_REF] Buchholz | Molecular and developmental analyses of thyroid hormone receptor function in Xenopus laevis, the African clawed frog[END_REF]). In the presence of TH, TH/TR binding triggers the removal of repressor-and the recruitment of activator-cofactors, which in turn enhances the expression of target

Figure I.9 | Thyroid hormones and deiodinase enzymes

Schematic representation of the biological role of deiodinase enzymes. T4 and T3 are the two active forms of TH that can bind TR. Dio: deiodinase enzyme. Adapted from Bianco and Kim (2006) and [START_REF] Holzer | Origin of thyroid hormone signalling in metazoans and implications in their metamorphosis[END_REF] metamorphosis genes (Figure I.10) [START_REF] Buchholz | Molecular and developmental analyses of thyroid hormone receptor function in Xenopus laevis, the African clawed frog[END_REF]. Therefore, in the context of metamorphosis, the surge of TH during climax induces an important transcriptional response that is responsible for the previously described morphological and physiological changes [START_REF] Buchholz | Molecular and developmental analyses of thyroid hormone receptor function in Xenopus laevis, the African clawed frog[END_REF]. There are actually two TR in tetrapods: TRα and TRβ, which are the products of the vertebrate-specific double whole-genome duplication [START_REF] Escriva | Analysis of lamprey and hagfish genes reveals a complex history of gene duplications during early vertebrate evolution[END_REF]Dehal and Boore 2005). In anurans, TRα is expressed early in the larval development, even before TH synthesis, and is responsible, in part, for blocking the metamorphic processes while waiting for optimal conditions to metamorphose (e.g., favorable environmental conditions, sufficient larval growth) [START_REF] Buchholz | Molecular and developmental analyses of thyroid hormone receptor function in Xenopus laevis, the African clawed frog[END_REF]. Unlike TRα, TRβ expression is controlled directly by TH [START_REF] Buchholz | Molecular and developmental analyses of thyroid hormone receptor function in Xenopus laevis, the African clawed frog[END_REF]. Therefore, peak expression of TRβ transmits the TH peak signal, particularly as TRβ is one of its own targeted genes (positive feedback loop) [START_REF] Tata | Autoinduction of nuclear hormone receptors during metamorphosis and its significance[END_REF]. In teleost fishes, there are two TRα: TRα-A and TRα-B, issuing from recent lineage-specific whole-genome duplications [START_REF] Escriva | Analysis of lamprey and hagfish genes reveals a complex history of gene duplications during early vertebrate evolution[END_REF]. At the end of the regulation loop, a negative feedback on both TRH and TSH syntheses, enables the system to stop after peaking (Figure I.10).

Figure I.10 | Thyroid hormone signaling

Environmental and metabolic cues can either induce or repress the release of TRH in the hypothalamus. TRH binds its receptors on the anterior pituitary, which then releases TSH. TSH binds its receptors on the thyroid, which then secretes TH. TH are transported to target cells where the deiodinase metabolism determines their faith. When T3 reaches TR in the nucleus, this activates the expression of metamorphosis genes (e.g., Klf9 gene, involved in brain development).

I.2.2. Metamorphosis in teleost fishes

In teleost marine fishes, it is now widely accepted that larvae metamorphose into morphologically, physiologically and ecologically distinct juvenile stages via the previously cited hormonally controlled processes (Pechenik et al. 1998;[START_REF] Youson | Fish Metamorphosis. Fish physiology: The physiology of developing fish, Part B, Viviparity and posthatching juveniles[END_REF]Paris and Laudet 2008;Laudet 2011;Dufour et al. 2012). However, for farming purposes, research has been almost exclusively conducted on temperate and commercial species, such as sea breams [START_REF] Campinho | Coordination of deiodinase and thyroid hormone receptor expression during the larval to juvenile transition in sea bream (Sparus aurata, Linnaeus)[END_REF]Nikolioudakis et al. 2010Nikolioudakis et al. , 2014)), sea basses (Pérez-Domínguez and Holt 2006), lampreys [START_REF] Manzon | Blocking of KC1O4-induced metamorphosis in premetamorphic sea lampreys by exogenous thyroid hormones (TH); effects of KC1O4 and TH on serum TH concentrations and intestinal thyroxine outerring deiodination[END_REF], tunas (Kawakami et al. 2008a(Kawakami et al. , 2008c)), flounders (Keefe and Able 1993;[START_REF] Tanaka | Development of the pituitary, thyroid and interrenal glands and applications of endocrinology to the improved rearing of marine fish larvae[END_REF][START_REF] Huang | Metamorphosis of summer flounder (Paralichthys dentatus): thyroid status and the timing of gastric gland formation[END_REF]Schreiber and Specker 1998;[START_REF] Soffientino | Age-dependent changes in the response of the stomach to thyroidal signaling in developmentally arrested larval summer flounder[END_REF] and other flatfishes [START_REF] Markle | Metamorphosis and an overview of earlylife-history stages in Dover sole Microstomus pacificus[END_REF][START_REF] Tanaka | Development of the pituitary, thyroid and interrenal glands and applications of endocrinology to the improved rearing of marine fish larvae[END_REF]Marchand et al. 2004;Ortiz Delgado et al. 2006;[START_REF] Galay-Burgos | Thyroid hormone receptor expression during metamorphosis of Atlantic halibut (Hippoglossus hippoglossus)[END_REF]Klaren et al. 2008;[START_REF] Manchado | Thyroid hormones down-regulate thyrotropin beta subunit and thyroglobulin during metamorphosis in the flatfish Senegalese sole (Solea senegalensis Kaup)[END_REF] In addition to the previously described metamorphic processes, studies in teleost fishes revealed some differences in TR expressions (Marchand et al. 2004;[START_REF] Galay-Burgos | Thyroid hormone receptor expression during metamorphosis of Atlantic halibut (Hippoglossus hippoglossus)[END_REF][START_REF] Manchado | Molecular characterization, gene expression and transcriptional regulation of thyroid hormone receptors in Senegalese sole[END_REF]. For instance, in the Atlantic halibut, TRα-A expression profile is similar to TRβ, while TRα-B expression profile is similar to the TRα expression profile observed in anurans [START_REF] Galay-Burgos | Thyroid hormone receptor expression during metamorphosis of Atlantic halibut (Hippoglossus hippoglossus)[END_REF] Peaks of TH levels and TR expressions are observed around the N and O developmental stages, which correspond to the stage where most changes are observed in the metamorphosing larvae. Adapted from Kawakami (2008a) also demonstrates another example in P. olivaceus. Additionally, the thyroid structure in teleost fish is actually not a true gland, but rather numerous and scattered follicles (but each with the same structure as the ones found in true glands) located in the subpharyngeal region (Figure I.13) [START_REF] Matthews | The thyroid gland of the Bermuda parrot fish, Pseudoscarus guacamaia[END_REF]Kawakami et al. 2008a).

I.2.3. Metamorphosis in coral reef fishes

In coral reef fishes, metamorphosis at the larva/juvenile transition has been acknowledged by researchers for more than 70 years (Breder 1944;[START_REF] Wenger | Suspended sediment prolongs larval development in a coral reef fish[END_REF]. However, our knowledge on what characterizes metamorphosis in coral reef fishes is scattered and biased toward morphological features (e.g., pigmentation, shape, skeletal development in A. triostegus metamorphosis, Figure I.14) (Breder 1944;Randall 1961;McCormick and Shand 1992;McCormick 1999;McCormick et al. 2002;[START_REF] Frédérich | Ontogenetic shape changes in Pomacentridae (Teleostei, Perciformes) and their relationships with feeding strategies: a geometric morphometric approach[END_REF]Frédérich et al. , 2010a;;McLeod et al. 2013). We still lack a precise description of the physiological triggers and molecular proceedings of metamorphosis in coral reef fishes. Indeed, there has been surprisingly little research conducted on metamorphosis triggering and the molecular pathway. To date and to my knowledge, only two studies have focused on the physiology of this process, by providing the first evidence that thyroid hormones may trigger metamorphosis in coral reef fishes (de Jesus et al. 1998;[START_REF] Trijuno | Effects of thyroxine and thiourea on the metamorphosis of coral trout grouper Plectropomus leopardus[END_REF], as expected from the literature on other non-reef teleost fishes. Moreover, the structure in follicles of the thyroid has been confirmed in the Bermuda parrot fish (Pseudoscarus guacamaia) [START_REF] Matthews | The thyroid gland of the Bermuda parrot fish, Pseudoscarus guacamaia[END_REF] and during this PhD in A. triostegus (Figure I.13). Nevertheless, the paucity of information concerning metamorphosis is particularly striking when considering the fact that this transformation occurs during the oceanic to reef transition (i.e., along settlement and larval recruitment), a step often referred to as a "critical period" in the life cycle of coral reef fishes (McCormick et al. 2002;Almany and Webster 2006;[START_REF] Hamilton | Larval history influences post-metamorphic condition in a coralreef fish[END_REF]). Indeed, mortality can reach up to 95 % during this transition [START_REF] Caselle | Early post-settlement mortality in a coral reef fish and its effect on local population size[END_REF]Planes and Lecaillon 2001;[START_REF] Spalding | Early postsettlement predation on three reef fishes: effects on spatial patterns of recruitment[END_REF]Doherty et al. 2004;[START_REF] Hamilton | Postsettlement survival linked to larval life in a marine fish[END_REF] demonstrating that metamorphosis can be a mortality bottleneck that shapes population dynamics.

Figure I.14 | Acanthurus triostegus metamorphosis

Immediately after their entry in the reef at night, metamorphosing and transparent-larvae (left) and metamorphosed and pigmented-juveniles (right) can be encountered in the same nursery areas. The vertical bars of black pigmentation can appear in less than two hours. Picture: M. Besson.

I.3. Coral reef fish metamorphosis and larval recruitment facing anthropogenic pressures

I.3.1. Anthropogenic pressures can degrade reefs and impair larval recruitment

In the current context of world-wide coral decline due to climate change and water pollution [START_REF] Gardner | Long-term region-wide declines in Caribbean corals[END_REF]Bellwood et al. 2004;[START_REF] Bruno | Regional decline of coral cover in the Indo-Pacific: timing, extent, and subregional comparisons[END_REF]Hughes et al. 2010), pressures upon coral reef fish populations and assemblages is no longer solely attributed to overfishing stressors but also replenishment issues [START_REF] Wilson | Multiple disturbances and the global degradation of coral reefs: are reef fishes at risk or resilient?[END_REF][START_REF] Wilson | Exploitation and habitat degradation as agents of change within coral reef fish communities[END_REF](Wilson et al. , 2010a(Wilson et al. , 2010b;;Munday et al. 2008;Pratchett et al. 2008). Indeed, the loss and degradation of coral habitats has been known for years to reduce the potential for attracting coral reef fish settlers (Lecchini 2005;Lecchini et al. 2007b[START_REF] Lecchini | Spatial and behavioural patterns of reef habitat settlement by fish larvae[END_REF][START_REF] Irisson | In situ observation of settlement behaviour in larvae of coral reef fishes at night[END_REF][START_REF] Wilson | Exploitation and habitat degradation as agents of change within coral reef fish communities[END_REF]Wilson et al. , 2010b;;Lecchini and Nakamura 2013;Dixson et al. 2014). Moreover, it has been recently emphasized that stressors such as oceanic acidification, sea level rise, boat noise and soil pollution could also directly affect coral reef fish larval development, growth, behavior and ability to settle. For instance, ocean acidification has been shown to disrupt olfactory, auditory and visual perception of cues usually involved in habitat selection in coral reef fishes (Munday et al. 2009(Munday et al. , 2014;;[START_REF] Simpson | Ocean acidification erodes crucial auditory behaviour in a marine fish[END_REF]Ferrari et al. 2012b;Leduc et al. 2013). Also, elevated sea temperature (according to predictions from [START_REF] Ganachaud | Observed and expected changes to the tropical Pacific Ocean[END_REF])) and subsequent reduced food availability to pelagic larvae [START_REF] Hays | Climate change and marine plankton[END_REF]Richardson 2008;[START_REF] Barton | The biogeography of marine plankton traits[END_REF]) have been proven to lower larval development rate, body condition at metamorphosis, and survival after metamorphosis, therefore diminishing larval recruitment success [START_REF] Hamilton | Larval history influences post-metamorphic condition in a coralreef fish[END_REF]Lo-Yat et al. 2011;McLeod et al. 2013). Furthermore, water pollutants such as anthropogenic boat noise and increasing events of soil and pesticide pollution (e.g. near river mouths, because of landclearing and increasing extreme climatic events, which cause massive rains and extensive erosionn), but also oil derivatives exposure, have been implicated in reducing larval recruitment [START_REF] Holles | Boat noise disrupts orientation behaviour in a coral reef fish[END_REF][START_REF] Wenger | Suspended sediment prolongs larval development in a coral reef fish[END_REF]O'Connor et al. 2016;Johansen et al. 2017;Lecchini et al. 2017). I participated to the latter study, which has been written and published during this PhD, contributing as sixth author (Lecchini et al. 2017). This study is available in this thesis (Appendix 3).

All these studies describe the impacts of climate change and water pollutants on larval life history traits and replenishment potential. When considering the extreme mortality experienced by coral reef fish at larval recruitment, small changes in body condition, growth, metamorphose success, settlement success and subsequent mortality rates, could have important replenishment consequences. Given that the replenishment of coral reef fish population is essentially dependent on larval recruitment (Doherty 2002), it is now well established that in degraded habitats, the decline in adult populations of coral reef fishes has more to do with larval recruitment failure than adult mortality (Jones et al. 2004).

I.3.2. Importance of metamorphosis in coral reef fish larval recruitment

The development of a larva into a juvenile, through metamorphosis, occupies a central role in larval recruitment. For instance, metamorphic changes are involved in (i) evading predators (e.g., by pigmentation change to the benthic coloration) (McCormick 1999;McCormick et al. 2002), (ii) diet shifts (e.g., through neoorientation of the mouth and remodeling of the guts) (Randall 1961;[START_REF] Labelle | Some aspects of the early life history of the redlip blenny, Ophioblennius atlanticus (Teleostei: Blenniidae)[END_REF]Nursall and Turner 1985;[START_REF] Green | Ontogeny of the digestive and feeding systems in the anemonefish Amphiprion melanopus[END_REF]McCormick et al. 2002) and (iii) finding a suitable microhabitat in the reef ecosystem (e.g., via the development of new sensory organs that promote greater awareness of the new environment and its broad range of novel sensory stimuli and selective forces) (McCormick and Shand 1992;[START_REF] Mccormick | Development and changes at settlement in the barbel structure of the reef fish, Upeneus tragula (Mullidae)[END_REF]Lecchini et al. 2007a;Mitchell et al. 2011;Frédérich et al. 2012). In fishes that metamorphose in the pelagic environment and settle to reef as a fully developed juvenile, such as the damselfish Abudefduf saxilitis (McCormick et al. 2002), metamorphic development of sensory organs is also likely to be required for the perception of multiple sensory cues enabling the return to the reef [START_REF] Leis | How Nemo finds home: the neuroecology of dispersal and of population connectivity in larvae of marine fishes[END_REF].

When facing the novel reef environment at settlement, coral reef fishes display innate preferences in their investigation for a suitable reef habitat (e.g., live coral odors, conspecifics visual cues, avoidance of predator odors) (Lecchini et al. 2007b;[START_REF] Vail | Metamorphosing reef fishes avoid predator scent when choosing a home[END_REF]Roux et al. 2016). I participated to the latter study, which has been written and published during this PhD, contributing as sixth author study (Roux et al. 2016). This study is available in this thesis (Appendix 4). Yet, larvae are also capable of learning and therefore adapting their behavioral responses to novel cues (e.g., recognition of a novel predator) [START_REF] Holmes | Smell, learn and live: the role of chemical alarm cues in predator learning during early life history in a marine fish[END_REF][START_REF] Manassa | Social learning improves survivorship at a lifehistory transition[END_REF]. This sensitive learning at the larval stage is called 'imprinting', and it has been recognized in limiting dispersal and facilitating the return to the reef in coral reef fish larvae (Gerlach et al. 2007;Paris et al. 2013). In clownfish, this imprinting process has been described in the context of the species-specific recognition between the fish and its anemone host [START_REF] Arvedlund | Host recognition and possible imprinting in the anemonefish Amphiprion melanoptus (Pisces -Pomacentridae)[END_REF][START_REF] Arvedlund | Host imprinting in anemonefishes (Pisces: Pomacentridae): does it dictate spawning site preferences?[END_REF]. In another teleost fish species such as the salmon Oncorhynchus kisutch, the TH are involved in the cellular proliferation within the olfactory epithelium during the time of imprinting (responsible for their homing behavior) (Dittman and Quinn 1996;[START_REF] Lema | Evidence that thyroid hormone induces olfactory cellular proliferation in salmon during a sensitive period for imprinting[END_REF]. This hormonally regulated process occurs during the surge of TH levels at the parr-smolt metamorphosis, which corresponds to the morphological and physiological changes supporting the freshwater to seawater transition. Since this phenomenon has been confirmed in other salmon species (Scholz et al. 1985;Hino et al. 2007) and also in a (non-teleost) sturgeon species [START_REF] Boiko | Effect of thyroid hormones on imprinting of chemical signals at early ontogenesis of the sturgeon Acipenser gueldenstaedti[END_REF], it would be particularly relevant to evaluate the impact of TH in the imprinting process of coral reef fishes. When considering the crucial role played by TH in neurodevelopment, particularly in auditory, olfactory and visual systems (Forrest et al. 2002), and the fact that (i) imprinting (Gerlach et al. 2007;Paris et al. 2013), (ii) neurodevelopment (Lecchini et al. 2014a), and (iii) integration of multiple sensory cues towards habitat selection (McCormick and Shand 1992;[START_REF] Mccormick | Development and changes at settlement in the barbel structure of the reef fish, Upeneus tragula (Mullidae)[END_REF]Lecchini et al. 2007a;Mitchell et al. 2011;Frédérich et al. 2012) occur at or around metamorphosis in coral reef fishes, we hypothesize that TH might play a key role in the recruitment success of coral reef fishes. A recent study demonstrated the disrupting impact of climate change, and in particular oceanic acidification, on coral reef fish ability to learn and imprint (Ferrari et al. 2012a). Additionally, several studies conducted on other teleost fishes and marine invertebrates illustrated the toxicity of endocrine disruptors such as pesticides, which affected not only survival and reproduction, but development and metamorphosis (Menendez and Ishimatsu 1993;[START_REF] Brown | Contaminant Effects on the Teleost Fish Thyroid[END_REF]Delgado et al. 2013). This is of particular interest when considering the substantial anthropogenic impacts on reefs through the worldwide increasing influx of chemical pollution (e.g., from pesticides) resulting from coastal agriculture and river runoff (Carvalho et al. 2002;Islam and Tanaka 2004;Fabricius 2005;Laabs et al. 2007;Leong et al. 2007;Lewis et al. 2009;[START_REF] Packett | Agricultural lands are hot-spots for annual runoff polluting the southern Great Barrier Reef lagoon[END_REF]Shaw et al. 2010;Roche et al. 2011;Kroon et al. 2012). Such pollutants are known to severely impact reef biodiversity and the biology and ecology of coastal marine organisms (Fabricius 2005;O'Connor et al. 2016). Among these sources of pollution, the Chlorpyrifos (CPF), an organophosphate insecticide widely used on tropical coastal crops (e.g., sugar-cane and rice crops in Australia and south-east Asia respectively (Cavanagh et al. 1999;Leong et al. 2007;Botté et al. 2012)), is one of the most common waterborne chemical pollutants encountered in coral reefs (Cavanagh et al. 1999;Botté et al. 2012). While several studies have demonstrated CPF neurotoxicity and endocrine disruption characteristics (Kavitha and Rao 2008;Botté et al. 2012;Deb and Das 2013;Slotkin et al. 2013), the negative impact on coral reef larval fish sensory abilities has also recently been acknowledged (Botté et al. 2012). Moreover, pesticides belonging to the same family have been discovered in considerable amounts in several fish and crustacean species in French Polynesia (Roche et al. 2011;Salvat et al. 2012). However, whether this pollutant affects metamorphosis itself remains unknown (Botté et al. 2012).

I.4. PhD project presentation

I.4.1. Background, problematics, and objectives

Altogether, climate change and water pollution thoroughly affect coral reef fish populations and assemblages. In particular, they can impact larval and metamorphic stages and subsequently impair larval recruitment success. The processes affected by such stressors are highly diversified and complex and may have a variety of molecular implications. However, due to their involvement in a broad range of developmental processes, TH appear to be powerful mediators, which could help us to characterize the interactions between environmental factors and metamorphic processes. In turn, given metamorphosis plays a central role in larval recruitment, characterizing metamorphic processes and how they impinge on the ecology of coral reef fishes during this important transition period is critical to understanding larval recruitment. This would enhance our understanding of coral reef fish replenishment, resilience and dynamics in regard to climate change and water pollution. This raises many questions:

Is coral reef fish metamorphosis triggered and controlled by TH, as it is in temperate and commercial non-reef teleost fishes?

How are larval recruitment and TH signaling coupled? Are there specific sensory organs and sensory abilities that develop at metamorphosis and what are their endocrine determinants?

Are there environmental factors that act as endocrine disruptors of TH signaling during coral reef metamorphosis and how would it subsequently affect larval recruitment success?

To answer these questions, this PhD thesis aims to:

(i) decipher the hormonal proceedings of coral reef fish metamorphosis; (ii) better characterize the importance of TH and metamorphosis in larval recruitment (e.g., 1/ development of sensory organs such as the eye retina, nostrils and lateral line, 2/ acquisition of sensory abilities such as the olfactory and visual recognition of predators and conspecifics, and 3/ avoidance of predation); and (iii) assess how environmental factors such as sea water temperature and CPF exposure can affect this key transition of the coral reef fish life cycle

To achieve this, the convict tang Acanthurus triostegus (

I.4.2. Model species and study site

A. triostegus is, to date, the most studied coral reef fish species in terms of metamorphosis (Figure I.13,Figure I.14) (Randall 1961;McCormick 1999;McCormick et al. 2002;Frédérich et al. 2010aFrédérich et al. , 2012)). Following their 44-60 day pelagic larval duration (Lo-Yat et al. 2011), the transparant A. triostegus larvae metamorphose into pigmented juveniles in a few days (Figure I.14) (McCormick 1999). During this transition, larval planktonic feeders turn into herbivorous juveniles, changing shape as their body height shortens, while their mouth migrates to a more vertical position (as in grazers), and their scales develop (McCormick 1999;McCormick et al. 2002;Frédérich et al. 2010aFrédérich et al. , 2012)). However, the molecular determinants of such spectacular processes remain unknown. Juveniles and adults feed on filamentous algae (i.e., turf) (Randall 1961). After reef colonization, young settlers are usually encountered in small groups of dozens of individuals, in shallow and quiet waters along the coastline. Contrarily, adults can be solitary but mostly form large aggregations (up to hundreds of individuals) around reef patches close to the barrier reef (Randall 1961).

A. triostegus is one of the most abundant and representative species in the Indo-Pacific (Figure I.6) (Randall 1961), and is particularly abundant in Moorea (French Polynesia) (Lecchini et al. 2004;Besson et al. 2017b). The island of Moorea lies 25 km to the North-West of Tahiti, at 17°30' South latitude, and 149°50' West longitude [START_REF] Galzin | Moorea Island Society Archipelago[END_REF]L'ORSTOM 1993). This volcanic island formed less than two millions years ago, originating from an oceanic hotspot [START_REF] Guillou | Age progression along the Society hotspot chain (French Polynesia) based on new unspiked K-Ar ages[END_REF]. It belongs to the Windward Islands of the Society Archipelago. Moorea has a triangular shape with 61 km of coastline surrounded by lagoon of 500 to 1.500 meters wide [START_REF] Galzin | Moorea Island Society Archipelago[END_REF]. The lagoon is delimited by a barrier reef that opens the lagoon to the ocean in twelve passes. Tidal fluctuations are very low in this region of the world, with tidal differences of a maximum of 40 cm (L'ORSTOM 1993).

I.4.3. Methodology

To achieve the objectives of this PhD, A. triostegus was sampled along its different metamorphic stages, from oceanic larvae to metamorphosed juveniles. Sampling was carried out using different techniques, from light-traps to hand nets and crest nets. All these fishing techniques are detailed in the next chapters, as well as in Appendix 2. After capture, fish were either transferred to aquaria at the CRIOBE research station, or kept in the lagoon in in situ cages, to study their metamorphosis under specific and controlled conditions. Different hormonal treatments, as well as differential temperature and CPF exposures were applied to metamorphosing individuals, to address the scientific questions above. I also generalized some of the observations made on A. triostegus, to other coral reef fish species belonging to other families, such as Chromis viridis, Chaetodon lunula, Ostorhinchus angustatus and Rhinecanthus aculeatus. Lastly, laboratory experiments resulting and following these in vivo experiments were conducted at the CRIOBE research station itself, but also at the Institut de Génomique Fonctionnelle in Lyon (France) and at the Observatoire Océanologique in Banyuls (France).

This study was carried out in accordance with the guidelines of the French Polynesia committee for animal ethics, and the experiments were approved by the CRIOBE-IRCP animal ethics committee. This study did not involve endangered or protected species. To achieve this, the fieldwork and analyzes presented in this chapter have been performed in close collaboration with Dr. G. Holzer. He was finishing his PhD while I was starting mine. Understanding the endocrine determinants that mediate larval recruitment in coral-reef fishes was somehow our common project, and the fruit of the collaboration between Dr. D. Lecchini and pr. V. Laudet. At least two years of my PhD were devoted to this chapter. Dr. G. Holzer and I ended up by writing a manuscript gathering all the results he obtained before I started my PhD, and all the results I obtained after he finished his. This manuscript has been submitted to eLife in April 2017, as a research article. To enlarge this work and to obtain a chance to get published in such journal, we have added some preliminary results on how metamorphosis could be prone to endocrine disruption in coral-reef fishes, with first clues about such a threat for coral-reef ecosystems. Consequently, this chapter also bring a few first answers to the two other questionsof my PhD, which are:

II.2. Manuscript information

Title: Fish larval recruitment to reefs is a thyroid hormone-mediated metamorphosis sensitive to the pesticide chlorpyrifos Authors: Guillaume HOLZER 1* , Marc BESSON 1,2,3* (equal first author), Anne LAMBERT 1 , Loïc FRANÇOIS 2 , Paul BARTH 1 , Benjamin GILLET 1 , Sandrine HUGHES 1 , Gwenaël PIGANEAU 3 , François LEULIER 1 , Laurent VIRIOT 1 , David LECCHINI 2,4,# and Vincent LAUDET 1,3,#,£ Affiliations:

II.3. Abstract

Larval recruitment, the transition of pelagic larvae into reef-associated juveniles, is a critical step for the resilience of marine fish populations but its molecular control is unknown. Here, we investigate whether thyroid-hormones (TH) and their receptors (TR) coordinate the larval recruitment of the coral-reef-fish Acanthurus triostegus. We demonstrate an increase of TH-levels and TR-expressions in pelagic-larvae, followed by a decrease in recruiting juveniles. We generalize these observations in four other coral reef-fish species. Treatments with TH or TR-antagonist, as well as relocation to the open-ocean, disturb A. triostegus larvae transformation and grazing activity.

Likewise, chlorpyrifos, a pesticide often encountered in coral-reefs, impairs A. triostegus TH-levels, transformation, and grazing activity, hence diminishing this herbivore's ability to control the spread of reef-algae. Larval recruitment therefore corresponds to a TH-controlled metamorphosis, sensitive to endocrine disruption. This provides a framework to understand how larval recruitment, critical to reefecosystems maintenance, is altered by anthropogenic stressors.

II.4. Introduction

Life history transitions are critical for many animal species and often correspond to concomitant developmental and ecological shifts (Bishop et al. 2006). Unfortunately, little is known about how internal and external cues act in concert during these events. The life cycle of most teleost reef fish include a major developmental and ecological transition. Adults reproduce in the vicinity of the reef, emitting eggs that disperse and hatch in the ocean, where the larvae grow (Leis and McCormick 2002). Larvae migrate back and enter reefs where they become juveniles, a step called larval recruitment (Leis and McCormick 2002). This step involves the perception of environmental cues for larvae to localize and settle in the reef, and is accompanied by major morphological changes (McCormick et al. 2002;Lecchini et al. 2005aLecchini et al. , 2005bLecchini et al. , 2013;;Barth et al. 2015). This transition of pelagic larvae into reef-associated juveniles is often referred to as metamorphosis and is critical for the maintenance of reef fish populations, but its molecular control remains largely unknown (Dufour and Galzin 1993;Doherty 2002;Leis and McCormick 2002;McCormick et al. 2002;Dixson et al. 2011;Barth et al. 2015). Since larval recruitment is an ecological event coupled to a morphological transformation, reminiscent of the situation in amphibians, it may correspond to a thyroid hormones (TH) controlled metamorphosis (Bishop et al. 2006;Brown and Cai 2007).

TH and their receptors (TR) trigger and coordinate metamorphosis of many species such as Xenopus (Brown and Cai 2007) or flatfish [START_REF] Solbakken | Thyroxine as a mediator of metamorphosis of Atlantic halibut , Hippoglossus hippoglossus[END_REF]). The thyroid gland produces mainly the thyroxine hormone (T4), which is peripherally transformed into triiodothyronine (T3), the active form (Chopra 1996). The transformation of T4 into T3, and the degradation of T4 and T3 are controlled by a family of enzymes called deiodinases (Bianco and Kim 2006b). TH levels peak at the climax of metamorphosis. Precocious treatment with TH triggers metamorphosis whereas goitrogen (TH synthesis inhibitors) treatment blocks it [START_REF] Tata | Amphibian metamorphosis as a model for the developmental actions of thyroid hormone[END_REF]). Similar mechanisms have been described in several teleost fishes, but in contexts disconnected from the natural environment (Brown 1997;de Jesus et al. 1998;[START_REF] Yamano | Differential gene expression of thyroid hormone receptor alpha and beta in fish development[END_REF]Kawakami et al. 2003;Marchand et al. 2004;McMenamin and Parichy 2013).

In the convict surgeonfish Acanthurus triostegus, larvae have a planktonic diet while juveniles and adults are herbivorous, and external morphological changes occurring at recruitment have been previously described (Randall 1961;McCormick 1999;McCormick et al. 2002;Frédérich et al. 2012). Interestingly, crest-captured larvae can delay their metamorphosis when relocated to the external slope of the reef (open ocean), showing an influence of the environment on their morphological transformation (McCormick 1999). Nevertheless, again, the molecular mechanisms remain unknown.

Here we report that A. triostegus larval recruitment to the reefs corresponds to metamorphosis with major pigmentation changes, remodeling of the digestive tract and behavioral modifications. Our results show that TH levels and TR expressions control this remodeling process. Furthermore, recruiting larvae can freeze their TH signaling when relocated on the external slope, demonstrating an influence of the environment on metamorphosis. Chlorpyrifos (CPF), a known reef pollutant and endocrine disruptor (Roche et al. 2011;Botté et al. 2012;Juberg et al. 2013;Slotkin et al. 2013), affects A. triostegus metamorphosis by impairing TH signaling, preventing intestine lengthening, and inhibiting fish grazing activity. CPF therefore impairs A. triostegus control on algae spreading, which is a major threat for reef conservation. We extend our observation upon TH levels to four other coral reef fish species. Our work provides a unifying framework that integrates the developmental, ecological and evolutionary perspectives of vertebrate life history transitions. The involvement of the TH signaling pathway in this key post-embryonic step, thus prone to endocrine disruption, provides an obvious entry point to study how anthropogenic stressors may affect reef fish populations.

II.5. Results

Acanthurus triostegus larvae undergo a major and rapid remodeling at recruitment

A. triostegus crest captured individuals (n = 297) experience an important weight loss between day 1 (0.71 ± 0.01 g) and day 8 (0.53 ± 0.01g) following their entry in the reef ( The intestine also goes through a drastic remodeling at recruitment. In particular, the gastro-intestinal tract lengthens from about 2.52 ± 0.07 cm in crest captured larvae to 7.38 ± 0.14 cm in day 8 juveniles ( To assess whether and to what extent this intestine remodeling was correlated to a diet shift at larval recruitment, we sequenced A. triostegus digestive tract content (18S mass sequencing) to identify its eukaryotic composition. We investigated crest captured individuals, day 8 juveniles and adults (Figure II.1W) using a blocking primer to minimize amplification of host sequences. Crest larval intestines are largely empty although we identified sequences of fungi and algae. In day 8 juveniles, the intestinal content is different with more brown and red algae, as well as few protostome sequences. This indicates a shift in the eukaryotic gut content between crest individuals and day 8 juveniles. Adults gut content is less diversified and is mainly composed of brown algae, red algae and alveolates, different from those found in day 8 juveniles (Figure II.1W). Using 16S metagenomic sequencing, we found differences in the relative quantity of bacterial community between the juveniles at day 2, 5 and 8 (Figure II.1 -figure supplement 1P). The 16S profile of the adult is also different from the juvenile profile, with the notable presence of Lachnospiraceae, among which the giant bacteria Epulopiscium fishelsoni is a well-known symbiont of Acanthuridae (Clements and Bullivant 1991).

Thyroid hormone signaling peaks at larval recruitment Given the role of TH in vertebrate metamorphosis (Laudet 2011) and the spectacular transformations that A. triostegus undergoes at larval recruitment, we analyzed TH levels in larvae captured in the far ocean (from 5 to 10 km offshore, n = 3), near ocean (2 km offshore, n = 5), crest-captured larvae and juveniles (raised in situ in lagoon cages, n = 297) up to day 8 after recruitment (

Thyroid hormone signaling and the environment control larvae transformation

To link the TH pathway, the environment and the metamorphic changes occurring at larval recruitment, we disturbed the TH signaling and the location of crest-captured larvae during their metamorphosis (from capture to up to day 8 after reef colonization).

In order to study how the environment controls the metamorphosis processes, we first relocated crest-captured larvae back to the external slope immediately after their reef entry. Indeed, mimicking oceanic condition has been acknowledged to delay metamorphosis (McCormick 1999) (Figure II.3A,upper panel). Similar to this earlier study, we observed a striking delay in the white pigmentation appearance between the vertical black stripes in day 2 relocated fish compared to their lagoon raised counterparts (Figure II.3A, lower panel). The levels of T4 and T3 in fish relocated on the external slope at day 2, day 5 and day 8 are overall higher than the levels of their control counterparts in the lagoon (Figure II.3B-C). The levels of T4, in particular, remain similar in day 2 to day 8 juveniles to the levels in the crest condition (Figure II.3B). The expressions of TRs and Klf9 in the relocated juveniles were also higher than in control juveniles at the same age (Figure II.3E). This suggests that the environment effectively controls the metamorphosis by modulating the TH signaling.

To assess the implication of the TH signaling pathway on metamorphic processes, we then raised crest-captured larvae in situ in the lagoon and injected them daily, in their ventral cavity, with 20 µl of different drug solutions that activate or disrupt their TH molecular pathway. Four drug treatments were applied: (i) solvent control (DMSO diluted 10.000 times in Phosphate Buffer Saline 1X, as all drugs were made soluble in DMSO and diluted 10.000 times in PBS 1X); (ii) T3 + iopanoic acid (IOP) both at 10 -6 M, IOP being used as an inhibitor of the deiodinase enzymes that therefore prevents the degradation of injected T3 (Galton 1989;Little et al. 2013;Lorgen et al. 2015); (iii) NH3 at 10 -6 M, NH3 being used as an antagonist of TR that prevents the binding of TH on TR (Lim et al. 2002;Renko et al. 2012) Given that teeth remodeling is a lengthy process that starts very early on in oceanic larvae and therefore before reef colonization, it is not surprising that the treatments performed on crest captured larvae do not affect (or are too late to affect) teeth development. Also, contrarily to individuals relocated on the outer slope, we did not observe any effect of T3+IOP nor NH3 treatments on skin pigmentation at day 2 and day 5 (Figure II.3 -figure supplement 2). This suggests that pigmentation during the larva to juvenile transition is strongly coupled to the environment but is not directly under TH control that may not sustain such rapid pigmentation changes. Taken together: (i) intestine lengthening and remodeling are mediated by TH signaling; and (ii) disturbing the TH pathway disrupts the normal development of fish after larval recruitment, showing that TH effectively controls some major aspects of A. triostegus metamorphosis.

Finally, we tested how TH signaling influences fish behavior. To achieve this, we used grazing activity (number of bites on algal turf), as A. triostegus larvae start to graze a couple of days after colonizing the reef, to investigate how TH signaling and metamorphosis completion influence the behavior of recruited fish. T3 + IOP treatment increases the grazing activity by around 50% in day 5 juveniles when compared to the control individuals (p-value = 0.004, Figure II.3G, blue lanes). On the contrary, NH3 treatment significantly decreases the number of bites at day 2 by 33% (p-value < 0.001, Figure II.3G, green lane). The grazing activity of fish relocated on the external slope (Figure II.3A) is also diminished by more than 4-fold at day 2 (p-value < 0.001, Figure II.3G, orange lanes). These data indicate that TH signaling promotes the biting activity of A. triostegus and subsequent grazing behavior adopted at larval recruitment. This shows that TH controls the transformations of the feeding process not only at the morphological and physiological levels but also at the behavioral level. Mean number of bites (on turf algae) per fish in a batch of 18 fish at day 2 (d2) and day 5 (d5) under different treatment conditions: solvent control (DMSO, grey shade), T3+IOP at 10 -6 µM (blue shade), NH3 at 10 -6 µM (green shade) or relocated to the external slope (orange shade). n = 3 to 6 independent bite counts (technical replicates) for each condition. Bite counting was not performed on crest captured individuals as A. triostegus larvae do not feed for at least 12 hours after colonizing the reef at night (MB pers. obs.). Statistics: same as in (E).

Thyroid hormone-mediated metamorphosis is conserved in coral reef fishes

To widen our understanding of coral reef fish metamorphosis, we investigated the TH signaling during larval recruitment in four other coral fish species (Rhinecanthus aculeatus, Chromis viridis, Chaetodon lunula and Ostorhinchus angustatus) from distant families (Near et al. 2012). In these species, both T4 and T3 levels drop between day 1 and day 8, up to 3-fold for T4 (in O. angustatus, p-value = 0.029) and up to 4-fold for T3 (in R. aculeatus, p-value = 0.05) (Figure II.4). This drop of TH levels after reef entry strongly suggests that TH are also key determinants for the metamorphosis of these species. T4 levels in R. aculeatus and T3 levels in C. viridis also rise between near ocean and day 1 individuals (Figure II.4), further confirming the model of a TH-mediated metamorphosis during coral reef fish larval recruitment. However, T4 levels are 3-fold higher in near ocean individuals than in day 1 individuals in C. viridis (Figure II.4). This suggests some potential species-specific variation in coral reef fish TH profiles at recruitment that would be extremely interesting to decipher in future studies on additional species and with regards to other aspects of coral reef fish ecology (e.g., diets, size and pigmentation status at recruitment).

Reef pollutant disrupts the transformation of Acanthurus triostegus

Chlorpyrifos (CPF) is an agricultural insecticide that is widely used on tropical coastal crops, therefore one of the most common waterborne chemical pollutants encountered in coral reef surrounding waters (Cavanagh et al. 1999;Leong et al. 2007;Roche et al. 2011;Botté et al. 2012). Given the endocrine determinants of coral reef fish metamorphosis (Figure II.2-3) and the CPF endocrine disruption characteristics (Juberg et al. 2013;Slotkin et al. 2013), we assessed the impact of CPF on A. triostegus metamorphosis. To achieve this, we used exposure doses previously reported in coral reef fish (1, 5 or 30 µg.l -1 of seawater) (Botté et al. 2012). CPF treatment at 30 µg.l -1 decreases the T3 levels by 50 % in day 2 juveniles (p-value = 0.021, Figure II.5A). No significant effect was observed on T4 levels regardless of CPF concentration and duration (p-value = 0.205 for day 2 and p-value = 0.496 for day 5, Figure II.5figure supplement 1A). We did not observe any effect of CPF treatments on fish pigmentation at day 2 and day 5 (Figure II.5 -figure supplement 1B). However, CPF exposure at 30 µg.l -1 prevents guts' lengthening at day 2 (p-value = 0.024, Figure II.5B left panel), and both exposure at 5 and 30 µg.l -1 prevent intestines' lengthening at day 5 (p-values = 0.047 and 0.029 respectively, Figure II.5B right panel). Strikingly, CPF exposure at 30 µg.l -1 also decreases the grazing activity of day 2 juveniles with 2.63 ± 0.09 bites per fish per min contrasting with 3.62 ± 0.28 bites per fish per min in the solvent control condition (p-value = 0.014, Figure II.5C). In day 5 juveniles, exposure at 5 and 30 µg.l -1 decreases the grazing activity with respectively 2.68 ± 0.18 and 2.63 ± 0.05 bites per fish per min compared to 4.43 ± 0.33 bites per fish per min for the solvent control fish (p-values = 0.001, Figure II.5C). Such a behavior in fish treated with

II.6. Discussion

In this study, we investigated the morphological, physiological and behavioral changes occurring during A. triostegus larval recruitment. These results highlight profound anatomical and physiological transformations of the skin and the whole digestive tract (Figure II.1B-W). In particular, the differences in 16S gut content are likely the consequence of a diet shift (carnivorous to herbivorous) and an environmental shift occurring during larval recruitment [START_REF] Sullam | Environmental and ecological factors that shape the gut bacterial communities of fish: a meta-analysis[END_REF]Frédérich et al. 2012;Baldo et al. 2015). We hypothesize that intestine remodeling (i.e. the loss of the villi and internal structures) accelerates the shift of the gut microbiota by allowing the establishment of a new bacterial community fitting the diet change. The presence of Lachnospiraceae in the adult but not in the larvae is consistent with the hypothesis that this family of bacteria is acquired through the environment in surgeonfishes (Miyake et al. 2016). Altogether, these observations reinforce the notion that the fish bacterial community depends on their diet and environment. These shifts of 16S and 18S gut contents, occurring between crest individuals and day 8 juveniles, strikingly coordinate with both the changes of the intestine and teeth morphologies observed during larval recruitment (Figure II.1M-V). All these changes from teeth to microbiota are consistent with A. triostegus change of diet after entering the reefs, conforming to previous observations (Randall 1961;McCormick 1999;Frédérich et al. 2012). This complete remodeling is associated with a severe weight loss ( triostegus with the TH-controlled post-embryonic process known in zebrafish (the major teleost fish developmental model, one of the few teleost fishes in which we have a functional knowledge of the metamorphosis process). Indeed, in both species, as well as in flatfish, another classical teleost model of metamorphosis, major pigmentation and craniofacial changes occur at metamorphosis (McMenamin and Parichy 2013;McMenamin et al. 2014). However, the appearance of dark melanophore stripes in zebrafish and A. triostegus is very likely to be controlled by different mechanisms. In A. triostegus this event is extremely rapid (as fast as 2 hours after reef entry, MB pers. obs.), strongly environmentally coupled, and probably not under direct TH control We demonstrated that the decrease of both T4 and T3 after reef entry can be generalized to phylogenetically distant coral fish species (Figure II.4). We propose that this decrease is valid for most if not all coral reef fishes, even if species-specific variations on this general theme can be observed, and that the larval recruitment of coral reef fish corresponds to a TH-controlled metamorphosis (Figure II.6A). Interestingly, variations in the magnitude of TH changes among the five coral reef fish species (Figure II.4) are reminiscent of the situation in stickleback, where differences in TH signaling between two ecotypes support life history trait variation (Kitano et al. 2010;Kitano and Lema 2013). We anticipate that this post-embryonic developmental step is shaped by evolution and varies according to species-specific life history traits such as pelagic larval duration, size at reef colonization and pigmentation status (i.e., metamorphic state of advancement) at recruitment. Given the dramatic diversity of life history strategies they propose, coral reef fish could be excellent models to decipher how these changes are elicited by changes in hormonal systems.

Among the endocrine disruptors already evidenced and encountered in coral reefs, CPF is known to be a thyroid disruptor with adverse effects on fish behavior (Sandahl et al. 2005;Kavitha and Rao 2008;Roche et al. 2011;[START_REF] Yang | Chlorpyrifos-oxon disrupts zebrafish axonal growth and motor behavior[END_REF]Botté et al. 2012;Deb and Das 2013;Juberg et al. 2013;Slotkin et al. 2013). We demonstrated that CPF exposure reduces A. triostegus physiological level of T3 at larval recruitment (Figure II.5A). Consistently with our observations on NH3 treated fish, exposure to CPF impairs metamorphosis, as evidenced with the repression of guts' lengthening (Figure II.5B), and the decrease in the juveniles' grazing activity (Figure II.5C). This latter point led to a reduced algal removal efficiency (Figure II.5D). As A. triostegus is one of the major herbivorous fish in coral reefs, this can severely impair the ability of coral reefs to recover after an algal invasion (e.g., after a bleaching event) and therefore contributes to the worldwide degradation of reef health (Russ 1984;Almany et al. 2007;Hughes et al. 2007Hughes et al. , 2014;;Ledlie et al. 2007)

II.7. Conclusion

Our results highlight how coral reef fish larval recruitment, the transition between the open ocean and the reef, is a TH-regulated metamorphosis at the crossroads of ecological, developmental, physiological and behavioral transformations (Holzer and Laudet 2015). These findings also demonstrate how coral reef fish TH signaling and larval recruitment processes can be altered by reef pollution. Since metamorphosis and larval recruitment are essential for the maintenance of fish populations and subsequent coral reef resilience, this study provides a general framework to better understand, at the molecular level, how global changes, water pollution and human activities can threaten reef ecosystems.

II.8. Materials and Methods

Fish capture, husbandry and conservation

Acanthurus triostegus, Ostorinchus angustatus, Chaetodon lunula, Chromis viridis and Rhinecanthus aculeatus larvae were captured at Moorea Island, French Polynesia. Larvae were captured during the night while colonizing the reef crest, using a crest net and hand nets (Dufour and Galzin 1993;Besson et al. 2017b). Larvae were then kept in cages located in situ in the lagoon and provided with continuous food from the water column supplemented with coral rubble and algal turf. For the clarity of this study, the age in day of the juvenile corresponds to the number of day spend in the reef in cages since capture, not the absolute age. Near ocean A. triostegus larvae were sampled at 1-2 km away from the reef crest using drifting light trap and hand nets from 0.5 m to 5 m depth. Far ocean A. triostegus larvae were also captured offshore by trawl haul more than 10 km away from the reef between 1 and 60 m deep. Larvae were euthanized in MS222 at 0.4 mg.ml -1 in filtered seawater. For TH dosage, larvae were frozen dry and conserved at -20°C prior to extraction. For RNA extraction, larvae were lacerated and kept in RNAlater (Sigma) 1 hour on ice and stored at -20°C. For histology, larvae were rinsed in PBS 1X, dissected and the intestines were kept in Bouin's fixative at room temperature. For µCT scan and metagenomics, Larvae were kept in 70 % ethanol at room temperature.

Fish treatments

Acanthurus triostegus captured at the crest were injected with 20 µl of drugs in the ventral cavity. Drugs tested were as follows: (i) solvent control (DMSO diluted 10.000 times in Phosphate Buffer Saline 1X, as all drugs were made soluble in DMSO and diluted 10.000 in PBS 1X); (ii) T3 + iopanoic acid (IOP) both at 10 -6 M,; (iii) NH3 at 10 -6 M; and (iv) T3 + IOP + NH3 all at 10 -6 M. IOP was used as an inhibitor of deiodinase enzymes, as evidenced in mammals and amphibians (Galton andHiebert 1987, 1988;Galton 1989;Simonides et al. 2008;[START_REF] Medina | The thyroid hormone-inactivating type III deiodinase is expressed in mouse and human β-cells and its targeted inactivation impairs insulin secretion[END_REF]Renko et al. 2012), and as routinely used in fish to prevent the immediate degradation of injected T3 (Little et al. 2013;Lorgen et al. 2015). NH3 is a known TR antagonist in vertebrates (Lim et al. 2002), a notion we confirmed in A. triostegus (Figure II.2 -figure supplement 1D). NH3 prevents the binding of TH on TR and impairs the binding of transcriptional coactivators to TR, which therefore remain in inactive conformation (Lim et al. 2002;Figueira et al. 2011). Fish were then kept in the lagoon in in situ cages and injected daily following the same protocol to maintain pharmacological treatment. Fish were sampled on the 2 nd and 5 th day after the beginning of this protocol. DMSO non toxicity was tested by comparing the pigmentation, the teeth development, the intestine length and internal structures between solvent-control individuals (treated with DMSO diluted 10.000 times in PBS 1X), and control individuals (simply raised in situ in lagoon cages as mentioned in the previous paragraph) (

Chlorpyrifos (CPF) exposure

After crest capture, A. triostegus larvae were transferred in aquaria (30 L x 20 W x 20 cm H) in groups of n = 10 fish. Each aquarium was filled with 9 liters of UV-sterilized and filtered -10 µm filter, and was equipped with an air stone. Fish were then immediately exposed to: nothing (control), acetone at a final concentration of 1:1.000.000 (solvent control, as CPF was made soluble in acetone) or CPF at a final concentration of 1, 5, and 30 µg.l -1 , as previously done in the literature in another coral reef fish (Botté et al. 2012). All water was replaced every day, ensuring the maintenance of water quality as well as a continuous concentration of the pesticide or solvent. Fish were sampled the 2 nd and 5 th day after the beginning of this protocol. Acetone non toxicity was tested by comparing the TH levels, the pigmentation, and the intestine length between solvent-control and control individuals (Figure II

Cloning of Acanthurus triostegus genes

Muscle from adult fish and whole larvae were used. Samples were cut using sterile scalpel blades and crushed in a Precelyss with Qiagen extraction buffer. RNA extractions were performed using the Macherey-Nagel RNA extraction kit following the manufacturer's instructions. Total pooled RNAs were retro-transcripted with the Invitrogen Super Script III enzyme following the manufacturer's instructions. For TR cloning, actinopterygian universal primers (Figure II.2 -figure supplement 2) were designed for TRα-A, TRα-B and TRβ to retrieve the full-length sequences of the genes. For polD2 and rpl7, actinopterygian universal primers were designed to retrieve partial sequences including at least one exon-exon junction. PCR amplicon were cloned in the Invitrogen PCR II plasmid following the manufacturer instruction for sequencing. TRα-A, TRα-B and TRβ were subcloned in pSG5 between EcoRI sites for in cellulo expression.

qPCR assay

Far ocean to day 8 juveniles were assayed. For each larvae 1 µg of RNA were used for retro-transcription using the Invitrogen Super Sript III following the manufacturer's instructions, including a DNAse I treatment. qPCR primers were designed to anneal on different exons, Rpl7 and Pold2 were used as normalization genes (Figure II.2 -figure supplement 2). qPCRs were performed in 96 well plate with the BioRad IQ Syber Green Super Mix in 10µl of final reaction per well following the manufacturer's ratios. qPCRs were assayed in a BioRad thermocyclers and analysed on BioRad CFX Manager software. Assays were performed on duplicates in at least two independent RNA extractions and retro-transcriptions. Klf9, which is known to respond to TH signaling, was used as a TH signaling reporter gene.

Phylogenetic analysis

The amino acid sequences of A. triostegus cloned TRs as well as available TR sequences (Figure II.2 -figure supplement 3) were aligned using MUSCLE software. Trees were generated using the Maximum Likelihood method with the Seaview 4 software under the JTT model with estimated gamma shape and eight rate categories (RRID:SCR_015059) (Gouy et al. 2010). Bootstrap analysis of 1000 replicates was carried out to support the tree.

Functional characterization of the receptors

Human embryonic kidney 293 cells (ATCC:CRL_11268, RRID:CVCL_0045) (Iwema et al. 2007;Gutierrez-Mazariegos et al. 2014;Sadier et al. 2015) were grown in Dulbecco's modified Eagle's medium supplemented with 10% of coal stripped foetal bovine serum and penicillin/streptomycin at 100µg.ml -1 . Cell were maintained at 37°C, 5 % CO2 and tests for mycoplasma contamination were negative. The transient transfection assays were carried out in 96-well plate with 30 000 cells per plate using Exgen500 according to the manufacturer's instructions. For each well, cells were transfected with 50ng of total DNA: 12,5 ng of full-length receptor encoding plasmid, 12,5 ng of reporter plasmid with four DR4 repeat in the luciferase promoter, 12,5 ng of β-galactosidase encoding plasmid and 12,5 ng of pSG5 empty plasmid. Drugs were incubated for 48 hours and cells were harvested using a passive lysis buffer and frozen at -20°C. On half of the lysate, luciferase activities were assayed with the luciferase reagent buffer from Promega on a Veritas Turner Biosystem luminometer.

On the other half of the lysate, the -galactosidase activity was measured using ONPG substrate and absorbance at 420 nm for normalization. Each assay was performed at least 3 times independently on well triplicates. Drugs from Sigma-Aldrich were diluted in DMSO à 10 -2 M then in sterile PBS1X prior treatment.

Thyroid hormone quantification TH were extracted from dry-frozen fish following an extraction protocol adapted from previous publications relating TH level variations in teleost fish (Tagawa and Hirano 1989;Einarsdottir et al. 2006;Kawakami et al. 2008a). Far ocean larvae to day 8 juveniles were assayed. At least three individuals per sampling point were used. Each fish was first crushed with a Precelyss in 500 µl methanol, centrifuged at 4°C and supernatant reserved, three times. Pooled supernatant were dried at 70°C. Hormones were re-extracted with 400 µl methanol, 100 µl chloroform and 100 µl barbital buffer twice from the first dried extract. Pooled supernatant was dried out and extract was reconstituted in 2 ml of PBS1X for quantification. The quantification was performed following the Roche ELICA kit on a Cobas analyser by a medical laboratory according to the manufacturer's standardized method.

Intestine histology

The intestines in Bouin's fixative were embedded in paraffin. Sections of 5 µm were performed in the proximal, medial and distal part using a microtome (Leica). The histological sections were stained with Hematoxylin and Eosin. Photography were taken on a Leica microscope and the mosaic reconstructed using Image J software.

Gut Metagenomics

For 16S mass sequencing, juveniles of 2, 5, 8 days after reef entry and adult A. triostegus gut were dissected in triplicates for a total of twelve samples. Total DNA was extracted with a Macherey-Nagel DNA extraction kit following the manufacturer's recommendations. 16S library were constructed for each individual using the Ion 16S Metagenomic kit from Life technologies following the manufacturer's recommendations. Sequencing of bacterial 16S was performed using a PGM Ion Torrent. Three controls for contaminations were performed. The environment control consisted of an open tube during the dissection. The extraction control monitored the DNA extraction process. The blank control monitored the library construction. The sequencing results were then analyzed using the Life technologies 16S pipeline. 18S mass sequencing was performed on crest larvae, 8 days juveniles and adults in triplicates. Intestine contents were extracted in the same condition as 16S. 18S library were constructed with the pre-amplified V7 region of the eukaryotic 18S (Figure II.2 -figure supplement 2). A blocking primer was designed to prevent the amplification of host sequences (Figure II.2 -figure supplement 2). The blocking primer was designed to partially overlap the 18S reverse primer and was modified with a C3 spacer. Sequencing was performed using a PGM Ion Torrent. The sequences were assembled and blasted against the PR2 database, completed with 18S sequenced from multicellular organisms for taxonomic affiliation.

µCT scan analysis Fish samples were conserved in 70 % ethanol, dehydrated in successive baths of 95 % ethanol, twice 100 % ethanol and in vacuum arena for at least 4 hours. Xray microtomographies were performed on a Phoenix Nanotom (General Electric) at 70 kV of tension, 100 mA of intensity with a tungsten filament. 3000 images per sample were taken at 500 ms of exposure per image and at a resolution ranging from 2.5 to 2.8 µm. 3D volumes were reconstructed and analysed with VGI studiomax software.

Grazing activity

For each condition (e.g. hormonal treatments, pesticide conditionings), 18 fish were placed in a 5 liter tank with coral rubbles covered with turf algae. After an acclimation period of 1 hour, we recorded the total number of bites made on turf algae during 10 min. This experiment was replicated with a new batch of fish 3 to 6 times depending on the condition.

Grazed turf biomass

For each pesticide exposition conditioning, we weighed (underwater) 3 pieces of coral rubble prior the introduction of a group of 20 crest captured fish (weight A), and after 5 days of grazing by this group of fish (weight B). For each piece of coral rubble, the weight of grazed algae was estimated through the difference weight B minus weight A.

Statistical information

All statistical analyses were conducted using the R-Cran project free software (http://www.rproject.org/, R-3.3.1). Mean comparisons were performed using Wilcoxon-Mann-Whitney U-test when comparing two means, and using univariate analysis of variance (ANOVA) followed by Tukey post-hoc test (should a significant difference be detected) for multiple comparisons. Prior ANOVA, normality of values (or residuals) and variance homogeneity were assessed using Shapiro and Bartlett tests. In qPCR analyses, comparisons of gene expression were performed through Student's t cumulative distribution functions, automatically computed by qPCR software CFX Biorad Manager, (CFX Manager 2017).

II.9. Additional information II.10.

Target gene Sens

Sequence (5' to 3') Annealing temperature (A) Cloning primers To achieve this, the intense fishing and fish husbandry efforts presented in this chapter have been performed in close collaboration with two interns that I supervised with Dr. D. Lecchini: I. Moniz and L. François. I then performed all the histological work, other analyses, and writing, during my last year. At least two years of my PhD were devoted to this chapter (one year in common with the previous chapter). Given the variety of processes addressed by this study, many other collaborators have been involved in the study elaboration as well as in the writing (see next sub-chapter).

TRα-A F CCTCAKTSTCCBGTGATTGGC 51°C TRα-A R TCABACYTCCTGGTCCTCRAASACC 56°C TRα-B F ATGGMACAMATGCCCRARGAGSAGG 56°C TRα-B R TCABACYTCCTGGTCCTCRAASACC 56°C TRβ F ATGTCAGAGCMAGSAGAVAAATGC 51°C TRβ R TCAGTCYTCRAASACYTCYAGGAAG 53°C rpl7 F CAGATCTTCAAYGGWGTNTTYGT 51°C rpl7 R GAADGGCCACAGGAAGTTGTTG 58°C pold2 F AGCTTCARYCGBCAGTATGC 51°C pold2 R CTGCTGTACYTCTVDATGTC 46°C klf9 F GVRCYHTRHRATGGYBGCVATGAT 60°C klf9 R TKVAKCATRCTKGGVTGGAAGYC 59°C (B) qPCR primers TRα-A F CAAAAGCCTCGCTCAGTTTAACCTG 60°C TRα-A R CTTGGCACTGCTCAATCTTCTCC 59°C TRα-B F GAAGAGGAAGAACAGCCAATGTTCG 60°C TRα-B R GTCACCACACACCACACATGG 60°C TRβ F GTCGCTGTCCTCCTTTAACCTGG 61°C TRβ R CCTCTTGGCAACGTTCGATTCG 60°C rpl7 F CAGGATTGCTGAGCCTTACATCG 59°C rpl7 R AATACGCTGTTTCCTCATCCTGC 60°C pold2 F CACCAAGAAGACTCAGGCTGG 59°C pold2 R GTGTAGTTGGTGGGGTCATACTGG 60°C klf9 F GCAAGTCGTCCCACCTCAAGG 59°C klf9 R GGAGAACTTCTTGCCACAGTCCG 61°C (C) Mass-seq 18S F GGCTTAATTTGACTCAACACG 53°C 18S R GGGCATCACAGACCTG 52°C blocking R ACCTGTTATTGCTCAATCTCGTGTGGCTG-C3-spacer 64°C
This chapter corresponds to a comprehensive and holistic analysis that tackle the two questions mentioned above, at the molecular, anatomical, behavioral and ecological levels. Given the novelty of the results conveyed by this work, we have decided (with the future manuscript co-authors) that a few additional experiments should be performed in order to maximize the chance to publish this research in a high impact-factor journal. More precisely, two small but important experiments have yet to be performed (see next sub-chapters). These experiments are currently being done, and hopefully part of the results should be available by September, and a first submission of this research should be done by the end of the year. To date, I wrote a first version of this manuscript, for a future submission to Nature (Letters). This explains the particular format of this chapter, with a short referenced summary of the study, followed by a short description and discussion of the results.

III.3. Referenced summary

The persistence and sustainability of coral-reef fish populations depends on the continued recruitment, i.e. arrival (settlement) and post-settlement survival of pelagic larvae into adult reef habitats (Jones et al. 2004;Almany et al. 2007). Due to the importance of recruitment, there is strong selective pressure for traits that increase an individual's likelihood of locating settlement habitats that maximize subsequent growth and fitness while minimizing mortality risk (Doherty 1987;McCormick and Hoey 2004). There is growing evidence that changes to water conditions due to global change and waterborne pollution can impair the sensory abilities of recruitment-stage fish, impacting habitat selection and decreasing survival rates (Besson et al. 2017a;Johansen et al. 2017). Recent research has identified that recruitment timing corresponds to a metamorphosis mediated by thyroid-hormones (TH) (Holzer et al. 2017). However, the role of TH signaling in sensory system maturation and the impact of environmental perturbations on this process remains largely unknown (Holzer et al. 2017). Here we show that the eye (vision), lateral-line (mechanosensory perception), and nostril (olfaction), all undergo rapid TH-controlled changes during recruitment in the convict surgeonfish Acanthurus triostegus. However, a +3°C temperature shift as well as exposure to chlorpyrifos (a common pesticide detected in coastal reef waters (Shaw et al. 2010)), decrease TH levels at a critical developmental stage, limiting sensory system maturation, impairing visual and olfactory abilities, and decreasing survival. Even small declines in survival during this sensitive recruitment stage could impair population replenishment (Jones et al. 2004;Almany et al. 2007). A reduced abundance of keystone algal herbivores, such as A. triostegus, may impair their ability to maintain or rescue coral-dominated habitats, further threatening the ecological and economic resilience of coral-reef ecosystems (Ledlie et al. 2007;Munday et al. 2008). In other vertebrate species, THs also integrate ecological, physiological, morphological and behavioral changes during critical life history stages (Brown and Cai 2007;Lorgen et al. 2015). As TH appear highly sensitive to ambient ecological conditions, identifying how they are affected by current and predicted disturbance is essential.

III.4. Results and discussion

Settlement, the transition from a pelagic larvae to a benthic-dwelling juvenile, is a particularly perilous step in the life history of most coral-reef fishes, with ~90 % mortality during this stage (Doherty et al. 2004). Any additional factors that increase this mortality rate could have dramatic consequences for population replenishment (Jones et al. 2004;Almany et al. 2007). One of the key factors influencing survival at recruitment is settlement-site selection, with larval fishes using acute sensory systems to locate appropriate benthic habitats (Lecchini et al. 2007a;Mitchell et al. 2011;Brooker and Dixson 2016). As coral-reefs are rapidly degrading globally due to anthropogenic activities (Hughes et al. 2003;Hoegh-Guldberg et al. 2007), a large number of studies have examined how larval sensory abilities (e.g., recognition of olfactory cues from conspecifics or predators) are impaired by factors such as global change and water pollution (Dixson et al. 2014;Johansen et al. 2017;Lecchini et al. 2017). However, to date no study has integrated molecular, anatomical, and behavioral approaches to identify the processes that impair larval sensory ecology and subsequent survival. Recent evidence that metamorphosis and recruitment in coralreef fishes are mediated by thyroid hormones (TH) prone to endocrine disruption (Holzer et al. 2017) provides a novel framework for examining how environmental perturbations could threaten this life history transition (Holzer et al. 2017). Thyroxine (T4), and more particularly its derivative triiodothyrorine (T3) are the two vertebrates TH, acting as key regulators of brain, olfactory, and visual system development in a number of teleost fishes and amphibians during metamorphosis (Forrest et al. 2002;Brown and Cai 2007;Hino et al. 2007;Lorgen et al. 2015). Given the similarities between the metamorphoses of coral-reef fishes and these other species (Holzer et al. 2017), TH appear to be likely candidates as controllers of sensory organ maturation in coral-reef fishes. If this is the case, endocrine disruption during recruitment could have unforeseen impacts on larval coral-reef fish sensory ecology and coral-reefs as a whole.

We used the convict surgeonfish, Acanthurus triostegus, as a model to determine the role of TH in sensory organ maturation in larval coral-reef fishes at recruitment. Larvae were collected just prior to settlement (i.e. 0 days post-settlement = d0) from reefs surrounding Moorea Island, French Polynesia. Larvae were then raised in cages placed in situ in a nursery area in the Moorea lagoon, until their second (d2), fifth (d5), eighth (d8), and thirtieth (d30) day post-settlement (Methods). Recruits completed metamorphosis within eight days post-settlement, as indicated by rapid pigmentation changes (Fig. To investigate the role of TH signaling in sensory organ maturation, we disturbed the TH pathway by injecting fish with different pharmacological drugs: (i) TH signal promotion = T3 treatment (T3 + iopanoic acid (IOP) both at 10 -6 M), and (ii) TH signal disruption = N3 treatment (NH3 at 10 -6 M). A third injection condition (TH signal undisturbed = CT control treatment) was performed to allow comparison with the maturation of sensory organs observed in control conditions at recruitment (Fig. . In addition, we also conducted another treatment where larvae were maintained from d0 in cages on the outer reef slope (OS treatment) (i.e. as opposed to the lagoon), as preventing access to inner reef is known to environmentally delay metamorphosis (Holzer et al. 2017) (Methods). The density of ggc did not vary between treatments or the control (Fig. III.2a), indicating that it is not controlled by TH. However, the T3 treatment induced an increased rate of maturation in the retina (elevated densities of prn and pes at d2, Among the many stressors affecting coral-reefs, elevated sea water temperature likely represents the greatest current threat to these ecosystems, with impacts ranging from habitat degradation, altered community composition, reduced biodiversity, and behavioral and physiological impairments in associated organisms (Nilsson et al. 2009;McLeod et al. 2013;Hughes et al. 2017). In addition to rising sea surface temperature, coral-reefs have also experienced increasing levels of chemical pollution (e.g., agricultural pesticides and petrogenic compounds) following intensive agricultural and industrial developments (Fabricius 2005;Burns 2014). Among these pollutants, the agricultural pesticide chlorpyrifos (CPF) is one of the most common encountered in coral-reef waters (Shaw et al. 2010). As CPF acts as an endocrine disruptor, coral-reef fishes are high sensitive to it during metamorphosis (Holzer et al. 2017). To determine the relative impact of thermal and chemical stressors on sensory organ maturation, we exposed d0 larvae to either a +0 (i.e. 28.5°C, ambient lagoon temperature), +1.5 (30.0°C), or +3.0 (31.5°C) temperature shift (following end-of-century projections for the tropical Pacific (IPCC 2014)), or to CPF at 1, 5, and 30 µg.l -1 . In addition, we also conducted a control (C) and solvent-control (SC) exposure, to control for aquarium and CPFsolvent (i.e. acetone) impacts (Methods). A temperature shift of +3°C reduced both T3 and T4 levels around twofold at d2 in comparison to fish exposed at 28.5°C ( at d2 (n=42, Kruskal-Wallis+Nemenyi), no difference at d5 (p=0.105, n=30, Kruskal-Wallis). b, T4 levels variations under different temperatures (d2 fish at 28.5°C as standards). A<B (p=0.001) at d2 (n=29, Kruskal-Wallis+Nemenyi), no difference at d5 (p=0.074, n=27, Kruskal-Wallis c, bpc density variations under different temperatures. A<B (p=0.034) at d2 (n=20, ANOVA+TukeyHSD) and no difference at d5 (p=0.375, n=20, ANOVA). d, prn density variations under different temperatures. No difference at d2 (p=0.637, n=20, ANOVA) and d5 (p=0.983, n=20, ANOVA). e, pes density variations under different temperatures. No difference at d2 (p=0.291, n=19, ANOVA) and d5 (p=0.177, n=20, Kruskal-Wallis). f, nostril lamellae number variations under different temperatures. No difference at d2 (p=0.609, n=29, Kruskal-Wallis) and d5 (p=0.626, n=20, Kruskal-Wallis). g, lateral line canal number variations under different temperatures. A<B (p=0.025) at d2 (n=29, ANOVA+TukeyHSD) and A<B (p=0.002) at d5 (n=20, ANOVA+TukeyHSD). h, T3 level variations under CPF exposure (d2 fish at 28.5°C as standards). A<B (p=0.039) at d2 (n=47, ANOVA+TukeyHSD), no difference at d5 (p=0.698, n=50, ANOVA). i, T4 levels variations under CPF exposure (d2 fish at 28.5°C as standards). No difference at d2 (p=0.255, n=42, ANOVA) d5 (p=0.578, n=50, ANOVA). j, bpc density variations under CPF exposure. A<B (p=0.049) at d2 (n=50, ANOVA+TukeyHSD) and A<B (p=0.004) at d5 (n=50, ANOVA+TukeyHSD). k, prn density variations under CPF exposure. No difference at d2 (p=0.993, n=50, ANOVA) and d5 (p=0.436, n=50, Kruskal-Wallis). l, pes density variations under CPF exposure. No difference at d2 (p=0.978, n=50, ANOVA) and A<B (p=0.022) at d5 (n=50, Kruskal-Wallis+Nemenyi). m, nostril lamellae number variations under CPF exposure. No difference at d2 (p=0.669, n=50, Kruskal-Wallis) and A<B (p=0.018) at d5 (n=49, Kruskal-Wallis+Nemenyi). n, lateral line canal number variations under CPF exposure. A<B (p=0.034) at d2 (n=93, ANOVA+TukeyHSD) and A<B (p=0.041) at d5 (n=56, ANOVA+TukeyHSD). h-j, adapted from Holzer (2017). (Methods).

Impaired sensory system maturation could reduce survival if it limits an individual's ability to identify and distinguish between critical sensory cues during recruitment (Johansen et al. 2017). We examined this by testing if the metamorphosis-, temperature-, and CPF-induced defects observed impacted the ability of A. triostegus larvae to identify predator sensory cues. Firstly, responses to chemical predator cues were assessed by comparing A. triostegus preferences for control seawater vs. seawater containing the odor of a common predator (Lutjanus fulvus) in two-channel Atema choice flume (Fig. III.4a,Methods). While A. triostegus showed no preference for either water source at d0, a clear preference for control seawater was observed in d2 and d5 CT-fish (Fig. III.4a). The same pattern was observed for T3 treated fish; however, N3and OS-treated fish showed no preference for control seawater over the predator treated seawater at d2 (Fig. III.4a). Secondly, responses to visual predator cues were examined by comparing reactions to L. fulvus in a choice aquarium (Fig. III.4c,Methods). At d2, T3 fish, along with CT fish and those held at ambient temperature (i.e. 28.5°C) avoided predator visual cues; however, N3 and OS individuals showed no particular attraction or aversion (Fig. III.4d). Thus, individuals whose sensory maturation was impaired by disruption of the TH pathway, either via pharmacological means or environmental conditions, are less able to identify important sensory cues that could increase survival. Finally, to test whether endocrine disruption could reduce recruitment success by increasing mortality rates, we conducted survival experiments in an in situ arena (Fig. III.4e,Methods). CT, T3, N3, and OS fish all exhibited different mortality rates when exposed to predators simultaneously. T3 individuals had higher survival rates than their control counterparts (CT) at both d2 and d5, while N3 fish exhibited an opposite pattern (Fig. III.4f). OS fish exhibited greater mortality rates than CT fish at d2, but similar rates to the control at d5 (Fig. III.4f). These differences in survival cannot be attributed to variation in fish size or condition between treatments, as these were similar between groups (III. Extended Data Fig 4). This research is a holistic analysis of molecular, morphological, histological and behavioral assays of multiple stressors affecting coral-reefs. It builds a mechanistic theory of the impacts of elevated temperature and water pollution during the critical recruitment phase in coral-reef fishes. This indicates the importance of proper endocrine function during coral-reef fish recruitment. A final experiment should test whether exposure to both +3.0°C and 30 µg.l -1 CPF also increase mortality rates, and whether performing T3 treatment on those fish improve their survival (i.e. by rescuing the TH signaling) (Fig. III.4g). This research highlights the need for a considerable diminution of anthropogenic pressures on coral-reefs, both locally (water pollution) and globally (climate change), as well as for a better understanding of the interactions between such stressors (Hale et al. 2017), more particularly at the brain level (Forrest et al. 2002).

III.5. Methods

Ethics statement

This study was carried out in accordance with the guidelines of the French Polynesia committee for animal ethics, and all experiments were approved by the CRIOBE-IRCP animal ethics committee. This study did not involve endangered or protected species.

Study site and species sampling

This study was conducted from February 2015 to August 2017, at Moorea Island, French Polynesia (S17°32'16.4589", W149°49'48.3018"). The convict surgeonfish Acanthurus triostegus (Linnaeus, 1758) has a pelagic larval duration of 53 ± 8 days (Randall 1961) after which oceanic larvae settle back to the reefs, essentially during new moon periods (Besson et al. 2017b). A. triostegus larvae were collected nightly (d0), while colonizing the reefs near Tema'e, on the north-east coast of the island (S17°29'49.7362", W149°45'13.899"). Sampling was performed using crest nets (Dufour and Galzin 1993). This species was chosen as our model species due to the high number of settling larvae available, and because of the previous studies that have started to tackle endocrine disruption at recruitment in this species (Besson et al. 2017a;Holzer et al. 2017).

Fish husbandry and measurements

Following their capture, young fish were immediately transferred to a nursery area on the north coast of the island (S17°29'26.5378", W149°53'29.2252") where they were raised in in situ cages (cylindrical cages, diameter: 50 cm, height: 70 cm, maximum of 10 fish per cage), permitting the study of their development in 'undisturbed' conditions. Cages were equipped with coral rubbles that were replaced daily, ensuring shelter and constant turf availability for fish (as A. triostegus feeds on algal turf after settlement (Randall 1961;Holzer et al. 2017)). Fish were then sampled on their second (d2), fifth (d5), eighth (d8), and thirtieth (d30) day of living within the reefs for the study of their pigmentation, behavior, and development. When necessary, fish were euthanized in freshly prepared MS222 at 0.4 mg.ml -1 in filtered seawater at 4°C, and instantly placed on ice to minimize animal suffering. Following euthanasia, all fish, except the ones reserved for histological analyses (to preserve tissue quality) were measured for weight (W), height (H) and standard length (SL), allowing the further calculation of the Fulton's condition factor (Fulton 1904) K = 100*(W/SL)^3 (with SL, in cm, and W, in g), as well as body depth BD = H/SL. Fish were also scaled and pictured for further measurements such as lateral line (LL) length.

Fish treatments

When tested for the role of their thyroid hormone signaling, A. triostegus at d0 were immediately injected after their capture, in their ventral cavity, each with 20 µl of different pharmacological treatments: (i) solvent control (CT, corresponding to DMSO diluted 10.000 times in Phosphate Buffer Saline (PBS) 1X, as the two pharmacological drugs were made soluble in DMSO and diluted 10.000 times in PBS 1X), (ii) triiodothyronine (T3) + iopanoic acid (IOP) both at 10 -6 M (T3 treatment), and (iii) NH3 at 10 -6 M (N3 treatment). IOP was used as an inhibitor of deiodinase enzymes, as evidenced in mammals and amphibians (Galton andHiebert 1987, 1988;Galton 1989;Simonides et al. 2008;[START_REF] Medina | The thyroid hormone-inactivating type III deiodinase is expressed in mouse and human β-cells and its targeted inactivation impairs insulin secretion[END_REF]Renko et al. 2012), and as routinely used in fish to prevent the immediate degradation of injected T3 (Little et al. 2013;Lorgen et al. 2015). T3 treatment was therefore applied to promote TH signaling. NH3 is a known TR antagonist in vertebrates (Lim et al. 2002) and A. triostegus in particular (Holzer et al. 2017). NH3 prevents the binding of thyroid hormones (THs) such as T3 on nuclear receptor (TR), therefore impairing the binding of transcriptional coactivators to TR, which therefore remain in an inactive conformation (Lim et al. 2002;Figueira et al. 2011). N3 treatment was thus applied to repress TH signaling by disrupting the TH pathway. Following these injections, fish were transferred to in situ cages in the same nursery area mentioned above. Before their sampling at d2 and d5, these fish were re-injected every morning to maintain the pharmacological treatments active. DMSO and NH3 non toxicity have been ensured in a previous study (Holzer et al. 2017).

Outer slope relocation

Immediately after their capture at d0, some A. triostegus larvae were transferred into in situ cages on the outer slope. Cages were not equipped with coral rubbles nor food to mimic oceanic conditions. This environmental relocation has been shown to delay some aspects of larval coral-reef-fish metamorphosis (McCormick 1999;Holzer et al. 2017). Outer slope relocation was therefore used as an additional 'delayed' metamorphosis condition, allowing comparisons with N3 treatment that pharmacologically represses TH signaling and hence represses metamorphosis processes more importantly.

Elevated temperature and chlorpyrifos (CPF) exposure After capture, d0 fish were immediately transferred to the CRIOBE marine research station. For elevated temperature exposure, fish were maintained in groups of 10 individuals in 70Lx24Wx45H cm aquaria filled with UV-sterilized and filtered (10 µm filter) seawater in a closed circulating system under 12:12 LD cycle (06:00, onset -18:00, offset). Aquaria were daily renewed with fresh coral rubble, providing shelter and constant turf availability for fish. Water temperature was maintained at either 28.5°C, 30.0°C or 31.5°C depending on the exposure condition. 28.5°C was chosen as the basal temperature as this was the mean temperature in Moorea lagoon at the time of the study, and as it corresponds to the mean annual lagoon temperature in this region. + 1.5°C and + 3.0°C temperature shifts were selected following projections for the end of the century for the tropical Pacific waters (IPCC 2014). For CPF exposure, fish were maintained in 20Lx11Wx15H cm aquaria filled with 3 liters of UV-sterilized and filtered (10 µm filter) seawater, and oxygenated with an air stone. Five different exposure conditions were applied: nothing (C, control condition), acetone at a final concentration of 1:1.000.000 (SC, solvent control condition, as CPF was made soluble in acetone), or CPF at a final concentration of 1, 5, or 30 µg.l -1 (1µg, 5µg, and 30µg conditions) as previously done in another study (Holzer et al. 2017). These concentrations are ecologically relevant and reflect appropriately the change of pesticide concentration larval fishes can encounter when settling, from the open ocean, in coral-reef coastal areas (NRA 2000; Leong et al. 2007;Bigot et al. 2016). Aquaria were not equipped with coral rubbles and all water was replaced every day, ensuring the maintenance of water quality as well as a constant concentration of the pesticide or solvent. The inability of fish to feed in this CPF exposure experiment may be one of the reasons explaining the developmental delays observed between control individuals of the CPF exposure experiment and the control individuals of the pharmacological treatment experiment. Comparison between CPF exposed fish with their relative control counterpart of this CPF exposure experiment allows for CPF-specific developmental effects to be quantified.

Eye retina histology

The retina is the light-sensitive tissue of the eye, and is composed of different cell-layers organized ventrally (vs) and dorsally (ds) around the optic nerve (on) (Fig. III.1b). To analyze retina cell layers, we first dissected fish around their eye region in a way allowing us to distinguish the right eye from the left eye, and the dorsal and ventral sides. Tissues were then fixed in Liquide de Bouin before being embedded in paraffin for microtome sectioning (5 µm). We chose cross sections of the retina that were done at similar depth by selecting sections performed at the level of the optic nerve (Fig. III.1b). Sections were then stained using Hematoxylin and Eosin. In A. triostegus from d0 to d8, these sections enabled us to identify different cell segments, types, and layers, among which (i) photocone-external segments (pes, perceiving light signals), (ii) photo-receptor-nuclei (prn, which are, in part, the internal and nuclear segments of the pes), (iii) bipolar cells (bpc, which integrate the synaptic signals originating from the photoreceptors) and (iv) ganglionar cells (ggc, which integrate signals from bpc and create action potential towards the optic nerve) were easily distinguishable (Fig. III.1c). In this study, we only scrutinized the right eye of A. triostegus fish, as this species was evidenced to be lateralized at recruitment, using mostly its right eye to examine predator stimuli (Besson et al. 2017a). Also, we only examined the dorsal side of the retina, as the ventral side undergoes no change at recruitment in another coral reef fish species (McCormick and Shand 1992)

Nostril histology

In fish, the olfactory organ is a paired structure composed of a left and a right nostril, which are each located on the bottom of their respective nasal cavity (Kasumyan 2004). In A. triostegus, these two nasal cavities are located on the dorsal surface, between the eye and the snout edge, and water circulates through them from an anterior to a posterior opening (Fig. III.1d). Each nostril is covered by an olfactory epithelium that can detect water chemicals, and is constituted of several olfactory lamellae forming an olfactory rosette (Kasumyan 2004) (Fig. III.1e). These pictures were obtained using SEM microscopy after a specific tissue preparation involving the dissection of the nostril cavities, fixation in a sodium cacodylate + glutaraldehyde solution for a week before washing and dehydrating samples, and lastly using HMDS to replace critical point drying. Samples were not metalized. SEM pictures were obtained using MiniMEB Hitachi TM3030 (University of French Polynesia). Following these observations and given the macroscopic size of nostrils in A. triostegus, lamellae counting was performed on tissues samples identically prepared, but observed later under a binocular stereomicroscope.

Lateral line histology

In fish, the lateral line (LL) enables individuals to detect weak water motions and pressure gradients, and is mainly composed of superficial and canal neuromasts, which are ciliary sensory structures located respectively on the fish skin or embedded in lateral line canals (Bleckmann and Zelick 2009). Using the same SEM protocol as for nostrils histology, we evaluated the LL system of A. triostegus essentially by scrutinizing its two curved lines starting from behind the eye to the base of the tail (Fig. III.1a), each located on the left and right body flanks. Most of the time, no superficial neuromasts were present on body flanks in d0 to d8 A. triostegus, whereas several LL canals, opening to the environment through a pore, were easily distinguished (Fig. III.1f-g). As for nostrils, LL canal counting was then performed under a binocular stereomicroscope. Counting was performed on either the right or left side of the body, as preliminary experiments revealed no significant difference between the numbers of LL canals present on the left or right flanks of the body. Given that fish body generally curves when critically dried in HMDS, this permitted us to select the best conserved and accessible side of the body for LL canal counting.

Thyroid hormone quantification TH were extracted from frozen-dry fish, following an extraction protocol adapted from previous studies relating TH levels in teleost fish (Tagawa and Hirano 1989;Einarsdottir et al. 2006;Kawakami et al. 2008b) and particularly in coral-reef fishes (Holzer et al. 2017). Fish were individually crushed in 500 µl of methanol using a Minilys and glass beads (3*30 secs, 5.000 rpm), centrifuged at 4°C and supernatant reserved, twice. Fish were grinded one last time with 400 µl of methanol, 100 µl of chloroform, and 100 µl of barbital buffer, centrifuged and supernatant reserved. Pooled supernatant were then dried at 70°C. TH were re-extracted with 400 µl of methanol, 100 µl of chloroform, and 100 µl of barbital buffer twice from the first dried extract. Pooled supernatant was re-dried and the extract reconstituted in 2 ml of PBS 1X for quantification (Roche Elica kit on a Cobas analyser, following the manufacturer's standardized method). TH levels in pg/g of fish were then transformed into relative levels, by selecting d2 fish exposed at the control temperature of 28.5°C as the baseline in the elevated temperature experiment, and by selecting control fish at d2 as the baseline in the CPF exposure experiment.

Behavioral tests

A two-channel Atema choice flume (Atema et al. 2002;Gerlach et al. 2007;Jutfelt et al. 2017) (25 x 5 cm) was used to assess preference of A. triostegus between two water inlets: control seawater (5 liters of UV-sterilized and filtered -10 µm filterseawater from the collection site, Ø) vs 'predator seawater' (5 liters of UV-sterilized and filtered -10 µm filter -seawater from the collection site in which five Lutjanus fulvus predators had been soaked for two hours prior to the experiment, P). Using flow meters, a slow 100 ml.min -1 flow rate was maintained in each water inlet (Fig. III.4a), allowing fish to swim without struggle again the current and ensuring that fish movements within the choice arena effectively reflect cue preference (Lecchini et al. 2017). Prior to each fish observation, dye tests were conducted to confirm laminar flow, without eddies or areas of water mixing, within the choice arena. After releasing the fish in the choice arena, a 2 min acclimation period was observed, then fish position was recorded every 2 sec for 5 min. Water inlets were then switched and another 2 min acclimation period followed by a 5 min period, during which fish position was again assessed every 2 sec, was observed. Preliminary experiments were conducted to ensure the absence of unanticipated biases for the two channel flume (e.g., fish preference for one side over the other, irrespective of water inlets). Fish preference for a water inlet was also ensured by comparing the first and second 5 min recorded periods, before and after water inlet switch. These experiments were performed under red light, to prevent potential visual perturbations such as the observer presence, and to allow comparison with d0 fish, whose preferences were tested at night, also under red light, right after their arrival and capture.

A double choice tank (Fig. III.4a) was used to assess preference of A. triostegus between two visual stimuli: an adjacent aquarium, equipped with an air stone, and containing a 10 cm (standard length) L. fulvus (P condition,Fig. III.4d) vs and adjacent aquarium, equipped with an air stone, and containing no fish (Ø condition,Fig. III.4d). Fish were first placed in the release area, without the ability to access and visualize the adjacent choice areas (opaque panels were positioned at the edges of the release area), for a 2 min acclimation period. Then, opaque panels were removed, leaving the fish able to visualize, through the two opposed transparent walls of the choice tank, the Ø and P visual stimuli, and to access the choice areas. Fish position was then assessed every two seconds during 10 min, using a camera to prevent any external visual disturbances such as an observer presence. Location of visual stimulus (left or right side of the double choice tank) were switched between each fish to ensure the absence of unanticipated biases for the double choice tank (e.g., fish global preference for one side over the other, irrespective of the presented visual stimuli).

Survival arena

Survival tests (Fig. III.4e-g) were conducted in an arena set up in situ in a quiet nursery lagoon area along the north-coast (S17°29'7.0272", W149°49'51.1166"). It consisted of a cubic meter cage (1mx1mx1m) with a hard bottom covered with sand and coral rubbles and four lateral walls equipped with fine mesh (5mm). Each group of fish was tagged separately with a specific color, using visible implant elastomer (Northwest Marine Technology), at least one hour before being released into the arena. All evaluated groups of fish were released simultaneously (8 to 16 fish per group) and allowed to acclimate for 30 min before the introduction of 5 to 8 L. fulvus (minimum 10 cm SL). After 4 hours, predators were removed, and survivors were identified to each initial group using their color tag. Overall survival rate in the arena (total number of survivors/total number of fish initially introduced) and survival rate in each group (number of survivor in group A/number of fish from group A initially introduced) were then calculated, permitting the final calculation of the relative survival rate of each group (survival rate of the group -overall survival rate in the arena). This standardization permitted a better comparison of survival rates among replicate runs that could have been influenced by differences due to the predators (e.g., numbers, behavior, hunger, etc.).

Statistical analyses

All statistical analyses were conducted using the R-cran project free software (R-3.3.1). Multiple means comparisons among fish groups were performed using univariate analysis of variance (ANOVA) followed by Tukey post hoc tests (should a significant difference be detected), when data (or linear model residuals) met assumptions of normality (Shapiro test) and homogenous variance (Bartlett test). When normality and homogenous variance were not verified, Kruskal-Wallis followed by Kruskal-Nemenyi post hoc tests were performed. Mean to mean comparisons were performed using Wilcoxon-Mann-Whitney U-test when n < 15.

III. Extended Data Fig 2 | Fulton's K condition factor variation along recruitment in Acanthurus triostegus

Fulton's K condition factor was assessed from day 0 (d0) to day 30 (d30) post-settlement. Fulton's K condition factor was calculated as follows: K = 100*(W/SL)^3 (with SL, in cm, and W, in g) (Fulton 1904)

IV.1. Foreword

The two previous chapters emphasized the importance of behavioral changes at recruitment in coral-reef fishes, underpinning the potential critical role of brain and sensory systems during recruitment. The study presented in Appendix 4 also demonstrates how brain lateralization processes are at play during metamorphosis and recruitment in a soldier coral-reef fish. However, such processes remain unknown in the model species of this PhD, Acanthurus triostegus. Moreover, the impacts of reef chemical pollutants such as the agricultural pesticide chlorpyrifos on such lateralized brain processes in coral reef fish have yet to be elucidated.

Consequently, this third chapter aims at bringing new information and new perspectives to the two last questions of my PhD, which are: 3 | Are there specific sensory organs and sensory abilities that develop at metamorphosis and what are their endocrine determinants? 4 | Are there environmental factors that act as endocrine disruptors of TH signaling during coral reef metamorphosis and how would it subsequently affect larval recruitment success?

To achieve this, the fieldwork and analyzes presented in this chapter have been performed in close collaboration with the former Master student C. Gache that I supervised with Dr. D. Lecchini during my PhD. I performed with C. Gache most of the field work, from fishing to fish husbandry, while he performed all the lateralization measures. I then conducted all the final analyzes and wrote the manuscript presented in this chapter. This study has been published in Scientific Reports on August, 22 nd 2017. This chapter corresponds to the final manuscript of this study, which is also available in its final pdf format in Appendix 6.

IV.3. Abstract

Lateralization, i.e. the preferential use of one side of the body, may convey fitness benefits for organisms within rapidly-changing environments, by optimizing separate and parallel processing of different information between the two brain hemispheres. In coral reef-fishes, the movement of larvae from planktonic to reef environments (recruitment) represents a major life-history transition. This transition requires larvae to rapidly identify and respond to sensory cues to select a suitable habitat that facilitates survival and growth. This 'recruitment' is critical for population persistence and resilience. In aquarium experiments, larval Acanthurus triostegus preferentially used their right-eye to investigate a variety of visual stimuli. Despite this, when held in in situ cages with predators, those larvae that previously favored their left-eye exhibited higher survival. These results support the "brain's righthemisphere" theory, which predicts that the right-eye (i.e. left-hemisphere) is used to categorize stimuli while the left-eye (i.e. right-hemisphere) is used to inspect novel items and initiate rapid behavioral-responses. While these experiments confirm that being highly lateralized is ecologically advantageous, exposure to chlorpyrifos, a pesticide often inadvertently added to coral-reef waters, impaired visuallateralization. This suggests that chemical pollutants could impair the brain function of larval fishes during a critical life-history transition, potentially impacting recruitment success.

IV.4. Introduction

Brain asymmetry and the preference to use one side of the body over the other to accomplish actions (termed lateralization), has been identified in a variety of vertebrate and invertebrate species (Bisazza et al. 1998;Vallortigara et al. 1999Vallortigara et al. , 2011;;Frasnelli et al. 2012;Rosa Salva et al. 2012). Lateralization processes have been characterized in adult organisms but also in early-life-stages, such as fish larvae (Barth et al. 2005;Dadda et al. 2010), fish juveniles (Roussigné et al. 2012;Roux et al. 2016), chicks (Vallortigara and Andrew 1991) (but see (Cooke 2004;Levin 2005;Güntürkün and Ocklenburg 2017) for reviews on embryonic and post-embryonic development of lateralized organs and acquisition of lateralized behaviors in several vertebrate and invertebrate species). Processes of lateralization can therefore be expected along the whole ontogeny of an animal organism. Brain lateralization is thought to increase cognitive abilities, in particular for decision making when facing novel multi-sensory signals, by enabling individuals to cope with divided attention and to partition, and optimize, the parallel processing of different types of information into the two separate brain hemispheres (Rogers 2000;Vallortigara 2000;Rogers et al. 2004;Sovrano et al. 2005;Vallortigara and Rogers 2005;Rosa Salva et al. 2012). With regards to habitat exploration and response to sensory cues, there are two main theories concerning brain lateralization in animals. The "brain's right hemisphere theory" assumes that the right hemisphere is predominantly used to process information regarding novel items and/or items requiring a rapid behavioral response, while the left hemisphere is used for categorizing stimuli and/or processing information that requires consideration of alternatives (MacNeilage et al. 2009;Rogers et al. 2013). Alternatively, the "valence theory" proposes that the right hemisphere is preferentially used to process information regarding negative stimuli, while the left hemisphere is used for the processing of information regarding positive stimuli (Hook-Costigan and Rogers 1998;Quaranta, A., Siniscalchi, M., Vallortigara 2007;Siniscalchi et al. 2013). While empirical evidence supports both theories, previous studies have generally only considered lateralized responses to social sensory stimuli under normal environmental conditions, and only in a few species of amphibians, birds, freshwater fishes, and mammals (Vallortigara and Andrew 1991;Sergent et al. 1992;Hook-Costigan and Rogers 1998;Sovrano et al. 1999Sovrano et al. , 2001;;Bisazza et al. 2002;Dadda et al. 2003;Sovrano 2004). Therefore, we still lack a comprehensive understanding of the ecological importance of sensory lateralization in marine fishes, as well as in the context of rapidly changing environments.

Among the most rapidly changing environments globally are coral reefs, one of the most biologically diverse, productive, and economically important ecosystems worldwide (Odum and Odum 1955;Connell et al. 1978;Moberg and Folke 1999). Over the past three decades, coral reefs have faced increasingly frequent and severe natural (e.g., cyclones, Acanthaster planci outbreaks) and anthropogenic perturbations (e.g., pollution from agricultural runoff, rising sea-water temperatures, overfishing) leading to global declines in habitat quality, biodiversity and associated ecosystem services (Moberg and Folke 1999;Hughes et al. 2003Hughes et al. , 2014Hughes et al. , 2017;;Bellwood et al. 2004;Hoegh-Guldberg et al. 2007). In the context of these rapidly changing conditions, reef fishes often play an important role in maintaining habitat structure, preserving associated biodiversity, and increasing ecosystem resilience (e.g., through the regulation of coral-algal interactions or by maintaining trophic chain sustainability) (Russ 1984;Hughes et al. 2007;Lecchini et al. 2007b). However, these species are often highly vulnerable to changing conditions, with individual health and population persistence impaired by both localized and global impacts (Hughes et al. 2014). The majority of reef fishes have a bipartite life cycle, consisting of a pelagic larval stage that facilitates dispersal followed by a largely sedentary reef-associated juvenile and adult stage (Leis and McCormick 2002). At the conclusion of the larval phase, the transition from the planktonic to reef environments is referred to as settlement (Leis and McCormick 2002). During this transition, reef-naïve larvae metamorphose into benthic dwelling juveniles and must rapidly identify and respond appropriately to a host of novel multi-sensory signals in order to select a suitable habitat that will facilitate their survival and growth (Planes and Lecaillon 2001;McCormick et al. 2002;Doherty et al. 2004;Lecchini et al. 2005aLecchini et al. , 2005b;;Almany and Webster 2006;Frédérich et al. 2012;Lecchini and Nakamura 2013). The selection of, and persistence in, an appropriate habitat is termed recruitment (Leis and McCormick 2002). Continued and consistent recruitment is essential to maintain reef fish populations and assist with the resilience and recovery of degraded reef fish communities (Doherty 2002). The ability to identify visual cues that indicate the identity and location of predators has shown to increase survival rates during recruitment in a number of coral reef fish species (Lecchini et al. 2005a(Lecchini et al. , 2005b;;Huijbers et al. 2012;Dixson et al. 2014;O'Connor et al. 2016).

The relationship between brain morphology, lateralization, social interactions, and anti-predator behaviors in early-life-stage coral reef fishes remains largely unknown (Lecchini et al. 2005a(Lecchini et al. , 2005b;;Dixson et al. 2014;Barth et al. 2015;Ferrari et al. 2015aFerrari et al. , 2015b;;Roux et al. 2016), with the role of behavioral lateralization during recruitment only initially assessed (Ferrari et al. 2015a(Ferrari et al. , 2015b)). These recent experiments have primarily used detour tests, which examine potential turning bias of individuals in a context of vigilance, and revealed that such lateralized behaviors can be associated with anti-predator response phenotypes and survival benefits (Nilsson et al. 2012;Ferrari et al. 2015aFerrari et al. , 2015b)). However, these studies were conducted on captive-bred larvae, larvae caught in light traps (which may selectively attract certain behavioral phenotypes), or wild caught post-settlement stage juveniles. Experimental use of individuals that have been reared, are from mixed larval, presettlement, and post-settlement stages, or are non-naïve and have past reef experience potentially adds biases to these findings. This prevents a complete understanding of the ecological importance of coral reef fish lateralization at the critical time of recruitment. In addition, current research has yet to examine the contribution of either brain hemisphere in accomplishing a specific task. Short-term exposure to environmental stressors, such as high predation risk or elevated CO2 levels, has been shown to alter lateralized anti-predator behaviors, which can lead to greater mortality rates (Nilsson et al. 2012;Ferrari et al. 2015b). These findings highlight the critical role of lateralization during the recruitment in coral reef fishes, and point towards potential population or ecosystem-level impacts when an environmental stressor is applied (Domenici et al. 2012(Domenici et al. , 2014;;Nilsson et al. 2012;Ferrari et al. 2015a). In particular, anthropogenic impacts on reefs, such as the worldwide increasing influx of chemical pollution (e.g., from pesticides) resulting from coastal agriculture and river runoff (Carvalho et al. 2002;Islam and Tanaka 2004;Fabricius 2005;Laabs et al. 2007;Leong et al. 2007;Lewis et al. 2009;[START_REF] Packett | Agricultural lands are hot-spots for annual runoff polluting the southern Great Barrier Reef lagoon[END_REF]Shaw et al. 2010;Roche et al. 2011;Kroon et al. 2012), are known to severely impact reef biodiversity and the biology and ecology of coastal marine organisms (Fabricius 2005;O'Connor et al. 2016). Among these sources of pollution, the Chlorpyrifos (CPF), an organophosphate insecticide widely used on tropical coastal crops (e.g., sugar-cane and rice crops in Australia and south-east Asia respectively (Cavanagh et al. 1999;Leong et al. 2007;Botté et al. 2012)), is one of the most common waterborne chemical pollutant encountered in coral reefs (Cavanagh et al. 1999;Botté et al. 2012). While several studies have demonstrated its neurotoxicity and endocrine disruption characteristics (Kavitha and Rao 2008;Botté et al. 2012;Deb and Das 2013;Slotkin et al. 2013), the negative impact on coral reef larval fish sensory abilities has also recently been acknowledged (Botté et al. 2012). Moreover, pesticides belonging to the same family have been discovered in important amounts in several fish and crustacean species in French Polynesia (Roche et al. 2011;Salvat et al. 2012). However, whether this pollutions affect or not behavioral lateralized processes remains unknown (Botté et al. 2012).

Here, we examined visual lateralization in the larvae of a common coral reef fish species, the convict surgeonfish Acanthurus triostegus (Randall 1961), during its recruitment phase. If settlement-stage larvae preferentially use their right eye to examine and categorize positive stimuli (e.g., conspecifics, such as in Myrispristis pralinia, a coral reef fish (Roux et al. 2016), or in freshwater fishes (Sovrano et al. 1999(Sovrano et al. , 2001(Sovrano et al. , 2005;;Sovrano and Andrew 2006)) and their left eye for negative stimuli or to inspect novel items and execute rapid responses (e.g., potential predators, such as in freshwater fishes (Sovrano et al. 1999(Sovrano et al. , 2001(Sovrano et al. , 2005;;Sovrano and Andrew 2006)), this would be good evidence for both the "brain's right hemisphere" and the "valence" theories. Using a range of aquarium based (Fig. IV.1) and in situ experiments we tested whether larvae exhibit a naïve preference for their left or right eye when inspecting both positive (mirror self-image, conspecific) or negative (predator) stimuli (Exp. 1) and whether visual lateralization increased or decreased vulnerability to predation (Exp. 2). Lastly, to examine the potential for coastal development to affect this mechanism we determined whether exposure to CPF, at ecologically relevant concentration (NRA 2000;Leong et al. 2007;Botté et al. 2012), would alter the degree of lateralization larvae exhibit (Exp. 3). The three dark gray walls represent the opaque wall of the aquarium, while the light blue wall corresponds to the transparent wall where the visual stimuli are presented. No eye preference was recorded when the fish was perpendicular to the transparent wall (i.e. binocular stimulation) or when it formed an angle larger than 180° with respect to the transparent wall (i.e. fish looking in the opposite direction).

IV.5. Results

For each individual within each experiment, a lateralization index (LI), reflecting the degree of preferential eye use, was calculated as: [(frequency of right eye use) / (frequency of right eye use + frequency of left eye use)] x 100. Values significantly higher than 50% indicate preference for right eye use while values significantly lower than 50% indicate preference for left eye use. Subsequent statistical tests were conducted using these values.

Exp. 1 tested whether larvae exhibit a naïve eye preference when inspecting visual cues from positive (conspecific) or negative (predator) stimuli. In the absence of any visual stimulus, A. triostegus larvae exhibited no eye preference (LI = 41.8 ± 4.6 %, Mann-Whitney U test, U = 32, n = 14, p-value = 0.364) (Fig. 2). However, larvae showed a significant preference when inspecting test stimuli (Kruskal-Wallis rank sum test, 𝜒 2 = 14.173, df = 3, p-value = 0.003), with LI values significantly higher when examining both conspecifics (Nemenyi post-hoc test with Tukey-Dist approximation, p-value = 0.006) or predator cues (p-value = 0.006). With regards to the eye preferred, larvae displayed a significant preference for their right eye when inspecting themselves in a mirror (LI = 57.3 ±2.4 %, Mann-Whitney U test, U = 93, n = 14, p-value = 0.009), when examining a group of five adult conspecifics (LI = 62.5 ± 5.5 %, U = 85, n = 14, p-n = 10, p-value = 0.004) or a predator (LI = 58.8 ± 2.4 %, U = 45, n = 10, p-value = 0.009) (Fig. 4). However, when exposed to CPF at 1 µg.l -1 for five days, larvae no longer exhibited any eye preference when inspecting either the conspecifics or predator (respectively LI= 49.0 ±1.9 % and 44.9 ± 3.7 %, U = 25 and 16, n = 10 and 10, pvalue = 0.846 and 0.262). In addition, when inspecting themselves in a mirror, CPF treated larvae now preferentially used of their left eye (LI = 43.3 ± 2.2 %, U = 1, n = 10, p-value = 0.004) (Fig. 4).

IV.6. Discussion

This study identified a strong preference for the right eye when inspecting stimuli from either a mirror, conspecifics, or a predator. While a preference for the using the right eye, or the left hemisphere of the brain, to categorize familiar stimuli (e.g. same size conspecifics) has also been seen in another coral reef fish: Myrispristis pralinia (Roux et al. 2016), our results suggest that this preference also extends to unfamiliar stimuli. Larvae used in this experiment were naïve without any prior experience of non-larval reef fishes, therefore adult conspecifics and predators are considered unfamiliar stimuli while mirror-images are considered as the image of a familiar conspecific (e.g. larva from the same larval cohort) (Sovrano 2004). The preference for the right eye is intriguing since it is opposite to the preference for the left eye generally observed during the inspection of mirror self-images in freshwater fishes and amphibians, and which conforms with the key role the right telencephalon hemisphere plays in social cognition (Vallortigara andAndrew 1994, 1991;Sergent et al. 1992;Hook-Costigan and Rogers 1998;Sovrano et al. 1999Sovrano et al. , 2001Sovrano et al. , 2016;;Bisazza et al. 2002;Dadda et al. 2003;Sovrano 2004;Sovrano and Andrew 2006;Zucca and Sovrano 2008). The difference suggests that patterns of laterality could be associated with habitat preference (e.g. freshwater vs. sea water) or evolutionary history, and thus driven by ecological pressures or phylogenetic constraints. For instance, the preferential use of the right eye could reflect a greater need for settlement-stage A. triostegus to initially categorize new sensory cues upon recruitment rather than establishing social interactions and aggregations (Lecchini et al. 2007a). We also observed that larvae that displayed a left-eye preference when inspecting a predator held in an adjacent aquarium in Exp. 1, experienced higher survival when exposed to predation risk (Exp. 2). This result could be attributed to a learning process, with right-brain lateralized individuals able to recognize previouslyexperienced threats faster than left-brain lateralized individuals when this has been examined in other fishes (Bibost and Brown 2014). Left-eye dominant larvae could potentially escape predation more efficiently. Even if it is unknown if the right or left eye dominated in Exp. 2 itself, it has been shown that an eye preference displayed during the first observation of a predator (as in Exp. 1) can influence behavioral responses (e.g. subsequent eye preference, turning rates) when facing for a second time that same, no longer unknown, stimulus (Cantalupo et al. 1995;Miklosi et al. 1997). Therefore, after the Exp. 1 conditioning, the left-eye users may adopt a behavioral response to predation that differs from the majority of their right-eye user and notlateralized conspecifics and that could favor their survival (e.g. by disorienting predators or by moving differently within their environment such as seen in mosquitofish (De Santi et al. 2001)). Overall, our results strongly support the "brain's right hemisphere" theory that predicts that the right-eye (i.e. left-hemisphere) is predominantly used to categorize stimuli, while the left-eye (i.e. right hemisphere) is dedicated to attending to events that require rapid behavioral responses, such as predation (MacNeilage et al. 2009;Rogers et al. 2013). In the context of larval settlement, where individuals face a variety of novel sensory cues and constantly high predation risk, our study suggests that there is a survival advantage to being lateralized, with the ability to optimize survival overcoming the obvious disadvantages of displaying predictable asymmetric behaviors. Future experiments brain lateralization in coral reef fishes should include juveniles that have been allowed to contact with older conspecifics, or that are no longer predator naïve, in comparison to others held in solitary confinement, to better understand how asymmetric behavioral patterns are adopted by a population and the importance of learning and selection in lateralization processes.

Our results show that the presence of chlorpyrifos (CPF), one of the most common waterborne pesticides encountered in coral reefs (Botté et al. 2012), can completely diminish or reverse critical lateralization patterns (Fig 4). Oceanic levels of chlorpyrifos contamination have only been assessed, to our knowledge, in the Southern Ocean, revealing levels up to 0.54 pg.l -1 (Bigot et al. 2016). This is much lower than the levels observed in Australian reef surface waters, where contamination have been shown to reach 1 µg.l -1 (NRA 2000), or than in certain Australian and Malaysian river mouths, where contamination levels can reach up to 26 µg.l -1 (NRA 2000; Leong et al. 2007). The concentration of 1 µg.l -1 is therefore ecologically relevant and reflects appropriately the change of pesticide concentration larval fishes can face when settling, from the open ocean, in coral reef coastal areas (NRA 2000; Leong et al. 2007;Botté et al. 2012;Bigot et al. 2016). This loss and reversal of lateralization in fish exposed to chlorpyrifos may further increase larval mortality and impact the subsequent recruitment and maintenance of reef ichthyologic diversity, as evidenced in Exp. 2 (Fig 3). Similar findings have been documented in the coral reef fish Pomacentrus wardi when juveniles were exposed to future ocean acidification conditions (Domenici et al. 2012). In addition, a turning bias, shifting from the right to the left, was observed in P. wardi populations exposed to elevated-CO2 concentrations (930 µatm) (Domenici et al. 2014). Lastly, the same study also revealed how increased temperatures (+ 3°C higher than ambient temperatures) can have a dampening effect on lateralization processes (Domenici et al. 2014). In the context of global degradation of coral reefs, it is striking to observe that pesticide pollution, oceanic acidification, and elevated water temperatures could have similar and potentially interactive effects in impairing lateralized behaviors of marine organisms and subsequent survival to predation, particularly in early developmental stages. With this study, we demonstrate how the worldwide increasing influx of pesticide onto coral reefs, due to expanding coastal agriculture, as well as changing patterns of land use upstream (Carvalho et al. 2002;Islam and Tanaka 2004;Laabs et al. 2007;Leong et al. 2007;Lewis et al. 2009;[START_REF] Packett | Agricultural lands are hot-spots for annual runoff polluting the southern Great Barrier Reef lagoon[END_REF]Shaw et al. 2010;Roche et al. 2011;Kroon et al. 2012), may lead to a loss or reversal of visual lateralization during a critical step of coral reef fish life cycle. This may potentially lead to greater larval mortality rates and reduce subsequent recruitment success. In addition, any potential reduction in the abundance of major herbivorous fishes in coastal marine ecosystems, such as A. triostegus in coral reefs (Randall 1961), due to chemical pollution from organophosphate components (Fulton and Key 2001), could impair the ability of reefs to cope with increasing macro-algal abundance and impact ecosystems recovery and resilience (Hughes et al. 2007;Ledlie et al. 2007).

While coral reefs are experiencing increasing perturbations (Hughes et al. 2014), persistence of species in degraded areas rely on the 'rescue' effect of recruitment (Doherty 2002). The potential for a population to be supplemented by recruits depends largely on pelagic larvae detecting an appropriate habitat, towards which they orientate in order to settle and persist (Doherty 2002). Successful recruitment of individuals could be partly jeopardized by the alteration of lateralized cognitive abilities of larvae caused by local stressors such as pesticides. An understanding of the underlying mechanisms of these physiological and behavioural disturbances requires further investigations.

IV.7. Materials and Methods

Sampling and study site Fish larvae were collected nightly from January to June 2016 using a crest net set on the north-east coast of Moorea Island, French Polynesia (17°29'52.19"S, 149°45'13.55"W). This crest net had a mesh size of 1 mm and was equipped with a rectangular mouth (width: 2 m, height: 1 m) oriented perpendicular to the water flow. Crest nets retain all settlement-stage larvae at the site as they move over the reef crest (Dufour and Galzin 1993;Dufour et al. 1996;Lecchini et al. 2004;Lo-Yat et al. 2011). This sampling technique ensures a non-selective capture (as opposed to light-traps) of wild larval fishes, precisely at the settlement stage, with no prior reef experience (as opposed to the capture of post-settlement juveniles within reefs using light traps or nets) (Besson et al. 2017b). While this method also permits the study of wild individuals caught in situ (as opposed to aquarium reared larval fish), it however does not ensure that the oceanic experience was the same for all larvae. As larval fish primarily recruit to reefs at night (Leis and McCormick 2002), the net was set up at 7 p.m. and larvae were collected at 6 a.m. the following day.

The convict surgeonfish, Acanthurus triostegus (Linnaeus, 1758) has a pelagic larval duration of 53 ± 8 days, after which larvae metamorphose and recruit in shoals on shallow sandy and rubble reef areas (Randall 1961;McCormick 1999;Frédérich et al. 2012). This species was chosen as our model due to the high number of larvae collected in the crest net (n = 278 larvae, size: 23 ± 2 mm). After removal from the crest net, larvae were transferred to CRIOBE research station where they were maintained in individual aquaria filled with UV-sterilized and filtered (10 µm filter) seawater before being tested. Water temperature was maintained at 28.5°C, under 12:12 LD cycle (06:00, onset -18:00, offset). For Exp. 1 and Exp. 2, tests were conducted immediately following collection (within 12 hours). Exp. 3 tests were conducted on the fifth day post-collection. Throughout their entire time in captivity fish were fed a commercial dry food twice daily.

Exp. 1: Do A. triostegus larvae exhibit a left-right visual preference when inspecting either negative (predator or positive (mirror self image, conspecific) stimuli?

Preferences for using the left or right eye to inspect visual stimuli was assessed using a protocol adapted from studies of brain lateralization in freshwater fishes (Sovrano et al. 1999(Sovrano et al. , 2001;;Sovrano 2004;Sovrano and Andrew 2006). The experimental aquarium was a rectangular glass tank (20 L x 11 W x 15 cm H) with all sides covered with white opaque screen except one of the long sides where stimuli were presented. The tank was lighted with a neon lamp (45 W) and filled with UV-sterilized, filtered (10 µm filter) seawater to 15 cm depth. A video camera was mounted above the experimental tank to record the fish position.

Four types of visual stimuli were presented to A. triostegus larvae: (i) Control: a white opaque screen was displayed on the transparent wall, so that all walls of the experimental tank were covered with white opaque screens. This control was conducted to account for any effect of the experimental procedure on fish behavior.

(ii) Mirror: a mirror was placed along the transparent wall, presenting larvae with a visual stimulus of their own reflection.

(iii) Conspecific: an aquarium (30 x 20 x 20 cm) containing juvenile conspecifics (n = 5, size: 80 ± 4 mm) was placed against the transparent wall. The conspecifics were collected with hand nets in shallow sandy areas surrounded by corals at Moorea Island.

(iv) Predator: similarly to (iii), a common reef predator of A. triostegus (Besson pers. obs.), Lutjanus fulvus, (n = 1, size: 162 mm) was presented in an aquarium placed against the transparent wall of the experimental aquarium.

For each stimulus, 14 biological replicates were conducted (one larva per replicate). Between each replicate, all water in the experimental tank was replaced. For each replicate, the use of the right or left monocular visual field (i.e. eye use) was assessed every two seconds for a total of 10 min. Discrimination between the use of the right or the left eye was determined by examining the angle between larva's body orientation and the transparent wall (where the visual stimulus was presented, Fig. 1), following the method established by Sovrano et al. (Sovrano et al. 1999) and used in various eye lateralization studies since (Sovrano et al. 2001(Sovrano et al. , 2005(Sovrano et al. , 2016;;Sovrano 2004;Sovrano and Andrew 2006). If the fish was perpendicular to the transparent wall (i.e. binocular stimulation), or if its body orientation to the transparent wall formed an angle larger than 180° with respect to the transparent wall (i.e. fish looking in the opposite direction), no eye preference was recorded (Sovrano et al. 2001(Sovrano et al. , 2016) ) (Fig. 1). In this study, binocular stimulation never occurred (in all three experiments). Moreover, when a visual stimulus was presented, the times when an eye use preference could not be established (because of the fish looking in the opposite direction of the stimulus) only represented 24.01 ± 2.25 % of the observations in Exp. 1, 23.78 ± 5.82 % of the observations in Exp. 2, and 24.20 ± 2.02 % in Exp. 3. Therefore, when examining a visual stimulus, fishes spent 75 % of the time examining this visual stimulus with either their left or their right eye, and the rest of the time looking in the opposite direction, but never examining the stimulus with both eyes. Observations of the left-eye or right-eye use (Fig. 1) resulted in a lateralization index (LI), reflecting relative eye use: LI = [(frequency of right eye use) / (frequency of right eye use + frequency of left eye use)] x 100 for each stimulus tested (Sovrano et al. 1999). Values significantly higher than 50 % indicate a preference for the right eye, whereas values significantly lower than 50 % indicate a preference for the left eye. For each stimulus, the difference between the measured LI and a 'no eye preference' case (LI = 50 %) was tested using a two-tailed Mann-Whitney U test, in order to identify any potential eye preference. Further statistical analyses to compare LI values between each stimulus were carried out by non-parametric analysis of variance (Wilcoxon rank sum test and Kruskall-Wallis test). For all stimuli, no significant difference was observed between the first 5 min of the trial and the last 5 min of the trial (Kruskal-Wallis test, 𝜒 2 = 92.8, df = 91, p-value = 0.42). To verify that our modified protocol (one single transparent window) did not introduce any behavioral bias compared to the more commonly used protocol (two transparent windows equipped with two mirrors), we performed an additional two-mirror stimulus where an aquarium was equipped with two transparent walls and two mirrors (Sovrano et al. 1999(Sovrano et al. , 2001;;Sovrano 2004). No significant difference was found when comparing LI values obtained in this twomirror protocol and the values obtained with the use of the one-mirror protocol (respectively LI = 59.8 % and 57.3 %, Wilcoxon rank sum test, W = 105, n = 14, p-value = 0.769), therefore we assume that our adapted method provides results comparable to conventional methods. All statistical analyses were conducted using the R-Cran software (R-3.3.1).

Exp. 2: Ecological importance of brain lateralization in larvae facing predation

Using a similar protocol as Exp. 1, larvae were tested individually to determine if they preferentially used the right eye, left eye, or displayed no lateralization when presented with a predator. Larvae were then classified and pooled into three groups according to their visual preference: 'left-eye dominant', 'right-eye dominant' and 'no eye dominant' individuals. Each group was tagged with a different color, using visible implant fluorescent filament (Northwest Marine Technology), at least one hour before being released simultaneously into a caged patch reef (1x1x1 m), built from rubble and live coral, and located in an open sandy area at 2 m depth. After a one hour habituation period, three predators, Lutjanus fulvus (n = 3, size: 165 ± 14 mm), were introduced into the cage. Prior to their introduction, the predators were held for 48 h in aquaria without food to ensure that they were motivated to feed. The experiment was stopped after two hours, predators were removed and the remaining larvae were counted and identified according to their color tag. The overall survival rate was recorded, as well as a survival rate for each group. For each group, a survival index (SI) was calculated: SI = survival rate of the group -overall survival rate in the cage. SI values allowed a comparison between the survivals of each replicate, regardless of the relative predation rate in each technical replicate. This experiment was replicated 6 times, and each time a different tag color was attributed to the groups to account for any potential predation bias towards one color. For each replicate, all larval groups contained the same number of fish (between 4 and 6 larvae per group depending on the replicate). Statistical analyses to compare SI values were conducted by using non-parametric analysis of variance (Wilcoxon rank sum test and Krukall-Wallis test, followed by Nemenyi post-hoc test should a significant difference be detected).

Exp. 3: Effect of chemical pollution on the visual lateralization of A. triostegus larvae

Preliminary experiments were conducted on three groups of n = 10 fish maintained in 30 L x 20 W x 20 cm H aquaria, which were exposed, after collection in the crest net, to chlorpyrifos (CPF) at 1, 30 and 100 µg.l -1 , as previously done in the literature in another coral reef fish (Botté et al. 2012). After 10 days of exposure, larvae at 30 µg.l -1 presented an irregular swimming behavior and most larvae at 100 µg.l -1 died after only one day of exposure. Larvae exposed to the 1 µg.l -1 concentration exhibited no behavioral abnormality and no mortality. Consequently, we tested the effect of a CPF pollution on larvae visual lateralization through two experimental conditions: (A) solvent control (9 liters of UV-sterilized and filtered -10 µm filterseawater + 9 µl of acetone) and (B) 1 µg.l -1 of CPF (9 liters of UV-sterilized and filtered -10 µm filter -seawater + 9 µg of CPF diluted in 9 µl of acetone).

For each condition, larvae were tested for their eye preference as in Exp. 1, when presented with the same visual stimuli as in Exp. 1 (control, mirror, conspecifics and predator), after a five day exposure (to CPF or to the solvent). Consequently, we performed n = 8 (2*4) experiments (control, mirror, conspecifics and predator visual stimulus with fish exposed to solvent vs. fish exposed to CPF at 1 µg.l - 1 ). For each of those 8 experiments, A. triostegus larvae were maintained, after collection in the crest net, in groups of n = 5 to 6 fish in 30 L x 20 W x 20 cm H aquaria. Each aquarium was equipped with an air stone. All water was replaced every day, ensuring the maintenance of water quality as well as a continuous concentration of the pesticide or solvent. A second technical replicate for these 8 experiments, with significantly similar results as in the first replicate, was performed. Consequently n = 10 to 11 biological replicates were conducted for each of these 8 experiments. Statistical tests for LI values analyses were performed as described in Exp. 1.

Ethical approval

All the experiments were approved by the CRIOBE-IRCP animal ethics committee and performed in accordance with the guidelines of the French Polynesia committee for publication and animal ethics.

V. Complete and rapid reversal of the body color pattern in juveniles of the surgeonfish Acanthurus triostegus at Moorea Island (French Polynesia)

V.1. Foreword I think one of the best part of science, and ecology in particular, is to have the chance to observe in situ an intriguing and original phenomenon, especially when it goes against our beliefs.

The convict surgeonfish Acanthurus triostegus is the model species of my PhD and has been largely described in the four first chapters. This species is remarkable for its gregarious behavior as juveniles and adults, and exhibit a relatively simple pigmentation pattern: black vertical bars on a whitish body. One of the most striking parts of A. triostegus's metamorphosis is the rapid pigmentation of the initially transparent larva. I have been looking at thousands of A. triostegus early-life-stage individuals while observing their pigmentation along metamorphosis, with always this consistent acquisition of black vertical bars in the first few hours after settlement, followed by the whitening of the rest of the body in a couple of days post-settlement. Therefore, my surprise was considerable when I observed in some of my aquaria young juveniles with the complete opposite pigmentation pattern: white vertical bars on a blackish body, and exhibiting an unusual aggressive and territorial behavior. I couldn't find anything related to that in the literature, and I made this observation several times, on different individuals, from different larval cohorts. I also checked and confirmed that such a pigmentation pattern can be found in situ, allowing me to think that this observation is somehow not specifically related to an aquarium stress.

To me, the other best part of science is probably to try to elucidate such intriguing phenomena, for example by deciphering their ecological relevance and understanding their mechanism. I really see that as the perfect mix between discovery, science, and ecological relevance, resulting in some sort of exciting challenges. This fifth chapter is therefore different from the three previous ones, as it is not a greatly elaborated research related to metamorphosis or pollutants during recruitment, but rather an observation of a counterintuitive and original phenomenon during larval recruitment in A. triostegus. This led me to discuss with Dr. P. Salis about the potential cellular mechanisms underlying this particular patterning and the fact that individuals can switch from one pigmentation to the other in less than a second. This collaboration resulted in a first description of this original phenomenon, and we have published it in Coral Reefs (2017-10-22). Three videos accompany this manuscript in the version submitted to Coral Reefs. These videos are available on demand by contacting me. This manuscript is available in Appendix 7.

Besson M., Salis P., Laudet V., and Lecchini D. 2017. Complete and rapid reversal of the body color pattern in juveniles of the surgeonfish Acanthurus triostegus at Moorea Island (French Polynesia). Coral Reefs 2017 https://doi.org/10.1007/s00338-017-1631-4.

V.3. Abstract

Color polymorphism is widespread in animals and can be associated with temporary adjustments to environmental variables (polyphenism). In teleost fishes, one of the most remarkable color polyphenism is the background-adaptation in flatfishes. However, such rapid and complete body color and pattern changes remain relatively unexplored in other species. In coral reef fish, Acanthurus triostegus is a gregarious species whose body color pattern consists of black vertical stripes on a whitish body. Here, we describe an entirely reverse body color pattern (white vertical stripes on a blackish body) that we observed in some A. triostegus juveniles in a nursery area at Moorea Island, French Polynesia. In aquaria, we determined that change from one color morph to the other is rapid, reversible, and corresponds to a color polyphenism associated with an unexpected aggressive and territorial behavior.

V.4. Introduction

The occurrence of two or more distinct and genetically influenced color morphs within a single interbreeding population, i.e. color polymorphism, is widespread across the animal kingdom (Endler 1980;Olendorf et al. 2006;Gray and McKinnon 2007). At the individual level, body colors and patterns (i.e. distributions of at least two colors across the body) can also be adjusted in response to environmental factors such as habitat background, temperature, food availability, population density and level of predation risk (for review, see Leimar 2007). This environmentally-influenced color plasticity within individuals of the same genotype is termed color polyphenism, and is highly adaptive when facing changing and selective environments (Gray and McKinnon 2007).

Color polyphenism has been particularly acknowledged in teleost fishes, and is well exemplified by the background-adaptation (camouflage) in flatfishes (Ramachandran et al. 1996;Höglund et al. 2002;Kelman et al. 2006). Body color and pattern adjustments can also be associated with temporary social status (Beeching 1995;Höglund et al. 2002), sexual display (Kodric-Brown 1998;Rhodes and Schlupp 2012), or response to a predation risk (Fuller and Berglund 1996). Changes in body colors and patterns are regulated by a variety of genetic, endocrine and cellular processes such as distribution, morphology, and density of pigment organelles and chromatophores (Fujii and Novales 1969;Sugimoto 2002;Olendorf et al. 2006;Miller et al. 2007). These changes can be fixed or reversible, and can occur within a few weeks or a few seconds, representing long-term or rapid adaptations to the environment (Sugimoto 2002;Sköld et al. 2013). For example, the dusky dottyback (Pseudochromis fuscus) can change its body coloration from yellow to brown and vice versa within two weeks, depending on the reef location and the type of prey available (Messmer et al. 2005;Cortesi et al. 2015), while the paradise whiptail (Pentapodus paradiseus) can switch its head and body reflective stripes from blue to red in less than one second for presumed communication matters (Mäthger et al. 2003).

Apart from the complex body color and pattern changes observed during the background-adaptation in flatfishes and agonistic interactions in cichlid fishes (Beeching 1995), rapid changes (within a few seconds) are generally discrete (e.g. apparition or change of a stripe's color) and relatively unexplored in other fish species (Mäthger et al. 2003;Rhodes and Schlupp 2012;Watson et al. 2014). In coral reef fishes, while long-term (within a few days or weeks) color and pattern changes have been described during ontogeny (Frédérich et al. 2010b), sexual maturation (Kodric-Brown 1998), and mimicry strategies (Cortesi et al. 2015), rapid changes remain largely unknown (Mäthger et al. 2003;Watson et al. 2014).

The convict surgeonfish Acanthurus triostegus is a common herbivorous reef-fish in the Indo-Pacific region (Randall 1961). Adults are encountered around reef patches close to the barrier reef where they live in large aggregations mostly feeding on turf algae (Randall 1961). The color pattern of reef-associated juveniles and adults consists of black vertical stripes on a whitish body (referred here as 'classical color pattern', CP, Fig V .1A) (Randall 1961;Frédérich et al. 2012). Here, in a nursery area of Moorea Island (Lecchini et al. 2005b), in French Polynesia, we observed some A. triostegus juveniles exhibiting a complete reversal of their color pattern: white vertical stripes on a blackish body (referred here as 'reversal of the color pattern', RP, Fig V .1B) and chasing their conspecifics. The aim of this study is to better characterize this RP color morph and to determine if it is associated with behavioral and/or developmental characteristics and corresponds to a color polyphenism. To do so, we collected large cohorts (n > 50) of reef-naïve A. triostegus larvae while they colonized the reef at Moorea Island, and raised them, in aquaria, until the appearance of the RP pattern. We demonstrated that the CP to RP color morph change is rapid and reversible, and corresponds to a color polyphenism associated with an unexpected territorial behavior.

V.5. Materials and Methods

Fish sampling and raising

Fish larvae were collected nightly in March 2015 using a crest net on the north-east coast of Moorea Island, French Polynesia (17°29'52.19"S, 149°45'13.55"W), close to the nursery area where RP juveniles were observed. The crest net had a mesh size of 1 mm and was equipped with a rectangular mouth (width: 2 m, height: 1 m) oriented perpendicular to the water flow. The crest net retains all settlement-stage larvae as they move over the reef crest and enter the net (Dufour and Galzin 1993). As larval fish primarily recruit to reefs at night, the net was set up at 7 p.m. and larvae were collected at 6 a.m. the following day. Three large cohorts of A. triostegus larvae were collected consecutively (n = 53, 60 and 69 respectively). After removal from the crest net, individuals were transferred to the CRIOBE research station where they were maintained, with only their respective larval cohort, in a 200 liters aquarium in an open water circuit (sea water was pumped from the lagoon area on the north shore of Moorea). Water temperature was maintained at 28.5°C, under 12:12 LD cycle (06:00, onset -18:00, offset), and fish were fed turf algae using live rocks and rubbles that were changed daily. At the end of the study, all CP and RP fish were released in the nursery area close to their collection site.

Juveniles' body color pattern, developmental stage, and behavior CP pattern was defined as black vertical stripes on a whitish body (Fig V .1A) (Randall 1961;McCormick 1999), while RP pattern was defined as the opposite color pattern: white vertical stripes on a blackish body (Fig V .1B). In the three cohorts studies, color morph respective prevalence (i.e. percentage of CP and RP fish in each of the three cohort) was assessed when RP juveniles were first observed, at day 7 postsettlement (Fig V .2A). The developmental stage of CP (n = 14) and RP (n = 10) fish was also assessed at that time, by measuring individual body depth (body height divided by standard length), as it is a good indicator of post-settlement development in A. triostegus (McCormick 1999;McCormick et al. 2002). Body depth measurements were then compared between RP and CP, as well as with measurements on A. triostegus juveniles from day 0 to day 11 post-settlement obtained in parallel to this study (raised in smaller groups of 20 individuals in lagoon in situ cages) (Fig V .2B). Lastly, aggressiveness of CP (n = 24) and RP (n = 24) fish was assessed at day 7 post-settlement, by measuring the percentage of time each spent with their dorsal fin erected (Fig V .2C), as evidenced in zebrafish and in a cod species (Brawn 1960;Gerlai 2003). A high percentage of time with the dorsal fin erected was considered as an aggressive and territorial behavior.

V.6. Results and Discussion

At day 7 post-settlement, 13.3 ± 2.5 % of A. triostegus juveniles had turned their color pattern from CP to RP (Fig V .1A-B, Fig V .2A). This color morph change can occur within a few seconds (Supplementary Video 1), and is also reversible, as RP juveniles turned instantly to CP and mixed up with the CP shoal when disturbed by a net or by the addition of a new rubble in the aquarium (Besson pers. obs.). This confirms that the CP to RP transition corresponds to a reversible, complete and rapid color pattern change. No difference in body depth was observed between CP and RP juveniles (Wilcoxon rank sum test, W = 67, p-value = 0.886) (Fig. V.2B). For both color morphs, the body depth did not exhibit any delay or advance compared to the body depth variations observed in A. triostegus juveniles that we raised in situ in smaller groups in parallel to this study (Fig V.2B). This suggests that the differences between the two morphs are not related to an ontogenetic difference. Actually, individuals turning from CP to RP were not predictable or did not show any prior detectable phenotypic difference. RP color morph was initially observed in situ in a dense nursery area of Moorea Island (Lecchini 2005), and was confirmed in aquaria in large juvenile groups (originating from the same larval cohort) during periods of intense foraging and decreasing resource availability. Introducing large amounts of turf algae and diminishing the number of juveniles in the aquarium delayed and even prevented the apparition of the RP color morph (Besson pers. obs.). Altogether, this suggests that this color plasticity corresponds to a color polyphenism, and that conditions of high juvenile density and low resource availability may be responsible for the apparition of the RP color morph.

RP CP

Interestingly, RP color morph was associated with a unique solitary, aggressive, and territorial behavior, with RP juveniles chasing their conspecifics (Supplementary Videos 1-3). This was exemplified by the significant difference between CP and RP juveniles in the percentage of time spent with the dorsal fin erected (7.6 ± 3.6 vs. 84.3 ± 4.0 % respectively, Wilcoxon rank sum test, W = 569, p-value < 0.001, Fig V .2C), which illustrates the aggressive behavior of RP individuals (Brawn 1960;Gerlai 2003). Consequently, the presence of the RP color morph resulted in a newly-organized fish community, with RP juveniles defending their territory while CP juveniles swam or foraged in groups above these guarded areas (Fig. V.1C, Supplementary Videos 2-3). Therefore, the CP to RP transition is a rare observation of a color morph change involving a rapid and complete reversal of the body color pattern that is not associated with camouflage (Ramachandran et al. 1996;Höglund et al. 2002;Kelman et al. 2006;Sköld et al. 2013;Watson et al. 2014). Besides, the RP color morph is also not related to a stressed or nocturnal pigmentation, which actually consists of the CP pattern with black rectangles between each stripes (see V. Supplementary Fig. 1). Actually, this is reminiscent of the situation in the cichlid fish Astronotus ocellatus, where agonistic interactions between males can lead to a rapid color pattern change (lightening of the body color and apparition of white vertical stripes) in individuals defeated in combat (Beeching 1995).

At the cellular level, body color and pattern changes can be divided in two categories: morphological and physiological color changes (MCC and PCC) (Fujii and Novales 1969;Leclercq et al. 2010). MCC often result from long-term changes in the distribution, morphology, and density of chromatophores (pigment cells) while PCC originate from rapid motile movements of pigment organelles (e.g. melanosomes) within the chromatophores (Sugimoto 2002;Leclercq et al. 2010;Sköld et al. 2013).

Here, the rapidity of the CP to RP color morph change suggests that the color and pattern changes result from a PCC, and that condensation/dispersion of melanosome in the melanophores may be responsible for the rapid and reversible black/white color reversal (Zhu and Thomas 1997). However, a cellular study of this process needs to confirm this hypothesis, supposing that one can sample the RP juveniles before they turn back their pattern to CP.

This study demonstrates color polyphenism in juveniles of A. triostegus, revealing fish ability to switch from one color pattern to its complete opposite within a few seconds. RP color morph was not observed in adults, suggesting that this color polyphenism may result from a juvenile-specific adaptive behavior to force some individuals to quit the cohort in case of low resource availability.

V.7. Additional information VI.1. Coral-reef fish larval recruitment is a TH-controlled metamorphosis

In this thesis, I have investigated the morphological, physiological, and behavioral changes occurring during A. triostegus recruitment. In particular, this research brings a new understanding of the changes encountered in the whole digestive system, from the dentition to the intestine remodeling, along with a precise description of the shifts in 16S and 18S gut content. Furthermore, this thesis also provides a first characterization of A. triostegus sensory system and their maturation during recruitment. There is a substantial amount of studies that have described other and complementary morphological features in A. triostegus recruitment (McCormick 1999;McCormick et al. 2002;Frédérich et al. 2010aFrédérich et al. , 2012)). However, none of them have related these transformations and developmental processes to any endocrine system. Coral-reef fish metamorphosis has been known for more than seventy years (Breder 1944). Given the similarities between the metamorphoses of coral-reef fish and other non-reef teleost fishes (McMenamin and Parichy 2013), the involvement of thyroid hormones (TH) was expected. Moreover, first evidences of their role was provided through the study of pigmentation and growth in larval coral reef fishes in aquaculture (de Jesus et al. 1998;[START_REF] Trijuno | Effects of thyroxine and thiourea on the metamorphosis of coral trout grouper Plectropomus leopardus[END_REF]. However, these studies did not permit deciphering the potential coupling between recruitment to the reefs (ecological event) and metamorphosis (endocrinological event), using wild captured larvae. Moreover, data from other teleosts are scarce and there is no general framework to understand the role of environment in teleost metamorphosis and how this event integrates in the fish ecology. Here, this research demonstrates that TH levels surge in larvae prior to reef entry, and decrease once they settle in the reef. This pattern has been observed not only in A. triostegus, but also in phylogenetically distant coral-reef fish species (e.g., Chromis viridis and Rhinecanthus aculeatus). As these TH profiles are reminiscent of the situation in amphibians and other non-reef teleost fishes, and given that the changes undergone by A. triostegus larvae at recruitment are controlled by these TH, this work provides the first evidence that larval recruitment of coral-reef fish corresponds to a TH-controlled metamorphosis.

Species-specific variations on this general theme could be observed, as this research did not demonstrate this pattern in all coral-reef fish families. To date, this project has examined TH profiles at recruitment in six species: A. triostegus, C. viridis, R. aculeatus, Chaetodon lunula, Ostorynchus angustatus and Platax orbicularis. TH level patterns were similar in these six species (i.e. TH surge before reef entry, TH decrease after reef entry), although the capture of oceanic larvae was not possible for some of them. The batfish species, P. orbicularis, was not sampled in the wild but in an aquaculture facility in Tahiti. While a peak of TH was observed at day 12 post hatching (corresponding to the time when most morphological changes are observed), further confirming the involvement of TH in P. orbicularis metamorphosis, this result was not included in this thesis as it could not be related to larval recruitment (as animals were raised and fed artificially). In a near future, TH profiles at recruitment of at least five more species (Zebrasoma scopas, Zebrasoma veliferum, Ctenochaetus striatus, Canthigaster bennetti and Epinephelus socialis) will be available. This will provide an extraordinary opportunity to study how variations in the magnitude of TH changes can be shaped by evolution and species-specific life history traits such as pelagic larval duration, pigmentation status at settlement (as some species' larvae colonize the reef fully pigmented while other are still transparent) and size at settlement (as some species are very small at recruitment, compare to the size of the adults, e.g. Serranids, while others are much bigger, e.g. Mullids) (McCormick et al. 2002). Given the considerable diversity of life history strategies they propose, coral reef fish could be excellent models to decipher how recruitment strategies are elicited by changes in hormonal systems. Interestingly, this is reminiscent of the situation in stickleback, where differences in TH signaling between two ecotypes support life history trait variations (Kitano et al. 2010;Kitano and Lema 2013).

VI.2. Coral-reef fish sensory ecology at recruitment

One of the key factors influencing survival at recruitment is settlement-site selection, with larval fishes using acute sensory systems to locate appropriate benthic habitats (Lecchini et al. 2007a;Mitchell et al. 2011;Brooker and Dixson 2016). As coralreefs are rapidly degrading globally due to anthropogenic activities (Hughes et al. 2003;Hoegh-Guldberg et al. 2007), a large number of studies have examined how larval sensory abilities (e.g., recognition of olfactory cues from conspecifics or predators) are impaired by factors such as global change and water pollution (Dixson et al. 2014;Johansen et al. 2017;Lecchini et al. 2017). However, to date no study has integrated molecular, anatomical, and behavioral approaches to identify the processes that impair larval sensory ecology and subsequent survival. In this context, this thesis brings first insights on the potential coupling between sensory organ maturation and subsequent sensory abilities. It demonstrates how an impaired sensory system maturation is correlated with (i) a limitation in individual's ability to identify and distinguish between critical sensory cues during recruitment, and (ii) reduced survival rates when facing predation. This is a very high topic in coral reef fish sensory ecology, as many efforts are devoted to examine the mechanisms by which larvae locate and select their settlement reef habitat (Dixson et al. 2014;Brooker and Dixson 2016;Johansen et al. 2017;Jutfelt et al. 2017;Lecchini et al. 2017). This thesis has evidenced that TH are key mediators for the maturation of sensory organs in A. triostegus. Further investigations are required to properly examine these maturation processes and their relevance in sensory abilities. For example, only variations among the different retina cell densities were examine during this thesis, while it would be more informative to compare ratios of densities between cell perceiving the visual signals (e.g. photoreceptors) and those that integrate these signals (e.g. bipolar cells) [START_REF] Shand | Ontogenetic changes in retinal structure and visual acuity: a comparative study of coral-reef teleosts with differing post-settlement lifestyles[END_REF]Lara 2001). Such comparisons should therefore be performed in the analyses presented in chapter II, before proceeding to its submission.

Histological analyzes of sensory organs represent a tremendous amount of work, and reaching the necessary conditions to properly assess larval fish sensory abilities can sometimes be difficult (Hale et al. 2017;Jutfelt et al. 2017). Therefore, I propose that simply monitoring TH levels in coral-reef fish recruits could be an easy and rapid alternative to these fastidious analyzes for characterizing the sensory states of coral-reef fish populations, for example when comparing a degraded vs. a pristine habitat.

VI.3. Coral-reef fish metamorphosis is prone to endocrine disruption

Among the many stressors affecting coral-reefs, increasing levels of chemical pollution (e.g., agricultural pesticides and petrogenic compounds), which followed the intensive agricultural and industrial developments (Fabricius 2005;Burns 2014), are sometimes neglected in comparison to the elevated sea water temperature threat (Nilsson et al. 2009;McLeod et al. 2013;Hughes et al. 2017). However, this chemical pollution is most often local, and should therefore be theoretically easier to control. Among these pollutants, the agricultural pesticide chlorpyrifos (CPF) is one of the most common encountered in coral-reefs waters (Shaw et al. 2010). CPF is known to be a thyroid disruptor with adverse effects on fish behavior (Sandahl et al. 2005;Kavitha and Rao 2008;Roche et al. 2011;[START_REF] Yang | Chlorpyrifos-oxon disrupts zebrafish axonal growth and motor behavior[END_REF]Botté et al. 2012;Deb and Das 2013;Juberg et al. 2013;Slotkin et al. 2013). In this thesis, I demonstrated that CPF exposure causes a variety of developmental and behavioral defects in A. triostegus at recruitment. Overall, CPF exposure reduces TH levels, delays intestine lengthening and remodeling, delays sensory organs maturation, inhibits grazing activity and impairs visual lateralization. As A. triostegus is one of the major herbivorous fish in coral reefs, this can severely impair the ability of coral reefs to recover after an algal invasion (e.g., after a bleaching event). Indeed, a reduced abundance of keystone algal herbivores, may impair their ability to maintain or rescue coral-dominated habitats, further threatening the ecological and economic resilience of coral-reef ecosystems (Russ 1984;Almany et al. 2007;Hughes et al. 2007Hughes et al. , 2014;;Ledlie et al. 2007;Munday et al. 2008;Pratchett et al. 2008). Oceanic levels of chlorpyrifos contamination have only been assessed, to my knowledge, in the Southern Ocean, revealing levels up to 0.54 pg.l -1 (Bigot et al. 2016). This is much lower than the levels observed in Australian reef surface waters, where contamination have been shown to reach 1 µg.l -1 (NRA 2000), or than in certain Australian and Malaysian reefs near river mouths, where contamination levels can reach up to 26 µg.l -1 (NRA 2000; Leong et al. 2007). The range of concentrations used in this thesis (1 to 30 µg.l -1 ) are therefore ecologically relevant and reflects appropriately the change of pesticide concentration larval fishes can face when settling, from the open ocean, in coral reef coastal areas (NRA 2000;Leong et al. 2007;Botté et al. 2012;Bigot et al. 2016). Altogether, this indicates that ambient levels of CPF pollution are already high enough to severely disrupt essential processes of coral-reef fish ecology. Considerable efforts should be made to reduce this pollution, especially as this could be handle locally, and with short-term effects.

Lastly, this thesis highlights the need for a better understanding of the combined effects of all the disturbances that affect coral-reef ecosystems. Assessing the impact of combined elevated temperature and CPF exposure on A. triostegus recruitment processus is something that is already planned, as mentionned in chapter III. In particular, given that both elevated temperature and CPF exposures separately repress metamorphic processes, I expect that their combination would amplify their impacts. Additionnaly, it would be particularly interesting to combine these stressors with acidification, another considerable threat to coral-reef ecosystems (Hoegh-Guldberg et al. 2007). Oceanic acidification has been evidenced to severely impair coral reef fish sensory abilities and brain function (Munday et al. 2009;[START_REF] Simpson | Ocean acidification erodes crucial auditory behaviour in a marine fish[END_REF]Ferrari et al. 2012aFerrari et al. , 2012b;;Leduc et al. 2013;[START_REF] Rossi | Lost at sea : ocean acidification undermines larval fish orientation via altered hearing and marine soundscape modification[END_REF]Lecchini et al. 2017). Unfortunately, I did not initially focus on the role of oceanic acidification on A. triostegus metamorphic processes during this thesis. While a few trials were performed at the end of my thesis, with preliminary results showing an interesting and counterintuitive trend with relatively increased TH levels in A. triostegus at day 2 and day 5 post-settlement, this topic definitely requires further investigations in a near future. While this thesis has shed some light on the importance of metamorphosis and TH in coral-reef fish larval recruitment facing anthropogenic pressures and endocrine disruption, most coral reef fish recruitment processes have yet to be elucidate. Additionnaly, given that coral organisms and marine invertebrates also possess pelagic larval stages that undergo metamorphosis when settling into the reefs (Burke 1983;Grasso et al. 2011), deciphering how ambient and predicted environmental conditions could disrupt their respective endocrine system during their vulnerable recruitment-stage, is essential.

VI.4. Conclusion

In this thesis, I have examined the recruitment phase of the convict surgeonfish Acanthurus triostegus, and determined that the ecological, morphological, physiological and behavioral changes occurring at recruitment correspond to a metamorphosis mediated by thyroid hormones (TH). Then, I investigated whether this metamorphosis is prone to endocrine disruption under anthropogenic disturbances such as elevated sea water temperature and agricultural pesticide pollution. I demonstrated that such pressures can reduce TH levels at a critical developmental stage in coral-reef fishes, impairing their metamorphic processes such as sensory organ maturation and sensory abilities acquisition, further increasing their mortality rates. Overall, this thesis is a holistic analysis that addresses molecular, morphological, histological, and behavioral assays of multiple stressors affecting coral-reef fish recruitment. It indicates the importance of a proper endocrine function during coralreef fish recruitment, highlighting the need for a better understanding of these processes for coral-reef conservation.

Abstract

The persistence and sustainability of coral-reef fish populations depends on the continued larval recruitment, i.e. successful settlement by pelagic larvae into adult reef habitats and postsettlement survival through metamorphosis to a juvenile stage. There is growing evidence that changes to water conditions due to global change and waterborne pollution can impair coral-reef fish sensory abilities to locate settlement habitats that maximize growth while minimizing mortality risk. However, the inner mechanisms of such impairments remain less well studied. In this thesis, I have examined the recruitment phase of the convict surgeonfish Acanthurus triostegus, and determined that the ecological, morphological, physiological, and behavioral changes occurring at recruitment correspond to a metamorphosis mediated by thyroid hormones (TH). Then, I investigated whether this metamorphosis is prone to endocrine disruption under anthropogenic disturbances such as elevated sea water temperature and agricultural pesticide pollution. I demonstrated that such pressures can reduce TH levels at a critical developmental stage in coral-reef fishes, impairing their metamorphic processes such as intestine remodeling, sensory organ maturation, and sensory abilities acquisition, further increasing their mortality rates. Overall, this thesis is a holistic analysis that addresses molecular, histological, anatomical, and behavioral assays of multiple stressors affecting coral-reef fish recruitment. It indicates the importance of a proper endocrine function during coral-reef fish recruitment, highlighting the need for a better understanding of these processes for coral-reef conservation.

Mots Clés

  Summary .............................................................................................................

Figure

  Figure I.1 | Coral reef types in French Polynesia A: Barrier reef (BR) and fringing reef (FR) in Maupiti Island. Fringing reefs can develop in shallow waters along the coasts while barrier reef corresponds to a coral reef growing parallel to the shoreline and separated from it by a lagoon. B: Tetiaroa Atoll. Atolls are rings of coral reefs and small islets surrounded by the ocean and surrounding a sandy lagoon where a volcano was standing before being submerged. Pictures: M. Besson.

Figure I. 7 |

 7 Figure I.7 | Settlement patterns or larval coral reef fishes in French Polynesia Green and dark green lines indicate relative abundance of settlers captured in two crest nets in Moorea, while light blue, blue, and dark blue lines indicate relative abundance of settlers captured in three nets in Nengo Nengo. Circular points indicate a crest net with a westward orientation, triangular points indicate a northward orientation and square points indicate a south-eastward orientation. Black and white circles above each panel indicate the lunar phases: new moon (black circle), first quarter, full moon (white circle) and last quarter. Adapted from Besson (2017).

Figure I. 13 |

 13 Figure I.13 | Thyroid follicles in the sub-pharyngeal region of Acanthurus triostegus Schiff reagent and hematoxylin staining of a 5 µm histological section of A. triostegus subpharyngeal region (primarily fixed in Liquide de Bouin). The numerous thyroid follicles are revealed in indigo. Scale bar indicates 50 µm.

Figure I. 14 |

 14 Figure I.14 | Acanthurus triostegus metamorphosisImmediately after their entry in the reef at night, metamorphosing and transparent-larvae (left) and metamorphosed and pigmented-juveniles (right) can be encountered in the same nursery areas. The vertical bars of black pigmentation can appear in less than two hours. Picture: M. Besson.

Figure

  Figure I.15 | Location of Moorea Island in French Polynesia A: Location of French Polynesia within the Pacific Ocean. B: Location of Moorea Island within French Polynesia. C: Moorea Island. Scale bar indicates 2 km. Adapted from Besson (2017).

Figure II. 1 |

 1 Figure II.1 | Changes in Acanthrurus triostegus during larval recruitment. Error bars represent standard errors. *: p-value < 0.05; **: p-value < 0.01; ***: p-value < 0.001. (A) Schematization of the reef colonization by a larva (left) turning into a juvenile (right). The background color shading centered into the reef crest is re-used in following figures. (B) Weight variation during larval recruitment. n = 3 biological replicates for 5-to-10 km offshore fish (far), n = 5 for 2 km offshore fish (near) and n = 297 for crest to day 8 (d8) fish. Statistics: mean comparisons (Wilcoxon-Mann-Whitney U-test). (C-G) Variation in pigmentation and morphology at different sampling points, (C) crest larva, (D) day 2 juvenile, (E) day 3 juvenile, (F) day 5 juvenile and (G) day 8 juvenile; scale bars indicate 0.5 cm. The white square represents the magnified region shown in the line below. These pictures are representative of n > 50 fish (biological replicates) per developmental stage, sampled at n > 20 different times during the year (technical replicates). (H-L) Close-up on the pigmented middle body band. Scale bars indicate 0.2 cm. These pictures are representative of n > 50 fish (biological replicates) per developmental stage, sampled at n > 20 different times during the year (technical replicates). (M-Q) X-Ray microtomography of larva, dentition A, B or C are highlighted respectively in red, blue or green. Scale bars indicate 0.5mm. These pictures are representative of n = 2 fish (biological replicates) per developmental stage. (R-V) Histological sections (5 µm width) of intestines with haematoxylin/eosin staining. Scale bars indicate 200µm. These pictures are representative of n = 6 sectioning technical replicates, performed twice (sampling technical replicates), on n = 2 fish (biological replicate). (W) Intestine length variation of A. triostegus during its larval recruitment. n = 9 to 14 biological replicates for each developmental stage (from n = 2 technical sampling replicates). Statistics: mean comparisons (Wilcoxon-Mann-Whitney U-test). (X) Heatmap clustering of the 18S sequences identified in the intestine content of crest individual (n = 3, left panel), day 8 juveniles (n = 3, middle panel) and adults (n = 3, right panel). Each panel is made of three columns corresponding to one individual. Each line corresponds to one taxonomic group found in the intestines: Spermatophyta (dark green), Conoidasisa (cyan), Dinophyceae (light yellow), Protostomia (light mink), Phaeophyceae (brown), Florideophycae (dark pink), Chlorophyta (light green), Stramenopiles (orange) and Ascomycetes (grey).

Figure II. 2 :

 2 Figure II.2: Thyroid hormone signaling during Acanthurus triostegus metamorphosis. Error bars represent standard errors. *: p-value < 0.05; **: p-value < 0.01; ***: p-value < 0.001. (A) T4 and (B) T3 dosage: Y-axis: T4 and T3 levels in pg.g -1 ; X-axis: sampling point. n = 3 for 5 to 10 km offshore fish (far), n = 5 for 2 km offshore fish (near), n = 297 for crest to day 8 (d8) fish. Statistics: mean comparisons (Wilcoxon-Mann-Whitney U-test). (C) Expression level of TRα-A, TRα-B, TRβ and Klf9 by qPCR, normalized with Pold2 and Rpl7. Y-axis; Relative

Figure II. 3 |

 3 Figure II.3 | Impact of the environment and thyroid hormone signaling on Acanthurus triostegus metamorphic processes.

Figure II. 4 |

 4 Figure II.4 | Thyroid hormone levels in distant coral reef fish at larval recruitment.Error bars represent standard errors. *: p-value < 0.05; **: p-value < 0.01; ***: p-value < 0.001. TH levels of: Rhinecanthus aculeatus (upper), Chromis viridis (middle up), Chaetodon lunula (middle low) and Ostorhinchus angustatus (lower). n = 3 to 5 biological replicates for each condition. First column is T4, second column is T3. Y-axis: T4 or T3 level in pg.g -1 . X-axis: sampling point (developmental stage). The phylogenic relationship between the species is plotted on the far left of the figure. Statistics: for each species, comparisons of each mean to the day 1 (d1) mean (Wilcoxon-Mann-Whitney U-test).

Figure

  Figure II.6 | Model of Acanthurus triostegus metamorphosis and disruption by chlorpyrifos. (A) Schematic representation of coral reef fish larval recruitment. TH level is plotted on the Y axis and development on the X axis. The onset of metamorphosis is in the open ocean and environmental influence upon development is plotted on the X-axis. (B) Consequences of undisturbed (upper panel) and CPF disturbed (lower panel) metamorphosis on grazing activity, and subsequent algal spreading and potential coral survival. Solid red bar indicates inhibition; dotted red line indicates lack of inhibition.

Figure

  Figure II.1 -figure supplement 1 | Changes in Acanthurus triostegus intestine and teeth during larval recruitment. (A-O) Complete guts histology of Acanthurus triostegus after reef colonization. First column: anterior part of the intestine (first cm), middle column: medium part (variable), last column: posterior part of the intestine (1 cm), (A-C): crest individual, (D-F): 2 days juvenile, (G-I): 3 days juvenile, (J-L): 5 days juvenile, (M-O): 8 days juvenile. Scale bars represent 200 µm. These pictures are representative of n = 6 section technical replicates for each of the proximal, middle and distal intestine, and were performed twice (sampling technical replicates), on n = 2 fish (biological replicates). (P) Heat map of bacterial gut community. Each column is one individual. Each row is bacterial taxa. Only the taxa with differential representations between the samples are shown. Sampling stages: day 2, day 5, day 8 and adult are indicated below the heat map. The shape of each of this developmental stage is schematized over the heat map. Each column is highlighted by a circle, the difference in shade between the circles emphasizes the stage difference. The color of each square in the heat map is proportional to the abundance of the taxa (row) in the sample (column). (Q) µCT scan of a crest larva, lateral view. Dental family C are highlighted in red and dental family B in blue. (R) µCT scan of an adult, frontal

Figure

  Figure II.2 -figure supplement 2 | Primers used in this study. (A) Degenerated primers used for the cloning of the A. triostegus genes in this study. (B) qPCR primers used in this study. (C) Primers used for 18S mass sequencing. The overlapping nucleotides between 18SR and the blocking primer are indicated in bold. The mean annealing temperature of each primer is indicated in the last column.

Figure II. 2 -

 2 Figure II.2 -figure supplement 3 | List of thyroid hormone receptors sequences used for the phylogeny. Species, genes, databases and accession numbers are indicated

Figure

  Figure II.5 -figure supplement 2: Pigmentation patterns and chlorpyrifos exposure in A. triostegus at metamorphosis (A) T4 level in pg.g -1 of day 2 (d2) and day 5 (d5) fish exposed to different conditions: solvent control (C, acetone, white bars), or exposed to CPF at 1 µg.l -1 (1, light yellow bars), 5 µg.l -1 (5, yellow bars) or 30 µg.l -1 (30, dark yellow bars). Exposure started right after crest capture. n = 10 fish in each condition.. See Figure 5 -source data 5 for detailed data and statistics. (B) Pigmentation patterns and in A. triostegus exposed to different conditions: solvent control, or CPF at 1 µg.l -1 , 5 µg.l -1 , or 30 µg.l -1 at day 2 (d2) and day (d5) during larval recruitment. These pictures are representative of n = 2 chlorpyrifos CPF exposure technical replicates performed on n = 10 fish (biological replicates) per condition and per day.

Figure III. 1 |

 1 Figure III.1 | Metamorphosis and sensory system changes at recruitment inAcanthurus triostegusa, Morphological and pigmentation changes in A. triostegus from day 0 (d0) to day 8 (d8) following their entry in the reefs. Scale bar indicates 1 cm. b, Cross section in the eye retina stained in hematoxylin/eosin, presenting the dorsal side (ds) the ventral side (vs) and the optical nerve (on). The dotted gray rectangle represents the magnified region shown in (c). Scale bar indicates 100 µm. c, Cross section in the eye retina stained in hematoxylin/eosin, presenting the ganglionar cell layer (GCL), the inner nuclear layer (INL) and the outer nuclear layer (ONL) that contain ganglionar cells (ggc), bipolar cells (bpc) and photoreceptors nuclei (prn). In the anterior part of the retina, photocone external segments (pes) are identified. Scale bar indicates 50 µm. Schematic representation of the four cell types are presented leftward to their legends. d, SEM picture localizing the right nasal cavity (dotted white rectangle) between the eye and the snout, where the right nostril is located. Scale bar indicates 1 mm. e, SEM picture of a right nostril rosetta, with 11 lamellae (light green dots). Scale bar indicates 100 µm. f, SEM picture of the right body flank where lateral line (LL) and LL canals are visible (light blue arrows). The dotted white rectangle represents the magnified region shown in (g). Scale bar indicates 1 mm. g, SEM picture of a lateral line canal (blue arrow) and its communication pore. Scale bar indicates 100 µm. h-m, Error bars indicate standard errors (error bars are not visible when standard errors are too small). Circles bordered in black represent means while alpha transparent circles in the background represent each data. h, Changes in ggc density along recruitment (no change from d0 to d8, p=0.986, n=57, ANOVA). i, Changes in bpc density along recruitment. A<B (p<0.001) and B<C (p=0.011) (n=57, ANOVA+TukeyHSD). j, Changes in prn density along recruitment. A<B (p=0.014) and B<C (p=0.025) (n=57, ANOVA+TukeyHSD). k, Changes in pes density along recruitment A<B (p=0.0.28) (n=57, A ANOVA+TukeyHSD). h-k, Densities are expressed in 10 -3 cell.µm -2 except for pes whose density corresponds to the number of pes in a 50 µm wide area. l-m, Changes in nostril lamellae numbers along recruitment. A<B (p=0.010), B<C (p=0.005) (n=42, ANOVA). m, Changes in the numbers of LL canal along recruitment. A<B (p<0.001), B<C (p=0.033), C<D (p=0.025) (n=54, ANOVA+TukeyHSD). (Methods).

Figure III. 3 |

 3 Figure III.3 | Impacts of elevated water temperature and chlorpyrifos exposure on thyroid hormone levels and sensory organ development during recruitment in Acanthurus triostegusa-n, Error bars indicate standard errors (error bars are not visible when standard errors are too small). Circles bordered in black represent means while alpha transparent circles in the background represent each data. d2 and d5 = day 2 and day 5 post-settlement, respectively. a, T3 level variations under different temperatures (d2 fish at 28.5°C as standards). A<B (p=0.021) at d2 (n=42, Kruskal-Wallis+Nemenyi), no difference at d5 (p=0.105, n=30, Kruskal-Wallis). b, T4 levels variations under different temperatures (d2 fish at 28.5°C as standards). A<B (p=0.001) at d2 (n=29, Kruskal-Wallis+Nemenyi), no difference at d5 (p=0.074, n=27, Kruskal-Wallis c, bpc density variations under different temperatures. A<B (p=0.034) at d2 (n=20, ANOVA+TukeyHSD) and no difference at d5 (p=0.375, n=20, ANOVA). d, prn density variations under different temperatures. No difference at d2 (p=0.637, n=20, ANOVA) and d5 (p=0.983, n=20, ANOVA). e, pes density variations under different temperatures. No difference at d2 (p=0.291, n=19, ANOVA) and d5 (p=0.177, n=20, Kruskal-Wallis). f, nostril lamellae number variations under different temperatures. No difference at d2 (p=0.609, n=29, Kruskal-Wallis) and d5 (p=0.626, n=20, Kruskal-Wallis). g, lateral line canal number variations under different temperatures. A<B (p=0.025) at d2 (n=29, ANOVA+TukeyHSD) and A<B (p=0.002) at d5 (n=20, ANOVA+TukeyHSD). h, T3 level variations under CPF exposure (d2 fish at 28.5°C as standards). A<B (p=0.039) at d2 (n=47, ANOVA+TukeyHSD), no difference at d5 (p=0.698, n=50, ANOVA). i, T4 levels variations under CPF exposure (d2 fish at 28.5°C as standards). No difference at d2 (p=0.255, n=42, ANOVA) d5 (p=0.578, n=50, ANOVA). j, bpc density variations under CPF exposure. A<B (p=0.049) at d2 (n=50, ANOVA+TukeyHSD) and A<B (p=0.004) at d5 (n=50, ANOVA+TukeyHSD). k, prn density variations under CPF exposure. No difference at d2 (p=0.993, n=50, ANOVA) and d5 (p=0.436, n=50, Kruskal-Wallis). l, pes density variations under CPF exposure. No difference at d2 (p=0.978, n=50, ANOVA) and A<B (p=0.022) at d5 (n=50, Kruskal-Wallis+Nemenyi). m, nostril lamellae number variations under CPF exposure. No difference at d2 (p=0.669, n=50, Kruskal-Wallis) and A<B (p=0.018) at d5 (n=49, Kruskal-Wallis+Nemenyi). n, lateral line canal number variations under CPF exposure.

  Figure III.4 | Impacts of thyroid hormone signaling, elevated water temperature and chlorpyrifos exposure on Acanthurus triostegus behavioral responses and survival when facing predation cues and predators

  . Error bars indicate standard errors (error bars are not visible when standard errors are too small). Circles bordered in black represent means while alpha transparent circles in the background represent each data. A<B (p=0.024) and B<C (p<0.001LL length was assessed from day 0 (d0) to day 8 (d8) post-settlement. Error bars indicate standard errors (error bars are not visible when standard errors are too small). Circles bordered in black represent means while alpha transparent circles in the background represent each data. No difference in lateral line length was observed from d0 to d8 (p=0.977, n=60, ANOVA) III. Extended Data Fig 4 | Fulton's K condition factor variation along with thyroid hormone (TH) signaling, temperature exposure (Temp), and chlorpyrifos (CPF) exposure during recruitment in Acanthurus triostegus

Figure

  Figure represents LI mean (±SE) values. Asterisks indicate significant differences (Mann-Whitney U tests, *: p < 0.05; **: p < 0.01) between Laterality Index (LI) values and the theoretical 50 % value (dotted line). 13 to 14 replicates (one fish per replicate) were conducted for each visual stimulus.

  Figure represents SI mean (±SE) values. The asterisk indicates a significant difference in survival index among the three groups (Kruskal-Wallis test, 𝜒 2 = 7.064, df = 2, p-value = 0.029) with 'left-eye dominant' experiencing high survival than both 'right-eye dominant' (Nemenyi post-hoc test with Tukey-Dist approximation, p-value = 0.01) and 'no eye dominant' larvae (Nemenyi post-hoc test with Tukey-Dist approximation, pvalue = 0.03). Six replicated were conducted. In each replicate, each group was made up with the same amount (four to six) of fish.

Fig. IV. 4 |

 4 Fig. IV.4 | Effect of pesticides on the visual preference of A. triostegus larvae Figure represents LI mean (±SE) values. Asterisks indicate significant differences (Mann-Whitney U tests, *: p < 0.05; **: p < 0.01) between Laterality Index (LI) values and the theoretical 50 % value (dotted line). Black asterisks above bars indicate a significant right-eye preference, while white asterisks below bars indicate a significant left-eye preference. 10 to 11 replicates (one fish per replicate) were conducted for each visual stimulus in both the pesticide exposure experiment (black bars) and the solvent-control exposure experiment (grey bars).

Fig. V. 1 |

 1 Fig. V.1 | Body color patterns and community organization of Acanthurus triostegus juveniles A: 'Classical color pattern' (CP) of a convict tang juvenile at day 7 post-settlement. B: 'Reversal of the color pattern' (RP) of two juveniles also erecting their dorsal fin. C: Shoal and community organization of CP (top) and RP (bottom-central) juveniles.

Fig. V. 2 |

 2 Fig. V.2 | Color morph ontogenetic stage, prevalence, and behavior of CP and RP juveniles at day-7 post-settlement Light gray: CP juveniles; Dark gray: RP juveniles. A: Color morph relative prevalence. B: Body depth (body height / standard length) of A. triostegus along settlement (day 0 to day 11), and of day 7 post-settlement (bold lines) CP and RP juveniles. C: Percentage of time spent with the dorsal fin erected in CP and RP juveniles. Errors bars indicate standard errors. ***: p-value < 0.001.

Figure I. 1 |

 1 Figure I.1 | Coral reef types in French Polynesia A: Barrier reef (BR) and fringing reef (FR) in Maupiti Island. Fringing reefs can develop in shallow waters along the coasts while barrier reef corresponds to a coral reef growing parallel to the shoreline and separated from it by a lagoon. B: Tetiaroa Atoll. Atolls are rings of coral reefs and small islets surrounded by the ocean and surrounding a sandy lagoon where a volcano was standing before being submerged. Pictures: M. Besson.

Figure I. 2 |

 2 Figure I.2 | Fishes in coral reefs A: Blacktail snappers (Lutjanus fulvus) on the foreslope reef of Fakarava Atoll (Tuamotu Archipelago, French Polynesia). B: Hawaiian bigeyes (Priacanthus meeki) on the foreslope reef of Rangiroa Atoll (Tuamotu Archipelago, French Polynesia). C: Yellowfin surgeonfishes (Acanthurus xanthopterus) in the channel pass of Rangiroa Atoll (Tuamotu Archipelago, French Polynesia). D: Convict tangs (Acanthurus triostegus) on patch reefs near the barrier reef of Tetiaroa Atoll (Society Archipelago, French Polynesia). E: Blue-green damselfishes (Chromis viridis) and Whitetail dascyllus (Dascyllus aruanus) in the lagoon of Moorea (Society Archipelago, French Polynesia). F: Bluegreen damselfishes (C. viridis) around their Pocillopora shelter in a fringing reef of Moorea Island (Society Archipelago, French Polynesia). Pictures: L. Thiault.

Figure I. 3 |

 3 Figure I.3 | Herbivorous fishes grazing on algae in a bleached coral reef A: Two herbivorous fishes: Chaetodon ephippium (front) and Acanthurus nigricans (back) feeding on turf algae that have recently grown on dead Pocillopora following a bleaching event in Moorea Island (French Polynesia). B: same observation with the line surgeonfish (Acanthurus lineatus). Pictures: L. Hedouin.

Figure I. 4 |

 4 Figure I.4 | Ten coral reef fishes from iconic coral reef fish families A: Acanthurus triostegus (Acanthuridae). B: Myripristis berndti (Holocentridae). C: Chaetodon humeralis (Chaetodontidae). D: Dascyllus trimaculats (Pomacentridae). E: Salarias fasciatus (Blenniidae). F: Apogonichthyoides pseudotaeniatus (Apogonidae). G: Stethojulis bandanensis (Labridae). H: Scarus spinus (Scaridae). I: Mulloidichthys flavolineatus (Mullidae). J: Carangoides ferdau (Carangidae). Adapted from FAO species identification guides (FAO 2001).

Figure I. 5 |

 5 Figure I.5 | The convict tang Acanthurus triostegus as a model for teleost coral reef fish characteristics n.: nostril, e.: eye, s.: scales, l.l.: lateral line, a.: anus, op.: operculum, p.op.: pre-operculum, m.: mouth. d.f.: dorsal fin, c.f.: caudal fin, a.f. : anal fin, pel.f. : pelvic fins, pec.f. : pectoral fin. SL: standard length, BH: body height, SnL: snout length. Picture: M. Besson.

  . For example, peaks of TH levels and TR expressions are observed during the metamorphosis of the Pacific Bluefin tuna (Thunnus orientalis) (Figure I.11) and during the metamorphosis of the Japanese flounder (Paralichtys olivaceus) (Figure I.12).

  Figure I.12

Figure I. 11 |

 11 Figure I.11 | Changes in TH levels (A) and TR expressions (B) during the development of the Pacific Bluefin tuna (Thunnus orientalis)

Figure I. 13 |

 13 Figure I.13 | Thyroid follicles in the sub-pharyngeal region of Acanthurus triostegus Schiff reagent and hematoxylin staining of a 5 µm histological section of A. triostegus sub-pharyngeal region (primarily fixed in Liquide de Bouin). The numerous thyroid follicles are revealed in indigo (see arrows). Scale bar indicates 50 µm.

Figure I. 12 |

 12 Figure I.12 | Changes in TH levels (A) and TR expressions (B) during the development of the African clawed frog (Xenopus laevis) and the Japanese flounder (Paralichtys olivaceus) TH (dotted lines) levels and TR expressions (solid and dashed lines) peak during metamorphosis of X. laevis and P. olivaceus. Contrarily, TRα-B in P. olivaceus (alternating dash-dot line) does not peak. Adapted from Shi (2000), Yamano and Miwa (1998) and figure originating from Schreiber (2013).

  Figure I.5, Figure I.6) (Linnaeus, 1758) was used as a model species, and all sampling and field experiments were conducted at the CRIOBE research station on Moorea Island (French Polynesia).

Figure I. 15 |

 15 Figure I.15 | Location of Moorea Island in French Polynesia A: Location of French Polynesia within the Pacific Ocean. B: Location of Moorea Island within French Polynesia. C: Moorea Island. Scale bar indicates 2 km. Adapted from Besson (2017).

  3 | Are there specific sensory organs and sensory abilities that develop at metamorphosis and what are their endocrine determinants? 4 | Are there environmental factors that act as endocrine disruptors of TH signaling during coral reef metamorphosis and how would it subsequently affect larval recruitment success?This manuscript has been published in October 2017, after my PhD defense, and is available in Appendix 5.Holzer G., Besson M. (equal first author), Lambert A., François L.,Barth P., Gillet B., Hughes S., Piganeau G., Leulier F., Viriot L., Lecchini D., Laudet V. 2017. Fish larval recruitment is a metamorphosis sensitive to reef pollutants. eLife 2017;6:e27595.

  Figure II.1A-B). Individuals also undergo extensive pigmentation changes (Figure II.1C-L), as exemplified by the very rapid appearance of black stripes (less than four hours, MB pers. obs.) after entering the reef (Figure II.1C-D, Figure II.1H-I). This is followed by the widening of these vertical black stripes and by the onset of the body's white pigmentation (Figure II.1D-G, Figure II.1I-L). Furthermore, using conventional X-Ray microtomography, we identified three different dental generations in crest larvae to day 8 juveniles (red, blue and green, Figure II.1M-Q). Crest larvae present tiny and poorly mineralized pointed teeth at the distal ends of the jaws (red, Figure II.1M, Figure II.1-figure supplement 1R) that are likely remnants of an oceanic larval dentition (dentition A). Other medium-sized and more mineralized teeth are in function in medial areas of the jaws (blue, Figure II.1M). These teeth display pointed cusps and belong to a more advanced dental generation (dentition B). Lastly, much larger teeth of the next generation (green, dentition C) are about to erupt from jaws in crest larvae (Figure II.1M) and replace A and B previous dentitions in day 2 to day 8 juveniles (Figure II.1N-Q). These teeth are highly similar to the shovel-shaped adult teeth (Figure II.1 -figure supplement 1S) that serve for grazing algae on hard substrata. The rapid formation of dentition C is consistent with a diet shift from planktivorous to herbivorous at recruitment. This shift is precisely organized, since it begins in the sagittal area of all individuals, and secondarily extends to the distal parts of the jaw (Figure II.1M-Q). The fact that crest captured larvae already have teeth of the C dentition about to erupt (Figure II.1M) indicates that fish are prepared for reef life conditions before reef entry.

  Figure II.1W). To characterize this change, we performed histological sections on the middle part of the intestine during the larval recruitment process (Figure II.1R-V). Intestines of crest larvae and day 2 juveniles have a thick wall with many muscle fibers and regular villi (Figure II.1R and S). At day 3, intestines undergo a spectacular remodeling with the disappearance of the epithelium, leaving only the muscular layer (Figure II.1T), while epithelium reformation occurs later at day 5 (Figure II.1U). In day 8 individuals, the intestine is fully remodeled: the epithelium is thicker and exhibits villi again (Figure II.1V). Such spectacular remodeling events are also observed in the proximal and distal portion of the intestines (Figure II.1 -figure supplement 1A-O).

Figure

  Figure II.1 | Changes in Acanthrurus triostegus during larval recruitment. Error bars represent standard errors. *: p-value < 0.05; **: p-value < 0.01; ***: p-value < 0.001. (A) Schematization of the reef colonization by a larva (left) turning into a juvenile (right). The background color shading centered into the reef crest is re-used in following figures. (B) Weight variation during larval recruitment. n = 3 biological replicates for 5-to-10 km offshore

  Figure II.2A-B). T4 levels are low in far ocean larvae and rise in fish located closer to the reef (near ocean larvae), before dropping 6-fold between the near ocean larvae and the day 8 juveniles (p-value = 0.001, Figure II.2A). This indicates a peak of T4 in near ocean and in crest larvae. Far ocean larvae exhibit a high level of T3 but with an important variability (Figure II.2B). Although these larvae live in the same ecological niche (i.e. open ocean) and are roughly of the same size (4.7, 5.5, and 6.3 mm), they were captured in different locations and are probably not at the same developmental stage. Near ocean and crest captured larvae have similar levels of T3 that are 6-fold higher than the level observed in day 8 juveniles (p-value < 0.001 Figure II.2B). We characterized the three TR of A. triostegus: TRα-A, TRα-B and TRβ. These receptors have between 86 % (TRα-A) and 96 % (TRβ) sequence identity with their zebrafish orthologs (Figure II.2 -figure supplement 1A). The phylogenetic analysis indicates that each of the three TR clusters with its respective teleosts orthologs (Figure II.2 -figure supplement 1B). Moreover, transactivation assays show that these receptors behave as genuine TR (Figure II.2 -figure supplement 1C). We thus assessed the expression levels of TRα-A, TRα-B, and TRβ during A. triostegus development (Figure II.2C), as well as Klf9, a conserved TR regulated gene(Denver and Williamson 2009). Far ocean larvae have two profiles: one with a very low TR and Klf9 expressions ("Far a" ocean) and one with very high levels of expression ("Far b" ocean)(Figure II.2C). This suggests that there are, at least, two distinct situations in far ocean larvae: larvae with low TH/TR levels and larvae in which TH/TR levels surge. Far b ocean, near ocean, and crest larvae have high expression levels of the all the TR as well as Klf9, indicating a strong activation of the TH signaling at these stages. We observed a drop of all TR expression between crest larvae and day 8 juveniles (pvalues = 0.001, 0.005 and 0.002 for TRα-A, TRα-B and TRβ respectively, Figure II.2C).

Figure II. 2 |

 2 Figure II.2 | Thyroid hormone signaling during Acanthurus triostegus metamorphosis. Error bars represent standard errors. *: p-value < 0.05; **: p-value < 0.01; ***: p-value < 0.001. (A) T4 and (B) T3 dosage: Y-axis: T4 and T3 levels in pg.g -1 ; X-axis: sampling point. n = 3 for 5 to 10 km offshore fish (far), n = 5 for 2 km offshore fish (near), n = 297 for crest to day 8 (d8) fish. Statistics: mean comparisons (Wilcoxon-Mann-Whitney U-test). (C) Expression level of TRα-A, TRα-B, TRβ and Klf9 by qPCR, normalized with Pold2 and Rpl7. Y-axis; Relative normalized expression X-axis: gene and sampling point.. Far ocean larvae are split in two groups: Far a (black stripes) and Far b (white). For each panel, the color shading is the same one as in Figure II.1A. For each sampling point 3 biological replicates and 3 technical replicates were performed. Statistics: comparisons of each mean to the Crest condition mean (Student's t cumulative distribution function, which was automatically computed by qPCR software CFX Biorad Manager, CFX Manager 2017).

  therefore disrupting A. triostegus TH pathway (Figure II.2 -figure supplement 1D); and (iv) T3 + IOP + NH3 all at 10 -6 M to ensure the non-toxicity of NH3. We used intestine total length, internal structure of guts (intestinal microvilli), dentition and pigmentation as markers of the advancement of metamorphosis since they strongly change after reef recruitment (Figure II.1). T3 + IOP treatment increases the length of the intestine in day 2 and day 5 juveniles (pvalues < 0.001, Figure II.3E, blue). On the contrary, NH3 treatment partially prevents the intestines lengthening occurring after recruitment (p-values < 0.001, Figure II.3E, green). Injections of T3 + IOP + NH3 result in an intestine length similar to the control individuals at day 2 and day 5 (p-values = 0.486 and 0.444 respectively, Figure II.3E). This shows that T3 and NH3 effectively compete, and confirms the non-toxicity of NH3. In parallel, intestines of fish relocated on the external slope are shorter than those of their lagoon counterparts at day 2 and day 5 (p-value < 0.001 and p-value = 0.042 respectively, Figure II.3E, orange). This shows that their development has been slowed down, similarly to what we observed with the NH3 treatment. A similar pattern was observed concerning the microvilli remodeling within guts. In the anterior part of the guts, while T3+IOP treatment accelerates the loss of the intestinal microvilli at day 2 and accelerates the development and the thickening of a new epithelium at day 5 (Figure II.3F, second column), NH3 treatment and external slope relocation prevent microvilli renewal at both day 2 and day 5 (Figure II.3F, third and fourth column). These observations can be extended to the anterior, medium and posterior sections of the guts (see Figure II.3 -figure supplement 1). These changes are consistent with the notion that TH control remodeling of the guts at metamorphosis in A. triostegus. We did not observe any effect on teeth development neither with T3+IOP or NH3 treatment, nor with external slope relocation (Figure II.3 -figure supplement 1).

Figure II. 3 |

 3 Figure II.3 | Impact of the environment and thyroid hormone signaling on Acanthurus triostegus metamorphic processes. Error bars represent standard errors. *: p-value < 0.05; **: p-value < 0.01; ***: p-value < 0.001. (A) Upper panel: schematization of the external slope experiment with control fish, in the lagoon (grey circle), and relocated fish, on the external slope (orange circle). Lower panel: comparison of the pigmentation between the relocated fish (left) and the control lagoon fish (right) at day 2. (B) T4 and (C) T3 dosage of crest captured larvae and day 2, 5 and 8 juveniles,

  CPF results in up to a 14-fold reduction in the biomass of turf grazed after 5 days (pvalue = 0.05, Figure II.5D).

Figure II. 4 |

 4 Figure II.4 | Thyroid hormone levels in distant coral reef fish at larval recruitment. Error bars represent standard errors. *: p-value < 0.05; **: p-value < 0.01; ***: p-value < 0.001. TH levels of: Rhinecanthus aculeatus (upper), Chromis viridis (middle up), Chaetodon lunula (middle low) and Ostorhinchus angustatus (lower). n = 3 to 5 biological replicates for each condition. First column is T4, second column is T3. Y-axis: T4 or T3 level in pg.g -1 . X-axis: sampling point (developmental stage). The phylogenic relationship between the species is plotted on the far left of the figure. Statistics: for each species, comparisons of each mean to the day 1 (d1) mean (Wilcoxon-Mann-Whitney U-test).

Figure II. 5 |

 5 Figure II.5 | Influence of chlorpyrifos exposure on Acanthurus triostegus thyroid hormone signaling, intestine length, grazing activity, and algal removal efficiency. Error bars represent standard errors. *: p-value < 0.05; **: p-value < 0.01; ***: p-value < 0.001. (A) T3 level in pg.g -1 of day 2 (d2) and day 5 (d5) fish exposed to different conditions: solvent control (C, acetone, white bars), or exposed to CPF at 1µg.l -1 (1, light yellow bars), 5 µg.l -1 (5, yellow bars) or 30 µg.l -1 (30, dark yellow bars). Exposure started right after crest capture. n = 10 fish in each condition. Statistics: ANOVA followed by Tukey posthoc tests, only significant differences with the solvent-control condition are indicated. (B) Intestine length in cm of fish at day 2 (d2) or day 5 (d5) under different conditions: solvent control (C, acetone, white bars), or exposed to CPF at 1µg.l -1 (1, light yellow bars), 5 µg.l -1 (5, yellow bars) or 30 µg.l -1 (30, dark yellow bars). Exposure started right after crest capture. n = 5 to 8 fish for each experimental condition. Statistics: same as in (A). (C) Mean number of bites (on turf algae) per fish in a batch of 18 fish on reef turf at day 2 (d2) or day 5 (d5), under different conditions: solvent control (C, acetone, white bars), or exposed to CPF at 1µg.l -1 (1, light yellow bars), 5 µg.l -1 (5, yellow bars) or 30 µg.l -1 (30, dark yellow bars). Exposure started right after crest capture. n = 3 independent bite counts for each condition. Statistics: same as in (A). (D) Mean turf biomass removed per fish in a batch of 10 fish, in 5 days following reef colonization, under different conditions: solvent control (C, acetone, white bars), or exposed to CPF at 1µg.l -1 (1, light yellow bars), 5 µg.l -1 (5, yellow bars) or 30 µg.l -1 (30, dark yellow bars). Exposure started right after crest capture. n = 3 independent measurements of grazed turf for each condition. Statistics: comparisons of each mean to the solvent-control (C) mean (Wilcoxon-Mann-Whitney U-test).

  Figure II.1B), suggesting that larval recruitment corresponds to a real metabolic challenge. The molecular investigation of TH signaling in the recruiting larvae indicates a surge of TH levels and TR expressions in near ocean larvae and crest individuals, and a drop in juveniles, after reef entry (Figure II.2A-C). This indicates that migration towards the reefs marks the onset of metamorphosis and starts very early on (Figure II.6A). Consequently, the transformation process is already well advanced when larvae reach the reef, suggesting that this event is triggered in the open ocean by, yet unknown, environmental cues (Figure II.6A). After reef entry, T3 treatment increases gut length while external slope relocation and NH3 treatment delay the metamorphosis, although they could not completely interrupt it (Figure II.3A-B). These results are consistent with the model of metamorphosis in amphibians (Brown and Cai 2007) and other fish species (McMenamin and Parichy 2013). It is interesting to relate the morphological and physiological changes we have observed in A.

(

  Figure II.3 -figure supplement 2). Contrarily, in zebrafish, this pigmentation process takes days and is part of a TH-controlled developmental program in which the triggering role of the environment is unknown (McMenamin et al. 2014). Altogether, our findings indicate that the onset and coordination of metamorphosis are two distinct events, both coordinated by TH and under environmental control. They further demonstrate that A. triostegus larval recruitment is a TH-controlled metamorphosis, as in amphibians and flatfishes (Brown and Cai 2007; Laudet 2011; McMenamin and Parichy 2013).

  (Figure II.6B).

Figure

  Figure II.6 | Model of Acanthurus triostegus metamorphosis and disruption by chlorpyrifos. (A) Schematic representation of coral reef fish larval recruitment. TH level is plotted on the Y axis and development on the X axis. The onset of metamorphosis is in the open ocean and environmental influence upon development is plotted on the X-axis. (B) Consequences of undisturbed (upper panel) and CPF disturbed (lower panel) metamorphosis on grazing activity, and subsequent algal spreading and potential coral survival. Solid red bar indicates inhibition; dotted red line indicates lack of inhibition.

  Figure II.3 -figure supplement 1, Figure II.3 -figure supplement 2).

  .5A-B, Figure II.5 -figure supplement 1A-B).

Figure

  Figure II.1 -figure supplement 1 | Changes in Acanthurus triostegus intestine and teeth during larval recruitment. (A-O) Complete guts histology of Acanthurus triostegus after reef colonization. First column: anterior part of the intestine (first cm), middle column: medium part (variable), last column: posterior part of the intestine (1 cm), (A-C): crest individual, (D-F): 2 days juvenile, (G-I): 3 days juvenile, (J-L): 5 days juvenile, (M-O): 8 days juvenile. Scale bars represent 200 µm. These pictures are representative of n = 6 section technical replicates for each of the proximal, middle and distal intestine, and were performed twice (sampling technical replicates), on n = 2 fish (biological replicates). (P) Heat map of bacterial gut community. Each column is one individual. Each row is bacterial taxa. Only the taxa with differential representations between the samples are shown. Sampling stages: day 2, day 5, day 8 and adult are indicated below the heat map. The shape of each of this developmental stage is schematized over the heat map. Each column is highlighted by a circle, the difference in shade between the circles emphasizes the stage difference. The color of each square in the heat map is proportional to the abundance of the taxa (row) in the sample (column). (Q) µCT scan of a crest larva, lateral view. Dental family C are highlighted in red and dental family B in blue. (R) µCT scan of an adult, frontal view (mouth open). (Q-R) The scale bar indicates 0.5 mm. These pictures are representative of n = 2 fish (biological replicates)

Figure

  Figure II.2 -figure supplement 2 | Primers used in this study. (A) Degenerated primers used for the cloning of the A. triostegus genes in this study. (B) qPCR primers used in this study. (C) Primers used for 18S mass sequencing. The overlapping nucleotides between 18SR and the blocking primer are indicated in bold. The mean annealing temperature of each primer is indicated in the last column.

Figure

  Figure II.2 -figure supplement 3 | List of thyroid hormone receptors sequences used for the phylogeny.

Figure II. 3 -

 3 Figure II.3 -figure supplement 1 | Thyroid hormone signaling and environment impact on the dentition and guts histology of Acanthurus triostegus at metamorphosis First column: X-Ray microtomography of larva, dentition A, B or C are highlighted respectively in red, blue or green. Scale bars indicate 0.5mm. These pictures are representative of n = 3 fish (biological replicates) per developmental stage and per treatment condition. Second to fourth columns: complete guts histology (5 µm wide microtome sections) of A. triostegus at day 2 and day 5 following reef colonization. First column: anterior part of the intestine (first cm), middle column: medium part (variable), last column: posterior part of the intestine (last cm). Haematoxylin and eosin staining. Scale bars indicate 200µm. These pictures are representative of n = 2 treatment technical replicates, and n = 2 microtome section and staining technical replicates, on n = 3 fish (biological replicates) per treatment and per day

Figure II. 5 -

 5 Figure II.5 -figure supplement 2: Pigmentation patterns and chlorpyrifos exposure in A. triostegus at metamorphosis (A) T4 level in pg.g -1 of day 2 (d2) and day 5 (d5) fish exposed to different conditions: solvent control (C, acetone, white bars), or exposed to CPF at 1 µg.l -1 (1, light yellow bars), 5 µg.l -1 (5, yellow bars) or 30 µg.l -1 (30, dark yellow bars). Exposure started right after crest capture. n = 10 fish in each condition.. See Figure 5 -source data 5 for detailed data and statistics. (B) Pigmentation patterns and in A. triostegus exposed to different conditions: solvent control, or CPF at 1 µg.l -1 , 5 µg.l -1 , or 30 µg.l -1 at day 2 (d2) and day (d5) during larval recruitment. These pictures are representative of n = 2 chlorpyrifos CPF exposure technical replicates performed on n = 10 fish (biological replicates) per condition and per day.

  III.1a), and decrease in body depth (III. Extended Data Fig1). Fulton's condition factor(Fulton 1904) rapidly declined from d0 to d8 (III. Extended Data Fig 2), indicating that metamorphosis constitutes a considerable metabolic challenge for fish. Histological analyses of the visual (retina, Fig. III.1b-c), olfactory (nostril, Fig. III.1d-e) and mechanosensory (lateral line (LL), Fig. III.1f-g) organs revealed rapid maturation post-settlement (d0 to d8) (Methods). Within the retina, the density of ganglionar cells (ggc) remained stable post-settlement (Fig. III.1h), but increased for bipolar cells (bpc), photoreceptor nuclei (prn), and photocone external segments (pes) (Fig. III.1i-k). The number of nostril lamellae and LL canals also increased, with a twofold surge in LL canals density between d0 and d2 (Fig. III.1l-m, III. Extended Data Fig 3).

Figure III. 1 |

 1 Figure III.1 | Metamorphosis and sensory system changes at recruitment in Acanthurus triostegus a, Morphological and pigmentation changes in A. triostegus from day 0 (d0) to day 8 (d8) following their entry in the reefs. Scale bar indicates 1 cm. b, Cross section in the eye retina stained in hematoxylin/eosin, presenting the dorsal side (ds) the ventral side (vs) and the optical nerve (on). The dotted gray rectangle represents the magnified region shown in (c). Scale bar indicates 100 µm. c, Cross section in the eye retina stained in hematoxylin/eosin, presenting the ganglionar cell layer (GCL), the inner nuclear layer (INL) and the outer nuclear layer (ONL) that contain ganglionar cells (ggc), bipolar cells (bpc) and photoreceptors nuclei

  Fig. III.2c-d), and in the nostril and LL (elevated number of lamellae at d5, Fig. III.2e, and a greater number of LL canals at both d2 and d5, Fig. III.2f). In contrast, N3-treated fish experienced repressed sensory organ maturation, particularly at d5 for the retina (Fig. III.2b-d), at d2 for the nostril (Fig. III.2e), and at both d2 and d5 for LL (Fig. III.2f). Finally, while the OS treatment caused delayed nostril maturation at d2 (Fig. III.2e), it had no effect on retina or LL (Fig. III.2a-d,f), suggesting environmental relocation had a relatively limited impact on their maturation.

Figure III. 2 |

 2 Figure III.2 | Importance of thyroid hormone signaling in sensory organ development at recruitment in Acanthurus triostegus a-f, Impacts of T3 (i.e. promoted TH signaling), N3 (i.e. disrupted TH signaling) pharmacological treatments on sensory organ development at day 2 (d2) and day 5 (d5) compared to control (CT) and outer slope relocated (OS, i.e. environmentally delayed recruitment) individuals. Error bars indicate standard errors (error bars are not visible when standard errors are too small). Circles bordered in black represent means while alpha transparent circles in the background represent each data. a, No difference between treatments in ggc density at d2 (p=0.582, n=51, ANOVA) and d5 (p=0.666, n=43, Kruskal-Wallis). b, A<B (p=0.018) and B<C (p<0.001) at d2 (n=51, ANOVA+TukeyHSD) and A<B (p=0.045) at d5 (n=43, ANOVA+TukeyHSD). c, A<B (p=0.002) at d2 (n=51, ANOVA+TukeyHSD) and no change at d5 (p=0.242, n=43, ANOVA+TukeyHSD). d, A<B (p=0.022) at d2 (n=47, Kruskal-Wallis+Nemenyi) and A<B (p=0.033) at d5 (n=43, Kruskal-Wallis+Nemenyi). e, A<B (p=0.005) at d2 (n=80, Kruskal-Wallis+Nemenyi) and A<B (p=0.044) at d5 (n=86, Kruskal-Wallis+Nemenyi). f, A<B (p<0.001) and B<C (p<0.001) at d2 (n=44, ANOVA+TukeyHSD) and A<B (p=0.002) at d5 (n=42, ANOVA+TukeyHSD). (Methods).

  Fig. III.3ab). Moreover, this +3°C increase also reduced bpc density by 7 % at d2 (Fig. III.3c), and LL canal number by 10 % at both d2 and d5 (Fig. III.3g). However, it had no impact on prn density, pes density, and nostril development (Fig. III.3d-f). CPF produced a twofold decrease in T3 at the highest concentration, but dit not affect T4 levels at any concentration (Fig. III.3h-i, adapted from Holzer, Besson, et al. (2017)). At both d2 and d5, reduced bpc densities and LL canals were observed in fish exposed to 30 µg.l -1 CPF (Fig. III.3j, Fig. III.3n), while reduced pes density and nostril lamellae were observed at d5 in individuals exposed to 30 µg.l -1 CPF (Fig. III.3l-m). Densities of prn exhibited no difference between CPF concentrations (Fig. III.3k-l).

Figure III. 3 |

 3 Figure III.3 | Impacts of elevated water temperature and chlorpyrifos exposure on thyroid hormone levels and sensory organ development during recruitment in Acanthurus triostegus a-n, Error bars indicate standard errors (error bars are not visible when standard errors are too small). Circles bordered in black represent means while alpha transparent circles in the background represent each data. d2 and d5 = day 2 and day 5 post-settlement, respectively. a, T3 level variations under different temperatures (d2 fish at 28.5°C as standards). A<B (p=0.021)

Figure III. 4 |

 4 Figure III.4 | Impacts of thyroid hormone signaling, elevated water temperature and chlorpyrifos exposure on Acanthurus triostegus behavioral responses and survival when facing predation cues and predators a, Design of the two-channel Atema choice flume apparatus used for olfactory preferences assessment in this study. Total length: 25 cm (laminating channel area = 15 cm, choice arena = 5 cm and drained exit area = 5 cm). Width: 5 cm. b, Olfactory preferences and TH signaling when facing olfactory cues from control seawater (Ø) vs seawater with predator Lutjanus fulvus odor (P). d2 and d5 = day 2 and day 5 post-settlement, respectively. *** indicates p<0.001 (n = 8 to 13 per treatment and per cue, Wilcoxon-Mann-Whitney). c, Design of the choice tank apparatus used for visual preference assessment in this study. Total length: 60 cm (release area = 30 cm, each choice area = 15 cm). Width: 15 cm. The tank is equipped with opaque walls expect for the two opposite walls where visual stimuli are presented. d, Impact of TH signaling, water temperature and CPF exposure (results in september) on visual preferences when facing visual cues from a 'fish-empty' tank (Ø) vs tank containing a L. fulvus live

  , and we selected the peripheral area of this dorsal side (Fig. III.1b) for comparison and posttreatment matters (densities were sometimes too high and thus impossible to count in the retina central area). Cell counting was performed in a 50 µm wide area perpendicular to the retina cell layers. The measure of density of photocone external segments corresponds to the number of counts in this 50 µm wide area, while the measures of densities of photoreceptors nuclei, bipolar and ganglionar cells correspond to the actual density per µm² after measuring the mean height of each cell layer (3 random measurements for each cell layer: GCL, INL and ONL, Fig. III.1c).

  Data Fig 1 | Body depth variation along recruitment in Acanthurus triostegus Body depth was assessed from day 0 (d0) to day 30 (d30) post-settlement. Body depth was calculated as follows: Body Height / Standard length. Error bars indicate standard errors (error bars are not visible when standard errors are too small). Circles bordered in black represent means while alpha transparent circles in the background represent each data. A<B (p<0.001) and B<C (p<0.001) (n=320, Kruskal-Wallis+Nemenyi).

  . Error bars indicate standard errors (error bars are not visible when standard errors are too small). Circles bordered in black represent means while alpha transparent circles in the background represent each data. A<B (p=0.024) and B<C (p<0.001) (n=320, Kruskal-Wallis+Nemenyi) III. Data Fig 3 | Lateral line (LL) length variation along recruitment in Acanthurus triostegus LL length was assessed from day 0 (d0) to day 8 (d8) post-settlement. Error bars indicate standard errors (error bars are not visible when standard errors are too small). Circles bordered in black represent means while alpha transparent circles in the background represent each data. No difference in lateral line length was observed from d0 to d8 (p=0.977, n=60, ANOVA) III. Expended Data Fig 4 | Fulton's K condition factor variation along with thyroid hormone (TH) signaling, temperature exposure (Temp), and chlorpyrifos (CPF) exposure during recruitment in Acanthurus triostegus Fulton's K condition factor was calculated as follows: K = 100*(W/SL)^3 (with SL, in cm, and W, in g) (Fulton 1904). Error bars indicate standard errors (error bars are not visible when standard errors are too small). Circles bordered in black represent means while alpha transparent circles in the background represent each data. At day 2 (d2) post-settlement, neither TH (p=0.976, n=62, ANOVA), temperature (p=0.849, n=129, Kruskal-Wallis), nor CPF (p=0.529, n=75, Kruskal-Wallis) affect Fulton's K condition factor in A. triostegus. Same observations were made at day 5 (d5) post-settlement: TH (p=0.715, n=92, Kruskal-Wallis); Temp (p=0.733, n=135, Kruskal-Wallis), and CPF (p=1, n=76, ANOVA). The exact same patterns have been observed for standard length, weight, height and body depth. (Methods).

Fig

  Fig. IV.1 | Schematic representation of the test apparatus for eye lateralization determination.

Fig. IV. 3 |

 3 Fig. IV.3 | Ecological importance of brain lateralization in larval survival facing direct predation. Survival index (SI) was calculated as follow: survival rate of the group minus the overall survival rate in the in situ cage. Figure represents SI mean (±SE) values. The asterisk indicates a significant difference in survival index among the three groups (Kruskal-Wallis test, 𝜒 2 = 7.064, df = 2, p-value = 0.029) with 'left-eye dominant' experiencing high survival than both 'right-eye dominant' (Nemenyi post-hoc test with Tukey-Dist approximation, p-value = 0.01) and 'no eye dominant' larvae (Nemenyi post-hoc test with Tukey-Dist approximation, pvalue = 0.03). Six replicated were conducted. In each replicate, each group was made up with the same amount (four to six) of fish.

Fig

  Fig. IV.4 | Effect of pesticides on the visual preference of A. triostegus larvae.

Figure

  Figure represents LI mean (±SE) values. Asterisks indicate significant differences (Mann-Whitney U tests, *: p < 0.05; **: p < 0.01) between Laterality Index (LI) values and the theoretical 50 % value (dotted line). Black asterisks above bars indicate a significant right-eye preference, while white asterisks below bars indicate a significant left-eye preference. 10 to 11 replicates (one fish per replicate) were conducted for each visual stimulus in both the pesticide exposition experiment (black bars) and the solvent-control exposition experiment (grey bars).

Fig. V. 1 |

 1 Fig. V.1 | Body color patterns and community organization of Acanthurus triostegus juveniles. A: 'Classical color pattern' (CP) of a convict tang juvenile at day 7 post-settlement. B: 'Reversal of the color pattern' (RP) of two juveniles also erecting their dorsal fin. C: Shoal and community organization of CP (top) and RP (bottom-central) juveniles.

Fig. V. 2 |

 2 Fig. V.2 | Color morph ontogenetic stage, prevalence, and behavior of CP and RP juveniles at day-7 post-settlement. Light gray: CP juveniles; Dark gray: RP juveniles. A: Color morph relative prevalence. B: Body depth (body height / standard length) of A. triostegus along settlement (day 0 to day 11), and of day 7 post-settlement (bold lines) CP and RP juveniles. C: Percentage of time spent with the dorsal fin erected in CP and RP juveniles. Errors bars indicate standard errors. ***: p-value < 0.001.

  bleaching on coral-reef fishes-ecological and economic consequences. Oceanogr Mar Biol An Annu Rev 46:251-296 Roche H, Salvat B, Ramade F (2011) Assessment of the pesticides pollution of coral reefs communities from French Polynesia. Rev d'écologie (Terre vie) 66:3-10 Rossi T, Nagelkerken I, Pistevos JCA, Connell SD, Nagelkerken I (2016) Lost at sea : ocean acidification undermines larval fish orientation via altered hearing and marine soundscape modification. Russ G (1984) Distribution and abundance of herbivorous grazing fishes in the central Great Barrier Reef. I. Levels of variability across the entire continental shelf . Mar Ecol Prog Ser 20:23-34 Sandahl JF, Baldwin DH, Jenkins JJ, Scholz NL, Andahl JAFS, Aldwin DAHB, Enkins JEJJ, Cholz NALS (2005) Comparative thresholds for acetylcholinesterase inhibition and behavioral impairment in coho salmon exposed to chlorpyrifos. Environ Toxicol Chem 24:136-45 Shand J (1997) Ontogenetic changes in retinal structure and visual acuity: a comparative study of coral-reef teleosts with differing post-settlement lifestyles. Environ Biol Fishes 49:307-322 Shaw M, Furnas MJ, Fabricius K, Haynes D, Carter S, Eaglesham G, Mueller JF (2010) Monitoring pesticides in the Great Barrier Reef. Mar Pollut Bull 60:113-122 Simpson SD, Munday PL, Wittenrich ML, Manassa RP, Dixson DL, Gagliano M, Yan HY (2011) Ocean acidification erodes crucial auditory behaviour in a marine fish. Biol Lett 7:917-20 Slotkin TA, Cooper EM, Stapleton HM, Seidler FJ (2013) Does thyroid disruption contribute to the developmental neurotoxicity of chlorpyrifos? Environ Toxicol Pharmacol 36:284-287 Trijuno DD, Yoseda K, Hirokawa J, Tagawa M, Tanaka M (2002) Effects of thyroxine and thiourea on the metamorphosis of coral trout grouper Plectropomus leopardus. Fish Sci 68:282-289 Yang D, Lauridsen H, Buels K, Chi LH, La Du J, Bruun DA, Olson JR, Tanguay RL, Lein PJ (2011) Chlorpyrifos-oxon disrupts zebrafish axonal growth and motor behavior. Toxicol Sci 121:146-159

1

  Institut de Génomique Fonctionnelle de Lyon; Université de Lyon; Université Claude Bernard Lyon1; UMR CNRS 5242; Ecole Normale Supérieure de Lyon, 46 Allée d'Italie, 69364 Lyon Cedex 07, France. 2 PSL Research University: EPHE-UPVD-CNRS, USR3278 CRIOBE, BP 1013, 98729 Papetoai, Moorea, French Polynesia 3 Observatoire Océanologique de Banyuls-sur-Mer, UMR CNRS 7232; Université Pierre et Marie Curie Paris;1, avenue Pierre Fabre, 66650 Banyuls-sur-Mer, France 4 Laboratoire d'Excellence «CORAIL», BP 1013, 98729 Papetoai, Moorea, French Polynesia * Equal first authors # Equal last authors £ To whom correspondence should be addressed: Observatoire Océanologique de Banyuls-sur-Mer, 1 avenue Pierre Fabre, 66650 Banyuls-sur-Mer, France. mail: vincent.laudet@obs-banyuls.fr

  Species, genes, databases and accession numbers are indicated.

	Species	Protein	Accession number	Database Species Protein	Accession number	Database
	Acanthopagrus schlegelii	TRα	ABQ96861.1	NCBI	Monodelphis domestica	TRα	NP_001184135.1	NCBI
	Acanthopagrus schlegelii	TRβ	ABQ96862.1	NCBI	Monodelphis domestica	TRβ	XP_007505233.1	NCBI
	Ambystoma mexicanum	TRα	AY174871.1	NCBI	Mus musculus	RARα	NP_033050.2	NCBI
	Ambystoma mexicanum	TRβ	AY174872.1	NCBI	Mus musculus	TRα	NM_178060.3	NCBI
	Anolis carolinensis	TRα	XP_008111558.1	NCBI	Mus musculus	TRβ	NM_001113417.1	NCBI
	Anolis carolinensis	TRβ	XP_008117165.1	NCBI	Necturus maculosus	TRα	Y16623.2	NCBI
	Branchiostoma floridae	TR	EF672344.1	NCBI	Necturus maculosus	TRβ	AY168331.1	NCBI
	Canis lupus familiaris	TRα	NP_001273791.1	NCBI	Oreochromis niloticus	TRα-A ENSONIT00000008144 Ensembl
	Canis lupus familiaris	TRβ	XP_862690.2	NCBI	Oreochromis niloticus	TRα-B ENSONIT00000022999 Ensembl
	Conger myriaster	TRα-A	AB183396.1	NCBI	Oreochromis niloticus	TRβ	ENSONIT00000012974 Ensembl
	Conger myriaster	TRα-B	AB183397.1	NCBI	Oryzias latipes	TRα	AB114860	NCBI
	Conger myriaster	TRβ-A	AB183394.1	NCBI	Oryzias latipes	TRβ	AB114861	NCBI
	Conger myriaster	TRβ-B	AB183395.1	NCBI	Paralichthys olivaceus	TRα-A	D16461.1	NCBI
	Danio rerio	TRα-A	NM_131396.1	NCBI	Paralichthys olivaceus	TRα-B	D16462.1	NCBI
	Danio rerio	TRα-B	XM_001920978	NCBI	Paralichthys olivaceus	TRβ	D45245.1	NCBI
	Danio rerio	TRβ	NM_131340.1	NCBI	Rattus norvegivus	TRα	NP_001017960.1	NCBI
	Epinephelus coioides	TRα-A	ABP62960.1	NCBI	Rattus norvegivus	TRβ	NM_012672	NCBI
	Epinephelus coioides	TRα-B	ABP62961.1	NCBI	Salmo salar	TRα	NM_001123628	NCBI
	Epinephelus coioides	TRβ	ABP62962.1	NCBI	Salmo salar	TRβ	NM_001123700	NCBI
	Eublepharis macularius	TRα	AB204861	NCBI	Sus scrofa	TRα	NM_214190	NCBI
	Eublepharis macularius	TRβ	AB204862	NCBI	Sus scrofa	TRβ	F1RS47_PIG	Ensembl
	Gallus gallus	TRα	NM_205313.1	NCBI	Xenopus laevis	TRα	NP_001081595	NCBI
	Gallus gallus	TRβ	NM_205447.1	NCBI	Xenopus laevis	TRβ	NP_001090182	NCBI
	Homo sapiens	TRα	NM_199334.3	NCBI	Xenopus tropicalis	TRα	NM_001045796.1	NCBI
	Homo sapiens	TRβ	NM_000461.4	NCBI	Xenopus tropicalis	TRβ	NM_001045805.1	NCBI

  RésuméLe maintien et le renouvellement des populations de poissons coralliens dépendent en grande partie du recrutement larvaire, c'est-à-dire de l'installation des larves pélagiques dans les habitats récifaux adultes, et de leur survie après s'être métamorphosées en juvéniles. De plus en plus d'études révèlent que les changements de composition de l'eau, causés par le changement climatique et la pollution, peuvent altérer les capacités sensorielles des poissons coralliens, diminuant leurs aptitudes à localiser des habitats propices (maximisant leur croissance et diminuant leur mortalité) lors de l'installation. Cependant, les mécanismes internes à l'origine de ces phénomènes sont méconnus. Lors de cette thèse, j'ai examiné le recrutement larvaire du poisson chirurgien bagnard Acanthurus triostegus et mis en évidence que les changements écologiques, morphologiques, physiologiques et comportementaux qui s'y déroulent correspondent à une métamorphose contrôlée par les hormones thyroïdiennes (HT). J'ai ensuite analysé comment des stress d'origine anthropique, tels que l'élévation des températures de surface et la pollution par un pesticide d'origine agricole, peuvent perturber sa métamorphose. Lors de cette étape clé de leur cycle de vie, ces perturbations diminuent les taux d'HT, altérant la maturation de leurs organes sensoriels, leurs capacités sensorielles, et augmentant leur mortalité. Cette thèse est donc une analyse holistique de l'impact des perturbations anthropiques sur les processus moléculaires, et les changements histologiques, anatomiques et comportementaux du recrutement larvaire des poissons coralliens. Elle souligne l'importance du système thyroïdien, et invite à une meilleure compréhension des processus endocriniens du recrutement larvaire, dans l'optique d'une amélioration de la conservation des récifs coralliens.

Acknowledgements (EN and FR)

10 Acknowledgment We thank M. Bronner, S. McMenamin, D. Hazlerigg and A. Schreiber for their comments that greatly improved this manuscript. We would also like to thank H. Jacob, C. Gache, A. Gilson, I. Moniz, C. Berthe, A. Savura, D. Van Osten and P. Schep for their help to catch fish larvae. We thank S. Planes, J. Burden, T. Lorin, M. Manceau, P. Salis and N. Roux for critical reviewing and for their constructive discussion concerning coral fish ecology and development. We are grateful to V. Domien for her fruitful advice on the design of the figures. We thank the association Te mana o te moana for the accommodation of our cages.

Acknowledgment We thank T. Lorin, and H. Dore for critical reviewing. and for their fruitful advice on the design of the figures. We thank the association Te mana o te moana for the accommodation of our cages.

Acknowledgment

We are grateful to Loïc François, Isadora Moniz and Anastasha Savura for their help to catch fish larvae. The IAEA is grateful to the Government of the Principality of Monaco for the support provided to its Environment Laboratories.

Acknowledgment

We are grateful to H. Jacob, A. Mignucci, L. François, I. Moniz and A. Savura for their help to catch fish larvae. We would like to thank B. Frédérich and T. Lorin for their helpful comments, which greatly improved this manuscript

V.2. Manuscript information

Title: Complete and rapid reversal of the body color pattern in juveniles of the surgeonfish Acanthurus triostegus at Moorea Island (French Polynesia)

Competing financial interests

The authors declare no competing financial interests.

IV. Exposure to agricultural pesticide impairs visual lateralization in a larval coral reef fish IV.2. Manuscript information

Title: Exposure to agricultural pesticide impairs visual lateralization in a larval coral reef fish Besson 1,2,*,# ,Camille Gache 1,3* ,Frédéric Bertucci 1,4 ,Rohan M. Brooker 5 ,Natacha Roux 1,2 ,,Hugo Jacob 1,6 ,Cécile Berthe 1,3 ,Valeria Anna Sovrano 7 ,Danielle L. Dixson 5 ,David Lecchini 1,3 Affiliations: value = 0.042), or when examining a predator (LI = 62.2 ± 3.6 %, U = 83, n = 13, p-value = 0.006) (Fig. 2). Exp. 2 tested whether visual lateralization increased or decreased vulnerability to predation. Individual larvae from Exp. 1 were classified and grouped as either 'no eye dominant', 'right-eye dominant', or 'left-eye dominant' based on the behavior displayed when exposed to the predator in Exp. 1. All three groups experienced significantly different survival rates (Kruskal-Wallis rank sum test, 𝜒 2 = 7.064, df = 2, p-value = 0.029) with 'left-eye dominant' larvae having a higher survival rate than both 'right-eye dominant' (Nemenyi post-hoc test with Tukey-Dist approximation, p-value = 0.01) and 'no eye dominant' larvae (p-value = 0.03) (Fig. 3).

Authors: Marc

Exp. 3 utilized the same protocol as in Exp. 1 and tested whether exposure to a waterborne organophosphate pesticide often encountered in coral reefs: chlorpyrifos (CPF), would alter the degree of lateralization larvae exhibit. Larvae exposed to CPF at 1 µg.l -1 (black bars in Fig. 4) and larvae only exposed to the CPF solvent (grey bars in Fig. 4) again showed no eye preference in the absence of visual stimuli (respectively LI = 52.5 ± 6.1 % and 52.3 ± 2.7 %, Mann-Whitney U test with respectively U = 33 and 35, n = 10 and 11, p-value = 0.625 and 0.476) (Fig. 4). However, a preference was seen when these larvae inspected a mirror (Wilcoxon rank sum test, W = 106, n = 10, p-value < 0.001), conspecifics (W = 87, n = 10, p-value = 0.004) or a predator (W = 85, n = 10, pvalue = 0.009). Similar to the trends seen in Exp. 1 (Fig. 2), solvent control larvae preferentially used their right eye when inspecting a mirror (LI = 59.2 ± 2.9 %, Mann-Whitney U test, U = 61.5, n = 11, p-value = 0.013), conspecifics (LI = 58.9 ± 2.