COMPONENT 3C - Project 3C8 New techniques of reef fi sh postlarvae capture

Mars 2010

MASTER INTERNSHIP REPORT

Seensorynsory aabilitiesbilities aandnd bbrainrain aanatomynatomy ooff ccoraloral rreefeef fi sshh aatt llarvalarval sstagetage ((FFrrenchench Poolynesialynesia)

AAuthor:uthor: RRynaeynae GGretareta LLanyonanyon The CRISP Coordinating Unit (CCU) was integrated into the Secretariat of the Pacifi c Community in April 2008 to insure maximum coordination and synergy in work relating to management in the region.

The CRISP Programme is implemented as part of the policy developed by the Secretariat of the Pacifi c Regional Environment Programme to contribute to the conservation and sustainable development of coral reefs in the Pacifi c.

he Initiative for the Protection and Management of This approach is articulated through a series of thematic T Coral Reefs in the Pacifi c (CRISP), sponsored by France objectives: and established by the French Development Agency Objective 1: Improved knowledge of the biodiversity, (AFD), is part of an inter-ministerial project that began in status and functioning of coral ecosystems. 2002. CRISP aims to develop a vision for the future of these Objective 2: Protection and management of coral unique ecosystems and the communities that depend on ecosystems on a signifi cant scale. Objective 3: Development of the economic potential them and to introduce strategies and projects to conserve represented by the use values and biodiversity of coral their biodiversity, while developing the economic and en- ecosystems. vironmental services that they provide both locally and Objective 4: Dissemination of information and know-le- globally. CRISP also, has a role in fostering greater integra- dge; and capacitybuilding and leadership with local, na- tion in this area between developed countries (Australia, tional and international networks. New Zealand, Japan, USA), French overseas territories and Pacifi c Island developing countries. The CRISP Programme comprises three major components: Component 1A: Integrated coastal management and The initiative follows a specifi c approach designed to: watershed management – associate networking activities and fi eldwork pro- – 1A1: Marine biodiversity conservation planning jects; – 1A2: Marine Protected Areas – bring together research, management and develop- – 1A3: Institutional strengthening and networking ment endeavours; – 1A4: Integrated coastal reef zone and watershed – combine the contributions of a range of scientifi c dis- management ciplines, including biology, ecology, economics, law Component 2: Development of coral ecosystems and social sciences; – 2A: Knowledge, benefi cial use and management – address the various land and marine factors aff ecting of coral ecosytems coral reefs (including watershed rehabilitation and – 2B: Reef rehabilitation management); – 2C: Development of active marine substances – avoid setting up any new body but supply fi nancial re- – 2D: Development of regional data base (ReefBase sources to already operational partners wishing to de- Pacifi c) velop their activities in a spirit of regional cooperation. Component 3: Programme coordination and development This is why the initiative was established on the basis – 3A: Capitalisation, value-adding and extension of of a call for proposals to all institutions and networks. CRISP programme activities – 3B: Coordination, promotion and development CRISP Coordinating Unit (CCU) of the CRISP programme Programme Manager: Eric CLUA – 3C: Support to alternative livelihoods SPC - PO Box D5 – 3D: Vulnerability of ecosystems and species 98848 Noumea Cedex – 3E: Economic task force New Caledonia Tel./Fax: (687) 26 54 71 E-mail: [email protected] www.crisponline.net

CRISP is funded by the following partners:

Ambassade de France à Fidji

UNIVERSITY OF THE SOUTH PACIFIC

School of Marine Studies, Laucala Campus, Fiji

Sensory abilities and brain anatomy of coral reef at larval stage

(French Polynesia)

By Rynae Greta Lanyon

Training course conducted from the 24th January to 27th February 2010

Supervisors: David LECCHINI (IRD - UR 227 CoReUs)

Milika SOBEY, Joeli VEITAYAKI & Edward LOVELL (USP)

Bernard MAIZERET (French Embassy at Fiji Islands)

Institut de recherche pour le développement

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REMERCIEMENTS

Au terme de ces deux mois de recherche, je tiens à remercier toutes les personnes qui, de près ou de loin, scientifiquement, financièrement ou moralement, ont contribué à l'aboutissement de ce rapport.

Je désire remercier David Lecchini, Chargé de recherche à l'Institut de Recherche pour le Développement (UR 128 Coreus) et Christophe Brié (Tropical Fish Tahiti) qui m'ont permis de réaliser ce stage. Je leur sais gré de m'avoir fait confiance tout au long de ce travail.

I would like to thank the Professors Milika Sobey,Edward Lovell and Joeli Veitayaki to select me to participe at this training course. Vinaka…

Je désire aussi remercier Mr. Bernard Maizeret, Conseiller de coopération et d’action culturelle à l’Ambassade de France à Fidji. Sans son aide, ce stage n’aura jamais vu le jour. Vinaka…

Ce travail a été réalisé au Centre de Recherche Insulaire et Observatoire de l’Environnement à Moorea. Je tiens à remercier très chaleureusement Serge Planes, Yannick Chancerelle et René Galzin d'avoir entrepris de nombreuses démarches pour le bon déroulement du stage et m'avoir permis de loger au Centre de Recherche. Un immense merci aussi à Pascal, Benoit et Franck pour leur aide de tous les jours.

Je tiens enfin à remercier mes collègues de Moorea : Viliame, Lindon, Cécile, Kevin, Moana … pour avoir mis la bonne ambiance au centre de recherche.

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FINANCEMENT DE L'ETUDE

* Financement par le programme CRISP (Coral Reef Initiative in the South Pacific) : L'étude a été financée par le programme CRISP : "Amélioration des techniques de capture des post-larves de poissons et de crustacés" (Composante C2A, R. Galzin & D. Lecchini; janvier 2010 / décembre 2010). L’initiative pour la protection et la gestion des récifs coralliens dans le Pacifique, engagée par la France et ouverte à toutes les contributions, a pour but de développer pour l’avenir une vision de ces milieux uniques et des peuples qui en dépendent ; elle se propose de mettre en place des stratégies et des projets visant à préserver leur biodiversité et à développer les services économiques et environnementaux qu’ils rendent, tant au niveau local que global. Elle est conçue en outre comme un vecteur d’intégration régionale entre états développés et pays en voie de développement du Pacifique. Le CRISP est un programme mis en œuvre dans le cadre de la politique développée par le Programme Régional Océanien pour l’Environnement afin de contribuer à la protection et la gestion durable des récifs coralliens des pays du Pacifique.

* Financement par l’Ambassade de France à Fidji : Mr. Bernard Maizeret a facilité grandement l’obtention de mon Visa.

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ABSTRACT

Over a period of four weeks samples of larvae were collected using crest nets. A total of 2313 larvae were collected consisting of 32 families and 75 species. Following identification the seven selected species were put into aquaria’s to be used for visual cue experiments. Three of each species were put into ten percent formal for twenty fours for fixation before the brain was dissected, thirty nine species were dissected and photographs of their brain taken. After the pictures were taken the fish, fish with open cranium and the brain were again put into ten percent formol foe preservation.

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1) INTRODUCTION

The biodiversity is declining, and habitat destruction and degradation are now commonplace. Examples of degradation can be found throughout marine ecosystems, including estuaries, saltmarshes, soft-bottoms, hard-bottoms, and coral reefs (Hughes et al. 2003). The degradation in coral ecosystems is usually characterized by coral mortality from natural and anthropogenic stressors (e.g., disease, hurricane damage, pollution, temperature-induced bleaching). This decrease of coral cover opens space on most reefs and causes substantial increases in cover and biomass of rapidly growing fleshy and filamentous macroalgae which, in turn, limits the recovery of coral populations and then modify fish and invertebrates communities (Edmunds & Carpenter 2001). Thus, areas experiencing perturbance often exhibit declines in adult populations, leading to a higher rate of extirpation than in pristine habitat, and the persistence of species in the area becomes reliant on the "rescue" effect of recruitment (Hanski & Gilpin 1997). The potential of the areas population to be supplemented by recruits, however, depends on whether pelagic larvae detect an appropriate habitat in that area and then settle and persist in that habitat (Sale 2002).

Thus, the abundance and diversity of coral reef fish depend mainly on the recruitment success (Sale 2002), which is characterized by important seasonal and inter-annual variations (Doherty & Williams 1998). Recruitment success depends, for a large part, on the number of survivors among pelagic larvae in the water column (Boehlert 1996). The life cycle of coral reef includes two major phases - a planktonic phase and a demersal or benthic phase. Coral reef fish spawn to produce eggs and larvae that disperse into the open ocean. After development as plankton, larvae recruit back to reefs. Most coral reef fishes have complex life cycles, involving the broadcast of eggs or larvae into the water column (Fontes et al. 2009), fertilized eggs disperse into the open ocean. Other species lay demersal eggs into nests, and then guard them until they hatch. Some species guard their young after hatching as well, but many simply allow the newly-hatched yolk sac larvae to disperse into the water column - where they enter the other major life cycle phase (planktonic larval phase). The planktonic phase is spent in the open ocean; often well away from any reefs. The planktonic phase usually lasts a few weeks to months. After the larval fish runs out of yolk, it eats plankton smaller than itself, such as diatoms, dinoflagellates, and copepods. Larval fish in turn are eaten by larger plankton, such as jellyfish, chaetognaths, and larval crabs. Assuming that the fish does not starve, get eaten, or become lost on the wrong ocean currents, near the end of its larval duration it begins searching for a suitable reef upon which to settle (or recruit).

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Coral reef fishes have a pelagic larval phase which ends with settlement onto the reef. Between hatching and settlement of fish larvae, growth and ontogenetic development result in a complete transformation of form and behavior. This development is associated with dispersal away from the reef and is followed at the end of the pelagic stage by the return and the settlement of fish on the reef (Dufour et al. 1996). The colonization process was observed to usually occur at night and in higher numbers during new moon periods than in full moon periods (Lecchini et al. 2004). There are several methods that can be used for sampling early and late larval stages in the field but the latest development and the method used in this research was a fixed net on the outer reef crest, which filters larvae from the water flowing from the ocean into the reef lagoon. This assumes that fish larvae are caught only during colonization onto the reef and not while moving back from the reef to the ocean or staying in one of the two environments. One of the great puzzles of coral reef fish ecology is how pelagic larvae locate the habitat in which they settle. At large spatial scales, some larvae detect the location of their settlement reefs by chemical cues emanating from the reefs (e.g. Atema et al. 2002, Huijbers et al. 2008) and/or reef noise (e.g. Montgomery et al. 2006, Simpson et al. 2008). After reaching the reef, individuals choose a settlement habitat based on the presence or absence of specific benthic substrates, conspecifics, and heterospecifics (e.g. Booth & Wellington 1998, Lecchini et al. 2005, 2007).

The ray-finned fishes represent the largest and most diverse vertebrate radiation, with an enormous range of variation in brain and behavior complexity, and specific adaptations. In consequence, knowledge of the cognitive capabilities and their neural basis in actinopterygian fishes could contribute to understanding not only their specific adaptations, but also the evolution of brain and behavior in vertebrates, including man (Broglio et al 2003). The main objective of the present study is to determine the visual abilities of coral reef fish larvae and to study the brain anatomy of the larvae. This was achieved by addressing the specific points: (1) coral reef fish larvae capture using crest net, (2) coral reef fish larvae identification, (3) the relative importance of visual cues from conspecifics versus heterospecifics, and (4) brain anatomy of coral reef fish larvae.

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2) MATERIALS AND METHODS

a) Study area

The present study was conducted on the west part of Moorea Island (Fig. 2). Moorea Island (17⁰30’ S, 149⁰5’ W) is a high volcanic island, 16 km away from Tahiti Island, in the Society Archipelago (French Polynesia). The island is encircled by a fringing reef which is located along the shore of the island. Further out is a barrier reef which is formed by the reef flat of 2 m depth and encloses a lagoon of about 800 to 1300 m wide. The average annual tidal range at Moorea is approximately 0.3 m. A constant flux of ocean water across the reef crest is created by waves breaking on the crest. The reef crest is intersected by passes connecting the open ocean with a lagoon channel through which lagoon water, which comes from waves breaking over the crest, returns into the ocean. The average residence time of the water in this system is 6hrs (Delesalle & Sournia 1992).

b) Tool capture

Samples of larvae were collected using a technique similar to Dufour and Galzin (1993). The net is 1.5 m wide x 0.75 m high x 5 m long and the mesh was of 1 mm size. Four hinged panels of 0.7 mm enlarged the mouth area of the net to 6 m (Fig. 1). The net was divided into three chambers: 1) the mouth where larvae enter, and the codends which is made of 2 parts; 2) the separator is designed in such a way that fish larvae are allowed to stay in sufficient water even if the water level is extremely low, it decreases mortality due to dessication and low oxygen level greatly; 3) and the collector where the larvae are captured. The whole structure is fastened secured by steel cables which are bolted firmly onto the reef- rock; this is to prevent the net from being swept away during times of strong current. The codend was attached during the evening as larval settlement usually occurs at night (Dufour & Galzin 1993). This method of collecting reef fish larvae was used over a period of four weeks (25/01/2010 – 18/02/2010).

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Upon collection of the larvae, the net is put in “off” mode, where the mouth of the net and its wings are laid down on the reef. The codend is detached from its base attachment and is released from the first chamber. It is important to ensure that no larvae are trapped within the first chamber before releasing the codend. All the contents of the codend are then emptied into a large cooler where any excess algae or rocks are removed. The nets were left on the crest without the codend and with the wing and net placed in “off” mode during the day.

Fig. 1. Schematic view and photo of crest net used at Moorea

c) Identification of coral reef fish at larval stage

At the laboratory, aerators were put into the cooler to supply oxygen to the larvae. The larvae were sorted into similar genera. Larval identification was undertaken using the meristics and morphology characteristics of the fish with the CRISP larvae identification guide handbook (Galzin et al. 2006) as an

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aid. The larvae were sorted alive as they would be used for laboratory experiment about visual cues and brain anatomy.

d) Laboratory experiment about visual cues

For this experiment, aquaria’s were set up as shown below (fig. 2). The first step of the experiment was to conduct a control experiment whereby tank 1 and 2 had no fish in them. The larvae were then placed in compartment A one at a time and the compartment in which it entered (B or C) was noted. For the second step heterospecific adults were put into tank 2 and again observations of which direction the larvae swam was noted. In the third step conspecific adults were put into tank1 and observation noted. After a certain number of larvae were observed tanks 1and 2 were switched and the experiment was conducted on the remaining larvae. This was done to confirm the attraction between larvae and adults.

B A C

2 Tank 2 Tank 1 0

2 30 0 c 20 cm 15 30 15 40 20

Fig. 2. Experimental choice chamber set up used to evaluate visual cues underlying habitat choice. The chamber consisted of an aquarium with three compartments (A, B and C) interconnected via funnels. Additional aquaria on either side of the choice chamber (labelled tank no. 1 and 2) were isolated from the choice chamber and mounted upon separate platforms to prevent the transfer of vibratory signals.

e) Brain anatomy

For each species, three individuals were put into a solution of MS 222. Once unconscious, the larvae were fixated in 10% formol for 24hrs. To get a solution of 10% formol, 37% formaldehyde was diluted with seawater using a ratio of 1:3 (formol:seawater). After 24hrs, the fish was removed and pictures were taken of the fish larvae, fish larvae without the cranium, dorsal view of the brain, ventral view of the brain, and two lateral views of brain. For each species, three individuals were fixated in formol: 1 larva (without dissection), 1 larva with open cranium, and 1 brain of larvae. The pictures were taken using a

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camera which was connected to a computer, and using the Camera Control Pro 2 software the light intensity and contrast were adjusted before the picture was taken.

3) RESULTS

Coral reef fish larvae capture with crest net - After four sampling weeks, a total of 2313 coral reef fish larvae belonging to 75 species, were collected (Fig 3, 4). Among the 75 species collected, 60 species were identified to the lowest taxonomist level. This was done with the help of Christophe Brie and a larval identification book by Maamaatuaiahutapu et al. (2006). The number is relatively low due to passing depression and cyclone flowed by huge waves and rough weather conditions, this prevented the net from being put up.

Fig 3. Larval species richness at colonization for the four week sampling period

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Fig 4. Coral reef fish larvae colonization and lunar cycle

The 5 most abundant families were Acanthuridae (10 species), Pomacentridae (9 species), Apogonidae (7 species), (7 species), and Chaetodontidae (6 species). The 5 most abundant species (Fig 7) were Stegastes fasciolatus (total abundance = 504), Chrysiptera leucopoma (total abundance = 218), Apogon yellow head (total abundance = 172), Scorpaenopsis diabolus (total abundance = 143), and microstoma (total abundance = 141).

Sensory abilities - Of the 7 species that were tested for visual cue, 4 species showed positive response to conspecific and also to heterospecific. The other 3 species showed no preference for either conspecific or heterospecific. This shows that the presence of conspecific can be a reliable indicator of suitable habitat but may also reflect a more competitive environment.

Brain anatomy – A total of 37 species of coral reef fish larvae were dissected and the brain removed and photographed and the anatomy studied. The morphology of the coral reef fish larvae brain varies. The five most important parts of the brain were present. Four of these parts (telecenphalon, optic tectum, cerebrellum, and hypothalamic lobe) were obvious in all species; the vagal lobe was not visible in most species unless observed carefully. Further studies on the brain of coral reef fish larvae will be continued at Seville University with Prof. Fernando Rodrίguez, Christina Broglio and Emilio Durang. I hope to conduct this study.

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4) DISCUSSION

This study shows that the colonization of the reef crest by coral reef fish larvae is mainly a nocturnal process. It has been shown that the settlement of the acronurus larvae of Acanthuridae occurs at night (Sale 1969, McFarland & Odgen 1985), as well as some Pomacentridae (Doherty 1981, Sweatman 1985, Robertson et al. 1988). As settlement is most probably an active process, it is assumed that most of the larvae and juvenile fishes are able to control the time in which they enter the lagoon. The precise timing of colonization seems consistent with the fact the specimens at these stages do not drift passively over the crest (Dufour & Galzin 1993).

My results support the hypothesis that most coral reef fish enter the reef at night. Records were not taken from the time of the full moon to the new moon due to a passing depression and cyclone which caused huge waves and rough seas. This prevented the net from being put up for fear that the codend would come loose or the ropes that fasten the net upright break causing collection of larvae to be impossible.

The extent to which larval stages of coral reef fish may aggregate in pelagic environments is relatively unknown, although recent evidence suggests that larvae may be dispersed pelagically in groups (Selkoe et al. 2006). In fish, some physiological studies have demonstrated that larvae have good visual abilities, but these abilities could improve after metamorphosis for some species (Kotrschal et al. 1990; Lara 2001). This could be the reason why the three species did not show a preference for either conspecific or heterospecific.

The first study to describe the brain' anatomy of coral reef fish at larval stage was conducted od several species. Thus, the "position" of brain in the cranium of coral reef fish larvae was found to be above the eyes for most and in others it lay behind the eyes. This first study is necessary before using the lesion methods or electrophysiological or optic techniques. About the extraction of the brain, we fixated the complete head (or the larvae) in a solution of 10% formol for several days before extraction. We used the same solution for preserving the brains. By this process, including the photographs, we will learn about the external morphology of the brain. Overall, for each species, we fixated three individuals: 1 larva (without dissection), 1 larva with open cranium, and 1 brain of larvae.

To conclude the results of this study support the hypothesis that most coral reef fish enter the reef at night. Visual cue is an important fact that contributes to the habitat in which the larvae will settle and

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visual preference may differ in different species. The brain of coral reef fish larvae has all of the five major parts put the morphology of the brain differs in different species.

5) REFERENCE Atema J, Kingsford MJ, Gerlach G. (2002) Larval Reef Fish Could use Odour for Detection, Retention and Orientation to Reefs. Mar Ecol Prog Ser 241:151-160

Boehlert GW. (1996) Larval Dispersal and Survival in Tropical Reef Fishes. In: Polunin NVC, Roberts CM (eds) Reef Fisheries. Chapman & hall, London, pp 61-84

Booth DJ, Wellington G (1998) Settlement Preferences in Coral-reef Fishes: Effects on patterns of Adults and Juvenile Distributions, Individual Fitness and Population Structure. Aust J Ecol 23:274-279

Broglio C, Rodriguez F, Salas C. (2003) Spatial Cognition and its Neural Basis in Teleost Fishes. Fish and Fisheries. 4:247-255

Delesalle, B,. Sournia, A. (1992). Residence Time of Water andPhytoplankton Biomass in Coral Reef Lagoons. Cont. Shelf Res. 12: 939-949

Doherty PJ (1981). Coral Reef Fishes: Recruitment-limited assemblages? Proc. 4th int. coral reef congr. 2:465-470

Doherty PJ, Williams DMB. (1998) The Replenishment of Coral Reef Fish Populations. Oceanogr Mar Biol Annu Rev 26:487-551

Dufour, V., Galzin, R. (1993) Colonization Patterns of Reef Fish Larvae to the Lagoon at Moorea Island, French Polynesia. Marine Ecology Progress Series 102, 143-52

Dufour, V., Riclet, E., Lo-Yat, A. (1996) Colonization of Reef Fishes at Moorea Island, French Polynesia: Temporal and Spatial Variation of the Larval Flux. Marine & Freshwater Resources, 47, 413-22

Fontes J, Cassele JE, Afonso P, Santos RS. (2009) Multi-scale Recruitment Patterns and Effects on Local Population Size of a Temperate Reef Fish. Journal of Fish Biology 75:1271-1286

Huijbers CM, Mollee EM, Nagelkerken I. (2008) Post-larval French Grunts (Haemulon flavolineatum) Distinguish Between Seagrass, Mangrove and Coral Reef Water: Implications for Recognition of Potential Nursery Habitat. J Exp Mar Biol Ecol 357:134-139

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Kotrschal K, Adam H, Branstatter R, Junger H, Taunreiter M, Goldschmid A (1990). Larval Size Constaints Determine Directional Ontogenetic Shifts in the Visual System of teleosts. Z Zool Syst Evolforsch 28 :166-182

Lara MR (2001). Morohology of the Eye and Visual Acuities in the Settlement-Intervals of Some Coral Reef Fishes (Labridae, Scaridae). Environ Biol Fish 62 :365-378

Lecchini D., Dufour V., Carleton J., Strand S. & R. Galzin, (2004). Study of the fish larval flux at Moorea Island: is the spatial scale significant? Journal of Fish Biology, 65: 1142-1146

Lecchini D, Shima J, Banaigs B, Galzin R. (2005) Larval Sensory Abilities and Mechanisms of Habitat Selection of a Coral Reef Fish During Settlement. Oecologia 143:326-334

Lecchini D, Osenberg CW, Shima JS, St Mary CM, Galzin R. (2007) Ontogenetic Changes in Habitat Selection During Settlement in a Coral Reef Fish: Ecological Determinants and Sensory Mechanisms. Coral Reefs 26:423-432

Maamaatuaiahutapu M, Remoissenet G, Galzin R. (2006). Guide d’identification des larves de poisons récifaux de Polynésie française. CRISP. Éditions Téthys 104p.

McFarland WN, Ogden JC (1985). Recruitment of Young Coral Reef Fishes From the Plankton. In: Reaka ML (ed). The Ecology of Coral Reefs. NOAA Symp. Ser. Undersea Res. 3:37-51

Montgomery JC, Jeffs A, Simpson SD, Meekan M, Tindle C. (2006) Sound as an Orientation Cue for the Pelagic Larvae of Reef Fishes and Decapod . Adv Mar Biol 51:143-196

Robertson DR, Green DG, Victor BC (1988). Temporal Coupling of Production and Recruitment of Larvae of a Caribbean Reef Fish. Ecology 69:370-381

Schmitt RJ, & Holbrook SJ. (2002) Correlates of Spatial Variation in Settlement of two Tropical Damselfishes. Marine and Freshwater Research 53:329-337

Sale PF (1969). PertinStimuli for Habitat Selection by the Juvenile Manini Acanthurus tristegus sandvicensis. Ecology 50:616-623

Sale PF (ed) (2002) Coral Reef Fishes: Dynamics and Diversity in a Complex Ecosystem. Academic Press, San Diego

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Salkoe KA, Gaines SD, Caselle JE, Warner RR (2006). Current Shifts and Kin Aggregation Explain Genetic Patchiness in Fish Recruits. Ecology 87:3082-3094

Simpson SD, Meekan MG, Jeffs A, Montgomery JC, McCauley RD. (2008) Settlement-Stage Coral Reef Fish Prefer the Higher-frequency Invertebrate-generated Audible Component of Reef Noise. Anim Behav 75:1861-1868

Sweatman HA (1985). The Timing of Settlement by Larvae Dascyllus aruanus: some Consequences for Larval Habitat Selection. Proc. 5th int. coral reef congr. 5:367-371

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ANNEX 1:

CORAL REEF FISH LARVAE CAPTURED WITH CREST NET

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FAMILY SPECIES ABD FAMILY SPECIES ABD

Acanthuridae Acanthurus triostegus 118 Scorpaenidae Scorpaenodes gaumensis 129 Acanthurus olivaceus 1 Scorpaenopsis diabolus 143 Acanthurus lineatus 2 Microdesmidae Ptereleotris micropelis 1 Acanthurus xanthopterus 2 ?? (Eel) 27 Ctenochaetus striatus 50 Scaridae ?? 8 Naso brevirostris 2 Tetraodontidae Canthigaster bennetti 3 Corythoichthys Naso vlamingii 5 Syngnathidae flavofasciatus 1 Naso lituratus 1 Blennidae sp1 (small) 7 Naso uniconis 1 sp2 (longest) 4 Zebrasoma scopas 1 sp3 (medium) 4 Apogonidae Apogon fraenatus 109 Balistidae Rhinecanthus aculeatus 7 Apogon exostigma 7 Fistulariidae Fistularia commersoni 3 Apogonichthys ocellatus 26 Gobiidae Valenciennea strigata 9 Apogon sp (long fin) 6 sp 72 (Apogon yellow head) 172 Serranidae Epinephelus merra 3 (Apogon black head) 32 Grammistes sexlineatus 2 Ostorhinchus angustatus 1 Synodontidae Saurida gracilis 39 Gymmapogon 14 sp 1 Holocentridae Myripristis berndti 3 Albulidae Albula glossodonta 9 Myripristis kuntee 12 Chaetodontidae Chaetodon citinellus 3 Myripristis pralinia 38 Chaetodon trifascialis 1 Sargocentron microstoma 141 Chaetodon lunulatus 1 Sargocentron spiniferum 15 Chaetodon auriga 5 Neoniphon argenteus 51 Chaetodon ephippium 4 Neoniphon sammara 7 Chaetodon unimacalutus 1 Pomacentridae Stegastes fasciolatus 504 Mullidae Parupeneus maltifasciatus 16 Stegastes lividis 2 Parupeneus barberinus 11 Stegastes nigricans 25 Labridae Thalassoma hardwicke 20 Chromis viridis 107 Bothidae Bothus mancus 13 Abudefduf sexfasciatus 1 Soleidae sp 2 Dascyllus aruanus 21 Lethrinidae Monotaxis grandoculis 12 Chrysiptera glauca 4 ?? Plereleotus sp 8 Chrysiptera leucopoma 218 Kuhliidae Kuhlia mugil 2 Pomacentrus pavo 17 Mugil mugil 2 Aulostomidae Aulostomus chinensis 2 ?? Carapus sp 2 Sphyraenidae Sphyraene barracuda 1 Zanclidae Zandus cornutus 5 Siganidae Siganus spinus 1 Lutjanidae Lutjanus kasmira 4 Muranidae sp 9

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ANNEX 2:

PICTURES OF BRAIN OF CORAL REEF FISH LARVAE

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Brain’ anatomy of coral reef fish larvae

In January / February 2010, fish larval was captured with crest nets during two months. So, a description of fish brain at larval stage was conducted: ‐ We took a picture of fish larvae; ‐ We opened the cranium of fish larvae and took a picture of the brain; ‐ Then, we took off the brain and took several pictures (under different view) of the brain. The study was carried out on several fish species. It is the first study to describe the brain' anatomy of coral reef fish at larval stage. Thus, we will know the "position" of brain in the cranium of coral reef fish larvae. This first study is necessary before using the lesion methods or electrophysiological or optic techniques. About the extraction of the brain, we fixated the complete head (or the larvae) in a solution of 10% formol for several days before extraction. We used the same solution for preserving the brains. By this process, including the photographs, we will learn about the external morphology of the brain. Overall, for each species, we fixated three individuals: 1 larva (without dissection), 1 larva with open cranium, and 1 brain of larvae.

Cb : Cerebrellum Tel : Telecenphalom Hyp : Hypothalamic lobe VL : Vagal lobe OT : Optic tectum

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Acanthurus lineatus Total length: 39 mm Is there scale on head: No Is there fat tissue between cranium and brain: Yes Additional information:

 Fat tissue can easily be removed with the cranium  The cerebrum is elongated and extends over the optic tectum  The cranium is thick

Picture of the fish larvae and the brain

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The study was conducted on 36 other species (see : ftp://ftp.ird.nc/outgoing/oceano/ in Lecchini file)

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