L A Lozano-Hernández

Benjamin Reig

Jean Baptiste Doucet

Ludovic Salvagnac

Stéphane Calvez

H Y Lee

C T Lee

Isabelle Séguy

Véronique Bardinal

Design and optimization of a blue fluorescent microcavity-organic light-emitting diode (MOLED) on AZO anode for an algae excitation light source application

Keywords: Blue OLED, MOLED, DBR, AZO, biosensor, microcavity OLED

à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

Since C.W. Tang and S. A. VanSlyke publication in 1987 [START_REF] Tang | Organic electroluminescent diodes[END_REF], organic light emitting diodes (OLEDs) have been attracted scientific and commercial attention because of the versatility of their optical and mechanical properties such as color intensity, high contrast, wide viewing angles, flexibility, lightness… [START_REF] Zou | Recent advances in organic light-emitting diodes: toward smart lighting and displays[END_REF][START_REF] Zhu | Recent advances in flexible and wearable organic optoelectronic devices[END_REF]. Nowadays, OLED are already widely commercialized in devices such as smartphones, TVs, smartwatches, etc. In addition, the advantages of OLED technology, allowing light source integration on many types of substrates, open up new and promising applications such as chemical and biological sensors [START_REF] Murawski | Emerging Biomedical Applications of Organic Light-Emitting Diodes[END_REF]. However, even if organic LEDs are already commercialized, their stability and lifetimes are not yet comparable to their inorganic counterparts, preventing their use in sensors where device stability is mandatory [START_REF] Huang | Micro-LED and OLED displays: present status and future perspectives[END_REF]. This lack of stability is partly due to Indium diffusion in organic layers. In this context, aluminum zinc oxide electrode (AZO) constitutes a low-cost, nontoxic and relative abundant alternative anode to ITO (indium tin oxide) since Zn diffusion is much lower [START_REF] Zhao | Organic light-emitting diodes with AZO films as electrodes[END_REF]. There was also increased research efforts on this issue, focusing on the synthesis of new molecules and on optimizing the extraction of emitted light [START_REF] Zou | Recent advances in organic light-emitting diodes: toward smart lighting and displays[END_REF]. With the development of different generation of emitting materials (such as fluorescent, phosphorescent, thermally activated delayed fluorescent -TADF and hyperfluorescent) OLEDs have now reached a higher efficiency, but the stability problem remains [START_REF] Hong | A Brief History of OLEDs-Emitter Development and Industry Milestones[END_REF]. Indeed, while fluorescent OLEDs can achieve lifetimes of the order of tens of thousands of hours, newest and more efficient systems as the hyperfluorescent are limited, in general, to just a few hours [START_REF] Monkman | Why Do We Still Need a Stable Long Lifetime Deep Blue OLED Emitter?[END_REF]. Blue fluorescent OLEDs (first generation of emitting materials) are still used in industry although they exhibit lower efficiency compared to the last materials generation [START_REF] Chan | Stable pure-blue hyperfluorescence organic light-emitting diodes with high-efficiency and narrow emission[END_REF]. As an additional improvement, several options to increase the outcoupling of light, the overall OLED efficiency and even tune the electroluminescence (EL) spectra of the device have been reported [START_REF] Zhang | Light Out-Coupling Management in Perovskite LEDs-What Can We Learn from the Past?[END_REF]. One of the most promising option is the microcavity-OLED (MOLED) based on the integration in the OLED stack of a distributed Bragg reflector (DBR) made of alternate layers of high and low refractive index [START_REF] Jordan | Efficiency enhancement of microcavity organic light emitting diodes[END_REF]. In a classic OLED, with organic semiconductors (OSCs) sandwiched between a transparent anode and a metallic cathode, only about 20% of the emitted light (internal quantum efficiency, IQE) is extracted. Around 50 % of this light is lost through the anode and the organic layers while another 30 % is lost in the substrate (wave guided mode) [START_REF] Huang | Ultrathin, lightweight and flexible organic light-emitting devices with a high light outcoupling efficiency[END_REF]. So, it is possible to infer that theoretically, only around 5 % of the injected electrons will be extracted as photons (external quantum efficiency, EQE) for a fluorescent OLED (IQEmax ~ 25 %) [START_REF] Pu | Optimizing the Charge Balance of Fluorescent Organic Light-Emitting Devices to Achieve High External Quantum Efficiency Beyond the Conventional Upper Limit[END_REF]. Due to microcavity effect, the EL emission of MOLEDs can be easily filtered at a design wavelength and light intensity can be significantly improved for this wavelength [START_REF] Lin | Microcavity-Enhanced Blue Organic Light-Emitting Diode for High-Quality Monochromatic Light Source with Nonquarterwave Structural Design[END_REF]. In this work we report on the design of a blue light source to be integrated in an optical biosensor dedicated to in situ monitoring of surface water quality. The system architecture consists of a fluidic structure with tanks and optical sensor (OLED and organic photodiode) located on its periphery (Fig. 1 (a)). The optical transducer will allow the detection of direct fluorescence emitted by green micro-algae at a wavelength of 680 nm (Fig. 1 (b)). The emission spectrum of classic OLEDs being wide, there could be significant spectral overlap between the signal to be detected and the emission tail of the OLED since the latter signal is of comparable intensity to that of algae fluorescence. So, the constraints related to algae fluorescence detection require the development of new device architecture for high efficiency (with a maximum emission matching algae absorption at 470 nm), reduced spectral width, and stable integrated deep blue OLED [START_REF] Cho | Microcavity two-unit tandem organic light-emitting devices having a high efficiency[END_REF].

To that extent, a MOLED based on MADN:DPAVBi emitting layer (EML) and (TiO2/SiO2)-based DBR was designed. Here, the microcavity effect was exploited as a tool to fit the FWHM (full width at half maximum) of the OLED emission peak to the absorbance bandwidth of the algae (30nm). It was simultaneously used to reduce the OLED emission range in the algae fluorescence bandwidth (650-700 nm). Considering these specific spectral limits, we optimized both optically and electrically the spacers in the organic stack. Moreover, the MOLED was fabricated on an AZO anode deposited on the DBR to avoid the indium diffusion problem, which has not been done before. First, we investigated by modeling the variation of the two key parameters for the design of the MOLEDs: the optical cavity length (resonance condition) and the EML position relative to the electric field intensity (antinode position) [START_REF] Cho | Microcavity two-unit tandem organic light-emitting devices having a high efficiency[END_REF]. A convergent solution for the optimal OLED and MOLED stacks, from the electronic and optical points of view, was found. Then, according to those results, OLED and MOLED devices were fabricated, characterized and compared.

Materials and methods

MOLEDs modelling was conducted by using a commercial software based on the matrix formalism [START_REF] Chakaroun | Experimental and theoretical study of the optical properties optimization of an OLED in a microcavity[END_REF]. Using this tool, it is possible to calculate the reflectivity and the transmission spectra of a multilayer stack if the refractive indices and thickness of each layer are known. To determine the real and imaginary refractive indices (n and k), ellipsometry measurements were conducted separately on each of the layers involved in the device and spectral models were derived from these data. To validate these models, full devices were then prepared on glass substrates coated with a 228 nmthick AZO (Al-doped zinc oxide) or DBR/AZO anode on glass for OLED and MOLED respectively. The dielectric Bragg mirror was formed by a stack of 80 nm-thick low index (n~1.5) SiO2 and 49 nm-thick high index (n~2.3) TiO2 alternate layers. These thicknesses are chosen to be quarter-wave optical layers at the aimed resonance wavelength (470nm). Enclosing the organic region between this DBR/AZO part and a metallic electrode playing the role of the top mirror leads to the formation of a vertical micro-cavity. In such a device, the emitted light is confined and transmitted out of the cavity only for the resonance wavelengths (see modeling section). SiO2 and TiO2 materials were chosen for their high relative index contrast and their low absorption in the visible range and were deposited on the glass substrate by sputtering. AZO was deposited on the DBRs by atomic layer deposition technique (ALD). The deposition conditions were optimized to obtain a sheet resistance of 54 /sq and a transmission of 79% [START_REF] Dugrenil | AZO electrodes deposited by atomic layer deposition for OLED fabrication[END_REF] The reflectivity spectra of the DBR/AZO/glass stacks and their uniformity along the wafers were measured before MOLED fabrication and found in good agreement with modelling (not shown here).

AZO was patterned by chemical etching using standard photolithography. The substrates were cleaned with acetone and isopropanol 10 min each in an ultrasonic bath. Subsequently, UV-ozone treatment was performed for 10 min in order to remove any carbon contamination [START_REF] Feng | Effect of deposition condition and UVozone post-treatment on work function of DC magnetron sputtered AZO thin films[END_REF]. The OSC layers were sequentially evaporated at 5.10 -7 torr with a rate of 0.03-0.05 nm/s in a Plassys MEB550B vacuum chamber installed in a glove box. The OLED/MOLED stack reported in Fig. 1 (a) is: 1,4,5,8,9,11-Hexaazatriphenylenehexacarbonitrile (HAT-CN) as hole injection layer (HIL), N,N'-Di(1-naphthyl)-N,N'-diphenyl-(1,1'-biphenyl)-4,4'-diamine (NPB) as hole transport layer (HTL), 9-Phenyl-3,6-bis(9-phenyl-9Hcarbazol-3-yl)-9H-carbazole (Tris-PCz) as electron blocking layer (EBL), 1,3-Bis(3,5-dipyrid-3ylphenyl)benzene (BmPyPhB) as electron transport layer (ETL), 8-Hydroxyquinolinolato-lithium (Liq) as electron injection layer (EIL). For the EML 2-methyl-9,10-bis(naphthalen-2-yl)anthracene (MADN) was used as the host material doped with 4,4'-Bis[4-(di-p-tolylamino)styryl]biphenyl (DPAVBi). This layer was obtained by the coevaporation of MADN and DPAVBi with a 4% doping level, controlled by the deposition rate of the two molecules (0.1 and 0.004 nm/s respectively). The 4% amount of DPAVBi has been set after a study of OLED efficiency vs doping ratio (not shown here). Aluminum was used as cathode and deposited at a rate of 1 nm/s. Finally, the devices were encapsulated with a cover glass sealed with epoxy adhesive inside the glove box.

After fabrication, J-L-V characterization and electroluminescence spectra were performed outside the glovebox by using a Keithley 2450 SourceMeter, an Irradian L203 photometer with a calibrated silicon photodetector and an Ocean Optics spectrometer respectively. External quantum efficiency calculations were made as reported by Shukla et. Al [START_REF] Shukla | Elementary approach to calculate quantum efficiency of polymer light emitting diodes[END_REF] by the J-L-V and EL spectra. The angular dependence of the EL intensity and corresponding spectra were also measured with a home-made set-up including a motorized goniometer.

Results and discussions

The Fig. 2 (a) displays the calculated reflectivity spectra of three different MOLEDs having 3 different numbers of pairs in the 470nm-centered DBR [START_REF] Dugrenil | AZO electrodes deposited by atomic layer deposition for OLED fabrication[END_REF]. For each of them, the thickness of the organic stack was finely tuned to get an optical cavity length fully resonant at 470 nm. As a result, a sharp dip at 470 nm is obtained for each of them in the middle of the high reflectivity band comprised between 400 nm and 600 nm, which means light transmission will be higher at 470 nm, as aimed. The FWHM (full width at half maximum) of the transmission peak will be also reduced compared to a standard OLED due to the microcavity effect. As seen, the FWHM is slightly varying with the number of pairs in the DBR and is equal to 26 nm for the 1.5 pairs-case. This spectral filtering is well suited to our pollution detection system application. One can also note that the reflectivity value at the resonance is not the same for the 3 cases, as it is mostly dependent of the balance between the bottom DBR reflectivity and the top aluminum electrode one. For the 1.5 pair case, this balance is almost perfect and the reflectivity at the resonance is close to zero. Consequently, light transmission through the transparent electrode will be optimal for this case and the MOLED efficiency is expected to be higher. A second reflectivity dip can be seen at 550nm~660nm, i.e. in a lower emission range of the OLED. It corresponds to the upper edge of the reflectivity stop band of the DBR and does not involve any amplification effect.The OLED stack was also optimized by carefully positioning the EML in such a way that it coincides to a maximum of the standing wave electric field in the multilayer stack (see Fig. 2 (b)) at an antinode position for which constructive interferences occur and light amplification is maximized. As observed, the magnitude and the distribution of the electrical field also depend on the number of pairs in the DBR. The most favorable case is again when a DBR with 1.5 pairs is used (MOLED 1.5: 44V/m) compared to the other cases (2.5 and 3.5, respectively 37V/m and 27V/m). The designed OLED stack, shown in Fig. 1 (a), was finely optimized to reach a solution meeting simultaneously the best OLED performances and MOLED optical design. In our case, according to the model results (Fig. 2 (b)), the latter condition is achieved for an EML layer located at 298 nm from the DBR mirror. In the literature, the most common selected method, was to adjust the cavity length by simply varying the thickness of the HTL, HIL or both. However, these devices architectures involve unusually thick layers of organic semiconductors that leads to a loss of performance and stability of the OLEDs [START_REF] Deng | Improving the Thermal Stability of Top-Emitting Organic Light-Emitting Diodes by Modification of the Anode Interface[END_REF]. Adachi et. Al [START_REF] Yamaguchi | A 1,4,5,8,9,11-hexaazatriphenylenehexacarbonitrile (HAT-CN) transport layer with high electron mobility for thick organic light-emitting diodes[END_REF] recently demonstrated that by modifying the HAT-CN layer from 10 nm to even 200 nm, it is possible to increase the overall OLED thickness while keeping the current density at the same value. Nevertheless, the EQE was significantly reduced from ~13 to ~9% corresponding to HAT-CN thicknesses of 10 and 200 nm respectively. Among these papers, one can note the use of a multi-layered HAT-CN(20 nm)/NPB(60 nm)/HAT-CN(15 nm)/NPB(60 nm) HTL [START_REF] Park | Enhanced light extraction efficiency and viewing angle characteristics of microcavity OLEDs by using a diffusion layer[END_REF] with a large thickness of 155 nm that fulfils the cavity length and at the same time enables an efficient hole transport by using two materials with excellent charge carrier mobility. In contrast, for a classic OLED the optimal thickness for HAT-CN and NPB is rather around 5-10 nm and 50 nm respectively [START_REF] Park | Electro-Optical Performance of Organic Thin-Film Using HAT(CN)6 between Anode and Organic Materials[END_REF][START_REF] Chu | Hole mobility of N,N′-bis(naphthalen-1-yl)-N,N′-bis(phenyl) benzidine investigated by using space-charge-limited currents[END_REF]. In this work, Tris-PCz was introduced as EBL (Fig. 1 (c)) instead of HTL (as is commonly used), to maintain not only an adequate charge balance in OLED (by choosing the NPB thickness value to the one usually used in classic OLED) but also to increase the number of layers for which the thickness can be slightly tuned (+/-5-10 nm), to exactly achieve the cavity length for MOLED optical optimization. In order to verify the electrical performances of this optically optimized stack, ITO-based OLED were fabricated and characterized. The obtained devices exhibited 4.7 % maximum external quantum efficiency (EQE) close to the 5% theoretical limit of fluorescent OLEDs (see table 1). The experimental EL spectra for the 4 designed AZO-based devices are depicted in Fig. 3 (a). As can be seen at the same applied current density (10 mA/cm 2 ), the peak for the MOLED 1.5 is around 3 times higher than the peak for the classic OLED. This significant improvement is in good agreement with the modelling results. The spectral filtering around 470 nm is also clear compared to the conventional OLED. The peak intensity for higher number of pairs (2.5 and 3.5) is also enhanced at 470 nm but to a lower extent. MOLED 2.5 presents a similar spectral behaviour, but a smaller intensity and a slightly red-shifted emission peak. Finally, a second peak is observed in the range [560-610 nm] for all MOLEDs. As above-mentioned, it corresponds to the upper limit of the DBR spectral band (see Fig. 2 (a)). The resulting parasitic emission in this range is not suited for our optical biosensor application. As expected, its amplitude increases with the number of pairs. Consequently, these measurements confirm that MOLED 1.5 case leads to the best results. As can be seen, the EL spectra of the MOLED 1.5 matches perfectly with the expected shape: a strong enhancement of the emission in the low reflectivity wavelengths around 470 nm and reduced emission in the 500-600 nm bandwidth. It is worth mentioning that the FWHM was reduced from around 74 nm for the classic OLED to 26 nm for the MOLED 1.5. In Fig. 3 (b), pictures of the OLED and MOLED 1.5 are shown, in which the change of the EL spectra can be appreciated simply by observing the color difference with a better color purity for the MOLED. Fig. 4 (a) and (b) display the angular dependence of the EL spectra for the standard OLED and MOLED 1.5 respectively. For the OLED the emission peak is slightly shifted and significantly reduced, while the emission intensity after 500 nm reduces but its contribution to the spectrum increases. On the other hand, the MOLED has a significant shift and reduction around its emission peak, while the emission in the 500-600 nm bandwidth remains similar (except at 90° where it is minimal). Fig. 4 (c) and (d) shows the experimental angular dependence of the normalized luminance and EL intensity for OLED and MOLED 1.5 respectively. It can be seen that, the OLED luminance is maximum at 40° and not at 0° as expected for an ideal Lambertian-like pattern emission. This is due to fact that the EL spectra for this blue OLED have a more significant contribution of green wavelengths (520 nm see Fig. 4 (a)) at oblique incidence. The angular distribution of the MOLED 1.5, which emits less in the green region (around 550 nm), has its maximum emission in the 0-20° range and a reduced emission for higher angles due to the microcavity effect. On the other hand, if we measure specifically the angular dependence of the EL intensity for the OLED and MOLED at 470 nm which is the wavelength target for the light excitation source (not taken into account any other wavelength), the effect of the microcavity is clear (Erreur ! Source du renvoi introuvable. (d)). This change in the light distribution for the MOLED is highly desirable from the sensor point of view because most of the light will be directed vertically (0°) to the algae.

On the other hand, as the emission is improved about 3 times in the MOLED 1.5 compared with the standard OLED, the current density needed to reach the same peak intensity will be lower. This reduction in the current applied is anticipated to extend the lifetime of the devices by reducing the excited states generated by recombining charges [START_REF] Chen | Mitigation of Electrical Failure of Silver Nanowires under Current Flow and the Application for Long Lifetime Organic Light-Emitting Diodes[END_REF]. In the Fig. 5 (b), EQE values as a function of the applied current density for the classic OLED and MOLED 1.5 are shown. MOLED 1.5 had a maximum improvement of 33 % compared to the classic OLED. Consequently, despite a reduced FWHM, the MOLED EQE is higher than the OLED one owing to the microcavity effect. Furthermore, the reduction of the necessary current density required to obtain a given luminescence level is expected to extend the lifetime of the devices for the sensor. In Table 1 the main performance values for the classic OLED and the MOLED 1.5 are compared. As expected, the turn-on voltage (3.3-3.4 V) and the wavelength for which the maximum emission is observed, λpeak (469 nm) are similar for OLED and MOLED (see (a)). On the other hand, the EQEmax is improved in the MOLED 1.5, from 3.4 to 3.7 %, compared to the value obtained for the OLED even when the luminance of the MOLED 1.5 is lower (5291 cd/m 2 vs 8294 cd/m 2 ). This is due to the fact the luminance is a measure of light that has a responsivity corresponding to the human eye and is formally known as the CIE photopic luminous efficiency function (V(λ)). Therefore V(λ) has its maximum response around the green wavelengths (555 nm), which are not favoured in the MOLED 1.5 case.

Conclusion

In this work, by designing a blue AZO-based MOLED for an optical biosensor, it has been obtained a light excitation source with improved emission in the blue range (470 nm) combined to a low emission in the green region in agreement with the requirements of the aimed algae fluorescence sensor. Exploiting a MOLED, the EQE was improved up to 33 % compared with the reference OLED, while at the same time the FWHM was reduced from about 74 nm to only 26 nm and the angular distribution decreased from 40° to 20°. Further work towards the integration of such light sources into biosensors will focus on the improvement of emission filtering around 600 nm by adding an internal filter in the structure and the study of the device stability and lifetime. In addition, the strategy here followed to find a simultaneous solution for the best OLED stack and the best MOLED design, could be applied in the future for newest and more efficient generation devices (phosphorescent, TADF, hyperfluorescent) once that their stability is comparable with the first generation (fluorescence).

Fig. 1 .

 1 Fig. 1. (a) Schematic representation of the optical bio-sensor with MOLED structure, (b) algae absorbance and fluorescence spectra and (c) energy diagram for the OLED stack.

Fig. 2 .

 2 Fig. 2. (a) Reflectivity and (b) electric field obtained by model for the 3 types of MOLED by using a DBR with 1.5, 2.5 and 3.5 pairs.

Fig. 3 .

 3 Fig. 3. (a) EL spectra for the classic OLED and the three different MOLEDs (1.5, 2.5 and 3.5) at 10 mA/cm 2 . All spectra are normalized with the peak of the classic OLED. (b) Pictures of the OLED and the MOLED 1.5.

Fig. 4 .

 4 Fig. 4. Angular dependence of (a) the normalized luminance and (b) the normalized EL peak intensity at 470 nm for the OLED and the MOLED 1.5. EL spectra at different angles for (c) the OLED and (d) the MOLED 1.5.

Fig. 5 .

 5 Fig. 5. (a) J-L-V characteristics and (b) the EQE for the classic OLED and the MOLED 1.5.

Table 1 .

 1 Performance of the OLED and MOLED devices. -on voltage at 0.1 cd/m 2 , Lmax= maximum luminance, EQEmax= maximum external quantum efficiency, EQE100= external quantum efficiency at 100 cd/m 2 , EQE1000= external quantum efficiency at 1000 cd/m 2 , λpeak= peak of emission

	Device	Von (V)	Lmax (cd/m 2 )	EQEmax (%)	EQE100	EQE1000	λpeak (nm)	FWHM
								(nm)
	ITO-OLED	3.1	20166	4.7	4.5	4	469	~74
	AZO-OLED	3.3	8294	3.4	3.1	2.4	469	~74
	AZO-MOLED 1.5	3.4	5291	3.7	3.7	3.2	469	26
	Von = turn

Acknowledgements

We acknowledge financial support by the Agence Nationale de la Recherche (ANR) under the project BELUGA (grant number: ANR-18-CE04-0007). This work was supported by LAAS-CNRS micro and nanotechnology platform, a member of the Renatech French national network. E. Scheid, J. Roul and B. Franc from LAAS-CNRS are also acknowledged for their help on sample characterizations.


Bastien Preuss
email: bastien.preuss@ird.fr

Dominique Pelletier

Laurent Wantiez

Yves Letourneur

Sébastien Sarramégna

Michel Kulbicki

René Galzin

Jocelyne Ferraris

Considering multiple-species attributes to understand better the effects of successive changes in protection status on a coral reef fish assemblage

Keywords: assessment model, coral reef ecosystem, fish assemblage, fishing effect, MPA effect

published or not. The documents may come

Considering multiple-species attributes to understand better the effects of successive changes in protection status on a coral reef fish assemblage

Introduction

A key issue in tropical areas is the impact of the huge diversity found on tropical reefs on the effect of management measures. In particular one may question the relative impact of such measures on the species composition of fish assemblages and their capacity of adaptation and their resilience knowing that these highly diverse assemblages are subject to a number of anthropogenic and natural disturbances [START_REF] Hughes | Catastrophes, Phase Shifts, and Large-Scale Degradation of a Caribbean Coral Reef[END_REF][START_REF] Connell | Disturbance and recovery of coral assemblages[END_REF][START_REF] Nyström | Spatial Resilience of Coral Reefs[END_REF]. In New Caledonia, recreational and subsistence fishing activities, mining and industrial activities, growing population and the development of tourism have all had a negative impact on the reef systems [START_REF] Labrosse | New Caledonia[END_REF]. MPAs are acknowledged as a major tool for biodiversity conservation and fisheries management [START_REF] Agardy | Advances in marine conservation: The role of marine protected areas[END_REF][START_REF] Sumaila | Addressing ecosystem effects of fishing using marine protected areas[END_REF] but they can also be seen as an instrument for an experimental approach aimed at improving our understanding of how communities respond to fishing. In this respect, MPAs are a tool for actively adaptive management in the sense of [START_REF] Walters | Adaptative control of fishing systems[END_REF]. Many studies have focused particularly on marine reserves and assessed their effects on fish assemblages and on marine organisms (see reviews by [START_REF] Roberts | Are marine reserves effective in management of reef fisheries?[END_REF][START_REF] Russ | Yet another review of marine reserves as reef fishery management tools[END_REF][START_REF] Halpern | The impact of marine reserves: do reserves work and does reserve size matter?[END_REF][START_REF] Pelletier | Designing indicators for evaluating the impact of Marine Protected Areas on coral reef ecosystems: a multidisciplinary standpoint[END_REF]. In most existing papers, the impact of reserve or fishing experiments was assessed on a taxonomic basis (usually families) (e.g. [START_REF] Alcala | Effects of marine reserves on coral fish abundances and yields of Philippine coral reefs[END_REF][START_REF] Jennings | Seychelles' protected areas: comparative structure and status of reef fish communities[END_REF][START_REF] Mcclanahan | Fishery Recovery in a Coral-Reef Marine Park and Its Effect on the Adjacent Fishery[END_REF][START_REF] Russ | Natural fishing experiments in marine reserves 1983-1993: Roles of history and fishing intensity in family responses[END_REF] or for species bearing particular importance in the context of the study (e.g. [START_REF] García-Rubies | Effects of total fishing prohibition on the rocky fish assemblages of Medes Islands marine reserve[END_REF][START_REF] Letourneur | Réponse des peuplements et populations de poissons aux réserves marines : le cas de l'île de Mayotte, Océan Indien occidental[END_REF][START_REF] Edgar | Effects of the declaration of marine reserves on Tasmanian reef fishes, invertebrates and plants[END_REF][START_REF] Johnson | The effectiveness of an existing no-take fish sanctuary within the Kennedy Space Center, Florida[END_REF]. Methods have classically tested differences in density, biomass and species richness between the reserve and a comparable zone. Few studies have evaluated reserve effects by grouping species other than at a taxonomic level; most have considered only a limited number of species or sometimes trophic groups (e.g. [START_REF] Russ | Marine reserves: rates and patterns of recovery and decline of large predatory fish[END_REF]. Assessment for a given species or species group does not provide a synoptic view of the impact of the reserve. Assessing the impact of a reserve at the fish assemblage level is more desirable in providing scientific elements for an ecosystem approach to management [START_REF] Botsford | The management of fisheries and marine ecosystems[END_REF][START_REF] Jennings | The effects of fishing on marine ecosystems[END_REF]. This is particularly true in coral ecosystem where diversity is particularly high (over 600 observable species in New Caledonia according to [START_REF] Kulbicki | Opening of an MPA to fishing: Natural variations in the structure of a coral reef fish assemblage obscure changes due to fishing[END_REF]. The structure of the assemblage can be analyzed based on taxonomic, ecological or economic grouping of species. This approach has been at the heart of several studies on the effects of MPAs on the reef fish assemblages of New Caledonia [START_REF] Ferraris | Assessing the impact of removing reserve status on the Abore Reef fish assemblage, New Caledonia[END_REF][START_REF] Amand | A step toward the definition of ecological indicators of the impact of fishing on the fish assemblage of the Aboré reef reserve (New Caledonia)[END_REF][START_REF] Kulbicki | Opening of an MPA to fishing: Natural variations in the structure of a coral reef fish assemblage obscure changes due to fishing[END_REF]. These first studies have demonstrated the advantage of multi-species groups over single species approaches in these highly diversified systems. The objective of this study is to assess the effects of successive changes in protection status on the fish assemblage of the Aboré reef reserve. Located in the South Lagoon of New Caledonia, SW Pacific Ocean, this MPA had been in place for three years, when part of it was open to fishing. Two years later, it was closed to fishing again. Fish assemblages were surveyed before and after the opening and the closure. We anticipated that changes in the fish assemblage would vary according to environmental factors and hypothesized that species attributes such as diet, species size, home range or interest for fishing would be important factors in this variation. For instance, highly targeted species may be more impacted by fishing than less valued ones, and mobile species may be less affected than sedentary ones.

Material and methods

The Aboré reef reserve MPA has been studied by [START_REF] Amand | A step toward the definition of ecological indicators of the impact of fishing on the fish assemblage of the Aboré reef reserve (New Caledonia)[END_REF]), Ferraris et al. (2005) and [START_REF] Kulbicki | Opening of an MPA to fishing: Natural variations in the structure of a coral reef fish assemblage obscure changes due to fishing[END_REF] and this present study contributes distinct data sets and time frames. Earlier studies used data from censuses where all fish species were recorded, resulting in more than four hundred species observed, but it could only be performed by highly trained divers and the number of replicates was necessarily low (69) as these censuses were time consuming. Data used in the present study pertain to a restricted list of species ( 213), but correspond to a much larger number of transects (212) and could thus be performed by less trained divers.

Several factors that might explain the variations of observed fish counts independently of protection status were investigated. Then, the impacts of opening the area to fishing and the second closure of the area were tested using a range of biological responses such as species richness, abundance, biomass and mean individual size. The fish assemblage was partitioned based on mobility, trophic regime, adult size and interest for fishing, and each response was calculated according to the different partitions. By crossing variables and partitions of fish assemblage the metrics per species group could be analyzed and the effects of changes in protection status at the fish assemblage level were assessed simultaneously. We were able to demonstrate the benefits of our approach, by comparing these results with an overall approach considering responses averaged over all species. The partitioning provided insights to better understand the effects of such changes on the fish assemblage.

Study area

The Nouméa lagoon, located in New Caledonia, SW Pacific Ocean (Figure 1), is a large lagoon seascape including coral reefs where several marine reserves (no take zones) were established in the 1980s to protect the coral reef ecosystem from the impacts of fishing. The present study took place in the Aboré reef reserve, located on a 25 km-long barrier reef, 20 km off Nouméa, and representing about 15 000 ha. The area was closed to fishing from 1988 to 1993, and then twothirds of the reef (area B) were again opened to fishing from August 1993 until July 1995 for a fishing experiment. The opening of the reserve immediately resulted in an intense fishing pressure; in the first two weeks, eight hundred boats were observed and the fish yield was 8.7 tons as estimated from a sampling of 57% of these boats [START_REF] Sarramégna | Contributions à l'étude des réserves marines du lagon sud-ouest de Nouvelle-Calédonie: influence des différents statuts de protection sur la structure des peuplements ichtyologiques[END_REF]. These levels more or less corresponded to what had previously been observed over an entire year [START_REF] Sarramégna | Contributions à l'étude des réserves marines du lagon sud-ouest de Nouvelle-Calédonie: influence des différents statuts de protection sur la structure des peuplements ichtyologiques[END_REF]. The whole reef has been closed to fishing since August 1995 (Figure 2; Table 1).

Sampling protocol

The impact of allowing fishing in the reserve and the restoration effect after the final closure on fish assemblage was monitored from 3 surveys. In July 1993, a survey was performed using 60 transects in 5 locations spaced along the reef (Figure 2), just before resumption of fishing on twothirds of the Aboré reef (area B, Figure 2), while one third remained closed (area A). In July 1995, a second survey of 110 transects was conducted on both the area A closed to fishing and the area B open to fishing. Area B was closed again to fishing in September 1995, resulting in the entire closure of the Aboré reef for the second time. Six years later, in 2001, a third survey of 42 transects was conducted on the Aboré reef MPA (Table 1). The experimental design stratified the reef into 2 geomorphological zones: the inner reef flat and the inner reef slope (Figure 2). The reef flat is a very shallow area ranging from 0.7 to 1.5 m, whereas the inner reef slope is an intermediate zone between the reef flat and the sandy bottom lagoon, with inner spurs and grooves [START_REF] Battistini | Eléments de terminologie récifale indopacifique[END_REF]. For each of the 3 years surveyed, the 5 sampling locations regularly spaced along the reef were selected to ensure a good longitudinal coverage where both geomorphological zones were present (Figure 2). At each location for each geomorphological zone, at least 2 transects, 500 m apart, were sampled (see Table 2 for sampling design by year, area and geomorphological zone). Our study is based on so-called commercial transects during which 213 species corresponding to 32 families from a restricted list were counted, including all fished species plus a number of species of scientific interest. Quantitative estimates of abundance of coral reef fishes using the 'distance sampling' method [START_REF] Buckland | Introduction to Distance Sampling: Estimating Abundance of Biological Populations[END_REF] were made using underwater visual census (UVC) [START_REF] Kulbicki | Comparison of density estimates derived from strip transect and distance sampling for underwater visual census: a case study of Chaetodontiidae and Pomacanthidae[END_REF]. Fifty m transects were marked by lines set on the bottom. A diver swam along the transect line and recorded fishes of the species list mentioned above. For each observation, the diver recorded the species and the size of the fish. Biomass of each fish was calculated through available length-weight relationships [START_REF] Kulbicki | A general approach to length-weight relationships for Pacific lagoon fishes[END_REF][START_REF] Kulbicki | Ecologie des poissons lagonaires de Nouvelle Calédonie[END_REF].

Partitioning the fish assemblage

Choosing criteria for constructing species groups raised the question of defining partitions of the fish assemblage that would be relevant to the impact of protection status. Four criteria were used: mobility, interest for fishing, trophic guild (feeding habits) and adult size. Following [START_REF] Grimaud | Influence de la distance à l'océan sur les peuplements ichtyologiques des récifs frangeants de Nouvelle-Calédonie[END_REF], four mobility groups were defined: (1) territorial species with a very restricted range (usually <10 m²), (2) sedentary species with a restricted range (10 to several 100 m²), (3) weakly mobile species often distributed over the entire reef area (up to several 1000 m²) and ( 4) highly mobile species usually foraging over very large areas. Species groups corresponding to distinct interests for fishing were defined based on Jollit et al. (unpublished): (1) highly targeted by spear fishing, (2) moderately targeted by spear fishing, (3) incidentally targeted by spear fishing, (4) highly targeted by line fishing, (5) moderately targeted by line fishing, (6) bycatch of line fishing, and ( 7) not fished. The trophic groupings were based on diet composition following the results of [START_REF] Ferraris | Assessing the impact of removing reserve status on the Abore Reef fish assemblage, New Caledonia[END_REF]: (1) piscivores, (2) macrocarnivores, (3) microcarnivores, (4) coral feeders, (5) herbivores, (6) microalgae feeders and detritivores, and (7) zooplankton feeders. Six size classes were defined based on adult sizes [START_REF] Kulbicki | Ecologie des poissons lagonaires de Nouvelle Calédonie[END_REF]: (1) 0-7 cm, (2) 8-15 cm, (3) 16-30 cm, (4) 31-50 cm, (6) larger than 50 cm.

Data analysis

We aimed at testing the impact on the whole fish assemblage of both the fishing effect after removal of reserve status and the definitive closure of the B area. Thus, we assessed these effects at two levels: i) overall variables per transect, namely species richness, abundance, and biomass summed over all species; and ii) metrics computed per species group, namely species richness, abundance, biomass, and mean size per species group. Both the criteria to group the species and the variables define a set of metrics. The methods used to assess the effects of changes in protection status upon the fish assemblage, involved both exploratory and inferential techniques. Exploratory techniques relied on the use of non-metric multidimensional scaling (MDS) to graphically display similarities between transects measured by the Bray-Curtis coefficient. The representativeness of the plots was evaluated by the stress value [START_REF] Clarke | Change in marine communities: an approach to statistical analysis and interpretation[END_REF]. Using the PRIMER software, we could visualize the influence of a factor of interest (like year or habitat) to explain differences among transects. Inferential techniques include one-way ANalysis Of SIMilarities (ANOSIM) which rests on a permutation procedure to test whether a factor significantly explains differences between groups of transects [START_REF] Clarke | Change in marine communities: an approach to statistical analysis and interpretation[END_REF]. To perform this test (using the PRIMER software), similarities between transects were calculated using the Bray-Curtis coefficient. ANOSIM tests were based on 999 permutations of the transects between factor levels. Aside from ANOSIM, Generalized Linear Models (GLM) [START_REF] Mccullagh | Generalized linear models[END_REF] were performed using R software, with suitable data distribution depending on the variable modelled to test and assess the effects of both removal and closure of area B in 1995, while accounting for other factors that were relevant to explain the variability of the fish assemblage. As the UVCs were performed by several divers, we first explored potential diver effects on variations in visual counts, using MDS plots, and ANOSIM applied to abundance, biomass and mean size data. Significant diver effects were detected, and thus an observer term was included as a factor in subsequent analyses in the way to consider the variability of counts brought by diversity of divers. In addition to the four factors considered for models of overall metrics (year, area, habitat and diver), models of metrics per species group also included the species group factor, with levels depending on the species grouping criterion as described earlier in this section i.e. mobility (4 levels), interest for fishing (7 levels), trophic regime (7 levels), and size class (6 levels). These models included first-order interactions between factors (except for the diver factor), and in addition second-order interactions between year, area and species group, in order to assess possible species-group-specific effects of protection status. For overall metrics, one model was fitted for each variable, while for metrics per species group, one model was fitted for each combination of variable and species grouping criterion. All metrics per species group were modelled in two steps: a binomial model for presence/absence and a lognormal model on non-zero values of corresponding metrics following the procedure proposed by [START_REF] Stefánsson | Analysis of groundfish survey data: combining the GLM and delta approaches[END_REF]. This method is suited for quantitative data with large proportions of zero values that makes them unable to meet the assumptions of regular GLMs. Modelling nonzero values led to sixteen model fits, crossing four metrics (abundance, biomass, mean size and species richness per species group) with four grouping criteria. The goodness-of-fit of each model was assessed through adjusted R² and global Fisher tests, and the conformity of model residuals to linear model assumptions was checked from standard residual plots and tests [START_REF] Venables | Modern Applied Statistics in Splus, 2nd edn. Springler[END_REF]. Once validated, models were selected to eliminate non-significant terms, based on the Akaike Information Criterion (AIC) [START_REF] Akaike | A new look at statistical model identification[END_REF]. The significance of each effect was evaluated through the analysis of variance table based on the Type III sums of squares. Regarding protection status, we were interested in both the fishing effect after removal of reserve status, and the definitive closure of B area. The effects of these changes in protection status were assessed and tested through the interaction between the year and the area (A/B) factors (year*area) for overall metrics, and in addition through the interaction between the year, area and species group factors (year*area*species group) for metrics per species group. When only the first order interaction between area and year was significant, all species groups responded in the same way to changes of protection status. The magnitude and direction of the effect was quantified by computing adjusted means per area, year and species group. Adjusted means correspond to predictions of the modelled metric based on the significant effects of the model, thus leaving aside residual variations. In the analysis, adjusted means were computed for the year, area and species group factors, while controlling for the diver effect. Multiple comparisons were performed using the Bonferroni correction for the following differences in adjusted means: (1) spatial difference between areas A and B in 1993, i.e. prior to the removal of reserve status; (2) temporal variation in area A and B between 1993, 1995 and 2001; (3) spatial difference between areas A and B in 1995 and 2001. When the second-order interaction between area, year and species group was significant, simultaneous confidence intervals were constructed per species group.

Results

Assessment of changes in protection status on overall metrics per transects.

Variations in species richness, abundance and biomass per transect depended on the area (Figure 3, Table 3). Mean species richness increased between 1993 and 1995 in area A then decreased in 2001, while in area B, it increased over the whole time period. Overall abundance per transect consistently decreased in both areas over the same period with a stronger decrease between 1993 and 1995 in area B and a stronger decrease between 1995 and 2001 in area A. Similarly, mean biomass decreased in both areas with a much more important decline in area A between 1993 and 1995. The observed trends were validated by significant models for overall metrics and for metrics per species group. In all models, residuals (not reported) conformed well to linear model assumptions, the global F-test was highly significant, although the variance explained by these models was quite low (Table 4). For these overall metrics, the diver effect was significant and well accounted for by the models (Table 4). There was no significant interaction between area and year, meaning that no effect of the 1993 fishing event, nor the 1995 closure could be detected from the species richness, abundance, or biomass data. In contrast, both abundance (p=0.0003) and biomass (p=0.0027) decreased over the years surveyed as observed previously (Table 4). Species richness varied by area (p=0.0003) (Table 4) and both biomass and species richness were significantly lower in the inner reel flat zone (p=0.0003 and p<0.0001, respectively).

Changes in protection status and species attributes

The probability of occurrence of all adult size groups that was lower in area A than in area B in 1993 while the difference was the other way around in 1995, showing a significant effect of protection status (p<0.02) (Table 5). In all presence/absence models, the species group factor was highly significant (p<0.01), illustrating differences in average occurrence between species groups irrespective of how they were defined. Variations in abundance and species richness were the metrics which were best described by the models in all species groups (Table 6). In order to rank factors according to their influence, we used the number of significant effects in the 16 models fitted (Table 7). Diver identity and species group effects were significant in all models, and thus explained more variance than spatial or temporal effects. First-order interactions involving the species group factor were also highly significant, pointing out the differences in response between groups to the effects of year, area and habitat. The change in protection status significantly affected mean size in the groups defined by fishing interest (p<0.01). Protection status also significantly affected abundance in the different mobility grouping (p<0.01), as well as biomass in the different trophic groupings (0.05<p<0.01). The adjusted means (Figures 456) based on year, area and species group effects (see Methods section) illustrate the species group effects and the additive diver effect (Table 5). Regarding the mobility criterion, between 1993 and 1995 (fishing period in area B), the abundance of sedentary species rose in area B and declined in area A, while the abundance of all other groups declined, particularly between 1995 and 2001 (Figure 4), but the only significant variation over time was the decline of highly mobile fishes in area B between 1993 and 1995, which can be explained by the fishing impact. The abundance of this group remained stable after 1995, corroborating a relative protection effect. Considering trophic groups (Figure 5), a decline in biomass between 1993 and 1995 was observed for all groups in both areas but zooplankton feeders. Yet, this decline was only significant for piscivores in area B in relation with the opening to fishing (note that 43% of piscivores were species highly or moderately targeted by both spear and line fishing). Macrocarnivores showed a global decline of biomass in both areas, with a biomass significantly lower in area B (fished) than in area A (no-take) in 1995. Between 1995 and 2001, all trophic groups tended to decline, except piscivores whose biomass increased slightly. Similar trends in abundance were observed also in area A. Zooplankton feeders display an inverse pattern in area B. Yet, almost half of the species in this group are highly or moderately targeted by spear fishing. Finally, mean size in the different fishing interest groups showed a variety of patterns over time in areas A and B (Figure 6). Between 1993 and 1995, mean size decreased for most groups in both areas, except for species moderately targeted by line fishing, bycatch of line fishing, and for unfished species. As expected, unfished species were not affected by the opening to fishing, and species moderately targeted by line fishing and bycatch of line fishing declined in B while remaining stable in A between 1993 and 1995. After the final closure of area B, between 1995 and 2001, the mean size of several groups further declined in both areas. The decline was mitigated in both areas for species highly targeted by line fishing, and in area B for bycatch and moderate targets of line fishing. For these two groups, the decline in mean size was larger in area A. The mean size of species caught by spear fishing declined but not significantly and over the three years in both areas. Note that no species group-specific protection status effect was found for the adult size criterion (Table 6).

Discussion

Spatio-temporal variations of the fish assemblage

First order interaction of year, area, and habitat with species groupings demonstrate that the complexity of variations in the measured abundance, species richness and biomass of the fish assemblage. The factors that determined significant single effects in the models were the year, habitat and diver factors. The significance level of the diver factor required the explicit inclusion of this factor in our models to control for observer variability. This aspect is often ignored or omitted in the literature. Regarding temporal variations, a significant effect involving at least the year factor was found in 18 out of the 23 fitted models in the whole study. Such variations corresponded to decreases over time of the studied metrics, a result that was also pointed by [START_REF] Kulbicki | Opening of an MPA to fishing: Natural variations in the structure of a coral reef fish assemblage obscure changes due to fishing[END_REF], although from distinct approaches. The causes of such temporal variations remain poorly understood and are obviously linked to factors that cannot be accounted for in such models. Undoubtedly, environmental fluctuations and events explain some of these variations and mask the effects of changes in protection status. For example, large scale oceanographic and climatic features such as El Niño Southern Oscillation (ENSO) events are known to largely influence water conditions (temperatures for example) and may seriously affect habitats and disrupt benthic populations and their reproductive success [START_REF] Allison | Ensuring persistence of marine reserves: catastrophes require adopting an insurance factor[END_REF]. Cyclonic events are also likely to occur in the studied area, and while no critical event took place during the period under study, consequences of such events may be observed on longer time scales. Other events like the strong winds that prevailed during the 1995 survey could also explain part of the variations under study. But without sampling outside of the MPA and over a long temporal series, it is difficult to detect effects of long term phenomena. Based on inferential models, we could assess the effects of changes of protection status through the interaction between year and area factors for a range of metrics. Overall abundance, biomass and species richness proved not be sensitive to changes in protection status. Regarding metrics per species group, a few metrics, namely piscivore biomass, the abundance of highly mobile species and the mean size of line fishing bycatch, displayed significant variations that were consistent with the changes in protection status endured by the fish assemblage. Several other metrics showed non-significant variations which tended to be consistent with these changes, e.g. the biomass of herbivores, the mean size of species moderately targeted by line fishing and species highly targeted by line fishing. Other variations could not be easily related to changes in protection status and overall there were few significant year*area interactions. In a number of cases, declining patterns observed in both A and B suggest a strong connection between these two adjoining areas. Such exchanges would inevitably reduce spatial differences in fish assemblage between A and B and therefore contribute to the lack of significant protection effects. The spillover of individuals from A to B would mitigate the decrease in B and/or the regeneration in A. Larval dispersion and larval settling depend on hydrodynamics that operate at scales larger than the MPA and may also contribute but are poorly known in this area. Fish movements may be particularly important in such coral reef formations consisting of linear barrier and multiple islands and coral patches. This point is confirmed by [START_REF] Chateau | Evidence of fish movements between a marine reserve and two unprotected reefs in a fragmented habitat in New Caledonia, as determined by acoustic telemetric[END_REF] who recently showed that fish mobility in the Caledonian lagoon is more important than previously considered. Habitat was a determining factor for explaining variations of the fish assemblage, even approximated at the scale of the geomorphological zone (significant factor in 14 models out of the 23 fitted). Geomorphological zones indeed correspond to distinct depth and coral type (Ferraris et al., 2005). Therefore environmental variations affecting coral cover and reef structure may ultimately be reflected in spatio-temporal variations of fish assemblages. Fine scale changes in habitat structure may help to improve the explanatory power of the models [START_REF] Ferraris | Assessing the impact of removing reserve status on the Abore Reef fish assemblage, New Caledonia[END_REF], but such data were not available for inclusion in our analysis. [START_REF] Ferraris | Assessing the impact of removing reserve status on the Abore Reef fish assemblage, New Caledonia[END_REF] showed that while accounting for fine-scale habitat data improved model fits, the degree of significance of the effects was only improved marginally. In addition, poaching may occur in the MPA that will mitigate the effect of protection. Another hypothesis is that the general decline of fish populations in the whole SW lagoon is related to the increase in human population (that occurs in Nouméa region) and the evolution of fishing methods. Fishes of the reserve migrate to zones out of the MPA as these other areas become depopulated. The simultaneous decline in abundance of most of the groups, fished or not, argues against the direct impacts of fishing.

Partitioning the fish assemblage

One of our objectives was to utilize species attributes to better understand the consequences of changes in protection status on the fish assemblage. Partitioning the fish assemblage according to a range of criteria provided a variety of insights, and is a step toward an ecosystem-approach to MPA assessment. In fact, the models of overall metrics displayed few significant effects, and none in relation to changes in protection status. In contrast, metrics per species groups revealed a larger number of significant effects, most of which included the species group factor, which means that for a given species grouping criterion, the variation of the metric modelled differed across the species groups. Accordingly, corresponding models explained a much larger fraction of variance (up to 77%) compared to models of overall metrics (less than 30%). Therefore, including species attributes in the models improved the assessment. This method revealed that piscivores and highly mobile species were groups who react the most to opening to fishing (figures 4 and 5). However, some of our results are not intuitive. Hence, significant effects of changes in protection status were detected for very mobile species, which a priori can move easily between areas A and B. This kind of effect is generally not expected. It is therefore important to note that 58% of the species of the highly mobile group are also species targeted or highly targeted by spear or line fishers. Yet, the abundance of these fishing groups was not significantly affected by changes in protection status. One could hypothesize that highly mobile species may have left area B after the opening to go to other reef areas with lower fishing pressure, including (but not exclusively) area A. Likewise, one could argue that partitioning species according to their interest for fishing gave few striking results, species strongly targeted by the main fishing gears showing little sensitivity to the opening of fishing. Beyond considerations about area connectivity, fish mobility and interest for fishing, these counter-intuitive results raise the question of defining species groups that are relevant to assess the effects of changes in protection status. One could thus contemplate to define groups based on the combination of two or more criteria.

Sampling considerations

Given the complexity of the data set, models appropriate to test the effects of changes in protection status had to include a relatively large number of explanatory factors: year, area, habitat, and diver. Although the number of observations was overall large (212 transects), it may not have been sufficient to unravel the variability attributable to these four factors. In other studies of the Aboré reef fish assemblage, [START_REF] Ferraris | Assessing the impact of removing reserve status on the Abore Reef fish assemblage, New Caledonia[END_REF], [START_REF] Amand | A step toward the definition of ecological indicators of the impact of fishing on the fish assemblage of the Aboré reef reserve (New Caledonia)[END_REF] and [START_REF] Kulbicki | Opening of an MPA to fishing: Natural variations in the structure of a coral reef fish assemblage obscure changes due to fishing[END_REF] used a different set of observations based on the census of all observed species for only two years, 1993 and 1995. The data set included only ca. 70 transects versus more than 200 (out of which 170 for both 1993 and 1995) in the present study. A larger number of significant interactions involving year and area were found in these two studies, including intuitive results. Although the data had been collected by several divers, subsequent variability was not problematic to evidence the effects at stake. Note that the above works only dealt with the 1993-1995 variation, a fishing effect that was probably more conspicuous than a restoration effect. Yet, in the present study, this fishing effect was not so obviously detected. This lack of significance raises the question of additional sources of variability, such as the diver effect (between-diver variability in our data set and difference in divers between the other references and the present study). It may also be related to the issue of the species list retained for the visual counts. Note that the stratification of the sampling scheme and the geographical range of stations were the same in both data sets. The present results lead us to conclude that additional sources of variation prevented us in this case from detecting the effects of changes in protection status (particularly the restoration effect), such as those mentioned hereabove in the discussion. When it comes to assessing restoration effects between 1995 and 2001, data is only available for 2001, with a lesser number of transects than the other two dates. Monitoring the restoration of the fish assemblage would require observations collected at several dates after the final closure. Assessing the response of fish assemblages to changes in protection status is a major issue for fisheries management. This work sheds original insight into the issue of designing adequate protocols for monitoring MPA in terms of conservation of biodiversity and sustainable exploitation of resources. In this respect, considering species attributes was useful and the partitioning criteria considered provided a variety of insights to better understand the effects of such changes on the fish assemblage. Although based on a coral reef ecosystem case, our findings may be applied to other contexts.

Tables

FiguresFigure 1 .

 1 Figures

Figure 2 .

 2 Figure 2. Sketch of the Aboré barrier reef, displaying i) the area A that has always been closed since 1990; ii) the area B that has been open to fishing between 1993 and 1995, and iii) the sampling locations crossing the two sampled habitats: inner reef flat and inner reef slope.

Figure 3 .

 3 Figure 3. Boxplots of overall metrics per area (columns) and year (X-axis): species richness (top), abundance (middle), and biomass in grams (bottom). The horizontal bold line is the median, the lower and upper hinges correspond to the first and second quartiles, and the lower and upper whisker framed the third and fourth quartile. Points located outside the whiskers are outlying values.

Figure 4 .

 4 Figure 4. Adjusted means for the factors year, area, and species group factors in the model of log(abundance) per mobility group. "**" (resp. "*") indicates a significant difference in adjusted mean at the p=0.01 (resp. p=0.05) level in multiple comparisons. Multiple comparisons were corrected using the Bonferroni method.

Figure 5 .

 5 Figure 5. Adjusted means for the factors year, area, and species group factors in the model of log(biomass) per trophic group. "**" (resp. "*") indicates a significant difference in adjusted mean at the p=0.01 (resp. p=0.05) level in multiple comparisons. Multiple comparisons were corrected using the Bonferroni method. Coral feeder group constituted of only one species did not allowed statistical comparisons.

Figure 6 .

 6 Figure 6. Adjusted means for the factors year, area, and species group factors in the model of mean size per fishing interest group. "**" (resp. "*") indicates a significant difference in adjusted mean at the p=0.01 (resp. p=0.05) level in multiple comparisons. Multiple comparisons were corrected using the Bonferroni method.

Table 1 .

 1 Evolution of the protection status on the Aboré reef.

		Area A	Area B
	1988 August	Closure to fishing	Closure to fishing
	1993 July (first survey)	Closed	Closed
	1993 September	Fishing closure maintained	Opened to fishing
	1995 July (second survey)	Closed	Opened
	1995 September	Fishing closure maintained	Closure to fishing
	2001 August (third survey)	Closed	Closed

Table 2 .

 2 Sampling scheme, reporting the number of transects per geomorphological zone, area (A and B) and year.

	A	B

Table 3 .

 3 Means of overall variable by area and year.

			A			B
		1993	1995	2001	1993	1995	2001
	Species
		22.8 ±7.5	24.2 ±6.9	21.0 ±4.4	19.2 ±6.1	20.6 ±6.55	21.2 ±5.6
	richness
	Abundance	200.8 ±76.3 192.5 ±96.5 132.6 ±69.9 220.0 ±107.4 195.1 ±109.2 162.7 ±64.57
	Biomass	45.3 ±48.8	23.6 ±19.8	13.4 ±12.6	26.6 ±19.5	21.8 ±17.0	15.2 ±11.9

Table 4 .

 4 Model results for overall metrics per transect: significant effects and adjusted R 2 . All effects are significant with p <0.01.

	Metric	Model	R²
	Species richness	area + habitat + diver	0.27
	Abundance	year + diver	0.21
	Biomass	year + habitat + diver	0.30

Table 5 .

 5 Models of presence/absence per species group, with goodness-of-fit statistics.

	Significativity of factors is express by bold: highly significant effects (p<0.01); underlined: significant
	effects (0.05>p>0.01); italics: for non-significant effects (p>0.05).
	Criterion	Effects retained	R²
	Mobility	year + diver + mobility	0.92
	Trophic	year + habitat + area + trophic	0.49
	Interest for fishing	habitat + area + fishing + diver +	0.53
		habitat:fishing + area:fishing
	Adult size	year + habitat + area + diver +	0.76
		adultsize + year:area + year:adultsize

Table 6 .

 6 Effects and goodness-of-fit (R 2 ) for models fit on non-zero values of metrics per species group. Bold: highly significant effects (p<0.01); underlined: significant effects (0.05>p>0.01); italics: for non-significant effects (p > 0.05).

	Criterion	Metric	Effects retained	R²
		Abundance	year + habitat + area + diver + mobility + year:mobility +	0.58
			area:mobility + habitat:mobility + year:area:mobility
		Biomass	year + habitat + area + diver + mobility + year:mobility +	0.48
	Mobility	Mean size	habitat:mobility + area:mobility year + habitat + area + diver + mobility + year:mobility +	0.34
			habitat:mobility
		Species	year + habitat + area + diver + mobility + area:mobility	0.56
		Richness
				0.74
			aire:trophic

Abundance year + habitat + area + diver + trophic + year:trophic + habitat:trophic + area:trophic 0.77 Biomass year + habitat + area + diver + trophic + year:trophic + habitat:trophic + area:trophic + year:area:trophic 0.60 Mean size year + habitat + diver + trophic + year:habitat + year:trophic 0.44 Trophic Species Richness year + habitat + area + diver + trophic + year:trophic + Abundance year + habitat + area + diver + fishing + year:fishing + habitat:fishing + area:fishing 0.67 Biomass year + habitat + area + diver + fishing +

year:fishing + habitat:fishing + area: fishing

	Interest for fishing	Mean size	year + habitat + area + diver + fishing + year:area + year:fishing + year:area:fishing	0.54 0.45
		Species	year +
		Richness

habitat + area + diver + fishing + year:fishing + habitat:fishing + area:fishing

				0.60
		Abundance	year + habitat + area + diver + adultsize + year:adultsize +	0.77
	Adult size	Biomass	habitat:adultsize + area:adultsize

year + habitat + area + diver + adultsize + year:adultsize + habitat:adultsize + area:adultsize 0.51

Acknowledgements

Data collection was funded by la Province Sud (DRN), by IRD, and by the Programme National pour l'Environnement Côtier (PNEC). The authors thank the other scientists who helped collecting the data in particular G. Mou-Tham (IRD Nouméa), P. Labrosse and E. Clua (CPS Nouméa), C. Chauvet (LERVEM, Université de Nouvelle-Calédonie). A special homage to Pierre Thollot who tragically died in a helicopter accident in November 2000. This work was made possible through a grant of the PNEC.