Sébastien Baekelandt

Baptiste Redivo

Syaghalirwa N M Mandiki

Thibaut Bournonville

Alexis Houndji

Najlae Benoît Bernard

El Kertaoui

Mélodie Schmitz

Pascal Fontaine

Jean-Noel Gardeur

Najlae El Kertaoui

Jean-Noël Gardeur

Yannick Ledoré

Patrick Kestemont

Multifactorial analyses revealed optimal aquaculture modalities improving husbandry fitness without clear effect on stress and immune status of pikeperch Sander lucioperca

Keywords: Brain neurotransmitters, stress, innate immunity, Pikeperch, fractional factorial design

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 __________________________________________________________________

Over the last decade, European inland aquaculture has shown weak increase in productivity despite the increase in the demand for fish products throughout the world (FAO, 2014). This low performance in fish production might be attributed to the low number of fish species that are cultivated in Europe. Indeed, European aquaculture is mainly focused on 5 fish species: Atlantic salmon (Salmo salar), rainbow trout (Oncorhynchus mykiss), common carp (Cyprinus carpio), European seabass (Dicentrarchus labrax) and gilthead seabream (Sparus aurata). Therefore, itis essential to diversify the fish species pool inorder to increase fish productivity throughout Europe and to tap into new niche markets. Pikeperch (Sander lucioperca) is one of the most promising freshwater fish species for diversification and an attractive alternative for inland aquaculture species according to its relatively fast growth compared to other, its high quality flesh and a favorable market acceptance leading to high economical expectations species [START_REF] Hilge | Aquaculture of fry and fingerling of pike-perch (Stizustediun Zuciuperca L.) -a short review[END_REF]; [START_REF] Barry | Proceedings of PERCIS III. The Third International Percid Fish Symposium[END_REF][START_REF] Wang | Effect of temperature and feeding frequency on growth performances, feed efficiency and body composition of pikeperch juveniles (Sander lucioperca)[END_REF][START_REF] Dalsgaard | Farming different species in RAS in Nordic countries :current status and future perspectives[END_REF].

However, the culture of pikeperch is still limited by an unpredictable high mortality rate and impairment in growth rate during both larval and juvenile stages with survival rate estimated between 8 and 30% [START_REF] Kestemont | Effect of weaning age and diet on pikeperch larviculture[END_REF][START_REF] Szkudlarek | Effect of stocking density on survival and growth performance of pikeperch, Sander lucioperca (L.), larvae under controlled conditions[END_REF][START_REF] Dalsgaard | Farming different species in RAS in Nordic countries :current status and future perspectives[END_REF]. A low welfare related to high stress level may be one of the major causes of high mortality rate observed for young pikeperch. While intensive aquaculture is well mastered for salmonid species such as Atlantic salmon and rainbow trout, aquaculture technology for percid fishes is not yet optimal. It has also been demonstrated that percid fish such as Eurasian perch (Perca fluviatilis) are more sensitive to some aquaculture stressors such as emersion and handling [START_REF] Jentoft | Effects of stress on growth, cortisol and glucose levels in non-domesticated Eurasian perch (Perca fluviatilis) and domesticated rainbow trout (Oncorhynchus mykiss)[END_REF]. For the latter species, it was also demonstrated that moderate hypoxia may considerably impair the behavior but also the physiological status such as the immune functions [START_REF] Strand | Effects of repeated disturbances on feed intake, growth rates and energy expenditures of juvenile perch, Perca fluviatilis[END_REF][START_REF] Douxfils | Influence of domestication process on immune response to repeated emersion stressors in Eurasian perch (Perca fluviatilis, L.)[END_REF]. Stress responsiveness has received little attention in pikeperch whatever its developmental stage. It has been reported that light characteristics such as light intensity and light spectrum have a huge impact on the pikeperch physiology and behavior due to some retinal histo-anatomical features (presence of tapetum lucidum and macroreceptors) which improve their vision in dim environment [START_REF] Kozlowski | Impact of light intensity on the results of rearing juvenile pikeperch, Sander lucioperca (L.), in recirculating aquaculture systems[END_REF][START_REF] Luchiari | Longer wavelengths of light improve the growth, intake and feed efficiency of individually reared juvenile pikeperch Sander lucioperca (L.)[END_REF][START_REF] Sarameh | Effects of different photoperiods and handling stress on spawning and reproductive performance of pikeperch Sander lucioperca[END_REF]. It was especially observed that pike-perch exhibited higher growth rate and food conversion rate under high light intensity (100 lux) of red spectrum than under white spectrum [START_REF] Luchiari | Longer wavelengths of light improve the growth, intake and feed efficiency of individually reared juvenile pikeperch Sander lucioperca (L.)[END_REF]. But there is no clear information about the optimal intensity or light spectrum. [START_REF] Luchiari | Light intensity preference of juvenile pikeperch Sander lucioperca (L.)[END_REF] also showed a clear preference of pikeperch for low light intensities. These findings are in agreement with the behaviour of juvenile and adult pikeperch in natural environments since this species is a crepuscular predator that is actively feeding during dusk and night [START_REF] Luchiari | Light intensity preference of juvenile pikeperch Sander lucioperca (L.)[END_REF][START_REF] Zingel | Effects of turbidity on feeding of the young-of-the-year pikeperch (Sander lucioperca) in fishponds[END_REF][START_REF] Dalsgaard | Farming different species in RAS in Nordic countries :current status and future perspectives[END_REF]. The effects of temperature conditions on pikeperch stress physiology are also reported inconsistently. It was reported that the optimal temperature for pikeperch is in the range from 10 to 27 °C [START_REF] Frisk | Thermal optimum for pikeperch (Sander lucioperca) and the use of ventilation frequency as a predictor of metabolic rate[END_REF], but fish size should be considered when optimizing temperature level in aquaculture production. According to some authors, better growth and feed utilization for ongrowing pikeperch are achieved under high temperature conditions ranging between 25 and 28 °C [START_REF] Rónyai | Effect of feeding regime and temperature on ongrowing results of pikeperch (Sander lucioperca L.)[END_REF][START_REF] Wang | Effect of temperature and feeding frequency on growth performances, feed efficiency and body composition of pikeperch juveniles (Sander lucioperca)[END_REF][START_REF] Dalsgaard | Farming different species in RAS in Nordic countries :current status and future perspectives[END_REF]. Nonetheless, [START_REF] Frisk | The effects of temperature on specific dynamic action and ammonia excretion in pikeperch (Sander lucioperca)[END_REF] reported that a smaller fraction of metabolic scope was utilized for digestion at 19 °C compared to 25 °C, indicating that low temperature conditions may be more suitable for pikeperch reared under intensive culture conditions. Extended and continuous photoperiods have been proposed to improve the growth performance in some fish species by increasing food intake [START_REF] Biswas | Combined effect of photoperiod and self-feeder on the growth performance of striped knifejaw, Oplegnathus fasciatus[END_REF], but no attempt has been done for pikeperch. Photoperiod manipulations should be appropriate to the feeding behaviour of targeted fish species to avoid a possible stress side effect.

The relationships between population parameters such as stocking density and physiological stress status or immune competence have been described in various fish species [START_REF] Barton | Stress in fishes : a diversity of responses with particular reference to changes in circulating corticosteroids[END_REF][START_REF] Pankhurst | The endocrinology of stress in fish: An environmental perspective[END_REF] whereas limited information is available for pike-perch. Preliminary observations reported that high stocking density has no marked effects on growth and food utilization of young pikeperch, and that pikeperch juveniles can be maintained at high densities comprised between 30 and 60 kg•m-3 without any increase in physiological stress response [START_REF] Molnár | The effect of initial stocking density on growth and survival of pike-perch fingerlings reared under intensive conditions[END_REF][START_REF] Steenfeldt | Further development of intensive pikeperch rearing in Denmark[END_REF][START_REF] Dalsgaard | Farming different species in RAS in Nordic countries :current status and future perspectives[END_REF]. On the contrary, another study reported that high density may increase the susceptibility to diseases for pikeperch juveniles [START_REF] Jensen | Susceptibility of pikeperch Sander lucioperca to a panel of ranavirus isolates[END_REF]. According to our observations, juveniles of pikeperch in intensive culture conditions are found to feed both on the ground and in the water column. Mid-floating feed has lower sinking velocity and better dispersion in the water which can improve feeding behavior in some fish species. No clear data are available on juvenile pikeperch and current practices are not yet standardized.

Numerous other stress factors can be encountered by cultured fish from water temperature to grading size and, as in any biological system, the overall performances are the result of interactions from multiple factors [START_REF] Trabelsi | Effects of 12 factors on burbot Lota lota (L., 1758) weaning performances using fractional factorial design experiment[END_REF]. Therefore, fractional factorial approach has been proposed to consider simultaneously the impact of a large number of interrelated aquaculture factors using a few number of experimental units [START_REF] Kobilinsky | Reparametrisation of interest in non-uniform factorial designs[END_REF][START_REF] Hamre | Antioxidant vitamins, minerals and lipid levels in diets for Atlantic salmon (Salmo salar, L.): effects on growth performance and fillet quality[END_REF][START_REF] Gardeur | Simultaneous effects of nutritional and environmental factors on growth and flesh quality of Perca fluviatilis using a fractional factorial design study[END_REF][START_REF] Teletchea | Effects of four factors on the quality of male reproductive cycle in pikeperch Sander lucioperca[END_REF].

It has been reported that a bi-directional communication between corticotropic axis and immune system is essential to maintain homeostasis in mammals and teleosts [START_REF] Tort | Stress and immune modulation in fish[END_REF][START_REF] Mathieu | In vivo response of some immune and endocrine variables to LPS in Eurasian perch (Perca fluviatilis, L.) and modulation of this response by two corticosteroids, cortisol and 11deoxycorticosterone[END_REF][START_REF] Nardocci | Neuroendocrine mechanisms for immune system regulation during stress in fish[END_REF]. Many pieces of evidence clearly support this close interaction since some of the immune and endocrine messengers belong to the same family of molecules and that the head kidney plays a central role in stress response regulation [START_REF] Tort | Stress and immune modulation in fish[END_REF]. In Eurasian perch, it has been shown that some corticosteroid hormones namely cortisol and 11-deoxycorticosterone are active mediators of the immune activity [START_REF] Mathieu | In vivo response of some immune and endocrine variables to LPS in Eurasian perch (Perca fluviatilis, L.) and modulation of this response by two corticosteroids, cortisol and 11deoxycorticosterone[END_REF]. However the relationships between stress and immunity in pikeperch have received little attention.

In aquaculture, prolonged, repeated and/or unavoidable other stressors are largely associated to maladaptive physiological effects including failures in immune functions and disease resistance [START_REF] Fast | Cortisol response and immune-related effects of Atlantic salmon (Salmo salar Linnaeus) subjected to shortand long-term stress[END_REF][START_REF] Douxfils | Does domestication process affect stress response in juvenile Eurasian perch Perca fluviatilis?[END_REF][START_REF] Tort | Stress and immune modulation in fish[END_REF]. The objectives of the present study were thus (a) to characterize the effects of major husbandry and environmental factors on growth related parameters and physiological status of cultured pikeperch and (b) to identify the optimal husbandry and environmental conditions which may induce mild physiological stress response, and thereby improve the growth and welfare of pikeperch in intensive culture conditions. To achieve these objectives, we selected 8 major husbandry and environmental factors according to the current pikeperch aquaculture practices and available information on potential stress impact of those factors on percid fish. These factors were compared in two modalities using a fractional factorial design.

Material and Methods __________________________________________________________

Experimental design

Taking into account current practices in major European pikeperch intensive farms and available data in the literature [START_REF] Luchiari | Light intensity preference of juvenile pikeperch Sander lucioperca (L.)[END_REF][START_REF] Teletchea | Effects of four factors on the quality of male reproductive cycle in pikeperch Sander lucioperca[END_REF][START_REF] Wang | Effect of temperature and feeding frequency on growth performances, feed efficiency and body composition of pikeperch juveniles (Sander lucioperca)[END_REF][START_REF] Sarameh | Effects of different photoperiods and handling stress on spawning and reproductive performance of pikeperch Sander lucioperca[END_REF][START_REF] Dalsgaard | Farming different species in RAS in Nordic countries :current status and future perspectives[END_REF], eight environmental factors at two levels (Table 1) were selected and tested in a multifactorial experiment based on a 2(8-4) reduced factorial design [START_REF] Kobilinsky | Reparametrisation of interest in non-uniform factorial designs[END_REF][START_REF] Torstensen | A factorial experimental design for investigation of e € ects of dietary lipid content and pro-and antioxidants on lipid composition in Atlantic salmon (Salmo salar) tissues and lipoproteins[END_REF][START_REF] Hamre | Antioxidant vitamins, minerals and lipid levels in diets for Atlantic salmon (Salmo salar, L.): effects on growth performance and fillet quality[END_REF][START_REF] Gardeur | Simultaneous effects of nutritional and environmental factors on growth and flesh quality of Perca fluviatilis using a fractional factorial design study[END_REF][START_REF] Mairesse | Effects of dietary factors, stocking biomass and domestication on the nutritional and technological quality of the Eurasian perch Perca fluviatilis[END_REF][START_REF] Blanchard | Ultrastructural features of hepatocytes in cultured Eurasian perch (Perca fluviatilis L.) as affected by nutritional and husbandry conditions[END_REF]. With a full factorial design study of 8 factors at 2 levels, 256 (28) treatments would be tested resulting in the calculation of the main effects and all the interactions. With a fractional factorial design study, the number of combinations (treatments) is reduced from 256 (28) to 16 (24). To generate such a design, an alias structure was selected (Table 2), determining which effects are confounded with others [START_REF] Hamre | Antioxidant vitamins, minerals and lipid levels in diets for Atlantic salmon (Salmo salar, L.): effects on growth performance and fillet quality[END_REF][START_REF] Trabelsi | Effects of 12 factors on burbot Lota lota (L., 1758) weaning performances using fractional factorial design experiment[END_REF]. Thus, itis possible to calculate main effects separated from each other and from the effects of two-factor interactions [START_REF] Kobilinsky | Reparametrisation of interest in non-uniform factorial designs[END_REF][START_REF] Hamre | Antioxidant vitamins, minerals and lipid levels in diets for Atlantic salmon (Salmo salar, L.): effects on growth performance and fillet quality[END_REF][START_REF] Gardeur | Simultaneous effects of nutritional and environmental factors on growth and flesh quality of Perca fluviatilis using a fractional factorial design study[END_REF]. The main advantage of this approach is that it considers simultaneously the impact of a large number of interrelated aquaculture factors using a limited number of experimental units. With a 2(8-4) reduced factorial design, each of the 16 combinations was tested once but each level of every factor was repeated eight times (Table 3).

Animals and rearing conditions

The present experiment was carried out in agreement with the European and French national legislations on animal welfare (protocol number: C54-547-1).

A stock of 3200 mixed-sex juveniles pikeperch (20-30 g) was provided by Asialor farm (Dieuze, France) and transferred to the URAFPA facilities at the University of Lorraine, France. Fish were first reared in 6 large tanks for acclimation and ongrowing until they reached 91 ± 5 g body weight. Working with fish around 90 g was relevant since high mortality rate is still observed at this developmental stage. During acclimation, they were maintained in constant conditions (temperature: 23 °C; light intensity: 10 lux; photoperiod: 12 D:12 L) and fed twice daily at 1.5% biomass with a sinking food. They were then distributed into 16 indoor 200 L-tanks and stocked at two densities (15 or 30 kg•m-3) according to Table 3. Each of the 16 experimental units was operating independently in a recirculating circuit (RAS). Rearing conditions, including light intensity, light spectrum, photoperiod, temperature, feed type, grading manipulations and oxygen saturation were then applied as indicated in Table 3. Temperature, light intensity and oxygen saturation were checked daily. Fish diets consisted in mid-floating or sinking feed (D-D Optibream 2P or 2P-Optobream, 4 mm, Skretting, France) containing the same contents of crude proteins (46%) and lipids (16%). Food fixed at 1.5% biomass was distributed during a photophase of either 24 h or 10 h depending on the tank photophase. Manipulations mimicking grading were applied every two weeks. For this purpose, fish were starved one day before handling. All fish were harvested from each tank by a handling-net, and put in one or two basins with water. Then, they were transferred to a fish size grader to simulate the sorting process. Duration of grading manipulations varied between 15-20 min per tank.

Sampling procedure

Fish were sampled on days 36 and 63 and were starved one day before. Six fish were removed randomly from each tank and anesthetized with MS-222 (150 mg/L). Blood was quickly collected by caudal vein puncture with heparinized syringes within 5 min and centrifuged at 7500g during 10 min at 4 °C. Plasma was stored at -80 °C until assayed. Fish were then rapidly killed by cervical dislocation to collect the whole brain and the anterior kidney. These organs were directly frozen in liquid nitrogen and stored at -80 °C.

Husbandry variables

Final individual weight (FIW), mortality rate (MR) and weight heterogeneity (CV) were all determined on day 63 for each experimental condition (Table 4). Specific Growth Rate (SGR) was also estimated at days 24 and 63.

Stress indicators

Cortisol and glucose assays

Plasma cortisol was assayed in triplicate using a cortisol ELISA kit (DRG, EIA-1887), following the manufacturer's instructions (BioSource, Belgium). The intra-assay coefficient of variation was 3.6%. The assay dynamic range was between 0 and 800 ng.ml-1 and the analytical sensitivity was 2.5 ng.ml-1. Plasma glucose was determined calorimetrically based on a glucose oxidase/per-oxidase method described by [START_REF] Trinder | Determination of glucose in blood using glucose oxidase with an alternative oxygen acceptor[END_REF].

Brain neurotransmitters

High Performance Liquid Chromatography (HPLC) was performed according the methods of [START_REF] Lepage | Differential Stress Coping in Wild and Domesticated Sea Trout[END_REF], with some modifications, to assess in whole brain serotonergic activity, expressed as serotonin (5-HT) / hydroxyl-indol-acetic acid ratio (5-HIAA) ratio.

For each fish, the whole brain tissue was weighed out and homogenized for 6 min in perchloric acid 4% (250 µL per 50 mg of tissue) containing 4 µM 2-3 dihydroxybenzoic acid (DHBA) as internal standard. The homogenate was sonicated for 20 s and then centrifuged at 21.000g for 20 min at 4 °C. The supernatant was transferred to a new tube, mixed with HPLC mobile phase (v/v; 75 mM NaH2PO4, 7 mM octane sulfonic acid (OSA, Sigma-Aldrich) and 10% MeOH adjusted to pH 3) and centrifuged at 21.000g for 20 min at 4 °C. The whole procedure was carried out on ice.

HPLC analysis was performed using a GP50 gradient pump (Dionex, Sunnyvale, USA) equipped with an autosampler FAMOS (LC packings). Neurohormones were monitored using a DC amperometry detector (Dionex, Sunnyvale, USA) with Glassy Carbon Working Electrode (0.80 V, Ag/AgCl -P/N 061677). The mobile phases were all degassed with helium. ChromeleonTM software (6.8) (Dionex, Sunnyvale, USA) was used for data acquisition and processing. The samples were individually applied (50 µl) on a 2.6 µm particle size (150 x 4.6 mm, I.D.) C18 analytical Kinetex column at 1 ml/min. The mobile phase consisted of 75 mM NaH2PO4, 7 mM octane sulfonic acid (OSA, Sigma-Aldrich) and 10% MeOH adjusted to pH 3.0. The column was reconditioned by washing with 95% MeOH during 10 min and then re-equilibrated with buffer during 20 min. The column was kept at 25 °C.

Purified 5-HT and 5-HIAA were obtained from Sigma-Aldrich. Standard solutions were treated similarly to samples. Concentrations of the compounds were calculated by interpolation of their respective standard curves. The intraassay coefficients of variation were 5.9% and 7.1% respectively.

Immune variables

Plasma lysozyme activity

Lysozyme activity was evaluated in plasma samples by the turbimetric method [START_REF] Siwicki | The phagocytic ability of neutrophils and serum lysozyme activity in experimentally infected carp. Cyprinus carpio L[END_REF]Douxfils et al., 2012). Plasma samples (10 µl) were mixed with 10 µl of Na2HPO4 0.05 M pH 6.2 and 130 µl Micrococcus lysodeikticus (Sigma-Aldrich) solution (0.6 g/L). This assay was performed in triplicate. Absorbance was measured at 450 nm every 5 min during 30 min at room temperature. Lysozyme activity (units) is defined as the amount of enzyme decreasing the turbidity of 0.001 OD per min.

. Plasma haemolytic activity of the alternative complement pathway

Plasma complement (ACH50) was assayed by measuring the haemolytic activity in plasma samples using rabbit erythrocytes as targets [START_REF] Sunyer | Natural haemolytic and bactericidal activities of sea bream Sparus aurata serum are effected by the alternative complement pathway[END_REF]. In brief, serial dilutions (from 15 to 160 times) of plasma samples were performed in veronal buffer (Biomerieux, Marcy-l'Etoile, France). Then 60 µl of each dilution were mixed with 10 µl of 3% rabbit erythrocytes suspended in veronal buffer. After incubation at 37 °C for 100 min and centrifugation at 2000g for 10 min at 4 °C, supernatants were collected and read at 405 nm. The spontaneous hemolysis was obtained by adding veronal buffer to 10 µl of rabbit erythrocytes and total lysis was obtained by mixing 10 µl of rabbit erythrocytes to distilled water (total volume = 70 µl). ACH50 was calculated by linear regression and corresponds to the lysis of 50% of the rabbit erythrocytes.

Expression levels of some immune genes in anterior kidney

For further immune markers, the expression level of two immune-related genes was determined in anterior kidney tissues, namely C-type lysozyme and complement component 3 (C3). Primer sequences are presented in Table 5. Total RNA from anterior kidney was extracted using TRIzol Reagant (ThermoFisher Scientific) according to manufacturer's instructions. Tissue samples were homogenized using a SpeedMill PLUS homogenizer (Ana-lytikJena, Germany) in tubes containing ceramic beads and TRIzol Reagant. Total RNA was resuspended in 50 µl of DPEC-treated water. RNA integrity and concentration was checked by denaturat-ing gel electrophoresis (1.2% agarose) and OD260/OD280 and OD260/OD230 nm absorption ratio using Nanodrop-1000 (Thermo-Fisher Scientific). Twelve µg of each RNA sample were treated with FreeDNA kit (Ambion, Austin, TX, USA) to remove genomic DNA. mRNA was then retrotranscribed with Reverse Transcription System kit (Promega, Wisconsin, USA) according to manufacturer's instructions. The cDNA was then 20 times diluted and aliquoted. qPCR was performed using Power SYBR® Green PCR Master Mix (Applied Biosystem, Warrington, UK), 2.5 µl of both right and left primers (5 µM) and 5 µl of the diluted cDNA. A four steps experimental run protocol was followed: denaturation (10 min at 95 °C), amplification (40 cycles, 15 s at 95 °C, 1 min at 60 °C), melting curve (40 to 95 °C, heating rate 0.10 °C s 1)*** and a final cooling step (4 °C) using a StepOne plus real time PCR machine (Applied Biosystem). Relative quantification of the target gene transcript was calculated following the Pfaffl method [START_REF] Pfaffl | A new mathematical model for relative quantification in realtime RT-PCR[END_REF], considering the Ct value and the primer efficiency. The relative expression levels of C-type lysozyme and complement C3 in each sample were normalized with the geometric mean of ef1-a and b-actin calculated by the relative standard curve method.

Statistical analyses

To determine the best combinations of factors-modalities, each experimental unit was assigned to a global score of interest. This global score was calculated using results of husbandry output variables and was based on the transformation of each output in centered reduced output [START_REF] Gardeur | Simultaneous effects of nutritional and environmental factors on growth and flesh quality of Perca fluviatilis using a fractional factorial design study[END_REF]. Principal component analyses (PCA) were also performed to analyze the global effect of combinations on husbandry output variables.

Main effects and two factor-interactions were then analyzed using Analys software [START_REF] Kobilinsky | Reparametrisation of interest in non-uniform factorial designs[END_REF]. This method is first based on the detection of potentially active effects using Daniel graphics [START_REF] Daniel | Use of half-normal plots in interpreting factorial two-level experiments[END_REF]. It is followed by ANOVA to test these potentially active effects. Significant results (p < 0.05) were finally confirmed by ANOVA (p < 0.05) using Statistica software version 10 (StatSoft Inc., France, 2011). Significant correlations were then calculated between growth parameters and stress and immune markers.

Results ______________________________________________________________________

Husbandry parameters

Global score results showed that the combination 15 (c15) resulted in the best husbandry performances (Table 3). High global scores of interest (>3) were also obtained for c1, c16, c9 and c3. All these experimental treatments resulted in the highest FIW (158-172 g), the lowest MR (3 to 13%) and the highest SGR (0.9 to 1.1%.d-1). The lowest weight heterogeneity was observed for c1, c4, c9, c13 and c15 and averaged 36%. The five best combinations were commonly influenced by the type of feed (sinking) as input variable. The worst husbandry outputs were observed with combinations c6, c7, c10 and c12 and they had in common the use of midfloating feed. Further ANOVA revealed no effect of feed type on SGR between D0 and D24 while sinking feed significantly improved SGR between D24 and D63 (p < 0.05).

Further comparison by PCA approach confirmed that only the feed type was the major influencing factor husbandry parameters but also with the light intensity (Fig. 1). Indeed, on the axis 1, c1, c3, c9, c15 and c16 were characterized by a high SGR, a high FIW and a low CV. The use of sinking feed mainly defined these combinations confirming that the use of such feed type led to significant higher SGR (Fig. 2a, p < 0.01) and FIW (Fig. 2b, p < 0.001) with values reaching respectively 0.86 ± 0.19%•d-1 and 157 ± 15 g. This type of feed also decreased the CV from 59 ± 8% to 39 ± 7% (Fig. 2c, p < 0.01).The opposite results were observed for c6, c7, c12 and c14. On the second axis, combinations c4, c10 and c13 displayed a high MR and they were mainly defined by the high light intensity vector. The opposite results were observed with the low light intensity for c2, c5 and c8. Lowest MR was observed under low light intensity (8 ± 7%) compared to high light intensity (22 ± 12%) (p < 0.05) (Fig. 2d).

Stress and immune parameters

The experimental combinations (c1, c3, c4, c5, c9, c13, c15 and c16) that included the sinking feed induced higher glucose level (392 ± 66 mg•ml-1) on day 36 (Fig. 3a, p < 0.05) but not on day 63. High temperature level (26 °C; combinations c2; c8; c9; c11; c12; c13; c15; c16) also increased glycaemia on day 63 (Fig. 3b, p < 0.05). No statistical influencing factor was observed for plasma cortisol level both on D36 and D63. Plasma cortisol values were nearly to basal level both on D36 and D63, except for C1 and C8 on D36; and a significant decrease was observed between D36 and D63 (p < 0.05). Therefore, non-significant coefficients of correlation could be calculated between cortisol or glucose values and those growth parameters on D36 and D63.

While no single factor influenced serotonergic activity in the brain, light spectrum and light intensity appeared clearly to act synergistically since the serotonergic activity increased when the red light was used with low light intensity (combinations c5, c12, c14 and c16) or when white light was combined with high light intensity (combinations c3, c9, c10 and c11) (Fig. 3c, p < 0.001). It is interesting to notice that the best three ranked combinations (c15, c1 and c16) in terms of husbandry performances were characterized by low values of serotonergic activity. Moreover, significant negative coefficient of correlation (R2 = 0.31; p < 0.05) was calculated between values of serotonergic activity on D63 and those of final body weight. So, red light spectrum at low intensity or white light spectrum at high intensity may induce both a higher stress status in pikeperch.

Low oxygen saturation significantly increased complement activity in plasma on D36 (p < 0.05, Fig. 4a) but not on D63, and no significant factor interaction was detected. Values for complement activity increased between D36 and D63 (p < 0.001). Light intensity and photoperiod acted in synergy on complement gene expression level in kidney on day 36 and such effect is not detected on D63. Significantly lower values were observed when a 10 lux intensity was combined with a long photoperiod than when combined with a short photoperiod (p < 0.05) (Fig. 4b). No effect on lysozyme activity and C-type lysozyme expression was observed.

Discussion ___________________________________________________________________

The present study aimed to define the main directive factormodalities for pikeperch aquaculture as well as the best combination of husbandry factors inducing the lowest stress status together with high husbandry performances and better welfare status. To our knowledge, very few studies have examined the optimization of aquaculture conditions for percid fish by taking into account a wide range of environmental and husbandry factors. Despite the loss of resolution compared to a full factorial design, fractional factorial approach has the main advantage of considering simultaneously the impact of a large number of inter-related aquaculture factors while traditional full factorial design rarely exceeds 3 factors [START_REF] Hamre | Antioxidant vitamins, minerals and lipid levels in diets for Atlantic salmon (Salmo salar, L.): effects on growth performance and fillet quality[END_REF][START_REF] Gardeur | Simultaneous effects of nutritional and environmental factors on growth and flesh quality of Perca fluviatilis using a fractional factorial design study[END_REF][START_REF] Trabelsi | Effects of 12 factors on burbot Lota lota (L., 1758) weaning performances using fractional factorial design experiment[END_REF].

In the present study, we have considered a 2 monthexperiment to better assess the effects of the 8 environmental factors on growth related parameters, stress markers and immune status. Only considering mortality, 3 of the 16 tanks exceeded 30% of mortality while 4 other tanks did not reached 5%. Such difference was not expected within 2 months but allowed us to consider more especially some factors as crucial for welfare and survival of pike-perch in intensive culture conditions.

Directive factors improving husbandry performances

The classification of the combinations and the PCA have revealed the type of feed and the light intensity as the two main directive factors for husbandry variables, which are regarded as valuable indicators for estimating the fitness in aquaculture conditions [START_REF] Moberg | The biology of animal stress: basic principles and implications for animal welfare[END_REF]. The use of sinking feed improved growth performances and decreased weight heterogeneity. Fish were fed with sinking feed before the experiment and no habituation to mid-floating feed was done. However, the change of feeding strategy and the lack of habituation do not have impacted feeding behavior during the first days since no effect of feed type was detected on SGR between D0 and D24 in contrast to differences observed on D63 at the end of the experiment. The use of mid-floating feed has thus impacted, independently of the lack of habituation, food intake resulting in decreased growth performances. It has been demonstrated that the use of sinking feed has a better effect on food intake and thereby on husbandry performances comparing to mid-floating feed in other fish species such as the Atlantic halibut Hippoglossus hippoglossus [START_REF] Kristiansen | Individual behaviour and growth of halibut (Hippoglossus hippoglossus L.) fed sinking and mid-floating feed :Evidence of different coping styles[END_REF].

The present study revealed that light intensity is a determining factor for pikeperch with better survival observed under low light intensities. Light intensity affects many behavioral and biological processes in fish, such as foraging and growth [START_REF] Fraser | The costs of becoming nocturnal: feeding efficiency in relation to light intensity in juvenile Atlantic Salmon[END_REF][START_REF] Trippel | Effects of photoperiod and light intensity on growth and activity of juvenile haddock (Melanogrammus aeglefinus)[END_REF][START_REF] Luchiari | Light intensity preference of juvenile pikeperch Sander lucioperca (L.)[END_REF]. The present study confirmed that pikeperch reared under conditions of low light intensity set at 10 lux displayed better husbandry performances than those submitted to 100 lux. Such preference of pikeperch for low light intensities has already been described by [START_REF] Luchiari | Light intensity preference of juvenile pikeperch Sander lucioperca (L.)[END_REF] since this species is a nocturnal and crepuscular predator. Sander species also possess a tapetum lucidum that is a specific anatomo-histological tissue of the retina which greatly amplifies the eye sensitivity to light [START_REF] Feiner | Environmental biology of Percid fishes[END_REF]. This preference for low light intensities can be related to an innate behavior to avoid possible harmful effects of light on light-sensitive eyes [START_REF] Sandström | Visual ecology of fisha review with special reference to percids[END_REF][START_REF] Luchiari | Light intensity preference of juvenile pikeperch Sander lucioperca (L.)[END_REF].

It is surprising that other tested factors such as grading were not demonstrated as directive influencing factors on husbandry variables in the context of the present study. Indeed, it has been reported that frequent manipulations markedly affect growth rate of young Eurasian perch [START_REF] Jentoft | Effects of stress on growth, cortisol and glucose levels in non-domesticated Eurasian perch (Perca fluviatilis) and domesticated rainbow trout (Oncorhynchus mykiss)[END_REF][START_REF] Strand | Effects of repeated disturbances on feed intake, growth rates and energy expenditures of juvenile perch, Perca fluviatilis[END_REF], a species biologically close to pikeperch. Perhaps, the frequency of grading every two weeks, and the relative manipulations were not so detrimental at the developmental stage used in the present experiment. Temperature level was not also found as main directive factor for husbandry performances, and the positive interactions with sinking feed were observed with the treatments which included the lowest temperature. So, our finding did not corroborate previous reports that high temperature promotes growth rate in pikeperch juveniles [START_REF] Rónyai | Effect of feeding regime and temperature on ongrowing results of pikeperch (Sander lucioperca L.)[END_REF][START_REF] Wang | Effect of temperature and feeding frequency on growth performances, feed efficiency and body composition of pikeperch juveniles (Sander lucioperca)[END_REF][START_REF] Dalsgaard | Farming different species in RAS in Nordic countries :current status and future perspectives[END_REF], but may support the hypothesis that more energy is spent for increased metabolic rates when pikeperch are reared at 25 °C [START_REF] Frisk | The effects of temperature on specific dynamic action and ammonia excretion in pikeperch (Sander lucioperca)[END_REF].

And considering that high temperature may weaken the immunocompetence and thereby increase pathogen outbreaks in intensive culture conditions [START_REF] Raida | Temperature-dependent expression of immunerelevant genes in rainbow trout following Yersinia ruckeri vaccination[END_REF][START_REF] Martins | Temperature effects on immune response and hematological parameters of channel catfish Ictalurus punctatus vaccinated with live theronts of Ichthyophthirius multifiliis[END_REF], we therefore conclude that temperature around 21 °C is more favourable for pikeperch culture.

Our results also support observations reporting that high stocking density has no marked effects on growth and food utilization of young pikeperch, and that small juveniles can be kept at high densities ranging between 15 and 30 kg•m-3 without any increase in physiological stress response [START_REF] Molnár | The effect of initial stocking density on growth and survival of pike-perch fingerlings reared under intensive conditions[END_REF]Steenfeldt etal., 2010;Dalsgaard etal., 2013). It was also reported that higher densities (30-60 kg•m-3) for larger pikeperch up to 2 kg are associated to good growth without any increase in crowding stress parameters [START_REF] Dalsgaard | Farming different species in RAS in Nordic countries :current status and future perspectives[END_REF].

Percid fish display sexual growth dimorphism as females grow faster than males [START_REF] Craig | Percid fishessystematics, ecology and exploitation, vol 3, Fish and aquatic resources series[END_REF]. Therefore, rearing all-female populations would improve production of these species and this would be achieved by hormonal sex reversal treatment [START_REF] Rougeot | Sex and ploidy manipulation in Percid fishes[END_REF]. The difference of growth performances between mixed-sex families and all-female families reaches 30% in juvenile Eurasian perch after 360 days of rearing (Rougeot and Mélard, 2016). However, the methods for allfemale production have not been yet optimized for pikeperch, and since sex-determination in not possible with juvenile pikeperch, a mixed-sex population was used in the present experiment.

Directive modality interactions for pikeperch physiological and immune status

In the present experiment, no effect of the different culture conditions on plasma cortisol level was observed. Cortisol is well known to be part of the stress response and to be a major stress indicator promoting the metabolic pathways that increase plasma glucose levels in response to energy expenditure [START_REF] Laiz-Carrión | Influence of Cortisol on Osmoregulation and energy metabolism in gilthead seabream Sparus aurata[END_REF][START_REF] Milla | Spleen immune status is affected after acute handling stress but not regulated by cortisol in Eurasian perch. Perca fluviatilis[END_REF][START_REF] Olivieira | Endocrine (plasma cortisol and glucose) and behavioral (locomotor and self-feeding activity) circadian rhythms in Senegalese sole (Solea senegalensis Kaup 1858) exposed to light / dark cycles or constant light[END_REF]. A decrease in cortisol release along exposure to long-lasting stressors is a mechanism that minimizes the deleterious effects of sustained cortisol elevation on biological functions [START_REF] Milla | Corticosteroids and the stress response in Percid fish[END_REF]. While the assessment of cortisol is used as a valuable indicator of first stress response, it has been debated whether such indicator is a reliable indicator of welfare status since itself is not predictive of the fish's ability to cope with a stress situation [START_REF] Ellis | Cortisol and finfish welfare[END_REF]. A previous experiment on pikeperch (unpublished data) showed that plasma cortisol level returned to basal level as soon as 1 h post-stress while glucose peak was sustained for more than 3 h. A similar rapid decrease in the amplitude of stress response was characterized in juvenile Eurasian perch following a single or multiple emersion stressor [START_REF] Douxfils | Influence of domestication process on immune response to repeated emersion stressors in Eurasian perch (Perca fluviatilis, L.)[END_REF]. These observations may suggest a habituation to stress and/or a rapid metabolism of cortisol indicating the interest for using various stress indicators to account for the stress responsiveness in pikeperch. While assaying cortisol from plasma samples has already been used in several fish species with good results when collected within 5 min to avoid handling-induced cortisol [START_REF] Wang | Temperature effect on the time course of cortisol release during acute stress in Eurasian perch (Perca fluviatilis)[END_REF][START_REF] Douxfils | Does domestication process affect stress response in juvenile Eurasian perch Perca fluviatilis?[END_REF], non-invasive methods have also been developed including direct fecal corticoid metabolites measurement and cortisol release measurement in water [START_REF] Ellis | Anon-invasive stress assay based upon measurement of free cortisol released into the water by rainbow trout[END_REF][START_REF] Cao | Establishment of anon-invasive method for stress evaluation in farmed salmon based on direct fecal corticoid metabolites measurement[END_REF]. These methods should be better considered for offering various advantages including the absence of fish disturbance and the reduction of the number of animals required [START_REF] Fanouraki | Water cortisol is a reliable indicator of stress in European sea bass, Dicentrarchus labrax[END_REF].

The significant impact of high temperature on plasma glucose observed in the present study may be related to a better feeding behaviour since the feed type influenced glycaemia. Higher temperature conditions ranging between 25 and 28 °C have been described to promote growth and feed utilization in pikeperch juveniles [START_REF] Rónyai | Effect of feeding regime and temperature on ongrowing results of pikeperch (Sander lucioperca L.)[END_REF][START_REF] Wang | Effect of temperature and feeding frequency on growth performances, feed efficiency and body composition of pikeperch juveniles (Sander lucioperca)[END_REF][START_REF] Dalsgaard | Farming different species in RAS in Nordic countries :current status and future perspectives[END_REF]. However, if this is convenient for juveniles, it cannot be extrapolated to larger pikeperch without investigation since several studies have shown that optimal temperature seems to decrease with fish weight as observed with the African catfish (Clarias gariepinus) or the Atlantic cod (Gadus morhua) [START_REF] Hogendoorn | Growth and production of the African catfish, Clarias lazera (C. & V.). II. Effects of bodyweight, temperature and feeding level in intensive tank culture[END_REF][START_REF] Björnsson | Growth model for Atlantic cod ( Gadus morhua): Effects of temperature and body weight on growth rate[END_REF][START_REF] Rónyai | Effect of feeding regime and temperature on ongrowing results of pikeperch (Sander lucioperca L.)[END_REF][START_REF] Wang | Effect of temperature and feeding frequency on growth performances, feed efficiency and body composition of pikeperch juveniles (Sander lucioperca)[END_REF]. In the present experiment, while the feeding behavior may have been improved by high temperature, this factor did not significantly affect growth performances. [START_REF] Frisk | The effects of temperature on specific dynamic action and ammonia excretion in pikeperch (Sander lucioperca)[END_REF] reported that a smaller fraction of metabolic scope was utilized for digestion at 19 °C compared to 25 °C, indicating that low temperature conditions are more favorable for pikeperch reared under intensive culture conditions. Even if this was not evidenced in the present study showing low cortisol levels on both D36 and D63, high temperature condition may induce along-term stress that leads to energy reallocation resulting in less energy available for some biological functions such as growth, reproduction or disease resistance [START_REF] Schreck | Effects of stress on fish reproduction, gamete quality, and progeny[END_REF][START_REF] Tort | Stress and immune modulation in fish[END_REF][START_REF] Segner | Health of farmed fish :its relation to fish welfare and its utility as welfare indicator[END_REF][START_REF] Milla | Corticosteroids and the stress response in Percid fish[END_REF].

Serotonergic activity has been described several times as a good indicator of acute and chronic stress in various fish species including the Senegalese sole Solea senegalensis and several salmonids [START_REF] Winberg | Roles of brain monoamine neurotransmitters in agonistic behaviour and stress reactions, with particular reference to fish[END_REF][START_REF] Gesto | The response of brain serotonergic and dopaminergic systems to an acute stressor in rainbow trout : a time course study[END_REF][START_REF] Gesto | A simple melatonin treatment protocol attenuates the response to acute stress in the sole Solea senegalensis[END_REF][START_REF] Conde-Sieira | Oral administration of melatonin counteracts several of the effects of chronic stress in rainbow trout[END_REF]. When considering serotonergic activity level in the presented study, red light spectrum at low intensity or white light spectrum at high intensity induced both a higher stress status. Environmental colors affect the vision of fishes, influencing for example food intake, signals for hierarchical status, reproduction, growth and even survival [START_REF] Downing | Impact of spectral composition on larval haddock, Melanogrammus aeglefinus L., growth and survival[END_REF][START_REF] Politis | Light impacts embryonic and early larval development of the European eel, Anguilla anguilla[END_REF][START_REF] Luchiari | Effects of ambient colour on colour preference and growth of juvenile rainbow trout Oncorhynchus mykiss (Walbaum)[END_REF]. Species-dependent preferences are a reflection of the photic environment the populations have evolved in [START_REF] Migaud | Evidence for differential photic regulation of pineal melatonin synthesis in teleosts[END_REF]. [START_REF] Luchiari | Longer wavelengths of light improve the growth, intake and feed efficiency of individually reared juvenile pikeperch Sander lucioperca (L.)[END_REF] showed an improved specific growth rate and feed efficiency under red light, only considering high light intensity. In parallel, [START_REF] Luchiari | Light intensity preference of juvenile pikeperch Sander lucioperca (L.)[END_REF] described a preference for low light intensities. Among the three stress indicators tested in the present study, only brain serotonergic activity showed plausible correlation with husbandry performances, indicating that high stress level is one of the main causes of low husbandry performances in pikeperch culture. By elsewhere, brain serotonergic activity maybe considered as a more reliable stress indicator in percid fish than plasma cortisol. It has been suggested that a low degree of responsiveness to stressors in cultured fish may result in better performances [START_REF] Pottinger | Genetic basis to the stress response[END_REF].

The converse may also be true considering the greater advantage of fish with a high degree of stress responsiveness, through a faster acclimation to environmental and social changes [START_REF] Pottinger | A comparison of plasma glucose and plasma cortisol as selection markers for high and low stress-responsiveness in female rainbow trout[END_REF]. While the latter authors have described no correlation between cortisol response and growth performances in rainbow trout, better growth was obtained in low cortisol responders [START_REF] Fevolden | Selection response of cortisol and lysozyme in rainbow trout and correlation to growth[END_REF]. And, as well as plasma cortisol level, it was reported that brain serotonergic, noradrenergic and dopaminergic activities are influenced by dominant-subordinate relationships in several fish species, including the rainbow trout (Overli et al., 1999). Social dominance was not followed in the present experiment but could have influenced the lack of relationship between growth and cortisol level since it has been shown in other fish species that socially defeated animals exhibit increased glucocorticoid secretion, sustained sympathetic activation and other physiological stress responses [START_REF] Winberg | Elevation of brain 5-HT activity, POMC expression, and plasma cortisol in socially subordinate rainbow trout[END_REF]Overli et al., 1999).

In terms of cortisol level, it is also surprising that grading manipulations did not induce higher stress status of pikeperch since grading practice has already been reported to markedly affect fish welfare [START_REF] Jentoft | Effects of stress on growth, cortisol and glucose levels in non-domesticated Eurasian perch (Perca fluviatilis) and domesticated rainbow trout (Oncorhynchus mykiss)[END_REF][START_REF] Strand | Effects of repeated disturbances on feed intake, growth rates and energy expenditures of juvenile perch, Perca fluviatilis[END_REF]. Before starting the experiment, the fish were size graded several times according to common practices of intensive pikeperch culture. Grading manipulations were then applied every two weeks according to the protocol. To explain the absence of the expected impact on cortisol level, it could be hypothesized that pikeperch submitted to such sequential stressors can exhibit a habituation on its cortisol response and an attenuation of the response as shown in several fish species [START_REF] Schreck | Accumulation and Long-Term Effects of Stress in Fish[END_REF][START_REF] Koakoski | Repeated stressors do not provoke habituation or accumulation of the stress response in the catfish Rhamdia quelen[END_REF]. However, the results on habituation and accumulation are highly dependent on the type of stressor, the length of time between discrete stressors and the number of repeated stressor [START_REF] Koakoski | Repeated stressors do not provoke habituation or accumulation of the stress response in the catfish Rhamdia quelen[END_REF]. In the present experiment, the purpose was not to characterize the stress response after fish manipulations but to characterize chronical effects of several husbandry practices and, therefore, the longterm impact on stress markers. It is worth noting that serotonergic activity appeared as more sensitive marker than cortisol since plausible correlation was established with growth rate.

Concerning immune status, humoral immune activities were slightly impacted by some tested factors during the 2-month period. The effects of low oxygen saturation on complement activity on day 36 were no longer observed on day 63, suggesting only a short-term effect. This decrease in the impact of low oxygen saturation may be account for the increase in ACH50 values between D36 and D63. On the other hand, the effect of low oxygen level on humoral immunity may be explained by the fact that pikeperch requires high oxygen saturation and recommendations are to maintain the oxygen level above 50% saturation to sustain growth and biofilter performance [START_REF] Dalsgaard | Farming different species in RAS in Nordic countries :current status and future perspectives[END_REF]. In contrast to what was observed for growth rate, long photoperiod combined with high light intensity, which was characterized as a stressful factor, led to an increase in the immune expression level of both tested genes. Long photoperiod increased the exposure time to stressful light conditions that may have resulted in a shortterm immune stimulation. However, since no single effect of photoperiod on stress and immune status was detected, the impact of such factor should be investigated in further experiments. The other factors tested in the present experiment were not revealed as directive variables for stress or immune status.

The lack of significant impact for high stocking density (30 kg•m-3) corroborates previous reports indicating that pikeperch juveniles from 10 to 50 g should be maintained at densities below 15-30 kg•m-3 while fish up to 2 kg can be kept around 30-60 kg•m-3 without any increase in crowding stress parameters, suppressed growth, or increase in feed conversion ratio [START_REF] Steenfeldt | Further development of intensive pikeperch rearing in Denmark[END_REF]. The lack of enough information on the imposing stress events for pikeperch in intensive aquaculture did not allow further discussion concerning the impact of the tested factors-modalities on immune status. So, further investigations are needed to explore potential effects of high stress responsiveness on immune status of pikeperch.

Conclusion __________________________________________________________________

This experiment was based on a factorial fractional design study which allowed simultaneously to study the effects of 8 common husbandry practices in two modalities on pikeperch growth and welfare. Several husbandry practices, including light intensity, feed type and temperature were revealed to be directive factors for husbandry and stress parameters in pikeperch aquaculture while immune status was mainly influenced by photoperiod and oxygen saturation level. Best husbandry performances were thus obtained with sinking feed and low light intensity, and no obvious relation was established between stress status and growth parameters. Further investigations focusing on light characteristics, temperature and feed type are needed to improve management strategies of pikeperch culture, and knowledge on the interrelations between stress and immunity in percid fish.

Variables

  Figure 1. Projections of husbandry outputs and combinations (1-16) on the two first axis of the principal component analysis. The plans 1 and 2 of the PCA explain 93% of the projected inertia. Axis 1 (inertia 66%) represents Specific growth rate (SGR), Final individual weight (FIW), final weight heterogeneity (CV) and the type of feed (Sinking S vs mid-floating F). Axis 2 (inertia 27%) represents mortality rate (MR) and light intensity (10 vs 100 lux).

Figure 2 .

 2 Figure 2. Effects of tested factors on (A) specific growth rate, (B) final individual weight, (C) final weight heterogeneity, and (D) mortality rate. Day 63. Data are presented as mean ± SEM (n = 8). Lowercase letters indicate a significant difference atp < 0.05.

Figure 3 .

 3 Figure 3. Effects of tested factors on (A) plasma glucose D36, (B) plasma glucose D63, and (C) serotonergic activity in brain. Data are presented as mean ± SEM (n = 8 (A); 8 (B); 4(C)). Lowercase letters indicate a significant difference at p < 0.05.

Figure 4 .

 4 Figure 4. Effects of tested factors on (A) plasma complement activity, and (B) relative C3 gene expression. Data are presented as mean ± SEM (n = 8 (A); 4 (B)). Lowercase letters indicate a significant difference at p < 0.05.

Table 1 . Selected modalities for the 8 environmental factors.

 1

	Factor -abbreviation	Level
	Light intensity (lux)	100	10
	Light spectrum	Industrial white Red (610 nm)
	Photoperiod (L:D)	24:00	10:14
	Density (kg•m -3 )	30	15
	Temperature (°C)	26	21
	Oxygen saturation (%)	90	60
	Feed type	Mid-floating	Sinking
	Fish grading	Yes	No

Table 2 . Aliasing structure considering the 8 environmental factors: light intensity (Int.), oxygen saturation (Oxy.); density (Dens.), light spectrum (Spec.), photoperiod (Photo.), water temperature (Temp.), grading and feed type.

 2

	Group	Aliased effects
	1	Int. * Oxy.; Dens. * Grading; Spec. * Temp.; Photo. * Feed
	2	Int. * Temp.; Dens. * Photo.; Spec. * Oxy.; Feed * Grading
	3	Int. * Dens.; Spec. * Feed; Photo. * Temp.; Grading * Oxy.
	4	Int. * Spec.; Dens. * Feed; Photo. * Grading; Temp. * Oxy.
	5	Int. * Grading; Dens. * Oxy.; Spec. * Photo.; Temp. * Feed
	6	Int. * Photo.; Dens. * Temp.; Spec. * Grading; Feed * Grading
	7	Int.

* Feed; Dens. * Spec.; Photo. * Oxy.; Temp. * Grading

Table 3 . Combinations (c1-c16) of the factors following a 2(8-4) reduced factorial design and results. Final weight heterogeneity (CV); Light spectrum: W = White, R = Red; Feed: S = Sinking, F = mid-Floating; Grading: Y = with manipulationsmimicking grading, N = without grading. Global score: note of interest for each combination, based on husbandry output variables. The grey lines correspond to the five best combinations according to the global score of interest.

 3

								Variables tested		Variables studied
	the factors	Combination of	(lux)	Light intensity	(kg•m -3 )	Density	Light spectra	Photoperiod (h)	(°C)	Temperature	Feed	Grading	saturation (%)	Oxygen	weight (g)	Final individual	(%)	Mortality Rate	CV (%)	rate (%d-1)	Specific growth	(D36, ng•mL -1 )	Plasma Cortisol	(D63, ng•mL -1 )	Plasma Cortisol	µg•mL -1 )	Glucose (D36,	Plasma	µg•mL -1 )	Glucose (D63,	Plasma	activity (D36)	Serotonergic	activity (D63)	Serotonergic	ACH50 (D36)	ACH50 (D63)	activity (D36, U)	Lysozyme	activity (D63, U)	Lysozyme (D36)	ef1+βactin	expression to	gene	Relative C3 (D36)	ef1+βactin	expression to	lysozyme gene	Relative	(D63)	ef1+βactin	expression to	gene	Relative C3 (D63)	ef1+βactin	expression to	lysozyme gene	Relative	interest	Global score of	global score	Rank of the
	c1 10 30 W 24 21 S	Y	90 168 4		37 0.9 84 31 457 318 0.84 0.61 98 202 17 19 0.011 0.018 0.00019 0.079 3.9 2
	c2 100 15 R	10 26 F		N	60 146 3		50 0.7 17 13 378 427 0.71 0.77 145 142 16 19 0.027 0.016 0.00034 0.027 1.3 6
	c3 100 15 W 24 21 S	N	60 172 13 40 1.0 20 15 355 371 0.94 0.74 162 215 16 18 0.014 0.045 0.00014 0.114 3.1 5
	c4 100 30 R	10 21 S	N	90 143 31 29 0.7 19 14 264 340 0.79 0.72 153 216 17 19 0.027 0.024 0.00014 0.042 0.4 8
	c5 10 15 R	10 21 S	Y	60 131 7		53 0.5 15 14 424 379 0.96 0.81 157 181 16 19	0.001 6	0.029 0.00026 0.025 -0.5 10
	c6 10 15 W 10 21 F		N	90 88 7		67 0		16 14 342 365 0.53 0.82 76 264 15 15 0.004 0.061 0.00050 0.090 -5.4 16
	c7 100 15 R	24 21 F		Y	90 113 24 61 0.3 17 13 273 343 0.69 0.78 140 210 14 19 0.021 0.024 0.00083 0.065 -4.0 15
	c8 10 15 W 24 26 F		Y	60 146 10 53 0.7 60 13 322 359 0.60 0.72 125 244 16 21 0.004 0.022 0.00049 0.030 0.4 7
	c9 100 15 W 10 26 S	Y	90 158 13 39 1.1 26 13 371 411 0.87 0.75 132 225 17 18 0.015 0.055 0.00248 0.088 3.1 4
	c10 100 30 W 10 21 F		Y	60 122 41 52 0.7 17 14 339 317 1.06 0.76 173 261 17 20 0.008 0.028 0.00005 0.011 -3.0 13
	c11 100 30 W 24 26 F		N	90 148 18 61 0.8 89 29 328 405 0.81 0.72 101 257 15 20 0.012 0.045 0.00100 0.015 -0.6 11
	c12 10 30 R	10 26 F		Y	90 114 24 57 0.3 23 13 304 325 1.19 0.68 96 250 11 17 0.006 0.061 0.00027 0.014 -3.6 14
	c13 100 30 R	24 26 S	Y	60 151 32 37 0.8 47 15 466 412 0.71 0.77 100 233 18 20 0.019 0.067 0.00059 0.021 0.3 9
	c14 10 30 R	24 21 F		N	60 117 4		72 0.4 17 13 271 337 0.94 0.75 132 298 15 16 0.012 0.033 0.00132 0.019 -2.8 12
	c15 10 30 W 10 26 S	N	60 167 3		36 0.9 27 15 420 373 0.67 0.73 124 249 16 16 0.027 0.049 0.00059 0.017 4.0 1
	c16 10 15 R	24 26 S	N	90 169 7		40 0.9 18 15 375 369 0.89 0.64 67 267 14 18 0.003 0.019 0.00012 0.010 3.4 3
	Me an													140 15 49 0.7 32 16 356 366 0.82 0.73 124 232 16 18 0.014 0.037 0.00058 0.042
	SD													24 12 13 0.3 24 6		63 35 0.17 0.06 31 38 2		2		0.008 0.017 0.00062 0.034

Table 4 . List of the husbandry variables and formulas. CV: Final weight heterogeneity.

 4

Acknowledgements ____________________________________________________________

This study has been supported under the framework of the European Union Seventh Framework Programme project DIVERSIFY (KBBE-2013-07 single stage, GA 603121) titled "Exploring the biological and socio-economic potential of new/emerging fish species for the expansion of the European aquaculture industry". The authors thank Prof. Pierre Van Cutsem (Unit of Research in Plant Cellular and Molecular Biology, Namur, Belgium) and Pierre Cambier for providing access to and guidance with their HPLC system.