N Limodin

J Y Buffière

J Réthoré

F Hild

S Roux

W Ludwig

Three-dimensional study of fatigue crack propagation using synchrotron X-ray microtomography

INTRODUCTION

Synchrotron X-ray microtomography imaging was used to study crack propagation taking into account the 3D nature of the crack morphology. A small loading device, which is directly mounted on the tomography set-up, enabled for in-situ loading and cycling of pre-cracked specimens. Scans were acquired during loading and unloading of the specimen to provide 3D images of the bulk in the vicinity of the crack at different fractions of the total propagation life. The tomographic images were subsequently treated by 3D Digital Image Correlation (DIC). DIC has already been used to measure Crack Opening Displacement (COD) and to estimate the corresponding stress intensity factor (SIF) value at the specimen surface [START_REF] Mekky | The fracture toughness of Ni/Al 2 O 3 laminates by digital image correlation I: Experimental crack opening displacement and Rcurves[END_REF]. However, up to now it has been performed in 2D only because 3D extensions of DIC are still new [START_REF] Roux | Three-dimensional image correlation from X-ray computed tomography of solid foam[END_REF]. In the present work, the application of 3D-DIC to tomographic data allowed rapid retrieving of the displacement field in the bulk of the cracked specimen. From this displacement field, both the crack geometry and COD could be obtained. The above mentioned method was applied to specimens cut from different regions of larger fatigue precracked samples made of nodular graphite cast iron to study the influence of the stress state in the larger sample on further crack growth in the smaller specimens.

MATERIAL AND METHODS

The studied material is a ferritic nodular graphite cast iron (3.65 wt% C, 3.2 wt% Si, 0.04 wt% Mg, <0.1 wt% Mn, <0.005 wt% S, 0.02 wt% P). After casting and heat treatment, the microstructure consists of a ferritic matrix with a homogeneous distribution of nearly spherical graphite nodules having an average diameter of 45 µm and a volume fraction of 14%. Young's modulus, yield stress and Poisson's ratio are equal to 175 GPa, 315 MPa, and 0.27, respectively. More details can be found in Ref. [START_REF] Adrien | Optimisation des cycles thermiques appliqués aux fontes G.S. ferritiques vis à vis des propriétés de fatigue[END_REF]. Carbon (nodules) and iron (matrix) have atomic numbers that are different enough to give a strong X-ray attenuation contrast. The nodules are thus easily imaged by tomography and can be used as natural markers for image correlation. Notched specimens with a 6 × 4-mm cross-section were pre-cracked in fatigue at a stress ratio of 0.1. The specimen faces were mirror polished to monitor crack growth with an optical travelling microscope. A loadshedding technique was used to maintain a value of the maximum Stress Intensity Factor (SIF) less than 12.8 MPa√m to make sure that the plastic zone size is less than 200µm at the crack tip (in plane strain). Fatigue cycling was stopped as soon as the crack had reached 1.5 mm in length. For the tomography experiment, two smaller dog-bone specimens with a square cross-section of 1.6 × 1.6 mm were spark cut inside two larger samples so that they enclose only a part of the crack tip. One small specimen was cut from one flat side of the larger sample so that one edge of the small specimen contains the crack tip at the former surface (close to a plane stress condition) while the other edge contains the crack tip that was at the centre of the larger specimen (close to plane strain). Another small specimen was cut from the mid-thickness of the large specimen (close to pure plane strain). These specimens will be referred to as side specimen and centre specimen hereafter.

The tomography experiments were performed on the ID19 beam line at the European Synchrotron radiation Facility (ESRF) in Grenoble, France. A monochromatic X-Ray beam (∆λ ⁄ λ = 10 -2 ) having a photon energy of 60 keV traverses the sample (Fig. 1) giving an incident-to-transmitted intensity ratio of about 10%. To obtain a complete scan of the specimen cross-section in the vicinity of the crack, six hundred radiographs were taken while the sample was rotating over 180 ° along its vertical axis; the scan time amounts to 42 minutes. A FReLoN (Fast Readout Low Noise) 14-bit CCD camera with a 2048 × 2048 pixel chip was used. A specially designed fatigue machine allowed for in-situ cycling and loading of the specimen. A PMMA tube, almost transparent to X-ray, is used as a load rig. The machine was directly mounted on the rotating stage (Fig. 1) of the beam line. Firstly, the specimen was progressively loaded to a maximum load, which is expected to result in a maximum stress intensity factor slightly smaller than the maximum value used in fatigue pre-cracking, and scans were acquired for different loading steps up to this maximum load. The specimen was then cycled at a stress ratio of 0.1 under constant amplitude to propagate the crack and scans were recorded at various fractions of the fatigue life. Reconstruction of the tomographic data was performed with a conventional filtered back-projection algorithm. It provides a 3D image with a greyscale colormap that is proportional to the local X-ray attenuation coefficient. A final region of interest of size 340 × 340 × 512 voxels with a voxel size of 5.06 µm was then focused on the crack to limit data size. DIC is a well established technique for computing displacement and strains at the specimen surface. In 3D, the same principle applies. Displacement fields are measured between a pair of images (here 3D reconstructed scans) of the same specimen at different loads. Images in grey level are characterized by f and g, their respective grey level valued function of pixel coordinates, x. The displacement field is obtained, using the so-called optical flow conservation, so that the image of the loaded sample, g, (i.e. the "deformed image") is matched to the reference image, f, when voxel location, x, is corrected for by the displacement field, u

f(x) = g(x + u(x)) (1)
A correlation algorithm based on a finite element kinematics [START_REF] Roux | Three-dimensional image correlation from X-ray computed tomography of solid foam[END_REF] was used. The image acquired at minimum load in the first cycle, i.e. at 14 and 24N for the side and centre specimens respectively, was used as the reference image, and correlation was sought between this reference image and the deformed image obtained at higher loads. A region of interest (ROI) of 288 × 288 × 288 voxels was centred on the original tomographic image. Eight-node cubic elements with an isotropic size of 16 voxels were chosen to mesh the ROI. Inside the cracked specimen, the presence of the crack discontinuity avoids matching the reference image to the corrected image. The difference between both images is the residual error map that gives invaluable information about the crack geometry as will be shown hereafter.

RESULTS

In the tomographic images, the apparent crack tip position can be measured via simple visual inspection using ImageJ software [4]. A plug-in allows for retrieving the coordinates of the pixel that is manually selected as the crack tip at each slice through the sample thickness. This enables one to detect variations of the crack front position with number of cycles for the side specimen (Fig. 2a) and the centre specimen (Fig. 2b). The crack lengths measured at the specimen surface with an optical microscope, at a better resolution than the tomographic images, are also plotted for comparison purposes. A difference in propagation behaviour is clearly visible between the two samples. On one hand, for the centre specimen, a crack advance is visible after only 2,000 cycles ("257N_2k") on the right hand side in Fig. 2b while the crack length on the other side remained almost unchanged until 3,000 cycles ("250N_3k") after which it grows at the same rate as other points along the crack front. On the other hand, in the side specimen, the crack remained arrested from the first cycle ("228N") to 40,000 cycles ("187N_40k") with a crack tip position that is close to that initially observed with a microscope (Fig. 2a). A crack advance finally appeared after 45,000 cycles ("186N_45k") with a crack growth rate that seems uniform along the crack front. Usually, tomographic images are thresholded to obtain a 3D rendering of the crack. However, in cast iron, the similarity of grey value between the crack and the nodules makes it impossible to visualize the crack alone. Therefore, the residual error map from DIC was thresholded using a 3D region growing algorithm to extract the voxels that belong to the crack from those corresponding to noise [START_REF] Rannou | Three dimensional experimental and numerical multiscale analysis of a fatigue crack[END_REF]. Figure 3 shows the crack geometries in the side and centre specimens after some propagation has occurred. The crack extent observed from the thresholded residual map matched the crack tip positions shown in Fig. 2 as soon as the crack tip is open enough, i.e. after 20,000 cycles for the side specimen and after 2,000 cycles for the centre specimen. The crack in the side specimen (Fig. 3a) is macroscopically flat with a slightly dissymmetric front that has maintained its shape during crack growth; dissymmetry can probably be attributed to a crack tunnelling effect with a crack that is longer on the side that was predominantly under plane strain condition in the larger pre-cracked specimen.

The crack surface in the centre specimen shows a step on the right hand side that has been inherited from the larger fatigue specimen (see detail of the crack surface highlighted in red in Fig. 3b). The crack on one side of the specimen has slowly departed from its mode I crack plane before reaching another straight crack path 600 µm away from the notch and about 150 µm above the main crack path. Reasons for this crack branching could not be inferred from surface observations and the microstructure (graphite nodule distribution) observed in the reconstructed image does not show any particular feature that may explain crack arrest. Due to this retardation effect, the initially symmetric and almost straight crack front shown in Fig. 2b has become dissymmetric during in situ cycling with a crack that is smaller on the right hand side in Fig. 3b where it was initially stopped.

a) b) Figure 3: Crack geometry in (a) the side specimen at 194N after 49,000 cycles and (b) in the centre specimen at 250N after 3,000 cycles (direction of crack growth is indicated by an arrow); a slice normal to the crack plane is extracted from the tomographic image to highlight the step in the crack path Image correlation provided the full displacement fields in the bulk of the specimen. Figure 4a shows an example of the displacement field, U z , along the loading axis at the maximum load of the first loading cycle in the centre specimen. The discontinuity due to the crack presence enables for the study of the mode I crack opening at a local scale. In Fig. 4b, a gradient of U z along a line indicated in blue in Fig. 4a is plotted for a position close to the crack mouth. The U z profile appears smooth and continuous away from the crack. Thus, the displacement jump on the crack surface, or COD, can be estimated from the difference between two slices on either side of the crack discontinuity in the U z field. The resulting COD map is plotted in the specimen cross-section in Fig. 4c; areas in light greys correspond to maximum opening. From this map, COD profiles are plotted at various locations in the sample thickness, for example along the red lines drawn in Fig. 4c, as a function of the distance to the crack mouth. Assuming a linear elastic behaviour, the crack tip displacement field in Mode I is known [START_REF] Anderson | Fracture Mechanics: fundamentals and applications[END_REF] and the SIF (Eq. ( 2)) is computed directly from the best fitting curve in Fig. 4d ( ) ( )

COD r E K u u I z z = = - - π π π 2 8
with r, the distance to the crack tip (2) A 2D map of COD values is shown in Fig. 5 together with the crack tip position reported in Fig. 2. In the side specimen (Fig. 5a), the crack tip opening was less than 0.3 voxel at the maximum load of the first cycle so that it can be considered closed. At 20,000 and 30,000 cycles, it has increased up to 0.4 voxel locally and at 40,000 cycles, it has reached an almost uniform value of 0.4 voxel while no significant growth has occurred. At 45,000 cycles and after, a further increase in COD occurred concomitantly with crack propagation.

Figure 5b shows the COD change with the number of cycles in the centre specimen from the first loading cycle to 3,000 cycles. At the first cycle, the crack was locally closed, i.e. with an opening less than 0.1 voxel, at the tip on the right hand side of the map whereas the opening amounted to 0.4 voxel on the left hand side. At 2,000 cycles, the crack has visibly grown on the left hand side while the crack tip position remained the same on the right hand side. Meanwhile, as the crack propagated on the left hand side, the COD increased on both sides of the specimen with still a much larger value on the side of the growing crack. After 3,000 cycles, the COD was about 2 voxels on the left and 1.4 voxel on the right hand side. The crack began to grow uniformly after 3,000 cycles.

DISCUSSION

Specimens with different stress state history at their surface have shown different behaviours under fatigue cycling conditions that were designed to induce similar ∆K values.

In the side specimen, the crack tip changes reported in Fig. 2a remain in the range of the crack length measured optically before tomography between 0 and 40,000 cycles of fatigue. Thus, the crack can be considered arrested and reasons for this retardation are likely to be due to crack closure at the tip. The COD maps in Fig. 5a remain fairly unchanged from the initial cycle up to 30,000 cycles with a value at the crack tip that increases very slightly. Only after 40,000 cycles does the COD increase noticeably (both at the tip and crack mouth) as compared to previous maps. This increase of COD results into an increase of the corresponding K I values obtained from COD maps, namely, at the first loading cycle, the SIF ranges from 7.6 to 5.5 MPa√m for, respectively, the long and short crack side then, this range rises up to 10.1 to 11.8 MPa√m at 40,000 cycles. Ogawa [START_REF] Ogawa | Near-threshold fatigue crack growth and crack closure in a nodular cast iron[END_REF] reported values for the opening SIF, K op , that are ranging from 6.5 to 9.5MPa√m for a similar material. The SIF values computed for the arrested crack are thus consistent with a crack that remained closed. Then, noticeable crack growth at 45,000 cycles results into a further larger increase in K I as expected (constant amplitude cycling). Initial crack closure might be attributed to the plastic zone size. Due to the small thickness of the tomography sample, it is possible that a large portion of the crack front is affected by the plastic zone size in plane stress inherited from the larger specimen. Once crack opening has reached a value high enough, the crack propagates at a rate in accordance with the da/dN-∆K curve reported in Ref. [START_REF] Nadot | A study of natural cracks initiated on casting defects by crack front marking[END_REF].

The crack in the centre specimen that has assumingly a plastic zone corresponding to plane strain condition all along its front was observed to propagate immediately when submitted to fatigue cycling again. This suggests that crack closure in plane strain is very small so that when submitted to conditions close to those used in pre-cracking, the crack opens immediately and almost the full ∆K range is available for crack growth. The measured values of K I at maximum load are 12.7 and 8.9 MPa√m, respectively on the growing and arrested crack side for the first loading cycle. Note that the 8.9 MPa√m value is in the range of the K op value in Ref. [START_REF] Ogawa | Near-threshold fatigue crack growth and crack closure in a nodular cast iron[END_REF]. Although plasticity induced crack closure is not expected to be important in plane strain conditions, close examination of the COD maps reveals that the crack seems partially closed on one side (Fig. 5b). The crack geometry observed in Fig. 3b shows that the area where the crack is closed at its tip and remains arrested during 3,000 cycles corresponds to a bifurcation of the crack path. A step of constant height from crack mouth to tip is visible on the right hand side of Fig. 3b. This crack branching could have induced crack retardation and subsequent roughness induced crack closure due to the presence of the asperity. The spatial resolution of the displacements fields along the x and y axis is not accurate enough to observe mixed mode loading occurrence at the asperity. However, computation of SIF along the crack front as in Ref. [START_REF] Rannou | Three dimensional experimental and numerical multiscale analysis of a fatigue crack[END_REF] may be able to catch such mixed mode occurrence at a local scale. After 2,000 cycles, the crack seems fully open under maximum load, i.e. at 257N, as shown on the COD maps in Fig. 5b and the corresponding K I values of 25.9 and 20.3 MPa√m are consistent with rapid crack growth, i.e. at a rate of about 10 -4 mm / cycle as reported in Ref. [START_REF] Nadot | A study of natural cracks initiated on casting defects by crack front marking[END_REF].

CONCLUSIONS

Application of 3D-DIC to crack propagation data obtained with tomography enables for the study of the influence of the loading history on crack growth. Crack under initial plane strain condition was observed to propagate as soon as the cyclic loading starts. However, it was proved sensitive to the roughness of the crack path that may induce crack growth retardation. To understand the roughness influence, the method described in [START_REF] Rannou | Three dimensional experimental and numerical multiscale analysis of a fatigue crack[END_REF] will soon be used to retrieve the SIF values along the crack front and to check if crack asperities induce mixed mode at the crack tip. When submitted to cyclic loading with a similar ∆K range, a crack with mixed plane stress to plane strain condition along its front was observed to remain arrested for more than 40,000 cycles. This retardation is thought to be due to plasticity induced crack closure. The combination of full 3D image analysis to visualize the crack, COD analysis and SIF computation along the crack front is the first step towards a complete understanding of 3D crack propagation knowing the crack history and geometry. Next step would be to model the crack propagation.

Figure 1 :

 1 Figure 1: X-Ray microtomography set up

Figure 2 :

 2 Figure 2: Crack tip position in the (a) side specimen and (b) centre specimen under maximum load at different numbers of cycles

Figure 4 :

 4 Figure 4: Determination of the (c) COD map in the specimen cross-section using the (b) discontinuity in the (a) 3D displacement field U z and subsequent K I computation from the (d) COD profile along the crack growth direction (the arrow indicates the crack growth direction)

Figure 5 :

 5 Figure 5: COD maps at maximum loads for various numbers of cycles in (a) the side specimen and (b) the centre specimen (colormap in voxels)

ACKNOWLEDGEMENTS

This work was carried out in the framework of the project "PROPAVANFIS" supported by the CETIM foundation, which is gratefully acknowledged. The authors are also grateful to ESRF for providing beam time on the ID19 beam line and to all staff members of the line for their help during the experiments.


Paula G Vissio
email: pvissio@gmail.com

Maria J Darias

María P Di

Daniela I Pérez Sirkin

Tomás H Delgadin

L Cal

Cerdá-Reverter P Suarez-Bregua

Fish skin pigmentation in aquaculture: the influence of rearing conditions and its neuroendocrine regulation

Keywords: Fish pigmentation, Chromatophore, Hormones, Aquaculture 16

Skin pigmentation pattern is a species-specific characteristic that depends on the 2 number and the spatial combination of several types of chromatophores. This feature 3 can change during life, for example in the metamorphosis or reproductive cycle, or as a 4 response to biotic and/or abiotic environmental cues (nutrition, UV incidence, 5 surrounding luminosity, and social interactions). Fish skin pigmentation is one of the 6 most important quality criteria dictating the market value of both aquaculture and 7 ornamental species because it serves as an external signal to infer its welfare and the 8 culture conditions used. For that reason, several studies have been conducted aiming to 9 understand the mechanisms underlying fish pigmentation as well as the influence 10 exerted by rearing conditions. In this context, the present review focuses on the current 11 knowledge on endocrine regulation of fish pigmentation as well as on the aquaculture 12 conditions affecting skin coloration. Available information on Iberoamerican fish 13 species cultured is presented.

Introduction 18

Aquaculture contributes significantly to global seafood supply, accounting for 46% of 19 the total production in 2018, and has had the fastest growth rate among major food 20 production sectors for several decades (FAO, 2020a). Today, aquaculture plays an 21 important role in income generation and food and nutrition security, particularly in 22 developing countries (Belton et al., 2018), and promotes local biodiversity and the 23 preservation of cultural traditions. Fish skin pigmentation is one of the most important 24 quality criteria dictating the market value of fish for human consumption and 25 ornamental use (Harpaz and Padowicz, 2007). For instance, colors in food are linked to 26 anticipated quality and are cues that allow consumers to make judgements about 27 desirability (Bjerkeng, 2008). However, aquaculture conditions can negatively affect the 28 natural skin coloration of fish (Lim et al., 2018), thus hampering successful 29 commercialization. Indeed, skin pigmentation in fish is regulated by both external 30 (biotic and abiotic) and internal (genetic, cellular, nervous, and hormonal) factors 31 [START_REF] Pittman | Fantastically plastic: fish larvae equipped for a new world[END_REF]. For that reason, several studies have been conducted aiming to 32 understand the mechanisms underlying fish pigmentation as well as the influence 33 exerted by rearing conditions.

34

Aquaculture in Iberoamerica (IA), the Spanish-and Portuguese-speaking nations of the 35 Americas and Europe, represents 3.05% of the world's global production with nearly 36 3.5 million tons produced in 2018 (FAO, 2020b), and several studies have addressed 37 skin pigmentation in fish species cultivated in these countries. Reared flatfish species 38 (Pleuronectiformes), which are known for their asymmetric pigmentation and their 39 ability to adapt to background color changes by adjusting their ocular-side pigmentation 40 all chromatophores are generated from a common chromatoblast (Bagnara et al., 1979) 89 through progressive fate-restriction processes (Le Douarin and Dupin, 2003;Dupin et 90 al., 2007;Kimura et al., 2014). Different chromatophores have been described in fish, 91 classified into light-absorbing chromatophores (melanophores, erythrophores, 92 xanthophores, and cyanophores) and light-reflecting chromatophores (leucophores and 93 iridophores) (Fujii, 2000;Sugimoto, 2002). While melanophores, iridophores, and 94 xanthophores are widely distributed in teleosts [START_REF] Schartl | What is a 1016 vertebrate pigment cell?[END_REF], leucophores are 95 less abundant [START_REF] Menter | Leucophores and iridophores of Fundulus 895 heteroclitus: biophysical and ultrastructural properties[END_REF]Iga and Matsuno, 1992;[START_REF] Nagao | Distinct interactions of Sox5 and Sox10 in fate 920 specification of pigment cells in medaka and zebrafish[END_REF]. In 96 addition, two cell types of melanophores and xanthophores, differing in size and 97 sequentially appearing during development, have been described in Japanese flounder,

98

Paralichthys olivaceus, and Senegalese sole [START_REF] Seikai | An association of 1021 melanophores appearing at metamorphosis as vehicles of asymmetric skin color formation with 1022 pigment anomalies developed under hatchery conditions in the Japanese flounder, Paralichthys 1023 olivaceus[END_REF][START_REF] Nakamura | Dual appearance of 928 xanthophores, and ontogenetic changes in other pigment cells during early development of Japanese 929 flounder Paralichthys olivaceus[END_REF] 99 Darias et al., 2013a). These two different cell types have been identified as larval and 100 adult melanophores and xanthophores in Japanese flounder [START_REF] Seikai | An association of 1021 melanophores appearing at metamorphosis as vehicles of asymmetric skin color formation with 1022 pigment anomalies developed under hatchery conditions in the Japanese flounder, Paralichthys 1023 olivaceus[END_REF] 101 [START_REF] Nakamura | Dual appearance of 928 xanthophores, and ontogenetic changes in other pigment cells during early development of Japanese 929 flounder Paralichthys olivaceus[END_REF], whereas in the case of Senegalese sole melanophores, the two 102 cell types rather correspond to different morphological stages of newly differentiated 103 and melanized melanophores at post-metamorphosis (Darias et al., 2013a).

104

Melanophores are the most common and studied chromatophores and are responsible 105 for the dorsal pigmentation in vertebrates (Aspengren et al., 2012). Melanophores are 106 dendritic shaped cells that extend their projections, containing the pigmented organelles 107 (melanosomes), almost parallel to the plane of the skin (Fujii, 2000;Nüsslein-Volhard 108 and Singh, 2017). In teleosts' skin, melanophores are mostly found in the dermis, 109 although they can also be observed in the epidermis (Fujii, 2000). et al., 1986).

131

Chromatophores density variation implies both differentiation and apoptosis processes.

132

It is believed that the number of melanophores increases through precursor cell 133 differentiation rather than the division of already differentiated melanophores 134 (Sugimoto et al., 2002). There is evidence that these precursors are neural crest-derived 135 stem cells that have been set aside in distinct niches, such as the ganglia of the 136 peripheral nervous system, the base of the fins, or in deep layers of the dermis, which 137 migrate and differentiate into adult-type pigment cells under specific stimuli (reviewed 138 in Sugimoto et al., 2002;[START_REF] Yamada | Origin of adult-type pigment cells forming the asymmetric 1124 pigment pattern, in Japanese flounder (Paralichthys olivaceus)[END_REF]Darias et al., 2013a;Frohnhöfer et al., 139 2013). This differentiation implies the molecular action of the chromathophore-140 differentiating genes that are also implicated in the regulation of skin pigment 141 concentration. In particular, microphthalmia-associated transcription factor (mitf) is 142 considered the master regulator of melanophore development and controls expression of 143 genes required for melanophore development, including dopachrome tautomerase (dct), 144 tyrosinase (tyr), tyrosinase related peptides (trp 1 and 2) and the receptor tyrosine kinase 145 (c-kit) (Steingrímsson et al., 2004, Darias et al., 2013a[START_REF] Nagao | Distinct interactions of Sox5 and Sox10 in fate 920 specification of pigment cells in medaka and zebrafish[END_REF]. Furthermore, 146 the sodium/potassium/calcium exchanger 5 (slc24a5) is crucial for proper melanin 147 synthesis. The paired box protein 3 (pax3) can promote or inhibit melanogenesis 148 through transcriptional regulation of mitf and cKit, the latter being necessary for 149 melanophore differentiation and responsible for the activation of tyr. Besides, pax3 can 150 also modulate the expression of trp1 and trp2 (see model in Darias et al., 2013a).

151

Although the molecular mechanisms of fish melanophore differentiation have been well 152 characterized, those of the other chromatophores have remained largely unknown 153 [START_REF] Otsuki | Identification of kit-ligand a as the Gene Responsible 958 for the Medaka Pigment Cell Mutant few melanophore[END_REF]. It was demonstrated that Sry-box transcription factor 5 (Sox5) acts 154 antagonistically against Sox10 in the specification of zebrafish chromatophores and in 155 melanophore and iridophore lineages in medaka. However, in this last species, The decrease in chromatophore number occurs by apoptosis stimulated by specific 161 factors (Sugimoto et al., 2002;Sköld et al., 2016) with the old and new actors involved in skin color regulation in fish. In addition, figure 182 1 shows the endocrine and nervous effect over the different chromatophores. In this 183 figure, the lack of studies on the regulation of some pigmentary cells is reflected. More (Grove, 1994;reviewed in Fujii, 2000).

192

Since Fujii and Oshima (1994), Fujii (2000) and Sköld et al. (2016) have reviewed this 193 issue in depth, we will only point out some aspects of this regulation that are relevant 194 for the following sections. It has been proposed that there is a constant rate of firing of 195 noradrenaline (NA) from nerve terminals that increases or decreases depending on 196 different stimuli (Fujii and Oshima, 1994). Other studies have demonstrated that ATP is 197 released as a co-transmitter together with NA (true-transmitter) and that, in the synaptic 198 cleft, it is dephosphorylated to adenosine, which survives longer and reverses NA 199 action, causing re-dispersion of pigment after the cessation of the stimulus (Fujii and 200 Oshima, 1994).

201

Noradrenaline interacts with ɑ-and β-adrenoreceptors, with ɑ2-adrenoreceptor subtype 202 being more effective than ɑ1-adrenoreceptor in producing pigment aggregation, while iridophores (Fujii and Oshima, 1994;reviewed in Fujii 2000;Sköld et al., 2016). The influence of nutrition in fish skin pigmentation has been widely reported; however, 247 fewer studies have dealt with its endocrine regulation. The association between nutrition 248 and pigmentation has been mostly studied in flatfish, which can present a high 249 incidence of pigmentation anomalies under aquaculture conditions, and in fish species 250 owing their skin coloration to dietary carotenoids.

251

In flatfish, larval nutrition has been proved to be essential for proper physiological and 252 morphological transformations occurring during the complex process of metamorphosis, 253 including pigmentation (Hamre et al., 2005;Boglino et al., 2013;Darias et al., 2013b).

254

Several studies have shown higher survival rates and better pigmentation when fish are 255 fed copepods than with any other live prey [START_REF] Seikai | Reduction in occurrence frequency of albinism in juvenile flounder Paralichthys 1018 olivaceus hatchery-reared on wild zooplankton[END_REF][START_REF] Shields | as feed for halibut larvae (Hippoglossus hippoglossus) in terms 1028[END_REF]Wilcox et 256 al., 2006). Several differences in nutrient composition between copepods and live preys 257 such as Artemia or rotifers have been suggested to account to the dissimilarities in the 258 pigmentation process of fish, such as the amount of docosahexaenoic (DHA) and 259 eicosapentaenoic acids (EPA) (and their ratios), polar lipids and amino acids, as well as 260 vitamin A (VA) and carotenoid composition [START_REF] Naess | A sensitive period during first feeding for the determination of pigmentation 916 pattern in Atlantic halibut, Hippoglossus hippoglossus L., juveniles: the role of diet[END_REF]. Kanazawa (1993) 261 suggested that albinism in flatfish resulted from the insufficiency of rhodopsin, the , 2008b;Eslamloo et al., 2015). Despite all the evidence, tank 358 characteristics are often under-considered in aquaculture, and, for instance, the color of 359 the rearing tanks is seldom described in the scientific literature.

360

Background adaptation is widely observed in fish and refers to the ability to modify 361 body color in response to environmental luminosity, as in the case of dark or bright 362 backgrounds. This ability is exploited in aquaculture to improve skin pigmentation. For 363 example, skin darkening in sparids, which negatively affects market value (Matsui et 364 al., 1992;Kolios et al., 1997;Lin et al., 1998;[START_REF] Rotllant | 996 Background colour influence on the stress response in cultured red porgy Pagrus pagrus[END_REF]Booth et al. 2004; 365 [START_REF] Van Der Salm | Differential release of α-melanophore 1082 stimulating hormone isoforms by the pituitary gland of red porgy, Pagrus pagrus[END_REF]Doolan et al., 2007), can be reversed by rearing these species 366 in white tanks (Doolan et al, 2008a, b). However, a white environment has been shown 367 to induce an increased stress response to overcrowding in P. pagrus, which may depend 368 on the size of the fish [START_REF] Rotllant | 996 Background colour influence on the stress response in cultured red porgy Pagrus pagrus[END_REF][START_REF] Van Der Salm | Differential release of α-melanophore 1082 stimulating hormone isoforms by the pituitary gland of red porgy, Pagrus pagrus[END_REF]. In tilapia,

369

Oreochromis mossambicus, white and grey backgrounds induce skin lightening, 370 whereas a black background induces skin darkening and a more stressful response (Van 371 der [START_REF] Van Der Salm | Background adaptation 1085 and water acidification affect pigmentation and stress physiology of tilapia, Oreochromis 1086 mossambicus[END_REF]. In goldfish, red and blue backgrounds are chronically stressful, 372 whereas a white background improves fish growth, but generates a skin color loss 373 (Eslamloo et al., 2015). In Lophiosilurus alexandri dark colored tanks promoted an 374 increase in plasma cortisol levels and a reduction in brightness of the skin, while the use 375 of light colors resulted in paler skin (Costa et al., 2017).

376

As previously mentioned, tank color not only affects fish pigmentation, but can also 377 cause other physiological changes. In several fish species, it has been observed that fish 378 adapted to a white background present better growth performance than those adapted to 379 other background color (Amiya et al. 2005;Karakatsouli et al. 2007;Strand et al. 2007;380 Takahashi et al., 2004;[START_REF] Yamanome | White background reduces the occurrence of staining, 1130 activates melanin-concentrating hormone and promotes somatic growth in barfin flounder[END_REF][START_REF] Pérez Sirkin | Melanin concentrating hormone (MCH) is involved in the regulation of 972 growth hormone in Cichlasoma dimerus (Cichlidae, Teleostei)[END_REF]Eslamloo et al., 381 2015). In part, this could be due to the high contrast between feed and background color 382 that improves the visibility of feed in the tanks (Jentoft et al.,2006;Strand et al. 2007;383 Eslamloo et al., 2015). Besides, white background induces high levels of Mch and,as 384 Mch has been proposed to play an orexigenic role in some species (Takahashi et al.,385 2014; [START_REF] Volkoff | The Neuroendocrine Regulation of Food Intake in Fish: A Review of Current 1100 Knowledge[END_REF], the increase in somatic growth could be interpreted as an increase 386 of food intake. On the other hand, in C. dimerus, it was demonstrated that Mch 387 stimulates Gh release in pituitary cultures, so the increase in fish growth could also be 388 related to this regulation [START_REF] Pérez Sirkin | Melanin concentrating hormone (MCH) is involved in the regulation of 972 growth hormone in Cichlasoma dimerus (Cichlidae, Teleostei)[END_REF].

389

The adaptation to black background results in the dispersion of pigment in 390 melanophores within a few hours, concomitant with an increase in plasma α-Msh levels 391 [START_REF] Mizusawa | Interrelation between 910 melanocyte-stimulating hormone and melanin-concentrating hormone in physiological body color 911 change: roles emerging from barfin flounder Verasper moseri[END_REF]. However, the involvement of this hormone in long-term 392 background adaptation has no consensus in fish (Cal et al., 2017). Despite what would 393 be expected, gilthead sea bream adapted to a white background for 15 days presented an 394 increase in plasma α-Msh levels compared to specimens adapted to grey or black 395 backgrounds [START_REF] Arends | [END_REF]. Similar results were observed in red porgy adapted 396 for one month to white or black backgrounds [START_REF] Rotllant | 996 Background colour influence on the stress response in cultured red porgy Pagrus pagrus[END_REF]. However, it has 397 been hypothesized that the regulation of α-Msh acetylation may be an important 398 mechanism for background adaptation, more than total amounts of α-Msh released into 399 the blood [START_REF] Arends | [END_REF].

400

The pioneering works of [START_REF] Zhu | Elevations of somatolactin in plasma and pituitaries and increased a-MSH cell 1142 activity in red drum exposed to black background and decreased illumination[END_REF], 1997, 1998) and [START_REF] Zhu | Cloning and phylogenetic relationship of 1150 red drum somatolactin cDNA and effects of light on pituitary somatolactin mRNA expression[END_REF] 401 introduced somatolactin (Sl) as a hormone involved in background color adaptation.

402

They suggested that Sl plays a role in background adaptation and possibly regulates 403 pigment movement in the chromatophores of sciaenid fishes. In C. dimerus, the long-404 term exposition to a black background produces an increase in the number and area of 405 SL immunoreactive cells (Cánepa et al., 2006), even from early stages of development 406 (Delgadin et al., 2020). Moreover, growth hormone receptor 1 (GhR1; probably the SL 407 receptor) was detected in the epidermis and dermis from fish scales (Cánepa et al.,408 2012). This receptor showed changes in its transcript level concomitant with changes in 409 melanophores, suggesting plausible evidence for the role of Sl and its receptor in the 410 regulation of chromatophores in C. dimerus (Cánepa et al., 2012). Furthermore, it was 411 determined that medaka larvae with biallelic mutations of the GhR1 receptor failed to 412 adapt to the background, unless at the beginning of development (Delgadin et al., 2020).

Social interactions 420

In many social species, skin pigmentation reflects the social hierarchy; for example, in 421 salmonids, social subordination is associated with skin darkening (Abbott et al., 1985;422 O'Connor et al., 1999;Höglund et al., 2000Höglund et al., , 2002)). Subordinate fish are subjected to 423 chronic stress induced by aggressive acts from dominant fish (Winberg and Lepage, 424 1998;[START_REF] Øverli | Short-term effects of fights for social dominance and the 961 establishment of dominant-subordinate relationships on brain monoamines and cortisol in rainbow 962 trout[END_REF]Höglund et al., 2000Höglund et al., , 2002)). As it was previously mentioned, 425 this leads to a chronic activation of the hypothalamic-pituitary-interrenal axis, and to an 426 increase of α-Msh that induces interrenal cortisol release and skin darkening (Fujii and 427 Oshima, 1986, Höglund et al., 2000). In Astatotilapia burtoni it was demonstrated that 428 yellow males are more aggressive than blue ones (Korzan and Fernald, 2007;Korzan et 429 al., 2008). Later on, it was demonstrated that blue males have higher cortisol levels than 430 yellow ones, indicated by an activation of the melanocortin system in the skin (Dijkstra ). Furthermore, a negative correlation was found between plasma cortisol levels 436 and dominance; fish of lower social hierarchy rank had higher plasma cortisol levels 437 than those in higher rank (Alonso et al., 2011(Alonso et al., , 2012)).

438

It is important to point out that the color of the tank can increase social agonist 439 encounters, besides inducing stress. In Nile tilapia, blue and brown tanks increased this 440 kind of behavior [START_REF] Merighe | Effect of background color on 898 the social stress of Nile tilapia (Oreochromis niloticus)[END_REF], whereas in Arctic char, Salvelinus alpinus, 441 white tanks induced a more aggressive behavior than black ones (Höglund et al., 2002). It is known that chromatophores respond directly to incident light. This "primary color 445 response" can be observed during embryonic and larval stages, when chromatophores 446 are not innervated or under endocrine control, as well as in adulthood regardless of the 447 presence of both regulatory systems (reviewed in Fujii, 2000;[START_REF] Oshima | Direct reception of light by chromatophores of lower vertebrates[END_REF]. In this 448 sense, cone opsin expression has been detected in melanophores (Chen et al., 2013) and 449 erythrophores (Ban et al., 2005;Chen et al., 2013) in Nile tilapia and in iridophores in 450 Neon tetra (Kasai and Oshima, 2006). Melanophores respond to wavelengths between 451 380-580 nm by dispersing melanosomes (Chen et al., 2013), and erythrophores 452 aggregate or disperse pigment depending on exposure to short or middle/long 453 wavelengths, respectively [START_REF] Sato | Direct effects of visible and UVA light on pigment migration in 1010 erythrophores of Nile tilapia[END_REF]Ban et al., 2005;Chen et al., 2013). The 454 photo-response of iridophores depends on light intensity, with the wavelength of 500 455 nm being the most effective one (Kasai and Oshima, 2006). Besides, the photic 456 environment affects fish pigmentation by modulating nervous and endocrine systems.

457

Unfortunately, there are few studies conducted on the impact of different wavelengths 458 on fish pigmentation. For example, adults of red porgy became paler when exposed to 459 blue light compared to individuals exposed to the full spectrum, with no observed 460 changes in melanin content or ɑ-Msh and cortisol levels (Szisch et al., 2002). These 461 authors proposed that these changes in fish coloration could be due to changes in 462 melanosome aggregation produced by the control of the nervous system. In addition, 463 Amano and Takahashi (2009) suggested that, since green light increased somatic 464 growth in barfin flounder, Verasper moseri [START_REF] Yamanome | Green light stimulates somatic 1136 growth in the barfin flounder Verasper moseri[END_REF], mch expression 465 and its secretion could be higher, so the skin color of those animals should be paler.

466

Unfortunately, the effect of green light on skin color was not analyzed in that study.

467

Light intensity has been shown to affect growth, behavior, physiology, and coloration in 468 some fish species, such as Paralichthys woolmani (Benetti, 1997;Venizelos and 469 Benetti, 1999;Han et al., 2005). [START_REF] Santos | The light intensity in growth, 1007 behavior and skin pigmentation of juvenile catfish Lophiosilurus alexandri (Steindachner)[END_REF] showed that light influences food 470 consumption and conversion, behavior, and plasma cortisol levels of Lophiosilurus 471 alexandri juveniles; however, no effect on skin pigmentation was observed.

472

Photoperiod can also induce alterations in skin color, given that melatonin not only acts 473 directly over chromatophores but also modifies other endocrine pathways that affect 474 skin pigmentation. For example, in Neon tetra, the red and brown colors produced by 475 erythrophores and melanophores were found to be markedly reduced at night, 476 suggesting the regulation of coloration by an endogenous circadian rhythm (Lythgoe 477 and Shand, 1983). Differences in skin pigmentation due to photoperiod were also chromatophores' physiology and thus modify fish pigment. The direct impact of some of 496 these stimuli on chromatophores is poorly studied, although, for example, it is accepted 497 that high temperatures aggregate chromatosomes, while lower temperatures disperse 498 them (Fujii and Oshima, 1994). In any case, these factors are generally considered as 499 stressors, promoting an acute or chronic stress response depending on the duration of 500 the stimuli (reviewed in Wendelaar Bonga, 1997). Promotes red and yellow skin coloration 45--48 --Weak aggregating effect on melanophores 45,46 .

--Dispersion of xanthosomes in Nile tilapia and the rose bitterling 46 , and of erythrosomes in Nile tilapia, swordtails and paradise gobies 47 .

Similar effects in two--spotted gobies and the mudsucker 48,49 Melatonin (Mt) --Pineal gland 50 --endocrine Seven transmembrane-domain--G--protein-coupled family: Mt1

Mt2 and Mel1c 51

Skin paling and transparency 49,52 --Aggregation of melanosomes 52 and erythrosomes 49,53 .

--Dispersion of erythrosomes and xanthosomes 49 .

--Increase between platelets spacing in iridophores 54,55 .

Thyroid hormones (Th) --Thyroid follicles, local conversion to T3 56--58 --endocrine transcription factors from the family of type--2 nuclear receptors: ɑ and β TR 58

Skin paling 59--62 --Prevent melanin synthesis 59,60 --Change the balance of melanophore stem cell proliferation/ differentiation in zebrafish 61,62 and Japanese flounder 63

--Decrease melanophore and increase xanthophore number Danio albolineatus

  vary through two different mechanisms. On the one hand, the 114 physiological color changes, which are rapid and transient, are produced by the motility 115 of pigment vesicles (chromatosomes) or by the movement of reflective structures within 116 their cells. On the other hand, the morphological color changes, which occur within 117 days and/or weeks, involve variations in skin pigment concentration or the density and 118 distribution of chromatophores in the integument (Leclercq et al., 2010). 119 The translocation of chromatosomes, which are characteristic of physiological color 120 changes, is coordinated by the microtubule and actin filament architecture of the 121 cytoskeleton and the associated motor proteins. When light-absorbing chromatophores 122 receive an input (such as hormones, neurotransmitters or environmental cues) that 123 produces the aggregation or dispersion of chromatosomes, the specific molecular motor 124 hauls pigment granules back toward the nucleus or to the cell periphery (Ligon and 125 McCartney, 2016). In general, decreases in cAMP levels and/or increases in Ca 2+ levels 126 within chromatophores trigger aggregation responses, while the opposite changes in 127 cAMP or Ca 2+ levels induce dispersion responses (reviewed in Fujii, 2000). Regarding 128 iridophores, which contain thin reflecting platelets in their cytoplasm, under certain 129 inputs they simultaneously change the distance between adjoining platelets, leading to a 130 shift in the spectral reflectance of the skin (Kasukawa

156

  xanthophores and leucophores developed from a shared progenitor Pax7a positive. This 157 progenitor differentiates in xantophore or leucophore depending on Sox5/Sox10. While 158 Sox5/10 promotes xanthophore specification, it represses leucophore formation (Kimura 159 et al., 2014; Nagao et al., 2018).

  160

  is mainly controlled by the sympathetic 189 postganglionic system. Chromatic information is captured by the eyes, processed in the 190 optic tectum and partly at the level of the motoneurons in the medulla, and sent to 191 chromatophores via direct nervous connections

  203 β2-adrenoreceptor subtype induces pigment dispersion. Concerning NA effects over 204 chromatosomes, its release induces aggregation of these vesicles in melanophores, 205 xanthophores and erythrophores, but disperses the light-scattering organelles in 206 leucophores, and also produces a change in the arrangement of reflecting platelets in 207

208

  [START_REF] Wendelaar Bonga | The stress response in fish[END_REF]. Furthermore, stressor-induced cortisol production has been associated 233 with disruption of the gut microbiome in fish(Uren Webster et al., 2020). Considering 234 the major role of the gut microbiome in the regulation of the physiology of the 235 organism, including the modulation of neuronal and endocrine pathways (Lerner et al., 236 2017), and the recent association found between the pseudo-albino phenotype and gut 237 microbiome modification in Senegalese sole (Pinto et al., 2019), deeper research 238 towards deciphering the molecular mechanisms and cellular processes of skin 239 pigmentation regulated by the gut microbiome and their link with other biological 240 processes will undoubtedly shed light into better understanding the intricate and 241 interlocked processes of physiological regulation in fish. 242 Finally, as more than one stressor can be present at the same time, different effects on 243 skin pigmentation can be observed depending on the hormonal response to each

  262production of which depends on VA, DHA and phospholipids, necessary to the 263 formation of melanin. According to this author, feeding Japanese flounder larvae fed a 264 diet deficient in those nutrients during the formation of the retina (at around 10 days 265 post hatching-dph) hampers the production of rhodopsin in the retina. The absence of 266 rhodopsin prevents the visual transmission from the retina to the central nervous system, 267 then the production of Msh is not triggered resulting in the interruption of black 268 pigment formation(Kanazawa, 1993). Copepods contain 5 times higher DHA content 269 than Artemia(Hamre et al., 2002), thus the involvement of DHA in vision development 270 and its importance to stimulate melanin synthesis might be the reason behind the 271 importance of DHA in pigmentation. Copepods also present higher levels of EPA.272Adequate levels of DHA and EPA and their ratios have shown to be necessary for the 273 correct development of skin pigmentation of turbot[START_REF] Reitan | Influence of lipid composition of live feed on growth, 987 survival and pigmentation of turbot larvae[END_REF][START_REF] Villalta | Arachidonic acid enriched live prey induces albinism in 1093 Senegal sole (Solea senegalensis) larvae[END_REF] 274Kanazawa, 1995), common sole, Solea solea (Heatch and Moore, 1997), Atlantic 275 halibut, Hipoglossus hipoglossus(Hamre et al., 2005), and California halibut,276Paralichthys californicus[START_REF] Vizcaíno-Ochoa | The effect of dietary 1096 docosahexaenoic acid (DHA) on growth, survival and pigmentation of California halibut Paralichthys 1097 californicus larvae (Ayres, 1810)[END_REF]. Besides, the lower iodine 277 content in Artemia compared to copepods has been suggested to decrease the level of 278 thyroid hormone (Th) in fish larvae(Hamre et al., 2005), which could in turn interfere 279 in the metamorphosis process, including pigmentation (Inui andMiwa, 2012; Wang et 280 al., 2019). VA also influences the fate of chromatophores in flatfish, high doses of retinoic acid stimulating the development of chromatophores in the blind side of flatfish 282[START_REF] Miwa | Retinoic acid stimulates development of adult-type chromatophores in the 901 flounder[END_REF]. It has been suggested that interactions between VA and 283 fatty acids, as well as between VA and Th at the nuclear receptor level are key in the 284 stimulation of normal pigmentation(Hamre et al., 2005). 285 Adequate dietary arachidonic acid (ARA) content has also been demonstrated to be 286 important for proper skin pigmentation of several flatfish species such as Yellowtail 287 flounder, Limanda ferruginea (Copeman and Parrish, 2002), common sole (Lund et al., 288 2008), turbot (Estévez et al., 1999), Japanese flounder (Estévez et al., 2001), Atlantic 289 halibut (Hamre et al., 2007) and Senegalese sole (Villalta et al., 2005; Darias et al., 290 2013b; Boglino et al., 2014). Pre-and pro-metamorphosis are the sensitive periods 291 during which nutrition exerts its greatest influence on pigmentation, coinciding with the 292 time in which chromatoblast differentiation takes place towards the adult type 293 chromatophores (Bolker et al., 2005; Darias et al., 2013b). Senegalese sole larvae fed 294 with high levels of ARA becoming pseudo-albinos at later stages developed 295 pigmentation in the same way as future normally pigmented specimens, but once 296 metamorphosed, the future pseudo-albinos began to show different relative proportions, 297 allocation patterns, shapes and sizes of skin chromatophores that progressively 298 disappeared during post-metamorphosis (Darias et al., 2013b). This process was mainly 299 attributed to the down-regulation of trp1 and slc24a5, which prevented melanin 300 production, and the involvement of pax3, mitf and asip1 (agouti signaling protein) in the 301 developmental disruption of the new post-metamorphic populations of melanophores, 302 xanthophores and iridophores (Darias et al., 2013b). Melanophores in pseudo-albino 303 specimens were less abundant and not so aggregated in patches as they were in normally 304 pigmented ones, whereas their shape differed (round vs. dendrite-like shape) suggesting 305 their inability to disperse melanin (Darias et al., 2013b). Besides, high amounts of 306 dietary ARA can produce imbalances in the relative content of EPA and DHA (Moren 307 et al., 2011), which in turn modify the relative concentrations of prostaglandin E of the 308 2 (ARA-derived) and 3 (EPA-derived) series (Boglino et al., 2014). In fact, EPA and 309 DHA compete as substrates for cyclo-and lipoxygenases, which are involved in 310 prostaglandin biosynthesis pathways. Prostaglandin E2 (PEG2) and PEG3 are potent 311 regulators of metabolism with opposing effects (Bell and Sargent, 2003); thus, the 312 balance in their synthesis from both series is dependent on a balanced dietary intake of 313 both ARA and EPA (Hamreet al., 2005). ARA-induced abnormally pigmented 314 individuals have shown to present higher levels of PGE2 than normally pigmented fish 315 fed with a control diet in both Senegalese sole(Villalta et al., 2005, Boglino et al., 316 2014) and common sole(Lund et al., 2010). Further, pseudo-albino specimens fed a 317 high ARA content diet displayed higher PGE2 concentrations than normally pigmented 318 fish fed the same diet(Boglino et al., 2014). In Senegalese sole, high dietary ARA 319 levels and altered PGE2 concentrations not only affected the pigmentation success, but 320 also disrupted the process of head remodeling during metamorphosis (Boglino et al., 321 2013).322Many fish species owe their bright coloration to carotenoids, which are the predominant 323 pigments in xanthophores and erythrophores. Fish are not able to biosynthesize 324 carotenoids de novo, and thus must obtain them from the diet(Bjerkeng, 2008). In 325 aquaculture, several cultured fish species require carotenoid supplementation in order to 326 avoid skin paleness(Bjerkeng, 2008). The effect of carotenoids on the endocrine system 327 as well as their mechanisms of action remain to be elucidated (De Carvalho and 328 Caramujo, 2017). However, it is known that carotenoid deposition in the skin is induced 329 during the breeding season in many fish species, and gonadal hormones such as 17ß-330 estradiol and 11-ketotestosterone have shown to play a role in carotenoid-based nuptial 331 coloration (reviewed inLeclercq et al., 2010; Lim et al., 2018).332Astaxanthin is the main carotenoid used in aquaculture feeds and is either obtained from 333 chemical synthesis or from natural resources such as algae, fungi, yeast and bacteria, 334(Lim et al., 2018). Besides a source of pinkish-red pigments, astaxanthin is known to 335 improve survival, growth performance, reproductive capacity, stress tolerance, disease 336 resistance and immune related gene expression (Lim et al., 2018). Several studies have 337 analyzed the effect of dietary astaxanthin on skin coloration of cultured fish species for 338 human consumption, such as Atlantic salmon, Salmo salar, rainbow trout, 339 Oncorhynchus mykiss, red porgy, gilthead sea bream, red sea bream, Pagrus major, 340 Japanese flounder or Australasian snapper, Pagrus auratus, as well as in ornamental 341 species, such as goldfish, Carassius auratus, kissing gourami, Helostoma temminckii, 342 false clownfish, Amphiprion ocellaris or koi carp, Cyprinus carpio, among others 343 (reviewed in Lim et al., 2018). Besides, other carotenoid sources have been also 344 assessed to enhance fish skin coloration, as for example the fucoxanthin-rich microalga 345 Phaeodactylum tricornutum (gilthead sea bream; Ribeiro et al., 2017), China rose 346 petals, Hibiscus rosa-sinensis (goldfish; Sinha and Asimi, 2007), annatto, Bixa orellana 347 (goldfish; Fries et al., 2014), sea lettuces Ulva rigida and Ulva lactuca (Nile tilapia; 348 Valente et al., 2016) or Spirulina sp. (yellow tail cichlid, Pseudotropheus acei; Guroy et 349 al., 2012), among others.

  rearing tanks are an important issue to consider in aquaculture,353since it has been demonstrated that they can induce stress(Ishibashi et al., 2013), affect 354 growth and survival (Martinez-Cardenas and Purser, 2015; Wang et al., 2017), induce 355 skeletal anomalies (Cobcroft and Battaglene, 2009), and alter fish behavior (Höglund et 356 al., 2002; Cobcroft and Battaglene, 2009) and skin pigmentation (van der Salm et al., 357 2005; Doolan et al.

413

  SL has been shown to be involved in different physiological processes, including 414 reproduction, stress responses, Ca2+ homeostasis, acid-base balance, growth, 415 metabolism, and immune responses (reviewed in Kawauchi et al., 2009); therefore, 416 changes in tank color can influence the general physiology in different ways, thus 417 affecting fish welfare.418 419

  dimerus, body color pattern is associated with different social status. A relation 433 between color and dominance was observed in territorial individuals, which had bright 434 body color patterns, while non-territorial individuals were opaque grey (Alonso et al., 435 2011

478

  observed in Japanese flounder after metamorphosis when comparing the effects of 479 continuous 24 h illumination (LL) to natural light conditions (15 h light: 9 h dark, LD) 480 during larval development. Itoh et al. (2012) found that larvae reared in LL presented 481 paler skin color, and a higher ratio of abnormal pigmentation after metamorphosis. 482 Moreover, LL conditions decreased tyrosine hydroxylase-1 (th1) in dopaminergic 483 neurons, but increased ɑ-msh levels in melanothrophs with no changes in mch 484 expression levels in the lateral tuberal nucleus (NLT). Authors concluded that there 485 could be an accumulation of ɑ-Msh in the melanothrophs because of the inhibitory 486 action of Mch over its protein secretion, causing a pale skin color. In another study, 487 Ginés et al. (2004) found that the skin luminosity of gilthead sea bream was higher 488 under the longer the photoperiod. Similar results were obtained by Lyon and Baker 489 (1993) in rainbow trout, who also described that Mch secretion reached a peak during 490 the light period and then, it gradually declined before night, when the lowest 491 concentrations were observed. These hormone variations were directly related to animal 492 skin paleness. 493 Other factors such as handling, crowding, transport, hydrostatic pressure, and variations 494 in temperature, oxygen, and salinity, can affect either directly or indirectly 495

  pattern in fish is species-specific and is given by the number and 504 spatial combination of several types of chromatophores. Research efforts have been 505 made to improve understanding of the underlying endocrine regulation of skin 506 pigmentation. In particular, recent studies have identified other actors besides the classic 507 color change regulators Mch and Msh, such as Sl, Asip, and Th, that seem to play an 508 important role in the regulation of pigmentation. In this sense, more studies are needed 509 to understand how these factors interact in a coordinated way to regulate skin color. 510 Furthermore, considering that most studies on the physiology of pigment cells focus on 511 melanophores and that some studies have demonstrated that cellular communication 512 between different types of chromatophores is essential in the pigmentation patterning 513 process, further research on the regulatory factors of all types of chromatophores and 514 the interactions among them is essential to understand the intricate mechanisms of skin 515 pigmentation as well as to identify the origin and the causes leading to pigmentation 516 disorders. Furthermore, as chromatophores derive from NCC, pigmentation anomalies 517 could be the visible sign of more complex physiological disruptions. 518 Skin pigmentation is one of the most important quality criteria dictating the market 519 value of fish for both human consumption and ornamental use. Rearing conditions such 520 as nutrition, tank coloration, UV incidence, surrounding luminosity, or social 521 interactions can negatively affect the natural skin coloration. In this sense, further 522 studies are needed to identify pigmentation-related endocrine factors that are being 523 modulated when fish are reared under suboptimal conditions. This knowledge will also 524 be useful to better understand the impact of rearing conditions on other biological 525 processes, as many endocrine signals affecting pigmentation are additionally regulating 526 processes such as growth, reproduction, or nutrition, among others. In this sense, skin 527 pigmentation could be considered an indicator of fish well-being. 528 New insights on the influence of stress in gut microbiome modulation and on the role of 529 gut microbiome in the regulation of skin pigmentation reinforces the need for a better 530 understanding of the influence of environmental conditions. Taken together, research on 531 the endocrine factors affecting pigmentation, the communication among different types 532 of chromatophores, the influence of nutrition and abiotic factor in the modulation of 533 these endocrine signals, and the role of gut microbiome in the regulation of these 534 physiological processes could contribute to identify the best rearing conditions for 535 species presenting pigmentation disorders and hence to improve their commercial

174 hormonal mechanism for skin color regulation (reviewed in Bertolesi et al., 2019). Two 175 hormones with opposite effects, skin lightening and darkening, have been proposed as 176 the main morphological and physiological color change regulators: the melanin-

  . Interestingly, xanthophores can 162 eliminate the surrounding melanophores, and vice versa, in zebrafish(Nakamasu et al.,

	163
	164	2009), and similarly, xanthophores in the skin of Senegalese sole pseudo-albinos
	165	seemed to be responsible for the degeneration of melanophores (Darias et al., 2013b).
	166	Besides, variation in melanophore morphology, which mainly implies variation in
	167	dendritic process elongation, has been described as another type of morphological color
	168	change (reviewed in Sugimoto et al., 2002).
	169	Since morphological color changes are preceded by physiological color changes, similar
	170	control mechanisms have been proposed to function both in the motile responses and in
	171	the chromatophore densities (reviewed in Sugimoto, 2002).
	172
	173	3.2 Hormone regulators
		Unlike other vertebrate taxa, it is generally accepted that teleost fish present a dual-
	177
	178	concentrating hormone (Mch) and the melanophore-stimulating hormone (Msh),
	179	derived from precursor Proopiomelanocortin. However, skin color regulation is more
	180	complex, and other regulatory factors have been identified in studies performed on the
	181	regulation over other chromatophores cells. Table 1 summarizes this complex scenario

https://doi.org/10.1016/j.aquaculture.2005.03.016

Academic Press, pp. 415-439. https://doi.org/10.1016/B978-012529650-2/50031-7

Acknowledgements 718 Agrár. 35, 3401-3414. https://doi.org/10.5433/1679-0359.2014v35n6p3401

This work was carried out with the collaboration of Red CYTED LARVAplus 540 (117RT0521) and was supported by Universidad de Buenos Aires (grant number: 541 20020160100110BA to PV), y 542 Técnicas (CONICET) (PIP 2014-2016:11220130100501CO to PV).

Author contribution: PGV, MJD, MPDY, and DIPS contributed equally to the design 545 and writing of the manuscript. THD contributed to the revision of the manuscript. All 546 authors approved the final version of the manuscript.

Bertolesi, G.E., Zhang, J.Z., McFarlane, S., 2019. Plasticity for colour adaptation in vertebrates explained 593 by the evolution of the genes pomc, pmch and pmchl. Pigment Cell Melanoma Res. 32, 510-527. 594 https://doi.org/10.1111/pcmr.12776 595 Bjerkeng, B., 2008. Carotenoids in aquaculture: fish and crustaceans, in: Britton, G., Liaaen-Jensen, S.,

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Pfander, H. (Eds.), Carotenoids. Birkhäuser,Basel, 597 Blanton, M.L., Specker, J.L., 2007. The hypothalamic-pituitary-thyroid (HPT)

601

High dietary arachidonic acid levels affect the process of eye migration and head shape in pseudo-

602

albino Senegalese sole Solea senegalensis early juveniles. J. Fish Biol. 83, 1302-1320.

603

https://doi.org/10.1111/jfb.12230 604 Boglino, A., Wishkerman, A., Darias, M.J., de la Iglesia, P., Andree, K.B., Gisbert, E., Estévez, A., 2014.

605

Senegalese sole (Solea senegalensis) metamorphic larvae are more sensitive to pseudo-albinism 606 induced by high dietary arachidonic acid levels than post-metamorphic larvae.