Miriam Rodríguez-Vázquez

Jennifer Falconi

Lisa Heron-Milhavet

Patrice Lassus

Charles Géminard

Alexandre Djiane
email: alexandre.djiane@inserm.fr

Fat body

Keywords: Glycolysis, inter-organ communication, adipose tissue, muscle wasting, Drosophila

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INTRODUCTION

In animals, complex exchanges between the different organs are central to their physiology and to their harmonious growth. In particular, coping with intermittent nutritional resources or developmentally evolving metabolic needs during growth and metamorphosis, reserve organs allow the storage and subsequent release of energy and nutrients when needed.

The fat body of Drosophila, or adipose tissue, represents the main organ for storing energy and nutrients in the form of sugars (glycogen), proteins, and lipids. These reserves are stored or released in response to external (dietary) and internal (hormones and cytokines) stimuli. In case of excess energy, high Insulin pathway in adipocytes promotes the storage of circulating carbohydrates (glucose or trehalose), as glycogen or lipids. Lipids stored in fat body cells come either from dietary lipids absorbed by the gut and transported to the adipocytes, or are synthesized within adipocytes through neo-lipogenesis. Core metabolism links glycolysis, the mitochondrial tricarboxylic acid (TCA) cycle, and neo-lipogenesis: pyruvate, the final product of glycolysis, enters the mitochondrion and the TCA cycle to produce citrate that will serve as precursor to produce fatty acids which are then incorporated into lipids. Alterations of the TCA cycle as observed in Seipin mutants [START_REF] Ding | Seipin regulates lipid homeostasis by ensuring calcium-dependent mitochondrial metabolism[END_REF] or in more recent studies tackling the function of mitochondrial activity by analyzing TFAM knock-down [START_REF] Sriskanthadevan-Pirahas | Adipose mitochondrial metabolism controls body growth by modulating systemic cytokine and insulin signaling[END_REF], show that lowering mitochondrial and TCA cycle activities results in lipid droplets and lipid storage shrinkage.

During larval stages, nutrient sensing and mTOR pathway regulation in adipocytes controls organismal growth [START_REF] Colombani | A nutrient sensor mechanism controls Drosophila growth[END_REF][START_REF] Géminard | Remote control of insulin secretion by fat cells in Drosophila[END_REF]. The evolutionary highly conserved mTOR pathway represents a key cellular metabolism integrator and regulator [START_REF] Bjedov | The Target of Rapamycin Signalling Pathway in Ageing and Lifespan Regulation[END_REF]. The mTor kinase, together with co-factors including Raptor, forms the TORC1 complex which then phosphorylates a wide array of substrates leading to metabolic adaptation (here increased metabolism in high aa availability; [START_REF] Bjedov | The Target of Rapamycin Signalling Pathway in Ageing and Lifespan Regulation[END_REF]). More specifically, TORC1 phosphorylates and activates Ribosomal protein S6 kinase (S6k) thus promoting ribosomal activity [START_REF] Saitoh | Regulation of an activated S6 kinase 1 variant reveals a novel mammalian target of rapamycin phosphorylation site[END_REF]. TORC1 also phosphorylates several transcription factors, but in Drosophila most of the transcriptional program downstream of the mTOR pathway is controlled by REPTOR.

TORC1 phosphorylates and inactivates the transcriptional co-activator REPTOR, preventing it from entering the nucleus and to associate with its transcriptional partner REPTOR-BP [START_REF] Tiebe | REPTOR and REPTOR-BP Regulate Organismal Metabolism and Transcription Downstream of TORC1[END_REF].

Besides its reserve function, the fat body of Drosophila constitutes thus a central endocrine organ.

In response to changes in their metabolism, fat body cells send different adipokines which will impact many organs and control remotely general organismal metabolism and growth [START_REF] Ahmad | Regulation of insulin and adipokinetic hormone/glucagon production in flies[END_REF][START_REF] Meschi | Adipokine and fat body in flies: Connecting organs[END_REF]. In particular, many adipokines control the release of insulin (insulin like peptides dilp2 and dilp5) from the insulin producing cells (IPCs) located in the larval brain [START_REF] Ahmad | Regulation of insulin and adipokinetic hormone/glucagon production in flies[END_REF][START_REF] Meschi | Adipokine and fat body in flies: Connecting organs[END_REF]. In response varying nutritional inputs and changes preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. Growth Blocking Peptides 1&2/Gbp1&2; upd2, and CCHamide-2/CCHa-2) or preventing (egr/TNFa) dilps production and secretion from IPCs [START_REF] Agrawal | The Drosophila TNF Eiger Is an Adipokine that Acts on Insulin-Producing Cells to Mediate Nutrient Response[END_REF][START_REF] Delanoue | Drosophila insulin release is triggered by adipose Stunted ligand to brain Methuselah receptor[END_REF][START_REF] Ingaramo | Fat Body p53 Regulates Systemic Insulin Signaling and Autophagy under Nutrient Stress via Drosophila Upd2 Repression[END_REF][START_REF] Koyama | Growth-Blocking Peptides As Nutrition-Sensitive Signals for Insulin Secretion and Body Size Regulation[END_REF][START_REF] Rajan | Drosophila cytokine unpaired 2 regulates physiological homeostasis by remotely controlling insulin secretion[END_REF][START_REF] Sano | The Nutrient-Responsive Hormone CCHamide-2 Controls Growth by Regulating Insulin-like Peptides in the Brain of Drosophila melanogaster[END_REF].

Interestingly, the control of Insulin pathway activity is not restricted to the regulation of by dilps secretion, but is also achieved by extracellular proteins such as ImpL2, dALS, and Sdr [START_REF] Alic | Lifespan extension by increased expression of the Drosophila homologue of the IGFBP7 tumour suppressor[END_REF][START_REF] Arquier | Drosophila ALS regulates growth and metabolism through functional interaction with insulin-like peptides[END_REF][START_REF] Okamoto | A secreted decoy of InR antagonizes insulin/IGF signaling to restrict body growth in Drosophila[END_REF]. These proteins can trap dilps in the hemolymph and are thought to act mainly as inhibitors. Upon reception by target cells, dilps activate the Insulin pathway to favor anabolic processes and the implementation of growth or repair programs. In Drosophila muscle cells, Insulin signaling play a role equivalent to IGF-1 signaling in mammals [START_REF] Yoshida | Mechanisms of IGF-1-Mediated Regulation of Skeletal Muscle Hypertrophy and Atrophy[END_REF] and prevent, at least in adults, muscle ageing and the formation of detrimental protein aggregates [START_REF] Demontis | FOXO/4E-BP signaling in Drosophila muscles regulates organismwide proteostasis during aging[END_REF][START_REF] Wessells | Insulin regulation of heart function in aging fruit flies[END_REF].

In this study we analyzed the role of glycolysis knock down on fat body biology and on the development of Drosophila larvae. Glycolysis shut down in adipocytes triggered a dramatic lipid reserve shrinkage and fat body atrophy. Fat body atrophy was also accompanied by a delay in pupariation, and in distant body wall muscle disorganization, suggesting the remote control by fat body cells of muscle cells integrity. Molecularly we further show that glycolysis shut-down in adipocytes resulted in low mTor activity and triggered the release of the egr/TNF-a adipokine, and the production of the dilps inhibitor ImpL2. We propose that the combined actions of increased egr/TNF-a and low Insulin are responsible, at least in part, for the muscle wasting triggered by distant fat body atrophy.

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RESULTS

Impaired glycolysis in adipose tissue leads to adipose tissue atrophy

To investigate the role of adipose tissue metabolism on the general development of the fly, we targeted glycolysis. Using lpp-Gal4 to drive inducible RNAi in fat body cells, we probed the function of different glycolysis enzymes (Fig. 1A) on the size and morphology of adipose tissue cells.

Knocking down the expression of the early enzymes of glycolysis did not have any significant effects, probably due to potential redundancy in enzyme function, or to existing alternative pathways such as the pentose shunt (Fig. 1A). However, knocking down the enzymes controlling the later steps of glycolysis, such Pglym78 or Enolase (Eno) resulted in smaller and atrophic fat body. First, Pglym78-RNAi or Eno-RNAi treated cells were significantly smaller than cells expressing a control RNAi (against the gene white). Average cell size were 55% and 75% smaller than controls, respectively (Fig. 1B&C). The knock down of the upper glycolysis enzyme Pgk had a weaker effect under these conditions (Fig. 1B&C). Then, both Pglym78-RNAi and Eno-RNAi cells had fewer and smaller lipid droplets than controls as monitored by bodipy stain (Fig. 1B&D). Similar results obtained knocking down two different steps of the glycolysis using two different RNAi constructs strongly argue that the effects observed were not unspecific effects of the tools used. Furthermore, similar results were obtained with ppl-Gal4, a second adipose-specific driver showing that upon glycolysis shut down, fat body cells undergo strong atrophy with smaller lipid reserves (Supplemental Fig. S1A-B). In lpp-Gal4> Pglym78-RNAi animals, carbohydrates reserves were not affected however, as shown by constant glycogen reserves in fat body cells and similar circulating sugar levels (trehalose; Supplemental Fig. S1C-D).

Pyruvate, the final product of glycolysis, represents an important source for Acetyl-CoA which fuels the mitochondrial TCA cycle. Citrate exiting the TCA cycle serves as precursor for fatty acid and lipid genesis. In order to support the link between glycolysis alteration and lipid droplets atrophy in our model, we then tested whether supplementing animals with pyruvate would bypass the effects of glycolysis knock down. Growing lpp-Gal4> Pglym78-RNAi larvae on a feeding media supplemented with 1% pyruvate restored lipid droplets content, albeit not completely (Fig. 1B-D). These results support the model in which lipid depletion and lipid droplets atrophy upon lower glycolysis knock down are a consequence of reduced pyruvate availability, for instance for TCA cycle derived lipogenesis, consistent with earlier reports showing that Seipin/SERCA knock down affected lipid reserves in Drosophila, in part through impaired glycolysis [START_REF] Ding | Seipin regulates lipid homeostasis by ensuring calcium-dependent mitochondrial metabolism[END_REF]. However, we cannot exclude that the supplied pyruvate could affect alternative metabolic pathways, or other organs important for fatty acids and lipids synthesis such as oenocytes, ultimately resulting in the sparing of lipid reserves in the larval fat body.

Adipose tissue atrophy promotes body wall muscle atrophy and disorganization

We then questioned what were the effects on larval growth of the adipose tissue atrophy after glycolysis shut down. Lpp-Gal4> Pglym78-RNAi larvae showed a developmental delay of around 48h as compared to controls, and larvae entered pupariation at day 7 rather than day 5 after egg laying (ael; Fig. 2A and Supplemental Fig. S2A). However, we did not observe dramatic changes in larval brain size or growth either at 5 days ael (compared to white RNAi controls), consistent with the existence of brain protective mechanisms under nutritional or energetic stress [START_REF] Cheng | Anaplastic lymphoma kinase spares organ growth during nutrient restriction in Drosophila[END_REF].

There was however a small reduction in the size of wing imaginal discs (Fig. 2B). Similar pupariation delay was also observed in Pgk-RNAi animals (Fig. 2A). Interestingly, in these animals the fat atrophy was much more subtle and we did not detect significant lipid droplets shrinkage (Fig. 1B-D), suggesting that either developmental delay and fat atrophy might be two independent processes, or that even subtle effects on lipid content might be sufficient to delay pupariation. Furthermore, when we compared similarly delayed Pgk-RNAi and Pglym78-RNAi animals at 6 days ael, we did not detect any difference in the size of brains and of wing discs, suggesting that fat atrophy does not directly control brain and discs growth in this model (Supplemental Fig. S2B). Animals depleted for Eno had the strongest developmental delay and never entered pupariation (Fig. 2A).

But while we did not detect alterations in the growth of brain and discs of Pglym78 depleted animals, body wall muscles were affected. Whole body wall muscle preparation, showed that upon Pglym78 or Eno knock-down in the larval fat body, several muscles showed abnormal morphology and appeared shorter and condensed, as evidenced by bright F-actin staining (Fig. 3A). Furthermore, the archetypical arrangements of muscles under the cuticle, either longitudinally or obliquely within segments, was perturbed in the affected animals. Focusing more specifically on the large VL3 and VL4 muscles located just laterally to the ventral midline in each larval segment, F-actin staining were weaker than in controls, suggesting a reduction in muscle fiber density (Fig. 3A). These different defects were not a consequence of poor handling of the larvae and dissection artifacts, since we could also document condensed muscle material (bright dots) using a transgenic line carrying a live GFP muscle marker (Zasp66::GFP protein trap fusion; Fig. 3A&B). These Zasp66::GFP features were reminiscent of the condensed severed muscles and of the dimmer fibers observed by F-actin staining on dissected and fixed cuticles.

Given the disorganization of body wall muscles, we next wondered whether muscular proteins could be found in the hemolymph. Collecting hemolymph from animals by gentle bleeding (see Materials & Methods), we could detect by western-blot analyses a-Actinin, and Zasp66::GFP in the blood of lpp-Gal4> Pglym78-RNAi larvae. These proteins are highly enriched in muscles, and were not found in the blood of control animals, (lpp-Gal4> w-RNAi or Pgk-RNAi for monitoring at 5 days or 6 days ael respectively), confirming that following fat body atrophy, muscle proteins were released in the circulation (Fig. 3C). Importantly, a-Actinin was not detectable by western-blot analyses on whole protein extracts from dissected fat body tissues (Supplemental Fig. S3A), further supporting the muscle origin of the muscular proteins detected in the hemolymph. It is noteworthy that the proteins detected were not due to a contamination by circulating cells (hemocytes) or large cellular debris, since they were still detected in the supernatant after high speed centrifugation of the collected hemolymph (Supplemental Fig. S3B). Together, these results show that impairing glycolysis in the adipose cells in the fat body of Drosophila larvae triggers 1) adipocyte atrophy, and 2) a nonautonomous distant muscle wasting, for which the release of a-Actinin in the blood could serve as a marker.

The mTOR effector REPTOR mediates fat body atrophy and distant muscle wasting

The mTOR kinase and the TORC1 complex, represent one of the major integrator and key regulator of cellular metabolism [START_REF] Bjedov | The Target of Rapamycin Signalling Pathway in Ageing and Lifespan Regulation[END_REF]. Upon glycolysis enzymes knock-down, we observed low TORC1 activity. First, using qPCR, we observed a major down-regulation of in the expression at the RNA level of the mTor kinase and of the key TORC1 activator Rheb [START_REF] Garami | Insulin activation of Rheb, a mediator of mTOR/S6K/4E-BP signaling, is inhibited by TSC1 and 2[END_REF][START_REF] Saucedo | Rheb promotes cell growth as a component of the insulin/TOR signalling network[END_REF][START_REF] Stocker | Rheb is an essential regulator of S6K in controlling cell growth in Drosophila[END_REF][START_REF] Zhang | Rheb is a direct target of the tuberous sclerosis tumour suppressor proteins[END_REF] in lpp-Gal4> Pglym78-RNAi fat body cells, suggesting that these cells were less prone to activate TORC1 (Fig. 4A). Second, the ribosomal activity regulator S6k was hypo-phosphorylated in the fat body of lpp-Gal4> Pglym78-RNAi animals compared to controls, further suggesting low TORC1 activity (Fig. 4B; [START_REF] Saitoh | Regulation of an activated S6 kinase 1 variant reveals a novel mammalian target of rapamycin phosphorylation site[END_REF]). Alternatively, this low phospho-S6k could be the consequence of higher phosphatase activity, but when combined with low Rheb expression, this result strongly suggest that TORC1 activity is impaired in the adipocytes of lpp-Gal4> Pglym78-RNAi.

During metabolic control in Drosophila, TORC1 acts mainly through its negative regulation of the transcription co-factor REPTOR. TORC1 phosphorylates REPTOR, thus promoting its cytoplasmic retention. When TORC1 activity is low, hypo-phosphorylated REPTOR translocates to the nucleus to turn on the transcription of target genes [START_REF] Tiebe | REPTOR and REPTOR-BP Regulate Organismal Metabolism and Transcription Downstream of TORC1[END_REF]. In lpp-Gal4> Pglym78-RNAi, low TORC1 signaling should thus translate in a higher REPTOR activity. Strikingly, knocking down REPTOR by RNAi in adipocytes very efficiently suppressed the fat body cells atrophy; and the average size of adipocytes was restored (compared to Pglym78 RNAi alone; Fig. 4C&E).

Consistently, fat body REPTOR RNAi also suppressed distant muscle wasting, as evidenced by properly shaped muscles and absence of severed muscles by whole muscle prep (Fig. 4D). Similarly, the intensity of F-actin staining in muscles, and in particular in the VL3 and VL4 muscles were restored, and a-Actinin protein levels in the circulating hemolymph were reduced (Fig. 4F).

Taken together, these results show that upon glycolysis shut down, adipocyte atrophy is mediated, at least in part, by increased REPTOR activity, leading to distant muscle disorganization.

Upd3 is expressed by adipose tissue but is not required nor sufficient for muscle wasting

We then asked what could be the molecular link that would mediate this inter-organ effect of adipose tissue atrophy on muscles. Adopting a candidate approach, we first investigated the potential role of upd3. Indeed, in a Drosophila adult model of cachexia and muscle wasting, upd3 has been shown to be secreted by yki-driven intestinal tumors and then act on distant muscles to command wasting [START_REF] Ding | Coordination of tumor growth and host wasting by tumorderived Upd3[END_REF]. Furthermore, in a larval model of neoplastic RasV12 / scrib-eye disc tumors, impairing the upd3 receptor domeless in body wall muscles significantly weakened the muscle wasting and disorganization [START_REF] Hodgson | Drosophila Larval Models of Invasive Tumorigenesis for In Vivo Studies on Tumour/Peripheral Host Tissue Interactions during Cancer Cachexia[END_REF]. These different arguments suggest that elevated upd3 levels could be an important mediator of muscle wasting, at least in the context of cachectic tumors.

Monitoring RNA expression level by qRT-PCR, revealed that upd3 was up-regulated in the atrophic fat body of lpp-Gal4> Pglym78-RNAi animals (Fig. 5A). It should be noted however that absolute levels might be low (high number of qPCR cycles required for detection), and that the folds increase in upd3 expression observed in the fat body of lpp-Gal4> Pglym78-RNAi animals stemmed from the absence of upd3 expression in control animals. To formally test the potential function of fat bodyderived upd3 we first performed loss-of-function experiments. We did not observe any modification of the fat body atrophy nor of the muscle disorganization and a-Actinin release in the blood, when driving in lpp-Gal4> Pglym78-RNAi larvae a RNAi directed against upd3 (Fig. 5B-D). These results suggest that upd3, produced by adipose cells after glycolysis shut-down, was not required for distant wasting of muscles. However, it remained possible that the critical source of upd3 were not the adipocytes (targeted by lpp-Gal4), but would have been the fat body resident hemocytes, and that upd3 would be required in these cells for muscle wasting. To address this possibility, we used the Cg-Gal4 driver which drives expression in both adipocytes and hemocytes. Driving Pglym78-RNAi alone using Cg-Gal4 led to similar fat body atrophy and distant muscle wasting as observed with lpp-Gal4 but, here again, we did not observe any rescue of the muscle wasting phenotype by targeting upd3 depletion in both adipocytes and hemocytes (Supplemental Fig. S4A&B). While, we cannot rule out that Pglym78-RNAi-mediated glycolysis shut-down in both adipocytes and hemocytes could trigger a different response and inter-organ messengers, the lack of modification of distant muscle wasting using lpp-Gal4 and Cg-Gal4 suggests that it was not mediated by upd3 expression in the fat body (adipocytes and hemocytes).

We next performed gain-of-function experiments to test whether upd3 could be sufficient to induce muscle wasting. We thus overexpressed upd3 using either a UAS-upd3 construct previously used [START_REF] Romão | The Upd3 cytokine couples inflammation to maturation defects in Drosophila[END_REF] or a fly line in which upd3 overexpression is achieved through the expression of a UAS controlled dCas9-SAM, combined with appropriate sgRNA located in the upstream vicinity of the upd3 gene (flySAM2.0 upd3; [START_REF] Jia | Next-generation CRISPR/Cas9 transcriptional activation in Drosophila using flySAM[END_REF]). Although high expression of upd3 could be preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

The copyright holder for this this version posted September 12, 2023. ; https://doi.org/10.1101/2023.09.09.556970 doi: bioRxiv preprint 9/23 achieved with the different tools used, the overexpression of upd3 in adipocytes (lpp-Gal4), or in the adipocytes and hemocytes (Cg-Gal4) did not promote muscle wasting, nor a-Actinin release in the blood (Supplemental Fig. S4C-E). Taken together these results strongly suggest that upd3 upregulated in fat body undergoing atrophy after glycolysis shut down did not mediate distant muscle disorganization.

Amongst the other upd family ligands, upd1 was not expressed in fat body cells, while upd2 was very modestly up-regulated following glycolysis shut-down (Fig. 5A). However, similarly to upd3, upd2 knock-down by RNAi did not modify fat body atrophy, nor distant muscle disorganization (Supplemental Fig. S5). These results further support that upd ligands secretion by the atrophic fat body did not mediate the distant muscle disorganization.

TNF-a/egr signaling is required for adipose-mediated muscle wasting

Having ruled out upd3, we then tested whether TNF-a/egr could be mediating the effects of the fat body atrophy on distant muscle wasting. Indeed, even though the link between TNF-a signaling and muscle wasting is complex, TNF-a has long been associated with inflammation, cachexia and sarcopenia in patients [START_REF] Patel | TNF-α and cancer cachexia: Molecular insights and clinical implications[END_REF]. Recent studies on Drosophila larval models of tumorinduced cachexia showed that knocking down in muscles wengen, one of the fly TNF-a receptor, partly suppressed muscle disorganization and wasting [START_REF] Hodgson | Drosophila Larval Models of Invasive Tumorigenesis for In Vivo Studies on Tumour/Peripheral Host Tissue Interactions during Cancer Cachexia[END_REF].

We first monitored the expression of egr and Tace, the gene coding for a metalloprotease required for the processing and release of the soluble active form of egr. The expression of egr was downregulated, but the expression of Tace was up-regulated at the RNA level following glycolysis shut down (Fig. 6A), suggesting that even though the egr gene was less expressed, there might be increased levels of active egr. We then tested whether egr signaling was required for the distant muscle wasting by interfering with the expressions of egr and Tace. Expressing RNAi against egr or Tace significantly rescued the muscle disorganization observed in lpp-Gal4> Pglym78-RNAi animals, as evidenced by normal F-actin staining (in particular at the level of the VL3 and VL4 muscles) and morphology of muscle fibers, without any bright accumulation, and a significant decrease in a-Actinin release in the blood (Fig. 6C-E). Importantly, this rescue of the muscles was observed without significant change to the fat body which remained atrophied (Fig. 6B). But if egr produced by adipocytes was required for distant muscle wasting, it was not sufficient. Indeed, the overexpression of an active egr did not promote muscle wasting or a-Actinin release in the blood (Fig. 6F), suggesting that the action of egr is dependent on context, and that egr represents a permissive rather

Muscle wasting is triggered by low Insulin signaling

One obvious candidate for the context dependent action of egr, is Insulin signaling. Indeed, previous studies showed that larval muscle size and organization is supported by Insulin receptor and Foxo activities [START_REF] Demontis | Integration of Insulin receptor/Foxo signaling and dMyc activity during muscle growth regulates body size in Drosophila[END_REF]. In Drosophila larvae, the Insulin producing cells in the larval brain represent the main source of circulating insulin-like peptides (dilps). However, the activity of these dilps is further controlled by secreted molecules that can bind and trap dilps, in particular ImpL2 [START_REF] Alic | Lifespan extension by increased expression of the Drosophila homologue of the IGFBP7 tumour suppressor[END_REF][START_REF] Honegger | Imp-L2, a putative homolog of vertebrate IGF-binding protein 7, counteracts insulin signaling in Drosophila and is essential for starvation resistance[END_REF]. ImpL2 expression was actually highly upregulated in fat body cells from lpp-Gal4> Pglym78-RNAi (Fig. 7A). We reasoned that this increase in ImpL2 could dampen circulating dilps, and thus block Insulin signaling in peripheral tissues, and more particularly in muscles.

Low circulating insulin levels were supported by the observation that lpp-Gal4> Pglym78-RNAi animals showed transcriptional characteristics of low insulin signaling with increased expression levels of InR and Ilp6 in body-wall muscles (Fig. 7A; [START_REF] Sriskanthadevan-Pirahas | Adipose mitochondrial metabolism controls body growth by modulating systemic cytokine and insulin signaling[END_REF]).

To test the relevance of ImpL2 expression in the fat body on the distant muscle disorganization, we invalidated it by RNAi. Inhibiting ImpL2 in adipocytes (lpp-Gal4) led to a dramatic rescue of the distant muscle: we observed better formed body wall muscles (with increased F-actin staining in VL3 and VL4 muscles in particular), and lower a-Actinin release in the blood (Fig. 7C-D). Similarly to what was observed for egr/Tace signaling, the muscle rescue by ImpL2 knock-down occurred without any change in the atrophy of the fat body (Fig. 7B).

Since REPTOR knock-down led to a dramatic rescue of both fat body atrophy and muscle disorganization, we wondered whether REPTOR activity would control the expression of the signaling molecules produced by the fat body and controlling muscle disorganization. Monitoring by qPCR, we only observed marginal changes in the expression of egr, Tace, or ImpL2 after REPTOR knock-down in Pglym78-RNAi (Fig. 7E). These results suggest that i) the expression of upd3, egr, and ImpL2 in the fat body after glycolysis knock-down is not controlled by REPTOR , and that ii) REPTOR controls the expression/release of other factors yet to be determined participating in the distant muscle disorganization.

Taken together, these results support a model in which glycolysis shut-down in adipocytes promotes REPTOR activity controlling lipid reserves atrophy. In parallel, it leads to the secretion of egr and ImpL2 which cooperate to trigger distant muscle disorganization (Fig. 7G).

DISCUSSION

In this study, impairing glycolysis in adipocytes through the down-regulation of Pglym78 or Eno enzymes, we uncovered a link between fat body atrophy and body wall muscle disorganization in Drosophila larvae. We further showed that this inter-organ communication relies on the cooperation of at least two signals: i) egr/TNF-a release by atrophic adipocytes, and ii) inhibition of circulating insulin signaling by ImpL2 (Fig. 7F). These two signals produced by the fat body were required to control muscle disorganization, but other yet unidentified signals might participate in this adipose > muscle communication. In this glycolysis shut-down fat body atrophy model, we could not find any involvement of upd ligands, and in particular of upd3, in the disorganization of muscles unlike what had been shown in the case of tumor-induced muscle wasting in adult Drosophila cachexia models [START_REF] Ding | Coordination of tumor growth and host wasting by tumorderived Upd3[END_REF]. It should be noted that, while we ruled out fat body-derived upd3, we did not formally test whether circulating upd ligands secreted by other organs (in response to the atrophied fat body) or whether Jak/Stat signaling in muscles played any role in muscle disorganization induced by fat atrophy.

Egr/TNF-a signaling was activated upon glycolysis shut-down in larval adipocytes as evidenced by its role for distant muscle disorganization. While the increased Tace expression suggests that more processed egr might be produced, the mechanisms behind this increased egr signaling remain to be explored, and could involve higher egr mRNA translation leading to increased protein levels, enhanced Tace cleaving activity, or stabilization of activated egr. Whether egr can act directly on muscles, as suggested by the role of its receptor wengen during tumour-induced muscle wasting [START_REF] Hodgson | Drosophila Larval Models of Invasive Tumorigenesis for In Vivo Studies on Tumour/Peripheral Host Tissue Interactions during Cancer Cachexia[END_REF], or indirectly, or through a combination of direct and indirect effects, remains however to be determined.

Since the overexpression of egr in fat body cells could not recapitulate the muscle disorganization observed upon Pglym78-RNAi, it suggests that egr signaling while required, must cooperate with other signals. One such signal is represented by the extracellular dilps inhibitor ImpL2 [START_REF] Alic | Lifespan extension by increased expression of the Drosophila homologue of the IGFBP7 tumour suppressor[END_REF][START_REF] Honegger | Imp-L2, a putative homolog of vertebrate IGF-binding protein 7, counteracts insulin signaling in Drosophila and is essential for starvation resistance[END_REF]. ImpL2 was indeed required for the distant muscle wasting when knocked down in adipocytes, suggesting that the extracellular trapping of dilps and thus dampened Insulin signaling was a major contributor to muscle disorganization in this system. This could reflect muscle growth problems since Insulin receptor and Foxo signaling are required for the growth of larval body wall muscles, including the VL3 and VL4 muscles [START_REF] Demontis | Integration of Insulin receptor/Foxo signaling and dMyc activity during muscle growth regulates body size in Drosophila[END_REF].

Alternatively, dilps/Foxo signaling has also been shown to prevent ageing of adult fly muscle and the formation of detrimental protein aggregates [START_REF] Demontis | FOXO/4E-BP signaling in Drosophila muscles regulates organismwide proteostasis during aging[END_REF][START_REF] Wessells | Insulin regulation of heart function in aging fruit flies[END_REF].

The disorganization observed in larval muscles in response to fat body atrophy might thus represent 12/23 processes related to adult muscle ageing. The critical role of extracellular Insulin inhibitors in muscle wasting was demonstrated previously in adult Drosophila cachexia models, in which either rasV12driven tumorous larval discs transplanted in adult flies, or yorkie-driven adult midgut tumors, induced muscle disorganization and wasting through the secretion of ImpL2 [START_REF] Figueroa-Clarevega | Malignant Drosophila tumors interrupt insulin signaling to induce cachexia-like wasting[END_REF][START_REF] Kwon | Systemic organ wasting induced by localized expression of the secreted insulin/IGF antagonist ImpL2[END_REF]. Even though the role of tumor derived ImpL2 in muscle wasting has not been formally tested in larval Drosophila models of cachexia, our results indicate that larval muscles are indeed sensitive to ImpL2 levels, at least in certain conditions.

The mechanisms by which glycolysis impairment leads to this elevated egr signaling and increased production of ImpL2 by the fat body cells remain however elusive. We could show that in response to Pglym78 knock-down, mTOR activity was inhibited and that REPTOR, the main transcriptional effector downstream of mTOR [START_REF] Tiebe | REPTOR and REPTOR-BP Regulate Organismal Metabolism and Transcription Downstream of TORC1[END_REF], was a major driver of fat body atrophy. However, REPTOR is unlikely to control egr, Tace and ImpL2 in the fat body since removing REPTOR in lpp-Gal4> Pglym78-RNAi cells, affected only marginally their expression levels (Fig. 7E). This is consistent with earlier studies showing that neither egr, Tace, or ImpL2 were controlled by mTOR/REPTOR in S2 cells or in whole larvae transcriptomic analyses [START_REF] Guertin | Functional genomics identifies TOR-regulated genes that control growth and division[END_REF][START_REF] Tiebe | REPTOR and REPTOR-BP Regulate Organismal Metabolism and Transcription Downstream of TORC1[END_REF]. However, REPTOR inhibition in the fat body, not only suppressed fat body atrophy, it also suppressed distant muscle disorganization. These observations suggest thus that REPTOR activity controls yet another unidentified signals affecting muscles (Fig. 7G). Two main models could be proposed here. In the first model, REPTOR controls the expression of an intercellular messenger that acts directly or indirectly cooperating with egr and ImpL2 leading to muscle disorganization. In the second model, the action of fat body REPTOR is not mediated by the production of an intercellular signaling molecule, but by its direct role on fat body atrophy. By promoting fat body atrophy, REPTOR makes the fat body unable to provide the nutrients necessary for muscle growth and/or maintenance of healthy muscles. This nutritional stress, combined with the action of egr and ImpL2 leads then to muscle disorganization. This second model is supported by the observation that, in adult muscles, REPTOR controls the balance between glycolysis and oxidative metabolism [START_REF] Saavedra | REPTOR and CREBRF encode key regulators of muscle energy metabolism[END_REF]. While the exact processes controlled by REPTOR in the larval fat remain to be studied, this study suggests that REPTOR might represent a major metabolic regulator controlling what nutrients could be stored or secreted by the fat body. Further studies are needed to distinguish between these different models.

Earlier reports showed that mTOR pathway inhibition in fat body cells, resulted in systemic effects with smaller overall organisms, in part through strong retention of dilps in the IPCs and thus lower peripheral insulin signaling [START_REF] Colombani | A nutrient sensor mechanism controls Drosophila growth[END_REF][START_REF] Delanoue | Drosophila insulin release is triggered by adipose Stunted ligand to brain Methuselah receptor[END_REF][START_REF] Géminard | Remote control of insulin secretion by fat cells in Drosophila[END_REF].

The tools used in these earlier studies (knock-down of the amino-acid transporter slimfast, or of the mTOR activator Rheb, or overpression of the mTOR inhibitor TSC1/2) led to a strong mTOR inhibition. After Pglym78 RNAi, we only observed subtle effects to the overall growth of the larva with slightly smaller wing discs but larvae with normal size (see Supplemental Fig. S2). These results suggest that at the small level of mTOR inhibition achieved here by Pglym78 RNAi, the remote control of organismal growth by mTOR sensing in the adipose tissue, is likely negligeable. However, we cannot exclude that it might contribute to the distant muscle disorganization observed.

How does Pglym78 knock-down control TORC1 activity remains to be explored. Our results showed that glycolysis shut-down resulted in low Rheb expression. Since Rheb is required for TORC1 activation, this low Rheb expression might contribute to the low TORC1 activity. Alternatively, AMPK signaling has been shown to antagonize mTOR signaling by phosphorylating and activating TSC2 [START_REF] Inoki | TSC2 integrates Wnt and energy signals via a coordinated phosphorylation by AMPK and GSK3 to regulate cell growth[END_REF], or by phosphorylating and inhibiting raptor [START_REF] Gwinn | AMPK phosphorylation of raptor mediates a metabolic checkpoint[END_REF]. Even though, the activity of AMPK has not been properly assessed after Pglym78 RNAi in adipocytes, blocking glycolysis should put cells under energetic stress, low ATP levels, and thus lead to higher AMPK activity. A recent report analyzing the role of adipocyte mitochondrial function in the regulation of organismal metabolism, showed that knocking down TFAM, the master transcription factor controlling mitochondrial genes, led to animals with increased general metabolism which developed faster than controls, shortening larval life. Interestingly, in these animals, the fat body cells produced less egr and less ImpL2 leading to an overall putative higher insulin signaling and faster metabolism [START_REF] Sriskanthadevan-Pirahas | Adipose mitochondrial metabolism controls body growth by modulating systemic cytokine and insulin signaling[END_REF]. This situation, impairing mitochondrial function, appears thus the opposite to the one we describe here knocking down glycolysis. Whether TFAM and mitochondrial function are involved in the effects of glycolysis shut down or vice versa, remains to be explored. Further studies should help clarify the link between glycolysis, mitochondrial function, AMPK and TORC1 regulation in adipocytes, and how this results in adipokine secretion and the control of developmental timing, and muscle maintenance.

Haemolymph extraction and trehalose measurement

3rd instar larvae were washed in water and then in 70% EtOH before being dried on filter paper.

Groups of ten larvae were placed on a piece of Parafilm on ice. Haemolymph was extracted by piercing carefully the cuticle with the forceps and then collected by pipetting. For Western-blot analyses, 5µl of collected haemolymph were immediately complemented by 5µl of 2X Laemmli buffer. 1.5µl of haemolymph was used to measure the glucose and trehalose according to the Glucose assay kit (Sigma-Aldrich #GAGO20). To remove cells and tissue debris, the haemolymph was centrifuged at 4C for 30min at 1000g and the supernatant was further centrifuged at 4C for 20min at 15000g.

Glycogen and glucose measurements in fat body

Dissected fat bodies pooled from 10 3rd instar larvae were homogenized for glycogen and glucose measurements according to the Glucose assay kit (Sigma-Aldrich #GAGO20).

Immunofluorescence

Tissues were dissected in PBS and fixed at room temperature (RT) in 4% formaldehyde for 30min.

After washing 3x 20min in PBT (PBS 0,2% Triton X-100), non-specific epitopes were blocked for 30min in PBT-BSA (PBT 0,5% BSA). Primary antibodies were incubated overnight at 4C in PBT-

Lipid droplets staining with BODIPY

Fat bodies from 3 rd instar larvae were dissected in PBS and fixed in 4% formaldehyde for 30min at RT. Tissues were then rinsed 2x in PBS and incubated in Bodipy FL Dye (1:500 dilution from a 1mg/mL stock, ThermoFisher) for 30min in PBS. They were rinsed 2x in PBS and immediately mounted in CitiFluor TM AF1 (Agar). Images were acquired on a Leica THUNDER microscope. Lipid droplet numbers and sizes were measured using FIJI (ImageJ) software.

Western blot

Western blot analyses were performed according to standard protocols. Primary antibodies used in this study: Mouse anti-a-actinin (1:2000; DSHB #2G3-3D7), Mouse anti-myosin (1:2000; DSHB #3E8-3D3), Rabbit anti-lipophorin II (1:15000; gift from J Culi), Rabbit anti-GFP (1:2000, Molecular Probes: A6455), anti-Histone H3 (1:100; CST #4499), Mouse anti-Tubulin (1:10000; Sigma-Aldrich #T6074), Rabbit anti-phospho-Drosophila p70 S6 Kinase (1:100; CST #9209). Secondary antibodies conjugated to HRP were from Jackson Labs Immuno Research (1:15000).

Quantitative RT-PCR

For each tissue, pooled groups of ten dissected tissues were lysed chemically with QIAzol Lysis Reagent (QIAGEN) and physically using a tissue disruptor Retsch MM400 (Verder Scientific). Total RNA was then purified using QIAGEN kits (QIAGEN RNeasy Lipid #74804 or RNeasy Plus #74134). Genomic DNA was removed by incubating with DNase (QIAGEN #79254), and cDNA was retro-transcribed using SuperScript III (Invitrogen #18080-044). Semi quantitative qPCR was performed on biological triplicates using SYBR Green I Master mix on a LightCycler ® 480 (Roche).

Fold change was estimated using the ∆∆CT approach.

Primers used:

  BSA. Samples were then washed 3x for 20min in PBT. Secondary antibodies were incubated 2h at RT in PBT-BSA. Samples were washed 3x in PBT and mounted in CitiFluor TM AF1 (Agar). Images were acquired on an upright Leica THUNDER microscope. Primary antibodies used in this study: Mouse anti-a-actinin (1:25; Developmental Study Hybridoma Bank -DSHB #2G3-3D7), Rat anti-ECadherin (1:25; DSHB #DCAD2), Rabbit anti-GFP (1:200; Torrey Pines Biolabs #TP401), Rat anti-dilp2 (1:200; Gift from P Leopold), Rabbit anti-dilp5 (1:400; Gift from P Leopold). Secondary antibodies used conjugated to Alexa Fluor 488, 555, 647, or to Cy3 were from Jackson Labs Immuno Research (1:200). DAPI was used at 1:1000 and Phalloidin-Rhodamine (Sigma-Aldrich #P1951) was used at 1:200.

Figure 1 .

 1 Figure 1. Lower glycolysis enzymes knock-down in adipose tissue results in fat body atrophy A. Schematic of the glycolysis pathway with the key enzymes in Drosophila. B. Fat body staining of the indicated genotypes showing nuclei (DAPI, blue), cell cortex (F-actin, red, white in B'), and lipid droplets (Bodipy, green); white bar: 50 µm. C-D. Quantification of average cell size (C) and lipid droplet area per cell (D) from images shown in B. One-way ANOVA (Kruskal-Wallis) statistical test, **** p<0.0001, ns not significant.

Figure 2 .

 2 Figure 2. Developmental delay after glycolysis enzymes knock-down in the fat body A. Pupariation curve of the indicated genotypes as cumulative percentage of pupae with time in hours after egg laying (ael).B. Size of brains and wing discs expressed in arbitrary units (pixels) in Pglym78-RNAi animals compared to w-RNAi controls at 5 days ael. Mann-Whitney test, * p=0.0175, ns not significant.

Figure 3 .

 3 Figure 3. Fat body Pglym78 or Eno knock-down promote body wall muscle disorganization A-B. Larval body wall muscles after glycolytic enzymes knock-down in fat body cells monitored using F-actin staining (white in A) on dissected fixed larvae, or the Zasp66:GFP reporter in live larvae (green in B). White bar: 200 µm in A and 750 µm in B. Red arrowhead in A indicate the VL3/VL4 muscles. Yellow stars highlight damaged muscles. In all panels anterior is up (A) or left (B).C. Western blot of whole protein extracts of hemolymph (5µl samples) from the larvae shown in A' and monitoring the presence of muscle proteins such as Myosin, a-Actinin, or Zasp66 (GFP). Apo-LppII is used as loading control.

Figure 4 .

 4 Figure 4. The mTOR target REPTOR mediates the fat atrophy and distant muscle disorganization in fat body Pglym78 knock-down larvae A. Expression of the TORC1 components mTor and raptor and of the main regulator Rheb in fat body cells monitored by qRT-PCR. Reference gene for normalization: rp49/RpL32. Error-bars show standard error of the mean (sem). One-way ANOVA statistical test. * p<0.05, p<0.01 B. Western blot of whole protein extracts of fat body tissues monitoring the TORC1 phosphorylation target S6k. Histone H3 is used as loading control. C. Fat body staining of the indicated genotypes showing nuclei (DAPI, blue in C), cell cortex (Factin, red in C and white in C'), and lipid droplets (Bodipy, green in C); white bar 50 µm.

  preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

	bioRxiv preprint	doi:	; https://doi.org/10.1101/2023.09.09.556970	this version posted September 12, 2023.	The copyright holder for this

  (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

	bioRxiv preprint	doi:	; https://doi.org/10.1101/2023.09.09.556970	this version posted September 12, 2023.	The copyright holder for this
					10/23

than instructive signals, or that egr must cooperate with other signals to promote distant muscle disorganization.

preprint

ACKNOWLEDGEMENTS

We thank R. Delanoue, M. Milán, P. Léopold, and A. Teleman, for sharing flies. We acknowledge the Bloomington Drosophila Stock Center (BDSC -NIH P40OD018537), the Vienna Drosophila Resource Center (VDRC), the Developmental Studies Hybridoma Bank, the Montpellier Drosophila facility, and FlyBase for providing reagents and tools critical for our research.

FUNDING

JF was supported by a PhD fellowship from the French Ministry for Education Research and Technology (MENRT). This project was supported by grants from the "Fondation ARC pour la recherche sur le cancer #PJA 20181207757", "GSO-Emergence", and "Agence Nationale de la Recherche #ANR-18-CE14-0041".

MATERIALS AND METHODS

Drosophila genetics

All crosses were cultured at 25C on standard food, except for the experiments with food supplemented with 1% of sodium pyruvate (Sigma Aldricht #P5280).

Stocks used are listed below:

Pupariation curve

Females were put to lay eggs for 4h, before removing all adults. The number of larvae that entered pupariation were counted at different time points after egg laying: 108, 114, 120, 131, 138, 145, 152, 161, 169 and 183h. The percentage of pupariation was calculated considering the total number of larvae that finally reached the pupal stage.

preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

AUTHOR CONTRIBUTIONS

Conceptualization: MRV, AD. Validation: MRV, JF, PM, CG. Formal analysis: MRV, JF, CG, AD.

Investigation: MRV, JF, LHM, PL, CG, AD. Writing -original Draft: AD. Writing -review and editing: CG, AD. Visualization: MRV, CG, AD. Supervision: AD. Project administration: AD.

Funding acquisition: CG, AD.

COMPETING INTERESTS

The authors declare no competing interest


J Xu

Yannick Guyot

A Nehari

A Pillonnet

G Ledoux

H Takeda

X Xiaodong

K Lebbou

Cr-doped Al 2 O 3 -YAG binary and Al 2 O 3 -YAG-ZrO 2 ternary eutectic materials crystallized by the micro pulling down technique and their characterization

Keywords: Eutectics, Cr, Color, Solidification, Microstructure, Luminescence

come

Cr-doped Al 2 O 3 -YAG binary and Al 2 O 3 -YAG-ZrO 2 ternary eutectic materials crystallized by the micro pulling down technique and their characterization

Introduction

Color eutectic ceramic materials have aroused a great attention in the innovation applications of Light-Emitting Diode (LED), watch pointer and jewelry decoration; it is required to produce higher quantity and performed more economic attributes [START_REF] Sai | Ce-doped Al 2 O 3 -YAG eutectic and its application for white LEDs[END_REF][START_REF] Song | Structure and white LED properties of Ce-doped YAG-Al2O3 eutectics grown by the micro-pulling-down method[END_REF][START_REF] Rocha | Silva1 Analysis and Improvement of Processes in the Jewelry Industry[END_REF][START_REF] Bertacchini | Multi-objective optimization and rapid prototyping for jewelry industry: methodologies and case studies[END_REF]. The colored (red, blue, yellow…) ceramics have the ability to be used in jewelry and luxury and can substitute some specific crystal such ruby. The eutectics ceramics materials offer technical characteristics in quality of hard material but also of scratch resistance, biocompatibility, insensitivity to any chemical attack, resistance to friction and abrasion, but also aesthetic advantages which intended for dressing applications [START_REF] Minford | Crystallography and microstructure of directionally solidified oxide eutectics[END_REF][START_REF] Benamara | Orientations and seed type effect on Al 2 O 3 -YAG-ZrO 2 eutectic microstructure solidified from the melt by the micro-pulling down technique[END_REF][START_REF] Benamara | Microstructure and crystallography of Al 2 O 3 -Y 3 Al 5 O 12 -ZrO 2 ternary eutectic oxide grown by the micropulling down technique[END_REF]. The Al 2 O 3 -YAG binary and Al 2 O 3 -YAG-ZrO 2 ternary systems [START_REF] Benamara | The impact of the composition and solidification rate on the microstructure and the crystallographic orientations of Al 2 O 3 -YAG-ZrO 2 eutectic solidified by the micro-pulling down technique[END_REF][START_REF] Stubican | Eutectic Solidification in Ceramic Systems[END_REF][START_REF] Wang | Self-thermal management YAG:Ce-Al 2 O 3 color converters enabling high-brightness laser-driven solid state lighting in a transmissive configuration[END_REF][START_REF] Kang | Thermal durability of YAG: Ce ceramic with containing Al 2 O 3 and its Raman analysis[END_REF] are among the eutectics ceramics materials that are identified to be the most important composites materials due to directional and rapid solidification from the melt [START_REF] Waku | High-temperature strength and thermal stability of a unidirectionally solidified Al 2 O 3 /YAG eutectic composite[END_REF][START_REF] Carroz | Tuning the sapphire EFG process to the growth of Al 2 O 3 /YAG/ZrO 2 : Y eutectic[END_REF]. The high melting temperature > 1500°C, allowing them to work stably in oxidizing environment due to inherent chemical-physical properties [START_REF] Mazerolles | Microstructures, crystallography of interfaces, and creep behavior of melt-growth composites[END_REF]. They present excellent mechanical properties that are heavily dependent on the microstructure, morphology and crystallographic orientations [START_REF] Laidoune | Yttria stabilized Al 2 O 3 -ZrO 2 eutectic crystal fibers grown by the laser heated pedestal growth (LHPG) method[END_REF][START_REF] Mesa | Microstructural stability and orientation relationships of directionally solidified Al 2 O 3 -Er 3 Al 5 O 12 -ZrO 2 eutectic ceramics up to 1600° C[END_REF] and are considered as potential engineering materials. It is well known that controlling uniform microstructure of the eutectic ceramics in the binary or ternary systems is related to the directionally solidification behavior that depended on the solidification methods and the specific composition in the equilibrium diagram [START_REF] Benamara | Shaped ceramic eutectic plates grown from the melt and their properties[END_REF][START_REF] Ingel | III, Lattice parameters and density for Y 2 O 3 -stabilized ZrO 2[END_REF][START_REF] Krogstad | Effect of yttria content on the zirconia unit cell parameters[END_REF][START_REF] Lee | Growth and characterization of Al 2 O 3 /Y 3 Al 5 O 12 /ZrO 2 ternary eutectic fibers[END_REF][START_REF] Lee | Microstructure of Y 2 O 3 doped Al 2 O 3 /ZrO 2 eutectic fibers grown by the micro-pulling-down method[END_REF]. Chromium oxides can be used in the ceramic because they are characterized by a large variety of colors. Therefore, chromium is an element of choice for obtaining colored minerals, since it colors many compounds in red, purple or green. It is in most cases in the form of Cr 3+ in octahedral symmetry. Chromium is a transition element of electronic configuration for the metal Cr 0 [Ar] 4s 2 3d 4 , which gives it a wide variety of possible degrees of oxidation from 0 to +6 [START_REF] Ester | Processing and microstructural study of surface laser remelted Al 2 O 3 -YSZ-YAG eutectic plates[END_REF]. However, oxidation states +3 and +6 are the most commonly encountered in minerals and glasses. The Cr 3+ gives rise to many colors, it is mainly found in octahedral geometry in minerals and is notably responsible for the green color of the emerald and the red color of ruby, but can also give purple as in stichtite [START_REF] Wang | Crystallography and interfacial structure in a directionally solidified Al 2 O 3 /Y 3 Al 5 O 12 /ZrO 2 eutectic crystal[END_REF][START_REF] Fu | Microstructure refinement approaches of melt-grown Al 2 O 3 /YAG/ZrO 2 eutectic bulk[END_REF]. The presence of Cr 3+ in more or less distorted octahedral geometry results in the presence of two main absorption bands in the visible range of its optical spectrum. The observed energy shifts of the absorption bands of the Cr 3+ between the different minerals explain the different colorations, since the transmission window in the visible range is modified with these displacements. By the analysis of the ruby and the emerald, Vauquelin et al [START_REF] Vauquelin | Déyeux et Bouillon[END_REF] discovers that chromium is responsible for the respective red and green colors of these two minerals. The synthesis of colored eutectics ceramics compounds is not well controlled. For example, despite all efforts, it has never before been possible to produce eutectics ceramics bright red in color to be used for jewelry and watch pivot. The aim of this paper is in particular to propose red colored Cr-doped Al 2 O 3 -YAG and Al 2 O 3 -YAG-ZrO 2 eutectic ceramic materials solidified by the micro-pulling down (µ-PD) technique allowing significantly expand the range of possible colors in the production of colored eutectic ceramics materials.

Experimental procedure

Crystal growth conditions

The starting materials were made from commercial high-purity (>99.99%), Cr 1. The mixture was sintered at 1500℃ for 24 hours in air atmosphere. Eutectic ceramic rods were solidified from the melt using the micro-pulling down technique presented in previous work [START_REF] Kang | Thermal durability of YAG: Ce ceramic with containing Al 2 O 3 and its Raman analysis[END_REF][START_REF] Carroz | Tuning the sapphire EFG process to the growth of Al 2 O 3 /YAG/ZrO 2 : Y eutectic[END_REF][START_REF] Laidoune | Yttria stabilized Al 2 O 3 -ZrO 2 eutectic crystal fibers grown by the laser heated pedestal growth (LHPG) method[END_REF][START_REF] Lee | Microstructure of Y 2 O 3 doped Al 2 O 3 /ZrO 2 eutectic fibers grown by the micro-pulling-down method[END_REF]. Iridium crucible is used, it is tapped in its lower part with a capillary die of 3mm in diameter. The crucible is enclosed in a chamber swept with an oxygen-free argon stream, and is charged with eutectic ceramic raw materials. The crucible is brought to a temperature of 1750°C2°C using a high frequency generator operating at 11 kHz and having a continuous power output of 25kw. When a pendant drops forms at the bottom of the capillary channel (Fig. 1), it is connected with a rod of eutectic ceramic of selected composition with diameter of 2mm to serve as the seed. When the drops weld to the seed, the seed is pulled down at a rate of 0.3mm.min -1 . A CCD camera was used to take inset a live picture through a small circular window (Fig. 1b). High-purity flowing argon was used to prevent oxidation of iridium crucible and after heater crucible support.

Eutectic seed

Capillary die

Liquid Crucible

After heater

Hole for CCD Camera

Drop liquid

Characterization methods

XRD phase analysis and Scanning electron microscopy

Room-temperature X-ray diffraction patterns were obtained using a Bruker D8 advanced diffractometer with Cu-Kα 1 and Cu-Kα 2 X-rays (λ=0.15406 and 0.15444 nm). The diffraction diagram is scanned over a 2θ range from 15°to 70° in steps of 0.02° with a counting time of 1 s per step and continuous rotation (20 rpm) for measurement. In order to investigate the formation and evolution of microstructures, the eutectic samples were cut and polished along the longitudinal and transversal, the pieces about 2 to 10 mm in length are embedded in organic resin. SEM observations of the polished samples were performed with a Phenom desktop scanning electron microscope (SEM) at 5 kV.

Spectroscopic characterizations

The fluorescence lines of ruby single crystals are used to evaluate the residual stresses in the solidified rods, the displacement of fluorescence line of chromium (Cr 3+ ) is measured. A 473 nm laser wavelength has been used as excitation source and focused on the surface of the polished samples through a 50X Olympus MIR plan objective with 0.25 NA in order to cover larger sample area. For each sample, a 121 point of measurement are taken on different areas of the polished transversal and longitudinal section, all the measured spectra are fitted using a Gaussian profile in order to determine the exact peak positions of Cr 3+ fluorescence lines. The emission spectra were registered at room (RT) and liquid nitrogen temperatures by exciting the crystals with an EKSPLA NT342B tunable OPO (Optical Parametric Oscillator) pumped by a pulsed and frequency-tripled Nd:YAG laser delivering 7 ns laser pulses. Emission signals were dispersed and detected in the visible range with the aid of a SHAMROCK 350 monochromator equipped with both 300 line/mm and 1200 line/mm gratings, and an ANDOR ICCD gated detector. The high temperature measurements were performed using a LINKAM SCIENTIFIC cell DSC600 with an analogous Photo-Luminescence set-up. The fluorescence decay measurements were performed by using the same laser excitation sources and a Jobin-Yvon HRS1 monochromator and by analyzing the signals with the help of R928 AsGa (RESEARCH Inc.) photomultiplier tubes coupled with a Wave Runner 64Xi LECROY digital scope, for the visible and NIR spectral ranges, respectively. The excitation spectra of Cr-doped Al 2 O 3 -YAG (binary system) and Al 2 O 3 -YAG-ZrO 2 (ternary system) eutectics in the range from 285 to 680 nm are recorded at room temperature with the help of an EDINBURGH Instruments FS5 spectrofluorimeter.

Result and discussion

Eutectic solidification and phase's characterization

As a function of the composition, the Cr doped binary (Al 2 O 3 -YAG) and ternary (Al 2 O 3 -YAG-ZrO 2 ) eutectic ceramics rods solidified from the melt are shown in Fig. 2. They were crystallized under stationary stable regime. Except the CrTE4 (0.4%Cr) sample, the Cr doped eutectic ceramics rods have a stable diameter of 3 mm. Whatever the system (binary or ternary), the red color contrast increase with increasing the Cr concentration. The variation in color coordinates (x,y) as presented in the CIE-1931 chromaticity diagram (Fig. 3) showing various red colors can be obtained by varying Cr concentration in the eutectic ceramics. The ceramics eutectics rods corresponding to high Cr concentration (1.9% Cr) have a strong red color close to the blood color. The surface of the samples is smooth without any visible macroscopic defects such as cracks or bubbles. Fig. 4a and4b showed the XRD diagram of the eutectic ceramics as a function of Cr concentration for the binary and the ternary systems. The grinding of the material makes it possible to overcome the phenomena of texturing and distortion of cells due to the internal stresses of the material at room temperature. The YAG is identified as the majority phase in both systems. In the case of the Cr-doped Al 2 O 3 -YAG binary system, only Al 2 O 3 and YAG phases are observed. Additional cubic ZrO 2 phase was crystalized in the case of the Cr-doped Al 2 O 3 -YAG-ZrO 2 ternary system. We did not registered the presence of YAlO 3 perovskite phase or inclusions belonging to the Al 2 O 3 , Y 2 O 3 , and Cr 2 O 3 oxides indicating that Cr ions doping did not create impurities phases or induce a significant changes in the structure architecture. Table 2 present the lattice parameters as a function of the Cr concentration. In case of the YAG phase, whatever the system (binary or ternary), we registered a light increasing of the lattice parameter (a). The YAG has a garnet structure with Y 3+ occupying a dodecahedral site and Al 3+ ions occupying octahedral and tetrahedral sites, respectively [START_REF] Song | Longitudinal cross-section microstructure of growth striation in Al 2 O 3 /Y 3 Al 5 O 12 /ZrO 2 directionally solidified eutectic ceramic prepared by laser floating zone[END_REF]. The cells contains eight

groups form Y 3 Al 2 Al 3 O 12 (X 3 Y 2 Z 3 O 12 )
. The X, Y, and Z sites cations respectively occupy the positions 24c, 16a and 24d, and the anions are located at the positions 96h.

The chromium (located on the Y sites) is therefore at oxidation state 3 and has a punctual symmetry 3 -or C3i in Schönflies notation. The Cr 3+ substitutes A1 3+ in a small trigonally distorted octahedral site with Cj symmetry [START_REF] Llorca | Directionally solidified eutectic ceramic oxides[END_REF]. The structure of Al 2 O 3 is the same as that of the Cr 2 O 3 . These two compounds form a complete solid solution, Al 2-x Cr x O 3 , by substitution of aluminum by chromium in octahedral site.

The solid solution changes color with the chromium concentration. The introduction of a few tens of ppm of chromium replacing aluminum produces a characteristic pink color. The color intensifies with increasing chromium concentration, and as soon as the latter exceeds a few 0.7at% for both binary and ternary systems, the compounds acquire a strong red component. The color change of compounds is mainly due to changes in the parameter of crystal field  0 [START_REF] Su | Rapid solidification of Al 2 O 3 /Y 3 Al 5 O 12 /ZrO 2 eutectic in situ composites by laser zone remelting[END_REF][START_REF] Reinen | Ligand-field spectroscopy and chemical bonding in Cr 3 +-containing oxidic solids[END_REF]. Chromium dopant involving in the coloration being essentially in the form Cr 3+ in more or less distorted octahedral symmetry. In addition, the broad absorption band peaking at 560 nm in the visible range is increasing with the Cr concentration, then for the highest Cr concentration the absorption at edge of this band for wavelength higher than 600 nm is also increasing giving to the sample a deep red color. Both binary and ternary eutectic ceramics rods contained Cr-doped α-Al 2 O 3 phase belonging to the solid solution Al 2-x Cr x O 3. Whatever the Cr concentration and the systems, all the solidified eutectic ceramic showed an increasing of a and c-lattices parameters in Cr-doped α-Al 2 O 3 phase. This is principally due to the difference of the Cr 3+ (61.5 pm) size which is considerably greater than the size of Al 3+ (53.3 pm) [START_REF] Burns | Mineralogical applications of crystal eld theory[END_REF]. The pics characteristics of Al 2-x Cr x O 3 are therefore progressively shifted towards the lowest values of 2 with Cr content (Fig. 4). In the case of the Cr-doped Al 2 O 3 -YAG-ZrO 2 system, we have not observed a variation of lattice parameter a in ZrO 2 phase. But the X-ray peaks of the cubic zirconia phase were shifted slightly because of some amount of Y 2 O 3 was dissolved.

On the basis of qualitative results under EDS, some amount of Y 2 O 3 was contained in the ZrO 2 phase. Fig. 5 and Fig. 6 show the SEM images of the transverse and the longitudinal sections cut from the Cr-doped Al 2 O 3 -YAG binary and Al 2 O 3 -YAG-ZrO 2 ternary eutectic ceramic rods. The precise knowledge of the solidification conditions, namely solidification rate and initial composition, makes it possible to study the influence of these different parameters on the morphology. The addition of the Cr in the binary Al 2 O 3 -YAG system do not strongly affect the architecture morphology of this system. Unlike the ternary system, the binary eutectic present a homogeneous microstructure for all the Cr concentration.

It is composed of two phases distinguished by their different sharps and colors, the black regions correspond to the α-Al 2 O 3 phase and the grey regions correspond to YAG (Y 3 Al 5 O 12 ) phase. The Al 2 O 3 is the matrix and the YAG phases are disconnected on transverse sections to the solidification direction. The shape and size of the YAG phase are irregular. The longitudinal sections (Fig. 6) show that the microstructure is irregular lamellar and correspond to Chinese Script microstructure. The lamellae are elongated along the solidification direction. The YAG size increase with Cr concentration increasing and reach a maximum average around 40 µm for 1.9at% Cr (Fig. 6d). The coarsening as a function of Cr concentration is quite homogeneous and the microstructure remained unchanged. In the binary system, the dimension, the homogeneity and the morphology of the microstructure are modified by varying the Cr concentration. A high Cr concentration promotes the contribution of the diffusion leading to an increase in the size of the microstructure. Increasing Cr concentration is similar to decreasing pulling rate in Al 2 O 3 -YAG system [START_REF] Song | Longitudinal cross-section microstructure of growth striation in Al 2 O 3 /Y 3 Al 5 O 12 /ZrO 2 directionally solidified eutectic ceramic prepared by laser floating zone[END_REF]. The microstructure corresponding to the lower Cr concentration (0.4at %) is much finer. In eutectic solidified with low Cr concentration smaller phase sizes, the diffusion was enhanced.

It is registered a significant coarsening of the fine microstructure. It seems that the coarsening process is Cr 3+ diffusion through Y 3 Al 5 O 12 and Al 2 O 3 .

The Cr-doped Al 2 O 3 -YAG-ZrO 2 system the microstructure is composed of three phases, Al 2 O 3 , YAG and ZrO 2 . The main phases are YAG and alumina, which respectively have volume fractions of 42 vol% and 40 vol% (Fig. 6). The zirconia phase is distributed mainly at the interface of the alumina and YAG phases or in the alumina phase with 1-5µm. Intragranular ZrO 2 with very small (0.1-0.8 µm) size rounded YAG phase. The longitudinal sections of the solidified samples show a "broken" or irregular lamellar microstructure. The lamellae are elongated along the solidification direction. In the ternary system, as a function of Cr concentration from 0.4 to 1.9 at%, we did not registered an increasing of YAG grains and magnification of the microstructure as observed in the Al 2 O 3 -YAG binary system. The microstructure of the ternary system is finer and with more curved interfaces than that of the Cr-doped Al 2 O 3 -YAG eutectic system solidified at the same rate. Whatever the Cr concentration (0.4-1.9at %), the homogeneous character disappears giving way to a colonies apparition but the microstructures are interconnected. Both YAG and ZrO 2 phases in the Cr-doped ternary eutectics showed different morphology than in their respective Al 2 O 3 -based binary eutectic system [START_REF] Pena | Processing, microstructure and mechanical properties of directionally-solidified Al 2 O 3 -Y 3 Al 5 O 12 -ZrO 2 ternary eutectics[END_REF]32,33], but Cr addition don't affect the shape of ZrO 2 particle and the script size of the YAG phase was found to be uniform for each cross section investigated. The SEM observations show that the Cr doping in the ternary system leads, in our case, to an excess of YAG (in grey) marked by the formation of large primary crystals. The Al 2 O 3 phase (black) is also slightly in excess compared to the garnet phase (grey).

Residual stress

The thermal elastic residual stresses of eutectics in the solidification process is caused by different thermal expansion mismatch between the α-Al 2 O 3 , YAG and ZrO 2 :Y phase [START_REF] Song | Longitudinal cross-section microstructure of growth striation in Al 2 O 3 /Y 3 Al 5 O 12 /ZrO 2 directionally solidified eutectic ceramic prepared by laser floating zone[END_REF]34].The fluorescence of Ruby single crystal is taken as reference to measure the residual stresses due to the electronic Cr 3+ levels is characterized by R1 an R2 luminescence lines, in most instances, the R2-line shift could be used to calculate approximate values of the hydrostatic stress σ h , the relation follows [START_REF] Mazerolles | New microstructures in ceramic materials from the melt for high temperature applications[END_REF][START_REF] He | Polarization Dependence of the Cr 3+ R-Line Fluorescence from Sapphire and Its Application to Crystal Orientation and Piezospectroscopic Measurement[END_REF]:

Δν 2 =7.61σ h ,
Where Δν 2 refers to the peak shift of R2-line (expressed in units of cm -1 ) and σ h present the hydrostatic stress given in units of GPa. Fig. 7 and Fig. 8 shows the fluoresce spectra of Cr 3+ present in the in the alumina phase of Cr doped Al 2 O 3 -YAG/Al 2 O 3 -YAG-ZrO 2 solidified eutectic. The evolution of the average hydrostatic stress measured on the transversal and longitudinal section of the samples is shown in Fig. 7 and Fig 8, the negative values of the hydrostatic stress indicate that the alumina phase is under compression, which is observed in references [START_REF] Benamara | Microstructure and crystallography of Al 2 O 3 -Y 3 Al 5 O 12 -ZrO 2 ternary eutectic oxide grown by the micropulling down technique[END_REF][START_REF] Pardo | Piezospectroscopic study of residual stresses in Al 2 O 3 -ZrO 2 directionally solidified eutectics[END_REF]. Whatever the transversal and longitudinal microstructure, the hydrostatic stress of binary eutectics less than that of ternary eutectics with same doped concentration due to distribution of ZrO 2 particles in the microstructure affected the residual stresses. The higher concentration of Cr-doped eutectic of Chinese script morphology is less compressed than that of lower concentration. In a word, the variation of the residual stresses of Cr doped Al 2 O 3 -YAG/Al 2 O 3 -YAG-ZrO 2 solidified eutectics, which not only depends on the cooling rate and time but also affected by the dopant and the concentration (Fig. 9).

Emission and excitation spectra

The room temperature (300K) emission spectra of Cr-doped Al 2 O 3 -YAG/Al 2 O 3 -YAG-ZrO 2 eutectics in the wavelength range of 600-900 nm are recorded under green (532 nm) and blue excitation light (450 nm) are shown in Fig. 10. Under the green excitation, Cr-doped Al 2 O 3 is clearly identified [START_REF] Sayir | The effect of the microstructure on mechanical properties of directionally solidified Al 2 O 3 /ZrO 2 (Y 2 O 3 ) eutectic[END_REF]. The sharp line centered at 694 nm corresponds to the forbidden transitions spin from 2 E to 4 A 2 level, referred to R-line, and the broad band centered at 774 nm is due to the phonon-assisted 4 T 2 to ground state 4 A 2 . This last transition was mostly observed when Cr-doped Al 2 O 3 is higher than 0.5 at%. Indeed, the relative intensity of broad band centered at 774 nm is increasing with the concentrations [START_REF] Sayir | The effect of the microstructure on mechanical properties of directionally solidified Al 2 O 3 /ZrO 2 (Y 2 O 3 ) eutectic[END_REF]. This effect is magnified in Cr-doped ternary eutectics. Moreover, under the blue excitation, an additional sharp line around 688 nm appears; it is attributed to the 2 E→ 4 A 2 transition of Cr-doped YAG phase [START_REF] Capeloto | Preparation, structural and spectroscopic study of sol-gel-synthesized Cr 3+ :Al 2 O 3 powder[END_REF]. The low temperature (77K) emission spectra of Cr-doped binary and ternary eutectics in the wavelength range of 680-850 nm were recorded also under excitation at 532 and 450 nm excitation as shown in Fig. 11. The broad emission band with peaks at 774 nm is observed due to 4 T 2 → 4 A 2 transition, in good agreement with R.C. Powell et al. [START_REF] Wamsley | The effect of pressure on the luminescence of Cr 3+ : YAG[END_REF]. At low temperature, the 4 T 2 emission band is more structured but remains as large as at RT, however, the effect of decreasing temperature strongly affects the structure of the spectra in the 690-710 nm range: splitting of the Cr:Al 2 O 3 R lines and magnification of the doublet observed in its edge around 700 nm. In order to analysis the rich luminescence spectrum in this range, the emission covering the spectral range from 680 nm to 710 nm is shown Fig. 12. Under green excitation at 532 nm, the first sharp lines lying at 693.4 nm and 692.0 nm are assigned to R1 E→ 4 A 2 and R2A→ 4 A 2 lines, respectively, due to the spin-orbit coupling and then, the splitting of the radiative transition 2 E→ 4 A 2 in Cr 3+ ions in octahedral crystal field [START_REF] Powell | Fluorescence studies of energy transfer between single and pair Cr 3+ systems in Al 2 O 3[END_REF][START_REF] Geller | Crystal chemistry of the garnets[END_REF]. Whereas, the second doublet of sharp lines at 704.0 nm and 700.9 nm corresponds to Cr 3+ -Cr 3+ pair lines which are designated N1 and N2 in Al 2 O 3 , respectively [START_REF] Wamsley | The effect of pressure on the luminescence of Cr 3+ : YAG[END_REF]. The ratio between the N and R-line intensity slightly increases in the ternary eutectics. In addition, under blue excitation (450 nm), a third doublet of sharp lines appears at 686.3 nm and 687.3 nm corresponding to the splitting of Cr R line in YAG. The relative intensity of N lines and R lines increases with the Cr concentration as expected. Under 532 nm excitation, no difference between ternary and binary eutectics emission spectra is observed whereas under 450 nm excitation at least two additional lines appear 689.5 nm and 696.7 nm in the ternary compound coming from Cr-doped YAG phase. Indeed under selective excitation in the 685-700 nm range, these additional lines only appear when excited in the two 687.3 and 686.0 nm Cr-YAG R lines. The high temperature emission spectra of eutectics from 315 to 673 K are shown in Fig. 13. Under 532 nm excitation of the Cr in the alumina phase, when the temperature is increasing, the 4 T 2 level becomes more and more thermally populated then the intensity of the broadband is increasing relatively to the line (694 nm) corresponding to the 2 E→ 4 A 2 emission. Under excitation of 450 nm, the line at 688 nm, 2 E→ 4 A 2 of Cr in YAG-phase, rapidly disappears (at already 100℃). Indeed when the temperature increases, the both 4 A 2 → 4 T 2 and 4 A 2 → 4 T 1 absorption or excitation bands, shown in the Fig. 14, become much broader and then, at 450 nm the excitation of Cr in alumina phase is enhanced. This result points out that the high temperature measurements corroborate that the majority of Cr 3+ ion entered favorably in the Al 2 O 3 phase than in the YAG phase. This behavior is observed in both ternary and binary eutectics.

Fluorescence decay times

The fluorescence decay curves of the chromium luminescence as a function of the Cr concentration were recorded under 532 and 450 nm excitation at 300K and 77K, then the lifetime constants are gathered in Table 3 and Table 4 respectively. As the decay curves become non-exponential for the two highest concentrations as shown in the fig. 15 we calculated the integrated lifetime. As expected, at RT, the long lifetimes of the Cr 3+ R-line 2 E→ 4 A 2 spin forbidden transition at 694.3 nm in Al 2 O 3 phase is decreasing from 3.84 ms to 0.94 ms when the concentration increases from 0.4 to 1.9 at. % Cr-doped for the binary eutectics. The concentration quenching effect in more pronounced in Cr-doped ternary, the lifetime being as short as 0.21 ms for 1.9 Cr at. %. This behavior is also noticeable when comparing luminescence decays of the broad band centered at 774.3 nm in the binary and ternary systems. For this wavelength, the decay curves are particularly non-exponential due to the competition of the relaxation process from the excited level to the emitting 4 T 2 level and the partial thermal equilibrium with the 2 E levels. However, this thermal coupling being less efficient at low temperature (77 K), it is observed that the values of decay times of the 4 T 2 → 4 A 2 allowed transition measured at 773.0 nm becomes shorter than at 300 K.

Finally, the decay time of Cr-doped binary eutectics is in good agreement with Cr 3+ :Al 2 O 3 powder [START_REF] Sayir | The effect of the microstructure on mechanical properties of directionally solidified Al 2 O 3 /ZrO 2 (Y 2 O 3 ) eutectic[END_REF], and Ruby bulk crystals [START_REF] Wamsley | The effect of pressure on the luminescence of Cr 3+ : YAG[END_REF] especially considering the lifetime of about 1ms for 2 at% of the Cr-Cr pair emission of the N-lines measured at 704nm under 532 nm at low temperature. It is interesting to note that the lifetime of the emission of Cr pairs is much shorter in the ternary 0.3ms than in the binary eutectics 0.8 ms.

Fig. 15 Fluorescence decay curves of the 694.3 nm emission under 532 nm excitation in Cr-doped binary eutectics at RT. Now considering the luminescence decay time of Cr-doped YAG phase at 687.3 nm recorded under 450 nm excitation , the lifetime at 77 K binary eutectic is measured about 7.28 ms for 0.4 at. % Cr-doped, then it is a little smaller than the 8.8 ms measured in 0.05% at 7 K [START_REF] Capeloto | Preparation, structural and spectroscopic study of sol-gel-synthesized Cr 3+ :Al 2 O 3 powder[END_REF][START_REF] Mykhaylyk | Multimodal non-contact luminescence thermometry with Cr-doped oxides[END_REF] but is slightly longer than that measured here in the Al 2 O 3 phase in the same sample. It can be explained by a lower intermediate ligand crystal field strength Dq/Bp <2.1 in YAG [START_REF] Vink | Meijerink Electron-phonon coupling of Cr 3+ in YAG and YGG[END_REF] than in Al 2 O 3 (Dq/B>2.33) [START_REF] Shen | Effect of pressure and temperature on the lifetime of Cr3+ in yttrium aluminum garnet[END_REF] while the Cr 3+ octahedral sites are slightly distorted [START_REF] Llorca | Directionally solidified eutectic ceramic oxides[END_REF] in YAG. At room temperature the lifetime 1.77 ms in both binary and ternary eutectics is in total agreement with the value given by Bray et al [START_REF] Capeloto | Preparation, structural and spectroscopic study of sol-gel-synthesized Cr 3+ :Al 2 O 3 powder[END_REF][START_REF] Vink | Meijerink Electron-phonon coupling of Cr 3+ in YAG and YGG[END_REF]. From table 2 and 3, one can note that the temperature quenching effect is stronger for the YAG phase than in ruby-phase while the phonon energy cutoff is rather the same , 865 cm -1 and 910 cm -1 [START_REF] Agachi | Luminescence spectroscopy of Cr 3+ in Al 2 O 3 polymorphs[END_REF] respectively. In the contrary the concentration quenching is larger in Al 2 O 3 than in the YAG phase, indeed the lifetime decreasing ten times between the highest and the lowest concentration at 77 K in Al 2 O 3 , and only three times in YAG. It suggests that the concentration of Cr in the YAG-phase should be lower than in Al 2 O 3 -phase due to a lower segregation coefficient in YAG .Finally, as for Cr in Al 2 O 3 phase, the decays become also shorter in the ternary compound for Cr in the YAG phase possibly because of the higher Cr concentration in ternary eutectics as already suggested from the analysis of the emission spectra or by the decay curves of the N-line emission of pairs. Finally, the dynamics of the new emission lines at 689 and 696 nm is the same as the Cr luminescence in the YAG-phase.

Conclusion

In this work, Cr-doped Al 2 O 3 -YAG binary and Al 2 O 3 -YAG-ZrO 2 ternary systems of eutectic ceramics were solidified from the melt by using the μ-PD technique. The coloration, the morphology and microstructure, the residual stress and the luminescence have been investigated. As a function of Cr-dopant concentration and the binary or ternary system, the relationship between the microstructure and the optical properties and, as a result, solidified eutectic ceramic with optimized microstructures have been elaborated The Cr-doped eutectics present a red color. Whatever the dopant concentration, mainly two phases (Al 2 O 3 -YAG) in case of binary system and three phases (Al 2 O 3 -YAG-ZrO 2 ) in case of ternary system are observed. The Chinese script microstructure, colonies and colony boundaries are observed in ternary eutectics. From the different size of YAG grains, the microstructure formation of binary eutectics depends on the chromium dopant concentration. Moreover, Al 2 O 3 and YAG phases show faceted morphologies, but for ZrO 2 phase, growth tends to a weakly faceted manner and forms rods or lamellae. Whatever the Cr dopant concentration, the hydrostatic stress of the binary eutectics are less than that of ternary eutectics.

The spectroscopic properties of the Cr-doped eutectic clearly show that Cr 3+ ions enter in both Al 2 O 3 and YAG phases in octahedral site. The increase of the intensity of the 4 T 2 → 4 A 2 emission band relatively to the 2 E→ 4 A 2 lines, in one hand, and the intensity ratio between the N-lines and R-lines recorded at 77K, in the other hand, shows that the Cr concentration in Al 2 O 3 phase should be higher in the ternary eutectic that in the binary eutectic. The analysis of the emission lifetime in both compound corroborate this observation: the luminescence quenching by concentration is emphasis in the ternary eutectics. Furthermore, the concentration of Cr in YAG-phase is smaller than in Al 2 O 3 phase. Finally, new lines, which are coupled to the YAG-phase, appear at low temperature in the highest concentrated ternary eutectics.

Finally, the luminescence, the spectroscopy of the doped Al2O3-YAG and Al2O3-YAG-ZrO2 systems are still in the beginning in comparison with chemical and mechanical properties and research efforts in this field are still very recent. However, from a study of the literature it can be observed that the combination of the oxide eutectic compositions for optics has not yet been explored completely and, we are probably still in the early stage of research in this field.

  2 O 3 , Al 2 O 3 , Y 2 O 3 and ZrO 2 powders. The powders were mixed in agate mortar at eutectic proportion according to the binary (80 mol%Al 2 O 3 /20mol%Y 2 O 3 ) and the ternary (65mol%Al 2 O 3 /16mol%Y 2 O 3 /19mol%ZrO 2 ) eutectic composition. The detailed compositions of different prepared materials and references are shown in Table

Fig. 1 .

 1 Fig. 1. Schematic illustration of the drop at the bottom of the capillary die (left), CCD camera vision of the growth initiation through the crucible and after heater (right)

Fig. 2 .Fig. 3 .

 23 Fig. 2. Cr-doped eutectic ceramics solidified from the melt by µ-PD (pulling rate 0.3 mm/min) CrBE1: Al 2 O 3 -YAG binary (Cr=1.9at%) CrBE2: Al 2 O 3 -YAG binary (Cr=1.5at%) CrBE3: Al 2 O 3 -YAG binary (Cr= 0.7at%) CrBE4: Al 2 O 3 -YAG binary (Cr=0.4at%) CrTE1: Al 2 O 3 -YAG-ZrO 2 Ternary (Cr=1.9at%) CrTE2: Al 2 O 3 -YAG-ZrO 2 Ternary (Cr=1.5at%) CrTE3: Al 2 O 3 -YAG-ZrO 2 Ternary (Cr= 0.7at%) CrTE4: Al 2 O 3 -YAG ZrO 2 Ternary (Cr=0.4at%)

Fig. 5 .

 5 Fig. 5. Transversal microstructure evolution as a function of concentration. (a) binary eutectic Cr 0.4at%, (b) binary eutectic Cr 0.7at%, (c) binary eutectic Cr 1.5at%, (d) binary eutectic Cr 1.9at%, (e) ternary eutectic Cr 0.4at%, (f) ternary eutectic Cr 0.7at%, (g) ternary eutectic Cr 1.5at%, (h) ternary eutectic Cr 1.9at%.

Fig. 6 .

 6 Fig. 6. Longitudinal microstructure evolution as a function of concentration. (a) binary eutectic Cr 0.4at%, (b) binary eutectic Cr 0.7at%, (c) binary eutectic Cr 1.5at%, (d) binary eutectic Cr 1.9at%, (e) ternary eutectic Cr 0.4at%, (f) ternary eutectic Cr 0.7at%, (g) ternary eutectic Cr 1.5at%, (h) ternary eutectic Cr 1.9at%.

Fig. 7 .

 7 Fig. 7.Ruby fluorescence (R1 and R2 bands) excited with a 473 nm laser in transversal sections of the eutectics as a function of concentration.

Fig. 8 .Fig. 9 .

 89 Fig. 8. Ruby fluorescence (R1 and R2 bands) excited with a 473 nm laser in longitudinal sections of the eutectics as a function of concentration.

Fig. 10 .

 10 Fig. 10. The room temperature (300K) emission spectra of eutectics (a) Cr-doped binary eutectics under excitation at 532 nm. (b) Cr-doped ternary eutectics under excitation at 532 nm. (c) Cr-doped binary eutectics under excitation at 450 nm. (d) Cr-doped ternary eutectics under excitation at 450 nm.

Fig. 11 .

 11 Fig. 11. The low temperature (77 K) emission spectrum of eutectics (a) Cr-doped binary eutectics under excitation at 532 nm. (b) Cr-doped ternary eutectics under excitation at 532 nm. (c) Cr-doped binary eutectics under excitation at 450 nm. (d) Cr-doped ternary eutectics under excitation at 450 nm.

Fig. 12 .

 12 Fig. 12. The low temperature (77 K) emission spectrum of eutectics range from 680 nm to 710 nm under excitation at 532 nm a), b); 450 nm c), d) and selective excitations at 694.2 and 687.3 nm e).

Fig. 13 .

 13 Fig. 13. High temperature emission spectrum, using a long-pass filter at 650 nm, of eutectics from 373 to 673 K (a) Cr-doped binary eutectic under excitation at 532 nm. (b) Cr-doped ternary eutectic under excitation at 532 nm. (c) Cr-doped binary eutectic under excitation at 450 nm. (d) Cr-doped ternary eutectic under excitation at 450 nm.

Fig. 14 .

 14 Fig. 14. Excitation spectrum of eutectics at room temperature (a) Cr-doped binary eutectics by monitoring the emission at 694 nm. (b) Cr-doped ternary eutectics by monitoring the emission at 694 nm. (c) Cr-doped binary eutectics by monitoring the emission at 688 nm. (d) Cr-doped ternary eutectics by monitoring the emission at 688 nm.

Table 1 . The different compositions used for melt solidification

 1

		Binary Eutectic ( Al 2 O 3 (80%) / Y 2 O 3 (20%) )
	Sample (Reference) Cr BE1 Cr BE2 Cr BE3 Cr BE4
	Dopant	1.9at% 1.5at%	0.7at% 0.4at%
	Ternary Eutectic ( Al 2 O 3 (65%) / Y 2 O 3 (16%) / ZrO 2 (19 %) )
	Sample (Reference)	Cr TE1 Cr TE2 Cr TE3 Cr TE4
	Dopant	1.9at% 1.5at% 0.7at%	0.4at%

Table 2 . Lattice parameters of Cr doped Al 2 O 3 -YAG/Al 2 O 3 -YAG-ZrO 2 as a function of the dopant concentration

 2

		Al 2 O 3 R-3c (Å)	Y 3 Al 5 O 12 Ia-3d (Å)	ZrO 2 Fm-3m(Å)
	PDF files	a= 4.7585,c= 12.9906	a=12.0020
	Cr BE1	a=4.7648, c=13.0074	a=12.0139
	Cr BE2	a=4.7624, c=12.9999	a=12.0110
	Cr BE3	a=4.7624, c=13.0017	a=12.0130
	Cr BE4	a=4.7608, c=12.9935	a=12.0094
	Cr TE1	a=4.7650, c=13.0072	a=12.0123	a=5.1605
	Cr TE2	a=4.7642, c=13.0037	a=12.0135	a=5.1619
	Cr TE3	a=4.7611, c=12.9957	a=12.0093	a=5.1625
	Cr TE4	a=4.7602, c=12.9955	a=12.0084	a=5.1628

Table 3 . The decay times (in ms) of various emission under excitation 450 nm and 532 nm at 77 K.

 3

	_____________________________________________________________________
		Excitation 532 nm	Excitation 450 nm
	_____________________________________________________________________
	Sample	694.3 nm	774.3 nm	688.7 nm
	Cr BE1	0.94	0.55	1.30
	Cr BE2	1.34	1.24	0.95
	Cr BE3	2.56	1.60	1.63
	Cr BE4	3.84	-	1.78
	Cr TE1	0.21	0.09	0.33
	Cr TE2	0.63	0.97	0.64
	Cr TE3	1.29	0.92	1.33
	Cr TE4	3.30	1.08	1.77
	_____________________________________________________________________

Table 4 . The decay times (in ms) of various emission under excitation 450 nm and 532 nm at 77 K.

 4

	___________________________________________________________________
		Excitation 532 nm		Excitation 450 nm
	____________________________________________________________________
	Sample	694.3 nm 704.0 nm 773.0 nm	687.3 nm
	Cr BE1	0.74	0.81	0.61	4.80
	Cr BE2	0.56	0.81	0.72	3.10
	Cr BE3	2.10	1.87	1.08	6.27
	Cr BE4	6.13	4.37	0.97	7.28
	Cr TE1	0.30	0.26	0.30	1.67
	Cr TE2	0.19	0.28	0.54	2.04
	Cr TE3	0.77	0.80	0.62	5.54
	Cr TE4	3.92	3.81	1.34	6.21
	_____________________________________________________________________