Romane Leboutet

Yanfang Chen

Renaud Legouis
email: renaud.legouis@i2bc.paris-saclay.fr

Emmanuel Culetto
email: authors.emmanuel.culetto@i2bc.paris-saclay.fr

Mitophagy during development and stress in C. elegans

Keywords: mitochondria, mitochondria homeostasis, mitophagy, Caenorhabditis elegans, paternal mitochondria elimination, aging, stress, heteroplasmy

Mitochondria is a key cellular organelle, which is tightly supervised by multiple oversight cellular mechanisms regulating mitochondrial biogenesis and mitochondria maintenance and/or elimination. Selective autophagy of mitochondria, id est mitophagy, is one of the cellular mechanisms controlling mitochondria homeostasis. The nematode Caenorhabditis elegans has recently emerged as a powerful model organism to study the roles and functions of mitophagy. We present here the current knowledge on cellular and molecular mechanisms underlying the selective elimination of mitochondria by autophagy in C. elegans in the context of developmental processes, aging and adaptive responses to various stresses

2 1) Mitochondria homeostasis is regulated through multiple supervision systems.

Mitochondrial functions

Mitochondria are double-membrane organelles originating from an endosymbiotic event whereby an alpha protobacteria has been domesticated by eukaryotic cell [START_REF] Sagan | On the origin of mitosing cells[END_REF]. This organelle possesses its own genome (mtDNA) [START_REF] Nass | Intramitochondrial fibers with DNA characteristics. Ii. Enzymatic and other hydrolytic treatments[END_REF] and integrates multiple catabolic pathways through oxidative phosphorylation to produce adenosine triphosphate (ATP) [START_REF] Hall | Oxidative phosphorylation, not glycolysis, powers presynaptic and postsynaptic mechanisms underlying brain information processing[END_REF]. This organelle shelters essential metabolic pathways such as the Krebs cycle and fatty acid β-oxidation, as well as the biosynthesis of heme and lipids and part of the urea cycle. Mitochondria is the major site for reactive oxygen species (ROS) production, which could act either as signaling molecule when produced at low doses or be detrimental for the cells when produced at higher dose [START_REF] Shadel | Mitochondrial ROS Signaling in Organismal Homeostasis[END_REF].

Mitochondria are involved in the early step of the signaling pathway of apoptosis and participate to cell calcium homeostasis. Mitochondria are dynamic organelles that consistently undergo fission and fusion events and move over the cell cytoskeleton tracks [START_REF] Summerhayes | Effect of microtubules and intermediate filaments on mitochondrial distribution[END_REF]. Such a dynamic is essential for the quantitative and qualitative adaptation of mitochondria to the cell physiology variations, and the participation to calcium regulation and spatial segregation of mtDNA and matrix components. This dynamic allows the specific localization of mitochondria to subcellular places where energy is required [START_REF] Sun | Motile axonal mitochondria contribute to the variability of presynaptic strength[END_REF], and eventually modulate the way mitochondria establish contact sites with other organelles.

Large mechanochemical GTPases from the Dynamin family are responsible for the fluidity of the mitochondria network reticulum by controlling fusion and fission of mitochondria [START_REF] Praefcke | The dynamin superfamily: universal membrane tubulation and fission molecules?[END_REF][START_REF] Van Der Bliek | Mechanisms of Mitochondrial Fission and Fusion[END_REF]. Because mitochondria are essential to the cell, its dysfunction could jeopardize cell homeostasis and trigger apoptosis, inflammation or senescence [START_REF] Bock | Mitochondria as multifaceted regulators of cell death[END_REF]. Indeed, numerous human diseases are associated with mitochondria dysfunction [START_REF] Alston | The genetics and pathology of mitochondrial disease[END_REF].

Mitochondrial maintenance

Mitochondrial homeostasis is the result of a constant equilibrium between biogenesis, reparation and degradation processes.

On the one hand, a series of mechanisms allow the building of new mitochondrion, increasing the mitochondrial mass. These processes include the replication of mtDNA, the expression of mitochondrial genes, the import of nuclear-encoded proteins and lipids, and mitochondrial dynamic, which encompasses transport, fusion and fission. Overall, these mechanisms constitute the mitochondrial biogenesis, which requires a tight crosstalk between two different genomes that is called the nucleo-mitochondrial interaction [START_REF] Grivell | Nucleo-mitochondrial interactions in mitochondrial gene expression[END_REF][START_REF] Melber | UPRmt regulation and output: a stress response mediated by mitochondrial-nuclear communication[END_REF].

On the other hand, there is a set of mechanisms involved in maintenance, reparation and eventually the removal of dysfunctional and damaged mitochondria. A first level of action is the regulation of protein degradation via mitochondrial proteases and the ubiquitinand proteasome-dependent OMMAD (outer mitochondrial membrane associated degradation) [START_REF] Xu | Mitochondrial E3 ubiquitin ligase MARCH5 controls mitochondrial fission and cell sensitivity to stress-induced apoptosis through regulation of MiD49 protein[END_REF]. A second mechanism is the mitochondrial unfolded protein response (UPR mt ), an evolutionary conserved adaptive transcriptional stress response, which promotes normal mitochondrial function recovery. UPR mt modifies nuclear genes transcription to foster protein folding and to prevent aggregate formation, to induce proteases and to limit ROS accumulation. Noticeably, UPR mt can also promotes mitochondrial biogenesis by stimulating the mtDNA replication and the mitochondrial import of lipids and proteins [START_REF] Naresh | Signaling and Regulation of the Mitochondrial Unfolded Protein Response[END_REF]). An additional maintenance mechanism relies on the formation of mitochondrial-derived vesicles that allows the selective elimination of mitochondrial damaged proteins and lipids in response to an oxidative stress [START_REF] Sugiura | A new pathway for mitochondrial quality control: mitochondrial-derived vesicles[END_REF]. It depends on the formation of buds, containing the jeopardized mitochondrial material, which are addressed to late endosomes for further degradation within the lysosomes [START_REF] Soubannier | A vesicular transport pathway shuttles cargo from mitochondria to lysosomes[END_REF].

Finally, the massive elimination of dysfunctional mitochondria could occur through jettisoned exosphere containing mitochondria [START_REF] Melentijevic | C. elegans Neurons Jettison Protein Aggregates and Mitochondria Under Neurotoxic Stress[END_REF], endosomal microautophagy [START_REF] Hammerling | Retinol as electron carrier in redox signaling, a new frontier in vitamin A research[END_REF] or mitochondrial-selective autophagy. Overall, these multiple mechanisms involved in quality control of mitochondria are modulated by intrinsic and environmental contexts. This review focusses on the functions and the molecular mechanisms of selective degradation of mitochondria by autophagy, id est mitophagy, in the model organism Caenorhabditis elegans.

2) Overview of the autophagy mechanisms

Autophagy allows the addressing of intracellular components to lysosome for degradation and recycling. It was first termed by de Duve [START_REF] De Duve | The lysosome[END_REF] to describe the cell degradation process taking place in the double-membrane organelle containing parts of cytosol and pieces of organelle structures. Indeed, three different types of autophagy co-exist within the cell depending on the mechanisms of delivery of cytoplasmic components to the lysosome: microautophagy, chaperone-mediated autophagy and macroautophagy [START_REF] Boya | Emerging regulation and functions of autophagy[END_REF]. Microautophagy (MI) is defined as the engulfment of cytosolic cargoes directly via invagination of the lysosomal/vacuolar membrane. It can degrade soluble proteins or even bigger cargoes such as portion of the nucleus, lipid droplets, mitochondria and endosomes [START_REF] Kissová | Selective and non-selective autophagic degradation of mitochondria in yeast[END_REF][START_REF] Tuttle | Selective autophagy of peroxisomes in methylotrophic yeasts[END_REF][START_REF] Tuttle | Divergent modes of autophagy in the methylotrophic yeast Pichia pastoris[END_REF][START_REF] Veenhuis | Degradation and turnover of peroxisomes in the yeast Hansenula polymorpha induced by selective inactivation of peroxisomal enzymes[END_REF][START_REF] Yokota | Formation of autophagosomes during degradation of excess peroxisomes induced by di-(2-ethylhexyl)phthalate treatment. II. Immunocytochemical analysis of early and late autophagosomes[END_REF]. MI depends on the activity of the Endosomal Sorting Complexes Required for Transport (ESCRT) machinery [START_REF] Oku | Evidence for ESCRT-and clathrin-dependent microautophagy[END_REF]. Interestingly a microautophagy related mechanism, named endosomal microautophagy (eMI), shares with MI many features but involves endosome [START_REF] Mukherjee | Selective endosomal microautophagy is starvation-inducible in Drosophila[END_REF][START_REF] Sahu | Microautophagy of cytosolic proteins by late endosomes[END_REF][START_REF] Uytterhoeven | Hsc70-4 Deforms Membranes to Promote Synaptic Protein Turnover by Endosomal Microautophagy[END_REF]. For example, in mouse embryonic fibroblasts, organelle such as mitochondria could be guided to the Rab5 positive early endosome and are engulfed in a ESCRT-0, -I and -II-dependent mechanism [START_REF] Hammerling | A Rab5 endosomal pathway mediates Parkindependent mitochondrial clearance[END_REF].

Chaperon-mediated autophagy (CMA) is another type of autophagy that directly imports substrates to lysosome for degradation [START_REF] Majeski | Mechanisms of chaperone-mediated autophagy[END_REF][START_REF] Massey | Pathophysiology of chaperone-mediated autophagy[END_REF]. In a similar way to eMI, CMA degrades protein, which interact with HSPA8 through a KFERQ like motif, and nearly 30% of cytosolic components could be degraded via CMA [START_REF] Dice | Peptide sequences that target cytosolic proteins for lysosomal proteolysis[END_REF]. Neither microautophagy nor CMA has been yet scrutinized in C. elegans, but while the former is evolutionary well conserved the latter is presumably not conserved in C. elegans, which is devoid of LAMP2A [START_REF] Eskelinen | Unifying nomenclature for the isoforms of the lysosomal membrane protein LAMP-2[END_REF][START_REF] Kaushik | The coming of age of chaperone-mediated autophagy[END_REF].

Macroautophagy (hereafter referred to as autophagy) is present in all eukaryotes and characterized by the formation of a double-membrane vesicle, the autophagosome, which is dedicated to cytoplasmic materials sequestration and delivery to the endo-lysosomal system.

Initially, autophagy has been studied in starvation condition and was considered a nonselective degradation process, during which autophagosomes engulfed indistinctly bulk part of the cytoplasm (Figure 1). However, a growing number of experimental results demonstrated that selective autophagy could target specific cargoes using a series of dedicated receptors. Autophagy receptors can specifically recognize the cargoes to be degraded and bridge them to autophagosomes by interacting with Atg8/LC3 [START_REF] Komatsu | Selective autophagy regulates various cellular functions[END_REF].

Based on the engulfed cargo, selective autophagy was further classified into mitophagy (mitochondria), pexophagy (peroxisome), nucleophagy (nucleus), ER-phagy (ER), lipophagy (lipid droplets), aggrephagy (protein aggregates), xenophagy (pathogens) and so forth [START_REF] Galluzzi | Molecular definitions of autophagy and related processes[END_REF].

Molecular machinery is well conserved between eukaryotes and Atg genes have been initially characterized in yeast [START_REF] Tsukada | Isolation and characterization of autophagy-defective mutants of Saccharomyces cerevisiae[END_REF]. However, Atg genes have been duplicated during evolution resulting in a more complex situation in mammals.

In the last 15 years, C. elegans has emerged as a powerful model to study autophagy [START_REF] Chen | Approaches for Studying Autophagy in Caenorhabditis elegans[END_REF] and genetic screens identified new autophagy genes some of which are absent in yeast [START_REF] Tian | C. elegans screen identifies autophagy genes specific to multicellular organisms[END_REF].

3) Mitophagy is a selective degradative process that contributes to mitochondria quality control

Generalities

Mitophagy is the process that selectively engulfs pieces or whole mitochondrion into autophagosomes, to form mitoautophagosome that subsequently fused with lysosomes for degradation. The term mitophagy was first coined by Lemaster [START_REF] Lemasters | Selective mitochondrial autophagy, or mitophagy, as a targeted defense against oxidative stress, mitochondrial dysfunction, and aging[END_REF] based on the work performed in yeast by Camougrand and colleagues [START_REF] Kissová | Uth1p is involved in the autophagic degradation of mitochondria[END_REF]. Since this initial molecular description of mitophagy in yeast, multiple in vivo evidences for the physiological importance of mitophagy have been put forward in other organisms. While in yeast, mitophagy is mediated by the receptor ATG32, C. elegans and mammals present several receptors and signalling pathways regulating mitophagy (Table1). Mitophagy is involved in multiple physiological processes, including programmed red blood cells maturation, apoptotic cell death, stress-responses (hypoxia, heat shock, etc…), and neurodegenerative diseases that challenge mitochondria functioning. The discovery of mitophagy and the detailed description of its various molecular mechanisms and underlying functions have been recently reviewed [START_REF] Montava-Garriga | Outstanding Questions in Mitophagy: What We Do and Do Not Know[END_REF][START_REF] Palikaras | Mechanisms of mitophagy in cellular homeostasis, physiology and pathology[END_REF][START_REF] Pickles | Mitophagy and Quality Control Mechanisms in Mitochondrial Maintenance[END_REF]. Therefore, we briefly state a general overview of the principles that lead to selective elimination of mitochondria via autophagy.

Mitophagy events covers three main categories: the basal, the stress-induced and the developmentally programmed mitophagy. The mitophagy flux can be divided into two major steps, the tagging of mitochondria to be eliminated and the subsequent formation of the mitoautophagosome.

Usually, mitophagy is triggered by modifications of the cell environment or stress conditions or developmental signalling that lead to mitochondria activity/parameters alteration and specific mitochondria tagging. It means that environmental or cellular stresses or other mitophagy stimuli are translated into a specific molecular modification of the outer mitochondrial membrane that primes mitochondria for degradation.

Thereafter, primed mitochondria recruits the autophagy machinery leading to the formation of an isolation membrane (IM), also called phagophore, nearby mitochondria. Finally, IM enwraps the targeted mitochondria and closes to form a mitoautophagosome, which subsequently fuses with the lysosome for degradation.

Tagging unwanted mitochondria

Overall, the selective elimination of mitochondria via autophagy requires the specific localization to the mitochondrial surface of a set of molecules recognized as cargoes for autophagosome (Table 1). These evolutionary conserved pathways include ubiquitin and autophagy receptors, both potentially harbouring post-translational modification such as polyubiquitination and/or phosphorylation. The best understood mechanism involves PTENinduced putative kinase protein 1 (PINK1) and the E3 ubiquitin ligase Parkin (Figure 2).

However, alternative E3-ubiquitine ligases could be involved such as Mul1 [START_REF] Rojansky | Elimination of paternal mitochondria in mouse embryos occurs through autophagic degradation dependent on PARKIN and MUL1[END_REF]. When mitochondria are damaged and loss their membrane potential, PINK1 is stabilized on the OMM and thus recruits Parkin, which subsequently ubiquitylates proteins at the OMM. Then, these multiple ubiquitin motifs are recognized by a set of autophagy receptor such as P62, NBR1, NDP52, Optineurin, TAXBP1 and ALLO-1 (Table 1). These receptors harbour both an ubiquitin binding site that interacts with ubiquitylated OMM proteins and a LC3 interacting region (LIR) motif that connects onto the IM, thus enabling the tethering between both organelles (Figure 2). Alternatively, a series of ubiquitin-independent mitophagy employ specific autophagy receptors directly localized to the mitochondrial membranes, such as Atg32, BCL2L13, DCT-1/NIX/BNIP3L, BNIP3, FUNDC1, FKBP8, PHB2 and AMBRA1 (Figure 2 and Table 1). In this case, the mitophagy stimulus triggers the increase of autophagy receptor density at the OMM by both transcriptional and posttranslational regulations to promote and strengthen direct interaction with Atg8/LC3 autophagy proteins. Additionally, alternative mechanisms allow the exposure of inner membrane of the mitochondria (IMM) autophagy receptor, PHB-2 and cardiolipin. This set of mitophagy receptors is differently employed depending on the organism, cellular context, developmental stage and environment. For example, when yeast, are submitted to nitrogen starvation the OMM protein Atg32, is upregulated and acts as an autophagy receptor, through its Atg8 interacting motif, leading to the specific recognition of mitochondria by forming autophagosome. Similarly, during the maturation of mammalian red blood cell, mitochondria are removed by a mechanism involving mitophagy via increased expression of the OMM protein NIX, which recruits isolation membranes to mitochondria through its LIR (Table 1).

Formation and maturation of the mitoautophagosome

The mitophagy receptors, mediate the interactions with the autophagy machinery to trigger autophagosome formation around mitochondria. Damaged mitochondria represent a platform for the multiple formation of mitophagosomes [START_REF] Dalle Pezze | ATG13 dynamics in nonselective autophagy and mitophagy: insights from live imaging studies and mathematical modeling[END_REF][START_REF] Zachari | Selective Autophagy of Mitochondria on a Ubiquitin-Endoplasmic-Reticulum Platform[END_REF]. Different receptors interact with the autophagosome nucleation UNC-51-Like Kinase 1 (ULK1) complex or the ATG8/LC3/GABARAP autophagosomal proteins. For example, ULK1 can interact directly with FUNDC1 and BCL2L13 whereas NDP52 contact FIP200 which is a ULK1 protein complex member to trigger mitochondria degradation [START_REF] Vargas | Spatiotemporal Control of ULK1 Activation by NDP52 and TBK1 during Selective Autophagy[END_REF]. Atg8/LC3/GABARAP are ubiquitin-like proteins, which localize to autophagosomal membrane through conjugation to phosphatidylethanolamine and mediate interactions with a number of mitophagy receptors possessing a LIR motif. Moreover, it has been shown that ATG8 homologues use a LIR motif to interact with ULK1 and could regulate autophagy (Alemu et al., 2012;[START_REF] Kraft | Binding of the Atg1/ULK1 kinase to the ubiquitin-like protein Atg8 regulates autophagy: Regulation of Atg1 kinase function[END_REF]. However, Atg protein including ULK1, WIPI-1, Atg14 and Atg16L1 can directly associate with depolarized mitochondria in the absence of LC3 [START_REF] Itakura | Structures containing Atg9A and the ULK1 complex independently target depolarized mitochondria at initial stages of Parkin-mediated mitophagy[END_REF]. In particular conditions, LC3/GABARAP homologs are dispensable for autophagosomes formation during PINK1/Parkin-dependent mitophagy but essential for autophagosomes-lysosome fusion [START_REF] Nguyen | Atg8 family LC3/GABARAP proteins are crucial for autophagosome-lysosome fusion but not autophagosome formation during PINK1/Parkin mitophagy and starvation[END_REF]. Additionally, studies in erythrocytes, suggest that the ubiquitin like conjugating system made of Atg5 and Atg7 is dispensable for mitophagy [START_REF] Nishida | Discovery of Atg5/Atg7-independent alternative macroautophagy[END_REF]. Overall, these studies question the importance of interactions between mitophagy receptors and Atg8/LC3 protein.

Importantly, recent publications showed that the extension of the autophagosomal membrane around the mitochondria is regulated by the Rab family of small GTPases and their canonical regulators Rab-GEF (GTP exchange factor) and Rab-GAP (GTPase activating protein) [START_REF] Tan | Rab7a and Mitophagosome Formation[END_REF]. During mitophagy, Rab7, whose activity is regulated by two Rab GAPs TBC1D15 and TBC1D17, is also recruited to the OMM where it mobilizes vesicles to build up autophagosomes. Both proteins are also recruited to the outer mitochondrial membrane via the OMM protein FIS and could interact via their LIR motifs with the nascent autophagosomal membrane containing LC3/GABARAP proteins. Inactivation of these GAPs lead to the accumulation around mitochondria of autophagosomal like membranes. It seems that these GAPs help to guide the extension of the autophagosomal membrane around mitochondria and spatially control the Rab7 activity which itself controls vesicle recruitment to the autophagosomal membrane [START_REF] Yamano | Mitochondrial Rab GAPs govern autophagosome biogenesis during mitophagy[END_REF]. Finally, once mitoautophagosome is formed it ultimately fuses with the lysosome for degradation. Before this ultimate step of mitophagy, mitoautophagosomes acquire different protein complexes to gain any capacity to fuse. These proteins complexes are tethering factors allowing the establishment of contact zones with lysosome and SNARE proteins to trigger membrane fusion between tethered organelles.

4) Tools to study mitophagy in C. elegans

In recent years, the model organism C. elegans has been a leading animal to study the physiological role of mitophagy (Figure 3). This organism allows addressing key biological questions regarding mitophagy functions during the ageing process and in the selective clearance of paternal mitochondria. C. elegans has multiple experimental advantages amongst them its genetic toolkit and its ease for cellular biology approaches. Specifically, strategies used to observe and analyse mitophagy in C. elegans are mainly based on imaging techniques employing combinations of photonic and electron microscopies. Immunostaining approach on fixed tissue to visualize both autophagosome and mitochondria has been developed.

Antibodies against

LGG-1 or LGG-2, the two homologues of the ubiquitin-like protein ATG8

that label the autophagosome membrane could be combined with marker of mitochondria. A colocalization analysis is indicative of possible mitophagy events or close contacts between both organelles. Hence, in C. elegans, live imaging of mitophagy events is often monitored by colocalization between fluorescent tagging of mitochondrial and autophagosomal markers (Figure 4). Multiple ways are available to visualize mitochondria, which have been recently

reviewed [START_REF] Rolland | How to analyze mitochondrial morphology in healthy cells and apoptotic cells in Caenorhabditis elegans[END_REF]. Briefly, multiple transgenic lines expressing different mitochondrial protein (either addressed to the OMM, IMM or the matrix) tagged with fluorophore are available. These strains include known C. elegans mitochondria autophagy receptors such as DCT-1/NIX or ALLO-1 [START_REF] Palikaras | Coordination of mitophagy and mitochondrial biogenesis during ageing in C. elegans[END_REF][START_REF] Sato | The autophagy receptor ALLO-1 and the IKKE-1 kinase control clearance of paternal mitochondria in Caenorhabditis elegans[END_REF].

Alternatively, mitochondria could be labelled with fluorescent dyes (red and deep red Mitotracker, tetramethylrhodamine ethyl ester (TMRE) and rhodamine 6G) [START_REF] Rolland | How to analyze mitochondrial morphology in healthy cells and apoptotic cells in Caenorhabditis elegans[END_REF].

C. elegans autophagosome are generally monitored in transgenic lines expressing either GFP or mCherry tagged LGG-1 and LGG-2 proteins. Interestingly, transgenic lines expressing autophagy proteins, LGG-1 and SQST-1, tagged with a novel fluorophore, CemOrange2 that is separable from GFP and far-red fluorescent proteins should facilitate multiplex imaging in the future [START_REF] Thomas | CemOrange2 fusions facilitate multifluorophore subcellular imaging in C. elegans[END_REF]. Several combination of organelle tags have been developed

by N. Tavernarakis and his colleagues. They created strains with a dual fluorescent reporter system that combines a mitochondria marker, the mitochondrial-targeted GFP, expressed along with an ubiquitously expressed LGG-1 tagged with DsRed. Additionally, they adapted the Rosella tool [START_REF] Rosado | Rosella: a fluorescent pH-biosensor for reporting vacuolar turnover of cytosol and organelles in yeast[END_REF] by constructing a C. elegans strain expressing the mitoRosella synthetic gene, to study mitophagy in C. elegans [START_REF] Palikaras | Coordination of mitophagy and mitochondrial biogenesis during ageing in C. elegans[END_REF]. The mitoRosella is a mitochondria-targeted pH sensitive GFP fused to the pH insensitive DsRed.

In the acidic autolysosome, the GFP part of the biosensor is quenched while the DsRed fluorophore is resistant. Thus, healthy/normal mitochondria could be observed thanks to both green and red signals they emitted. Subsequently, once mitophagy events occur, autophagosome engulfs mitochondria and then fuses with lysosome, which results in the quenching of the GFP signal and the reduction of the GFP/DsRED ratio indicative of the mitophagy flux. This tool has been originally adapted to study mitophagy in the large body wall muscle cells of C. elegans but also adapted to the nervous system (Fang et al., 2019). In addition, the overall mitochondrial homeostasis could be analysed through its mass modification. These observations could be assessed in C. elegans by quantifying the overall fluorescence signal from mitochondrial targeted GFP specifically expressed in intestinal cells under the control of the ges-1 promoter [START_REF] Palikaras | Coordination of mitophagy and mitochondrial biogenesis during ageing in C. elegans[END_REF]. Additionally, addressing the Timer protein to the mitochondria (MitoTimer), allows to monitor mitochondrial biogenesis and mitophagy [START_REF] Laker | A novel MitoTimer reporter gene for mitochondrial content, structure, stress, and damage in vivo[END_REF]. The dual emission fluorescent "Timer" probe initially fluoresces in green then shifts irreversibly to red fluorescence when it mature following a form of oxidation (dehydrogenation) of the Tyrosine 67 residue [START_REF] Terskikh | Fluorescent timer": protein that changes color with time[END_REF]. The MitoTimer Red/Green fluorescence ratio does not depend on the overall expression of MitoTimer nor in changes in mitochondria pH. On one hand, the green fluorescence level indicates newly synthetized MitoTimer protein addressed to the mitochondrial matrix and reflects the increase of MitoTimer production or import and mitochondrial biogenesis. On the other hand, the level of red fluorescence, which indicates older mitochondria, mainly depends on degradative pathways such as autophagy, mitochondrial proteases or the ubiquitin proteasome system. Overall, measurements of red fluorescence over the green signal emitted by MitoTimer allows collection of quantitative data that mirrors multiple parameters controlling the mitochondrial homeostasis and therefore should be interpreted cautiously for monitoring mitophagy. In C. elegans, MitoTimer has been so far only expressed in mechanosensory neuron but it is a promising complementary tool to monitor mitochondria biogenesis in other cells and tissues. Alternatively, a dual Fluorescent Protein tag fused to the mitochondrial protein TOMM-7 has been proposed to monitor the mitophagy flux by western blot [START_REF] Chapin | Tissue-specific autophagy responses to aging and stress in C. elegans[END_REF]. Finally, direct observation of mitoautophagosome could be performed in C. elegans with electron microscopy (EM) (Al Rawi et al., 2011;[START_REF] Meléndez | Autophagy genes are essential for dauer development and life-span extension in C. elegans[END_REF]. This technic allows visualization of autophagosome, possibly at different maturation steps [START_REF] Manil-Ségalen | The C. elegans LC3 Acts Downstream of GABARAP to Degrade Autophagosomes by Interacting with the HOPS Subunit VPS39[END_REF]. Additionally, EM analysis of autophagosomes from wild type and mutant strains could provide insights into the mechanism defects that are associated with genome alteration [START_REF] Wang | Kinetics and specificity of paternal mitochondrial elimination in Caenorhabditis elegans[END_REF]. It helps to monitor and distinguish normal mitochondria from modified ones, targeted by autophagosome, with altered structure such as matrix aggregates or cristae disappearance [START_REF] Wang | Kinetics and specificity of paternal mitochondrial elimination in Caenorhabditis elegans[END_REF][START_REF] Zhou | Mitochondrial endonuclease G mediates breakdown of paternal mitochondria upon fertilization[END_REF].

Interestingly, EM and direct observation of fluorescent reporter construct labelling either distinct autophagosomal or mitochondrial proteins could be combined in C. elegans by applying the Correlative Light and Electron Microscopy (CLEM) technique. This technique has been used to analyse the mechanism underlying apoptotic corpse elimination through consecutive recruitment of LGG-1 and LGG-2 during C. elegans development [START_REF] Largeau | Correlative Light and Electron Microscopy to Analyze LC3 Proteins in Caenorhabditis elegans Embryo[END_REF] and could be adapted to further study mitophagy in C. elegans.

5) Physiological and developmental mitophagy in C.elegans

The model organism C. elegans, due to its intrinsic multiple experimental advantages and specific autophagy tools developed by the research community, has lead the way to the discovery of key developmental and physiological roles for mitophagy. Hereafter, we will present and discuss mitophagy mechanisms involved in C. elegans development and stress response.

Elimination of paternal mitochondria

In humans, as in most animals, the mtDNA is maternally inherited [START_REF] Birky | The inheritance of genes in mitochondria and chloroplasts: laws, mechanisms, and models[END_REF], with exceptional pathological cases in which paternal mtDNA could be transmitted to the offspring [START_REF] Luo | Biparental Inheritance of Mitochondrial DNA in Humans[END_REF](Luo et al., , 2013)) 5). MO are sperm specific organelles that derived from the Golgi apparatus and probably play a role similar to the vertebrate acrosome [START_REF] Nishimura | Spermatogenesis[END_REF]. Electron microscopy analyses of the fertilized oocyte revealed that paternal mitochondria are small and rounded whereas maternal ones are thin and tubular.

Paternal mitochondria lose their membrane potential, have no cristae but present thicker membranes containing holes and dark speckles within the matrix. In the meantime, ubiquitination events take place on paternal mitochondria and MO [START_REF] Sato | The autophagy receptor ALLO-1 and the IKKE-1 kinase control clearance of paternal mitochondria in Caenorhabditis elegans[END_REF] and the depletion of the E1 ubiquitin activating E1 enzyme UBA-1 or the E2 ubiquitin conjugating enzymes UBC-16 and UBC-18 alters the PME [START_REF] Molina | Ubiquitination is required for the initial removal of paternal organelles in C. elegans[END_REF]. This ubiquitination is at least partially Parkin-independent since pink-1 and pdr-1 mutants display no defect in PME [START_REF] Lim | Fndc-1 contributes to paternal mitochondria elimination in C. elegans[END_REF]. Probably, two parallel but independent mechanisms contribute to PME.

One is dependent on ubiquitination of the OMM proteins and involves both proteasome and mitophagy, the other is ubiquitination-independent and involved FNDC-1, the orthologue of FUNDC1 (FUN14 domain containing 1) [START_REF] Lim | Fndc-1 contributes to paternal mitochondria elimination in C. elegans[END_REF]. The paternal mitochondria ubiquitination triggers the recruitment of the mitophagy receptor ALLO-1, which upon phosphorylation by the IKKE-1 kinase interacts with LGG-1, via a LIR domain, and participates to the formation of autophagosome around the paternal mitochondria [START_REF] Sato | The autophagy receptor ALLO-1 and the IKKE-1 kinase control clearance of paternal mitochondria in Caenorhabditis elegans[END_REF]). An additional mitophagy receptor, the prohibitin PHB-2, is localized to the IMM and becomes accessible for allophagy following alteration of the OMM [START_REF] Wei | Prohibitin 2 Is an Inner Mitochondrial Membrane Mitophagy Receptor[END_REF] (Figure 5). The initial alteration of the OMM could be due to the process OMMAD because depletion of the two proteasomal subunits RPN-10 and RAD-23 affects PME [START_REF] Molina | Ubiquitination is required for the initial removal of paternal organelles in C. elegans[END_REF][START_REF] Zhou | Elimination of paternal mitochondria through the lysosomal degradation pathway in C. elegans[END_REF].

Several studies reported differential functions for LGG-1 and LGG-2, the homologues of the mammalian GABARAP and LC3, respectively, during allophagy. After fertilization, they both localize in clusters containing paternal mitochondria and MOs, which degradation is completed at the 8-cell stage (Al Rawi et al., 2011;[START_REF] Sato | Degradation of paternal mitochondria by fertilization-triggered autophagy in C. elegans embryos[END_REF][START_REF] Zhou | Elimination of paternal mitochondria through the lysosomal degradation pathway in C. elegans[END_REF].

Genetic studies revealed that the allophagic clusters are completely abolished in lgg-1 null mutant but persist for longer time in lgg-2 mutants, due to a delay in the formation of autolysosome. These data illustrate that lgg-1 is essential for autophagosome formation, whereas lgg-2 has a role in autophagosome fusion with lysosome. Moreover, LGG-2 interacts with VPS-39, an Homotypic fusion and vacuole Protein Sorting (HOPS) complex subunit, to mediate the tethering and acidification of autophagosomes with lysosomes downstream of Rab5-Rab7 [START_REF] Djeddi | Sperm-inherited organelle clearance in C. elegans relies on LC3-dependent autophagosome targeting to the pericentrosomal area[END_REF][START_REF] Manil-Ségalen | The C. elegans LC3 Acts Downstream of GABARAP to Degrade Autophagosomes by Interacting with the HOPS Subunit VPS39[END_REF].

Mitophagy and iron metabolism

Several proteins of the mitochondrial electron transport chain need iron as a key element to accomplish their functions, and iron deregulation is often detrimental for mitochondria functions. Recently, a study on C. elegans identifies that the induction of mitophagy is part of an adaptive iron starvation response induced as a protective mechanism against mitochondrial stress [START_REF] Schiavi | Iron-Starvation-Induced Mitophagy Mediates Lifespan Extension upon Mitochondrial Stress in C. elegans[END_REF]. Mitochondrial respiration is dependent of iron-sulfur (Fe-S) clusters, which biosynthesis is stimulated by the mitochondrial protein Frataxin (FXN) [START_REF] Gervason | Physiologically relevant reconstitution of iron-sulfur cluster biosynthesis uncovers persulfide-processing functions of ferredoxin-2 and frataxin[END_REF]. Mutations of fxn result in dysfunctions of the respiratory chain and lead to multiple symptoms observed in Friedreich's ataxia. In C.

elegans, the moderate depletion using RNAi, of FXN homologue FRH-1, induces lifespan extension [START_REF] Ventura | Caenorhabditis elegans mitochondrial mutants as an investigative tool to study human neurodegenerative diseases associated with mitochondrial dysfunction[END_REF]. Interestingly, mitophagosome formation and mitophagy are induced in frh-1 RNAi worms, and lifespan extension depends on PDR-1, PINK-1, DCT-1, as well as on the autophagy receptor p62/SQST-1. EM analysis of frh-1 worms revealed the accumulation of electron-dense iron deposits within mitochondrial matrix, which is consistent with data in mouse [START_REF] Puccio | Mouse models for Friedreich ataxia exhibit cardiomyopathy, sensory nerve defect and Fe-S enzyme deficiency followed by intramitochondrial iron deposits[END_REF]. Frh-1 (RNAi) mimics a hypoxia state through cytosolic iron deprivation, which activates the hypoxia-inducible factors HIF-1 and AHA-1, to modulate longevity [START_REF] Zhang | The HIF-1 hypoxiainducible factor modulates lifespan in C. elegans[END_REF]. Moreover, frh-1 RNAi can also upregulate the expression of dct-1 and a series of globin genes, while it downregulates the expression of sqst-1/p62. Globin genes encode iron-containing proteins that regulate the cellular redox status in response to hypoxia [START_REF] Hoogewijs | From critters to cancers: bridging comparative and clinical research on oxygen sensing, HIF signaling, and adaptations towards hypoxia[END_REF]. Upon iron deprivation, using the iron chelator 2,2'-dipyridyl,the worms present a mitophagy-dependent increased longevity, which overlaps the FRH-1 depletion.

Interestingly, C. elegans infection by Pseudomonas aeruginosa also induces an iron related mitophagy. Amongst the virulence factors produced by P. aeruginosa, siderophores, such as pyoverdin, retrieve the iron from its host. Consequently, C. elegans infected by P.

aeruginosa experiments an acute iron sequestration similar to the slight decrease of frh-1 mRNA level. This iron sequestration induced a mitochondrial turnover through a protective mitophagy process. For example, pink-1 and pdr-1 depletion sensitize animals to P.

aeruginosa [START_REF] Kirienko | Mitophagy confers resistance to siderophore-mediated killing by Pseudomonas aeruginosa[END_REF] indicating that mitophagy is necessary for C. elegans resistance to the infection, which probably impairs mitochondrial function through iron chelation and damages mitochondria. Therefore, iron chelation-induced mitophagy eliminates damaged mitochondria to preserve energy homeostasis during this pathogen infection.

Dual effect of mitophagy on heteroplasmy maintenance.

In most animals, mtDNA is maternally inherited and typically, individuals harbour a unique mtDNA genotype defining the homoplasmic state. However, germline and somatic mutations can lead to the coexistence of a mixture of mutant and normal mtDNA within the same individual, qualified as heteroplasmy and responsible of numerous human mitochondrial diseases [START_REF] Wallace | Mitochondrial DNA genetics and the heteroplasmy conundrum in evolution and disease[END_REF]. Several C. elegans heteroplasmic strains have been isolated [START_REF] Konrad | Mitochondrial Mutation Rate, Spectrum and Heteroplasmy in Caenorhabditis elegans Spontaneous Mutation Accumulation Lines of Differing Population Size[END_REF][START_REF] Tsang | Stable heteroplasmy but differential inheritance of a large mitochondrial DNA deletion in nematodes[END_REF]. These stains combined with powerful novel technical possibilities such as purification of mitochondria from specific tissues (muscle, neuron, intestine, epidermis, gonad) even from a single worm make C. elegans a powerful model for studying mechanisms regulating heteroplasmy state maintenance (Ahier et al., 2018). The C. elegans heteroplasmic strain, harbouring a 3.1kilobase deletion (uaDf5) has twice mtDNA molecules compared to the wild type. Dissection of worms shows that uaDf5 content varies within tissues of individual worms [START_REF] Liau | A persistent mitochondrial deletion reduces fitness and sperm performance in heteroplasmic populations of C. elegans[END_REF]. Interestingly, PDR-1/Parkin is required for maintaining low levels of uaDF5 mtDNA in C. elegans suggesting that this key protein involved in triggering mitophagy could have a beneficial role [START_REF] Valenci | Parkin modulates heteroplasmy of truncated mtDNA in Caenorhabditis elegans[END_REF]. However, the mechanism underlying heteroplasmic status maintenance seems much more complicated. Two recent reports have shown that the heteroplasmic strain constitutively induces UPR mt , which promotes proliferation of the uaDF5. Interestingly, the loss of the UPR mt transcription factor ATFS-1 facilitates mitophagy activity and induces a decrease of mtDNA copy-number [START_REF] Gitschlag | Homeostatic Responses Regulate Selfish Mitochondrial Genome Dynamics in C. elegans[END_REF][START_REF] Lin | Maintenance and propagation of a deleterious mitochondrial genome by the mitochondrial unfolded protein response[END_REF]. The expression of an error prone mtDNA polymerase in C. elegans generates multiple mtDNA deletions with a variable heteroplasmic state, and displays multiple phenotype defects such as neuromuscular dysfunction. Surprisingly, a genetic screen to identify modulators of this mutator phenotype reveals the paradox that a reduced mitophagy may be beneficial for this worm [START_REF] Haroon | Multiple Molecular Mechanisms Rescue mtDNA Disease in C. elegans[END_REF]. Altogether, these reports point out a very complex implication of mitophagy in the regulation of the heteroplasmic state in C. elegans.

6) Mitophagy during aging and neurodegeneration in C. elegans

Mitophagy in aging and stress conditions

A recent work revealed a novel physiological role for mitophagy, which allows mitochondria clearance all along the aging process in C. elegans (Figure 6). The progressive accumulation of the mitochondrial mass, observed during aging of the worm, could induced some persistent cell malfunctions and be responsible of the decline of health associated with aging [START_REF] Palikaras | Coordination of mitophagy and mitochondrial biogenesis during ageing in C. elegans[END_REF]. Interestingly, this phenotype is further enhanced when key autophagy gene or the mitophagy Pink/parkin pathway are depleted, demonstrating that the mitochondria homeostasis is partly modulated via mitophagy during aging. The mitophagy receptor DCT-1 (DAF-16/FOXO Controlled, germline Tumour affecting-1), homologue of NIX/BNIP3L and BNIP3, is crucial during this process and its depletion results in the accumulation of abnormal mitochondria, similarly to pdr-1and pink-1 animals. DCT-1 is localized at the OMM, and contains a LIR motif, which mediates its interaction with LGG-1 [START_REF] Palikaras | Coordination of mitophagy and mitochondrial biogenesis during ageing in C. elegans[END_REF]. The overexpression of DCT-1 allows worm to better adapt to various stressful condition such as heat shock, UV exposure and pharmacological treatment challenging mitochondria (FCCP, paraquat). The ubiquitination of the lysine 26 of DCT-1 increases upon mitophagy induction, in a PDR-1 and PINK-1-dependent manner and the three mutants are more sensitive to mitochondrial stress. DCT-1 participates with PINK-1 and PDR-1 to the extension of lifespan dependent on both the insulin pathway (daf-2 mutant) and mitochondrial proteins (clk-1 and isp-1 mutants).

This study also showed that dct-1 gene expression and mitophagy are both regulated by the transcription factor SKN-1, the homologue of the mammalian NRF2/NFE2L2. SKN-1 is activated under oxidative stress conditions [START_REF] Ghose | Anoxiareoxygenation regulates mitochondrial dynamics through the hypoxia response pathway, SKN-1/Nrf, and stomatin-like protein STL-1/SLP-2[END_REF][START_REF] Ploumi | Mitochondrial biogenesis and clearance: a balancing act[END_REF] and promotes mitochondrial biogenesis by up-regulating several mitochondria genes. SKN-1 depletion leads to the modification of mitochondrial morphology, an increase of cytoplasmic Ca 2+ and a mitochondrial membrane depolarization and blocks DCT-1 mediated mitophagy.

By coupling mitophagy and mitochondrial biogenesis, SKN-1 is a key player of a loop which regulation is important during the aging of animals (Figure 6). Interestingly, an additional mechanism of control of mitophagy during aging in C. elegans relies on the specific interaction of two mRNAs, from puf-8 and mff-1 genes. MFF-1 is an OMM protein that recruits the key mitochondrial fission factor DRP-1 to mitochondria. In old adult worms, puf-8 mRNA inhibits the translation of mff-1 mRNA, decreasing the mitochondrial fission and also the mitophagy, which, ultimately results in the accumulation of altered mitochondria [START_REF] D'amico | The RNA-Binding Protein PUM2 Impairs Mitochondrial Dynamics and Mitophagy During Aging[END_REF].

Finally, a recent report studied the involvement of mitophagy in mev-1 mutants that are highly sensitive to environmental stresses. This study also demonstrates that the mitophagy genes dct-1 and pdr-1 but pink-1 are required for mev-1 to survive to heat shock [START_REF] Civelek | Inhibition of mitophagy decreases survival of Caenorhabditis elegans by increasing protein aggregation[END_REF]. This conclusion is further supported by results indicating that mitophagy deficient animals are more sensitive to the ultraviolet C radiation [START_REF] Hartman | Genetic Defects in Mitochondrial Dynamics in Caenorhabditis elegans Impact Ultraviolet C Radiation-and 6-hydroxydopamine-Induced Neurodegeneration[END_REF].

Overall, these results indicate that mitophagy is a key cellular process that helps C. elegans to cope with stressful environmental context.

Pharmacological modulation of mitophagy protects from age-induced decline

Three molecules that specifically modulate mitophagy in C. elegans have been identified: Urolithin A (UA), tomatidine and nicotinamide adenine dinucleotide (NAD + ). UA, a part of the organic compounds known as benzo-coumarins, is the first natural compound shown to induces mitophagy in worm as well as rat and mammalian cells [START_REF] Ryu | Urolithin A induces mitophagy and prolongs lifespan in C. elegans and increases muscle function in rodents[END_REF].

The UA precursor, ellagitannin, is found in multiple fruits and is processed in UA by the microbiota. UA can extend lifespan and improve worm fitness by inducing mitophagy. In young adult, UA treatment induces mitochondria fragmentation and leads to the reduction of mitochondrial content. The latter phenotype depends on the mitophagy genes pink-1, dct-1, skn-1 or the autophagy genes bec-1, sqst-1, vps-34, which depletion inhibits the lifespan extension following UA exposure. Indeed, UA treatment induces autophagy, which is detected by the increased LGG-1 positive autophagosomes. In older worms, UA exposure leads to mitochondrial content increase and to a higher transcription rate of respiratory chain subunit genes, compared to control animals. During the UA long-term treatment, the mitochondrial biogenesis is facilitated by the initial mitophagy induction observed in young adult. The enhanced mitochondrial biogenesis improves mitochondrial function through stabilizing ATP content and increasing the respiration capacity, and therefore leads to a prolonged locomotion and pharyngeal pumping activities of treated worms. Interestingly, this result suggest that UA is capable to uncouple mitophagy and mitochondrial biogenesis during aging; stimulating first mitophagy then mitochondrial biogenesis. Interestingly, most of the effects of UA on C. elegans are also observed in mammalian muscle and intestinal cells and, mice and rat aging models treated with UA show improvement of their muscle activity. This molecule shows a high potential to be used as a nutritional supplement to improve mitochondria function and muscle quality in human elderly people and clinical trials aiming to test the effect of UA on human health have been undertaken [START_REF] Andreux | The mitophagy activator urolithin A is safe and induces a molecular signature of improved mitochondrial and cellular health in humans[END_REF].

Tomatidine is a steroid like compound enriched in unripe tomatoes. This molecule have beneficial effects in mouse cells for anti-inflammatory [START_REF] Chiu | Tomatidine inhibits iNOS and COX-2 through suppression of NF-kappaB and JNK pathways in LPS-stimulated mouse macrophages[END_REF] and antitumorigenic effects [START_REF] Fujiwara | Tomatidine, a tomato sapogenol, ameliorates hyperlipidemia and atherosclerosis in apoE-deficient mice by inhibiting acyl-CoA:cholesterol acyltransferase (ACAT)[END_REF]. In mouse skeletal muscle cells, tomatidine promotes muscle fitness partially through increasing mitochondrial mass [START_REF] Dyle | Systems-based discovery of tomatidine as a natural small molecule inhibitor of skeletal muscle atrophy[END_REF].

Interestingly, tomatidine treatment extends worm lifespan, as well as health span [START_REF] Fang | Tomatidine enhances lifespan and healthspan in C. elegans through mitophagy induction via the SKN-1/Nrf2 pathway[END_REF], as manifested by the improvement of swimming and pharyngeal pumping in aged animals. Interestingly, tomatidine induces moderate ROS production, which in turn activates SKN-1/Nrf-2 pathway. SKN-1, in turn, promotes PINK-1/DCT-1-dependent mitophagy and mitochondrial biogenesis to keep mitochondria healthy. Additionally, tomatidine action seems to be dependent on the key UPR mt gene atfs-1 and its beneficial effect has been further extended to primary rat cortical neurons and human HEK-203 cells [START_REF] Fang | Tomatidine enhances lifespan and healthspan in C. elegans through mitophagy induction via the SKN-1/Nrf2 pathway[END_REF]. This study further provides experimental evidences indicating that somehow pharmacological treatment, activating mitophagy through SKN-1 pathway, may provide new approaches to improve healthy aging. It is highly possible that UA induced mitophagy and lifespan extension shares the same pathway triggered by tomatidine treatment.

NAD + is an important coenzyme linked to the cell energy homeostasis. It is part of the redox couple NAD + /NADH, which provides the mitochondria respiratory chain with electrons and protons. NAD + is also a coenzyme for the nuclear deacetylase sirtuin 1 that modulates chromatin structure and regulates multiple cellular pathways including aging. In C. elegans, NAD + depletion inhibits mitophagy while its replenishment stimulates mitophagy by upregulating DCT-1 through the sirtuin homolog SIR-2.1 and DAF-16 dependent mechanism [START_REF] Fang | Tomatidine enhances lifespan and healthspan in C. elegans through mitophagy induction via the SKN-1/Nrf2 pathway[END_REF]. Interestingly, it has been also shown that NAD + depletion is a major driver of the metabolic defects observed in the premature aging autosomal recessive disease Werner syndrome (WS). Both in WS model worm, wrn-1 mutant, and in patient cells, a depletion of NAD + is associated with mitochondria dysfunction and defective mitophagy. The NAD + replenishment improves the mitochondrial function by restoring mitophagy in a DCT-1-and UNC-51 dependent mechanism (Fang et al., 2019), which opens future therapeutic strategy for several diseases [START_REF] Connell | NAD+ metabolism as a target for metabolic health: have we found the silver bullet?[END_REF].

Overall, C. elegans has been a leading model organism to put forward drug that selectively modulate mitophagy and contribute to set up numerous pre-clinical and clinical trials aiming to analyse putative translation of these mitophagy inducers drug beneficial effect to human.

Mitophagy and neurodegenerative processes

Alzheimer's disease (AD), Parkinson's disease (PD) and amyotrophic lateral sclerosis associated with frontotemporal dementia (ALS/FTD) are the major diseases that affect the functioning of the brain in the human population. These pathologies have currently no effective treatment and result in a progressive destruction of the nervous system leading to a loss of: memory, cognitive functions, sensory abilities, speech and locomotion.

AD, PD and ALS/FTD share similar cellular dysfunctions regarding calcium homeostasis, lipid metabolism, energy production and recycling of cellular waste products, related to mitochondria and autophagy, and which are mostly linked to mitochondrial associated membranes function [START_REF] Area-Gomez | On the Pathogenesis of Alzheimer's Disease: The MAM Hypothesis[END_REF][START_REF] Paillusson | There's Something Wrong with my MAM; the ER-Mitochondria Axis and Neurodegenerative Diseases[END_REF].

Several C. elegans models of neurodegenerative diseases have been used to challenge the possible causal link between these disease-associated gene mutations and nervous system dysfunctions [START_REF] Alexander | Use of Caenorhabditis elegans as a model to study Alzheimer's disease and other neurodegenerative diseases[END_REF]. The exploration of the link between mitophagy and neurodegeneration, in animal models of AD, indicates that conserved mitochondrial stress response signature emerges comprising UPR mt and mitophagy pathways [START_REF] Sorrentino | Enhancing mitochondrial proteostasis reduces amyloid-β proteotoxicity[END_REF]. Mitophagy induction by NAD + boosting compound attenuates Aβ toxicity in C.

elegans. The stimulation of the UPR mt and mitophagy can decrease protein aggregation, increase worm fitness and delays progression of symptoms. By adapting for neural expression the genetic tools for studying mitophagy (see section 4), a recent report provided additional evidences for mitophagy involvement in neurodegenerative diseases in C. elegans (Fang et al., 2019). The treatment of worms with UA (see section 6) triggers a dct-1, pink-1 and pdr-1 dependant mitophagy flux in nerve cells without inducing bulk autophagy. The authors also tested the beneficial effect of mitophagy in two C. elegans models of AD, the pan-neuronal expression of the human Aβ1-42 protein [START_REF] Voglis | A synaptic DEG/ENaC ion channel mediates learning in C. elegans by facilitating dopamine signalling[END_REF] and the neuronal expression of the pro-aggregant F3ΔK280 tau fragment [START_REF] Fatouros | Inhibition of tau aggregation in a novel Caenorhabditis elegans model of tauopathy mitigates proteotoxicity[END_REF]. The pharmacological stimulation of mitophagy is beneficial for both models and restore the cognitive defects observed on learning and memory. A similar effect has been reported in mice model of AD, suggesting a conserved role of mitophagy in neurodegenerative diseases (Fang et al., 2019).

Multiple C. elegans models of PD, including genetic and pharmacological induced models have been reported in the literature [START_REF] Cooper | Modeling Parkinson's Disease in C. elegans[END_REF]. Mutations in parkin and Pink1 cause autosomal recessive form of PD and the depletion of both genes in C.

elegans lead to accumulation of defective mitochondria. pink-1 null mutant presents mitochondrial cristae defect both in muscle and neuron, as well as increased sensitivity to oxidative stresses and neuron dysfunction [START_REF] Sämann | Caenorhabditits elegans LRK-1 and PINK-1 act antagonistically in stress response and neurite outgrowth[END_REF]. Surprisingly, the defects in pink-1 mutant could be suppressed by depleting lrk-1, the homologue of LRRK2 which is associated with PD in human [START_REF] Biskup | Localization of LRRK2 to membranous and vesicular structures in mammalian brain[END_REF]. LRK-1 functions in axonal-dendritic polarity of synaptic vesicles in neurons of C. elegans [START_REF] Sakaguchi-Nakashima | LRK-1, a C. elegans PARK8-related kinase, regulates axonal-dendritic polarity of SV proteins[END_REF].

LRK-1 and PINK-1 act antagonistically in neurite outgrowth, suggesting that the mitophagy defect in pink-1 mutant could affect neuron outgrowth defect. Finally, patients with ALS/FTD accumulate TDP-43 (Tar DNA-binding protein 43) in degenerating neuron [START_REF] Neumann | Ubiquitinated TDP-43 in frontotemporal lobar degeneration and amyotrophic lateral sclerosis[END_REF]. Interestingly, knockdown of TDP-43 enhances autophagosome and lysosome biogenesis indicating somehow a link between ALS/FTD and autophagy. [START_REF] Xia | TDP-43 loss of function increases TFEB activity and blocks autophagosome-lysosome fusion[END_REF].

When overexpressed in the C. elegans nervous system, the human TDP-43 protein accumulates in the nucleus and results in an uncoordinated locomotion associated with abnormal synapse structure [START_REF] Ash | Neurotoxic effects of TDP-43 overexpression in C. elegans[END_REF]. The full exploitation of these worm models regarding any defect in mitophagy and the implication on ALS/FTD is still pending.

7) CONCLUSION

Recent researches performed with the nematode C. elegans have clearly contributed to the better understanding of the molecular mechanisms of mitophagy and its physiological roles at the scale of a whole animal. The most representative inputs from these studies concerns the fields of developmental biology, aging, adaptation to stress and neurodegenerative pathologies.

Studies in C. elegans revealed that the maternal restricted transmission of mtDNA requires the selective elimination of paternal mitochondria by mitophagy and led to the identification of an original mechanism that is independent of Pink/parkin and relies on the mitophagy receptor ALLO-1. These data, supported by studies in the model Drosophila melanogaster paved the way for understanding this essential question in mammals (see Box 1). Additionally, the worm C. elegans provided the first clue on a regulatory mechanism that coordinates mitochondrial biogenesis and mitophagy during aging and involved the master regulator SKN-1. It also contributed to demonstrate that an inner membrane protein, Prohibitin 2, could act as a mitophagy receptor [START_REF] Wei | Prohibitin 2 Is an Inner Mitochondrial Membrane Mitophagy Receptor[END_REF], and to identify the links between iron deficiency and mitophagy [START_REF] Schiavi | Iron-Starvation-Induced Mitophagy Mediates Lifespan Extension upon Mitochondrial Stress in C. elegans[END_REF]. Finally, C. elegans studies have been important to better understand the relationships between neurodegenerative diseases and mitophagy and allowed to test and validate novel molecules that could be further developed as therapeutics to enhance mitophagy and improve healthy aging.

Several technological improvements for studying mitophagy and mitochondrial biogenesis have been possible using C. elegans, taking advantage of its transparency to developed the fluorescent biomarkers Rosella [START_REF] Palikaras | Coordination of mitophagy and mitochondrial biogenesis during ageing in C. elegans[END_REF] and MitoTimer [START_REF] Laker | A novel MitoTimer reporter gene for mitochondrial content, structure, stress, and damage in vivo[END_REF][START_REF] Terskikh | Fluorescent timer": protein that changes color with time[END_REF]. The power of genetic approaches and engineering of transgenic nematodes using molecular biology approaches strongly suggests that additional live-imaging tools will appear soon in C. elegans. Nevertheless, the mitochondrial degradation per se often lacks clear experimental demonstration in C. elegans, and would strongly benefit of the development of biochemical tools for the quantification of the delivery/ degradation of mitochondrial cargos into the lysosome [START_REF] Chapin | Tissue-specific autophagy responses to aging and stress in C. elegans[END_REF].

One can expect that in the near future, studies in C. elegans will bring new insights on molecular mechanism controlling mitophagy, and identify the roles for mitophagy in many biological processes. The important question of the basal mitophagy is particularly difficult to address, which explains why most of the studies that identified the mitophagy mechanisms are generally done in the context of a massive and non-physiological stress of the mitochondria.

C. elegans could be useful to identify whether the signalling events, the mitophagy receptors and the regulation of the basal mitophagy are different from the developmental or pathological processes. By the simplicity of its anatomy and the presence of multiple tissues, C. elegans is also a powerful system to decipher the variations of autophagy/mitophagy functions between organs and during the whole life cycle. Neurology and aging pathologies are one obvious target but the differentiation and physiology of other cell types could be studied as well as trans-differentiation processes that have been described in the nematode. C.

elegans could also open the study of autophagy/mitophagy in very complex biological processes such as circadian rhythm, the sleep-like state lethargus or very long starvation survival period.

Finally, another key point in the field of mitophagy is to understand the mechanisms of cooperation and coordination between the various mitochondria quality control pathways.

How UPR mt , mitophagy, proteasome and other processes like the exophers jettison [START_REF] Melentijevic | C. elegans Neurons Jettison Protein Aggregates and Mitochondria Under Neurotoxic Stress[END_REF] are globally regulated to cope with a dysfunctional mitochondria network remains unclear and C. elegans could be useful to address such a question.

Funding source and acknowledgements

Our apologies to authors whose works have not been cited due to space restriction. We would like to thank Céline Largeau for the electron microscopy image. YC and RoL are supported by fellowships from the "China Science Council" and the Fondation pour la Recherche Médicale (ECO20170637554), respectively. Work in ReL team is supported by the Fondation ARC (SFI20111203826), la Ligue Contre le Cancer (5FI13659TYKB).

Box 1. Convergence of struggles to eliminate paternal mitochondria

Multiple mechanisms, depending on the animal, act before or just after fertilization during the early embryogenesis, to eliminate paternal mitochondria. Early work from Sutovsky and colleagues showed that paternal mitochondria are ubiquitinated for an efficient degradation mediated by proteasome in fertilized cow and monkey eggs [START_REF] Sutovsky | Ubiquitinated sperm mitochondria, selective proteolysis, and the regulation of mitochondrial inheritance in mammalian embryos[END_REF]. Nishimura and colleagues then showed, in the fish medaka (Oryzias latipes), that the paternal mtDNA number is progressively reduced at the end of the spermatogenesis and then is specifically and progressively digested just after fertilization [START_REF] Nishimura | Active digestion of sperm mitochondrial DNA in single living sperm revealed by optical tweezers[END_REF] 'Farrell, 2012). This mechanism is similar to the one observed in C. elegans whereby the endonuclease CPS-6 originally localized to the intermembrane space has access to the mtDNA, after rupture of the IMM by an unknown mechanism. This step seems to be important for the subsequent PME steps [START_REF] Zhou | Mitochondrial endonuclease G mediates breakdown of paternal mitochondria upon fertilization[END_REF]. Moreover, an additional active mechanism exists also in drosophila eggs to get rid of paternal mitochondria [START_REF] Politi | Paternal mitochondrial destruction after fertilization is mediated by a common endocytic and autophagic pathway in Drosophila[END_REF]. This mechanism uses non conventional autophagy and endocytic pathways to eliminate sperm mitochondria within the eggs even if these sperm mitochondria is devoid of DNA. Paternal mitochondria destruction (PMD) initiates with vesicles resembling MVB that put mitochondria into small fragments that are subsequently sequestered in autophagosomes that finally fuse with lysomes. P62 and ubiquitination are involved but this mechanism is not Parkin dependent [START_REF] Politi | Paternal mitochondrial destruction after fertilization is mediated by a common endocytic and autophagic pathway in Drosophila[END_REF]. In mice, the situation is debated. One report indicates that most motile sperm eliminate their mtDNA and leave only vacuolar mitochondria before fertilization. Then the sperm mitochondria persist until morula stage, even they are observed to colocalize with autophagy proteins p62 and LC3 just after fertilization. Nonetheless, their clearance is independent of autophagy pathway (Luo et al., 2013). However, this conclusion is challenged by a functional analysis showing that paternal mitochondria are maintained in 60% of the embryos depleted for Parkin and Mul1.

Therefore, the authors concluded that in mice, PMD required a Parkin and Mul1 dependent autophagy [START_REF] Rojansky | Elimination of paternal mitochondria in mouse embryos occurs through autophagic degradation dependent on PARKIN and MUL1[END_REF]. Sutovsky and colleagues addressed the PME question in another vertebrate, the porcine zygote, and provided evidences for another type of active paternal mitochondria destruction mechanism. This mechanism is based on a P62/SQSTM1dependent autophagy, VCP-mediated dislocation of ubiquitinated mitochondria protein and presentation to the 26S proteasome [START_REF] Song | Autophagy and ubiquitin-proteasome system contribute to sperm mitophagy after mammalian fertilization[END_REF]. Overall, multiple different systems have been selected during the evolution to specifically eliminate paternal mitochondria in most animals suggesting that PME step is critical for the subsequent animal development.

Bibliography

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Legends of figures

Figure 1 .

 1 Figure 1. The three types of autophagy in most eukaryotic cells (A) Macroautophagy, which is commonly named autophagy, is characterized by the sequestration of cargos within a double membrane structure, called the autophagosome. In mammals, the autophagy starts by the formation of a flat membranous structure, the phagophore, which elongates and close to generate a double membrane vesicle. Finally, the external membrane of the sealed autophagosome fused with the lysosome to generate an acidic and degradative compartment named the autolysosome, allowing recycling of autophagic cargoes. The core autophagy machinery forms various complexes that acts in four major steps. the ULK1/2 (unc-51 like autophagy activating kinase 1/2) complex is involved in the initiation step of the process. The class III phosphatidylinositol 3-kinase (PI3K) complex induces locally PI3P (phosphatidylinositol 3 phosphate) synthesis and allows the nucleation of the phagophore by recruiting the PI3P binding complex. The Atg12 and the Atg8 conjugation systems are controlling the elongation of the phagophore to form the autophagosome through lipidation of Atg8s to phosphatidylethanolamine (PE). (B) Membrane invagination of either the endosome or the lysosome allows the direct sequestration of cargoes within degradative compartments by endosomal microautophagy or microautophagy, respectively. (C) Chaperone-mediated autophagy (CMA) targets proteins containing the KFERQ motif that is recognized by several chaperones, including Hsp70 (Heat shock protein 70kDa). During CMA, LAMP2 (Lysosomal-Associated Membrane Protein) mediates the transfer of Hsp70protein aggregates within the lysosome.

Figure 2 .

 2 Figure 2. Ubiquitin-dependent or independent mitophagy pathways involve multiple receptors. Two main mitophagy pathways allow the selective degradation of mitochondria by autophagy in physiological and stress induced conditions. (A) On one hand, mitochondrial receptors mediate mitophagy through the direct interaction with Atg8/LC3 proteins. Such mitochondrial receptors are outer mitochondrial membrane proteins NIX NBIP3 (NIX-NIP-3-Like Protein X / BNIP3L-BNIP3 like), FUNDC1 (FUN14 Domain Containing 1), BCL2L13 (Bcl2-like protein 13), and inner membrane protein prohibitin or the phospholipid cardiolipin. (B) On the other hand, the Pink1 (PTEN-induced putative kinase 1)-Parkin dependent mitophagy pathway requires cytosolic protein adapters. Upon mitochondrial damage, PINK1, that is usually cleaved and processed, is stabilized at the outer mitochondrial membrane where it phosphorylates both ubiquitin residues on mitochondrial protein and the E3 ubiquitin ligase Parkin. Activation of Parkin results in the high density of phospho-ubiquitin chains linked to substrates, which triggers the mitochondrial recruitment of the receptors OPTN (Optineurin), NDP52(Nuclear dot protein 52 kDa), SQSTM1(sequestosome 1), NBR1 (Neighbor of BRAC1) and TAX1BP1 (Tax1-binding protein) and their interaction with the autophagy machinery. In both pathways, Atg8/LC3s can interact with ULK1 (unc-51 like autophagy activating kinase 1) to induce autophagosome formation.

Figure 3 .

 3 Figure 3. Schematic representation of C. elegans life cycle indicating autophagy and mitophagy characterized processes. The development cycle of C. elegans is rapid and stereotyped (3.5 days at 20 °C), and consists of an embryonic stage and four larval stages before producing an adult animal that can live several weeks. In response to environmental stress an alternative developmental pathway results in the formation of a diapause stage called dauer larva. The figure only shows hermaphrodite animals, which are fertile for several days in absence of cross-fertilization by males. The various types of autophagy and the associated cargo are indicated in orange.

Figure 4 .

 4 Figure 4. Detection of mitophagy by microscopy in C. elegans (A) Live imaging confocal microscopy of mitophagy events (yellow arrows) monitored by the colocalization between mitochondria stained with the fluorescent Mitotracker (red) and autophagosomes labelled with GFP::LGG-1 (green), the C. elegans homolog of GABARAP. (B) Transmission electron microscopy image of muscle cells showing regular mitochondria

  . In

	Drosophila melanogaster, sperm mtDNA is eliminated during spermatogenesis, by two
	mechanisms. The first one occurs at the elongation stage of postmeiotic sperm development,
	during which, mitochondrial nuclease, Endonuclease G (EndoG), eliminates only mtDNA

rather than the complete mitochondria. Later on during spermatogenesis, the residual mtDNA is eliminated during spermatid individualization stage, through a cellular remodelling process that modify spermatid tail (DeLuca and O

(m) and a stressed-mitochondria containing dark inclusions, engulfed by an autophagosome (yellow arrow). The mitochondrial homeostasis depends on the balance between mitochondrial biogenesis and degradation. In physiological condition, a basal mitophagy flux regulates the mitochondrial content. Mitochondrial damages result in the increase of cytosolic Ca 2+ and ROS causing the releasing of several transcription factors, including SKN-1 (SKiNhead-1). The mitochondrial quality control is first ensured by UPR mt (Unfolded Protein Response in mitochondria), an adaptive transcriptional response that promotes protein refolding and mitochondrial recovery.

When dysfunctions are persistent and the UPR mt overwhelmed, elimination of the defective mitochondria occurs through mitophagy. The transcriptional factor SKN-1 promotes the activation of nuclear genes coding for mitochondrial proteins and mitophagy components, such as DCT-1 (Daf-16/ FOXO controlled germline tumor affecting-1) which is localized at the outer mitochondrial membrane and can interact with LGG-1 to mediate mitophagy. E3 ubiquitin ligase Cytosolic protein [START_REF] Narendra | Parkin is recruited selectively to impaired mitochondria and promotes their autophagy[END_REF][START_REF] Schiavi | Iron-Starvation-Induced Mitophagy Mediates Lifespan Extension upon Mitochondrial Stress in C. elegans[END_REF] SQSTM1/p62 -sequestosome 1

SQST-1

Ubiquitin-binding receptor protein Cytosolic protein [START_REF] Geisler | PINK1/Parkin-mediated mitophagy is dependent on VDAC1 and p62/SQSTM1[END_REF][START_REF] Zhang | The composition of a protein aggregate modulates the specificity and efficiency of its autophagic degradation[END_REF] TAX1BP1 -Tax 1 binding protein 1 n.d Ubiquitin-binding receptor protein Cytosolic protein [START_REF] Whang | The Ubiquitin Binding Protein TAX1BP1 Mediates Autophagasome Induction and the Metabolic Transition of Activated T Cells[END_REF]


domain: spi chim phys

text:
Jian Liu

Qingsong Song

Jie Xu

Jun Guo

Yuxin Pan

Na Li

Dongzhen Li

Peng Liu

Xiaodong Xu

Jun Xu
email: xujun@mail.shcnc.ac.cn

Kheirreddine Lebbou
email: kheirreddine.lebbou@univ-lyon1.fr

Up-conversion properties of Yb,Tb:YAG single crystals grown by the micro-pulling-down method

Keywords: Up-conversion, Micro-pulling-down technique, Visible laser, Energy transfer

Yb,Tb:YAG crystals with various Yb 3+ and Tb 3+ concentration were grown by the micro-pulling-down technique (µ-PD). The absorption spectra, fluorescence spectra and fluorescence decay curves of Yb,Tb:YAG crystals were measured at room temperature. Under 980 nm excitation, Yb,Tb:YAG crystals exhibited intense visible up-conversion luminescence due to the cooperative energy transfer from two Yb 3+ ions to one Tb 3+ ion. Under 4850 nm excitation, Yb,Tb:YAG crystals yielded near-infrared down-conversion emission, ascribing to the cooperative absorption of pumping photons and cooperative energy transfer from one Tb 3+ ion to two Yb 3+ ions. The energy transfer processes from Yb 3+ ions to Tb 3+ ions were discussed, and the energy transfer efficiency was calculated. The results show that Yb,Tb:YAG crystal is promising for visible laser output when pumped by InGaAs laser diode (LD).

Introduction

Nowadays, much attention has been paid to rare-earth doped materials for their visible emission, which can be used in iatrology, biomedical diagnosis, astronomy and telecommunication [START_REF] Kränkel | Out of the blue: semiconductor laser pumped visible rare-earth doped lasers[END_REF][START_REF] Mainster | Wavelength Selection in Macular Photocoagulation: Tissue Optics[END_REF][START_REF] Duering | Generation of tunable 589 nm radiation as a Na guide star source using an optical parametric amplifier[END_REF][START_REF] Juncar | Absolute Determination of the Wavelengths of the Sodium D1 and D2 Lines by Using a CW Tunable Dye Laser Stabilized on Iodine[END_REF]. Tb 3+ -doped materials could emit the sharp and intense fluorescence in the yellow-green range, which are considered to be promising materials for practical use. Especially, the yellow emission around 587 nm matches well with the D2 absorption line of sodium atoms, which can be used to excite the sodium layer at attitude of ~ 90 km in the laser guide system [START_REF] Max | Image improvement from a sodium-layer laser guide star adaptive optics system[END_REF][6][START_REF] Beletic | Designer detectors: a new paradigm for instrument development[END_REF][START_REF] Zhang | Versatile Raman fiber laser for sodium laser guide star[END_REF][START_REF] Wei | First light for the sodium laser guide star adaptive optics system on the Lijiang 1.8m telescope[END_REF]. Unfortunately, the relevant pumping source around 485 nm is immature, the price of the pumping device is expensive. Normally, the pump radiation was offered by a frequency-doubled optically pumped semiconductor laser (2ω-OPSL) [START_REF] Metz | Efficient green and yellow lasers in Tb 3+ -doped LiYF4, LiLuF4, and KY3F10 crystals[END_REF][START_REF] Metz | Efficient continuous wave laser operation of Tb 3+ -doped fluoride crystals in the green and yellow spectral regions[END_REF][START_REF] Metz | Performance and wavelength tuning of green emitting terbium lasers[END_REF][START_REF] Hernändez | Efficient directly emitting high-power Tb 3+ :LiLuF4 laser operating at 587.5 nm in the yellow range[END_REF][START_REF] Chen | Efficient visible laser operation of Tb:LiYF4 and LiTbF4[END_REF][15].

In the Yb 3+ /Tb 3+ co-doped system, Yb 3+ ions are used as sensitizer ions. Yb 3+ ion was considered as a sensitized ion since 1960s for its large absorption cross section, and was widely used with commercially application of the 976 LD pump sources. Up-conversion is an anti-Stokes process to convert near infrared light to visible light. A lot of efforts have been focused on the fluoride materials doped with different rare earth ions, such as Ho 3+ , Tm 3+ and Er 3+ as activators and Yb 3+ ions as sensitizers [15][START_REF] Jin | Enhancing multiphoton upconversion from NaYF4: Yb/Tm@NaYF4 core-shell nanoparticles via the use of laser cavity[END_REF][START_REF] Gnanasammandhan | Near-IR photoactivation using mesoporous silica-coated NaYF4: Yb, Er/Tm upconversion nanoparticles[END_REF]. For the reason that the low phonon energy of the fluorides is beneficial for the high up-conversion efficiency achievement. However, fluorides exhibit relatively poor stability, and in comparison, YAG single crystal is an excellent laser gain medium due to its high thermal conductivity and excellent physical and chemical properties [START_REF] Xu | Structural, thermal and luminescent properties of Yb-doped Y3Al5O12 crystals[END_REF], which play a crucial role in practical application. Compared with Yb 3+ /Ho 3+ , Yb 3+ /Tm 3+ , Yb 3+ /Er 3+ co-doped materials, the energy transfer in Yb 3+ /Tb 3+ co-doped materials are different. There do not exist immediate level available for the energy transfer. For the Yb 3+ /Tb 3+ dopants, the mechanism of up-conversion is called cooperative energy transfer (CET), two excited Yb 3+ (sensitizer) ions simultaneously transfer their energy to one Tb 3+ (activator) ion, and the Tb 3+ is excited to the 5 D4 level. In previous literatures, some works had been done in Yb 3+ /Tb 3+ co-doped materials [START_REF] Arai | Upconversion properties of Tb 3+ -Yb 3+ codoped fluorophosphates glasses[END_REF][START_REF] Qiao | Intense ultraviolet upconversion luminescence of Yb 3+ and Tb 3+ co-doped glass ceramics containing SrF2 nanocrystals[END_REF][START_REF] Grzy | Energy migration in YBO3:Yb 3+ ,Tb 3+ materials: Down-and upconversion luminescence studies[END_REF][START_REF] Huang | Luminescence, energy transfer, and up-conversion mechanisms of Yb 3+ and Tb 3+ co-doped LaNbO4[END_REF]. However, Yb 3+ /Tb 3+ co-doped YAG crystal has never been reported. It is necessary to explore the potential of Tb 3+ ions in YAG crystal for visible laser output through up-conversion process.

The micro-pulling-down (μ-PD) technique allows the growth of fiber-shaped single crystals with high pulling rate. It has many advantages over the conventional crystal growth techniques, such as controllable crystal shape and low cost that includes raw materials and time saving. The crystals grown by the μ-PD technique showed good laser performance [START_REF] Wang | High-performance Ho:YAG single-crystal fiber laser in-band pumped by a Tm-doped all-fiber laser[END_REF][START_REF] Xue | Spectral properties and laser performance of Ho:CNGG crystals grown by the micro-pulling-down method[END_REF][START_REF] Zhao | 35 W continuous-wave Ho:YAG single-crystal fiber laser[END_REF][START_REF] Xue | Spectroscopic and laser properties of Tm:CNGG crystals grown by the micro-pulling-down method[END_REF][START_REF] Cai | High power and energy generation in a Nd:YAG single-crystal fiber laser at 1834 nm[END_REF].

In this paper, Yb 3+ /Tb 3+ co-doped YAG single crystals were grown by the μ-PD technique. We mainly report the cooperative sensitized up-conversion of the Yb 3+ /Tb 3+ co-doped YAG single crystals. The spectroscopic properties including the absorption spectra, fluorescence spectra, fluorescence decay curves were studied.

Experimental

The Yb,Tb:YAG single crystals were grown by the μ-PD method. The high purity powders Tb4O7 (99.99%), Yb2O3 (99.999%), Al2O3 (99.999%), Y2O3 (99.999%) were used as raw materials and weighed according to the formula (Y1-x-yTbxYby)3Al5O12 (x=0-0.16; y=0-0.16). The powders were pressed into bulks and sintered at 1300 °C for 20 h in the air. Then the polycrystalline materials were loaded into an iridium crucible for crystal growth. The experimental model of the micro-pulling-down technique (μ-PD) is described as Figure .1. The <111> oriented pure YAG crystal was used as the seed and pulled down continuously with a pulling rate 0.3 mm/min. The molten zone, the meniscus and part of the grown crystal were directly observed and monitored with a CCD camera through the insulating hole. The detail of the crucible part was shown in Figure . 1 (b). The Yb:Tb codoped YAG melts wet well the lip of the crucible, it is fixed at the bottom of the capillary die. Adjustment of fiber diameter was realized by controlling the RF power supply and the pulling rate. Increase of the crucible temperature and pulling rate results in decrease of the fiber diameter. The higher RF power will be accompanied by larger thickness of the melt film between the crucible and the fiber. The as-grown crystals were shown in Fig. 2(a). The crystals are transparent with green color, which was due to the oxygen vacancies and formed Re-F color center in Yb,Tb:YAG crystals [START_REF] Liu | Spectroscopic properties of Tb:Y 3Al5O12 crystal for visible laser application[END_REF]. After annealed in the air for 12 hours at 1250 °C, the crystals changed from green to colorless, as shown in Fig. 2(b). Powders crushed from the grown crystal was used to determine the crystal structure. The phase compositions of all the samples were characterized using an X-ray diffraction (XRD) machine equipped with a copper target X-ray tube (D2 Avance, Bruker, Karlsruhe, Germany) in the scanning range of 10°-90°. The morphology of the polished Yb,Tb:YAG crystals were recorded on a scanning electron microscope (SEM, JSM-6510, JEOL, Kariya, Japan). The absorption spectra of Yb,Tb:YAG crystals were measured using a spectrophotometer (Lambda 950, Perkin -Elmer UV-VIS-NIR) in the wavelength range of 300-3000 nm. The emission properties of the Yb,Tb:YAG crystals were recorded by an Edinburgh Fluorescence under 485 nm and 980 nm excitation. All the measurements were performed at room temperature.

Results and discussion

The XRD patterns of the as-grown Yb,Tb:YAG crystals for the different Yb 3+ and Tb 3+ concentrations, together with the standard pattern of pure YAG crystal, are shown in Fig. 3. As can be seen, the observed peaks fit well with the pure YAG phase, which indicates that the synthesized Yb,Tb:YAG crystals are isostructural with YAG crystal and the high concentration dopant did not cause any significant impact on the phase structure. Not any new secondary phase could be observed in the grown Yb, Tb codoped yttrium aluminium garnet. The lattice parameters for the doped crystals were calculated using the JADE software. The lattice constants of Yb,Tb:YAG crystals were calculated to be 1.1919, 1.1954, 1.1977, 1.1995 and 1.2001 nm for the Yb0.16, Yb0.16Tb0.02, Yb0.16Tb0.04, Yb0.16Tb0.08 and Yb0.16Tb0.12 crystals, respectively. The lattice parameters were smaller than that of YAG crystal (1.2008 nm). As it can be seen, the size of the unit cell monotonously increases as Tb 3+ -doping concentration level increases. This expansion of the unit cell can be attributed to the larger radius of Tb 3+ ions. The room temperature absorption spectra of Yb,Tb:YAG crystals in the range of 300-2500 nm were shown in Fig. 4. The observed absorption bands are assigned to the transitions from the ground states of Tb 3+ : 7 F6 or Yb 3+ : 2 F7/2 to the excited ones. Because of the low Tb concentration, the absorptions are intense and narrower, it is related to the ions which are located in more defined coordination sites within the garnet structure. As shown in the inset of Fig 4(a), the weak band at 486 nm related to the 7 F6→ 5 D4 transition of Tb 3+ , which matches well with the emission wavelength of frequency-doubled optically pumped semiconductor laser, is usually used for pumping of Tb 3+ laser. The absorption coefficient at 486 nm was calculated to be 0.04, 0.08, 0.17 and 0.21 cm -1 for the Yb0.16Tb0.02, Yb0.16Tb0.04, Yb0.16Tb0.08 and Yb0.16Tb0.12 crystals, respectively. The intense absorption in the 850-1050 nm wavelength region is due to the absorption of Yb 3+ ions, which is attributed to the 2 F7/2 → 2 F5/2 transition. As the Yb 3+ doping concentration increases from 2 at.% to 16 at.% with the same Tb 3+ doping concentration of 8 at.%, the absorption coefficient at 940 nm increases linearly from 2.0 cm -1 to 16.1 cm -1 as shown in the inset of Fig. 4(b). Fig. 5 displays the up-conversion spectra of the Yb,Tb:YAG crystals under 980 nm excitation at room temperature. Most of the typical emission lines from the f-f transitions of Yb 3+ and Tb 3+ , can be detected. The Yb,Tb:YAG crystals exhibit intense visible luminescence and three distinguished bands are corresponding to the transitions from 5 D4 energy level to 7 F5, 7 F4 and 7 F3 energy levels of Tb 3+ ions, respectively. The characteristic positions of the emission peaks were nearly the same among the nine Yb,Tb:YAG samples. The emission spectra of Tb 3+ at different concentration are shown in Fig. 5(a). For comparison, we did not register the emission of Yb:YAG crystal within the test wavelength range. When the concentration of Tb 3+ 8 at.%, the emission intensity of 5 D4 reaches the maximum. In order to investigate the energy transfer from Yb 3+ to Tb 3+ , we grew a series of Yb,Tb:YAG crystals with different Yb 3+ concentration. Fig. 5(b) presents the emission spectra of Tb 3+ with different Yb 3+ concentration. The trend in the variation of the up-conversion intensity at 543 nm, 587 nm and 623 nm due to the influence of the Yb 3+ concentration is shown in the inset of Fig. 5(b). The emission intensity increases gradually with the increase of Yb 3+ concentration from 2 at.% to 8 at.%, because the distance between Yb 3+ and Tb 3+ ions is shortened by the increasing concentration of Yb 3+ , which inducing the improved energy transfer efficiency between rare earth ions and enhanced emission intensity. Then the emission intensity decreases with the further increase of the concentration of Yb 3+ , which is due to quenching effect resulting from the pairing or aggregation of Yb 3+ ions [START_REF] Huang | Luminescence, energy transfer, and up-conversion mechanisms of Yb 3+ and Tb 3+ co-doped LaNbO4[END_REF]. Fig. 6 illustrates the simplified energy levels of Yb 3+ and Tb 3+ in Yb,Tb:YAG crystal and the up-conversion mechanisms under 980 nm excitation are indicated. The difference in the energy transfer efficiencies between Tb 3+ and Yb 3+ in YAG can be explained by the nature of the transitions in Tb 3+ and Yb 3+ ions. Pumping at 980 nm, the Yb 3+ ions are excited from ground state 2 F7/2 to the excited state 2 F7/2, then efficient cooperative sensitization takes place easily from two Yb 3+ ions to their neighbor Tb 3+ ion, and the excited state 5 D4 of Tb 3+ is populated largely. The cooperative energy transfer can be described as follows: Yb 3+ : 2 F5/2 + Yb 3+ : 2 F5/2 + Tb 3+ : 7 F6 → Yb 3+ : 2 F7/2 + Yb 3+ : 2 F7/2 + Tb 3+ : 5 D4. The cooperative energy transfer process produce the 543 nm, 587 nm and 623 nm emission originated from 5 D4 → 7 FJ (J= 5, 4, 3) transitions. The room temperature emission spectra of Yb,Tb:YAG crystals excited by 485 nm were also recorded, as shown in Fig. 7. The visible emission intensity increases gradually with the increase of Tb 3+ concentration, indicating that no concentration quenching occurs even the Tb 3+ concentration is as high as 12 at.%. The characteristic emission bands located at 900-1100 nm due to the 2 F5/2 → 2 F7/2 transition of Yb 3+ are observed for both Yb 3+ singly doped and Yb 3+ , Tb 3+ doped YAG crystals. The two Yb 3+ ions were exited together from the ground state 2 F7/2 to the excited state 2 F5/2 by means of cooperative absorption of pumping photons and or/ by cooperative energy transfer process from 5 D4 level of Tb 3+ to Yb 3+ : Tb 3+ : 5 D4 + Yb 3+ : 2 F7/2 + Yb 3+ : 2 F7/2 → Tb 3+ : 7 F6 + Yb 3+ : 2 F5/2 + Yb 3+ : 2 F5/2 [START_REF] Liu | Cooperative downconversion and near-infrared luminescence of Tb 3+ -Yb 3+ codoped lanthanum borogermanate glasses[END_REF]. The cooperative absorption process is dominant to the excitation of Yb 3+ : 2 F5/2 level by cooperative absorption because Yb 3+ has a much large absorption coefficient around 970 nm than Er 3+ around 485 nm. When the concentration of Tb 3+ increased to 12 at.%, the emission intensity at 1029 nm reached the maximum, showing the effective cooperative down-conversion process from one Tb 3+ ion to two Yb 3+ ions. The fluorescence decay curves corresponding to the 2 F5/2 → 2 F7/2 transition are shown in Fig. 8, which were monitored at 1050 nm and excited at 980 nm. The fluorescence lifetime for the 2 F5/2 multiplet were estimated and listed in Table 1. Owing to radiative trapping [START_REF] Sumida | Effect of radiation trapping on fluorescence lifetime and emission cross section measurements in solid-state media[END_REF], the measured fluorescence lifetime of Yb:YAG crystal is longer than the intrinsic fluorescence lifetime (0.951 ms). The decay lifetime decreases with the increasing of Tb 3+ content, which can be explained by the cooperative energy transfer from Yb 3+ : 2 F5/2 to Tb 3+ : 5 D4. The cooperative energy transfer efficiency from Yb 3+ to Tb 3+ in Yb,Tb:YAG crystals can be calculated by using the formula ηET=1-τx/τ0 [START_REF] Ye | Infrared quantum cutting in Tb 3+ ,Yb 3+ codoped transparent glass ceramics containing CaF2 nanocrystals[END_REF]. Where τ0 is the fluorescence lifetime of the singly Yb 3+ doped YAG crystal, τx stands for the fluorescence lifetime of Yb 3+ ions in the presence of Tb 3+ ions. As listed in Table 1, the energy transfer efficiency from Yb 3+ to Tb 3+ ions was calculated to be 3.4%, 16.3%, 27.8%, and 45.4% for the Tb0.02Yb0.16, Tb0.04Yb0.16, Tb0.08Yb0.16, and Tb0.12Yb0.16 samples, respectively. All the results show that Yb,Tb:YAG crystal could be a promising visible laser medium when pumped by InGaAs laser diode (LD).

Conclusion:

Tb 3+ and Yb 3+ co-doped YAG single crystals were grown using the micro-pulling-down method. The room temperature XRD showed that the high concentration dopant did not change the garnet phase structure and do not provide impurities. The effect of Yb 3+ and Tb 3+ concentration on absorption spectra, emission spectra and fluorescence lifetimes of Yb,Tb:YAG crystals were investigated at room temperature. The absorption coefficient showed the concentration dependence. Under 980 nm excitation, Yb,Tb:YAG crystals exhibited intense visible up-conversion luminescence due to the cooperative energy transfer from two Yb 3+ ions to one Tb 3+ ion. The cooperative energy transfer mechanism between Yb 3+ and Tb 3+ ions was discussed and the energy transfer efficiency was calculated. Moreover, the cooperative down-conversion process from one Tb 3+ ion to two Yb 3+ ions was also studied. The results show that Yb,Tb:YAG crystal is promising for visible laser output when pumped by InGaAs laser diode (LD).

Figure 1 .Figure 2 .

 12 Figure 1. Schematic illustration of the micro-pulling-down technique.

Figure 3 .

 3 Figure 3. Room temperature XRD spectra of Yb,Tb:YAG crystals with different dopants concentration.

Figure 4 .

 4 Figure 4. Room temperature absorption spectra of Yb,Tb:YAG crystals (a) Yb0.16Tbx (x=0, 0.02, 0.04, 0.08, 0.12):YAG crystals (b) YbyTb0.08 (y=0.02, 0.04, 0.08, 0.12, 0.16):YAG crystals.

Figure 5 .

 5 Figure 5. Room temperature emission spectra of Yb,Tb:YAG crystals excited at 980 nm (a) Yb0.16Tbx (x=0, 0.02, 0.04, 0.08, 0.12):YAG crystals (b) YbyTb0.08 (y=0.02, 0.04, 0.08, 0.12, 0.16):YAG crystals.

Figure 6 .

 6 Figure 6. Simplified energy levels of Yb 3+ and Tb 3+ in Yb,Tb:YAG crystal and transition mechanisms

=Figure 7 .

 7 Figure 7. Room temperature emission spectra of Yb0.16Tbx (x=0, 0.02, 0.04, 0.08, 0.12):YAG crystals excited at 485 nm

Figure 8 .

 8 Figure 8. Decay curves of 2 F5/2 multiplet of Yb,Tb:YAG crystals under excitation of 980 nm.

Table 1 .

 1 Measured lifetime of 2 F7/2 multiplet of Yb 3+ and cooperative energy transfer efficiency from Yb 3+ to Tb 3+ in Yb0.16Tbx:YAG x=(0, 0.02, 0.04, 0.08, 0.12) crystals

	Yb0.16Tbx:YAG	τYb (ms)	ηET (%)
	0	1.61	-
	0.02	1.55	3.4
	0.04	1.34	16.3
	0.08	1.15	27.8
	0.12	0.87	45.4

Acknowledgements

This work is partially supported by National Natural Science Foundation of China (No.61805177 and No. 61621001), Fund of Key Laboratory of Optoelectronic Materials Chemistry and Physics, Chinese Academy of Sciences (No. 2008DP173016), MOE Key Laboratory of Advanced Micro-Structured Materials, "Qinglan Project" of the Young and Middle-aged Academic Leader of Jiangsu College and University.


The emergence of Tomato Leaf Curl New Delhi Virus in France

(atiwich.patthamapornsirikul@inrae.fr) S1-P7

The Mediterranean basin and Mainland France are facing the risk of Tomato leaf curl New Delhi virus or ToLCNDV (genus Begomovirus, family Geminiviridae) transmitted by the whitefly Bemisia tabaci. In Mediterranean countries, ToLCNDV was detected in 2012 in Spain (1) then throughout Southern Europe and North Africa (2). In September 2020, symptoms reminiscent to ToLCNDV were observed on zucchini in the Bouches-du-Rhône and Gard regions of France. The presence of ToLCNDV was confirmed using RCA (Rolling Circle Amplification) and PCR (Polymerase chain reaction) (3). Complete genome sequencing confirmed that the French isolates belonged to the "Mediterranean" ToLCNDV clade. However, molecular diversity studies suggested that at least two different variants, differing by 1-2%, have been introduced, possibly from different origins. Isolates from the two variants were cloned and characterized biologically, displaying contrasted severities in cucurbits with a recovery phenotype associated with one variant on susceptible melon and zucchini. Experiments are underway to characterize the molecular determinants of the phenotypic differences, the potential tradeoff with other properties (host range in cultivated and wild hosts, transmission specificity and efficiency) and the risks of durable establishment of the virus in the environment.

A

  . Patthamapornsirikul , E. Verdin, and C. Desbiez Pathologie Végétale, INRAE, Avignon, France.


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Cancer incidence in primary Sjögren's syndrome: Data from the French hospitalization database

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