Gonzalo Quiroga-Artigas
email: gonzalo.quiroga-artigas@crbm.cnrs.fr

María Moriel-Carretero

Storage cell proliferation during somatic

Keywords: tardigrade, storage cell, eutely, EdU, cell proliferation, DNA replication

INTRODUCTION

Tardigrades, often called water bears, are tiny animals (0.1-1mm long) that belong to the superphylum Ecdysozoa (Fig. 1A). This clade is characterized by the presence of an exoskeleton or a cuticle, which acts as a protection for their bodies [START_REF] Hickman | Integrated Principles of Biology[END_REF]. Tardigrades' cuticle plays pivotal roles in their life cycle, enabling them to respond and adapt to diverse environmental challenges [START_REF] Czerneková | The tardigrade cuticle[END_REF]. Like other ecdysozoans, tardigrades must undergo a process called ecdysis (i.e., molting), which involves producing a new cuticle and shedding the old one (known as exuvium), in order to grow in size [START_REF] Hickman | Integrated Principles of Biology[END_REF][START_REF] Czerneková | The tardigrade cuticle[END_REF]. Tardigrades are renowned for their exceptional capacity to withstand extreme conditions, leading to a significant surge in research on this subject in recent years [START_REF] Møbjerg | New insights into survival strategies of tardigrades[END_REF][START_REF] Kasianchuk | The biomedical potential of tardigrade proteins: A review[END_REF]. However, a more comprehensive understanding of tardigrade biology under physiological conditions is also crucial, as it is necessary to really understand the changes they undergo when exposed to harsh scenarios. Among tardigrades, Hypsibius exemplaris (Fig. 1B) is established as an emerging model organism in evolutionary developmental biology and extreme tolerance research [START_REF] Goldstein | The Future of Cell Biology: Emerging Model Organisms[END_REF][START_REF] Goldstein | Tardigrades and their emergence as model organisms[END_REF].

Like many other tardigrades, it exhibits parthenogenesis and synchronizes its molting period with oviposition to provide additional physical protection to the developing embryos through the exuvium [START_REF] Goldstein | Tardigrades and their emergence as model organisms[END_REF][START_REF] Altiero | Reproduction, development and life cycles. Water Bears: the Biology of Tardigrades[END_REF]. H. exemplaris hatch from their eggshells upon completion of embryonic development, measuring about 125 µm in length, and resembling small adults. They progressively increase in size through multiple molts, eventually reaching a fully-grown adult state measuring approximately 240 µm in length ((8), this work). Subsequently, they continue to molt at a relatively constant rate until their death, although these subsequent molting events no longer contribute to additional growth (Fig. 1C). H. exemplaris start producing eggs (i.e., reach sexual maturity) shortly after hatching, long before reaching their fully-grown size (Fig. 1D; [START_REF] Altiero | Reproduction, development and life cycles. Water Bears: the Biology of Tardigrades[END_REF][START_REF] Vasanthan | Life history traits for the freshwater Tardigrade Species Hypsibius exemplaris reared under laboratory conditions[END_REF]). To simplify the terminology, we named 'adults' those tardigrades that had both reached sexual maturity and their fully-grown size, and 'juveniles' those that were still growing, irrespective of sexual maturity (Fig. 1C-D).

Tardigrades feature a particular cell type, the storage cells, found floating freely throughout their body cavity fluid ( [START_REF] Gross | X-ray imaging of a water bear offers a new look at tardigrade internal anatomy[END_REF][START_REF] Hyra | Body cavity cells of Parachela during their active life[END_REF]; Fig. 1B). Their primary function is to assist in tardigrade nutritional maintenance by storing and distributing energy in the form of protein, glycogen, and fat [START_REF] Hyra | Body cavity cells of Parachela during their active life[END_REF][START_REF] Reuner | Comparative studies on storage cells in tardigrades during starvation and anhydrobiosis[END_REF]. They also participate in vitellogenesis [START_REF] Hyra | Body cavity cells of Parachela during their active life[END_REF][START_REF] Poprawa | Ultrastructural changes of the storage cells during oogenesis in Dactylobiotus dispar (Murray, 1907) (Tardigrada: Eutardigrada)[END_REF], and potentially contribute to immunity [START_REF] Volkmann | Ultrastructural localization of tyrosinase in the tardigrade cuticle[END_REF]. A recent study has shown increased expression of genes belonging to the SAHS (secretory abundant heat-soluble) family in storage cells [START_REF] Sae Tanaka | In vivo expression vector derived from anhydrobiotic tardigrade genome enables live imaging in Eutardigrada[END_REF], hinting at their potential involvement in desiccation tolerance. In H. exemplaris, lipids constitute the primary reservoir material within these cells [START_REF] Hyra | Body cavity cells of Parachela during their active life[END_REF]. These lipids can be stained with vital dyes such as BODIPY ((11, 16), see methods), revealing abundant fat in the form of lipid droplets inside the cytoplasm of storage cells (Fig. 1E-F). Storage cells can be isolated from the organism through dissection, enabling independent experimentation with them, separate from the whole organism ((17); Fig. 1F).

Eutely refers to a biological phenomenon observed in certain organisms where somatic cell division ceases once embryonic development is complete, implying a relative constancy in cell numbers throughout an animal's growth [START_REF] Van Cleave | Eutely or cell constancy in its relation to body size[END_REF]. This suggests that subsequent growth involves an increase in cell size rather than an increase in cell count. The prevailing belief, both in peer-reviewed scientific literature [START_REF] Hickman | Integrated Principles of Biology[END_REF][START_REF] Vasanthan | Life history traits for the freshwater Tardigrade Species Hypsibius exemplaris reared under laboratory conditions[END_REF][START_REF] Milo | Cell Biology by the Numbers[END_REF][START_REF] Beltrán-Pardo | Tolerance to gamma radiation in the tardigrade hypsibius dujardini from embryo to adult correlate inversely with cellular proliferation[END_REF] and in tardigrade biology outreach websites, is that tardigrades are eutelic animals. However, over 50 years ago, variations in organ cell numbers and potential somatic mitoses among different tardigrade species were already documented [START_REF] Bertolani | Mitosi somatiche e costanza cellulare numerica nei Tardigradi[END_REF][START_REF] Bertolani | Variabilitá numerica cellulare in alcuni tessuti di Tardigradi[END_REF]. More recently, two studies pointed at cell proliferation in two different somatic cell types. One study captured a limited number of storage cells presenting condensed chromosomes using DNA staining approaches in the tardigrade Richtersius coronifer [START_REF] Czernekova | Mitosis in storage cells of the eutardigrade Richtersius coronifer[END_REF]. Another study showed molecular evidence of cell proliferation at the anterior and posterior ends of the midgut in the tardigrade H. exemplaris, a process involved in replacing the cells lining the gut rather than in increasing the overall cell numbers [START_REF] Gross | Detection of cell proliferation in adults of the water bear Hypsibius dujardini (Tardigrada) via incorporation of a thymidine analog[END_REF].

Discrepancies regarding which cell types proliferate were noted by the authors compared to previous claims, possibly due to the starvation conditions in which the experiments were performed [START_REF] Gross | Detection of cell proliferation in adults of the water bear Hypsibius dujardini (Tardigrada) via incorporation of a thymidine analog[END_REF]. In a recent biology book [START_REF] Giribet | The invertebrate tree of life[END_REF], the authors wrote 'facts as basic as whether or not tardigrades are eutelic required considerable investigation (i.e. active bibliographic search) from our side'. Further, in a recent tardigrade review [START_REF] Jönsson | Cell Biology of the Tardigrades: Current Knowledge and Perspectives[END_REF] the question 'Are tardigrades eutelic animals?' was discussed, and the authors reasoned that this was not the case, due to the isolated indications of cell divisions and the cell number inconstancy studies mentioned above.

The ongoing debate surrounding whether tardigrades are genuinely eutelic or not, and the apparent difficulty for both the broader scientific community and the general public to reach a consensus on this matter, underscores the need for a more definitive characterization of the cell types that proliferate under normal physiological conditions within this enigmatic phylum.

In this study, we used H. exemplaris to determine whether somatic cell proliferation and an increase in cell numbers occur during tardigrade post-embryonic growth. Our findings reveal that overall cell numbers increase when comparing hatchlings to adults, mainly attributed to a prominent rise in storage cell numbers. Notably, EdU, a molecular tool for monitoring DNA replication, incorporates into storage cells at significantly higher rates during tardigrade growth than in fully-grown adults. We also show that starvation arrests growth and blocks EdU incorporation in storage cells, altogether linking storage cell proliferation to somatic growth. Moreover, we found a direct association between storage cell DNA replication and ecdysis, and reveal that hampering DNA replication provokes animal death during molting. Our results offer a comprehensive insight into DNA replication patterns during tardigrade growth, decisively demonstrating that tardigrades cannot be categorized as eutelic animals.

RESULTS

Tardigrade growth is accompanied by an increase in storage cell number

We first collected and fixed animals at both extremes of body size: hatchlings and fullygrown adults (Fig. 1C). We utilized the fluorescent DNA marker DAPI (4',6-diamidino-2phenylindole) to quantify the number of cell nuclei present at each growth stage (see methods; Fig. 2A; SI Appendix, Video S1). Our analysis revealed approximately 1140 ± 60 nuclei in hatchlings and approximately 1400 ± 118 in adults, pointing at a significant increase in cell numbers as the animals attained larger sizes (Fig. 2A-B). As we compared adult animals to hatchlings, we observed a notable increase in the area stained by the lipid vital dye BODIPY (Fig. 2C, in green). This led us to hypothesize that this change could be associated with an augmented number of storage cells. Leveraging the nuclei's position within the animal, their small size, and the intense BODIPY staining surrounding these nuclei, we successfully identified and quantified the number of storage cells in both hatchlings and adult tardigrades.

While hatchlings contained 72 ± 10 storage cells, the counts for adult H. exemplaris showed they bear 281 ± 64 storage cells, indicating a significant rise in storage cell numbers (Fig. 2C-D; SI Appendix, Video S2). Accordingly, we occasionally observed storage cells that appeared to be in the process of division (SI Appendix, Fig. S1). Intriguingly, the difference in the total number of cells, and the difference in the total number of storage cells between hatchlings and adults was similar, suggesting that the overall increase in cell number primarily results from storage cell proliferation. Our results show that the number of cells increases during tardigrade somatic growth and suggest that storage cells are the main cell type contributing to this raise.

Storage cells predominantly proliferate during animal growth

To investigate cell proliferation at the molecular level, we monitored DNA replication via the incorporation of the thymidine analog EdU, a marker previously employed successfully in tardigrades [START_REF] Gross | Detection of cell proliferation in adults of the water bear Hypsibius dujardini (Tardigrada) via incorporation of a thymidine analog[END_REF]. Initially, we collected hatchlings and incubated them in EdU for three weeks (Fig. 3A), approximately the duration required for a hatchling to develop into a fully-grown adult when kept at 15°C and fed ad libitum (Fig. 1C). This approach had the potential of allowing us to identify any DNA replication that may take place during H. exemplaris growth. We detected EdU incorporation in storage cells, germ cells inside the ovary, and gut cells (Fig. 3B-B').

Interestingly, there was an absence of EdU incorporation in any other organ, including epidermal cells, brain cells, ganglia, buccal apparatus, Malpighian tubules, and claw glands (Fig. 3B). While it is known that gut cells incorporate EdU in H. exemplaris [START_REF] Gross | Detection of cell proliferation in adults of the water bear Hypsibius dujardini (Tardigrada) via incorporation of a thymidine analog[END_REF], our observation represents the first molecular evidence demonstrating DNA replication, and thus cell proliferation, in tardigrade storage cells.

Tardigrade germ cells reside in the anterior end of the ovary, and derive from primordial germ cells that are specified during embryonic development [START_REF] Heikes | The embryonic origin of primordial germ cells in the tardigrade Hypsibius exemplaris[END_REF]. They form germ cell clusters by undergoing mitosis followed by incomplete cytokinesis [START_REF] Poprawa | Germ cell cluster organization and oogenesis in the tardigrade dactylobiotus parthenogeneticus bertolani, 1982 (Eutardigrada, murrayidae)[END_REF][START_REF] Jezierska | Oogenesis in the tardigrade Hypsibius exemplaris Gąsiorek[END_REF], thus being expected to incorporate EdU. Within these clusters, the cell completing meiosis develops into an oocyte, while the remaining differentiate into trophocytes (nourishing cells) [START_REF] Poprawa | Germ cell cluster organization and oogenesis in the tardigrade dactylobiotus parthenogeneticus bertolani, 1982 (Eutardigrada, murrayidae)[END_REF][START_REF] Jezierska | Oogenesis in the tardigrade Hypsibius exemplaris Gąsiorek[END_REF]. Accordingly, in addition to EdU incorporation in germ cells, we observed EdU signal in trophocytes and oocytes of H. exemplaris (SI Appendix, Fig. S2), corroborating the notion that these cell types are the progeny of germ cell clusters.

Next, we explored whether storage cell proliferation varied between (growing) juveniles and (fully-grown) adults. Juveniles and adults were incubated in EdU for seven days (Fig. 3C), a duration sufficient for all animals to experience at least one molting event when kept at 15°C. We observed that growing juveniles incorporated EdU in a large number of storage cells, whereas we could only detect a limited number of EdU + storage cells in adults (Fig. 3D -green asterisks), rendering the differences highly significant (Fig. 3E). Juveniles' EdU incorporation in a substantial number of storage cells occurred regardless of the animals' reproductive status at the time of fixation (non-gravid -Fig. 3D; gravid, or laying eggs -SI Appendix, Fig. S3). Our findings first show that the majority of storage cell number expansion takes place during tardigrade growth. Second, they evidence that storage cell proliferation also happens, although to a much-lessened extent, after tardigrades have reached their fully-grown status.

Starvation halts animal growth and suppresses storage cell proliferation

Tardigrades exhibit remarkable starvation tolerance, enduring several weeks without food [START_REF] Reuner | Comparative studies on storage cells in tardigrades during starvation and anhydrobiosis[END_REF]. Although prior studies noted that starvation reduces storage cell size [START_REF] Reuner | Comparative studies on storage cells in tardigrades during starvation and anhydrobiosis[END_REF], little is known about its potential effects on tardigrade growth and cell proliferation. To assess this, we first collected small juveniles of H. exemplaris and transferred them to dishes with and without Chlorococcum algae ("Fed" and "Starved" conditions). We observed that the majority of starved animals entered a contracted, resting state (Fig. 4A) shortly after food deprivation.

Upon reintroducing algae after one week, they resumed activity within minutes, and their guts were filled with algae within 24 hours (SI Appendix, Fig. S4A), indicating reversibility of the 'starvation state'. We also noticed a marked reduction in lipid staining inside the storage cells of starved individuals after one week (SI Appendix, Fig. S4B), suggesting that the fat reserves of storage cells were utilized to sustain the animals during starvation. Additionally, we collected small juveniles again, measured their length, and exposed them to the aforementioned conditions. After seven days, fed individuals exhibited significant growth, while starved ones showed no substantial increase in size (Fig. 4B-C), implying that starvation induces growth arrest in tardigrades.

To assess cell proliferation during food deprivation, we simultaneously exposed juveniles and adults to EdU and starvation for seven days (Fig. 4D). In both cases, we found a complete absence of EdU incorporation in storage cells, with rare exceptions where a few storage cells were EdU + (Fig. 4E-F). Essentially, in most individuals of either growth stage we could only detect EdU + gut cells (Fig. 4E -red asterisks). These results align with a previous study, which showed that starved adult H. exemplaris incubated with EdU for up to four days only exhibited EdU incorporation in gut cells [START_REF] Gross | Detection of cell proliferation in adults of the water bear Hypsibius dujardini (Tardigrada) via incorporation of a thymidine analog[END_REF]. Altogether, these findings demonstrate that starvation arrests tardigrade growth and inhibits EdU incorporation in storage cells, unambiguously linking storage cell proliferation to growth.

EdU incorporation in storage cells takes place during molting

To investigate whether DNA replication in storage cells occurs at specific stages of H. exemplaris post-embryonic development or is a continuous process throughout growth, we conducted 24-hour EdU incubations in juvenile animals, including those in proximity to molting (Fig. 5A). The vast majority of animals that did not molt during the 24-hour EdU incubation exhibited no EdU incorporation in storage cells (Fig. 5B-C); only gut cells displayed EdU labeling (red asterisks -Fig. 5B). In contrast, juveniles fixed during molting displayed a significant number of EdU + storage cells (green asterisks in Fig. 5B; Fig. 5C), in addition to EdU + gut and germ cells (red and white asterisks, respectively; Fig. 5B). These results strongly associate storage cell proliferation with the molting process (Fisher's exact test: p < 0.0001; SI Appendix, Table S1). Hence, our findings indicate that storage cell (and germ cell) proliferation does not occur constantly during tardigrade growth but rather in bursts at each ecdysis.

Hampering DNA replication progression results in tardigrade death during molting

This prompted us to assess the potential relevance of DNA replication for the molting process. To this purpose, we used hydroxyurea (HU), a drug known to effectively slow down the progression of replication forks and, consequently, EdU incorporation, in other aquatic invertebrates [START_REF] Quiroga-Artigas | GNL3 is an evolutionarily conserved stem cell gene influencing cell proliferation, animal growth and regeneration in the hydrozoan Hydractinia[END_REF][START_REF] Ramon-Mateu | Regeneration in the absence of a blastema requires cell division but is not tied to wound healing in the ctenophore Mnemiopsis leidyi[END_REF]. First, to ensure that HU successfully reduces EdU incorporation in replicative cells of H. exemplaris, we simultaneously exposed juvenile animals to HU and EdU for seven days (Fig. 5D). We used a 20mM HU dose, intermediate between the doses typically used in yeast (~100mM) and human cells (1-5mM) [START_REF] Ovejero | Coordination between phospholipid pools and DNA damage sensing[END_REF]. We observed an overall decrease in EdU incorporation in animals subjected to 20mM HU compared to control animals (Fig. 5E). Quantification of the number of EdU + storage cells, gut cells, and germ cells (including their progeny: trophocytes and oocytes) in both control and HU-treated condition revealed a highly significant reduction in all cases for animals under HU treatment (Fig. 5F-H). Having confirmed that HU disrupts replication progression in H. exemplaris by significantly reducing EdU incorporation in various replicative cell types, we then turned to assess its effects on molting.

In these first experiments, we already observed that, by day 7, a significant number of animals had died. To gain a more comprehensive insight into tardigrade survival under HU exposure, we incubated H. exemplaris specimens in 20mM HU and monitored mortality for up to 13 days. After nine days of incubation, allowing sufficient time for all treated animals to molt, we found that 96% ± 4% of the HU-treated animals had perished (Fig. 5I). Remarkably, all dead animals died during ecdysis, irrespective of their size or whether they had undergone egg laying during the process (Fig. 5E), indicating that ecdysis-associated deaths were unrelated to oviposition or egg viability during molting. Altogether, we conclude that hindering DNA replication leads to molting-related fatalities, emphasizing the critical role of accurate DNA replication in allowing effective tardigrade molting and survival.

DISCUSSION

This study addresses the controversy in the current literature regarding whether tardigrades maintain a constant number of cells during post-embryonic development, and we demonstrate unequivocally that this is not the case. Using the emerging model H. exemplaris, we illustrate how tardigrade storage cells primarily undergo proliferation during animal growth, leading to an increase in their numbers. We reveal that food deprivation induces a reversible 'starvation state' in H. exemplaris, during which both growth and storage cell proliferation are halted. Our 24-hour EdU pulse experiments establish a clear association between molting and storage cell DNA replication, and allowed us to illustrate a comprehensive representation of the DNA replication events occurring during tardigrade post-embryonic life for the three main replicative cell types we identified (Fig. 6). Furthermore, we establish that proper DNA replication is essential for tardigrade survival, as its disruption results in mortality during molting, the sole phase in which gut, germ, and storage cells undergo replication simultaneously (Fig. 6).

H. exemplaris does not present a defined number of cells, typical of eutelic animals, as its cell count increases with age and body growth (Fig. 2B). This is reinforced by the observed variability in the total number of cells among individuals of the same species (Fig. 2B). While we have demonstrated that this increase in cell number is primarily attributed to storage cell proliferation (Fig. 2D; Fig. 3), we also detected cell proliferation in the gut and ovary. However, these proliferative events are involved in cell replacement and the formation and sustenance of oocytes, respectively, rather than in overall cell number increase [START_REF] Gross | Detection of cell proliferation in adults of the water bear Hypsibius dujardini (Tardigrada) via incorporation of a thymidine analog[END_REF][START_REF] Poprawa | Germ cell cluster organization and oogenesis in the tardigrade dactylobiotus parthenogeneticus bertolani, 1982 (Eutardigrada, murrayidae)[END_REF][START_REF] Jezierska | Oogenesis in the tardigrade Hypsibius exemplaris Gąsiorek[END_REF]. In contrast to observations from decades ago in various species of Macrobiotus, where some ganglion and claw gland cells were interpreted as mitotic [START_REF] Bertolani | Mitosi somatiche e costanza cellulare numerica nei Tardigradi[END_REF], our investigations did not reveal any evidence of cell proliferation in any other organs or tissues of H. exemplaris (Fig. 3B, SI Appendix, Fig. S2). Thus, storage cells represent the primary cell type in H. exemplaris that undergoes proliferation and results in a cell number expansion.

Based on our findings, H. exemplaris cannot be categorized as entirely eutelic.

However, the absence of cell proliferation in specific organs, such as the epidermis and the nervous system, suggests that some degree of cell constancy may exist in various tardigrade organs. To gain a more comprehensive understanding, it would be valuable to reevaluate the proliferative capacities of different cell types in a range of tardigrade species using contemporary molecular techniques like EdU incorporation. This approach could provide insights into whether the absence of cell proliferation in certain organs is a common feature shared across the phylum.

The presence of cell proliferation in a limited number of cell types implies that the growth of most tardigrade organs primarily occurs through an increase in cell size rather than through cell proliferation. In this context, an increment in the number of storage cells may serve as a mechanism to support a larger body size and the associated higher demand for stored materials. This hypothesis has been previously suggested [START_REF] Czernekova | Mitosis in storage cells of the eutardigrade Richtersius coronifer[END_REF], and our data offer two pieces of evidence to support it. First, we show that growth arrest induced by starvation in juvenile tardigrades leads to the inhibition of storage cell proliferation (Fig. 4). Second, in adult H. exemplaris, where full growth has been achieved, the majority of storage cells within the animal no longer undergo proliferation (Fig. 3 D-E). From another perspective, considering that storage cells also contribute to vitellogenesis [START_REF] Hyra | Body cavity cells of Parachela during their active life[END_REF][START_REF] Poprawa | Ultrastructural changes of the storage cells during oogenesis in Dactylobiotus dispar (Murray, 1907) (Tardigrada: Eutardigrada)[END_REF], and given that a larger body size allows for a greater number of eggs to develop in the ovary, we postulate that an increase in storage cell numbers throughout tardigrade growth could further enhance the capacity to produce and nourish a larger number of eggs per individual. This aligns with our unreported observations that adult animals consistently produce more eggs than juveniles.

While anterior and posterior midgut cells maintain a constant rate of proliferation (Fig. 6, ( 24)), germ and storage cells appear to be arrested in G1 or in Go, only resuming cycling to undergo DNA replication during molting (Fig. 5B-C; Fig. 6). Correspondingly, a similar association between storage cell mitotic activity and molting has been reported in the tardigrade species R. coronifer [START_REF] Czernekova | Mitosis in storage cells of the eutardigrade Richtersius coronifer[END_REF]. These observations would imply that a specific cue, received at the time of molting, prompts these cell types to resume the cell cycle towards DNA replication. What could be the nature of such a signal? Tardigrades share orthologs of several genes associated with arthropod molting processes, including the ecdysone receptor and early-activated genes driven by ecdysone [START_REF] Schumann | Halloween genes in panarthropods and the evolution of the early moulting pathway in Ecdysozoa[END_REF]. This indicates a certain level of conservation of molting determinants within the Panarthropoda [START_REF] Schumann | Halloween genes in panarthropods and the evolution of the early moulting pathway in Ecdysozoa[END_REF][START_REF] De Oliveira | Ancient origins of arthropod moulting pathway components[END_REF]. Notably, recent research has shown that low ecdysone levels can stimulate cell proliferation in Drosophila wing imaginal discs [START_REF] Perez-Mockus | The ecdysone receptor promotes or suppresses proliferation according to ligand level[END_REF]. Downstream of ecdysteroid hormones, a cascade of evolutionarily ancient ecdysis-related neuropeptides (ERNs) plays crucial roles during ecdysis [START_REF] De Oliveira | Ancient origins of arthropod moulting pathway components[END_REF]. Originally responsible for regulating life cycle transitions in both molting and non-molting phyla, these ERNs have been co-opted to orchestrate the molting process in ecdysozoans [START_REF] De Oliveira | Ancient origins of arthropod moulting pathway components[END_REF][START_REF] Zieger | Ancestral Role of Ecdysis-Related Neuropeptides in Animal Life Cycle Transitions[END_REF]. Some of the genes encoding these ERNs are conserved in tardigrades, including eclosion hormone (EH) and crustacean cardioactive neuropeptide (CCAP), which are upregulated at hatching [START_REF] Zieger | Ancestral Role of Ecdysis-Related Neuropeptides in Animal Life Cycle Transitions[END_REF].

Intriguingly, H. exemplaris presents five EH and two CCAP paralogs [START_REF] Zieger | Ancestral Role of Ecdysis-Related Neuropeptides in Animal Life Cycle Transitions[END_REF][START_REF] Koziol | Precursors of neuropeptides and peptide hormones in the genomes of tardigrades[END_REF]. Along these lines, a neuropeptide-receptor couple has been found to induce meiotic progression in jellyfish oocytes arrested at prophase I [START_REF] Artigas | A G protein-coupled receptor mediates neuropeptideinduced oocyte maturation in the jellyfish Clytia[END_REF], suggesting that ERN-receptor couples could also regulate cell cycle progression. Collectively, we propose that ecdysteroid-type molting hormones and/or ERNs may serve as triggers for the resumption of the germ and storage cell cycle in tardigrades.

The question emerges as to why HU kills H. exemplaris during molting, irrespective of whether they are juveniles or adults. Indeed, even though adult tardigrades exhibit minimal storage cell proliferation (Fig. 3D-E; Fig. 6), they still perish during molting when exposed to HU treatment. Tardigrade molting events are likely the most energy-demanding stages of their post-embryonic development, since they involve replacing the buccal apparatus, claws, and the entire cuticle (2), synchronization with egg laying [START_REF] Altiero | Reproduction, development and life cycles. Water Bears: the Biology of Tardigrades[END_REF], and bursts of storage and germ cell proliferation (Fig. 5B-C). Given that gut cells undergo permanent renewal [START_REF] Gross | Detection of cell proliferation in adults of the water bear Hypsibius dujardini (Tardigrada) via incorporation of a thymidine analog[END_REF], animals' death during molting could reflect a profound impact of disrupting gut cell proliferation, consequently affecting overall energy absorption. In this scenario, the decrease in gut cell proliferation would have a more deleterious effect on survival than that of storage cells.

Alternatively, yet not exclusively, molting-associated fatalities could be ascribed to more direct effects of HU. HU slows down the progression of replication forks and inhibits the firing of late origins [START_REF] Kliewer | A Mec1-and Rad53-dependent checkpoint controls latefiring origins of DNA replication[END_REF]. Consequently, the coordination of the replicative program with other DNA-related processes, such as transcription, will be perturbed [START_REF] Hoffman | Break-seq reveals hydroxyurea-induced chromosome fragility as a result of unscheduled conflict between DNA replication and transcription[END_REF]. As shown in other ecdysozoans, ecdysis is a highly transcriptionally-active process [START_REF] Schumann | Halloween genes in panarthropods and the evolution of the early moulting pathway in Ecdysozoa[END_REF]. Moreover, it has been recently demonstrated that successful molting in the nematode Caenorhabditis elegans requires rhythmic accumulation of transcription factors, which, in turn, relies on rhythmic transcription [START_REF] Meeuse | elegans molting requires rhythmic accumulation of the Grainyhead/ LSF transcription factor GRH -1[END_REF]. Therefore, HU treatment could enhance tardigrade lethality during molting due to profound alterations in the execution of the ecdysis transcriptional schedule.

Additionally, HU induces DNA damage through oxidative stress [START_REF] Davies | Hydroxyurea Induces Hydroxyl Radical-Mediated Cell Death in Escherichia coli[END_REF], and also leads to DNA break accumulation when replication fork stalling is long-lasting [START_REF] Ian | Hydroxyurea: Induction of breaks in template strands of replicating DNA[END_REF], two DNA damageassociated signals that trigger a robust DNA damage response [START_REF] Ciccia | The DNA Damage Response: Making It Safe to Play with Knives[END_REF]. In the context of H. exemplaris, where there is no storage cell proliferation between molting cycles, DNA damage signals may be managed or controlled. However, they may translate into a systemic death signal when this insult occurs during a burst of storage cell proliferation. In agreement, DNA damage in proliferative cell types has recently been shown to trigger an alert in the whole animal that accelerates inflammatory and aging outputs [START_REF] Matos-Rodrigues | In vivo reduction of RAD51 -mediated homologous recombination triggers aging but impairs oncogenesis[END_REF].

In summary, our study provides the first molecular evidence of storage cell proliferation in a tardigrade species. It determines that tardigrades do not present cell constancy during post-embryonic development, contrarily to several previous claims, thus resolving a longstanding controversy in the field. Moreover, our data shed new light on tardigrade cell biology and establish a clear connection between tardigrade ecdysis and cell cycle transitions. The finding that storage cells undergo replication opens new possibilities for in vitro culture of this cell type and suggests its potential as a valuable model for studying cell cycle dynamics, responses to replication stress, and DNA damage control in tardigrades. With the advent of transgenesis [START_REF] Sae Tanaka | In vivo expression vector derived from anhydrobiotic tardigrade genome enables live imaging in Eutardigrada[END_REF] and CRISPR [START_REF] Kumagai | Application of CRISPR/Cas9 system and the preferred no-indel end-joining repair in tardigrades[END_REF] technologies in tardigrades, along with the established RNAi methods [START_REF] Tenlen | RNA interference can be used to disrupt gene function in tardigrades[END_REF][START_REF] Boothby | Tardigrades Use Intrinsically Disordered Proteins to Survive Desiccation[END_REF], Hypsibius exemplaris is increasingly becoming a promising genetic experimental model, altogether marking an exciting era in tardigrade research.

MATERIAL AND METHODS

Animal husbandry and drug treatment H. exemplaris (Z151 strain) husbandry was conducted as previously described [START_REF] Roszkowska | Tips and tricks how to culture water bears: simple protocols for culturing eutardigrades (Tardigrada) under laboratory conditions[END_REF], with minor modifications. In brief, tardigrades were maintained in an incubator at 15 °C, placed inside 55 mm diameter plastic Petri dishes filled halfway with spring water (Volvic) filtered through a 0.2 µm mesh. To facilitate tardigrade movement, the bottom of the dishes was scratched with sandpaper. Photoperiod and relative humidity inside the incubator were not monitored. Animals were fed ad libitum with Chlorococcum algae, cultivated in 50 ml tubes with BG-11 Growth Media (Gibco, A1379901). Water changes were performed every two weeks. All experimental incubations lasting ≥24 hours were carried out under these same conditions in 35 mm diameter dishes, placed inside cardboard boxes to avoid degradation of potentially photosensitive compounds.

Tardigrades were subjected to incubation with the ribonucleotide reductase inhibitor hydroxyurea (HU; Sigma-Aldrich, H8627) at a concentration of 20 mM, dissolved in filtered spring water (FSW) for the duration of the experiment. Renewal of HU took place every 2-3 days to ensure its continued effectiveness.

DAPI and BODIPY staining

Animals were collected and filtered through a 40 µm mesh, then transferred to a glass depression slide within a humid chamber using a glass Pasteur pipette. Whenever possible, samples were kept in darkness for the duration of the experiment. To label neutral lipids, live specimens were incubated in 10 µg/ml BODIPY (Difluoro{2-[1-(3,5-dimethyl-2H-pyrrol-2ylidene-N)ethyl]-3,5-dimethyl-1H-pyrrolato-N}boron; Sigma-Aldrich, 790389) diluted in FSW for 30 minutes. Subsequently, samples were rinsed three times with FSW and fixed in 4% PFA in 1x PBS for 1 hour at room temperature (RT). After fixation, three 15-minute washes in 1x PBS were performed. Samples were then incubated in the DNA fluorescent stain DAPI (4',6-diamidino-2-phenylindole; Sigma-Aldrich, D9542) at a concentration of 1 µg/ml in 1x PBS for 30 minutes. Following this step, the samples were washed three times in 1x PBS for 15 minutes, transferred to a slide, and mounted in ProLong Gold Antifade Mountant (Invitrogen, P36930) prior to confocal imaging.

EdU experiments

To detect DNA replication, animals were incubated in 100 µM EdU (EdU Click-iT™ Cell Proliferation Kit for Imaging, Alexa Fluor 488 Dye; Invitrogen, C10337) diluted in FSW containing algae (except during experimental starvation) for specific durations (24 hours, 7 days, or 3 weeks). In the 3-week experiment, EdU was refreshed weekly. Following EdU exposure, tardigrades were collected, transferred to a glass depression slide, and fixed with 4% PFA in 1x PBS + 1% Triton X-100 (PTx) for 1 hour at RT. After fixation, the samples underwent three 15-minute washes in PTx. Subsequently, they were blocked for 2 hours in a 0.2 µm-filtered blocking solution containing 10% Bovine Serum Albumin (BSA) in 1x PBS.

The Click-iT EdU detection reaction was carried out for 1 hour at RT according to the manufacturer's instructions. After the detection reaction, four 10-minute PTx washes were conducted. DAPI was used to stain nuclei, and samples were mounted in ProLong Gold for confocal imaging, as described above. In some cases, tardigrades were euthanized with 10% EtOH before fixation to prevent the animals from contracting during exposure to PFA.

Imaging, length measurements, cell counting and statistics

To capture live images of tardigrades, animals were anesthetized using 20mM levamisole hydrochloride (MedChemExpress, HY-13666) in mqH2O and mounted on slides, covered with coverslips. Clay was positioned in each corner of the coverslip to prevent compression, except for the intentionally compressed specimen in Fig. 1B. Differential interference contrast (DIC) color images were taken with a Leica K3C digital color camera attached to a compound light microscope (Leica Thunder). For measuring the body length of tardigrades, measurements were conducted using ImageJ [START_REF] Schneider | NIH Image to ImageJ: 25 years of image analysis[END_REF]. The measurement was taken from the head to the juncture on the posterior-most segment with legs [START_REF] Vasanthan | Life history traits for the freshwater Tardigrade Species Hypsibius exemplaris reared under laboratory conditions[END_REF]. For starved tardigrades, measurements were obtained immediately after animals came back to an active state, ensuring their full body extension.

Fluorescently labeled images were acquired using a Zeiss LSM980 confocal microscope, equipped with an Airyscan2 module. To ensure consistency when comparing samples within a given experiment, identical scanning parameters were applied to all conditions in each independent experiment. Confocal Z-stacks and projections were processed and adjusted for brightness and contrast in ImageJ, using K. Terretaz's visualization toolset (https://github.com/kwolbachia/Visualization_toolset).

To count the total number of cells in hatchlings and adults, Imaris software (Oxford instrument) was employed. Nuclei were highlighted using custom thresholding to allow for quantification. Quantifications of BODIPY-stained storage cells and EdU + cells were performed manually in ImageJ. In all instances, z-stacks encompassing the entire depth of the animals were utilized. Supplementary videos S1 and S2 were assembled using Imaris and ImageJ software, respectively. Tardigrade schematics and figure compilation were generated using Adobe Illustrator.

To determine statistical significance, normality of all datasets was first assessed using the Shapiro-Wilk test. Two-tailed Student's t-test was conducted for the dataset concerning total number of storage cells (Fig. 2D). For all other comparisons, Mann-Whitney U nonparametric tests were employed for two-way comparisons, and the Kruskal-Wallis test was used for statistical analyses in Fig. 4C. Fisher's exact test was selected for the association assessment, based on a 2 × 2 contingency table. Statistical significance for all quantitative comparisons is represented as ****, where p-value ≤ 0.0001. Graphs and statistical analyses were performed using GraphPad Prism 9. The number of replicative storage cells decreases markedly as tardigrades reach their fullygrown adult size, highlighting that most storage cell proliferation occurs during somatic growth.

FIGURES AND

Figure 1 .

 1 Figure 1. The tardigrade Hypsibius exemplaris and its storage cells. (A) A simplified animal phylogeny, highlighting the position of tardigrades (Tardigrada) within the Ecdysozoa. Tardigrades share common ancestry with the Onychophora + Arthropoda group, collectively forming the Panarthropoda clade (after Schultz et al. (51), Wu et al. (52), Laumer et al. (53), Dunn et al. (54), and Campbell et al. (55)). Certain groups like Placozoa and Xenacoelomorpha are omitted for simplicity. Images sourced from Phylopic.org. (B) Anatomy of H. exemplaris. Internal organs are revealed in a smushed specimen. The cuticle refracts light and envelops the animal, while the green patch inside the midgut indicates the digestion of Chlorococcum algae. (C) Schematic representation of H. exemplaris post-embryonic development. (D) Images of two gravid juveniles at different sizes and a gravid fully-grown

Figure 2 .

 2 Figure 2. Somatic growth involves overall cell number increase, mainly driven by storage cell number expansion. (A) Representative images of a hatchling (top) and an adult (bottom) of H. exemplaris, showing nuclei (DAPI, blue) and software-assisted nuclei recognition (in gray; see methods). Rightmost panels show merged images. The full depth of the animals was captured in confocal z-stacks, and the images shown are maximum projections. (B) Violin plots displaying the total number of nuclei in hatchlings (n = 25) and adults (n = 22). Center yellow lines show the medians; discontinuous yellow lines show the quartiles; the width of the violin at any given point represents the density of data points at that value; each quantified sample is represented by an empty circle. (C) Representative images of a hatchling (top) and an adult (bottom) of H. exemplaris, showing DNA (DAPI, blue) and neutral lipids (BODIPY, green). Insets show magnifications of the regions outlined in C, corresponding to the antero-dorsal region of the tardigrade body, were a big proportion of storage cells are normally found (Fig. 1E; (10)). Nuclei in insets are shown in gray. Individual images are shown, rather than maximum projections, to facilitate storage cell visualization within the animals. (D) Violin plots depicting the number of storage cells in hatchlings (n = 26) and adults (n = 27). Violin plot description as in (B). ****, p ≤ 0.0001. Scale bars: 50 µm in (A, C); 10 µm in (C insets).

Figure 3 .

 3 Figure 3. Storage cell proliferation predominantly occurs during somatic growth. (A) Schematic depicting the 3-week EdU incubation experiment, concerning (B). (B) Representative image of tardigrade upon 3-week exposure to EdU showing nuclei (DAPI, blue) and EdU + cells (orange). (B') Magnification of regions outlined in (B), highlighting the three main replicative cell types labeled with EdU (gray). (C) Experimental schematics for 7day EdU exposure (D-E). (D) Representative images of juvenile (top) and adult (bottom) after 1-week EdU exposure, displaying nuclei (DAPI, blue) and EdU + cells (orange). Right images show only EdU + cells (gray). (E) Violin plots showing the number of EdU + storage cells in juveniles (n = 95) and adults (n = 90). Plotted values belong to three independent experiments. Each quantified animal is represented by a colored dot, with distinct colors corresponding to individual experiments. Larger dots represent each experiment's mean. Plot description as in Fig. 2B. Images shown are maximum projections of the animals' full depth, except for (B'), which focuses on specific cell types using sub-stack projections. Asterisks denote EdU + cell types (storage cells, green; germ cells, white; gut cells, red). ****, p ≤ 0.0001. Scale bars: 100 µm in (B, D); 5 µm in (B').

Figure 4 .

 4 Figure 4. Impact of starvation on growth and storage cell proliferation. (A) Representative image of H. exemplaris in the described 'starvation state'. (B) Representative images of H. exemplaris juveniles at day 0 and at day 7 under starved and fed conditions. (C) Violin plots illustrating tardigrade length in juveniles at day 0 (n = 172) and at day 7 under starved (n = 92) and fed (n = 80) conditions. Plot description follows Fig. 2B. (D) Experimental schematics for 7-day EdU exposure in starved juveniles and adults (E-F). (E) Representative images of starved juvenile (top) and starved adult (bottom) after 1-week EdU exposure, showing nuclei (DAPI, blue) and EdU + cells (orange). Right images display only EdU + cells (gray). Red asterisks indicate EdU + gut cells. The full depth of the animals was captured in confocal z-stacks, and the images shown are maximum projections. (F) Violin plots showing the number of EdU + storage cells in starved juveniles (n = 115) and starved adults (n = 112). Plotted values belong to three independent experiments. Plot conventions are consistent with Fig. 3E. Red tardigrade schematics indicate starved condition. ns = nonsignificant; **** = p ≤ 0.0001. Scale bars: 50 µm.

Figure 5 .

 5 Figure 5. Storage cells undergo replication during molting, while blocking replication results in molting-related fatalities. (A) Experimental schematics for 24-hour EdU exposure (B-C). (B) Representative images of non-molting (top) and molting (bottom) tardigrades upon 24-hour EdU exposure, illustrating animal morphology (DIC, gray) and EdU + cells (orange). Arrowhead indicates the old cuticle being molted. Right images show only EdU + cells (gray). (C) Violin plots displaying the number of EdU + storage cells in nonmolting (n = 452) and molting animals (n = 76). Plotted values belong to four independent experiments. (D) Schematic illustrating the 7-day experiment exposing tardigrades to EdU and HU simultaneously, concerning (E-H). (E) Representative images of control (-HU) and 20 mM HU-treated animals after 1-week EdU exposure, showing animal morphology (DIC, gray) and EdU + cells (orange). Arrowhead indicates a laid egg that has degraded. Right images display only EdU + cells (gray). Maximum projections of the animals' full depth are shown. Asterisks as in Fig. 3. Scale bars = 50 µm. (F-H) Violin plots showing the number of EdU + storage cells (F), gut cells (G), and germ cells (including their progeny: oocytes and

Figure 6 .

 6 Figure 6. Overview of DNA replication events in tardigrade post-embryonic development. Replicative gut cells are represented as red circles and ovals. Replicative germ cells are represented as blue circles. Replicative storage cells are represented as small green circles. In summary, gut cells in the anterior and posterior parts of the midgut replicate continuously, while germ and storage cells only undergo replication at each molting event.

ACKNOWLEDGEMENTS

We thank Simon Galas and Myriam Richaud for providing us with H. exemplaris and Chlorococcum samples, and for giving us advice on tardigrade manipulation. We also thank Benjamin Lacroix for helping us to establish the model by sharing his equipment, and Kseniya Samardak and Sylvain Kumanski for their help with tardigrade husbandry. We are grateful to Kevin Terretaz for his valuable tips and macros on ImageJ. We also express gratitude to the CRBM direction for its support. We acknowledge the imaging facility MRI, member of the France-BioImaging national infrastructure supported by the French National Research Agency (ANR-10-INBS-04, «Investments for the future»). This research was supported by the I-SITE MUSE (Montpellier Université d'Excellence).

AUTHOR CONTRIBUTIONS


Elisabeth Hornberger
email: elisabeth.hornberger@tu-berlin.de

Thomas Merzdorf

Henrike Schmies

Jessica Hübner

Malte Klingenhof

Ulrich Gernert

Matthias Kroschel

Björn Anke

Martin Lerch

Johannes Schmidt

Arne Thomas

Raphaël Chattot

Isaac Martens

Jakub Drnec

Peter Strasser
email: pstrasser@tu-berlin.de

Impact of Carbon N-Doping and N-Pyridinic Content on the Fuel Cell Performance and Durability of Carbon Supported Pt Nanoparticle Catalysts

E.H. performed the synthesis, XRD, ZP and RDE experiments. T.M

Introduction

Green hydrogen generated from water electrolysis using renewable energy power sources is a clean and sustainable option to meet the fossil fuel free energy demands. The conversion from the chemical energy stored in the hydrogen bond into usable electricity finds application in the low temperature proton exchange membrane fuel cell (PEMFC), which is already utilized in automotive, heavy-duty vehicles or stationary devices. The extensive commercialization of such PEMFC devices is limited by cost, power density and durability issues when comparing with competing technologies such as traditional combustion engines or battery systems. [START_REF] Gröger | Review-Electromobility: Batteries or Fuel Cells[END_REF][START_REF] Cunanan | A Review of Heavy-Duty Vehicle Powertrain Technologies: Diesel Engine Vehicles, Battery Electric Vehicles, and Hydrogen Fuel Cell Electric Vehicles[END_REF] Efforts have been made to reduce the amount of Pt in the catalyst material while maintaining the performance and improving the stability of the cathode catalytic layer (CCL). [START_REF] Kongkanand | The Priority and Challenge of High-Power Performance of Low-Platinum Proton-Exchange Membrane Fuel Cells[END_REF][START_REF] Orfanidi | The Key to High Performance Low Pt Loaded Electrodes[END_REF] The cathode side of the PEMFC where the oxygen reduction reaction (ORR) takes place is one of the key concerns due to the sluggish kinetics of the ORR that cause large overpotential losses. The catalytic activity and the power density are strongly depending on the realization of the triple phase boundary: Pt catalyst, oxygen gas and ionomer electrolyte interface. The ionomer in low temperature PEMFC, such as the commonly used Nafion polymer, not only provides protons, but also free sulfonate groups that can adsorb on the active Pt surface and attenuate the performance in the low current density region. [START_REF] Subbaraman | Three Phase Interfaces at Electrified Metal-Solid Electrolyte Systems 1. Study of the Pt(hkl)-Nafion Interface[END_REF][START_REF] Subbaraman | Oxygen reduction reaction at three-phase interfaces[END_REF][START_REF] Kodama | Effect of the Side-Chain Structure of Perfluoro-Sulfonic Acid Ionomers on the Oxygen Reduction Reaction on the Surface of Pt[END_REF] Inhomogeneously distributed ionomer, however, limits the oxygen transport towards the Pt surface and reduces the performance in the high current density region. [START_REF] Suzuki | Model for investigation of oxygen transport limitation in a polymer electrolyte fuel cell[END_REF][START_REF] Weber | Unexplained transport resistances for low-loaded fuel-cell catalyst layers[END_REF][START_REF] Jinnouchi | Molecular Dynamics Simulations on O 2 Permeation through Nafion Ionomer on Platinum Surface[END_REF][START_REF] Kudo | Humidity and Temperature Dependences of Oxygen Transport Resistance of Nafion Thin Film on Platinum Electrode[END_REF] One solution for improved ionomer-catalyst interaction was proposed to be N-doped carbon materials. [START_REF] Orfanidi | The Key to High Performance Low Pt Loaded Electrodes[END_REF][START_REF] Ott | Ionomer distribution control in porous carbon-supported catalyst layers for highpower and low Pt-loaded proton exchange membrane fuel cells[END_REF] Surface -NH x groups can be introduced to the carbon support, e.g. via ammonolysis, which develop Coulombic interactions with the -SO 3 -groups of the ionomer beneficially influencing the ionomer distribution towards an increased homogeneity. [START_REF] Arrigo | Tuning the Acid/Base Properties of Nanocarbons by Functionalization via Amination[END_REF] Orfanidi et al. applied this concept to a solid Vulcan XC 72R (moderate surface area (SA), higher degree of graphitization) and Ott et al. used a mesoporous Ketjenblack EC-300J (internal pores, high SA) as carbon material for the ammonolysis. [START_REF] Orfanidi | The Key to High Performance Low Pt Loaded Electrodes[END_REF][START_REF] Ott | Ionomer distribution control in porous carbon-supported catalyst layers for highpower and low Pt-loaded proton exchange membrane fuel cells[END_REF] Both studies showed an improved H 2 /air performance in membrane electrode assembly (MEA) testing that was directly attributed to an improved ionomer distribution and thus an improved oxygen mass transport at high current densities. The latter study demonstrated an impressive performance that was further credited to highly accessible pores. The concept of accessible pores refers to porous carbon materials with preferred pore openings of 4-7 nm that can host Pt particles in the internal pores protected from the ionomer poisoning but are still accessible by protons and oxygen. [START_REF] Yarlagadda | Boosting Fuel Cell Performance with Accessible Carbon Mesopores[END_REF] Such catalysts with accessible pores can facilitate both high ORR activity and reactant transport properties without adversely affecting each other.

The durability of Pt/C catalysts proves to be an issue in fuel cell application. [START_REF] Kongkanand | The Priority and Challenge of High-Power Performance of Low-Platinum Proton-Exchange Membrane Fuel Cells[END_REF][START_REF] Banham | Ultralow platinum loading proton exchange membrane fuel cells: Performance losses and solutions[END_REF][START_REF] Pan | Current challenges related to the deployment of shapecontrolled Pt alloy oxygen reduction reaction nanocatalysts into low Pt-loaded cathode layers of proton exchange membrane fuel cells[END_REF] Degradation phenomena in relation to the Pt particles of the catalyst have been identified as a) particle growth accompanied with decreased electrochemical active surface area (ECSA) due to coalescence/Ostwald ripening and aggregation/agglomeration processes, b) Pt dissolution and c) particle migration/detachment. [START_REF] Shao-Horn | Instability of Supported Platinum Nanoparticles in Low-Temperature Fuel Cells[END_REF] The carbon support material is prone to degradation phenomena due to carbon corrosion via electrochemical oxidation in the presence of water following equation 1 at potentials above 0.207 V 18 :

1 However, the kinetics of the carbon oxidation reaction are poor under typical fuel cell operating conditions. Several studies reported that minor carbon corrosion develops for potentials above 0.6 V, and becomes severe at potentials above 1.4 V in situations of start-up/shutdown and/or local fuel starvation at the anode. [START_REF] Maass | Carbon support oxidation in PEM fuel cell cathodes[END_REF][START_REF] Maillard | An EC-FTIR study on the catalytic role of Pt in carbon corrosion[END_REF][START_REF] Macauley | Carbon Corrosion in PEM Fuel Cells and the Development of Accelerated Stress Tests[END_REF][START_REF] Reiser | A Reverse-Current Decay Mechanism for Fuel Cells[END_REF] Reiser et al. [START_REF] Reiser | A Reverse-Current Decay Mechanism for Fuel Cells[END_REF] rationalized that at these conditions when the anode is partially exposed to both, hydrogen and oxygen, the electrolyte potential drops, resulting in a current flow of opposite direction at the oxygen exposed region (compared to the normal fuel cell mode) that causes a raised cathode interfacial potential with a high oxidative impact in the carbon.

Furthermore, the presence of Pt particles is believed to further promote the carbon corrosion in the electrochemical environment. [START_REF] Willisau | The influence of Pt-activation on the corrosion of carbon in gas diffusion electrodes -A DEMS study*[END_REF][START_REF] Siroma | Electrochemical Corrosion of Carbon Materials in an Aqueous Acid Solution[END_REF] ORR catalysts with durable Pt and carbon compounds are crucial for a successful application of the PEMFC.

In an earlier work, we prepared a set of N-doped Vulcan carbons via ammonolysis at elevated temperature and quantitatively investigated the degradation due to carbon corrosion with hightemperature differential electrochemical mass spectrometry (HT-DEMS) at 140 °C. [START_REF] Schmies | Impact of Carbon Support Functionalization on the Electrochemical Stability of Pt Fuel Cell Catalysts[END_REF] MEAs fabricated of unmodified and N-doped Vulcan carbons were subjected to increasing potentials from 0.06 V up to 1.05 V and the volatile products of the cathode were analyzed using the HT-DEMS. The direct comparison of unmodified and N-doped Vulcan revealed a sharp decrease of the corrosion current accompanied by a comparable strong decrease in mass ion current with m/z = 44 for CO 2 for the Ndoped Vulcan. We concluded from this that the N-functionalization has a beneficial influence on the carbon corrosion durability of the pure Vulcan material. This and a remarkable rotating disk electrode (RDE) durability with minor ORR activity losses of the platinized N-doped carbon catalyst (Pt/N-Vulcan) suggests that N-doped carbon could act as promising support material for the PEMFC cathode with high carbon corrosion resistance durability and improved ionomer distribution.

However, detailed investigation with MEA single cell measurements was missing to complete the presented results.

In the present work, unmodified and N-doped carbon were prepared and uniformly decorated with Pt nanoparticles of narrow particle size distribution using a customized fluidized bed reduction reactor. [START_REF] Hornberger | Design and Validation of a Fluidized Bed Catalyst Reduction Reactor for the Synthesis of Well-Dispersed Nanoparticle Ensembles[END_REF] The set of catalysts were physico-chemically characterized and investigated towards their ORR performance and catalyst durability with focus on Pt stability and carbon corrosion using MEAbased fuel cell measurements, in addition to RDE. To further elucidate the effects of the N-doping, operando high energy X-ray diffraction (HE-XRD) experiments were conducted. With these investigations, we help to understand the degradation phenomena towards improved ORR catalysts for the low temperature PEMFC.

Results and Discussion

Physico-chemical Characterization

For this study, a set of Pt-based ORR catalysts were prepared using commercial Vulcan XC 72R

(denoted as V) carbons and from-it-derived N-doped Vulcan carbons. Typically, the N-doped carbons were functionalized via pre-oxidation with HNO 3 and ammonolysis at the elevated temperatures of 400 °C and 800 °C as published before (denoted as N-V-XXX, with XXX representing the ammonolysis temperature, synthesis details in supporting information). [START_REF] Ott | Ionomer distribution control in porous carbon-supported catalyst layers for highpower and low Pt-loaded proton exchange membrane fuel cells[END_REF][START_REF] Arrigo | Tuning the Acid/Base Properties of Nanocarbons by Functionalization via Amination[END_REF][START_REF] Schmies | Impact of Carbon Support Functionalization on the Electrochemical Stability of Pt Fuel Cell Catalysts[END_REF] The pre-oxidation step of the synthesis leads to an increase of O-moieties at the carbon surface in form of carboxylic, hydroxylic and NO x groups for oxidized Vulcan (O-V, see Table 1). [START_REF] Ott | Ionomer distribution control in porous carbon-supported catalyst layers for highpower and low Pt-loaded proton exchange membrane fuel cells[END_REF] These oxygenated surface groups are then successively reduced during the ammonolysis steps to nitrogenated surface groups. The elemental bulk composition was determined using elemental analysis of carbon, hydrogen, nitrogen, and sulfur (CHNS analysis). The N-doped carbons show a low degree of N-incorporation into the C matrix of 2.7 and 1.4 at-% N for N-V-400 and N-V-800, respectively, indicating a decomposition of less stable N-and/or O-groups with higher temperature.

The unmodified V carbon contains no bulk N-content. The remaining O-content was determined to be 3.7 and 2.2 at-% O for N-V-400 and N-V-800, respectively. N-V-400 therefore has the highest degree of heteroatom-doping. and cylindrical pore model (pore diameter >2 nm). The size of micropores is defined as < 2 nm and of mesopores as 2-50 nm. [START_REF] Mcnaught | IUPAC. Compendium of Chemical Terminology[END_REF] The total surface area of both methods yield in comparable values, considering that results from BET measurements are prone to overestimate the surface area while results from QSDFT often underestimate the surface area due to density differences of bulk nitrogen and nitrogen adsorbed in micropores. [START_REF] Orfanidi | Ink Solvent Dependence of the Ionomer Distribution in the Catalyst Layer of a PEMFC[END_REF] The BET SA of the unmodified V carbon is 229 m 2 •g -1 . After the ammonolysis, the N-doped carbons show a decreased SA when being ammonolysed at 400 °C (199 m 2 •g -1 ) and an increased SA when being treated at 800 °C (245 m 2 •g -1 ). The pore size analysis reveals that both N-doped carbons have an increased amount of mesopores of ~26-29 %. This could be explained by ammonia etching for both carbon materials at the present mesopores. [START_REF] Jaouen | Non-noble electrocatalysts for O2 reduction: how does heat treatment affect their activity and structure? Part I. Model for carbon black gasification by NH3: parametric calibration and electrochemical validation[END_REF][START_REF] Sherwook | Hydrogen cyanide: Synthesis from elements and from ammonia and carbon[END_REF] The micropore ratio of N-V-400 drastically decreases by 52 %. This decrease of nitrogen gas accessible micropores could be explained by a blockage of these micropores by surface groups. [START_REF] Orfanidi | The Key to High Performance Low Pt Loaded Electrodes[END_REF][START_REF] Toupin | Spontaneous Functionalization of Carbon Black by Reaction with 4-Nitrophenyldiazonium Cations[END_REF] For N-V-800, these blocked pores might open up again due to the decomposition/incorporation of the surface groups at higher temperature. Therefore, the micropore/total ratio is like V. The solid character of the N-doped V materials is preserved after the carbon functionalization. The surface area stays at a moderate level and the pores remain external and no internal pores are created. Thermogravimetric The dispersion behavior of the carbon materials in aqueous media was examined via the measurement of the surface zeta potential (ZP, Table 2). High absolute values of the ZP suggest increased surface charges that result in a low degree of aggregation due to strong repulsive forces within the dispersion. [START_REF] Zhang | The morphology, structure and electrocatalytic ability of graphene prepared with different drying methods[END_REF] This is of interest for the ink formulation that commonly use a mixture of water and organic solvents as ethanol or isopropanol and following preparation of homogeneous electrode layers. The ZP is 27.8 mV for V, decreases for N-V-400 to 16.4 mV and increases again to 28.9 mV for N-V-800. From the results a similar good dispersion behavior is expected for V and N-V-800, whereas N-V-400 might show less stable dispersion.

The crystalline structure of the carbon materials was studied with XRD, and the collected patterns The unmodified and N-doped carbons were decorated with Pt particles using a fluidized bed reduction reactor. [START_REF] Hornberger | Design and Validation of a Fluidized Bed Catalyst Reduction Reactor for the Synthesis of Well-Dispersed Nanoparticle Ensembles[END_REF] The carbon materials were impregnated in a slurry approach with a Pt salt (H 2 PtCl 6 ), homogenized using a horn sonifier, quenched in liquid nitrogen and dried in vacuo. The impregnated dry powders were transferred to the fluidized bed reduction reactor and reduced at 200 °C for 2 h in hydrogen atmosphere (4 % H 2 in Ar). The collected catalyst powders have a nominal low Pt loading of 20 wt-% Pt (Table S 1) and show broad face-centered cubic (fcc) Pt phase XRD reflections suggesting small crystallite sizes (Figure 1a). The Pt particle size is extracted from analysis of TEM images (Table S 1). The Pt particles are of small size (~2 nm) and uniformly distributed on the carbon supports. The Pt deposition method using the slurry approach with an excess volume of solvents and the solid character of the V carbon led to a distribution of Pt particles on the external surface of the carbon for the majority of the particles. This can be qualitative visualized by combining scanning electron microscopy (SEM) and transmission SEM (TSEM) images. SEM imaging exposes the Pt particles deposited on the exterior surface only, while the bright field TSEM imaging displays the Pt particles on both the exterior and interior surface. S 2). XPS is very surface sensitive, therefore it is of no surprise that Pt/V reveals a very small N 1s peak even though no bulk N content was found in CHNS analysis. The N-content could derive from the unknown industrial production processes of Vulcan XC 72R. The large percentage of N moieties for all three catalyst is pyridinic-N and pyrrolic-N. As shown before, the fraction of pyridinic-N increases with increased ammonolysis temperature while the fraction of pyrrolic-N decreases. [START_REF] Ott | Ionomer distribution control in porous carbon-supported catalyst layers for highpower and low Pt-loaded proton exchange membrane fuel cells[END_REF][START_REF] Schmies | Impact of Carbon Support Functionalization on the Electrochemical Stability of Pt Fuel Cell Catalysts[END_REF] This is in agreement with an incorporation of N atoms into the carbon matrix while pyrrolic-N decompose at elevated temperatures under release of HCN to form pyridinic-N. [START_REF] Arrigo | Dynamic surface rearrangement and thermal stability of nitrogen functional groups on carbon nanotubes[END_REF]

Electrochemical Characterization

To investigate the initial ORR performance, the catalysts were tested using the rotating disk electrode (RDE) technique. As reference, a widely known commercial 20 wt-% Pt/C with Vulcan XC 72R as carbon support (HISPEC3000, Johnson Matthey) was investigated in parallel. Figure 1a includes the fcc-Pt phase pattern and Figure 1e the morphology of HISPEC3000. Interestingly, the Pt particle size appear somewhat larger when analyzed the TEM micrographs (~3 nm, Table S and Pt/N-V-800 increases after the C-AST suggesting an additional cleaning and roughening of the Pt particles surface. For Pt/N-V-400, the changes of the ECSA H-upd are within the error. More interestingly, the C-AST reveals no signs of carbon corrosion for all three catalysts in terms of evolution of a chinon-hydrochinon redox pair (~0.5-0.75 V RHE ) in the CV. This suggests a good carbon corrosion durability for this set of catalysts. Yet it must be noted, that the RDE C-AST stability test in liquid electrolyte at room temperature do not necessarily translate toward full cell measurements. [START_REF] Qiao | Advanced Nanocarbons for Enhanced Performance and Durability of Platinum Catalysts in Proton Exchange Membrane Fuel Cells[END_REF] For the MEA setup, key parameters of the system change, e.g. electrode preparation, proton and oxygen transport, or water management. Therefore, a detailed analysis of the carbon corrosion in MEA environment is presented in the upcoming section.

The Pt-AST reveals a good and comparable retention of the mass-activity for Pt/V and Pt/N-V-800, while for Pt/N-V-400 it is only fair. This behavior follows the evaluation trends of the ECSA H-upd . Only small losses of the active Pt surface area during potential cycling are found for Pt/V and Pt/N-V-800.

TEM images and the particle size distributions suggest that this is due to low degree of particle growth via Ostwald ripening processes and/or agglomeration (Figure S 10), while the Pt particles overall remain uniform distributed on the carbon support. The loss of the active Pt surface area is more severe for Pt/N-V-400 even though TEM images exhibit no significantly pronounced particle growth or agglomeration compared to the other samples. However, TEM imaging generates only very local information. In summary, we prepared a set of Pt-based catalysts on unmodified and Ndoped carbon supports using the fluidized bed reduction reactor that shows good retention of mass activity, ECSA H-upd and no carbon corrosion when tested in RDE towards durability (C-AST: 5,000 CV, 1-1.5 V RHE , 500 mV•s -1 , Pt-AST: 10,000 SWV, 0.6 V RHE and 0.95 V RHE , 3 s at each potential).

Operando HE-XRD Experiments

For a more global and detailed investigation of the structural stability of the catalysts, operando HE-XRD experiments were performed at the Extremely Brilliant Source of the European Synchrotron Radiation Facility (ESRF-EBS, Grenoble, France) at beamline ID31. Because the availability of beam time and the total experimental time is limited, we decided to subject the catalyst to a combined Ptand C-AST with a degradation protocol consisting of 5,000 square wave cycles (steps between 0.6 V RHE (3 s) and 1.1 V RHE (3 s), denoted as durability (D-)AST). Note that half a Pt-AST consisting of only 5,000 cycles between 0.6 and 0.95 V RHE at a scan rate of 100 mV•s -1 did not trigger any measurable degradation for any of the catalysts. [START_REF] Schmies | Impact of Carbon Support Functionalization on the Electrochemical Stability of Pt Fuel Cell Catalysts[END_REF] Figure S 11 shows the evolution of the X-ray diffraction pattern as excerpt of the Pt[220] Bragg reflection. To further elucidate the structural evolution, a detailed analysis of the diffraction patterns using the Rietveld refinement approach was conducted. [START_REF] Rietveld | A profile refinement method for nuclear and magnetic structures[END_REF] For the acquired operando diffraction patterns, it was found that one fcc-Pt phase was adequate for a good fit of the crystalline material, herein the crystalline Pt particles. 3a). This behavior could be explained by a cleaning of the Pt surface as reported in literature during the first 100 cycles of the D-AST. [START_REF] Harzer | Cathode Loading Impact on Voltage Cycling Induced PEMFC Degradation: A Voltage Loss Analysis[END_REF] Prior to the D-AST, a conditioning procedure was performed for 100 cycles between 0.05 and 1 V RHE at 100 mV/s. The D-AST now has a more oxidative upper potential limit of 1.1 V RHE , therefore producing cleaner surface. The conditioning was continued and a maximum value for the ECSA H-upd was recorded for all three homemade samples the stage of 100 cycles and 10 cycles for HISPEC3000. The strongest relative increase of the ECSA H-upd is found for Pt/V, followed by HISPEC3000, Pt/N-V-800 and Pt/N-V-400, respectively. The lattice parameter a, however, is showing a contrary behavior (Figure 3b). The strongest relative contraction of the lattice is found for Pt/N-V-400, followed by Pt/N-V-800 and Pt/V, respectively. Previous works on Pt nanoparticles strain dynamics revealed that lattice parameter contraction is associated to desorption of various charged species from the surface. [START_REF] Martens | Probing the Dynamics of Platinum Surface Oxides in Fuel Cell Catalyst Layers Using in Situ X-Ray Diffraction[END_REF][START_REF] Chattot | Electrochemical Strain Dynamics in Noble Metal Nanocatalysts[END_REF] HISPEC3000 shows no such contraction.

After the beneficial cleaning of the Pt surface over the first 100 cycles, the degradation phenomena of particle growth due to Ostwald ripening and agglomeration, Pt dissolution and/or particle detachment start to take over with increasing cycle number. The ECSA H-upd values are decreasing and in the HE-XRD patterns the Pt reflections start to sharpen up (Figure S 11). Figure 3c shows the continuously increase of the coherent length as indicator of particle growth. Interestingly, the Pt/N-V-400, Pt/N-V-800 and HISPEC3000 samples degrade much faster in ECSA H-upd and coherent length than Pt/V. Further, the shape evolution of the CVs reveals a drastic degree of carbon corrosion visible in the evolution of a chinon-hydrochinon redox pair around 0.5-0.75 V RHE for Pt/N-V-800 and somewhat lower degree of carbon corrosion for Pt/N-V-400 (Figure S 13). Pt/V and HISPEC3000 have only very little formation of a chinon-hydrochinon redox pair in the CV. Both Pt/N-V catalysts exhibit an enhanced carbon corrosion of the N-doped carbon supports. This interferes with the presumption of enhanced carbon corrosion durability as predicted from earlier work [START_REF] Schmies | Impact of Carbon Support Functionalization on the Electrochemical Stability of Pt Fuel Cell Catalysts[END_REF] and the preliminary RDE investigation, where none of the catalyst indicated any pronounced carbon corrosion when subjected to the C-AST (5000 cycles, 1-1.5 V RHE , 500 mV•s -1 ). In summary, the incorporation of N via pre-oxidation and ammonolysis in the C matrix seems to result rather in an introduction of weak spots then in a stabilization of the C matrix.

It can be concluded that the fluidized bed reactor is an effective tool for the synthesis of Pt-based catalysts. The uniform distribution of the homemade Pt particles and the carbon corrosion resistance of V support exhibit good structural stability for Pt/V, especially in the direct comparison with the Pt/V reference HISPEC3000. Even though Pt/N-V-400 and Pt/N-V-800 were platinized using the same method, the two catalysts show less carbon corrosion resistance and consequently much higher degradation in the operando HE-XRD experiment.

MEA-based Single Cell Measurement

To further investigate the catalyst properties in a more realistic application, MEA-based single cell measurements were conducted with a reproduced synthesis batch of Pt/V, Pt/N-V-400, and Pt/N-V-800. Each deposition of Pt particles on the different carbon materials is subjected to fluctuations and therefore resulting in a unique Pt loading, Pt surface area and particle size distribution. The individual ORR performances of the catalysts determined using RDE experiments reveal small variations within the error bar (Table S 4). Fraction of the N-moieties from deconvolution of N 1s XP spectra are depicted in Figure S 14 and Table S 5. MEAs for a 10 cm 2 single-cell were prepared for Pt/V, Pt/N-V-400, Pt/N-V-800 and HISPEC3000 with a geometric Pt mass loading of 0.15 mg Pt •cm -2 and an ionomer to carbon ratio (I/C) of 0.6 at the cathode using a long chain ionomer. The anodes were kept identical to minimize fluctuation and prepared using a commercial 50 wt-% Pt/C catalyst (Umicore) with a geometric Pt mass loading of 0.1 mg Pt •cm -2 . Figure 4a illustrates the beginning of life (BOL) H 2 /air performance at 100 % relative humidity (RH) (measurement conditions are described in the supporting information). Each polarization curve was corrected for the monopolar plate and conduct resistance. The measurement for this determination was conducted with an MEA without catalyst layer, therefore each part of the system and the cell, except the catalyst layers, is subtracted from the overall contribution.

The BOL H 2 /air performance is similar for all three homemade catalysts. At low current densities, Pt/N-V-800 has a slightly higher intrinsic activity than Pt/V and Pt/N-V-400. However, at high current densities a larger potential drop is observed for Pt/N-V-800 indicating oxygen mass transport limitations. This corresponds well with similar observations by Ott et al. who reported larger potential drop at high current densities when using N-doped Ketjenblack ammonolyzed at elevated temperature (600 °C) as carbon support for the Pt/C cathode at 200 kPa abs , suggesting that further improvement of the cell components (flow field, gas diffusion layer, etc.) are necessary for optimized oxygen supply. [START_REF] Ott | Ionomer distribution control in porous carbon-supported catalyst layers for highpower and low Pt-loaded proton exchange membrane fuel cells[END_REF] When comparing the BOL H 2 /air performance of the homemade Pt/V and the commercial Pt/C of HISPEC3000 (Figure S 15), both catalyst reveal a comparable performance even though the homemade Pt/V has a smaller Pt particle size compared to HISPEC3000as derived from analysis of TEM images, indicating that the preparation of the catalyst materials using the fluidized bed reduction reactor results in good Pt utilization competing with commercial catalysts. Following the preliminary RDE investigations and the suggested DoE protocols, two AST were conducted: Pt-AST (30,000 square wave voltammograms (SWV) between 0.6 and 0.95 V with 3 s holding time at each potential) and C-AST (5,000 CV, 1-1.5 V, 500 mV•s -1 ). [START_REF]Fuel Cell technical Roadmap[END_REF] One metric to evaluate the evolution of the polarizations curves is the investigation of the voltage loss at certain current densities.

Figure 4b and Table 3 illustrate the voltage loss at 0.8 A•cm -2 for Pt-AST. All three homemade catalyst present a comparable degradation behavior. Each catalyst has a drastic voltage loss over the first 5,000 cycles, followed by further moderate voltage losses over the next 25,000 cycles. This results in comparable total voltage losses for Pt/V and Pt/N-V-800 and slightly higher voltage losses for Pt/N-V-400. This trend is accompanied by the evolution of the ORR mass activity where Pt/N-V-400 has a lower retention than Pt/V and Pt/N-V-800 (Figure S 16). However, the overall performance of Pt/N-V-800 is better over the course of the Pt-AST. Figure 4c and Table 3 , respectively. Further research is needed is to explain this behavior because several components in the CCL could be accountable.

During the Pt-AST, all catalysts suffer from severe loss of the ECSA H-upd , highest for Pt/N-V-400 with 68 % and somewhat lower for Pt/V and Pt/N-V-800 with ~60 %. These results indicate that the catalyst degradation of Pt/V and Pt/N-V-800 is comparable, with the latter having a better overall performance during the Pt-AST, thus making it an overall better catalyst for the ORR. Pt/N-V-400 reaches only a moderate performance, leading to the conclusion, that the different material properties of Pt/N-V-400 and Pt/N-V-800 have a significant influence on the performance of the catalysts. Those could be attributed to the different ammonolysis temperatures of 400 °C and 800 °C.

It could be deduced that a maximized N content is no guarantor for improved MEA performance. The environment of the N species seems to be crucial. Here, Pt/N-V-800 consists of a larger fraction of pyridinic-N moieties (Figure S 14c and Table S 5). The basic character of such pyridinic-N that could in combination act with the acidic sulfonic groups of the Nafion ionomer as Brønsted acid-base pair resulting in a better distributed ionomer and thus better MEA performance. It must be noted that these effects become more prominent when measurements are performed in dry conditions (reduced RH values) but these are beyond the scope of this study. Another beneficial impact could be the increased surface area and broadened pore structure of the carbon when treated at 800 °C, resulting in an improved mass transport. [START_REF] Ott | Impact of Carbon Support Meso-Porosity on Mass Transport and Performance of PEMFC Cathode Catalyst Layers[END_REF] Altogether, the various indicators meet in a sweet spot for an optimized modified carbon support material for improved MEA performance, that will need further investigation to disentangle the various lose ends.

For the C-AST, the potential loss at 0.6 A•cm -2 is investigated and depicted in Figure 4d and Table 3.

Both Pt/N-V catalysts show better initial performance at this current density, however after the first 200 cycles a significantly higher potential loss is observed. While Pt/V shows no significant potential drop, Pt/N-V-400 and Pt/N-V-800 reveal a potential loss of 115 mV and 66 mV, respectively. Over the course of the 5,000 cycles of the C-AST, all catalysts end up with severe degradation of the catalyst layer under MEA operating conditions. This is in contrast with the assumptions drawn from TGA measurements that suggested higher thermal stability of the N-doped carbon supports (Figure S 2).

The carbon corrosion was observed with a non-dispersive infrared (NDIR) analyzer located at the cathode gas exhaust, measuring the gas concentrations of CO and CO 2 online (Figure S 17). This method has the significant advantage over the conventional mass spectrometer measurements that the volatile carbon corrosion products can be continuously measured during the C-AST without the need to take aliquots. Though, only specific gases can be measured, in this case O 2 , CO and CO 2 . The measured combined concentrations of CO and CO 2 (CO x ) can then be integrated and converted into a carbon mass. Factoring in the carbon loading of the catalyst layer, the relative loss of carbon can be extracted (Figure 4e). This carbon mass originates from the whole single-cell measurement setup and all carbon-based materials must be considered when attributing the carbon loss: catalyst material of the cathode (Pt/C), ionomer (Nafion membrane and additional ionomer in the catalyst layer), gas diffusion layer, graphite flow field, gaskets. All single cell components were kept identical besides the cathode catalyst material. Therefore, the C-AST was also performed with an MEA without cathode catalyst layer, indicating no significant carbon corrosion during the 5000 cycles, leading to the conclusion, that the measured CO and CO 2 are mainly evolved carbon from the catalyst layer. The carbon corrosion of all three catalysts over the first 1,000 cycles is similar, indicating that the V carbon and its N-doped derivates have the same carbon degradation pattern independent of the synthesis procedure (Figure 4e). This contrasts with the performance trends observed as the potential loss at 0.6 A•cm -2 over the course of the C-AST. The data suggests that while the total amount of oxidized carbon is equal for Pt/V, Pt/N-V-400 and Pt/N-V-800, the influence on the MEA performance is much more detrimental, giving rise to significative differences. A possible explanation for this could be that the integration of defects, in form of nitrogen (and oxygen) atoms, into the support matrix facilitates pore collapsing after high potential excursions by destabilizing the carbon structure, which would then lead to the decreased performance of the N-doped carbons.

Table 3: BOL and EOL MEA-based fuel cell performance (at 0.8 and 0.6 A cm -2 for the for the Pt-AST and C-AST protocol, respectively), ECSA , catalytic (massbased) activity evaluated at 0.9 V for the Pt-AST protocol (30,000 SWV between 0.6 (3 s) and 1.1 V (3 s)) and for the C-AST protocol (5,000 CV between 1.0 and 1.5 V at a scan rate of 500 mV•s -1 ).

Conclusion

In this study, Pt electrocatalysts on three different Vulcan-based carbon supports for the ORR were prepared using a customized fluidized bed reduction reactor. One support material was a commercial Vulcan used as reference for this study (Pt/V). The other two support materials were N-doped Vulcans with different ammonolysis temperatures (Pt/N-V-400 and Pt/N-V-800). The three homemade catalysts were compared to a commercial Pt/C (HISPEC3000). The physicochemical characterization of the supports disclosed the importance of the synthesis temperature: Pt/N-V-400 has the highest level of heteroatom-doping, the lowest ZP as well as the smallest fraction of micropores. The other two catalysts, Pt/V and Pt/N-V-800, have comparable fractions of micropores and a similar ZP yet a higher level of heteroatom-doping for Pt/N-V-800. In RDE measurements, all three catalysts have initially similar massbased activity and ESCA H-upd values and maintain them after both Pt and carbon corrosion degradation protocol testing, showing no enhanced influence on the performance of the N-doping of the supports in acidic electrolyte. The catalysts were subjected to a more severe stability test and investigated using operando HE-XRD experiments at beamline ID31 at the ESRF to discover a link for the evolution of structural and electrochemical characteristics. The experiments revealed a superior stability of the Pt/V sample in liquid acidic media that outperformed the commercial Pt/C (HISPEC3000) reference material.

However, Pt/N-V-400 and Pt/N-V-800 exhibited a proneness towards degradation phenomena, especially carbon corrosion, within the D-AST. MEA measurements were conducted to test the catalysts in a PEMFC environment. The different support materials had a minor impact on the durability during Pt-AST, Pt/N-V-800 and Pt/V revealed similar degradation behavior, whereas Pt/N-V-400 had slightly higher performance losses over the course of the Pt-AST. However, Pt/N-V-800 demonstrated a better overall performance compared to Pt/V, suggesting a higher activity under operating conditions for the N-doped carbon support. We suggested that this could be due to an improved ionomer distribution due to higher amounts of pyridinic-N moieties in the carbon matrix. For the C-AST, the heteroatom-doping of the support material seems detrimental as the performance loss is severely higher compared to the unmodified carbon, while all catalysts emit similar amounts of carbonous gases as observed with NDIR measurements. This leads us to the conclusion, that the heteroatom-doping and thereby the incorporation of defects into the carbon matrix does not benefit the carbon corrosion durability under high potential cycling (1-1.5 V) but can favor the performance under operating conditions (0.6-0.9 V).

The investigation of the carbon corrosion is an important tool to gain further insight into the degradation behavior of the support material occurring while optimizing the performance of ORR catalysts in PEMFCs. N-doped carbons used as support for Pt-based ORR electrocatalysts represent a promising option to further tweak the three-phase boundary at the active sites by improving the ionomer distribution on the support material.

  analysis (TGA) measurements were performed to investigate the thermal stability of the carbon materials in Ar atmosphere and revealed a high thermal stability for the pure carbon supports up to >500 °C. (FigureS2).

  are typical for V-based materials and indicate no drastically enhanced crystallinity of the graphitic character after the ammonolysis, because of the relatively low ammonolysis temperatures (Figure S 3).

Figure 1 :

 1 Figure 1: Structural and morphological characterization of the pristine catalysts in terms of a) XRD patterns and TEM images of b) Pt/V, c) Pt/N-V-400, d) Pt/N-V-800 and e) commercial Pt/C (HISPEC3000).

Figure S 4

 4 illustrates an exemplary set of SEM and TSEM images for Pt/V. To further elucidate the N environment of the final catalyst, X-ray photoelectron spectroscopy (XPS) analysis of the N 1s peak was performed after the Pt deposition because the introduction of Pt nanoparticles could have an influence of the N environment compared to the pure carbon materials. Based on earlier work, the presence of five N moieties were identified and deconvoluted: pyridinic-N, pyrrolic-N, quaternary-N, graphitic-N, and N-oxide (Figure S 5 and Table

  1) than suggested by the broad reflections in the XRD pattern. This could be explained by Pt particles that are composed of crystallites with smaller domain sizes prepared in the yet unrevealed synthesis process of the manufacturer. The size distribution of the Pt particles of HISPEC3000 is less uniformly and narrow then for the homemade Pt/(N-)C samples prepared in the fluidized bed reduction reactor.

Figure 2 :

 2 Figure 2: Electrochemical characterization during potential cycling following the carbon corrosion C-AST and the Pt stability Pt-AST protocols using RDE in terms of the evolution of a) mass-based activity, b) specific activity and c) ECSA H-upd .

Figure 2 and

 2 Figure 2 and Table S 3 depict the RDE results in terms of Pt mass-based activity and ECSA H-upd , andthe Pt mass-free specific activity. The ORR Pt mass-based activities of all catalysts (homemade and commercial) were evaluated as ~0.2 A mg -1 at 0.9 V versus reversible hydrogen electrode (RHE). This is a typical value for spherical pure Pt particles on a solid carbon and confirm that the N-doping of the

Figure S 12 illustrates

 12 the evolution of the CVs during potential cycling and allows to extract the ECSA H-upd during potential cycling. Figure3illustrated the evolution of ECSA H-upd , lattice parameter and coherence length as function of the number of cycles of the D-AST.

Figure 3 :

 3 Figure 3: Electrochemical and structural response of operando liquid cell HE-XRD experiments (5,000 SWV between 0.6 (3 s) and 1.1 V RHE (3 s) in 0.1 M HClO 4 ) of Pt/V (grey), Pt/N-V-400 (red), Pt/N-V-800 (blue) and comm. Pt/C (HISPEC3000, green). Evolution of a) ECSA H-upd as fraction of the maximum value b) lattice parameter a, c) coherence length.

Figure 4 :

 4 Figure 4: Fuel cell performance of Pt/V (grey), Pt/N-V-400 (red) and Pt/N-V-800 (blue) under potential cycling. a) Beginning of life polarization curve of 10 cm² MEA. b) Evolution of voltage @ 0.8 A•cm -2 of Pt-AST protocol (30,000 SWV between 0.6 (3 s) and 0.95 V (3 s)). c) BOL and EOL ECSA of Pt-AST. d) Evolution of voltage @ 0.6 A•cm -2 of C-AST protocol (5,000 CV between 1.0 and 1.5 V at a scan rate of 500 mV•s -1 ). 3) Relative fraction of carbon MEA loss as determined via NDIR of C-AST.

Table 1 :

 1 Elemental composition of the unmodified and N-doped carbons.

					at-% O
	Sample	at-% N	at-% C	at-% H	(calculated residual
					to 100 at-% total)
	V (unmodified)	0.0 ± 0.0	98.4 ± 0.4	0.2 ± 0.3	1.4
	O-V	0.0 ± 0.0	89.9 ± 0.1	0.0 ± 0.0	10.1
	N-V-400	2.7 ± 0.2	93.6 ± 0.5	0.1 ± 0.0	3.6

Table 2 :

 2 Surface characterization of the unmodified and N-doped carbons in terms of surface area, pore structure and surface charge.

	Sample	BET SA / m²/g	QSDFT SA / m²/g micropores mesopores	total	Ratio of micropores/total / %	Surface zeta potential / mV
	V (unmodified) 229	91	103	195	47	27.8
	N-V-400	199	44	130	174	25	16.4
	N-V-800	245	87	133	221	40	28.9

  report the ECSA evolution during the Pt-AST. Pt/V exhibits with 72 ± 2 cm 2 •g Pt -1 the highest ECSA H-upd measured in the MEA setup.

	Interestingly, both Pt/N-V-400 and Pt/N-V-800 expose a significantly lower ECSA H-upd of only 48 and
	45 m 2 •g Pt -1

Acknowledgement

This project received financial support from the German Federal Ministry of Education and Research (BMBF) through grant 03XP0251 ("KorrZellKat") and grant 03SF0531B ("HT-linked"). We thank Juana Krone for the elemental analysis and Astrid Müller-Klaucke for ICP-OES measurements. We thank the Zentraleinrichtung Elektronenmikroskopie of Technische Universität Berlin and Stefanie Kühl for their support with electron microscopy measurements. We thank Detre Teschner and the Department of Inorganic Chemistry of the Fritz-Haber-Institut of the Max-Planck-Gesellschaft for providing access to the XPS instrument. We acknowledge the European Synchrotron Radiation Facility for provision of synchrotron radiation facilities.