Aurélie Babin
email: aurelie.babin@hotmail.fr

Jérôme Moreau

Yannick Moret
email: yannick.moret@u-bourgogne.fr

Storage of carotenoids in crustaceans as an adaptation to modulate immunopathology and optimize immunological and life-history strategies

Keywords: carotenoids, immune cost, resistance, tolerance, ageing, invertebrate

Why do some invertebrates store so much carotenoids in their tissues? Storage of carotenoids may not simply be passive and dependent on their environmental availability, as storage variation exists at various taxonomic scales, including among individuals within species.

While the strong antioxidant and sometimes immune-stimulating properties of carotenoids might be beneficial enough to cause the evolution of features improving their assimilation and storage, they may also have fitness downsides explaining why massive carotenoid storage is not universal. Here, we examine the functional and ecological implications of carotenoid storage for the evolution of invertebrate innate immune defences, especially in crustaceans, which massively store carotenoids for unclear reasons. We propose three testable hypotheses about the role of carotenoid storage in immunological (resistance and tolerance) and lifehistory strategies (with a focus on ageing), which may ultimately explain the storage of large amounts of these pigments in a context of host-pathogen interactions.

Introduction

Carotenoids are natural lipid-soluble bioactive pigments, produced by photosynthetic organisms (plants, bacteria, and algae) in which they absorb light for photosynthesis and provide photoprotection. [1] Apart from the rare exceptions involving symbiotic interactions with microorganisms, [2,3] animals cannot produce carotenoids themselves and must acquire them from their diet. [1,4] There are over 600 carotenoids known as xanthophylls and carotenes when containing or not oxygen, respectively. [4] Despite such a great diversity of carotenoids in nature, only a few of them are absorbed and stored in animal tissues and fluids. [5][6][7] This suggests that they are either selectively accumulated by animals and/or metabolically transformed for their storage. [8] The amounts of carotenoids stored by animals vary across taxa [9,10] and within species, [11] suggesting variability in their physiological importance in addition to fluctuations in their environmental availability.

Because they are involved in many biological functions, [4,5] carotenoids may have strong implications in evolutionary biology. Their storage was suggested as indicative of individual quality, such that carotenoid-colored traits could be sexually selected. [12] They have been found to have beneficial effects on survival, growth, and immunity, although this is still strongly debated, especially in the bird literature from which most of the studies were conducted. [5,[13][14][15][16][17] Non oxygenated carotenoids, such as beta-carotene can be important precursors to vitamin A in many animals, whereas oxygenated ones, also called xanthophyll carotenoids, such as astaxanthin and lutein, are not. [18][19][20] Astaxanthin, mainly produced by fungi and algae, is massively stored by aquatic animals. [8] Specifically, most crustaceans accumulate massive amounts of astaxanthin as circulating lipid droplets in the haemolymph and in the eggs, and as esterified forms in their tissues. [7,[START_REF] Castillo | General survey of the carotenoids in crustacea[END_REF][START_REF] Dembitsky | [END_REF][23] This pigment, rarely found in large amounts in vertebrates except for reports in salmonid fish [8,[24][25][START_REF] San-Jose | [END_REF] , is essentially produced from the metabolism of other carotenoids acquired in the diet [27,28] and is among the carotenoids having the highest antioxidant potential. [29] The precise reasons for such substantial pigment accumulation in crustaceans remain unclear. However, its accumulation has not been related so far to sexual signalling. Furthermore, while the involvement of carotenoids as antioxidants in birds appears inconclusive (Koch et al. 2018) We propose that the strong antioxidant and the frequent immune-stimulating roles of astaxanthin may limit the immunopathology cost associated with the immune response of crustaceans. [11,30,31] Parasitism is an important source of stress for organisms. While the immune defences protect from parasitic and pathogenic agents (here after pathogens), inflammatory responses from the innate immune system also cause immunopathological disorders. Inflammation is a phenomenon known from both vertebrates and invertebrates, corresponding to a fast but nonspecific response characterised by the delivery of fluids, cytotoxic chemicals and cells to damaged and infected tissues, in order to fend off infectious agents and initiate tissue repair.

The cytotoxic chemicals released by the respiratory burst comprise highly reactive oxygen species (ROS) and reactive nitrogen species (RNS) toxic to both pathogens and hosts' tissues, leading to immunopathology. [32] When damaged tissues are not fully repaired and homeostasis is not fully restored, inflammation can further develop into a chronic condition, with inevitable long-term debilitating consequences, such as increased rates of morbidity and mortality at older age. [32] Organisms have evolved physiological processes that reduce the impact of inflammatory injury, thus limiting the immunopathology costs. The integrated antioxidant defence system controls the balance between the production and the detoxification of cytotoxic free radicals, produced by basal metabolic activity and the immune response. This system combines endogenous antioxidant enzymes, such as superoxide dismutase, catalase and glutathione peroxidase, with exogenous (diet-acquired) antioxidants, such as vitamin E (tocopherol), vitamin C (ascorbate), polyphenolic antioxidants, and carotenoids. [33,34] Among these compounds, carotenoids may scavenge free radicals produced by immune activity because of their long carbon skeleton containing conjugated double bonds and, in some cases, oxygenated end rings. [35][36][37][38] They also have the potential to interact with endogenous antioxidant enzymes. [31,39,40] Here, we emphasize that the environmental availability of carotenoids and the ability of organisms to assimilate and store these pigments, may impact the evolution of innate immunity, by improving the defences against pathogens while reducing the fitness costs associated with the inflammation response. We particularly focus on crustaceans for several reasons, developed in the dedicated section below: their immunity relies on innate immune systems alone, thus leading to immunopathology [START_REF] Schmid-Hempel | Evolutionary parasitology[END_REF] ; they are known to accumulate naturally large amounts of astaxanthin, a carotenoid with a particular high antioxidant ability [29] ,which is also frequently reported to enhance their immunity. [31,[START_REF] Pan | [END_REF][43][44][45] ; and carotenoid storage in crustaceans has not been involved so far in communication displays, which simplifies the investigation of its functions. We first briefly review the mechanisms underlying the invertebrate inflammatory response, with a special focus on the production of ROS and RNS.

After describing the integrated antioxidant defence system that includes dietary carotenoids, we discuss the role of astaxanthin in modulating immunopathology, immunological strategies (tolerance versus resistance) and ageing. Finally, we explore whether crustaceans could have evolved the ability to store large amounts of astaxanthin in their tissues to maintain higher levels of innate immune defences at lower immunopathological costs in response to local pathogens.

Innate immunity and immunopathology in invertebrates

Invertebrate innate immunity is a costly system of protection that detects and eliminates a wide variety of pathogens and unhealthy tissues. It comprises constitutive defences relying on immune cells (haemocytes or coelomocytes) and several rapidly inducible enzymatic cascades (e.g. the prophenoloxidase cascade) at the core of the inflammatory response. [46] Upon infection, recruited phagocytic immune cells increase their oxygen consumption during the respiratory burst that leads to ROS production (Fig. 1), enhancing pathogen killing within phagocytic cells and in the vicinity of extracellular bound pathogens. [47] ROS are produced by the activity of the membrane bound NADPH oxidase enzymatic complex, which catalyses the production of NADP + and cytotoxic superoxide anions (O 2 .-). Superoxide anions can then be transformed into other damaging and unstable molecules, such as hydroxyl radicals OH -, and hydrogen peroxide H 2 O 2 forming hypochlorous acid HOCl in reaction with chloride. [48] Recruited immune cells can also generate RNS, such as nitric oxide (NO). [49,50] In immune cells, the synthesis of NO by inducible nitric oxide synthase, iNOS, has a broad antipathogenic action (Fig. 1). NO can react with ROS yielding other highly reactive radicals (Fig. 1): combined with hydrogen peroxide, it yields hydroxyl radicals; [48,51] combined with superoxide anions, it yields oxidant and nitrating peroxynitrite radicals, ONOO-, that can subsequently damage a large range of the cell molecules, inducing apoptotic or necrotic cell death. [48,52] A major response of invertebrate immunity accompanying the rapid aggregation of the immune cells with each other and with the pathogens is the melanisation reaction catalysed by phenoloxidase enzymes (PO), which heal wounds through clotting and clear invading pathogens through nodulation or encapsulation. [53] POs are present as inactive proenzymes (prophenoloxidase or proPO) in immune cells. They are released and converted into their active form in the haemolymph upon wounding or infection. In crustaceans, infection may further generate the cleavage of a fragment of the hemocyanin, a respiratory protein, which also has PO activity. [54] Melanin synthesis is accompanied by the production of ROS and RNS, helping to kill intruders. [50,53] However, excessive PO cascade activity can subsequently damage the host's own tissues and organs (Fig. 2). [55,56] Immunopathological effects upon encapsulation and clotting are likely to be frequent in invertebrates since the occurrence and abundance of wounding can be extremely high. [57][START_REF] Stoks | Dragonflies and damselflies -Model organisms for ecological and evolutionary research[END_REF][START_REF] Siva-Jothy | Insect infection and immunity[END_REF] Repeated wound clotting and multiple melanisation reactions in response to repeated infections may be then harmful, as it exposes the host organs to cumulative cytotoxic radicals in the open circulating system.

Carotenoids as part of the integrated antioxidant defence system

The accumulation of cytotoxic ROS and RNS is limited by the detoxifying activity of the integrated antioxidant system, including endogenous enzymes and non-enzymatic exogenous compounds. [33,[START_REF] Chaudière | [END_REF] Three major antioxidant enzymes detoxify free radicals (see Fig. 1).

Superoxide dismutase transforms highly reactive superoxide anions O 2 .

-into hydrogen peroxide. Then catalase converts hydrogen peroxide into water. Hydrogen peroxide detoxification can also be catalysed by peroxidase when ROS are associated with substrates that can be oxidised. [33,61] In addition to these three major enzymes, crustaceans possess peroxinectins, proteins with multiple roles in immune defences, including peroxidase activity.

Upon immune challenge, peroxinectins are released by haemocytes after the activation of the proPO cascade, bound to extracellular superoxide dismutase, thus helping to detoxify the hydrogen peroxide produced by superoxide dismutase activity. [62,63] However, peroxinectins seem to have lower peroxidase activity than catalase upon immune challenge. [31] Non-enzymatic exogenous compounds with antioxidant properties are acquired through dietary intake and presumably help antioxidant enzymes to keep the oxidative burden under control. These compounds act either as hydrogen donors (chemical traps), or as free radical scavengers (physical traps). [33,[START_REF] Chaudière | [END_REF]64] Non-enzymatic antioxidants are divided into hydrophilic compounds, present in aqueous cellular compartments (e.g. vitamin C, glutathione), and lipophilic compounds (e.g. carotenoids), which can be included in cell membranes (Fig. 1) and associated with lipoproteins. [33,[START_REF] Chaudière | [END_REF] All these exogenous antioxidant compounds being acquired from the diet, their environmental availability can be limited and limiting. Hence, this environmental availability, as well as the ability to assimilate these antioxidant compounds, will be a crucial parameter influencing the potential synergy with endogenous antioxidant enzymes and in the general antioxidant capacity. Among these compounds, carotenoids are powerful antioxidants [35,36] and relatively abundant in natural food sources. [65] Unlike other dietary antioxidants, they can be stored in large amounts in animal tissues. [7,66] Hence, carotenoids potentially play a major role in the regulation of immunopathology, and therefore in the modulation and the evolution of immune defences.

Crustaceans as systems for studying physiological and evolutionary implications of carotenoid storage

Along with their biological proprieties, carotenoids are involved in several functions including in communication displays in birds, amphibians, and reptiles. Carotenoid-based coloration of the skin and plumage has been found to be an honest secondary signal of resistance to oxidative stress, body condition, and immunocompetence. [5,25] Several hypotheses on the molecular mechanisms underlying signal honesty have been formulated, including the allocation trade-offs and shared molecular pathways, leading to specific expected relationships between stored amounts of carotenoids and other physiological parameters. [15,67,68] Nevertheless, it has been suggested that stored carotenoids may be minor antioxidants for birds, preferentially used for sexual signalling, while other non-pigmentary antioxidants (e.g. melatonin) may be used primarily for detoxification during immune activity. [69] In these bird examples, carotenoid-based coloration might have been under strong selection, leading to diversion of these pigments for sexual signalling. Therefore, taxa that do not use these pigments for communication displays would be more straightforward to study the role of carotenoids in alleviating oxidative stress in immunopathology, avoiding interferences with signalling.

In invertebrates, aquatic crustaceans are among the greater animal reservoirs of carotenoids by accumulating mostly astaxanthin in their tissues, cuticle, haemolymph, and eggs. [7,9,[START_REF] Dembitsky | [END_REF]28] They can obtain this pigment in their diet by feeding on various algae and microbes that are producing them. [8][9][10] However, the most common carotenoids contained in their food are typically beta-carotene, zeaxanthin, lutein, and other less abundant carotenoids that crustaceans are oxidizing to produce the astaxanthin they accumulate. [27,28] They can also directly obtain astaxanthin by consuming animals that have already performed the bioconversion of other carotenoids into astaxanthin and stored it. Astaxanthin has the highest antioxidant activity among natural carotenoids: it is 10 times more effective than ß-carotene and over 100 times more effective than vitamin E. [70][71][72] However, the presence of salt lowers the relative oxidant activity of astaxanthin. [73] Assuming that animals store astaxanthin mainly for its antioxidant potential, it may not be surprising that some marine organisms (crustaceans, but also some fish, see the introduction) store them in large amounts to compensate for its reduced antioxidant activity in salty water, although this needs to be formally tested. Astaxanthin also has unique other biochemical properties making it able to form chelate complexes with metals that could otherwise impair mitochondrial function. [73] Its ability to be esterified allows a better stabilization of the free form and prevents its aggregation that would otherwise favour pro-oxidant effects. [29] The latter phenomenon could be further prevented by the ability of the pigment to form proton loss neutral radicals. [29] Together, these properties likely enable the accumulation of large amounts of astaxanthin with limited negative effects, making it an efficient protective agent against environmental stressors.

The natural storage of large quantities of astaxanthin seems to be the healthy/normal situation for crustaceans, whereas the food depletion in carotenoids seems to be unusual; the astaxanthin storage rate depending on the pigment nature and availability. [28] In freshwater and marine crustaceans, the storage of astaxanthin was associated to numerous beneficial physiological effects, including immunity [74][75][76] and antioxidant defences [77,78] but has never been reported to be involved to sexual signalling, either directly or indirectly. By contrast with vertebrate and insect immunity, crustacean immunity relies strongly on hemocytes, the cytotoxic activity of the proPO cascade and on the production of ROS/RNS. The large panel of antimicrobial peptides described in insects, allowing them to efficiently control microbial infections without generating self-damage, has no equivalent, so far, in crustaceans. [START_REF] Cerenius | Invertebrate Immunity[END_REF] Crustacean immunity is therefore strongly dependent on self-reactive processes to combat infection by pathogens. Furthermore, crustaceans have an open circulatory system frequently exposed to microbes often in high concentrations in their natural habitat. [START_REF] Nagasawa | [END_REF][81][82] All these elements suggest that storage of carotenoids has a central role in the physiology of crustaceans, which make then an important model system to study the role of carotenoids in the detoxification of the immune response.

Hypothesis 1: carotenoids modulate immunological strategies of crustaceans by limiting immunopathology

Animals may survive infection through two non-mutually exclusive immunological strategies: resistance and tolerance. [83,84] Resistance reduces pathogen burden by the action of the host's immune system, but leads to significant immunopathology, and hence to fitness costs (see above). Alternatively, the host may restrain its immune response and tolerate a certain pathogen burden, limiting the immunopathology associated with the immune response. These two immunological strategies could be closely linked, as limiting immunopathology (tolerance strategy) may allow the host's immune system to be more efficient at eliminating pathogens (resistance strategy). [85] Therefore, any mechanism limiting immunopathology is expected to have profound implications for the evolution of tolerance and/or resistance, [32,86] and for host-pathogen interactions. [85] Here, we propose that the immune-stimulant and antioxidant activities of carotenoids could influence the crustacean's resistance and tolerance immunological strategies, by enhancing immune response efficiency while limiting immunopathology. This would translate into a very efficient and lower-cost immune function resulting from the storage of the storage of large amounts of astaxanthin. [30,31] Carotenoids could improve resistance as they were reported to stimulate immune defences of vertebrate animals [e.g. 5,15,16,87] , although this is not always the case. [14,37,38,88] In marine and freshwater crustaceans, immunity is frequently improved by dietary carotenoids. [31,[START_REF] Pan | [END_REF][43][44][45] For instance, in the amphipod crustacean Gammarus pulex, experimental dietary supplementation with carotenoids enhanced immune activity and increased resistance to microbial infection (Fig. 2). [30] Dietary supplementation with carotenoids increases phenoloxidase activity and total haemocyte count in the giant freshwater prawn Macrobrachium rosenbergii. [89] Furthermore, the injection of astaxanthin in the same species increases total haemocyte count and survival when exposed to the pathogenic bacterium Lactococcus garvieae. [90] Likewise, a dietary supplementation with astaxanthin increased the total haemocyte count of the marine whiteleg shrimp Litopenaeus vannamei juveniles. [43] Similarly, a yeast-rich diet containing carotenoids increased total haemocyte count and phenoloxidase activity in the marine Indian prawn Fenneropenaeus indicus. [44] The mechanisms through which carotenoids directly or indirectly enhanced basal levels of immune effectors are still unknown. Nevertheless, recent findings suggest that astaxanthin exhibits antimicrobial activity against a large range of microbes in vitro through yet unknown mechanisms. [91,92] Storage of this pigment could therefore provide a direct advantage upon microbial infection.

Carotenoids could also improve tolerance in several ways. They may prevent oxidative stress-mediated damage resulting from the immune response by scavenging cytotoxic free radicals (Fig. 1), [35] helping in their detoxification, [36] hence limiting the immunopathology cost (Fig. 3). This might be particularly important in crustaceans whose immunity strongly relies on auto-reactive immune responses in an open circulating system where the organs bathe in the haemolymph. These processes are suspected to occur in carotenoid-supplemented G. pulex, which do not suffer from additional survival costs compared to control gammarids, despite exhibiting stronger immune activity and resistance to microbes. [30] From a functional perspective, improved tolerance mediated by carotenoids in G. pulex might result from the take-over of the activity of the antioxidant enzyme superoxide dismutase in the detoxification of the superoxide anions of the innate immune response, while the activity of the other antioxidant enzyme catalase is promoted. [31] Nevertheless, further studies are needed to better understand the functional role of carotenoids in the detoxification process in association with endogenous antioxidant enzymes.

Carotenoid-mediated tolerance may also result from interactions between the pigments and the regulation of the host's metabolism. [19,93] Infections can have dramatic metabolic consequences, characterised by the loss of energetic reserves. This results in elevated circulation of glucose, amino acids and fatty acids, which can cause morbidity and death. [94,95] Carotenoids, especially astaxanthin, have hypolipidemic and hypoglycemic effects in rodents after supplementation. [19,96] Moreover, there is evidence that astaxanthin can interact with nuclear receptors of the peroxisome proliferator-activated receptor (PPAR) superfamily, which regulates lipid and glucose metabolism. [97] It is therefore possible that astaxanthin may prevent metabolism dysregulation upon infection through interaction with PPARs. If so, carotenoids may not only improve tolerance by preventing wasting of metabolic reserves, but also impact resource trade-offs involving the immune function upon immune challenge.

Beyond the functional characterisation of the interactions of carotenoids with the host physiology, testing the above hypothesis relies on determining the phenotypic consequences of the dietary supplementation with the pigments. Especially, under the above hypothesis, the supplementation of the food of crustaceans with carotenoids is expected to improve the survival and reproductive success of hosts exposed to infectious or non-infectious immune challenges if the pigments enhance immunity or tolerance, respectively.

Hypothesis 2: carotenoids modulate ageing in crustaceans by limiting immunopathology

Ageing is the progressive deterioration of physiological functions with age, leading to agedependent decline in fecundity and increased mortality. [START_REF] Rose | Evolutionary Biology of Aging[END_REF] Several non-mutually exclusive hypotheses have been put forward to explain ageing, and many involve cumulative oxidative damage by biological processes. [START_REF] Kaszubowska | [END_REF]100] Mechanisms such as antagonistic pleiotropy, when the expression of genes is beneficial early in life but becomes adverse at older age, [101,102] and damage by ROS and RNS, all contribute to ageing. [103,104] Ageing and inflammation-mediated immunopathology are intrinsically linked, as ageing is characterised by a prolonged proinflammatory status as a result of an imbalance between pro-and anti-inflammatory status. As stated above, ROS and RNS accumulation is a prominent theory of ageing, [103,104] and their regulation in the host when interacting with pathogens appears critical in the ageing process. [32,56] It has been proposed that early-life inflammation could have long-term or delayed pathological costs, yielding increased morbidity and mortality at older age. [105,106] In line with this, a non-pathogenic immune response experimentally elicited in early life in the mealworm beetle, T. molitor, reduces adult life span. [32] In this insect, activation of the prophenoloxidase cascade and the release of ROS and RNS by haemocytes in response to non-self have an immunopathological effect on Malpighian tubules (insect 'kidneys'), leading to a lower secretion efficiency. Their function is impaired on the long term, [55] causing ageing acceleration [56] and shortening lifetime reproductive success. Preventing immunopathology early in life may reduce ageing acceleration and therefore improve lifetime reproductive success. The enzymatic and non-enzymatic antioxidant scavengers, together with a variety of molecular repair processes, can help to limit immunopathology, and therefore ageing.

Given all these elements, we hypothesise that, in the context of ageing, fitness benefits from the storage of antioxidant carotenoids in crustaceans, especially astaxanthin for crustaceans, in dealing with inflammation consequences such as immunopathology, should be greater at early age than at older age. Given its biological properties mentioned earlier, this would be particularly the case for the storage of astaxanthin after its stabilisation as esterified forms for storage in crustacean tissues. [7,8,24] This might also be particularly relevant in animals whose immunity relies almost exclusively on inflammatory responses, like again in crustaceans. After infection at early stages, carotenoids are expected to help keep the balance between pro-and anti-inflammatory status under control for a longer period of time, preventing ageing acceleration and preserving lifetime reproductive success (Fig. 3). Hence, maternal transfer of carotenoids into the eggs might be highly advantageous to prevent ageing acceleration in offspring in septic environments. [107,108] In the particular case of crustaceans, we therefore predict that stored astaxanthin will have a greater positive effect on lifespan and early reproductive success early in life. In line with this, small gammarid crustaceans, that are the youngest, store higher quantities of carotenoids in their haemolymph than larger individuals, which are the oldest. [30] Whether this age-related pattern of carotenoid storage is adaptive in the context of ageing is not yet known. Its consequences on lifetime reproductive success and ageing have to be tested.

In crustaceans, ageing is also likely further prevented by the unique properties of the astaxanthin they accumulate. In addition of its high antioxidant activity, astaxanthin is able to form chelate complexes with metal ions, such as Zn 2+ , which is known to inhibits the mitochondrial complex I (NADH: ubiquinone oxidoreductase) [109] , that is central for energy transduction and whose dysfunction is implicated in neurodegenerative and muscular diseases and in ageing. [29,73] Hypothesis 3: carotenoids storage is adaptive to fight pathogens Some organisms, including crustaceans, have evolved carotenoproteins (i.e. carotenoids in association with proteins) [76,[START_REF] Britton | Natural functions[END_REF] that allow the storage of large amounts of carotenoids in their

tissues for yet unknown reasons, and its involvement in sexual selection has never been reported so far (see the above section presenting crustaceans as relevant models systems to address the proposed hypotheses). Most crustaceans feed on decaying organic matter, probably full of obligate and opportunistic pathogens. [START_REF] Macneil | [END_REF] Crustaceans also often live in microbes-rich aquatic environments that almost permanently expose their open circulatory system to microbial infections. [START_REF] Nagasawa | [END_REF][81][82] From a host-pathogen interaction perspective, given the beneficial roles of carotenoids, especially astaxanthin, in the regulation of the immune function and associated immunopathology, [31,43,75] we propose that, in these organisms, the storage of large amounts of carotenoids [30] and the maternal transfer of carotenoids [7,28] could have evolved to deal with pathogen attacks, using the more efficient inflammatory response provided by carotenoid enrichment. On the one hand, selection imposed by pathogens should favour resistance, giving rise to a more efficient immune response and higher levels of immune defences. [112,113] On the other hand, since antioxidant defences are crucial to deal with the oxidative stress associated with the immune response and with the pathogens themselves, [114] improved tolerance should be favoured simultaneously, yielding more efficient antioxidant defences through increased antioxidant enzyme production for instance.

Hence, interactions with pathogens should increase broad sense immune costs, i.e. direct costs due to resource consumption by the immune response, and indirect costs resulting from immunopathology and the use of enzymatic antioxidant defences. [55,[114][115][116] The storage of immune-stimulant and antioxidant carotenoids should alleviate all these operating costs, providing crustaceans with a very efficient and lower-cost immune function. [30,31] In this context, we hypothesise that there should be detectable correlations between the pathogen prevalence and the quantities of stored carotenoids among crustacean populations. Crustacean populations exhibiting higher pathogen prevalence, i.e. crustacean populations that are exposed to more frequent pathogen attacks, should evolve greater capacity of carotenoid storage in the tissues and/or in the haemolymph. While, on the one hand, such a relationship is likely driven by the potential benefits of storing large amounts of carotenoids under high pathogen pressure, on the other hand, it might also be driven by potential costs associated to carotenoids storage under low pathogen pressure.

Indeed, the above elements indicate that carotenoid storage is highly advantageous.

However, natural variation in the amounts of stored carotenoids persist at each taxonomic level in animals, among phyla, [9] species, [10] and populations within species. [11] This variation could reveal local differences in the environmental availability of carotenoids. It could also result from fitness costs associated with carotenoid storage. Under the hypothesis that carotenoid storage evolved to support the immune system in fighting pathogens, the expression of its costs should be maximal in the absence of pathogens. Moreover, the fact that carotenoid storage occurs despite bearing fitness costs would strongly support its adaptive nature. While detrimental effects of carotenoids have been suggested, [12] the potential fitness costs associated with their storage remain poorly investigated even in animals storing naturally these pigments. In copepod crustaceans, carotenoid reserves decrease the infection risk but increase the predation risk by making them conspicuous to predators. [117][118][119] In birds, negative effects of high doses of carotenoids were reported on skeletal muscles and reproduction, mainly under relatively non-stressful conditions, suggesting context-dependent effects of carotenoids. [120,121] Similarly, in the mealworm beetle, T. molitor, carotenoid supplementation provided larvae with a survival benefit under immune challenge, while it was associated with a survival reduction and prolonged larval development without immune challenge. [122] Furthermore, at high concentrations, carotenoids may potentially reduce the immune response efficiency, through their high capacity to scavenge the free radicals released by the immune system, whose function is to kill pathogens. In some invertebrates, carotenoids may also potentially down-regulate the immune response by interfering with the cellular NO signalling, known as a major regulator of the insect immune response. [123][124][125][126] Indeed, when NO stimulates both cellular and humoral immunity of insects, the strong antioxidant power of carotenoids may scavenge a fraction of circulating NO, thus down-regulating basal immune activity. In addition, certain carotenoids, such as astaxanthin, were reported to inhibit the enzymatic activity of the nitric oxide synthase responsible for NO production from L-arginin (Fig. 1), which activity is normally increased upon immune challenge. [19,124] Such a general immune depressive effect of carotenoids was recently evidenced in T. molitor, increasing the susceptibility to entomopathogenic infections; [122] It contrasts available evidence showing that carotenoids improve the immune response success in crustaceans. [30, 31, 43-45, 89, 90] However, since crustaceans have evolved specialized features to store large amounts of carotenoids in their tissues in contrast to insects, [76] these pigments might be of special importance in their physiology, including in immunity. This suggests that dietary carotenoids may be challenging for biological systems, at least for those that did not evolved specialized features to store them, and that beneficial and detrimental effects resulting from carotenoid diet supplementation might be host specific and context-dependent. Carotenoid storage might also have undiscovered costs, leading to spatial and temporal natural variation within and among invertebrate populations. It is therefore necessary to study the origins of natural variation in carotenoid content to know whether it is adaptive and to understand the conditions of carotenoid storage evolution.

Another important element for the storage of carotenoids to be adaptive is that its expression should be genetically or epigenetically encoded and therefore heritable so that natural selection can act on it. So far, no available studies have investigated whether the capacity to store carotenoids is heritable. Under the hypothesis that carotenoids storage is adaptive to fight pathogens, genetic variance and heritability for this trait might therefore be expected. Ample additive genetic variance and heritability have been found for components of the innate immune response in invertebrates. [127] Investigating the genetic basis of carotenoid storage by measuring its heritability and estimating potential genetic correlations with components of the innate immune response as well as other life-history traits appears of primary importance. In particular, in addition to providing information on how much selection could act on the expression of carotenoid storage, this approach may also reveal to what extent it is associated to the expression of the innate immune response to pathogens.

Conclusions and perspectives

Despite its benefits against infections, the inflammatory immune response, through releasing oxidative cytotoxic molecules, has inevitable immunopathology costs, which decrease lifetime reproductive success and longevity. [32] Endogenous antioxidant enzymes coupled with exogenous antioxidant compounds, including dietary carotenoids, help to limit immunopathology. [105] Carotenoids are powerful antioxidants [35,36] able to limit the oxidativemediated self-harm resulting from the immune response. Hence in addition to directly or indirectly stimulate immunity in some organisms [30, 42-45, 87,8 2, 90] , carotenoids may limit the persistence of a pro-inflammatory status causing age-associated morbidity and death. [103,104] Furthermore, carotenoids such as astaxanthin, may prevent metabolic wasting upon infection by influencing the host's metabolism regulation. [96,97,128] Unlike other dietary antioxidants, carotenoids can be stored in animal tissues and, for yet unknown reasons, aquatic crustaceans exhibit particularly impressive capacities to store astaxanthin compared to other taxa. [7,9,66] From these observations, we infer that greater carotenoid storage in crustaceans could be adaptive against pathogens.

To examine this possibility, we propose three testable hypotheses:

(1) As astaxanthin is antioxidant and immune-stimulant, the evolution of the crustacean immune function and enzymatic antioxidant defence system may have occurred in a particular context, because of their capacity to store large amounts of carotenoids. Large astaxanthin storage should therefore promote both resistance and tolerance to infection, providing crustaceans with a highly efficient immune response, but at low immunopathological costs.

(2) As inflammation-associated immunopathology is closely linked to ageing, storage of carotenoids should prevent ageing acceleration resulting from early-life infection and therefore enhance lifetime reproductive success. In this context, maternal transfer of carotenoids into the eggs and the storage of these pigments early in life should be advantageous in a septic environment.

(3) By promoting resistance and tolerance, carotenoids may interfere in host-pathogen interactions, which would lead to correlations between these pigments storage and the pathogen prevalence among crustacean populations. Therefore, high pathogen pressure should favour higher capacity to store carotenoids.

These above roles of carotenoid storage might be particularly important in aquatic crustaceans, which live in microbes-rich environments constantly challenging their immune system, compare to other animals [START_REF] Nagasawa | [END_REF][81][82] , at least for two main potential reasons. First, the Alternatively, pathogen clearance and wound healing may not be entirely successful, leading to increased self-harm, chronic inflammation and immunological oxidative stress (dashed arrows).

  , astaxanthin in crustaceans, by contrast, may act as an important antioxidant (Ambati et al., 2014; Snoeijs and Häubner, 2014; Schneider et al., 2016; de Carvalho and Caramujo, 2017; Weaver et al. 2018).

Figure 1 .

 1 Figure 1. Diagrammatic representation of the respiratory burst during phagocytosis by

Figure 2 .

 2 Figure 2. Induction of the immune response (phagocytosis and prophenoloxidase cascade)

Figure 3 .

 3 Figure 3. Schematic view of the effects of the immune response on resistance to pathogens

Figure 1

 1 Figure 1.

Figure 3

 3 Figure 3.

Acknowledgements

We thank T. Rigaud for comments, and C. Chateau Smith for proof-reading our manuscript.

Funding

This work was supported by the French National Research Agency (ANR-08-JCJC-0006), the CNRS, and the Conseil Régional de Bourgogne.

crustacean immune response strongly relies on hemocytes and their cytotoxic activity resulting from the prophenoloxidase system and ROS/RNS production, and marginally on the production of non self-reactive immune effectors such as the insect antimicrobial peptides and the vertebrate antibodies of the acquired immune system. [START_REF] Cerenius | Invertebrate Immunity[END_REF] Second, aquatic crustaceans have relatively large access to carotenoids in their habitat. [8][9][10] One may speculate that such a large carotenoid availability may have prevented the evolution of immune innovations allowing a reduced use of ROS/RNS, like in insects.

Author's contributions Y.M. conceived the ideas. A.B., J.M., and Y.M. performed the literature search and led the writing of the manuscript.

Competing interests

We have no competing interests.


Naiane Naidek

Kai Huang

George Bepete

Maria Luiza

M Rocco

Alain Pénicaud

Aldo J G Zarbin

Elisa S Orth
email: elisaorth@ufpr.br

Anchoring conducting polymeric monomers on single-walled-carbon nanotubides: towards covalently linked nanocomposites

Keywords: covalent functionalization, nanocomposites, carbon nanotubes, conducting polymers, polypyrrole

The functionalization of carbon nanotubes (CNTs) has long been a challenge, due to the low reactivity of CNTs. Herein we present a novel approach to functionalize CNT with three different monomers of conductive polymers, all by means of a covalent bond directly on the carbon surface. A polymeric nanocomposite covalently linked, derived from polypyrrole (Ppy) was also obtained. Firstly, highly reactive negatively charged single wall CNTs (SWCNT) salts were functionalized with monomers of conductive polymers: 3-Bromothiophene, 3-Acetylthiophene and 1-(2-Bromoethyl)-1Hpyrrole, leading to three CNT-functionalized materials. After the functionalization, a "grafted from" approach was used to polymerize the pyrrole-derived CNT and obtain a final, covalently linked, polymeric nanocomposite of SWCNTs/Ppy. All samples were characterized by X-ray photoelectron spectroscopy, thermogravimetric analysis, scanning electron microscopy, infrared and Raman spectroscopy, which confirmed the functionalization and the nanocomposite polymerization. Overall, the results evidence the efficiency of the covalent functionalization directly on the skeleton of the CNTs, followed by the polymerization and formation of a novel covalently linked nanocomposite of Ppy and SWCNTs. These materials are particularly interesting since their synergistic effect can benefit future optimal applications such as supercapacitors and artificial muscles.

Introduction

Carbon nanotubes (CNT) comprise a very interesting class of materials with unique morphologies and a structure providing extraordinary mechanical properties with a high Young modulus (around 1 TPa) and tensile strength (up to 300 GPa), extraordinary electrical and thermal properties. Promising applications are foreseen for CNTs in several areas such as electronics, medicine and automotive among others. [START_REF] Ferreira | Carbon nanotube functionalized with dodecylamine for the effective dispersion in solvents[END_REF] However, their poor dispersibility in any solvents constitutes a limitation towards further applications. The preparation of modified CNT containing attached molecules allows to overcome the lack of CNT dispersibility. Several compounds may interact with CNT by different interactions and the main approaches to modify the CNTs are based on the (i) covalent and non-covalent functionalization of the surface of the CNTs and on (ii) filling of the empty cavity of the CNT with molecules or metal oxides. [START_REF] Pénicaud | Spontaneous Dissolution of a Single-Wall Carbon Nanotube Salt[END_REF][3][START_REF] Husmann | Design of a Prussian Blue Analogue/Carbon Nanotube Thin-Film Nanocomposite: Tailored Precursor Preparation, Synthesis, Characterization, and Application[END_REF][START_REF] Schnitzler | One-step route to iron oxide-filled carbon nanotubes and bucky-onions based on the pyrolysis of organometallic precursors[END_REF] Seeking for new and optimized properties, CNT surface functionalization is a perfect way to overcome the drawbacks of the lack of dispersibility and maximize CNT potential applications. The non-covalent functionalization of CNTs is an interesting approach, and can occur by the adsorption or wrapping of functional molecules, surfactants or polymers through van der Waals interactions, π-π stacking and hydrogen bonding, among others. [3,[START_REF] Bilalis | Non-covalent functionalization of carbon nanotubes with polymers[END_REF][START_REF] Vaisman | The role of surfactants in dispersion of carbon nanotubes[END_REF] However, these are weak interactions and the groups are not effectively linked to the CNT surface, becoming easily susceptible to leaching, which can sometimes compromise the application. Another approach for functionalization is the covalent bonding onto the CNT surface that can be realized by several organic reactions such as halogenation, hydrogenation, cycloaddition, radical additions, electrophilic addition, among others. Indeed, these reactions allow the inclusion of organic molecules by a covalent bond on the CNT surface avoiding leaching. They also allow the additional insertion of different functional groups or polymers on functionalized CNTs. With hexagonal rings of carbon sp² hybridization, [START_REF] Yaragalla | Structural and optical properties of functionalized multi-walled carbon nanotubes[END_REF] the addition of molecules on the CNTs surface alters the extended π system due the formation of sp 3 carbons, thus changing their electronic properties that could open the field of new applications possibilities [START_REF] Voiry | Stoichiometric control of single walled carbon nanotubes functionalization[END_REF].

Covalent functionalization is limited by CNT surface reactivity, which is close to the reactivity of alkenes. The functionalization reactions usually require high temperatures (60° up to 500°C) and several reactions steps. Chemical functionalization allows to add functional groups such as oxygenated, amino or alkane groups directly on the CNTs surface. Oxygenated groups such as carboxylic, hydroxyl and epoxy are also promising targets to carry out site-directed functionalization, for example leading to ester and amide linkage or epoxy opening. Moreover, CNT can be functionalized with monomers that can further be polymerized, forming covalently attached polymeric nanocomposites derived from CNTs, yielding synergistic effects between CNTs and polymers [START_REF] Ferreira | Carbon nanotube functionalized with dodecylamine for the effective dispersion in solvents[END_REF][START_REF] Spitalsky | Carbon nanotube-polymer composites: Chemistry, processing, mechanical and electrical properties[END_REF].

Covalent and non-covalent surface functionalization have shown great results in several applications [START_REF] Qiu | Ferrocenyl-functionalized carbon nanotubes with greatly improved surface reactivity for enhancing electrocapacitance[END_REF][START_REF] Lee | Functionalized carbon nanotubes for adsorptive removal of water pollutants[END_REF][START_REF] Manoj | Biomass-derived, activated carbon-sulfur composite cathode with a bifunctional interlayer of functionalized carbon nanotubes for lithium-sulfur cells[END_REF][START_REF] Ali | Capacitive performance of cysteamine functionalized carbon nanotubes[END_REF][START_REF] Habibizadeh | Preparation and characterization of PEGylated multiwall carbon nanotubes as covalently conjugated and non-covalent drug carrier: A comparative study[END_REF]. However, approaches for covalent functionalization usually require several steps or high temperature reactions, especially for obtaining polymeric nanocomposites. Some examples are summarized in Figure 1. One of the simplest functionalization reactions is the halogenation of CNT, which may happen by the fluorination of CNTs (Figure 1A). The reaction takes part with fluorinated compounds from room to high temperatures (500°C) and can be extended to chlorine and bromine. [3,[START_REF] Adamska | Fluorination of Carbon Nanotubes -A Review[END_REF][START_REF] Zhang | Carbon nanofibres fluorinated using TbF4 as fluorinating agent[END_REF]] Also, CNTs have been subjected to glow discharge and atomic hydrogen providing hydrogenated CNT, shown in Figure 1B. [START_REF] Khare | Functionalization of Carbon Nanotubes Using Atomic Hydrogen from a Glow Discharge[END_REF] In another study, CNTs were functionalized with alkylhalides (Br, I and Cl) through an electrophilic addition (Figure 1C), under microwave conditions. [START_REF] Xu | Microwave-induced electrophilic addition of single-walled carbon nanotubes with alkylhalides[END_REF] In fact, covalent functionalization can provide new properties, and the efficiency of covalent functionalization was demonstrated by Polyakov et al through a covalent link between single wall CNT (SWCNT) and a molecule containing zinc and phthalocyanine (ZnPc, Figure 1D). The nanocomposite was used as sensor, detecting low concentrations of ammonia, CO2 and other gas. [START_REF] Polyakov | Effect of covalent and non-covalent linking of zinc(II) phthalocyanine functionalised carbon nanomaterials on the sensor response to ammonia[END_REF][START_REF] Kadem | Effect of covalent and non-covalent linking on the structure, optical and electrical properties of novel zinc(II) phthalocyanine functionalized carbon nanomaterials[END_REF] Both covalent and noncovalent functionalization showed great improvement on gas response sensing, and the covalently anchored nanocomposite shows a higher sensor response than the noncovalent nanocomposite. CNT functionalization through a free radical addition of a sulfur derivative species has also been reported [START_REF] Mao | Thiol functionalized carbon nanotubes: Synthesis by sulfur chemistry and their multi-purpose applications[END_REF] in two steps: free radical functionalization with sulfur powder and UV irradiation, followed by the reduction with NaBH4.(Figure 1E). The obtained CNT-SH was able to be further functionalized with epoxy, alkene and thiol compounds. In another approach Yao et al [START_REF] Yao | Polymerization from the Surface of Single-Walled Carbon Nanotubes -Preparation and Characterization of Nanocomposites[END_REF] have reported a strategy to obtain polymeric nanocomposites which involves the functionalization of CNTs with cycloalkanes (in acetone, trimethylamine (TEA), dimethylfuran (DMF) and dimethylsulfoxide (DMSO)). For the functionalization, the first step requires a 1,3dipolar cycloaddition followed by an esterification with 2-bromoisobutyryl bromide (Figure 1F). After the second reaction, the functionalized CNT was polymerized with methyl methacrylate forming the polymerized nanocomposite. Covalently linked polymeric nanocomposites of CNT also may occur by 3 steps as shown in (Figure 1G), (i) formation of the acid-functionalized MWCNT (MWCNT-COOH) followed by (ii) reaction with SOCl2 and Br derivatives and (iii) reaction based on the reversible addition fragmentation chain-transfer (RAFT polymerization) with the RAFT agent PhC(S)SMgBr. [START_REF] Cui | Functionalization of multiwalled carbon nanotubes by reversible addition fragmentation chain-transfer polymerization[END_REF] The resulting material was polymerized with the assistance of 2,2′-Azobis(2-methylpropionitrile) (AIBN). Herein, we propose a more straightforward method for functionalizing CNT with monomers and further obtaining a polymeric nanocomposite covalently linked. Specifically, we targeted reduced nanotube salts, which are negatively charged and highly reactive in reductive functionalization. Previous reports by Voiry et al have shown a stoichiometric control of SWCNT functionalization using reduced nanotube salts with 1-bromo-decanoic acid and 4-(5-bromopentyloxy)-40-cyanobiphenyl while Hof et al studied the functionalization of oxygenated SWCNT with thiophene coupled derivatives through ester linkages [START_REF] Voiry | Stoichiometric control of single walled carbon nanotubes functionalization[END_REF][START_REF] Hof | New Basic Insight into Reductive Functionalization Sequences of Single Walled Carbon Nanotubes (SWCNTs)[END_REF][START_REF] Voiry | Portrait of carbon nanotube salts as soluble polyelectrolytes[END_REF][START_REF] Catheline | Graphene solutions[END_REF][START_REF] Pénicaud | Deconstructing Graphite: Graphenide Solutions[END_REF][START_REF] Pénicaud | Concentrated solutions of individualized single walled carbon nanotubes[END_REF][START_REF] Voiry | Dissolution and alkylation of industrially produced multi-walled carbon nanotubes[END_REF]. Moreover, this functionalization directly on the carbon backbone has not been reported yet with monomers of conducting polymers, to the best of our knowledge. Herein we report a novel SWCNT-derived nanocomposite with polypyrrole (Ppy) covalently linked by a "grafted from" approach for the growth of the polymers, Figure 2. Firstly, we anchored the monomers 3bromothiophene (BTI), 3-acetylthiophene (BATI) and 1-(2-bromoethyl)-1H-pyrrole (BPI) on the CNT surface, using reductive functionalization, with the functionalization directly on the surface of SWCNT. This gave three materials, namely: SWCNT-BTI, SWCNT-BATI and SWCNT-BPI, respectively. Further, as a proof of concept, the sample functionalized with BPI was polymerized using FeCl3 in order to obtain a covalently linked nanocomposite of SWCNT and Ppy, giving Poly-SWCNT-BPI. The samples were fully characterized by X-Ray photoelectron spectroscopy (XPS), scanning electron microscopy (SEM), infrared (FTIR) and Raman spectroscopy, and thermogravimetric analysis (TGA). Overall, we have successfully obtained the proposed functionalized materials, which are promising candidates for a wide spectrum of applications.

Experimental details

The reactants employed in the synthesis of the nanocomposites were BTI (97%), BPI (97%) and BATI (97%), dimethyl sulfoxide (DMSO) (97%), FeCl3.6H2O (≥ 98%) obtained from Sigma-Aldrich and pyrrole (99%) from Acros organics. The reduced nanotubes salt was obtained from a SWCNT (CoMoCAT®) using a previously described method, using potassium. [START_REF] Pénicaud | Spontaneous Dissolution of a Single-Wall Carbon Nanotube Salt[END_REF][START_REF] Voiry | Stoichiometric control of single walled carbon nanotubes functionalization[END_REF]

Preparation of SWCNT salt

The potassium salt of SWCNTs was prepared by reacting potassium with SWCNT powder on a hot plate, under an inert argon-filled glovebox environment as has been reported before for nanotubes and for graphite intercalation. [START_REF] Hof | New Basic Insight into Reductive Functionalization Sequences of Single Walled Carbon Nanotubes (SWCNTs)[END_REF][START_REF] Bepete | Hydroxide Ions Stabilize Open Carbon Nanotubes in Degassed Water[END_REF][START_REF] Bepete | Eau de graphene" from a KC8 graphite intercalation compound prepared by a simple mixing of graphite and molten potassium[END_REF] Stoichiometry was chosen as KC10 to have a sufficiently high functionalization upon later exposure to the monomers BTI, BATI and BPI. [START_REF] Schäfer | On the Way to Graphane-Pronounced Fluorescence of Polyhydrogenated Graphene[END_REF] In a typical reaction, 50 mg of freshly cut potassium metal was added to a 20 mL glass vial containing a 150 mg SWCNT sample. The mixture of potassium and SWCNT sample contained in a glass vial was heated on a hot plate at 180 °C with continuous mixing for 10 min using a stainless-steel spatula. The vial was left on the hot plate, and the mixture was mixed with the stainless-steel spatula at every 10 min interval for 1 h and then every 30 min for 4 h. The product was allowed to cool to room temperature, and the vial was tightly capped for later use.

Functionalization of SWCNT salt

To avoid oxidation of SWCNT salt, the entire procedure was carried out in a glovebox under inert atmosphere. [START_REF] Voiry | Stoichiometric control of single walled carbon nanotubes functionalization[END_REF][START_REF] Voiry | Stoichiometric control of single walled carbon nanotubes functionalization[END_REF] For comparison purposes, the synthesis of bare Ppy was carried out under the same polymerization conditions described above, however without the presence of SWCNT-BPI. Also, an oxidized sample, namely SWCNT-Ox was synthetized by exposing the reduced carbon nanotubes to air, just the same way as the SWCNT-BPI was exposed to air after synthesis. Finally, a non-covalently linked nanocomposite between SWCNT-Ox and Ppy was synthetized in order to compare. A procedure similar to that described for obtaining the Poly-SWCNT-BPI was used, albeit using SWCNT-Ox instead of SWCNT-BPI, as initial precursor. The non-covalent nanocomposite was named SWCNT-Ox-Ppy. After synthesis, all samples were separated by vacuum filtration, washed with distilled water and dried in an oven at 50 °C. Figure 2 presents an overall summary of all samples synthesized. The FTIR spectra were obtained in a spectrophotometer Bomem with 64 accumulations using KBr pellets. SEM images were acquired on a Tescan SEM, by deposition of the powder dispersed in isopropanol on a silicon substrate. TGA analyses were performed on a TA instrument SDT Q600 model with a heating rate of 5 °C.min -1 varying the temperature from ambient to 1000 ° C, using synthetic air atmosphere.

Results and discussion

The functionalization targeted herein lead to various samples that were thoroughly investigated by several techniques. It is important to note that the first indication of the successful functionalization is the precipitation during the reaction. All samples (reduced SWCNT salts and functionalized SWCNT salts) were handled under inert atmosphere (glovebox) during the synthesis. At the end of the synthesis the samples were exposed to air (outside the glovebox) and they oxidized as a consequence of the high reactivity of the SWCNT salts [START_REF] Voiry | Stoichiometric control of single walled carbon nanotubes functionalization[END_REF]. Note that even after functionalization, it has been reported that some unreacted charges remain on the CNTs, leading to hydrogenation and hydroxylation. [START_REF] Hof | New Basic Insight into Reductive Functionalization Sequences of Single Walled Carbon Nanotubes (SWCNTs)[END_REF] Hence, along with raw SWCNTs, the analogous oxidized SWCNT-Ox will be used for comparison throughout the characterization.

Functionalization of the SWCNTs with BPI, BTI and BATI was first confirmed by the XPS measurements presented in Erreur ! Source du renvoi introuvable.3. XPS survey spectra show two main peaks, at 285.0 eV for C1s and at 532.0 eV for O1s [START_REF] Gholami | Morphology and electrical properties of electrochemically synthesized pyrrole-formyl pyrrole copolymer[END_REF]. One indication of functionalization of CNT with BPI, BTI and BATI is the low amount of oxygen presented in the XPS spectra of the functionalized samples. Furthermore, for the SWCNT-BPI spectrum, a new peak is observed at 400.0 eV which refers to the N1s core level and thus to the nitrogen bond [START_REF] Gholami | Morphology and electrical properties of electrochemically synthesized pyrrole-formyl pyrrole copolymer[END_REF]. The quantities of C, N and O for the SWCNT-BATI, SWCNT-BTI and SWCNT-BPI samples are shown in Table 1. The presence of 2.43% of N and the decrease of O (down to 5.39%) for SWCNT-BPI confirms the functionalization of the CNT. Regarding the XPS survey spectra of the nanocomposites (Figure 3), C 1s, N 1s, O 1s and Cl 2p core level peaks are present in the spectra of both covalent and noncovalently linked polymerized nanocomposites. Indeed, the quantity of C, N, O and Cl (Table 1) are very similar for both nanocomposites. High resolution N1s core level peaks in the spectra of the polymerized nanocomposites, Poly-SWCNT-BPI (covalent) and SWCNT-Ox-Ppy (non-covalent) agree with the presence of Ppy and the nanocomposite formation, as will be discussed in the following. For the covalently and non-covalently linked nanocomposites (Poly-SWCNT-BPI and SWCNT-Ox-Ppy), the C1s high resolution spectra are presented in Figure 5.

The peak assignments are very similar to the C1s high resolution spectra of SWCNT-BPI. [START_REF] Ratso | Enhanced oxygen reduction reaction activity of iron-containing nitrogen-doped carbon nanotubes for alkaline direct methanol fuel cell application[END_REF][START_REF] Ji | In-situ reduction of Ag nanoparticles on oxygenated mesoporous carbon fabric: Exceptional catalyst for nitroaromatics reduction[END_REF] Regarding the N1s spectra, they were deconvoluted using three Gaussian/Lorentzian peaks: the major peak centered at 400.0 eV is related to the aminelike structure (-NH-) whereas the peak >401 eV is attributed to the positively charged nitrogen atoms (-N + ) of the Ppy, evidencing its oxidized species, and the peak at 398.1

eV is assigned to the imine-like structure of nitrogen species (=N-) [START_REF] Tabačiarová | Study of polypyrrole aging by XPS, FTIR and conductivity measurements[END_REF][START_REF] Lim | In situ XPS study of thermally deposited aluminium on chemically synthesized polypyrrole films[END_REF]. The obtained results evidence the formation of the nanocomposites and the oxidized form of the polymer due to the presence of charged nitrogen atoms. In order to further characterize the carbon-like structure, the Raman spectra is presented in Figure 6 and shows features providing general information about the CNT.

The peaks in the low frequency range below 400 cm -1 are known as the radial breathing modes (RBM) of the SWCNT. At higher frequencies, the high intensity peak at 1580 cm -1 (G + -band) and the low intensity peak at 1532 cm -1 (G --band) are the two main components of the G-band feature for SWCNT. The first one related to the longitudinal optical mode whereas the latter is related to the transversal optical mode. [START_REF] Dresselhaus | Raman spectroscopy of carbon nanotubes[END_REF] Raman bands intrinsic to the functionalized groups were not observed on the Raman spectra of SWCNT-BPI, SWCNT-BATI and SWCNT-BTI, which may be associated to a low degree of functionalization. Even for significant degrees of functionalization (~30% of the carboxylic acid converted to amides in the case of graphene oxide) [START_REF] Hostert | Imidazole-derived graphene nanocatalysts for organophosphate destruction: Powder and thin film heterogeneous reactions[END_REF] no bands associated to the anchored moieties are usually observed due to the characteristic intense bands of the resonant carbon nanomaterials, which dominate the spectra. However, nanotube surface modifications may be evaluated by the D/G ratio. The band at 1300 cm -1 (D-band) is related to defects of the sp² structure. [START_REF] Dresselhaus | Raman spectroscopy of carbon nanotubes[END_REF] The ratio of the intensity of the D vs G bands (obtained from the area of the deconvoluted bands) is used to estimate the functionalization degree of carbon nanotubes and higher ID/IG values is attributed to greater functionalization. [START_REF] Voiry | Stoichiometric control of single walled carbon nanotubes functionalization[END_REF][START_REF] Hof | New Basic Insight into Reductive Functionalization Sequences of Single Walled Carbon Nanotubes (SWCNTs)[END_REF] Indeed, the calculated ID/IG relation of the samples shows an increase as follows:

SWCNT<SWCNT-Ox<SWCNT-BTI<SWCNT-BATI<SWCNT-BPI (Figure 6), indicating a higher degree of defects at the surface of the functionalized materials. As anticipated by XPS, this may be associated with the presence of the BTI, BATI and BPI functional groups onto the surface of SWCNT [START_REF] Voiry | Stoichiometric control of single walled carbon nanotubes functionalization[END_REF]. The deconvoluted spectra are shown on SI.

Raman spectroscopy of the polymeric Poly-SWCNT-BPI and SWCNT-Ox-Ppy (Figure 6) was used to identify the polymer formation on the nanocomposites covalently and non-covalently linked. New bands are observed in the spectra which are a clear indication of the presence of Ppy. The bands at 926 and 1044 cm -1 are correlated to the C-H out-of-plane and the CH stretching of neutral species of Ppy, respectively, [START_REF] Crowley | In situ resonance Raman spectroelectrochemistry of polypyrrole doped with dodecylbenzenesulfonate[END_REF] whereas the band at ca 1340 cm -1 is derived from a superposition of the Ppy bands in the nanocomposite with the SWCT bands. The Ppy spectrum has already been reported in the literature [START_REF] Crowley | In situ resonance Raman spectroelectrochemistry of polypyrrole doped with dodecylbenzenesulfonate[END_REF][START_REF] Zuo | One-step electrochemical preparation of sulfonated graphene/polypyrrole composite and its application to supercapacitor[END_REF] [48] and Table 2 summarizes the main bands of Ppy. The Raman features of SWCNT are still present in these spectra; meanwhile the G and D-band are overlapped by the Ppy Raman bands, making impracticable the ID/IG analysis of the nanocomposites. However, the 2D (also called G') band is still visible and not overlapped in the Poly-SWCNT-BPI and SWCNT-Ox-Ppy. Additionally, FTIR analysis (spectra on Figure 7) shows significant differences when comparing the functionalized samples and raw SWCNTs. For raw SWCNTs, the band at 1640 cm -1 is due to the vibrational modes of C=C and the band at 3450 cm -1 is due to O-H groups that can be related to atmospheric moisture attached to SWCNTs. [START_REF] Chen | Chemical modification of pristine carbon nanotubes and their exploitation as the carbon hosts for lithium-sulfur batteries[END_REF][START_REF] Chouhan | Biotransformation of multi-walled carbon nanotubes mediated by nanomaterial resistant soil bacteria[END_REF] Similar features are observed on the SWXNT-Ox spectra. The formation of new bands in the spectra e.g. at 1200 cm -1 , 1730 cm -1 (C=O) for SWCNT-BATI and 1431 cm -1 for SWCNT-BPI and 1030 cm -1 for both, indicates functionalization. The band at 1431 cm -1 in the SWCNT-BPI spectra may be attributed to the stretching of C-N from pyrrole ring [START_REF] Idris | Influence of gamma irradiation on polymerization of pyrrole and glucose oxidase immobilization onto poly (pyrrole)/poly (vinyl alcohol) matrix[END_REF][START_REF] Costa | Synthesis and characterization of conducting polypyrrole/SBA-3 and polypyrrole/Na-AlSBA-3 composites[END_REF] and at 1030 cm -1 to C-N stretching related to the amine groups [START_REF] Gholami | Morphology and electrical properties of electrochemically synthesized pyrrole-formyl pyrrole copolymer[END_REF]. Upon polymerization, FTIR analysis of Poly-SWCNT-BPI and SWCNT-Ox-Ppy shows the typical bands of Ppy. [START_REF] Naidek | Covalently linked nanocomposites of polypyrrole with graphene: Strategic design toward optimized properties[END_REF] Focus is given to the presence of the band at 960 cm -1 attributed to the C-C out-of-plane vibrations. This is a characteristic signature of Ppy polymerization. Overall, the FTIR analysis provides additional evidence of SWCNT functionalization and successful polymerization of the nanocomposites. The SWCNT-BATI sample also presents two weight loss events, the first one at 176 °C associated with evaporation of the remaining solvent (DMSO) and the second at As widely discussed in the literature [START_REF] Kasprzak | Functionalization of graphene: does the organic chemistry matter?[END_REF][START_REF] Schirowski | Fundamental Insights into the Reductive Covalent Cross-Linking of Single-Walled Carbon Nanotubes[END_REF][START_REF] Bottari | Chemical functionalization and characterization of graphenebased materials[END_REF] covalent functionalization of carbon nanomaterials nowadays continue to be a challenge, despite their many promising applications. Pursuing novel routes or improvements of known methodologies is thus important. Overall, in this paper we have synthesized and characterized three different SWCNT functionalized materials, covalently attached on the carbon skeleton structure of SWCNT with different monomers, employing mild reaction conditions. Moreover, the functionalized material was susceptible of polymerization, resulting in a nanocomposite of SWCNT with a covalently attached polymer. As a proof of concept, we pursued the polymerization of the pyrrole-derived material and the functionalized SWCNT yielded a polymeric nanocomposite for which we expect a synergistic effect between SWCNTs and polypyrrole. Regarding the polymerization process, we believe that the monomers anchored on the surface of SWCNTs take part in the process of chemical oxidative polymerization, providing a polymer anchored on the surface of the SWCNT (Figure 10). [START_REF] Naidek | Covalently linked nanocomposites of polypyrrole with graphene: Strategic design toward optimized properties[END_REF]. Likewise, the polymer formed is comprised of monomers freely available (pyrrole from the reaction medium) and also the pyrrole derivative attached to the CNT, leading to a highly interconnected polymer, strongly linked to the CNT. Thereby, the polymer is wrapping the SWCNT-BPI through a covalent bond providing a homogeneous morphology. In contrast, for the non-covalent nanocomposites, polymer wrapping is not homogeneously distributed and regions containing only Ppy are observed.

Conclusions

The complexity of organic chemistry reactions on carbon structures limits the carbon functionalization, nowadays presented as a strategic tool for development of new materials with promising applications. Herein, we have functionalized SWCNTs

Figure 1 .

 1 Figure 1. Summary of some reported functionalization of CNTs and formation of

  100 mg of SWCNT salt and 50 mL of ultra dry DMSO (dried through an Inert TM column directly delivering dry solvent inside the box) were left stirring overnight in a round-bottom flask and centrifuged 2 times, at 4000 rpm for 30 min. The undissolved material was separated from the solution. The covalent functionalization was carried out with monomers: BATI, 0.73 g (3.55 mmol), BTI, 400 µL (4.3 mmol) and BPI, 500 µL (4.1 mmol) solubilized in 2 mL ultra dry DMSO. The monomers were separately added at once to 30 mL of SWCNT salt solution and left to stir for 24 h. A precipitate was obtained, indicative of functionalization. The suspensions of reacted SWCNTs were exposed to air, out of the glovebox, washed with HCl (2 mol L -1 ) deionized water and then with ethanol, by centrifugation. The final solids were dried in an oven under vacuum at 50 °C for 19 h and the samples were named SWCNT-BATI, SWCNT-BTI and SWCNT-BPI. 2.3 Chemical polymerization of SWCNT-BPI Polymerization of the covalently functionalized nanotube (SWCNT-BPI) was achieved by the addition of 3.5 µL of a pyrrole monomer solution (0.05 mmol) in a round-bottom flask containing 4 mL (0.4 mg.mL -1 ) of SWCNT-BPI in aqueous solution. The dispersion was kept stirring while 5 mL of the oxidizing agent FeCl3.6H2O (0.25 mol.L -1 , 1.25 mmol) in aqueous solution was added slowly. The dispersion was then stirred at room temperature for 6 hours. The final solid Poly-SWCNT-BPI, was separated by vacuum filtration, washed with distilled water and dried in oven at 50 °C.

Figure 1 .

 1 Figure 1.Summary of the functionalization scheme

Figure 3 .

 3 Figure 3. XPS survey spectra of SWCNT-Ox, SWCNT-BPI, SWCNT-BATI, SWCNT-BTI and the covalently and non-covalently linked nanocomposites.

Figure 4

 4 Figure4shows the deconvolution of the C1s spectra of the samples SWCNT-Ox,

Figure 4 .

 4 Figure 4. High-resolution C1s XPS spectra of SWCNT-Ox, SWCNT-BATI, SWCNT-BPI and SWCNT-BTI and high-resolution N1s XPS of SWCNT-BPI.

Figure 5 .

 5 Figure 5. High-resolution C1s and N1s XPS spectra of Poly-SWCNT-BPI and SWCNT-Ox-Ppy.

Figure 6 .

 6 Figure 6. Representative Raman spectra (a) of the samples with excitation at 632.8 nm, showing the ID/IG value increases from SWCNT<SWCNT-Ox<SWCNT-BTI<SWCNT-BATI<SWCNT-BPI; the inset shows a representative SWCNT-BPI Raman deconvolution. Also are shown the representative Raman spectra (b) of the covalent and non-covalent nanocomposites.

Figure 7 .

 7 Figure 7. FTIR transmission spectra of functionalized samples (SWCNT-Ox, SWCNT-BATI, SWCNT-BPI) and the covalently and non-covalently linked nanocomposites (Poly-SWCNT-BPI and SWCNT-Ox-Ppy).

Figure 8

 8 Figure 8 presents the TGA data of the functionalized SWCNTs (SWCNT-BPI,

Figure 8 .

 8 Figure 8.TGA of the samples of the raw and functionalized SWCNTs in air.

Figure 9 .

 9 Figure 9. SEM images of the oxidized and functionalized SWCNTs, covalent and noncovalent linked nanocomposite.

Figure 10 .

 10 Figure 10.Schematic representation of the reaction mechanism and covalently and non-

  with three different monomers by a one-step and room temperature reaction. Also, we have synthetized covalent and non-covalent nanocomposites of SWCNT and Ppy, the former based on a covalently anchored monomer polymerization forming nanocomposites of conductive polymer on the carbon skeleton of the SWCNT and the latter based on π-π interactions. The covalent attachment of the monomers on the surface of SWCNT was confirmed by XPS measurements, Raman spectroscopy, and other techniques. Regarding the covalent and non-covalent polymerized nanocomposites, both presented similar chemical properties and, in both composites, the Ppy was in the conductive form. Nevertheless, SEM images show a more homogeneous coverage of SWCNTs with Ppy on the covalently linked nanocomposite (Poly-SWCNT-BPI). Overall, the challenge of carbon functionalization has been successfully accomplished. The development of different routes to obtain new materials, e.g. decreasing the number of reactions steps and favoring mild conditions, is an advance in the functionalization chemistry of carbon structures. In this paper, using a one-step reaction under mild conditions, the CNT functionalization has been simplified. The strategy adopted here allows the functionalization of CNTs with several other molecules and potentiates new application possibilities of CNT nanocomposites.

Table 1 .

 1 Surface chemical composition of the functionalized and polymerized nanocomposites, atomic percentage obtained by XPS.

			Sample	C 1s N 1s O 1s S 2p	Cl 2p Si 2p
			SWCNT-Ox	75.20	0	21.45	0		0	3.35
			(%)
			SWCNT-BPI	88.74 2.43	5.39	2.00	0	0.88
			(%)
	covalent		(%) SWCNT-BATI	80.78	0	15.12	1.70	0	2.40
			SWCNT-BTI	83.18 0.91	11.64	1.95	0	1.59
			(%)
			Poly-SWCNT-	69.27 11.8	17.18	0		1.59	0
			BPI (%)		0
	non-	covalent	Ppy (%) SWCNT-Ox-	6 70.84 11.1	14.80	0		1.75 1.60

406 404 402 400 398 396 394 398.4 eV C=N-C (imine) 400.5 eV C-NH-C (pyrrole) Intensity, a.u. Binding Energy, eV Raw data Mo3p 3/2

	a.u.	C 1s SWCNT-ox Background Raw Data		284.8 eV C=C	a.u.	C 1s SWCNT-BATI Background Raw data	284.8 eV C=C
	Intensity,		288.3 eV COOR	286.6 eV C-O			Intensity,	288.3 eV COOR	286.4 eV C-O	285.6 eV C-S
		292	290 Binding energy, eV 288 286 284	282		292	290 Binding Energy, eV 288 286 284	282
		C 1s SWCNT-BPI		284.8 eV C=C				C 1s SWCNT-BTI		284.8 eV C=C
		Raw data						Raw data
	a.u. Intensity,	Background 286.3 eV C-O 287.2 eV C=O CO -2 3	285.5 eV C-N	a.u. Intensity,	Background 288.3 eV COOR	286.6 eV C-O	285.5 eV C-S
		Shake-up	COOH	COOR
		292	290	288	286	284	282		292	290	288	286	284	282
			Binding Energy, eV					Binding Energy, eV
											Background
						N 1s
						SWCNT-BPI

294 292 290 288 286 284 282 280

		Raw Data
		Background	284.6 eV
				C=C
	a.u.	C 1s Poly-SWCNT-
	Intensity,	290.9 eV 288.8 eV O-C=O	286.5 eV C-O 285.5 eV C-N
		Shake-up
			287.8 eV
			C=O
		Binding Energy, eV

BPI 294 292 290 288 286 284 282 280 290.9 eV Shake-up 288.8 eV O-C=O Intensity, a.u. Binding Energy, eV

								Raw Data Background		284.6 eV C=C
							C1s
							SWCNTox-Ppy
										286.5 eV
									C-O 287.8 eV C=O	285.5 eV C-N
		Raw Data	400 eV				Raw Data	400 eV	N1s
		Background	-NH-				Background	-NH-	SWCNTox-Ppy
	a.u.			N1s Poly-SWCNT-BPI	a.u.
	Intensity,	401.8 eV -N +	398.01 eV =N-		Intensity,	401.5 eV -N +		398.1 eV =N-
	406	404	402 Binding Energy, eV 400 398	396	394	406	404	402 Binding Energy, eV 400 398	396	394

Table 2 .

 2 Raman bands of Ppy.[START_REF] Crowley | In situ resonance Raman spectroelectrochemistry of polypyrrole doped with dodecylbenzenesulfonate[END_REF][START_REF] Zuo | One-step electrochemical preparation of sulfonated graphene/polypyrrole composite and its application to supercapacitor[END_REF][START_REF] Naidek | Covalently linked nanocomposites of polypyrrole with graphene: Strategic design toward optimized properties[END_REF]

	Band position (cm -1 )
	1581	C=C stretching (carbon skeleton)
	1385	antisymmetric C-N stretching
	1324	C-C stretching of the neutral species
	1235	antisymmetric C-H (plane) bending
	1080	C-H (plane) bending of oxidized species
	1045	C-H (plane) bending of neutral species
	970	cationic ring deformation of Ppy
	928	ring deformation associated to dication


or in ambient conditions depending on the electric field intensity as well as on the used gas (mixtures of N2, O2, Ar, He, methane etc.). Cold plasmas are complex mixtures of neutral species, electrons, particular, when generated in oxygen-nitrogen mixtures (air), multiple reactive oxygen and nitrogen species (ROS, RNS) are present in the gas phase. Because of their diverse and intense reactivities, and relative ease for production, cold plasmas reveal themselves as excellent approaches to treat surfaces in order to modify their physical (cleaning, roughening) and chemical (functional groups) states.

In this context, we applied cold plasmas to modify the reactivity of metal and carbon electrode surfaces and studied the resulting effects on the detection of compounds involved by biosensors. 1,2 We first treated platinum and black platinum modified electrodes by 100 % Oxygen-plasmas produced by radio-frequency modulated electric fields (plasma cleaner setup) and observed lower capacitive currents, more stable faradaic responses, decreased standard potentials and improved sensitivity for H2O2 detection. These properties were related (XPS analyses) to a change of ratio between Pt oxides, which improve the surface hydrophilicity and initial adsorption of ROS. Similarly, the reactivity of glassy carbon electrodes could be homogenized and improved for the detection of both inner-or outer-sphere electron transfer species when treated by plasmas, allowing to prevent from polishing electrode surfaces for their cleaning or activation. Moreover, plasma effects were stable for periods longer than one week.

Reciprocally, such electrochemical sensors were involved to decipher on the nature of species produced by cold atmospheric pulsed plasmas when used to expose physiological fluids, cells or tissues. [3][4][5] Plasmas raise exponential interests for biomedical applications because of their strong reactivities. However, the true nature of reactive species involved in biocidal or oxidative bio-activation remains widely unsolved. To decipher on the in situ reactivity within solutions exposed to plasmas, we developed shielded microelectrodes to detect ROS and RNS appearing in solution, at millimetric distances from the plasma flow and electric source (nanosecond pulsed discharges). The concentration rises of hydrogen peroxide, nitrite as well as of superoxide anion were monitored with an unprecedented spatial and temporal resolutions. 6 Consequently, we have demonstrated that electrochemical sensors can benefit from plasma treatments and vice versa.

Using Cold Plasmas to Improve Electrochemical Sensors and vice versa

  K20.Colin Camille, a Badets Vasilica, a Girard Fanny, a,b Lefrançois Pauline, a Botsos-Margerit Ugo, a Zigah Dodzi, a Blanc Sylvie, b Sojic Neso, a Clément Franck, b Arbault Stéphane a a Univ. BORDEAUX, ISM, Bordeaux INP, CNRS UMR 5255, NSysA Group, Pessac, France b UPPA, IPREM, CNRS UMR 5254, Pau, 64000, France stephane.arbault@u-bordeaux.fr Cold plasmas are weakly ionized gases, generated by applying High-Voltage electrical discharges in a gaseous environment. They can be produced by sinusoidal or pulse power supplies, at either low pressures

  Girard, M. Peret, N. Dumont, V. Badets, S. Blanc, K. Gazeli, C. Noël, T. Belmonte, L. Marlin, J. P. Cambus, G. Simon, N. Sojic, B. Held, S. Arbault and F. Clément, Phys. Chem.Chem. Phys., 2018, 20, 9198-9210. 6 F. Girard-Sahun, V. Badets, P. Lefrançois, N. Sojic, F. Clément and S. Arbault, Anal. Chem., 2019, 91, 8002-8007.

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