ۙۛۛ ﮠۣۢۨۗ۩ۣۘۦۤ

ﮞۺۣۣۛ۠ۨۧٷۦٷێ ۙ۠ۖٷ۠ٷ۪ﯠ

ۣۢ ۍﯣﯙ

ۨۗۍ ھڽڼھ

R Ruiz Daniels

S Bel Tran

R Poulin

C Lagrue

Do parasites adopt different strategies in different intermediate hosts? Host size, not host species, influences Coitocaecum parvum (Trematoda) life history strategy, size and egg production

Keywords: life cycle abbreviation, intermediate hosts, Coitocaecum parvum, Paracalliope fluviatilis, Paracorophium excavatum

Host exploitation induces host defence responses and competition between parasites, resulting in individual parasites facing highly variable environments. Alternative life strategies may thus be expressed in context-dependent ways, depending on which host species is used and intra-host competition between parasites. Coitocaecum parvum (Trematode) can use facultative progenesis in amphipod intermediate hosts, Paracalliope fluviatilis, to abbreviate its life cycle in response to such environmental factors. Coitocaecum parvum also uses another amphipod host, Paracorophium excavatum, a species widely different in size and ecology from P. fluviatilis. In this study, parasite infection levels and strategies in the two amphipod species were compared to determine whether the adoption of progenesis by C. parvum varied between these two hosts. Potential differences in size and/or egg production between C. parvum individuals according to amphipod host species were also investigated. Results show that C. parvum life strategy was not influenced by host species. In contrast, host size significantly affected C. parvum strategy, size and egg production. Since intra-host interactions between co-infecting parasites also influenced C. parvum strategy, size and fecundity, it is highly likely that within-host resource limitations affect C. parvum life strategy and overall fitness regardless of host species.

INTRODUCTION

Phenotypic plasticity is often seen as an adaptive strategy in highly variable environments [START_REF] Anurag | Phenotypic plasticity in the interactions and evolution of species[END_REF]. Host exploitation by parasites generally results in rapid evolutionary responses of hosts for defence purposes, resulting in a situation where parasites face a constantly changing environment and have to co-evolve with their hosts [START_REF] Van Valen | Molecular evolution as predicted by natural selection[END_REF]. Furthermore, the fitness of a parasite completely depends upon host-parasite interactions; infection and reproduction rates in a particular host-determining parasite success [START_REF] Paterson | Frontiers in host-parasite ecology and evolution[END_REF]. Parasite phenotypic plasticity and alternative growth strategies should thus be expressed in precise context-dependent ways, depending on the parasite's environment/host (Thomas et al. 2002a;[START_REF] Parker | Optimal growth strategies of larval helminths in their intermediate hosts[END_REF].

The environment of a parasite is often not restricted to a single host since multiple species, parasites and/or hosts, are frequently encountered in a parasite life cycle [START_REF] Fredensborg | Larval helminths in intermediate hosts: does competition early in life determine the fitness of adult parasites[END_REF][START_REF] Gower | Intraspecific competition and the evolution of virulence in a parasitic trematode[END_REF][START_REF] Rauch | How a complex life cycle can improve a parasite's sex life[END_REF]. This is especially true for trematodes, their complex life cycles involving up to 4 widely different hosts and countless potential intra-and interspecific parasite competitors [START_REF] Poulin | Evolutionary Ecology of Parasites. 2nd Edn[END_REF]. Although in the majority of trematodes a three-host life cycle is maintained, several species have lost a host from their cycle [START_REF] Poulin | Trematode life cycles: short is sweet?[END_REF]. This life-cycle abbreviation is often realized by dropping the definitive host, maturing and reproducing in the second intermediate host through progenesis and self-fertilization. In some species, life-cycle abbreviation remains facultative and both strategies are observed concurrently in parasite populations [START_REF] Poulin | Trematode life cycles: short is sweet?[END_REF].

For example, the digenean trematode Coitocaecum parvum is able to adaptively adopt progenesis or the normal 3-host life cycle in response to different environmental factors [START_REF] Macfarlane | Life cycle of Coitocaecum anaspidis Hickman, a New Zealand digenetic trematode[END_REF]Holton, 1984a, b;Lagrue andPoulin, 2007, 2008a). Adult C. parvum colonize the digestive tract of fish definitive hosts where they sexually reproduce. Eggs are released with fish feces and hatch into freeswimming miracidiae. After entering the first intermediate host, the snail Potamopyrgus antipodarum, they develop into cercariae-producing sporocysts.

Cercariae leave the snail and must infect an amphipod second intermediate host where they encyst as metacercariae. The parasite can then adopt one of two alternative life strategies; either wait for a fish definitive host to consume the infected amphipod host or undergo progenesis while still inside the amphipod intermediate host and reproduce by selfing [START_REF] Poulin | Trematode life cycles: short is sweet?[END_REF][START_REF] Lefèbvre | Progenesis in digenean trematodes: a taxonomic and synthetic overview of species reproducing in their second intermediate hosts[END_REF]. Progenetic individuals produce viable eggs released into the environment upon the death of the amphipod host. A previous study showed no evidence for heritability of progenesis or negative effects of selfing on offspring fitness (Lagrue and Poulin, 2009a). However, many factors influence adoption of the abbreviated life cycle by individual C. parvum. For example, progenesis rate increases when fish definitive hosts are absent from the environment [START_REF] Lagrue | Life cycle abbreviation in the trematode Coitocaecum parvum: can parasites adjust to variable conditions?[END_REF]. Time spent in the amphipod host also increases the likelihood of progenesis adoption (Lagrue and Poulin, 2009b). Finally, co-infecting parasites and intra-host competition in the second intermediate host (the amphipod Paracalliope fluviatilis) strongly affect the adoption of progenesis in C. parvum (Lagrue and Poulin, 2008a;Lagrue et al. 2009).

Although C. parvum uses 2 different amphipod species as intermediate hosts, how its life-history strategy (abbreviated or normal 3-host cycle) may differ between these two hosts remains unknown. The two amphipod species often co-exist in the field despite profound ecological differences. While Paracalliope fluviatilis is demersal and freeswimming, Paracorophium excavatum is a purely benthic, sedentary, burrow-dwelling amphipod; this may increase its exposure to the non-swimming cercariae of C. parvum (Holton, 1984a). Paracorophium excavatum is also larger, may provide more resources to trematode parasites and thus affect the adoption of progenesis by C. parvum and/or egg production in progenetic individuals. Most studies have only considered P. fluviatilis as C. parvum intermediate host even though P. excavatum was early documented as a potential host in which progenesis occurred (Holton, 1984a;[START_REF] Luque | Larval Hysterothy-lacium sp. (Nematoda, Anisakidae) and trematode metacercariae from the amphipod Paracorophium excavatum (Corphiidae) in New Zealand[END_REF]. However, potential differences in progenesis rates and egg production between C. parvum individuals infecting co-existing P. fluviatilis and P. excavatum are still unknown.

While intra-host (intra-and interspecific) competition among parasites is known to affect both life strategy and egg production in C. parvum, these effects have been studied only in P. fluviatilis (Lagrue and Poulin, 2008a). Intraspecific competition between co-infecting C. parvum individuals strongly affects progenesis rates and decreases egg production in P. fluviatilis, the smaller of the two amphipod host species (Lagrue and Poulin, 2008a). It is possible that such effects are dampened by the larger size and higher available resources of P. excavatum hosts.

Similarly, Microphallus sp. is known to infect both amphipods species [START_REF] Luque | Larval Hysterothy-lacium sp. (Nematoda, Anisakidae) and trematode metacercariae from the amphipod Paracorophium excavatum (Corphiidae) in New Zealand[END_REF]. It is also capable of manipulating amphipod host behaviour to enhance its transmission to bird definitive hosts [START_REF] Hansen | Impact of a microphallid trematode on the behaviour and survival of its isopod intermediate host: phylogenetic inheritance[END_REF][START_REF] Coats | The consequences of parasitic infections for host behavioural correlations and repeatability[END_REF]. Because C. parvum and Microphallus sp. do not share definitive hosts, conflicting interests over transmission routes and/or developmental strategy are likely to arise [START_REF] Brown | Cooperation and conflict in host-manipulating parasites[END_REF]Thomas et al. 2002b;[START_REF] Poulin | Host sharing and host manipulation by larval helminths in shore crabs: cooperation or conflict[END_REF]. Coitocaecum parvum might thus benefit from adopting progenesis in the presence of Microphallus sp.; its transmission probabilities being likely reduced by Microphallus sp. host manipulation. Microphallus sp. prevalence and abundance are also much higher in P. excavatum than P. fluviatilis [START_REF] Luque | Larval Hysterothy-lacium sp. (Nematoda, Anisakidae) and trematode metacercariae from the amphipod Paracorophium excavatum (Corphiidae) in New Zealand[END_REF]). Furthermore, P. excavatum is host to a large fish nematode (Hedruris spinigera) while P. fluviatilis is not [START_REF] Luque | Larval Hysterothy-lacium sp. (Nematoda, Anisakidae) and trematode metacercariae from the amphipod Paracorophium excavatum (Corphiidae) in New Zealand[END_REF]Lagrue and Poulin, 2008a, b). Metacercariae of C. parvum infecting P. excavatum are consequently more likely to compete with other parasites than conspecifics in P. fluviatilis. Natural prevalence and/or abundance variations in parasitic infections between P. excavatum and P. fluviatilis may consequently influence progenesis adoption in C. parvum in its two intermediate host species.

In this study, we examined parasite prevalences and abundances in the two amphipod host species and gave particular attention to the frequency of progenesis in C. parvum infecting P. excavatum and P. fluviatilis to detect possible species-specific intermediate host-induced effects on the parasite strategy. For C. parvum individuals that adopted progenesis, we determined whether parasite size and/or egg production was higher in P. excavatum than in the smaller P. fluviatilis. Finally, we examined potential effects of interactions with co-infecting parasites on size and egg production of progenetic C. parvum.

MATERIALS AND METHODS

Study site and animal sampling

Naturally infected Paracalliope fluviatilis and Paracorophium excavatum amphipods were collected in Lake Waihola (46°01′S, 170°05′E), South Island, New Zealand, using dip nets (500 μm nylon mesh). All amphipods were collected during February and April 2012 and both species were collected at the same time and location to avoid potential spatial or temporal effects on parasite prevalences and/or abundances. Paracorophium excavatum were captured by dredging the fine surficial sediment where this species burrows. The retained sediment with associated organisms was placed in sorting trays from which P. excavatum individuals were collected [START_REF] Schnabel | Population genetic structure of two New Zealand corophiid amphipods and the presence of morphologically cryptic species: implications for the conservation of diversity[END_REF]. Paracalliope fluviatilis were collected from the same area by dragging dip nets in patches of macrophytes (Elodea canadensis).

Amphipods were separated from macrophytes using a 5 mm mesh sieve and collected in sorting trays. All amphipods were kept alive in plastic containers filled with local water and brought back to the laboratory for later dissection. Amphipods were dissected within 3 days of capture.

Parasite prevalences and abundances, and

Coitocaecum parvum strategy, size and egg production Amphipods were killed in 70% ethanol and rinsed with distilled water to allow for easy manipulation (measurement and dissection) without affecting data collection on parasites [START_REF] Lefèbvre | Progenesis in digenean trematodes: a taxonomic and synthetic overview of species reproducing in their second intermediate hosts[END_REF]. Before dissection, amphipods were identified (species) and measured (total body length) to the closest 0•1 mm under a binocular microscope (× 20 magnification) as host size can influence parasite prevalence and abundance in naturally infected amphipods (Lagrue and Poulin, 2008b). For each amphipod, the species (C. parvum, Microphallus sp. and/or H. spinigera) and number of parasites were recorded to determine parasite prevalences (proportion of infected hosts) and abundances (number of parasites per host). Metacercariae of C. parvum were also classified as either normal (non-egg producing) or progenetic (egg producing) according the presence of eggs in the cyst or still in utero.E a c h metacercaria was measured (total length) and its eggs counted.

Statistical analysis

Potential differences in parasite prevalences and/or proportion of progenetic C. parvum between the two amphipod species were tested in a pair-wise manner using Fisher's exact tests. Effects of intermediate host species (P. fluviatilis and P. excavatum) on specific parasite abundances (C. parvum, Microphallus sp. and H. spinigera) and possible differences in host size between the two amphipod species and between infected and uninfected hosts were examined using non-parametric tests (Mann-Whitney U test), as data did not follow normal distributions. Potential relationships between host size, parasite abundance, and C. parvum size and egg production were also tested using linear regressions. Non-parametric tests and linear regressions described above were performed using STATISTICA Software 6.0 (StatSoft Inc., France).

General Linear Models (GLM) were used to test for the effects of multiple factors on the probability of C. parvum metacercariae adopting progenesis, and on the size and/or egg production of progenetic metacercariae. Factors potentially affecting the probability of progenesis in C. parvum and included in the models were host species, host size, number of coinfecting Microphallus sp., number of co-infecting non-egg producing C. parvum metacercariae and/or presence of the fish nematode H. spinigera. The dependent variable was treated in a binomial manner with C. parvum metacercariae coded either as 1 (progenetic/egg-producing) or 0 (non-egg producing). GLMs were also performed to determine the factors affecting the size and egg production of progenetic metacercariae. Host species, host size, number of co-infecting Microphallus sp., number of co-infecting non-egg producing C. parvum metacercariae and/or presence of H. spinigera were included as factors in those GLMs. The size of progenetic metacercariae was also included as a factor when egg production was the response variable. In GLMs testing for the factors affecting C. parvum egg production, only amphipod hosts containing eggproducing C. parvum were included. Log-transformation before analyses was used when necessary to normalize the data. All factors were initially included in the GLMs to determine the most appropriate model using the Akaike Information Criterion (AIC), with the model awarded the lowest AIC having the highest explanatory power for the observed data. Only factors remaining in the best models are presented in the GLM result tables. In all models, effect size was determined using t-values and degrees of freedom. GLMs were performed using R Software 2.15.0 (R Development Core Team, 2011).

RESULTS

Host species, host size and parasite prevalences and abundances

Overall, 812 amphipods were measured and dissected, 219 P. excavatum and 593 P. fluviatilis. 1).

Host size was significantly different between the two amphipod species (Mann-Whitney U test, Z = 15•916, P < 0•0001); P. excavatum being larger than P. fluviatilis (Table 2). Amphipod size was also different between infected and uninfected individuals. In P. excavatum, infected individuals were significantly larger than their uninfected conspecifics for the three parasites species (Mann-Whitney U test, Z=-3•479, -2•42 and -3•046, P = 0•0005, 0•016 and 0•002 for C. parvum, Microphallus sp. and. H. spinigera, respectively; see Table 2 for details). The same trend was observed in P. fluviatilis with C. parvum and Microphallus sp. infected amphipods being significantly larger than uninfected individuals (Mann-Whitney U test, Z = -5•393 and -3•059, P < 0•0001 and P = 0•002, respectively; Table 2); note that H. spinigera was never found infecting P. fluviatilis. There was also a significant effect of host size on parasite abundances (number of parasites per individual host). Larger P. excavatum individuals tended to be more heavily infected than their smaller conspecifics (r = 0•338, 0•436 and 0•207, n = 219, P<0•0001, P < 0•0001 and P = 0•002 for C. parvum, Microphallus sp. and H. spinigera, respectively; Fig. 1). Again, similar trends were detected in P. fluviatilis; parasite abundances were significantly correlated to amphipod host size (r = 0•208 and 0•180, n = 593, both P <0•0001 for C. parvum and Microphallus sp., respectively; Fig. 1).

Coitocaecum parvum strategy, size and egg production

A total of 488 C. parvum metacercariae were recovered; 311 found in P. excavatum and 177 in P. fluviatilis. The proportion of progenetic metacercariae was not significantly different between the two host species (12•8% and 14•7% for P. excavatum and P. fluviatilis, respectively; Fisher's exact test, χ 2 = 0•32, P = 0•570). This is consistent with results from GLMs showing that only host size had a significant effect on C. parvum strategy (Table 3). Host species and the number of co-infecting nonprogenetic C. parvum were included in the model with the lowest AIC score although their effect size was small and not statistically significant (Table 3).

The presence of Microphallus sp. metacercariae had no detectable effect on C. parvum strategy.

Similarly, host species did not influence the size of progenetic metacercariae (Table 4). The only factor significantly influencing C. parvum size was the host size; larger amphipod hosts tending to harbour larger egg-producing C. parvum metacercariae, regardless of host species (r = 0•559, n = 57, P < 0•0001; Fig. 2). Again, host species had no effect on C. parvum egg production. The number of eggs produced by progenetic C. parvum was clearly influenced by the size of the metacercaria (Table 5); egg production increasing with C. parvum size, regardless of host species (r = 0•826, n = 57, P < 0•0001; Fig. 3). Intrahost interactions between parasite species seemed to play a major role on C. parvum egg production (Table 5). Furthermore, these effects are likely to be species-specific. Progenetic C. parvum egg production was clearly higher in the presence of Microphallus sp. (mean egg production ± S.E. = 306•9 ± 50•2 and 151•7 ± 38•4 with and without co-infecting Microphallus sp., respectively; Mann-Whitney U test, Z = -1•993, P = 0•046). However, there was no significant effect of the presence of nonprogenetic C. parvum (241•4 ± 50•4 and 219•9 ± 41•9 with and without co-infecting non-progenetic C. parvum, respectively; Mann-Whitney U test, Z = 0•176, P = 0•868), possibly because of a densitydependent effect, rather than just presence or absence (Table 5; Lagrue and Poulin, 2008a). Contrastingly, co-infection with H. spinigera significantly reduced C. parvum egg production in Table 1. Prevalences (proportion of infected amphipods) and abundances (mean number of parasites ± S.E. per individual amphipod) for the three species of parasites (Coitocaecum parvum, Microphallus sp. and Hedruris spinigera) in the two amphipod host species (Paracorophium excavatum and Paracalliope fluviatilis) sampled in Lake Waihola (When prevalence was 0, parasite abundance could not be calculated and is shown as not available (na).)

Host

•0 ± 0•1 4•2 ± 0•1 3•8 ± 0•1 4•1 ± 0•1 3•7 ± 0•1 4•6 ± 0•2 3•9 ± 0•1 P. fluviatilis 2•8 ± 0•0 3•1 ± 0•1 2•7 ± 0•0 2•9 ± 0•1 2•8 ± 0•

DISCUSSION

The main goal of the study was to explore potential factors influencing the frequency of progenesis in C. parvum metacercariae in their amphipod second intermediate hosts. In particular, we tested whether the parasite adjusted its life strategy to the species of amphipod host. Results show that host species per se did not influence C. parvum's strategy; the proportion of progenetic metacercariae was similar in P. excavatum and P. fluviatilis, the two amphipod host species. Furthermore, intermediate host species per se had no effect on progenetic C. parvum size or egg production. Overall, P. excavatum was more heavily infected than P. fluviatilis. Both prevalence and abundance of the three parasite species were higher in P. excavatum. Whether this difference was due to the larger size of this species compared to P. fluviatilis or different biological characteristics between the two amphipod species (benthic versus demersal and/or sedentary burrowing versus free-swimming, respectively) cannot be determined with certainty from our study. However, since larger individuals tend to be more heavily infected than smaller individuals in both amphipod species and P. excavatum is significantly larger than P. fluviatilis, it is likely that host size plays a significant role in parasite infection levels. Nevertheless, C. parvum cercariae cannot swim and instead crawl on the substrate to find a suitable intermediate host while H. spinigera is transmitted through the ingestion of the parasite eggs by amphipod hosts. It is thus possible that the benthic and detritivore life style of P. excavatum leads to higher exposure to infections in this species compared to P. fluviatilis. Information on Microphallus sp. transmission mode is lacking and interpretation of our results is difficult but similar trends are plausible.

Larger host size may also be linked to higher levels of within-host resources availability to parasites allowing for higher parasite abundance, growth and/or egg production [START_REF] Saad-Fares | Abundance/host size relationship in a fish trematode community[END_REF][START_REF] Grutter | Intraspecific and interspecific relationships between host size and the abundance of parasitic larval gnathiid isopods on coral reef fishes[END_REF][START_REF] Johnson | Correlated evolution of host and parasite body size: tests of Harrison's rule using birds and lice[END_REF]. Although host species per se did not affect the likelihood of C. parvum adopting progenesis, our results show that host size and thus possibly withinhost space/resources significantly affected parasite abundances and C. parvum strategy, size and egg production. The size of C. parvum progenetic metacercariae, and subsequently egg production, are known to be correlated with intermediate host size [START_REF] Lagrue | Life cycle abbreviation in the trematode Coitocaecum parvum: can parasites adjust to variable conditions?[END_REF]. Since P. excavatum is significantly larger than P. fluviatilis, C. parvum metacercariae adopting progenesis in P. excavatum are on average larger and produce more eggs, a result consistent with an effect of host resource availability on C. parvum size and egg production.

Alternatively, parasite infections may induce differential mortality rates in the two host species. Interspecificd i fferences in parasite prevalences and abundances between the two amphipod hosts may be explained by a higher parasite-induced mortality in P. fluviatilis following infection and/or parasite accumulation. Our results tend to show that differential mortality is unlikely since parasite abundances increased with host size, indicating that both species accumulate parasites as they grow with no apparent increased mortality [START_REF] Thomas | Differential mortality of two closely related host species induced by one parasite[END_REF][START_REF] Rousset | Inference of parasite-induced mortality from distributions of parasite loads[END_REF]. Larger, hence older, individual hosts thus contain higher parasite abundances, because of longer time for infections to accumulate [START_REF] Read | The ecology of genetically diverse infections[END_REF]. They may also contain older progenetic metacercariae that have had more time for growth and/or egg production. Several factors tend to indicate that host size rather than host age influences C. parvum growth and egg production. First, there is no seasonal variation in C. parvum prevalence, proportion of progenetic metacercariae or size/age distribution in amphipod hosts (Lagrue and Poulin, 2008b). Infection by C. parvum is most likely constant over time and host age/size; large/old amphipod hosts should not necessarily contain older metacercariae. Second, growth and egg production can be very rapid in C. parvum; individuals adopting progenesis can quadruple in size in 5 weeks, having produced over 20 eggs by that time and egg production then increasing exponentially with parasite size, unless limited by within-host resources (Lagrue andPoulin, 2007, 2009b). Coitocaecum parvum metacercariae of the same age also show a clear bimodal distribution in size, progenetic individuals being much larger than normal ones while individuals of intermediate sizes are rare (Lagrue and Poulin, 2009b). Size and/or egg production of C. parvum metacercariae is thus unlikely to depend solely on the size of individual parasites. Finally, for an equivalent size, female amphipods are older than males but C. parvum prevalence, proportion of progenetic metacercariae, as well as egg production, are similar in these hosts (Lagrue and Poulin, 2008a). Amphipod host size, rather than age, likely drives C. parvum strategy, size and egg production although host age may eventually have some influence on C. parvum strategy (Lagrue and Poulin, 2009b).

Coitocaecum parvum and Microphallus sp. were also found in higher prevalence and abundance in P. excavatum than in P. fluviatilis and the nematode H. spinigera was found to infect P. excavatum only. Intra-host competition is thus more common and probably more intense in P. excavatum. The presence of co-infecting parasites can have variable effects on C. parvum life strategy, size and/or egg production, possibly due to species-specific and abundance related intra-host competition effects. Intra-and interspecific parasite competition can apply to transmission routes and/or host resources (Lagrue and Poulin, 2008a). In P. fluviatilis, the presence of Microphallus sp. did not induce higher progenesis rates but clearly increased egg production in C. parvum. Microphallus sp. manipulates intermediate host behaviour to reach bird definitive hosts while C. parvum must infect a fish host or use progenesis to reproduce [START_REF] Hansen | Impact of a microphallid trematode on the behaviour and survival of its isopod intermediate host: phylogenetic inheritance[END_REF]Lagrue and Poulin, 2008a). Conflicts over transmission pathways may thus induce progenetic C. parvum to produce as many eggs as possible, eventually killing its host, before Microphallus sp. host manipulation succeeds. Our results suggest that this trend is similar and probably exacerbated since Microphallus sp. abundance, and hence host manipulation [START_REF] Brown | Field evidence for density-dependent effects in the trematode Microphallus papillorobustus in its manipulated host, Gammarus insensibilis[END_REF], is much higher in P. excavatum. In contrast, co-infection with H. spinigera or non-progenetic C. parvum, both needing to reach a fish definitive host, seemed to reduce C. parvum size and/or egg production. Since there is no conflict over transmission in these situations, host resource limitations most likely induce reductions in size and egg production in progenetic individuals (Lagrue and Poulin, 2008a). Although all parasites probably use similar host resources, results indicate that intra-host competition with H. spinigera induces the most drastic reduction in C. parvum egg production. This is not surprising since H. spinigera larvae are very large compared to progenetic C. parvum (around 8 times larger) and host size [START_REF] Luque | Larval Hysterothy-lacium sp. (Nematoda, Anisakidae) and trematode metacercariae from the amphipod Paracorophium excavatum (Corphiidae) in New Zealand[END_REF][START_REF] Luque | New evidence on a cold case: trophic transmission, distribution and host specificity in Hedruris spinigera (Nematoda: Hedruridae)[END_REF]. Hedruris spinigera likely uses more host resources than the proportionally smaller non-progenetic C. parvum or Microphallus sp. metacercariae (around 33% and 20% of progenetic C. parvum size, respectively; Lagrue and Poulin, 2008b), thus exerting stronger competition pressure on co-infecting progenetic C. parvum. Coitocaecum parvum's strategy, size and egg production seemed to be influenced by the presence of other parasites in species-and intensity-specific ways. Resource availability in general, be it within-host space or energy, is likely to have strong influences on the size and egg production of progenetic metacercariae.

Overall, the frequency of progenesis did not vary between amphipod host species. However, host size had a small but significant effect on the probability of metacercariae becoming progenetic, regardless of host species. Similarly, host species per se did not strongly influence egg production compared to host size, and thus possibly host age. Disentangling host size and host age effects on C. parvum strategy, growth and/or egg production could be achieved by experimentally infecting amphipod hosts of equivalent age but different size, either males and females of the same species or individuals of the two species used in our study. Alternatively, amphipods of similar size and thus contrasting age could be infected using the same methods [START_REF] Lagrue | Life cycle abbreviation in the trematode Coitocaecum parvum: can parasites adjust to variable conditions?[END_REF].

Within-host resource availability is probably an important factor influencing parasite life strategy, size and egg production and this is largely determined by host size. Since P. excavatum hosts are significantly larger than P. fluviatilis, apparent host species effects on C. parvum strategy, size and egg production are most likely due to interspecific size differences between intermediate host species. However, further experiments should look at the relative effects of within-host resource (i.e. energy, nutrients) and space (host size) limitations on C. parvum metacercariae. This may be tested by experimentally infecting amphipod hosts of similar sizes that are subsequently fed with diets of contrasting nutritive value, or hosts of contrasting sizes fed on an energetically equivalent diet.

In conclusion, results suggest that factors other than host species have more important effects on C. parvum's strategy. These include previously documented factors such as definitive host presence or time since infection, and factors tested herein like intermediate host size or intra-host interactions between co-infecting parasites. Overall, the plasticity offered by facultative progenesis may allow parasites to adjust their developmental strategy in a contextdependent way in response to an array of environmental variables (host size/resources, species-specific intra-host competition, transmission probability) but regardless of host species. Since progenesis acts as a reproductive insurance, allowing individuals to produce at least a few eggs, what maintains progenesis as a facultative strategy is not yet known and would deserve further investigation.

  0 na na P. excavatum (61•2 ± 19•1 and 367•2 ± 54•3 with and without co-infecting H. spinigera, respectively; Mann-Whitney U test, Z = 2•724, P = 0•004); P. fluviatilis was not considered here since it was never infected by H. spinigera.

Fig. 1 .

 1 Fig. 1. Abundances (mean ± S.E.) of Coitocaecum parvum (top graphs), Microphallus sp. (middle) and Hedruris spinigera (bottom graph) in relation to host size (amphipod length class) in Paracorophium excavatum (left graphs) and Paracalliope fluviatilis (right). Sample size (i.e. number of amphipods) per host size class is indicated above each data point on the top graphs. Note that P. fluviatilis was never infected by H. spinigera.

Table 4 .

 4 General Linear Model testing for the effects of host species (Paracorophium excavatum and Paracalliope fluviatilis), host size (total body length), number of co-infecting parasites (Microphallus sp. and/ or non-progenetic Coitocaecum parvum metacercariae) and presence of the nematode Hedruris spinigera on the size of progenetic C. parvum metacercariae (body length) (Results shown originate from the model awarded the lowest AIC (Akaike Information Criterion) score. Significant P values are indicated by *.)

Fig. 2 .

 2 Fig. 2. Relationship between amphipod host size (body length in mm) and the size of progenetic Coitocaecum parvum metacercariae (body length in mm) in the two amphipod species, Paracorophium excavatum and Paracalliope fluviatilis. Line of best fit and coefficient of determination are shown on the figure.

Fig. 3 .

 3 Fig. 3. Relationship between the size of progenetic Coitocaecum parvum metacercariae (body length in mm) and egg production in the two amphipod host species, Paracorophium excavatum and Paracalliope fluviatilis. Line of best fit and coefficient of determination are shown on the figure.

  Parasite prevalences were significantly higher in P. excavatum than P. fluviatilis (Fisher's exact tests, χ 2 = 57•64, 47•65 and 224•54, all P < 0•0001 for C. parvum, H. spinigera and Microphallus sp., respectively; see Table 1 for details). Similarly, parasite abundances were significantly higher in P. excavatum than P. fluviatilis (Mann-Whitney U test, Z = 8•284, 7•056 and 17•396, all P < 0•0001 for C. parvum, Microphallus sp. and H. spinigera respectively; Table

Table 2 .

 2 Host size (mean amphipod body length in mm ± S.E.) for the two amphipod species (Paracorophium excavatum and Paracalliope fluviatilis) sampled in Lake Waihola according to their infection status (infected or uninfected (i.e. uninf.)) (Data are shown for the three species of parasites, Coitocaecum parvum, Microphallus sp. and Hedruris spinigera. When parasite prevalence was 0, mean infected host size is shown as not available (na).)

		P. fluviatilis			P. excavatum
	Parasite	Prevalence (%)	Abundance (± S.E.)	Prevalence (%)	Abundance (± S.E.)
	C. parvum	18•2	0•30 ± 0•09	44•3		1•42 ± 0•25
	Microphallus sp.	22•8	0•46 ± 0•14	80•4		8•66 ± 0•96
	H. spinigera	0•0	na		8•2		0•08 ± 0•06
	Parasite		C. parvum		Microphallus sp.	H. spinigera
	Host size (mm ± S.E.)	Total	Infected	Uninf.	Infected	Uninf.	Infected	Uninf.
	P. excavatum	4

Table 3 .

 3 General Linear Model testing for the effects of host species (Paracorophium excavatum and Paracalliope fluviatilis), host size (total body length), number of co-infecting parasites (Microphallus sp. and/ or non-progenetic Coitocaecum parvum metacercariae) and presence of the nematode Hedruris spinigera on C. parvum life strategy (occurrence of progenetic individuals) in amphipod hosts (Results shown originate from the model awarded the lowest AIC (Akaike Information Criterion) score. Significant P values are indicated by *.)

	Factors	Estimate	S.E.	t-value	P	Effect size
	Host species	0•562	0•404	1•390	0•165	0•049
	Host size	1•348	0•217	6•221	< 0•0001*	0•213
	Non-progenetic C. parvum	0•093	0•067	1•379	0•168	0•048

Table 5 .

 5 General Linear Model testing for the effects of host species (Paracorophium excavatum and Paracalliope fluviatilis), host size (total body length), number of co-infecting parasites (Microphallus sp. and/ or non-progenetic Coitocaecum parvum metacercariae), presence of the nematode Hedruris spinigera and C. parvum size (body length) on the number of eggs produced by progenetic metacercariae in amphipod hosts (Results shown originate from the model awarded the lowest AIC (Akaike Information Criterion) score. Significant P values are indicated by *.)

	Factors	Estimate	S.E.

Do parasites adopt different strategies in different hosts?

R. Ruiz Daniels and others

ACKNOWLEDGEMENTS

This project was funded by an Erasmus Mundus Program 'European Master in Applied Ecology' (EMAE) grant to R.R.D. The authors would also like to thank Richard Sydney Taylor (University of Bristol) for his help during the process and Mathieu Sicard (University of Poitiers) for his administrative assistance. We thank an anonymous reviewer for constructive comments on a previous version of the manuscript.


Christian Beauger
email: christian.beauger@mines-paristech.fr

Guillaume Lainé

Alain Burr

Aurélie Taguet

Belkacem Otazaghine

Arnaud Rigacci

Nafion®-sepiolite composite membranes for improved Proton Exchange Membrane Fuel Cell performance

Keywords: Sepiolite, Nafion, membrane, low humidity, PEMFC 1

come

Nafion®-sepiolite composite membranes for improved proton exchange membrane fuel cell performance

Introduction

Due to their wide range of possible applications (transport, mobile or stationary), Proton Exchange Membrane Fuel Cells (PEMFC) are amongst the most studied fuel cells (FC). Nevertheless, despite numerous advanced research studies, they still suffer from limitations. One of them concerns the electrolytic membrane employed so far which impedes the fuel cell to operate at high temperature. Different strategies have been followed to overcome the limitation of Nafion®, the perfluorinated ionomer widely chosen as the standard for PEMFC [START_REF] Bonnet | Hybrid organic-inorganic membranes for a medium temperature fuel cell[END_REF][START_REF] Jones | Advances in the Development of Inorganic-Organic Membranes for Fuel Cell Applications[END_REF][START_REF] Li | Approaches and recent development of polymer electrolyte membranes for fuel cells operating above 100 degrees[END_REF]. Non-fluorinated polymers such as PEEK [START_REF] Bae | Properties of selected sulfonated polymers as proton-conducting electrolytes for polymer electrolyte fuel cells[END_REF][START_REF] Gosalawit | Sulfonated montmorillonite/sulfonated poly(ether ether ketone) (SMMT/SPEEK) nanocomposite membrane for direct methanol fuel cells (DMFCs)[END_REF][START_REF] Kreuer | On the development of proton conducting polymer membranes for hydrogen and methanol fuel cells[END_REF], PSU [START_REF] Kerres | Development and characterization of crosslinked ionomer membranes based upon sulfinated and sulfonated PSU -Crosslinked PSU blend membranes by alkylation of sulfinate groups with dihalogenoalkanes[END_REF][START_REF] Lufrano | Sulfonated polysulfone ionomer membranes for fuel cells[END_REF] or PBI [START_REF] Staiti | Membranes based on phosphotungstic acid and polybenzimidazole for fuel cell application[END_REF][START_REF] Lee | Water-stable crosslinked sulfonated polyimide-silica nanocomposite containing interpenetrating polymer network[END_REF][START_REF] Peron | Solution sulfonation of a novel polybenzimidazole. A proton electrolyte for fuel cell application[END_REF][START_REF] Jones | Recent advances in the functionalisation of polybenzimidazole and polyetherketone for fuel cell applications[END_REF] and their sulfonated counterparts have been extensively studied as alternatives. Another strategy consists in incorporating inorganic fillers such as metal oxides [START_REF] Watanabe | Self-humidifying polymer electrolyte membranes for fuel cells[END_REF][START_REF] Antonucci | Investigation of a direct methanol fuel cell based on a composite Nafion (R)-silica electrolyte for high temperature operation[END_REF][START_REF] Adjemian | Silicon oxide Nafion composite membranes for proton-exchange membrane fuel cell operation at 80-140 degrees C[END_REF][START_REF] Aricò | Comparison of ethanol and methanol oxidation in a liquid-feed solid polymer electrolyte fuel cell at high temperature[END_REF][START_REF] Sacca | Nafion-TiO 2 hybrid membranes for medium temperature polymer electrolyte fuel cells (PEFCs)[END_REF]18,[START_REF] Zoppi | Hybrids of perfluorosulfonic acid ionomer and silicon oxide by sol-gel reaction from solution: Morphology and thermal analysis[END_REF][START_REF] Mauritz | Organic-inorganic hybrid materials: perfluorinated ionomers as sol-gel polymerization templates for inorganic alkoxides[END_REF][START_REF] Miyake | Evaluation of a sol-gel derived Nafion/silica hybrid membrane for polymer electrolyte membrane fuel cell applications -II. Methanol uptake and methanol permeability[END_REF], acids [START_REF] Peled | A novel proton-conducting membrane[END_REF][START_REF] Staiti | Hybrid Nafion-silica membranes doped with heteropolyacids for application in direct methanol fuel cells[END_REF][START_REF] Shao | Hybrid Nafion-inorganic oxides membrane doped with heteropolyacids for high temperature operation of proton exchange membrane fuel cell[END_REF][START_REF] Helen | Synthesis and characterization of composite membranes based on alpha-zirconium phosphate and silicotungstic acid[END_REF][START_REF] Shanmugam | Synthesis and characterization of silicotungstic acid based organic-inorganic nanocomposite membrane[END_REF], phosphates or phosphonates [START_REF] Alberti | Inorgano-organic proton conducting membranes for fuel cells and sensors at medium temperatures[END_REF][START_REF] Alberti | Novel Nafion-zirconium phosphate nanocomposite membranes with enhanced stability of proton conductivity at medium temperature and high relative humidity[END_REF][START_REF] Costamagna | Nafion (R) 115/zirconium phosphate composite membranes for operation of PEMFCs above 100 degrees C[END_REF] into the Nafion® matrix to make the composite or hybrid membrane more mechanically stable, less permeable to reactant and more hygroscopic. Different clays, montmorillonite [START_REF] Mura | Study on the conductivity of recast Nafion (R)/montmorillonite and Nafion (R)/TiO 2 composite membranes[END_REF][START_REF] Lin | A novel composite membranes based on sulfonated montmorillonite modified Nafion® for DMFCs[END_REF][START_REF] Jung | Preparation and performance of a Nafion (R)/montmorillonite nanocomposite membrane for direct methanol fuel cell[END_REF] or laponite [START_REF] Bebin | Nafion (R)/clay-SO 3 H membrane for proton exchange membrane fuel cell application[END_REF] for instance, have also been evaluated as possible fillers. Palygorskite and sepiolite are fibrous clays known to be very hygroscopic and whose acicular morphology may help to make composite membranes more mechanically resistant. Palygorskite has already been used as promising filler in Nafion® [START_REF] Xu | Mineral nanofibre reinforced composite polymer electrolyte membranes with enhanced water retention capability in PEM fuel cells[END_REF]. In comparison with pristine Nafion® membrane, the composite realized with Nafion® and palygorskite has shown several advantages such as a higher water uptake, both improved mechanical properties and proton conductivities at 0 %RH. However the composite membrane was not tested in a membrane-electrode assembly.

Similarly to palygorskite, the structure of sepiolite is highly porous. This hydrated magnesium silicate (Si 12 Mg 8 O 30 (OH) 4 (H 2 O) 4 , 8H 2 O) is based on SiO 4 tetrahedra layers, with an inversion of the apical ends every six units (Figure 1). These layers are interconnected by MgO 6 octahedra, thus creating nanochannels of 3.5x10.6 Ų in cross-section [START_REF] Tartaglione | Thermal and morphological characterisation of organically modified sepiolite[END_REF]. Two types of water molecules are present in the structure: water coordinated to Mg 2+ ions at the edges of the octahedral layers (H 2 O coord. ) and zeolithic water in the channels (H 2 O zeol. ), hydrogen-bonded to coordinated water molecules. [START_REF] Chivrac | Starch nano-biocomposites based on needle-like sepiolite clays[END_REF] with permission from Elsevier.

Sepiolite, seldomly used as fillers in proton exchange membranes [START_REF] Fernandez-Carretero | Hybrid ion-exchange membranes for fuel cells and separation processes[END_REF][START_REF] Fernandez-Carretero | Preparation and Characterization of Hybrid Membranes based on Nafion (R) using Partially Sulfonated Inorganic Fillers[END_REF][START_REF] Fernandez-Carretero | PEMFC Performance of MEAS Based on Nafion (R) AND sPSEBS Hybrid Membranes[END_REF], was chosen to improve the performance of Nafion®. Different composites membranes have been prepared, characterized and used to form Membrane Electrodes Assemblies (MEA). These MEA were then tested in single cell under PEMFC specific operating conditions in order to clearly show the advantage of sepiolite for high temperature and low relative humidity. The crossinfluences of the relative humidity of the feeding gases and the operating temperature of the cell were evaluated on the MEA behaviour.

Experimental

Cast Membranes preparation

The membranes were prepared by evaporation of the solvent from a Nafion® dispersion. Starting from a 20 wt% dispersion (ION POWER, DE2021), a 5 wt% dispersion was prepared, using 1-propanol (Fluka 98%), and used to realize both pure and composite membranes. In the case of composites, the sepiolite (Tolsa PangelS9) was added to the dispersion and dispersed with ultrasounds (Bandelin UW2200, 70 W for 1 min, 3 times). The 13x13 cm² mold containing the dispersion was first heated at 80 °C for 2 h and then at 120 °C for 1 h. The obtained membrane was recovered by immersion of the glass support in deionised water. It was finally washed and protonated as follows : 1 h in boiling 0.5 M nitric acid (HNO 3 Riedel-de Haën 69%), 1 h in boiling 5% hydrogen peroxide (H 2 O 2 , Fluka 30%) and 1 h in boiling 0.5 M sulphuric acid (H 2 SO 4 , Aldrich 95-98%). Before being characterized and tested each membrane was stored in deionised water. Two membranes of each composition were elaborated, one for characterization, the other one for single-cell test.

Sepiolite sulfonation and characterization

Due to the discontinuity of the layers, numerous OH groups are available for condensation reaction. Tartaglione et al. [START_REF] Tartaglione | PP and PBT composites filled with sepiolite: Morphology and thermal behaviour[END_REF] have reported that the specific number of OH groups equals 2.2 per 100 Ų. Based on the specific surface area provided by Tolsa for sepiolite Pangel S9 (S BET = 320 m²/g), the maximum number of OH groups per gram of sepiolite can be estimated to 1.2 mmol/g. The sulfonation of sepiolite was realized following two protocols. The first one (#1) was described previously by Fernandez-Carretero et al. [START_REF] Fernandez-Carretero | Hybrid ion-exchange membranes for fuel cells and separation processes[END_REF]. In this protocol, the first step consisted in grafting phenyl-silane groups on sepiolite through condensation reactions between the hydroxyl groups present at the surface of the sepiolite and triethoxyphenylsilane. Hydrochloric acid (3 ml) and isopropanol (17 ml) were added to sepiolite (1 g) under magnetic stirring. Triethoxyphenylsilane (0.6 g) was added after ultrasonic homogenization (Bandelin UW2200, MS73 microtip, 70 W, 90 s). The mixture was heated 15 h at 65 °C, filtered and washed successively with methanol (50 ml), methanol and water (50 ml 50/50) and water (50 ml). The modified sepiolite was finally dried at 90 °C for 2 h and ground in an agate mortar. The second step consisted in the sulfonation of the phenyl group. The modified sepiolite was treated with fuming sulfuric acid (50 g) under magnetic stirring at 40 °C for 24 h, filtered and washed twice with water at room temperature and then washed three times with water at 50 °C. The so-obtained modified sepiolite (sepiolite#1) was finally dried again at 90 °C for 2 h and grounded in an agate mortar. The second protocol (#2) is based on a new procedure developed in our laboratory. The first step of this procedure consisted in the functionalization of fillers by an epoxy group. 10 g of sepiolite, 1 g (4.2×10 -3 mol) of (3-glycidyloxypropyl)trimethoxysilane and 100 ml of an ethanol/water (90/10) solution were introduced in a 250 ml flask fitted with a condenser. The mixture was then stirred and heated at solvent reflux for 15 hours. The mixture was next centrifuged (speed: 400 rpm) to eliminate the liquid phase and washed three times with acetone. The epoxy modified sepiolite was dried under vacuum. The second step consisted in the introduction of the -SO 3 Na group on the epoxy functionalized sepiolite. 10 g of previously epoxy modified sepiolite, 1 g (5.1×10 -3 mol) of sodium 4-aminobenzenesulfonate and 80 ml of methanol were introduced in a 250 ml flask fitted with a condenser. The mixture was then stirred and heated at methanol reflux for 15 hours. The mixture was next centrifuged (speed: 400 rpm) to eliminate the liquid phase. The treated filler was washed two times with deionised water to remove non reacted sodium 4aminobenzenesulfonate and two times with acetone. The -SO 3 Na functionalized sepiolite was dried under vacuum. The last step was the modification of the -SO 3 Na functionalized sepiolite into a -SO 3 H functionalized sepiolite. 5 g of a -SO 3 Na functionalized sepiolite and 200 ml of deionised water were introduced in a 500 ml flask. The mixture was stirred and the pH was adjusted to 3 with a 0.01 M H 2 SO4 solution. The mixture was centrifuged (speed: 400 rpm) to isolate the treated filler and then washed three times with deionised water and two times with acetone. The -SO 3 H functionalized fillers (sepiolite#2) was dried under vacuum. Both modified sepiolite obtained from protocol 1and 2 are represented on Figure 2. The functionalization has been checked by thermogravimetric analysis (TGA), infrared spectroscopy, pyrolysis coupled to Gas Chromatography and Mass Spectroscopy (Py-GC/MS) and finally titration. The modification protocol#2 was validated on silica nanoparticles (SIDISTAR® from Elkem).

Thermal characterization was carried out by thermogravimetric analysis (Perkin Elmer Pyris-1 instrument) on 10 mg of samples, under nitrogen. Samples were first heated at 10 °C/min from 25 to 110 °C, followed by an isotherm at 110°C for 10 min, in order to evacuate all adsorbed water molecules. They were then heated again from 110 to 900°C, at 10 °C/min, in order to eliminate grafted groups.

Infrared spectroscopy was performed on a Bruker tensor 27 in ATR mode.

The Py-GC/MS analytical setup consisted of an oven pyrolyzer connected to a GC/MS system. A Pyroprobe 5000 pyrolyser (CDS Analytical) was used to flash pyrolyze the samples in a helium environment. This pyrolyzer is supplied with an electrically heating platinum filament. One coil probe enables the pyrolysis of samples (less than one mg) placed in quartz tube between two pieces of rockwool. The sample was heated at 900 °C for 15 s then the gases were drawn to the gas chromatograph for 5 min. The pyrolysis interface was coupled to a 450-GC gas chromatograph (Varian) by means of a transfer line heated at 270 °C. In this oven the initial temperature of 70 °C was held for 0.2 min, and then raised to 250 °C at 10 °C/min. The column is a Varian Vf-5ms capillary column (30 m × 0.25 mm) and helium (1 ml/min) was used as the carrier gas, a split ratio was set to 1:50. The gases were introduced from the GC transfer line to the ion trap analyzer of the 240-MS mass spectrometer (Varian) through the direct-coupled capillary column. Identification of the products was achieved comparing the observed mass spectra to those of the NIST mass spectral library. Titration has been carried out following a standard protocol. The fully protonated sepiolite was immersed in 50 ml of 0.001 M NaOH (solution prepared from Acros organics pellets) and 0.1 M NaCl (Sigma Aldrich S3014) in order for the Na + ions to replace the H + of the sulfonated groups, which thus make the solution pH increased. The remaining HO -ions were then titrated with 0.001 M HCl (prepared from 1M HCl Fluka 84425), thus allowing to calculate the number of protons exchanged. The IEC is expressed as the number of millimoles of protons exchanged per gram of dry sepiolite (meq/g).

Membrane Electrodes Assembly

In order to be tested in fuel cell operating conditions, the Nafion®/sepiolite composite membranes were integrated in a Membrane Electrodes Assembly (MEA). To do so, the membrane was hot pressed between two electrodes (Paxitech, 50 cm², 0.6 mgPt/cm²) according to the following procedure: 2 metric tons (i.e. 40 kg/cm²) at 100 °C for 15 min and 10 tons (i.e. 200 kg/cm²) at 120 °C for 10 min, free cooling at 10 metric tons (press CARVER model 3850).

Membranes characterizations

Water uptake

The water uptake (W ut ) was measured from the difference of the weight of a membrane stored in water at room temperature W w , and that of the same membrane dried out at 80 °C for 15 h, W d , according to the following formula, W ut = (W w -W d ) /W d ×100.

Swelling

In the presence of water, membranes tend to swell, leading to mechanical constraints in the fuel cell, all the more important as more MEAs are stacked. The swelling (S i ) was measured as the percentage of size increase in the direction i of the membrane (i=1 for side 1, 2 for side 2 and th for the thickness). These percentages were calculated by difference between the size measured after equilibration of the membrane in water at room temperature (RT) on one hand and boiling water (BT) on the other hand. Hence, S i the swelling percentage in the direction i was calculated as followed: S i =100×[(x i ) BT -(x i ) RT ] / (x i ) RT , where (x i ) BT is the length (in cm) of the membrane in the direction i in boiling water and (x i ) RT is the length (in cm) of the membrane in the direction i in water at room temperature. For the thickness swelling, we have S th = 100× (th BT -th RT )/th RT , th being the thickness measured in µm.

Ion Exchange Capacity (IEC)

The IEC was measured by titration according to the procedure described in 2.2. The fully protonated membrane was immersed in 0.1 M NaOH (20 ml solution prepared from Acros organics pellets) The remaining HO -ions were titrated with 0.01 M HCl (prepared from 1M HCl Fluka 84425).

Microscopies (SEM)

Microscopic observations were realized on FEI XL30 ESEM equipped with a qualitative chemical analysis probe (EDS) to evaluate the sepiolite dispersion within the Nafion® matrix.

Mechanical tests

Mechanical tests were performed in order to control the influence of the fillers on the mechanical resistance of the membrane, which is closely related to its durability in FC operating conditions. Dynamic Mechanical Analysis, performed on a TRITEC 2000 in tensile mode, allowed to determine the Elastic modulus (E). The measurements were realised at 1Hz and at a heating rate of 2 °C/min between 25 °C and 250 °C.

MEA characterizations

MEAs were tested on our Fuel Cell test bench, controlled by a Biologic potentiostat HCP 803. This Lab equipment allows testing 50 cm² active surface area MEA up to 100 °C, at different stoechiometry and various relative humidity. The polarization curves (U=f(j)) were performed at 75 and 100 °C and between 25 and 100% relative humidity (RH) as described in an earlier publication [START_REF] Ouattara-Brigaudet | Carbon Aerogels as Catalyst Supports and First Insights on Their Durability in Proton Exchange Membrane Fuel Cells[END_REF]. These tests also allowed having access to the MEA resistance and the membrane hydrogen crossover.

First of all, the MEA needed to be conditioned to reach their optimum performance. Generally speaking, conditioning tests consist in applying specific operating cycles to the MEA, controlling either the voltage or the intensity. In the context of this study, we chose to apply the following cycle:  3 round trips at 5 mV/s, from the Open Circuit Voltage (OCV) down to 0.3 V  One step at 0.5 V from OCV (5 mV/s), during 1.5 h (no significative evolution of the current afterwards)

Results and discussion

Sepiolite characterization

In order to check to what extent sepiolite has been modified, pristine sepiolite, sepiolite#1 and #2 have been characterized by TGA, IR spectroscopy, Py-GC/MS and titration.

As detailed by several authors [START_REF] Tartaglione | Thermal and morphological characterisation of organically modified sepiolite[END_REF][START_REF] Kuang | Nanostructured hybrid materials formed by sequestration of pyridine molecules in the tunnels of sepiolite[END_REF], the heating of pristine sepiolite reveals a multi-step dehydration process, corresponding to the loss of zeolitic water first and then of coordinated water. The zeolitic water is lost in one step, between room temperature and 100 °C, while the coordinated water is lost in two steps, between 100 and 300 °C first and then between 300 and 600 °C. Another step is then observed, from 800°C, corresponding to the dehydroxylation of sepiolite anhydride which loses its structure, resulting in the formation of enstatite and silica.

Thermal characterization of pristine sepiolite and both sulfonated sepiolites (8.25 mg for sepiolite#1 and 11.7 mg for sepiolite#2) was carried out by TGA, following the protocol described in the experimental section. The isotherm step at 110 °C was used to dry samples so as to avoid any disturbance of the results by the weight loss due to the zeolitic water (Figure 3.a). During this step, pristine sepiolite undergoes an 8.4 % weight loss (zeolitic water loss). In the case of organo-modified sepiolites, following protocol 1 and 2, the weight loss is lower compared to pristine sepiolite: 7.2 % and 3.8 %, respectively.

This can be explained by (i) a lower amount of zeolitic water in the organo-modified sepiolite and/or (ii) a higher molecular weight of modified sepiolite due to the presence of organic molecules. Considering that all the samples are in the same hydration state after the first step (loss of the zeolitic water), the comparison of their behavior between 110 °C and 900 °C (Figure 3.b) allows checking to what extent the sepiolites have been modified.

The first two steps observed, between 110 °C and 800 °C on Figure 3.b, for pristine sepiolite (straight line) corresponds to a weight loss of 7.5 wt%. They can be attributed to the loss of coordinated water (dehydration). Then, after 800 °C, dehydroxylation occurs.

Between 110 °C and 800 °C, the modified sepiolites #1 and #2 show significantly different behaviors from that of pristine sepiolite but also one from the other. Their behavior is however similar to that of pristine sepiolite above 800 °C, probably corresponding also only to dehydroxylation.

Between 110 and 800 °C the total weight loss of sepiolites #1 and #2 is roughly 11 wt%, i.e. 3.5 wt% more than that of pristine sepiolite. This is a first indication that the sepiolites have been modified. Such a weight loss increase corresponds to 0.3 mg for sepiolite#1 and 0.4 mg for sepiolite#2, ie 0.2 mmol of functional groups per gram of sepiolite#1 and 0.1 mmol of functional groups per gram of sepiolite#2. The calculation was performed assuming that only the silane function of the grafted molecule remains on the mineral filler and that all the organic part was decomposed without char formation.

In order to clarify whether the precursors used are grafted at the surface of sepiolite or trapped in its porosity, both modified sepiolites have been analysed by FTIR spectroscopy and their spectra compared to that of pristine sepiolite (Figure 4). On the spectrum of pristine sepiolite (Figure 4.a) OH stretching vibrations from Mg 3 OH classically appear in the 3600 cm -1 region. The bands at 980, 1010 and 1209 cm -1 are ascribed to Si-O bonds within silica tetrahedra [START_REF] Alkan | FTIR and zeta potential measurements of sepiolite treated with some organosilanes[END_REF]. Their shift in both modified sepiolites is an indication of an interaction between the grafted group and sepiolite. The band around 1650 cm -1 is attributed to water molecules hydrogen bonded to the surface. Finally the bands appearing at 786, 688 and 644 cm -1 are also attributed to OH vibrations from Mg 3 OH (deformation at 786 cm -1 and bending at 688 and 644 cm -1 ) [START_REF] Wan | Synthesis and characterization of biomimetic hydroxyapatite/sepiolite nanocomposites[END_REF].

Sepiolite#1 shows an additional infrared absorption band around 1080 cm -1 (Figure 4.c), which could be attributed to the new Si-O bond coming from the grafted phenyl silane. It may also result from the formation of silica. The bands at 738 cm -1 observed for the precursor is also present in sepiolite#1, at 743 cm -1 and may be ascribed to the Si-phenyl bond. In the same time, the absorption bands corresponding to the C-H vibrations of the ethoxy groups, at 2887, 2927 and 2975 cm -1 , disappeared in the spectrum of sepiolite#1. Since these ethoxy groups from the precursor are expected to be eliminated during the condensation reaction, and considering the weight losses observed in TGA, we concluded that the phenylsilane has been grafted on sepiolite. Concerning sepiolite#2, the FTIR spectrum shows also an additional absorption band around 1080 cm -1 , similar to that observed for sepiolite#1 but weaker. This is probably due to a smaller functionalization, consistent with TGA results. As for sepiolite#1, there is no absorption band in the region of methoxy C-H vibrations, showing that the precursor, here (3glycidyloxypropyl)trimethoxysilane has reacted with sepiolite OH groups.

In both cases, the absence of C-H vibration bands in the region [2900-3000 cm -1 ] is a clear indication that the modifications observed cannot come from trapped precursor molecules.

Py-GC/MS analysis has been carried out in order to confirm again the expected surface modifications.

The Py-GC/MS analysis shows for both protocols the release of organic molecules resulting from the decomposition of groups expected to be grafted on sepiolite (Figure 5). As expected, the pyrolysis at 900°C of the sepiolite sulfonated with the protocol 1 shows mainly a release of benzene which is a clear signature of the group grafted on sepiolite#1.

The chromatogram obtained for the sepiolite 2 shows large differences in comparison to sepiolite #1. The pyrolysis of this modified sepiolite gives a greater variety of molecules. Most of them contain aromatic rings which are formed during the degradation process at high temperature. The presence of nitrogen in some of them is a good indication of a proper functionalization.

Since i) FTIR signal is quite weak for sepiolite#2, probably due to a limited functionalization, and ii) both TGA and Py-GC/MS results could also result from precursor molecules trapped in the sepiolite channels, the procedure of surface modification with protocol#2 has been validated with spherical silica nanoparticles (SIDISTAR® from Elkem).

The chromatogram obtained for silica#2, pyrolised at 900 °C, is very similar to that of sepiolite #2, which proves the formation of the same products during the pyrolysis step of the analysis. Since the precursor could not be trapped in any silica porosity, the detected organic fragments do result from the degradation of groups grafted at the surface of silica. The grafting protocol is thus validated on silica and we can reasonably assume that it is also efficient for sepiolite.

The degree of sulfonation has finally been checked by titration for each modified sepiolite.

The results are reported in table 1, along with the data obtained from TGA analysis.

* concentration expressed in mmol/g to be compared to the IEC of the membranes

The concentration of -SO 3 H groups obtained for both modified sepiolites are consistent with those obtained with TGA, validating again the two grafting protocols.

The characterisations performed on both modified sepiolites allowed to demonstrate that the grafting protocols were successful. They also revealed that sepiolite#1 is more sulfonated than sepiolite#2 with -SO 3 H concentration respectively of 0.2 mmol/g and 0.1 mmol/g.

Membrane preparation

Membranes were prepared with a targeted thickness of 50 µm. Pure Nafion® membranes from the ION POWER dispersion, M112, and commercial N112 from Dupont, were considered as our reference to study the influence of the sepiolite incorporation in the composites. The pure Nafion® membrane labelled M112 was compared to the commercial N112 membrane to evaluate the influence of our protocol of membrane preparation. Composite membranes were also prepared with pristine and sulfonated sepiolites (according to both protocols 1 and 2). Table 2 details the different membranes realized for this study.

Membrane characterization

Our first concern was to evaluate the dispersion state of the sepiolite in the membrane. The micrograph of composite membrane prepared with pristine sepiolite does not show any visible sepiolite segregation within the thickness of the membrane (Figure 6.b). Despite the use of a microtip to prepare the M112SxS series (sulfonation protocol 1), segregation has clearly been observed with the sulfonated sepiolite (Figure 6.c). On the contrary, the sulfonation protocol 2 developed for this study allowed avoiding such a segregation (M112SxSH series, Figure 6,d,). These observations are confirmed with the qualitative chemical analysis (EDS) performed across the thickness of the membranes. Silicon, for sepiolite, and fluorine, for Nafion®, have been probed and the Si/F atomic ratio was calculated in order to check the sepiolite distribution within the membrane thickness (Table 3). The Si/F atomic ratio is roughly constant across both M112S10 and M112S10SH membranes (points 1 to 4), thus confirming the good dispersion of fillers within the polymer matrix. On the contrary, the Si/F atomic ratio is highly inhomogeneous for M112S10S membrane, in agreement with SEM observation showing a strong segregation. The same phenomenon was observed with M112S20S (20 wt% of sepiolite).

The dispersion state of sepiolite has a direct influence on the mechanical resistance of the membrane. This has been checked by mechanical characterization. It was assumed that the introduction of sepiolite could improve the mechanical resistance of the composite and thus its durability in FC operating conditions. The main results of the Dynamics Mechanical Analysis performed on our membranes are reported in Figure 7. The elastic modulus (E) is representative of the membrane tensile stiffness. M112 is significantly less stiff than N112, due to its elaboration process. In composites prepared with pristine sepiolite (M112Sx), the higher the sepiolite content the higher the stiffness. This confirms our assumption on the role of sepiolite in the composite, in good agreement with reported data on composite polymers [START_REF] Fernandez-Carretero | Hybrid ion-exchange membranes for fuel cells and separation processes[END_REF]. This result is also consistent with the rather good dispersion of the sepiolite within the Nafion® matrix. On the contrary, the poor dispersion of the sulfonated sepiolite in the M112SxS series (sulfonation protocol #1) results in mechanical properties which remain roughly similar to that of pure Nafion®. As already mentioned, the second sulfonation protocol (#2), developed for this study, allowed to homogeneously disperse the modified sepiolite within the Nafion® matrix. As a result, the elastic modulus of the M112SxSH series is increasing with sepiolite content thus confirming the better dispersion of sepiolite observed in SEM.

As expected the reinforcing effect has been observed only with a satisfying dispersion of fillers within the matrix. When this condition is satisfied, the reinforcing occurs from 10 wt% of sepiolite in the composite.

The table 4 summarizes characterization results of the different membranes described in this study. First of all, the thicknesses of the membranes are quite close to the target value, considering that the commercially available N112 is already 10% thicker than expected. During the membrane preparation, the amount of Nafion® dispersion has been adjusted for the M112SxSH series in order to reach the targeted 50 µm thickness.

It is noteworthy that all the features of our pure Nafion® membrane (M112), are slightly better than those of N112. In agreement with some reported data [START_REF] Dimitrova | Modified Nafion®-based membranes for use in direct methanol fuel cells[END_REF][START_REF] Shao | Preparation and characterization of hybrid Nafionsilica membrane doped with phosphotungstic acid for high temperature operation of proton exchange membrane fuel cells[END_REF][START_REF] Dimitrova | Transport properties of ionomer composite membranes for direct methanol fuel cells[END_REF], the IEC and the water uptake are higher for M112 compared to N112, while the swelling is lower whatever the dimension of the membrane. This is probably due to differences in elaboration routes, the commercial N112 being prepared by extrusion.

The introduction of sepiolite in Nafion® reinforces mechanically our composites membranes. Another expected impact is the increase of the hygroscopic nature of the composites which will help the proton conduction. This should even be emphasized thanks to sepiolite functionalization.

As expected, the water uptake continuously increases with the sepiolite content in the composites. The same trend has been observed for the membranes swelling, which is, for all compositions, larger in the thickness dimension. The increase of water uptake due to the introduction of sepiolite does not depend on the type of sepiolite. The water uptake is indeed very similar for all the composite membranes prepared with the same amount of sepiolite, regardless the type of sepiolite, modified or not. This means that the modification of sepiolite does not alter its ability to retain water in the composite. The gain in water uptake corresponds actually to roughly three times the amount of sepiolite (expressed in wt%) added in the membrane. For instance, an addition of 10 wt% of sepiolite leads to a 30 % increase of water uptake.

The IEC of composite membranes prepared with pristine sepiolite is decreasing with an increasing percentage of sepiolite. This effect is mainly due to a dilution effect, since the sepiolite is not proton conductive. The sulfonation of sepiolite, whatever the protocol, allowed increasing the IEC of composites membranes prepared with sepiolite#1 or #2. The IEC of the M112SxS series is notably higher than that of the M112SxSH one, probably because of the higher degree of sulfonation resulting from protocol#1.

Both the water uptake and the IEC increase, respectively due to the introduction of sepiolite and its sulfonation, are expected to improve the performances of MEAs prepared with composite membranes.

MEA single cell tests

The different membranes were used to realize MEAs which were then tested at 75 °C and 100 °C and between 25 % and 100 % RH. The different polarization curves are shown on Figures 8 to 12.

N112 was first tested to get some reference data. It is clear from Figure 8 that at 75 °C (solid lines) the performances decrease slightly with relative humidity whereas at 100 °C (dashed lines) the drop is much more significant. Indeed, at 75 % RH the performances are very similar at 75 °C and 100 °C. On the contrary, at 50 % RH a noticeable drop is observed at 100 °C compared to 75 °C. So, for N112, at low relative humidity, the performances are much lower at 100 °C than at 75 °C. This is consistent with different data reported for PFSA membranes in the literature, on the evolution, with temperature and relative humidity, of the proton conductivity [START_REF] Doyle | High-temperature proton conducting membranes based on perfluorinated ionomer membrane-ionic liquid composites[END_REF][START_REF] Kreuer | Short-side-chain proton conducting perfluorosulfonic acid ionomers : Why they perform better in PEM fuel cells[END_REF] and also of the cell performances [START_REF] Tang | Self-assembled Nafion-silica nanoparticles for elevated-high temperature polymer electrolyte membrane fuel cells[END_REF].

Concerning M112, in agreement with the results obtained on recast membranes e.g. by Adjemian et al. [START_REF] Adjemian | Silicon oxide Nafion composite membranes for proton-exchange membrane fuel cell operation at 80-140 degrees C[END_REF], the performances at 75°C are slightly better than those of N112 (Figures 9 and13). Such a behavior is related to the already noticed better features of this membrane: higher IEC and better water uptake. A decrease of performances is here also observed with the decrease of the relative humidity, whatever the temperature. The aforementioned better features of M112 compared to those of N112 (IEC and water uptake), are however not strong enough levers to keep higher MEA performances at 100 °C. They are actually significantly lower. The preparation process (casting vs extrusion) may be responsible of the poorer performances of M112 observed at 100 °C. From Figures 10 to 12, it can be noticed that the introduction of sepiolite in a Nafion® matrix has a significant impact on the performances of the corresponding MEA at 100 °C, whereas the impact at 75 °C is rather limited. The sulfonation of sepiolite was really beneficial since it allowed improving again the performances, especially at high temperature and low relative humidity.

Comparing Figure 9 (M112) and Figure 10 (M112S10), it is clear that the introduction of pristine sepiolite leads already to higher performances at 100 °C whereas the performances are similar at 75 °C. The sulfonation of sepiolite following the protocol #1 allowed improving the performances at 75 °C and 100 °C, especially at low relative humidity for the higher temperature. The sulfonation protocol developed for this study (#2) allowed reaching even much better performances at high temperature and low relative humidity. This improvement compared to M112S10S could also result from a thinner membrane. However, the improvement at 100 °C comparatively to 75 °C is more pronounced for M112S10SH than for M112S10S.

Provided that the different polarization curves do not cross each other, a convenient way to compare the results obtained here is to look at the current densities at a given voltage. Such a comparison was performed at 0.6 V and is illustrated on Figure 13. Generally speaking, up to 75 % RH, the lower the relative humidity, the lower the current density at 0.6 V. This effect is more pronounced at high temperature.

At 75 °C, all recast membranes showed better performances than N112, even if, M112S10SH excepted, the membranes are thicker. At 100 °C, as discussed previously, the unloaded recast membrane showed poorer performances than N112, but the introduction of sepiolite improves significantly its performances. Indeed, at 75 % RH, the current density at 0.6 V for M112S10 showed a 40 % increase compared to M112 and 12 % compared to N112. We assume this is due to the hygroscopic nature of sepiolite which retains water in the membrane, thus helping the proton conduction process. The sulfonation of sepiolite, whatever the protocol (1 for M112S10S and 2 for M112S10SH), led to a further improvement of the performances (current density at 0.6 V), especially at high temperature and low relative humidity. Indeed, compared to M112, a 50% increase of the current density at 0.6 V was observed for M112S10S, at 100 °C and 50 % RH (60% for M112S10SH and 20% for M112S10 in the same conditions). At 100 °C and 25 % RH the increase is even larger: 70 % and 130 % respectively for M112S10S and M112S10SH, compared to M112. The water uptake of these composites is higher than that of M112, which could partially accounts for the better performance observed. Nevertheless the water uptake is similar for all 10 wt% composites and thus cannot be the unique responsible for the improvement observed. The different IEC (lower for M112S10 compared to M112 and higher for M112S10S and M112S10SH) may also partly explain the observed evolutions. Indeed, the sulfonation of sepiolite is supposed to favor the proton conduction through the membrane.

So both the hygroscopic nature of sepiolite and its sulfonation helped improving the membrane performances. These impacts are more pronounced at high temperature and low relative humidity.

A deeper analysis is necessary to discriminate the important membrane features which could impact the MEA performances. So the difference of performances discussed hereabove have been analysed based on complementary data obtained during the MEA single cell testing, namely the MEA resistance and the membrane hydrogen crossover. The MEA resistance (Figure 14.a) is directly linked to the membrane conductivity while the hydrogen crossover impacts the open circuit voltage (Figures 14.b andc). Thus low MEA resistance and low hydrogen crossover will lead to higher performances.

Among other operating conditions, the MEA resistance has been measured at open circuit voltage, under flowing H 2 at the anode and N 2 at the cathode.

As expected, whatever the temperature, the lower the relative humidity, the higher the resistance. No big difference was observed between 75 °C and 100 °C, keeping the same relative humidity. The introduction of sepiolite, pristine or sulfonated, in the membrane, entails only a very slight increase of resistance. As the performances of the corresponding MEA are generally improved with the introduction of sepiolite, it can be stated that such an increase of resistance is not limiting. It is however interesting to notice that in severe conditions, namely 100 °C and 25 % RH, M112S10 exhibited a higher resistance, probably because the pristine sepiolite is not proton conductive. On the contrary the lower resistance of M112S10S and M112S10SH may be due to the proton conductivity of sepiolite induced by the sulfonation. The lower resistance of M112S10SH compared to that of M112S10S comes from the fact that the membrane is thinner. Since it was prepared with a less sulfonated sepiolite, we would indeed expect a higher resistance than M112S10S. These results are consistent with the evolution of the current density at 0.6 V previously discussed and confirm the beneficial effect of the sulfonation of sepiolite. Three parameters have to be considered here: the sulfonation degree of sepiolite, its distribution within the membrane and the thickness of the membrane. In M112S10S, the sepiolite#1 is less evenly distributed than sepiolite#2 in M112S10SH but its sulfonation degree is higher than that of sepiolite#2. With the same thickness, M112S10SH would have a higher resistance than M112S10S. So the sulfonation degree seems to be a major feature since, with comparable thickness, it would allow M112S10S to have a lower resistance than M112S10SH despite a poorer distribution of sepiolite within the membrane.

Another important parameter to be considered in performances analysis is the hydrogen crossover (Figure 14.b).

It is clear from Figures 14.b, and c that the higher the hydrogen crossover, the lower the open circuit voltage. The hydrogen crossover corresponds indeed to a loss of hydrogen for the electrochemical conversion. It is hence important to reduce the hydrogen crossover as much as possible.

It is noteworthy that for the majority of the composites, the sepiolite introduced in the Nafion® matrix entails a decrease of hydrogen crossover (increase of open circuit voltage) compared to that of M112, as already observed for different systems [START_REF] Adjemian | Investigation of PEMFC operation above 100˚C employing perfluorosulfonic acid silicon oxide composite membranes[END_REF]. The resulting hydrogen crossover is even for some cases really close to that of N112. This is quite remarkable since recast membranes are known to be more permeable to hydrogen than commercial ones, accounting for the higher crossover observed for M112 compared to N112. So the better performances of MEAs may also partly come from the decrease of hydrogen crossover entailed by the introduction of sepiolite. The behavior observed for M112S10SH is different since a larger hydrogen crossover is observed. This is again partly due to the fact that the membrane is thinner. It could also result from a poorer interaction of sepiolite with Nafion. The function added on sepiolite following protocol #2 is indeed different both in size and chemical nature than that of protocol #1. We assume that this may be responsible for a weaker interaction with the polymer matrix, thus impacting the membrane "porosity" and so the hydrogen crossover. The interface between the polymer matrix and the fillers plays indeed a major role on hydrogen crossover [START_REF] Tang | Self-assembled Nafion-silica nanoparticles for elevated-high temperature polymer electrolyte membrane fuel cells[END_REF]. Nevertheless, considering the FC tests results, especially at high temperature and low relative humidity, such a high crossover is not that detrimental and is compensated by a good distribution, contributing to a better conductivity, and a reduced thickness.

The membrane resistance and the hydrogen crossover should be both lowered by optimizing the thickness of the membrane, the sulfonation degree of sepiolite and by improving the compatibility between the filler, here sepiolite, and the polymer matrix, Nafion®.

Conclusion

Composite Nafion-sepiolite membranes realized in this study showed better performances than the pure Nafion membranes, especially at high temperature and low relative humidity. Two protocols have been used in order to functionalize sepiolite and make it proton conductive. They gave rise to two different concentrations of sulfonic acid groups: 0.1 and 0.2 mmol/g. The introduction of pristine sepiolite in Nafion® has shown some beneficial effects. On the one hand, the water uptake and the mechanical properties have been improved. On the other hand, the IEC has been decreased. Sulfonation of sepiolite to make it proton conductive has allowed limiting the decrease of IEC. Moreover, it led to improved fuel cell performances compared to composites prepared with pristine sepiolite. This is especially true at high temperature low relative humidity, despite, in some cases, either a rather poor dispersion of the fillers in the Nafion® matrix (M112S10S) or a high resulting H 2 crossover (M112S10SH). The compatibility between sulfonated sepiolite and Nafion® should thus be optimized so as to increase the MEA performances (output power and durability through better mechanical resistance).

In the case of the M112Sx series, the expected increase of the hygroscopic nature of only one side of the membrane (the 30 % Si side), due to sepiolite segregation, may however be valorized to retain water at the anode side. This gradient of properties could both lower the electro-osmotic drag and increase the water diffusion from the cathode side where it is produced to the anode side.

Figure 1 :

 1 Figure 1: schematic representation of the sepiolite structure after Chivrac et al.. Reprinted from[START_REF] Chivrac | Starch nano-biocomposites based on needle-like sepiolite clays[END_REF] with permission from Elsevier.

Figure 2 :

 2 Figure 2: Sulfonated sepiolites obtained by protocol 1 described by Fernandez-Carretero et al. [37] (left) and developed in our laboratory 2 (right).

Figure 3 :

 3 Figure 3: TGA under nitrogen of pristine sepiolite and sulfonated sepiolites (protocols 1 and 2), a) from 25 to 110°C at 10°C/min followed by an isotherm at 110°C for 10 min, b) from 110 to 900°C at 10°C/min.

Figure 4 :

 4 Figure 4: ATR-FTIR spectra of a) pristine sepiolite, b) triethoxyphenylsilane, c) sepiolite#1 and d) sepiolite#2

Figure 5 :

 5 Figure 5: Py-GC/MS chromatograms obtained for sulfonated sepiolites (protocols #1 &2) and silica (protocol #2).

Figure 6 :

 6 Figure 6: SEM micrographs of cryofractured membranes

Figure 7 .

 7 Figure 7. Elastic modulus measured for the different membranes by Dynamics Mechanical Analysis at 25 °C and 1 Hz.

Figure 8 .

 8 Figure 8. Polarization curves obtained for N112, at 75 °C (solid lines) and 100 °C (dashed lines), between 25 % and 100% relative humidity.

Figure 9 .

 9 Figure 9. Polarization curves obtained for M112, at 75 °C (solid lines) and 100 °C (dashed lines), between 25 and 100% relative humidity.

Figure 10 .

 10 Figure 10. Polarization curves obtained for M112S10, at 75 °C (solid lines) and 100 °C (dashed lines), between 25 and 100% relative humidity.

Figure 11 .

 11 Figure 11. Polarization curves obtained for M112S10S, at 75 °C (solid lines) and 100 °C (dashed lines), between 25 and 100% relative humidity.

Figure 12 .

 12 Figure 12. Polarization curves obtained for M112S10SH, at 75 °C (solid lines) and 100 °C (dashed lines), between 25 and 100% relative humidity.

Figure 13 .

 13 Figure 13. Influence of the temperature and the relative humidity (RH) on the current density at 0.6 V for the different membranes: N112 (black), M112 (white), M112S10 (light grey), M112S10S (dark grey) and M112S10SH (gradation), from left to right.

Figure 14 .

 14 Figure 14. Influence of the temperature and the relative humidity (RH) on the MEA resistance (a), on the H 2 crossover (b) and on the open circuit voltage (c), for the different membranes [from the left to the right: N112 (black), M112 (white), M112S10 (light grey), M112S10S (dark grey) and M112S10SH (gradation)].

Table 1 :

 1 TGA and titration results used to calculate the concentration of -SO 3 H groups grafted at the surface of sepiolite following protocol#1 and #2.

				-SO 3 H concentration
		Weight loss	Weight loss	(mmol/g)*
	Sample	due to the drying step	attributed to the grafted part	Calc. from TGA	Calc. from
		(wt%)	(wt%)	degradation	titration
				step
	Pristine sepiolite	8.4	7.5	-	0.03
	Sepiolite #1	7.2	11	0.2	0.25
	Sepiolite #2	3.8	11	0.1	0.13

Table 2 :

 2 Membranes prepared for the study

			Sepiolite
	Membrane label
		Type	Amount (wt%)
	N112	-	0
	M112	-	0
	M112Sx	pristine	x = 05, 10, 20
	M112SxS	Sepiolite#1	x = 05, 10, 20
	M112SxSH	Sepiolite#2	x = 02, 05, 10

Table 3 :

 3 Si/F atomic ratio (%) measured with EDS analysis at different position through the membrane.

	Position		Si/F atomic ratio (%)
		M112S10	M112S10S	M112S10SH
	1	0.5	0.3	0.3
	2	0.4	0.5	0.4
	3	0.3	0.8	0.5
	4	0.4	30	0.4

Table 4 .

 4 Membrane characterization results (th = thickness, IEC = Ion Exchange Capacity, W ut = water uptake and S = swelling)

		th +/-1	IEC+/-10%	W ut +/-0.5		S +/-0.5
	Membrane	(µm)	(meq/g)	(wt%)		(%)
					s 1	s 2 th.
	N112	56	0.9	25.3	6 5,3	8
	M112	68	1.1	30	4	3	4,6
	M112S02SH	48	1.2	32.2	3.8 3.7 4.2
	M112S05	63	1.1	35	5,6 5,7 8,6
	M112S05S	66	1.3	33,5	6,2 6,2 6,5
	M112S05SH	44	1.2	34.3	4.8 4.9 6.8
	M112S10	63	0.9	38,4	5,9 6,1 10,5
	M112S10S	79	1.2	38,6	5,1 5,1 6,8
	M112S10SH	48	1.2	38.4	6.5 6.3 10.4
	M112S20	62	0.7	45,5	9	9	12,7
	M112S20S	76	1.2	43,8	6,4 6,1 8,6

Acknowledgements

The authors wish to thank the CARNOT MINES Institute and NANOMINES for financing this study and TOLSA SA for having provided sepiolite S9 and valuable technical data. They are also grateful to Patrick Leroux (CEP), Pierre Ilbizian (CEP), Michel-Yves PERRIN (CEMEF) and Suzanne JACOMET (CEMEF) for technical support.