Vincent Prié

Joaquin Soler

Rafael Araújo

Xavier Cucherat

Laurent Philippe

Nicolas Patry

Benjamin Adam

Nicolas Legrand

Philippe Jugé

Nina Richard

Rafael Araujo

Karl M Wantzen

Elodie Hugues

Guillaume Métayer

Frédérique Moinot

Olivier Guerri

Challenging exploration of troubled waters: a decade of surveys of the giant freshwater pearl mussel Margaritifera auricularia in Europe

Keywords: Distribution, museum collections, historical data, scuba diving surveys, large rivers, conservation

come

Introduction

Freshwater ecosystems are the most threatened ecosystems worldwide [START_REF] Dudgeon | Freshwater biodiversity: importance, threats, status and conservation challenges[END_REF]) and freshwater bivalves rank amongst the most threatened animals in the world (Lydear et al. 2004;[START_REF] Lopes-Lima | Conservation status of freshwater mussels in Europe: state of the art and future challenges[END_REF].

One of them, the Giant Freshwater Pearl Mussel Margaritifera auricularia (Spengler, 1793), ranges amongst the most imperilled bivalve species. Although it was considered widespread in most of the western Europe rivers at the beginning of the 20th century, it is now considered as critically endangered by the IUCN (Araujo & Ramos, 2001;[START_REF] Prié | Inventaire de la Grande mulette Margaritifera auricularia dans le Sud-Ouest de la France (I)[END_REF]. The Giant Freshwater Pearl Mussel had become so rare during the 20 th century that it was not even considered when the European Habitat Directive species lists have been established. Indeed, the Giant Freshwater Pearl Mussel is difficult to observe: it lives downstream in large rivers, a habitat that is difficult to survey due to deepness, turbidity, current and often navigation. Hence, not surprisingly, it has been overlooked by malacologists of the 20 th century. However, it nowadays still survives as a few populations in south-west

France and eastern Spain.

The species was first rediscovered in Spain in 1985 [START_REF] Altaba | The last known population of the freshwater mussel Margaritifera auricularia (Bivalvia, Unionoida): A conservation priority[END_REF] and in France in 2000 [START_REF] Cochet | Redécouverte d'une population vivante de la Grande Mulette, Margaritifera auricularia, sur la Vienne et la Creuse[END_REF]. Since 1998, the biology, distribution and lifecycle of the Giant Freshwater Pearl Mussel in Spain were described (Araujo & Ramos, 1998a, b;Araujo & Ramos, 2000 a, b;Araujo & Ramos, 2000;[START_REF] Grande | The gonads of Margaritifera auricularia (Spengler, 1793) and Margaritifera margaritifera (L. 1758) (Bivalvia: Unionoidea)[END_REF]Araujo et al., 2001;[START_REF] Araujo | Glochidium metamorphosis in the endangered freshwater mussel Margaritifera auricularia (Spengler, 1793): A histological and scanning electron microscopy study[END_REF][START_REF] Nienhuis | The rediscovery of Spengler's freshwater pearlmussel Pseudunio auricularius (Spengler, 1793) (Bivalvia, Unionoidea, Margaritiferidae) in two river systems in France, with an analysis of some factors causing its decline[END_REF][START_REF] Gómez | Channels and ditches as the last shelter for freshwater mussels. The case of M. auricularia and other naiads at the mid Ebro River basin, Spain[END_REF]. Since then, some few news of the species in Spain have been released in national and international congresses (i. e. [START_REF] Nakamura | The first year of Margaritifera auricularia (Spengler, 1793)[END_REF]; Online Resource 1), but, apart from [START_REF] Araujo | Influence of flow diversions on Giant freshwater pearl mussel populations in the Ebro River, Spain[END_REF] there are no new scientific results published since 2008. In France, focused surveys have led to the rediscovery of many populations since 2007, but most of these results are unpublished (but see [START_REF] Prié | Evaluation de l'impact d'un projet de canal sur les naïades de l'Oise (France) et découverte de valves récentes de Margaritifera auricularia (Spengler, 1793) (Bivalvia : Margaritiferidae)[END_REF][START_REF] Prié | Une population majeure de la très rare Grande Mulette Margaritifera auricularia (Bivalvia ; Margaritiferidae) dans la Charente (France)[END_REF][START_REF] Prié | Nouvelles populations de Grande Mulette Margaritifera auricularia (Spengler, 1793) (Bivalvia, Margaritiferidae) découvertes dans le sud-ouest de la France[END_REF] or available only as grey literature (Online Resource 1).

An extensive review of all available data on Margaritifera auricularia's distribution is provided here for the first time, together with new data from museum collections and recent field surveys. This article clarifies the past and present distribution of the species, presents the results of the last ten years' surveys in France and Spain and discusses conservation perspectives.

Material and Methods

Bibliography review

The bibliography since 1793 (species description date) has been extensively reviewed. Local publication and grey literature were also consulted when available. Bibliographic data was generally imprecise, but allowed figuring a broad image of the original distribution and ecology of M. auricularia (Fig. 1). Bibliography review thus provided the first indications for where to look for this species.

Museum collections

A first review of museum collections had been performed by Araujo & Ramos (2000a) at a global scale. This review mostly aimed at large national museum collections and included also Margaritifera marocana (Pallary, 1918), a species living only in Morocco (Araujo et al., 2009a). We then inventoried all the regional museum and Universities collections in France. Fifty-eight local natural history collections were identified. Each of them was contacted and questioned about the presence of malacological collections, freshwater bivalves and eventually M. Specimens collected since 2000, year of the re-discovery of the species in France, were not included in the results presented here.

Field surveys and population sizes

Numerous field surveys aiming at freshwater mussels have been performed in France and Spain (Fig. 1, Table 1).

These dedicated surveys aimed at places most likely to host the species, i.e. places identified by literature data, museum collection data or, for France, species habitats modelling (Prié et al., 2014). Moreover, some surveys took place into the frame of impact studies. These impact studies were triggered when M. auricularia was living -or when available data suggested that it could still be living -in an area impacted by a development project. The results of these impact studies are generally not published, consisting only in various cryptic reports (but see [START_REF] Prié | Evaluation de l'impact d'un projet de canal sur les naïades de l'Oise (France) et découverte de valves récentes de Margaritifera auricularia (Spengler, 1793) (Bivalvia : Margaritiferidae)[END_REF][START_REF] Prié | Une population majeure de la très rare Grande Mulette Margaritifera auricularia (Bivalvia ; Margaritiferidae) dans la Charente (France)[END_REF]Araujo & Alvarez-Cobelas, 2016). We here summarize for the first time all the grey literature related to M. auricularia in France and Spain (Online Resource 1).

M. auricularia mainly lives in downstream ecosystems. Surveying this habitat is challenging because it is often deep, turbid, strongly flowing and navigable. In the Ebro historic channels, sampling depends on the hydraulic works made by the Confederación Hidrográfica del Ebro; it is necessary to decrease the water level in order to wade the channel bottom to find the specimens [START_REF] Gómez | Channels and ditches as the last shelter for freshwater mussels. The case of M. auricularia and other naiads at the mid Ebro River basin, Spain[END_REF]. In France, some populations are readily accessible, living in the banks (Vienne River) or in shallow waters (Creuse, Luy or Arros River). For those populations, snorkelling or wading with viewing glasses allowed efficient surveys. However, cumbersome methods based on a team of scuba-divers were needed in most cases. For some surveys, a boat was used to shuttle the divers from a place to another. For others, divers dove from the river banks and sampling plans were then constrained by river accessibility.

Population sizes given here were estimated based on exhaustive counts of observed living individuals (Luy, Creuse and Vienne Rivers); statistical analyses (Ebro, Arros and Charente Rivers), or in the worst case, by a subjective appreciation based on the density of specimens observed (Dronne, Adour and Save Rivers).

The Seine (downstream) and Eure Rivers could only be surveyed by dredging. The dredger used had an aperture of 50 cm, a 25-mm mesh, weighted 11 kg and was propelled by a 30-horsepower engine Zodiac by means of a 30 m long rope. In the Eure River, different biotopes and flow facies were aimed at (mud, sand, stones, riffles, vegetation). In the Seine River, water was up to 6 meters deep and too troubled for operators to see the river bed.

Catches were then randomly positioned. Catches were 8 to 10 m long in the Eure River, and up to 40-50 m long in the Seine River. Sediment collected by the dredger was pulled up and sorted out on the boat. Wading surveys were adopted upstream the Seine River.

In the Somme River, a boat was used to shuttle divers and 82 bank to bank transects were sampled on a 26 km long river stretch. In the Oise River, the divers were also transported by boat from a spot to another, but diving plans were constrained by river condition (from very strong current to muddy bottoms). Areas with very strong current were sampled combining scuba-diving and climbing technics, with a 100 m long static rope secured on a tree on the bank. The diver used a climbing harness and caving equipment in addition to scuba diving gear to progress on the rope. Fins were used to go from side to side in the current, allowing to cover a ca.90 m long cone-shaped surface on the river bottom. Altogether, 115 dives have been carried out on a 35 km long stretch of the river, from the confluence with the Aisne River downstream to a few kilometres upstream the town of Sempigny. Upstream this stretch of river, surveys were carried out wadding randomly in suitable habitats.

In the Charente River, the population was estimated based on scuba-diving transects surveys. A boat was used to shuttle the divers from a transect to another. A 20 m long line was settled down on the bottom of the river and scuba divers counted every living specimen left and right of the line at a distance of 2 m. Each sample then covered 80 m 2 . Transects were repeated every kilometer in the river stretch where mussels were present, and then every three kilometers downstream and upstream the population's distribution limits. A total of 43 transects were repeated on a stretch of 60 km. Detection probability has been estimated at 75% using iterated observations analysed with the software MARK [START_REF] White | Program MARK: Survival estimation from populations of marked animals[END_REF]. Geographical statistics [START_REF] Anselin | The Moran Scatterplot as an ESDA Tool to Assess Local Instability in Spatial Association[END_REF] were performed using GeoDa software [START_REF] Anselin | GeoDa: An introduction to spatial data analysis[END_REF]. Suitable habitat length in the whole river was delimited downstream by the limit of the mud cover due to the influence of the Saint-Savinien's impoundment, upstream by the limit of the living population. Between these limits, the substrate and general ecological quality of the river was very homogenous. In this stretch of favourable habitat, live specimens have been observed wherever we have dived between 2010 and 2016, thus confirming that the population is uniformly distributed.

Fourteen sampling surveys were undertaken between September 2000 and June 2006 in the Ebro River, totalising 25 km, wading in shallow waters and with a team of divers in the deeper parts of the river. Divers used submerged ropes to perform bank to bank or longitudinal transects (survey methods were detailed and reported in [START_REF] Gómez | Channels and ditches as the last shelter for freshwater mussels. The case of M. auricularia and other naiads at the mid Ebro River basin, Spain[END_REF][START_REF] Araujo | Influence of flow diversions on Giant freshwater pearl mussel populations in the Ebro River, Spain[END_REF].

In the Dronne and Isle Rivers, about 100 km stretch of each river upstream their confluence was surveyed, both by wading and scuba-diving from the banks. The estimation of the population size was based on author's appreciation only, and is likely underestimated: over 50 specimens have been observed during the surveys, with a subpopulation of 30 specimens in the lower location (exhaustive count). We estimate that about half of the living individuals have been observed during surveys, which is unlikely given the detection probability in this large river. About 60 km of the Save River was surveyed by wading and scuba-diving, aiming at an exhaustive count of the few remaining specimens which were found only in the lower section of the river. Most of the sampling in the Adour River was undertaken by wading and snorkelling, with scuba divers requested only for a few deeper places. As for the Dronne River, few specimens were found in isolated places, with biggest subpopulation numbering about ten specimens. Population size is estimated based on experts' appreciation only. The Arros River is highly impacted by agriculture practices. The remaining favourable habitats were found isolated between the numerous impoundments' influences. A first survey was conducted by scuba divers, but the deepest places did not have suitable habitats. A more intensive survey was then organized by a team wading with viewing glasses. The total length of river stretches having suitable habitats was 54 km. Within these 54 km, sixteen sites were sampled. On each sampled site, stretches of 100 m to 1 km were exhaustively surveyed.

Population size was estimated based on average densities observed during surveys, multiplied by favourable habitat's surface. In the Luy River, divers explored the deepest pools while most of the river can be explored by wading. The main population is found in a very shallow place, and exhaustive counts were performed three times (years 2010, 2011, 2012) by five persons wading in a line, about one meter apart, ensuring efficient scanning of every single place of the river bed. However, detection probability is never 100%. Some specimens may spend some time completely buried in the sediment and are overlooked (see below the results for the Luy River). The results of these assumed exhaustive counts are therefore likely underestimated.

The most intensive surveys took place in the Vienne and Creuse Rivers. The surveys aimed at providing exhaustive counts of all living specimens. Observers with viewing glasses and divers (depending on the depth) were lined one meter apart and moved forward upstream, ensuring efficient scanning of every single place of the river bed. Sampling was reiterated several time between 2009 and 2016 using the same methods.

In this study, when shells only have been collected, we considered "ancient shells" those that were worn and uncomplete, without periostracum nor ligament remains. "Recent shells" include shells with at least periostracum and ligament remains. We consider as "juveniles" specimens with shell length lower that 11 cm, "subadults" specimens from 11 to 14 cm. Occasionally, some adult specimens had very short shells, especially in the Charente River, but these were obviously very old given the growth lines density and shell wear.

Genetic analyses

Tissue samples have been collected from ten specimens from the Ebro River in Spain, and ten specimens from the Vienne River (Loire coastal drainage), two specimens from the Luy River (Adour River coastal drainage), two specimens from the Charente River and one specimen from the Save River (Garonne River coastal drainage) in France. Foot tissue samples were snipped in the field and preserved in 90° ethanol for molecular analysis.

For Spanish specimens, DNA was extracted using CTAB protocol: tissue samples, preserved in ethanol or frozen, were ground to a powder in liquid nitrogen before adding 600 m L of CTAB lysis buffer (2% CTAB, 1.4 M NaCl, 0.2% b-mercaptoethanol, 20 mM EDTA, 0.1 M TRIS [pH = 8]) and subsequently digested with proteinase K (100 mg.ml -1 ) for 2-5 h at 60° C. Total DNA was extracted according to standard phenol/chloroform procedures (Sambrook & Maniatis, 1989). For French specimen DNA was extracted using the Nucleospin Tissue Kit (marketed by Macherey-Nagel), following the manufacturer's protocol. Extractions, amplifications and sequencing were performed by Genoscreen (France).

To test genetic variability between populations, we examined fragments of two mitochondrial genes, COI and 16S, used previously by Huff et al. (2004); these showed the greatest phylogenetic resolution power for relationships among margaritiferids. 28S nuclear gene fragments were also amplified, but different fragments were targeted for French and Spanish specimens. The COI, 16S and 28S gene were amplified by polymerase chain reaction (PCR) using the protocol described by [START_REF] Prié | Molecular phylogeny, taxonomy and distribution of French Unio species (Bivalvia, Unionidae)[END_REF] for French specimens, and described by Machordom et al. (2003) and Araujo et al. (2016) for Spanish specimens. The amplified fragments were purified by ethanol precipitation prior to sequencing both strands using BigDye Terminator kits (Applied Biosystems, ABI). Products were electrophoresed on an ABI 3730 genetic Analyser (Applied Biosystems). The forward and reverse DNA sequences obtained for each specimen were aligned and checked using the Sequencer program (Gene Code Corporation) after removing primer regions. Sequences were automatically aligned using ClustalW multiple alignments implemented in BioEdit 7.0.5.3 [START_REF] Hall | BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT[END_REF]. The accuracy of automatic alignments was confirmed by eye. Genebank accession numbers are provided in Table 2.

Results

Bibliography

Available literature provided valuable data, although generally without precise location nor date. Nevertheless, a first historical distribution map could be drawn from ancient literature data. Margaritifera auricularia is known from the Netherland, England and Germany from fossil records only. However, some shells collected in the Unstrut River in Germany are very well preserved and probably date back to historical times, at least until the early Middle Ages [START_REF] Bössneck | Die Große Flussperlmuschel (Pseudunio auricularius Spengler, 1793) in Mitteldeutschland: Neue Befunde zur historischen und fossilen Verbreitung (Bivalvia: Margaritiferidae)[END_REF]. Fossil data in Spain includes a Mediterranean Quaternary river in Yecla (Murcia) with 129.000-140.000 years old specimens [START_REF] Andrés | Margaritifera auricularia (Spengler, 1793): primeras evidencias sobre el antiguo cuaternario de Yecla (Murcia)[END_REF] and many other Atlantic rivers with 5.000 years old specimens [START_REF] Araujo | Former Iberian distribution of Margaritifera auricularia (Spengler) (Bivalvia: Margaritiferidae)[END_REF]. In France, fossil data near Marseille (coming from archaeological excavation) and in Massif Central (found amongst fossils collected in a cave) were presumably a result of human transportation.

According to historical data collected, Margaritifera auricularia was only found in large rivers, in a calcareous substrate, in France, Spain and Italy. In France, historical data mainly comes from the Atlantic and Channel sea coastal drainages, with only one occurrence in the Mediterranean coastal drainages, in the Saône River (Rhône tributary). In Italy and Spain, the species is historically known from two Mediterranean coastal drainages, the Po and Ebro Rivers (Araujo & Ramos, 2000a). In Spain M. auricularia lived in two historic channels from the Ebro River, the Canal Imperial and the Canal de Tauste, where there were about 5000 live specimens. The more recent data published about these Spanish populations were recorded in Araujo & Ramos (2000b), [START_REF] Gómez | Channels and ditches as the last shelter for freshwater mussels. The case of M. auricularia and other naiads at the mid Ebro River basin, Spain[END_REF] and [START_REF] Araujo | Influence of flow diversions on Giant freshwater pearl mussel populations in the Ebro River, Spain[END_REF].

Museum collections

The Museum collections have been examined first by Araujo & Ramos (2001) Among them, 332 were localized at a river drainage scale. A third of the specimens came from the Garonne drainage, 19 % from the Saône River (half of them coming from a single batch collected by Coutagne in 1879) and 17% from the Ebro River (Fig. 2B). Other drainages represent less than 30% of the Museum collections specimens. About 80% of the specimens dated were collected before the beginning of the 20 th century.

Field surveys and populations sizes

A total of 2.500 km of rivers has been surveyed for M. auricularia in France and Spain during the last ten years (see bibliography and Online Resource 1 for details). These surveys covered most of the river stretches for which literature or museum collections data was available. Eleven populations could be identified, eight in France and three in Spain, plus a single individual found recently in the Ebro River (pers. comm. from R. Álvarez-Halcón to R. Araujo) upstream Zaragoza (Fig. 3, Table 3). In Spain, the main population, with 5.000 live specimens, live at the Canal Imperial in Aragón. Although there have been some recent mortalities, some young specimens probably under ten years old have been observed during the last years (pers. comm. from J. Guerrero to R. Araujo). The other two Spanish populations, on the Canal de Tauste and the lower Ebro River, are today practically testimonials (pers. com. of the Generalitat of Catalonia to R. Araujo). See [START_REF] Gómez | Channels and ditches as the last shelter for freshwater mussels. The case of M. auricularia and other naiads at the mid Ebro River basin, Spain[END_REF], [START_REF] Araujo | Margaritifera auricularia[END_REF] and [START_REF] Araujo | Influence of flow diversions on Giant freshwater pearl mussel populations in the Ebro River, Spain[END_REF] for more information.

In France, field surveys allowed finding ancient shells in the Seine, in the Vesle and in the Aisne Rivers; in the Saône River (Rhône drainage) near Pontailler-sur-Saône and in the Garonne River near Agen, findings which corroborate historical data. We believe the species was extirpated long time ago in those rivers. In the Oise River (Seine drainage), very recent shells have been found in 2007 and 2008, some of them still embedded in their natural position, suggesting that the species became extirpated very little time before the surveys took place.

The populations of the Creuse and Vienne Rivers (Loire drainage) are the most studied in France. They live in shallow and clear water, allowing regular surveys using viewing glasses or snorkelling. Although these populations are rather small (about 250 specimens altogether), over 40 juveniles were found in the Vienne and Creuse Rivers, which represent about 15% of the population.

Three sites with a few tens of live specimens were discovered in the Dronne River, including one juvenile of about ten cm. Additionally, some isolated individuals were also observed, suggesting the population is scarce but relatively widespread. In the Save River, only 5 live specimens were observed. Sampling conditions are difficult, with variable depth and current strength, and very low visibility. We can therefore suppose that our detection probability is low. But based on survey results, we estimate that the population should not exceed a few tens of living individuals. It is likely rapidly declining given the bad condition of the river and the large number of recent shells collected compared to the very few living specimens observed. The Adour drainage rivers were known to host M. auricularia from both literature and Museum collections data. In the Adour mainstream, the population is now highly fragmented, with only three sites where live specimens could be found. One of them, the most upstream, is now extirpated [START_REF] Prié | Nouvelles populations de Grande Mulette Margaritifera auricularia (Spengler, 1793) (Bivalvia, Margaritiferidae) découvertes dans le sud-ouest de la France[END_REF]. The total population is estimated to be about 300 specimens in the total length of the Adour mainstream, but we still need a better estimation based on an appropriate sampling protocol. On the Luy tributary, a population of about 150 specimens is found in a very small stretch of river. Interestingly, although this River is very shallow (from 30 cm to 1,5 m), clear and easy to survey (hence detection probability is optimal), successive counts of 2010, 2011 and 2012 lead to respectively 110, 96 and 145 specimens. We suppose that a significant part of the population lives buried in the sediment, which biases the results of the counts. The Arros River had been overlooked by literature review and field surveys up to 2016. Following the findings in Museum collections, dedicated field surveys were conducted in 2016, allowing the rediscovery of a living population. This population's size was estimated to about 200 individuals on the 54 km of favourable habitat. The Charente River was known from the ancient literature to host an important population of M. auricularia [START_REF] Bonnemère | Les mollusques des eaux douces de France et leurs perles[END_REF]. Shell fragments and very few live specimens had been found by naturalists since 2003 [START_REF] Nienhuis | The rediscovery of Spengler's freshwater pearlmussel Pseudunio auricularius (Spengler, 1793) (Bivalvia, Unionoidea, Margaritiferidae) in two river systems in France, with an analysis of some factors causing its decline[END_REF]P. Jourde pers. com.). Intensive field surveys performed in 2007, 2010, 2016 led to the discovery of the largest population worldwide. Geographical statistics based on scuba-diving transects showed that the population was not aggregated. Hence the total population size could be estimated by multiplying the average density by the total surface of suitable habitat in the stretch of river inhabited by M. auricularia. The population size in the Charente River was estimated to be about 100.000 (80.000 -120.000) individuals, between the towns of Cognac upstream to Port-d'Envaux downstream.

Genetic diversity

Margaritifera auricularia is genetically remarkably homogenous. The specimens from France and Spain all shared the same 16S and COI haplotype, but two specimens from Spain: specimens vouchered FW1238-14 and FW1238-12, with for COI T-> A in position 37, T->A in position 50 and G->C in position 73; and for 16S T-> C in position 176. The French and Spanish specimens could not be compared for 28S as different gene fragments were amplified. But within France, all specimens shared the same haplotype and within Spain, all specimens shared the same haplotype.

Discussion

Historical and actual data

The number of specimens found in the various regional museum collections was unexpected. Margaritifera auricularia is a large species that retained collector's attention. Most data from museum collections corresponded to the literature data, excepted those from the Arros and Vezere Rivers in France. Surprisingly, most French specimens came from the Garonne and Saône Rivers, were the species is now believed to be extirpated or very rare. In contrast, very few specimens came from the Charente River, where the largest population is found nowadays, and where industrial fisheries were established to make nacre shirt buttons [START_REF] Bonnemère | Les mollusques des eaux douces de France et leurs perles[END_REF]. Similarly, museum collections host no specimen from the Vienne or Creuse Rivers, where healthy populations live in shallow and clear waters. In the Seine drainage, most shells came from upstream and the Aisne tributary, while the Oise tributary seems to have host the last population.

The historical review confirmed that M. auricularia was once present as far as the Thames in England and Netherlands and Germany where fossil specimens have been found and studied (Araujo & Ramos, 2001). On historical times, we found museum records (recent shells) from the Rhine in France or Germany (precise location being unknown), the Seine and the Rhône in France, the Pô in Italy and the Tajo in Spain, where the species is now believed to be extirpated (Araujo & Ramos, 2001). Today, Margaritifera auricularia is considered restricted to five coastal drainages: from north to south the Loire drainage (two close populations in the Vienne and Creuse Rivers), the Charente drainage, the Garonne drainage (two very isolated populations, in the Dronne and Save Rivers), the Adour drainage (at least three isolated populations, one in the Adour itself, one in the Luy and one in the Arros) and the Ebro River (three populations, two in channels and a small one remaining in the Ebro itself). As has been previously estimated (Prié et al., 2014), Margaritifera auricularia's range contraction has probably reached about 90% in the last two centuries.

Surveying downstream ecosystems

Large rivers are amongst the most difficult ecosystems to sample. Deepness, turbidity and water current are challenging conditions. In addition, large rivers are subject to navigation, which makes scuba-diving potentially hazardous. Nevertheless, scuba diving appears to be the most efficient way to produce data for species such as M. auricularia: despite malacological surveys undertaken with canoes and dredging, only a few shell fragments had been collected in the Charente River before scuba diving sampling had been set up. Scuba divers met hundreds of shells and living specimens there. Similarly, scuba-divers collected the few living specimens, that today are probably dead by now, in the main Ebro River in Spain [START_REF] Araujo | Influence of flow diversions on Giant freshwater pearl mussel populations in the Ebro River, Spain[END_REF]. In the Oise River, a few ancient shell fragments had been collected on the banks by amateur malacologists, but scuba-diving allowed finding numerous shells in most of the river stretches investigated. In the Garonne River mainstream, a malacologist spent about 20 days wading and searching for shells on the gravelled banks. In two days, a team of three divers found four shell fragments.

While bivalve surveys have been conducted in the Saône River (ex. [START_REF] Mouthon | Effects of the 2003 heatwave and climatic warming on mollusc communities of the Saône: a large lowland river and its two main tributaries (France)[END_REF], no shell fragments had ever been collected before 2016's scuba-diving prospections. The advances in the distribution knowledge of M. auricularia in France and Spain are directly linked to new investigation methods and scuba diving is so far the most efficient mean of survey for this species.

Conservation and further perspectives

Main threats

While overfishing may have contributed to the species decline in the past [START_REF] Bonnemère | Les mollusques des eaux douces de France et leurs perles[END_REF]Prié et al., 2011;[START_REF] Araujo | Influence of flow diversions on Giant freshwater pearl mussel populations in the Ebro River, Spain[END_REF], it is obviously river management and agriculture impacts that nowadays cause the most important threats to the Giant Freshwater Pearl Mussel. Both causes are linked together, at least in the southern part of the species distribution area: river management aims at providing freshwater for corn culture, especially in summer. Hence, numerous dams are built, even in small rivers, to maintain pools for pumping water in the dry season. These dams produce lotic and silty conditions unsuitable for the Giant Freshwater Pearl Mussel. Altogether, these small dams can affect about than 70% of a given rivers stretch. In the Dronne, Arros and Save Rivers in France for example, the Giant Pearl Freshwater Mussel populations survive in the form of dashed lines, only in riffles (shallow parts of streams where water flows brokenly) with gravel or stony bottoms, between long portions of lotic conditions. Moreover, these dams constitute obstacles for potential fish hosts. The presumed natural host fish of the Giant Freshwater Pearl Mussel, the European Sturgeon Acipenser sturio, has been extirpated from almost all European rivers mainly because of dams [START_REF] Lepage | Threatened fishes of the world: Acipenser sturio Linnaeus, 1758 (Acipenseridae)[END_REF][START_REF] Gesner | Acipenser sturio. The IUCN Red List of Threatened Species[END_REF]. River management has been an important threat in Spain too. Water regulation and the replacement of natural bottoms with concrete have been responsible for a massive death of Naïads. Recently, there has been an unusual high mortality of adults in the Imperial Channel (pers. com. from the Diputación General de Aragón to R. Araujo), but the causes are unknown.

Although a probable cause of recruitment failure, moderate levels of pollution and eutrophication have not demonstrated to be a significant threat to adult specimens. Some population survive in highly human-impacted waters. For example, one of the highest Giant Freshwater Pearl Mussel densities spot lies just downstream the Saintes sewage system in the Charente River. The same kind of conditions occurs at the Canal Imperial in Aragón with the water coming from the Ebro River, which is highly polluted. Overall, the species survives in rivers that are highly impacted by agriculture and domestic effluents. But we still don't know how these eutrophic and polluted waters may impact juvenile survival [START_REF] Augspurger | Advances and opportunities in assessing contaminant sensitivity of freshwater mussel (Unionidae) early life stages[END_REF][START_REF] Strayer | Causes of recruitment failure in freshwater mussel populations in southeastern New York[END_REF][START_REF] Archambault | Survival and behaviour of juvenile unionid mussels exposed to thermal stress and dewatering in the presence of a sediment temperature gradient[END_REF].

Invasive species probably add to the threats M. auricularia is facing. Widespread invasive species such as Corbicula fluminea probably affect the freshwater mussels of Europe like it has been demonstrated for other species in North America (eg. [START_REF] Soussa | Invasive bivalves in fresh waters: impacts from individuals to ecosystems and possible control strategies[END_REF]. However, no clear impacts have been described for M. auricularia, and the healthiest populations survive in rivers largely colonized by Corbicula. The zebra mussel Dreissena polymorpha attaches to the valves of M. auricularia in the Ebro, probably affecting filtration efficiency. This phenomenon has not been observed in France, where the zebra mussel remains at low densities in the rivers of the Atlantic coast.

Habitat management

Contrarily to the Freshwater Pearl Mussel M. margaritifera, for which experiments of habitat managements have proved to be successful [START_REF] Altmüller | Erfolgreiche Artenschutzmaßnahmen für die Flussperlmuschel Margaritifera margaritifera L. durch Reduzierung von unnatürlichen Feinsedimentfrachten -Erfahrungen im Rahmen des Lutterprojekts[END_REF], the Giant Freshwater Pearl Mussel lives in downstream ecosystems. Attempts to implement broad scale drainage system management are therefore unrealistic. However, some realistic management objectives can be achieved to improve the habitat quality locally, in a short or middle term. The deconstruction of the numerous impoundments (many of them being disused) seems the most efficient way to restore suitable riverbed conditions for the Giant Freshwater Pearl Mussel. Although the negative impact of pollution and eutrophication are not clearly known, they are for sure not needed for the species survival.

Improving water quality through reasonable agricultural practices, with buffer strips or grass strips along waterways, should be a middle-term objective.

Farming projects

Breeding farms have been established for many endangered mussel species. In Europe, there is an abundant literature dealing with M. margaritifera breeding farms. Some trials are also ongoing for U. crassus and for various Unio species in Spain [START_REF] Araujo | Conservation of two endangered European freshwater mussels. A three-year semi-natural breeding experiment[END_REF]. Regarding the Giant Freshwater Pearl Mussel, attempts of ex-situ breeding have been performed in Spain [START_REF] Nakamura | The first year of Margaritifera auricularia (Spengler, 1793)[END_REF], and a LIFE project is ongoing in France to artificially breed the species in controlled conditions. Juveniles have been successfully produced [START_REF] Nakamura | The first year of Margaritifera auricularia (Spengler, 1793)[END_REF], but we still face obstacles in the rearing of these juveniles (although some juveniles are still alive, Nakamura com. pers.).

Genetic diversity

The very low genetic diversity for the mitochondrial genes studied was unexpected as (i) the Giant Freshwater Pearl Mussel populations are geographically isolated for a long time; and (ii) strong morphological differences are found between populations (Fig. 4). (i) The populations from France belong to the Atlantic drainage and the population from Spain to the Mediterranean drainage, two geographically isolated bioregions. Strong genetic divergences are observed for other freshwater mussel species from the Iberian Peninsula: U. delphinus from the "pictorum" lineage and U. tumidiformis from the "crassus" lineage were recently considered as distinct species based on molecular divergences (Reis & Araujo, 2009;Araujo et al., 2009b). But on the other hand, some species do not show significant genetic divergences (ex. U. mancus, Prié et al., 2012;Potomida littoralis, Araujo et al., 2016;[START_REF] Froufe | Phylogeny, phylogeography, and evolution in the Mediterranean region: News from a freshwater mussel (Potomida, Unionida)[END_REF]. (ii) The different populations known today have obvious morphological differences in shell size and shape (Fig. 4). The specimens from the Charente River population have a peculiarly small and conspicuously ear-like shell shape, contrasting to the Vienne and Dordogne Rivers populations, which are larger and more elongated; and to the Arros and Save Rivers populations, which are remarkable with their huge sizes. Some populations live in deep coastal rivers (ex. Ebro, Vienne and Charente populations) while others seem to be confined to shallow riffle sections of the upstream rivers (ex Save and Adour populations), but these ecological traits are not linked to shell morphological differences.

Margaritiferidae are known to have very low mitochondrial DNA evolution rates (Araujo et al., 2016;[START_REF] Bolotov | Multi-locus fossil-calibrated phylogeny, biogeography and a subgeneric revision of the Margaritiferidae (Mollusca: Bivalvia: Unionoida)[END_REF]. Population genetics based on microsatellites allowed to differentiate evolutionary units within the related species Margaritifera margaritifera [START_REF] Geist | Drainage-independent genetic structure and high genetic diversity of endangered freshwater pearl mussels (Margaritifera margaritifera) in northern Europe[END_REF]Stoekle et al., 2016) and M. marocana (Soussa et al., submitted). But first studies using microsatellites based on M. margaritifera primers have failed to reveal any population structure in France (Prié, unpublished data). If the ex-situ breeding projects are successful, the population genetics question will become unavoidable.

The fish host issue

The known host fish of Margaritifera auricularia are sturgeon species Acipenser sturio, A. nacari and A. baeri, the River Blenny Salaria fluviatilis and the Eastern Mosquitofish Gambusia holbrooki (Araujo & Ramos, 1998b;Araujo et al., 2000;Araujo et al., 2001;[START_REF] Altaba | Experimental demonstration of viability for the endangered giant pearlmussel Margaritifera auricularia (Bivalvia: Unionoida) in its natural habitat[END_REF]Lopez & Altaba, 2005;[START_REF] Lopez | The European sturgeon Acipenser sturio is a suitable host for the glochidia of the freshwater pearl mussel Margaritifera auricularia[END_REF].

The only native Acipenser species in the area of occurrence of Margaritifera auricularia is the European sturgeon A. sturio. This species became extirpated from most European Rivers during the 20th century.

Nowadays, it is almost extinct, with last documented natural reproduction dating back to 1994 in the Garonne

River. The River Blenny is a Mediterranean species whose range does not overlap with the French populations of M. auricularia. The Eastern Mosquitofish, an introduced species, lives in shallow and standing to slowflowing waters. It is not usually found in places favoured by Margaritifera auricularia. Reported success as host fish for M. auricularia glochidia was questionable. Experiments with other common fish species that occur within the distribution range of M. auricularia (Anguilla Anguilla, Barbus graellsii, Barbus haasi, Parachondrostoma toixostoma, Cobitis paludicola, Liza aurata, Mugil cephalus, Alburnus alburnus, Carassius auratus, Cyprinus carpio, Gobio gobio, Scardinus erythrophthalmus and Tinca tinca) failed to produce juveniles (Araujo et al., 2001;Lopez & Altaba, 2005).

The actual knowledge on M. auricularia host fish cannot explain the recruitment observed recently in the Atlantic drainage rivers. We therefore suspect an overlooked host fish species. For example, Alosa species, which are migratory fish and still breed in the drainages were M. auricularia produces juveniles, are good candidates [START_REF] Llorente | Las náyades (Mollusca, Unionoida) del Calcolítico de Camino de las Yeseras[END_REF]. But there must be another fish host to explain reproduction in the Dronne and Charente Rivers, which are isolated from the sea by impoundments; or in very upstream populations such as those of the Arros or Aisne Rivers, were migratory fishes do not breed. The other hypothesis could be that reproduction occurred in France periodically taking advantage of accidental releases of A. baeri, a common species in French fish farms (but not in Spain). We have recently succeeded in completing the full cycle on the three-spined stickleback Gasterosteus aculeatus in controlled conditions (Soler et al., in prep.). As this species is widespread within the range of M. auricularia and tolerant to brackish waters, it could also be a good candidate as a natural fish host. To find the natural host fish species of M. auricularia in France is now vital for the survival and conservation of this freshwater bivalve.

Conclusion

Margaritifera auricularia has become very rare in the last century, with an estimated range contraction of 90%.

Only three populations were known worldwide before 2007. Intensive surveys in the last decade allowed rediscovering nine more. Given the magnitude of the efforts allocated to surveying the species in its historical range, we now believe that there are very little chances to rediscover unnoticed populations (excepted maybe in north-east France).

Although some juveniles were found recently, they remain very scarce and most extant populations seem to live on a borrowed time. Within the time lapse of this study, some populations already became extirpated in the Ebro and Adour Rivers. The status of the species therefore remains worrying. Priority populations for conservation are the Charente River's population, because it is by far the largest worldwide; the Vienne and Creuse population, because it has the higher level of natural recruitment; the Adour drainage populations, because they form an important and unique metapopulation; and the Ebro population because it is now the only remaining one in the Mediterranean drainage. Conservation challenges for the next years are (i) an appropriate management of the rivers which host the priority populations; (ii) the development of farming projects, in order to reinforce existing populations; (iii) research on fish hosts, for a better comprehension of the species' threats; ecological requirements, to understand which are the habitat factors driving the species' recruitment success; population genetics to plan conservation efforts according to the genetic diversity of the remaining populations; (iv) a wide scale development of modern survey methods such as scuba diving and environmental DNA in order to discover the potentially remaining unnoticed populations.

Despite these efforts, we may fail to save the Giant Freshwater Pearl Mussel from extinction. However, current researches help to shed light on the obscure river downstream ecosystems' ecological functions and threats, as well as to develop exploring methods for this challenging environment.

  auricularia specimens. When M. auricularia specimens were recorded in the inventories or discovered in the collection by the curator, pictures were sent to us to confirm identification. Eventually, some of the most important collections (Musée des Confluences in Lyon, Museum d'Histoire Naturelle in Bordeaux, Museum d'Histoire Naturelle in Toulouse, Museum national d'Histoire naturelle in Paris, Museum d'Histoire Naturelle in Lille, Museum d'Histoire Naturelle in Nantes, Museum d'Histoire naturelle in Orleans, University of Rennes, University of Montpellier) were visited by one of us.

  at a wide scale, focusing mainly on national museums worldwide. Prié et al. (unpublished data, Online Resource 1) have focused on French regional collections only. Out of the 58 collections identified, 25 had at least one specimen of M. auricularia (Fig. 2A): Musée du Château in Annecy, Musée des Confluences in Lyon, Museum of Perpignan, Musée zoologique of Strasbourg, Muséum -Aquarium of Nancy, Museum of Auxerre, Muséum d'histoire naturelle in Bordeaux, Muséum d'histoire naturelle in Bourges, Muséum d'histoire naturelle in Grenoble, Museum d'Histoire Naturelle in Nantes, Muséum d'Histoire Naturelle in Toulouse, Museum d'Histoire Naturelle Victor Brun in Montauban, Museum d'Histoires Naturelles in Colmar, Muséum of Orléans, Muséum of Dijon, Muséum Lecoq in Clermont-Ferrand, Muséum national d'Histoire naturelle in Paris, Muséum national d'histoire naturelle in Lille, Paraclet center of ONEMA in Boves, Pôle muséal of Troyes, Université of Bourgogne in Dijon, Université of Montpellier I, Université of Rennes I, Museum d'histoire naturelle in la Rochelle, Museum of Cherbourg-Octeville. Part of the data from Museum collections were fossil specimens. A total of 400 non-fossil specimenswere found in Museum collections, including the 37 specimens already found byAraujo & Ramos (2001).

Figure 1 :

 1 Figure 1: fossil (black crosses) and historical data (white dots for precise locations, blue lines for rivers names only) collected from the literature and Museum collections; and subsequent intensive field surveys locations (polygons).

Figure 2 :

 2 Figure 2: A: location of main museum collection investigated (dot size according to number of M. auricularia specimens). B: number of specimens held in Museum collections per main coastal drainages (the Saône River is actually a tributary of the Rhône, but all the specimens are located in the Saône and none elsewhere in the Rhône).

Figure 3 :

 3 Figure 3: results of last ten years' field surveys and known past and actual distribution of M. auricularia. Fossil data (black crosses), historical data (white dots), shells collected in the last 10 years (orange dots) and still living populations (red dots).

Figure 4 :

 4 Figure 4: morphological variability of Margaritifera auricularia. A: Vienne River; B-C: Charente River; D: Dronne River; E: Save River; F: Luy River; G: Arros River, H: Ebro River.

Table 1 :

 1 summary of the methods used in France for surveying M. auricularia. For Spain, survey methods were detailed and reported in[START_REF] Gómez | Channels and ditches as the last shelter for freshwater mussels. The case of M. auricularia and other naiads at the mid Ebro River basin, Spain[END_REF]

	670
	671	Álvarez-Cobelas, 2016.
	672
		Coastal			Use of			Authors
		drainage	River	Year	a boat	Scuba-diving	Other methods	involved
		Somme	Somme River	2011	X	4 divers, 5 days		XC, VP
			Seine River (downstream)	2011	X		Dredging (2 persons for 4 days)	XC
			Seine River (upstream)	2015			Snorkeling (2 persons for one days)	XC
				2007-				VP, LP,
			Oise River	2008	X	3 divers, 20 days		XC
		Seine	Aisne River	2011		2-4 divers, 5 days		XC, VP
								VP, LP,
								NL, NP,
		Rhône	Saône and Doubs Rivers	2016		4 divers, 5 days	Wading with viewing glasses (2 persons, 5 days)	BA
			Loire, Indre and Cher	2010-		2-3 divers, about 5	Wading with viewing glasses (2-4 persons, estimated to about 10	LP, VP
			Rivers	2011		days	days altogether)
				2009-		2 to 6 divers, over	Wading with viewing glasses and snorkeling (2 to 6 persons,	VP, XC,
		Loire	Vienne and Creuse Rivers	2016		20 days altogether	estimated to over 30 days altogether)	LP
				2007-		2 to 3 divers, about		VP
		Charente	Charente River	2016	X	20 days altogether
				2012-		2 divers, about 10	Wading with viewing glasses (1-2 persons, about 5-10 days	VP
			Dronne and Isle Rivers	2014		days altogether	altogether)
						2 divers, 3 days		VP
			Dordogne River	2016	X	altogether	Wading with viewing glasses (1 person, about 5 days)
								VP, LP,
								NP, BA,
			Vézère River	2016		3 divers, 3 days	Wading with viewing glasses and snorkeling (2 persons 3 days)	NL
			Garonne River	2016		3 divers, 2 days	Wading with viewing glass (1 person, 30 days)	VP, NL
		Garonne	Save River	2009		2 divers, 1 day	Wading with viewing glass and snorkeling (1 person, 5 days)	VP, BA
				2012-		2 divers approx. 9	Wading with viewing glasses and snorkeling (2 to 6 persons, 10	VP, BA,
			Adour River	2014		days	days)	NL
								VP, BA,
		Adour	Arros River	2016		3 divers, 3 days	Wading with viewing glasses (6 persons, 4 days)	NL
	673

This work was conducted within the scope of the LIFE project "Life13BIOFR001162 Conservation of the Giant Pearl Mussel in Europe". We thank Dominique Tesseyre from the Adour-Garonne Water Agency; Julie Marcinkowsky and Gérard Tardivo from the DREAL Centre as well as the DREAL Picardie for providing financial support for large-scale surveys of M. auricularia in France;

28


Sullivan Renouard
email: sullivan.renouard@univ-orleans.fr

Christophe Hano

Pierre Ouagne

Jo € El Doussot

Jean-Philippe Blondeau

Eric Lain

Cellulose coating and chelation of antibacterial compounds for the protection of flax yarns against natural soil degradation

Keywords:

Natural cellulosic fibres such as flax fibres present interesting mechanical properties as well as biodegradability, and by-products, such as short flax fibres, could be used to produce geotextiles to stabilise soils. Today, geotextiles are often made of coir fibres, which have a high lignin percentage leading to their slow degradation in soil. Fibres with a high cellulosic content, such as those of flax, exhibit lower resistance to soil degradation. This study investigates solutions to improve this parameter with a view to increasing their service life and therefore their credibility compared to coir fibres for geotextile applications.

For this purpose, a cellulose coating of yarns made of short flax fibres was performed and its stability under a water flow was assessed. The ability to form a cellulose sheath was estimated by chromaticity measurements of flax fibres after applying a dye specific to lignin. Infrared spectrometry analysis to monitor the level of protection against degradation by cellulolytic enzymes was also carried out. It appears that the cellulose coating provides an efficient physical protection, preventing access of these enzymes to their fibrous substrate. Then, the possibility of conferring antibacterial properties on the cellulose coating by chelating phytoalexin molecules such as gramine on it was assayed and proven to be effective against soil cellulolytic bacteria such as Cellvibrio fulvus and Cellvibrio vulgaris. This study therefore establishes that coating flax yarns with cellulose associated with antibacterial molecules could contribute to obtaining a longer service life in soil for geotextiles manufactured from flax fibres.

Introduction

Flax fibre is a natural fibre used in the textile industry or as a reinforcement for crude earth constructions and composite materials due to its high mechanical properties and relatively low cost [1e10]. Short flax fibres are a by-product of the textile industry and a renewable resource considered fully biodegradable making them an interesting candidate for manufacturing geotextiles. Nevertheless, although biodegradability is required for such products, it has to be quite slow for geotextile applications. Today, most geonets are manufactured from coir fibres. Coir and flax fibres are mainly composed of cellulose, hemicellulose, pectin and lignin but coir fibres have a higher lignin percentage [START_REF] Yan | Flax fiber and its composites e a review[END_REF][START_REF] Rencoret | Del Río, Structural characterization of lignin isolated from coconut (Cocos nucifera) Coir fibers[END_REF]. Lignin is known for its resistance to degradation as lignin-degrading organisms are less widespread than cellulolytic ones. Consequently, when submitted to natural soil degradation by microbial populations [START_REF] Hu | Microbial dynamics associated with multiphasic decomposition of 14C-labeled cellulose in soil[END_REF], flax fibres are likely to show greater sensitivity than coir fibres. To overcome this higher degradability, flax fibres could be coated to increase their service life by providing physical protection. However, the coating must keep the environmental interest of flax fibre geotextiles and therefore also be biodegradable since a slowing down not an abolition of degradation is required. Cellulose seems a credible and interesting candidate because it has already been used for coating [START_REF] Offord | Method for Producing Cellulosic Sheaths Around Fibers of Textiles and Textiles Produced Thereby[END_REF][START_REF] Miyauchi | Cellulose Sheathed Nanotube Fibers[END_REF] and this compound, composed of b-1,4 glucosidic linked D-glucopyranose units, is a renewable, biodegradable and the most abundant biopolymer on earth [START_REF] Klemm | Cellulose: fascinating biopolymer and sustainable raw material[END_REF]. On the other hand, flax fibre yarn is very flexible so any coating applied should be at least as flexible to remain in place. As flax fibre flexibility is due to its main component, cellulose, this seems to be one of the rare biodegradable candidates with the needed flexibility. Moreover, knowing the high sensitivity of cellulose to biodegradation, we have considered a combined protection approach: antimicrobial molecules could be associated with a cellulose coating in order to increase its resistance toward biological attacks, as already done on coir fibres using oil [START_REF] Sumi | Effect of antimicrobial agents on modification of coir[END_REF].

This work investigated the feasibility of a cellulose coating on flax fibre yarns and an antibacterial compound bound to the cellulose coating to assess the efficiency of such treatment against degradation processes, mimicking natural soil degradation. For this purpose, the ability of the cellulose coating to prevent enzymatic degradation using a cellulase cocktail from Aspergillus niger [START_REF] Renouard | Protection of flax fiber-based yarns against natural soil degradation by chitosan[END_REF][START_REF] Sharma | A biodegradation study of forest biomass by Aspergillus Niger F 7 : correlation between enzymatic activity, hydrolytic percentage and biodegradation index[END_REF] was analysed. Polyquaternium-10, a polymeric quaternary ammonium salt of hydroxyethyl cellulose currently used in shampoo to coat hairs due to its ability to form a film easily, was used here as a positive control for coating ability and was assayed to provide a basis for comparison. Then, the possibility of associating antibacterial compounds with flax fibre yarns (previously coated or not with cellulose) was explored using antibacterial phytoalexins. This chemical protection was then evaluated against the soil-borne cellulolytic bacteria Cellvibrio [START_REF] Blackall | Cellulolytic and dextranolytic gram-negative bacteria: revival of the genus cellvibrio[END_REF], a microorganism already used in previous experiments [START_REF] Renouard | Protection of flax fiber-based yarns against natural soil degradation by chitosan[END_REF]. Our aim was to investigate the possibility of using such natural products (with low environmental impact) to increase the potential service life of flax fibre-based geotextiles with (and without) a cellulose coating.

Materials and methods

Biological material

Twisted yarns (90 turns/m) made of short flax fibres (1 cm long) produced from tows originating from various flax fibre cultivars were provided by Groupe Depestele (France).

Cellvibrio fulvus and Cellvibrio vulgaris strains were purchased from the BCCMTM/LMG bacteria collection, Gent University (Belgium). They were cultivated in a tryptic soy broth medium in the presence of flax fibre yarn (raw or previously treated as described below). To measure the antibacterial effect, growth was monitored by spectrophotometry at 630 nm after 12 h at 28 C with shaking (300 rpm). The antibacterial compounds gramine or saponin were bound to the yarns using the metal chelation process described by [START_REF] Kennedy | Active insolubilized antibiotics based on cellulose-metal chelates[END_REF] [START_REF] Kennedy | Active insolubilized antibiotics based on cellulose-metal chelates[END_REF]. This cellulose-metal chelation protocol uses iron III chloride as the chelating metal.

Chemicals and enzyme

Cellulose (C6288), p-nitrophenyl b-D-glucopyranoside (N7006), sodium carbonate (57795), sodium phosphate monobasic (58282), sodium phosphate dibasic (57907), iron III chloride (157740), gramine (G10806), saponin (47036) and Aspergillus niger cellulase (22178) were purchased from Sigma. Polyquaternium-10; JR-400 was purchased from Wenlin.

Coating of yarns with cellulose or Polyquaternium-10

1% w/v cellulose or Polyquaternium-10 solutions were prepared by suspending the powder in boiling distilled water. Flax yarns were then immersed and shaken in cellulose or Polyquaternium-10 suspension for 8 h at 25 C. The impregnated flax fibre yarns were then dried in an oven at 37 C for 7 days.

Enzymatic degradation and activity

Flax fibre yarn degradation using Aspergillus niger cellulase from Sigma was optimised in a previous work: Renouard et al. [START_REF] Renouard | Protection of flax fiber-based yarns against natural soil degradation by chitosan[END_REF] demonstrated that optimal degradation occurred at a pH of around 7, an incubation temperature of 37 C, an incubation duration of at least 8 h and a cellulase concentration of at least 5 U/ ml. Here, the optimal conditions were used with 5 U/ml as the cellulase concentration and 16 h as the incubation duration.

To measure enzymatic activity, a protocol derived from Renouard et al. [START_REF] Renouard | Protection of flax fiber-based yarns against natural soil degradation by chitosan[END_REF] was used. An enzymatic cocktail (50 ml of a 5 U/ ml solution in 0.2 M pH 7 phosphate buffer) with p-nitrophenyl b-D-glucopyranoside (50 ml of a 0e10 mM solution in 0.2 M pH 7

phosphate buffer) was applied on cellulose or Polyquaternium-10 (100 ml of 1% w/v cellulose, Polyquaternium-10 solutions or water as the control) for 30 min at 25 C and then stopped by applying 100 ml of 1 M sodium carbonate solution. The amount of p-nitrophenolate released by the enzyme activity was measured by spectrophotometry at 405 nm. The results were expressed in arbitrary units of absorbance per min (AU/min). As p-nitrophenyl b-D-glucopyranoside digestion induces glucose release, the glucose inhibition curve on Aspergillus niger cellulase from Sigma was established prior to the enzymatic assays and showed that no significant activity difference was detected up to 0.05% glucose. In our conditions (0.045% was the maximal glucose concentration), glucose inhibition did not have to be considered (Supplementary Fig. 1). The Lineweaver-Burk plot was used to estimate the Michaelis constant, Km, and the maximum reaction rate, Vmax. Using this method, the y axis-intercept is related to 1/Vmax and the slope to Km/Vmax.

Fibre coloration analysis

Yarns were placed for 5 min in a phloroglucinol solution (phloroglucinol 1% w/v in ethanol 95%) and then for 5 min in a 5 M HCl solution. This led to the pink coloration of the lignin promoted by the phloroglucinol solution. The fibres' final colour was determined using a Minolta Chroma CR-200 colorimeter. The results are presented on a chromacity diagram: this system, called a CIE diagram, can describe any colour through the use of only three elementary colours (red, green and blue).

ATR-FTIR analysis

Reflectance spectra were recorded using a Bruker V70 interferometer working under a dehydrated airflow in reflectivity mode, with an ATR (Attenuated Total Reflectance) accessory containing a gold crystal. The measurements were performed for wave numbers situated in the middle of the infrared range (between 600 cm À1 and 4500 cm À1 ). The instrument resolution was about 4 cm À1 and measurements were averaged over 64 scans. As previously described [START_REF] Renouard | Characterization of ultrasonic impact on coir, flax and hemp fibers[END_REF], ATR-FTIR ratios were used to quantify crystalline cellulose (1375 cm À1 /1595 cm À1 ), amorphous cellulose (897 cm À1 / 1595 cm À1 ), xylan (1089 cm À1 /1595 cm À1 ), xyloglucan (1078 cm À1 / 1595 cm À1 ) and pectin (1610 cm À1 /1595 cm À1 ) using the ligninspecific band (1595 cm À1 ) as the reference since lignin is not degraded by cellulase treatment.

Statistical treatment of data

All data presented in this study are the mean and standard deviation of at least 3 independent replicates. A comparative statistical analysis of groups was carried out using Student's test. Statistical tests were considered to be significant at p<0.05, the pvalue being the probability of finding the observed, or more extreme, results when the null hypothesis is true. Graphical and statistical processing was performed using Microsoft EXCEL 2010 software.

Results and discussion

Coating ability of cellulose

Based on the coating protocol of [START_REF] Hamzeh | Pre-extraction of hemicelluloses from bagasse fibers: effects of dry-strength additives on paper properties[END_REF] [START_REF] Hamzeh | Pre-extraction of hemicelluloses from bagasse fibers: effects of dry-strength additives on paper properties[END_REF], flax fibre yarns were coated using a 1% cellulose suspension.

Weight gain of flax fibre yarns after cellulose coating

The yarns exposed to the cellulose suspension showed a weight gain of 13.91%, demonstrating the coating ability of cellulose. According to Hubbe (2006) [START_REF] Hubbe | Bonding between cellulosic fibers in the absence and presence of dry-strength agentseA review[END_REF], water can be considered a solvent for segments of macromolecules such as hemicellulose and cellulose, and these segments tend to mix with each other. Thus, a random process of molecular motions would be expected to result in interpenetration and tangling. This mutual diffusion process at facing surfaces explains the cellulose coating ability on flax fibre.

Property of cellulose coating

The high positive correlation (0.982) between the weight gain and the cellulose percentage of the coating solution (Fig. 1) suggests cellulose stacking. The mutual diffusion process describe by Hubbe (2006) [START_REF] Hubbe | Bonding between cellulosic fibers in the absence and presence of dry-strength agentseA review[END_REF] can also explain it, considering the possibility of tangling between several cellulose fibres.

Cellulose coating stability

Coated flax fibre yarns were submitted to water movement during 14 days to test the stability of the cellulose coating (Fig. 2A). This information is crucial if this protective coating is to be used to increase the service life of geotextiles exposed to moisture and/or rain flow. No significant weight loss was observed after water agitation treatment on flax fibre yarns coated with cellulose (Fig. 2B). The interpenetration and tangling process due to the mutual diffusion at facing surfaces described by Hubbe (2006) [START_REF] Hubbe | Bonding between cellulosic fibers in the absence and presence of dry-strength agentseA review[END_REF] enables flax fibre yarn to be coated with cellulose and this coating presents a credible stability. cellulase was used [17] [18]. ATR-FTIR enables quantitative analysis of the flax fibre composition with or without enzymatic treatment by using ratios with lignin as a reference [START_REF] Renouard | Characterization of ultrasonic impact on coir, flax and hemp fibers[END_REF]. To achieve a direct fibre analysis, the coatings were removed before spectrometry measurements by shaking the yarns in boiling water. FTIR analysis showed that the enzymes applied to the flax yarns did not have any effect on the flax fibre main components: cellulose (crystalline or amorphous), hemicellulose (xylan, xyloglucan, etc.) and pectin when the yarns were coated with cellulose or Polyquaternium-10. On the contrary, raw yarns (which were not coated) underwent degradation, revealed by the drop in the intensity of the specific band ratios of their components (Fig. 3, Supplementary Fig. 2) [START_REF] Renouard | Protection of flax fiber-based yarns against natural soil degradation by chitosan[END_REF].

Protective ability of cellulose and

To sum up, a cellulose coating confers a protection on flax fibre yarns against enzymatic attacks.

Characterisation of cellulose protection against enzymatic degradation

In an attempt to understand how the protection provided by the coating works, further experiments were performed to test two hypotheses. The first was that the coating products can affect the ability of the enzyme to degrade its cellulose substrate. In this case, the enzymatic parameter would undergo a change revealing the kind of enzymatic activity inhibition. The results are shown in Fig. 4 (Supplementary Fig. 3): Polyquaternium-10 had no impact on enzymatic activity, but cellulose inhibited the cellulase activity as expected logically since cellulose is the natural substrate of cellulase. Enzymatic parameter measurements indicated that cellulose induced only an increase in the Michaelis constant (Km) without modification of the maximum reaction rate (Vmax). In other words, there was competition between nitrophenyl b-D-glucopyranoside, the substrate provided in the experiment to measure the cellulase activity, and cellulose itself. This seems logical as cellulose acts as a lure but does not inhibit the enzyme's intrinsic ability to act on its substrate. Thus, neither cellulose nor Polyquaternium-10 will change the capacity of cellulase to attack the fibre by classic enzymatic degradation.

Consequently, the protection provided to flax fibre yarns by the coating could result from the formation of a sheath that covers them completely and prevents access of cellulase to the fibres. Polyquaternium-10, which can form a film, does not change the enzyme activity and therefore the ability to attack the cellulose. It is used here as a positive control to observe what may happen when a physical barrier, such as a coating that hinders the access of enzyme to its substrate, is applied.

To investigate this second hypothesis, i.e. the ability of a cellulose coating to form a complete physical protection for flax fibre yarns, phloroglucinol coloration (pink coloration) of the accessible lignin of the flax fibres was performed. The chromaticity diagram shows that uncoated flax fibre yarns reveal pink coloration whereas yarns coated with cellulose or Polyquaternium-10 do not (Fig. 5). Consequently, it can be deduced that cellulose, as well as Polyquaternium-10, forms a protective sheath around flax fibre yarns thus establishing a physical barrier that hinders the access of phloroglucinol to the lignin of the flax fibres. One can suppose that the coating will also prevent the access of the enzymes to the fibres. Consequently, it can be concluded that, similarly to chitosan used in a previous work [START_REF] Renouard | Protection of flax fiber-based yarns against natural soil degradation by chitosan[END_REF], cellulose, as well as Polyquaternium-10, can form a sheath that completely covers the flax fibre yarns and efficiently protects them against enzymatic attacks.

A B A A A B A A A B A A A B A A A B A A 0 0.5 1

Antibacterial chelating possibility

To prevent coir fibre geotextile degradation, Sumi et al. (2016) [START_REF] Sumi | Effect of antimicrobial agents on modification of coir[END_REF] applied antifungal lipids on them, which reduced the microbial activity. For our flax fibres, a process specifically adapted to cellulose was used on coated and uncoated flax yarns to bind phytoalexins, which are biodegradable natural compounds produced by plants to fight their pathogens. [START_REF] Kennedy | Active insolubilized antibiotics based on cellulose-metal chelates[END_REF] [START_REF] Kennedy | Active insolubilized antibiotics based on cellulose-metal chelates[END_REF] developed a protocol to chelate antibacterial compounds on cellulose using iron III as the mediator to convert cellulose into a more reactive form: the cellulose-metal form. Metal ions can then be replaced by electron-donating groups of antibacterial molecules, leading to antibacterial compound chelation on cellulose. This protocol was tested here to chelate the saponin and gramine phytoalexins, which possess antibacterial activity [START_REF] Hong | Gramine-induced growth inhibition, oxidative damage and antioxidant responses in freshwater cyanobacterium Microcystis aeruginosa[END_REF] [25] [START_REF] Soetan | Evaluation of the antimicrobial activity of saponins extract of Sorghum Bicolor L, Moench[END_REF], on cellulosic flax fibre yarns. The inhibitory effect of phytoalexins on Cellvibrio fulvus and Cellvibrio vulgaris, which are soil bacteria that produce enzymes causing flax fibre degradation [START_REF] Renouard | Protection of flax fiber-based yarns against natural soil degradation by chitosan[END_REF] [START_REF] Blackall | Cellulolytic and dextranolytic gram-negative bacteria: revival of the genus cellvibrio[END_REF], was then tested on cellulose-coated and raw flax yarns.

To estimate the antibacterial activity conferred by the chelated phytoalexins on cellulose coating or flax fibre yarns, bacteria were quantified compared to the corresponding control without phytoalexins using the absorbance at 630 nm, which is directly related to the bacterial amount. The antibacterial assay principle is that antibacterial activity will slow down bacterial growth so to investigate bacterial growth retardation, a difference in bacterial amount is measured. Therefore, the experiment was carried out after 12 h, corresponding to the exponential phase of the two Cellvibrio strains (Supplementary Fig. 4), during which the most sensitive difference in bacterial amount can be measured to provide better evidence of the difference in bacterial growth.

For both Cellvibrio strains, the same results were observed (Fig. 6). When applied on flax fibre yarns without prior cellulose coating, no antibacterial activity was conferred by the phytoalexin treatment (Fig. 6). On the contrary, with prior cellulose coating on flax fibre yarns, gramine provided a significant inhibition of bacterial growth for both Cellvibrio strains compared to the control samples (Fig. 6). As expected, treatment with saponin did not lead to bacterial growth inhibition since saponin only acts against Grampositive bacteria [START_REF] Soetan | Evaluation of the antimicrobial activity of saponins extract of Sorghum Bicolor L, Moench[END_REF] and Cellvibrio bacteria are Gram-negative. This clearly shows that it is possible to chelate antibacterial compounds to the cellulose coating of the fibre but not directly on the fibre. These results also demonstrate that the antibacterial activity observed is due only to the specific activity of the chelated molecule.

To understand why there is no antibacterial activity without prior cellulose coating, the phytoalexins that remained unbound after treating the flax fibre yarns were quantified: this showed that without prior cellulose coating, there was no phytoalexin chelation (Supplementary Fig. 5). One can hypothesise that lignin prevents the access of the phytoalexin compound to the flax fibre cellulose due to its hydrophobicity. Another possibility is a repulsion of iron III cations (needed for chelation of antibacterial molecules) by cations such as calcium, which are part of the flax fibre composition [START_REF] Rihouey | Calcium and acidic pectin distribution in flax cell walls: evidence for different kinds of linkages in the cell junction and middle lamella of the cortical parenchyma of flax hypocotyl[END_REF].

To summarise this section, the observations show that antibacterial chelation is only effective if a cellulose coating is previously applied to the flax yarns. The bacterial growth inhibition by a natural compound such as gramine could contribute to slowing down the natural degradation of the cellulose-based yarns while preserving the biodegradable aspect, since phytoalexins themselves can be degraded as they are natural plant compounds.

Conclusions

In this work, it was established that flax fibre yarns can be coated in cellulose by dipping. This coating was also shown to be resistant to a water flow. The cellulose coating provides a complete physical protection to flax fibre yarns, which hinders their degradation by the cellulase of one telluric microorganism. Moreover, the presence of this coating offers the possibility of binding natural antibacterial compounds such as gramine or saponin since the binding of an antibacterial phytoalexin active against Gramnegative bacteria was effective at preventing the development of cellulolytic bacteria such as Cellvibrio fulvus and Cellvibrio vulgaris cultivated in the presence of flax fibre yarns treated in this way. These treatments, only carried out using natural compounds, could therefore be used to increase the service life of flax fibre-based geonets.

Polyquaternium- 10 3. 2 . 1 .Fig. 1 .Fig. 2 .

 102112 Fig.1. Flax fibre yarn weight gain induced (by cellulose coating) evolution according to the cellulose percentage of the soaking solution completed with the correlation line (grey line) and associated correlation coefficient (R 2 ). Measurements associated with different letters differ significantly from each other at p < 0.05 using Student's test.

Fig. 3 .Fig. 4 .

 34 Fig.3. ATR-FTIR data. Each bar is the relative absorbance normalised using the lignin band as the reference). Measurements associated with different letters differ significantly from each other at p < 0.05 using Student's test.

Fig. 5 .Fig. 6 .

 56 Fig. 5. Coloration of fibres with phloroglucinol presented on a chromaticity diagram.

Acknowledgments

This project was funded by the "R egion Centre e Val de Loire" (GEFIL programme).

Appendix A. Supplementary data

Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.polymdegradstab.2017.02.006.