Souty-Grosset

M C Trouilhe

F Ricard

C Souty-Grosset

MANAGEMENT OF THE WHITE-CLAWED CRAYFISH (AUSTROPOTAMOBIUS PALLIPES) IN WESTERN FRANCE: ABIOTIC AND BIOTIC FACTORS STUDY

Keywords: Austropotamobius pallipes, water quality, biotic index, macroinvertebrates, Principal Component Analysis (PCA)

Management of the white-clawed crayfish (Austropotamobius pallipes) in Western France: Abiotic and biotic factors study

GESTION DE L'ÉCREVISSE À PATTES BLANCHES (AUSTROPOTAMOBIUS PALLIPES) DANS L'OUEST DE LA FRANCE : ÉTUDE DES FACTEURS BIOTIQUES ET ABIOTIQUES

Résumé

En France, la distribution de l'écrevisse à pattes blanches, Austropotamobius pallipes (Lereboullet, 1858), est réduite, fragmentée et principalement située en tête de bassin. Afin de préserver cette espèce indigène, il est nécessaire de caractériser ses exigences écologiques (qualité de l'eau et de l'habitat). Dans ce but, une étude de deux ans est menée dans le département des Deux-Sèvres (Ouest de la France) depuis novembre 2002. Neuf ruisseaux sur quatre bassins hydrographiques différents sont régulièrement visités. Deux sites de prélèvements par ruisseau sont prospectés, le premier site est localisé au niveau de la population d'écrevisses et le second 2 à 3 km en aval. Les paramètres physico-chimiques (18) sont mesurés deux fois par mois et des facteurs biotiques sont estimés bi-annuellement. Dans cette étude, le protocole de l'I.B.G.N. (Indice Biologique Global Normalisé) basé sur la détermination de macroinvertébrés a été utilisé comme facteur biotique pour estimer la qualité biologique de l'eau. Les résultats de cette étude préliminaire sur deux ruisseaux (Thouet et Verdonnière) montrent que les données physico-chimiques et biologiques prises séparément ne fournissent pas d'informations fiables quant aux exigences écologiques d'A. pallipes. Cependant, l'utilisation d'analyses multivariées (Analyse des Composantes Principales) pour combiner les facteurs abiotiques et biotiques révèle une bonne corrélation entre ces paramètres. Ainsi, il semble que la matière organique soit un meilleur facteur discriminant que la matière minérale en ce qui concerne la présence ou l'absence de l'écrevisse à pattes blanches.

Mots-clés :

Austropotamobius pallipes, qualité de l'eau, indice biotique, macroinvertébrés, Analyse des Composantes Principales (ACP).

INTRODUCTION

The white-clawed crayfish, Austropotamobius pallipes (Lereboullet, 1858), has a widespread distribution in Western Europe [START_REF] Holdich | Distribution of crayfish in Europe and some adjoining countries[END_REF]. A. pallipes mainly occurs in headwaters and, over the last few decades, the distribution of this species has become restricted and fragmented due to several factors such as crayfish plague and degradation of water quality influenced by human activities [START_REF] Holdich | The white-clawed crayfish, Austropotamobius pallipes, in Great Britain and Ireland with particular reference to its conservation in Great Britain[END_REF][START_REF] Vigneux | Les introductions de crustacés décapodes d'eau douce en France. Peut-on parler de gestion ?[END_REF]. In France, this species appears to be demanding in terms of water and habitat quality [START_REF] Grandjean | Distribution and proposal for the conservation of the indigenous freshwater crayfish species, Austropotamobius pallipes pallipes, in a French department[END_REF][START_REF] Grandjean | Life history and population biology of the white-clawed crayfish, Austropotamobius pallipes pallipes, in a brook from The Poitou-Charentes region (France)[END_REF]. Since 1983, A. pallipes has been listed as vulnerable on the red list of threatened animals of the International Union for the Conservation of Nature and Natural Resources [START_REF] Groombridge | IUCN Red list of threatened animals[END_REF]. The white-clawed crayfish is also listed in annexes II and V of European Community Directives for the Conservation of Natural Habitats and Wild Flora and Fauna (92/43/EEC and 97/62/EU). This requires the establishment of management plans to conserve both threatened species and their biotope [START_REF] Vigneux | Les introductions de crustacés décapodes d'eau douce en France. Peut-on parler de gestion ?[END_REF].

Over the last two decades, there have been several French studies to characterize the physico-chemical or biological requirements of A. pallipes [START_REF] Arrignon | Population of the crayfish Austropotamobius pallipes pallipes (Lereboullet) in a brook of Corsica (France)[END_REF][START_REF] Grandjean | Distribution and proposal for the conservation of the indigenous freshwater crayfish species, Austropotamobius pallipes pallipes, in a French department[END_REF][START_REF] Grandjean | Life history and population biology of the white-clawed crayfish, Austropotamobius pallipes pallipes, in a brook from The Poitou-Charentes region (France)[END_REF][START_REF] Grandjean | Biological water quality assessment in running waters sites harboring the endangered crayfish, Austropotamobius pallipes based on macroinvertebrates and its potential in habitat selection for restocking[END_REF]NEVEU, 2000a, b;[START_REF] Broquet | Distribution and habitat requirements of the white-clawed crayfish, Austropotamobius pallipes, in a stream from the Pays de Loire region, France: an experimental and descriptive study[END_REF].

Nevertheless, the use of only physico-chemical or biological parameters provides an oversimplified understanding of water quality. The recent combination of chemical and biological methods constitutes a better approach for an integrated assessment of freshwater quality [START_REF] Lazaridou-Dimitriadou M | Contribution to the ecological quality of Aliakmon river (Macedonia, Greece): a multivariate approach[END_REF]. Indeed, ecological indices may have several purposes and give complementary information to those obtained with physico-chemical data. They can be used to assess the condition of the environment or to monitor trends in condition over time. They can provide an early warning signal of changes in environment and they can also be used to diagnose the cause of an environmental problem [START_REF] Dale | Challenges in the development and use of ecological indicators[END_REF]. In this study, the French I.B.G.N. (Indice Biologique Global Normalisé, AFNOR 1992) was chosen to assess water quality based on the occurrence of macroinvertebrates.

The aim of this study is to characterize the ecological requirements (habitat and water quality) of A. pallipes in order to explain its current restricted and fragmented distribution in the Deux-Sèvres French department. Multivariate analyses (Principal Component Analysis) are used to examine the relationships between abiotic and biotic parameters. This paper presents preliminary results conducted on two brooks from this department (Thouet and Verdonnière) since November 2002.

MATERIAL AND METHODS

Study sites

The study was conducted in two French brooks (Thouet and Verdonnière, Deux-Sèvres department) closely located in the Thouet catchment (Figure 1). Two sampling sites were monitored per brook: the first being where the population of white-clawed crayfish was present and the second about 2 km downstream of the population. The Thouet site harbouring crayfish (named site 1) was located 2 km from the source: banks were overgrown by alders (Alnus sp.). The surrounding vegetation was quite important, providing shade. The Thouet site without crayfish (site 2) also had similar vegetation and shade. In the Verdonnière site with crayfish (site 3), the vegetation of the right bank consisted of hazel trees while no vegetation was found on the left bank, thus the stream was more exposed to sunlight than both Thouet sites. The Verdonnière site without A. pallipes (site 4) is located near the confluence of the two studied brooks. The site was fairly sunny, banks were not altered and the vegetation was mainly of alders.

Stones, shingle and coarse gravel were the major substrate in the Thouet site with crayfish. In the Thouet site without crayfish, the substrate was essentially composed of leaf litter, roots and coarse gravel. In the Verdonnière site harbouring the whiteclawed crayfish, stones, shingle and coarse gravel were also predominant. The last site, Verdonnière without crayfish, presented the most heterogeneous substrate with a codominance of litter, roots, stones, shingles, coarse gravel, sand and silt. All these data were summarized in Table I.

Physico-chemical parameters

For each site, eighteen physicochemical parameters were measured twice monthly since November 2002. Methods and analytic technologies applied in this study were normalized. In situ measurements of temperature (T), dissolved oxygen (O 2 ), pH, conductivity (Cond) and redox potential (E) were recorded using a digital meter (WTW) with the appropriate probes. Water turbidity (Turb) was also measured using a HACH Pocket Turbidimeter Cat. No. 52600-00

In the laboratory, water samples were filtered through a 0.45 mm "millipore" membrane and were analysed for Sodium (Na), Potassium (K), Chlorures (Cl), Nitrates (NO 3 ), Nitrites (NO 2 ) and Sulphates (SO 4 ). Total Organic Carbon (TOC), UV (254 nm), fluorimetry (Fluo) and Ammonium (NH 4 ) analyses were carried out on unfiltered samples. Total Suspended Solids (TSS) were measured as the weight of material retained by the fibreglass filters.

Use of Principal Component Analysis (PCA)

Multivariate analyses were applied using Principal Component Analysis (PCA). The analyses were carried out with the SPSS 11.0 for Windows statistical package (SPSS Inc., Microsoft Co.).

Macroinvertebrate sampling and biotic scores

The French IBGN (Indice Biologique Global Normalisé) method (AFNOR, Norm NF T 90-350, 1992), which is based on the occurrence of macroinvertebrates, was adopted to estimate biological water quality. In March 2003, eight samples were taken at each of the four sites using a standard Surber net (surface: 500 cm 2 and mesh size: 500 µm) following [START_REF] Verneaux | Une nouvelle méthode pratique d'évaluation de la qualité des eaux courantes. Un indice biologique de qualité générale (IBG)[END_REF]. The reference-sampling table was used to sample the habitats of each site taking into account the nature of the substrate and the speed of the water. The order of sampling followed the substrate list order, which is based on the abilities of the different substrates to contain organisms ranging from bryophytes first to algae and clay last (Table I). Samples were preserved in 10% formaldehyde. In the laboratory, the macroinvertebrates were sorted using 500 µm mesh sieves and identified to families (with the exception of Hydrozoa, Hydracarina and Oligochaeta).

The I.B.G.N. score for a site ranges on a scale from 0 to 20 where 0 indicates a high level of pollution and 20 no pollution. This score is based on a faunal list restricted to 138 macroinvertebrate taxa of which 38, in nine indicator groups, are indicators of water quality; group 9, including Plecoptera, is the most sensitive to pollution and group 1, including worms is the most tolerant. The score depends on both the number of taxa and the indicator groups recorded from the eight samples.

Substrates Superficial speed V (cm.s -1 > 150 150 > V > 75 75 > V > 25 25 > V 5 V < 5 Bryophyles 9 3 Submerged spermatophyles 8 Litter, roots 7 1 (× 2)-2 (× 3)-4 (× 2) Stones, shingle 250 mm > d > 25 mm 6 1 ( × 3)-3 (× 3)-4 4 Coarse gravel 25 mm > d > 2,5 mm 5 2 ( × 2)-3 (× 2)-4 1 (× 2)-2 4

Comparisons of macroinvertebrate fauna

The Shannon's diversity index (H) was used to estimate habitat diversity at each site [START_REF] Charvet | Les méthodes biologiques d'évaluation de la qualité des eaux basées sur les macro invertébrés benthiques[END_REF]. This index ranged from 0.5 for the least diversified habitat to 4.5 for the most. This index was calculated as follows:

H = (1/N)[N i (Ln N/Ln 2 -Ln N i /Ln 2)]
where N was the total number of individuals of a sampling site and N i the number of individuals of each taxon. The Jaccard's similarity index (i) was used to assess the similarity of macroinvertebrate fauna between two sites [START_REF] Charvet | Les méthodes biologiques d'évaluation de la qualité des eaux basées sur les macro invertébrés benthiques[END_REF]. This index was not only applied to all recorded taxa but also to the most pollution sensitive taxa (Ephemeroptera, Trichoptera and Plecoptera). The Jaccard's similarity index was calculated as follows:

I = 100 × n c /(n i + n j -n c )
where n c was the number of common taxa between two sites i and j, n i the number of taxa of site i and n j the number of taxa of site j.

Trophic guilds

The macroinvertebrate fauna was classified into 8 trophic guilds -shredders, collectors, scrapers, detritivore shredders, grazers, substrate feeders, suckers and predators -to provide insights into the nature of stream disturbance [START_REF] Merrit R | An introduction to the Aquatic Insects of North America[END_REF].

The macroinvertebrate fauna was also classified according to two functional feeding patterns: autochthonous nutrition which was represented by taxa feeding on macroinvertebrates, macrophytes or algae (predators, grazers and substrate feeders) and allochthonous nutrition which was typical of taxa feeding on external supplies (collectors, shredders, detritivorous shredders).

RESULTS

Physico-chemical parameters

In situ and laboratory measurements

Table II summarises chemical and physical measurements for both sites. Averages of each parameter were computed from twice monthly measurements from November 5 th 2002 to May 7 th 2003. Average values of the different parameters were quite similar during the study period from a site to another one. Nevertheless some differences occurred into four parameters: Turbidity, NH 4 , TSS and UV. In both cases, the sites harbouring crayfish presented much lower values than the ones recorded on the site without crayfish. The Verdonnière samples from the site with crayfish presented a lower average for the four considered parameters than did those from Thouet with the white-clawed crayfish. Thouet and Verdonnière sites without A. pallipes showed higher Turbidity, NH 4 , TSS and UV mean values than the sites harbouring crayfish.

Table II

Averages All the analytical variables were well represented. The PCA showed that the first two principal components represented up to 53% of the total variance (PC 1 34%; PC 2 19%) of the observations. Figure 2 gives the projection of the various analytical variables in the principal plane. Component 1 was clearly built from the mineral variables (Na, SO 4 , Cl, Alkalinity, Conductivity, pH, etc...) in accordance with the geological substrates, while component 2 was largely built from the organic variables, except for potassium and for ammonium that was an indicator of domestic and/or agricultural pollution.

Macroinvertebrate fauna

Table III lists the macroinvertebrates found at the four studied sites.

Thouet

The Thouet site containing crayfish yielded 3694 macroinvertebrates from 28 taxa (Table IV). Gammaridae (Crustacea) were dominant and represented 82% of the total number of individuals collected at this site. Six families of Diptera were represented. The E.T.P. (Ephemeroptera-Trichoptera-Plecoptera) group was represented by 10 taxa: 4 families of Ephemeroptera, 5 of Trichoptera and 1 of Plecoptera. Among these orders, Baetidae and Limnephilidae were the most numerous (Table III). In the 28 taxa, 13 were rare (only represented by 1 or 2 individuals). The sample from the site without crayfish on Thouet included 4 095 individuals representing 23 taxa (Table IV). Gammaridae was still the dominant taxon and represented 86% of the total number of individuals of the site. The highest diversity was recorded within the Diptera represented by 6 families. 8 families of Ephemoptera and Trichoptera (4 of each) were found and were represented by 149 individuals. Between these two orders, Baetidae and Limnephilidae were the most numerous (Table III). 7 of the 23 were rare.

Table III List

Verdonnière

The Verdonnière samples from the site with the white-clawed crayfish contained 2 013 individuals within 21 taxa (Table IV). Chironomidae (Diptera) represented 43% of the individuals. The order with the greatest family representation was the Trichoptera with 6 families. The resource of E.T.P. was composed of 12 taxa: 5 families of Ephemeroptera, 6 of Trichoptera and 1 of Plecoptera. Baetidae, Nemouridae and Heptageniidae were the best-represented families (Table III). Only 2 taxa of the 21 recorded were rare. Downstream the white-clawed crayfish population of Verdonnière, 1146 individuals were recorded among 23 taxa (Table IV). Chironomidae were the most numerous taxon and represented 49% of the sampled macroinvertebrates. The highest diversity was again observed within the Diptera (6 families). This site harboured 4 and 6 families of Ephemeroptera and Trichoptera respectively. Within the E.T.P. group, Baetidae, Psychomyiidae and Sericostomatidae were the most sampled (Table III). Among the 23 recorded taxa, 12 were rare.

Table IV

Summary of the main results from macroinvertebrate study from the 4 sites.

Tableau IV Résumé des principaux résultats obtenus par l'étude des macroinvertébrés sur les 4 sites.

Comparisons of macroinvertebrate fauna

The values of Shannon's diversity index applied to Thouet were quite low: 1.22 and 1.02 for site harbouring A. pallipes and site without crayfish respectively (Table IV). Values of 2.36 and 2.1 were recorded on similar sites on Verdonnière (Table IV).

Percentages similarities ranged from 37.50 to 53.33 when Jaccard's similarity index was applied to all taxa (Table V). The two sites without crayfish (53.33%) showed the highest similarity and the two sites of Verdonnière (37.50%) showed the lowest one. Water quality Good Good Good Fair When Jaccard's index was applied to E.T.P. only, higher similarity percentages were recorded (Table V). The highest similarity was 69.23% between the two sites harbouring the white-clawed crayfish. The lowest recorded values were observed between the two sites on the same brook (50% and 46.67% for Thouet and Verdonnière respectively). IV). According to the I.B.G.N. protocol, the two sites of Thouet and the site harbouring crayfish of Verdonnière had good water quality whereas the Verdonnière site without crayfish had fair water quality. For both site of Thouet an indicator group of 7 was recorded; Goeridae (Trichoptera) and Leptophlebiidae (Ephemeroptera) were the most pollution sensitive taxa for Thouet and Verdonnière respectively (Table IV). For the Verdonnière site-harbouring crayfish the most pollution sensitive taxa was Philopotamidae (Trichoptera), which represented an indicator group of 8. The Verdonnière site without crayfish showed an indicator group of 6 and Sericostomatidae (Trichoptera) was the most pollution sensitive taxa.

Trophic guilds

The distribution of individuals by trophic guild is illustrated for each site in Figure 3. Both Thouet sites showed a similar distribution of trophic groups, the dominant group being represented by shredders due to the high number of Gammaridae (85% and 88% respectively).

Percentages of functional feeding groups were more balanced for Verdonnière (Figure 3). In the site harbouring the white-clawed crayfish, three major guilds were distinguished: collectors (46%, Chironomidae, Diptera), grazers (25%, Baetidae, Ephemeroptera) and shredders (24%, Gammaridae, Crustacea). On the downstream site, three major guilds were also observed: collectors (51%, Chironomidae), suckers (29%, Oligochaeta) and shredders (15%, Gammaridae).

The distribution of individuals according to functional feeding groups (autochthonous or allochthonous) is illustrated in Figure 4. For each brook, most macroinvertebrates had an allochthonous feeding pattern due to a high proportion of Gammaridae for Thouet and Chironomidae for Verdonnière.

Figure 3

Distribution of trophic guilds from the macro invertebrate samples in the 4 sites. Figure 5 shows a PCA plot of the variables measured for each sampling point during the study period. Each factor score has been associated to the corresponding I.B.G.N. score. The I.B.G.N. score decreased (arrow) in the direction of an increase in the organic character and to a lesser extent of the mineral character. Figure 6 shows under the same conditions the factor scores in relation to the presence or absence of crayfish. The limit of presence seems to be in the vicinity of an I.B.G.N. score of 13.

Discussion

Both sites harbouring the white-clawed crayfish showed good to fair water quality based on macroinvertebrates with I.B.G.N. scores ranging from 14 to 13. These results are lower than I.B.G.N. scores previously reported by [START_REF] Grandjean | Biological water quality assessment in running waters sites harboring the endangered crayfish, Austropotamobius pallipes based on macroinvertebrates and its potential in habitat selection for restocking[END_REF] for 5 brooks located nearby in the Deux-Sèvres department. These relatively low scores are explained by low taxon diversities of only 28 and 21 taxa. The absence of individuals of indicator groups 8-9 in the Thouet site harbouring the white-clawed crayfish also partly explains the score recorded on this site. This good but not optimal water quality estimate suggests that A. pallipes can live in brooks having sub-optimal water quality. [START_REF] Grandjean | Life history and population biology of the white-clawed crayfish, Austropotamobius pallipes pallipes, in a brook from The Poitou-Charentes region (France)[END_REF][START_REF] Grandjean | Biological water quality assessment in running waters sites harboring the endangered crayfish, Austropotamobius pallipes based on macroinvertebrates and its potential in habitat selection for restocking[END_REF] have already demonstrated this phenomenon in the same area, [START_REF] Broquet | Distribution and habitat requirements of the white-clawed crayfish, Austropotamobius pallipes, in a stream from the Pays de Loire region, France: an experimental and descriptive study[END_REF] The high number of rare taxa (13) on the Thouet site harbouring the white-clawed crayfish shows that this environment is precarious because the score would drop to 11 if these rare taxa disappeared. In 2000 the situation was similar because [START_REF] Delvalle | Étude biologique du Thouet Amont[END_REF] had estimated an I.B.G.N. score of 15 on this site with 15 rare taxa out of 31 total taxa. In the same way, the Verdonnière site without crayfish appears to be precarious presenting 12 rare taxa out of 23. In addition, a high number of Asellidae was recorded indicating a high level of dissolved organic matter. This observation is corroborated by the high values of Turbidity, TSS and TOC measured on this site. On the contrary, Verdonnière site with crayfish shows a very low rate of rare taxa (2/21) indicating a stable state of the environment.

The Shannon diversity index is always higher for sites harbouring A. pallipes. However, there is variation between these sites. Thus, Verdonnière Shannon index value is twice as high as the one recorded in the Thouet (2.36 and 1.22 respectively). The Jaccard's similarity index shows strong taxa similarity between sites of the order of 50%. The similarity rate increases to 69% between the two sites harbouring A. pallipes, when the Jaccard's index is applied to E.T.P. only. This high similarity can be explained by a habitat analogy between these two sites dealing both with superficial speed ranges and substrates (coarse gravel).

Comparisons between the list of taxa of each site show that in both Thouet and Verdonnière Plecoptera, which are very sensitive to organic pollution, are only recorded at sites harbouring the white-clawed crayfish. As in the study of [START_REF] Grandjean | Biological water quality assessment in running waters sites harboring the endangered crayfish, Austropotamobius pallipes based on macroinvertebrates and its potential in habitat selection for restocking[END_REF] (i) sites harbouring A. pallipes contain families of Plecoptera, (ii) there is higher family and individual richness in Trichoptera, Ephemeroptera and Diptera, particularly at sites harbouring the white-clawed crayfish than where A. pallipes is missing and (iii) moreover, all the sites are located near the source and so run through forested areas which would explain the high proportion of allochthonous nutrition. Thus, the occurrence of Plecoptera seems to represent a good criterion for the selection of A. pallipes habitat. Nevertheless, it is difficult to assume that allochthonous organic production characterizes crayfish habitats since, as a general rule, allochthonous production characterizes headstream environments.

Average values of the different physico-chemical parameters were quite similar during the study period (from November 2002 to May 2003). It is now well known that, in France, A. pallipes habitats are generally small forest streams with running water and with a high density of shelters such as stones, gravels and roots [START_REF] Arrignon | Population of the crayfish Austropotamobius pallipes pallipes (Lereboullet) in a brook of Corsica (France)[END_REF][START_REF] Grandjean | Distribution and proposal for the conservation of the indigenous freshwater crayfish species, Austropotamobius pallipes pallipes, in a French department[END_REF][START_REF] Grandjean | Life history and population biology of the white-clawed crayfish, Austropotamobius pallipes pallipes, in a brook from The Poitou-Charentes region (France)[END_REF]NEVEU, 2000a, b). The width of the brooks is often less than 2 m and the depth not more than 1 m. Dissolved oxygen concentrations range from 7 to 10 mgL -1 , water temperatures do not exceed 18 ˚C during summer, pH values are around 8 and waters present high conductivity from 200 to 600 µScm -1 [START_REF] Grandjean | Biological water quality assessment in running waters sites harboring the endangered crayfish, Austropotamobius pallipes based on macroinvertebrates and its potential in habitat selection for restocking[END_REF]. As far as depth and width are concerned, the sites, which were studied here, fit with the ranges noted above. The pH values for sites harbouring the white-clawed crayfish are a bit lower than the ones measured by FOSTER (1995) (7.27-8.64) in central Wales (Great Britain) or even those obtained by GARCIA-ARBERAS and RALLO (2000) (7.95-8.45) in Basque Country (Spain) and by BROQUET et al. (2002) (7.45) in the Pays de Loire région. Even if it is difficult to compare dissolved oxygen measurements from different studies because this parameter is related to water temperature depending on time of day, season etc., our findings agree with those previously recorded [START_REF] Foster | Factors influencing the distribution and abundance of the crayfish Austropotamobius pallipes (Lereboullet) in Wales and the Marches, UK[END_REF][START_REF] Broquet | Distribution and habitat requirements of the white-clawed crayfish, Austropotamobius pallipes, in a stream from the Pays de Loire region, France: an experimental and descriptive study[END_REF]. It seems also to be difficult to compare conductivity to previous studies because of its tight relation to geological substrate. Nevertheless, our conductivity values are lower than the optimal range 200-600 µScm -1 . Our TSS values are also higher than the ones presented by [START_REF] Broquet | Distribution and habitat requirements of the white-clawed crayfish, Austropotamobius pallipes, in a stream from the Pays de Loire region, France: an experimental and descriptive study[END_REF] (7.0 mgL -1 ). TOC values appear to be higher than those measured by FOSTER (1995) (2.4-3.28 mgL -1 ). Values of TH obtained on the two sites harbouring the white-clawed crayfish are higher than the ones recorded by FOSTER (1995) (36.3 mgL -1 CaCO 3 ) on such sites. NH 4 concentrations are about twice as high in the Thouet site with crayfish than in Verdonnière similar site owing to the supply of leaves from the banks. Nevertheless, these concentrations are much higher than the ones recorded by FOSTER (1995) (0.007 mgL -1 ) and lower than the ones presented by GARCIA-ARBERAS and RALLO (2000) (0.17 ± 0.19 mgL -1 ). As far as anions (Cl, SO 4 , NO 3 and NO 2 ) are concerned, it is noticeable that in the case of Verdonnière NO 3 concentration is higher in the site with crayfish. Cl and NO 3 concentrations are much higher than the ones found by FOSTER (1995) (0.58-37.3 mgL -1 and 0.57-4.20 mgL -1 ) and by [START_REF] Garcia-Arberas | Survival of natural populations of Austropotamobius pallipes in rivers in Biscay, Basque country (north of Iberian peninsula)[END_REF]. NO 2 are low in all references. SO 4 values are a bit lower than the ones recorded by FOSTER (1995) (8.77-23.6 mgL -1 ). Na and K concentrations in the sites harbouring A. pallipes appear similar to those surveyed in Basque Country by [START_REF] Garcia-Arberas | Survival of natural populations of Austropotamobius pallipes in rivers in Biscay, Basque country (north of Iberian peninsula)[END_REF] (10.34 ± 3.53 mgL -1 and 2.44 ± 2.96 mgL -1 respectively) but K concentrations are higher than K concentrations presented by FOSTER (1995) (0.25-2.5 mgL -1 ).

Our results, as well as those of other authors [START_REF] Laurent | Une station d'écrevisses à pieds blancs: Austropotamobius pallipes Lere. (Decapoda-Astacidae) en zone périurbaine[END_REF][START_REF] Foster | Toxicity of field simulated farm waste episodes to the crayfish Austropotamobius pallipes (Lereboullet): elevated ammonia and reduced dissolved oxygen concentrations[END_REF][START_REF] Troschel | Distribution and ecology of Austropotamobius pallipes in Germany[END_REF][START_REF] Broquet | Distribution and habitat requirements of the white-clawed crayfish, Austropotamobius pallipes, in a stream from the Pays de Loire region, France: an experimental and descriptive study[END_REF], show that it is difficult to establish links between the physico-chemical parameter levels and the presence or absence of crayfish. However, our results examined by PCA, reveal distinctly the predominance of crayfish according to these physico-chemical variables. The combination of chemical and biological methods constitutes a recent approach for an integrated assessment of freshwater quality. Greek studies [START_REF] Lazaridou-Dimitriadou M | Contribution to the ecological quality of Aliakmon river (Macedonia, Greece): a multivariate approach[END_REF][START_REF] Kampa | Ecological quality of the River Axios (N. Greece) during spring and summer[END_REF] have already shown that correlations between abiotic and biotic parameters using multivariate analyses could give a good and global assessment of freshwater quality. Finally, the clusters corresponding to the various sites are ordered according to I.B.G.N. score. This highlights, for the first time, the relationship existing between the physiochemical characteristics of water on the one hand, and the biological characteristics of the aquatic environment on the other hand.

CONCLUSION

As A. pallipes presence could be predicted in part by factors related to water quality but their absence or abundance could not be connected to water quality, it is becoming more and more difficult to consider this species as a bio indicator [START_REF] Demers | The water quality requirements of white-clawed crayfish, Austropotamobius pallipes (Lereboullet)[END_REF]. This subject has also been discussed in a roundtable summarized in this present issue. It appears that a good correlation could exist between abiotic and biotic parameters and the study of more brooks could show that I.B.G.N. scores could be predicted using a PCA analysis. These first results have also shown that mineral matter did not seem to be a discriminating factor in A. pallipes presence or absence whereas organic matter appeared to be a better discriminating factor. Nevertheless, these assumptions will have to be confirmed by the study of seven other brooks from the same area actually in progress.

  Figure 1Location of the two brooks sampled in the Deux-Sèvres département (France). The 4 sampled sites are reported as follows: 1, Thouet with crayfish; 2, Thouet without crayfish; 3, Verdonnière with crayfish and 4, Verdonnière without crayfish.

Figure 1

 1 Figure 1 Localisation des deux cours d'eau étudiés dans le département des Deux-Sèvres (France). Les 4 sites échantillonnés sont reportés comme suit : 1, Thouet avec écrevisses ; 2, Thouet sans écrevisse ; 3, Verdonnière avec écrevisses et 4, Verdonnière sans écrevisse.

  (± standard deviation) and ranges of physical and chemical parameters measured on each sites between November 5 th 2002 and May 7 th 2003. Tableau II Moyennes (± écarts-types) et étendues des paramètres physiques et chimiques mesurés sur chacun des sites entre le 5 novembre 2002 et le 7 mai 2003.

Figure 2

 2 Figure 2Plotting, with the use of PCA, of 16 experimental variables measured to the plane defined by the first two components.

Figure 2

 2 Figure 2 Représentation, à l'aide d'une ACP, de 16 variables expérimentales mesurées sur les ruisseaux du Thouet et de la Verdonnière selon le plan défini par les deux premières composantes.

  of macroinvertebrates (8 samples) leading to the calculation of I.B.G.N. score for each site. IG = Indicator Group. Table III Liste des macroinvertébrés (8 prélèvements) servant au calcul de la note I.B.G.N. pour chacun des sites. IG = Groupe Indicateur.

  faune de macro invertébrés entre les 4 sites à l'aide de l'indice de similarité de Jaccard. Les chiffres en gras ne tiennent compte que de la faune d'Éphéméroptères, de Trichoptères et de Plécoptères. Les valeurs sont exprimées en pourcentages. I.B.G.N. scores I.B.G.N. scores ranged from 12 to 14 (Table

  Figure 3Répartition des macroinvertébrés selon les différentes guildes trophiques dans les 4 stations.

Figure 4

 4 Figure 4Distribution of macro invertebrates from the 4 sites according to their functional feeding groups: allochthonous or autochthonous.

Figure 4

 4 Figure 4Répartition des macroinvertébrés des 4 stations en fonction de leur mode de nutrition allochtone ou autochtone.

  in the Pays de Loire région (France) and DEMERS and REYNOLDS (2002) in Ireland.

Figure 5

 5 Figure 5 Plotting, with the use of PCA, of I.B.G.N. scores of the Thouet and Verdonnière brooks according to the plane defined by the first two components. The arrow direction shows an increase in the organic matter character. Figure 5 Représentation, à l'aide d'une ACP, des notes I.B.G.N. sur les ruisseaux du Thouet et de la Verdonnière selon le plan défini par les deux premières composantes. La direction de la flèche indique une augmentation du facteur matière organique.

Figure 6

 6 Figure 6 PCA plot of the I.B.G.N. scores for the Thouet and Verdonnière brooks in accordance with the white-clawed crayfish presence or absence.

Figure 6

 6 Figure 6 ACP des notes I.B.G.N. des ruisseaux du Thouet et de la Verdonnière en fonction de la présence ou de l'absence de l'écrevisse à pattes blanches.

Table I Experimental protocol of sampling according to the I.B.G.N. method. The 8 samples per sites are reported in this table for the 4 studied sites: 1, Thouet with crayfish; 2, Thouet without crayfish; 3, Verdonnière with crayfish and 4, Verdonnière without crayfish. Numbers in brackets indicate the number of samples taken from a same substrate for each site. Tableau I Protocole expérimental de prélèvement selon la méthode de l'I.B.G.N. Les 8 prélèvements pour les 4 sites sont représentés dans ce tableau : 1, Thouet avec écrevisses ; 2, Thouet sans écrevisse ; 3, Verdonnière avec écrevisses et 4, Verdonnière sans écrevisse. Les chiffres entre parenthèses indiquent le nombre de prélèvements effectués sur un même substrat pour chacun des sites.

 I

Principal Component Analysis(PCA)

ACKNOWLEDGEMENTS

Special thanks are due to Julian Reynolds for his useful advice and suggestions. This work was supported by a doctoral fellowship provided by the European Programme LEADER + (La Gâtine, Château d'eau du Poitou) and was funded by the CPER (Contrat de Plan État Région) entitled "Programme transversal Eaux", University de Poitiers.


F Grandjean
email: frederic.grandjean@univ-poitiers.fr

BIOLOGICAL WATER QUALITY ASSESSMENT OF THE WHITE-CLAWED CRAYFISH HABITAT BASED ON MACROINVERTEBRATE COMMUNITIES: USEFULNESS FOR ITS CONSERVATION

Keywords: Austropotamobius pallipes, macroinvertebrates, biotic indices, rivers, water quality

published or not. The documents may come     Biological water quality assessment of the white-clawed crayfish habitat based on macroinvertebrate communities: Usefulness for its conservation

ÉTUDE DE LA QUALITÉ BIOLOGIQUE DE L'EAU DE L'HABITAT DE L'ÉCREVISSE À PATTES BLANCHES, BASÉE SUR LES COMMUNAUTÉS DE MACROINVERTÉBRÉS :

UTILITÉ POUR SA CONSERVATION

RÉSUMÉ

Une étude des macroinvertébrés de trois cours d'eau hébergeant des populations d'écrevisse à pattes blanches a été menée dans le département de la Haute-Loire (France). Son but est d'améliorer notre compréhension de ces écosystèmes pour aider à la conservation d'A. pallipes. Ces ruisseaux traversent des zones pastorales, bordés par une ripisylve bien développée qui offre un ombrage important. Les valeurs concernant la température de l'eau, la concentration d'oxygène dissous, le pH et la conductivité se placent dans la gamme de tolérance de cette espèce. Un total de 34, 31, 29 taxons et 1 502, 1 364, 2 707 individus ont été collectés pour les stations d'Holme, de Besque et de Bellecombe, respectivement. Les résultats ont montré une qualité de l'eau de bonne a très bonne avec des notes IBGN comprises entre 15 (Bellecombe) et 17 (Holme et Besque), reflétant un impact limité des perturbations d'origine anthropique. La diversité taxonomique est forte pour les stations d'Holme et Besque avec un indice de Shannon autour de 3,2 traduisant une grande hétérogénéité d'habitat et un peuplement faunistique équilibré. Bellecombe a montré une diversité limitée avec un indice de Shannon de 1,42, résultant du nombre important de Chironomidae. Ce ruisseau souffre probablement d'une légère pollution organique laquelle pourrait être reliée aux faibles vitesses de courant observées pendant l'échantillonnage. Les tests de similarité selon l'indice de Jaccard ont montré de forts pourcentages de taxons en commun parmi les ETP (Ephémères-Trichoptères-Plécoptères) entre tous les sites. Cette forte similarité des communautés de macroinvertébrés benthiques pourrait être un critère utile pour identifier des ruisseaux en vue de repeuplement.

Mots-clés :

Austropotamobius pallipes, macroinvertébrés, indices biotiques, rivière, qualité d'eau.

INTRODUCTION

The white-clawed crayfish Austropotamobius pallipes lives in clean, well-oxygenated calcium-rich water that do not contains too much sediment. They are considered to be sensitive to physical and chemical alterations of their habitat [START_REF] Verneaux | Une nouvelle méthode pratique d'évaluation de la qualité des eaux courantes. Un indice biologique de qualité générale (IBG)[END_REF]. As a result of degradation of water and habitat quality, A. pallipes, which was formely widespread in Europe and particularly in France, is now confined to headwater with welldeveloped riparian cover [START_REF] Vigneux | Les introductions de crustacés décapodes d'eau douce en France. Peut-on parler de gestion ?[END_REF][START_REF] Holdich | Distribution of crayfish in Europe and some adjoining countries[END_REF]. For example, among the 178 populations of A. pallipes recorded within Poitou-Charentes rivers system (France) in 1978, only 38 persist at the present time (Authors, unpublished data). Because of its decline, A. pallipes has been listed as vulnerable on the red list of threatened animals of the International Union for the Conservation of Nature and Natural Resources (I.U.C.N.) [START_REF] Groombridge | IUCN Red list of threatened animals[END_REF], and is also listed in annexes II and IV of European Community Directives for the Conservation of Natural Habitats and Wild Flora and Fauna (92/43/EEC and 97/62/EU) as a species requiring special conservation measures. Among the conservation measures listed by [START_REF] Dieguez-Uribeondo | A plan of restoration in Navarra for the native freshwater crayfish species of Spain, Austropotamobius pallipes[END_REF], the preservation and the re-establishment of the populations of A. pallipes within its habitat range is a necessary prerequite to its conservation. This requires a good knowledge of crayfish habitat in terms of water quality but also in terms of ecological condition. Indeed, knowledge of physical and chemical characteristics provides little information about the ecological state of rivers and does not provide a consistent assessment of river condition. Characterizing aquatic ecosystems using biological criteria enables a more robust interpretation of ecological condition than using simple water chemical quality alone [START_REF] Lazaridou-Dimitriadou M | Contribution to the ecological quality of Aliakmon river (Macedonia, Greece): a multivariate approach[END_REF]. In freshwater ecosystems, a wide variety of biotic groups is used for biomonitoring the state of ecosystems. According to [START_REF] Mandaville | Limnology-eutrophication and chemistry, carrying capacities, loadings, benthic ecology and comparative data; Project F-1, soil & water Conservation society of Metro Halifax[END_REF], the macroinvertebrates are the most popular group. There are many reasons which explain this craze. Benthic macroinvertebrates are found in all aquatic environments from thermal springs to salt lakes. These communities include worms, snails, water mites, leeches, small crustacean, and insect larvae, inhabit the sediment or live on or in other bottom substrates in the aquatic environment [START_REF] Klemm | Macroinvertebrate field and laboratory methods for evaluating the biological integrity of surface waters[END_REF]. They are an important food source for aquatic predator such as fish, amphibians and crayfish. They exploit the physical characteristics of streams to obtain their food (MERITT and CUMMINS, 1996) and affect nutrient availability and plant productivity in the water. Their community structure responds to water quality perturbations in a predictable fashion. The presence of some species is indicative of clean, unpolluted waters, while other species thrive in polluted waters. Because of these characteristics, benthic invertebrates are commonly used to evaluate the ecological integrity of streams and as indicators of water quality [START_REF] Barbour | Rapid bioassessment protocols for use in streams and wadeable rivers: Periphyton, Benthic macroinvertebrates, and fish[END_REF].

Recently, a number of studies have evaluated the suitability of macroinvertebrates to characterize the water quality and habitat of A. pallipes brooks by the use of specific French biotic index called IBGN (see materials & methods) [START_REF] Grandjean | Distribution and proposal for the conservation of the indigenous freshwater crayfish species, Austropotamobius pallipes pallipes, in a French department[END_REF][START_REF] Grandjean | Life history and population biology of the white-clawed crayfish, Austropotamobius pallipes pallipes, in a brook from The Poitou-Charentes region (France)[END_REF][START_REF] Grandjean | Biological water quality assessment in running waters sites harbouring the endangered crayfish, Austropotamobius pallipes based on macroinvertebrates and its potential in habitat selection for restocking[END_REF][START_REF] Broquet | Distribution and habitat requirements of the white-clawed crayfish, Austropotamobius pallipes, in a stream from the Pays de Loire region, France: an experimental and descriptive study[END_REF]. They reported that crayfish, formely considered as bio-indicator, can live in brooks having sub-optimal water quality.

In this study, the taxonomic composition of benthic macroinvertebrates in three brooks harbouring A. pallipes is determined. The aim of study is to help for its conservation in three ways (1) to watch the evolution of crayfish sites over time by monitoring of the global water quality of these ones; (2) to prevent the risk of disappearance of crayfish population in pointing out a habitat degradation and (3) to collect informations which could be used to select suitable habitat in restocking plans in a given ecoregion.

MATERIALS AND METHODS

Physical characteristics of studied sites

Samples were collected in three brooks that runs through the Haute-Loire department. The crayfish sites were in first order streams located less than 3km from the stream source (Table I). A description of few physical and chemical parameters of each sites was made (habitat description, water temperature, dissolved oxygen concentration, pH, and conductivity).

Macroinvertebrate sampling

Sampling followed the I.B.G.N. protocol [START_REF] Afnor | Qualité de l'eau, Recueil des Normes Françaises[END_REF]. Eight samples of macroinvertebrates were taken at each of the 3 sites using a standard Surber sampler (surface 500 cm 2 , mesh size 500 µm) according to the protocol established by [START_REF] Verneaux | Une nouvelle méthode pratique d'évaluation de la qualité des eaux courantes. Un indice biologique de qualité générale (IBG)[END_REF]. Samples were placed in a plastic bag and preserved in 10% formaldehyde. The habitats at each site were sampled according to a reference sampling table taking into account the nature of the substrate and water speed (Table II). Substrates are classified according to their ability to contain organisms (Table II) from bryophytes first, to algae and clay, last. The order of sampling followed the substrate list order. In the laboratory, the animals were sorted using a 500 µm mesh sieve and identified taxonomically to family level (apart from Hydrozoa, Hydracarina and Oligochaeta). Algae or clay 0 Biological Indices Various biotic index system have been developed which give numerical scores to specific indicator organisms at particular taxonomic level [START_REF] Armitage | The performance of a new biological water quality score system based on macroinvertebrates over a wide range of unpolluted running water sites[END_REF]. The following indices were computed.

Shannon-Wiener diversity index

The Shannon-Wiener diversity index is one of the most widely used species diversity indices for examining overall community characteristics. This diversity measure is based on information theory; simply, the measure of order (or disorder) within a particular system.

In the present study, this order could be characterized by the number of species and/or the number of individuals in each species, within the sample plots.

s H′ = -Σ (p i ) (log 2 p i ) i=1
where H′ = Index of species diversity; s = Number of species; pi = Proportion of total sample belonging to ith species; When properly manipulated, it will always result in a diversity value (H') ranging between 0 (indicating low community complexity) and 4 (indicating high community complexity). This index was performed for all sites.

Jaccard similarity inde

Using Jaccard's Index with species richness data yields values ranging from 0 to 1, reflecting the proportion of common species between the two samples. The Jaccard Index was also used which is a measure of similarity. This equation is: J = W/(A + B -W). Where A = number of taxa in community A; B = number of taxa in community B; W = number of taxa in common. This index was computed using the most sensitive taxa to pollution: Ephemeroptera, Plecoptera and Trichoptera.

ETP index

The Ephemeroptera, Plecoptera and Trichoptera (EPT) index displays the taxa richness within the insect groups which are considered to be sensitive to pollution and therefore should increase with increasing of water quality. The EPT index is equal to the total number of families represented within these three orders in the sample.

Ratio of ETP/C

The abundance of EPT and C (Chironomidae) indicates the balance of the community, since EPT are considered to be more sensitive and Chironomidae less sensitive to environmental stress [START_REF] Plafkin | Rapid bioassessment protocols for use in streams and rivers: Benthic macroinvertebrates and fish[END_REF]. The EPT/C index is calculated by dividing the sum of the total number of individuals classified as Ephemeroptera, Plecoptera, and Trichoptera by the total number of individuals classified as Chironomidae.

Trophic guilds

The animals were assigned to a particular functional feeding group according to the classification given by MERRITT and CUMMINS (1996).

Biotic index

The I.B.G.N. biotic index, developed for French streams, is the most appropriate biotic index of water quality for this study. The I.B.G.N. faunal list is restricted to 138 taxa of which 38, in nine indicator groups, are indicators of water quality. Group 9, including Plecoptera, is the most sensitive to pollution; group 1, including worms which can live in highly polluted, turbid waters with little oxygen, is the most tolerant to pollution. The invertebrate samples reflect the biological water quality on a scale of 0-20, where 0 indicates pollution and 20 no pollution. The I.B.G.N. score for a site depends on both the number of taxa and the indicator groups recorded from the 8 samples.

RESULTS

Sites description

Physical and chemical characteristics are given in the Table I. The three brooks are located in similar geomorphologic and hydrological settings. They run through pastoral environment with a riparian zone well developed offering important shade. The river beds do not have aquatic macrophytes. Bryophytes are present in all sites. In summer, the surface flow is low, especially for Bellecome (Table II). The water temperatures are cold for all brooks, falling within the range of 12.1 to 14.1 ˚C. The water is well oxygenated with dissolved oxygen concentration of 8.1, 8.8 and 9 mg/l O 2 for Besque, Holme and Bellecombe, respectively. The streams have similar characteristics in terms of depth and width (Table I). The substrat is relatively diversified for all of them (Table II). Stones are dominant in Holme and Bellecombe (70% of recovery) whereas sand is the substrate the more represented in Besque (60%).

Taxonomic composition of benthic macroinvertebrates

The number of individuals and taxa range from 1 364 to 2 750, and from 29 to 34, respectively (Table III). A total of 1 502 specimens represented 34 taxa in Holme, with Chironomidae (Diptera: 598 individuals) the dominant taxon. The Diptera, Trichoptera and Ephemeroptera orders were the most family-rich, with 9, 7 and 5 families respectively (Table IV). Among the most sensitive taxa were two families of Plecoptera, Leuctridae and Nemouridae. The sample from Bellecombe included 2 750 individuals representing 29 taxa. The family of Chironomidae was the most frequently represented, with 2207 individuals. The orders with the greatest family representation were the Diptera and Ephemeroptera with 5 families each (Table IV). Two families of Plecoptera, Leuctridae and Nemouridae were also recorded. The Besque samples contained 1 364 individuals with 381 Chironomidae, 312 Gammaridae (Crustacea) and 132 Baetidae (Ephemeroptera) representing 31 taxa. The Diptera had the greatest number of families with 8, followed by Ephemeroptera with 5 (Table IV). A high diversity of plecopteran families was also recorded with Nemouridae (104 ind.), Leuctricidae (11 ind.), Perlidae (4 ind.) and Perlodidae (1 ind.) (Table IV).

Biological indices

Shannon diversity values for the three sites are given in the Table III. This index values ranged from 1.42 to 3.29 for all sites harbouring crayfish. High diversity were observed within Besque and Holme, with a value of 3.29 and 3.19 respectively whereas Bellecombe showed a low diversity with a value of 1.42 suggesting the presence of

DIPTERA Anthomyidae 3 Atherycidae X 1 1 Ceratopogonidae X 1 3 Chironomidae 1 598 2207 381 Dixidae X 1 Dolichopodidae X 1 Empididae X 10 4 Limoniidae X 18 1 37 Psychodidae X 16 1 Ptychopteridae X 1 Simuliidae X 33 3 107
Tabanidae X 4 dominant taxa. Indeed the most abundant taxa, Chironomidae, comprised > 80% of the macroinvertebrate assemblage in Bellecombe. The similarity test according to the Jaccard index varied greatly from 0.59 to 0.79. Holme and Besque have 79% of common taxa whereas percentages were 70 and 59 with Bellecombe respectively. The ETP values are ranged from 11 to 14. Bellecombe presented the lower number of ETP taxa with 11. Besque and Holme harboured 13 and 14 ETP taxa. The ETP/C ratio was high for Besque and Bellecombe more than 0.76 and low for Bellecombe with 0.13. The distribution of individuals into functional feeding groups is illustrated in Figure 1. Shredders (mainly Gammaridae), collectors (Hydropsychidae, Simulidae and Chironomidae) and grazers (mainly Baetidae) were well represented in Holme and Besque. Gathering collectors were major components at Bellecombe representing 83% of total guilds. These consumers were 4 and 6 times more abundant in Bellecombe than Besque and Holme respectively. III). Following the I.B.G.N. protocol, the Holme and Besque sites had very good water quality whereas Bellecombe had good quality water.

DISCUSSION

The three sites harbouring the white-clawed crayfish showed good to excellent water quality based on macroinvertebrate sampling, with IBGN scores ranging from 15-17, reflecting an absence or limited impact of the anthropogenic disturbances. These results fell within the ranges of IBGN value generally reported for others French crayfish sites [START_REF] Grandjean | Distribution and proposal for the conservation of the indigenous freshwater crayfish species, Austropotamobius pallipes pallipes, in a French department[END_REF][START_REF] Grandjean | Life history and population biology of the white-clawed crayfish, Austropotamobius pallipes pallipes, in a brook from The Poitou-Charentes region (France)[END_REF][START_REF] Grandjean | Biological water quality assessment in running waters sites harbouring the endangered crayfish, Austropotamobius pallipes based on macroinvertebrates and its potential in habitat selection for restocking[END_REF][START_REF] Broquet | Distribution and habitat requirements of the white-clawed crayfish, Austropotamobius pallipes, in a stream from the Pays de Loire region, France: an experimental and descriptive study[END_REF]. These other studies confirmed that A. pallipes can live in brooks having sub-optimal water quality. The high I.B.G.N. scores seem to indicate that these crayfish population are not actually threatened by degradation of water quality because they are largely higher than the lowest value of 13 estimated for several French brooks [START_REF] Grandjean | Life history and population biology of the white-clawed crayfish, Austropotamobius pallipes pallipes, in a brook from The Poitou-Charentes region (France)[END_REF][START_REF] Grandjean | Biological water quality assessment in running waters sites harbouring the endangered crayfish, Austropotamobius pallipes based on macroinvertebrates and its potential in habitat selection for restocking[END_REF][START_REF] Broquet | Distribution and habitat requirements of the white-clawed crayfish, Austropotamobius pallipes, in a stream from the Pays de Loire region, France: an experimental and descriptive study[END_REF]. Recently, GRANDJEAN (unpublished data) estimated a IBGN score of 10 for a brook harbouring the white-clawed crayfish. This low value is explained both by the low taxa diversity and the absence of high indicator groups (9-8-7). At the sampling date, this population seemed to be in good health (no pathogens) with large individuals reflecting a good growth rate (126 mm of total size for the larger male). The occurrence of this species in waters of fair quality was considered surprising. However, four months later, field operations have revealed the disappearance of the population. The brook suffered probably of organic pollution revealed by IBGN before the crash of crayfish population. This dramatic example showed nevertheless that IBGN could provide an warning signal of changes in environment. Moreover, it is suggested that a IBGN score of 10 was not enough to maintain the crayfish population.

The analysis of several metrics such as Shannon diversity and Jaccard indices, ETP taxa richness showed that Besque and Holme have similar faunal characteristics. They have a high macroinvertebrates diversity around 3.2 resulting from equilibrated faunal community. A high number of macroinvertebrates were collected from Bellecombe, but diversity among the taxa was much lower around 1.4. These results could be related to the lack of heterogeneity of habitats. Even if the number of subtrates sampled in this study is relatively similar among brooks, the water flow was more heterogenous in Holme and Besque than in Bellecombe where 7 samples have been made in the same water speed (V < 5 cm s-1 ). As reported by STATZNER and HIGLER (1986), the hydrologic regime highly affected the presence of taxa clearly bound to water flow. For example Hydropsychidae was absent in Bellecombe but present in Holme and Besque. The Jaccard index on ETP taxa is ranged from 0.59 between Bellecombe and Besque to 0.79 among Holme and Besque. This high value of similarity indices based on the most intolerant macroinvertebrates in crayfish streams should allow them to be used to select habitats for restocking. This results are not in accordance with the considerable variation in macroinvertebrate community found by [START_REF] Grandjean | Biological water quality assessment in running waters sites harbouring the endangered crayfish, Austropotamobius pallipes based on macroinvertebrates and its potential in habitat selection for restocking[END_REF] among five white-clawed crayfish sites located in Poitou-Charentes region. These authors found a lowest Jaccard index around 0.27. However, this low similarity of benthic community could be explained by the difference of water quality among the sites because one presented a IBGN score of 13 and the other 19. In this study, the IBGN score were similar or close for the three sites.

As expected in the headwater stream with well-developed riparian cover as studied here, where detritiral inputs were a significant food source, the shredding macroinvertebrates (Gammaridae, Nemouridae, Limnephilidae) are very abundant in Holme and Besque. This results are in accordance with those obtained by [START_REF] Grandjean | Life history and population biology of the white-clawed crayfish, Austropotamobius pallipes pallipes, in a brook from The Poitou-Charentes region (France)[END_REF][START_REF] Grandjean | Biological water quality assessment in running waters sites harbouring the endangered crayfish, Austropotamobius pallipes based on macroinvertebrates and its potential in habitat selection for restocking[END_REF] which found percentages of shredders ranged from 37% to 77% on six French brooks. A higher density of gathering collectors (Oligochaeta, Chironomidae) was particularly observed in Bellecombe with a ratio EPT/C extremely low compared to the others. According to [START_REF] Bis | Effects of catchment properties on hydrochemistry, habitat complexity and invertebrate community structure in a lowland river[END_REF], a high proportion of collectors is related to the presence of fine particulate organic matter which were probably an important source of energy and nutrients in Bellecombe and therefore strongly influenced the functional organization of macroinvertebrate community. This result suggested that Bellecombe is affected by weak organic pollution which could be related to the low water flow observed in this brook.

In general context to select suitable habitat for restocking, ours results confirmed the hypotheses drawn by [START_REF] Grandjean | Biological water quality assessment in running waters sites harbouring the endangered crayfish, Austropotamobius pallipes based on macroinvertebrates and its potential in habitat selection for restocking[END_REF], namely that the presence of Plecoptera seems to be an excellent criterion for the selection of suitable sites for restocking crayfish. The three sites harboured at least two families of Plecoptera; The heterogeneity of substrates seem to be also a key characteristic of good crayfish habitat (NEVEU, 2000a, b). The high number of both Ephemeroptera and Diptera families also reflected the good quality of sediments. The shredders are well represented in crayfish brook. This result indicates that crayfish habitats are characterized by allochthonous organic production from beyond the edge of the stream. All these streams, near their sources, run through forest which contributes much organic leaf material. To conclude, even if the status of A. pallipes as bioindicator is questioned (see roundtable), its presence requires nevertheless a preserved environment to human activities with a good quality of water and habitat.

  the IBGN method. Eight samples are taken per site: 1, Holme; 2, Bellecombe and 3, Besque. Tableau II Grille d'échantillonnage de la méthode IBGN. 8 prélèvements par site sont effectués : 1, station d'Holme ; 2, station de Bellecombe et 3, station de Besque.

Figure 1

 1 Figure 1Distribution of trophic guilds within the samples of macroinvertebrates from three studied sites.

  Figure 1Distribution des guildes trophiques dans les échantillons de macroinvertébrés des trois sites étudiés.

Table I Main physical and chemical parameters of studied sites. Tableau I Principaux paramêtres physiques et chimiques des sites étudiés. Holme Besque Bellecombe

 I

	Stream order	I	I	I
	Depth (m)	0.05-0.40	0.05-0.60	0.05-0.40
	Width mean (m)	2.3	2.28	2.9
	Water temperature (˚C)	12.4	14.1	12.9
	Bryophytes	present	present	present
	Sand	20%	60%	10%
	Gravels	10%	10%	10%
	Stones	70%	30%	70%
	Aquatic macrophytes	none	none	none
	Canopy cover	50-80%	50-80%	50-80%
	Fine sediment "Mud"	none	none	10%
	Surrounding	pastoral area	pastoral area	pastoral area

Table III Values of biologial and biotic indices used in this study. Tableau III Valeurs des indices biologiques et biotiques utilisés dans cette étude. Holme Bellecombe Besque

 III

	Total taxa richness	34	29	31
	Number of individuals	1 502	2 750	1 364
	EPT taxa richness	14	11	13
	EPT abundance	0.26	0.106	0.25
	EPT/C	0.76	0.13	0.9
	Shannon diversity	3.19	1.42	3.29
	IBGN score	17	15	17
	Water quality	very good	good	very good

Table IV Faunal lists of the main invertebrate orders found in the 3 studied sites (IG : Indicator Group for each taxa). Tableau IV Listes faunistiques des principaux ordres de macroinvertébrés trouvés dans les 3 stations étudiées (GI : Groupe Indicateur de chaque taxon).

 IV

		IG	Holme	Belle	Besque
	PLECOPTERA
	Leuctridae	7	2 0	2 5	2 5
	Nemouridae	6	7 3	7	3
	Perlodidae	9			1
	Perlidae	9			4
	TRICHOPTERA
	Brachycentridae	X	4
	Hydropsychidae	3	2 8		9
	Rhyacophilidae	4	1 7	5	1 3
	Polycentropodidae	4	4	1 2
	Sericostomatidae	6	4	2
	Glossosomatidae	7			1
	Philopotamidae	8	1
	Limnephilidae	3	6	7	3
	EPHEMEROPTERA
	Baetidae	2	177	99	132
	Leptophlebiidae	7	6	95	18
	Heptageniidae	5	1	16	7
	Ephemeridae	6	4 6	7	1 2
	Ephemerellidae	3	7	19	28


Dandan Yao

Vincent Hugues

Mireille Blanchard-Desce
email: m.blanchard-desce@ism.u-bordeaux1.fr

Olivier Mongin

Christine O Paul-Roth
email: christine.paul@insa-rennes1.fr

Frédéric Paul
email: frederic.paul@univ-rennes1.fr.

Dendritic Molecular Assemblies for Singlet Oxygen Generation: meso-Tetraphenylporphyrin-Based Biphotonic Sensitizers with Remarkable Luminescence

Four new TPP-based chromophores (1-4) peripherally functionalized with dendrons containing 2-fluorenyl groups were studied for their potential to serve in photodynamic therapy. Their linear and nonlinear optical properties were derived. With significant TPA cross-sections at 790 nm, good singlet oxygen generation capabilities and relatively large intrinsic fluorescence, sensitizers such as 1, might become particularly appealing for theranostics.

Due to their large occurrence in nature and biological importance for life, porphyrins have attracted considerable attention as chromophores for various uses connected with living organisms. 1 In particular, their capability to generate singlet oxygen ( 1 O 2 ) upon photoexcitation has been abundantly studied for applications in photodynamic therapy (PDT), [2][3][4] leading for instance to the clinical use of selected porphyrin derivatives such as photofrin ® or visudyne ® . 5,6 More recently, advances in this field focused in the development of chromophores possessing larger two-photon absorption (TPA) cross-sections and likely to be excited with NIR lasers, thereby allowing for enhanced penetrability in tissues of the exciting beam, better spatial control of the 1 O 2 generation and overall lower photodamages to organisms compared to visible excitation. 5-9, # In this connection, porphyrin arrays with TPA cross-sections and impressive photosensitizing activities have been reported. [14][15][16][17] However, for these compounds the TPA enhancement mostly results from quasi-resonance of the exciting beam with the one-photon absorbing (OPA) Q state at low energy. [17][18][19] In order to obtain photosensitizers without any interfering OPA state, ‡ another strategy was also explored. It consisted in functionalizing the porphyrin core with good two-photon absorbers at the periphery. 20 For such derivatives, the porphyrin-centered excited state active for oxygen photo-sensitizing can then be populated by resonant energy transfer (FRET) following biphotonic excitation of the TPA chromophores. Molecular assemblies with [21][22][23][24][25][26] dyadic [27][28][29] or even more complex architectures [30][31][32][33] were successfully tested in this context.

1 A C C E P T E D M A N U S C R I P T dendritic,
An even more recent concern in this field is to simultaneously perform imaging and curing of living tissues in a dual therapic and diagnostic (also called "theranostic") approach. 20,34,35 This turned out to be a quite challenging task to achieve with porphyrin-based photosensitizers, since the lowest (Q) excited states of porphyrin (which actually sensitizes oxygen) exhibit often only a modest to negligible fluorescence. 36 Apart from few porphyrin arrays, 14 most of the porphyrin-based sensitizers obtained so far are poorer luminophores than H 2 TPP and are therefore only of modest interest for imaging purposes. 37 Thus, the development of analogues of these compounds exhibiting enhanced luminescence remains an appealing and challenging task. 20,38 Scheme 1. Selected H 2 TPP-based Photosensitizers.

In this respect, we wondered about the performances of peripherally functionalized meso-tetraphenylporphyrins (H 2 TPP) derivatives such as 1-4. Fluorene is indeed a powerful fluorophore, 39 giving rise to compounds with fairly high TPA cross-sections, especially when extended conjugation is present. 40 Accordingly, we could previously show that related dendritic structures present a significantly improved fluorescence compared to that of H 2 TPP, due to an efficient energy transfer taking place from the peripheral fluorene units to the central TPP core. 36,41,42 In comparison, preservation of some § -overlap between the peripheral fluorenyl units and the central TPP core in 1-4 might even be more beneficial to both TPA and luminescence. 43,44 Furthermore, given that TPP derivatives are often able to efficiently generate 1 O 2 upon excitation, we also wondered about the potential of such

2 A C C E P T E D M A N U S C R I P T
structures for photodynamic therapy. In line with our expectations, we now show that these new TPP-based dendritic derivatives constitute quite promising structures for such uses.

The new compounds 1-4 were obtained in 28-13% yield in one step from the corresponding functional aldehydes and pyrrole in propionic acid under typical Adler-Longo conditions. 45 The functional aldehydes where themselves obtained from the brominated aldehydes and aryl the ad hoc arylethynyl precursors following a classic multistep synthetic approach based on Sonogashira couplings. The resulting porphyrins were completely characterized by elemental analysis, HRMS, 1 H and 13 C NMR (see ESI). Their linear optical properties were subsequently studied. In line with data previously gathered on related structures, 36, 41, 42 1-4 display the typical features of the porphyrin core such as an intense Soret band near 426 nm and four weaker Q-bands in the 517-650 nm range along with a strong structured band near 320 nm corresponding to the fluorene-based * transition. As expected, the intensity of the latter increases when progressing from 1 to 4 (i.e. with the number of fluorenyl groups in the compound) to eventually become the most intense UV-vis absorption in 4 (Figure 1). When excited at this particular wavelength (320 nm), all these molecules fluoresce in the red, corresponding to an emission from the Qstates which are the lowest singlet excited states, with essentially no fluorescence stemming from the fluorenyl-based * state. Comparison between the absorption spectra and normalized excitation spectra allows pointing out a very efficient energy transfer to the lowest singlet energy state (above 90 % efficiency) for all compounds. Also, the fluorescence quantum yields were derived for 1-4 and were shown to be larger or comparable than for H 2 TPP (Table 1) or related TPP-based fluorophores. 39 In this respect, two fluorenyl units at the meta-positions appear to be less effective than one fluorene unit in para-position on the meso phenyl groups of the central TPP core (compare  F for 1 and 3). This suggests that 1,4conjugation is an important parameter for enhancing luminescence. Otherwise a reverse TPA cross-sections were then measured for 1-4 by two-photon excitation fluorescence (TPEF). In line with our expectations, the presence of fluorenyl groups leads to a much larger effective TPA at 790 nm than for H 2 TPP. The later shows as strict quadratic dependence on the power of the incoming laser beam, in line with an instant biphotonic excitation (see ESI), and opens thereby access to minute spatial control of this phenomenon with 1-4 for any applied use. Again, comparison between 1 and 3 reveals that one 2-alkynylfluorenyl group in para positions to each peripheral meso-phenylene groups of the TPP core is much better for TPA than two such groups in meta-positions, further outlining the importance of crossconjugation through the meso-phenylene groups. Albeit -conjugation between the dendrons and the porphyrin core must be somewhat limited in the most stable (non planar) conformers, § this observation nevertheless emphasizes the importance of that particular feature for TPA. Albeit much lower than for 1-4, the fluorescence of many commercial porphyrin-based photosensitisers is often used for one-photon imaging during clinical trials. 37 2 max factor relative to that for H2TPP. f Data from lit. 47 While two-photon excitation has been demonstrated for many of them, 5,6 the corresponding cross-sections compare usually to that of H 2 TPP (i.e. are typically at least one order of magnitude below these found for 1-4). In this respect, the presence of large TPA cross-sections allied to relatively large fluorescence quantum yields in 1-4 constitutes presently a quite appealing property for imaging purposes.

Finally, the quantum yields for singlet oxygen generation were determined for 1-4 (ESI). Quite interestingly, we found that these are better or compare to that of H 2 TPP, revealing

A C C E P T E D M A N U S C R I P T
that fluorescence is not obtained at the expense of singlet oxygen generation for these compounds. For 1, this leads to a remarkable 37-fold enhancement of the  2 max factor taken as a figure of merit for two-photon singlet oxygen generation over TPP, while 16-to 23-fold enhancements were obtained for 2-4 (Table 1), outlining the interest of these compounds as biphotonic sensitizers for photodynamic therapy. These enhancements evidence the beneficial role of the peripheral fluorene-containing dendrons partly conjugated with the central TPP core for biphotonic photosensitization of oxygen. Note that for 1-4, taking  as a lower bound for the yield of intersystem crossing, the sum  F +  is close to unity. With this kind of structure, this leaves little room for further improvement of either fluorescence or singlet oxygen generation subsequent to monophotonic excitation. This also means that for these luminophores, non radiative processes such as internal conversion contribute less than fluorescence for regenerating the ground state.

In conlusion, we have reported here the photochemical study of four new TPPbased dendritic chromophores (1-4) possessing 4 to 16 ethynylfluorenyl groups at their periphery. In comparison to H 2 TPP, we show that these derivatives possess a much improved 1 O 2 generation capability upon biphotonic excitation and also overall better fluorescence yields. Furthermore, a strict quadratic dependence of the twophoton excited fluorescence (TPEF) intensity on the fluence of the exciting laser beam was established in all cases, allowing optimal spatial resolution to be expected from these chromophores for imaging and therapy. Thus, 1, 2 and 4 present TPA crosssections at 790 nm and also a specific red fluorescence which supersede those of many TPP-based photosensitizers developed so far for two-photon photodynamic therapy. These performances can be traced back to their unique structures in which the peripheral fluorenyl preserve some -overlap with the central TPP core at the meso positions, allowing some of them to remain partly conjugated, without strongly red shifting the lowest porphyrin-based transitions. These peculiar structural features essentially preserve the linear optic signature of the TPP core in absorption and emission and thus afford an optimal transparency window, while inducing an overall faster radiative decay and boosting the TPA cross-section around 800 nm. In conclusion, this work emphasizes the remarkable photochemical properties of molecular assemblies incorporating TPP and 2-fluorenyl moieties in "semidisconnected" 20 conjugates well known their robustness and photostablity. § § In regard to the simplicity and synthetic versatility of their structure, when suitably solubilized in physiological media by proper functionalization, 26 compounds such as 1, likewise to recently reported chlorines, 52 might become quite promising photosensitizers for theranostics. Work along these lines and also devoted to study the energy transfer process in these remarkable compounds is underways.

A C C E P T E D M A N U S C R I P T Experimental
General procedure for synthesis of 1-4. A mixture of the corresponding aldehyde (1.72 mmol) and propionic acid (6.5 mL) was heated to 120 o C. After dropwise addition of a solution of pyrrole (1 eq) in propionic acid (1.0 mL), the reaction medium was further kept refluxing for 1.5-5.5 h (see ESI). After cooling at room temperature, MeOH was added to the reaction mixture and the precipitate was filtered. The residue could be purified by repeated chromatography on silica using petroleum ether/CH 2 Cl 2 mixtures as eluant. The title compounds were isolated in 28%-13% yield. ‡ The interference between OPA and TPA, can limitate achievement of an optimal 3D-spatial resolution in some cases. Indeed, it has been shown with some of these compounds that the generation of the excited state on the laser fluence is not always strictly quadratic at ambient temperatures in certain cases. 16,18 § In H 2 TPP, the dihedral angle between the phenyl rings and the macrocycle is not 90° but closer to 60°, so -conjugation is not fully disrupted. 46 § § In this connection, they were often used as active molecular components for optical limitation puposes. 48

Notes

Figure 1 .

 1 Figure 1. Absorption and Normalized Emission Spectra of 1-4 in CH 2 Cl 2 .

  trend than observed between 1 and 3 should have been stated based on the absorption spectra of these compounds.

#

  For recent examples of TPA-PDT, see for instance refs10-13.

Table 1 .

 1 Photophysical data in dichloromethane of selected porphyrin derivatives. a

	Cpnd	fluorenes/ porphyrin	abs max (nm) UV band	abs max (nm) Soret	bs max (nm) Q-bands	em (nm)	F a	 b	1-F- c	2PA max (nm)	2 max (GM) d	Enhanc ement factor e
	H2TPP	0	/	417	514, 549, 590, 647	650, 716	0.11	0.60	0.29	790	12 f	1
	1	4	324	427	518, 555, 592, 650	657, 722	0.20	0.70	0.10	790	380	37
	2	8	330	425	520, 555, 592, 650	655, 721	0.19	0.65	0.16	790	190	17
	3	8	326	427	517, 552, 589, 646	650, 718	0.11	0.59	0.30	790	200	16
	4	16	327	426	517, 552, 590, 646	651, 716	0.13	0.56	0.31	790	290	23

a Fluorescence quantum yield determined relative to H2TPP in toluene, upon excitation at the Soret band. b 1 O2 formation quantum yield determined relative to H2TPP in CH2Cl2 (Δ[TPP] = 0.60) upon excitation in the Soret band of the compounds (425 nm). c Upper bound yield of non radiative decay processes other than intersystem crossing. d Intrinsic TPA cross-sections measured by TPEF in the fs regime; a fully quadratic dependence of the fluorescence intensity on the excitation power is observed and TPA responses are fully non-resonant. e

Acknowledgments

DY thanks the China Scholarship Council for a PhD grant. The CNRS is acknowledged for financial support.