P Laffaille
email: plaffaille@hotmail.com

J Pe ´tillon

E Parlier

L Vale ´ry

F Ysnel

A Radureau

E Feunteun

J.-C Lefeuvre

Does the invasive plant Elymus athericus modify fish diet in tidal salt marshes?

Keywords: invasive species, fish nursery, food resources, salt marsh function, Amphipoda

The invasion of Mont-Saint-Michel Bay salt marshes (France) by a grass species (Elymus athericus) has led to important changes in vegetation cover, which is likely to modify the habitat for many invertebrates. Some of them constitute the main food items for several fish species, such as young sea bass (Dicentrarchus labrax) and sand goby (Pomatoschistus minutus), that feed in salt marsh creeks during high tides. As a result, fish nursery functions of salt marshes could be modified by the E. athericus invasion. In order to test this hypothesis, gut contents of the two most abundant fish species (sea bass and sand goby) were compared before and after E. athericus invasion in the same salt marsh creek and using the same methodology. The accessibility and availability of the main food item, the semi-terrestrial amphipod Orchestia gammarella, were estimated and compared between invaded (dominated by E. athericus) and original areas (dominated by Atriplex portulacoides). Gut content analysis showed a significantly greater percentage of fish leaving with empty guts from E. athericus areas than from A. portulacoides areas. The sea bass diet composition study showed a major shift in the relative importance of the main food items: before E. athericus invasion, diets were dominated by the semi-terrestrial species O. gammarella, whereas after the E. athericus invasion they were dominated by a marine mysid Neomysis integer. The same trend was found for sand gobies, with a shift of the main food item from O. gammarella before invasion to the polychaete Hediste diversicolor after invasion. These trophic changes may be explained by the lower accessibility and availability of O. gammarella in invaded communities than in natural ones. The E. athericus invasion, observed throughout northern Europe, is thus likely to disturb trophic function of natural salt marshes for fish. This preliminary study of the E. athericus invasion is also an illustration that invasive species are an urgent problem in conservation biology.

Introduction

Salt marshes are complex ecotones between terrestrial and marine systems and are among the most productive systems of the biosphere [START_REF] Lefeuvre | European salt marshes diversity and functioning: the case study of the Mont-Saint-Michel bay, France[END_REF]. Since the introduction of the outwelling concept [START_REF] Odum | Energy flow in ecosystems: a historical review[END_REF], which states that tidal events export detrital organic matter produced in salt marshes to marine waters, strong evidence has also indicated that the export of organic matter from terrestrial to aquatic systems supports secondary production [START_REF] Loneragan | Seagrass and mangroves as carbon sources for food webs supporting penaeid prawns in a tropical Australian estuary: a stable isotope study[END_REF][START_REF] Kurata | Stable isotope analysis of food sources for salt marsh snails[END_REF]. Consequently, salt marshes generally have a very high conservation value, directly as a result of their species complement and because of their role as a feeding ground for migrant birds [START_REF] Norris | The density of redshank Tringa totanus breeding on the saltmarshes of the Wash in relation to habitat and its grazing management[END_REF][START_REF] Lefeuvre | European salt marshes diversity and functioning: the case study of the Mont-Saint-Michel bay, France[END_REF], and fish, especially juveniles [START_REF] Boesch | Dependence of fishery species on salt marshes: the role of food and refuge[END_REF][START_REF] Laffaille | Feeding ecology of 0-group sea bass Dicentrarchus labrax in salt marshes of Mont-Saint-Michel bay (France)[END_REF][START_REF] Laffaille | Can thin-lipped mullet directly exploit the primary and detritic production of European macrotidal salt marshes? Estuarine[END_REF].

Thirty fish species colonize and forage in the salt marshes of the Mont-Saint-Michel Bay (France) during the flood tide (Laffaille et al., 2000a). The habitats of the intertidal salt marshes of the Mont-Saint-Michel Bay are highly suitable for an abundant fish community. Marine straggler species (Clupeidae, Gadidae, etc.) are infrequently found. Marine estuarine dependent species (Mugilidae, Serranidae, Pleuronectidae, etc.) and estuarine species (Gobiidae, etc.) are more abundant and dominate the fish community, especially Pomatoschistus spp., Liza spp. and Dicentrarchus labrax L. Anadromous and catadromous species (especially the European eel, Anguilla anguilla L.) migrate between the marine and freshwater environment and colonize salt marshes and reclaimed marshes [START_REF] Laffaille | Habitat preferences of different European eel size classes in a reclaimed marsh: a contribution to species and ecosystem conservation[END_REF]. In contrast, freshwater species (Cyprinidae, Esocidae, etc.) are infrequent and generally die rapidly in this environment where abiotic conditions (salinity, temperature, turbidity, etc.) change rapidly.

During high tide, young sea bass (Dicentrarchus labrax L.) and sand gobies (Pomatoschistus minutus Pallas) exploit salt marsh production by feeding on resident invertebrates, mainly Orchestia gammarella [START_REF] Laffaille | Compe ´tition alimentaire entre deux espe `ces de gobies, Pomatoschistus lozanoi (de Buen) et P. minutus (Pallas), dans un marais sale ´macrotidal[END_REF][START_REF] Laffaille | Feeding ecology of 0-group sea bass Dicentrarchus labrax in salt marshes of Mont-Saint-Michel bay (France)[END_REF]. Pomatoschistus minutus is among the most abundant fish species of coastal waters of western Europe [START_REF] Costa | Changes in the Tagus nursery function for commercial fish species: some perspectives for management[END_REF], and occurs frequently at high densities in a large European intertidal salt marsh (Laffaille et al., 2000a;[START_REF] Salgado | Feeding ecology of the gobies Pomatoschistus minutus (Pallas, 1770) and Pomatoschistus microps (Kroyer, 1838) in the upper Tagus estuary, Portugal[END_REF]. The sand goby is an important prey for fish-eating vertebrates [START_REF] Moreira | Trophic relationships in the community of the upper Tagus Estuary (Portugal): a preliminary approach[END_REF][START_REF] Hamerlynck | The food of Pomatoschistus minutus (Pisces, Gobiidae) in Belgian coastal waters, and a comparison with the food of its potential competitor P. lozanoi[END_REF]. Dicentrarchus labrax is one of the most abundant commercial species of the Atlantic coast and in the Mont-Saint-Michel Bay (Laffaille et al., 2000a), where young fish, especially young of the year (0C), exploit tidal salt marshes [START_REF] Laffaille | Feeding ecology of 0-group sea bass Dicentrarchus labrax in salt marshes of Mont-Saint-Michel bay (France)[END_REF].

The present research team has surveyed the progression and transformation processes of the Mont-Saint-Michel Bay salt marshes since 1984 when the first vegetation map was produced [START_REF] Bouchard | Progression des marais sale ´s a `l'ouest du Mont-Saint-Michel entre 1984 et[END_REF][START_REF] Vale ´ry | Impact of the invasive native species Elymus athericus on carbon pools in a salt marsh[END_REF]. This long-term survey has highlighted radical changes of the vegetation cover in the salt marsh due to a rapid invasion by Elymus athericus (Z Elytrigia atherica). Formerly confined to the high marsh, this native species is progressively invading the middle and low salt marshes where it often forms a dense monospecific stand [START_REF] Van Wijnen | Nitrogen accumulation and plant species replacement in three salt marsh systems in the Wadden Sea[END_REF][START_REF] Bockelmann | Competitive exclusion of Elymus athericus from a high-stress habitat in a European saltmarsh[END_REF][START_REF] Bouchard | Sheep grazing as management tool in Western European saltmarshes[END_REF]. In some areas of the Mont-Saint-Michel Bay, this species has progressively extended its area threefold in only 10 years and has replaced the middle marsh Atriplex portulacoides communities [START_REF] Vale ´ry | Impact of the invasive native species Elymus athericus on carbon pools in a salt marsh[END_REF]. During the past 10 years, E. athericus has spread at a rate of 4 ha yr ÿ1 , while the overall area of the salt marsh only increased by 1.5 ha yr ÿ1 . The percent of marsh occupied by this species has increased from 37% to 73% between 199173% between and 200173% between (Vale ´ry et al., 2004)). Although this species spread along creeks in 1991 (i.e. it was exclusively distributed along the banks of drainage creeks), its distribution in 2001 was no longer limited to the channel creeks but was filling in between the creeks network. The increase of salt marsh area occupied by E. athericus occurred to the detriment of A. portulacoides, which covered nearly 1/3 of the marsh in 1991 [START_REF] Bouchard | Progression des marais sale ´s a `l'ouest du Mont-Saint-Michel entre 1984 et[END_REF] but only 1/8 of the marsh in 2001 [START_REF] Vale ´ry | Impact of the invasive native species Elymus athericus on carbon pools in a salt marsh[END_REF].

Such a progression is characteristic of invasive species [START_REF] Cronk | Plant Invaders: The Threat to Natural Ecosystems[END_REF], common in European salt marshes [START_REF] Bockelmann | Competitive exclusion of Elymus athericus from a high-stress habitat in a European saltmarsh[END_REF]. Biological invasions are ongoing worldwide and have become recognized as a major factor resulting in ecosystem process changes [START_REF] Vitousek | Biological invasion by Myrica faya alters ecosystem development in Hawaii[END_REF][START_REF] Kourtev | Exotic plant species alter the microbial community structure and function in the soil[END_REF]. Invasive species decrease the functional biodiversity, which could explain their impact on ecosystem functions such as primary productivity [START_REF] Naeem | Biodiversity and plant productivity in a model assemblage of plant species[END_REF][START_REF] Tilman | Productivity and sustainability influenced by biodiversity in grassland ecosystems[END_REF], organic nutrient matter cycles [START_REF] Hooper | The effects of plant composition and diversity on ecosystem processes[END_REF][START_REF] Vale ´ry | Impact of the invasive native species Elymus athericus on carbon pools in a salt marsh[END_REF], hydrology and pedogenesis [START_REF] Vitousek | Biological invasions and ecosystem processes: towards an integration of population biology and ecosystem studies[END_REF]. In coastal areas, ecological functioning might be disturbed by Elymus athericus, in particular because the organic matter flux processes between salt marshes and coastal waters may be modified and the whole or part of the food web might also be unbalanced. Several functions of the salt marshes may therefore be modified by such an invasion: the export capacity of the salt marsh could be modified and in turn could affect mussel and oyster culture with resulting economic effects; the habitat value for Anatidae and other game birds could be changed [START_REF] Lefeuvre | Biodiversity in salt marshes: from patrimonial value to ecosystem functioning. The case study of the Mont-Saint-Michel bay[END_REF][START_REF] Vale ´ry | Impact of the invasive native species Elymus athericus on carbon pools in a salt marsh[END_REF]. The role of invasive plants on the alteration of trophic function for larvae and juveniles of fish and crustaceans has been studied along the North American Atlantic coast with common reed Phragmites australis (see for example [START_REF] Able | Effects of common reed (Phragmites australis) invasion on marsh surface macrofauna: response of fishes and decapod crustaceans[END_REF][START_REF] Wainright | Relative importance of benthic microalgae, phytoplankton, and the detritus of smooth cordgrass (Spartina) and the common reed (Phragmites) to brackish marsh food webs[END_REF][START_REF] Hanson | Nekton use of a Phragmites australis marsh on the Hudson river[END_REF]. In comparison, the effects of E. athericus on trophic function for European fish have rarely been studied, although European salt marshes play important roles for fish communities.

The aim of this study is to examine the impact of Elymus athericus on trophic function for fish communities, one of the connotations of the nursery concept [START_REF] Beck | The identification, conservation, and management of estuarine and marine nurseries for fish and invertebrates[END_REF]. Because natural salt marshes dominated by Atriplex portulacoides are highly productive and provide large quantities of organic matter (from 1080 to 1990 g dry weight of net aerial primary production m ÿ2 yr ÿ1 ), they provide habitat refuges and food resources to sustain high densities of Orchestia gammarella [START_REF] Lefeuvre | European salt marshes diversity and functioning: the case study of the Mont-Saint-Michel bay, France[END_REF]. It is therefore hypothesized that the new habitat created by E. athericus is likely to reduce food resources for young fish during high tides and to disturb salt marsh trophic and consequently nursery functions. To test this, the feeding and diet of Dicentrarchus labrax (sea bass) and Pomatoschistus minutus (sand goby) were compared in salt marshes of the Mont-Saint-Michel Bay before and after E. athericus invasion. In order to study the effects of Elymus on fish food resources, accessibility and availability of their main food item in natural salt marshes between invaded and natural plots were also compared.

Material and methods

Study site

The Mont-Saint-Michel Bay (France) is a wide littoral zone located in the NormaneBreton Gulf (latitude 48 40# N, longitude 1 35# W) extending over 500 km 2 (Fig. 1). This area is a macrotidal system characterized by a high tidal range (mean tidal range Z 10e11 m, maximum Z 16 m). The intertidal area covers 220 km 2 and contains 180 km 2 of mud and sand flats and 40 km 2 of salt marshes. Salt marshes are drained by a dense creek system [START_REF] Lefeuvre | European salt marshes diversity and functioning: the case study of the Mont-Saint-Michel bay, France[END_REF] and are flooded during 43% of tides when the tidal range is greater than 11.25 m. Flooding lasts on average 2 h per tide. The rest of the time the salt marsh, including tidal creeks and vegetated tidal flats, is emerged. Thus, this complex is colonized by fish and marineeestuarine invertebrates such as mysids, amphipods and decapods only for very short periods (1e2 h) during the spring tides when the vegetated creeks and the vegetated intertidal surface are accessible [START_REF] Laffaille | Role of fish communities in particulate organic matter fluxes between salt marshes and coastal marine waters in the Mont-Saint-Michel bay[END_REF](Laffaille et al., , 2000a)). Other macro-invertebrates are semi-terrestrial (e.g. the amphipod Orchestia gammarella) or adapted to the submersioneinundation cycles such as the polychaete Hediste diversicolor. These invertebrates are resident in salt marshes.

Fish sampling

Sea bass and sand gobies were, respectively, caught in 1997 and 1998 before the Elymus athericus invasion in one salt marsh creek [START_REF] Laffaille | Compe ´tition alimentaire entre deux espe `ces de gobies, Pomatoschistus lozanoi (de Buen) et P. minutus (Pallas), dans un marais sale ´macrotidal[END_REF][START_REF] Laffaille | Feeding ecology of 0-group sea bass Dicentrarchus labrax in salt marshes of Mont-Saint-Michel bay (France)[END_REF] and in 2002 in the same salt marsh creek after E. athericus invasion. During this period, only the vegetation has changed; the tidal hydrology (flooding frequency and duration), the water chemistry (temperature, conductivity, nitrate, phosphate, salinity, etc.), altitudinal ranges and the climatic conditions are the same. The epifaunal composition was almost the same in Atriplex portulacoides vegetation in 1986 and in 2004, except for some very mobile arthropods (e.g. cursorial spiders) that tended to enter natural areas from the invaded surroundings [START_REF] Pe ´tillon | Impact of an invasive plant (Elymus athericus) on the conservation value of tidal salt marshes in western France and implications for management: responses of spider populations[END_REF].

The study creek is situated 2.5 km from the coastline on a 10 m wide creek, which drains a 5.7 ha watershed. Fish were caught monthly from spring (recruitment period in the bay) to autumn when sea bass and sand gobies colonized intertidal marshes with maximum abundances in the Mont-Saint-Michel Bay. A minimum of eight samples were collected each year. Each month, one or two samples were collected (if the first fish sample size was small) in spring time. Samplings were made during the evening ebb in order to analyse diet composition after residence in salt marsh creeks [START_REF] Laffaille | Compe ´tition alimentaire entre deux espe `ces de gobies, Pomatoschistus lozanoi (de Buen) et P. minutus (Pallas), dans un marais sale ´macrotidal[END_REF][START_REF] Laffaille | Feeding ecology of 0-group sea bass Dicentrarchus labrax in salt marshes of Mont-Saint-Michel bay (France)[END_REF]. Samplings were conducted using 4 mm mesh fyke nets set across the creek (see [START_REF] Laffaille | Role of fish communities in particulate organic matter fluxes between salt marshes and coastal marine waters in the Mont-Saint-Michel bay[END_REF]. All samples were stored immediately in a refrigerated box (about 0 C) in salt marsh and in a deep freezer (ÿ18 C) at the laboratory until examination of the gut contents. The time between sample collection and deposition was about 2e3 h. Fish were individually measured to the nearest mm [fork length (FL) for sea bass and total length (TL) for goby].

Gut content analysis

The entire digestive tract was removed from each fish. The number of empty stomachs was counted, and a vacuity index (V, proportion of empty stomachs) was determined as an index of feeding intensity. V was compared using a Chi-square test with Yates correction.

Prey items were identified for each fish species and were weighed to the nearest 1 mg. For each sample, the percentage of occurrence (F i , percentage of full stomachs where prey i was present), the percentage abundance (N i , numerical percentage of prey i in the food item community) and the biomass composition (B i , biomass percentage of prey i in the food item community) were calculated. In order to analyse and compare the diets at the population level, the Main Food Index (MFI) modified by [START_REF] Laffaille | Compe ´tition alimentaire entre deux espe `ces de gobies, Pomatoschistus lozanoi (de Buen) et P. minutus (Pallas), dans un marais sale ´macrotidal[END_REF] was used for each food item i:

MFI i Z h ½B i ðN i CF i Þ=2 1=2 X ðMFIÞ
i !100:

MFI were compared using a G test or likelihood ratio test as modified by [START_REF] Williams | Improved likelihood ratio tests for complete contingency tables[END_REF].

Orchestia gammarella availabilities and accessibilities

Both availability and accessibility of the main food item were estimated and compared between habitats invaded by Elymus athericus and the natural habitat of middle salt marshes (i.e. Atriplex portulacoides communities). The invaded and uninvaded areas had similar altitudinal ranges. The availability of Orchestia gammarella was estimated by density (number m ÿ2 ) measurements using a depletion method. Within each plant community, four 1 m 2 quadrats were randomly installed and sampled by hand catches and pitfall traps until there were no more individuals. Final hand collections were carried out to collect potential remaining individuals. Previous studies showed the need for 10 sampling days to empty a quadrat (Pe ´tillon et al., unpublished data). All densities were corrected according to the vertical distribution of O. gammarella (estimated by vertical soil samplings, 1 per quadrat). The procedure was carried out three times during summer 2003.

The accessibility of Orchestia gammarella was estimated by abundance measurements (number day ÿ1 trap ÿ1 ). Orchestia gammarella abundances were sampled by pitfall traps (circular plastic, 10 cm in diameter, 17 cm depth and ethyleneglycol for preservative) in the two vegetation groups studied, i.e. Atriplex portulacoides and Elymus athericus communities. The number of animals caught depends on both population size and activity [START_REF] Greenslade | Pitfall trapping as a method for studying populations of Carabidae (Coleoptera)[END_REF] and pitfall traps provide an estimation of accessibility of this invertebrate (moving individuals) to fish species.

In order to characterize the habitat formed by each plant community, the following parameters were measured within a radius of 1 m around each pitfall trap: litter depth and vegetation height of each plant species.

All measurements were compared using unpaired t test with Welch's correction. This test does not assume similar variances. However, this test assumes that data are sampled from populations that have a normal distribution. This assumption was tested using KolmogoroveSmirnov's method (Lillefors option).

Results

Sea bass diet

A total of 792 sea bass (mean LF G sd Z 32.2 G 8.5 mm, range between 18 and 67 mm) were analysed in 1997e1998; 121 sea bass (mean LF G sd Z 28.2 G 5.5 mm, range between 19 and 43 mm) were caught and analysed in 2002. Differences of vacuity index were very significant before (1997e1998) and after (2002) Elymus invasion (c 2 Z 139.57, p ! 0.0001): generally very few sea bass had empty stomachs (mean V Z 1.4%) when fish left salt marshes during ebb in 1997e1998, whereas 25.5% of sampled fish in 2002 had empty stomachs. There was no significant difference between 1997 and 1998 before Elymus invasion (c 2 Z 19.7, p O 0.75). Before Elymus invasion, the main food item of sea bass was Orchestia gammarella (mean MFI Z 64%), which occurred in 79% of the stomachs (Table 1). This amphipod dominated the prey community (N Z 58% and B Z 73%). All other prey were of lesser importance (MFI ! 25%), although mysids occurred in 29% of the stomach contents. During these years (before Elymus invasion), there was no significant difference between the diets (G adj. Z 11.2, p O 0.75).

In 2002 (after Elymus invasion), seven food items were identified in sea bass stomachs (Table 1); however, Neomysis integer was the most common (this mysid was present in 81% of stomachs containing food) and was the dominant prey (MFI Z 61%). Orchestia gammarella was now a secondary prey (MFI Z 32%) and was only present in 23% of sea bass stomachs. The other food items were scarcer (individual MFI ! 3%). These differences in sea bass diet were significant (G adj. Z 48.7, p ! 0.005) between 1997e1998 and 2002.

Sand goby diet

A total of 886 sand gobies (mean TF G sd Z 42.0 G 6.1 mm, range between 20 and 69 mm) were analysed in 1997; 184 sand gobies (mean TF G sd Z 40.5 G 7.2 mm, range between 21 and 60 mm) were caught and analysed in 2002. Pomatoschistus minutus diet composition followed the same trend noted for sea bass diet. In fact, 24% of sand gobies caught in 2002 had empty stomachs, whereas only 13% in 1997. Differences of vacuity index were significant before (1997) and after (2002) Elymus invasion (c 2 Z 103.73, p ! 0.0001). Among the 13 food items identified in sand goby stomachs in 1997, Orchestia gammarella dominated (N Z 72% and B Z 76%) and occurred in 72% of individual diets (Table 2). Eight food items were identified in 2002. Hediste diversicolor was the main food item (MFI Z 56%), whereas O. gammarella and Neomysis integer were secondary (MFI Z 18% and 11%, respectively). These differences in sand goby diet were very significant (G adj . Z 51.3, p ! 0.005).

Orchestia gammarella availabilities and accessibilities

The amphipod Orchestia gammarella availabilities (Fig. 2) were significantly different (unpaired t test with Welch correction, Welch's approximate t Z 3.567, p Z 0.004) between areas dominated by Atriplex portulacoides (mean G sd Z 650 G 456 O. gammarella per m 2 ; range: 229e1786) and areas dominated by Elymus athericus (mean G sd Z 171 G 92 O. gammarella per m 2 ; range: 42e355). Accessibilities followed the same trend (Fig. 3), because O. gammarella was significantly more abundant (Welch's approximate t Z 3.218, p Z 0.008) in natural areas (i.e. A. portulacoides community) than in invaded areas (i.e. E. athericus community), mean G sd Z 1260 G 1139 O. gammarella per day and per trap (range: 146e4287) and mean G sd Z 190 G 170 O. gammarella per day and per trap (range: 31e472), respectively.

Vegetation height and litter depth

The Community dominated by Atriplex portulacoides and the community dominated by Elymus athericus were significantly different in terms of their vegetation height and litter depth (Welch's approximate t Z 7.095 and 8.124, both p ! 0.0001, respectively). The community dominated by A. portulacoides was shorter (mean vegetation height G sd Z 25.4 G 5.8 cm) than that of E. athericus (65.4 G 18.6 cm) and provided smaller amounts of litter (mean litter depth G sd Z 0.5 G 0.3 cm and 3.5 G 1.2 cm, respectively, for A. portulacoides and for E. athericus).

Discussion

The Atriplex portulacoides and Elymus athericus communities are highly productive, generating a large quantity of organic matter [START_REF] Lefeuvre | European salt marshes diversity and functioning: the case study of the Mont-Saint-Michel bay, France[END_REF] that can provide habitat and food for dense communities of marsh dwelling detritivorous crustaceans such as Orchestia gammarella [START_REF] Cre ´ach | Stable isotopes and gut analyses to determine feeding relationships in saltmarsh macroconsumers[END_REF]. However, both availabilities and accessibilities of O. gamarella were significantly lower in invaded habitats than in natural habitats. Although these populations are likely to vary during a year and between years [START_REF] Aksissou | Biode ´mographie et dynamique d'une population d'Orchestia gammarellus (Crustacea, Amphipoda, Talitridae) du littoral me ´diterrane ´en du Maroc[END_REF], the comparison of this food resource between natural and invaded areas is representative of a general decrease in population dynamics in invaded habitats (also analysed by the relative abundance of age classes: Pe ´tillon et al., unpublished data). This trend can be related to a lower quality of food for this decomposer (presence of phytoliths and high , 2004), which prefers to feed upon soft food items [START_REF] Moore | Some observations on food and feeding of the supralittoral beach-hopper Orchestia gammarellus (Pallas) (Crustacea: Amphipoda)[END_REF]. The reduction in total densities of O. gammarella is, however, not enough to explain such a decrease of this food resource for sea bass and sand goby in invaded salt marshes by E. athericus. In fact, it can be argued that O. gammarella was very dense compared to other food resources in both natural and invaded sites, and therefore still available for fish even in the E. athericus community.

Furthermore, this amphipod is known to be greatly consumed in the Atriplex portulacoides community by Pomatoschistus minutus [START_REF] Laffaille | Compe ´tition alimentaire entre deux espe `ces de gobies, Pomatoschistus lozanoi (de Buen) et P. minutus (Pallas), dans un marais sale ´macrotidal[END_REF] and Dicentrarchus labrax [START_REF] Laffaille | Feeding ecology of 0-group sea bass Dicentrarchus labrax in salt marshes of Mont-Saint-Michel bay (France)[END_REF]. The decrease of the amphipod as food would therefore appear to be related to lower prey accessibility for fish in the Elymus athericus community than in A. portulacoides. One hypothesis is that the high quantity of litter produced by the tall grass E. athericus reduces Orchestia gammarella accessibility for fish communities. An increase of habitat complexity has already been proved to reduce foraging efficiency (e.g. [START_REF] Corona | Epibenthic amphipod abundance and predation efficiency of the pink shrimp Farfantepenaeus duorarum (Burkenroad, 1939) in habitats with different physical complexity in a tropical estuarine system[END_REF][START_REF] Bush | Food limitation in a nursery area: estimates of daily ration in juvenile scalloped hammerheads, Sphyrna lewini (Griffith and Smith, 1834) in K ane'ohe Bay, O'ahu, Hawai'i[END_REF]. Thus, Elymus invasion may be responsible for a reduction of both availability and accessibility of the main item O. gammarella, leading to a shift in food resources from this main item to the marine mysid Neomysis integer for sea bass and to the polychaete Hediste diversicolor for sand goby.

Orchestia gammarella is a semi-terrestrial crustacean with poor swimming capabilities. By comparison, the other invertebrates are more difficult to catch for aquatic predators either because they are better swimmers (e.g. Neomysis integer) or benthic invertebrates strongly associated with the substratum (e.g. Hediste diversicolor). It can be suggested that fish caught these difficult ''substitution prey'' mainly because they occur at very high densities in salt marsh creeks (see [START_REF] Holland | Linkages between tidal creeks ecosystems and the landscape and demographic attributes of their watersheds[END_REF] for polychaetes' density calculations), representing the main food item for other young fish species (e.g. N. integer for stickleback: [START_REF] Søndergaard | Neomysis integer in a shallow hypertrophic brackish lake: distribution and predation by three-spined stickleback (Gasterosteus aculeatus)[END_REF]. Feeding activity varied mainly according to the dominant vegetation, and sand gobies and young sea bass appeared to have an opportunistic feeding behaviour response to the invasion of Elymus athericus in the salt marshes. Such a shift due to a decrease of the main food item was already shown in sheep grazed salt marshes of the Mont-Saint-Michel Bay by Laffaille et al. (2000b). Once more, the semi-terrestrial O. gamarella was replaced by N. integer in sea bass diets.

Elymus invasion also resulted in lower food consumption (increase in vacuity index after Elymus athericus invasion) observed in Pomatoschistus minutus and Dicentrarchus labrax and supported by the non-significant difference between 1997 and 1998 (before Elymus invasion) for sea bass. The fact that diets of fish from uninvaded marsh were similar in 1997 and 1998 suggests, but clearly does not prove, that they would have been similar in 2002. But, differences between before and after Elymus invasion were great and certainly greater than between 1997e1998 and 2002 in an uninvaded marsh that does not now exist.

Elymus athericus invasion is thus likely to reduce the unique landesea exchanges of organic matter via the fish vector. In fact, only a few fish species are able to assimilate salt marsh primary production directly or indirectly; this is the case for mullets Liza and Mugil spp., sand gobies Pomatoschisus spp. and sea bass Dicentrarchus labrax in the Mont-Saint-Michel Bay [START_REF] Laffaille | Role of fish communities in particulate organic matter fluxes between salt marshes and coastal marine waters in the Mont-Saint-Michel bay[END_REF][START_REF] Laffaille | Can thin-lipped mullet directly exploit the primary and detritic production of European macrotidal salt marshes? Estuarine[END_REF]. Salt marsh primary production is essentially assimilated by many invertebrates, and especially by Orchestia gammarella and mysids in European salt marshes [START_REF] Zagursky | Macrophyte detritus in the winter diet of the estuarine mysid, Neomysis americana[END_REF][START_REF] Cre ´ach | Stable isotopes and gut analyses to determine feeding relationships in saltmarsh macroconsumers[END_REF]. Tidal creeks seem to act as a drainage area where the secondary production (i.e. invertebrates) of the whole salt marsh system gets concentrated. A previous study on the exploitation of the macrotidal salt marshes by the most abundant fish species (Laffaille et al., 2000a), confirmed the general assumption that nekton migrate into intertidal creeks and vegetated intertidal areas during flood tide to feed. Fish exploit this secondary production (i.e. invertebrates) and, indirectly, the large primary production of the macrotidal salt marshes during spring tides when vegetated intertidal surface is accessible. Following Elymus invasion, the primary production of invaded marsh areas is very high and similar to natural areas [START_REF] Vale ´ry | Impact of the invasive native species Elymus athericus on carbon pools in a salt marsh[END_REF]. The reduction in secondary production export (mainly Amphipoda) by fishes could then lead to: (1) an accumulation of organic matter stocks in invaded salt marshes associated with very large quantities of litter [START_REF] Vale ´ry | Impact of the invasive native species Elymus athericus on carbon pools in a salt marsh[END_REF] present study) and (2) a disappearance of salt marsh trophic and nursery functions in the long term. Even if the trophic function of creeks in invaded systems is reduced, it is probable that young fishes will still frequent them because of their refuge function (e.g. [START_REF] Bush | Food limitation in a nursery area: estimates of daily ration in juvenile scalloped hammerheads, Sphyrna lewini (Griffith and Smith, 1834) in K ane'ohe Bay, O'ahu, Hawai'i[END_REF][START_REF] Paterson | The fishes associated with three associated intertidal salt marsh creeks in a temperate southern African estuary[END_REF].

The invasion pattern of Elymus athericus [START_REF] Vale ´ry | Impact of the invasive native species Elymus athericus on carbon pools in a salt marsh[END_REF] is similar to the invasion of Phragmites australis (common reed) in salt marshes along the North American Atlantic coast (see for example [START_REF] Bart | Environmental determinants of Phragmites australis expansion in a New Jersey salt marsh: an experimental approach[END_REF]. In fact, P. australis in North American salt marshes has changed recently from a species with a limited distribution to an invasive species producing dense monospecific stands. This expansion has converted mosaics of vegetation into dense monotypic stands [START_REF] Chambers | Expansion of Phragmites australis into tidal wetlands of North America[END_REF], potentially changing the functions of these ecosystems. Some research has focused on whether P. australis stands to serve as a valuable wetland habitat and foraging areas for marsh nekton [START_REF] Hanson | Nekton use of a Phragmites australis marsh on the Hudson river[END_REF]. Moreover, there is evidence supporting the view that P. australis does not have a deleterious effect on the ability of marshes to function as habitat for fauna and especially nekton. [START_REF] Fell | Does invasion of oligohaline tidal marshes by reed grass, Phragmites australis (Cav.) Trin. ex Steud., affect the availability of prey resources for the mummichog, Fundulus heteroclitus[END_REF] and [START_REF] Warren | Rates, patterns, and impacts of Phragmites australis expansion and effects of experimental Phragmites control on vegetation, macroinvertebrates, and fish within tidelands of the lower Connecticut River[END_REF] reported that fish foraging and the abundance of invertebrates were not affected by the expansion of P. australis. Other authors have found that fish species composition was also not affected by common reed invasion [START_REF] Meyer | Comparison of nekton use of Phragmites australis and Spartina alterniflora marshes in the Chesapeake Bay, USA[END_REF] or that nekton densities are similar to those in some non-P. australis dominated wetlands [START_REF] Hanson | Nekton use of a Phragmites australis marsh on the Hudson river[END_REF]. Moreover, [START_REF] Wainright | Relative importance of benthic microalgae, phytoplankton, and the detritus of smooth cordgrass (Spartina) and the common reed (Phragmites) to brackish marsh food webs[END_REF] provided evidence that P. australis may contribute to the food chain in tidal marsh systems. Other studies showed differences, such as [START_REF] Posey | Benthic communities of common reed Phragmites australis and marsh cordgrass Spartina alterniflora marshes in Chesapeake Bay[END_REF], who reported a slightly higher abundance of fauna in Spartina alterniflora marshes than in P. australis marshes. The reasons for these differences included the presence of shallow pools around S. alterniflora and the lack of them around P. australis, possible differences in food availability and differences in stem density and/or canopy thickness [START_REF] Fell | Comparison of fish and macroinvertebrate use of Typha angustifolia, Phragmites australis, and treated Phragmites marshes along the lower Connecticut River[END_REF].

Increasing human pressure and activities have led to strong disturbances, functional changes and destruction of habitats, especially in wetland ecosystems [START_REF] Lefeuvre | Biodiversity in salt marshes: from patrimonial value to ecosystem functioning. The case study of the Mont-Saint-Michel bay[END_REF]. Throughout the world, most wetlands are exploited for agricultural purposes. One of the most common is the exploitation of the primary production by domestic stock grazing [START_REF] Bakker | Nature management by grazing and cutting[END_REF]. The impacts of herbivores on salt marsheseestuarine coastal systems are quite well known [START_REF] Kiehl | Impact of different sheep grazing intensities on saltmarshes in northern Germany[END_REF]Laffaille et al., 2000b;[START_REF] Lefeuvre | European salt marshes diversity and functioning: the case study of the Mont-Saint-Michel bay, France[END_REF]. However, one recent and important impact on coastal wetland is that of invasive plants such as Elymus athericus [START_REF] Bockelmann | Competitive exclusion of Elymus athericus from a high-stress habitat in a European saltmarsh[END_REF] and Spartina spp. [START_REF] Callaway | The introduction and spread of smooth cordgrass (Spartina alterniflora) in south San Francisco Bay[END_REF]. Similar to the spread of other invasive species, such as Lythrum salicaria [START_REF] Mal | Time-dependant competitive displacement of Typha angustifolia by Lythrum salicaria[END_REF] in freshwater wetlands, E. athericus (in Europe) and Phragmites australis (in North America) in coastal wetlands, invasive species tend to reduce species richness. In the case of Elymus, invaded habitats tend to present higher densities and species richness of terrestrial arthropods than natural habitats [START_REF] Pe ´tillon | Impact of an invasive plant (Elymus athericus) on the conservation value of tidal salt marshes in western France and implications for management: responses of spider populations[END_REF], except for amphipods (present study). The massive loss of species and ecosystems, and especially wetlands and associated organisms [START_REF] Gibbs | Wetland loss and biodiversity conservation[END_REF], has made it imperative to investigate the roles of species in the functioning of ecosystems [START_REF] Schwartz | Linking biodiversity to ecosystem function: implications for conservation ecology[END_REF] and more generally shifts in ecosystem functioning provoked by many invasive species. The conservation implications of the link between species richness and ecosystem function have recently been intensified [START_REF] Hector | Conservation implications of the link between biodiversity and ecosystem functioning[END_REF] and management of invasive species is an urgent problem in conservation biology. In particular, it has been emphasized that invasions by E. athericus remain limited in salt marshes that are grazed by sheep [START_REF] Bakker | Nature management by grazing and cutting[END_REF][START_REF] Kiehl | Impact of different sheep grazing intensities on saltmarshes in northern Germany[END_REF][START_REF] Bouchard | Sheep grazing as management tool in Western European saltmarshes[END_REF].

Following the Elymus athericus invasion, the tidal salt marsh of the Mont-Saint-Michel Bay seems to have changed its trajectory and now fulfils a new function: initially a source of organic matter, it might become a carbon sink [START_REF] Vale ´ry | Impact of the invasive native species Elymus athericus on carbon pools in a salt marsh[END_REF]. In the long term, this C sequestration is likely to impact the marine food webs by reducing available resources, especially for fish species. This study shows that E. athericus invasion alters the trophic function for European fish and consequently the nursery function according to the definition of [START_REF] Beck | The identification, conservation, and management of estuarine and marine nurseries for fish and invertebrates[END_REF]. Bioenergetics and isotopic studies should urgently be conducted to determine the new trophic pathways leading to dominant fish production and the effects of the shift in dominant prey resources on trophic transfer dynamics.

Fig. 1 .

 1 Fig. 1. Location of the Mont-Saint-Michel Bay.

Table 1

 1 Food items identified in the stomach contents of young sea bass caught during ebb in 1997e1998 and in 2002. N: numeric frequency. B: biomass frequency.

F: frequency of occurrence. MFI: main food index 1997e1998 2002

Table 2

 2 Food items identified in the stomach contents of sand goby caught during ebb in 1997 and in 2002. N: numeric frequency. B: biomass frequency. F: frequency of occurrence. MFI: main food index

	Vegetable	0.0 0.1 0.1 0.1 e	e	e	e

Fig. 3. Abundances (number of individuals m ÿ2 ) of Orchestia gammarella in invaded (Elymus athericus) and natural (Atriplex portulacoides) habitats (mean G sd, N Z 4 per date).

lignin content: Vale ´ry et al.

Acknowledgements

We thank M. Ar Gouilh, A. Jan and C. Aubert for help in counting and identifying amphipods. This study was funded by: ( 1) the CNRS Program 'Environnement, Vie et Socie ´te ´s: Zone Atelier de la Baie du Mont-Saint-Michel et ses Bassins Versants', (2) Ministe `re de l'Ecologie et du De ´veloppement Durable Program 'Invasions Biologiques' and (3) Institut Franc ¸ais de la Biodiversite ´Program 'Usages et Biodiversite ´s dans les Marais Sale ´s'.

We thank the reviewers for many helpful suggestions.


S Brosse

P Laffaille

S Gabas

European species. S Lek

Is scuba sampling a relevant method to study fish microhabitat in lakes? Examples and comparisons for three European species

We compared fish microhabitat use patterns in the littoral zone of a lake using a new direct method (point abundance sampling by scuba, PASS) and the widely used point abundance sampling by electrofishing technique (PASE). We collected microhabitat data for age 0π roach (Rutilus rutilus L.), perch (Perca fluviatilis L.), and pike (Esox lucius L.). The two methods yielded different results for fish assemblage structure and microhabitat patterns. Using PASE, fish were mainly found in ''shel-Key words: sampling methods; fish ter habitats'' such as shallow waters and dense vegetation. It is likely microhabitat; scuba diving; lake; Rutilus rutilus; Perca fluviatilis; Esox lucius that this behavior is caused by the disturbance of the observer stamping around. Using PASS, fish escapement behavior was rarely observed.

Introduction

The cornerstone of many ecological studies is an accurate estimation of population abundance and density in various biotic and abiotic environments [START_REF] Begon | Ecology: indi-viduals, population, and communities[END_REF]. This is easily achieved when a complete enumeration of the populations is possible; however, very few animal populations can be completely enumerated and most of the ecological explanations come from samples. Many sampling methods have been set up to try to achieve reliable estimates according to the studied organisms and communities, but each sampling method has its own shortcomings [START_REF] Verner | Assessment of counting techniques[END_REF][START_REF] Krebs | Ecological methodology[END_REF][START_REF] Alexander | Application of mark-recapture models to estimation of the population size of plants[END_REF]. Such biases are emphasized in aquatic systems, where sampling is often more complex than in terrestrial ecosystems. Numerous methods are available for estimating fish biomass, density and habitat in natural environments (Ever-hart et al. 1975;[START_REF] Nielsen | Fisheries techniques[END_REF][START_REF] Meador | Methods for sampling fish communities as part of the national waterquality assessment program[END_REF][START_REF] Murphy | Fisheries techniques[END_REF] but these methods have frequently maintained a somewhat narrow perspective that is usually limited by the data provided by the sampling approach employed. However, reliably assessing population habitats and community structure requires a sampling approach with sample units comparable at all levels of ecological perception, from a large spatial scale (i.e. macrohabitat) to a narrower one (i.e. microhabitat) [START_REF] Krebs | Ecological methodology[END_REF].

In freshwater ecosystems, although fish may be collected using a large number of sampling methods to assess density or population size (e.g. gillnets [START_REF] Hamley | Review of gillnet selectivity[END_REF]; toxicans (Davies & Sheldon 1983); electrofishing [START_REF] Cowx | Fishing with electricity, applications in freshwater fisheries management[END_REF]; and hydroacoustics (MacLennan & Simmonds 1992)), microhabitat studies require effective sampling, numerous small-scale samples and measurements of the habitat features for each sample [START_REF] Nelva | Une nouvelle me ´thode d'e ´tude des peuplements ichtyologiques dans les grands cours d'eau par e ´chantillonnage ponctuel d'abondance[END_REF]). To reach this goal, the point abundance sampling theory was set up. [START_REF] Blondel | La me ´thode des indices ponctuels d'abondance (IPA) ou des releve ´s d'avifaune par ''stations d'e ´coute[END_REF] originally developed it for studying nesting birds. It is based on the statistical theory that many small sample units provide more precise results than a few large samples. Using this theory, two major kinds of sampling methods can be employed to define fish microhabitat. i) Sampling methods that require catching fish, such as electrofishing, are now routinely used in aquatic ecology [START_REF] Cowx | Fishing with electricity, applications in freshwater fisheries management[END_REF]. One of the most common methods, known to be efficient in freshwater research, used to define fish microhabitat, is the point abundance sampling by electrofishing (PASE) technique [START_REF] Nelva | Une nouvelle me ´thode d'e ´tude des peuplements ichtyologiques dans les grands cours d'eau par e ´chantillonnage ponctuel d'abondance[END_REF]. Even though this method provides reproducible samples, it is known to be size-selective [START_REF] Copp | Electrofishing for fish larvae and juveniles: equipment modifications for increased efficiency with short fishes[END_REF]. ii) Underwater visual methods, such as diving, have been primarily applied in marine waters. These methods are currently very common and recognized for studying the relationships between reef fish assemblages and habitat characteristics [START_REF] Brock | A preliminary report on a method of estimating reef fish populations[END_REF][START_REF] Mattheus | A comparative study of habitat use by young-of-the-year, subadult, and adult rockfishes on four habitat types in central Puget Sound[END_REF][START_REF] Tupper | Effects of habitat on settlement, growth, and postsettlement survival of Atlantic cod (Gadus morhua)[END_REF][START_REF] Charton | Correlation between habitat structure and a rocky reef fish assemblage in the southwest Mediterranean[END_REF]. In continental hydrosytems, these methods were mainly employed in North American streams to study salmonid behavior [START_REF] Ellis | Diving and photographic techniques for ob-serving and recording salmon activities[END_REF][START_REF] Keenleyside | Skin-diving observations of Atlantis salmon and brook trout in the Miramichi River, New Brunswick[END_REF]) and microhabitat [START_REF] Beecher | Predicting microdistributions of steelhead (Oncorhynchus mykiss) parr from depth and velocity preference criteria: test of an assumption of the instream flow incremental methodology[END_REF]. Scuba diving, however, has been little used in lakes. In Europe, the work of [START_REF] Rossier | Spatial and temporal separation of littoral zone fishes of Lake Geneva (Switzerland -France)[END_REF] is probably the first habitat study using diving in a lake, but it covered a large geographic area and microhabitat was never taken into account. To address this deficiency, we developed a direct visual fish observation method, point abundance sampling by scuba (PASS) and applied it to the littoral zone of a lake to study fish population on a microhabitat scale.

This article reports how the PASS method was set up and aims to assess the capabilities of this technique compared with PASE. We therefore investigated the microhabitat use of three fish species (Rutilus rutilus L., Perca fluviatilis L. and Esox lucius L.) during the first months of their biological cycles. The results were compared with a standard electrofishing sampling strategy (PASE). These results led us to discuss the advantages and disadvantages of direct (i.e. PASS) and indirect (i.e. PASE) sampling methods to define a species assemblage and the microhabitat use of some fish populations.

Material and methods

Study area

The study was undertaken during summer 1998 in Lake Pareloup (France). This reservoir is located in southwestern France, near the town of Rodez. It covers a total area of 1250 ha for a volume of about 168¿10 6 m 3. The maximum depth is 37 m and the average depth is 12.5 m. Lake Pareloup is a warm monomictic lake that is submitted to summer thermal stratification with low oxygen content below the thermocline (located about 10 m below the surface from early June to mid-September), which prevents the fish from colonizing deep water during this period [START_REF] Richeux | Inventaire piscicole de la retenue hydroe ´le ´ctrique de Pareloup (Aveyron, France) lors de la vidange de Juin 1993. Essai d'un nouveau syste `me d'acquisition et de traitement des signaux d'un e ´chosondeur[END_REF]. In order to obtain comparable samples we chose two restricted areas of the lake that present the same environmental and topographical characteristics. The two sampling areas were chosen for their topographical heterogeneity providing numerous kinds of habitats. Considering the environmental features of the two sites, [START_REF] Pujo | Leve ´bathyme ´trique de la retenue de Pareloup[END_REF] showed that they are identical topographically. Moreover, the fish community was studied in the two bays during summer 1997 using PASE, and no statistical difference between the relative abundance of each species within the two bays was found by Wilcoxon's nonparametric paired test (ZΩª0.78, PΩ0.43), testifying to the identical faunistic characteristics of the two selected sites.

Sampling

Eleven transects were defined in each location (i.e. PASE in the first bay and PASS in the second). Their lengths depended on the depth and varied between 4 and 32 m. Each transect was defined by a 2-m-wide observation lane from the bank across to 1.5 m depth, marked off every 1 m. This gave 201 sampling points. For each sampling point, nine environmental variables were measured in order to assess fish microhabitat, which included: two topographical variables expressed in meters (distance from the bank and depth), one biological variable, the flooded vegetation cover expressed as the percentage of cover on the surface of each sampling point and six substratum variables expressed as the percentage of boulders, pebbles, gravel, sand, silt and mud. Both fish sampling techniques were performed weekly from spawning (early June) to the juvenile period (late August) for the three studied species [START_REF] Brosse | Habitat, dynamique spatiale et structure des communaute ´s pisciaires en milieu lacustre[END_REF] in the littoral zone of the lake (i.e. 12 weeks). In this approach, three young fish populations were taken into account to assess the ability of PASE and PASS to define fish microhabitat. These species were i) the roach (Rutilus rutilus L.), the most common fish in the lake [START_REF] Angelibert | Changes in roach (Rutilus rutilus L.) population structure induced on draining a large reservoir[END_REF], belonging to the omnivorous trophic guild, ii) the perch (Perca fluviatilis L.), a small predator known to be zooplanktivorous and usually ichthyophagous, and iii) the pike (Esox lucius L.) a top predator fish. The two sampling pro-cedures were tested on these three species belonging to different trophic guilds with various behavioral traits. Moreover, young fish were chosen, as they are more abundant in the littoral areas than adults [START_REF] Brosse | Habitat, dynamique spatiale et structure des communaute ´s pisciaires en milieu lacustre[END_REF], thus allowing a better estimation of their microhabitat use and therefore a more accurate comparison between the two sampling methods.

In the first site, PASE [START_REF] Nelva | Une nouvelle me ´thode d'e ´tude des peuplements ichtyologiques dans les grands cours d'eau par e ´chantillonnage ponctuel d'abondance[END_REF]) as modified by [START_REF] Copp | Electrofishing for fish larvae and juveniles: equipment modifications for increased efficiency with short fishes[END_REF] was employed to evaluate the density of each species and the microhabitat of several populations. Fish were collected using a backpack electroshocker fitted with a small 10-cm ring anode. This method is known to provide reproducible and quantifiable point samples and is efficient for the entire fish community, even if the field of attraction differs according to fish size. This technique consists of a discreet approach by foot to the sampling point. The use of a rubber-rowing dinghy was discarded because of its inability to sample shallow, gently slopping, muddy areas with this method. Upon arrival at the chosen point, the anode was swiftly immersed about 0.5 m into the water and any shocked fish were then collected with a fine-mesh dipnet. Fish specimens were preserved in 4% formaldehyde. This sampling design requires the presence of three people: the first with the electroshocker, the second with the dipnet and a third on the bank to note the results.

In the second site, PASS was set up. The swimming along of transects by a free diver was used to obtain approximate fish numbers. Fish counts were made while swimming along the transect, and the results obtained were expressed as densities or through extrapolation as estimates of total number of individuals of each species occurring in each sample according to [START_REF] Sale | Assemblages of fish on patch reefs -predictable or unpredictable ?[END_REF]. Censusing begins with the diver located far from the bank, in open waters; he then moves closer, swimming towards the bank. The observer swam in each lane, covering the full area of each point. Water clarity was sufficient to determine and count all the fish for each 2-m-wide and 1-m-long point. For each fish species, individuals recorded were sorted into two classes: young (0π) and older (Ø1π) fishes. When dense fish shoals were observed (mainly young fish), counting was limited to a subsample and then extrapolated over the entire volume of the sample. This last procedure has been applied to 199 samples out of the total of 1310 PASS samples. According to [START_REF] Northcote | Underwater census of stream fish populations[END_REF] and [START_REF] Eklov | Effects of habitat complexity and prey abundance on the spatial and temporal distributions of perch (Perca fluviatilis) and pike (Esox lucius)[END_REF], a constant speed of about 5 m/min was used in order to minimize disturbance of the fish community. This sampling design required the presence of only one person, who noted all the information on a waterproof board.

Data processing

The two data matrices (i.e. PASE and PASS) were used to develop microhabitat preference indices for each environmental variable as a measurement of habitat use by the fish population vs. habitat availability, based on the method of [START_REF] Ivlev | Experimental ecology of the feeding of fishes[END_REF] and improved by [START_REF] Beecher | Predicting microdistributions of steelhead (Oncorhynchus mykiss) parr from depth and velocity preference criteria: test of an assumption of the instream flow incremental methodology[END_REF]. Similar improvements of Ivlev's electivity index are commonly used for fish habitat use studies [START_REF] Copp | Shifts in the microhabitat of larval and juvenile roach, Rutilus rutilus L. in a floodplain channel[END_REF][START_REF] Copp | An empirical model for predicting microhabitat of 0π juvenile fishes in a lowland river catchment[END_REF][START_REF] Poizat | Multi-scale approach to specieshabitat relationships: juvenile fish in a large river section[END_REF]. Preference was calculated as a normalized ratio of utilization to availability for different intervals of each environmental variable. Preference indexes were obtained after dividing each variable into several modalities. Their number was defined according to the range of variation of each variable. The following formula was used:

IΩ(Ob/Ex)/(Ob/Ex) max ª0.5
where Ob is the number of fish observed for the modality, Ex is the expected number of fish for a theoretical random distribution and (Ob/Ex) max is the maximum value of (Ob/Ex) for the modality. I varies between ª0.5 and π0.5. Positive values indicate preference and negative values indicate avoidance for a given variable. Therefore, values between ª0.1 and 0.1 can be considered as revealing indifference; from ª0.30 to ª0.11 and from 0.11 to 0.30 illustrate slight avoidance or preference, respectively; and from ª0.5 to ª0.31 and 0.31 to 0.5 reveal strong avoidance or preference, respectively. To estimate any significant differences between the two methods (i.e. PASE and PASS), we used the Wilcoxon nonparametric test according to [START_REF] Sokal | Biometry. The principles and practice of statistics in biological research[END_REF], which compared the two paired groups to find out whether their difference was significantly different from a nil value. This test was computed using SPSS release 8 for Windows. In the same way, the relative abundance and occurrence of every individual of each species, collected using the two sampling methods, were compared using the Wilcoxon nonparametric statistical test according to [START_REF] Sokal | Biometry. The principles and practice of statistics in biological research[END_REF].

Results

Within the whole data matrices, 1480 and 1310 point samples were collected, 21,049 fish were caught and 177,698 observed using PASE and PASS, respectively. The discrepancy between the amount of fish numbered using the two methods is due to the different sizes of the samples collected using each method (i.e., ca. 0.2 m 2 for PASE and 2 m 2 for PASS) and do not induce bias in the analyses, as we considered the relative values of fish abundance and occurrence. The mean abun- dance of fish estimated were 71.1 and 67.8 fish per m 2 using PASE and PASS, respectively. For each species, young (0π) and adult fish (1π and older) were considered as different populations to avoid bias due to behavioral and habitat differences between young and adult individuals. Moreover, this study favors the whole range of sizes of 0π fish and therefore satisfies possible habitat changes occurring during the development of each studied species, providing in this way an overview of the main habitat characteristics of the populations considered.

Of the 15 fish species colonizing the lake [START_REF] Brosse | Influence of some topographical variables on the spatial distribution of lake fish during summer stratification[END_REF], 10 were recorded in our study by both sampling methods (Fig. 1). The study of the relative occurrence (i.e. percentage of samples where the considered population is captured) and abundance (i.e. numerical percentage of the considered population relative to the total number of fish captured) of each species within the fish community, using the two sampling methods, revealed almost the same results with both sampling methods (Fig. 1). The Wilcoxon's nonparametric statistical paired test showed no significant differences between the two methods for the estimation of the relative oc-currence (ZΩª0.50, PΩ0.62) and abundance (ZΩ ª0.20, PΩ0.85) of the different fish species within the community. A more precise study of Fig. 1 showed that several populations of adult fishes (e.g. adult perch and adult roach) were found to occur more frequently using PASS than PASE (Fig. 1A). In the same way, young perch and young bleak (Alburnus alburnus L.) were found to be more abundant using PASE, whereas the abundance of young pike and young tench (Tinca tinca L.) was higher using PASS (Fig. 1B).

Concerning microhabitat data, the Pearson correlation matrix showed a strong correlation between the variables silt and sand (ª0.61 for PASE and ª0.86 for PASS) and between silt and vegetation cover (ª0.75 for PASE and ª0.65 for PASS). To avoid colinearity between variables, the percentage of silt was removed from the two data matrices. Thus, all the statistical analyses were performed on a set of eight variables (distance from the bank, depth, percentage of mud, sand, gravel, pebbles, boulders, and flooded vegetation cover). Wilcoxon's nonparametric test showed a significant difference between PASE and PASS for the microhabitat preference of each of the three young fish populations studied (ZΩª4.20, PϽ0.01 for roach, ZΩª4.29, PϽ0.01 for perch and ZΩª4.29, PϽ0.01 for pike). Thus, the fish microhabitat estimated using PASE and PASS gave different results.

Using electrofishing, roach were found to live close to the bank and generally avoided distances from the bank, which were further than 5 m (Fig. 2A). With PASS, roach were observed further from the bank (5-10 m), but they avoided distances over 15 m (Fig. 2B). The results obtained by PASE showed that roach also avoided deep water (more than 0.5 m), whereas with scuba diving, a lot of roach were observed at depths greater than 0.5 m. Muddy substratum was avoided with PASS, whereas roach collected with PASE were found on mud. Finally, considering flooded vegetation cover, PASS revealed a preference for intermediate vegetation cover (i.e. 25-75%), whereas PASE revealed a preference for dense cover.

The general trends in perch microhabitat were quite similar for the two topographical variables using both methods (Fig. 3). The same microhabitat profiles were obtained for distance from bank and depth, characterized by a preference for intermediate distances (5-10 m) and depths (0.5-1 m). Nevertheless, PASE showed that perch microhabitat choice is hardly affected by large substrata such as pebbles and boulders, whereas PASS showed perch that clearly avoided these substrata. In the same way, using PASE, perch were found inside vegetation, whereas using PASS, they were found in both vegetated areas and in open water. The two remaining substrata (mud and gravel) did not exhibit a clear difference between the two sampling methods.

For pike (Fig. 4), the microhabitat profiles showed that, using PASE, fish were always collected less than 10 m from the bank in shallow water (less than 0.5 m), whereas PASS results gave a narrower range of habitat features (between 2.5 and 10 m from the bank and between 0.25 and 0.5 m deep). Similarly, pike were found to colonize the whole range of vegetated areas using PASE, whereas PASS results showed that they colonized only intermediate vegetation cover. Finally, PASE habitat profiles revealed that pike tended to avoid large substrata, whereas such a trend was not clear from PASS.

Discussion

The relative occurrence and abundance showed that both methods can reliably sample the whole fish community. Nevertheless, the techniques differ due to the biases each introduces. Compared with PASS, the underestimation by PASE of the occurrence of some adult fish populations is thought to be due to the observer stamping around driving the most mobile species away. Such a bias in the electrofishing methods has already been reported [START_REF] Bain | A quantitative method for sampling riverine microhabitats by electrofishing[END_REF][START_REF] Copp | Electrofishing for fish larvae and juveniles: equipment modifications for increased efficiency with short fishes[END_REF]. Likewise, PASE underestimated the abundance of some young fish populations known to colonize highly vegetated areas (e.g. tench and pike), as the collection of stunned fish is limited by dense vegetation [START_REF] Dewey | Effectiveness of a drop net, a pop net and an electrofishing frame for collecting quantitative samples of juveniles fishes in vegetation[END_REF]. PASS also exhibits its own shortcomings; young perch abundance could be underestimated due to errors concerning fish counts when very dense shoals were encountered. Similarly, young bleak (Alburnus alburnus L.) abundance was underestimated by PASS. Such errors could be due to the inability of the observer to discriminate between young bleak and roach or rudd when dense shoals with both species were encountered. Thus, we can hypothesize that a large part of the young bleak population was counted as other young cyprinids.

Considering microhabitat studies, fish microhabitat profiles assessed using the two sampling methods revealed different patterns for each species. Using PASE, roach were found on muddy substrata, which is unusual, as mud does not constitute a food item for young roach [START_REF] Dubois | Re ´gime et comportement alimentaire du gardon (Rutilus rutilus L.) et de la perche (Perca fluviatilis L.) de la retenue de Pareloup. I. Les alevins[END_REF][START_REF] Angelibert | Changes in roach (Rutilus rutilus L.) population structure induced on draining a large reservoir[END_REF]). This species is usually strongly associated with sandy substratum [START_REF] Copp | Shifts in the microhabitat of larval and juvenile roach, Rutilus rutilus L. in a floodplain channel[END_REF], and medium depths (about 0.75 m). However, habitat features concerning flooded vegetation obtained with PASS were in accordance with previous studies, revealing firstly, that roach feeding efficiency is maximal with low vegetation density and open water [START_REF] Persson | Predator-mediated competition in prey refuges: the importance of habitat dependent prey resources[END_REF][START_REF] Eklov | The response of prey to the risk of predation: proximate cues for refuging juvenile fish[END_REF]) and secondly, that the spatial occupancy of roach located close to shelters can be related to a predation avoidance of adult perch and young pike [START_REF] Mittelbach | Foraging efficiency and body size: a study of optimal diet and habitat use by bluegills[END_REF][START_REF] Persson | Juvenile competitive bottlenecks: the perch (Perca fluviatilis)-roach (Rutilus rutilus) interaction[END_REF][START_REF] Eklov | Piscivore efficiency and refuging prey: the importance of predator search mode[END_REF][START_REF] Eklov | Species-specific antipredator capacities and prey refuges: interactions between piscivorous perch (Perca fluviatilis), and juvenile perch and roach (Rutilus rutilus)[END_REF].

Concerning perch, according to Eklov & Hamrin (1989), structurally complex habitats sustain higher macroinvertebrate biomasses, which constitute one of the main feeding items for perch [START_REF] Persson | Predator-mediated competition in prey refuges: the importance of habitat dependent prey resources[END_REF][START_REF] Eklov | Effects of habitat complexity and prey abundance on the spatial and temporal distributions of perch (Perca fluviatilis) and pike (Esox lucius)[END_REF]. Consequently, perch, were found close to the open waters at intermediate depths and distances from the bank using both sampling methods. Thus, perch microhabitat was reliably assessed with both sampling methods even if a significant difference was found between PASE and PASS, these general trends in perch distribution are in accordance with previous ecological studies [START_REF] Diehl | Foraging efficiency of three freshwater fish: effects of structural complexity and light[END_REF][START_REF] Macchiusi | Prey behaviour and sizeselective predation by fish[END_REF]. The preference of muddy bottoms and the avoidance of large substratum can be related to the search for food associated with mud (chironomids and other macroinvertebrates) and the avoidance of boulders and pebbles, which are usually colonized by large predators such as pike-perch (Stizostedion lucioperca L.) [START_REF] Brabrand | Habitat shift in roach (Rutilus rutilus) induced by the introduction of pike-perch (Stizostedion lucioperca)[END_REF] and thus could be considered as predator avoidance. On the contrary, the results obtained by PASE for large substrata seem quite irrelevant.

Concerning pike, according to [START_REF] Turner | Use of visual census for estimating population size in northern pike (Esox lucius)[END_REF] and [START_REF] Eklov | Effects of habitat complexity and prey abundance on the spatial and temporal distributions of perch (Perca fluviatilis) and pike (Esox lucius)[END_REF], PASS results showed that young pike require at least 30% vegetation cover and are associated with dense vegetation and shallow water. This behavior is due to the feeding habits of pike; which is an ambushing predator waiting for its prey close to the bottom, inside the vegetation. Moreover, young pike colonize vegetated areas to avoid cannibalism from the larger pike [START_REF] Eklov | Effects of habitat complexity and prey abundance on the spatial and temporal distributions of perch (Perca fluviatilis) and pike (Esox lucius)[END_REF]). In the same way, PASS showed a preference for vegetated areas located at intermediate distances and depths. This habitatuse, assessed with PASS, can be considered as a trade-off between prey availability and predation avoidance, whereas PASE revealed that pike were found close to the bank in very shallow water. Such a habitat could characterize a refuge habitat encouraged by the sampling design. With PASS, if the observer moved slowly, the pike retreated, swam slowly around the observer and ended up at the same location as when first encountered. According to [START_REF] Turner | Use of visual census for estimating population size in northern pike (Esox lucius)[END_REF], no apparent fleeing reactions to divers were exhibited.

General trends in fish microhabitat use were reliably assessed with both PASE and PASS methods, but as shown above, fish microhabitat was assessed better using PASS than PASE. Using PASE, fishes are mainly found in ''shelter habitats'' such as shallow water and dense vegetation. Such behavior is probably caused by the environmental disturbance of the sampling method. This hypothesis is supported by the study of substrate occupancy by the three species. Using the PASE method, substrate occupancy was usually irrelevant, as the fish escaped from their usual areas such as feeding zones (for which substrate composition is determinant) to shelters. According to [START_REF] Bain | A quantitative method for sampling riverine microhabitats by electrofishing[END_REF] and [START_REF] Dewey | Effectiveness of a drop net, a pop net and an electrofishing frame for collecting quantitative samples of juveniles fishes in vegetation[END_REF], PASE probably induces bias due to the indirect nature of the observation: i.e., the use of a catching design and the escapement caused by the observer stamping around. However, electrofishing can be used in a wider range of situations than PASS and is less affected by turbidity and wind-induced waves, which make visual observations by scuba divers impossible (Table 1). Moreover, with PASE fish are captured and thus the number of fish and the developmental stage or length of each individual are determined accurately. Nevertheless, this catching method is inoperative in areas deeper than 1 m and is limited by water conductivity (e.g. low or high conductivity for mountain lakes or brackish waters) and by accessibility to the shore (e.g. sheer bank slope or large woody debris) [START_REF] Cowx | Fishing with electricity, applications in freshwater fisheries management[END_REF]). In the same way, temporal studies with repetitive samples during a short period are biased due to the damage caused to the sampling area (vegetation cover, substratum) by repeated stamping of the observer [START_REF] Hammer | Feeding behaviour of roach (Rutilus rutilus) larvae and the fry of perch (Perca fluviatilis) in Lake Lankau[END_REF]. Using PASS, fish escapement behavior was rarely observed; moreover, this technique does not affect the environment or the welfare of the fish. Nevertheless, a major of the census estimate is that the total number of fish observed constitutes a measure of the actual number present. The validity of this assumption depends upon various observational variables: crypticity of the fish (or the screening effect of the habitat), hiding dispersion of alerted individuals before the arrival of the diver, varying visibility of different size classes, duplicate counts, experience and speed of the observer and conditions related to weather and turbidity [START_REF] Brock | A preliminary report on a method of estimating reef fish populations[END_REF][START_REF] Northcote | Underwater census of stream fish populations[END_REF][START_REF] Keast | Strip counts as a means of determining densities and habitat utilization patterns lake fishes[END_REF][START_REF] Helfman | Underwater methods[END_REF]). According to [START_REF] Sale | Precision and accuracy of visual census technique for fish assemblages on coral patch reefs[END_REF], visual censuses can display high repeatability, but they seldom (if ever) completely sample the fish present at a site especially with dense fish shoals such as young roach and young perch. Nevertheless, with good underwater visibility, divers should be able to obtain reliable estimates of fish abundance.

As shown in Table 1, which comprises a synthesis of our own results and from the literature concerning electrofishing [START_REF] Bain | A quantitative method for sampling riverine microhabitats by electrofishing[END_REF][START_REF] Barrett | Effects of direct current electrofishing on the mottled sculpin[END_REF][START_REF] Copp | Electrofishing for fish larvae and juveniles: equipment modifications for increased efficiency with short fishes[END_REF][START_REF] Cowx | Fishing with electricity, applications in freshwater fisheries management[END_REF][START_REF] Dewey | Effectiveness of a drop net, a pop net and an electrofishing frame for collecting quantitative samples of juveniles fishes in vegetation[END_REF][START_REF] Perrow | Sampling fish communities in shallow lowland lakes: pointsample electric fishing vs electric fishing with stop-nets[END_REF] and scuba sampling [START_REF] Ellis | Diving and photographic techniques for ob-serving and recording salmon activities[END_REF][START_REF] Sale | Precision and accuracy of visual census technique for fish assemblages on coral patch reefs[END_REF][START_REF] Helfman | Underwater methods[END_REF][START_REF] Turner | Use of visual census for estimating population size in northern pike (Esox lucius)[END_REF], PASS seems better suited than indirect sampling designs (e.g. PASE) to determine fish microhabitat in the littoral areas of lakes because of the accuracy of the results provided by a method that avoids physically disturbing the assemblage. We can therefore recommend this method for ecological studies of fish populations and communities. This approach appears to be relevant and more suitable than other techniques especially considering rare and endangered species due to its nondestructive nature.

Resumen 1. Hemos desarrollado un nuevo me ´todo directo por escafandra (i.e. muestreo de abundancia puntual por observacio ´n desde escafandra, PASS) para estudiar microhabitats de peces en zonas litorales de lagos. Nuestro fin concreto fue ´evaluar la relevancia de este me ´todo en relacio ´n a muestreos esta ´ndar de abundancia puntual con pesca ele ´ctrica (PASE). 2. Los muestreos se llevaron a cabo en 2 zonas litorales del Lago Pareloup (Francia) de similares caracterı ´sticas ambientales que fueron utlizadas para comparar las caracterı ´sticas de habitat obtenidas utilizando dos te ´cnicas de muestreo distintas. Tres especies de peces representativas del grupo ecolo ´gico y, especialmente, de la cadena tro ´fica, fueron considerados; juveniles (Oπ) de Rutilus rutilus, Perca fluviatilis, y Esox lucius. Las dos matrices de datos (i.e. PASE y PASS) fueron utilizadas para desarrollar indices de preferencias de microhabitat para 8 variables ambientales como medida del uso de habitat por la poblacio ´n de peces vs. disponibilidad de habitat. 3. Test no-parame ´tricos de Wilcoxon revelaron diferencias significativas entre los dos me ´todos para cada poblacio ´n. Adema ´s, la estructura de la comunidad de peces y los perfiles de microhabitat que fueron evaluados utilizando ambos me ´todos revelaron patrones diferentes para cada poblacio ´n. Utilizando PASE, los peces fundamentalmente encontrados sobre habitats de ''shelter'' tales como aguas someras y vegetacio ´n densa. Tal comportamiento podrı ´a estar causado por las molestias ocasionadas por el propio observador. Utilizando PASS, comportamiento de escape por parte de los peces fue raramente observado. 4. Concluimos que esta te ´cnica de muestreo directa y no destructiva puede ser utilizada para obtener estimas precisas de microhabitat de comunidades de peces que asumimos ma ´s apropiada que PASE para estudios de habitat.

Fig. 1 .

 1 Fig. 1. Relative occurrence (a) and abundance (b) expressed as a percentage for each of the 19 fish populations (young and adults) belonging to 10 species, using the two sampling techniques. The ten species were the following: roach (Rutilus rutilus L.), rudd (Scardinius erythrophtahlmus L.), perch (Perca fluviatilis L.), bleak (Alburnus alburnus L.), bream (Abramis brama L.), gudgeon (Gobio gobio L.), pike (Esox lucius L.), tench (Tinca tinca L.), carp (Cyprinus carpio L.) and pumpkinseed (Lepomis gibbosus L.).

Fig. 2 .

 2 Fig. 2. Microhabitat profile of young roach calculated for the eight environmental variables (see text for details) using the two sampling techniques. (a). PASE, (b). PASS. Dis: distance from the bank, Dep: depth, San: sand, Mud: mud, Gra: gravel, Peb: pebbles, Bou: boulders, Veg: flooded vegetation; pres: presence, abs: absence.

Fig. 3 .

 3 Fig. 3. Microhabitat profile of young perch calculated for the eight environmental variables (see text for details) using the two sampling techniques. (a). PASE. (b). PASS. Dis: distance from the bank, Dep: depth, San: sand, Mud: mud, Gra: gravel, Peb: pebbles, Bou: boulders, Veg: flooded vegetation; pres: presence, abs: absence.

Fig. 4 .

 4 Fig. 4. Microhabitat profile of young pike calculated for the eight environmental variables (see text for details) using the two sampling techniques. (a). PASE. (b). PASS. Dis: distance from the bank, Dep: depth, San: sand, Mud: mud, Gra: gravel, Peb: pebbles, Bou: boulders, Veg: flooded vegetation; pres: presence, abs: absence.

Table 1 .

 1 Overview of the range conditions of use and environmental disturbances caused by direct (i.e. PASS) and indirect (i.e. PASE) sampling methods for ecological studies of fish populations and communities.

	PASE	PASS

πππ highly affected, ππ affected, π hardly affected, 0 not affected.

Acknowledgments

We are grateful to H. Collier for correcting the English and we thank G. D. Grossman, who provided helpful comments that improved the manuscript.