Claudia Gérard
email: claudia.gerard@univ-rennes1.fr

Emilie Lance

Decline of freshwater gastropods exposed to recurrent interacting stressors implying cyanobacterial proliferations and droughts

Keywords: Gastropoda, co-occurring multiple stressors, toxic blooms, water withdrawal, invasive species, trematodes

published or not. The documents may come

Decline of freshwater gastropods exposed to recurrent interacting stressors implying cyanobacteria proliferations and droughts

Introduction

Deterioration of freshwaters is increasing worldwide due to anthropogenic activities and global change [START_REF] Relyea | Assessing the ecology in ecotoxicology: a review and synthesis in freshwater systems[END_REF][START_REF] Sumpter | Protecting aquatic organisms from chemicals: the harsh realities[END_REF]. Therefore, freshwater biota increasingly suffer from exposure to multiple abiotic and biotic stressors that threaten biodiversity (e.g., Vinebrooke et al. 2004;[START_REF] Dewson | A review of the consequences of decreased flow for instream habitat and macroinvertebrates[END_REF][START_REF] Gérard | Long-term dynamics and community structure of freshwater gastropods exposed to parasitism and other environmental stressors[END_REF][START_REF] Strayer | Alien species in fresh waters: ecological effects, interactions with other stressors, and prospects for the future[END_REF][START_REF] Nunes | Pathways and gateways of freshwater invasions in Europe[END_REF][START_REF] Botana | Toxicological perspective on climate change: aquatic Toxins[END_REF]. Stressors can be punctual, recurrent or continuous; consequently, organisms usually undergo combined mixtures of interacting stressors, which effects may be synergistic, but are often unknown or difficult to predict [START_REF] Holmstrup | Interactions between effects of environmental chemicals and natural stressors: a review[END_REF][START_REF] Lemm | Identification and interaction of multiple stressors in central European lowland rivers[END_REF].

Because of the health hazard for biota induced by exposure to multiple stressors, there is a growing interest on this research topic [START_REF] Fischer | The toxicity of chemical pollutants in dynamic natural systems: The challenge of integrating environmental factors and biological complexity[END_REF][START_REF] Lemm | Identification and interaction of multiple stressors in central European lowland rivers[END_REF]. Unfortunately, we still poorly understand to what extent interactive effects of multiple recurrent and concomitant stressors (and related stresses) influence the dynamics of community structure, potentially inducing resistance, resilience or decline of the populations depending on their sensitivity [START_REF] Connell | On the evidence needed to judge ecological stability or persistence[END_REF].

Freshwater gastropods are efficient bioindicators of ecosystem health (Tallarico 2016). They constitute a substantial part of the freshwater biomass and, as primary consumers, an important link between primary producers (e.g., potentially toxic cyanobacteria) and higher consumers (leeches, crayfish, insects, fish, waterfowl, and mammals) [START_REF] Habdija | The contribution of gastropod biomass in microbenthic communities in a karstic river[END_REF][START_REF] Dillon | The Ecology of Freshwater Molluscs[END_REF]. They can represent an intoxication route to predators in food web as demonstrated for microcystins (MCs) [START_REF] Lance | Evidence of trophic transfer of microcystins from the gastropod Lymnaea stagnalis to the fish Gasterosteus aculeatus[END_REF], well-known hepatotoxins distributed worldwide and produced by some strains of cyanobacteria [START_REF] Chorus | Toxic Cyanobacteria in Water. A Guide to Public Health Consequences, Monitoring and Management[END_REF]. Freshwater gastropods can be intoxicated via direct ingestion of toxic cyanobacteria as grazers, and via oral and/or transtegumental absorption of cyanotoxins dissolved in water and/or adsorbed on particles [START_REF] Zurawell | Influence of lake trophic status on the occurrence of microcystin-LR in the tissue of pulmonate snails[END_REF][START_REF] Zhang | Bioaccumulation of the hepatotoxic microcystins in various organs of a freshwater snail from a subtropical Chinese lake, Taihu Lake, with dense toxic Microcystis blooms[END_REF]. Recurrent proliferations of toxic cyanobacteria, typically occurring during summer and autumn in temperate regions, may lead to a strong decrease in gastropod abundance and species richness [START_REF] Gérard | Long-term dynamics and community structure of freshwater gastropods exposed to parasitism and other environmental stressors[END_REF][START_REF] Lance | Impact of toxic cyanobacteria on gastropods and microcystin accumulation in a eutrophic lake (Grand-Lieu, France) with special reference to Physa (= Physella) acuta[END_REF]. Freshwater gastropods, typically inhabiting shallow littoral zones, also frequently experience drought with consequent water withdrawal and habitat loss, potentially representing a severe environmental stressor [START_REF] Schwarz | Temporary aquatic habitats: constraints and opportunities[END_REF][START_REF] Strzelec | The gastropod fauna of an unstabilised dam reservoir in Southern Poland[END_REF][START_REF] Dewson | A review of the consequences of decreased flow for instream habitat and macroinvertebrates[END_REF]).

The increase of predator pressure and interspecific competition exerted by various taxa of macroinvertebrates and vertebrates due to the restricted habitat area lead to the elimination of most gastropods, but new generations typically appear after the water level rises again [START_REF] Dudgeon | The effects of water fluctuations on a gently shelving marginal zone of Plover Cove Reservoir, Hong Kong[END_REF]Gérard 2001a;[START_REF] Gérard | Long-term dynamics and community structure of freshwater gastropods exposed to parasitism and other environmental stressors[END_REF]. Larval trematodes frequently parasitize freshwater gastropods and constitute a biotic stressor that decreases host fitness [START_REF] Esch | Snail-trematode interactions and parasite community dynamics in aquatic systems: a review[END_REF][START_REF] Combes | Interactions Durables. Ecologie et Evolution du Parasitisme[END_REF] for reviews). Parasites interact with natural and anthropogenic stressors to increase mortality and reduce animal health in myriad ways in a wide spectrum of host and parasite taxa [START_REF] Morley | Interactive effects of infectious diseases and pollution in aquatic molluscs[END_REF]Marcogliese and Pietrock 2011 for reviews). The combined effects of parasites and other stressors can reduce either resistance or tolerance to infection. However, most ecotoxicological studies completely neglected trematodes (and other parasites) despite their potential confounding factor in the responses of gastropods exposed to stresses [START_REF] Morley | Parasitism as a source of potential distortion in studies on endocrine disrupting chemicals in molluscs[END_REF][START_REF] Marcogliese | Combined effects of parasites and contaminants on animal health: parasites do matter[END_REF][START_REF] Martiguez | Parasitism can be a confounding factor in assessing the response of zebra mussels to water contamination[END_REF], and despite trematodes are indicators of environmental changes such as decrease of free-living biodiversity in aquatic ecosystems over time (e.g., [START_REF] Keas | The prevalence of cercariae from Stagnicola emarginata (Lymnaeidae) over 50 years in Northern Michigan[END_REF][START_REF] Morley | Anthropogenic pressure on a molluscan-trematode community over a long-term period in the Basingstoke Canal, UK, and its implications for ecosystem health[END_REF].

Here, we conducted a monthly one-year monitoring (2013) of a gastropod community submitted to multiple interacting stressors in a French eutrophic dam reservoir. In this site, gastropods, including the invasive North American Physa acuta, experienced recurrent blooms since at least 1997 and punctuated droughts (as in 2004) [START_REF] Gérard | Influence of toxic cyanobacteria on community structure and microcystin accumulation of freshwater molluscs[END_REF], and some of them harbored larval trematodes (Gérard, pers. obs.). We examined the temporal fluctuations of two major abiotic parameters: water temperature, which is crucial for ectotherms such as gastropods and which can correlate with other physicochemical parameters or with cyanobacterial blooms, and depth, which reflects the availability of habitat/space resources. We investigated the cyanobacterial occurrence in water and the concentration of intracellular MCs in cyanobacteria. We measured the intoxication of gastropods by toxic cyanobacteria through the accumulation of MCs in their tissues. We also examined the monthly occurrence of parasitic trematodes in gastropods in the reservoir. Finally, we recorded the monthly occurrence of macroinvertebrate taxa other than gastropods because of their potential impact on gastropods as predators or competitors, and their own response to environmental stressors. We compared data collected during the year 2013 with data obtained during some months of 2004 [START_REF] Gérard | Influence of toxic cyanobacteria on community structure and microcystin accumulation of freshwater molluscs[END_REF]Gérard, pers. com.). We aimed to check long-term changes at 9-year interval in the composition of gastropods, under the influence of exposure to the following interacting stressors: cyanobacteria proliferations, droughts, trematode parasites, and invasive non-native P. acuta.

Material and methods

Study-site

The Frémur River (western France, 48°32' N, 2°04' W), representative of many small coastal catchments in Western Europe, is scattered with many dam obstructions that reduce the average velocity and increase the water depth. As a result, the total extent of the catchment is 5 ha of running water (streams) and 75 ha of still waters (ponds and reservoirs). The most important dam (14 m high) creates a 3 x 10 6 m 3 reservoir for drinking water.

Winters are generally mild and quite rainy. During summer, cyanobacterial scum regularly occurred on the water surface since at least the year 1997, and measurements made in September 2003 revealed a density of 221 000 cells.mL -1 , dominated by Microcystis aeruginosa (Acou et al. 2008).

We conducted monitoring at the station "Les Rues", a eutrophic reservoir below dam obstructions. According to [START_REF] Gérard | Influence of toxic cyanobacteria on community structure and microcystin accumulation of freshwater molluscs[END_REF], high densities of cyanobacteria occurred in this station from May to October 2004 including 90% of genera potentially producing toxins among them MCs [i.e., Anabaena, Aphanizomenon, Microcystis, Planktothrix, Trichodesmium (Skulberg et al. 1993)]. Moreover, in August 2004[START_REF] Gérard | Influence of toxic cyanobacteria on community structure and microcystin accumulation of freshwater molluscs[END_REF] detected MCs in water samples with intracellular MCs of 17.05 µg L -1 MC-LR equivalent (MC-LReq).

Samplings and measurements

We made monthly samplings from January to December 2013. On each occasion, we measured water temperature (°C) and maximum water depth (cm) in a littoral area of 20 m length by 2 m width, and we sampled 500 mL of water in the uppermost part of the water column with a bailer to measure intracellular MC concentrations. We also visually qualified the intensity of cyanobacterial proliferations in four classes: 0 (no visible cyanobacteria), 1 (some cyanobacteria visible), 2 (some patches of cyanobacteria accumulation at the surface and/or on the shore line), 3 (continuous layer of cyanobacteria at the surface, 'green' water). Then, we sampled gastropods (and other macroinvertebrates) with a pond-net (nylon mesh: 1 mm, square aperture: 0.5 × 0.5 m) trawled from the surface sediment through the water column for three minutes along the 20 m long × 2 m wide area, as in [START_REF] Gérard | Influence of toxic cyanobacteria on community structure and microcystin accumulation of freshwater molluscs[END_REF].

In the laboratory, we harvested phytoplankton cells by filtration (1 µm Cyclopore track etched membrane, Whatman, Maidstone, UK) of the water sample and we froze the filter (-80°C) before MC analysis. We carefully examined the pond-net contents in tap water, and we counted and identified all the gastropods following [START_REF] Glöer | Süsswassermollusken. Deutscher Jugendbund für Naturbeobachtung[END_REF]. The descriptors used to characterize the gastropod community were species richness (S), abundance (A), frequency (F, relative abundance of a species in the community), and occurrence frequency (FO, percentage of months a species was collected in the 12 months sampled). We also noted the general occurrence of other macroinvertebrate taxa in the pond-net contents according to [START_REF] Tachet | Invertébrés d'eau douce : systématique, biologie, écologie[END_REF], but without specific identification.

We measured the shell size (shell height for conic shells and diameter for discoid shells) of each gastropod to the nearest 0.1 mm to establish the age structure of populations and to discriminate juveniles and adults according to [START_REF] Glöer | Süsswassermollusken. Deutscher Jugendbund für Naturbeobachtung[END_REF]. Then, we removed each gastropod from its shell under stereoscopic microscope to detect parasitic infection by larval trematodes (sporocysts or rediae, and cercariae). If present, we observed parasites alive under light glass coverslip pressure using bright-field and phase-contrast microscopy. We used trematode prevalence as parasitic descriptor [i.e., number of hosts infected with a particular parasite species / number of examined hosts [START_REF] Bush | Parasitology meets ecology on its own terms: Margolis et al. revisited[END_REF]]. Finally, we froze and weighted gastropod tissues (-80°C) before MC analysis. For each species, we quantified MCs in gastropods using one or several individuals depending on body fresh mass (FM). In total, we realized 36 MC-analyses corresponding to 268 gastropods belonging to seven species.

To compare data at a 9-year interval in the same study-site, we used data published in [START_REF] Gérard | Influence of toxic cyanobacteria on community structure and microcystin accumulation of freshwater molluscs[END_REF] and we provided unpublished data obtained in June 2004 and October 2004: water temperature and maximal depth, mean shell size of gastropods, trematode prevalence, and MC concentrations in healthy versus parasitized gastropods.

MC analysis

We used the High Pressure Liquid Chromatography (HPLC) to measure the MC concentration (in the cyanobacterial biomass) of the water samples. The intracellular MCs contained in the filtered phytoplanktonic biomass were extracted with 2 mL of 100% methanol, placed in an ultrasound water bath for 15 min at 35 kHz, and then subjected to probe sonication (Hielscher Ultrasonics GmbH, Germany) with a cycle of 0.5 s at 50% of amplitude during 1 min. The supernatant was analysed by HPLC as described in [START_REF] Lance | Interactions between Cyanobacteria and Gastropods. I. Ingestion of toxic Planktothrix agardhii by Lymnaea stagnalis and kinetics of microcystin bioaccumulation and detoxification[END_REF]. The MC detection limit (DL) in cyanobacterial samples was 0.04 µg.L -1 . Below this level, the identification process can be uncertain due to the poor quality of spectra. We expressed the monthly intracellular MC concentration in water in µg.L -1 MC-LReq using three standards (MC-RR, MC-YR and MC-LR).

We performed MC analysis on frozen tissues of gastropods by immuno-assay method with ELISA Microcystin Plate Kit (Envirologix INC) which detects free MCs from a 0.05 µg.L -1 -threshold to the nearest 0.01 µg.L -1 [START_REF] Codd | The evaluation of Envirogard Microcystin Plate and Tube Kits[END_REF][START_REF] Gilroy | Assessing potential health risks from microcystin toxins in blue-green algae dietary supplements[END_REF]. After an elution with 1mL of 80 % MeOH, samples were placed in an ultrasound water bath for 15 min at 35 kHz, and next subjected to three cycles of centrifugation-probe sonication (Hielscher Ultrasonics GmbH, Germany) with a probe sonication cycle of 0.5 s at 70% of amplitude during 1 min. After the last centrifugation, the supernatant was kept and diluted to ensure the presence of less than 10% methanol in wells of the ELISA tests, according to the manufacturer's protocols (Abraxis LLC, Warminister, PA, USA). Fluorescence was measured at 450 nm using an absorbance microplate reader (Tecan, Männedorf, Switerland). We expressed MC concentration in gastropods (MCCG) in MC-LReq ng.g -1 FM.

Data analyses

We performed analyses using R software (R Core Team 2014). Differences were regarded as significant when P ≤ 0.05. The data were log-transformed to meet the assumptions of normality and homogeneity of variance. We used two-way analysis of variance (ANOVA) to test for differences in MCCG according to the sampling date, the gastropod species, the developmental stage (juvenile vs adult), and the presence of larval trematodes. We fit linear regression to examine the relationship between MCCG and snail size.

We reported mean values followed by 95% confidence interval (CI). For percentages (i.e., F, FO, and prevalence), we calculated CI using the score method [START_REF] Newcombe | Two-sided confidence intervals for the single proportion: comparison of seven methods[END_REF].

Results

1) Temporal fluctuations in environmental conditions in 2013: water temperature, maximal water depth, cyanobacteria occurrence and intracellular MC concentration

During the year 2013, the water temperature fluctuated from 0.5°C in March, corresponding to a snowy episode with ice of 3 mm thick covering the littoral area, to 22.0°C in July (Table 1, mean monthly temperature of 13 ± 4°C).

The maximal water depth in the sampling area fluctuated from 20 cm in August 2013 to 65 cm in January 2013 (Table 1, mean monthly maximal depth of 48 ± 9 cm). Because of the summer drought, the depth decreased by half between July and August, and remained ≤ 39 cm until the end of the year (Table 1). Due to the consequent water withdrawal, the littoral area became dry and the distance between the initial shore and the water edge was comprised between 8 and 14 m from August to December 2013; therefore, neither vegetation nor organic debris were available as habitat/refuge and food for the snails.

We observed the occurrence of cyanobacteria (class 1 in abundance) for the first time in June 2013, and then increasing proliferations (classes 2 and 3) occurred from August to October 2013 (Table 1). During this period (August-October), we detected high concentrations of intracellular MCs, i.e., from 7.8 to 41.3 µg.L -1 , compared to the low values (< 0.3 µg.L -1 or < DL) recorded at the other months of the year (Table 1, mean monthly intracellular MC concentration of 5.4 ± 7.7 µg.L -1 ).

2) Monitoring of the gastropod community in 2013

In total, we sampled 2490 gastropods over the year 2013 belonging to seven species among five families (in decreasing abundance: Physidae, Planorbidae, Lymnaeidae, Bithyniidae, and Ancylidae) (Table 2).

The exotic pulmonate Physa acuta was the highly dominant species and the most occurrent species of the gastropod community, each other species representing less than 3% in abundance frequency (Table 2).

The monthly abundance of gastropods greatly varied depending on the sampling date, from 0 in September, October and December to 1258 individuals in July (Table 1). The mean monthly abundance and species richness were respectively 207.50 ± 275.69 gastropods and 2.33 ± 1.32 species. We collected 91.41% of the gastropods during June and July, whereas only 0.08% from August to December 2013. The disappearance of gastropods coincided with the increase of intracellular MCs in the water samples (Table 1).

Concerning P. acuta, based on its relative size-frequency distributions (Fig. 1), two or even three generations were observed in most months with two periods of recruitment in May and July, corresponding to a decrease in the mean shell size, respectively 4.0 ± 0.4 and 3.0 ± 0.1 mm (Table 3).

According to the mean shell size of each species per month (Table 3), the gastropod community was mainly composed of juveniles in June and July, when cyanobacteria began to proliferate and water depth decreased (Table 1).

No gastropods infected by larval trematodes occurred in 2013 among the 2490 gastropods sampled (prevalence = 0.00%, CI = 0.00-0.15%).

3) MC accumulation in gastropods in 2013

The MC-analyses made in 2013 revealed the presence of MCs in all gastropod tissues whatever the gastropod species and the sampling date (Table 4). MCCG values were extremely variable and ranged from 59 to 4149 MC-LReq ng.g -1 FM (both values corresponding to P. acuta tissues) with a mean of 1085 ± 411 ng.g -1 FM.

Accumulation of MCs significantly differed depending on the sampling date (F = 3.419; P = 0.027). The greatest values of MCCG occurred in February (3109 ± 1075 ng.g -1 FM) and the lowest in spring and summer (May-August, 385 ± 168 ng.g -1 FM) (Table 4). MCCG was not significantly different according to the gastropod species (F = 0.094; P = 0.996), the shell size (F = 2.725; P = 0.108) and the developmental stage (F = 2.606; P = 0.121).

4) Temporal fluctuations of the occurrence of macroinvertebrates other than gastropods in 2013

then Bivalvia (Table 5).

Depending on taxa considered, the frequency of occurrence greatly varied from 8.33% (CI = 1.49-35.39%) to 100% (CI = 75.75-100%) (Table 5). Chironomidae, Ephemeroptera, and Trichoptera occurred all along the year whatever environmental conditions (FO ≥ 75%), whereas Limnadidae, Asellidae and Corixidae were frequent only before the stressful period (August-December 2013), and then completely disappeared after August 2013.

The taxa diversity doubled from April to May (13 taxa) and reached a peak in July (17 taxa), and then, strongly decreased from August (seven taxa) to the end of the study (three taxa in December) (Table 5).

5) Comparison at a 9-year interval with data collected in June and October 2004

Water temperatures in June and October 2004, respectively 18.5°C and 15°C, were on the same order than in June and October 2013 (Table 1).

The maximum water depths in June and October 2004, respectively 55 and 35 cm, were similar to those in 2013 (Table 1), as well as the water withdrawal recorded in October 2004 due to summer drought, i.e., 15 m between the initial shore and the water edge.

In June 2004, we sampled the same five species of gastropods than in June 2013, mainly at a juvenile stage based on their mean shell size (Tables 1,3,6). However, the total gastropod abundance was 13-fold higher in June 2013 than in June 2004. In particular, the dominant non-native species, P. acuta, was 20-fold more abundant in June 2013 than in June 2004, representing 93.52% of the gastropod community (vs 63.16% in June 2004). The native pulmonates Radix ovata and Planorbis planorbis were respectively thrice and twice more abundant in June 2013 (vs 2004) but represented a lower part of the community, respectively 1.18% (vs 5.26% in 2004) and 4.62% (vs 25.00%). No gastropods infected by larval trematodes occurred in June 2004 among the 76 gastropods sampled (prevalence = 0.00%, CI = 0.00-4.81%).

In October 2004, we collected seven individuals of P. acuta and eleven individuals of R. auricularia at an adult stage, whereas we found no gastropods in October 2013 (Tables 1,6). Three snails among 18 were infected by rediae and patent cercariae of a species of Echinostomatidae (prevalence = 16.67%, CI = 5.84-39.22%). These infected snails belonged to R. auricularia and were adults with shell size of 16.7, 18.4 and 21.7 mm (mean size of 18.9 ± 4.7 mm), thus greater than that of uninfected R. auricularia varying from 9.2 to 18.0 mm (mean size of 13.4 ± 2.5 mm).

The comparison with MCCG values recorded previously by [START_REF] Gérard | Influence of toxic cyanobacteria on community structure and microcystin accumulation of freshwater molluscs[END_REF] showed that MCCG was highly superior in 2013 than in 2004 (F = 57.502; P = 1.404e -9 ) (Tables 4,7). Moreover, MC accumulation was significantly higher in October 2004 than in June 2004 (F = 11.240; P = 0.008) (Table 7). When larval trematodes occurred in October 2004, MCCG was not different between infected and healthy snails (F = 0.154; P = 0.715) (Table 7).

Discussion

Disentangle intricate effects of multiple interacting stressors, and related stresses, on freshwater biota over time in the field is challenging and of growing interest for scientists [START_REF] Fischer | The toxicity of chemical pollutants in dynamic natural systems: The challenge of integrating environmental factors and biological complexity[END_REF][START_REF] Lemm | Identification and interaction of multiple stressors in central European lowland rivers[END_REF]. Our field survey contributes to study responses of gastropods to two frequent combined stressors recurrent in stagnant waters, i.e., toxic cyanobacteria proliferations and droughts, typically occurring during summer in most temperate regions [START_REF] Chorus | Toxic Cyanobacteria in Water. A Guide to Public Health Consequences, Monitoring and Management[END_REF][START_REF] Lake | Disturbance, patchiness, and diversity in streams[END_REF][START_REF] Gérard | Long-term dynamics and community structure of freshwater gastropods exposed to parasitism and other environmental stressors[END_REF]). In addition, we take into account concomitantly the potential stress induced by i) the often-neglected parasitism by larval trematodes, ii) the non-native invasive gastropod Physa acuta originated to North America, and iii) the macroinvertebrates commonly acting as predators or competitors of gastropods [START_REF] Dillon | The Ecology of Freshwater Molluscs[END_REF]. The disappearance of nearly all freshwater gastropods (and most other macroinvertebrate taxa) from August to December 2013 reveals stressful conditions in our study-site, probably resulting from negative impact of interacting multiple stressors and their various related stresses.

1) Impact of the main stressors and their related stresses on gastropods

Cyanobacteria proliferations

In addition to toxin production by some strains leading to health hazards, proliferations of cyanobacteria result in related stresses such as disequilibria in phytoplankton representing food resources for freshwater gastropods, but also ammonia release and hypoxia / anoxia [START_REF] Chorus | Toxic Cyanobacteria in Water. A Guide to Public Health Consequences, Monitoring and Management[END_REF][START_REF] Zurawell | Hepatotoxic cyanobacteria: a review of the biological importance of microcystins in freshwater environments[END_REF][START_REF] Paerl | Harmful cyanobacterial blooms: causes, consequences, and controls[END_REF] Otten 2013 for reviews). Ammonia (unionized NH3) is toxic for most aquatic biota with different tolerance degree between species [START_REF] Richardson | Acute ammonia toxicity for eight New Zealand indigenous freshwater species[END_REF][START_REF] Alonso | Short-term toxicity of ammonia, nitrite, and nitrate to the aquatic snail Potamopyrgus antipodarum (Hydrobiidae, Mollusca)[END_REF], and abundance of gastropods decreases with increasing ammonia concentrations due to toxicity [START_REF] Cabuk | Species composition and seasonal variations of the gastropoda in Upper Sakarya River system (Turkey) in relation to water quality[END_REF]. Depletion of dissolved oxygen may also be detrimental for gastropods using exclusive gill breathing such as Bithynia tentaculata (recorded in our study), and in a lesser extent for pulmonates potentially aerial breathing with differential sensitivity [START_REF] Aldridge | Physiological ecology of freshwater prosobranchs[END_REF]McMahon 1983 for reviews). For instance, P. acuta is among the most tolerant macroinvertebrates to hypoxic-anoxic conditions [START_REF] Pardo | Water abstraction in small lowland streams: unforeseen hypoxia and anoxia effects[END_REF].

Concerning the cyanotoxins, at least some strains of cyanobacteria produced MCs during our study in 2013, in particular from August to October (from 8 to 41 µg.L -1 vs < 0.3 µg.L -1 for the other months), and with higher concentrations than in 2004. Indeed, from May to October 2004, intracellular MCs were detected only in August (17 µg.L -1 ) in water column samples [START_REF] Gérard | Influence of toxic cyanobacteria on community structure and microcystin accumulation of freshwater molluscs[END_REF]. In other waterbodies submitted to toxic blooms, the highest intracellular MC concentration recorded in phytoplankton was lower or on the same order (7 µg.L -1 and 9 µg.L -1 in French lakes, 11 µg.L -1 in Canadian lakes), even if surface scum samples could contain considerably higher MC concentrations (400 µg.L -1 at the peak of the bloom) [START_REF] Kotak | Cyanobacterial toxins in Canadian freshwaters: a review[END_REF][START_REF] Gérard | Influence of toxic cyanobacteria on community structure and microcystin accumulation of freshwater molluscs[END_REF][START_REF] Lance | Impact of toxic cyanobacteria on gastropods and microcystin accumulation in a eutrophic lake (Grand-Lieu, France) with special reference to Physa (= Physella) acuta[END_REF]. Experiments in controlled conditions showed that realistic MC intoxications via ingestion of toxic cyanobacteria or exposure to dissolved MCs (from 3 to 50 µg.L -1 ) negatively affect gastropod performance, e.g., fecundity decrease (Gérard et al. 2005;[START_REF] Gérard | Variation in the response of the invasive species Potamopyrgus antipodarum (Smith) to natural (cyanobacterial toxin) and anthropogenic (herbicide atrazine) stressors[END_REF][START_REF] Lance | Interactions between Cyanobacteria and Gastropods. II. Impact of toxic Planktothrix agardhii on the life-history traits of Lymnaea stagnalis[END_REF][START_REF] Lance | Consumption of toxic cyanobacteria by Potamopyrgus antipodarum (Gastropoda, Prosobranchia) and consequences on life traits and microcystin accumulation[END_REF].

Such fecundity decrease can negatively influence the dynamic of gastropod populations [START_REF] Lance | Impact of microcystin-producing cyanobacteria on reproductive success of Lymnaea stagnalis (Gastropoda, Pulmonata) and predicted consequences at the population level[END_REF]). In the field, recurrent toxic proliferations of cyanobacteria may result in profound changes and even decline of gastropod communities [START_REF] Gérard | Long-term dynamics and community structure of freshwater gastropods exposed to parasitism and other environmental stressors[END_REF][START_REF] Gérard | Influence of toxic cyanobacteria on community structure and microcystin accumulation of freshwater molluscs[END_REF][START_REF] Lance | Impact of toxic cyanobacteria on gastropods and microcystin accumulation in a eutrophic lake (Grand-Lieu, France) with special reference to Physa (= Physella) acuta[END_REF], decline also observed during our one-year monitoring in 2013.

MC accumulation occurred in all gastropod tissues whatever species and sampling date in 2013, implying exposure to toxic cyanobacteria throughout the year and/or partial detoxification of MCs, not degraded, or metabolized at a slower rate than intoxicated. The concentration of MCs in gastropod tissues is correlated with toxin in the phytoplankton, likely resulting of grazing herbivory [START_REF] Zurawell | Influence of lake trophic status on the occurrence of microcystin-LR in the tissue of pulmonate snails[END_REF], and is assessed to be higher in gastropods from highly vs lowly MC-contaminated waters [START_REF] Gérard | Influence of toxic cyanobacteria on community structure and microcystin accumulation of freshwater molluscs[END_REF][START_REF] Lance | Impact of toxic cyanobacteria on gastropods and microcystin accumulation in a eutrophic lake (Grand-Lieu, France) with special reference to Physa (= Physella) acuta[END_REF].

Therefore, higher values of gastropod MC accumulation in 2013 (vs 2004) reflect higher contamination of the study-site over time.

During 2013, mean monthly MC accumulation in gastropods greatly fluctuated depending on sampling date, and highest values occurred in February when we recorded no cyanobacteria and no intracellular MCs in the water. It may be surprising but: i) lowest values recorded in spring and summer 2013 corresponded mostly to neonates and juveniles, much less exposed to MCs during their short life than adults, and ii) intracellular MC concentrations in the water were lower in spring and beginning of summer compared to late summer and autumn. Highest values of mean monthly MC accumulation in winter may suggest MC intoxication of gastropods by overwintering vegetative filaments of potentially toxic cyanobacteria such as Aphanizomenon flos aquae and Planktothrix agardhii observed in temperate lakes [START_REF] Head | An assessment of the influence of recruitment from the sediment on the development of plaktonic populations of cyanobacteria in a temperate mesotrophic lake[END_REF][START_REF] Briand | A perennial bloom of Planktothrix agardhii (Cyanobacteria) in a shallow eutrophic French lake: limnological and microcystin production studies[END_REF]Mankiewicz-Boczek et al. 2011) and dominant in our study-site [START_REF] Gérard | Influence of toxic cyanobacteria on community structure and microcystin accumulation of freshwater molluscs[END_REF].

According to some studies, extremely high inter-and intraspecific variations in the MC contents are commonly observed in molluscs, and MC accumulation is on average four times greater in pulmonates than in prosobranchs and bivalves [START_REF] Zurawell | Influence of lake trophic status on the occurrence of microcystin-LR in the tissue of pulmonate snails[END_REF][START_REF] Gérard | Influence of toxic cyanobacteria on community structure and microcystin accumulation of freshwater molluscs[END_REF][START_REF] Lance | Impact of toxic cyanobacteria on gastropods and microcystin accumulation in a eutrophic lake (Grand-Lieu, France) with special reference to Physa (= Physella) acuta[END_REF]. Surprisingly, in our study, no statistically significant differences appeared in MC accumulation between gastropod species. This result may be due to insufficient and biased sampling effort (36 MC-analyzes among them 19 for P. acuta at different developmental stages and 17 for the six other species). However, the maximal values of MC accumulation were always reached in the non-native invasive P. acuta among various co-occurring gastropod species as shown in other studies (up to 24268 ng.g -1 FM for P. acuta vs 3523 ng.g -1 FM for other gastropods) [START_REF] Gérard | Influence of toxic cyanobacteria on community structure and microcystin accumulation of freshwater molluscs[END_REF][START_REF] Lance | Impact of toxic cyanobacteria on gastropods and microcystin accumulation in a eutrophic lake (Grand-Lieu, France) with special reference to Physa (= Physella) acuta[END_REF], suggesting inter-species differences in MC metabolization capacities and detoxification pathways.

Further investigations are needed on the possible relationships between inter-species differences of MC accumulation and physiological stress sensitivity to MCs (and other cyanotoxins), and the consequences on the dynamics of gastropod community structure exposed to toxic blooms.

Droughts

As the water level decreased in our study-site, the littoral area formerly inhabited by freshwater gastropods dried out as observed both in 2004 and 2013. It resulted in a restricted habitat area, depleted in vegetation and organic debris potentially used as habitat/refuge and food (i.e., decrease of resource availability).

Reduction and fragmentation of habitat space due to drought periods generally result in increased biotic interactions such as predation and competition, deterioration of water quality, increased sedimentation, high water temperatures, and hypoxia (< 4 mg 02/l) or even anoxia [START_REF] Schwarz | Temporary aquatic habitats: constraints and opportunities[END_REF][START_REF] Lake | Disturbance, patchiness, and diversity in streams[END_REF][START_REF] Dewson | A review of the consequences of decreased flow for instream habitat and macroinvertebrates[END_REF]; Pardo and García 2016 for reviews). Most freshwater gastropods exposed to drought disappear (e.g., [START_REF] Dudgeon | The effects of water fluctuations on a gently shelving marginal zone of Plover Cove Reservoir, Hong Kong[END_REF]Gérard 2001a) as in our study, despite adaptive strategies such as aestivation and desiccationresistant eggs to survive drying conditions [START_REF] Aldridge | Physiological ecology of freshwater prosobranchs[END_REF][START_REF] Macmahon | Physiological ecology of freshwater pulmonates[END_REF] for reviews). Nevertheless, gastropod communities are resilient to drought stressor since recolonization occurs with gradual restoration of aquatic habitat [START_REF] Dudgeon | The effects of water fluctuations on a gently shelving marginal zone of Plover Cove Reservoir, Hong Kong[END_REF]Gérard 2001a).

2) Larval trematodes acting as biotic stressors and/or stress bioindicators

Helminth parasites such as trematodes provide information on host populations, and more generally, on free-living biodiversity and changes in ecosystem structure and functioning; a healthy ecosystem being one that is rich in parasite species [START_REF] Keas | The prevalence of cercariae from Stagnicola emarginata (Lymnaeidae) over 50 years in Northern Michigan[END_REF][START_REF] Hudson | Is a healthy ecosystem one that is rich in parasite species?[END_REF][START_REF] Morley | Anthropogenic pressure on a molluscan-trematode community over a long-term period in the Basingstoke Canal, UK, and its implications for ecosystem health[END_REF][START_REF] Hechinger | Can parasites be indicators of free-living diversity? Relationships between species richness and the abundance of larval trematodes and of local benthos and fishes[END_REF][START_REF] Marcogliese | Parasites of the superorganism: are they indicators of ecosystem health?[END_REF][START_REF] Marcogliese | The Distribution and Abundance of Parasites in Aquatic Ecosystems in a Changing Climate: More than Just Temperature[END_REF]. The heteroxenous life cycle of most trematodes comprises three obligate hosts including a vertebrate as definitive host and a mollusc as first intermediate host (harboring rediae or sporocysts producing cercariae), the second intermediate host (with metacercariae) generally being an invertebrate (Esch and Fernandez 1994 for review). Larval trematodes frequently infect freshwater gastropods as first intermediate hosts, with detrimental effects on their life-traits, e.g., survival decrease, partial or total parasitic castration [START_REF] Combes | Interactions Durables. Ecologie et Evolution du Parasitisme[END_REF] for review), and thus may be considered as a biotic stressor. Prevalence may vary from 0 to 100% in a same site, depending on gastropod species and sampling date, with a mean monthly prevalence around 6% for the whole gastropod community as for instance in the Combourg Lake [START_REF] Gérard | Long-term dynamics and community structure of freshwater gastropods exposed to parasitism and other environmental stressors[END_REF]).

In our study-site, none of the 2490 snails sampled in 2013, among them 156 belonging to six native species, were parasitized by larval trematodes. This total absence of trematodes in 2013 may be indicative of a loss of free-living diversity (including intermediate and definitive hosts) and of a stressed ecosystem where vulnerable/sensitive stages of trematodes (e.g., free larval miracidia and cercariae) exposed to various environmental stressors probably died. Indeed, survival of larval trematodes often decreases following stress exposure [START_REF] Mackenzie | Parasites as indicators of water quality and the potential use of helminth transmission in marine pollution studies[END_REF][START_REF] Morley | Pollution toxicity to the transmission of larval digeneans through their molluscan hosts[END_REF] for reviews), and trematode prevalence correlates inversely with perturbation degree [START_REF] Huspeni | Using larval trematodes that parasitize snails to evaluate a salt marsh restoration project[END_REF][START_REF] King | Impacts of agriculture on the parasite communities of northern leopard frogs (Rana pipiens) in southern Quebec, Canada[END_REF][START_REF] Merlo | Testing temporal stability of the larval digenean community in Heleobia conexa (Mollusca: Cochliopidae) and its possible use as an indicator of environmental fluctuations[END_REF]. Moreover, local biomasses of all trematode life stages correlate positively with those of their hosts [START_REF] Lagrue | The scaling of parasite biomass with host biomass in lake ecosystems: are parasites limited by host resources?[END_REF], and both diversity and frequency of trematode species also correlate positively with diversity of gastropod species acting as first intermediate hosts (Gérard 2001b;[START_REF] Gordy | A comprehensive survey of larval digenean trematodes and their snail hosts in central Alberta, Canada[END_REF]). In the eutrophic Combourg Lake, 15 of the 17 species of gastropods present at the beginning of the long-term study played the role of first intermediate host for eleven morphotypes of cercariae (Gérard 2001b;[START_REF] Gérard | Long-term dynamics and community structure of freshwater gastropods exposed to parasitism and other environmental stressors[END_REF]. The recurrent exposure to toxic blooms and summer droughts induced the total disappearance of larval trematodes in this lake due to the decline of the gastropod community, and possibly, due to their own stress sensitivity [START_REF] Gérard | Long-term dynamics and community structure of freshwater gastropods exposed to parasitism and other environmental stressors[END_REF]). The single trematode species found only in 2004 in the study-site was an echinostomatid at the redial and cercarial stages infecting the native lymneid R. auricularia. No other gastropod species was infected [even by metacercariae generally characterized by low host-specificity [START_REF] Esch | Snail-trematode interactions and parasite community dynamics in aquatic systems: a review[END_REF][START_REF] Żbikowski | Invaders of an invader -Trematodes in Potamopyrgus antipodarum in Poland[END_REF]]. The cercarial morphotype was previously recorded in the Combourg Lake and named "echinostome 1" in absence of specific identification [START_REF] Gérard | Long-term dynamics and community structure of freshwater gastropods exposed to parasitism and other environmental stressors[END_REF]. In this lake, redial and cercarial stages of "echinostome 1" mainly infected R. auricularia, but also Radix peregra, Planorbis planorbis and Gyraulus albus [START_REF] Gérard | Long-term dynamics and community structure of freshwater gastropods exposed to parasitism and other environmental stressors[END_REF]. In general, members of Echinostomatidae are allogenic, i.e., temporary resident with the life cycle not entirely within the lake, with molluscs as first and second intermediate hosts, and a waterfowl as definitive host [START_REF] Esch | Snail-trematode interactions and parasite community dynamics in aquatic systems: a review[END_REF][START_REF] Gérard | Long-term dynamics and community structure of freshwater gastropods exposed to parasitism and other environmental stressors[END_REF]. Thus, recolonization of the water body by "echinostome 1" we recorded in 2004 may occur via passive dispersion of its molluscan hosts and frequentation of the site by waterfowl definitive hosts, but its maintenance may fail due to sensitivity to stressful environmental conditions and/or absence of one host species in the life cycle.

Finally, larval trematodes did not constitute a biotic stressor for gastropods in our study-site, and their absence may be indicative of a poor environmental quality and a lack of free-living biodiversity.

3) The invasive non-native Physa acuta as a biotic stressor exposed to multiple combined stressors

The freshwater snail, P. acuta, is an exceptionally efficient invader native to North America, introduced mainly via aquarium trade in Europe during the 19th century, and then established in many regions of Africa, Asia, Australia, New Zealand, and South America [START_REF] Albrecht | Invasion of ancient Lake Titicaca by the globally invasive Physa acuta (Gastropoda: Pulmonata: Hygrophila)[END_REF][START_REF] Bousset | Neither variation loss, nor change in selfing rate is associated with the worldwide invasion of Physa acuta from its native North America[END_REF] for reviews). In North American waters, P. acuta occupied every habitat from highly ephemeral pools to deep permanent lakes, and its exceptional habitat breadth stands in contrast to distributional studies of other freshwater taxa [START_REF] Turner | Hydroperiod, predators and the distribution of physid snails across the freshwater habitat gradient[END_REF]. In contrast with other hermaphroditic invasive species, worldwide invasion of P. acuta is not associated with a genetic variation loss nor a change in outcrossing mating system towards more selfing [START_REF] Bousset | Neither variation loss, nor change in selfing rate is associated with the worldwide invasion of Physa acuta from its native North America[END_REF]. The expansion of its invaded range is partly attributed to efficient passive dispersal using animal vectors (waterbirds and large mammals) and off-road vehicles (Kappes and Haase 2012;[START_REF] Van Leeuwen | How did this snail get here? Several dispersal vectors inferred for an aquatic invasive species[END_REF][START_REF] Banha | Dispersal of two freshwater invasive macroinvertebrates, Procambarus clarkii and Physella acuta, by off-road vehicles[END_REF]. Successful invasion of P. acuta is also explained by high reproductive output and high tolerance to water quality decrease (e.g., sewage, pollutants such as heavy metals, cyanobacterial proliferations, hypoxia/anoxia) and changing environmental conditions (e.g., depth fluctuations, temperature increase) [START_REF] Brackenbury | Recolonization of the Umsindusi River, Natal, South Africa, by the invasive gastropod, Physa acuta (Basommatophora, Physidae)[END_REF][START_REF] Blakely | Longitudinal patterns in benthic communities in an urban stream under restoration[END_REF][START_REF] Gérard | Long-term dynamics and community structure of freshwater gastropods exposed to parasitism and other environmental stressors[END_REF][START_REF] Albrecht | Invasion of ancient Lake Titicaca by the globally invasive Physa acuta (Gastropoda: Pulmonata: Hygrophila)[END_REF][START_REF] Turner | Hydroperiod, predators and the distribution of physid snails across the freshwater habitat gradient[END_REF][START_REF] Zukowski | Freshwater snails in competition: alien Physa acuta (Physidae) and native Glyptophysa gibbosa (Planorbidae) in the River Murray, South Australia[END_REF][START_REF] Lance | Impact of toxic cyanobacteria on gastropods and microcystin accumulation in a eutrophic lake (Grand-Lieu, France) with special reference to Physa (= Physella) acuta[END_REF][START_REF] Pardo | Water abstraction in small lowland streams: unforeseen hypoxia and anoxia effects[END_REF].

Moreover, in contrast to native species, P. acuta is not or rarely parasitized in introduced areas such as Europe, Africa and Australia [START_REF] Brackenbury | Recolonization of the Umsindusi River, Natal, South Africa, by the invasive gastropod, Physa acuta (Basommatophora, Physidae)[END_REF][START_REF] Gérard | Long-term dynamics and community structure of freshwater gastropods exposed to parasitism and other environmental stressors[END_REF][START_REF] Mitchell | Sharing the load: a survey of parasitism in the invasive freshwater pulmonate, Physa acuta (Hygrophila: Physidae) and sympatric native snail populations[END_REF], in accordance with enemy release hypothesis [START_REF] Torchin | Introduced species and their missing parasites[END_REF]. For all these reasons, P. acuta is often the dominant species in stressed ecosystems [START_REF] Brackenbury | Recolonization of the Umsindusi River, Natal, South Africa, by the invasive gastropod, Physa acuta (Basommatophora, Physidae)[END_REF][START_REF] Blakely | Longitudinal patterns in benthic communities in an urban stream under restoration[END_REF]Gérard et al. 2008;[START_REF] Albrecht | Invasion of ancient Lake Titicaca by the globally invasive Physa acuta (Gastropoda: Pulmonata: Hygrophila)[END_REF][START_REF] Turner | Hydroperiod, predators and the distribution of physid snails across the freshwater habitat gradient[END_REF][START_REF] Lance | Impact of toxic cyanobacteria on gastropods and microcystin accumulation in a eutrophic lake (Grand-Lieu, France) with special reference to Physa (= Physella) acuta[END_REF], as shown in our study. In its introduced area, some authors demonstrated that the generalist and tolerant P. acuta may outcompete native gastropod species, and thus constitutes a biotic stressor for these latter with consequent changes in community structure and potential decline of some native species [START_REF] Zukowski | Freshwater snails in competition: alien Physa acuta (Physidae) and native Glyptophysa gibbosa (Planorbidae) in the River Murray, South Australia[END_REF][START_REF] Höckendorff | Biotic interactions under climate warming: temperature-dependent and species-specific effects of the oligochaete Chaetogaster limnaei on snails[END_REF].

However, stress exposure may also lead to the disappearance of P. acuta as in Lithuanian inland waters (Europe) where the locally established snail was not recorded since 1950s, possibly due to long term application of herbicides and consequent depletion of oxygen and/or food resources [START_REF] Buktus | Distribution and current status of non-indigenous mollusc species in Lithuanian inland waters[END_REF]).

In our study, the predominance of P. acuta in the gastropod community clearly increased in 2013 compared to 2004 despite stressful environmental conditions and increased MC contamination. As for other gastropods, P. acuta disappeared from August onwards, probably due to acute combined stress exposure (mainly high MC contamination of water and drought). Nevertheless, we can suppose successful recolonization of the study-site by immigration mainly due to efficient passive dispersal that can also benefit to other gastropod species (Kappes and Haase 2012;[START_REF] Van Leeuwen | How did this snail get here? Several dispersal vectors inferred for an aquatic invasive species[END_REF][START_REF] Banha | Dispersal of two freshwater invasive macroinvertebrates, Procambarus clarkii and Physella acuta, by off-road vehicles[END_REF]. Despite high MC level in its tissues (as mentioned previously), P. acuta survived and reproduced (two recruitment periods in May and July) in the study-site as also shown in other studies [START_REF] Gérard | Influence of toxic cyanobacteria on community structure and microcystin accumulation of freshwater molluscs[END_REF][START_REF] Lance | Impact of toxic cyanobacteria on gastropods and microcystin accumulation in a eutrophic lake (Grand-Lieu, France) with special reference to Physa (= Physella) acuta[END_REF]. It may suggest an adaptive strategy of limited investment in energy-consuming detoxification, allowing saving energy allocated to maintenance and life traits, and consequently leading to better competitive ability compared to native gastropod species. We need further investigations in controlled conditions to verify this hypothesis by determining if MCtoxicity threshold is higher for P. acuta than for native gastropod species, and exploring relations between gastropod ability to cope to multiple stressors and to detoxify MCs.

4) Macroinvertebrates acting as predators or competitors for gastropods (biotic stressors) and/or stress bioindicators

Many taxa of freshwater macroinvertebrates recorded in our study-site may prey on gastropods (Astacidae, Notonectidae, Nepidae, Gerridae, Naucoridae, Pleidae, Anisoptera and Zygoptera) or compete with them for food and space (Enchytraeidae, Sphaeridae, Limnadidae, Asellidae, Gammaridae and Corixidae) [START_REF] Dillon | The Ecology of Freshwater Molluscs[END_REF]. Based on our one-year monitoring study, predatory and competitive pressures potentially exerted by macroinvertebrates on gastropods differ among taxa according to their occurrence frequency. Further investigations including specific identification and monthly abundances are needed to determine which macroinvertebrate species can be considered as biotic stressors for gastropods in our study-site. Nevertheless, the impact of predation and competition by macroinvertebrates appears limited over time since nearly all macroinvertebrates disappear after August 2013. Exposure to combined toxic blooms and droughts and their related stresses probably explain the disappearance of most macroinvertebrates, consequently which may be rather considered as stress bioindicators (vs gastropod biotic stressors). Indeed, drought commonly induces decrease of macroinvertebrate richness due to decrease of habitat diversity [START_REF] Gérard | Dynamics and structure of a benthic macroinvertebrate community in a lake after drought[END_REF][START_REF] Dewson | A review of the consequences of decreased flow for instream habitat and macroinvertebrates[END_REF][START_REF] Pardo | Water abstraction in small lowland streams: unforeseen hypoxia and anoxia effects[END_REF]. Increases in cyanobacterial toxicity generally coincide with decreases in total abundance and richness in macroinvertebrate communities [START_REF] White | Impacts of a toxic Microcystis bloom on the macroinvertebrate fauna of Lake Elphinstone, Central Queensland, Australia[END_REF]. Intensive blooms lead to the dying out of most benthic macroinvertebrates, except some taxa less sensitive to cyanotoxin exposure and becoming more abundant [START_REF] Krzyzanek | The effect of water blooms caused by blue-green algae on the bottom macrofauna in the Goczalkowice Reservoir (southern Poland) in 1992[END_REF][START_REF] Oberholster | The influence of a toxic cyanobacterial bloom and water hydrology on algal populations and macroinvertebrate abundance in the upper littoral zone of Lake Krugersdrift, South Africa[END_REF]. Moreover, ammonia exposure and hypoxia/anoxia related to both toxic blooms and droughts may have adverse effects on macroinvertebrates [START_REF] Richardson | Acute ammonia toxicity for eight New Zealand indigenous freshwater species[END_REF][START_REF] Pardo | Water abstraction in small lowland streams: unforeseen hypoxia and anoxia effects[END_REF].

Among the macroinvertebrate taxa recorded in our study-site, three taxa, i.e., Chironomidae, Ephemeroptera and Trichoptera, maintain themselves throughout the year despite stressful conditions. Future studies are necessary to identify the species belonging to the three persistent taxa, and to test their stress tolerance in order to understand how they cope to combined toxic blooms and droughts and their related stresses.

Conclusion

According to our results and previous studies on stress effects, the main stressors acting on gastropods in our study-site are almost certainly toxic blooms and droughts. The combination of both stressors and their related stresses result in extremely stressful conditions for native freshwater gastropods, i.e., decrease of space and food availability, intoxication by ammonia, MCs and probably other cyanotoxins, increase of predation and competition pressures, hypoxia/anoxia, warming. These interacting stresses which effects may be synergistic seem to induce profound and long-lasting effects on taxonomic richness and abundance of gastropods, leading to the elimination of gastropods, as also shown through a long-term study on 10 year-interval in a eutrophic lake submitted to recurrent toxic blooms and droughts [START_REF] Gérard | Long-term dynamics and community structure of freshwater gastropods exposed to parasitism and other environmental stressors[END_REF]).

On the contrary, the biotic stressors studied here: parasitic trematodes, non-native invasive P. acuta, predatory and competitive macroinvertebrates, have probably no or minor impact on gastropods, because they are themselves affected by the above-mentioned combined stressors and almost all disappear following stress exposure, indicating adverse environmental conditions. Despite the fact that gastropods tend to disappear at the end of summer due to acute stressful conditions, they showed extremely high reproductive effort in late spring-early summer 2013. Highly efficient breeding may be an adaptive response to environmental fluctuations and recurrent exposure to multiple interacting stressors and related stresses [START_REF] Sulmon | Abiotic stressors and stress responses: what commonalities appear between species across biological organization levels?[END_REF]. Both high breeding capacities of gastropods and successful recolonization of the stressed ecosystem, probably by efficient passive dispersion from healthier surrounding waterbodies [START_REF] Kappes | Slow, but steady: dispersal of freshwater mollusks[END_REF], suppose a relative resilience of gastropods with alternation of local extinctions vs recolonizations. On a long term, recurrent and increasing stresses could induce a loss of diversity to the detriment of the most sensitive species and to the benefit of the most resistant or tolerant. This hypothesis is supported by the increasing dominance at 9-year interval of the invasive non-native P. acuta, a species commonly dominant in stressed ecosystems and which can outcompete native gastropods, in relation with its better ability to cope with co-occurring stresses and its higher capacity of recolonization compared to native species (see numerous references detailed previously). Further long-term investigations on population dynamics are needed to understand how recurrent and stressful environmental conditions may influence interspecific competitive interactions.

In the future, we also need to include parasites in ecotoxicological studies as already suggested by several authors [START_REF] Morley | Interactive effects of infectious diseases and pollution in aquatic molluscs[END_REF][START_REF] Marcogliese | Combined effects of parasites and contaminants on animal health: parasites do matter[END_REF], and to obtain more data on parasites and their complex interactions with hosts and other stressors, as well as on responses of parasites to multiple interacting stressors. In our study, the absence of trematode parasites due to acute stressful conditions and decline of gastropod host species, but also decline of macroinvertebrates potentially acting as second intermediate hosts, confirms their potential use as indicators of poor free-living biodiversity and stressed ecosystem (e.g., [START_REF] Keas | The prevalence of cercariae from Stagnicola emarginata (Lymnaeidae) over 50 years in Northern Michigan[END_REF][START_REF] Morley | Anthropogenic pressure on a molluscan-trematode community over a long-term period in the Basingstoke Canal, UK, and its implications for ecosystem health[END_REF]. One question asks about potential differences in toxin accumulation between infected and uninfected snails, with what consequences on toxin transfer through the food web since trematodes generally favor predation of parasitized molluscs by vertebrate definitive hosts [START_REF] Combes | Interactions Durables. Ecologie et Evolution du Parasitisme[END_REF]. Some authors demonstrated changes in pollutant (xenobiotics and cadmium) accumulation in freshwater and marine bivalves due to larval trematode parasitism [START_REF] Heinonen | The effects of parasites and temperature on the accumulation of xenobiotics in a freshwater clam[END_REF][START_REF] Baudrimont | Evidence of an altered protective effect of metallothioneins after cadmium exposure in the digenean parasite-infected cockle (Cerastoderma edule)[END_REF], but studies on this subject are scarce and do not investigate trematodes-gastropods associations.

Figure caption

Fig. 1 Relative size-frequency distributions and mean shell size (± CI) of the invasive gastropod Physa acuta each month from January to August 2013 in the station "Les Rues" of the Frémur River (mean size and size classes in mm, relative abundance from 0 to 30% with 10%-step graduation). We sampled no P. acuta from September to December 2013. Sample sizes are indicated in Table 1.

  Figure 1

Table 1 . Environmental conditions, i.e., water temperature (°C), maximal water depth (cm), intensity of cyanobacteria occurrence (class from 0 to 3) and intracellular MCs (µg.L -1 ), and abundance and species richness of gastropods sampled in the Frémur during 2013. DL = detection limit (0.04 µg.L -1 ).

 1

		Jan	Feb	Mar	Apr	May	Jun	Jul	Aug	Sep	Oct	Nov	Dec	Total
	Water temperature (°C)	5	3	0.5	11	15	16	22	21	17	17	9.5	7
	Max water depth (cm)	65	65	60	60	60	55	40	20	38	38	39	37
	Cyanobacteria occurrence	0	0	0	0	0	1	0	2	3	3	0	0
	Intracellular MCs (µg.L -1 )	≤ DL	≤ DL	≤ DL	≤ DL	0.260 0.274 0.192 7.819 41.320 14.249 0.171 0.073
	Caenogastropoda
	Bithynia tentaculata	1				2	2	6						11
	Heterobranchia
	Physa acuta	34	44	33	14	30	952	1226	1					2334
	Planorbis planorbis	15	3	5		2	47							72
	Hippeutis complanatus	21	4	3			5	12						45
	Radix ovata					1	12	11						24
	Radix auricularia							2				1		3
	Ancylus fluviatilis							1						1
	Total abundance	71	51	41	14	35	1018	1258	1	0	0	1	0	2490
	Species richness	4	3	3	1	4	5	6	1	0	0	1	0	7

Table 2 . Structure of the gastropod community in the Frémur from January to December 2013. A: abundance, F: frequency ± CI (%), FO: mean frequency of occurrence ± CI among the 12 sampled months (%), CI: confidence interval. Abbreviations of gastropod species in parentheses.

 2

		A	F (CI)	FO (CI)
	Caenogastropoda
	Bithynia tentaculata (Bte)	11	0.44 (0.25-0.79)	33.33 (13.81-60.94)
	Heterobranchia
	Physa acuta (Pac)	2334	93.73 (92.71-94.62)	66.67 (39.06-86.19)
	Planorbis planorbis (Ppl)	72	2.89 (2.30-3.63)	41.67 (19.33-68.05)
	Hippeutis complanatus (Hcom)	45	1.81 (1.35-2.41)	41.67 (19.33-68.05)
	Radix ovata (Rov)	24	0.96 (0.65-1.43)	25.00 (8.89-53.23)
	Radix auricularia (Rau)	3	0.12 (0.04-0.35)	16.67 (4.70-44.80)
	Ancylus fluviatilis (Afl)	1	0.04 (0.01-0.23)	8.33 (1.49-35.39)

Table 3 . Mean shell size ± confidence interval (CI) of each gastropod species from January to December 2013 in the Frémur. We sampled no gastropods in September, October and December.

 3

		January	February	March	April	May	June	July	August	November
	Bithynia tentaculata	8.5				8.15 ± 0.45 8.45 ± 0.65 2.78 ± 0.98
	Physa acuta	5.32 ± 0.32 5.59 ± 0.28 5.58 ± 0.32 5.64 ± 0.40 3.98 ± 0.42 4.05 ± 0,12 2.98 ± 0,07	6.0
	Planorbis planorbis	4.73 ± 0.35 3.73 ± 0.99 5.60 ± 0.33		5.60 ± 0.40 2.86 ± 0.26
	Hippeutis complanatus	2.63 ± 0.08 2.73 ± 0.09 2.77 ± 0.15			3.94 ± 0.65 2.40 ± 0.19
	Radix ovata					3.5	4.85 ± 1,99 5.96 ± 1,55
	Radix auricularia							13.00 ± 0.43		14.3
	Ancylus fluviatilis							4.3

Table 4 . Abundance (A), frequency (F) ± CI (%) and mean shell size ± CI of gastropods in June and October 2004 in the Frémur (CI: confidence interval).

 4 [START_REF] Gérard | Influence of toxic cyanobacteria on community structure and microcystin accumulation of freshwater molluscs[END_REF] observed cyanobacterial occurrence from May to October 2004 with a peak in July, but they did not detect intracellular MCs excepted in August 2004 (17.05 µg.L -1 ).

			June 2004			October 2004
		A	F (CI)	Size ± CI	A	F (CI)	Size ± CI
	Bithynia tentaculata	3	3.95 (1.35-10.97)	4.08 ± 5.83
	Physa acuta	48	63.16 (51.92-73.12)	4.78 ± 0.66	7	38.89 (20.30-61.38)	8.66 ± 1.32
	Planorbis planorbis	19	25.00 (16.63-35.78)	4.09 ± 0.71
	Hippeutis complanatus	2	2.63 (0.72-9.10)	2.25 ± 0.00
	Radix ovata	4	5.26 (2.07-12.77)	4.50 ± 2.44
	Radix auricularia				11	61.11 (38.62-79.70) 14.93 ± 2.54

Table 7 . Monthly occurrence and frequency of occurrence (FO%, confidence interval CI) of macroinvertebrates other than gastropods in the Frémur during 2013. Abundance is indicated in parentheses when only some individuals were recorded.

 7

		Jan	Feb	Mar	Apr	May	Jun	Jul	Aug	Sep	Oct	Nov	Dec	FO% (CI)
	Annelida
	Enchytraeidae					x	x	x				x		33.33 (13.81-60.94)
	Piscicolidae					x	x	x						25.00 (8.89-53.23)
	Erpobdellidae							x						8.33 (1.49-35.39)
	Bivalvia
	Sphaeriidae					x (1)		x (1)						16.67 (4.70-44.80)
	Crustacea
	Limnadidae			x	x	x	x	x	x					50.00 (25.38-74.62)
	Asellidae	x	x	x	x	x	x	x	x					66.67 (39.06-86.19)
	Gammaridae	x	x (1)											16.67 (4.70-44.80)
	Astacidae							x (1)	x (4)		x (1)			25.00 (8.89-53.23)
	Hexapoda
	Chironomidae	x	x	x	x	x	x	x	x	x	x	x	x (1)	100.00 (75.75-100.00)
	Corixidae	x	x	x		x	x	x	x					58.33 (31.95-80.67)
	Notonectidae		x				x	x						25.00 (8.89-53.23)
	Nepidae					x		x						16.67 (4.70-44.80)
	Gerridae					x								8.33 (1.49-35.39)
	Naucoridae							x						8.33 (1.49-35.39)
	Pleidae									x (1)				8.33 (1.49-35.39)
	Trichoptera	x	x	x	x	x			x	x	x	x		75.00 (46.77-91.11)
	Anisoptera						x	x						16.67 (4.70-44.80)
	Zygoptera	x	x				x	x						33.33 (13.81-60.94)
	Ephemeroptera	x	x	x	x	x	x	x	x	x	x	x	x (4)	100.00 (75.75-100.00)
	Coleoptera					x	x	x		x (1)			x (1)	41.67 (19.33-68.05)
	Arachnida
	Hydracarina		x (1)	x (10)	x	x	x	x				x		58.33 (31.95-80.67)

Acknowledgments. We thank Valérie Briand for bibliographical assistance.

analyses using the tissues of one or several individuals depending on their body fresh weight, number of individuals per sample in parentheses; juveniles in italics, adults in bold). See abbreviations of gastropod species in

Table 2. Jan Feb Mar Apr May Jun Jul Aug Nov Bte 1237.26 (1)

Conflict of interest