Parasitism can be a confounding factor in assessing the response of zebra mussels to water contamination

Environmental Pollution 162 (2012) 234e240

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Parasitism can be a confounding factor in assessing the response of zebra mussels to water contamination Laëtitia Minguez a, Thierry Buronfosse b, Jean-Nicolas Beisel a, Laure Giambérini a, * a

Université Paul Verlaine e Metz, Laboratoire des Interactions Ecotoxicologie, Biodiversité, Ecosystèmes (LIEBE), CNRS UMR 7146, Campus Bridoux, Rue du Général Delestraint, F 57070 Metz, France b Université de Lyon, Laboratoire d’endocrinologie, Ecole Nationale Vétérinaire de Lyon, Avenue Bourgelat, 69280 Marcy l’Etoile, France

a r t i c l e i n f o

a b s t r a c t

Article history: Received 17 August 2011 Received in revised form 25 October 2011 Accepted 2 November 2011

Biological responses measured in aquatic organisms to monitor environmental pollution could be also affected by different biotic and abiotic factors. Among these environmental factors, parasitism has often been neglected even if infection by parasites is very frequent. In the present field investigation, the parasite infra-communities and zebra mussel biological responses were studied up- and downstream a waste water treatment plant in northeast France. In both sites, mussels were infected by ciliates and/or intracellular bacteria, but prevalence rates and infection intensities were different according to the habitat. Concerning the biological responses differences were observed related to the site quality and the infection status. Parasitism affects both systems but seemed to depend mainly on environmental conditions. The influence of parasites is not constant, but remains important to consider it as a potential confounding factor in ecotoxicological studies. This study also emphasizes the interesting use of integrative indexes to synthesize data set. Ó 2011 Elsevier Ltd. All rights reserved.

Keywords: Dreissena polymorpha Microparasites Biological responses Environmental monitoring

1. Introduction Zebra mussels (Dreissena polymorpha) are freshwater bivalves used worldwide to monitor environmental pollution. Their advantages relate to terms of sampling facility, sessile habits, great sensitivity to xenobiotics and high filtering rate (Guerlet et al., 2007; Kraak et al., 1991; review in Kwan et al., 2003). Generally, biological responses of bivalves are a reflection of the water quality but this relationship is affected by a number of biotic and abiotic factors, like reproductive condition or food availability (Bochetti and Regoli, 2006; Domouhtsidou and Dimitriadis, 2001; Guerlet et al., 2007; Klerks and Fraleigh, 1997; Kraak et al., 1991). Among these environmental factors, parasitism has often been neglected in such studies even if infection by parasites is very frequent. Indeed, more and more studies report parasites as confounding factors disturbing the physiological status of their hosts and particularly their endocrine system, their defence systems, or their survival (Marcogliese and Pietrock, 2011; Morley, 2006, 2010; Paul-Pont et al., 2010; reviews in Sures, 2004, 2008a). D. polymorpha is not spared from infections since over 45 endosymbiont species have been described using it as host in their

* Corresponding author. E-mail address: [email protected] (L. Giambérini). 0269-7491/$ e see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.envpol.2011.11.005

life cycles (Mastitsky, 2004; Mastitsky and Gagarin, 2004; Mastitsky and Samoilenko, 2005; Molloy et al., 1997, 2001, 2005). Moreover, a few studies have shown that parasitism can affect the bioaccumulation of some metals by zebra mussels (Kraak and Davids, 1991) or also their biological responses in the face of environmental pollution (Minguez et al., 2009). After our previous results (see Minguez et al., 2009), we broadened our studies to other sites with different chemical contamination and particularly a case study up- and downstream from a waste water treatment plant (WWTP). We also tested additional biomarkers at cellular, tissue and individual organism levels. The biological responses currently studied are commonly used in biomonitoring. Four cellular responses in the digestive gland were assessed by histochemistry and image analysis: the structural changes of the lysosomal and peroxisomal systems and the accumulation of neutral lipids and lipofuscin granules. The lysosomal system is involved both in a huge number of normal physiological processes such as intracellular digestion or immune responses, and in detoxification/excretion processes of toxic xenobiotics (Cajaraville et al., 1995; Etxeberria et al., 1995; Moore, 1988). The peroxysomal system consists of organelles containing enzymes (e.g. catalase) which mainly participate in the b-oxidation of fatty acids and antioxydant processes (Cancio and Cajaraville, 2000). Neutral lipids represent an important energy source for bivalve molluscs (Calvaletto and Gardner, 1999; Olsen, 1999). Protein, glycogen and

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triglyceride levels available in gonads were also quantified. Energy reserves are closely linked to environmental conditions and the reproductive cycle (Stoeckmann and Garton, 2001). The aim of the present study was to assess (1) the infection level related to the zebra mussel depth of attachment in order to determine if one of the microhabitats present less infected organisms than the other, and (2) the influence of parasitism on the physiology of D. polymorpha from a specific geographic situation (i.e. up- and downstream from the WWTP of Metz, Northeastern France). The obtained results can be important for the use of zebra mussels in the assessment of freshwater quality, if no information about possible infections by parasites is available. 2. Materials and methods 2.1. Sampling and tissue preparation Two sampling sites were selected on northeastern France up- and downstream from the WWTP of Metz, on the Moselle River (49
080 06.5500 N 06
100 51.9800 E and
49 100 46.4500 N 06
11056.7100 E, respectively). Moreover, the second site is also located downstream from the confluence of the Moselle River with the Seille River (Fig. 1). Water and sediment were collected to determine main physico-chemical characteristics (e.g. trophic conditions, main organic and metallic burdens, see Table 1) and transported to the laboratory in 2 L polyethylene tanks. The following water analyses were made: cations were determined by flame AAS (PerkineElmer Aanalyst 100) after water acidification (1% HNO3). Only NH4þ was analyzed by graphite furnace AAS (Varian Spectra-300). Anion concentrations were measured by ion exchange chromatography (Dionex). The chemical oxygen demand, 5-day biochemical oxygen demand (COD and BOD5) and suspended matter were evaluated, respectively, by volumetric, oxymetric and gravimetric methods. The sediment analyses were performed after sieving (4 mm) according to the standards EN 13346/ISO 11885 for metals and XP X 33012 for PAHs. Two groups of zebra mussels were collected, on May 2008, related to their habitats, either on the banks or in the river channel, in the two study sites. For the first group organisms were handpicked between 80 and 120 cm depth on the rocky shores (i.e. 100 organisms per study site) whereas drag samplings were conducted to collect mussels from the channel (400 cm depth). For mussels of channels, a sampling effort was made, and 40 or 45 organisms were collected up- and downstream the WWTP, respectively. The shell lengths for each experimental group were (mean  standard deviation): upstream bank: 16.8  1.1 mm, channel: 17.1  1.8 mm, downstream bank: 18.4  1.3 mm, channel: 19.3  2.1 mm. All shells were thoroughly cleaned for any surface deposits or epibionts, and the maximum length (L), height (h) and width (w) were measured with a calliper rule to the nearest 0.01 mm. The soft tissues were then removed, the excess body fluid absorbed with absorbent paper, and then tissues were weighted on a Pioneer PA214CM balance to the nearest 0.1 mg. A condition index (CI) expressed in g cm3, was calculated as follows: CI ¼ m/(L  h  w), giving an indication of the mussel’s nutritional status and stress. For organisms sampled on banks, a part of the gonad (also weighed) and the digestive gland were then excised and used to quantify energetic reserves and

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Table 1 Results of the physico-chemical analysis carried out on sediment and water of each sampling sites (May 2008). Parameters

Upstream

Downstream

WWTP of Metz (Moselle river) Sediment (mg/kg dry matter) Cr Cu Hg Ni Pb Zn Acenaphtene Anthracene Benzo(a)anthracene Benzo(a)pyrene Benzo(b)fluoranthene Benzo(e)pyrene Benzo(ghi)perylene Benzo(k)fluoranthene Chrysene Dibenzo(ah)anthracene Fluoranthene Fluorene Indeno(1.3cd)pyrene Naphthalene Phenanthrene Pyrene Water (mg/L) pH Conductivity 25
C (mS/cm) Coliforms (n/100 mL)a Enterococcus (n/100 mL)a Escherichia coli (n/100 mL)a NH4þ NO2 NO 3 N kjeldahl  PO4 P total Cl SO2 4 Chlorophyll a Phaeopigment Suspended matter Ca2þ Mg2þ Naþ Kþ COD BOD5 a

47 37 <1 31 102 312 <0.02 0.34 1.00 0.53 0.86 1.98 0.73 0.38 1.01 0.05 2.18 0.03 0.85 0.18 0.79 1.58

149 57 <1 89 118 300 0.04 0.11 0.23 0.13 0.25 0.50 0.24 0.10 0.23 0.02 0.61 0.08 0.19 0.10 0.41 0.54

7.90 1640 3000 40 3000 0.07 0.03 1.54 0.58 0.05 0.18 432 71 2.22E-03 5.75E-03 11.5 170 10.5 110.0 6.31 43 1.3

7.95 1720 3000 200 3000 0.01 0.02 1.31 0.65 0.01 0.11 422 65 1.56E-02 1.26E-02 24.3 180 11.9 116.6 6.10 21 2.0

Data from the French water agency (2010).

cellular responses, respectively (see below). All the remaining tissues were used for the parasite inventory. No analyses of biological responses were performed on zebra mussels collected from channels, since there were not enough infected individuals to do biological analyses (i.e. 40e45 individuals). 2.2. Parasite inventory The procedure for parasite inventory is described by Minguez et al. (2009). Briefly, after classical histological techniques, 30e40 sections of zebra mussel (5 mm) were studied microscopically for the presence of parasites. On each section, all the organs are visible. The level of infection was assessed using standard epidemiological parameters (Bush et al., 1997): prevalence (percentage of infected organisms) and mean intensity (mean number of a parasite per infected organism, data were given as mean values  standard deviation (SD)). The study of biological responses was performed only on zebra mussels sampled on the banks. After the inventory, several experimental groups of 3e5 individuals were formed according to the parasite species or the association of parasites. When possible, the host sex was also taken into account. The experimental groups were then used for the assessment of the cellular defences and energetic reserves. 2.3. Determination of gonadal index Fig. 1. Location of the sampling sites in the Moselle River. WWTP: waste water treatment plant.

A mean gonadal index (GI) was calculated for each experimental group by microscopic observation of the slides. This index allows the determination of the

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gonad maturity of zebra mussels (Tourari et al., 1988). Mussels were classified in one of six successive stages of gonad maturation, common or not to both sexes: an apparent sexual rest (stage 0), gametogenesis initiation (stage Ia), early gametogenesis (stage Ib for males and stage IaPrS for females), advanced gametogenesis (stage II for males and stage IaS for females), sexual maturity (stage III for males and stage IaPostS for females) and spawning (stageE). An arbitrary score from 0 to 5 was attributed to each stage, and the following formula was used to calculate gonadal index: GI ¼ (S ni$si)/N where ni is the number of individuals in each stage, si the score of the stage and N the total number of individuals. 2.4. Histochemistry and stereology on the digestive gland The removed digestive glands were prepared as described in Giambérini and Cajaraville (2005) and used to measure four cellular responses: the structural changes of the lysosomal system, the peroxisomal catalase activity, and the accumulation of neutral lipids and lipofuscin granules. According to Giambérini and Cajaraville (2005), the digestive lysosomal system was located by the revelation of b-glucuronidase activity in unfixed cryostat sections. Unsaturated neutral lipids were demonstrated by oil red O staining (Moore, 1988), and lipofuscin granules were stained by the Schmorl reaction (Pearse, 1972). To reveal the peroxisomal catalase, we used the methods described by Guerlet et al. (2006). These four cellular biomarkers were quantified on digestive tissue sections (8 mm) by image analysis (Cell*, Olympus) using a Sony DP 50 color video camera connected to an Olympus BX 41 microscope with a 100x objective. Five fields of view were randomly analysed on one section per individual. Areas not belonging to digestive tissues were discarded from analysis. The stereological parameters used in this study to simplify the dataset are the volume density of the lysosomal and peroxysomal system, and the surface densities of neutral lipid droplets and lipofuscin granules (VvL ¼ VL/VC; VvP ¼ VP/VC; SvNL ¼ SNL/VC; SvLF ¼ SLF/VC, where C ¼ digestive cell cytoplasm, L ¼ lysosomes, P ¼ peroxisomes, NL ¼ neutral lipids, LF ¼ lipofuscin, S ¼ surface, V ¼ volume) (Lowe et al., 1981). 2.5. Energetic reserves in gonads The excised pieces of gonad were used to quantify proteins, triglycerides and glycogen levels. Samples were homogenized in 200 mL PBS 1x and energetic reserves directly measured using a KonelabÒ (Thermo Fisher Scientific, Cergy Pontoise). Measurements were based on colorimetric methods (Burtis and Ashwood, 2001). Briefly, the glycerol obtained after the hydrolysis of triglycerides by a lipase was measured at 510 nm. The bond between proteins and red pyrogallolemolybdate complexes was measured at 600 nm. To measure the glycogen levels, it was extracted and digested by an amiloglucosidase (CAS 9032-08-0, Sigma Aldrich, France). Then, glucose was oxidized by a glucose-oxydase leading to a chromogen whose concentration was measured at 510 nm. Finally, glycogen concentrations were calculated using oyster glycogen as calibrating control (CAS 9005-683-8, SigmaeAldrich, France). 2.6. Data analysis All the statistical analyses were performed using Statistica software version 7.1. (Statsoft, USA). Differences were considered significant at p < 0.05. The c2 test was used to evaluate differences in prevalence rates. Mean infection intensities and physiological responses were examined with the one-way analysis of variance (ANOVA) followed by Duncan’s post-hoc tests after testing for normality and variance homogeneity of the data. Thus, measurements of the intensity of infection and the biological responses were log-transformed to meet these assumptions. The correlation between biological responses was assessed with the Spearman correlation coefficient. In multiple biomarker surveys, the use of integrative data treatment brings a better comprehension of complex sets of results in a synthetic and more relevant way than the individual analysis of each single biomarker response. Therefore, to evaluate the impact of parasite species and water quality, we applied the “Integrated Biomarker Response”, which combines all the measured biomarker responses into one general “stress index” (IBR, Beliaeff and Burgeot, 2002). Three types of IBRs were calculated: (1) a general index with all tested biomarkers and the condition index. Triglycerides in the gonad were found to correlate with neutral lipids in the digestive gland and so were removed to not over-represent the response of lipid contents in the final index value. Protein levels bringing no information were also removed, since the contents were not different between experimental groups. (2) An IBR with the energetic reserves in the gonad (i.e. glycogen, triglycerides, proteins) and (3) an IBR with only the four cellular responses measured in the digestive gland were also determined (i.e. lysosomes, perosysomal catalase, lipofuscines, neutral lipids). The IBR calculation of Beliaeff and Burgeot (2002) was slightly modified: individual areas Ai connecting the ith and the (iþ1) th radius coordinates of the star plot were obtained in a simpler way, according to the following formula (Guerlet et al., 2010): Ai ¼ 1/2 sin(2p/n)SiSiþ1 where Si and Siþ1 represent the individual biomarker scores (calculated from standardized data) and their successive star plot radius coordinates and n represent the number of radii corresponding to the biomarkers used in the survey. We chose to range the biomarkers according to their order in the hierarchy of the

biological organization, from the subcellular to the individual level, as follows: VvL < VvP < SvLF < SvNL < Glycogen level < CI, in the ‘general IBR’.

3. Results 3.1. Study sites Results of physico-chemical analysis carried out on the sediment and water of both sites are reported in Table 1. The upstream station showed a poor quality according to the quality thresholds defined in the French System of Evaluation of waterways “Water Quality, SEQ-Eau v2” particularly for PAHs in sediments which concentrations were at least two-times higher than in the other study site (MEDD and Agences de l’Eau, 2003). As for the downstream station, it displayed a very poor quality due to their higher metallic contamination with chrome and nickel concentrations almost three times higher than those observed at the upstream station. Concerning the water samples, the two sites showed on average the same conductivity but mainly differed by higher concentrations of nitrogen, phosphorus, and the COD in the upstream site whereas more suspended matter, chlorophyll a/phaeopigments and bacteria (e.g. Enterococci) were found in the downstream station. 3.2. Parasite assemblages and microhabitats The inventory of parasites in the different sites and microhabitats revealed that zebra mussels were only infected by Ophryoglena spp. (Oph), ciliates in the digestive gland lumina, and intracellular bacteria in digestive cells, Rickettsiales-like organisms (RLOs). No macroparasite species were observed. Fig. 2 shows the prevalence rates (A and B) and mean infection intensities (C and D) of each parasite. The intracellular bacteria RLOs were predominant in the downstream station with two times more infected zebra mussels (Fig. 2A) with almost two times more bacterial inclusions (Fig. 2C). No differences were observed related to the microhabitats, i.e. the bank or the channel. In contrast, prevalence rates (Fig. 2B) and mean infection intensities (Fig. 2D) of Ophryoglena spp. displayed different patterns related to the site and the microhabitats. In the upstream station, more zebra mussels were infected in the banks than in the channel whereas in the downstream the opposite pattern was observed. Co-infections with RLOs and Ophryoglena spp. tended to display the same profile (Fig. 3). Differences were observed only between the channel (prevalence ¼ 15.56%) and the bank (prevalence ¼ 6.93%) of the downstream station or the channel of the upstream station (prevalence ¼ 3.23%) (p < 0.10). 3.3. Biological responses The Integrated Biomarker Responses are shown in Fig. 4. All the tested biomarkers were taken into account in the first IBR to synthesize the effects of station quality and parasitism on the physiological status of zebra mussels (Fig. 4A). The two other IBRs were calculated to more closely examine the stress induced in the storage of energetic reserves in the gonad (Fig. 4B) or in cellular responses measured in the digestive gland (data not shown). Mussels from upstream the WWTP seemed to be more stressed (higher IBR values) than mussels from the downstream station, without effect of the infection status. In contrast, parasitism tended to enhance the stress associated with the station in organisms from downstream the WWTP (Fig. 4A). Looking more precisely at the energetic reserves quantified in the gonad, organisms sampled upstream the WWTP displayed lesser energetic reserves (higher IBR values, Fig. 4B) especially in glycogen (ANOVA, p < 0.05). Protein contents were strictly the same in mussels from the two

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Fig. 2. Parasitological parameters of the two studied microparasites: Prevalence (A) and mean infection intensities (S.D.) (C) of intracellular bacteria Rickettsiales-like organisms (RLOs), and prevalence (B) and mean infection intensities (D) of ciliates Ophryoglena spp. Different letters indicate significant differences between groups (p < 0.05).

stations (data not shown). However these differences do not affect the gonad maturity since mussels from both sites displayed almost the same gonadal index (GI z 4.5). Cellular responses measured in the digestive gland displayed an opposite pattern, with higher IBR values for downstream organisms, particularly infected ones (data not shown). These higher values were related to a more developed lysosomal system, an accumulation of neutral lipids and lipofuscin granules (data not shown). Moreover, it should be noted that females and males did not respond in the same way to environmental factors, like pollution and parasitism (Fig. 4). For example, females displayed fewer energetic reserves than males particularly upstream WWTP (Fig. 4B). Biological responses taken individually showed that the effect of the predominant parasite of each shallow area, alone, prevailed in cases of co-infections (data not shown). Ophryoglena spp. dominated in zebra mussels from upstream WWTP, whereas RLOs infections did in those of the downstream station.

Fig. 3. Prevalence of coinfections with RLOs and Ophryoglena spp. in the four microhabitats. The p-values were indicated when less than p < 0.10.

4. Discussion The present investigation in the framework of environmental quality assessment brings new information on the system formed by zebra mussels and its endoparasites, through the study of parasite infra-communities and the host biological responses observed up- and downstream from a perturbation, i.e. a waste water treatment plant. 4.1. Parasites and microhabitats The interactions between pollution and parasitism can vary and mainly depend on the level/sort of pollution, the parasite species and habitat (Morley, 2010; Poulin, 2007; Sures, 2008b). In the present study, only microparasites, intracellular bacteria and ciliates, were observed in the digestive gland. No bivalve was infected by macroparasites (e.g. trematodes, nematodes). These macroparasites classically need several hosts in their life cycles and possess free-living stages. Their absence in the two stations could be due to a higher susceptibility of infected (vs healthy) hosts to pollution or the absence of one of them, or also the negative effect on free-living stages (miracidium larvae, cercariae) (Lei and Poulin, 2011; Morley et al., 2001, 2003; Pietrock et al., 2002; Siddall and Clers, 1994). In contrast, investigations on microparasites highlighted that their prevalence rates tended to increase with the pollution (Chu et al., 2002; Jacobson et al., 2010; Kim et al., 1998; Minguez et al., 2011; Moles, 1999). The zebra mussel is a filter feeder fixed on hard substrates at different depths. Therefore we wondered if there were differences in infection between organisms sampled on the banks or in the channels. This information can be useful if we want to minimize the number of sampled infected mussels and thus, the potential associated bias in ecotoxicological studies. A sampling effort was made in the channels to get enough zebra mussels, but only 40

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with a higher amount of pollution in water. This contamination could weaken zebra mussels thus more susceptible to infections. In contrast, in channel samples ciliates were more numerous in zebra mussels from the downstream station. This pattern could be explained by sediment toxicity. Indeed, the upstream station seemed to be more impacted and the lower prevalence rate of Ophryoglena spp. observed in the up-channel suggest a stronger mortality of infected mussels which cannot be thus sampled any more. The toxicity level of downstream sediment would promote ciliate infections without leading to the death of the hosts. Microhabitats with higher levels of Ophryoglena infection tended to also display more coinfections. To date, any competitive exclusion has been highlighted between Ophryoglena sp. and RLOs in zebra mussels, so in the micro-habitats where ciliates or RLOs were abundant, the probability of coinfections is increased. In the channel of the downstream site where ciliates and RLOs prevalence were higher, the rate of co-infections tended to be the highest. 4.2. Biological responses

Fig. 4. Integrated Biomarker Response (IBR) of each infection status x study site couple, with (A) energetic reserves and cellular responses (i.e. health status), and (B) energetic reserves in the gonad only. NI: non-infected, Oph: infected by Ophryoglena spp., RLOs: infected by Rickettsiales-like organisms, Oph-RLOs: co-infection by both parasites, M: male, F: Female.

individuals per channel have been collected. Nonetheless, even if all the biological responses could not be measured it remains interesting to compare the parasite communities related to the microhabitat and the site quality. Zebra mussels from the downstream station of the WWTP of Metz were more infected by RLOs than those from the upstream one. However, no differences were observed between banks and channels, which could be explained by the number of individuals in each population. The downstream site was defined by a very poor quality related to higher concentrations of chromium and nickel. This difference in infection parameters was consistent with previous results showing a positive correlation between RLOs prevalence and the level of nickel contamination, in Crassostrea spp. and Dreissena polymorpha (Kim et al., 1998; Minguez et al., 2011). Moreover, downstream, fecal bacteria were more abundant. Reible (1999) pointed out that Rickettsiale-like bacteria were often associated with the decomposition of fecal matter and thus related to rural wildlife or municipal discharges. Concerning the infection by Ophryoglena spp., different patterns were observed between the up- and downstream of the WWTP and between the bank and channel. First, looking at bank samples, zebra mussels were more infected in the up-station. This site is characterized by a two-fold higher chemical oxygen demand (COD) (level 3 according to the “Water Quality, SEQ-Eau v2”) than in the downstream site (level 1), linked

More and more studies have shown the interest in multi-marker approaches for monitoring both environmental quality and the health of organisms, since a single biological response does not provide information on the general health status (Binelli et al., 2010; Brown et al., 2004; Guerlet et al., 2006, 2007; Zorita et al., 2006). Nonetheless, results of such studies where each biomarker is separately interpreted are difficult to share with managers, and currently, we more often use multivariate techniques, like principal component analyses, ‘Integrative Biomarker Response’ index (IBR) or the DISAV expert system (Beliaeff and Burgeot, 2002; Broeg and Lehtonen, 2006; Dagnino et al., 2007; Damiens et al., 2007; Guerlet et al., 2010). In the present investigation, we performed three IBR to integrate all the tested biological responses into one ‘stress index’. An index was calculated for each studied tissue, one with the energetic reserves in the gonad and another with cellular responses in the digestive gland. The last index, i.e. ‘health status IBR’, was obtained with the responses measured at different biological levels, i.e. cellular (defence systems), tissular (energetic reserves) and individual (CI) and characterized the general health status of organisms. Zebra mussels sampled upstream displayed less energetic reserve (data not shown), highlighted here with higher IBR values. This difference can be explained by the food quantity/quality more important in the down-station (i.e. chl a/phaeopigment concentrations) which could come from the sowing of the Moselle River by the Seille River. Intersex differences were also observed in energetic reserves. For example, females displayed higher triglyceride content in gonads whatever their infection status or the site. This may be explained by the accumulation of lipid nutrients in the oocytes whereas males cannot store lipid reserves in the spermatocystes (Mouneyrac et al., 2008; Palais et al., 2011). Parasitism seemed to have a significant effect on energetic reserves, increasing the stress associated with site quality and this was particularly true for females. Indeed, in both sites, infection by Ophryoglena spp. and/or RLOs involved glycogen depletion in female gonads compared with non-infected females. The gonad is an important storage area of glycogen for the gametogenesis (Jokela et al., 1993). However, a part of this energy could be allocated to maintenance in infected females, glycogen being quickly mobilized. Concerning the cellular responses, differences between sites were less marked. The activation of cellular systems measured in organisms sampled upstream would be related with defence mechanisms against pollution. In contrast, the responses of zebra mussels from the down-site, like a more developed lysosomal system and an accumulation of neutral lipids, were related to food intake since the lysosomal system is

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involved in intra- and extracellular digestion and allow reserve storage (Guerlet et al., 2007; Moore, 1988). Parasitism would have less effect on these responses, except for mussels coming from the downstream where single infections by Ophryoglena spp. or RLOs seemed to be an additional stress. Here, co-infections by both parasites have a lesser influence on tested biological responses except for females from downstream. However, in our previous study, co-infections observed in a site impacted by industrial activities, were associated with lysosome enlargement and neutral lipid depletion (Minguez et al., 2009). The parasitism influence on biological responses seemed not to be simple to look ahead and would depend on the environmental conditions. In the present investigation, the general health-status index was particularly defined by the lysosomal system and the energetic reserves. The influence of parasitism was more pronounced for mussels from the downstream station. Moreover, females seemed to be more sensitive to multi-stress conditions, as previously seen with tissuespecific IBR. Damiens et al. (2007) underlined the importance of biomarker number in the IBR calculation: the more markers there are, the more the weight of an individual factor is markedly reduced. Thus, the general IBR could be sufficient and brought a general view of the health status of the organisms infected or not in each station. However, the use of the two other indexes more specific to a tissue and a function also provide interesting results on the biological responses undergone by the various organs. 5. Conclusion The specific purpose of this study was to investigate the potential bias associated with the use of infected zebra mussels in ecotoxicological studies. The first part of the study focuses on the parasite infra-community related to the micro-habitat, in order to determine if one of them can display less infected organisms. In both habitats zebra mussels were parasitized, but depending on the study site the prevalence of Ophryoglena spp. was different between the bank and the channel. In the second part, we investigate the physiological status of zebra mussels in terms of energetic reserves and cellular defences related to environmental quality and the infection status. The influence of parasitism on biological responses seemed to be strongly dependent on environmental conditions and suggest that it is not simple to predict. Nevertheless, it remains important to consider that parasitism could be a confounding factor in ecotoxicological studies. The use of integrative indexes brought here a more synthetic view of the set of results. Acknowledgement We acknowledge Philippe Wagner for his work in the field, Philippe Rousselle for physico-chemical analyses. We thank also the Endocrinology Laboratory of the National Veterinary School of Lyon (Prof. F. Garnier) for the financial grant of biochemical analysis. Céline Dussard is gratefully acknowledged for technical help in the use of the Konelab, and Sharon Kruger for English corrections. Financial support was provided by CNRS-INSU (programme EC2CO) and CPER LorraineZAM (Contrat Projet Etat Région Lorraine, Zone Atelier Moselle). References Beliaeff, B., Burgeot, T., 2002. Integrated Biomarker Response: a useful tool for ecological risk assessment. Environ. Toxicol. Chem. 21 (6), 1316e1322. Binelli, A., Cogni, D., Parolini, M., Provini, A., 2010. Multi-biomarker approach to investigate the state of contamination of the R. Lambro/R. Po confluence (Italy) by zebra mussel (Dreissena polymorpha). Chemosphere 79, 518e528. Bochetti, R., Regoli, F., 2006. Seasonal variability of oxidative biomarkers, lysosomal parameters, metallothioneins and peroxisomal enzymes in the Mediterranean mussel Mytilus galloprovincialis from Adriatic Sea. Chemosphere 65, 913e921.

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