Interactions between parasitism and biological responses in zebra mussels (Dreissena polymorpha): Importance in ecotoxicological studies

ARTICLE IN PRESS Environmental Research 109 (2009) 843–850

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Interactions between parasitism and biological responses in zebra mussels (Dreissena polymorpha): Importance in ecotoxicological studies ¨ Laetitia Minguez a, Antoinette Meyer a, Daniel P. Molloy b, Laure Giambe rini a, a

Universite Paul Verlaine—Metz, Laboratoire des Interactions, Ecotoxicologie, Biodiversite , Ecosyste mes (LIEBE), CNRS UMR 7146, Campus Bridoux, Rue du Ge ne ral Delestraint, F-57070 Metz, France b Division of Research and Collections, New York State Museum, Albany, NY 12230, USA

a r t i c l e in fo

abstract

Article history: Received 3 March 2009 Received in revised form 20 July 2009 Accepted 27 July 2009 Available online 18 August 2009

Given that virtually all organisms are hosts for parasites, the investigation of the combined effects of contamination and parasitism is important in the framework of aquatic bioindication procedures. To assess the impact of such multistresses at the host cellular level, we sampled parasitized zebra mussel (Dreissena polymorpha) populations from two sites in northeast France that presented different levels of contamination. Experimental groups were formed based on parasite species and host gender and tested by histochemistry and automated image analysis for biological responses, such as structural changes of the lysosomal system and neutral lipid accumulation. Infected organisms displayed smaller and more numerous lysosomes compared with uninfected congeners, and infection further elevated the effect of the chemical contamination on this biomarker. In contrast, co-infection of females with selected parasites did produce inverse results, i.e. a more developed lysosomal system and neutral lipid depletion. Our data, therefore, suggest that parasitism in zebra mussels represents a potential confounding factor in ecotoxicological studies and must be taken into account in environmental risk assessment studies. & 2009 Elsevier Inc. All rights reserved.

Keywords: Dreissena polymorpha Biomarkers Parasites Environmental contamination

1. Introduction Aquatic ecosystems undisturbed by human activities are rare. The need to detect and assess the impact of pollution on environmental quality has led to the development of biological markers (i.e. biomarkers). However, many environmental factors other than pollution (e.g. stage of development, reproduction, food availability, season, etc.) can influence biomarker response and make the interpretation of results difficult (Domouhtsidou and Dimitriadis, 2001; Bocchetti and Regoli, 2006; Guerlet et al., 2007). Parasites can induce some host physiological changes in their favor such as an energetic reallocation for their own development (Plaistow et al., 2001) or a weakening of the host immune system (Rigaud and Moret, 2003). In this multistress context, parasitism could represent a confounding factor interacting with other stress factors (Sures, 2004). Moreover, given that virtually all organisms are hosts for parasite species, the interaction between contamination and parasitism could have serious implications for environmental risk assessment. The trematodes Bunodera luciopercae and Schistosoma mansoni can influence immune and endocrine systems of their mollusc host

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E-mail address: [email protected] (L. Giambe rini). 0013-9351/$ - see front matter & 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.envres.2009.07.012

(i.e. Pisidium amnicum and Biomphalaria glabrata, respectively) (Heinonen et al., 2001; Morley et al., 2006), and thus if they are not excluded from field or laboratory studies, they can lead to false-positive or false-negative conclusions. Likewise, parasitized individuals of the freshwater bivalve P. amnicum had an increased tolerance towards contaminants such as polychlorobiphenylates (Heinonen et al., 2001). Freshwater invertebrates have also been shown to be more susceptible to environmental perturbation when infected (Gammarus pulex/aluminum or ammonium, Cerastoderma edule/hypoxia) (McCahon and Poulton, 1991; Wegeberg and Jensen, 1999; Prenter et al., 2004). Besides survival processes, other physiological mechanisms such as cellular defense or endocrine regulation can be influenced by parasites. The activities of the antioxidant system (i.e. catalase, glutathione reductase, glutathione s-transferase or lipidic peroxidase) have been recorded to increase in infected fishes (Dautremepuits et al., 2002; Marcogliese et al., 2005). When infected by the trematode Labratrema minimus, the cockle C. edule showed a decrease of metallothionein synthesis involved in the homeostasis and detoxification of metals (Baudrimont et al., 2005). Parasites, particularly those that induce castration, can also interfere with the neuroendocrine system and distort the results of endocrine disruptors studies (Morley, 2006). Zebra mussels (Dreissena polymorpha), a well-known bioindicator species (Kraak et al., 1991), can be parasitized by several

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species of trematodes, ciliates and intracellular bacteria using it as first or second intermediate host (Molloy et al., 1997, 2001, 2005; Laruelle et al., 2002). Some of these parasites (e.g. Bucephalus polymorphus, Phyllodistomum folium or haplosporidians) are believed to cause severe host pathology, i.e. castration, gill deformation and connective tissue destruction, respectively (Molloy et al., 1997; Karatayev et al., 2002; Laruelle et al., 2002). There are relatively few investigations examining the interaction between ecotoxicology and parasitology in freshwater ecosystems (e.g. Heinonen et al., 2000, 2001; Marcogliese et al., 2005). Historically parasitism has been primarily studied in organisms of commercial interest, such as marine bivalves and fishes (Mackenzie, 1999; Baudrimont et al., 2005). Parasites can be additional stressors affecting the physiological homeostasis of their hosts and thus can represent a potential source of data distortion, a confounding factor, in ecotoxicological studies. Therefore, in the present investigation we hypothesized that parasitism could affect biomarkers used in the study of the freshwater bivalve D. polymorpha. In particular, our research goal was to test the influence of parasitism on two biological responses commonly used in biomonitoring and related to the general physiological status of the freshwater bivalve D. polymorpha: the digestive lysosomal system responses and the lysosomal accumulation of neutral lipids (Giambe rini and Cajaraville, 2005; Guerlet et al., 2007). The lysosomal system is involved both in normal functions as food uptake/digestion and detoxification/excretion processes of toxic metals and organic pollutants. Its alterations following organism exposure have been classified into membrane destabilization, variation in structural parameters (number and/or size) or enzyme activity and composition changes (e.g. neutral lipid contents) (Viarengo et al., 1987; Moore, 1988; Holtzman, 1989; Cajaraville et al., 1995). Neutral lipids represent an important energy source for bivalve molluscs (Calvaletto and Gardner, 1999; Olsen, 1999) and an accumulation in tissues is often associated with lysosomal system damages.

2.2. Tissue preparation A part of the digestive gland was excised from each organism and served to measure the biological responses by histochemistry prepared as described in Giambe rini and Cajaraville (2005). All the remaining tissues were fixed for 48 h in Bouin’s Fixative, rinsed in water and embedded in paraffin after dehydration in graded series of ethanol–toluene. The tissue sections (5 mm thick) were stained in Gill II hematoxylin/eosin and examined using light microscopy (r1000  ) for parasite identification and gonadal index. 2.3. Inventory of parasite species For each individual, 30–40 tissue sections were observed to identify parasites. The different parasite/commensal species identified in this study were those previously described in D. polymorpha. They are found (i) in the digestive gland like Rickettsiales-like organisms (rlo, intracellular bacteria) (Molloy et al., 2001) or Ophryoglena sp. (oph, ciliates) (Molloy et al., 2005), (ii) in gills like Conchophthirus acuminatus (commensal ciliates), Sphenophrya dreissenae (ciliates), or P. folium (Trematoda) and (iii) near or in gonadal tissue like Echinoparyphium sp. (echino, Trematoda), B. polymorphus (Trematoda) and Aspidogaster sp. (Trematoda) (Molloy et al., 1997; Laruelle et al., 2002). The level of infection was assessed using standard epidemiological parameters: prevalence and mean intensity (authors, unpublished data). After the inventory, several experimental groups of 5–8 mussels were formed according to parasite species or the association of parasites. When possible, a distinction between males and females was made. The experimental groups were then used for the assessment of the biomarker responses. To determine if the infection state influenced the mussels’ biological responses, more non-infected individuals were selected for the control group in order to avoid an infected group being over-represented. 2.4. Determination of gonadal index Gonad maturity was assessed by microscopic observation of the slides, through the determination of a mean gonadal index (GI) for each experimental group (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 (stage E). An arbitrary score from 0 to 5 was attributed to each stage, and the following formula was used to calculate gonadal index: P ni s i GI ¼ 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. Materials and methods 2.5. Histochemistry and stereology 2.1. Sampling Zebra mussels were collected twice in two rivers in northeast France and examined microscopically for the presence of parasites. According to the parasite species and host gender, experimental groups were constituted and tested by histochemistry and automated image analysis for biological responses such as structural changes of the lysosomal system and neutral lipid accumulation. Two sampling sites presenting different levels of contamination were selected in northeastern France: Commercy (C) on the Meuse River (481450 21.2900 N, 051360 25.3700 E) was chosen as a reference site because of its low levels of urbanization and industry and Sierck-les-Bains (S) on the Moselle River (491260 32.8200 N, 06121013.2400 E) was selected as a site impacted by salts, steel and industrial activities. Mussels were collected in January during the resting period (n ¼ 196 (C); n ¼ 128 (S)) and April 2007 before the spawning period (n ¼ 120 (C); n ¼ 110 (S)). Detailed results are presented only for April (except for PCA), the results of winter investigations showing no significant difference between experimental groups (authors, unpublished data). The shell lengths of the mussels throughout the samplings were 15–24 and 20–28 mm for Sierckles-Bains and Commercy, respectively. During each sampling date, water and sediment were collected to determine main organic and metallic burdens and transported to the laboratory in 2 L polyethylene tanks. The sediment analyses were performed after sieving (4 mm) according to the standards EN 13346/ISO11885 for metals and XP X 33012 for PAH. The following water analyses were made: cations were determined by flame AAS (Perkin-Elmer Aanalyst 100) after water acidification (1% HNO3). Only NH+4 were 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 digestive lysosomal system was located by the revelation of b-glucuronidase activity in unfixed cryostat sections according to Cajaraville et al. (1991) and adapted to freshwater organisms by Giambe rini and Cajaraville (2005). Unsaturated neutral lipids were demonstrated by oil red O staining (Moore, 1988). Cellular biomarkers were quantified on digestive tissue sections (8 mm thick) by image analysis (Analysis pro 3.2, Olympus) using a Sony DP 50 color video camera connected to an Olympus BX 41 microscope with a 100  objective. Five fields of view were randomly analyzed on one section per individual in each experimental group (total sampling area per organism: 63172 mm2). Only areas belonging to digestive tissues were analyzed. One to four stereological parameters were calculated: the surface density for the two biomarkers (SvL ¼ SL/VC and SvNL ¼ SNL/VC), and only for lysosomes the volume density (VvL ¼ VL/VC), the surface to volume ratio (S/VL ¼ SL/VL, interpreted as the inverse of lysosome size) and the numerical density (NvL ¼ NL/VC) where C ¼ digestive cell cytoplasm, L ¼ lysosomes, NL ¼ neutral lipids, N ¼ number, S ¼ surface, V ¼ volume. 2.6. Statistical analysis Statistical analyses were undertaken with STATISTICA software version 7.1, Statsoft, USA. The non-parametric Kruskal–Wallis test was used to evaluate the effect of the infection status on biomarker responses, followed by the Mann–Whitney U-test with Bonferroni correction. Significant differences (Po0.05) between experimental groups formed in function of the parasite species were studied using one-way analysis of variance (ANOVA) followed by Duncan’s post-hoc test after testing for normality and homogeneity of the data. In order to achieve homoscedasticity (assessed by Brown–Forsythe test), VvL and NvL were log-transformed before the analysis. Principal component analysis (PCA) was performed on the mean values of three cellular and physiological biomarkers

ARTICLE IN PRESS L. Minguez et al. / Environmental Research 109 (2009) 843–850 (SvL, SvNL and GI) for each experimental group at each sampling period (i.e. Commercy, C and Sierck-les-Bains, S, on January 1 and April 4).

3. Results 3.1. Water physico-chemistry and contaminant burdens Results of physico-chemical analysis carried out on the sediment and water of both sites are reported in Table 1. The sediment data showed elevated organic and metallic contamination at Sierck-les-Bains with some pollutant concentrations (i.e. Pb, Zn or fluoranthene) five times higher than those observed at Commercy. Water from Sierck-les-Bains showed an enhanced conductivity in agreement with higher minerals (see + Cl, SO2 4 and Na ). 3.2. Effect of the infection status on biological responses When considering only the infection status without taking parasite species into account, lysosomal volume density (VvL) was Table 1 Results of the physico-chemical analysis carried out on sediment and water of each sampling sites. Parameters

Sediment: (mg kg1 dried 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(1,3 cd)pyrene Naphthalene Phenanthrene Pyrene Water: (mg L1) PH Conductivity 25 1C (mS cm1) NH4+ NO2 NO3– N kjedahl PO4– P total Cl– SO2– 4 Suspended matter 2+ Ca Mg2+ Na+ K+ COD BOD5

Sierck-les-bains on Moselle River

Commercy on Meuse River

45.572.1 49.072.8 o1 29.074.2 107.5723.3 375.0729.7 o0.5 1.17 1.871.1 1.670.7 1.970.6 4.371.9 1.570.5 0.870.3 1.971.1 o0.5 4.672.3 o0.5 1.370.6 o0.5 1.771.2 3.572.0

28.077.1 16.071.4 o1 19.574.9 19.074.2 78.574.9 o0.5 o0.5 0.7 0.56 0.66 0.9 0.59 o0.5 0.78 o0.5 1.471.1 o0.5 0.54 o0.5 1.2 1.83

8.270.2 1295792

8.170.0 501765

0.1470.01 0.0270.01 2.570.6 1.270.3 0.0470.05 0.2170.07 260.0728.3 89.5724.7 26.4717.6 153.579.2 14.772.4 71.1711.7 5.470.5 9.072.8 2.970.7

0.0870.01 0.0270.0 2.870.1 0.770.3 0.0470.05 0.1970.15 13.571.6 35.6714.0 32.4738.3 88.9712.5 8.871.6 7.170.7 1.671.3 12.577.8 2.270.1

Results are given as mean7SD (parameters determined on two dates) or as single value (parameter below detection limit at one date).

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significantly lower in infected mussels at both study sites (Fig. 1A). The surface density (SvL) followed the same pattern except in mussels from Sierck-les-Bains (Fig. 1B). Infected mussels showed smaller (higher S/VL ratio) and more numerous lysosomes (Fig. 1C, D). Moreover, comparing study sites, infected mussels from the more impacted one, Sierck-les-Bains, exhibited the same lysosomal structure (no difference in VvL) as infected zebra mussels from Commercy, but with smaller and more numerous lysosomes. No significant difference was observed between infected and non-infected mussels in terms of neutral lipid contents, but organisms from Commercy displayed higher levels of neutral lipids (Fig. 1E). 3.3. Effect of parasite species on biological responses Different experimental groups were formed at each site as outlined in the parasite inventory (Table 2). Only one group was formed for mussels parasitized by Echinoparyphium sp. since there were not enough mussels of each gender. The influence of parasite species on biological responses is presented in Figs. 2 and 3. At Commercy, the infected mussels displayed a less-developed lysosomal system (lower VvL and SvL) (Fig. 2A, B) with smaller and more numerous lysosomes (Fig. 2C, D). The species of parasite (Ophryoglena sp., RLOs or Echinoparyphium sp.) did not influence these general lysosomal responses. No significant difference was observed between experimental groups for neutral lipid contents, but we did note a tendency of infected males to accumulate more neutral lipids than non-infected ones (Fig. 2E). All experimental groups showed a mean gonadal index close to 2 corresponding to gonads in early gametogenesis, without marked difference between males and females (Fig. 2F). The single-infected mussels from Sierck-les-Bains also had a less-developed lysosomal system (lower VvL and SvL) compared to their non-infected congeners (Fig. 3A, B), and again the species of parasite did not influence these general lysosomal responses. In contrast, in the case of co-infection by Ophryoglena sp. and RLOs (such double-infection prevalence rate was 11%), the lysosomal parameters (VvL and SvL) were significantly higher compared to single infection by either one of these species. This increase of volume and surface densities in co-infected mussels was due to a significant increase in lysosome size in females (Fig. 3C) and a rise in lysosome number in males (Fig. 3D). A difference between males and females was observed with females showing fewer lysosomes (Fig. 3D). Such co-infected mussels tended to display fewer lipidic reserves than non-infected ones or single-infected, and the difference was significant in infected versus non-infected females (Fig. 3E). The later results confirmed the tendencies observed in mussels from Commercy on January (authors, unpublished data). Gonadal index values showed that females had more developed gonads (GIE2) than males in apparent sexual rest (GIr1) (Fig. 3F). Moreover, females co-infected with Ophryoglena sp. and Rickettsiales-like organisms showed a lower mean gonadal index than other females (none or single infection) due to a delay of gonad maturation (Fig. 3F). 3.4. Principal component analysis To further synthesize results, a multivariate analysis using mean values of biomarkers (GI, SvL and SvNL) was carried out (Fig. 4). In the case of the parasite inventory made in January at Commercy, no uninfected organisms were observed. Mussels showed a double infection of Ophryoglena sp.-RLOs, infection by Echinoparyphium sp., P. folium or by only Ophryoglena sp. with high

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Fig. 1. Structural changes of lysosomal digestive system and neutral lipid contents in D. polymorpha in function of the infection status in April (Commercy and Sierck-lesBains). Plots show the median, 25th–75th percentile (within box), minimum/maximum value (error bar). For lysosomes: (A) volume density VvL, (B) surface density SvL, (C) surface to volume ratio S/VL, (D) numerical density NvL, for neutral lipids: (E) surface density SvNL. Different letters indicate significant differences between groups (Kruskal–Wallis ANOVA, Bonferroni correction of p-value).

intensity. We chose a limit at 10 Ophryoglena sp. per organisms to distinguish the intensity of infection. Factors 1 and 2 explain over 95% of the total variance in the data matrix. Factor 1 explains 76% of total variance and is mainly characterized by seasons relative to neutral lipid contents and gonadal index, with a contrast between spring (negative loads: high neutral lipid reserves and GI) and winter (positive loads: low neutral lipid reserves and GI). Factor 2 explains 19% of total variance and could correspond to the intensity of the lysosomal system responses (higher values corresponding to positive coordinates), associated with the intensity of infection (in case of C1-values with co-infected groups with higher values) and the gender of the host (in case of C1-values and S4-values, with the same experimental group females displaying higher lysosomal responses). Discrimination between both study sites were also observed on the F1-axis for spring values and on the F2-axis for winter values.

4. Discussion In the present field investigation, parasitized zebra mussels displayed a less-developed digestive lysosomal system as evidenced by smaller and more abundant lysosomes compared with their non-infected congeners, irrespective of the environment quality (i.e. level of chemical contamination at the collection site). The reduction of the lysosomal volume density associated with an increase of their number could be the result of a fission process of larger ones or more likely the synthesis of new lysosomes ´ (i.e. changes in the lysosomal turnover) (Marigomez, and BaybayVillacorta, 2003). Indeed, Ockroy et al. (2002) and Cˆonsoli et al. (2005) have observed in other host–parasite systems (e.g. Pieris brassicae–Cotesia glomerata/bacteria and Heliothis virescens– Toxoneuron nigriceps, respectively) that infected insects had higher total protein concentration in hemolymph compared to control hosts. However, a change in the eating behavior of mussels such as

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Table 2 Experimental groups obtained following the parasite inventory and number of mussels in each group (N), in both study sites, from April collection. Experimental groups

Non-infected # (NI M) Non-infected ~ (NI F) # infected by Ophryoglena sp. (oph M) ~ infected by Ophryoglena sp. (oph F) # infected by RLOs (rlo M) ~ infected by RLOs (rlo F) # infected by Ophryoglena sp. and RLOs (oph-rlo M) ~ infected by Ophryoglena sp. and RLOs (oph-rlo F) Infected by Echinoparyphium sp. (echino)

Sierck-les-Bains on Moselle River N

Commercy on Meuse River N

8 8 7 7 6 6 6

8 8 8 8 7 – –

6

–

5

5

847

a slight decrease in the food intake cannot be dismissed as a contributing factor to lysosome size reduction. Indeed, a decrease in the filtration rate and the food intake has been observed in Crassostrea virginica parasitized by Haplosporidium nelsoni (Barber et al., 1988). Parasitism seemed to be an additional stress factor triggering even a larger lysosomal response than site contamination alone. Comparing both aquatic sampling sites, infected organisms showed the same lysosome structure (the same VvL), but parasitized mussels from the most impacted one, Sierck-les-Bains, had smaller and even more numerous lysosomes. This is in agreement with other field and laboratory studies where mussels from polluted sites or exposed to contaminants also displayed an activation of the digestive lysosomal system compared to their congeners in control situation (Marigomez et al., 1996; Zorita et al., 2006; Koukouzika and Dimitriadis, 2008). The activation of the lysosome system is considered as a general stress response in bivalves and may be due to an enhanced exocytosis associated

Fig. 2. Stereological parameters of lysosomes and neutral lipids in the digestive gland cells of D. polymorpha coming from Commercy, in April. For lysosomes: (A) volume density, (B) surface density, (C) surface to volume ratio, (D) numerical density; for neutral lipids: (E) surface density. (F) Percentages of mussels at each gamete developmental stage and gonadal index values (black dots). Organisms are males (M) or females (F), non-infected (NI), infected by Ophryoglena sp. (oph), RLOs (rlo) or Echinoparyphium sp. (echino). Histogram bars represent mean+SD. Significant differences (Duncan’s test, po0.05) between pairs of means are indicated by stars in the matrix.

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Fig. 3. Stereological parameters of lysosomes and neutral lipids in the digestive gland cells of D. polymorpha coming from Sierck-les-Bains, in April. For lysosomes: (A) volume density, (B) surface density, (C) surface to volume ratio, (D) numerical density; for neutral lipids: (E) surface density. (F) Percentages of mussels at each gamete developmental stage and gonadal index values (black dots). Organisms are males (M) or females (F), non-infected (NI), infected by Ophryoglena sp. (oph), RLOs (rlo), Ophryoglena and RLOs (oph-rlo) or Echinoparyphium sp. (echino). Histogram bars represent mean+SD. Significant differences (Duncan’s test, po0.05) between pairs of means are indicated by stars in the matrix.

with the synthesis of new lysosomes (Marigomez et al., 1996; Guerlet et al., 2006). As previously discussed, we similarly suspect the increase in lysosomes in parasitized zebra mussels to be the result of the synthesis of new lysosomes rather than lysosome fission. Mussels from Sierck-les-Bains displayed less lipidic reserves than those from Commercy, without infection status influence. However, in most ecotoxicological studies organisms in the most contaminated sites showed an increase in their neutral lipid contents (Moore, 1988; Domouhtsidou and Dimitriadis, 2001; Bocchetti and Regoli, 2006). The reduction in lipidic reserves observed in our study could be due to a relocation of reserves towards defense mechanisms (Zorita et al., 2006; Guerlet et al., 2007). However, we cannot discard the possibility that this lipid reduction could be due to an alteration in the quantity and/or quality of eaten food (Guerlet et al., 2007). Our results suggest that parasite species and the infection intensity may be important factors to take into account. Mussels

from both sites parasitized by RLOs or Ophryoglena sp. showed a less-developed lysosomal system compared with non-infected ones, and no difference between parasite species influences was observed. In contrast, a co-infection by both above parasites led to inverse results and seemed to activate the lysosomal system (i.e. more voluminous lysosomes). An enlargement of lysosomes has been observed in molluscs (Moore and Halton, 1973; Johnston et al., 1982) and in fishes infected by pathogenic trematodes (Broeg et al., 1999; Schmidt et al., 2003). In our study mussels co-infected by RLOs and Ophryoglena sp. displayed lipidic depletion compared to their non-infected congeners. These parasites associated together seemed to involve a relatively substantial energetic cost for the host. As observed by Robledo et al. (1995) in serum components (i.e. total and reducing carbohydrates) of the mussel Mytilus galloprovincialis, the effect of several types of parasites together can be more pronounced than that due to one type of parasite. Two mechanisms could be involved in the lysosome enlargement and the lipidic depletion in

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Fig. 4. Principal components analysis based on mean values of gonadal index (GI) and surface density of lysosomes (SvL) and neutral lipid contents (SvNL) determined in D. polymorpha from two sites (C: Commercy, S: Sierck-les-Bains) and at 2 months (1: January, 4: April), for all experimental groups.

co-infected mussels: an autophagic process responsible for the break of macromolecules like fats into lower molecular weight compounds that are then used to build new cell materials through the cell’s own lysosomal machinery (Moore and Halton, 1973), or a process of apoptosis involving the degradation of cells into apoptotic bodies and then their phagocytosis by surrounding cells (Pipe and Moore, 1985). However, we cannot differentiate these two mechanisms on the basis of lysosome morphology, and both could occur under stress conditions like parasitism or contaminant exposure (Pipe and Moore, 1985). Co-infected female mussels showed less-developed gonads compared to their congeners uninfected or infected by one parasite species only. The available energy of an organism is finite and divided between maintenance, growth and reproduction according to the demand for maintaining each function (Kern et al., 2001). One hypothesis would be that a multiple infection in females involved the use of neutral lipids for survival processes (e.g. autophagy) instead of sexual maturation or growth. Indeed, Taskinen (1998) has shown that infected Anodonta piscinalis grew less than unparasitized ones. However, we cannot exclude that a part of these lipidic reserves normally used for gamete production and/or growth could be diverted by the parasites for their own development (Jokela et al., 1993). The two genders seemed not to have the same susceptibility to stress, with females responding more strongly than males to environmental factors. This could be due to different reproduction involvement of each sex, since females have to use more energy for the egg yolk synthesis.

procedures. The effect of parasitism was studied in terms of infection status (i.e. infected or not) and parasite species. Our results indicate that in sites of different environmental quality (i.e. chemical contamination) the presence of a single parasite modified the zebra mussel’s digestive lysosomal system response (intra-site difference between infected and non-infected organisms) and even elevated the effect of environment quality (intersite difference between infected organisms). In addition, we observed that the infection intensity and the host gender were also important factors for ecotoxicological monitoring. Indeed, in one type of co-infection (i.e. Ophryoglena sp. and RLOs) structural modifications of the lysosomal system (more developed system) and of neutral lipid contents (depletion) were observed more markedly in females. Thus, if the samples used to assess environment quality had unknowingly included a high proportion of infected mussels (e.g. prevalence of Ophryoglena sp. at Commercy on January was 97%), the estimated level of site contamination could be considerably in error. This investigation has thus demonstrated that parasitism in zebra mussels can be considered as an additional stress factor and thus a potentially important confounding factor in ecotoxicological studies. Our study suggests that biomonitoring studies using, in their biomarker battery, the structural modifications of the digestive lysosomal system and the neutral lipid accumulation should take into account the presence of infectious organisms and host gender. These two additional parameters add further complexity and effort to the interpretation of ecotoxicological data, but increase the likelihood of successful data analysis and accuracy of research conclusions.

5. Conclusions Acknowledgments This study was initiated to better understand the interaction of ecotoxicology and parasitology. Its specific purpose was to investigate if biological responses of zebra mussels could be influenced by parasites and therefore interfere with bioindication

We thank Philippe Rousselle for physico-chemical analysis on water samples and the CPER Lorraine-ZAM (Contrat Plan Etat Re gion Lorraine, Zone Atelier Moselle) for the materiel support to

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the laboratory. Sharon Kruger is gratefully acknowledged for her English corrections. The authors wish to thank the reviewers for their helpful suggestions which improve the manuscript. References Barber, B.J., Ford, S.E., Haskin, H.H., 1988. Effects of the parasite MSX (Haplosporidium nelsoni) on oyster (Crassostrea virginica) energy metabolism—II. Tissue biochemical composition. Comp. Biochem. Physiol. A 91 (3), 603–608. Baudrimont, M., de Montaudouin, X., Palvadeau, A., 2005. Impact of digenean parasite infection on metallothionein synthesis by the cockle (Cerastoderma edule). A multivariate field monitoring. Mar. Pollut. Bull. 52 (5), 494–502. Bocchetti, 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, 913–921. Broeg, K., Zander, S., Diamant, A., Korting, W., Kruner, G., Paperna, I., von Westernhagen, H., 1999. The use of fish metabolic, pathological and parasitological indices in pollution monitoring. I-North Sea. Helgoland Mar. Res. 53, 171–194. Cajaraville, M.P., Abascal, I., Etxberria, M., Marigomez, I., 1995. Lysosomes as cellular markers of environmental pollution: time- and dose-dependent responses of the digestive lysosomal system of mussels after petroleum hydrocarbon exposure. Environ. Toxicol. Water Qual. 10, 1–8. ´ Cajaraville, M.P., Marigomez, J.A., Angulo, E., 1991. Automated measurement of lysosomal structure alterations in oocytes of mussels exposed to petroleum hydrocarbons. Arch. Environ. Contam. Toxicol. 21, 395–400. Calvaletto, J.F., Gardner, W.S., 1999. Seasonal dynamics of lipids in freshwater benthic invertebrates. In: Arts, M.T., Waiman, B.C. (Eds.), Lipids in Freshwater Ecosystems. Springer, New York, pp. 109–131. Cˆonsoli, F.L., Brandt, S.L., Coudron, T.A., Vinson, S.B., 2005. Host regulation and release of parasitism-specific proteins in the system Toxoneuron nigriceps–Heliothis virescens. Comp. Biochem. Physiol. B 142, 181–191. Dautremepuits, C., Betoulle, S., Vernet, G., 2002. Antioxidant response modulated by copper in healthy or parasitized carp (Cyprinus carpio L.) by Ptychobothrium sp. (Cestoda). Biochim. Biophys. Acta 1573, 4–8. Domouhtsidou, G.P., Dimitriadis, V.K., 2001. Lysosomal and lipid alterations in the digestive gland of mussels, Mytilus galloprovincialis (L.) as biomarkers of environmental stress. Environ. Pollut. 115, 123–137. Giambe rini, L., Cajaraville, M.P., 2005. Lysosomal responses in the digestive gland of the freshwater mussel, Dreissena polymorpha, experimentally exposed to cadmium. Environ. Res. 98, 210–214. Guerlet, E., Ledy, K., Giambe rini, L., 2006. Field application of a set of cellular biomarkers in the digestive gland of the freshwater snail Radix peregra (Gastropoda, Pulmonata). Aquat. Toxicol. 77, 19–32. Guerlet, E., Ledy, K., Meyer, A., Giambe rini, L., 2007. Towards a validation of a cellular biomarker suite in native and transplanted zebra mussels: a 2-year integrative field study of seasonal and pollution-induced variations. Aquat. Toxicol. 81, 377–388. Heinonen, J., Kukkonen, J.V., Holopainen, I.J., 2000. Toxicokinetics of 2,4,5trichlorophenol and benzo(a)pyrene in the clam Pisidium amnicum: effects of seasonal temperatures and trematode parasites. Arch. Environ. Contam. Toxicol. 39 (3), 352–359. Heinonen, J., Kukkonen, J.V.K., Holopainen, I.J., 2001. Temperature- and parasiteinduced changes in toxicity and lethal body burdens of pentachlorophenol in the freshwater clam Pisidium amnicum. Environ. Toxicol. Chem. 20 (12), 2778–2784. Holtzman, E., 1989. In: Lysosomes. Plenum Press, New York, London p. 439. Johnston, B.R., Halton, D.W., Moore, M.N., 1982. Bucephaloides gracilescens: a quantitative study of the lysosomal cellular stress response induced by sporocysts and developing cercariae within the white furrow shell, Abra abra. Z. Parasitenkd. 67, 31–36. Jokela, J., Uotila, L., Taskinen, J., 1993. Effect of the castrating trematode parasite Rhipidocotyle fennica on energy allocation of freshwater clam Anodonta piscinalis. Funct. Ecol. 7 (3), 332–338. Karatayev, A.Y., Burlakova, L.E., Molloy, D.P., Volkova, L.K., Volosyuk, V.V., 2002. Field and laboratory studies of Ophryoglena sp. (Ciliata: Ophryoglenidae) infection in zebra mussels, Dreissena polymorpha (Bivalvia: Dreissenidae). J. Invertebr. Pathol. 79, 80–85. Kern, S., Ackermann, M., Stearns, S.C., Kawecki, T.J., 2001. Decline in offspring viability as a manifestation of aging in Drosophila melanogaster. Evolution 55, 1822–1831. Koukouzika, N., Dimitriadis, V.K., 2008. Aspects of the usefulness of five marine pollution biomarkers, with emphasis on MN and lipid content. Mar. Pollut. Bull. 56, 941–949. Kraak, M.S.H., Scholten, M.C.T., Peeters, W.H.M., de Kock, W.C., 1991. Biomonitoring of heavy metals in the western European rivers Rhine and Meuse using the freshwater mussel Dreissena polymorpha. Environ. Pollut. 74, 101–114. Laruelle, F., Molloy, D.P., Roitman, V.A., 2002. Histological analysis of trematodes in Dreissena polymorpha: their location, pathogenicity, and distinguishing morphological characteristics. J. Parasitol. 88 (5), 856–863.

Mackenzie, K., 1999. Parasites as pollution indicators in marine ecosystems: a proposed early warning system. Mar. Pollut. Bull. 38, 955–959. Marcogliese, D.J., Brambilla, L.G., Gagne , F., Gendron, A.D., 2005. Joint effect of parasitism and pollution on oxidative stress biomarkers in yellow perch Perca flavescens. Dis. Aquat. Org. 63, 77–84. ´ Marigomez, I., Baybay-Villacorta, L., 2003. Pollutant-specific and general lysosomal responses in digestive cells of mussels exposed to model organic chemicals. Aquat. Toxicol. 64, 235–257. Marigomez, I., Orbea, A., Olabarrieta, I., Etxberria, M., Cajaraville, M.P., 1996. Structural changes in the digestive lysosomal system of sentinel mussels as biomarkers of environmental stress in mussel-watch programmes. Comp. Biochem. Phys. C 113, 291–297. McCahon, C.P., Poulton, M.J., 1991. Lethal and sublethal effects of acid, aluminium and lime on Gammarus pulex during repeated simulated episodes in a Welsh stream. Freshwater Biol. 25, 169–178. Molloy, D.P., Giambe rini, L., Morado, J.F., Fokin, S.I., Laruelle, F., 2001. Characterization of intracytoplasmic prokaryote infections in Dreissena sp. (Bivalvia: Dreissenidae). Dis. Aquat. Org. 44 (3), 203–216. Molloy, D.P., Karatayev, A.Y., Burlakova, L.E., Kurandina, D.P., Laruelle, F., 1997. Natural enemies of zebra mussels: predators, parasites, and ecological competitors. Rev. Fish. Sci. 5 (1), 27–97. Molloy, D.P., Lynn, D.H., Giambe rini, L., 2005. Ophryoglena hemophaga n. sp. (Ciliophora: Ophryoglenidae): a parasite of the digestive gland of zebra mussels Dreissena polymorpha. Dis. Aquat. Org. 65, 237–243. Moore, M.N., 1988. Cytochemical responses of the lysosomal system and NADPHferrihemoprotein reductase in molluscan digestive cells to environmental and experimental exposure to xenobiotics. Mar. Ecol. Prog. Ser. 46, 81–89. Moore, M.N., Halton, D.W., 1973. Histochemical changes in the digestive gland of Lymnaea truncatula infected with Fasciola hepatica. Z. Parasitenk. 43, 1–16. Morley, N.J., 2006. Parasitism as a source of potential distortion in studies on endocrine disrupting chemicals in molluscs. Mar. Pollut. Bull. 52, 1330–1332. Morley, N.J., Lewis, J.W., Hoole, D., 2006. Pollutant-induced effects on immunological and physiological interactions in aquatic host–trematode systems: implications for parasite transmission. J. Helminthol. 80, 137–149. Ockroy, K.S., Cole, T.J.W., Trenczek, T.E., Dorn, S., 2002. Comparison of parasitism by Cotesia glomerata with bacterial infection and wounding in Pieris brassicae: induction of new haemolymph polypeptides and changes in humoral immune response. J. Invertebr. Pathol. 81, 12–18. Olsen, Y., 1999. Lipids and essential fatty acids in aquatic food webs: what can freshwater ecologists learn from mariculture. In: Arts, M.T., Wainman, B.C. (Eds.), Lipids in Freshwater Ecosystems. Springer, New York, pp. 109–131. Pipe, R.K., Moore, M.N., 1985. Ultrastructural changes in the lysosomal-vacuolar system in digestive cells of Mytilus edulis as a response to increased salinity. Mar. Biol. 87, 157–163. Plaistow, S.J., Troussard, J.-P., Ce zilly, F., 2001. The effect of the acanthocephalan parasite Pomphorhynchus laevis on the lipid and glycogen content of its intermediate host Gammarus pulex. Int. J. Parasitol. 31, 346–351. Prenter, J., MacNeil, C., Dick, J.T.A., Riddell, G.E., Dunn, A.M., 2004. Lethal and sublethal toxicity of ammonia to native, invasive, and parasitised freshwater amphipods. Water Res. 38, 2847–2850. Rigaud, T., Moret, Y., 2003. Differential phenoloxidase activity between native and invasive gammarids infected by local acanthocephalans: differential immunosuppression?. Parasitology 127, 571–577. Robledo, J.A.F., Santare m, M.M., Gonza lez, P., Figueras, A., 1995. Seasonal variations in the biochemical composition of the serum of Mytilus galloprovincialis Lmk. and its relationship to the reproductive cycle and parasitic load. Aquaculture 133, 311–322. ¨ Schmidt, V., Zander, S., Korting, W., Broeg, K., v. Westernhagen, H., Dizer, H., Hansen, P., Skouras, A., Steinhagen, D., 2003. Parasites of flounder (Platichthys flesus L.) from the German Bight, North Sea, and their potential use in biological effects monitoring. Helgol. Mar. Res. 57C (3–4), 262–271. Sures, B., 2004. Environmental parasitology: relevancy of parasites in monitoring environmental pollution. Trends Parasitol. 20 (4), 170–177. Taskinen, J., 1998. Influence of trematode parasitism on the growth of a bivalve host in the field. Int. J. Parasitol. 28, 599–602. Tourari, A.L., Crochard, C., Pihan, J.C., 1988. Action de la tempe rature sur le cycle de reproduction de Dreissena polymorpha (Pallas). Etude in situ et en laboratoire. Haliotis 18, 85–98. Viarengo, A., Moore, M.N., Mancinelli, G., Mazzucotelli, A., Pipe, R.K., Farrar, S.V., 1987. Metallothioneins and lysosomes in metal toxicity and accumulation in marine mussels: the effect of cadmium in the presence and absence of phenanthrene. Mar. Biol. 94, 251–257. Wegeberg, A.M., Jensen, K.T., 1999. Reduced survivorship of Himasthla (Trematoda, Digenea)-infected cockles (Cerastoderma edule) exposed to oxygen depletion. J. Sea Res. 42, 325–331. Zorita, I., Ortiz-Zarragoitia, M., Soto, M., Cajaraville, M.P., 2006. Biomarkers in mussels from a copper site gradient (Visnes, Norway): an integrated biochemical, histochemical and histological study. Aquat. Toxicol. 78, 109–116.