Interactive effects of metal contamination and pathogenic organisms on the introduced marine bivalve Ruditapes philippinarum in European populations

Environmental Pollution 158 (2010) 3401e3410

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Interactive effects of metal contamination and pathogenic organisms on the introduced marine bivalve Ruditapes philippinarum in European populations Ika Paul-Pont a, *, Xavier de Montaudouin a, Patrice Gonzalez a, Florence Jude a, Natalie Raymond a, Christine Paillard b, Magalie Baudrimont a a b

Université Bordeaux 1, UMR 5805 CNRS, Station Marine d’Arcachon, place du Dr. Peyneau, Arcachon 33120, France Université de Bretagne OccidentaledIUEM, LEMAR UMR 6539 CNRS, Place Nicolas Copernic, Technopôle Brest Iroise, 29280 Plouzané, France

Co-infection by opportunistic pathogens affects metal accumulation and some defense-related activities in the Manila clam Ruditapes philippinarum.

a r t i c l e i n f o

a b s t r a c t

Article history: Received 12 December 2009 Received in revised form 15 July 2010 Accepted 21 July 2010

In natural environment, marine organisms are concomitantly exposed to pollutants and multiple disease agents resulting in detrimental interactions. The present study evaluated interactive effects of metal contamination (cadmium) and pathogenic organisms (trematode parasites Himasthla elongata and pathogenic bacteria Vibrio tapetis) singularly and in combination on the bivalve Ruditapes philippinarum, an introduced species to Europe, under laboratory controlled conditions. After 7 days, metal bioaccumulation and pathogen load were analyzed as well as metallothionein (MT) response and hemocyte concentrations and activities. Results showed that infection by opportunistic pathogens affects metal accumulation, leading to maximal Cd accumulation in co-infected clams. Among stressors only V. tapetis induced significant effects on immune parameters whereas a particular interaction “trematode-bacteria” was shown on MT responses. Despite low trematode infection in agreement with the resistant status of R. philippinarum to these macroparasites, significant interaction with bacteria and metal occurred. Such results highlight the necessity of taking pathogens into account in ecotoxicological studies. Ó 2010 Published by Elsevier Ltd.

Keywords: Manila clam Pathogens Cadmium Metallothionein Immune defense

1. Introduction Coastal marine organisms are at risk of exposure to a wide variety of both natural and anthropogenic stressors. Coastal zones are endangered by anthropogenic inputs of contaminants due to their extensive use in agricultural, chemical and industrial processes. Metals such as cadmium (Cd) have long been recognized as major pollutants of the marine environment, constituting a hazard to marine organisms (Cheung et al., 2003; Roesijadi, 1994). There is growing concern of the environmental effects of metal pollution in terms of disease and disease susceptibility (Morley, 2010; Pipe and Coles, 1995). In many invertebrates, immunity is carried out by effector molecules as well as proliferation and activities of non-specific cells, the hemocytes. These components are contained in the hemolymph, an open circulatory system in direct contact with nervous and endocrine systems maintaining homeostasis in invertebrate (Pipe and Coles, 1995). This interdependence makes the immune processes particularly sensitive to

* Corresponding author. E-mail address: [email protected] (I. Paul-Pont). 0269-7491/$ e see front matter Ó 2010 Published by Elsevier Ltd. doi:10.1016/j.envpol.2010.07.028

environmental stressors, including metals. Pollutants could also directly affect pathogenic organisms and consequently their occurrence, distribution and virulence in a positive or negative way depending on an unknown number of interactive variables, e.g. host pollutant metabolism or parasite exposure (Sures, 2008). In contrast, infectious agents could interfere with the bioaccumulation of toxic compounds (Cross et al., 2003; Evans et al., 2001). Therefore, concomitant exposure to metal and pathogens may lead to different kinds of interactions and are more complex than a simple sum of both of them (Sures, 2008). Exploited marine bivalves are often used as sentinel organisms in contaminant monitoring programs due to their ability to filter large quantities of water leading to the accumulation of contaminants from seawater and food (Beliaeff et al., 2002; Nicholson, 2003). During feeding, bivalves may ingest many species of bacteria, providing substantial carbon and nitrogen contribution (McHenery and Birkbeck, 1986). Some species of bacteria are pathogenic. Among those, species from the genus Vibrio are an important cause of diseases in cultured fish and shellfish (Jeffries, 1982; McGladdery, 1999). Numerous reports of Vibriosis and associated mass mortalities have been reported in the Manila clam Ruditapes philippinarum in Japan, Korea and the European Atlantic

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coastline (England, Ireland, Italy, Spain and Norway) (Paillard et al., 2008). The disease was caused by the Gram-negative, non-sporulating motile bacterium Vibrio tapetis (Paillard, 2004). In a context of stressful environmental condition, several studies demonstrated an increase of susceptibility to Vibrio spp. infections in different hosts in relation to environmental perturbation such as modifications of temperature or salinity (Paillard et al., 2004; Reid et al., 2003). Finally, co-infection with parasites could also increase the susceptibility of the host to Vibrio spp. infections (Rajkumar et al., 2007; Soudant et al., 2004). Thus, in the context of metal pollution, we could wonder if such portals of entry, locally increasing bacterial load, might interfere with metal accumulation in host tissues. The aim of this work was thus to assess potential interactions between cadmium contamination, bacterial challenge (V. tapetis) and trematode parasite infestation (Himasthla elongata) on the clam R. philippinarum through a three factors experimental design. Previous studies showed that the introduced species Manila clam was resistant to trematodes (Dang et al., 2009). However, there are some reports of such infestation in the native area (Lee et al., 2001; Ngo and Choi, 2004) with high prevalence of infested clams (7e42.9%) (Park et al., 2008). Particularly, H. elongata is not encountered in the native area of R. philippinarum (Japan, Korea, Philippines), but is sympatric in this species in European ecosystems and is considered as an opportunistic trematode species with many potential bivalve second intermediate hosts (¼euryxenic parasite) (Lauckner, 1983). The status of introduced species gives this study another dimension. Indeed, many of these species were deliberately introduced due to their competitive performance in terms of growth, survival and resistance. In a context of multiple stressors, particular sensitivity could be present in comparison to that observed in native species (Karatayev et al., 2009; Morley, 2008; Paul-Pont et al., 2010a). Thus, it is relevant to assess whether the apparent resistance of Manila clams to native trematode remained the same in association with the highly pathogenic bacteria V. tapetis and/or metal exposure. Laboratory experiment was performed with these sources of stress applied in single, double and triple combinations during seven days. At the end of the experiment, Cd accumulation, bacterial load, parasite infestation and several physiological parameters were assessed. MT concentration was determined in gills and visceral mass in relation to its role in detoxification of toxic metals and its ability to respond to inflammatory processes (Gagné et al., 2007). Finally, analysis of hemocytes concentration and activities (phagocytosis, oxidative burst, viability, adherence capability) were performed in order to assess the modulation of immune function in relation to multiple environmental stressors. 2. Material and methods 2.1. Bivalve treatments Manila Clams Ruditapes philippinarum (38.5  0.6 mm shell length, mean  SD) were collected in March 2007 at Andernos, Arcachon Bay, France (44 42’N, 18’W). The sampling site consists of an intertidal Zostera noltii seagrass bed that is under the influence of freshwater input (Leyre River) (Lassalle et al., 2007). Salinity ranges from 22 to 32 psu and temperature ranges from 1  C in winter to 25 C in summer. The median grain size is 142 mm corresponding to fine sand (Lassalle et al., 2007). The experimental procedure followed a three-factor design with three treatments (C: Cadmium; H: Himasthla elongata; V: Vibrio tapetis) and the four possible interactions (CH; CV; HV; CHV). Clams were acclimatized for three days prior to the beginning of the experiment. This experiment was conducted in laboratory controlled conditions using glass aquaria of 12  12  24 cm filled with 1 L of synthetic seawater (Instant Ocean, salinity: 30.3  0.2 psu, mean  SD), aerated by a diffuser system. Plastic coverings were placed over the inside walls to avoid cadmium contamination and to minimize cadmium adsorption on walls. The temperature was fixed at 15  C (0.7  C, mean  SD), pH was regularly monitored and remained stable (8.1  0.3, mean  SD) and the photoperiod was fixed at 12 h using a timer and artificial light sources. No sand was added in order to minimize bacterial contamination as the bacteria may adsorb to the sediment surface and so

may bias bacterial load calculation at the end of the experiment. Four clams were introduced into each experimental unit (EU) and three EU replicates were carried out for each experimental condition. Therefore a total of 24 EU were running simultaneously for a period of seven days. 2.2. Contamination protocol Vibrio tapetis (CTC4600) was isolated from diseased R. philippinarum (Borrego et al., 1996). Bacterial contamination was achieved by adding a culture of V. tapetis to the water column to reach a concentration of 1 109 bacteria mL1 at T0. This recreation of natural conditions was selected rather than injection of the culture into the palleal cavity or directly into the muscle to minimize stress. In order to keep a relatively constant load of V. tapetis, concentration of 1 109 bacteria mL1 was also adjusted after 3 days. Himasthla elongata cercariae were collected from infected periwinkles (Littorina littorea). Infected snails were kept at 15  C and fed with macro algae (Ulva spp.). For the experiment, periwinkles were placed at 20  C under artificial light source. This condition induces cercariae release into the water from which batches of ten were immediatly transferred (cercariae age < 1 h) into the EU until each EU contained 100 cercariae. To maintain a constant pressure of infection, cercariae were added at T0 and after 1, 3 and 4 days. A single nominal concentration of Cd at 133 nM (corresponding to 15 mg Cd L1 added as CdCl2) was selected. After the first contamination (T0), metal quantification in the water of the experimental units was performed everyday to ensure that metal concentration remained constant throughout the experimental period. Any decrease in metal concentration due to adsorption and absorption mechanisms was compensated for daily by the addition of aqueous Cd solution. 2.3. Sampling procedure After seven days of exposure (T7), one clam per EU was removed. Hemolymph was withdrawn from the adductor muscle of each individual and was observed under a stereomicroscope to ensure the quality of samples (no gamete, no dilution with seawater, no cell debris, etc.). Hemolymph was kept on ice until immunological analyses. Foot and mantle were dissected and squeezed between two sterilized glass slides under a stereomicroscope. Then, H. elongata metacercariae were rapidly counted. The remaining tissues were dissected to separate gills and visceral mass. A small section (>50 mg) of both organs was used for MT quantification and was placed into polyethylene bags (Whirl-Pak) under N2 atmosphere at 80  C to minimize MT oxidation. The rest of both organs were kept at 80  C until cadmium quantification. 2.4. Metals and metallothionein quantification Cadmium quantification was carried out in the water and the different organs of each clam. The Sampledwater was acidified at 2% with nitric acid (Fluka; Buchs, Switzerland. 65% HNO3) before Cd analysis. Tissues were digested in 1e1.5 mL (depending on weight of tissue) of nitric acid at 100  C for 3 h to dissolve metals in the liquid for quantification. After a six-fold dilution of digestates with ultrapure water (MilliQÒ, Bedford, MA, USA), Cd concentrations were determined by electrothermic atomic absorption spectrophotometry with Zeeman correction, using a graphite furnace (M6 Solar AA spectrometer, Thermoptec). The detection limit was 0.1 mg Cd L1. Analytical methods were simultaneously validated for each sample series by the analysis of standard biological reference materials (Tort-2: Lobster hepatopancreas and Dolt-3: Dogfisher liver from National Research Council of Canada, Ottawa). Throughout our cadmium analyses, mean values of Tort-2 and Dolt-3 were 27.3  0.4 mg g1 and 18.9  0.6 mg g1, respectively. These values were in certified ranges of Tort-2 and Dolt-3. To quantify MT, an inorganic mercury saturation method followed by total mercury determination using flameless atomic absorption spectrometry (LECO AMA 254, ALTEC, Prague, Czech Republic) was used. This technique was first described by Baudrimont et al. (1997b). Inorganic mercury saturation relies on mercury’s affinity to MT and its ability to displace other heavy metals which can also fix to MT. The amount of mercury detected in each sample is proportional to MT concentration. As the exact quantity of Hg-binding sites per MT molecule is unknown for R. philippinarum, MT concentration cannot be expressed in mol g1 (wet weight, ww). MT concentrations were expressed as nmol Hg-binding sites per g (ww): [(ng Hg in sample)/(mL of supernatant)]  [(tissue dilution)/(Hg molar mass)]. The detection limit was estimated at 1 ng Hg. Three reference samples or “blanks” were prepared to monitor the Hg complexation efficiency of the hemoglobin. The mean of the three blank values at each analytical run was deduced from the Hg burdens measured in each sample. A recovery percentage from purified rabbit liver MT (Alexis biochemicals ALX202-071) was systematically determined. This “internal standard” enabled us to determine the ratio between the binding sites measured after Hg saturation and the potential binding sites indicated by the supplier and previously verified by Cd and Zn determinations on purified MT solution samples. Throughout our MT analyses, the mean recovery percentage was 94.3  2.8%. This value was consistently within the certified ranges (100  20%) of the method.

I. Paul-Pont et al. / Environmental Pollution 158 (2010) 3401e3410 2.5. Immunological analyses

2.5.1. Viability Hemocyte viability was determined by the addition of 100 mL of anti-aggregant solution for bivalve hemocytes (AASH; Auffret and Oubella, 1994) and 50 mL of filtered sterile seawater (FSSW) to50 mL of hemolymph from each individual. Samples were incubated for 2 h at 18  C in dark conditions with 2 mL of SYBR green I and 2 mL of propidium iodide (PI; 10 mg mL1). Analysis by flow cytometry allowed us to estimate precisely the percentage of dead cells in each sample (Delaporte et al., 2003). 2.5.2. Total hemocyte count and hemocyte adhesion Total hemocyte count (THC) was measured by the fixation of 50 mL of each hemolymph sample with 100 mL of a 6% formalin solution and 50 mL of FSSW. Samples were incubated with 2 mL of SYBR green I in darkness at room temperature for 30 min before flow-cytometric measurement. THC was calculated according to a protocol described by Lambert et al. (2007). To calculate the percentage of hemocyte adhesion, a subsample of each hemolymph was distributed (100 mL) in 24-well microplates and 2 fold diluted with 100 mL of FSSW. After 3 h of incubation at 18  C, the supernatant containing cells not adhering was transferred into a flow cytometer tube and fixed by addition of 200 mL of a 6% formalin solution. After 30 min of incubation with SYBR Green I, cell concentration was then evaluated as described above. 2.5.3. Phagocytosis An aliquot of 100 mL of hemolymph, primarily diluted with 200 mL of FSSW, was brought in contact with 30 mL of the solution of 2 mm diameter latex fluorescent beads. Tubes were incubated for 2 h at 18  C in dark condition. Analysis by flow cytometry allowed the detection of hemocytes containing fluorescent beads on the FL1 detector. Phagocytic activity was calculated as the percentage of hemocytes that have ingested three fluorescent beads or more (Delaporte et al., 2003). 2.5.4. Reactive oxygen species production A 100 mL aliquot of hemolymph was diluted with 200 mL of FSSW and 4 mL of the 20 70 -dichlorofluorescein diacetate (DCFH-DA) solution (final concentration of 0.01 mM) was added to each tube. Tubes were then incubated for 2 h at 18  C. After the incubation period, DCF fluorescence, quantitatively related to the ROS production of hemocytes, was measured at 500e530 nm by flow cytometry. Results are expressed as the mean geometric fluorescence (in arbitrary units, AU). 2.6. Statistical analyses All variables were analyzed using a three-way ANOVA in order to determine possible interactive effects between Cd contamination, parasite and bacterial infection on clams’ responses (Sokal and Rohlf, 1981). Previously, normality was assumed and homogeneity of variance was verified with Cochran’s test (data were log-transformed when homogeneity of variance was not achieved). Percentages of phagocytic, adhered and dead hemocytes were arcsin-transformed to follow homogeneity requirements. Whenever ANOVA was significant, differences between treatments were separated by a Fisher test of means comparison. Statistical analysis was performed using STATISTICA 7.1 software for Windows.

7

Number of cyst per individual

Hemolymph was withdrawn from the adductor muscle, using a 1-mL plastic syringe (25-gauge needle) and was filtered through an 80-mm mesh and then transferred into an individual micro-centrifuge tube held on ice to minimize cell clumping prior to analysis. Immunological analyses were performed using a FACScalibur flow cytometer (BD Biosciences, San Jose, CA). Hemocyte viability, phagocytic activity and reactive oxygen species production were measured according to protocols described by Delaporte et al. (2003). The total hemocyte count and the percentage of hemocyte adhesion were measured according to protocols described by Lambert et al. (2007).

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6 5 4 3 2 1

0 Control

H

V

HV

C

CH

CV

CHV

Experimental conditions Fig. 1. Mean number of trematode cysts in tissues of Ruditapes philippinarum (mean  SE) after 7 days of exposure. H: parasitized with Himasthla elongata; V: infected by Vibrio tapetis; C: cadmium contaminated; HV, CH, CV, CHV: interactions between stressors.

colonies in TCBS medium could not allow us to discriminate V. tapetis from all other Vibrio spp. Consequently, analyses of results were considered in a “binary” sense i.e. either EUs were infected by V. tapetis or not. 3.3. Cadmium exposure Cd concentrations in both organs of non-exposed clams were similar between treatments (i.e. control, H, V and HV) and remained significantly lower than Cd-exposed clams (Fig. 2). In non-exposed clams, digestive gland tissue showed higher Cd concentration (330  63 ng g1, dry weight (dw), mean  SE) than gill tissue (99  19.7 ng g1, dw) (mean  SE, p ¼ 0.0027). In Cdcontaminated clams, all treatments induced significant effects on Cd concentration in gills (Fig. 2a, Table 1). In addition, the interaction Cd  bacteria  parasite (p ¼ 0.023) highlighted the higher Cd accumulation found in clams infected by both pathogens (CHV: 1229  166 ng g1, dw) compared to clams only infected by one pathogen (CH: 408  55 ng g1, dw and CV: 589  79 ng g1, dw) (Fig. 2a, Table 1). In the visceral mass tissue such interaction was also found in relation to the significantly higher Cd accumulation observed in triple stressed clams (CHV: 2135  181 ng g1, dw) (Fig. 2b, Table 1). Finally, when pooling all Cd conditions, visceral mass was significantly more contaminated (1473  153 ng g1, dw) than gills (651 112 ng g1, dw) (p ¼ 0.0004). 3.4. Metallothionein response

Trematode infection remained low in clams after 7 days of exposure nevertheless the average number of metacercariae in tissues (1.33  0.38, mean  SE) of experimentally infected clams was significantly higher than those of non-infected clams (Fig. 1, Table 1). No effect of cadmium or V. tapetis contamination, alone or in combination, was shown on trematode infection in clams (Table 1).

High MT concentration was found in control individuals, but lower in gills than in visceral mass tissue (10.9  4.3 and 59.5  14.8 nmol sites Hg g1 wet weight (ww), respectively (mean  SE)) (Fig. 3a, b). Parasites had a significant effect on MT concentrations (p < 0.05) both in the gills and in the visceral mass (Table 1). A significant interaction between bacteria and trematodes on MT responses was noticed in both organs suggesting that the effect of a first pathogen on MT synthesis was modulated by the presence of the second infectious agent, whatever the Cdcontamination treatment was. In fact, Vibrio treatment tended to induce a decrease of MT concentration compared to the control, whereas the double infection HimasthlaeVibrio led to an increase in MT concentration. This trend was shown in both the noncontaminated and Cd-exposed group, especially in the gills.

3.2. Bacterial challenge

3.5. Immune responses

No quantitative results were available for the bacterial concentration in the body tissue of individuals as count of bacterial

No significant effects of cadmium, parasites and bacterial challenge, combined or not, was shown on phagocytosis activity

3. Results 3.1. Trematode infection

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Table 1 Results of the three-way ANOVA comparing the effect of Cd contamination, parasite and bacterial infection (fixed factors) on different variables measured in Ruditapes philippinarum after 7 days of experiment. Variable

Source of variation

DL

Trematode infestation

Cd Bacteria Parasite Cd  Bacteria Cd  Parasite Bacteria  Parasite Cd  Bacteria  Parasite

1 1 1 1 1 1 1

F 1.735 0.048 4.819 0.434 1.735 0.048 0.434

p 0.206 0.829 0.043 0.520 0.206 0.829 0.520

Variable

Source of variation

DL

F

p

Adhesion

Cd Bacteria Parasite Cd  Bacteria Cd  Parasite Bacteria  Parasite Cd  Bacteria  Parasite

1 1 1 1 1 1 1

0.244 1.496 0.297 1.731 0.236 11.060 1.428

0.628 0.239 0.594 0.207 0.634 0.004 0.250

Cd Gills

Cd Bacteria Parasite Cd  Bacteria Cd  Parasite Bacteria  Parasite Cd  Bacteria  Parasite

1 1 1 1 1 1 1

100.848 23.564 9.768 20.751 8.876 9.258 6.282

< 0.001 < 0.001 0.007 < 0.001 0.009 0.008 0.023

Granulocytes

Cd Bacteria Parasite Cd  Bacteria Cd  Parasite Bacteria  Parasite Cd  Bacteria  Parasite

1 1 1 1 1 1 1

0.002 3.379 0.483 0.170 0.220 0.076 3.727

0.967 0.045 0.497 0.686 0.645 0.787 0.071

Cd Visceral mass

Cd Bacteria Parasite Cd  Bacteria Cd  Parasite Bacteria  Parasite Cd  Bacteria  Parasite

1 1 1 1 1 1 1

89.219 2.911 8.938 0.354 5.943 5.966 6.145

< 0.001 0.110 0.010 0.561 0.029 0.028 0.027

Hyalinocytes

Cd Bacteria Parasite Cd  Bacteria Cd  Parasite Bacteria  Parasite Cd  Bacteria  Parasite

1 1 1 1 1 1 1

2.450 2.595 0.600 1.033 0.123 1.372 0.001

0.137 0.127 0.450 0.325 0.731 0.259 0.981

MT Gills

Cd Bacteria Parasite Cd  Bacteria Cd  Parasite Bacteria  Parasite Cd  Bacteria  Parasite

1 1 1 1 1 1 1

0.110 0.521 6.194 1.115 0.247 6.869 2.835

0.744 0.481 0.024 0.307 0.626 0.019 0.112

Viability

Cd Bacteria Parasite Cd  Bacteria Cd  Parasite Bacteria  Parasite Cd  Bacteria  Parasite

1 1 1 1 1 1 1

4.013 9.766 0.029 0.058 0.108 0.841 2.198

0.062 0.007 0.866 0.813 0.747 0.373 0.158

MT Visceral mass

Cd Bacteria Parasite Cd  Bacteria Cd  Parasite Bacteria  Parasite Cd  Bacteria  Parasite

1 1 1 1 1 1 1

0.120 4.020 6.128 0.402 0.930 6.426 0.981

0.734 0.062 0.025 0.535 0.349 0.022 0.337

Phagocytosis

Cd Bacteria Parasite Cd  Bacteria Cd  Parasite Bacteria  Parasite Cd  Bacteria  Parasite

1 1 1 1 1 1 1

1.017 3.532 0.315 0.635 0.432 0.476 0.010

0.328 0.079 0.583 0.437 0.521 0.500 0.922

THC

Cd Bacteria Parasite Cd  Bacteria Cd  Parasite Bacteria  Parasite Cd  Bacteria  Parasite

1 1 1 1 1 1 1

0.068 2.718 0.016 0.237 0.172 0.276 1.486

0.798 0.119 0.902 0.633 0.684 0.607 0.240

Oxidative burst

Cd Bacteria Parasite Cd  Bacteria Cd  Parasite Bacteria  Parasite Cd  Bacteria  Parasite

1 1 1 1 1 1 1

0.144 0.996 1.388 3.594 0.111 2.167 0.690

0.710 0.335 0.258 0.079 0.744 0.163 0.420

(37.7  2.0%, mean value  SE), oxidative burst (142.6  14.2 A.U., mean value  SE), total hemocyte count (THC) (711 333  60 137 cell mL1, mean value  SE) and hyalinocyte concentration (H) (210 190  22 306 cell mL1, mean value  SE) after 7 days of exposure (Table 2). However, a significant effect of bacteria on granulocyte concentration appeared after this time (Fig. 4a, Table 1). The granulocyte concentration decreased in clams infected with Vibrio compared to non-Vibrio infected corresponding treatment (HV vs. H/CV vs. C/CHV vs. CH), except in V treatment (Fig. 4a, Table 2). This bacterial challenge also led to a significant decrease of hemocyte viability (Fig. 4b, Tables 1 and 2), whereas trematode parasites had no effect on this parameter (Table 1). Finally, the percentage of hemocyte adhesion was not significantly modified in clams by Vibrio or Himasthla alone, although there was a significant interaction “Bacteria  Parasite” (p ¼ 0.004, Table 1) on this immune parameter. 4. Discussion Even though R. philippinarum constitutes a resistant species to trematode infection, this study showed that co-infection with the

pathogenic bacteria V. tapetis can interact with metal accumulation and some defense-related activities in the Manila clam. Although Cd exposure did not lead to strong effects on physiological parameters, interaction between trematodes and bacteria was not negligible and bacterial infection showed the strongest effects on immune parameters. In non-contaminated clams, Cd concentrations were higher in the visceral mass than in gills. Clams came from a site exhibiting traces of Cd in the sediment (Paul-Pont et al., 2010b). Since, the digestive gland reflected metal exposure via the trophic route, acting as a storage compartment, a trophic contamination of the clam population via ingested particles could occur in the field. Indeed, with the application of a biokinetic model (Luoma and Rainbow, 2005; Wang and Fisher, 1999; Wang et al., 1996), it is now well established that the trophic route is often an important pathway for metal accumulation in marine suspension and deposit feeders. Cd concentrations of experimentally contaminated bivalves were also three fold higher in the visceral mass than in the gills, despite the fact that Cd contamination was introduced directly into the water (direct route) instead of by a trophic route. Daily

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a

a

1600

[MT] in nmol sites Hg.g-1, ww

[Cd] in ng.g-1 (dw)

1400 1200 1000

800 600 400

3405

25

20

15

10

5

200 0 Control

H

V

HV

C

CH

CV

0

CHV

Control

H

V

Experimental conditions

b

2500

[MT] in nmol sites Hg.g-1, ww

b

2000 [Cd] in ng.g-1 (dw)

HV

C

CH

CV

CHV

CH

CV

CHV

Experimental conditions

1500

1000

500

80

70 60 50 40 30

20 10

0

0

Control

H

V

HV

C

CH

CV

CHV

Experimental conditions

Control

H

V

HV

C

Experimental conditions

Fig. 2. Cadmium concentrations in ng g1, dw (mean  SE) in gills (a) and visceral mass (b) of clams after 7 days of exposure. H: parasitized with Himasthla elongata; V: infected by Vibrio tapetis; C: cadmium contaminated; HV, CH, CV, CHV: interactions between stressors.

Fig. 3. Metallothionein concentrations in nmol Hg-binding sites g1, ww (mean  SE) in gills (a) and visceral mass (b) of cockles after 7 days of exposure. H: parasitized with Himasthla elongata; V: infected by Vibrio tapetis; C: cadmium contaminated; HV, CH, CV, CHV: interactions between stressors.

quantification of Cd concentrations in the water of each EU, filtered or not (B 0.2 mm), confirmed that algae did not adsorb Cd fraction (data not shown). For metals, the digestive gland acts as a storage compartment in addition to its main detoxification roles (Raspor et al., 2004). Such transfer of toxic compounds from gills to the visceral mass corresponds to detoxification processes as it has already been suggested by Smaoui-Damak et al. (2004) and Bebianno et al. (1994) in relation to high MT content in this organ. Shi and Wang (2005) demonstrated a close relationship between Cd body burden, MT content and Cd assimilation efficiency. Thus, high MT concentration in the visceral mass of clams may be responsible for the high Cd accumulation. In addition, specific adaptation to metal contamination may be considered in relation to the status of introduced species for this bivalve. Indeed, different Cd accumulation patterns and MT responses were observed in the native cockle Cerastoderma edule exposed to the same stressors (Paul-Pont et al., 2010a). In Particular, cockles displayed lower MT concentrations in the visceral mass related to lower Cd accumulation in this tissue. However, cockles came from a different and uncontaminated site inside the Arcachon Bay (Paul-Pont et al., 2010a). These different locations of native (C. edule) and introduced (R. philippinarum) bivalves introduced a bias to directly compare both populations. Further studies were required to discriminate the part of native/introduced status from the part of the origin (life history in each sampling site) in explaining differences between both populations (Paul-Pont et al., 2010b). Another important difference between native and introduced bivalves exposed to multiple stressors concerned the effects of pathogens on metal accumulation (Paul-Pont et al., 2010a). In

introduced clams, metal accumulation was modified by V. tapetis and, to a lesser extent trematode infection (Table 1) (Fig. 5-(1)). The effect of H. elongata was probably indirect since it did not increase Cd accumulation in single stress conditions (Fig. 2a, b) whereas in association with V. tapetis it showed a synergistic effect on Cd bioaccumulation both in the gills and visceral mass of clams. Due to the low number of cysts of H. elongata in the Manila clams, it was not intuitive to hypothesize that such low infection levels could modify metal bioaccumulation. However, V. tapetis quantification in clams tissues being unavailable, we might envisaged an increase in bacterial concentration following co-infection with parasite as has already been shown in other host-pathogen models (Pylkkö et al., 2006; Evans et al., 2007; Rajkumar et al., 2007). Such increases in V. tapetis infection could severely damage clams to a point where pollutant exposure and accumulation were enhanced. Under the same experimental protocol, native cockles exposed to Cd and one pathogen (bacteria or trematode) displayed lower Cd accumulation than cockles submitted to Cd single exposure. In addition, an antagonistic effect of both pathogens was shown since Cd concentrations in co-infected cockles remained similar to Cd single exposed cockles (Paul-Pont et al., 2010a). These discrepancies between cockles and clams may be due to specific sensitivity to pathogens of both bivalve species. Success of experimental trematode infection in Manila clams was effective. However, only a few trematodes cercariae succeeded in encysting in clams (from 1 to 7 cysts) as is mentioned by Dang et al. (2009) who suggested that clam flesh was too hard for native cercarial penetration. In addition, Dang et al. (2009) demonstrated similar infection levels in the native clam Ruditapes

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Table 2 Hemocyte counts and activities (mean  standard error) in hemolymph after 7 days of experiment. Immune parameters

Experimental conditions Control

Total hemocyte count (cell mL1)

Mean SE Mean SE Mean SE Mean SE Mean SE Mean SE Mean SE

Granulocytes (cell mL1) Hyalinocytes (cell mL1) Adhesion (%) Viability (%) Phagocytosis (%) Oxidative burst (A.U.)

6.24 2.25 2.36 1.42 1.39 2.15 88.2 7.2 91.6 2.0 34.0 5.2 172.3 29.7

H      

105 105 105 105 105 104

V

9.12 3.02 3.59 1.32 2.40 8.54 96.8 1.3 87.0 2.2 41.3 7.5 129.5 23.9

     

105 105 105 105 105 104

6.98 5.90 2.85 3.35 1.65 7.04 98.3 0.9 82.6 2.0 40.5 4.4 78.3 5.5

HV      

105 104 105 104 105 104

5.46 1.05 1.34 5.37 1.62 4.28 87.0 3.9 80.3 2.6 42.8 6.1 170.8 52.3

C      

105 105 105 104 105 104

9.24 5.16 4.13 4.35 2.67 4.75 93.2 2.4 86.2 1.8 43.1 4.0 93.5 50.7

CH      

105 104 105 104 105 104

8.00 2.72 2.39 1.10 3.36 1.13 97.2 0.9 85.4 4.1 29.0 0.6 119.1 54.5

CV      

105 105 105 105 105 105

5.68 1.22 1.71 4.85 2.03 2.27 92.6 1.8 86.9 2.9 31.9 5.8 159.7 31.3

CHV      

105 105 105 104 105 104

6.19 7.57 2.02 5.88 1.71 1.22 86.1 6.0 79.5 1.5 39.3 7.8 223.0 6.6

     

105 104 105 104 105 104

6.E+05 5.E+05

[Granulocytes] cell.mL

a -1

Abbreviations: H: parasitized with Himasthla elongata; V: infected by Vibrio tapetis; C: cadmium contaminated; HV, CH, CV, CHV: interactions between stressors.

4.E+05 3.E+05 2.E+05 1.E+05 0.E+00 Control

H

V

HV

C

CH

CV

CHV

CV

CHV

CV

CHV

Experimental conditions

b

100

Viability (%)

90 80 70 60 50 Control

H

V

HV

C

CH

Experimental conditions

c

110

Adhesion (%)

100 90 80 70 60

50 Control

H

V

HV

C

CH

Experimental conditions Fig. 4. Response of immune parameters in function of experimental conditions (mean  SE) after 7 days of exposure: (a) granulocyte concentration (cell/mL); (b) Adhesion capability (%); (c) Hemocytes viability (%). H: parasitized with Himasthla elongata; V: infected by Vibrio tapetis; C: cadmium contaminated; HV, CH, CV, CHV: interactions between stressors.

decussatus, suggesting that the low success of H. elongata infection in Manila clams was not due to its status of introduced species in Arcachon Bay. Thus, higher trematode infection found in cockles (Paul-Pont et al., 2010a) was certainly more due to higher sensitivity of this species to trematode parasite than to its status of native species. The experimental infection efficiency remained similar irrespective of the presence or not of other sources of stress, i.e. metal and V. tapetis contaminations (Fig. 5-(2)). This result revealed that Cd did not impact on the infection capabilities of H. elongata cercariae. This is contrary to the findings of other investigations where an increase in pollutant levels has affected parasites in such a way that they are less able to infect their hosts (Cross et al., 2003; Lafferty and Kuris, 1999; Morley et al., 2003a,b). In the same way, the presence of V. tapetis did not seem to enhance trematode infection in the Manila clam. Similar results were found in a study conducted by Pylkkö et al. (2006) in which bacterial infection (Aeromonas salmonicida) did not increase parasite infestation (Diplostomum spathaceum) in the European grayling (Thymallus thymallus). Finally, the similar infection efficiency between treatments also indicated that there was not a weakening of bivalves due to cadmium contamination to such a point where infection by H. elongata was promoted. Unfortunately, in this study we could not assess the effective bacteria load in clams at the end of the experiment. No quantitative results of bacterial concentration in fluids and tissues of clams were available due to the lack of V. tapetis specific primers allowing us to quantify it by real-time quantitative PCR. Since V. tapetis constitutes one of the most pathogenic bacteria for the Manila clam, it would have been very interesting to assess potential facilitation mechanisms due to an infection with trematode parasites or cadmium exposure (Fig. 5-(3)). Cusack and Cone (1986) concluded that parasites acted as vectors to other pathogens by creating portals of entry (tissue lesions). Recent studies have confirmed this hypothesis in fish (Evans et al., 2007; Pylkkö et al., 2006; Shoemaker et al., 2008) and others studies have demonstrated a decreased host resistance following parasite infection linked to increased host susceptibility to other pathogens (Bowers et al., 2000; Tully and Nolan, 2002). In addition, immunotoxicity of metal contamination could also lead to an increase in bacterial infection. One of the most important heavy metal homeostasis and detoxification mechanisms in marine invertebrates involves the sequestration of metal compounds by MT (Coyle et al., 2002; Viarengo and Nott, 1993). Cd is known to be one of the powerful inducers of MT (Stillman, 1995). However, in our experiment no effect of Cd, alone or in combination was found in MT concentrations both in gills and visceral mass (Fig. 5-(4)). This lack of MT

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Fig. 5. Synopsis of the interactive effects of cadmium exposure, trematode and bacterial infections in some defense-related activities of the Manila clam Ruditapes philippinarum.

induction following Cd exposure could be explained by the presence of other efficient intracellular ligands such as high molecular mass protein, enzymes, amino acids and peptides like glutathione, which can interfere with the process of metal sequestration (Langston et al., 1998; Viarengo et al., 1989). The important role of insoluble fractions (metal-rich granules, cellular debris and organelles) in detoxification of Cd was also demonstrated in R. philippinarum (Ng and Wang, 2004). In addition, control clams displayed high basal MT concentration in the visceral mass and to a lesser extent in gills. This high baseline level could be sufficient to respond to metallic stress and could consequently mask any effect of Cd exposure. Even if the digestive gland of molluscs is often selected as a target tissue for the analysis of MT as a biomarker of metal exposure related to its main metabolic and storage roles for metals (Bebianno and Langston, 1991; Geffard et al., 2005), it could not be sensitive enough for trace metal exposure. In fact, the basal

MT pool in the digestive gland of molluscs is high in relation with the homeostasis of essential metals (Geffard et al., 2001) or other metabolic processes such as reproduction or nutrition. Numerous studies reported that physiological changes caused by gonadal development (Baudrimont et al., 1997a) (Baudrimont et al., 2006) and food abundance (Raspor et al., 2004) contribute more to changed MT levels than bioavailable toxic metal concentrations. Thus, trace metal exposure at sublethal concentrations might not reach a level which induces additional MT synthesis. In both organs, trematode parasites caused significant effects on MT responses (Table 1; Fig. 5-(5)). It is possible that few established metacercariae, or even cercarial penetration attempts, have a significant detrimental effect on a species that is not used to trematode attack. Penetration and encystment of parasites as well as bacteria colonization may lead to tissue damages and an inflammatory state (Canesi et al., 2002; Desclaux, 2003; Jensen

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et al., 1999) and it is now well documented that MT was induced in inflammatory states related to wounds, infections or diseases (Gagné et al., 2007). Co-infection leads to additional effects on MT responses with MT concentrations twofold in combined parasite  bacteria (HV) treatment compared to parasite (H) and bacteria (V) single exposures in gill tissue (Fig. 5-(6)). Once more, it suggests possible facilitation mechanisms as described above or at least interaction between both parasite and bacteria. Thus, infection by opportunistic pathogens as well as co-infection may not only modify pollutant accumulation but also detoxification processes such as MT synthesis (Baudrimont et al., 2006). Such interactions have to be taken into consideration in the context of ecotoxicological studies (Morley, 2010). In our experiment neither cadmium exposure nor trematode infection induced significant changes in hemocyte counts and activities (Fig. 5-(7)). Even if the low efficiency of trematode infection in clams tallied with the lack of any significant effect, modulation of the immune system following significant Cd accumulation in exposed clams should have been expected. Cadmium toxicity on bivalve immune parameters has been widely reported in literature in numerous species (Auffret and Oubella, 1994; Boutet et al., 2002; Brousseau et al., 2000; Coles et al., 1995). However, strongest effects of cadmium were mainly reported within in vitro studies although in vivo experiment have lead to none or low variations of hemocytes parameters after Cd exposure (Bouilly et al., 2006). In vitro studies working on cellular defenses of R. philippinarum after experimental exposure to V. tapetis mainly revealed that this bacterium caused a decrease in hemocyte viability, phagocytic activity (Allam and Ford, 2006), adhesion (Choquet et al., 2003) and lysosome concentrations (Allam et al., 2000). In vivo experiments, realized by injection of V. tapetis suspensions in adductor muscle, pallial and extrapallial cavities, confirmed these findings and demonstrated the positive correlation between the percentage of granulocytes and phagocytosis with a consistent resistance to Brown Ring Disease, whereas total hemocyte counts showed no consistent associations (Allam et al., 2001). To our knowledge the present experiment is the first report of V. tapetis infection by addition of bacterial suspension directly in water column. Bacterial suspension used in this experiment (109 cfu mL1) was higher than concentrations of suspensions used in pallial/extrapallial injections (5  107 to 5  108 cfu mL1) due to the indirect way of infection. Clams infection by balneation was chosen to minimize stress in animals and to approach as far as possible more realistic conditions of infection in natural environment. This kind of experimental infection might explain the lower intensity of hemocyte responses to V. tapetis exposures, alone or in combination. No significant effect of this bacterium was shown on total hemocyte count, phagocytosis and oxidative burst after 7 days of infection (Fig. 5(7)). No changes in THC and phagocytic activity were also reported in the hemolymph after infection by V. tapetis in a study conducted by Reid et al. (2003). These authors suggested no direct role for such increases in total hemocyte densities in reduced disease levels. Despite similar THC, a negative effect of bacteria was revealed on granulocyte concentration (Fig. 5-(8); Table 2). Changes in proportion of cell types in infected individuals without any modification of THC have been reported in oysters Ostrea edulis infected by Bonamia ostreae (Cochennec-Laureau et al., 2003). These authors correlated this modification of granulocytes concentration to both degranulation or degradation of granulocytes and specific hemocyte type recruitment from hemolymph to site of infections. Specific hemocyte type recruitment to infection site was suspected in the present experiment. Since bacteria initially colonized mantle and periostracal lamina (Paillard, 2004), cellular components of

extrapalleal fluids are known to play a crucial role in defense processes during the first step of infection (Allam et al., 2000). Granulocytes are considered to be the most phagocytic hemocytes in Ruditapes spp. related to their high phagocytic activities (López et al., 1997) and their wide production of hydrolytic enzymes and antimicrobial substances (López-Cortes et al., 1999), and are consequently considered as the main hemocytes mobilized following bacterial challenge (Allam et al., 2006). In addition, hemocytes can be mobilized towards hemolymph and extrapalleal fluid in relation to the origin of infection (Allam et al., 2006). Infiltration of hemocytes to damaged mantle or extrapallial compartments has been already reported (Bricelj et al., 1992; Allam et al., 2000). Therefore, specific recruitment of granulocytes from hemolymph to extrapalleal fluid following bacterial infection was suggested to explain the decrease of such hemocyte types in the hemolymph. No direct effect of bacteria was revealed on hemocyte adhesion (Table 2) in compariston to the findings of Choquet et al. (2003) which showed a decrease of hemocyte adhesion following V. tapetis exposure. However, a significant interaction between bacteria and parasite on this parameter was observed (Fig. 5-(9)) and might be linked with an increase of bacterial concentration due to co-infection with trematodes as described above. An important decrease in hemocyte viability by V. tapetis infection was demonstrated in this study (Fig. 5-(10); Table 2) in accordance with previous works (Paillard, 2004). V. tapetis possesses thermosensitive cytotoxic factors that are able to kill in vitro hemocytes of R. philippinarum (Allam et al., 2006). Finally, it is important to note that the same experiment performed on native cockles from Arcachon Bay revealed very different responses in terms of detoxication processes (MT concentration) and immune responses (Paul-Pont et al., 2010a). Such results may give some pointers in assessing particular adaptations of introduced species compared with native species. However, some restrictions have to be considered in the direct comparison of both studies. Cockles and clams constitute different species living in different areas with different environmental conditions. Thus, in order to carefully assess the role of the introduced status in the adaptation of clams to multiple stressors, it would be essential to perform the same experiment in the native clam R. decussatus coming from a similar sampling site. Indeed, interactions between pathogens and pollutants in introduced or invasive species, compared to native species, are poorly understood and require specific attention (Morley, 2008). 5. Conclusion Infection by opportunistic pathogens affects metal accumulation and leads to maximal Cd accumulation in co-infected clams. Such result highlights the necessity to take into account pathogens in ecotoxicological studies. Among stressors only V. tapetis induced significant effects on immune parameters whereas a particular interaction “trematode-bacteria” was shown on MT responses. Such modulation was highlighted with an apparent non-deleterious pathogen (i.e. trematode) for Manila clams. Thus, heavy alterations would have been expected with more pathogenic organisms. Future work will concentrate on determining valid methods to accurately quantify V. tapetis in clams fluids and tissues. Such further work constitutes an essential step to assess potential enhancement of bacterial infection following trematode penetration. Overall, particular features appeared in triple stress conditions, in terms of metal detoxification and immune processes, demonstrating the necessity of considering the impact of multiple factors in coastal ecosystems in assessing the health status of organisms.

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Acknowledgments This work was carried out with the financial support of the “Région Aquitaine” and the French National Research Agency (ANR e “Multistress project”). Authors are grateful to Francis Prince, Pascal Lebleu, Henri Bouillard and Frances Haynes for their help and their technical support before and during the experiment. Authors thank Sarah Culloty, University of Cork (Ireland) for improving the manuscript. References Allam, B., Paillard, C., Auffret, M., 2000. Alterations in hemolymph and extrapallial fluid parameters in the Manila clam, Ruditapes philippinarum, challenged with the pathogen Vibrio tapetis. Journal of Invertebrate Pathology 76, 63e69. Allam, B., Ashton-Alcox, K.A., Ford, S.E., 2001. Haemocyte parameters associated with resistance to brown ring disease in Ruditapes spp. clams. Developmental and Comparative Immunology 25, 365e375. Allam, B., Ford, S.E., 2006. 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