(Perca flavescens) in the St. Lawrence River, Canada

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of pollution in the St. Lawrence River, Quebec, Canada, to examine the effects of the ... brevis Ransom, 1920 and genus Diplostomum von Nordmann, 1832.
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Interactions between parasites and pollutants in yellow perch (Perca flavescens) in the St. Lawrence River, Canada: implications for resistance and tolerance to parasites David J. Marcogliese, Claire Dautremepuits, Andre´e D. Gendron, and Michel Fournier

Abstract: Parasites were examined in yellow perch, Perca flavescens (Mitchill, 1814), from four localities ranging in degree of pollution in the St. Lawrence River, Quebec, Canada, to examine the effects of the most prevalent parasite species on expression of biomarkers of oxidative stress. Various biomarkers appeared to be affected by the infection levels of Apophallus brevis Ransom, 1920 and genus Diplostomum von Nordmann, 1832. For certain biomarkers, interactions between infection level and pollution type were detected for A. brevis, Diplostomum spp., and genus Ichthyocotylurus Odening, 1969. Activity of glutathione reductase in gill tissue decreased with increasing numbers of A. brevis, but only at the two most polluted localities. Catalase activity in kidney increased with numbers of Diplostomum spp. at the polluted localities, but not at the two least contaminated sites. Results suggest that parasites may affect expression of biomarkers of pollution and that pathogenicity of parasites may be enhanced under polluted conditions. Exposure to contaminants appears to reduce tolerance, but not resistance, to parasites in yellow perch in this system. This type of immunosuppression may be widespread in polluted ecosystems. Re´sume´ : Dans le but d’explorer l’influence des principaux parasites sur l’expression des marqueurs de stress oxydatif chez les poissons, des perchaudes, Perca flavescens (Mitchill, 1814), ont e´te´ re´colte´es a` quatre localite´s du fleuve Saint-Laurent (Que´bec, Canada) repre´sentant diffe´rents degre´s de pollution, pour ensuite faire l’objet d’un examen parasitologique. Les analyses montrent que les niveaux de plusieurs des biomarqueurs mesure´s dans les tissus des perchaudes sont significativement affecte´s par les niveaux d’infection par Apophallus brevis Ransom, 1920 et le genre Diplostomum von Nordmann, 1832. Pour certains biomarqueurs, les analyses montrent des interactions entre les niveaux d’infection par A. brevis, Diplostomum spp. et le genre Ichthyocotylurus Odening, 1969 et le degre´ de pollution. Par exemple, l’activite´ de la glutathion-re´ductase dans les branchies diminue avec l’augmentation du nombre d’A. brevis, mais uniquement aux deux sites les plus pollue´s. De son coˆte´, l’activite´ de la catalase dans le rein augmente avec le nombre de Diplostomum spp. aux sites pollue´s, mais non aux deux sites les moins contamine´s. Les re´sultats sugge`rent que les parasites peuvent exercer une influence sur l’expression des biomarqueurs de pollution et que la pathoge´nicite´ de certains parasites peut eˆtre exacerbe´e dans des conditions de contamination. L’exposition aux contaminants semble re´duire la tole´rance mais non la re´sistance des perchaudes aux parasites dans ce syste`me. Il est possible que cette forme d’immunosuppression soit re´pandue dans les e´cosyste`mes pollue´s.

Introduction Aquatic contaminants can have profound effects on parasite species abundance, composition, and richness (MacKenzie 1999; Lafferty 1997; Marcogliese 2005). However, in recent years, there have been appeals for multidisciplinary studies combining the use of parasites as bioindicators of pollution with the examination of the effects of parasites on physiological biomarkers of pollution (Sures 2004, 2006, 2007, 2008; Marcogliese et al. 2005, 2009; Morley et al. 2003; VidalMartı´nez 2007).

Typically, biomarkers are used in ecotoxicology as functional measures of exposure to an environmental insult. They may include molecular, biochemical, and physiological endpoints that operate at a suborganismal level of organization, and are used to indicate exposure to a stressor such as contaminants (Adams 2002). Given that biomarkers respond to one or more stressors, it would not be surprising to discover that in some cases parasites can induce a biomarker response. Yet, aside from immunological measurements that are often independently used by parasitologists, ecotoxicologists, and ecologists, few studies have examined

Received 21 July 2009. Accepted 2 December 2009. Published on the NRC Research Press Web site at cjz.nrc.ca on 13 February 2010. D.J. Marcogliese1 and A.D. Gendron. Fluvial Ecosystem Research Section, Aquatic Ecosystem Protection Research Division, Water Science and Technology Directorate, Science and Technology Branch, Environment Canada, St. Lawrence Centre, 105 McGill Street, 7th Floor, Montre´al, QC H2Y 2E7, Canada. C. Dautremepuits2 and M. Fournier. INRS-Institut Armand Frappier, Universite´ du Que´bec, 531 Boulevard des Prairies, Laval, QC H7V 1B7, Canada. 1Corresponding 2Present

author (e-mail: [email protected]). address: Clair’Environnement, Parc Gouraud, 02200 Soissons, France.

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doi:10.1139/Z09-140

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the effects of parasitism on common ecotoxicological endpoints. For example, cortisol production is linked with exposure to environmental stress and is often used in ecotoxicology. However, exposure of European eels, Anguilla anguilla (L., 1758), to the nematode Anguillicoloides (= Anguillicola) crassus (Kuwahara et al., 1974) also was shown to increase cortisol production (Sures et al. 2006). In addition, infections of laboratory rats with Moniliformis moniliformis (Bremser, 1811) affected cortisol homeostasis (Sures et al. 2002). The protein metallothionein is routinely used as a biomarker of exposure to heavy metals, but recently it has been demonstrated that its synthesis was modulated by the digenean Labratrema minimus (Stossich, 1887) in the cockle Cerastoderma edule (L., 1758) (Baudrimont et al. 2003, 2006; Baudrimont and de Montaudouin 2007). The acanthocephalan Polymorphus minutus (Goeze, 1782) inhibited the production of heat shock proteins, another common biomarker of stress, in the amphipod Gammarus roeseli Gervais, 1835 (Sures and Radszuweit 2007). The occurrence of pigmented macrophages in various fish tissues such as spleen and liver is a commonly used indicator of exposure to pollution. However, numbers of pigmented macrophages also increased in spleens of spottail shiners, Notropis hudsonius (Clinton, 1824), infected with the digenean Plagioporus sinitsini Mueller, 1934, but only at polluted localities (Thilakaratne et al. 2007). Exposure to toxic substances and inflammation leads to the production of reactive oxygen species (ROS), which can damage DNA, lipids, and proteins (Storey 1996; Sorci and Faivre 2009). Organisms including fish possess enzymatic defense mechanisms to cope with ROS (Winston and Di Giulio 1991). However, should production of ROS exceed defensive capacity, oxidative stress results. Digeneans, cestodes, nematodes, and crustaceans infecting fish and invertebrates have been shown to affect antioxidant metabolism or induce oxidative stress (Bello´ et al. 2000; Neves et al. 2000; Dautremepuits et al. 2002a, 2002b, 2003; Marcogliese et al. 2005). In this study, helminth parasites from yellow perch, Perca flavescens (Mitchill, 1814), from four localities representing a gradient in contamination levels were examined to evaluate whether or not the parasites found in yellow perch affect the expression of enzymatic and nonenzymatic biomarkers of antioxidant metabolism. To answer this question, biomarkers and parasites were quantified in the same individual fish.

Materials and methods Sampling localities and fish collections The St. Lawrence River is a major waterway in eastern North America that connects the Great Lakes to the Gulf of St. Lawrence. Four sampling localities in the St. Lawrence River were chosen based on sediment contamination data and their position relative to municipal sewage effluents and other sources of pollution (Fig. 1; Dautremepuits et al. 2009). Beauharnois, at the mouth of the St. Louis River (45819.051’N, 738 53.020’W), is considered the most contaminated of the four with very high levels of mercury and PCBs (Loiselle et al. 1997; Marcogliese et al. 2005; Dautremepuits et al. 2009). The next most contaminated, Iˆlet Vert (45842.230’N, 73827.143’W), is 4 km downstream from the sewage outflow from the Island of Montre´al. This

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locality is characterized by high levels of certain metals, most notably chromium, and high fecal coliform counts, ranging from >1000 to 7600(100 mL)–1 (Marcogliese et al. 2006; Dautremepuits et al. 2009). The ˆIles aux Sables (46807.027’N, 73801.611’W) are located farther downstream in Lake St. Pierre. Moderate levels of certain metals and high levels of chromium have been detected there (Dautremepuits et al. 2009; Pelletier 2008). ˆIle Dorval (45826.018’N, 73844.257’W) is found upstream from the Island of Montre´al in Lake St. Louis. This locality has the lowest contamination levels of the four (Loiselle et al. 1997; Marcogliese et al. 2005, 2006; Dautremepuits et al. 2009). At all four localities, only metals surpass recommended federal and local probable effects and toxic effects sediment guidelines, with the exception of PCBs at Beauharnois (Dautremepuits et al. 2009). Localities are ranked Beauharnois > ˆIlet Vert > ˆIles aux Sables > ˆIle Dorval in terms of overall contamination (Dautremepuits et al. 2009). Immature yellow perch of age 1+ (8.80 ± 0.23 g and 86.58 ± 0.70 mm) were sampled with a beach seine (22.6 m  1.15 m; 3 mm mesh) in June 2004. Juvenile fish were used to ensure that the fish from each locality were local. The number of fish caught at each locality was 21 at ˆIlet Vert, 30 at Beauharnois, 21 at ˆIles aux Sables, and 28 at ˆIle Dorval. Fish were sacrificed in the field with a blow to the head. Head kidney and gill tissues were removed immediately, then stored on ice and brought to the laboratory. Subsequently, tissues and fish were stored at –80 8C until further analysis of their biomarkers and parasites. Parasite analyses Fish were examined using a stereomicroscope for macroparasites using standard parasitological techniques. Prior to dissection, fork length was measured to the nearest millimetre and each fish was weighed to the nearest 0.1 g. Fish were examined externally. Eyes were removed and dissected. The body cavity and viscera were examined. Organs (brain, liver, gall bladder, heart, urinary bladder) were removed, squashed between glass plates, and examined. The stomach and intestine were opened longitudinally and examined. They were then squashed between glass plates to detect worms in the tissue. The skin was removed from the flesh, which was thin-sliced, squashed between glass plates, and examined. The kidneys and gills were not examined, as they were required for biomarker analysis. Parasites were identified using the keys in Arai (1989), Moravec (1994), Gibson (1996), and Hoffman (1999). Biomarker analyses All samples were kept on ice throughout the preparation and analyses. Samples were processed and analyzed according to the procedures outlined in Dautremepuits et al. (2009). Frozen samples of head kidney tissue were homogenized by suspending 0.2 g of tissue in 3 mL of phosphate-buffered saline (PBS) (Dulbecco’s; Sigma, St. Louis, Missouri, USA) in a potter-pestle homogenizer (Sigma). Gill tissue was homogenized in 1:10 (m/v) cold PBS (Dulbecco’s) containing KCl (1.17%), using a potter – Fisher Scientific Dyna-Mix (Suwanee, Georgia, USA) homogenizer. Homogenates were centrifuged at 4000 rev/min (3450g) for 30 min at 4 8C. The supernatants were collected and immediately analysed for Published by NRC Research Press

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Fig. 1. Map of the St. Lawrence River, Quebec, Canada, showing the location of the four sampling localities for yellow perch (Perca flavescens) in June 2004. Inset shows the location of the sampling area in eastern Canada.

antioxidant enzyme activities. Analyses of protein content, thiols (SH), glutathione S-transferase (GST; EC 2.5.1.18), and glutathione reductase (GRd; EC 1.8.1.7) activity were performed on gill and head kidney tissue. Analyses of catalase (EC 1.11.1.6), ceruloplasm, and lysozyme activity were performed on head kidney tissue only. GST breaks down lipid hydroperoxides, GRd participates in the turnover of reduced glutathione, and catalase breaks down hydrogen peroxide (Wilhelm Filho 1996; Mourente et al. 2002). Lysozyme is a nonspecific humoral factor that acts against disease and responds to stress (Bols et al. 2001; Fatima et al. 2007). Ceruloplasmin is an acute-phase protein that functions as a scavenger of ROS and is considered an indicator of disease or tissue damage (Stadnyk and Gauldie 1991; Floris et al. 2000). Total protein content (mgmL–1) of each sample was measured with a Bio-Rad DC protein assay kit (Bio-Rad Laboratories, Mississauga, Ontario, Canada). SH groups were measured using the DTNB method in a spectrophotometer. Four microlitres of sample were added to 46 mL of PBS (0.2 molL–1, pH 6.8) and 50 mL of PBS containing 1 mmolL–1 DTNB (5,5’-dithiobis-2-nitrobenzonic

acid; Sigma) in a 96-well tissue-culture plate. Absorbance was measured at 412 nm after 10 min of incubation at room temperature. GSH (reduced glutathione) commercial solution (Sigma) was used as a standard. Measurement of GST activity was adapted from Habig et al. (1974). The reaction mixture consisted of 100 mL PBS (0.1 molL–1, pH 6.5), 50 mL reduced glutathione (Sigma) (1 mmolL–1), 25 mL H2O, 10 mL 1-chloro-2,4-dinitrobenzene (CDNB; Sigma) (1 mmolL–1), and 15 mL of sample in a total volume of 200 mL. The change in absorbance was recorded at 340 nm during 5 min and the enzyme activity calculated as micromole of CDNB formed per minute per milligram of protein using a molar extinction coefficient of 9.6  103 Lmol–1cm–1. GRd activity was measured using reduced nicotinamide adenine dinucleotide phosphate (NADPH) (Sigma) and oxidized glutathione (GSSG) (Sigma) as substrate according to Kno¨rzer et al. (1996) but modified to our material. One unit of GRd was defined as the NADPH consumed per minute that catalysed the reduction of 1 mmolL–1 of GSSG. An extinction coefficient of 6.22 Lmmol–1cm–1 was used for NADPH. Enzyme concentration (unitsmL–1) was calculated Published by NRC Research Press

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Table 1. Summary statistics and site of parasite infections in yellow perch (Perca flavescens) from four localities in the St. Lawrence Ile Dorval (N = 28) Species

Site

Digenea Genus Apatemon Szidat, 1928 Azygia angusticauda* (Stafford, 1904) Bunodera luciopercae* (Mu¨ller, 1776) Apophallus brevis Cryptogonimus chili Osborn, 1903 Clinostomum complanatum (Rudolphi, 1814) Diplostomum spp. Ichthyocotylurus spp. Genus Neochasmus Van Cleave and Mueller, 1932 Genus Ornithodiplostomum Dubois, 1936 Phyllodistomum superbum* Stafford, 1904 Genus Posthodiplostomum Dubois, 1936 Rhipidocotyle papillosa (Woodhead, 1929) Tylodelphys scheuringi (Hughes, 1929) Uvulifer ambloplitis (Hughes, 1927) Unidentified metacercaria

Muscle, body cavity Stomach, intestine Stomach, intestine Muscle, fins, skin, operculum Muscle, liver Muscle, mouth, operculum Humor, lens Body cavity Muscle, optic nerve, liver Body cavity, brain Urinary bladder Body cavity, muscle Muscle, liver Humor Muscle, skin, fins

Abundance (mean ± SE)

Prevalence (%)

0.03±0.03 0.20±0.10 0.07±0.05 22.63±4.16 0.03±0.03 0.20±0.07 18.27±3.50 2.10±0.42 0.07±0.07 0.33±0.17 1.77±0.43 0 0 0 0 0

3 13 7 100 3 20 100 70 3 17 63 0 0 0 0 0

Cestoda Genus Proteocephalus Weinland, 1858

Intestine

0.80±0.26

33

Nematoda Dichelyne cotylophora* (Ward and Magath, 1917) Genus Hysterothylacium Ward and Magath, 1917 Philometra cylindracea* (Ward and Magath, 1917) Rhaphidascaris acus

Intestine Body cavity Body cavity Liver

0.37±0.09 0 0 0.97±0.50

77 0 0 27

Acanthocephala Neoechinorhynchus rutili* Stiles and Hassall, 1905

Intestine

0

Hirudinea Genus Myzobdella* Leidy, 1851 Genus Placobdella* Blanchard, 1893

Fins, skin Fins

0.03±0.03 0.10±0.06

0 3 10

*Adult parasites.

as (Dabsorbance of sample – Dabsorbance of blank)  (dilution factor) / 6.22 Lmmol–1cm–1 (for NADPH). A commercial GRd solution (Sigma) was used as a standard. The catalase activity in the head kidney was assayed following the method of Claiborne (1985) and Giri et al. (1996). The assay mixture consisted of 190 mL PBS (0.05 molL–1, pH 7.0), 100 mL hydrogen peroxide (Prolabo, San Diego, California, USA) (0.01 molL–1) and 10 mL of sample in a final volume of 300 mL. Change in absorbance was recorded at 240 nm. Catalase activity was calculated as nanomole of H2O2 consumed per minute per milligram of protein using a molar extinction coefficient of 43.6 Lmol–1cm–1. The lysozyme activity in the head kidney was determined by the turbidimetric assay (Studnicka et al. 1986). A 10 mL sample was added to 200 mL of Micrococcus lysodeikticus (0.2 gL–1 in 0.05 molL–1 of PBS at pH 6.2) suspension and the decrease in absorbance was recorded at 450 nm by spectrophotometry (PowerWave X; Bio-Tek Instruments, Winooski, Vermont, USA) for 30 min in a 96-well tissue culture plate (Sarstedt, Newton, North Carolina, USA). One unit of lysozyme activity was defined as the amount of enzyme that catalysed a decrease in absorbance of 0.001 min–1. A commercial solution of lysozyme (Sigma, Oakville, Ontario, Canada) was used as a standard.

Ceruloplasmin activity in head kidney samples was measured as p-phenylenediamine (PPD) oxidase activity (Sigma, Canada) (Pelgrom et al. 1995). Fifteen microlitres of biological sample or standard of ceruloplasmin (Sigma, Canada) were mixed with 100 mL acetate buffer (1.2 molL–1, pH 5.5) containing 0.1% PPD as substrate in a 96-well tissueculture plate (Sarstedt). Each sample was incubated in the presence of 100 mL NaN3 (0.5%) (azide blank) (Sigma, Canada) for 30 min at 37 8C. The reaction was stopped by addition of 100 mL NaN3 (0.5%). One unit of ceruloplasmin was defined as the amount of oxidase that catalysed a decrease in absorbance of 0.001 min–1 recorded at 550 nm with a microplate spectrophotometer (PowerWawe X; Bio-Tek Instruments). Statistical analysis To determine if parasites affected the expression of biomarkers in each of the two tissues and whether there was an interaction with pollution, criteria were established for further analyses. To ensure adequate sample size at each locality, only parasites with a minimum prevalence of 40% at each locality were considered further. These included Apophallus brevis Ransom, 1920, genus Diplostomum von Nordmann, 1832, and genus Ichthyocotylurus Odening, Published by NRC Research Press

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River, Quebec, Canada, in June 2004. Iles aux Sables (N = 21)

Ilet Vert (N = 21)

Beauharnois (N = 30)

Abundance (mean ± SE)

Prevalence (%)

Abundance (mean ± SE)

Prevalence (%)

Abundance (mean ± SE)

Prevalence (%)

2.71±0.73 0.08±0.06 10.25±2.94 9.50±2.79 0.25±0.21 0 1.54±0.60 4.92±1.79 2.67±0.98 0.46±0.13 0.08±0.08 4.08±1.17 0.25±0.11 0.46±0.19 0.04±0.04 0.13±0.07

71 8 71 96 8 0 42 96 63 38 4 88 21 29 4 13

0 0 0 3.33±0.57 0 0.05±0.05 6.52±1.59 2.62±0.73 0.33±0.16 1.81±0.55 0.33±0.14 0 0.14±0.10 0 0 0

0 0 0 95 0 5 86 67 24 67 24 0 10 0 0 0

0.03±0.03 0 0.23±0.13 6.23±0.67 0.07±0.07 0.90±0.21 24.00±3.71 4.73±0.94 0.03±0.03 0.60±0.16 0.37±0.16 0 0.13±0.09 0.20±0.09 0.73±0.54 0

3 0 10 93 3 53 97 80 3 37 23 0 7 17 17 0

3.17±1.03

54

0.05±0.05

5

1.07±0.23

53

0.13±0.07 0.04±0.04 0.04±0.04 0.83±0.20

13 4 4 54

0.05±0.05 0 0 0.10±0.07

5 0 0 10

0.20±0.07 0 0 0.80±0.18

27 0 0 53

0

0.20±0.09

17

4 10

0.03±0.03 0

3 0

0

0

0

0 0

0 0

0.04±0.04 0.14±0.10

1969. For the purposes of analyses, localities were combined into polluted (Beauharnois and ˆIlet Vert) and reference (Iˆles aux Sables and ˆIle Dorval) categories based on the degree of pollution. Fish were characterized as lightly or heavily infected for each of the three parasite species based on the relative distribution of parasites among hosts. For A. brevis, Diplostomum spp., and Ichthyocotylurus spp., light and heavy infections were defined as £5 and >5,