Intestinal Nematodes Affect Selenium Bioaccumulation, Oxidative

Article pubs.acs.org/est

Intestinal Nematodes Affect Selenium Bioaccumulation, Oxidative Stress Biomarkers, and Health Parameters in Juvenile Rainbow Trout (Oncorhynchus mykiss) Olesya Hursky†,§ and Michael Pietrock*,†,‡,∥ †

Toxicology Centre, University of Saskatchewan, 44 Campus Drive, Saskatoon, Saskatchewan, Canada S7N 5B3 Department of Veterinary Biomedical Sciences, University of Saskatchewan, 52 Campus Drive, Saskatoon, Saskatchewan, Canada S7N 5B3

‡

S Supporting Information *

ABSTRACT: In environmental studies, parasites are often seen as a product of enhanced host susceptibility due to exposure to one or several stressors, whereas potential consequences of infections on host responses are often overlooked. Therefore, the present study focused on effects of parasitism on bioaccumulation of selenium (Se) in rainbow trout (Oncorhynchus mykiss). Joint effects of biological (parasite) and chemical (Se) stressors on biomarkers of oxidative stress (glutathione-Stransferase (GST), superoxide dismutase (SOD)), and fish health (condition factor (K), hepatosomatic index (HSI), gross energy) were also examined. Fish of the control group received uncontaminated food, while test fish, either experimentally infected with the nematode Raphidascaris acus or not, were exposed to dietary selenomethionine (Se-Met) at an environmentally relevant dose over 7 weeks. Selenium bioaccumulation by the parasite was low relative to its host, and parasitized trout showed slowed Se accumulation in the muscle as compared to uninfected fish. Furthermore, GST and SOD activities of trout exposed to both Se-Met and parasites were generally significantly lower than in fish exposed to Se-Met alone. Gross energy concentrations, but not K or HSI, were reduced in fish exposed to both Se-Met and R. acus. Together the experiment strongly calls for consideration of parasites when interpreting effects of pollutants on aquatic organisms in field investigations.

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INTRODUCTION

stressors can increase, decrease, or may cause no alterations in stress responses of infected organisms.1 By definition, parasites are considered detrimental to hosts in one way or another.7 Recent research, however, suggests that outcomes of parasitic infections could also be advantageous to parasitized individuals. For example, intestinal macroparasites, specifically cestodes and acanthocephalans, of terrestrial and aquatic organisms have repeatedly been shown to bioaccumulate metals and trace elements to concentrations orders of magnitude higher than their infected hosts.8−11 Hence it has been proposed that parasites may play a beneficial role by removing metals from the host’s intestinal lumen and/or body, thereby potentially reducing both amount of metal ions available for uptake and risk of metal-induced pathologies to the host.12 Based on the parasites’ potential to bioaccumulate contaminants to extremely high concentrations, use of cestodes and acanthocephalans in environmental monitoring as bioaccumulation indicators and sentinel species has been reviewed13 and successfully implemented.14−16 However, despite the growing number of studies using parasites as bioindicators of environmental pollution, it must not be

Throughout their life wild animals are exposed to manifold stressors of natural and anthropogenic origin which can compromise animal health. In this context, parasites (and other pathogens) have received increasing attention as they may interact with anthropogenic stressors, such as pollutants, subsequently leading to health impairments.1−3 In a wide spectrum of host−parasite systems, lethal and sublethal effects of parasitic infections in conjunction with environmental stress have previously been studied. For example, Kelly et al.4 investigated survival of the freshwater fish Galaxias anomalus infected by the digenean Telogaster opisthorchis and simultaneously exposed to the common herbicide glyphosate. Increased mortalities were noticed relative to the controls when fish were exposed to both stressors, while exposure to either stressor alone had no effect on G. anomalus survival.4 At sublethal level, reduced condition factor and attenuated antioxidant responses, respectively, were found in winter flounder (Pseudopleuronectes americanus)5 infected with the protozoan Trypanosoma murmanensis and common carp (Cyprinus carpio)6 infected with the cestode Ptychobothrium sp., respectively, compared to uninfected individuals when exposed to a chemical contaminant. Effects of parasitic infections on hosts, however, are context-dependent, and it has been demonstrated that parasites in combination with other © XXXX American Chemical Society

Received: October 7, 2014 Revised: January 14, 2015 Accepted: January 15, 2015

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DOI: 10.1021/es5048792 Environ. Sci. Technol. XXXX, XXX, XXX−XXX

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Environmental Science & Technology

fish condition,29,30 were calculated to potentially demonstrate the consequences of Se-Met exposure and parasitic infection on overall health of rainbow trout.

overlooked that the current knowledge on contaminant uptake by parasites almost exclusively originates from field investigations, which inherently lack information on, e.g., exact exposure period of host and parasite species, contaminant concentration throughout exposure, and contamination of parasites prior to host infection, among other factors. Laboratory experiments which address these issues, therefore, are highly desirable and have repeatedly been called for to further the understanding of the role of parasites in stressed environments.13,17,18 Selenium (Se) is an essential micronutrient required for all vertebrates including fish. Industrial activities such as metal mining, coal combustion, oil refining, and agriculture can result in elevated Se concentrations in the environment. Due to its narrow margin between safety and toxicity along with the propensity to bioaccumulate in aquatic food chains, Se has considerable biological importance as even a slight increase in environmental Se concentrations can be harmful to biota.19 In fish, for example, Se exposure has been shown to elicit stress responses, reduce body condition, cause deformities, and increase mortality, among other effects.20,21 Therefore, Se contamination of the environment and its consequences on biological systems has gained increasing scientific attention in the past decade. In the aquatic environment, Se uptake by organisms can be through water and/or diet, with dietary exposure being the most important pathway of Se accumulation in fish.22 Selenomethionine (Se-Met) is the major dietary Se source available to fish and has high trophic transfer potential relative to other Se forms.23 Three major mechanisms have been suggested for Se toxicity: membrane and protein damage caused by Se-generated reactive oxygen species (ROS), substitution of Se for sulfur during protein assembly, and inhibition of Se methylation metabolism resulting in the accumulation of toxic hydrogen selenide.24,25 Until recently, oxidative damage as a potential mechanism of Se toxicity in fish has received very little notice. Palace et al.26 reported that the metabolism of Se-Met in rainbow trout (Oncorhynchus mykiss) embryos entailed generation of ROS. Furthermore, exposure of trout to waterborne sodium selenite led to an induction of enzymatic antioxidants (superoxide dismutase (SOD) and glutathione peroxidase (GPx)),27 as well as altered concentration of hepatic glutathione (GSH),25 which is one of the most important nonenzymatic antioxidants. The induction of catalase (CAT), SOD, and GPx activities were also observed in primary cultures of rainbow trout hepatocytes exposed to selenite in vitro.28 Together these findings suggest that oxidative stress can be an important factor in Se toxicity to fish. In order to obtain insights into host−parasite relationships in polluted environments, a laboratory experiment on combined effects of a natural (parasite) and anthropogenic (Se-Met exposure) stressor on juvenile rainbow trout was initiated. The specific objectives were to determine to what extent rainbow trout and its gut-dwelling nematode Raphidascaris acus bioaccumulate Se from dietary exposure of the fish to an environmentally relevant Se-Met concentration. Second, activities of two enzymes involved in antioxidant response (glutathione-S-transferase (GST), SOD), were assessed to investigate joint effects of parasitism and Se-Met exposure on oxidative stress in fish. Additionally, nonspecific biomarkers such as Fulton’s condition factor (K), hepatosomatic index (HSI), and gross energy (GE) content, which are widely used to evaluate integrated effects of multiple stressors on individual

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MATERIALS AND METHODS Chemicals. Seleno- L-methionine (purity >98%) was purchased from Sigma-Aldrich (Oakville, ON, Canada). Test Species. Female rainbow trout (O. mykiss) approximately 1.5 year of age were randomly selected from an inhouse stock raised from eggs obtained from a commercial supplier (Troutlodge, Sumner, WA, U.S.A.). Prior to initiation of the study, trout were reared in a 1666-L flow-through tank receiving dechlorinated Saskatoon City water at approximately 6 °C and maintained under a 12L:12D photoperiod. Until beginning of Se-Met exposure, all fish were fed a commercially available trout feed (Martin Classic Sinking Fish Feed, Martin Mills Inc., Elmira, ON, Canada) once daily at a rate of approximately 2% body weight. Control fish were fed this uncontaminated feed throughout the length of the experiment. Experimental Protocol. The study was approved by the University of Saskatchewan’s Animal Research Ethics Board (# 20090085) and adhered to the Canadian Council of Animal Care guidelines for humane animal use. For the experiments, 232 female rainbow trout were randomly assigned to four 719 L tanks supplied continuously with running water at a flow rate of 4 L/min and maintained at approximately 6 °C under 12L:12D photoperiod. The mean weight (±SD) of trout at initiation of the exposure was 94.6 ± 5.6 g. One of the tanks was randomly designated for control trout, and three tanks were randomly designated for test fish. Of these, trout of one tank were exposed to Se-Met only (Se-Met group) and, to secure sufficient numbers of infected fish, trout of two tanks were exposed to Se-Met and parasites (Se-Met + parasites groups). Raphidascaris acus is a common parasitic nematode of freshwater fish, including rainbow trout. Infective larvae (L3), which are acquired by eating infected intermediate hosts, require approximately 21 days undergoing two moults into the adult stage in the intestine of its definitive host. Complete development of the nematode in the definitive host from the time of acquiring infection until egg production takes approximately two months.31 As a consequence, 28 days prior to onset of Se-Met exposure, rainbow trout from the two Se-Met + parasites groups were fed pieces of lake whitefish (Coregonus clupeaformis) intestines containing cysts with larval (L3) R. acus32 to produce mean infection levels of 25−30 parasites/fish. This infection method has been verified previously in rainbow trout during a pilot study, obtaining 75% infection rate. Throughout the experiment, all trout were fed approximately 1.5% bodyweight of uncontaminated (control group) or Se-Met laced feed 6 days per week, split between 2 daily feedings to ensure complete consumption of the food. For details on preparation of Se-Met contaminated food, see the Supporting Information. On day 0, 5 fish were sampled randomly from the tanks to determine pre-exposure antioxidant enzyme activities and health parameters of rainbow trout. Subsequent sampling was conducted weekly over a period of 7 weeks. Sample size was 6 fish each from control and the Se-Met group, and 8 fish from each of the two Se-Met + parasites groups. For sampling the net was moved slowly through the individual tanks, and groups of fish were carefully netted of which individual trout were randomly taken until required sample size was reached. B


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in a microplate spectrophotometer every minute for 10 min at room temperature. The activity of SOD was measured as units/ mg protein, where 1 unit of enzyme activity refers to 50% inhibition of the change in absorbance. Total protein was measured according to Bradford34 adapted to a microplate spectrophotometer using the Total Protein Assay Kit (Sigma, MO, U.S.A.). Human albumin in saline with 0.1% sodium azide was used as standard and absorbance measured at 595 nm. Fish Health Parameters. As an indication of general body condition, Fulton’s condition factor (K) = [weight/length3 × 100] as well as the hepatosomatic index (HSI) = [(liver weight)/(body weight) × 100] were calculated for control and test fish. Additionally, gross energy (GE) contents (MJ/kg) were calculated for each fish sampled, using the formula GE = [0.0253 × dry matter (%)1.6783].35 Statistical Analyses. All data are presented as mean ± standard error of mean (SEM). A total of 16 fish per sampling event was taken from the two Se-Met + parasites groups for determination of health parameters and Se analysis. However, activities of enzymes involved in antioxidant response were measured only in rainbow trout that actually exhibited R. acus infection upon dissection. In view of the comparatively low infection rate obtained and to increase statistical power, infected fish of the two Se-Met + parasites groups were combined for analysis of GST and SOD data. Significant differences among the fish groups were analyzed by one-way analysis of variance (ANOVA) followed by Tukey’s HSD post hoc test or by Games Howell post hoc test if homogeneity of variance was not assumed (SPSS, version 18 for Windows). Normality of distribution and homogeneity of variances were verified using the Shapiro−Wilk and Levene’s tests, respectively. Data that did not meet the assumptions were log 10 transformed. Significant differences in cases where only two groups were present were analyzed by Student’s t-test, and homogeneity of variance was tested by Levene’s test. Condition factor and HSI were analyzed using nonparametric Kruskal− Wallis test followed by Bonferroni-corrected pairwise Mann− Whitney U-test (as the data did not meet normality and homogeneity of variance requirements for ANOVA). A p ≤ 0.05 was considered significant.

Subsequently, trout were immediately euthanized with 150 mg/ L MS-222, which induced euthanasia within 2 min. Fish mass (g) and length (cm) were recorded for calculation of condition factor (K), and livers were weighed for calculation of the hepatosomatic index (HSI). Head kidney and approximately 0.2 g of liver tissue were excised for enzyme analyses and processed as described below. Muscle tissue (approximately 1.0 g, without skin and bones) was taken from the area posterior to the dorsal fin and frozen at −20 °C until later Se analysis and determination of gross energy (GE) levels. Intestines of fish of the Se-Met + parasites groups were dissected and examined for gross pathological changes and parasitic infection. Nematodes were counted, removed from the intestine, and rinsed with 0.68% saline solution. Nematode samples were frozen at −20 °C until Se analysis; some R. acus samples were transferred into 4% formalin for species identification.31 Selenium Analyses. Details on determination of Se in control and spiked food, R. acus cysts (to obtain information on Se concentration of the larval parasites prior to infection of rainbow trout), nematodes, and rainbow trout muscle are given in the Supporting Information. Selenium concentration in rainbow trout muscle was determined on days 7, 28, 35, and 49. Nematode samples (combined according to sampling day) were analyzed on days 7, 14, and 35 as only those days provided sufficient parasite material for determination of Se concentration. Total concentrations of Se were measured by use of inductively coupled plasma mass spectrometry (ICP-MS X SeriesII, Thermo Electron Corp.) at the Toxicology Centre (University of Saskatchewan, Saskatoon, SK, Canada). A limit of quantification (LOQ) of 0.5 mg Se/kg DW food was determined from method blanks. Recovery of Se was ascertained using certified reference material (Lobster Hepatopancreas Reference Material for Trace Metals (TORT-2), National Research Council, Ottawa, ON, Canada). Measurement of GST and SOD Activities, Protein Assay. Detailed information on determination of oxidative stress enzyme activities is given in the Supporting Information. Briefly, liver (approximately 200 mg) and head kidney samples (60−80 mg) were taken from each collected fish and rinsed with phosphate buffered saline (PBS) solution to remove any red blood cells and clots. Organ samples from individual fish were combined and jointly homogenized on ice in 1.0 mL of phosphate buffer and centrifuged at 15 000 g to remove nuclei and cell debris. The resulting supernatants were collected separately for each fish and stored at −80 °C until further analysis. For analysis, supernatant samples were pooled for each time point and test group. The GST activity was determined using the GST Assay Kit (Sigma-Aldrich, U.S.A.) utilizing 1chloro-2,4 dinitrobenzene (CDNB) as substrate.33 Measurements were conducted using a microplate spectrophotometer (Specta Max190, Molecular Devices Corporation, U.S.A.) at 25 °C during 6 min as the change in absorbance at 340 nm. One unit of GST was defined as μmol CDNB conjugate formed/ min/mg protein using molar extinction coefficient of 5.3 mM−1 (path length: 0.552 cm). The SOD activity was measured in pooled supernatants (see above) using a SOD kit (Enzo Life Sciences, U.S.A.) where relative SOD activity of the experimental sample was determined from percent inhibition of the rate of formation of WST-1 formazan. The reaction mixture consisted of 10X SOD Buffer, WST-1 Reagent, Xanthine Oxidase, and distilled water. The reaction was initiated by adding 1X Xanthine Solution to all the wells. Absorbance at 450 nm was measured

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RESULTS Fish Survival and Parasite Infection. There were no mortalities in either the control group, Se-Met exposed group, or fish exposed to both Se-Met and nematodes for the duration of the experiment. Infected fish were found at every time point of sampling, except on day 49. Infection levels were relatively low: in total, 18 out of 112 fish examined from the two Se-Met + parasites groups were infected with R. acus, resulting in overall prevalence of 16%. Mean intensity (number of parasites/number of infected fish, ±SEM) was 9.0 (±3.0) nematodes/infected fish. Dissections revealed no gross pathological changes in intestines of infected fish. Selenium Analyses. Total Se concentration in nonspiked (control) food (±SEM) was 2.03 (±0.01) mg Se/kg DW, while that of the Se spiked food was 8.47 (±0.24) mg Se/kg DW. Differences in Se concentration between these two diets were statistically significant (p < 0.05). Selenium concentration of the cysts taken from lake whitefish and fed to rainbow trout for infection purposes was 1.26 (±0.10) mg Se/kg DW. Mean Se concentration (mg Se/kg DW) in the rainbow trout muscle is presented in Figure 1. Concentrations of Se in fish C


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Figure 1. Muscle selenium (Se) concentration of rainbow trout (Oncorhynchus mykiss) either infected or not with the intestinal nematode Raphidascaris acus. Fish were fed 1.5% bodyweight/day of uncontaminated (gray, control group) or seleno-L-methionine spiked (8.47 mg Se/kg DW) food (dark, Se-Met group; hatched, Se-Met + parasites groups), respectively. Bars are means ±SEM of pooled samples; groups with different letters are significantly different (p < 0.05) within sampling date.

Figure 2. Glutathione S-transferase (GST) activity in combined liver/ head kidney samples of rainbow trout (Oncorhynchus mykiss) either infected or not with the intestinal nematode Raphidascaris acus. Fish were fed 1.5% bodyweight/day of uncontaminated (gray, control group) or seleno-L-methionine spiked (8.47 mg Se/kg DW) food (dark, Se-Met group; hatched, Se-Met + parasites groups), respectively. One unit of GST activity is defined as μmol 1-chloro2,4 dinitrobenzene (CDNB) conjugate formed/min/mg protein. Bars are means of triplicates ± SEM of pooled samples; groups with different letters are significantly different within sampling date (p < 0.05).

muscle of control fish ranged between 1.05 and 3.29 mg Se/kg DW. For the first 7 days of the exposure there was no significant difference in Se concentrations between control, SeMet group, and Se-Met + parasites groups. However, after 28 days, Se concentrations were significantly lower in control group compared to Se-Met exposed trout for the remaining duration of the experiment (p < 0.05). Selenium concentration in muscle tissue of the Se-Met group fish appeared to reach a plateau at approximately 5 mg Se/kg DW after 28 days of exposure and did not change thereafter. In contrast, a slow but continuous increase (p < 0.05) in Se concentration with time was recorded in fish of the Se-Met + parasites groups. Mean muscle Se concentration of fish from the Se-Met + parasites groups was generally significantly lower than in the Se-Met group (p < 0.05), aside from day 49, when Se concentrations in fish from both groups were not statistically different. Selenium concentration of R. acus was below detection limits after first 7 days of Se-Met exposure but increased with time, reaching 0.025 mg Se/kg DW on day 14 and 0.47 mg Se/kg DW on day 35. Effects of Selenium and Parasite Exposures on Oxidative Stress Biomarkers. Significant differences between GST activity in control and Se-Met group were observed at the different time points. However, GST activity fluctuated, and comparison of these two groups did not yield a consistent pattern over time (Figure 2). In contrast, GST activity of fish exposed to both Se-Met and parasites was generally significantly lower than in the fish exposed to Se-Met alone (p < 0.05). Furthermore, a nonsignificant pattern between GST activity in control and Se-Met + parasites groups was found, with fish exposed to both stressors tending to have lower GST activity than control fish. Moreover, a significantly increased SOD activity relative to control fish was recorded in trout exposed to Se-Met (Figure 3). In contrast, SOD activity of trout exposed to both stressors (Se-Met + parasites) was always significantly lower than in fish exposed to Se-Met only, but frequently not significantly different from control group levels (p < 0.05). Parameters of Fish Health. Mean Fulton’s condition factor (±SEM) ranged from 1.01 (±0.11) to 1.53 (±0.18) and showed no temporal pattern nor significant differences between groups (except on day 35 when mean K of the Se-Met +

Figure 3. Superoxide dismutase (SOD) activity in combined liver/ head kidney samples of rainbow trout (Oncorhynchus mykiss) either infected or not with the intestinal nematode Raphidascaris acus. Fish were fed 1.5% bodyweight/day of uncontaminated (gray, control group) or seleno-L-methionine spiked (8.47 mg Se/kg DW) food (dark, Se-Met group; hatched, Se-Met + parasites groups), respectively. One unit of enzyme activity refers to 50% inhibition of the changes of absorbance. Bars are means of triplicates ± SEM of pooled samples; groups with different letters are significantly different within sampling date (p < 0.05).

parasites groups was significantly higher than of the other groups). Mean HSI (±SEM) varied from 0.93 (±0.03) to 1.89 (±0.14). With the exception of the Se-Met + parasites groups having a statistically significantly higher HSI than the other groups on day 28, there were no significant differences in HSI between groups at all time points. Gross energy content (Figure 4) was not significantly different between control fish and trout from the Se-Met group. However, fish from the Se-Met + parasites groups had significantly lower GE values than did control fish (p < 0.05), except on day 35. As well, GE contents of fish exposed to SeMet and parasites were lower than those exposed to Se-Met alone. However, these differences were statistically significant on days 7 and 28 only. D


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than corresponding levels in the fish. Raphidascaris acus may therefore not be very suitable as bioindicator of environmental Se pollution, at least when sampled from rainbow trout. The current results thus experimentally confirm indications from field research which suggest that nematodes are poor bioaccumulators of metals relative to acanthocephalans or cestodes.13 For example, Anguillicoloides crassus, a swim bladder nematode of eels (Anguilla spp.), accumulates virtually no lead,41 and its concentrations of cadmium, chromium, and other elements were lower than those of its host.42 On the contrary, the nematode Philometra ovata sampled from the body cavity of cyprinid fish had significantly higher concentrations of lead, cadmium, and chromium than its host.43 Still, in that same study the ratio between metal concentration in the nematode and concentration in host muscle was lower than those known from fish−acanthocephalan host−parasite systems,13 corroborating the comparatively low metal bioaccumulation capacity of nematodes. Other experimental data on biotic (e.g., developmental stage, microhabitat) and abiotic (e.g., other contaminants, exposure conditions) variables, however, would be desirable to comprehensively understand the relevance of nematodes and other parasite taxa for contaminant uptake of their hosts. Oxidative Stress Response. GST is an important family of multifunctional intracellular enzymes and has commonly been used as a biomarker for assessing oxidative stress in organisms exposed to, e.g., organic and inorganic pollutants (reviewed in Van der Oost et al.44). The initial increase of GST activity relative to control fish noted in the Se-Met group could be a result of increase in GSH conjugation in response to Se-Met exposure; however, as levels of GSH were depleted with time via oxidation to GSSG, the activity of GST decreased. Varying GST levels in stressed organisms are not uncommon, however, and it is possible that time dependency of enzymatic activity may also be a main reason for inconsistent GST activities subsequent to pollutant exposure measured in manifold field studies.44 Interestingly, GST activity in fish exposed to both SeMet and parasites was usually significantly lower than in the fish exposed to Se-Met alone. Experimental studies have shown that infection of vertebrates with digeneans (Dicrocoelium dendriticum, Fasciola sp.) or cestodes (Schistocephalus solidus, Ligula intestinalis) cause a reduction in GST activity45−47 which, among others, has been attributed to the parenchymal inflammation and associated increased secretion of inflammatory factors characteristic of the host’s immune response during parasitosis.46 Notably, the current findings suggest that parasitism-linked reduction in GST activity (relative to the Se-Met group) persists also under polluted conditions. In the view that parasitic lifestyle is widespread in nature,48 the present results are of prime importance for ecotoxicological investigations on wild animals as parasitism may lead to reduced GST concentrations in infected hosts and thus obscure negative effects resulting from pollutant exposure. Nonetheless, interactions between parasitism, pollution stress, and GST response remain complex, especially given that parasites themselves have been shown to secrete antioxidants (like GST) to evade host defenses.49 Unlike GST, SOD activity in rainbow trout liver/head kidney was significantly increased in fish exposed to Se-Met alone when compared to control and parasitized fish for the entire exposure period. Most probably, increase of SOD activity in trout fed Se-Met diet has occurred as a direct response to Se induced superoxide anion production, as primary antioxidant

Figure 4. Gross energy values of rainbow trout (Oncorhynchus mykiss) either infected or not with the intestinal nematode Raphidascaris acus. Fish were fed 1.5% bodyweight/day of uncontaminated (gray, control group) or seleno-L-methionine spiked (8.47 mg Se/kg DW) food (dark, Se-Met group; hatched, Se-Met + parasites groups), respectively. Gross energy was calculated according to Schreckenbach et al.35 Bars are means ±SEM; groups with different letters are significantly different within sampling date (p < 0.05).

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DISCUSSION Selenium Uptake. A 7-week dietary exposure of female rainbow trout juveniles to Se-Met concentrations similar to those found in prey organisms collected from Se affected sites22,23,36,37 led to muscle tissue concentrations below the tissue-based Se criterion to protect aquatic life (7.9 mg/kg DW) proposed by the United States Environmental Protection Agency.38 Interestingly, while at the end of the exposure period muscle Se concentrations were similar (around 5 mg/kg DW) in infected and noninfected fish, infection with R. acus influenced temporal course of Se bioaccumulation in rainbow trout. In Se-Met exposed (but noninfected) fish, Se concentrations increased for the first 28 days but then remained relatively constant until end of the experiment. Commonly, after uptake Se-Met is either metabolized directly to reactive Se forms or is stored in body proteins.39 Distribution and incorporation of Se into the various tissues then depend on the presence of selenoproteins, which can become oversaturated with the substrate over time. The results presented herein suggest that, due to oversaturation of selenoproteins, further Se bioaccumulation in the Se-Met group was no longer dependent on the concentration of Se-Met in the diet, leading to constant Se levels in the muscle. This steady state in Se levels, therefore, may reflect a change from concentrationdependent acute toxicity to subchronic toxicity. In contrast, a slower but continuous buildup of Se levels in trout muscle was recorded in fish infected with R. acus. The exact mechanism(s) behind this pattern is not clear. However, as parasites, including nematodes, need Se for growth and metabolism,40 it is likely that some Se-Met supplied to trout via diet was taken up by R. acus and thus was not available for (re)uptake (see Sures and Siddall12) by the fish host. As the current study demonstrated, Se concentration in R. acus increased with time confirming continuous Se-Met uptake by the parasite. Moreover, it seems plausible that increased Se demand of trout for, e.g., production of immune cells, peptides, proteins, and molecules to fight off parasite infection also may have contributed to slowed Se deposition and accumulation in muscle of infected fish relative to noninfected individuals. Although nematodes were able to accumulate Se in their tissues, accumulation capacity was low, as whole body concentrations of Se in the parasites were significantly lower E


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spottail shiner (Notropis hudsonius) collected downstream of a Se-releasing uranium mill when compared to fish from a reference lake.56 In contrast, other studies have reported both decreased K and HSI in fish exposed to environmental Se.57 Obviously, in field settings K and HSI are influenced by plenty of factors, such as season, sex, or life stage,30 respectively, which in their sum can compensate for potentially negative effects caused by Se pollution. Furthermore, the dependence of these two indicators on multitude of variables reveals their limitation as diagnostic tool in environmental research. Gross energy content of fish (i.e., energy released as heat when completely combusted58) is often assessed as a measure of general health and condition,32,59 and, in theory, it reflects total energy available to the fish to cope with energydemanding situations. Healthy fish should reveal GE concentrations above 4 MJ/kg body weight to withstand long-lasting stressful conditions.35 In the current study GE values in all rainbow trout samples were above that benchmark and therefore reflected a “good health state” of the test individuals. Nonetheless, statistical differences between control fish and parasitized trout exposed to Se-Met were found, as trout exposed to combined stressors had significantly lower GE values than the controls. Although it cannot be excluded that decreased GE stores in Se + parasites groups were impacted solely by infection with R. acus, diminished energy stores in parasitized fish exposed to dietary Se-Met denote that Se-Met exposure combined with parasitic infection can generate higher energetic demands in the host than Se-Met exposure alone. Environmental Implications. Effects of nematode parasitism on metal bioaccumulation as well as uptake of metals (or trace elements) by parasitic nematodes has, to the best of our knowledge, never been investigated in laboratory-infected fish. The present experiments, therefore, for the first time provide information on both temporal course and capacity of contaminant accumulation in parasitized fish and a parasitic nematode under standardized conditions. Whether or not removal of toxicants by nematodes and other parasite taxa has substantial beneficial effects on wildlife health remains to be shown. In the current study, infection levels were relatively low, but almost identical values have been found in salmonids from different European waters.60 However, as presented herein, even comparatively mild parasitism can significantly alter responses of biomarkers which are commonly used to assess oxidative stress, health, and condition of wild animals in polluted environments. As virtually all free-living organisms harbor one or several parasite species which each can be comprised of a few or up to several hundreds of individuals per host, the current study calls for putting a closer and, in particular, more detailed attention to parasitic organisms in ecotoxicological research.

role of SOD is to remove the superoxide anion radical to form oxygen and hydrogen peroxide.50 Misra and Niyogi28 and Misra et al.51 have also reported induction of SOD activity in rainbow trout hepatocytes by sodium selenite exposure in vitro. The decrease in SOD activity after day 35 can be related to lower GST activity and concomitant depletion of cellular GSH levels. Reduction of Se-Met to CH3Se− (which produces O2•−) requires two molecules of GSH,26 and thus it is possible that GSH depletion contributed to a decrease in O2•− production, and subsequently to reduced SOD activity due to substrate limitation. Similar relationship between GSH and SOD activity was also observed by Misra and Niyogi28 in vitro. In addition, in the present experiment, parasitized trout exposed to Se-Met revealed significantly lower SOD activity than the Se-Met group fish. This difference in enzyme activity persisted throughout the entire exposure period. As SOD is directly required for dismutation of O2•−, parasitism-linked reduction of its activity suggests a decreased capacity of infected trout to prevent cellular damage produced by ROS associated with Se-Met exposure. Put into a field setting, infected fish inhabiting polluted waters may, therefore, be at greater risk of oxidative damage than their levels of SOD activity suggest. Limited data is available about interactions between parasitic infection and antioxidant enzyme production in fish from polluted environments. Research has shown that responses to multiple stressors (e.g., parasitism and pollution) vary between hosts, parasites, types of chemical stressor, tissues, and other factors. For example, Marcogliese et al.52 reported the number of Apophallus brevis, a digenean metacercaria that causes blackspot disease in fish, under polluted conditions to be negatively correlated with the activity of glutathione reductase in the gills of yellow perch (Perca f lavescens), while the activity of CAT in head kidney was positively correlated with the number of eyeflukes (Diplostomum spp.). Furthermore, Dautremepuits et al.6 measured higher GST activity in carp (Cyprinus carpio) exposed to copper and parasitized by the cestode Ptychobothrium sp. compared with exposed but uninfected fish. Therefore, as emphasized by Marcogliese and Pietrock,1 due to different effects that different parasite species may have on (oxidative) stress response of their hosts under polluted conditions, it is essential not to treat all parasite species as equals which is the case when different parasite taxa are combined as a “total parasite load” in testing for effects of parasitism. A representative example is the study about parasitism and health biomarkers in bullfrogs (Rana catesbeiana) sampled along a gradient of agriculturally impacted land where it was found that certain parasites had effects on selected biomarkers while total parasite loads did not.53 More importantly, potential influences of parasitism on host responses should not be ignored at all. Effects of Stressors on General Fish Health Parameters. Exposure to Se-Met did not alter K and HSI values of the fish, suggesting that consuming Se-Met contaminated food with a concentration of 8.47 mg Se/kg DW does not impact these two biomarkers of general condition and liver energy accumulation. Experimental work on trout exposed to waterborne selenite25 and zebrafish (Danio rerio) fed Se-Met enriched food, respectively,54 yielded similar results and showed no alteration in K and HSI values either. Field evidence, however, is contradictory: Kelly and Janz55 have found no significant differences in K and HSI between northern pike (Esox lucius) from Se-contaminated and clean lakes. Likewise, no difference in K but reduced HSI was recorded in juvenile

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ASSOCIATED CONTENT

S Supporting Information *

Details on preparation of Se spiked food, Se analysis, and determination of enzyme activities. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Phone: 49-(0)3320140616. Fax: 49-(0)33201-40640. F


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Environmental Science & Technology Present Addresses

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Saskatchewan Research Council, 125-15 Innovation Boulevard, Saskatoon, SK, Canada, S7N 2X8. ∥ Institut für Binnenfischerei Potsdam-Sacrow, Im Königswald 2, 14469 Potsdam, Germany. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors thank Noland Henderson (Montreal Lake Cree Nation) for collecting lake whitefish. We also express gratitude to Dr. Xiaofeng Wang and Michael Kautzman and Dr. Jason Raine (all University of Saskatchewan) for their help with chemical analyses and support in the lab. We are also thankful to Larisa Matwee, JJ Kim, and Gary Sun who helped with the fish dissections. The Aquatic Toxicology Research Facility at the Toxicology Centre, University of Saskatchewan, is acknowledged for providing space and support for the husbandry of rainbow trout and the exposure portion of the study. Valuable comments by the anonymous reviewers are greatly appreciated. The project was financially supported by a Natural Sciences and Engineering Research Council of Canada (NSERC) Discovery Grant to M.P. (grant # 371538-2009).

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Intestinal Nematodes Affect Selenium Bioaccumulation, Oxidative

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