Chemosphere 139 (2015) 541–549

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Antioxidant responses of triangle sail mussel Hyriopsis cumingii exposed to harmful algae Microcystis aeruginosa and hypoxia Menghong Hu a,b,d, Fangli Wu a, Mingzhe Yuan a, Qiongzhen Li c, Yedan Gu a, Youji Wang a,b,d,⇑, Qigen Liu a,b,⇑ a

College of Fisheries and Life Science, Shanghai Ocean University, Shanghai 201306, China Key Laboratory of Exploration and Utilization of Aquatic Genetic Resources, Ministry of Education, Shanghai 201306, China Guangxi Academy of Fishery Science, Nanning 530021, China d Department of Integrative Ecophysiology, Alfred-Wegener-Institute Helmholtz Center for Polar and Marine Research, 27570 Bremerhaven, Germany b c

h i g h l i g h t s  The combined effects of toxic cyanobacteria and hypoxia on mussels were determined.  Interactive effects of cyanobacteria and hypoxia on biomarkers were observed.  Toxic algae plays more important role on haemolymph response than that of hypoxia.  Toxic M. aeruginosa or hypoxia exposure history shows latent effects on mussels.

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Article history: Received 22 April 2015 Received in revised form 29 June 2015 Accepted 26 July 2015 Available online 27 August 2015 Keywords: Harmful algae Microcystis aeruginosa Hypoxia Triangle sail mussel Hyriopsis cumingii Antioxidant responses

a b s t r a c t Bloom forming algae and hypoxia are considered to be two main co-occurred stressors associated with eutrophication. The aim of this study was to evaluate the interactive effects of harmful algae Microcystis aeruginosa and hypoxia on an ecologically important mussel species inhabiting lakes and reservoirs, the triangle sail mussel Hyriopsis cumingii, which is generally considered as a biomanagement tool for eutrophication. A set of antioxidant enzymes involved in immune defence mechanisms and detoxification processes, i.e. glutathione-S-transferases (GST), glutathione (GSH), superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPX), lysozyme (LZM) in mussel haemolymph were analyzed during 14 days exposure along with 7 days depuration duration period. GST, GSH, SOD, GPX and LZM were elevated by toxic M. aeruginosa exposure, while CAT activities were inhibited by such exposure. Hypoxia influenced the immune mechanisms through the activation of GSH and GPX, and the inhibition of SOD, CAT, and LZM activities. Meanwhile, some interactive effects of M. aeruginosa, hypoxia and time were observed. Independently of the presence or absence of hypoxia, toxic algal exposure generally increased the five tested enzyme activities of haemolymph, except CAT. Although half of microcystin could be eliminated after 7 days depuration, toxic M. aeruginosa or hypoxia exposure history showed some latent effects on most parameters. These results revealed that toxic algae play an important role on haemolymph parameters alterations and its toxic effects could be affected by hypoxia. Although the microcystin depuration rate of H. cumingii is quick, toxic M. aeruginosa and/or hypoxia exposure history influenced its immunological mechanism recovery. Ó 2015 Elsevier Ltd. All rights reserved.

1. Introduction Eutrophication following enhancement of human activity has increased in frequency, intensity and geographical distribution of ⇑ Corresponding authors at: College of Fisheries and Life Science, Shanghai Ocean University, Shanghai 201306, China. E-mail addresses: [email protected] (Y. Wang), [email protected] (Q. Liu). 0045-6535/Ó 2015 Elsevier Ltd. All rights reserved.

toxic blooms during the last several decades (Derwent et al., 1998; Ibelings et al., 2007). One of the more serious impacts of eutrophication on aquatic ecosystems is the disappearance of submerged macrophytes and the shift to a phytoplankton-dominated state (Korner, 2001). Over the last several decades, many regions throughout the world have experienced harmful algal blooms, which were caused by a variety of microalgal species (Karim et al., 2002; Aguiar et al., 2011). Toxic Microcystis aeruginosa, is a


M. Hu et al. / Chemosphere 139 (2015) 541–549

well-known bloom forming cyanobacteria species, which can produce several types of microcystins (Sabatini et al., 2011). Microcystins are potent inhibitors of serine and threonine phosphatases, and influence intracellular signalling, cell growth, and differentiation processes (Takenaka and Otsu, 1999). Aquatic animal species can accumulate bloom forming algae and their toxins by filtering water containing those harmful aglae for their food, while less amounts are directly absorbed in dissolved form (Burmester et al., 2011). In most cases, eutrophication associated with subsequent harmful algal blooms has been identified as the main cause for hypoxia due to the oxygen depletion for decomposing the dead cells by bacteria (Diaz, 2001). Hypoxia, the condition where dissolved oxygen (DO) levels in water is less than 2.8 mg O2 L 1 (Diaz and Rosenberg, 1995), has been observed in different water bodies around the world during the last several decades (Diaz, 2001). Although it can be a natural phenomenon caused by vertical stratification, such as formation of haloclines and thermoclines (Rosenberg et al., 1991), eutrophication makes hypoxia situations worse and more frequently (Gray et al., 2002; Aguiar et al., 2011). Bloom forming algae and hypoxia are considered to be two main stressors which are associated with eutrophication (Karim et al., 2002; Aguiar et al., 2011). However, very few studies documented the combined effects of the two stressors on aquatic animals. Mussels and their reactions to eutrophication attained research interests because they have a long history as sentinel organisms for contaminant monitoring in aquatic environments (Goldberg et al., 1978). Responses of some mussel species to toxic algal toxins have been previously described (Pflugmacher et al., 1998; Contardo-Jara et al., 2008; Burmester et al., 2011; Vareli et al., 2012; Kwok et al., 2012), but the interactive effects of harmful algae and hypoxia exposure on the triangle sail mussel Hyriopsis cumingii have not been well characterized beyond controlled toxicity bioassays. Condition of haemolymph system is crucial for disease emergence and organism survival, so the evaluation of immunological defense mechanisms can provide important early warning signals of the chronic effects of toxic or environmental stressors and the susceptibility of animals to infectious diseases (Hannam et al., 2009). The main functions of antioxidant and immune systems including superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPX), glutathione-S-transferases (GST), glutathione (GSH) and lysozyme (LZM), which are responsible to eliminate the active oxygen (O2 ) and foreign particles. Antioxidant responses may enhance when the production rate of reactive oxygen species is fast, leading the organism to suffer from oxidative stress (Pan et al., 2006). At present, antioxidant enzyme activities in invertebrate bivalve animals were usually used as biomarkers of oxidative press and damage in order to reflect the health status of the organisms (Solé et al., 1995; Pan et al., 2006; Vareli et al., 2012). The pollutants firstly entered into the gills along the water current from prosopyle, and then reached tissues and organs especially the digestive gland through haemolymph circulation. The changes in the biomarkers of toxicology in the haemolymph can reflect the process of oxidative damage of whole organisms caused by the pollutants and so do the metabolism of the pollutants in the organisms (Pan et al., 2006). Hereby, examining an organism’s antioxidant or defence system may be useful for assessing the chronic, sublethal effects of harmful algae on aquatic organisms. The present study aimed to assess the combined effects of harmful algae M. aeruginosa and hypoxia on the antioxidant responses in a widely distributed freshwater pearl mussel species, triangle sail mussel H. cumingii (Lea 1852) which is generally considered as a bio-management tool species for eutrophication (Fei et al., 2005; Zhang et al., 2007; Hu et al., 2013). Previous studies have shown that harmful algae can induce oxidative stress and change antioxidant capacity (e.g., catalase (CAT), glutathione

(GSH), superoxide dismutase (SOD), glutathione peroxidase (GPX), glutathione-S-transferase (GST), glutathione reductase (GR) and lipid peroxidation (LPO)) in mussels (Manfrin et al., 2012), such as in the blue mussel Mytilus galloprovincialis (Gorbi et al., 2012) and the green mussel Perna virids (Kwok et al., 2012). The tested parameters in this study, i.e. glutathione-Stransferases (GST), glutathione (GSH), superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPX), lysozyme (LZM) were assumed to be sensitive to toxic algae and hypoxia. We hypothesized that toxic M. aeruginosa would stimulate the antioxidant responses, and hypoxia would influence the immune responses of mussels to harmful algae with some interactive effects; and as a bioremediation tool species, the immune conditions of triangle sail mussel H. cumingii could recover rapidly after toxic M. aeruginosa and hypoxia exposure. The present study could contribute to a better assessment of ecotoxicological risk of algal blooms. 2. Materials and methods 2.1. Experimental mussels Two years old triangle sail mussels H. cumingii (95.22 ± 6.35 mm shell length, 89.67 ± 7.13 g wet weight with shell) which did not reach sexual maturity, were collected from Jinhua Weiwang Aquaculture Farm, Zhejiang Province, China. In the present study, the sex ratio of the mussels was unclear because it was almost impossible to determine sex of the mussels by their appearance. The mussels were lively transported to Shanghai Ocean University, Shanghai, China, where the experiment was performed. 2.2. Algal cultures Green microalga Chlorella vulgaris (clone FACHB-8), commonly provided as an aquaculture food for mussels, was bought from Freshwater Algae Culture Collection of Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan, China and was used as a control and a complementary diet for the experiment. C. vulgaris were cultured in 0.45 lm filtered, UV-sterilized and autoclaveated freshwater containing Watanbe medium (Watanabe, 1960) in 100 L closed tanks at 25 °C with a 12:12 h light: dark cycle. Batch cultures of C. vulgaris were harvested usually at a cell density approaching 5–8  106 cells mL 1. Harmful algae M. aeruginosa (clone FACHB-905) was also originally obtained from Freshwater Algae Culture Collection of Institute of Hydrobiology, which is known to produce microcystins (Sabatini et al., 2011). The FACHB-905 strain of M. aeruginosa grows as single cell with a total content of 3.60 lg mg 1 (Gan et al., 2010). This strain was grown in BG11 medium with 0.45 lm filtered, UV-sterilized and autoclaveated freshwater (Stanier et al., 1971), and cultures were maintained at 25 °C with a 12:12 h light: dark cycle in 100 L closed tanks as C. vulgaris. Cells were harvested in stationary phase, usually at a cell density approaching 1  107 cells mL 1. Algal cell densities were determined by counts using haemocytometer under a light microscope. Each cultured strain was used in single cell suspensions to feed the mussels. Cells were harvested during the late exponential growth phase. Algal cells were collected by nylon mesh gentle filtration and resuspended in 0.45 lm filtered freshwater prior to experimentation. 2.3. Experimental design Five-hundred and forty mussels were distributed randomly in twenty-seven 20 L tanks (20 mussels per tank). Before starting

M. Hu et al. / Chemosphere 139 (2015) 541–549

the experiment, mussels were acclimated for one week, fed 27 g C. vulgaris daily (around 1% of their dry tissue weights, above the maintenance requirements of the animals) in a 500 L fibre-glass tank with a filtering system at 25 °C, which can standardize the physiology of mussels (Pan et al., 2006). Acclimation was followed by 14 days of exposure to nine different treatments (3  3 factorial design), using three levels of DO (1.0 ± 0.05 mg O2 L 1 as severe hypoxic, 3.0 ± 0.06 mg O2 L 1 as moderate hypoxic, and 6.0 ± 0.08 mg O2 L 1 as normoxic) and three different diet treatments (see below), with three replicates (i.e., 3 tanks) per treatment. Following this, a depuration period of seven days was applied during which mussels were fed only C. vulgaris at 50.08 ± 1.95 mg L 1 and under 6.0 ± 0.08 mg O2 L 1. DO levels were regulated by digital DO controllers (ColeParmer, Illinois, model No. 01972-00). The experimental diets, which particulate organic matters were adjusted to approximately 50 mg L 1 (50.24 ± 1.73 mg L 1, dry weight), were prepared. The ratios of cell biomass of toxic M. aeruginosa in the three diet treatments were 0%, 50% and 100%. Non-toxic C. vulgaris was used as a supplement to maintain the same algal biomass (dry weight) in the water among the nine treatments, thus avoiding the potential influence of food abundance on the physiological performance of mussels. Water renewal and microalgae additions were performed twice a day (08:00 and 20:00). Five mussels per tank were sampled at the middle and the end of exposure (T = 7, 14 d); and at the end of depuration (T = 21 d). For each replicate, three mussels were sampled for immunological indexes tests, and the other two mussels were used for toxin accumulation analysis per sampling time.


incubation at room temperature. GSH content was determined by a standard curve generated with GSH at different concentrations. SOD activity was measured using the colorimetric assay of xanthine/xanthine oxidase activity (SOD determination kit, FlukaSwitzer-land) at 450 nm using a calibration curve established with commercially available horse radish SOD (Sigma-Aldrich, Germany). CAT activity was measured by the H2O2 decomposition in 0.1 M Tris buffer (pH 8.0) containing 0.5 mM EDTA and 10 mM H2O2 (Beutler, 1975) and expressed as unit (U) where one unit is defined as the amount of enzyme that decompose 1 mM of H2O2 per minute. The enzymatic assay was performed for 1 min at 240 nm at 30 °C using standard curves. GPX activity was assayed by following the rate of NADPH oxidation at 340 nm by the coupled reaction with glutathione reductase (Lawrence and Burk, 1976). The assay mixture included 600 ll buffer (50 mM potassium phosphate, 1 mM EDTA, 1 mM NaN3, pH 7.5), 100 ll 0.2 mM reduced glutathione (GSH), 100 ll 0.1 mM NADPH, 8 ll glutathione reductase and haemolymph sample (20 ll). After 5 min of preincubation (25 °C), the reaction was initiated by adding 100 ll 0.25 mM H2O2. The specific activity was determined using the extinction coefficient of 6.22 mM 1 cm 1. Lysozyme (LZM) was determined as described previously (Luna-Acosta et al., 2010). The lysozyme assay was done in triplicate for each sample and compared to hen egg white lysozyme standards (2.5–20 lg mL 1), in the presence of Micrococcus lysodeikticus (Sigma-Aldrich, France). One unit of lysozyme corresponds to the amount of enzyme that diminishes absorbance at 450 nm by 0.001 per minute at pH 7.0, at 25 °C.

2.4. Haemolymph collection 2.6. Toxin accumulation Haemolymph samples were taken by inserting the pointed end of the 22-gauge needle to the sinus of the posterior adductor muscle after breaching the shell with pincers. 2 mL of haemolymph was collected from each mussel. The samples were transferred into 15 mL centrifuged tubes (Terumo) and held on ice until all extractions were completed. For each replicate, three mussels were sampled and pooled to reduce individual variation and to provide sufficient haemolymph for the assays. Three replicates were prepared for each treatment. Haemolymph samples were centrifuged at 3500g for 10 min at 4 °C in order to separate the cellular (haemocytes) fraction from the plasma. The haemolymph samples were stored at 80 °C before analysis. Haemolymph protein concentrations were determined according to the procedure of Bradford (1976) using a calibration curve with bovine serum albumin (BSA, Sigma Aldrich, Germany) for quantification. Enzyme activities are related to the protein concentration in the sample and expressed as U mg protein 1. All subsequent determinations of samples were done in duplicate. 2.5. Determination of biochemical parameters GST activity was measured by the technique of (Habig and Jakoby, 1981), using 2 mM 1-chloro-2,4-dinitrobenzene (CDNB) and 2 mM reduced glutathione in 0.1 M potassium phosphate buffer, pH 7.0. The absorbance was monitored for 2 min at 340 nm at 30 °C for a better detection in the microplate reader. One unit (U) of GST activity is the amount of enzyme which catalyzes the conjugation of 1 mM of substrate per minute. GSH levels were measured following the Anderson (1985) procedure, with some modifications. One hundred ll haemolymph were acidified with 50 mL of 10% sulfosalicylic acid. After centrifugation at 8000g for 10 min, supernatant (acid-soluble GSH) aliquots were mixed with 6 mM 5,5-dithiobis-(2-nitrobenzoic) acid (DTNB) in 0.143 M sodium sulfate buffer (pH7.5), containing 6.3 m MEDTA. Absorbance at 412 nm was measured after 30 min

Whole soft mussel tissue was dissected for free dissolved microcystin extraction. Mussel tissues were freeze-dried. All the samples were weighed before and after drying. Microcystin extraction was performed as Vareli et al. (2012) described. To assess the effectiveness of the extraction procedure, spiked recovery test was carried out on non intoxicated mussel tissue. The homogenized samples were spiked with 0.8 lg L 1 of microcystin-LR (Beacon Analytical Systems, Inc., Portland, ME, USA). Samples were then extracted and analyzed using ELISA kit (Beacon Analytical Systems, Inc., Portland, ME, USA). Extraction efficiency in whole soft mussel tissue was found to be 91% (0.73 lg L 1 ± 0.03). The microcystin contents (MC) in the samples was measured by the highly sensitive ELISA test kit (0.1 lg microcystin equiv. l 1) with a detection range of 0.1–2 lg microcystin L 1 (Beacon Analytical Systems, Inc., Portland, ME, USA). This assay uses a polyclonal antibody that binds both microcystins and a microcystin–enzyme conjugate. The Beacon Microcystin Plate Kit is not able to differentiate among different microcystin variants. For the ELISA measurements, appropriate volumes of each sample were evaporated to dryness in a vacuum concentrator (Eppendorf Vacuum Concentrator, Model 5301) at low temperatures and the residues were then dissolved in 100 mL of milliQ water. Total microcystin contents (lg) per the test samples (g) were obtained. 2.7. Statistical analyses Statistical analysis was performed using the software SPSS version 16.0 (SPSS Inc., Chicago, IL, USA). The data of the tested parameters (MC, GST, GSH, SOD, CAT, GPX and LZM) were expressed as mean ± SD. Prior to statistical analysis, results were initially tested for normality (Shapiro–Wilk’s test on residues with 1% risk) and equality of variance (Levene’s test, 5% risk). When needed, data were transformed prior to analysis to meet homoscedasticity and normality assumptions. GST data were log transformed.


M. Hu et al. / Chemosphere 139 (2015) 541–549

Three-way ANOVA was used to analyze DO, harmful algae and time course effects on MC, GST, GSH, SOD, CAT, GPX, LZM. Tukey’s honestly significant difference (HSD) post multiple range tests were used to compare treatment means in cases where factors did not significantly interact. When interaction between or among factors was significant, the influence of each factor on treatment means was tested at fixed levels of the other factor. The existence and

strength of relationships between immune parameters were determined by parametric Pearson’s correlation analysis. Principal component analysis (PCA) was performed to discriminate the different treatments. Both of Pearson’s correlation analysis and PCA were conducted by XLSTATÒ2014 (Addinsoft Inc., New York, NY, USA). Differences were considered significant at P < 0.05. 3. Results


Degrees of freedom

Mean square




2 2 2 4 4 4 8

640.028 8741.348 815.814 260.570 30.690 502.165 29.179

896.615 12250.000 1143.000 365.033 42.993 703.483 40.877

Antioxidant responses of triangle sail mussel Hyriopsis cumingii exposed to harmful algae Microcystis aeruginosa and hypoxia.

Bloom forming algae and hypoxia are considered to be two main co-occurred stressors associated with eutrophication. The aim of this study was to evalu...
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