Immune responses to combined effect of hypoxia and high temperature in the green-lipped mussel Perna viridis

Marine Pollution Bulletin 63 (2011) 201–208

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Marine Pollution Bulletin journal homepage: www.elsevier.com/locate/marpolbul

Immune responses to combined effect of hypoxia and high temperature in the green-lipped mussel Perna viridis Youji Wang a, Menghong Hu a, Paul K.S. Shin a,b, Siu Gin Cheung a,b,⇑ a b

Department of Biology and Chemistry, City University of Hong Kong, Tat Chee Avenue, Kowloon, Hong Kong State Key Laboratory in Marine Pollution, City University of Hong Kong, Tat Chee Avenue, Kowloon, Hong Kong

a r t i c l e Keywords: Hypoxia Temperature Perna viridis Immune response Biomarker

i n f o

a b s t r a c t Flow cytometry was used to examine immune responses in haemocytes of the green-lipped mussel Perna viridis under six combinations of oxygen level (1.5 mg O2 l1, 6.0 mg O2 l1) and temperature (20 °C, 25 °C and 30 °C) at 24 h, 48 h, 96 h and 168 h. The mussels were then transferred to normoxic condition (6.0 mg O2 l1) at 20 °C for further 24 h to study their recovery from the combined hypoxic and temperature stress. Esterase (Est), reactive oxygen species (ROS), lysosome content (Lyso) and phagocytosis (Pha) were reduced at high temperatures, whereas hypoxia resulted in higher haemocyte mortality (HM) and reduced phagocytosis. For HM and Pha, changes were observed after being exposed to the stresses for 96 h, whereas only a 24 h period was required for ROS and Lyso, and a 48 h one for Est. Recovery from the stresses was observed for HM and Pha but not other immune responses. Ó 2011 Elsevier Ltd. All rights reserved.

1. Introduction Hypoxia, the condition where dissolved oxygen (DO) levels in water is less than 2.8 mg O2 l1 (Diaz and Rosenberg, 1995), has been observed in numerous estuarine and coastal ecosystems around the world; today, over 400 such areas in the world are known as dead zones, covering more than 245 000 km2 of sea bottom (Diaz and Rosenberg, 2008). In most cases, eutrophication caused by excessive anthropogenic input of nutrients and organic matter into water bodies with poor circulation has been identified as the main cause for hypoxia (Diaz, 2001), although it can be a natural phenomenon caused by vertical stratification, such as formation of haloclines and thermoclines (Rosenberg et al., 1991). Major ecological impacts include mass mortality of marine animals, alteration of the benthic community and decline in aquaculture production (Diaz and Rosenberg, 1995). More and more scientific evidence has been accumulated showing that water temperature has risen during the past 15–20 years (MacKenzie and Schiedek, 2007), likely with negative consequences on the marine ecosystem (Conley et al., 2009). The ability to adapt to new temperature regimes, and variations amongst aquatic species in their present thermal tolerance limits, will be strong determining factors in the success of populations to meet the stress of rising temperature (Pörtner and Knust, 2007). In this ⇑ Corresponding author at: Department of Biology and Chemistry, City University of Hong Kong, Tat Chee Avenue, Kowloon, Hong Kong. Tel.: +852 27887749; fax: +852 27887406. E-mail address: [email protected] (S.G. Cheung). 0025-326X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.marpolbul.2011.05.035

regard, recent warming already exceeds the physiological ability of some local species to adapt, which consequently may lead to major changes in the structure, function and services of ecosystems. Additionally, high water temperatures reduce oxygen solubility and increase biological oxygen demand, often producing extremely hypoxic, or even anoxic, conditions. Temperature increases also enhance stratification, thereby reducing the supply of oxygen to bottom waters by vertical mixing, particularly in the summer period. The internal defence of bivalves against diseases, parasites and environmental stresses consists of cellular and humoral immunity. Cellular defence involves haemocytosis, phagocytosis, encapsulation and nacresation (Gosling, 2003). During phagocytosis, reactive oxygen species (ROS), such as hydrogen peroxide (H2O2), hydroxyl radical (OH) and superoxide anion (O2), are secreted. This process is known as respiratory burst. For humoral defence, biologically active molecules are secreted by haemocytes or other cells into the haemolymph. For example, hydrolytic lysozyme, in lysosomes, is shown to exert microbiocidal effects on bacterial membranes (Cheng, 1996). Esterase is responsible for the hydrolysis of a number of choline esters, including acetylcholine, playing an important role in immunological defence in mussels (Pretti and CognettiVarriale, 2001). Changes in temperature and DO have been reported to affect immune responses in bivalves, including circulating haemocytes, antioxidase activity, respiratory burst and phagocytosis (Fisher et al., 1987; Pampanin et al., 2002; Cheng et al., 2004a, b; Monari et al., 2007). However, all of these studies only focused on the single effects of either DO or temperature; yet, environmental stresses

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rarely occur in isolation. For example, hypoxia may exacerbate under high temperatures. When stresses occur concurrently, their combined effects may be different from those of individual stresses, as combined effects may be additive, synergistic or antagonistic. In view of this, Kidwell et al. (2009) proposed the importance and necessity of quantification of the interactive effect of hypoxia with other environmental stressors. The green-lipped mussel Perna viridis is widely distributed in the Indo-Pacific region (Siddall, 1980). It is extensively cultured as a protein source in S.E. Asia (Chalermwat and Lutz, 1989) and an important bio-indicator for pollution monitoring (Richardson et al., 2008). In Hong Kong, P. viridis is predominant in sheltered harbours, where seawater experiences extensive hypoxia (Environmental Protection Department, 2008) and high temperatures in summer (Thiyagarajan and Qian, 2003). The aim of this study was to assess the combined effects of temperature and hypoxia on immune defences in P. viridis. Mussels were exposed to hypoxia for a period of 168 h under different temperatures (20 °C, 25 °C and 30 °C) and allowed to recover under normoxia at 20 °C for 24 h. Haemocyte mortality (HM), phagocytosis (Pha), activity of esterase (Est), reactive oxygen species (ROS) and lysosomal content (Lyso) in the haemolymph were evaluated using flow cytometry to assess if these immune parameters can be regarded as biomarkers of temperature and hypoxic stresses.

2. Materials and methods

2.3. Haemolymph collection Haemolymph was collected using a syringe (1 ml) equipped with a needle (0.75  38 mm) from the sinus of the posterior adductor muscle after breaching the shell with pincers. 1.0– 1.5 ml of haemolymph was collected from each mussel. The haemolymph was stored on ice until processed to reduce spontaneous aggregation (Chen et al., 2007a). For each treatment, six mussels were sampled and pooled to reduce individual variation and to provide sufficient haemocytes for the assays. Three replicates were prepared for each treatment. 2.4. Cell analysis by flow cytometry A BD FACSCalibur flow cytometer (BD Biosciences) with an air cooled argon laser, providing an excitation at 488 nm, was used. A FSC threshold was defined in order to eliminate cell debris and bacteria. Data were depicted as cell cytograms indicating the relative size (FSC value), the granularity (SSC value) and the fluorescence channels corresponded to the fluorescent marker used. A total of 20 000 events were acquired for each measurement and stored in the list mode data format. The fluorescence frequency distribution histogram of the haemocyte population was subsequently obtained. The type of fluorescence recorded depended on the parameter monitored: phagocytosis, esterase, ROS and lysosomal content were measured at FL1, and haemocyte mortality was evaluated at FL2. Data were analysed using BD CellQuest™ Pro software (BD Biosciences, USA).

2.1. Experimental animals 2.5. Parameter measurements Adult mussels (shell length: 79.5 ± 7.5 mm; wet weight: 31.2 ± 8.6 mg) were collected from a sheltered bay in Yung Shue O, Hong Kong. Upon returning to the laboratory, they were maintained in a fibre-glass tank (500 L) equipped with a filtering system and air supply and fed with the brown alga Thalassiosira pseudonana (concentration: 5.0  105 cells ml1) once every 2 days. The seawater was maintained at 20 °C and 30‰ during acclimation, and individuals of P. viridis were allowed to acclimate to laboratory conditions for one week prior to experimentation.

2.2. Experimental design To examine the combined effects of hypoxia and temperature, the experiment was set up in a factorial design, using two levels of dissolved oxygen (1.57 ± 0.08 mg l1 as hypoxic and 6.26 ± 0.22 mg l1 as normoxic) and three levels of temperature (20.22 ± 0.03 °C, 25.16 ± 0.03 °C and 30.30 ± 0.01 °C) for a total of six treatments. The ranges of DO and water temperature selected were within the ranges experienced by P. viridis in Hong Kong waters in summer (Wu, 1982; Thiyagarajan and Qian, 2003). Each treatment consisted of three replicates with 48 mussels per replicate (aquarium 20 L). The system, which was adopted from Wang et al. (2010), comprised an experimental tank, a digital DO controller (ColeParmer, Illinois, model No. 01972-00), a cylinder of compressed nitrogen and an air pump. The DO level in each experimental tank was monitored automatically by the oxygen probe of the controller. When the desired DO level deviated from the pre-set value, the DO controller would send a signal to the valves connecting to the nitrogen gas tank or air pump so as to restore the desired DO level by delivering either nitrogen or air into the experimental tank. The mussels were exposed to the above conditions for 168 h, followed by a recovery period of 24 h under 6.26 mg O2 l1 and 20.22 °C. All immune parameters were evaluated at 0 h, 24 h, 48 h, 96 h, 168 h and 196 h (with a 24 h recovery period). Dead mussels were removed as encountered.

2.5.1. Haemocyte mortality Haemocyte mortality (HM) was quantified using 400 ll of haemolymph according to a modified procedure of Delaporte et al. (2003). Haemocytes were incubated in the dark for 30 min before a flow cytometer analysis at 4 °C with 10 ll of propidium iodide (PI, 1.0 mg ml1, Sigma Aldrich) for a final concentration of 50 lg ml1. Percentage of haemocyte mortality was calculated as the percentage of haemocytes showing PI fluorescence relative to total haemocyte counts. 2.5.2. Phagocytosis Phagocytosis (Pha) was measured in vitro as the proportion of cells that had ingested three or more fluorescent beads (Xue et al., 2001; Gagnaire et al., 2003). 400 ll of haemolymph were incubated for 1 h in the dark at an ambient temperature with 10 ll of a 1/10 dilution of FluorospheresÒ carboxylate-modified microspheres (diameter 1.0 lm, yellow–green fluorescent, Invitrogen). The final concentration of beads was 108 beads ml1 and the final ratio of beads to haemocytes was 100:1. 2.5.3. Esterase Esterase (Est) activity was evaluated using Fluorescein Diacetate (FDA, Sigma). Initial FDA stock solutions were prepared by dissolving FDA in dimethyl sulphoxide (DMSO) to a concentration of 0.04 mM and stored at 20 °C. A working solution of FDA (400 lM) was prepared by diluting (1/10) the FDA stock solution using filtered sterile seawater. 400 ll of haemolymph were incubated with 2 ll of FDA solution in the dark at ambient temperature for 15 min. Percentage of cells presenting the enzymatic activity was defined on the basis of fluorescent cells amongst all cells (Gagnaire et al., 2008). 2.5.4. Reactive oxygen species (ROS) ROS was evaluated using 20 70 -dichlorofluorescein diacetate (DCFH-DA, Sigma). The enzymatic activity was defined on the basis

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of fluorescent cells amongst all cells. A 10 mM DCFH-DA stock solution was prepared in DMSO and then diluted to 10% in filtered sterile seawater as working solution. Each analysis required 400 ll of haemolymph and 4 ll of DCFH-DA working solution. Haemocytes were incubated in the dark, at ambient temperature, for 15 min for ROS detection (Delaporte et al., 2003). 2.5.5. Lysosomal content The lysosomal content (defined as the relative amount of LysoTracker fluorescence observed) was determined using a commercial kit (LysoTrackerÒ Yellow HCK-123, 1 mM in DMSO, Invitrogen). 1 ll of a LysoTracker aliquot was added to 400 ll haemolymph. Cells were then incubated for 2 h for lysosome presence. Incubations took place in the dark, at room temperature, for 2 h and the reaction was stopped using ice (Gagnaire et al., 2008). Mean intensity of LysoTracker fluorescence exhibited by all the haemocytes was expressed in arbitrary units (A.U.). 2.6. Statistical analysis Each immune parameter was measured three times to get an average value. Prior to the analysis, normality of the data was evaluated by using the Shapiro–Wilk’s W test and homogeneity of variances, checked by Levene’s test using the statistical software SPSS 16.0. Percentage data were arcsine transformed. The effects of DO, temperature and their interactions were analysed using two-way analysis of variance (ANOVA). When an interaction was observed, significant effects of temperature were analysed with one way ANOVA at each fixed DO level, and Tukey’s HSD post hoc multiple range tests were performed to determine which treatments were different. Similarly, significant effects of DO at each time point were determined for each temperature using a Student t test. 3. Results At the end of the experiment, cumulative mortality of P. viridis was significantly affected by DO and temperature, but not by the interaction between DO and temperature (Fig. 1). The mortality rate at 30 °C was significantly higher than that at 20 °C and 25 °C (p < 0.001), but there was no significant difference between 25 °C and 20 °C. The mortality at DO 1.5 mg l1 was significantly higher than that under DO 6.0 mg l1 (t = 4.861, p = 0.001). All immune parameters from all treatments showed no significant differences at 0 h (Table 1). Table 1 shows the effects of DO and temperature on the immune responses of P. viridis at 24 h, 48 h, 96 h, 168 h and 192 h (recovery), respectively. Haemocyte mortality (HM) was relatively insensitive to temperature, except at 48 h (Table 1). The effect of DO, however, was

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significant at the latter part of the experiment at 96 h and 168 h, with higher mortality being recorded under hypoxia (Fig. 2). The interaction between DO and temperature was also significant at 96 h, at which HM was significantly higher at 1.5 mg O2 l1 than at 6.0 mg O2 l1, at both 20 °C (t = 4.32, p < 0.05) and 25 °C (t = 15.01, p < 0.005). During recovery, the effect of DO and temperature on HM became insignificant. Phagocytosis (Pha) was affected by DO, temperature and the interaction between DO and temperature at 168 h (Table 1). Pha at 20 °C was significantly higher than that at 25 °C and 30 °C under both DO levels (1.5 mg O2 l1: F = 49.456, p < 0.001; 6.0 mg O2 l1: F = 137.182, p < 0.001) (Fig. 3). Pha at 6.0 mg O2 l1 was significantly higher than that at 1.5 mg l1 at 20 °C (t = 6.59, p = 0.022, Fig. 3). The effect of DO and temperature on Pha was transient, with full resumption of phagocytic activity within the 24 h recovery period. Esterase (Est) was relatively independent of DO (except at 168 h), but was significantly reduced at higher temperatures starting at 48 h (Fig. 4). The interaction between DO and temperature was insignificant. No recovery from high temperature stress was observed during the recovery period (Table 1). ROS was relatively independent of DO (except at 96 h, when ROS at 6.0 mg O2 l1was higher than at 1.5 mg O2 l1), but was significantly reduced at higher temperatures starting at 24 h (Fig. 5). The interaction between DO and temperature was insignificant, except at 96 h. No recovery from high temperature stress was observed during the recovery period (Table 1). Lysosomal content (Lyso) was significantly affected by DO, temperature and the interaction between DO and temperature (except for DO at 24 h and 192 h) (Table 1). In general, higher values of Lyso were obtained at lower temperatures except for 1.5 mg O2 l1 at 24 h and 6.0 mg O2 l1 at 96 h, as well as during recovery (Fig. 6). At 48 h, Lyso at 1.5 mg O2 l1 was significantly lower than that at 6.0 mg O2 l1 at 20 °C (t = 19.894, P < 0.05) but the value at 6.0 mg O2 l1 was lower than at 1.5 mg O2 l1 at 30 °C (t = 15.108, p < 0.05). At 96 h, Lyso at 1.5 mg O2 l1 was significantly higher than that at 6.0 mg O2 l1 at both 20 °C (t = 19.19, p < 0.05) and 25 °C (t = 16.36, p < 0.05). At 168 h, Lyso at 6.0 mg O2 l1 was significantly higher than that at 1.5 mg O2 l1 at 20 °C (t = 18.273, p < 0.05). 4. Discussion The present study has demonstrated a higher mortality of P. viridis under hypoxia and high temperature. Mass mortality of P. viridis was observed in summer in Hong Kong, especially in sheltered and eutrophic harbours. This was suggested to be a result of aerial exposure at midday when air temperature can reach 30 °C or above

Fig. 1. Mortality of P. viridis exposed to six combinations of DO (1.5 mg O2 l1, 6.0 mg O2 l1) and temperature (20 °C, 25 °C, 30 °C) with two-way ANOVA results.

28.305 1.553 0.269 17.777 50.751 <0.001 27.972 43.73 <0.001 353.857 103.482 <0.001 12.411 11.89 <0.01 137.664 83.448 <0.001

(Cheung, 1993). These harbours, however, experienced hypoxia in summer and resulted in defaunation of the benthic community (Wu, 1982). The findings of the present study, therefore, have highlighted the possibility of hypoxia and high water temperature as additional stresses contributing to the summer mortality rate. Mortality of the clam Chamelea gallina was also higher in summer in Italy (Monari et al., 2007). Increases in mortality in bivalves with increases in temperature are probably owing to temperature-induced immunosuppression in these animals (Ford and Tripp, 1996). Although bivalves are more resistant to hypoxia, as compared with fish and other invertebrates (Gray et al., 2002), resistance to hypoxia in bivalves is species specific (de Zwaan et al., 1992). Bivalves generally can defy mortality for short time periods at low DO but are vulnerable to prolonged oxygen deficiencies (de Zwaan, 1977; Chen et al., 2007b).

3.761 0.206 0.818 80.542 229.938 <0.001 97.561 152.526 <0.001 127.797 37.373 <0.001 125.751 120.473 <0.001 196.655 119.206 <0.001 3.774 0.207 0.661 2.012 5.743 0.054 20.654 3.23E + 01 <0.001 1267.113 370.555 <0.001 40.309 38.617 <0.001 6.695 4.058 0.091 0.007 0.051 0.95 0.024 2.553 0.119 0.008 0.616 0.556 0.033 4.451 <0.05 0.015 1.005 0.395 0.018 1.182 0.34

Lyso

DO 1 DO  T 2

T 2

DO  T 2

Y. Wang et al. / Marine Pollution Bulletin 63 (2011) 201–208

0.035 0.258 0.777 0.302 32.66 <0.001 0.372 27.91 <0.001 0.223 30.041 <0.001 0.254 16.551 <0.001 0.103 6.814 <0.05

4.2. Pha

0.006 0.044 0.838 0.026 0.311 0.588 0.026 3.074 0.105 0.014 3.191 0.099 0.036 6.982 <0.05 0.004 0.34 0.571 7.69E-05 0.002 0.998 0.027 2.179 0.156 0.001 0.079 0.925 1.85E-05 0.005 0.995 0.024 186.482 <0.001 0.001 0.134 0.876

0.001 0.038 0.963 0.002 0.194 0.826 0.002 0.228 0.799 0.008 2.389 0.134 0.005 38.488 <0.001 0 0.045 0.956

0.012 0.081 0.923 0.099 1.175 0.345 0.338 40.592 <0.001 0.26 59.145 <0.001 0.402 76.991 <0.001 0.081 7.28 <0.01

0.037 0.254 0.78 0.005 0.054 0.947 0.013 1.592 0.244 0.014 3.247 0.075 0.006 1.063 0.376 0.031 2.811 0.1

0.017 0.124 0.731 0.015 1.633 0.226 0 0.022 0.884 0.049 6.662 <0.05 0.035 2.291 0.156 0.023 1.517 0.242

The low HM values (less than 5%) obtained in all treatment groups in this study agreed with previous studies on bivalves, such as the Eastern (Atlantic) oyster Crassostrea virginica (Ashton-Alcox and Ford, 1998) and Pacific oyster Crassostrea gigas (Delaporte et al., 2003). It is because bivalves are characterised by having the debris of dead cells effectively removed by surviving haemocytes instead of having them accumulate in the haemolymph (Hégaret et al., 2003). HM, however, was enhanced under hypoxic stress, probably owing to cytolysis, induced by oxidative inhibition of membrane Na+–K+ ATPase activity (Li and Jackson, 2002), and a reduction in the capacity for surviving haemocytes to remove the dead cells. Similar results were obtained for the venus clams C. gallina (Pampanin et al., 2002) and the scallop Chlamys farreri (Chen et al., 2007a). HM in P. viridis was relatively insensitive to temperature except at 48 h, when HM increased with temperature. An increase of haemocyte mortality at elevated temperatures was also observed in the Eastern oyster C. virginica (Hégaret et al., 2003).

192 h (recovery)

168 h

96 h

48 h

24 h

The bold values are those of which the P values are statistically significant.

0.001 0.032 0.861 0.004 0.351 0.565 0.003 0.374 0.552 0.011 3.152 0.101 0.002 17.462 0.001 0.001 0.07 0.796 5.37E-05 1.046 0.381 3.01E-05 0.489 0.625 5.20E-05 1.922 0.189 0 11.173 <0.005 6.31E-05 2.919 0.093 2.43E-06 0.249 0.783 1.19E-05 0.231 0.797 5.11E-06 0.083 0.921 0 10.835 <0.005 4.17E-05 3.033 0.086 2.80E-05 1.295 0.309 2.85E-05 2.921 0.093 1.3E-05 0.252 0.625 5.80E-05 0.942 0.351 2.54E-05 0.939 0.352 0 31.99 <0.001 0 17.555 0.001 1.87E-07 0.019 0.892

DO 1 df

MS F P MS F P MS F P MS F P MS F P MS F P 0h

ROS Est

DO 1

Pha

T 2

DO  T 2 HM

DO 1

T 2

DO  T 2

T 2

DO  T 2

DO 1

T 2

4.1. Hm

Source

Table 1 Summary of two-way ANOVA results on effects of dissolved oxygen (DO) and temperature (T) on haemocyte mortality (HM), phagocytosis (Pha), esterase (Est), reactive oxygen species (ROS) and lysosome (Lyso). Dissolved oxygen: 1.5 and 6.0 mg O2 l1; temperature: 20 °C, 25 °C and 30 °C.

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Although Pha in P. viridis was lower upon exposure to hypoxic and/or high temperature stress, such responses were only observed after the mussels were exposed to the stressful conditions for 168 h. Similar responses to temperature stress were also observed in other bivalves such as C. virginica (Hégaret et al., 2003) and C. gigas (Gagnaire et al., 2006). Contrasting results with Pha being enhanced at high temperatures, however, were found in the Eastern oyster, C. virginica (Foley and Cheng, 1975) and European flat oyster Ostrea edulis (Fisher et al., 1987). Pha is considered as one of the sensitive immunomarkers to hypoxic stress (Pampanin et al., 2002), with Pha being reduced under hypoxic stress (Matozzo et al., 2005). Several hypotheses have been proposed to explain changes in Pha in response to hypoxic stress. Firstly, it has been demonstrated that bivalves possess a primitive form of a catecholaminergic stress-response system involving noradrenaline and dopamine (Lacoste et al., 2001a). Noradrenaline, one of the most important catecholamines in the mollusc haemocyte released into haemolymph during stress, has a dose-dependent inhibition on Pha of bivalve haemocytes via b-adrenergic/cAMP signalling pathway (Lacoste et al., 2001b). This dose-dependent inhibition effect of noradrenaline under stress may explain the significant difference in the Pha observed during aerial exposure stress (Lacoste et al., 2001a). Secondly, Pha of bivalve haemocytes is also modulated by specific membrane receptors, such as integrins for transmission of suitable signals into the cytoplasm, which may be altered under hypoxic stress (Terahara et al., 2006).

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Fig. 2. Haemocyte mortality (HM) of P. viridis exposed to six combinations of DO (1.5 mg O2 l1, 6.0 mg O2 l1) and temperature (20 °C, 25 °C, 30 °C).

Fig. 3. Phagocytosis (Pha) of P. viridis exposed to six combinations of DO (1.5 mg O2 l1, 6.0 mg O2 l1) and temperature (20 °C, 25 °C, 30 °C).

Fig. 4. Esterase (Est) of P. viridis exposed to six combinations of DO (1.5 mg O2 l1, 6.0 mg O2 l1) and temperature (20 °C, 25 °C, 30 °C).

4.3. Est Est was sensitive to high temperature stress, with a reduction in activity occurring after P. viridis were exposed to the stress for 48 h. No recovery, however, was observed after P. viridis were returned to the normal condition. In contrast, Est was relatively insensitive to hypoxia. Esterase activity in mussels was inhibited by neuro-

toxic compounds, such as organophospate, carbamate pesticides (Ozretica and Krajnovic Ozretic, 1992; Tsangaris et al., 2008) and heavy metals (Najimi et al., 1997). Therefore, Est is considered an useful biomarker of pollutants. These results, however, indicated that caution should be taken as temperature may be a confounding factor in long-term monitoring programs for contaminants, e.g., Mussel Watch Program.

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Fig. 5. Reactive oxygen species (ROS) of P. viridis exposed to six combinations of DO (1.5 mg O2 l1, 6.0 mg O2 l1) and temperature (20 °C, 25 °C, 30 °C).

4.4. Ros Respiratory burst, the release of reactive oxygen species (ROS) by haemocytes, is a critical step in the innate immune response. It is an important mechanism by which potential pathogens and parasites are eliminated following phagocytosis in bivalves (Bayne 1990; Pipe, 1992). As P. viridis under high temperatures suffered from higher mortality and lower metabolism, probably some enzymes which are responsible for producing ROS are inhibited (Lushchak 2011) and, hence, resulted in lower ROS production. The effect of DO on ROS, however, was mixed. Many authors have pointed out that, exposure to environmental hypoxia reduces the ability of mollusc haemocytes to produce ROS (Boyd and Burnett, 1999; Cheng et al., 2004b), suggesting a kind of mechanism for HIF-induced metabolic adjustments (Michiels et al., 2002), this was also the case in the present study. The effect of DO was only observed at 96 h in P. viridis, with a higher ROS being obtained at a higher DO. This agreed with the general understanding that ROS increases with oxygen availability and metabolic rate (Ross et al., 2001). It is known that ROS can be produced by incomplete reduction of molecular oxygen on mitochondria electron transport chain (Chandel and Schumacker, 2000). Although progress has been made in understanding the transcriptional mechanisms activated to deal with hypoxia, but the underlying mechanism of oxygen sensing needed for this activation is not well understood (Wu 2002). 4.5. Lyso Lysosomes are cellular organelles. They contain hydrolytic enzymes, including lysozymes, for intracellular degradation and host

defence (Olsen et al., 2003). Lysosomal content decreased when the softshell clam Mya arenaria was infected by the bacterium Vibrio splendidus (LGP32-GFP) and may be caused by degranulation of actively phagocytosing cells (Mateo et al., 2009). In general, values of Lyso were reduced at higher temperatures in P. viridis. This indicated that immunity was lowered under temperature stress. The relationship between DO and lysosomal content, however, was weak. No study has been reported upon the effect of environmental stress on lysosomal content but lysosomes were found to accumulate a diverse range of chemical contaminants, which can lead to membrane damage resulting in leakage of their contents into the cytosol and damage to cells (Lowe and Fossato, 2000). 4.6. Recovery The rate of recovery from hypoxic and/or temperature stress varied amongst the immune responses in P. viridis. In fact, there seemed to be a correlation between sensitivity to stress and the rate of recovery. Immune responses which were less sensitive to the stresses had the observable effects being found later in the experiment (96 h for HM under temperature stress and 168 h under hypoxic and temperature stress for Pha). Their rates of recovery, however, were faster and occurred within 24 h. In contrast, Est, Per and Lyso were much more sensitive to the stresses, with observable effects occurring 24 h after being exposed to the stresses for Per and Lyso, 48 h for Est and no recovery effects were observed until the end of the recovery period. Pha and haemocytic infiltration are the primary cellular responses in bivalves, serving as the first line of defence against foreign substances, while a multitude of haemolymph factors, such as enzymes, constitutes the

Fig. 6. Lysosomal content of P. viridis exposed to six combinations of DO (1.5 mg O2 l1, 6.0 mg O2 l1) and temperature (20 °C, 25 °C, 30 °C).

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