Toxic Dinoflagellate Alexandrium tamarense Induces Oxidative Stress and Apoptosis in Hepatopancreas of Shrimp (Fenneropenaeus chinensis)

LIANG Zhongxiu, LI Jian,, LI Jitao, TAN Zhijun, REN Hai and ZHAO Fazhen

1) College of Fisheries and Life Science, Shanghai Ocean University, Shanghai 201306, P.R. China

2) Key Laboratory of Sustainable Development of Marine Fisheries of Ministry of Agriculture; Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Qingdao 266071, P. R. China

Toxic Dinoflagellate Alexandrium tamarense Induces Oxidative Stress and Apoptosis in Hepatopancreas of Shrimp (Fenneropenaeus chinensis)

LIANG Zhongxiu1),2), LI Jian2),*, LI Jitao2), TAN Zhijun2), REN Hai1), and ZHAO Fazhen2)

1) College of Fisheries and Life Science, Shanghai Ocean University, Shanghai 201306, P.R. China

2) Key Laboratory of Sustainable Development of Marine Fisheries of Ministry of Agriculture; Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Qingdao 266071, P. R. China

This study investigated the inductive effect of Alexandrium tamarense, a toxic dinoflagellate producing paralytic shellfish poison, on oxidative stress and apoptosis in hepatopancreas of Chinese shrimp, Fenneropenaeus chinensis. The individuals of F. chinensis were exposed to 200 and 1000 cells mL-1of A. tamarense with their superoxide dismutase (SOD), glutathione S-transferase (GST) activities, malonyldialdehyde (MDA) concentration, and caspase gene (FcCasp) expression in hepatopancreas determined at 12, 24, 48, 72 and 96 h. In addition, apoptosis in hepatopancreas of F. chinensis at 96 h after exposure was determined through terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay. The hepatopancreatic SOD and GST activities of F. chinensis exposed to 1000 cells mL-1of A. tamarense showed a bell-shaped response to exposure time. The hepatopancreatic MDA concentration of F. chinensis exposed to 1000 cells mL-1of A. tamarense increased gradually from 48 to 96 h, and such a trend corresponded to the decrease of GST activity. The hepatopancreatic FcCasp transcript abundance of F. chinensis exposed to 1000 cells mL-1of A. tamarense was positively and linearly correlated to MDA concentration. Results of TUNEL assay showed that exposure to 1000 cells mL-1of A. tamarense induced apoptosis in the hepatopancreas of F. chinensis. Our study revealed that A. tamarense exposure influenced the antioxidative status of F. chinensis and caused lipid peroxidation and apoptosis in the hepatopancreas of shrimp.

Alexandrium tamarense; oxidative stress; apoptosis; Fenneropenaeus chinensis

1 Introduction

Harmful algal blooms can damage shrimp due to their oxygen consumption and toxin production (Alonso-Rodríguez and Páez-Osuna, 2003). The toxic dinoflagellate Alexandrium tamarense is known to produce paralytic shellfish poisoning toxins (PSTs) and non-PSP toxic substances such as hemolytic toxin (Yan et al., 2003; Tan, 2006). These toxins impose a far-reaching impact on the health of aquatic organisms and humans (García et al., 2004). In natural environments, aquatic organisms are exposed to toxic A. tamarense during bloom formation and decay mainly through the ingestion of Alexandrium cells and/or the bioaccumulation of water-borne toxins (Tan et al., 2007; Costa et al., 2010).

The Chinese shrimp Fenneropenaeus chinensis is widely cultured in China (Wang et al., 2011). This species is ideally suited for culture because of its rapid growth, resistance to low temperature, excellent nutritional properties, and easiness of adaptation to artificial culture conditions (Li et al., 2006). Concomitant with the growth of this industry has been an increase in the importance of looking at diseases, mostly those provoked by infectious agents. Previous research has focused on the most important diseases of cultured crustacean associated with viral, bacterial, and fungal infections. In recent decades, the effects of environmental stress and toxins have gained increasing research attention (Stephan et al., 2005; Wang et al., 2011). In ponds where prawns are cultured, dinoflagellate blooms frequently occur because of the shallow water and high nutrient loading (Su et al., 1993; Lin, 1996). The issue of the potential hazard of A. tamarense to prawns arose in 1989 when Su et al. (1993) suspected that A. tamarense could cause mortality in prawns. In shrimp farms in some areas of China, the observed effects are morbidity and a delay in the growth of shrimp that turns into an economic loss (Zhu, 1993).

Reactive oxygen species (ROSs) and the resulting oxidative stress to living organisms have been reported to be involved in the pollution-mediated mechanism of toxicity, which can damage macromolecules such as DNA and proteins and cell membrane (Skulachev, 1998; Living-stone, 2001). Antioxidant enzymes are able to eliminate highly reactive intermediate ROSs induced by pollutants to maintain cell homeostasis. Superoxide dismutase (SOD) and glutathione S-transferases (GST) are among the major antioxidant enzymes. Malonyldialdehyde (MDA) is an end point of lipid peroxidation (LPO), whose formation is regarded as a general indicator of LPO (Winston, 1991). ROS can be involved in apoptosis which is well conserved in terms of both morphological features and genes controlling the process (Armstrong, 2006; Fleury et al., 2002). Caspases are the critical central molecules of apoptotic pathways (Thornberry, 1998). Previous results indicated that overexpression of caspase gene resulted in apoptotic morphological features in shrimp cells (Leu et al., 2008; Wang et al., 2011).

The impact of toxic strains of A. tamarense on the survival, growth, fecundity and nutritional status of shrimp has been studied (Tan et al., 2002). In addition, the toxic dinoflagellate may induce oxidative stress as shown by enhanced LPO and depressed antioxidant defense system of aquatic organisms (Estrada et al., 2007). However, few studies have dealt with A. tamarense-induced apoptosis via the production of free radicals in F. chinensis.

The aim of this study was to evaluate the toxicity of A. tamarense to F. chinensis. First, A. tamarense-induced oxidative stress to the shrimp was measured through determination of the antioxidant enzyme activities and MDA concentration. Second, A. tamarense-induced apoptosis in hepatopancreas of the shrimp was evaluated by determining FcCasp transcript abundance with terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay. The results will aid to gaining valuable information on shrimp antioxidative system and the physiology of hepatopancreas under A. tamarense stress.

2 Materials and Methods

2.1 Shrimp Culture

Chinese shrimp (F. chinensis), 6.8 cm ± 0.3 cm in length and 3.6 g ± 0.2 g in weight, were collected from Baorong Aquatic Product Technology Co., Ltd. (Qingdao, China). Prior to the experiment, the shrimp were reared in 200-L polyvinyl chloride polymer tanks containing aerated seawater (salinity 30 and temperature 23℃ ± 1℃) for seven consecutive days to acclimate them to laboratory conditions. During acclimation, the shrimp were fed with commercial prawn pellets, three times each day, and seawater was exchanged by 30% every day.

2.2 Algal Preparation

The toxic dinoflagellate A. tamarense ATHK was obtained from Institute of Oceanology, Chinese Academy of Sciences, and cultured in f/2 medium prepared with filtered (0.45 μm) seawater. Strain ATHK was grown in monoalgal culture in 5-L glass flasks on a 14 h:10 h light:dark photocycle at 20℃. Algal cells at exponential growth phase were harvested and used for the experiments. According to Tan et al. (2007), the PSTs produced by strain ATHK were mainly composed of N-sulfocarbamoy1 toxins (C, B1) and gonyautoxin (GTX1/4, GTX2/3). The GTX toxins accounted for the highest proportion of the total toxins (54.84%). The total concentration of PSPs produced by ATHK was 5.538 pg STX Equal/cell.

2.3 A. tamarense Exposure and Sampling

After acclimation, 270 shrimp were randomly put into nine 100-L plastic containers, 30 each. The experimental shrimp were randomly divided into three groups, three replicates each. Diluted culture of A. tamarense with fresh seawater was used for the exposure treatment, and fresh seawater was used as control. According to preliminary experimental results, the 96-h median lethal concentration of A. tamarense was 10000 cells mL-1. In order to get stressful response with low mortality rate, the concentration of A. tamarense was adjusted to be 200 and 1000 cells mL-1in this experiment. During the testing period, the shrimp were fed commercial prawn pellets, three times each day, and each test solution was renewed daily based on a static renewal method for toxicity test (Buikema et al., 1982). Hepatopancreases were collected from six shrimp each group after 12, 24, 48, 72 and 96 h of exposure and separately preserved in liquid nitrogen for RNA extraction and biochemical determinations. In addition, hepatopancreases were collected from two shrimp each group at the end of the exposure period and fixed with Davidson’s solution (95% ethanol 330 mL, formalin 220 mL, acetic acid 115 mL and H2O 335 mL) for 24 h, then transferred to 70% ethyl alcohol for storage until further analysis.

2.4 Biochemical Analysis

Hepatopancreas samples were prepared as 10% (w:v) homogenates with normal saline. The homogenates were centrifuged at 4000 r min-1and 4℃ for 10 min. The supernatant was recovered and frozen at -80℃ until biochemical determinations. The SOD and GST activities and MDA concentration were determined with commercial test kits (Jiancheng, Ltd, Nanjing, China) following the manufacturer’s instructions. The protein concentration was determined with a Coomassie Brilliant Blue protein assay kit (Jiancheng, Ltd, Nanjing, China).

2.5 RNA Extraction and cDNA Synthesis

Total RNA was extracted from hepatopancreas using TRIzol Reagent (Invitrogen, Carlsbad, CA, USA) following manufacturer’s protocol. Contaminant DNA was removed from RNA extract using RQ1 RNase-Free DNase (Promega, Madison, WI, USA). First-strand cDNA synthesis was performed using M-MLV reverse transcriptase (Promega Madison, WI, USA) according to the manufacturer’s instructions.

2.6 Quantification of mRNA Abundance of FcCasp Gene

FcCasp gene-specific primers (FcCAS-F and FcCAS-R) were designed to amplify a 127-bp product, and 18S rRNA gene-specific primers (18S-F and 18S-R) were designed to amplify a 218-bp fragment (Table 1). Nuclease-free water was used instead of cDNA templates as PCR control.

The mRNA abundance of FcCasp in the hepatopancreas of F. chinensis after A. tamarense exposure was measured with quantitative real-time PCR (qRTPCR) on an ABI 7500 real-time PCR machine (Applied Biosystems, Foster City, CA, USA) as described by Marisa and Juan (2005). All PCR reactions were repeated in triplicate and the 20-μL qRTPCR reaction mixture contained 10 μL of 2×SYBR Premix Ex Taq α (TaKaRa), 1 μL of cDNA (1:10 diluted with PCR-grade H2O), 0.8 μL each of 10 μmol L-1forward and reverse primers, 0.4 μL of Rox Reference Dye, and 7 μL of PCR-grade water. The qRTPCR was set as 95℃ for 30 s, followed by 40 cycles of 95℃for 5 s and 60℃ for 34 s. Melting curve analysis of amplification products was performed at the end of each PCR reaction to confirm that only one PCR product was amplified and detected. The fluorescent real-time PCR data were analyzed using the 7500 System SDS Software (Applied Biosystems, Foster City, CA, USA).

Table 1 Primers used in this study

2.7 TUNEL Assay

DNA fragmentation analysis was conducted using the DeadEndTMColorimetric TUNEL System (Promega, USA) according to manufacturer’s instructions. The tissue sections were deparaffinized, rehydrated, and treated with protein digestion buffer at room temperature for 15 min, followed by incorporation of biotinylated nucleotide mix in the presence of working-strength terminal deoxynucleotidyl transferase (TDT) at 37℃ for 60 min. The reactions were terminated by immersing the slides in 2×saline sodium citrate (SSC) at room temperature for 15 min. Then, the tissue samples were developed with dimethylaminoazobenzene at room temperature for 5 min, and the slides were mounted in an aqueous mounting medium followed by examination under a light microscope. One positive control test was included in this experiment. The rehydrated paraffin sections of hepatopancreas tissues were incubated with 10 U mL-1DNase (Promega, Madison, WI, USA) for 10 min and then washed thoroughly with phosphate buffered saline prior to TUNEL assay. A negative control was prepared by immersing a rehydrated paraffin tissue section into the labeling solution without TDT and then directly used for TUNEL assay.

2.8 Statistical Analysis

Data are expressed as means ± standard error. After testing the homogeneity of variance, statistical difference between treatments and controls were determined by single-factor one-way ANOVA. LSD multiple comparison tests were carried out when the variances were homogeneous. Values were calculated using SPSS 16.0 software. For all tests, the level of significance was set at P < 0.05.

3 Results

3.1 Hepatopancreatic SOD and GST Activities and MDA Concentration

The hepatopancreatic SOD activity of shrimp exposed to 1000 cells mL-1of A. tamarense was significantly (P < 0.05) higher than that of control at all sampling points and showed a bell-shaped response to the increase of exposing time. The maximum hepatopancreatic SOD activity (3.9-folds of control) was observed at 48 h post treatment (Fig.1a). However, the hepatopancreatic SOD activity of shrimp exposed to 200 cells mL-1of A. tamarense was similar to each other (P > 0.05) except for those observed at 48 h.

Likewise, the hepatopancreatic GST activity of shrimp exposed to 1000 cells mL-1of A. tamarense showed a bell-shaped response to the increase of exposing time and reached the maximum (2.8-folds of control) at 48 h post treatment. Thereafter, the hepatopancreatic GST activity decreased to the minimum (0.6-fold of control) at 96 h post treatment (Fig.1b). Similar to the hepatopancreatic SOD activity, the hepatopancreatic GST activity of shrimp exposed to 200 cells mL-1of A. tamarense did not change significantly throughout the experiment (P > 0.05). The hepatopancreatic MDA concentration of shrimp exposed to 1000 cells mL-1of A. tamarense increased gradually from 48 to 96 h (Fig.1c), whereas no significant differences were observed in hepatopancreatic MDA concentrations between 200 cells mL-1of A. tamarense treatment and the control group (P > 0.05).

3.2 Expression Profile of FcCasp Gene

As compared to that of control, the hepatopancreatic FcCasp transcript abundance of shrimp exposed to 1000 cells mL-1of A. tamarense was significantly inhibited in the first 12–48 h (P < 0.05). Thereafter, the hepatopancreatic FcCasp transcript abundance increased immediately and peaked at 96 h (1.6-folds of control) post treatment (Fig.2). However, the hepatopancreatic FcCasp transcript abundance of shrimp exposed to 200 cells mL-1of A. tamarense showed no significant increase compared with that of control (P > 0.05).

Fig.1 The hepatopancreatic superoxide dismutase (SOD, a) and glutathione S-transferase (GST, b) activities and malonyldialdehyde (MDA, c) concentration of F. chinensis exposed to A. tamarense. * Means are significantly different from that of control.

Fig.2 The expression profile of FcCasp in hepatopancreas of F. chinensis exposed to A. tamarense.

3.3 TUNEL Observation

TUNEL-stained cells showed the characteristic ultrastructural features of DNA damage (Fig.3). Apoptotic cells were stained brown by TdT. Brownish nuclei were observed in hepatopancreas of 1000 cells mL-1of A. tamarense treatment (Fig.3D), being similar to the TUNEL-positive nuclei found in positive control (Fig.3B). Conversely, TUNEL-positive nuclei were not observed in control (Fig.3C) or 200 cells mL-1of A. tamarense group (Fig.3E).

3.4 Correlation Between MDA Concentration and FcCasp Transcript Abundance in Hepatopancreas

As clear apoptosis was shown in hepatopancreas of shrimp under 1000 cells mL-1of A. tamarense treatment (Fig.3), the correlation between MDA concentration and FcCasp transcript abundance in hepatopancreas of F. chinensis in 1000 cells mL-1group was analyzed. Results showed that the FcCasp transcript abundance had a positive correlation with MDA concentration in hepatopancreas of F. chinensis in 1000 cells mL-1of A. tamarense treatment (R2= 0.8606) (Fig.4).

Fig.3 Microscopic images (×40 magnification) of TUNEL assay of hepatopancreas in F. chinensis after exposure to 1000 cells mL-1of A. tamarens for 96 h. A, negative control; B, positive control, indicating DNA cleavage; C, control group; D, 1000 cells mL-1of A. tamarense treatment; E, 200 cells mL-1of A. tamarense treatment. Arrows indicate apoptotic cell (brown).

Fig.4 Correlation between malonyldialdehyde (MDA) concentration and FcCasp transcript abundance in hepatopancreas of F. chinensis exposed to 1000 cells mL-1of A. tamarense.

4 Discussion

There is a dynamic balance between the production and elimination of ROSs. Cells possess antioxidant defense systems to prevent the formation of ROSs, and the SOD activity plays an important role in antioxidant protection of invertebrates (Livingstone, 2001; Livingstone, 2003). It is known that superoxide anion (O2-) is one of the major ROSs. SOD is the first enzyme to respond to O2-and offers the greatest response to oxidative stress (Trezado et al., 2006). The increase in hepatopancreatic SOD activity of shrimp exposed to 1000 cells mL-1of A. tamarense indicated that O2-was formed during the exposure treatment. In contrast, the hepatopancreatic SOD activity of shrimp under 200 cells mL-1of A. tamarense treatment was not effectively stimulated within the exposure time except at 48 h. This observation indicated that the level of O2-produced under 200 cells mL-1of A. tamarense was not sufficient to effectively increase the SOD activity. The hepatopancreatic SOD activity of shrimp under 1000 cells mL-1of A. tamarense treatment decreased at 72 h post treatment, probably due to the poisonous effect of excessive O2-(Sun et al., 2007). The bell-shaped response pattern of hepatopancreatic SOD activity of F.chinensis in the present study was consistent with the reported in perch Lateolabrax japonicus (Tan, 2006).

GSTs are a multicomponent family of phase II biotransformation enzymes that detoxify diverse electrophilic endogenous and xenobiotic substrates by means of conjugation with GSH to produce less toxic and more water-soluble compounds (Ron et al., 2003), each of which plays a vital role in protecting tissues from oxidative stress (Wang and Ballatori, 1998). The increase in hepatopancreatic GST activity of shrimp exposed to 1000 cells mL-1of A. tamarense indicated that the presence of A. tamarense generated stress to the cellular metabolism of shrimp. The toxic dinoflagellate A. tamarense ATHK is known to produce PSTs, mainly N-sulfocarbamoyl toxins (C, B1) and Gonyautoxin (GTX1/4, GTX2/3) (Tan et al., 2007). The GSTs catalyze the conjugation of PSTs with GSH, promoting the excretion of those compounds (Costa et al., 2012). The transformation of C-11 sulfated carbamate analogues (e.g., GTX2+3 and GTX1+4) into STX and NEO involved the GSH conjugation using GST as catalyst (Sakamoto et al., 2000; Sato et al., 2000). Induction of the GST activity has also been reported in Atlantic salmon and puffer fish exposed to PSTs (Gubbins et al., 2000; Jeon et al., 2008; Costa et al., 2012). The GST activity under 1000 cells mL-1of A. tamarense first increased and then decreased with the increase of exposing time, indicating that more ROSs were produced and that the GST activity was more effectively enhanced at higher dose. Meanwhile, extra ROSs inactivated the GST activity by interacting with the enzyme (Wang and Ballatori, 1998). The relatively altered GST activity at the lowest level under 200 cells mL-1of A. tamarense within the exposure time might have been caused by the low level of oxidative stress that could not effectively induces GST activity in shrimp.

Because of the presence of antioxidants including SOD and GST, the hepatopancreatic MDA concentration of shrimp under 1000 cells mL-1of A. tamarense did not vary significantly within 24 h. However, the antioxidant response was not sufficient to prevent oxidative damage with increasing exposure time. The hepatopancreatic MDA concentration under 1000 cells mL-1of A. tamarense treatment was significantly greater than that of control from 48 to 96 h, and displayed a time-response relationship. The increasing trend of hepatopancreatic MDA concentration was in accordance with the decreasing trend of GST activity. Thus, it is predicted that the MDA concentration would increase while the GST activity would be inhibited if shrimp are chronically exposed to 1000 cells mL-1of A. tamarense. The increased hepatopancreatic MDA concentration of shrimp exposed to 1000 cells mL-1of A. tamarense indicated that the critical balance between oxidants and antioxidants was disrupted and that the defense mechanism of antioxidants was declined in shrimp under A. tamarense stress. Similarly, a previous study reported that Alexandrium could induce oxidative stress as shown by the enhancement of MDA production (Chen, 2008). Estrada et al. (2007) revealed that the toxic dinoflagellate Gymnodinium catenatum could induce oxidative stress as shown by enhanced LPO production and depressed antioxidant defense system of giant lions-paw scallop Nodipecten subnodosus.

The peroxidation products accumulated in hepatopancreas could result in changes in cellular ultrastructure and induce apoptosis (Xu et al., 2009). Caspases are the critical central molecules of apoptotic pathways (Thornberry, 1998). Leu et al (2008) noted that overexpression of Penaeus monodon caspase resulted in apoptotic morphological features in SF-9 cells. In the present study, the hepatopancreatic FcCasp transcript abundance of shrimp under 1000 cells mL-1of A. tamarense was significantly induced from 72 to 96 h and apoptotic cells were observed at 96 h post treatment. These observations demonstrated that A. tamarense induced apoptosis in hepatopancreas. A positive correlation was observed between the hepatopancreatic MDA concentration and the FcCasp transcript abundance, which confirmed that LPO is related to the development of apoptosis in hepatopancreas of shrimp (Buttke and Sandstrom, 1994).

5 Conclusions

Results of the present study showed that exposure to 1000 cells mL-1of A. tamarense influenced the antioxidative status of F. chinensis, and caused LPO and apoptosis in the hepatopancreas of shrimp. Future studies are needed to analyze the ROS level for directly proving the oxidative stress caused by A. tamarense. The current results couldn't prove that PSTs were the main cause of oxidative stress and apoptosis in the hepatopancreas of shrimp. Therefore, further studies are necessary to examine the effect of PSTs on above-mentioned indices.

Acknowledgements

This study was supported by Earmarked Fund for Modern Agro-industry Technology Research System of China (Grant No. CARS-47), Special Fund for Agroscientific Research in the Public Interest of China (Grant No. 201103034) and the National ‘863’ Project of China (Grant No. 2012AA10A409).

Alonso-Rodríguez, R., and Páez-Osuna, F., 2003. Nutrients, phytoplankton and harmful algal blooms in shrimp ponds: A review with special reference to the situation in the Gulf of California. Aquaculture, 219: 317-336.

Armstrong, J. S., 2006. Mitochondrial membrane permeabilization: The sine qua non for cell death. Bioessays, 28: 253-260.

Buikema, A. L., Niederlehner, B. R., and Cairns, J. J., 1982. Biological monitoring, part IV-toxicity testing. Water Research, 16: 239-262.

Buttke, T. M. and Sandstrom, P. A., 1994. Oxidative stress as a mediator of apoptosis. Immunology Today, 15: 7-10.

Chen, Y., 2008. Toxic effects and mechanisms of Diarrhetic Shellfish Poisoning (DSP) and other HAB toxins on mammalian cells. Ph.D thesis. Chinese Academy of Sciences, 60-69.

Costa, P. R., Botelho, M. J., and Lefebvre, K. A., 2010. Characterization of paralytic shellfish toxins in seawater and sardines (Sardina pilchardus) during blooms of Gymnodinium catenatum. Hydrobiologia, 655: 89-97.

Costa, P. R., Pereira, P., Guilherme, S., Barata, M., Nicolau, L., Santos, M. A., Pacheco, M., and Pous?o-Ferreira, P., 2012. Biotransformation modulation and genotoxicity in white seabream upon exposure to paralytic shellfish toxins produced by Gymnodinium catenatum. Aquatic Toxicology, 106-107, 42-47.

Estrada, N., Romero, M. J., Campa-Córdova, A., Luna, A., and Ascencio, F., 2007. Effects of the toxic dinoflagellate, Gymnodinium catenatum on hydrolytic and antioxidant enzymes, in tissues of the giant lions-paw scallop Nodipecten subnodosus. Comparative Biochemistry and Physiology, 146: 502-510.

Fleury, C., Mignotte, B., and Vayssiere, J. L., 2002. Mitochondrial reactive oxygen species in cell death signaling. Biochimie, 84: 131-141.

García, C., Bravo, M. C., Lagos, M., and Lagos, N., 2004. Paralytic shellfish poisoning: Post mortem analysis of tissue and body fluid samples from human victims in the patagonia fjords. Toxicon, 43: 149-158.

Gubbins, M. J., Eddy, F. B., Gallacher, S., and Stagg, R. M., 2000. Paralytic shellfish poisoning toxins induce xenobiotic metabolishing enzymes in Atlantic salmon (Salmo salar). Marine Environmental Research, 50: 479-483.

Jeon, J. K., Lee, J. S., Shim, W. J., Aarakawa, O., Takatani, T., Honda, S., and Noguchi, T., 2008. Changes in activity of hepatic xenobiotic-metabolizing enzymes of tiger puffer (Takifugu rubripes) exposed to paralytic shellfish poisoning toxins. Journal of Environmental Biology, 29: 599-603.

Leu, J. H., Wang, H. C., Kou, G. H., and Lo, C. F., 2008. Penaeus monodon caspase is targeted by a white spot syndrome virus anti-apoptosis protein. Developmental and Comparative Immunology, 32: 476-486.

Li, Y. Q., Li, J., and Wang, Q. Y., 2006. The effects of dissolved oxygen concentration and stocking density on growth and non-specific immunity factors in Chinese shrimp, Fenneropenaeus chinensis. Aquaculture, 256: 608-616.

Lin, Y. S., 1996. Red tide caused by a marine toxic dinoflagellate, Alexandrium tamarensis (Lebour) Baleon, in shrimp ponds in Xiamen. Taiwan Strait, 15: 16-18 (in Chinese).

Livingstone, D. R., 2001. Contaminant-stimulated reactive oxygen species production and oxidative damage in aquatic organisms. Marine Pollution Bulletin, 42: 656-666.

Livingstone, D. R., 2003. Oxidative stress in aquatic organisms in relation to pollution and aquaculture. Revue De Medecine Veterinaire, 154: 427-430.

Marisa, L. W., and Juan, F. M., 2005. Real-time PCR for mRNA quantitation. Biotechniques, 39: 75-85.

Ron, V. D. O., Jonny, B., and Nico, P. E. V., 2003. Fish bioaccumulation and biomarkers in environmental risk assessment: A review. Environmental Toxicology and Pharmacology, 13: 57-149.

Sakamoto, S., Sato, S., Ogata, T., and Kodama, M., 2000. Formation of intermediate conjugates in the reductive transformation of gonyautoxins to saxitoxins by thiolcompounds. Fisheries Science, 66: 136-141.

Sato, S., Sakai, R., and Kodama, M., 2000. Identification of thioether intermediates in the reductive transformation of gonyautoxins into saxitoxins by thiols. Bioorganic & Medicinal Chemistry Letters, 10: 1787-1789.

Skulachev, V. P., 1998. Cytochromec in the apoptotic and antioxidant cascades. Febs Letters, 423: 275-280.

Stephan, P., Claudia, W., Stephanie, W., Helge, S., and Harri, K., 2005. Activity and substrate specificity of cytosolic and microsomal glutathione S-transferase in Australian black tiger prawns (Penaeus monodon) after exposure to cyanobacterial toxins. Environmental Toxicology, 20: 301-307.

Su, H. M., Liao, I. C., and Chiang, Y. M., 1993. Mass mortality of prawn caused by Alexandrium tamarense blooming in a culture pond in southern Taiwan. In: Toxic Phytoplankton Blooms in the Sea. Smayda, T. J., and Shimizu, Y., eds., Elsevier Science Publishers B V, Amsterdam, 329-333.

Tan, Z. J., Yan, T., Zhou, M. J., Li, J., Yu, R. C., and Wang, Y. F., 2002. The effects of Alexandrium tamarense on survival, growth and reproduction of Neomysis awatschensis. Acta Ecologica Sinica, 22: 1635-1639.

Sun, Y., Yin, G., Zhang, J., Yu, H., and Wang, X., 2007. Bioaccumulation and ROS generation in liver of freshwater fish, goldfish Carassius auratus under HC Orange No.1 exposure. Environmental Toxicology, 22: 256-263.

Tan, Z. J., 2006. Toxic effects and its mechanism of dinoflagellate Alexandrium tamarense on perch Lateolabrax japonicus. Ph.D thesis. Chinese Academy of Sciences, 24-34.

Tan, Z. J., Yan, T., and Yu, R. C., 2007. Transfer of paralytic shellfish toxins via marine food chains: A simulated experiment. Biomedical and Environmental Sciences, 20: 235-241.

Thornberry, N. A., 1998. Caspases: Key mediators of apoptosis. Chemistry and Biology, 5: 97-103.

Trezado, C., Hidalgo, M. C., García-Gallego, M., Morales, A. E., Furne, M., Domezain, A., Domezain, J., and Sanz, A., 2006. Antioxidant enzymes and lipid peroxidation in sturgeon Acipenser naccarii and trout Oncorhynchus mykiss. A comparative study. Aquaculture, 254: 758-767.

Wang, W., and Ballatori, N., 1998. Endogenous glutathione conjugates: Occurrence and biological functions. Pharmacological Reviews, 50: 335-352.

Wang, Y., Li, J., Liu, P., Li, J. T., Zhang, Z., Chang, Z. Q., He, Y. Y., and Liu, D. Y., 2011. The responsive expression of a caspase gene in Chinese shrimp Fenneropenaeus chinensis against pH stress. Aquaculture Research, 42: 1214-1230.

Winston, G. W., 1991. Oxidants and antioxidants in aquatic animals. Comparative Biochemistry and Physiology Part C Comparative Pharmacology, 100: 173-176.

Xu, W. N., Liu, W. B., and Liu, Z. P., 2009. Trichlorfon-induced apoptosis in hepatocyte primary cultures of Carassius auratus gibelio. Chemosphere, 77: 895-901.

Yan, T., Zhou, M. J., Fu, M., Yu, R. C., Wang, Y. F., and Li, J., 2003. Effects of the dinoflagellate Alexandrium tamarense on early development of the Scallop Argopectan irradians concentricus. Aquaculture, 217: 167-178.

Zhu, M. Y., 1993. Red tide in shrimp ponds along the Bohai Sea. In: Toxic Phytoplankton Blooms in the Sea. Smayda, T. J., and Shimizu, Y., eds., Elsevier Science Publishers B V, Amsterdam, 363-367.

(Edited by Qiu Yantao)

(Received May 8, 2013; revised May 30, 2013; accepted July 8, 2014)

? Ocean University of China, Science Press and Spring-Verlag Berlin Heidelberg 2014

* Corresponding author. Tel: 0086-532-85830183

E-mail: lijian@ysfri.ac.cn