Ecotoxicology DOI 10.1007/s10646-015-1477-x

Isolated and combined exposure to ammonia and nitrite in giant freshwater pawn (Macrobrachium rosenbergii): effects on the oxidative stress, antioxidant enzymatic activities and apoptosis in haemocytes Yufan Zhang1 • Chaoxia Ye1 • Anli Wang1 • Xuan Zhu1 • Changhong Chen1 Jianan Xian1 • Zhenzhu Sun1



Accepted: 29 April 2015  Springer Science+Business Media New York 2015

Abstract The residual contaminators such as ammonia and nitrite are widely considered as relevant sources of aquatic environmental pollutants, posing a great threat to shrimp survival. To study the toxicological effects of ammonia and nitrite exposure on the innate immune response in invertebrates, we investigated the oxidative stress and apoptosis in haemocytes of freshwater prawn (Macrobrachium rosenbergii) under isolated and combined exposure to ammonia and nitrite in order to provide useful information about adult prawn immune responses. M. rosenbergii (13.44 ± 2.75 g) were exposed to 0, 5, and 25 mg/L total ammonia-N (TAN) and 0, 5, and 20 mg/L nitrite-N for 24 h. All ammonia concentrations were combined with all nitrite concentrations, making a total of nine treatments studied. Following the exposure treatment, antioxidant enzyme activity, reactive oxygen species (ROS) generation, nitric oxide (NO) generation, and apoptotic cell ratio of haemocytes were measured using flow cytometry. Results indicated that ROS generation was sensitive to the combined effect of ammonia and nitrite, which subsequently affected the Cu–Zn SOD activity. In addition, CAT showed the highest activity at 5 mg/L TAN while GPx decreased at 5 mg/L TAN and returned towards baseline at 25 mg/L. NO generation synchronized with the apoptotic cell ratio in haemocytes, indicating that NO & Chaoxia Ye [email protected] & Anli Wang [email protected] 1

Key Laboratory of Ecology and Environmental Science in Guangdong Higher Education, Key Laboratory of Safe and Healthy Aquaculture in Guangdong Province, College of Life Science, South China Normal University, Guangzhou 510631, People’s Republic of China

production was closely associated with programmed cell death. Both NO production and apoptotic ratios significantly decreased following 25 mg/L TAN, which may be due to the antagonistic regulation of NO and GPx. We hypothesized that the toxicological effect of nitrite exhibited less change in physiological changes compared to that of ammonia, because of the high tolerance to nitrite exposure in mature M. rosenbergii and/or the competitive effects of chloride ions. Taken together, these results showed that ammonia and nitrite caused a series of combined oxidative stress and apoptosis in M. rosenbergi, but further studies are of great need to explain the mechanisms. Keywords Macrobrachium rosenbergii  Ammonia  Nitrite  Innate immunity  Apoptosis  Flow cytometry

Introduction In view of the booming aquaculture development and the increasing demands for the environmental protection, the use of totally or partly recirculated water in aquaculture industry may possibly result in a sharply increased accumulation level of ammonia that can be rapidly oxidized to nitrite, and these two potential metabolic waste products are the most common toxicants, posing a great threat to the survival of aquatic animals (Xian et al. 2011). The increasing accumulation level of ammonia and nitrite in water is highly associated with the bacterial denitrification and nitrate ammonification (Cheng et al. 2013). Total ammonia in water is present as two forms, ammonium (NH4?) and unionized ammonia (NH3). In general, the elevated level of unionized ammonia (NH3) may attenuate the metabolism and lower the innate immunity in pawn, resulting in a series of physiological malfunctions such as ionic imbalance, slower growth, molting failure,

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nervous disorder, respiratory metabolism impairment, and finally a significantly increased mortality (Meinelt et al. 2010; Alcaraz et al. 1997). In contrast, Nitrite derived from the rapidly oxidated ammonia can accumulates in shrimp ponds and the nitrite concentration can reach approximately 20 mg/L in recirculated water (Tacon et al. 2002). In addition, the major toxicological effect of nitrite in the crustacean is to rapidly decrease the methemocyanin amount and thus lowering oxyhemocyanin levels in haemocytes, which may depress the physiological response by hypoxia, decrease the innate immunity and increase its susceptibility to bacterial infection (Xian et al. 2011; Chen and Cheng 1995). Recent studies indicate that the modern immune defense system is found in vertebrates, while the invertebrates only harbor the non-specific innate immunity such as prophenoloxidase-activating defense system, serine protease clotting processes, the action of endogenous antimicrobial peptides and phagocytosis of foreign materials or invading pathogen (Du et al. 2013) During these processes, NADPH oxidase can be activated via the mitochondria redox sensitive signal cascades and thus induce ROS generation such as superoxide anion (O2-) and reactive oxygen intermediates (ROI), playing an indispensable role in the immune defense against the toxicological effect from various stimuli (Chiu et al. 2010), while the excessive level of stress-induced ROS is also a key driving force behind the direct or indirect damage to the biomacromolecules within the host, including DNA damage and lipid peroxidation, leading to the cell dysfunction. Evidences are emerging that antioxidant enzymes (including Cu–Zn SOD, CAT, and GPx) and non-enzymatic soluble antioxidants (including vitamin-E and vitamin-C) are playing an important role in the immune defense as professional regulators capable of mediating the clearance of stress-induced ROS (Cheng et al. 2006; Dandapat et al. 2003). In addition, Cu– Zn SOD converts O2- into H2O2 (which is considered as a less-damaging molecule), which is subsequently converted into H2O by CAT and GPx (Yeh et al. 2009). In some previous studies, it has been demonstrated that the antioxidant enzymes in shrimp haemocytes are sensitive to stress-induced NO production following the exposure to nitrite or heavy metal (Xian et al. 2010, 2011). Furthermore, the invading pathogens can stimulate ROS overproduction and thereby cause the mutations in mtDNA and cell apoptosis (Orrenius et al. 2007). Therefore, ROS and NO production, antioxidant defense mechanisms, and apoptosis all could contribute to a better understanding of the innate immunity of crustaceans. The giant freshwater prawn (Macrobrachium rosenbergii) is a fast-growing and nutrition-rich species with high economic values (Tidwell et al. 2000). With the devastating disease problem in the white shrimp (Litope-

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naeus Vannamei) aquaculture industry (Zhang et al. 2013), M. rosenbergii has become a popular aquaculture species in China. The culture area of M. rosenbergii in China has increased at a surprising rate to thirteen thousands hectares in 2012, with a total production level of 130,000 tons primarily concentrated in the Yangtze River Delta and the Pearl River Delta region (Liu and Li 2013). The tolerance of M. rosenbergii to ammonia and nitrite has been studied before. Armstrong et al. (1978, 1976) reported that the 24 h LC50 of TAN (NH4??NH3) on M. rosenbergii larvae is 115 mg/L and the 24 h LC50 of nitrite-N is 130 mg/L. Alternatively, Cavalli et al. (2000) reported that the 24 h LC50 of TAN (NH4??NH3) ranged from 50 to 150 mg/L, depending on the amount of highly unsaturated fatty acids in the experimental diets. In addition, Chen and Lee (1997) studied the effects of nitrite on mortality of M. rosenbergii at different external chloride concentrations and found that the 96 h LC50 of nitrite-N in juvenile M. rosenbergii was as low as 12.87 mg/L. These differences in M. rosenbergii tolerance to ammonia and nitrite have numerous causes, such as various stages of growth (Dandapat et al. 2003), different water pH (Meinelt et al. 2010) and the amount of chloride ion in water (Alcaraz et al. 1997). Although several studies on the immune responses of M. rosenbergii to environmental stressors have previously been conducted (Cheng and Chen 2002; Cheng et al. 2003; Naqvi et al. 2007), there is limited data on the combined effect of ammonia and nitrite on ROS production and apoptosis in adult M. rosenbergii. In this study, the aims were to comprehensively study the oxidative stress and apoptosis in haemocytes of adult M. rosenbergii following the exposure to ammonia and nitrite. This research is intended to provide useful information about a relatively coherence between innate immune parameters in pawn and the toxicological effects of ammonia and nitrite exposure.

Materials and methods Animals Macrobrachium rosenbergii (13.44 ± 2.75 g) were obtained from a commercial farm on Panyu Seagull Island (Guangzhou, Guangdong Province, P.R. China) and acclimated in plastic tanks for 2 weeks prior to experiments. Fresh water was supplied to each tank in a circulating-filtered system following dechlorination. During the acclimation period, shrimp were fed twice daily with shrimp diet (42 % protein, 5.4 % fat, 4.8 % fiber, and 15 % ash, supplied by Haid Group, Guangdong, China) until 24 h before the experimental treatments began. Only shrimp visually healthy and in the intermolt stage were used for the study.

Isolated and combined exposure to ammonia and nitrite in giant freshwater pawn (Macrobrachium…

Ammonia and nitrite exposure

ROS production determination

Exposure experiments were conducted in triplicate with eight shrimp per plastic tank in 30 L water (22 ± 1 C, pH 7.6–7.7) aerated continuously using an air stone and exposed to 0, 5, and 25 mg/L TAN and 0, 5, and 20 mg/L nitrite-N. All ammonia concentrations were combined with all nitrite concentrations, giving a total of nine exposure groups. The desired nitrite and ammonia concentrations were achieved by adding dissolved NaNO2 and NH4Cl. The designed and test concentrations of TAN and nitrite-N in the experimental water are shown in Table 1. The amount of Un-ionized NH3 was calculated using the following equation:

To analysis levels of ROS, 200 lL of dilute haemolymph was incubated with 10 lM cell permeant probe 20 ,70 dichlorofluorescein diacetate (DCFH-DA, Sigma) for 30 min in the dark at room temperature. Subsequently, fluorescent cells were measured by flow cytometry (Becton–Dickinson FACSCalibur). ROS production was expressed as mean fluorescence of DCF.

Un-ionized ammonia =

1:124  TAN  10PH ½ePH=ð273þTÞ  þ 10PH

T is the temperature (C) (Kim et al. 2008). Preparation of haemocyte suspensions After 24 h of exposure, six shrimp were randomly sampled from each tank. Haemolymph was extracted from the ventral sinus of each shrimp by a 25 gage needle and 2.5 mL syringe containing an equal volume of ice-cold anticoagulant solution (AS, glucose 20.5 g/L, sodium citrate 8 g/L, sodium chloride 4.2 g/L, pH 7.5) (Xian et al. 2011). The diluted haemolymph from each shrimp was transferred into a separate tube held on ice. Fifty microliters of hemolymph were removed for flow cytometry analysis. The remaining hemolymph was centrifuged at 800g for 10 min at 4 C and the supernatant was removed and used as a plasma preparation.

Measurement of the antioxidant enzymatic activity in haemocytes The plasma extracted from the centrifuged hemolymph was used to assay for antioxidant enzyme activity. Cu–Zn SOD activity was estimated according to the method of Das et al. (2000) where superoxide radicals react with hydroxylamine hydrochloride to produce nitrite, which in turn produces diazonium compounds that form a measurable red azo compound with absorption maxima of 543 nm. Mn-SOD was inactivated by KCN and Cu–Zn SOD eliminates other superoxide radicals produced by riboflavin. The specific activity of Cu–Zn SOD was expressed as NU/mL (Nitrite unit). CAT activity was calculated by measuring the optical intensity of a yellow complex produced by molybdate and H2O2 (Du et al. 2013). Ammonium molybdate was added to terminate the H2O2 degradation catalyzed by catalase and the absorbance was detected at 405 nm. Specific activity of 1 U CAT activity was expressed as 1 lmol H2O2/ s/mL. GPx activity was measured by using a Nanjing Jiancheng A005 (Nanjing, Jiangsu, China) following the manufacturer’s instructions. GPx catalyzes the oxidation of

Table 1 Experimental water analysis Designed treatment Total Ammonia-N (mg/L) 0

5

25

Parameters Nitrite-N (mg/L)

Total Ammonia-Na (mg/L)

NH3 (Un-ionized)b (mg/L)

Nitrite-Na (mg/L)

0

1.53 ± 0.28

0.031

0.02 ± 0.02

5

1.1 ± 0.19

0.022

5.22 ± 0.56

20

1.34 ± 0.33

0.027

21.32 ± 0.55

0

5.51 ± 0.64

0.111

0.07 ± 0.03

5

5.66 ± 0.59

0.114

5.83 ± 0.48

20

6.25 ± 0.44

0.126

20.67 ± 0.75

0 5

24.34 ± 1.27 25.06 ± 1.82

0.489 0.504

0.02 ± 0.03 6.23 ± 0.44

20

24.28 ± 1.34

0.488

20.34 ± 0.72

a

Means ± SD (standard deviation) represent values of the tested concentrations of TAN and nitrite-N of three replicates

b

The concentrations of Un-ionized NH3 were calculated using the equation mentioned above

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glutathione in the presence of glutathione reductase and NADPH; the oxidized form of glutathione is converted with a synchronous oxidation of NADPH to NADP?. The decrease in absorbance at 340 nm was measured. Specific activity was expressed as GPx U/mL. Measurement of NO production in haemocytes The fluorescent probe 4-amino-5-methylamino-20 ,70 difluorofluorescein diacetate (DAF-FM DA, Molecular Probes) was used to detect intracellular NO production (Xian et al. 2012). After reacting with NO inside the cells, DAF-FM is converted to DAF-FM triazole (k-excitation = 495, k-emission = 515), whose fluorescence quantum efficiency is 160-fold greater. Haemocyte suspensions were incubated with DAF-FM DA for 60 min at room temperature, shielded from light and measured with a FL1 detector of flow cytometer. Results are given as the mean of DAF-FM fluorescence, in arbitrary FL1 units.

Results ROS generation in haemocytes The effect of ammonia and nitrite exposure on ROS production is presented in Table 2; Fig. 1. After 24 h exposure to ammonia and nitrite stress, nitrite did not statistically affect ROS generation and the interaction between ammonia and nitrite was also not significant (P [ 0.05). However, ROS was significantly enhanced in haemocytes with increasing ammonia concentrations (P \ 0.05). Cu–Zn SOD activity in haemocytes Without the addition of nitrite, Cu–Zn SOD activity did not change even when the concentration of TAN increased from 0 to 25 mg/L (Table 3; Fig. 2). However, under the combined exposure of ammonia and nitrite, Cu–Zn SOD activity levels significantly increased as ammonia increased.

Determination of apoptotic cell ratio in haemocytes CAT activity in haemocytes The haemocytes apoptotic cell ratio was detected using an Annexin V-FITC/PI apoptosis detection kit (Invitrogen), following the manufacturer’s instructions and a previous publication (Xian et al. 2010). Fifty microliters of hemolymph was diluted with anticoagulant to make a suspension of 1 9 106 cells per mL hemocyte, centrifuged, and resuspended at 3 9 106 cells per mL in 1 9 Annexin V binding buffer (10 mM HEPES/NaOH, pH 7.4, 140 mM NaCl, 2.5 mM CaCl2). We stained 100 lL of haemocyte sample with 5 lL of Annexin V-FITC and 10 lL of 50 lg/ mL PI working solution for 15 min in the dark at room temperature. After the addition of 400 lL 1 9 Annexin V binding buffer, the cells were immediately analyzed by flow cytometry. Results are expressed as Annexin V-FITC/ PI dot plot. Live cells were negative for both probes. Early apoptotic cells stained positive with Annexin V-FITC and negative with PI while end stage apoptotic, necrotic, and/or dead cells stained positive with both Annexin V-FITC and PI. Statistical analyses Statistical analyses were performed using SPSS version 18. All data were expressed as mean ± SD (standard deviation). Two-way analysis of variance (ANOVA) were used to determine the significant interaction between factors (ammonia and nitrite). A multiple-comparison (Duncan) test was conducted to examine the significant differences among all treatments. Statistically significant differences were set at P \ 0.05.

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Nitrite did not significantly alter the activity of CAT nor was the interaction between nitrite and ammonia (Table 3; Fig. 3). However, CAT activity increased when the concentration of TAN reached 5 mg/L, indicating that ammonia significant promotes the activity of CAT. GPx activity in haemocytes Ammonia and nitrite significantly affect the activity of GPx in hemolymph (Table 3; Fig. 4). Once again, nitrite did not result in an increase in GPx activity, as ammonia did. Alternatively, the activity of GPx declined significantly when the concentration of TAN reached 5 mg/L and recovered at 25 mg/L. NO production in haemocytes The amount of NO in haemocytes was significantly affected by stresses (Table 2; Fig. 5). Like the activity of CAT, the NO production index significantly increased at 5 mg/L TAN groups and then decreased as TAN increased. Neither nitrite concentration nor the interaction between ammonia and nitrite caused significant changes in NO production. Apoptotic cell ratio Neither the nitrite concentration nor the interaction between ammonia and nitrite caused significant changes in

Isolated and combined exposure to ammonia and nitrite in giant freshwater pawn (Macrobrachium… Table 2 Flow cytometric analysis of haemocytes Parametersa

Designed treatment Total Ammonia-N (mg/L)

Nitrite-N (mg/L)

0

5

Apoptotic cell ratio (%)

ROS (DCF fluorescence)

NO (DAF fluorescence)

0

19.37 ± 4.75ab

35.96 ± 3.99ab

29.40 ± 11.5ab

5

ab

20

10.71 ± 2.15ab

0

21.24 ± 4.88

ab

29.71 ± 7.83

b

5 25

Factorial ANOVA: P values

13.15 ± 6.13

a

27.26 ± 7.24

23.31 ± 0.74a

34.89 ± 9.40ab

17.19 ± 3.69a

abc

85.51 ± 12.68d

abc

64.58 ± 8.71

37.91 ± 5.50 39.62 ± 9.09

ab

84.62 ± 12.82d

c

20

17.87 ± 12.28

0 5

a

46.53 ± 13.81

5.74 ± 1.89 11.68 ± 6.25ab

54.38 ± 10.11 54.13 ± 7.84c

68.85 ± 10.11 cd 52.11 ± 9.35bc

20

10.13 ± 2.20ab

55.74 ± 13.06c

63.40 ± 28.88

cd

b

Total Ammonia-N

0.04

0.01

Nitrite

0.57

0.49

0.09

TAN 9 Nitrite

0.62

0.82

0.59

a

cd

bc

0.01

Means ± S.D. of three replicates and values within the same column with different superscripts are significantly different (P \ 0.05)

b

P values of of TAN, nitrite and the interaction between them of flow cytometric parameters are presented in corresponding columns by twoway ANOVA

Fig. 1 ROS production of haemocytes in Macrobrachium rosenbergii exposed to ammonia and nitrite. Different letters indicate significant (P \ 0.05) differences among all sets of groups. *The table represents the P values of TAN, Nitrite-N and the interaction between them by two-way ANOVA. The P values lower than 0.05 indicate significant differences were found among treatments caused by TAN, Nitrite-N and/or the interaction between them

the apoptotic cell ratio (Table 2; Fig. 6). However, the apoptotic cell ratios at the 5 mg/L TAN groups increased and then declined to control levels at 25 mg/L TAN. The lowest apoptotic cell ratios occurred in the group incubated in 25 mg/L TAN combined with 0 mg/L nitrite.

Discussion In the present experiment, we chose to use concentration gradients for TAN and nitrite-N of 0, 5, 25 mg/L and 0, 5, 20 mg/L respectively. Considering that excessive mortality

may influence the experiment results, the highest levels of ammonia and nitrite stress were designed lower than the LC50 mentioned above (Armstrong et al. 1978, 1976). Practical external culture circumstances and concentration designs of former scholars were also referenced when choosing our experimental values (Guo et al. 2013; Cavalli et al. 2000). External ammonia is known to disturb the ionic equilibrium in haemocytes by taking up excessive level of Na? or NH4? but losing the K? iron, leading to functional disturbances in nerves (Vedel et al. 1998). Although the main toxicological mechanism of nitrite

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Y. Zhang et al. Table 3 Antioxidant enzymes activity Treatment

Parameters

Total Ammonia-N (mg/L)

Nitrite-N (mg/L)

0

0

5

Factorial ANOVA: P values

CAT (lmol H2O2/s/ml)

GPx (U/ml)

68.01 ± 2.55bc

3.92 ± 1.42ab

1703.40 ± 236.33bc

a

5

40.72 ± 9.49

20

54.51 ± 7.61b

3.89 ± 0.50

ab

3.30 ± 0.63a bc

0

67.50 ± 17.60

5

75.97 ± 6.50

c

67.25 ± 4.11

bc

20 25

Cu–Zn SOD (NU/ml)

1807.43 ± 170.76c 1784.63 ± 5.94c

5.86 ± 0.71

c

1305.43 ± 117.38a

5.16 ± 1.07

bc

1385.10 ± 290.49ab

5.03 ± 1.35

abc

1508.90 ± 145.16abc

abc

0 5

c

69.70 ± 3.17 71.19 ± 5.09c

4.92 ± 0.73 4.39 ± 0.59abc

1665.27 ± 80.60bc 1485.27 ± 306.16abc

20

75.93 ± 4.97c

4.56 ± 0.72abc

1654.62 ± 67.93abc

a

Total Ammonia-N

0.01

0.01

Nitrite

0.29

0.41

0.01 0.50

TAN 9 Nitrite

0.01

0.94

0.62

Means ± S.D. of three replicates and values within the same column with different superscripts are significantly different (P \ 0.05) a

P values of of TAN, nitrite and the interaction between them of antioxidant enzymes are presented in corresponding columns by two-way ANOVA

Fig. 2 SOD activity of hemolymph in Macrobrachium rosenbergii exposed to ammonia and nitrite. Different letters indicate significant (P \ 0.05) differences among all sets of groups. *The table represents the P values of TAN, Nitrite-N and the interaction between them by two-way ANOVA. The P-values lower than 0.05 indicate significant differences were found among treatments caused by TAN, Nitrite-N and/ or the interaction between them

exposure is to increase the methemoglobin amount by oxidizing hemoglobin in haemocytes and thereby cause the hypoxia, nitrite can also interfere with the electrolyte balance in crustaceans by competing with chloride uptake or disturbing the K? balance (Cheng et al. 2013; Meinelt et al. 2010). The ionic fluctuation in membranes is highly associated with a series of physiological changes, including the activation of NADPH oxidase (Chanock et al. 1994). In general, the activated NADPH oxidase can produce free radicals and ROS (Babior 1999), which is widely considered as an indispensable mediator capable of regulating the signaling cascades for synchronizing the

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innate immunity with the adaptive immune response. In the present study, ROS amount in haemocytes increased significantly with the highest levels of ROS production occurring at 25 mg/L TAN combined 20 mg/L nitrite-N group following elevated levels of ambient ammonia. Similarly, previous studies indicate that a sharp increase of ROS production is detected in tiger prawn, implying that the toxicological effects of ammonia and nitrite may be related to ironic imbalance in invertebrates (Xian et al. 2011). Despite ROS is playing a regulatory role in the immune defense system, the excessive level of stress-induced ROS

Isolated and combined exposure to ammonia and nitrite in giant freshwater pawn (Macrobrachium… Fig. 3 CAT activity of hemolymph in Macrobrachium rosenbergii exposed to ammonia and nitrite. Different letters indicate significant (P \ 0.05) differences among all sets of groups. *The table represents the P values of TAN, Nitrite-N and the interaction between them by two-way ANOVA. The P values lower than 0.05 indicate significant differences were found among treatments caused by TAN, Nitrite-N and/ or the interaction between them

Fig. 4 GPx activity of hemolymph in Macrobrachium rosenbergii exposed to ammonia and nitrite. Different letters indicate significant (P \ 0.05) differences among all sets of groups. *The table represents the P values of TAN, Nitrite-N and the interaction between them by two-way ANOVA. The P values lower than 0.05 indicate significant differences were found among treatments caused by TAN, Nitrite-N and/ or the interaction between them

Fig. 5 NO production of haemocytes in Macrobrachium rosenbergii exposed to ammonia and nitrite. Different letters indicate significant (P \ 0.05) differences among all sets of groups. *The table represents the P values of TAN, Nitrite-N and the interaction between them by two-way ANOVA. The P values lower than 0.05 indicate significant differences were found among treatments caused by TAN, Nitrite-N and/ or the interaction between them

may damage tissues and biomacromolecules, as well as lead to the oxidative stress (Wang et al. 2009). Antioxidant enzymes such as SOD, CAT and GPx are responsible for

eliminating the excess ROS (Dandapat et al. 2003; Liao et al. 2012). In the current paper, the Cu–Zn SOD activity in M. rosenbergii increased after the exposure to ascending

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Y. Zhang et al. Fig. 6 Apoptotic cell ratio of haemocytes in Macrobrachium rosenbergii exposed to ammonia and nitrite. Different letters indicate significant (P \ 0.05) differences among all sets of groups. *The table represents the P values of TAN, Nitrite-N and the interaction between them by two-way ANOVA. The P values lower than 0.05 indicate significant differences were found among treatments caused by TAN, Nitrite-N and/ or the interaction between them

ammonia concentration. Although the Cu–Zn SOD activity was not statically affected by nitrite exposure, the interaction between ammonia and nitrite on the activity of Cu– Zn SOD was significant. These results are not consistent with few other reports, which did not find significant interactions between ammonia and nitrite on hemolymph iron concentration and enzyme activity (Cheng et al. 2013; Vedel et al. 1998). These differences may be due to the differences between species and parameters selected. However, Vedel et al. (1998) validate and identify the additive toxicological effects of nitrite and ammonia exposure on muscle K? in shrimp. Moreover, Zbigniew and Pokora (2006) indicated that ambient pollutants such as cadmium and anthracene can trigger ROS accumulation, thus increasing SOD activity in cells. Thus, taken together with the previous studies, the ionic imbalance in invertebrate following the exposure to ammonia and nitrite may cause a combined effect on ROS generation, thus further stimulating the Cu–Zn SOD activity. From this perspective, the mechanism of nitrite toxicity should be re-evaluated in a more comprehensive way. CAT and GPx are involved in the H2O2 breakdown process (Yeh et al. 2009). Despite the similar function of CAT and GPx, in this study, we found that CAT and GPx may harbor different functionality in response to ammonia and nitrite exposure in a diverse activated manner or a regulated mechanism. CAT activity increased following increasing TAN exposure, then falling slightly towards control levels at 25 mg/L TAN exposure. In contrast, the trend of GPx activity was completely opposite to that of CAT activity. This result is consistent with Dandapat et al. (2003), who also observe an increase in CAT activity that occurred simultaneously with a decrease in the GPx activity in M. rosenbergii. Furthermore, GPx has been reported to have a higher affinity for H2O2 than that of CAT,

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and it can also eliminate H2O2 production in a more effective manner (Guo et al. 2013), thus playing a more important role in preventing damage/death from toxicity in early larval stages of crustaceans (Dandapat et al. 2003). Thus, the activities of CAT and GPx would change accordingly after the exposure to the critical points of external ammonia and nitrite, meaning that the induced CAT activity was taking priority when the threat level was low (5 mg/L of TAN), but as the external threat increased (25 mg/L of TAN) the GPx activity enhanced correspondingly. In haemocytes, NO is produced mainly by the activation of nitric oxide synthase (NOS), which is regarded as a crucial signaling modulator exerting a number of effects in physiological responses and biological changes in marine animals, including apoptosis (Palumbo 2005). In the current paper, changing trends of NO production under different treatments were identical with the apoptotic ratio of haemocytes. Significantly higher indices were found in the middle concentration of ammonia (5 mg/L TAN) and declined at the highest concentration (25 mg/L TAN). Cooper and Giulivi (2007) report that NO can disturb the mitochondrial respiration by inhibiting cytochrome oxidase activity and thus may stimulate the leakage of superoxide and H2O2 production from mitochondria, which in turn causes oxidative stress to cells and induces apoptosis (Borutaite and Brown 2006). Apoptosis induced by NO has been previously proven (Brown 2010), which may help to explain the observed synchronization between NO and the trends of apoptotic ratio in this research. Furthermore, NO inactivates glutathione reductase (GR) and therefore reduces the substrate amounts of GPx (Savvides et al. 2002), suggesting that the contrary functions of NO and GPx likely causes an antagonistic regulation between them, which may help explain why both NO producton and the

Isolated and combined exposure to ammonia and nitrite in giant freshwater pawn (Macrobrachium…

apoptotic ratio in haemocytes decreased at 25 mg/L TAN while GPx activity is increased. However, NO generation is regulated by numerous factors with new regulators seemingly published on a regular basis (Kleinert et al. 2004). The primary regulation method of NO generation is still unclear and need further researches. Recent findings indicate that nitrite exhibits less toxicological effects in prawn than ammonia, and the high tolerance to nitrite exposure is observed in mature M. rosenbergii (Armstrong et al. 1976). Additionally, nitrite also exerts a competitive effect in chloride ions uptake in fresh water (Alcaraz et al. 1997). Nevertheless, the same concentrations of nitrite-N exposure can exhibit a more significantly toxicological effect in white shrimp and tiger shrimp (Xian et al. 2011; Guo et al. 2013), indicating that the toxicological effects of nitrite and ammonia exposure may vary depending on differences in shrimp species. In conclusion, 24 h exposure to ammonia and nitrite stimulated the oxidative stress and apoptosis in haemocytes of adult M. rosenbergii. High doses of ammonia and nitrite exposure promoted ROS production at different levels, which in turn induced antioxidant responses and stimulated activation of antioxidant enzymes. Due to the similar toxicity on ionic equilibrium, ammonia significantly enhanced the Cu–Zn SOD activity by interacting with nitrite. In addition, the activation order of CAT and GPx was opposite; CAT was activated to manage lower levels of environmental stress while the more efficient GPx was reinforced as the stress became more intense. NO production exhibited great synchronization with apoptotic haemocytes ratios, indicating that NO plays an important role in apoptosis. Interestingly, NO production and apoptotic cell ratios in haemocytes significantly declined under a more acute intense stress. The rising GPx levels may have antagonistic effects on NO activity. However, nitrite did not affect the oxidative stress and apoptosis to the same level as ammonia did, which may be due to a higher tolerance nitrite exposure in adult M. rosenbergii and/or the competitive effects of chloride ions. To sum up, the oxidative stress and apoptosis in haemocytes of adult M. rosenbergii were significant stimulated by the combined effect from ammonia and nitrite exposure. The non-specific immune indices which we studied in this paper demonstrated a series of sequential immune responses. However, some mechanisms of these immune reactions are still unclear, and further works should be focus on the answers. Acknowledgments This research was supported by the National Natural Science Foundation of China (31100296), Guangdong Provincial Natural Science Foundation (S2011020003256), Scientific and Technological Planning Project of Guangdong Province (2012B020307004), and the Scientific and Technological Planning Project of Guangzhou City.

Conflict of interest of interest.

The authors declare that they have no conflict

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Isolated and combined exposure to ammonia and nitrite in giant freshwater pawn (Macrobrachium rosenbergii): effects on the oxidative stress, antioxidant enzymatic activities and apoptosis in haemocytes.

The residual contaminators such as ammonia and nitrite are widely considered as relevant sources of aquatic environmental pollutants, posing a great t...
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