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Effects of chronic ammonia exposure on ammonia metabolism and excretion in marine medaka Oryzias melastigma
Fish & Shellfish Immunology 65 (2017) 226e234
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Effects of chronic ammonia exposure on ammonia metabolism and excretion in marine medaka Oryzias melastigma Na Gao a, b, Limei Zhu a, Zhiqiang Guo a, Meisheng Yi c, **, Li Zhang a, * a
Key Laboratory of Tropical Marine Bio-resources and Ecology, Guangdong Provincial Key Laboratory of Applied Marine Biology, South China Sea Institute of Oceanology, Chinese Academy of Sciences, Guangzhou, 510301, China b University of Chinese Academy Sciences, Beijing, 100049, China c Guangdong Provincial Key Laboratory of Marine Resources and Coastal Engineering, Sun Yat-sen University, Guangzhou, Guangdong, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 27 October 2016 Received in revised form 1 March 2017 Accepted 16 April 2017 Available online 18 April 2017
Ammonia is highly toxic to aquatic organisms, but whether ammonia excretion or ammonia metabolism to less toxic compounds is the major strategy for detoxification in marine fish against chronic ammonia exposure is unclear to date. In this study, we investigated the metabolism and excretion of ammonia in marine medaka Oryzias melastigma during chronic ammonia exposure. The fish were exposed to 0, 0.1, 0.3, 0.6, and 1.1 mmol l1 NH4Cl spiked seawater for 8 weeks. Exposure of 0.3e1.1 mmol l1 NH4Cl had deleterious effects on the fish, including significant reductions in growth, feed intake, and total protein content. However, the fish could take strategies to detoxify ammonia. The tissue ammonia (TAmm) in the 0.3e1.1 mmol l1 NH4Cl treatments was significantly higher than those in the 0 and 0.1 mmol l1 NH4Cl treatments after 2 weeks of exposure, but it recovered with prolonged exposure time, ultimately reaching the control level after 8 weeks. The amino acid catabolic rate decreased to reduce the gross ammonia production with the increasing ambient ammonia concentration. The concentrations of most metabolites remained constant in the 0e0.6 mmol l1 NH4Cl treatments, whereas 5 amino acids and 3 energy metabolism-related metabolites decreased in the 1.1 mmol l1 NH4Cl treatment. JAmm steadily increased in ambient ammonia from 0 to 0.6 mmol l1 and slightly decreased when the ambient ammonia concentration increased to 1.1 mmol l1. Overall, marine medaka cope with sublethal ammonia environment by regulating the tissue TAmm via reducing the ammonia production and increasing ammonia excretion. © 2017 Elsevier Ltd. All rights reserved.
Keywords: Oryzias melastigma Chronic exposure Toxicity Ammonia excretion Amino acid catabolism
1. Introduction There are two forms of ammonia in water, unionized (NH3) and ionized (NHþ 4 ). Since the late 20th century, the ammonia concentration in natural waters has been widely elevated by an influx of ammonia from multiple anthropogenic activities, including the discharge of sewage effluent and industrial waste, and the overuse of chemical fertilizer in agriculture [6]. Additionally, the ammonia
Abbreviations: HEA, high environment ammonia; SGR, specific growth rate; IR, feed intake rate; TAmm, total ammonia (NHþ 4 þ NH3); TUrea, total urea; MO2, oxygen consumption rate; JAmm, ammonia excretion rate; JUrea, urea excretion rate; GSase, glutamine synthetase. * Corresponding author. ** Corresponding author. E-mail addresses: [email protected] (M. Yi), [email protected] (L. Zhang). http://dx.doi.org/10.1016/j.fsi.2017.04.010 1050-4648/© 2017 Elsevier Ltd. All rights reserved.
concentration can be elevated in intensive fish farming operations due to fish excretion, biological degradation of uneaten feed, and restricted water flow. For instance [5], reported that the concentration of ammonia reached 46 mg l1 in an intensive breeding aquaculture system. Excessive ammonia in water is often a threat to fish health, although ammonia is the major end product of protein catabolism (>50%) in teleost fish [10]. High environmental ammonia (HEA) could lead to reduction of growth rate, physical stamina [9], disruption of the ionic balance [36], increased vulnerability to disease [1], histopathological changes in gill epithelia [4], and oxidative damage [35] in fish. Acute ammonia exposure usually greatly influences ammonia metabolism and excretion in many fish species [23,48,55]. Acute ammonia exposure leads to an increase in the total tissue/plasma ammonia concentration upon initial exposure, which subsequently plateaus or decreases in later exposure. For example, the plasma TAmm significantly increased from about 80 to 200 mmol l1 after 4 h
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and then slightly increased to about 250 mmol l1 at 12 h - 24 h of exposure for seawater acclimated rainbow trout (Oncorhynchus mykiss) exposed to 1000 mmol l1 NH4HCO3 [47]. The plasma TAmm of increased 100-fold to 5000 mmol l1 at 24 h and recovered to control level after 24e48 h in Pacific hagfish (Eptatretus stoutii) exposed to 20 mmol l1 ammonia [8]. Many fish have developed various defense strategies against acute HEA. Firstly, converting ammonia into less toxic nitrogenous compounds such as glutamine and urea is an important detoxification strategy against acute ammonia exposure [3,37,44] and chronic ammonia exposure [15]. For example, glutamine synthetase (GSase) that catalyzes ammonia and glutamate to glutamine works in many tissues including brain, liver, intestine, and muscle to detoxify ammonia in rainbow trout [3]. The high ammonia tolerance of gulf toadfish was dependent on its ability to convert ammonia to urea [44]. Secondly, increasing ammonia excretion is crucial for defending against both endogenous and exogenous ammonia [8,20]. Lastly, decreasing endogenous ammonia production is another important strategy to detoxify ammonia [17]. By this strategy, muderskippers could detoxify endogenous ammonia under aerial exposure [24]. To date, most of these studies have been conducted in freshwater fish at acute exposure. However, it is not clear which strategy plays the major role during chronic ammonia exposure (>1 month) in marine fish. In realistic situations, fish are usually chronically subjected to HEA at sublethal ammonia levels in natural or agricultural waters. The findings obtained from acute ammonia exposure studies have yielded limited information on chronic ammonia exposure. It is therefore more environmentally relevant to examine the toxic effects and ammonia metabolism in chronic ammonia exposure. Moreover, marine fish could have different strategies from those of freshwater fish when subjected to HEA [47,54]. For example, rainbow trout acclimated to seawater had faster recovery of ammonia excretion than that acclimated to freshwater after being exposed to 1000 mmol l1 ammonia for 24 h [47]. It is therefore important to study the toxicity and detoxification mechanisms of marine fish under chronic ammonia exposure. Recently, the marine medaka (Oryzias melastigma) has been strongly proposed as a new model fish for marine ecotoxicological research [19]. Yet few studies have investigated the ecotoxicology of ammonia in this species. According to the above studies, we hypothesize that the metabolism and excretion of ammonia are the strategies in the marine fish to counteract chronic HEA. The objectives of the present study were therefore to examine the chronic toxicity, metabolism and excretion of ammonia in marine medaka. Specifically, the sublethal ammonia toxicity was evaluated through the SGR, IR, total protein, and MO2 of the fish. Additionally, ammonia metabolism (tissue TAmm, amino acid catabolism, metabolites content, GSase activity) and nitrogen excretion (JAmm and JUrea) were determined in order to demonstrate the ammonia detoxification mechanisms. 2. Materials and methods 2.1. Laboratory-reared fish Marine medaka had been raised in our laboratory since 2012 (more than five generations). They were maintained in aerated seawater (composed of recrystallized sea salt; Landebao Co., China) at a salinity of 30 ± 1 ppt (mean ± SD), pH of 8.0 ± 0.1, temperature of 25 C ± 1, and photoperiod of 12 h light:12 h dark. Marine medaka in 0.04 ± 0.004 g wet weight, (1.67 ± 0.12 cm in length, 6months old) were selected for experiments and fed every day (3%e 5% ration relative to body mass) with a ground commercial diet (crude protein, >¼ 44%; crude ash, <¼15%; crude fiber, <¼ 5%;
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moisture, <¼12%; total phosphorus, 0.5%e3%; calcium, 0.5%e3%; NaCl, 0.3%e3%; lysine, <¼2%, Foshan Shunde Fenghua Feedstuff Industry Co., Ltd., Foshan, China). All procedures were approved by the Animal Research Ethics Board of the Chinese Academy of Sciences and were in accordance with the Guidelines of the Chinese Council on Laboratory. 2.2. Ammonia exposure Ten glass tanks (50 29 29 cm) with 15 L seawater were prepared as two replicates for five ammonia exposure treatment. Two hundred fish were transferred into each tank and exposed to different concentrations of NH4Cl for 8 weeks. A 4 mol l1 NH4Cl (CNW; Shanghai, China) stock (adjusted to pH 8.0 with NaOH) was used to prepare solutions with five concentrations of ambient ammonia, nominal 0, 0.1, 0.3, 0.6 and 1.1 mmol l1 (actual concentrations: 0.02 ± 0.01, 0.10 ± 0.01, 0.31 ± 0.01, 0.61 ± 0.03 and 1.11 ± 0.02 mmol l1 respectively, sampled before and after renewing water everyday and measured by the indophenol blue method [18]). The pH of the water was also checked every day throughout the experiment using a pH electrode (Ohaus, Starter 3C). The water pH could maintain at a constant value of 8.0 ± 0.1 for the buffer system of CO2/HCO 3 in the seawater. The 96-h LC50 of marine medaka was approximately 2.6 mmol l1 NH4Cl based on our preliminary 96-h lethal concentration test. A concentration of 1.1 mmol l1 NH4Cl, calculated as 40% of the 96-h LC50 [32], was chosen as the chronic LC50 of NH4Cl. A concentration of 0.1 mmol l1 NH4Cl is an environmentally relevant concentration that occurs frequently in freshwater and marine environments [25]. The fish fed once daily and uneaten pellets were collected 1 h after feeding. Half of the seawater was renewed daily and an extra amount of NH4Cl was added to maintain the ammonia concentration during the exposure. 2.3. Specific growth rate, feed intake and oxygen consumption rate measurements To determine the specific growth rate (SGR), fifteen fish from each treatment were randomly selected at the start of the exposure and reared in parallel. Each fish was weighed at the start and end of the exposure, and the SGR was calculated as follows: SGR ¼ [lnWf e lnWi] / days 100
(1)
where Wi and Wf are the initial and final mean body weight during the experimental period, respectively. After 8 weeks of exposure, twenty-five fish were randomly selected from each treatment to measure the feed intake rate (IR). The fish in each treatment were held in a glass tank (30 19 16 cm) containing 2 L water with corresponding HEA concentration for 7 days. They were fed every day with weighed pellets, and the uneaten pellets were collected 1 h after the feeding, dried and re-weighed. The fish in each treatment were weighed, and the IR, (body weight day1) was measured daily during a 7-day experimental period. To determine the MO2, three replicates of fifteen fasted fish (24 h of fast) were selected randomly from each treatment after eight weeks of ammonia exposure. The fifteen fish were batchweighed and placed into a plastic box (700 ml) that could maintain a good seal, with the probe of the oxygen meter inserted into the box. A magnetic stirring rotor was placed into the box, and a perforated metal sheet was used to separate the magnetic stirring rotor from the above water column in the box. During measurement of the oxygen consumption rate, the box was kept on the magnetic stirrers (RCT basic, IKA) with the magnetic stirring rotor
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working to homogenize the oxygen in the water. Before the oxygen consumption measurement, the fish were acclimated in the box for 1 h. After acclimation, data were recorded from the oxygen meter (DO500, Clean) every 15 min for a period of 1 h. The MO2 (mg g1h1) was calculated as follows: MO2 ¼ d(DO) / dt V / W
(2)
Where DO is the dissolved oxygen during the 1 h (mg l1); t is time (h); W is the weight of the 15 fish in the seal box (g); V is the volume of water in the seal box (l). 2.4. Measurement of total ammonia in tissues After 24 h of fast, six fish were randomly netted from each treatment, flash-frozen in liquid nitrogen and stored at 80 C for measurement of the TAmm in tissues when the fish were exposed for 2, 4 and 8 weeks, as described by Ref. [53]. The tissue TAmm was measured using an ammonia assay kit (Sigma, AA0100, MO, USA) based on the glutamate dehydrogenase/NAD method [43]. Briefly, each fish was weighed, pounded into a powder with liquid nitrogen, transferred to a 1.5 ml cold centrifuge tube with 0.25 ml of cold PCA/EDTA, and then centrifuged (4 C, 3000 rpm) for 30 s. Subsequently, 0.2 ml of the supernatant was withdrawn into a new 1.5 ml tube. The pH of the supernatant was adjusted to 6.8e8.0 with 0.2 ml of KOH. The supernatant was centrifuged (4 C, 3000 rpm) again, and the new supernatant was withdrawn and stored at 80 C for further tissue TAmm measurements following the kit procedure. 2.5. Amino acid catabolism rate determination After 24 h of fast, eight fish were randomly netted from each treatment to determine the amino acid catabolism rate after exposure for 8 weeks. The amino acid catabolism rate was measured using the radioisotopic tracing method [28]. The fish were anaesthetized in MS-222 (0.05 g l1) and injected intraperitoneally (i.p.) with 3.7 KBq L-[4, 5-3H(N)]-leucine (37 MBq ml1) (Perkin Elmer, MA02118; BSN, USA) at a dose of 1 ml using a 5 ml micro-fine syringe. Then, each fish was placed into 50 ml of aerated seawater for 24 h for depuration. After depuration, each fish was sacrificed by an anesthetic overdose, MS-222, and moved into a new scintillation vial. One milliliter of 2 mol l1 NaOH was added into the new scintillation vial to digest the fish at 80 C overnight. Scintillation solution (4 ml) was added into the digested fish, separately. The radioactivity of 3H in the fish (Sf) and the solution injected into the fish (St) were measured using a liquid scintillation counter (Beckman, L6500). The amino acid catabolism rate (%, d1) could therefore be calculated from the equation described in Ref. [28] with modification, as follows: Amino acid catabolism rate ¼ 1 e Sf / St
10 min. The methanol/water layer containing the polar metabolites was transferred to a glass vial and dried in a centrifugal concentrator. The extracts of the whole fish were dissolved in 600 ml of phosphate buffer (100 mmol l1 NaH2PO4 and Na2HPO4, including 0.5 mmol l1 TSP (NORELL), pH 7.0) in D2O (Sigma-Aldrich; St. Louis, USA), vortexed and centrifuged at 3000 g for 5 min at 4 C. The supernatant (550 ml) was then transferred into a 5 mm NMR tube for 1H NMR spectroscopic analysis on a Bruker AV 500 NMR spectrometer at 500.18 MHz (at 25 C), as described by Ref. [26]. All 1 H NMR spectra were phased, baseline-corrected, and calibrated (TSP at 0.0 ppm) manually using TopSpin (version 2.1, Bruker BioSpin, Canada). The peaks of the NMR spectra were assigned according to the chemical shift tables [12,40]. The metabolites were identified and quantified by Chenomx Suite software (Evaluation Version, Chenomx Inc., Canada). 2.7. GSase activity and total protein in tissues At 4 weeks and 8 weeks of exposure, five fasted fish (24 h of fast) were randomly netted from each treatment, flash-frozen in liquid nitrogen and stored at 80 C for measurement of the GSase activity and total protein content in tissues, as described by Ref. [52]. Each fish was washed in cold Cortland saline, weighed and homogenized in ice-cold Cortland saline (1 ml). Then, each homogenized sample was centrifuged (4 C, 2000 rpm), and the supernatant was withdrawn for the total protein content and GSase activity measurements. The total protein content was measured by the bicinchoninic acid method using a commercial kit (BioTeke; BJ, China). The GSase activity was assayed using a Glutamine Synthetase Kit (Nanjing Jiancheng Bioengineering; Nanjing, China) based on the production of g-glutamyl hydroxamate [34]. 2.8. Ammonia and urea excretion rate determination The JAmm were determined at 4 and 8 weeks of exposure, and the JUrea were determined at 8 weeks of exposure as described by Ref. [56] (see eqs. (3) and (4) for the calculation of JAmm and JUrea, respectively). The two parameters were measured for fed fish and fasted fish (24 h of fast), and five replicates were performed in each treatment. The fed fish were fed in the original tank for 1 h, and ten fish from each treatment were randomly netted for the measurements. The fasted fish were fasted for 24 h, and ten fish from each treatment were then randomly netted for the measurements. The ten fish were placed into a 100 ml glass beaker with 50 ml of aerated seawater. Then, water samples were taken from the glass beaker at 0 and 12 h to determine TAmm and TUrea in the water. At the end of all the experiments, the fish were weighed individually. All water samples were stored at 20 C. The TAmm and TUrea in water were measured by the indophenol blue method [18] and diacetyl-monoxime method [29], respectively. JAmm (mmol g1 h1) and JUrea (mmol g1 h1) were calculated as follows:
(3) JAmm ¼ ([TAmm]f - [TAmm]i) V / (t M)
(4)
JUrea ¼ ([TUrea]f - [TUrea]i) V / (t M)
(5)
2.6. Metabolomic analysis The change in metabolites in the fish after ammonia exposure for 8 weeks was analyzed by the NMR-based metabolomic method described by Ref. [49]. Ten fish from each treatment were selected, ground in liquid nitrogen, individually transferred into bullet tubes with 50 ceramic beads (1 mm diameter), and thoroughly homogenized in 400 ml of methanol and 85 ml of water. A total of 500 ml of homogenate was withdrawn and vortexed after 440 ml of chloroform was added. The mixture was allowed to stand on ice for
where [TAmm]f and [TAmm]i are the final and initial concentrations of ammonia in water during the sampling periods in the experiment; [TUrea]f and [TUrea]i are the final and initial concentrations of urea in water; and V, t and M are the volume (l), time (h), and mean mass (g), respectively. 2.9. Statistics Data are expressed as the means ± S.E. The SGR, IR, total protein
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content, amino acid catabolism rate, metabolites content and MO2 at different ammonia exposure concentrations were evaluated using one-way analysis of variance (ANOVA) followed by the LSD test. The JAmm, JUrea, GSase, tissue TAmm at different ammonia exposure concentrations and exposure time were evaluated using two-way analysis of variance (ANOVA) followed by the LSD test. A significance level of P < 0.05 was employed throughout the analyses. All statistical analyses were performed in SPSS (vs.16, SPSS Inc., Chicago, USA) and Sigmaplot 12.0 (vs.12, Sigmaplot Inc., California, USA).
The dissolved oxygen concentration (mg l1) at the beginning of the measurement were 6.03 ± 0.19 mg l1 in all the treatments and 5.71e5.28 mg l1 at the ending of the measurement. The dissolved oxygen concentration during the measurement periods was not hypoxic to the fish. The MO2 generally increased with the ammonia level and increased 21% and 27% in 0.6 mmol l1 and 1.1 mmol l1 treatments (P < 0.05, Fig. 1D).
3. Results
The tissue TAmm increased by approximately 2-fold when the fish were exposed to 0.3, 0.6 and 1.1 mmol l1 NH4Cl for 2 weeks compared to the control (P < 0.05, Fig. 2). After 4 weeks of exposure, the tissue TAmm of the 0.6 and 1.1 mmol l1 treatments were also higher than the control by 65% and 57%, respectively. However, the tissue TAmm in all treatments showed no significant difference after 8 weeks of exposure (P > 0.05, Fig. 2). The amino acid catabolism rate of the fish exposed to 0.6 mmol l1 NH4Cl was slightly lower than that of the 0e0.3 mmol l1 treatments (P > 0.05, Fig. 3). The fish exposed to 1.1 mmol l1 NH4Cl had significantly lower amino acid catabolism rates compared to the control (P < 0.05). 26 metabolites were identified and quantified in marine medaka, including amino acids, energy metabolism-related metabolites and organic osmolytes (Table 1). All the metabolites in 0.1, 0.3, 0.6 mmol l1 treatments had no significant difference to the control. 5 amino acids (b-alanine, arginine, glutamate, glutamine, histidine), 3 energy metabolism-related metabolites (glucose, fumarate, lactate), and hyppotaurine, choline decreased
3.1. Chronic toxicity: growth, feed intake, oxygen consumption and total protein content The SGR of the fish decreased significantly when the ambient NH4Cl concentration exceeded 0.3 mmol l1 after 8 weeks of exposure (P < 0.05, Fig. 1A). The SGR of the treatments exposed to 0.3 and 0.6 mmol l1 NH4Cl declined to 57% of the control, and that of the 1.1 mmol l1 treatment was even reduced to negative values (P < 0.05). The IR of the fish declined by 11% and 44% when exposed to 0.6 and 1.1 mmol l1 NH4Cl relative to the control, respectively (P < 0.05, Fig. 1B). There was a decreasing trend in the IR when the NH4Cl level increased from 0 to 1.1 mmol l1. The total protein content in the fish exposed to 1.1 mmol l1 NH4Cl decreased by 20% relative to the control (P < 0.05, Fig. 1C). A gradual decrease with increasing ammonia levels was found.
3.2. Ammonia metabolism: tissue ammonia concentration, amino acid catabolism, metabolites and GSase activity
Fig. 1. Specific growth rate (SGR) (A), feed intake rate (FI) (B) (N ¼ 15), total protein content (C) (N ¼ 5) and oxygen consumption rate (MO2) (D) (N ¼ 3) of marine medaka exposed to 0, 0.1, 0.3, 0.6 and 1.1 mmol l1 NH4Cl for 8 weeks. Values are the means ± S.E. The means with different letters indicate significant differences among the treatments (P < 0.05).
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generally increased with the NH4Cl level up to 0.6 mmol l1 and then slightly decreased at 1.1 mmol l1 (Fig. 5B). The fed fish had the lowest JUrea in the 1.1 mmol l1 treatment (P < 0.05), and there was no significant difference among all the treatments for JUrea of the fasted fish (P > 0.05, Fig. 6). Moreover, the percentage of urea in the total metabolic nitrogen excretion was less than 20%. 4. Discussion 4.1. Chronic toxicity of ammonia to marine fish
Fig. 2. Tissue TAmm of marine medaka exposed to 0, 0.1, 0.3, 0.6 and 1.1 mmol l1 NH4Cl for 2 weeks, 4 weeks and 8 weeks. Values are the means ± S.E. (N ¼ 6). The means with different letters are significantly different among the treatments at the same exposure time (P < 0.05).
significantly (P < 0.05) in the 1.1 mmol l1 NH4Cl treatment compared to the control. The glutamine synthetase (GSase) activity did not vary significantly in any treatments at 4 weeks (P > 0.05, Fig. 4). At 8 weeks, however, the GSase activity was significantly lower in the 0.6 and 1.1 mmol l1 treatments than in the 0 and 0.1 mmol l1 treatments (P < 0.05). 3.3. Ammonia and urea excretion After 4-week HEA exposure, the JAmm of the fed fish significantly increased at 0.1 mmol l1 NH4Cl (P < 0.05, Fig. 5A) and then decreased with the increase of ammonia levels, reaching the lowest level at 1.1 mmol l1 NH4Cl (P < 0.05). The JAmm of the fasted fish was highest in the 0.6 mmol l1 treatment (P < 0.05), followed by the 0.3 mmol l1 treatment (P < 0.05), and was similar among the other treatments (P > 0.05, Fig. 5A). After 8-week HEA exposure, all treatments had similar JAmm when the fish were fed (P > 0.05, Fig. 5B). The JAmm of the fasted fish
The growth of marine medaka was significantly inhibited when the NH4Cl levels were within 0.3e1.1 mmol l1 in this study. Growth reduction in chronic HEA exposure has been widely demonstrated in fish, such as the European sea bass (Dicentrarchus labrax) [9], Nile tilapia (Oreochromis niloticus L) [11], and common carp [39]. A decrease in feed intake under HEA exposure is suggested to be one of the main causes of a reduced growth rate [14,33]. Our findings confirm that the feed intake decreased in parallel with the growth rate of the fish when the NH4Cl levels were above 0.3 mmol l1. Similar results were also observed by Ref. [9]; who estimated that 90% of the reduction in growth rate was attributed to the decrease in feed intake in European sea bass exposed to 0.3 mmol l1 NH4Cl for 20 days. It was reported that the decrease of growth under HEA indicated that fish requires extra energy to cope with HEA [39]. The elevated JAmm could involve in the energy-dependent pathways in the HEA treatments coincident with the elevated MO2 in the 0.6 and 1.1 mmol l1 NH4Cl treatments. These results suggested that marine medaka allocate more energy to excrete ammonia instead of growth under HEA. Similar to our results, MO2 increased with increased ambient ammonia under chronic ammonia exposure was also observed in juvenile big bellied seahorse [2] and European seabass Dicentrarchus labrax [22]. That paradox of inadequate energy supply for the low feed intake and higher energy demand to deal with HEA existed in marine medaka under chronic HEA exposure. This, consequently, led to the over-consumption of energy metabolism-related metabolites. In this study, glucose, fumarate, and inosine decreased in the 0.6 and 1.1 mmol l1 NH4Cl treatments which were in line with above elaboration. In addition, fish exposed to the ambient NH4Cl level of 0.1 mmol l1, as an environmentally realistic concentration, had similar growth performance as the control, indicating that marine medaka could acclimate to the low level of ammonia [13]. also reported no significant effect of a chronic low ammonia level (0.2 mmol l1 NH4Cl) on the growth of juvenile spotted wolf fish (Anarhichas minor). However [27], found that the growth rate of walleye (Sander vitreus) significantly increased after 56 days of exposure to a low ammonia concentration (100e300 mmol l1 NH4Cl). These authors suggested that the low ammonia might enhance the nitrogen accessibility and thus improve the protein retention in the fish body [45]. also found that rainbow trout had a higher growth rate and more rapid protein production when exposed to low ammonia (70 mmol l1 TAmm) relative to the control after exposure for 71 days. The proposed mechanism by which exogenous ammonia stimulates growth involves an increase in protein synthesis for the incorporation of ammonia into amino acids [45]. 4.2. Regulation of ammonia metabolism in chronic ammonia exposure
Fig. 3. Amino acid catabolism rate (ACR) of marine medaka exposed to 0, 0.1, 0.3, 0.6 and 1.1 mmol l1 NH4Cl for 8 weeks. Values are the means ± S.E. (N ¼ 8). The means with different letters indicate significant differences among the treatments (P < 0.05).
The tissue TAmm in the 0.3e1.1 mmol l1 treatments increased to the highest level in the first two weeks and then decreased to comparable levels as the control at 8 weeks, suggesting that marine
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Table 1 The content of main metabolites in marine medaka exposed to 0, 0.1, 0.3, 0.6 and 1.1 mmol l1 NH4Cl for 8 weeks (mmol/g). Metabolite Amino acid b-alanine Arginine Glutamate Glutamine Histidine Aspartate Glycine Isoleucine Leucine Phenylalanine Threonine Tyrosine Valine Energy metabolism-related Creatine phosphate Glucose Fumarate Lactate Organic osmolytes Hyppotaurine Dimethylamine Inosine Taurine Others Choline Malonate O-acetylcholine O-phosphocholine 4-aminobutyrate
C0.1
C0 0.13 2.09 2.03 0.88 2.06 0.69 6.33 0.34 0.51 0.59 0.88 0.27 0.39
± ± ± ± ± ± ± ± ± ± ± ± ±
0.02a 0.24a 0.13a 0.06a 0.23a 0.06 0.53ab 0.04 0.07 0.1 0.1ab 0.03ab 0.04
0.12 2.03 2.03 0.88 1.95 0.67 6.02 0.31 0.59 0.55 0.91 0.35 0.47
C0.3 ± ± ± ± ± ± ± ± ± ± ± ± ±
0.01ab 0.19ab 0.15a 0.04a 0.25a 0.05 0.40ab 0.05 0.07 0.08 0.08a 0.05a 0.06
0.10 1.82 2.00 0.95 1.85 0.59 6.20 0.28 0.62 0.51 0.97 0.28 0.38
C0.6 ± ± ± ± ± ± ± ± ± ± ± ± ±
0.01ab 0.22ab 0.13a 0.08a 0.14a 0.04 0.35ab 0.04 0.08 0.08 0.15a 0.03ab 0.06
0.09 1.63 1.60 0.79 1.48 0.68 6.67 0.29 0.64 0.46 0.69 0.27 0.43
C1.1 ± ± ± ± ± ± ± ± ± ± ± ± ±
0.01ab 0.19ab 0.15ab 0.06ab 0.26ab 0.07 0.69a 0.04 0.08 0.07 0.07ab 0.04ab 0.06
0.09 1.46 1.44 0.67 1.10 0.63 4.83 0.27 0.62 0.37 0.60 0.24 0.46
± ± ± ± ± ± ± ± ± ± ± ± ±
0.01b 0.24b 0.20b 0.09b 0.26b 0.07 0.66b 0.05 0.09 0.06 0.09b 0.03b 0.09
5.96 ± 0.21ab 62.13 ± 9.13a 0.03 ± 0.003ab 1.73 ± 0.19ab
5.71 ± 0.34a 70.19 ± 10.51a 0.04 ± 0.004a 2.06 ± 0.20a
5.65 ± 0.24a 52.53 ± 8.80ab 0.03 ± 0.002ab 1.52 ± 0.08b
6.71 ± 0.43ab 50.94 ± 13.11ab 0.02 ± 0.005b 1.39 ± 0.16b
6.10 ± 0.46b 27.47 ± 8.11b 0.02 ± 0.004c 0.93 ± 0.14c
0.94 ± 0.09a 0.07 ± 0.01 1.45 ± 0.16 10.89 ± 1.01
0.92 ± 0.08a 0.07 ± 0.004 1.57 ± 0.07 11.37 ± 0.81
0.81 ± 0.06a 0.08 ± 0.002 1.49 ± 0.08 11.50 ± 0.41
0.80 ± 0.07a 0.08 ± 0.005 1.38 ± 0.09 11.31 ± 0.54
0.57 0.08 1.29 9.68
± ± ± ±
0.06b 0.01 0.07 1.21
0.01b 0.04b 0.01 0.11 0.02
0.04 0.25 0.11 0.07 0.25
± ± ± ± ±
0.01b 0.05ab 0.01 0.01 0.02
0.07 0.30 0.12 0.11 0.29
± ± ± ± ±
0.01ab 0.04ab 0.01 0.01 0.02
0.08 0.20 0.14 0.12 0.29
± ± ± ± ±
0.01a 0.02a 0.01 0.01 0.03
0.06 0.27 0.12 0.19 0.24
± ± ± ± ±
0.01ab 0.03ab 0.01 0.08 0.02
0.05 0.34 0.13 0.20 0.25
± ± ± ± ±
Values are the means ± S.E. (N ¼ 10). The means in the same metabolites with different letters indicate significant differences among different treatments (P < 0.05).
Fig. 4. Glutamine synthetase (GSase) activity in marine medaka exposed to 0, 0.1, 0.3, 0.6 and 1.1 mmol l1 NH4Cl for 4 and 8 weeks. Values are the means ± S.E. (N ¼ 5). The means with different letters indicate significant differences among the treatments at the same exposure time (P < 0.05).
medaka acclimated to HEA after chronic exposure. Similar findings were reported in Atlantic salmon parr (Salmo salar L.). The plasma ammonia of that was significantly increased from 1.5 mmol l1 to 1.7 mmol l1 the first 22 days, and then declined to 0.7 mmol l1 with no significantly difference with the control level at the 105 days exposure of 480 mmol l1 HEA exposure [21]. This demonstrates that fish could maintain a relatively stable tissue TAmm under chronic ammonia exposure. Contrary to chronic exposure, fish often increase their plasma TAmm to a steady level during acute HEA exposure but do not recover to the control level [8,47]. Based on the
results of the present study, the decrease in TAmm in the fish tissue resulted from the decrease in endogenous ammonia and the increase in the output of ammonia, including decreasing ammonia absorption from food (i.e., decrease in feed intake, Fig. 1B), decreasing amino acid catabolism (Fig. 3), and increasing ammonia excretion (Fig. 5A and B). Regulating the tissue TAmm due to decreased feed intake was referred to above (in section 4.1). Here, we also determined the amino acid catabolism rate, and the results demonstrated that this rate apparently decreased when the ambient NH4Cl levels increased from 0.3 to 1.1 mmol l1, suggesting that the regulation of amino acid catabolism also plays a critical role in ammonia avoidance. Reduction of endogenous ammonia production by decreasing the amino acid catabolism rate is suggested to be an effective strategy for ammonia avoidance in fish [24]. Mudskipper (Periophthalmodon schlosseri), for instance, is an air-breathing fish with limited gill ventilation that can easily build-up ammonia in the body. These fish decrease their proteolysis rate and amino acid catabolism rate to avoid ammonia intoxication from elevating endogenous ammonia level in their tissues [24]. Loach (Misgurnus anguillicaudatus), another air-breathing fish, accumulated 14.5 mmol/g hepatic ammonia, which is higher than the average ammonia level (1e10 mmol/g) of most air-tolerant teleosts [7]. These fish were also capable of decreasing amino acid catabolism to reduce endogenous ammonia production and thus stabilize the tissue TAmm in HEA exposure [7]. The increase in GSase activity is frequently found to be an important strategy for ammonia detoxification in fish, both under acute and chronic HEA exposure [3,50]. For example, GSase activity increased in various organs (liver, intestine, muscle) of the Chinese black sleeper (Bostrichthys sinensis) when exposed to 15 mmol l1 NH4Cl for 48 h [3] [15]. reported that the GSase activity at cellular level increased 3~4-fold in Nile tilapia brain after HEA
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treatments at 4 weeks of exposure and significantly decreased in the 0.3e1.1 mmol l1 treatments at 8 weeks of exposure. This suggested that marine medaka could provide more than enough GSase capacity and did not need to increase GSase activity. This was in line with the study of [53]; where the GSase activity changed little in the brain of rainbow trout exposed to 250 mmol l1 NH4(SO4)2 for 1 þ month. On the other hand, in the present study, we observed that marine medaka had adequately controlled the stable TAmm in tissue and detoxified ammonia by increasing their ammonia excretion (discussed below). As both the reaction catalyzed by GSase and ammonia excretion against ammonia gradient are ATP request the steady GSase activity in marine medaka was benefit for reducing their energy expenditure and reserve more energy for ammonia excretion. In this study, few changes in the amino acid levels were observed in the 0.1e0.6 mmol l1 ammonia treatments. This suggests that marine medaka can maintain balanced amino acid levels under chronic NH4Cl exposure (<0.6 mmol l1). However, threonine, arginine, glutamate, glutamine, b-alanine, glycine, tyrosine, and histidine in the 1.1 mmol l1 NH4Cl had lower levels than the other treatments. This may attributed to the declined feed intake and the low protein content after chronic HEA exposure. In fact, different types of amino acids had different responses in different fish under HEA exposure. For example, when juvenile Nile tilapia were exposed to 0.3 mmol l1 NH4Cl for 70 days, glutamine, glycine, alanine, histidine and serine increased and glutamate and aspartate decreased [15]. The fish tend to increase the level of total free amino acids to reduce endogenous ammonia to alleviate ammonia toxicity. However, marine medaka under chronic HEA exposure did not adopt this strategy but rather maintained steady GSase activity and balanced amino acids, which indicates an adaptation to chronic ammonia for marine medaka. 4.3. Regulation of nitrogen excretion at chronic ammonia exposure Fig. 5. Ammonia excretion rate (JAmm) of the fed and fasted marine medaka exposed to 0, 0.1, 0.3, 0.6 and 1.1 mmol l1 NH4Cl for 4 weeks (Fig. 6A) and 8 weeks (Fig. 6B). Values are the means ± S.E. (N ¼ 5). The means with different letters indicate significant differences among the treatments at the same exposure time (P < 0.05).
Fig. 6. Urea excretion rate (JUrea) of the fed and fasted marine medaka exposed to 0, 0.1, 0.3, 0.6 and 1.1 mmol l1 NH4Cl for 8 weeks. Values are the means ± S.E. (N ¼ 5). The means with different letters indicate significant differences among the treatments (P < 0.05).
exposure (5 or 10 mg l1 total ammonia nitrogen) for 70 days. In contrast, we found that the GSase activity differed little among all
We demonstrated that regulation of the ammonia excretion rate was an important strategy to maintain a relatively stable tissue TAmm in marine medaka under HEA exposure. The present study demonstrated that JAmm was generally maintained or significantly increased in the fed and fasted fish in different HEA exposure treatments, suggesting ammonia excretion was elevated to counteract chronic HEA exposure. Even in the highest ammonia exposure treatment (1.1 mmol l1), although JAmm in the fed fish was reduced after 4-week exposure, it was recovered to the control level after 8-week exposure. These results could explain why the tissue TAmm at 1.1 mmol l1 HEA treatment was initially elevated but recovered to the control level at the end of 8-week exposure. Similar to the present study, many fish can excrete ammonia actively against an inwardly directed ammonia gradient, such as Clarias gariepinus [17], rainbow trout [30,47], Alcolapia grahami [20], E. stoutii [8], and spiny dogfish shark Squalus acanthias [31]. The mechanism of that may involve rhesus glycoprotein with other transporters (V-type Hþ-ATPase, Naþ/Hþ exchanger, Naþ/KþATPase, carbonic anhydrase) [30,51,47,38]. At acute HEA exposure, JAmm is usually initially reversed, but will be reestablished and greater than control level following a few hours of exposure [8,30,47]. However, no net-uptake of ammonia was observed in marine medaka after the fish exposed for 8 weeks. It seems that marine medaka had acclimated the HEA and directly stepped into the stage of excreting ammonia. Obviously, our results revealed different patterns of ammonia excretion between acute and chronic HEA exposure. The marine medaka could acclimate to the ammonia environment during the long-term exposure, and apply to elevate ammonia excretion as one of the important mechanisms to reduce body ammonia level.
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Ambient ammonia did not affect the JUrea of marine medaka indicating that marine medaka was not able to avoid ammonia toxicity by increasing urea excretion. Similar results have also been reported in the ammonotelic Pacific hagfish and killifish, which did not convert ammonia into urea nor significantly increase the JUrea in response to HEA [8,16]. Besides, marine medaka excrete only about 20% nitrogenous waste as urea which was likely to come from purine degradation or argininolysis [41,42]. This ureogenesis way was metabolically costly, for which synthesizing 1 mol urea requires at least 2 mol of ATP [46]. Therefore, it is not a common strategy for most ammonotelic fish handling ammonia. 4.4. Conclusions Our study demonstrated that chronic exposure to >0.3 mmol l1 ammonia had clear toxic effects on marine medaka. The fish maintained a constant tissue ammonia level at chronic ammonia exposure, which showed a different pattern from that at acute ammonia exposure. The maintenance of the tissue ammonia level in marine medaka mainly involved in decrease of the amino acid catabolism rate, and increase of the ammonia excretion rate. On the other hand, marine medaka allocate more energy to deal with HEA by increasing oxygen consumption. However, the conversion of ammonia into amino acids or urea might not be essential for the detoxification of ammonia in this fish at chronic ammonia exposure.
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We thank the two anonymous reviewers for their constructive comments on this work. This research was supported by the State Key Development Program for Basic Research of China (2015CB452904), National Natural Science Foundation of China (41376161, 31501862), Research Fund Program of Guangdong Provincial Key Laboratory of Marine Resources and Coastal Engineering (201503) and Science and Technology Planning Project of Guangdong Province, China (2014B030301064). References [1] P.A. Ackerman, B.J. Wicks, G.K. Iwama, D.J. Randall, Low levels of environmental ammonia increase susceptibility to disease in Chinook salmon smolts, Physiol. Biochem. Zool. 79 (2006) 695e707. [2] M.B. Adams, M.D. Powell, G.J. Purser, Effect of acute and chronic ammonia and nitrite exposure on oxygen consumption and growth of juvenile big bellied seahorse, J. Fish. Biol. 58 (2001) 848e860. [3] P.M. Anderson, M.A. Broderius, K.C. Fong, K.N.T. Tsui, S.F. Chew, Y.K. Ip, Glutamine synthetase expression in liver, muscle, stomach and intestine of Bostrichthys sinensis in response to exposure to a high exogenous ammonia concentration, J. Exp. Biol. 205 (2002) 2053e2065. € €ksal, A. Ozkul, [4] A.Ç.K. Benli, G. Ko Sublethal ammonia exposure of Nile tilapia (Oreochromis niloticus L.): effects on gill, liver and kidney histology, Chemosphere 72 (2008) 1355e1358. [5] J.C. Chen, P.C. Liu, Y.T. Lin, C.K. Lee, Super intensive culture of red-tailed shrimp Penaeus penicillatus, J. World Aquacult. Soc. 19 (1988) 127e131. [6] C.H. Cheng, F.F. Yang, R.Z. Ling, S.A. Liao, Y.T. Miao, C.X. Ye, A.L. Wang, Effects of ammonia exposure on apoptosis, oxidative stress and immune response in pufferfish (Takifugu obscurus), Aquat. Toxicol. 164 (2015) 61e71. [7] S.F. Chew, Y. Jin, Y.K. Ip, The loach Misgurnus anguillicaudatus reduces amino acid catabolism and accumulates alanine and glutamine during aerial exposure, Physiol. Biochem. Zool. 74 (2001) 226e237. [8] A.M. Clifford, G.G. Goss, M.P. Wilkie, Adaptations of a deep sea scavenger: high ammonia tolerance and active NH4þ excretion by the Pacific hagfish (Eptatretus stoutii), Comp. Biochem. Physiol. A Mol. Integr. Physiol. 182 (2015) 64e74. [9] A. Dosdat, J. Person-Le Ruyet, D. Coves, G. Dutto, E. Gasset, A. Le Roux, , Effect of chronic exposure to ammonia on growth, food utilisation G. Lemarie and metabolism of the European sea bass (Dicentrarchus labrax), Aquat. Living Resour. 16 (2003) 509e520. [10] F.B. Eddy, Ammonia in estuaries and effects on fish, J. Fish. Biol. 67 (2005) 1495e1513. [11] S.A. El-Shafai, F.A. El-Gohary, F.A. Nasr, N.P. van der Steen, H.J. Gijzen, Chronic
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Full length article
Effects of chronic ammonia exposure on ammonia metabolism and excretion in marine medaka Oryzias melastigma Na Gao a, b, Limei Zhu a, Zhiqiang Guo a, Meisheng Yi c, **, Li Zhang a, * a
Key Laboratory of Tropical Marine Bio-resources and Ecology, Guangdong Provincial Key Laboratory of Applied Marine Biology, South China Sea Institute of Oceanology, Chinese Academy of Sciences, Guangzhou, 510301, China b University of Chinese Academy Sciences, Beijing, 100049, China c Guangdong Provincial Key Laboratory of Marine Resources and Coastal Engineering, Sun Yat-sen University, Guangzhou, Guangdong, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 27 October 2016 Received in revised form 1 March 2017 Accepted 16 April 2017 Available online 18 April 2017
Ammonia is highly toxic to aquatic organisms, but whether ammonia excretion or ammonia metabolism to less toxic compounds is the major strategy for detoxification in marine fish against chronic ammonia exposure is unclear to date. In this study, we investigated the metabolism and excretion of ammonia in marine medaka Oryzias melastigma during chronic ammonia exposure. The fish were exposed to 0, 0.1, 0.3, 0.6, and 1.1 mmol l1 NH4Cl spiked seawater for 8 weeks. Exposure of 0.3e1.1 mmol l1 NH4Cl had deleterious effects on the fish, including significant reductions in growth, feed intake, and total protein content. However, the fish could take strategies to detoxify ammonia. The tissue ammonia (TAmm) in the 0.3e1.1 mmol l1 NH4Cl treatments was significantly higher than those in the 0 and 0.1 mmol l1 NH4Cl treatments after 2 weeks of exposure, but it recovered with prolonged exposure time, ultimately reaching the control level after 8 weeks. The amino acid catabolic rate decreased to reduce the gross ammonia production with the increasing ambient ammonia concentration. The concentrations of most metabolites remained constant in the 0e0.6 mmol l1 NH4Cl treatments, whereas 5 amino acids and 3 energy metabolism-related metabolites decreased in the 1.1 mmol l1 NH4Cl treatment. JAmm steadily increased in ambient ammonia from 0 to 0.6 mmol l1 and slightly decreased when the ambient ammonia concentration increased to 1.1 mmol l1. Overall, marine medaka cope with sublethal ammonia environment by regulating the tissue TAmm via reducing the ammonia production and increasing ammonia excretion. © 2017 Elsevier Ltd. All rights reserved.
Keywords: Oryzias melastigma Chronic exposure Toxicity Ammonia excretion Amino acid catabolism
1. Introduction There are two forms of ammonia in water, unionized (NH3) and ionized (NHþ 4 ). Since the late 20th century, the ammonia concentration in natural waters has been widely elevated by an influx of ammonia from multiple anthropogenic activities, including the discharge of sewage effluent and industrial waste, and the overuse of chemical fertilizer in agriculture [6]. Additionally, the ammonia
Abbreviations: HEA, high environment ammonia; SGR, specific growth rate; IR, feed intake rate; TAmm, total ammonia (NHþ 4 þ NH3); TUrea, total urea; MO2, oxygen consumption rate; JAmm, ammonia excretion rate; JUrea, urea excretion rate; GSase, glutamine synthetase. * Corresponding author. ** Corresponding author. E-mail addresses: [email protected] (M. Yi), [email protected] (L. Zhang). http://dx.doi.org/10.1016/j.fsi.2017.04.010 1050-4648/© 2017 Elsevier Ltd. All rights reserved.
concentration can be elevated in intensive fish farming operations due to fish excretion, biological degradation of uneaten feed, and restricted water flow. For instance [5], reported that the concentration of ammonia reached 46 mg l1 in an intensive breeding aquaculture system. Excessive ammonia in water is often a threat to fish health, although ammonia is the major end product of protein catabolism (>50%) in teleost fish [10]. High environmental ammonia (HEA) could lead to reduction of growth rate, physical stamina [9], disruption of the ionic balance [36], increased vulnerability to disease [1], histopathological changes in gill epithelia [4], and oxidative damage [35] in fish. Acute ammonia exposure usually greatly influences ammonia metabolism and excretion in many fish species [23,48,55]. Acute ammonia exposure leads to an increase in the total tissue/plasma ammonia concentration upon initial exposure, which subsequently plateaus or decreases in later exposure. For example, the plasma TAmm significantly increased from about 80 to 200 mmol l1 after 4 h
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and then slightly increased to about 250 mmol l1 at 12 h - 24 h of exposure for seawater acclimated rainbow trout (Oncorhynchus mykiss) exposed to 1000 mmol l1 NH4HCO3 [47]. The plasma TAmm of increased 100-fold to 5000 mmol l1 at 24 h and recovered to control level after 24e48 h in Pacific hagfish (Eptatretus stoutii) exposed to 20 mmol l1 ammonia [8]. Many fish have developed various defense strategies against acute HEA. Firstly, converting ammonia into less toxic nitrogenous compounds such as glutamine and urea is an important detoxification strategy against acute ammonia exposure [3,37,44] and chronic ammonia exposure [15]. For example, glutamine synthetase (GSase) that catalyzes ammonia and glutamate to glutamine works in many tissues including brain, liver, intestine, and muscle to detoxify ammonia in rainbow trout [3]. The high ammonia tolerance of gulf toadfish was dependent on its ability to convert ammonia to urea [44]. Secondly, increasing ammonia excretion is crucial for defending against both endogenous and exogenous ammonia [8,20]. Lastly, decreasing endogenous ammonia production is another important strategy to detoxify ammonia [17]. By this strategy, muderskippers could detoxify endogenous ammonia under aerial exposure [24]. To date, most of these studies have been conducted in freshwater fish at acute exposure. However, it is not clear which strategy plays the major role during chronic ammonia exposure (>1 month) in marine fish. In realistic situations, fish are usually chronically subjected to HEA at sublethal ammonia levels in natural or agricultural waters. The findings obtained from acute ammonia exposure studies have yielded limited information on chronic ammonia exposure. It is therefore more environmentally relevant to examine the toxic effects and ammonia metabolism in chronic ammonia exposure. Moreover, marine fish could have different strategies from those of freshwater fish when subjected to HEA [47,54]. For example, rainbow trout acclimated to seawater had faster recovery of ammonia excretion than that acclimated to freshwater after being exposed to 1000 mmol l1 ammonia for 24 h [47]. It is therefore important to study the toxicity and detoxification mechanisms of marine fish under chronic ammonia exposure. Recently, the marine medaka (Oryzias melastigma) has been strongly proposed as a new model fish for marine ecotoxicological research [19]. Yet few studies have investigated the ecotoxicology of ammonia in this species. According to the above studies, we hypothesize that the metabolism and excretion of ammonia are the strategies in the marine fish to counteract chronic HEA. The objectives of the present study were therefore to examine the chronic toxicity, metabolism and excretion of ammonia in marine medaka. Specifically, the sublethal ammonia toxicity was evaluated through the SGR, IR, total protein, and MO2 of the fish. Additionally, ammonia metabolism (tissue TAmm, amino acid catabolism, metabolites content, GSase activity) and nitrogen excretion (JAmm and JUrea) were determined in order to demonstrate the ammonia detoxification mechanisms. 2. Materials and methods 2.1. Laboratory-reared fish Marine medaka had been raised in our laboratory since 2012 (more than five generations). They were maintained in aerated seawater (composed of recrystallized sea salt; Landebao Co., China) at a salinity of 30 ± 1 ppt (mean ± SD), pH of 8.0 ± 0.1, temperature of 25 C ± 1, and photoperiod of 12 h light:12 h dark. Marine medaka in 0.04 ± 0.004 g wet weight, (1.67 ± 0.12 cm in length, 6months old) were selected for experiments and fed every day (3%e 5% ration relative to body mass) with a ground commercial diet (crude protein, >¼ 44%; crude ash, <¼15%; crude fiber, <¼ 5%;
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moisture, <¼12%; total phosphorus, 0.5%e3%; calcium, 0.5%e3%; NaCl, 0.3%e3%; lysine, <¼2%, Foshan Shunde Fenghua Feedstuff Industry Co., Ltd., Foshan, China). All procedures were approved by the Animal Research Ethics Board of the Chinese Academy of Sciences and were in accordance with the Guidelines of the Chinese Council on Laboratory. 2.2. Ammonia exposure Ten glass tanks (50 29 29 cm) with 15 L seawater were prepared as two replicates for five ammonia exposure treatment. Two hundred fish were transferred into each tank and exposed to different concentrations of NH4Cl for 8 weeks. A 4 mol l1 NH4Cl (CNW; Shanghai, China) stock (adjusted to pH 8.0 with NaOH) was used to prepare solutions with five concentrations of ambient ammonia, nominal 0, 0.1, 0.3, 0.6 and 1.1 mmol l1 (actual concentrations: 0.02 ± 0.01, 0.10 ± 0.01, 0.31 ± 0.01, 0.61 ± 0.03 and 1.11 ± 0.02 mmol l1 respectively, sampled before and after renewing water everyday and measured by the indophenol blue method [18]). The pH of the water was also checked every day throughout the experiment using a pH electrode (Ohaus, Starter 3C). The water pH could maintain at a constant value of 8.0 ± 0.1 for the buffer system of CO2/HCO 3 in the seawater. The 96-h LC50 of marine medaka was approximately 2.6 mmol l1 NH4Cl based on our preliminary 96-h lethal concentration test. A concentration of 1.1 mmol l1 NH4Cl, calculated as 40% of the 96-h LC50 [32], was chosen as the chronic LC50 of NH4Cl. A concentration of 0.1 mmol l1 NH4Cl is an environmentally relevant concentration that occurs frequently in freshwater and marine environments [25]. The fish fed once daily and uneaten pellets were collected 1 h after feeding. Half of the seawater was renewed daily and an extra amount of NH4Cl was added to maintain the ammonia concentration during the exposure. 2.3. Specific growth rate, feed intake and oxygen consumption rate measurements To determine the specific growth rate (SGR), fifteen fish from each treatment were randomly selected at the start of the exposure and reared in parallel. Each fish was weighed at the start and end of the exposure, and the SGR was calculated as follows: SGR ¼ [lnWf e lnWi] / days 100
(1)
where Wi and Wf are the initial and final mean body weight during the experimental period, respectively. After 8 weeks of exposure, twenty-five fish were randomly selected from each treatment to measure the feed intake rate (IR). The fish in each treatment were held in a glass tank (30 19 16 cm) containing 2 L water with corresponding HEA concentration for 7 days. They were fed every day with weighed pellets, and the uneaten pellets were collected 1 h after the feeding, dried and re-weighed. The fish in each treatment were weighed, and the IR, (body weight day1) was measured daily during a 7-day experimental period. To determine the MO2, three replicates of fifteen fasted fish (24 h of fast) were selected randomly from each treatment after eight weeks of ammonia exposure. The fifteen fish were batchweighed and placed into a plastic box (700 ml) that could maintain a good seal, with the probe of the oxygen meter inserted into the box. A magnetic stirring rotor was placed into the box, and a perforated metal sheet was used to separate the magnetic stirring rotor from the above water column in the box. During measurement of the oxygen consumption rate, the box was kept on the magnetic stirrers (RCT basic, IKA) with the magnetic stirring rotor
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working to homogenize the oxygen in the water. Before the oxygen consumption measurement, the fish were acclimated in the box for 1 h. After acclimation, data were recorded from the oxygen meter (DO500, Clean) every 15 min for a period of 1 h. The MO2 (mg g1h1) was calculated as follows: MO2 ¼ d(DO) / dt V / W
(2)
Where DO is the dissolved oxygen during the 1 h (mg l1); t is time (h); W is the weight of the 15 fish in the seal box (g); V is the volume of water in the seal box (l). 2.4. Measurement of total ammonia in tissues After 24 h of fast, six fish were randomly netted from each treatment, flash-frozen in liquid nitrogen and stored at 80 C for measurement of the TAmm in tissues when the fish were exposed for 2, 4 and 8 weeks, as described by Ref. [53]. The tissue TAmm was measured using an ammonia assay kit (Sigma, AA0100, MO, USA) based on the glutamate dehydrogenase/NAD method [43]. Briefly, each fish was weighed, pounded into a powder with liquid nitrogen, transferred to a 1.5 ml cold centrifuge tube with 0.25 ml of cold PCA/EDTA, and then centrifuged (4 C, 3000 rpm) for 30 s. Subsequently, 0.2 ml of the supernatant was withdrawn into a new 1.5 ml tube. The pH of the supernatant was adjusted to 6.8e8.0 with 0.2 ml of KOH. The supernatant was centrifuged (4 C, 3000 rpm) again, and the new supernatant was withdrawn and stored at 80 C for further tissue TAmm measurements following the kit procedure. 2.5. Amino acid catabolism rate determination After 24 h of fast, eight fish were randomly netted from each treatment to determine the amino acid catabolism rate after exposure for 8 weeks. The amino acid catabolism rate was measured using the radioisotopic tracing method [28]. The fish were anaesthetized in MS-222 (0.05 g l1) and injected intraperitoneally (i.p.) with 3.7 KBq L-[4, 5-3H(N)]-leucine (37 MBq ml1) (Perkin Elmer, MA02118; BSN, USA) at a dose of 1 ml using a 5 ml micro-fine syringe. Then, each fish was placed into 50 ml of aerated seawater for 24 h for depuration. After depuration, each fish was sacrificed by an anesthetic overdose, MS-222, and moved into a new scintillation vial. One milliliter of 2 mol l1 NaOH was added into the new scintillation vial to digest the fish at 80 C overnight. Scintillation solution (4 ml) was added into the digested fish, separately. The radioactivity of 3H in the fish (Sf) and the solution injected into the fish (St) were measured using a liquid scintillation counter (Beckman, L6500). The amino acid catabolism rate (%, d1) could therefore be calculated from the equation described in Ref. [28] with modification, as follows: Amino acid catabolism rate ¼ 1 e Sf / St
10 min. The methanol/water layer containing the polar metabolites was transferred to a glass vial and dried in a centrifugal concentrator. The extracts of the whole fish were dissolved in 600 ml of phosphate buffer (100 mmol l1 NaH2PO4 and Na2HPO4, including 0.5 mmol l1 TSP (NORELL), pH 7.0) in D2O (Sigma-Aldrich; St. Louis, USA), vortexed and centrifuged at 3000 g for 5 min at 4 C. The supernatant (550 ml) was then transferred into a 5 mm NMR tube for 1H NMR spectroscopic analysis on a Bruker AV 500 NMR spectrometer at 500.18 MHz (at 25 C), as described by Ref. [26]. All 1 H NMR spectra were phased, baseline-corrected, and calibrated (TSP at 0.0 ppm) manually using TopSpin (version 2.1, Bruker BioSpin, Canada). The peaks of the NMR spectra were assigned according to the chemical shift tables [12,40]. The metabolites were identified and quantified by Chenomx Suite software (Evaluation Version, Chenomx Inc., Canada). 2.7. GSase activity and total protein in tissues At 4 weeks and 8 weeks of exposure, five fasted fish (24 h of fast) were randomly netted from each treatment, flash-frozen in liquid nitrogen and stored at 80 C for measurement of the GSase activity and total protein content in tissues, as described by Ref. [52]. Each fish was washed in cold Cortland saline, weighed and homogenized in ice-cold Cortland saline (1 ml). Then, each homogenized sample was centrifuged (4 C, 2000 rpm), and the supernatant was withdrawn for the total protein content and GSase activity measurements. The total protein content was measured by the bicinchoninic acid method using a commercial kit (BioTeke; BJ, China). The GSase activity was assayed using a Glutamine Synthetase Kit (Nanjing Jiancheng Bioengineering; Nanjing, China) based on the production of g-glutamyl hydroxamate [34]. 2.8. Ammonia and urea excretion rate determination The JAmm were determined at 4 and 8 weeks of exposure, and the JUrea were determined at 8 weeks of exposure as described by Ref. [56] (see eqs. (3) and (4) for the calculation of JAmm and JUrea, respectively). The two parameters were measured for fed fish and fasted fish (24 h of fast), and five replicates were performed in each treatment. The fed fish were fed in the original tank for 1 h, and ten fish from each treatment were randomly netted for the measurements. The fasted fish were fasted for 24 h, and ten fish from each treatment were then randomly netted for the measurements. The ten fish were placed into a 100 ml glass beaker with 50 ml of aerated seawater. Then, water samples were taken from the glass beaker at 0 and 12 h to determine TAmm and TUrea in the water. At the end of all the experiments, the fish were weighed individually. All water samples were stored at 20 C. The TAmm and TUrea in water were measured by the indophenol blue method [18] and diacetyl-monoxime method [29], respectively. JAmm (mmol g1 h1) and JUrea (mmol g1 h1) were calculated as follows:
(3) JAmm ¼ ([TAmm]f - [TAmm]i) V / (t M)
(4)
JUrea ¼ ([TUrea]f - [TUrea]i) V / (t M)
(5)
2.6. Metabolomic analysis The change in metabolites in the fish after ammonia exposure for 8 weeks was analyzed by the NMR-based metabolomic method described by Ref. [49]. Ten fish from each treatment were selected, ground in liquid nitrogen, individually transferred into bullet tubes with 50 ceramic beads (1 mm diameter), and thoroughly homogenized in 400 ml of methanol and 85 ml of water. A total of 500 ml of homogenate was withdrawn and vortexed after 440 ml of chloroform was added. The mixture was allowed to stand on ice for
where [TAmm]f and [TAmm]i are the final and initial concentrations of ammonia in water during the sampling periods in the experiment; [TUrea]f and [TUrea]i are the final and initial concentrations of urea in water; and V, t and M are the volume (l), time (h), and mean mass (g), respectively. 2.9. Statistics Data are expressed as the means ± S.E. The SGR, IR, total protein
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content, amino acid catabolism rate, metabolites content and MO2 at different ammonia exposure concentrations were evaluated using one-way analysis of variance (ANOVA) followed by the LSD test. The JAmm, JUrea, GSase, tissue TAmm at different ammonia exposure concentrations and exposure time were evaluated using two-way analysis of variance (ANOVA) followed by the LSD test. A significance level of P < 0.05 was employed throughout the analyses. All statistical analyses were performed in SPSS (vs.16, SPSS Inc., Chicago, USA) and Sigmaplot 12.0 (vs.12, Sigmaplot Inc., California, USA).
The dissolved oxygen concentration (mg l1) at the beginning of the measurement were 6.03 ± 0.19 mg l1 in all the treatments and 5.71e5.28 mg l1 at the ending of the measurement. The dissolved oxygen concentration during the measurement periods was not hypoxic to the fish. The MO2 generally increased with the ammonia level and increased 21% and 27% in 0.6 mmol l1 and 1.1 mmol l1 treatments (P < 0.05, Fig. 1D).
3. Results
The tissue TAmm increased by approximately 2-fold when the fish were exposed to 0.3, 0.6 and 1.1 mmol l1 NH4Cl for 2 weeks compared to the control (P < 0.05, Fig. 2). After 4 weeks of exposure, the tissue TAmm of the 0.6 and 1.1 mmol l1 treatments were also higher than the control by 65% and 57%, respectively. However, the tissue TAmm in all treatments showed no significant difference after 8 weeks of exposure (P > 0.05, Fig. 2). The amino acid catabolism rate of the fish exposed to 0.6 mmol l1 NH4Cl was slightly lower than that of the 0e0.3 mmol l1 treatments (P > 0.05, Fig. 3). The fish exposed to 1.1 mmol l1 NH4Cl had significantly lower amino acid catabolism rates compared to the control (P < 0.05). 26 metabolites were identified and quantified in marine medaka, including amino acids, energy metabolism-related metabolites and organic osmolytes (Table 1). All the metabolites in 0.1, 0.3, 0.6 mmol l1 treatments had no significant difference to the control. 5 amino acids (b-alanine, arginine, glutamate, glutamine, histidine), 3 energy metabolism-related metabolites (glucose, fumarate, lactate), and hyppotaurine, choline decreased
3.1. Chronic toxicity: growth, feed intake, oxygen consumption and total protein content The SGR of the fish decreased significantly when the ambient NH4Cl concentration exceeded 0.3 mmol l1 after 8 weeks of exposure (P < 0.05, Fig. 1A). The SGR of the treatments exposed to 0.3 and 0.6 mmol l1 NH4Cl declined to 57% of the control, and that of the 1.1 mmol l1 treatment was even reduced to negative values (P < 0.05). The IR of the fish declined by 11% and 44% when exposed to 0.6 and 1.1 mmol l1 NH4Cl relative to the control, respectively (P < 0.05, Fig. 1B). There was a decreasing trend in the IR when the NH4Cl level increased from 0 to 1.1 mmol l1. The total protein content in the fish exposed to 1.1 mmol l1 NH4Cl decreased by 20% relative to the control (P < 0.05, Fig. 1C). A gradual decrease with increasing ammonia levels was found.
3.2. Ammonia metabolism: tissue ammonia concentration, amino acid catabolism, metabolites and GSase activity
Fig. 1. Specific growth rate (SGR) (A), feed intake rate (FI) (B) (N ¼ 15), total protein content (C) (N ¼ 5) and oxygen consumption rate (MO2) (D) (N ¼ 3) of marine medaka exposed to 0, 0.1, 0.3, 0.6 and 1.1 mmol l1 NH4Cl for 8 weeks. Values are the means ± S.E. The means with different letters indicate significant differences among the treatments (P < 0.05).
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generally increased with the NH4Cl level up to 0.6 mmol l1 and then slightly decreased at 1.1 mmol l1 (Fig. 5B). The fed fish had the lowest JUrea in the 1.1 mmol l1 treatment (P < 0.05), and there was no significant difference among all the treatments for JUrea of the fasted fish (P > 0.05, Fig. 6). Moreover, the percentage of urea in the total metabolic nitrogen excretion was less than 20%. 4. Discussion 4.1. Chronic toxicity of ammonia to marine fish
Fig. 2. Tissue TAmm of marine medaka exposed to 0, 0.1, 0.3, 0.6 and 1.1 mmol l1 NH4Cl for 2 weeks, 4 weeks and 8 weeks. Values are the means ± S.E. (N ¼ 6). The means with different letters are significantly different among the treatments at the same exposure time (P < 0.05).
significantly (P < 0.05) in the 1.1 mmol l1 NH4Cl treatment compared to the control. The glutamine synthetase (GSase) activity did not vary significantly in any treatments at 4 weeks (P > 0.05, Fig. 4). At 8 weeks, however, the GSase activity was significantly lower in the 0.6 and 1.1 mmol l1 treatments than in the 0 and 0.1 mmol l1 treatments (P < 0.05). 3.3. Ammonia and urea excretion After 4-week HEA exposure, the JAmm of the fed fish significantly increased at 0.1 mmol l1 NH4Cl (P < 0.05, Fig. 5A) and then decreased with the increase of ammonia levels, reaching the lowest level at 1.1 mmol l1 NH4Cl (P < 0.05). The JAmm of the fasted fish was highest in the 0.6 mmol l1 treatment (P < 0.05), followed by the 0.3 mmol l1 treatment (P < 0.05), and was similar among the other treatments (P > 0.05, Fig. 5A). After 8-week HEA exposure, all treatments had similar JAmm when the fish were fed (P > 0.05, Fig. 5B). The JAmm of the fasted fish
The growth of marine medaka was significantly inhibited when the NH4Cl levels were within 0.3e1.1 mmol l1 in this study. Growth reduction in chronic HEA exposure has been widely demonstrated in fish, such as the European sea bass (Dicentrarchus labrax) [9], Nile tilapia (Oreochromis niloticus L) [11], and common carp [39]. A decrease in feed intake under HEA exposure is suggested to be one of the main causes of a reduced growth rate [14,33]. Our findings confirm that the feed intake decreased in parallel with the growth rate of the fish when the NH4Cl levels were above 0.3 mmol l1. Similar results were also observed by Ref. [9]; who estimated that 90% of the reduction in growth rate was attributed to the decrease in feed intake in European sea bass exposed to 0.3 mmol l1 NH4Cl for 20 days. It was reported that the decrease of growth under HEA indicated that fish requires extra energy to cope with HEA [39]. The elevated JAmm could involve in the energy-dependent pathways in the HEA treatments coincident with the elevated MO2 in the 0.6 and 1.1 mmol l1 NH4Cl treatments. These results suggested that marine medaka allocate more energy to excrete ammonia instead of growth under HEA. Similar to our results, MO2 increased with increased ambient ammonia under chronic ammonia exposure was also observed in juvenile big bellied seahorse [2] and European seabass Dicentrarchus labrax [22]. That paradox of inadequate energy supply for the low feed intake and higher energy demand to deal with HEA existed in marine medaka under chronic HEA exposure. This, consequently, led to the over-consumption of energy metabolism-related metabolites. In this study, glucose, fumarate, and inosine decreased in the 0.6 and 1.1 mmol l1 NH4Cl treatments which were in line with above elaboration. In addition, fish exposed to the ambient NH4Cl level of 0.1 mmol l1, as an environmentally realistic concentration, had similar growth performance as the control, indicating that marine medaka could acclimate to the low level of ammonia [13]. also reported no significant effect of a chronic low ammonia level (0.2 mmol l1 NH4Cl) on the growth of juvenile spotted wolf fish (Anarhichas minor). However [27], found that the growth rate of walleye (Sander vitreus) significantly increased after 56 days of exposure to a low ammonia concentration (100e300 mmol l1 NH4Cl). These authors suggested that the low ammonia might enhance the nitrogen accessibility and thus improve the protein retention in the fish body [45]. also found that rainbow trout had a higher growth rate and more rapid protein production when exposed to low ammonia (70 mmol l1 TAmm) relative to the control after exposure for 71 days. The proposed mechanism by which exogenous ammonia stimulates growth involves an increase in protein synthesis for the incorporation of ammonia into amino acids [45]. 4.2. Regulation of ammonia metabolism in chronic ammonia exposure
Fig. 3. Amino acid catabolism rate (ACR) of marine medaka exposed to 0, 0.1, 0.3, 0.6 and 1.1 mmol l1 NH4Cl for 8 weeks. Values are the means ± S.E. (N ¼ 8). The means with different letters indicate significant differences among the treatments (P < 0.05).
The tissue TAmm in the 0.3e1.1 mmol l1 treatments increased to the highest level in the first two weeks and then decreased to comparable levels as the control at 8 weeks, suggesting that marine
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Table 1 The content of main metabolites in marine medaka exposed to 0, 0.1, 0.3, 0.6 and 1.1 mmol l1 NH4Cl for 8 weeks (mmol/g). Metabolite Amino acid b-alanine Arginine Glutamate Glutamine Histidine Aspartate Glycine Isoleucine Leucine Phenylalanine Threonine Tyrosine Valine Energy metabolism-related Creatine phosphate Glucose Fumarate Lactate Organic osmolytes Hyppotaurine Dimethylamine Inosine Taurine Others Choline Malonate O-acetylcholine O-phosphocholine 4-aminobutyrate
C0.1
C0 0.13 2.09 2.03 0.88 2.06 0.69 6.33 0.34 0.51 0.59 0.88 0.27 0.39
± ± ± ± ± ± ± ± ± ± ± ± ±
0.02a 0.24a 0.13a 0.06a 0.23a 0.06 0.53ab 0.04 0.07 0.1 0.1ab 0.03ab 0.04
0.12 2.03 2.03 0.88 1.95 0.67 6.02 0.31 0.59 0.55 0.91 0.35 0.47
C0.3 ± ± ± ± ± ± ± ± ± ± ± ± ±
0.01ab 0.19ab 0.15a 0.04a 0.25a 0.05 0.40ab 0.05 0.07 0.08 0.08a 0.05a 0.06
0.10 1.82 2.00 0.95 1.85 0.59 6.20 0.28 0.62 0.51 0.97 0.28 0.38
C0.6 ± ± ± ± ± ± ± ± ± ± ± ± ±
0.01ab 0.22ab 0.13a 0.08a 0.14a 0.04 0.35ab 0.04 0.08 0.08 0.15a 0.03ab 0.06
0.09 1.63 1.60 0.79 1.48 0.68 6.67 0.29 0.64 0.46 0.69 0.27 0.43
C1.1 ± ± ± ± ± ± ± ± ± ± ± ± ±
0.01ab 0.19ab 0.15ab 0.06ab 0.26ab 0.07 0.69a 0.04 0.08 0.07 0.07ab 0.04ab 0.06
0.09 1.46 1.44 0.67 1.10 0.63 4.83 0.27 0.62 0.37 0.60 0.24 0.46
± ± ± ± ± ± ± ± ± ± ± ± ±
0.01b 0.24b 0.20b 0.09b 0.26b 0.07 0.66b 0.05 0.09 0.06 0.09b 0.03b 0.09
5.96 ± 0.21ab 62.13 ± 9.13a 0.03 ± 0.003ab 1.73 ± 0.19ab
5.71 ± 0.34a 70.19 ± 10.51a 0.04 ± 0.004a 2.06 ± 0.20a
5.65 ± 0.24a 52.53 ± 8.80ab 0.03 ± 0.002ab 1.52 ± 0.08b
6.71 ± 0.43ab 50.94 ± 13.11ab 0.02 ± 0.005b 1.39 ± 0.16b
6.10 ± 0.46b 27.47 ± 8.11b 0.02 ± 0.004c 0.93 ± 0.14c
0.94 ± 0.09a 0.07 ± 0.01 1.45 ± 0.16 10.89 ± 1.01
0.92 ± 0.08a 0.07 ± 0.004 1.57 ± 0.07 11.37 ± 0.81
0.81 ± 0.06a 0.08 ± 0.002 1.49 ± 0.08 11.50 ± 0.41
0.80 ± 0.07a 0.08 ± 0.005 1.38 ± 0.09 11.31 ± 0.54
0.57 0.08 1.29 9.68
± ± ± ±
0.06b 0.01 0.07 1.21
0.01b 0.04b 0.01 0.11 0.02
0.04 0.25 0.11 0.07 0.25
± ± ± ± ±
0.01b 0.05ab 0.01 0.01 0.02
0.07 0.30 0.12 0.11 0.29
± ± ± ± ±
0.01ab 0.04ab 0.01 0.01 0.02
0.08 0.20 0.14 0.12 0.29
± ± ± ± ±
0.01a 0.02a 0.01 0.01 0.03
0.06 0.27 0.12 0.19 0.24
± ± ± ± ±
0.01ab 0.03ab 0.01 0.08 0.02
0.05 0.34 0.13 0.20 0.25
± ± ± ± ±
Values are the means ± S.E. (N ¼ 10). The means in the same metabolites with different letters indicate significant differences among different treatments (P < 0.05).
Fig. 4. Glutamine synthetase (GSase) activity in marine medaka exposed to 0, 0.1, 0.3, 0.6 and 1.1 mmol l1 NH4Cl for 4 and 8 weeks. Values are the means ± S.E. (N ¼ 5). The means with different letters indicate significant differences among the treatments at the same exposure time (P < 0.05).
medaka acclimated to HEA after chronic exposure. Similar findings were reported in Atlantic salmon parr (Salmo salar L.). The plasma ammonia of that was significantly increased from 1.5 mmol l1 to 1.7 mmol l1 the first 22 days, and then declined to 0.7 mmol l1 with no significantly difference with the control level at the 105 days exposure of 480 mmol l1 HEA exposure [21]. This demonstrates that fish could maintain a relatively stable tissue TAmm under chronic ammonia exposure. Contrary to chronic exposure, fish often increase their plasma TAmm to a steady level during acute HEA exposure but do not recover to the control level [8,47]. Based on the
results of the present study, the decrease in TAmm in the fish tissue resulted from the decrease in endogenous ammonia and the increase in the output of ammonia, including decreasing ammonia absorption from food (i.e., decrease in feed intake, Fig. 1B), decreasing amino acid catabolism (Fig. 3), and increasing ammonia excretion (Fig. 5A and B). Regulating the tissue TAmm due to decreased feed intake was referred to above (in section 4.1). Here, we also determined the amino acid catabolism rate, and the results demonstrated that this rate apparently decreased when the ambient NH4Cl levels increased from 0.3 to 1.1 mmol l1, suggesting that the regulation of amino acid catabolism also plays a critical role in ammonia avoidance. Reduction of endogenous ammonia production by decreasing the amino acid catabolism rate is suggested to be an effective strategy for ammonia avoidance in fish [24]. Mudskipper (Periophthalmodon schlosseri), for instance, is an air-breathing fish with limited gill ventilation that can easily build-up ammonia in the body. These fish decrease their proteolysis rate and amino acid catabolism rate to avoid ammonia intoxication from elevating endogenous ammonia level in their tissues [24]. Loach (Misgurnus anguillicaudatus), another air-breathing fish, accumulated 14.5 mmol/g hepatic ammonia, which is higher than the average ammonia level (1e10 mmol/g) of most air-tolerant teleosts [7]. These fish were also capable of decreasing amino acid catabolism to reduce endogenous ammonia production and thus stabilize the tissue TAmm in HEA exposure [7]. The increase in GSase activity is frequently found to be an important strategy for ammonia detoxification in fish, both under acute and chronic HEA exposure [3,50]. For example, GSase activity increased in various organs (liver, intestine, muscle) of the Chinese black sleeper (Bostrichthys sinensis) when exposed to 15 mmol l1 NH4Cl for 48 h [3] [15]. reported that the GSase activity at cellular level increased 3~4-fold in Nile tilapia brain after HEA
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treatments at 4 weeks of exposure and significantly decreased in the 0.3e1.1 mmol l1 treatments at 8 weeks of exposure. This suggested that marine medaka could provide more than enough GSase capacity and did not need to increase GSase activity. This was in line with the study of [53]; where the GSase activity changed little in the brain of rainbow trout exposed to 250 mmol l1 NH4(SO4)2 for 1 þ month. On the other hand, in the present study, we observed that marine medaka had adequately controlled the stable TAmm in tissue and detoxified ammonia by increasing their ammonia excretion (discussed below). As both the reaction catalyzed by GSase and ammonia excretion against ammonia gradient are ATP request the steady GSase activity in marine medaka was benefit for reducing their energy expenditure and reserve more energy for ammonia excretion. In this study, few changes in the amino acid levels were observed in the 0.1e0.6 mmol l1 ammonia treatments. This suggests that marine medaka can maintain balanced amino acid levels under chronic NH4Cl exposure (<0.6 mmol l1). However, threonine, arginine, glutamate, glutamine, b-alanine, glycine, tyrosine, and histidine in the 1.1 mmol l1 NH4Cl had lower levels than the other treatments. This may attributed to the declined feed intake and the low protein content after chronic HEA exposure. In fact, different types of amino acids had different responses in different fish under HEA exposure. For example, when juvenile Nile tilapia were exposed to 0.3 mmol l1 NH4Cl for 70 days, glutamine, glycine, alanine, histidine and serine increased and glutamate and aspartate decreased [15]. The fish tend to increase the level of total free amino acids to reduce endogenous ammonia to alleviate ammonia toxicity. However, marine medaka under chronic HEA exposure did not adopt this strategy but rather maintained steady GSase activity and balanced amino acids, which indicates an adaptation to chronic ammonia for marine medaka. 4.3. Regulation of nitrogen excretion at chronic ammonia exposure Fig. 5. Ammonia excretion rate (JAmm) of the fed and fasted marine medaka exposed to 0, 0.1, 0.3, 0.6 and 1.1 mmol l1 NH4Cl for 4 weeks (Fig. 6A) and 8 weeks (Fig. 6B). Values are the means ± S.E. (N ¼ 5). The means with different letters indicate significant differences among the treatments at the same exposure time (P < 0.05).
Fig. 6. Urea excretion rate (JUrea) of the fed and fasted marine medaka exposed to 0, 0.1, 0.3, 0.6 and 1.1 mmol l1 NH4Cl for 8 weeks. Values are the means ± S.E. (N ¼ 5). The means with different letters indicate significant differences among the treatments (P < 0.05).
exposure (5 or 10 mg l1 total ammonia nitrogen) for 70 days. In contrast, we found that the GSase activity differed little among all
We demonstrated that regulation of the ammonia excretion rate was an important strategy to maintain a relatively stable tissue TAmm in marine medaka under HEA exposure. The present study demonstrated that JAmm was generally maintained or significantly increased in the fed and fasted fish in different HEA exposure treatments, suggesting ammonia excretion was elevated to counteract chronic HEA exposure. Even in the highest ammonia exposure treatment (1.1 mmol l1), although JAmm in the fed fish was reduced after 4-week exposure, it was recovered to the control level after 8-week exposure. These results could explain why the tissue TAmm at 1.1 mmol l1 HEA treatment was initially elevated but recovered to the control level at the end of 8-week exposure. Similar to the present study, many fish can excrete ammonia actively against an inwardly directed ammonia gradient, such as Clarias gariepinus [17], rainbow trout [30,47], Alcolapia grahami [20], E. stoutii [8], and spiny dogfish shark Squalus acanthias [31]. The mechanism of that may involve rhesus glycoprotein with other transporters (V-type Hþ-ATPase, Naþ/Hþ exchanger, Naþ/KþATPase, carbonic anhydrase) [30,51,47,38]. At acute HEA exposure, JAmm is usually initially reversed, but will be reestablished and greater than control level following a few hours of exposure [8,30,47]. However, no net-uptake of ammonia was observed in marine medaka after the fish exposed for 8 weeks. It seems that marine medaka had acclimated the HEA and directly stepped into the stage of excreting ammonia. Obviously, our results revealed different patterns of ammonia excretion between acute and chronic HEA exposure. The marine medaka could acclimate to the ammonia environment during the long-term exposure, and apply to elevate ammonia excretion as one of the important mechanisms to reduce body ammonia level.
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Ambient ammonia did not affect the JUrea of marine medaka indicating that marine medaka was not able to avoid ammonia toxicity by increasing urea excretion. Similar results have also been reported in the ammonotelic Pacific hagfish and killifish, which did not convert ammonia into urea nor significantly increase the JUrea in response to HEA [8,16]. Besides, marine medaka excrete only about 20% nitrogenous waste as urea which was likely to come from purine degradation or argininolysis [41,42]. This ureogenesis way was metabolically costly, for which synthesizing 1 mol urea requires at least 2 mol of ATP [46]. Therefore, it is not a common strategy for most ammonotelic fish handling ammonia. 4.4. Conclusions Our study demonstrated that chronic exposure to >0.3 mmol l1 ammonia had clear toxic effects on marine medaka. The fish maintained a constant tissue ammonia level at chronic ammonia exposure, which showed a different pattern from that at acute ammonia exposure. The maintenance of the tissue ammonia level in marine medaka mainly involved in decrease of the amino acid catabolism rate, and increase of the ammonia excretion rate. On the other hand, marine medaka allocate more energy to deal with HEA by increasing oxygen consumption. However, the conversion of ammonia into amino acids or urea might not be essential for the detoxification of ammonia in this fish at chronic ammonia exposure.
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We thank the two anonymous reviewers for their constructive comments on this work. This research was supported by the State Key Development Program for Basic Research of China (2015CB452904), National Natural Science Foundation of China (41376161, 31501862), Research Fund Program of Guangdong Provincial Key Laboratory of Marine Resources and Coastal Engineering (201503) and Science and Technology Planning Project of Guangdong Province, China (2014B030301064). References [1] P.A. Ackerman, B.J. Wicks, G.K. Iwama, D.J. Randall, Low levels of environmental ammonia increase susceptibility to disease in Chinook salmon smolts, Physiol. Biochem. Zool. 79 (2006) 695e707. [2] M.B. Adams, M.D. Powell, G.J. Purser, Effect of acute and chronic ammonia and nitrite exposure on oxygen consumption and growth of juvenile big bellied seahorse, J. Fish. Biol. 58 (2001) 848e860. [3] P.M. Anderson, M.A. Broderius, K.C. Fong, K.N.T. Tsui, S.F. Chew, Y.K. Ip, Glutamine synthetase expression in liver, muscle, stomach and intestine of Bostrichthys sinensis in response to exposure to a high exogenous ammonia concentration, J. Exp. Biol. 205 (2002) 2053e2065. € €ksal, A. Ozkul, [4] A.Ç.K. Benli, G. Ko Sublethal ammonia exposure of Nile tilapia (Oreochromis niloticus L.): effects on gill, liver and kidney histology, Chemosphere 72 (2008) 1355e1358. [5] J.C. Chen, P.C. Liu, Y.T. Lin, C.K. Lee, Super intensive culture of red-tailed shrimp Penaeus penicillatus, J. World Aquacult. Soc. 19 (1988) 127e131. [6] C.H. Cheng, F.F. Yang, R.Z. Ling, S.A. Liao, Y.T. Miao, C.X. Ye, A.L. Wang, Effects of ammonia exposure on apoptosis, oxidative stress and immune response in pufferfish (Takifugu obscurus), Aquat. Toxicol. 164 (2015) 61e71. [7] S.F. Chew, Y. Jin, Y.K. Ip, The loach Misgurnus anguillicaudatus reduces amino acid catabolism and accumulates alanine and glutamine during aerial exposure, Physiol. Biochem. Zool. 74 (2001) 226e237. [8] A.M. Clifford, G.G. Goss, M.P. Wilkie, Adaptations of a deep sea scavenger: high ammonia tolerance and active NH4þ excretion by the Pacific hagfish (Eptatretus stoutii), Comp. Biochem. Physiol. A Mol. Integr. Physiol. 182 (2015) 64e74. [9] A. Dosdat, J. Person-Le Ruyet, D. Coves, G. Dutto, E. Gasset, A. Le Roux, , Effect of chronic exposure to ammonia on growth, food utilisation G. Lemarie and metabolism of the European sea bass (Dicentrarchus labrax), Aquat. Living Resour. 16 (2003) 509e520. [10] F.B. Eddy, Ammonia in estuaries and effects on fish, J. Fish. Biol. 67 (2005) 1495e1513. [11] S.A. El-Shafai, F.A. El-Gohary, F.A. Nasr, N.P. van der Steen, H.J. Gijzen, Chronic
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