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Aquaculture 434 (2014) 325–333
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Aquaculture
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Effect of dietary vitamin C on non-specific immunity and mRNAexpression of three heat shock proteins (HSPs) in juvenile Megalobramaamblycephala under pH stress
Jinjuan Wan a,b,1, Xianping Ge a,⁎, Bo Liu a,⁎⁎, Jun Xie a, Suli Cui a, Ming Zhou a,c, Silei Xia a, Ruli Chen a
a Key Laboratory of Genetic Breeding and Aquaculture Biology of Freshwater Fishes, Ministry of Agriculture, Freshwater Fisheries Research Center, Chinese Academy of Fishery Sciences,9 Shanshui East Road, Wuxi 214081, Chinab Freshwater Fisheries Research Institute of Jiangsu Province, 79 Chating East Street, Nanjing 210017, Chinac Shanghai Ocean University, 999 Hucheng Loop Road, Shanghai 201306, China
⁎ Correspondence to: X.P. Ge, No. 9 Shanshui East RoaChina. Tel./fax: +86 510 85557892.⁎⁎ Correspondence to: B. Liu, No. 9 Shanshui East RoaChina. Tel./fax: +86 510 85551454.
E-mail addresses: [email protected] (J. Wan), gex(B. Liu).
1 Tel.: +86 25 86581562; fax: +86 25 86618250.
http://dx.doi.org/10.1016/j.aquaculture.2014.08.0430044-8486/© 2014 Elsevier B.V. All rights reserved.
a b s t r a c t
a r t i c l e i n f oArticle history:Received 13 April 2014Received in revised form 27 August 2014Accepted 28 August 2014Available online 4 September 2014
Keywords:Megalobrama amblycephalaVitamin CNon-specific immunityHSPs mRNA expressionpH stress
This study determined the effect of dietary vitamin C on non-specific immunity and onmRNAexpression of threeHSPs in juvenileMegalobrama amblycephala under pH stress. Six diets were formulated to contain 0.2, 33.4, 65.8,133.7, 251.5 and 501.5 mg/kg of vitamin C. Each diet was fed to triplicate groups of fish in cylindrical tanks(220-L, N = 25 fish/tank). After 90 days of feeding, 20 fish per tank were exposed to pH stress (pH ≈ 9.5) for10 days. Serum alanine aminotransferase (ALT), alkaline phosphatase (ALP), total protein (TP), complementC3 (C3), complement C4 (C4), cortisol, hepatic superoxide dismutase (SOD), antisuperoxide anion free radical(ASAFR), malondialdehyde (MDA) and the relative mRNA expression of heat shock protein 60 (HSP60), 70(HSP70), and 90 (HSP90) were investigated. The results showed that after pH stress, serum ALP, TP, C3, C4,hepatic SOD, and ASAFR levels were significantly reduced (P b 0.05) while serum ALT, cortisol, hepatic MDAand HSP60 and HSP70 mRNA expression levels were significantly increased (P b 0.05). On the other hand, sup-plementation with vitamin C significantly reduced levels of serum cortisol (65.8–501.5 mg/kg vitamin C dietgroups), ALT (33.4 mg/kg vitamin Cdiet group) and hepaticMDA (133.7 and 251.5 mg/kg vitamin Cdiet groups).Supplemented groups had increased serum ALP activity (all treated groups) as well as increased relative mRNAexpression of hepatic HSP60 (133.7–501.5 mg/kg vitamin C diet groups) and HSP70 (133.7 and 251.5 mg/kgvitamin C diet groups) (P b 0.05). These results indicate that ingestion of a basal diet supplemented with133.7–251.5 mg/kg vitamin C could enhance resistance against pH stress in M. amblycephala to some degree.
© 2014 Elsevier B.V. All rights reserved.
1. Introduction
In aquaculture, fish often encounter high temperature, environmen-tal pollutants, high stocking density, invasion of bacteria and viruses,and human interference. All these adverse factors cause a stress re-sponse in fish. In freshwater ponds, pH levels fluctuate from 6.6 to10.2 because of carbon dioxide consumption (Boyd, 1990; Murray andZeibell, 1984). High pH levels cause a stress response in fish, resultingin oxidative stress (Liu et al., 2007), disturbances in electrolytic balance(Wilkie andWood, 1991, 1994), slowgrowth (Panet al., 2007) and a de-cline in survival (Wang et al., 2002), which are sometimes accompaniedby serious disease or even mass death (Li and Chen, 2008).
d, Wuxi PC 214081 FFRC CAFS
d, Wuxi PC 214081 FFRC CAFS
[email protected] (X. Ge), [email protected]
Vitamin C, also known as L-ascorbic acid, is an essential micronutrientfor normal growth andphysiological function infish. It plays an importantrole in growth (Ai et al., 2004; Bae et al., 2012), collagen formation (Eoand Lee, 2008), reproduction (Emata et al., 2000; Lee and Dabrowski,2004), minimizing toxicity by water contaminants (Li and Lovell, 1985),immune response (Sobhana et al., 2002; Ai et al., 2006; Nayak et al.,2007) and response to stressors (Ortuno et al., 2003; Chen et al., 2004;Trenzado et al., 2007; Ming et al., 2012). An exogenous source of vitaminC is required infish diet becausemost teleostfish are unable to synthesizevitamin C (Fracalossi et al., 2001). Inadequate supply of dietary vitamin Cusually results in a number of deficiency symptoms such as spinal defor-mation, retarded growth and depressed immunity, which causes signifi-cant losses in practical fish farming, especially during the sensitive startperiod (Tewary and Patra, 2008).
Wuchang bream (Megalobrama amblycephala Yih) is a major fresh-water species cultured in China. However, in recent years, there wasan increase in disease in M. amblycephala especially diseases causedby stress. Various immunostimulants (i.e., glucans, vitamins, essentialfatty acids) have beenwidely used in aquaculture, and their stimulating
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effects on disease and stress resistance have been documented (Boshraet al., 2006). In a previous study, we evaluated the effect of differentlevels of dietary vitamin C on growth performance, hematology andmuscle physiochemical indices, and mortality of M. amblycephala. Ourresults indicated that weight gain (WG) and specific rate (SGR)increased with the increase of dietary vitamin C. For optimal WG, theminimum dietary vitamin C level was 150 mg/kg (Wan et al., 2013).However, to our knowledge, no information has been published thatevaluates the effect of dietary vitamin C levels on the anti-pH stress re-sponse for M. amblycephala. Therefore, we sought to study the effect ofvitamin C on non-specific immunity and on mRNA expression of threeHSPs in juvenile M. amblycephala under high pH stress conditions(pH ≈ 9.5). Our results provide suggestions for the prevention of dis-ease and alleviation of stress.
2. Materials and methods
2.1. Fish, vitamin C, and diet
We obtained 450 healthy juvenile M. amblycephala that were ofsimilar size (mean weight 6.40 ± 0.05 g) from the Nanquan fish farmof the Freshwater Fisheries Research Center, Chinese Academy ofFishery Science, China. The fish were placed in 18 cylindrical tanks(220-L, N = 25 fish/tank) and were acclimated for 22 days. Followingacclimation, we randomly divided the 18 tanks into six groups (N = 3tanks/group): the control group was fed a basic diet (Table 1) and fivetreatment groups were fed a basal diet supplemented with 33.4, 65.8,133.7, 251.5 and 501.5 mg/kg of vitamin C, respectively.
The basal diet was formulated to contain about 32.12% crude proteinand 6.18% lipid. Six diets were formulated to contain 0.0, 30.0, 60.0,120.0, 240.0 and 480.0 mg ascorbic acid per kg, respectively. Coated-ascorbic acid (CAA) (95% ascorbic acid equivalent, Roche, Switzerland)was used as the vitamin C source. However, the analyzed ascorbic acidlevels were 0.2, 33.4, 65.8, 133.7, 251.5 and 501.5 mg kg−1 for the sixdiets. Various feedstuffs were separately pulverized and screenedthrough a size 60 mesh sieve and first mixed with calcium dihydrogenphosphate, soy lecithin, choline chloride, ethoxyquin, mineral andvitamin premix (vitamin C free), then evenly mixed with vitamin C,and finally evenly mixed with bulk feed ingredients. The feed wasthen pelleted into 1 mm granular feed using a pelletizer (4-2 style,Xinchang Machinery Ltd, China) and dried in a ventilated oven at40 °C for 12 h. All pellets were stored at 4 °C until use.
Table 1Formulation and composition of experimental diet.
Ingredients (%) Proximate composition (%)
Casein (vitamin C free) 27.50 Crude protein 32.12Gelatin 6.50 Crude lipid 6.68Calcium dihydrogen phosphate 2.75 Nitrogen-free extractc 37.91Soybean oil 6.00 Lysine 2.26Soy lecithin 1.00 Methionine 0.79Choline chloride (50%) 0.15Vitamin premix (vitamin C free)a 0.50Mineral premixb 0.50Dextrin 10.00α-Starch 25.00Microcrystalline cellulose 9.05Carboxyl-methyl cellulose 11.00Ethoxyquin 0.05Total 100.00
a Vitamin premix (IU or per kg premix): vitamin A, 900,000 IU; vitamin D, 250,000 IU;vitamin E, 4500 mg; vitamin K 3220 mg; vitamin B1, 320 mg; vitamin B2, 1090 mg;vitamin B6, 5000 mg; vitamin B12, 116 mg; pantothenate, 1000 mg; folic acid, 65 mg;choline, 60,000 mg; biotin, 50 mg; Inositol, 15,000 mg; Niacin acid, 2500 mg, Vitamin C,20,000 mg.
b Mineral premix (per kg premix): CuSO4·5H2O, 2.5 g; FeSO4·7H2O, 28 g; ZnSO4·7H2O,22 g; MnSO4·4H2O, 9 g; Na2SeO3, 0.045 g; KI, 0.026 g; CoCl2·6H2O,0 .1 g.
c Nitrogen-free extract, % = 100%-(Moisture + CP + EE + CF + Ash)%, and theothers are measured according to the hFeed Industry Standard of China.
2.2. Rearing management
Wecultured thefish in the aquariumwith an automatic temperaturecontrol recirculating system. The tanks were supplied with aeratedrecycled water at a rate of 2 L min−1. The fish were fed by hand threetimes a day (8:00–9:00, 11:00–12:00, and 15:00–16:00) at a feedingrate of 2.0%–4.0% body weight during the experiment. The amount offeed was adjusted every two weeks to account for increasing bodyweight. During the experiment, we measured the water temperatureat 8:00 and 16:00 each day and checked the water quality once aweek. The mean water quality indices were: water temperature rangedfrom 26 °C to 28 °C, DO N 6 mg L−1, NH3 b 0.05 mg L−1, and pH 7.20–7.60. After 90 days, fish from each tank were counted and weighed.
2.3. pH challenge experiment
Upon completion of the rearing experiment and according to themethods described in Li and Chen (2008), after the first sampling(0 d), 20 fish of similar size were sampled from each tank and subjectedto a pH stress (high pH level: 9.5) test for 10 days in cylindrical tanks(220-L) with running water. Water temperature ranged from 26 °C to28 °C, and the flow rate was 2 L/min, DO N 6 mg/L, NH3 b 0.05 mg L−1.Adding 4 N NaOH adjusted the water pH level twice a day (8:00,16:00). During the stress experiment, there was no feeding andminimalhuman interference to prevent additional stress.
2.4. Sampling and processing
Upon completion of the rearing experiment, blood samples werecollected from three fish per tank before stress (0 d) and on days 1, 5or 10 after high pH stress, respectively. Fish were netted quickly andanesthetized with MS-222 (Tricaine methanesulfonate, Sigma, USA) ata concentration of 150 mg/L, and then blood was sampled from thecaudal vein using a 2-mL medical syringe, and was allowed to clot at4 °C for 1–2 h. Following centrifugation (3000 rpm, 10 min, 4 °C), theserum was removed and frozen at −20 °C until use. The abdominalcavity of fish was cut open immediately after blood collection. About0.1 g of liver was frozen in liquid nitrogen and stored at −80 °C fordetermination of gene expression. Another piece of liver was stored at−20 °C to measure antioxidant capacity.
2.5. Analysis and measurement
2.5.1. Serum alanine aminotransferase (ALT), alkaline phosphatase (ALP)and total protein (TP) measurement
The level of ALT, ALP and TP were measured by an automatic bio-chemical analyzer Mindary BS-400 (Shenzhen, China) using assay kitspurchased from Shenzhen Mindary Bio-medical Electronics Co., Ltd.
2.5.2. Serum complement C3 (C3), complement C4 (C4) and cortisolmeasurement
The levels of C3 and C4 were measured using the immuno-turbidimetric method and the kits were purchased from ZhejiangYilikang Biotech Co., Ltd. The level of cortisol was measured by theautomatic chemiluminescence immunoassay analyzer MAGLUMI 1000(Shenzhen, China) using assay kits purchased from Shenzhen NewIndustries Biomedical Engineering Co., Ltd.
2.5.3. Hepatic superoxide dismutase (SOD), antisuperoxide anion freeradical (ASAFR) and malondialdehyde (MDA) measurement
Hepatic samples were homogenized in ice-cold phosphate buffer(1:10 dilution) (phosphate buffer: 0.064 M, pH 7.4). The homogenatewas then centrifuged for 10 min (4 °C, 4000 ×g) and aliquots of thesupernatant were used to quantify hepatic SOD, ASAFR and MDA.Hepatic SOD activity, ASAFR activity and MDA content were measuredusing the xanthine oxidase method (Granelli et al., 1995), xanthine
327J. Wan et al. / Aquaculture 434 (2014) 325–333
oxidase method and barbituric acid colorimetry (Mihara and Uchiyarma,1978), respectively. We measured the hepatopancreas protein contentusing the Folinmethod (Lowry et al., 1951), with bovine serumalbuminas the standard. These kits were purchased fromNanjing Jiancheng Bio-engineering Institute of China.
2.5.4. Real-time PCR measurement of hepatic HSP60, HSP70 and HSP90M. amblycephala cDNA sequences in GenBank were used to design
the primers for HSP60 (accession no. KC521465), HSP70 (accessionno. FJ375325), HSP90 (accession no. KC521466) and Beta-actin(accession no. AB037865) (Wan et al., 2014). The primers were:(1) 5′-TGCTGTCTACTGCTGAAGCCGTTGT-3′ and 5′-CCATCACTCAGTTTCGGCAGGTTT-3′ for HSP60 cDNA; (2) 5′-CGACGCCAACGGAATCCTAAAT-3 and 5′-CTTTGCTCAGTCTGCCCTTGT-3′ for HSP70 cDNA;(3) 5′-TGCGGGACAACTCCACCAT-3′ and 5′-TCCAATGAGAACCCAGAGGAAAGC-3′ for HSP90 cDNA and (4) 5′-TCTGCTATGTGGCTCTTGACTTCG-3′ and 5′-CCTCTGGGCACCTGAACCTCT-3′ for Beta-actincDNA. All primers were synthesized by Shanghai Biocolor, BioScience &Technology Company, China. The PCR products were 123–152 bp long.
Total RNA was extracted from 50 to 100 mg of liver tissue usingTrizol reagent (Dalian Takara Co. Ltd., China). The purified RNA general-ly had an OD260/OD280 ratio of 1.8–2.0. RNA samples were treated withRQ1 RNase-Free DNase (Dalian Takara Co. Limited, China) to avoidgenomic DNA amplification. We generated cDNA from 500 ng DNase-treated RNA using an ExScript™ RT-PCR Kit (Dalian Takara Co. Ltd.,China). The reverse transcription PCR reaction solution consisted of500 ng RNA, 2 μL 5× buffer, 0.5 μL dT-AP Primer (50 mM), 0.25 μLExScript™ RTase (200 U μL−1), and DEPC H2O, up to a final volume of10 μL. The reaction conditions were as follows: 42 °C for 40 min, 90 °Cfor 2 min, and 4 °C thereafter.
Real-timequantitative PCRwas used to determinemRNA levelswitha SYBR Green one-step fluorescence kit (Ming et al., 2012) and wasperformed in a Mini Opticon Real-Time Detector (Bio-Rad, USA). Thefluorescent quantitative PCR reaction solution consisted of 12.5 μLSYBR® premix Ex Taq™ (2×), 0.5 μL PCR Forward Primer (10 μM),0.5 μL PCR Reverse Primer (10 μM), 2.0 μL RT reaction mix (cDNAsolution), and 9.5 μL dH2O. The reaction conditions were as follows:95 °C for 10 s, followed by 45 cycles consisting of 95 °C for 5 s, 62 °Cfor 15 s, 72 °C for 10 s, plate read, and final step at 72 °C for 3 min.After the program finished, the Ct values of the target genes (threeHSPs) and a chosen reference gene (Beta-actin) were obtained fromeach sample. The standard equation and correlation coefficient weredetermined by constructing a standard curve using a serial dilutionof cDNA; HSP60: Y = −0.310x + 10.65, R2 = 0.991; HSP70: Y =−0.361x + 13.38, R2 = 0.995; HSP90: Y = −0.314x + 10.29,R2 = 0.996; Beta-actin: Y = −0.304x + 9.817, R2 = 0.990; Y is thelogarithm of the starting template to base 10 and x is the Ct values.The relative expression level of the gene could be calculated bydouble-standard curve method (Tang and Jia, 2008).
2.5.5. Dietary ascorbic acid measurementEach diet (five samples per group) was homogenized in ice-cold
phosphate buffer (1:10 dilution) (phosphate buffer: 0.064 M, pH 7.4).The homogenate was then centrifuged for 10 min (4 °C, 4000 ×g) andaliquots of the supernatant were used to quantify the correspondinglevels of dietary ascorbic acid. Determination of ascorbic acid in the dietwas taken by spectrophotometer and the Fe (II)-1,10-Phenanthroline-BPR system was used. The assay kits were purchased from NanjingJiancheng Bioengineering Institute of China.
2.6. Data statistics and analysis
Statistical analysis was performed using SPSS 16.0 for Windows.Data were expressed as mean ± SEM and subjected to a one-wayANOVA followed by Duncan's multiple range test. P value b 0.05 wasconsidered to be significant.
3. Results
3.1. The effect of vitamin C on serum ALT, ALP and TP in juvenileM. amblycephala
Fig. 1A shows that serum ALT activity in all groups increased underpH stress. Before stress (0 h), the ALT levels in fish fed a diet with 133.7and 501.5 mg/kg vitamin C were significantly lower than those of thecontrol (P b 0.05). After pH stress, the ALT activity in fish fed the33.4 mg/kg vitamin C diet was still significantly lower than that of thecontrol group 10 days after pH stress (P b 0.05), while the difference be-tween the other treatments and the control group was not significant(P N 0.05).
Fig. 1B indicates that serum ALP activity in all groups decreasedunder pH stress. Before stress, the differences among the ALP levels ofall groupswere not significant (P N 0.05). After pH stress, the ALP activityin fish fed the 33.4 mg/kg vitamin C diet group was significantly higherthan that of the control 10 days after pH stress. ALP activity in othertreated groups were significantly higher than that of the control 5 daysand 10 days after pH stress (P b 0.05), while the difference betweenthe other treatments and the control group was not significant(P N 0.05).
Serum TP levels in all groups declined under pH stress (Fig. 1C).Before stress, the TP levels in all groups were not significantly different(P N 0.05). After stress, the TP levels in fish fed the diet with 133.7 mg/kgvitamin C was significantly higher than that of the control 10 days afterpH stress (P b 0.05), while the difference between the other treatmentsand the control group was not significant (P N 0.05).
3.2. The effect of vitamin C on serum C3, C4 and cortisol on juvenileM. amblycephala
Fig. 2A indicates that serum C3 content in juvenile M. amblycephalain all groups decreased under pH stress. Before pH stress, the C3 contentin fish fed the diet with 501.5 mg/kg vitamin C was significantly higherthan that in other groups (P b 0.05). After stress, the C3 content in fishfed the 501.5 mg/kg vitamin C diet was significantly higher than thatof the control 1 day after pH stress, C3 content in fish fed all treatmentdiets was significantly higher than that of the control 5 days after pHstress (P b 0.05), while the difference between the other treatmentsand the control group was not significant (P N 0.05).
Serum C4 content in all groups also decreased under pH stress(Fig. 2B). Before stress, the C4 content in fish fed the diet with133.7 mg/kg vitamin C was significantly higher than that of the othergroups (P b 0.05). After stress, the C4 content in fish fed the 65.8 mg/kgvitamin C diet was significantly higher than that of the control5 days after pH stress. C4 content in fish fed the 133.7, 251.5 and501.5 mg/kg vitamin C diets was significantly higher than that of thecontrol 10 days after pH stress (P b 0.05), while the difference betweenthe other treatments and the control group was not significant(P N 0.05).
Under pH stress, serum cortisol content in all groups tended to in-crease at first and then decrease (Fig. 2C). Before stress, the differencesamong all groups were not significant (P N 0.05). After stress, the corti-sol levels in fish fed the diet with 33.4 mg/kg of vitamin C were signifi-cantly lower than those of the control 5 days after pH stress. Serumcortisol levels in the other treated groups were significantly lowerthan those of the control 1 day and 5 days after pH stress (P b 0.05),while the difference between the other treatments and the controlgroup was not significant (P N 0.05).
3.3. The effect of vitamin C on hepatic anti-oxidization enzyme activity injuvenile M. amblycephala
As shown in Fig. 3A, hepatic SOD activity in all groups tended to in-crease at first and then decrease under pH stress. Before pH stress, the
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Fig. 1. Effect of various levels of vitamin C on serum ALT (A), ALP (B) and TP (C) levels of juvenileM. amblycephala under pH stress. Notes: Different capital letters above the bars indicatesignificant differences (P b 0.05) at different time points in the same group in Duncan's test; different small letters above the bars indicate significant differences (P b 0.05) in differentgroups at the same time point in Duncan's test; data are expressed as mean ± SEM (n = 9); the same below.
328 J. Wan et al. / Aquaculture 434 (2014) 325–333
SOD activity in fish fed a diet with 65.8, 133.7 and 251.5 mg/kg ofvitamin C was significantly higher than that of the control. After stress,the SOD activity in fish fed the 65.8 mg/kg vitamin C diet was signifi-cantly higher than that of the control 1 day and 5 days after pH stress.SOD activity in fish fed the 133.7, 251.5 and 501.5 mg/kg vitamin Cdiets was significantly higher than that of the control 1, 5, and 10 daysafter pH stress (P b 0.05), while the difference between the other treat-ments and the control group was not significant (P N 0.05).
Fig. 3B shows that hepatic ASAFR activity in all groups also appearedto increase at first and then decrease under pH stress, similar to thechange of SOD. Before stress, ASAFR activities in treated groups weresignificantly higher than those of the control (P b 0.05). After stress, theASAFR activity in fish fed the 65.8 mg/kg vitamin C diet was significantlyhigher than that of the control 1day and5days after pH stress, andASAFR
activity infish fed the 133.7 mg/kg vitamin Cdietwas significantly higherthan that of the control 1 day after pH stress. ASAFR activity in treatedgroups was significantly higher than that of the control 10 days afterpH stress (P b 0.05), while the difference between the other treatmentsand the control group was not significant (P N 0.05).
It can be seen in Fig. 3C that hepatic MDA content in all groupstended to increase under pH stress. Before stress, there were no signifi-cant differences among the MDA levels in all groups (P N 0.05). Afterstress, compared with the control group, the MDA content in fish fedthe diet with 133.7 mg/kg vitamin C decreased significantly 5 days and10 days after pH stress. The MDA content in fish fed the 251.5 mg/kgvitamin C diet decreased significantly 10 days after pH stress (P b 0.05),while the difference between the other treatments and the controlgroup was not significant (P N 0.05).
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Fig. 2. Effect of various levels of vitamin C on serum C3 (A), C4 (B) and cortisol (C) levels of juvenileM. amblycephala under pH stress. Note: Data are expressed as mean ± SEM (n= 9).Legends are the same as in Fig. 1.
329J. Wan et al. / Aquaculture 434 (2014) 325–333
3.4. The effect of vitamin C on the relative level of hepatic HSP60, HSP70 andHSP90 mRNA in juvenile M. amblycephala
Fig. 4A shows that under pH stress, the expression levels of hepat-ic HSP60 mRNA in all groups tended to increase at first and then de-crease. Before stress, the HSP60 mRNA expression levels in the fishfed a diet with 133.7 and 251.5 mg/kg of vitamin C were significantlyhigher than those of the other groups (P b 0.05). After pH stress, theHSP60 mRNA expression level in the fish fed the 133.7 mg/kg vita-min C diet was significantly higher than that of the control 1 dayand 5 days after pH stress. HSP60 mRNA expression level in the fishfed the 251.5 mg/kg vitamin C diet was significantly higher thanthat of the control 1 day and 10 days after pH stress. The expressionlevels of HSP60 mRNA in the fish fed the 501.5 mg/kg vitamin C dietwas significantly higher than those of the control 1 day after pH stress(P b 0.05), while the difference between the other treatments and thecontrol group was not significant (P N 0.05).
The expression levels of hepatic HSP70 mRNA in all groups also ap-peared to increase at first and then decrease under pH stress, similarto the change inHSP60 (Fig. 4B). Before stress, theHSP70mRNAexpres-sion level in the fish fed the diet with 251.5 mg/kg vitamin Cwas higherthan that of the other groups (P b 0.05). After stress, the expressionlevels of HSP70mRNA in fish fed the 133.7, 251.5 and 501.5 mg/kg vita-min C diets were significantly higher than those of the control by day 1and day 5. On the other hand 10 days after pH stress, the expressionlevel of HSP70 mRNA in the fish fed the diet with 501.5 mg/kg vitaminC was significantly higher than that of the control (P b 0.05), while thedifference between the other treatments and the control group wasnot significant (P N 0.05).
Fig. 4C indicates that the expression levels of hepaticHSP90mRNA inall groups increased at first and then decreased, and by day 10, the ex-pression level of HSP90 mRNA returned to a similar level before stress.After pH stress, the expression level of HSP90 mRNA in fish fed thediet with 133.7 mg/kg vitamin C was significantly higher than that of
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AS
AF
R l
evel
/(U
/mg p
rot)
0.2
33.4
65.8
133.7
251.5
501.5
Before stress Days after stress
B
aC
aBC aB
aA
aCaC
abB
abA
aC aC
abB
abA
aCaBC bB
bA
aC
aBC abB
bA
aCaBC
abB
abA
0
10
20
30
40
0d 1d 5d 10d
MD
A
acti
vit
y/(
nm
ol/
mg p
rot)
0.2
33.4
65.8
133.7
251.5
501.5
Before stress Days after stress
C
Fig. 3.Effect of various levels of vitamin C on hepatic SOD (A), ASARF (B) andMDA (C) levels of juvenileM. amblycephalaunder pH stress. Note: Data are expressed asmean±SEM(n=9).Legends are the same as in Fig. 1.
330 J. Wan et al. / Aquaculture 434 (2014) 325–333
the control 10 days after pH stress. HSP90 mRNA expression levels infish fed the 251.5 mg/kg vitamin C diet were significantly higher thanthose of the control on days 1, 5 and 10 after pH stress. The expressionlevel of HSP90 mRNA in fish fed the 501.5 mg/kg vitamin C diet wassignificantly higher than that of the control at 5 days and 10 days afterpH stress (P b 0.05), while the difference between the other treatmentsand the control group was not significant (P N 0.05).
4. Discussion
Like other vertebrates, stressed fish exhibit a generalized stress re-sponse that is characterized by changes at the biochemical, physiologi-cal and organismal levels as well as an increase in stress hormones.Such a generalized stress response also occurs at the cellular level andhas been called cellular stress response (Iwama et al., 1999). Sinceblood parameters are usually kept stable despite normal environmentalvariations, their disturbance is a good indicator of altered physiology(Davis, 2004; Groff and Zinkl, 1999).
ALT is a ubiquitous aminotransferase found in the mitochondria offish, and it plays an important role in indicating hepatopancreas func-tion and damage (Ozaki, 1978). ALP is an important enzyme that regu-lates a number of essential functions in all living organisms. Serum TP isinvolved in maintaining normal osmotic pressure, constant pH, and thetransport of lipid acids and bilirubin. Therefore, they are often used asindicators of a fish's response to stress and health (Guo et al., 2010).Hegazi et al. (2014) reported that ALT activity increased significantlyunder high density stress in Nile tilapia (Oreochromis niloticus L.) finger-lings. Rao (2007) reported that ALP activity at high pH (pH = 8.5) wassignificantly lower than that of the control and Das et al. (2006) report-ed that serum TP levels at pH 9.0 were significantly lower than those ofthe control (pH 7.4) in three Indianmajor carps. Similarly, in this exper-iment, the supplemented diet (especially for the 133.7 mg/kg vitamin Cdiet) could effectively increase serum ALP and TP levels and decreaseserum ALT levels in fish under pH stress. This indicates that vitamin Ccould enhance the immunity of fish under stress, which is similar to re-ports from Menezes de et al. (2006) and Gursu et al. (2004).
bB
cA bAbAB
bC
bcA abA
abBbB
bcA bAabABaB
bAaA
bB
aB
aA
bBaB
bC
aA
bB
abBC
0
1
2
3
4
5
0d 1d 5d 10d
HS
P6
0/β
-act
in m
RN
A
0.2
33.4
65.8
133.7
251.5
501.5
Before stress Days after stress
A
bc
bA
bBC bBCbC
bA
abBbBC
bC
bA abB
bBCbB
aA
aA
bBaB
aA
aB
bB
bD
aA
aBaCD
0
1
2
3
4
0d 1d 5d 10d
HS
P7
0/β
-act
in m
RN
A
0.2
33.4
65.8
133.7
251.5
501.5
Before stress Days after stress
B
bB
bA
cB cBbB
bA
cB bcBbAB
bA
cB bcABbB
abA
bcB abA
aA
aA
aA aA
bC
bA
bB
aA
0
1
2
3
4
5
0d 1d 5d 10d
HS
P9
0/B
eta-
acti
n m
RN
A
0.2
33.4
65.8
133.7
251.5
501.5
Before stress Days after stress
C
Fig. 4. Effect of various levels of vitamin C on relative expression levels of hepatic HSP60 (A), HSP70 (B) and HSP90 (C) mRNA of juvenileM. amblycephala under pH stress. Note: Data areexpressed as mean ± SEM (n = 9). Legends are the same as in Fig. 1.
331J. Wan et al. / Aquaculture 434 (2014) 325–333
The complement system plays a key role in innate and adaptive im-munity and involves more than 35 soluble plasma proteins (Hollandand Lambris, 2002). C3 is the central component of the complementsystem, and C4 plays an integral role in the activation of the classicand lectin pathways (Boshra et al., 2006). Ortuno et al. (2001) reportedthat short-term crowding stress (100 kg/m3 for 2 h) induced an imme-diate depressive effect on serum complement activity in the giltheadseabream (Sparus aurata). Similarly, other stressors can also dramatical-ly decrease complement activity in fish (Sunyer et al., 1995; Ortunoet al., 2002). Since complement is down-regulated in many situationsof stress, it has been proposed that complement activity could be agood indicator of fish immuno-competence in stressed animals (Tortet al., 1996). In this experiment, all groups under pH stress showed asimilar trend for serum C3 and C4 activity. The stimulating effect ofvitamin C on complement activity has been demonstrated in variousfish species (Boshra et al., 2006). In our study, serum C3 and C4 activityin the vitamin C treated groupswere significantly higher than that of the
control in juvenile M. amblycephala under pH stress. These findingssuggest a stress-resistant role for vitamin C in fish.
Cortisol, a widely used indicator of stress response in fish (Hsiehet al., 2003), is a stress hormone, and has been shown to enhanceglucose production in fish and play an important role in hyperglycemicresponses associated with stress (Vijayan et al., 1997). In this experi-ment, the serum cortisol levels in all groups under pH stress increasedat first and then decreased. At first, stress induces a number of phys-iological changes in teleosts, including strengthening hypothalamus–pituitary–interrenal axis vitality that leads to an increase in thesecretion of catecholamine, and causes high levels of serum cortisol.Then there is a decrease in cortisol because the fish adapt to the pHstress. Our study also showed that higher-doses of vitamin C couldeffectively decrease serum cortisol levels, relieving the adverse effectsof high pH stress, which is similar to observations in gilthead seabream(S. aurata L.) under crowding stress (Montero et al., 1999) andmultiplestressors (Ortuno et al., 2003). The above studies suggest that fish fed a
332 J. Wan et al. / Aquaculture 434 (2014) 325–333
diet with vitamin C can better tolerate a wider range of pH stress thanthe control group. The reason vitamin C affects cortisol levels might bethe inhibitory action of vitamin C on steroidogenesis, which is mediatedby controlling the peroxidation of unsaturated fatty acids to prevent theproduction of cholesterol, thereby further preventing the formation ofcortisol (Ming et al., 2012).
In the course of normal metabolism in organisms, the productionand elimination of reactive oxygen species (ROS) maintain a dynamicbalance (Ming et al., 2012). SOD and ASAFR are part of the critical anti-oxidant enzyme system in the body, which can catalyze the reaction ofsuper anion transforming to H2O2 and O2, so it can scavenge the superanion in the tissue. Therefore, they play an important role in theself-defense system and possess a vital function in the immune system(Yu and Gu, 2007; Zhou et al., 2012). However, MDA, the main compo-nent of lipid peroxides, has a strong biotoxicity andwill damage the cellstructure and function (Freeman and Crapo, 1982).
Ha et al. (2009) reported that SOD activity was significantlydecreased 3 days after pH stress (pH 9.0–9.2) occurred in “HuanghaiNo. 1” (Fenneropenaeus chinensis). Similarly, Li and Chen (2008) report-ed that SOD activity was significantly decreased 6–24 days after pHstress (pH 10.1) occurred in white shrimp (Litopenaeus vannamei). Inthis experiment, hepatic SOD levels tended to increase on the first dayafter pH stress and then decrease for the rest of the time, which mightbe related to the activation of the induced enzyme in M. amblycephalaunder acute pH stress. Later the decline of SOD was probably causedby the fact that the levels of ROS surpassed the clear threshold of theirown. ASAFR showed the same trend. However, there was increasedhepatic MDA content in all groups under pH stress, which is similar tothat in the report by Rao (2007). All results showed that pH stressleads to oxidative damage. In our study, under pH stress, hepatic SODand ASAFR activity in the treated groups were higher than that of thecontrol in different levels, whereas MDA content was lower. This issimilar to the report on the levels of SOD, ASAFR and MDA in diabeticrats under oxidative stress (Aksoy et al., 2005). It indicates that vitaminC has an antioxidant function, which may be due to the fact that it canscavenge free radicals itself, and resume the activities of glutathioneperoxidase and vitamin C groups so as to revive the function of anti-free radicals (Ming et al., 2012). Our study supported this fact, especiallyin the 133.7 and 251.5 mg/kg vitamin C diets.
Studies show that HSPs are involved in numerous cellular functionsand the immune response. The HSP family consists of HSP60, HSP70,HSP90, HSP100, and other low-molecular mass HSPs. HSP60, HSP90,and especially HSP70, play an important role in the response to variousstresses (Lindquist and Graig, 1998; Georgopoulos and Welch, 1993;Fink, 1999; Hartl and Hayer-Hartl, 2002; Zheng et al., 2010).
Martín et al. (1998) reported that HSP60 and HSP70 showed higherexpression 24 h after 33 °C hyperthermic stress and 4 h after acidicstress in four different teleostean fish species (gourami, carp, goldfishand trout). Dong et al. (2008) reported that HSP70 levels increasedand reached peak levels 6 h after thermal stress occurred in sea cucum-ber (Apostichopus japonicus Selenka). Lif and Zhangm (2009) reportedthat HSP90 mRNA levels, in both hemocytes and gill, were induced at2 h and depressed at 8 h during hypoxia stress in Chinese shrimp(Fenneropenaeus chinensis). Similarly, in our study, the expression levelsof hepatic HSP60, HSP70 and HSP90 mRNA in all groups tended to rap-idly increase 1 day after pH stress, and then decrease. Increased levels inHSPs induce tolerance of cells, tissues, and the whole fish to subsequentstressors, which suggests that itmay be possible to develop strategies toenhance tolerance to stressors by inducing the cellular stress response(Iwama et al., 1999). However, when the stress intensity is too high orthe stress lasts too long, it may cause mutation in cell membrane struc-ture and protein composition of the liver, which stops the transcriptionof HSPs (Qiang et al., 2012). In this experiment, before and after stress,the expression levels of HSP60, HSP70 and HSP90 mRNA in the treatedgroups, especially the higher dosage of vitamin C groups (133.7, 251.5and 501.5 mg/kg vitamin C diets), were higher than those of the control
group. Similarly, Ming et al. (2012) found that compared to those of thecontrol, the expression levels of HSC70 and HSP70 mRNA increased inthe Vitamin C group after high temperature stress. But Mahmoud(2004) reported that vitamin C could delay the generation of HSPsthrough antioxidant radicals in poultry. These differences might beassociated with tissue specificity of different species. The specific mech-anism needs further study.
5. Conclusions
In conclusion, a basal diet supplemented with vitamin C(133.7 mg/kg–251.5 mg/kg diet) could increase non-specific immu-nity, antioxidant capacity and mRNA expression level in three HSPsin juvenileM. amblycephala, and enhance resistance to high pH stressin fish.
Acknowledgments
This work was supported by the Modern Agriculture IndustrialTechnology System special project of the National Technology Systemfor Conventional Freshwater Fish Industries (CARS-46), by the SpecialFund for Agro-scientific Research in the Public Interest (20100302)and by the National Nonprofit Institute Research Grant of FreshwaterFisheries Research Center, Chinese Academy of Fishery Sciences(2014A08XK02) and the Three New Projects of Fishery in Jiangsu prov-ince (D2013-5),
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