- Email: [email protected]
Proteome modifications of fingerling rainbow trout (Oncorhynchus mykiss) muscle as an effect of dietary nucleotides
Aquaculture 324–325 (2012) 79–84
Contents lists available at SciVerse ScienceDirect
Aquaculture journal homepage: www.elsevier.com/locate/aqua-online
Proteome modifications of fingerling rainbow trout (Oncorhynchus mykiss) muscle as an effect of dietary nucleotides Saeed Keyvanshokooh ⁎, Ahmad Tahmasebi-Kohyani Department of Fisheries, Faculty of Marine Natural Resources, Khorramshahr University of Marine Science and Technology, Khorramshahr, Khouzestan, Iran
a r t i c l e
i n f o
Article history: Received 27 July 2011 Received in revised form 8 October 2011 Accepted 11 October 2011 Available online 19 October 2011 Keywords: Rainbow trout Dietary nucleotides Growth Proteomics
a b s t r a c t A feeding study was conducted to determine the effect of dietary nucleotides (NT) on growth performance and muscle proteome profile of rainbow trout fingerlings. Five experimental diets according to different levels of supplemented nucleotides (0, 0.05, 0.1, 0.15 and 0.2%) were assayed in rainbow trout for 8 weeks. Each diet was randomly allocated to triplicate groups of fish with initial average weight of approximately 23 g. The percentage of body weight gain (WG) and feed efficiency (FE) of fish were better when the fish were fed 0.15–0.2% diets. At the end of feeding trial, fish fed the basal and 0.2% diets were subjected to proteomic analysis. Proteins of the muscle tissue were analyzed using two-dimensional electrophoresis and MALDI-TOF/TOF mass spectrometry. Dietary NT caused differential expression of muscle metabolic proteins including glyceraldehyde-3-phosphate dehydrogenase, creatine kinase, adenylate kinase, nucleoside diphosphate kinase, and triosephosphate isomerase. In addition to metabolic enzymes, troponin-T-1 as a structural protein was found to increase in abundance in the treated fish. The altered expression of both metabolic and structural proteins in fish fed NT may be related to higher growth rate in rainbow trout. These findings provide basic information to understand possible mechanisms of dietary NT contribution to better growth in rainbow trout. © 2011 Elsevier B.V. All rights reserved.
1. Introduction Nucleotides are low molecular weight intracellular compounds which play key roles in diverse essential physiological and biochemical functions including encoding genetic information, mediating energy metabolism and signal transduction (Carver and Allan Walker, 1995). Dietary nucleotides have been reported to be beneficial for humans and animals (Gil, 2002) since they positively influence lipid metabolism, immunity, and tissue growth, development and repair (reviewed in Gil, 2002). Heightened attention on nucleotide supplementation was stimulated by the reports of Burrells et al. (2001b), which indicated enhanced resistance of salmonids to viral, bacterial and parasitic infections as well as improved efficiency of vaccination and osmoregulation capacity. Dietary supplementation of nucleotides in some aquatic species has been revealed to improve growth performance (Adamek et al., 1996; Ramadan et al., 1991), immune responses (Burrells et al., 2001b; Leonardi et al., 2003; Li and Gatlin, 2004; Ramadan et al., 1994; Sakai et al., 2001) and disease resistance (Burrells et al., 2001a,b; Li and Gatlin, 2004; Sakai et al., 2001). Regarding the development of dietary nucleotides in fish nutrition research, the application of novel tools in molecular biology will be necessary to detect various aspects of metabolism and influences on
⁎ Corresponding author. Tel.: + 98 632 4234725; fax: + 98 632 4233322. E-mail address: [email protected] (S. Keyvanshokooh). 0044-8486/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.aquaculture.2011.10.013
physiological responses. Cutting edge technologies such as proteomics, genomics and metabolomics are probably the best approaches that offer a comprehensive method to study biochemical systems by expanding the level of investigation from single biomolecules to a wide range of molecules present in a cell or tissue at once, in terms of their presence and relative abundance, without a priori knowledge (Alves et al., 2010). Current annual production of farmed rainbow trout in Iran is approximately 50,000 tons. The recent accessibility to a large number of salmonid expressed sequences within public databases such as GenBank has made proteomic research on salmonids much more practical (Vilhelmsson et al., 2004). The present study was conducted to determine the effects of dietary nucleotides on muscle proteome of fingerling rainbow trout (Oncorhynchus mykiss). 2. Materials and methods 2.1. Fish and experimental conditions 675 rainbow trout fingerlings were obtained from a commercial rainbow trout farm in Lordegan, Iran. The fish were transported to the laboratory, acclimated to laboratory conditions and fed the basal experimental diet without dietary nucleotides for 2 weeks. At the end of the acclimation period, fish with an average weight of approximately 23 g were randomly selected and stocked in 15 1000-L tanks (triplicate groups per dietary treatment) at a density of 45 fish/tank.
80
S. Keyvanshokooh, A. Tahmasebi-Kohyani / Aquaculture 324–325 (2012) 79–84
The mean water quality parameters were maintained as follows: temperature 13.3 °C, dissolved oxygen 8.2 mg L − 1 and pH 7.7. 2.2. Feed and feeding Basal diet formulation and proximate composition analysis are shown in Table 1. The experimental diet was formulated to have 0% (basal), 0.05%, 0.1%, 0.15%, and 0.2% of “Optimun” supplements (Chemoforma, Augst, Switzerland). This product contained inosine monophosphate (IMP), adenosine monophosphate (AMP), guanosine monophosphate (GMP), uridine monophosphate (UMP), cytidine monophosphate (CMP) and ribonucleic acid (RNA). Dietary ingredients were thoroughly mixed in a mixer, made into pellets and air-dried at room temperature. The pellets were broken into small pieces, packed and stored in a freezer until used. Fish were fed by hand to apparent satiation five times per day (at hours: 8, 11, 14, 16 and 18) with one of the five experimental diets over 8 weeks. 2.3. Growth measurements At the end of feeding experiment, the fish were weighed from each tank. Percentage of body weight gain (WG) and feed efficiency (FE) were calculated for each group as follows: WG ¼ ½100 ðf inal body weight−initial body weight Þ=initial body weight
FE ¼ ½ðf inal body weight−initial body weight Þ=feed intake
Table 1 Ingredients composition of test diets fed by rainbow trout. Ingredient (% dry weight)
Basal diet
0.05%
0.10%
0.15%
0.20%
Fish meal a Wheat, white Fish oil a Soybean oil e Soybean meale Mineral premix d Vitamin premix c Vitamin C e Anti fungus e Di calcium phosphate Cellulose b Optimun g
43 18.65 3 3 26 1.5 1.5 0.1 0.25 1 2.0 0
43 18.65 3 3 26 1.5 1.5 0.1 0.25 1 1.95 0.05
43 18.65 3 3 26 1.5 1.5 0.1 0.25 1 1.90 0.10
43 18.65 3 3 26 1.5 1.5 0.1 0.25 1 1.85 0.15
43 18.65 3 3 26 1.5 1.5 0.1 0.25 1 1.80 0.20
Analyzed proximate composition (% dry weight) Crude protein 48.25 48.25 Crude lipid 18.86 18.86 h Carbohydrate 8.23 8.23 Ash 12.25 12.25 Dry matter % 87.59 87.59 Gross energy (Kj g− 1 diet) 20.24 20.24
48.25 18.86 8.23 12.25 87.59 20.24
48.25 18.86 8.23 12.25 87.59 20.24
48.25 18.86 8.23 12.25 87.59 20.24
f
a Pars Kilka Corporation., Iran, based on 100% dry weight included 72% crude protein, 16.2% crude lipid. b Merck Corporation, Germany. c Unit kg− 1 of mixture: Vitamin: retinol acetate (A), 1,200,000 IU; Cholecalciferol (D3), 400,000; DL-α-tocopheryl acetate (E), 30 IU; menadione sodium bisulfite (K3), 1200 mg; L-ascorbic acid (C), 5400 mg; D-biotin (H2), 200 mg; thiamin mononitrate (B1) ,200 mg; riboflavin (B2), 3600 mg; calcium d-pantothenate (B3), 7200 mg; niacinamide (B5), 9000 mg; pyridoxine hydrochloride (B6), 2400 mg; folic acid (B9), 600 mg; cyanocobalamin (B12), 4 mg; antioxidant 500 mg; Carrier up to 1 kg. d Unit kg− 1 of mixture: Mineral: Fe, 4500 mg; Cu, 500 mg; Co, 50 mg; Se, 50 mg; Zn, 6000 mg; Mn, 5000 mg; I, 150 mg; choline chloride, 150,000 mg; Carrier up to 1 kg. e Khorak-dam Abzian corporation, Iran. f Garmab shimi corporation, Iran. Contains: phosphor 17%, calcium 23.27%, moist 3%, fluorine 0.17%. g Chemoforma, Augst, Switzerland. h Carbohydrate calculated based on: 100 − (crude protein + crude lipid + ash).
2.4. Fish sampling and protein extraction Based on growth measurements (Table 2), fish fed diets with 0 and 0.2% NT were selected for proteomic analysis. Nine fish from each group (three fish per tank) were sacrificed. Muscle tissue samples were taken from behind the head and above the lateral line, within 5 min of slaughter, snap frozen in liquid nitrogen and stored at −80 °C until further analysis. In order to minimize the effects of individual variation, the muscle tissues of (each) three individuals were mixed before proteome extraction so that three pools were prepared for each experimental group. The tissue samples were washed in 40 mM Tris–HCl buffer (pH = 7), before being mechanically homogenized in cold lysis buffer containing 7 M urea, 2 M thiourea, 4% CHAPS, 50 mM DTT, 50 mM Tris (pH = 7), 0.2% carrier ampholyte, 1 mM PMSF, 0.25% RNase, and 1% DNase. Each sample was maintained for 1 h at room temperature for protein release. Cellular debris were removed by centrifugation at 12,000 ×g for 10 min, supernatants were collected and their protein content was determined according to the method of Bradford (Bradford, 1976) with BSA as standards. 2.5. Two dimensional gel electrophoresis 17 cm Immobilized pH gradient (IPG) strips with linear pH range of 3–10 (BioRad, USA) were passively rehydrated for at least 16 h with 1.2 mg protein in 300 μL of rehydration buffer containing 8 M urea, 4% CHAPS, 50 mM DTT, 0.2% ampholyte and bromophenol blue traces. Strips were focused at 20 °C with the following program: 20 min with a linear ramp (0–250 V), 5 h with a linear ramp (250– 10,000 V) and 50,000 Vh with a rapid ramp. After isoelectric focusing the strips were equilibrated in the first step with 6 M urea, 50 mM Tris–HCl pH 8.8, 20% glycerol, 2% SDS and 2% DTT for 20 min; and the second step with the same buffer but 2.5% iodoacetamide instead of DTT for another 20 min. Each IPG strip was then laid onto a 12% SDS-PAGE gel for second dimension electrophoresis. The running conditions were 16 mA/gel for 30 min and 24 mA/gel for approximately 5 h until the bromophenol blue front was 1 cm above the bottom of the gel. The gels were stained using colloidal Coomassie brilliant blue G-250 procedure (Candiano et al., 2004). To confirm the reproducibility of the results, three 2-DE runs were performed for each experimental condition. 2.6. Analysis of 2-D gels The gels were scanned using the Densitometer GS-800 scanner (BioRad, USA) and stored as TIF files. Spot detection and matching were performed using the PG200 software (Nonlinear Dynamics, UK). Each set of gel replicates was combined into average gels, which represented spots that were reproducibly present on both sets of the replicate gels. The software automatically selected the gel with greatest number of spots as the image for the reference gel and added unmatched spots from the other gels to this image to give a comprehensive reference gel
Table 2 Final weight, weight gain and feed efficiency (FE) of rainbow trout fed different levels of dietary nucleotides for 8 weeks. Experimental diets (%) 0 0.05 0.1 0.15 0.2
Final weight (g)
Weight gain (%)
Feed efficiency (FE)
46 ± 0.7a 46 ± 0.5a 58 ± 0.4b 66 ± 0.7c 67 ± 0.8c
98 ± 8a 99 ± 6a 151 ± 9b 187 ± 7c 192 ± 5c
0.62 ± 0.21a 0.73 ± 0.18 b 0.76 ± 0.24 b 0.85 ± 0.26 c 0.95 ± 0.17d
Values are mean±S.E.M. of three replicate groups. Mean values with different superscripts are significantly different from each other (significance level is defined as P b 0.05).
S. Keyvanshokooh, A. Tahmasebi-Kohyani / Aquaculture 324–325 (2012) 79–84
for matching spots on the different gels. The spots detected automatically by the software were visually inspected. Spot filtering and editing were performed manually to remove artifacts and to correct for spots that did not split correctly or were not detected by the software's automatic spot detection process. After the filtering and editing steps, the average gels were recreated, and the spots on the average gels were matched, using the comprehensive reference gel as a map to link the spots on different gels. The software subtracted a background value for each spot based on the entire surface area of the gel. Spot volumes were normalized, based on the total volume of all of the spots on each gel, and both of these operations were performed recursively every time spots were edited on any of the gels. Finally, normalized volume (NV) of the matched protein spots that were found to vary in expression between the diets were analyzed for statistical significance using Student's t-test, P b 0.05. Each spot that met this criterion was then visually verified to see if it was correctly detected, and the accuracy of the matching of the spots between the replicate sets was confirmed. Induction and repression factors (IF and RF) correspond to the ratio: NV nucleotide/NV controls. NV coefficients of variability (CV) were calculated for each spot between all the gels of the same condition, using the formula: CV = standard deviation × mean − 1 × 100. 2.7. In-gel trypsin digestion, MALDI-TOF/TOF MS and database searching Gel pieces were washed two times with 50% (v:v) aqueous acetonitrile containing 25 mM ammonium bicarbonate, then once with acetonitrile and dried in a vacuum concentrator for 20 min. Sequencing-grade, modified porcine trypsin (Promega) was dissolved in the 50 mM acetic
81
acid supplied by the manufacturer, then diluted 5-fold by adding 25 mM ammonium bicarbonate to give a final trypsin concentration of 0.02 μg/μL. Gel pieces were rehydrated by adding 10 μL of trypsin solution, and after 30 min enough 25 mM ammonium bicarbonate solution was added to cover the gel pieces. Digests were incubated overnight at 37 °C. A 1 μL aliquot of each peptide mixture was applied directly to the ground steel MALDI target plate, followed immediately by an equal volume of a freshly-prepared 5 mg/mL solution of 4-hydroxy-αcyano-cinnamic acid (Sigma) in 50% aqueous (v:v) acetonitrile containing 0.1%, trifluoroacetic acid (v:v). Positive-ion MALDI mass spectra were obtained using a Bruker ultraflex III in reflectron mode, equipped with a Nd:YAG smart beam laser. MS spectra were acquired over a mass range of m/z 800–4000. Final mass spectra were externally calibrated against an adjacent spot containing 6 peptides (des-Arg1-Bradykinin, 904.681; Angiotensin I, 1296.685; Glu1Fibrinopeptide B, 1750.677; ACTH (1–17 clip), 2093.086; ACTH (18–39 clip), 2465.198; ACTH (7–38 clip), 3657.929.). Monoisotopic masses were obtained using a SNAP averagine algorithm (C 4.9384, N 1.3577, O 1.4773, S 0.0417, H 7.7583) and a S/N threshold of 2. For each spot the ten strongest peaks of interest, with a S/N greater than 30, were selected for MS/MS fragmentation. Fragmentation was performed in LIFT mode without the introduction of a collision gas. The default calibration was used for MS/MS spectra, which were baseline-subtracted and smoothed (Savitsky-Golay, width 0.15 m/z, cycles 4); monoisotopic peak detection used a SNAP averagine algorithm (C 4.9384, N 1.3577, O 1.4773, S 0.0417, H 7.7583) with a minimum S/N of 6. Bruker flexAnalysis software (version 3.3) was used to perform the spectral processing and peak list generation for both the MS and MS/MS spectra.
Fig. 1. 2-DE map of muscle proteins of rainbow trout (Oncorhynchus mykiss), prepared by linear wide-range immobilized pH gradients (pH 3–10, 17 cm; BioRad, USA) in the first dimension and on 12% SDS-PAGE for the second dimension. Proteins were stained with colloidal Coomassie brilliant blue G-250. Labeled spots indicate proteins with significant altered expression profile after dietary nucleotides treatment (see Table 3).
82
S. Keyvanshokooh, A. Tahmasebi-Kohyani / Aquaculture 324–325 (2012) 79–84
Tandem mass spectral data were submitted to database searching using a locally-running copy of the Mascot program (Matrix Science Ltd., version 2.1), through the Bruker ProteinScape interface (version 2.1). Search criteria included: Enzyme, Trypsin; Fixed modifications, Carbamidomethyl (C); Variable modifications, Oxidation (M); Peptide tolerance, 250 ppm; MS/MS tolerance, 0.5 Da; Instrument, MALDITOF-TOF. 3. Results and discussion 3.1. Growth performance There was no fish mortality during the 8-week feeding trial among the five treatment groups. Fish fed 0.15–0.2% diets had highest WG, followed by fish fed the 0.1% diet, and lower in fish fed 0–0.05% diets. After 8 weeks of feeding, FEs of fish fed 0.05–0.2% diets were significantly higher than those of fish fed the NT-free basal diet (Table 2). There was an obvious trend of higher WG and FE with an increase in nucleotides in the diet, indicating that dietary supplementation of 0.15–0.2% nucleotides in diet exerts a positive effect on growth performance of rainbow trout fingerlings. Similarly, a growth-promoting effect was noted in Atlantic salmon (Salmo salar) fed nucleotides at level of 0.25% (Burrells et al., 2001b). Lin et al. (2009) also reported that dietary supplementation of 1.5 g mixed-NT kg− 1 exerted a positive effect on weight gain of grouper (Epinephelus malabaricus). Although the administration of dietary nucleotides has been reported to enhance the growth performance of cultured species such as tilapia (Ramadan et al., 1991), red drum (Li et al., 2007a) and white shrimp (Li et al., 2007b), the growthpromoting effect of nucleotides has not been observed in hybrid striped bass (Li and Gatlin, 2004) and juvenile red drum (Li et al., 2005). Based on current knowledge, it seems that growth enhancing effect of nucleotides may vary in different species and depends on feeding period, life stages and the type of nucleotide supplement (Li and Gatlin, 2006).
proliferation, development and differentiation. NDPK plays an important role in early embryonic development of Atlantic salmon (Murphy et al., 2000). NDPK is also important for the normal development of Drosophila. In shrimp, NDPK acts as a defense-related enzyme and plays roles in the anti-viral innate immune reaction (Zhao et al., 2007). The expression of NDPK was also up-regulated in hemolymph of scallop after bacterial challenge (Shi et al., 2008). Borda et al. (2003) presumed that an exogenous supply of nucleotides may promote growth of fish and crustaceans in early stages to meet their high rate of cell replication. Also, in the present study, the fish fed on 0.2% diet had higher levels of IgM, lysozyme and alternative complement activity (ACH50) (Tahmasebi-Kohyani et al., 2010). These results suggest that NDPK may play roles in the higher rate of cell proliferation and innate immune responses of fish fed dietary nucleotides. Adenylate kinase (AK) showed a decreased expression in fish fed nucleotides. AK is reported to play an important role in energy metabolism and in efficient energy transfer (Dzeja and Terzic, 2003). Ak interconverts Mg + 2 ATP (or Mg + 2 GTP) and free AMP with Mg + 2 ADP (or Mg + 2 GDP) and free ADP by catalyzing the addition of a phosphate to the β-position of AMP using a phosphate of the γposition of ATP (or GTP) as a phosphoryl donor, thereby resulting in the production of two molecules of ADP (or ADP and GDP) (Noda,
3.2. Proteomics The objective of this study was to examine the effects of dietary nucleotides on muscle proteome of rainbow trout. Muscles of control and treated groups were subjected to proteome analysis by 2-DE. Using the spot finding protocol of PG200 software, an average of 710 protein spots per gel were observed, with Mr ranging between 10 and 250 kDa and pI between 3.5 and 9.5. Most protein spots were located in the pI range between 5 and 8 (Fig. 1). Normalized volumes (vol. %) of the matched spots in replicates of basal and NT-fed samples were statistically analyzed by Student's ttest. Eight spots were significantly changed in abundance between the control and treated fish, and 6 of these were increased while the others were decreased in the NT-fed fish (Fig. 2). The differences in abundance between the two groups ranged from −2 to + 2.64-fold. All the spots were matched to the proteins of S. salar, and none of them could be matched to rainbow trout proteins since the genome of rainbow trout is not completely sequenced. Dietary NT caused differential expression of muscle metabolic proteins including glyceraldehyde-3-phosphate dehydrogenase, creatine kinase, adenylate kinase, nucleoside diphosphate kinase A, and triosephosphate isomerase. In addition to metabolic enzymes, troponin-T-1 as a structural protein was found to increase in abundance in the treated fish (Table 3). The expression of nucleoside diphosphate kinase (NDPK) was increased in fish fed dietary nucleotides. NDPK is a key metabolic enzyme that catalyzes the synthesis of non-adenine nucleoside triphosphate (NTP) by transferring the terminal phosphate between nucleoside diphosphate (NDP) and NTP (Shi et al., 2008). This enzyme is widely distributed and is believed to play a role in the general homeostasis of cellular nucleoside triphosphate pools. In addition to this metabolic role, a number of other functions have been assigned to NDPK such as
Fig. 2. Comparison of 2-DE spots with significant altered expression level (P b 0.05, student t-test) between control and NT-fed rainbow trout. The 2-D pattern of control is on the left and that of NT treated fish is on the right.
S. Keyvanshokooh, A. Tahmasebi-Kohyani / Aquaculture 324–325 (2012) 79–84
83
Table 3 Identification of differentially expressed proteins in the muscle tissue of rainbow trout fed dietary nucleotides (NT) for 8 weeks. Spot number
Protein name
1 2 3 4 5 6 7 8
Glyceraldehyde-3-phosphate dehydrogenase-1 Fast myotomal muscle troponin-T-1 Fast myotomal muscle troponin-T-1 Creatine kinase Adenylate kinase Nucleoside diphosphate kinase A Triosephosphate isomerase 1b Triosephosphate isomerase 1b
Accession number
Ms/ Ms score
Organism
gi|197632425 gi|197632601 gi|197632601 gi|197632231 gi|213511412 gi|213511196 gi|213515400 gi|213515400
47 125 113 96 107 106 336 500
Salmo salar S. salar S. salar S. salar S. salar S. salar S. salar S. salar
Expression
Overexpressed Overexpressed Overexpressed Overexpressed Underexpressed Overexpressed Underexpressed Overexpressed
IFa
2.64⁎ 2.60⁎ 2.06⁎ 2.52⁎ 1.7⁎ 1.55⁎
RFa
2⁎ 1.46⁎
Mean NV
CV (%)
NT/control
NT/control
0.127/0.048 0.138/0.053 0.066/0.032 0.043/0.017 0.013/0.026 0.041/0.024 0.097/0.142 0.502/0.323
15/23 8/21 20/16 5/9 23/23 16/13 21/10 14/17
⁎ P b 0.05. a The induction (IF) and repression (RF) factors are the ratios between normalized volumes in treated and control.
1973). The NDPK and adenylate kinase enzymes are both involved in the metabolism of nucleotides within the cell. AK belongs to the nucleoside monophosphate kinase class of enzymes, and differs from the NDPKs in its substrate specificity. While NDPK equilibrates nucleoside di- and triphosphates (Hooks et al., 1989), AK equilibrates adenylates and free Mg + 2 (Igamberdiev and Kleczkowski, 2003) in the reversible ATP-dependent phosphorylation of AMP to ADP and dAMP to dADP (Hooks et al., 1994). The decrease of AK in our study may be as a result of NDPK overexpression as it is shown that AK stimulated NDPK activity, whereas NDPK inhibited the activity of AK (Johansson et al., 2008). However, further studies have to be performed in order to understand how the interaction between NDPK and AK is regulated. The fish fed on nucleotide-supplemented diet exhibited an increased expression of glyceraldehyde-3-phosphate dehydrogenase (GAPDH). GAPDH is a key glycolytic enzyme that catalyzes the reversible conversion of glyceraldehyde-3-phosphate to 1,3-bisphosphoglycerate. Owing to its role in the glycolysis pathway, GAPDH has been routinely considered as a housekeeping gene. However, there is increasing evidence that GAPDH has gained several other new functions such as nuclear RNA export, DNA replication, DNA repair, membrane fusion and microtubule bundling (Sirover, 1999), as well as the apoptotic cascade (Chuang et al., 2005). The biochemical analysis of chicken muscle proteome dynamics under conditions of rapid growth has revealed drastic expression changes in various glycolytic enzymes, including GAPDH (Doherty et al., 2004; Teltathum and Mekchay, 2009). In analogy, the proteomic profiling of ovine muscle hypertrophy showed increased levels of GAPDH during muscle growth (Hamelin et al., 2006). In contrast, weight loss is accompanied by differential effects on numerous glycolytic enzymes, such as enolase, aldolase and GAPDH, as well as lactate dehydrogenase (Almeida et al., 2010a,b). The results of our study support the above outlined studies suggesting that GAPDH can be useful for the future establishment of a biomarker signature of muscle growth and weight gain in rainbow trout. Interestingly, two isoforms of triosephosphate isomerase (TPI) were detected in the present study, an acidic one (spot 7) and a less acidic one (spot 8). The TPI in spot 7 was down-regulated and the TPI in spot 8 was up-regulated. The proteomic analysis of chicken muscle under conditions of rapid growth has revealed increased levels of glycolytic enzymes including TPI (Doherty et al., 2004; Teltathum and Mekchay, 2009). Also, glycolytic enzymes such as TPI increased during muscle growth in sheep (Hamelin et al., 2006). Moreover, atrophying muscle showed decreased abundance of three successive enzymes in the intermediate steps of the glycolytic pathway, fructose-bisphosphate aldolase, TPI and GAPDH in rainbow trout (Salem et al., 2010). Although the abundance of both spots was affected by dietary supplementation of NT, the total TPI levels (spot 7+ spot 8) increased (Table 3), suggesting the role of glycolytic enzymes in better growth of fish fed dietary NT. Creatine kinase (CK) is an enzyme catalyzing the conversion of adenosine diphosphate (ADP) to adenosine triphosphate (ATP), consuming phosphocreatine and generating creatine. CK is typically found in tissues
with high energy demand like cardiac and skeletal muscle, brain, retina and primitive-type spermatozoa (Wallimann and Hemmer, 1994). The expression of CK was increased in fish fed dietary nucleotides. Reddish et al. (2008) reported the association of aldolase, enolase, pyruvate kinase, creatine kinase, and their fragments with body mass and length in Perca flavescens. Variability in expression levels of aldolase, enolase, and CK was also documented by other authors in association with development in zebrafish (Bosworth et al., 2005) and in trout (Morzel et al., 2006). In addition, Salem et al. (2010) reported the downregulation of CK in atrophying muscle. The over-expression of CK observed in this study could be related to higher growth rate in fish fed on NT-supplemented diet. Of the spots that were shown to increase with dietary NT, two were identified as the fast myotomal muscle troponin-T-1. In fish, muscle growth is commonly reported to occur by a combination of hyperplasia and hypertrophy, that is, through an increase in both myocyte number and size (Weatherley and Gill, 1985). Addis et al. (2010) related the expression of structural proteins including troponin with sea bream muscle growth. Structural proteins are the main constituents of sarcomere, the contractile unit of muscular fibers. The over-expression of both metabolic and structural proteins in fish fed NT may be related to the contribution of hyperplasia and hypertrophy to muscle growth in rainbow trout. In conclusion, we represented some changes in the muscle proteome of rainbow trout fed dietary nucleotides. The altered expression of both metabolic and structural proteins in fish fed NT may be related to higher growth rate in rainbow trout. These findings provide basic information to understand possible mechanisms of dietary NT contribution to better growth in fish. Acknowledgment This work was funded by grant P-1/4-12 from Khorramshahr University of Marine Science and Technology. We thank Dr. Behrouz Vaziri and Fatemeh Zandi (Pasteur Institute of Iran) for their technical and supportive assistance. We also thank Nemat Mahmoudi (Tarbiat Modares University) for his assistance. References Adamek, Z., Hamackova, J., Kouril, J., Vachta, R., Stibranyiova, I., 1996. Effect of Ascogen probiotics supplementation on farming success in rainbow trout (Oncorhynchus mykiss) and wels (Silurus glais) under conditions of intensive culture. Krmiva (Zagreb) 38, 11–20. Addis, M.F., Cappuccinelli, R., Tedde, V., Pagnozzi, D., Porcu, M.C., Bonaglini, E., Roggio, T., Uzzau, S., 2010. Proteomic analysis of muscle tissue from gilthead sea bream (Sparus aurata, L.) farmed in offshore floating cages. Aquaculture 309, 245–252. Almeida, A., Campos, A., Francisco, R., Van Harten, S., Cardoso, L., Coelho, A., 2010a. Proteomic investigation of the effects of weight loss in the gastrocnemius muscle of wild and NZW rabbits via 2D electrophoresis and MALDI TOF MS. Animal Genetics 41, 260–272. Almeida, A., Van Harten, S., Campos, A., Coelho, A., Cardoso, L., 2010b. The effect of weight loss on protein profiles of gastrocnemius muscle in rabbits: a study using
84
S. Keyvanshokooh, A. Tahmasebi-Kohyani / Aquaculture 324–325 (2012) 79–84
1D electrophoresis and peptide mass fingerprinting. Journal of Animal Physiology and Animal Nutrition 94, 174–185. Alves, R.N., Cordeiro, O., Silva, T.S., Richard, N., de Vareilles, M., Marino, G., Di Marco, P., Rodrigues, P.M., Conceição, L.E.C., 2010. Metabolic molecular indicators of chronic stress in gilthead seabream (Sparus aurata) using comparative proteomics. Aquaculture 299, 57–66. Borda, E., Martinez-Puig, D., Cordoba, X., 2003. Well-balanced nucleotide supply makes sense. Feed Mix 11, 24–26. Bosworth, I., Charles, A., Chou, C.W., Cole, R.B., Rees, B.B., 2005. Protein expression patterns in zebrafish skeletal muscle: initial characterization and the effects of hypoxic exposure. Proteomics 5, 1362–1371. Bradford, M.M., 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein–dye binding. Analytical Biochemistry 72, 248–254. Burrells, C., Williams, P., Forno, P., 2001a. Dietary nucleotides: a novel supplement in fish feeds: 1. Effects on resistance to disease in salmonids. Aquaculture 199, 159–169. Burrells, C., Williams, P., Southgate, P., Wadsworth, S., 2001b. Dietary nucleotides: a novel supplement in fish feeds: 2. Effects on vaccination, salt water transfer, growth rates and physiology of Atlantic salmon (Salmo salar L.). Aquaculture 199, 171–184. Candiano, G., Bruschi, M., Musante, L., Santucci, L., Ghiggeri, G.M., Carnemolla, B., Orecchia, P., Zardi, L., Righetti, P.G., 2004. Blue silver: A very sensitive colloidal Coomassie G 250 staining for proteome analysis. Electrophoresis 25, 1327–1333. Carver, J.D., Allan Walker, W., 1995. The role of nucleotides in human nutrition. The Journal of Nutritional Biochemistry 6, 58–72. Chuang, D.M., Hough, C., Senatorov, V.V., 2005. Glyceraldehyde-3-phosphate dehydrogenase, apoptosis, and neurodegenarative diseases. Annual Review of Pharmacology and Toxicology 45, 269–290. Doherty, M.K., McLean, L., Hayter, J.R., Pratt, J.M., Robertson, D.H.L., El Shafei, A., Gaskell, S.J., Beynon, R.J., 2004. The proteome of chicken skeletal muscle: changes in soluble protein expression during growth in a layer strain. Proteomics 4, 2082–2093. Dzeja, P.P., Terzic, A., 2003. Phosphotransfer networks and cellular energetics. The Journal of Experimental Biology 206, 2039. Gil, A., 2002. Modulation of the immune response mediated by dietary nucleotides. European Journal of Clinical Nutrition 56, S1. Hamelin, M., Sayd, T., Chambon, C., Bouix, J., Bibé, B., Milenkovic, D., Leveziel, H., Georges, M., Clop, A., Marinova, P., 2006. Proteomic analysis of ovine muscle hypertrophy. Journal of Animal Science 84, 3266. Hooks, M.A., Clark, R.A., Nieman, R.H., Roberts, J.K.M., 1989. Compartmentation of nucleotides in corn root tips studied by 31P-NMR and HPLC. Plant Physiology 89, 963. Hooks, M.A., Shearer, G.C., Roberts, J.K.M., 1994. Nucleotide availability in maize (Zea mays L.) root tips (estimation of free and protein-bound nucleotides using 31Pnuclear magnetic resonance and a novel protein–ligand-binding assay). Plant Physiology 104, 581. Igamberdiev, A.U., Kleczkowski, L.A., 2003. Membrane potential, adenylate levels and Mg2 + are interconnected via adenylate kinase equilibrium in plant cells. Biochimica et Biophysica Acta (BBA)-Bioenergetics 1607, 111–119. Johansson, M., Hammargren, J., Uppsäll, E., MacKenzie, A., Knorpp, C., 2008. The activities of nucleoside diphosphate kinase and adenylate kinase are influenced by their interaction. Plant Science 174, 192–199. Leonardi, M., Sandino, A., Klempau, A., 2003. Effect of a nucleotide-enriched diet on the immune system, plasma cortisol levels and resistance to infectious pancreatic necrosis (IPN) in juvenile rainbow trout (Oncorhynchus mykiss). Bulletin of the European Association of Fish Pathologists 23, 52–59. Li, P., Gatlin, D.M., 2004. Dietary brewers yeast and the prebiotic Grobiotic (TM) AE influence growth performance, immune responses and resistance of hybrid striped bass (Morone chrysops M. saxatilis) to Streptococcus iniae infection. Aquaculture 231, 445–456. Li, P., Gatlin III, D.M., 2006. Nucleotide nutrition in fish: current knowledge and future applications. Aquaculture 251, 141–152.
Li, P., Burr, G.S., Goff, J., Whiteman, K.W., Davis, K.B., Vega, R.R., Neill, W.H., Gatlin III, D.M., 2005. A preliminary study on the effects of dietary supplementation of brewers yeast and nucleotides, singularly or in combination, on juvenile red drum (Sciaenops ocellatus). Aquaculture Research 36, 1120–1127. Li, P., Gatlin III, D.M., Neill, W.H., 2007a. Dietary supplementation of a purified nucleotide mixture transiently enhanced growth and feed utilization of juvenile red drum, Sciaenops ocellatus. Journal of the World Aquaculture Society 38, 281–286. Li, P., Lawrence, A.L., Castille, F.L., Gatlin III, D.M., 2007b. Preliminary evaluation of a purified nucleotide mixture as a dietary supplement for Pacific white shrimp Litopenaeus vannamei (Boone). Aquaculture Research 38, 887–890. LIN, Y.H., Wang, H., SHIAU, S.Y., 2009. Dietary nucleotide supplementation enhances growth and immune responses of grouper, Epinephelus malabaricus. Aquaculture Nutrition 15, 117–122. Morzel, M., Chambon, C., Lefèvre, F., Paboeuf, G., Laville, E., 2006. Modifications of trout (Oncorhynchus mykiss) muscle proteins by preslaughter activity. Journal of Agricultural and Food Chemistry 54, 2997–3001. Murphy, M., Harte, T., McInerney, J., Smith, T.J., 2000. Molecular cloning of an Atlantic salmon nucleoside diphosphate kinase cDNA and its pattern of expression during embryogenesis. Gene 257, 139–148. Noda, L., 1973. 8 adenylate kinase. The Enzymes, 8, pp. 279–305. Ramadan, A., Atef, M., Afifi, N., 1991. Effect of the biogenic performance enhancer (Ascogen “S”) on growth rate of tilapia fish. Acta Veterinaria Scandinavica. Supplementum 87, S304–S306. Ramadan, A., Afifi, N.A., Moustafa, M., Samy, A., 1994. The effect of ascogen on the immune response of tilapia fish to Aeromonas hydrophila vaccine. Fish & Shellfish Immunology 4, 159–165. Reddish, J.M., St Pierre, N., Nichols, A., Green Church, K., Wick, M., 2008. Proteomic analysis of proteins associated with body mass and length in yellow perch, Perca flavescens. Proteomics 8, 2333–2343. Sakai, M., Taniguchi, K., Mamoto, K., Ogawa, H., Tabata, M., 2001. Immunostimulant effects of nucleotide isolated from yeast RNA on carp, Cyprinus carpio L. Journal of Fish Diseases 24, 433–438. Salem, M., Kenney, P.B., Rexroad III, C.E., Yao, J., 2010. Proteomic signature of muscle atrophy in rainbow trout. Journal of Proteomics 73, 778–789. Shi, X.Z., Zhao, X.F., Wang, J.X., 2008. Molecular cloning and analysis of function of nucleoside diphosphate kinase (NDPK) from the scallop Chlamys farreri. Biochemistry 73, 686–692. Sirover, M.A., 1999. New insights into an old protein: the functional diversity of mammalian glyceraldehyde-3-phosphate dehydrogenase. Biochimica et Biophysica Acta 1432, 159–184. Tahmasebi-Kohyani, A., Keyvanshokooh, S., Nematollahi, A., Mahmoudi, N., PashaZanoosi, H., 2010. Dietary administration of nucleotides to enhance growth, humoral immune responses, and disease resistance of the rainbow trout (Oncorhynchus mykiss) fingerlings. Fish & Shellfish Immunology 30, 189–193. Teltathum, T., Mekchay, S., 2009. Proteome changes in Thai indigenous chicken muscle during growth period. International Journal of Biological Sciences 5, 679. Vilhelmsson, O.T., Martin, S.A.M., Médale, F., Kaushik, S.J., Houlihan, D.F., 2004. Dietary plant-protein substitution affects hepatic metabolism in rainbow trout (Oncorhynchus mykiss). The British Journal of Nutrition 92, 71–80. Wallimann, T., Hemmer, W., 1994. Creatine kinase in non-muscle tissues and cells. Molecular and Cellular Biochemistry 133, 193–220. Weatherley, A., Gill, H., 1985. Dynamics of increase in muscle fibers in fishes in relation to size and growth. Cellular and Molecular Life Sciences 41, 353–354. Zhao, Z.Y., Yin, Z.X., Weng, S.P., Guan, H.J., Li, S.D., Xing, K., Chan, S.M., He, J.G., 2007. Profiling of differentially expressed genes in hepatopancreas of white spot syndrome virusresistant shrimp (Litopenaeus vannamei) by suppression subtractive hybridisation. Fish & Shellfish Immunology 22, 520–534.
Contents lists available at SciVerse ScienceDirect
Aquaculture journal homepage: www.elsevier.com/locate/aqua-online
Proteome modifications of fingerling rainbow trout (Oncorhynchus mykiss) muscle as an effect of dietary nucleotides Saeed Keyvanshokooh ⁎, Ahmad Tahmasebi-Kohyani Department of Fisheries, Faculty of Marine Natural Resources, Khorramshahr University of Marine Science and Technology, Khorramshahr, Khouzestan, Iran
a r t i c l e
i n f o
Article history: Received 27 July 2011 Received in revised form 8 October 2011 Accepted 11 October 2011 Available online 19 October 2011 Keywords: Rainbow trout Dietary nucleotides Growth Proteomics
a b s t r a c t A feeding study was conducted to determine the effect of dietary nucleotides (NT) on growth performance and muscle proteome profile of rainbow trout fingerlings. Five experimental diets according to different levels of supplemented nucleotides (0, 0.05, 0.1, 0.15 and 0.2%) were assayed in rainbow trout for 8 weeks. Each diet was randomly allocated to triplicate groups of fish with initial average weight of approximately 23 g. The percentage of body weight gain (WG) and feed efficiency (FE) of fish were better when the fish were fed 0.15–0.2% diets. At the end of feeding trial, fish fed the basal and 0.2% diets were subjected to proteomic analysis. Proteins of the muscle tissue were analyzed using two-dimensional electrophoresis and MALDI-TOF/TOF mass spectrometry. Dietary NT caused differential expression of muscle metabolic proteins including glyceraldehyde-3-phosphate dehydrogenase, creatine kinase, adenylate kinase, nucleoside diphosphate kinase, and triosephosphate isomerase. In addition to metabolic enzymes, troponin-T-1 as a structural protein was found to increase in abundance in the treated fish. The altered expression of both metabolic and structural proteins in fish fed NT may be related to higher growth rate in rainbow trout. These findings provide basic information to understand possible mechanisms of dietary NT contribution to better growth in rainbow trout. © 2011 Elsevier B.V. All rights reserved.
1. Introduction Nucleotides are low molecular weight intracellular compounds which play key roles in diverse essential physiological and biochemical functions including encoding genetic information, mediating energy metabolism and signal transduction (Carver and Allan Walker, 1995). Dietary nucleotides have been reported to be beneficial for humans and animals (Gil, 2002) since they positively influence lipid metabolism, immunity, and tissue growth, development and repair (reviewed in Gil, 2002). Heightened attention on nucleotide supplementation was stimulated by the reports of Burrells et al. (2001b), which indicated enhanced resistance of salmonids to viral, bacterial and parasitic infections as well as improved efficiency of vaccination and osmoregulation capacity. Dietary supplementation of nucleotides in some aquatic species has been revealed to improve growth performance (Adamek et al., 1996; Ramadan et al., 1991), immune responses (Burrells et al., 2001b; Leonardi et al., 2003; Li and Gatlin, 2004; Ramadan et al., 1994; Sakai et al., 2001) and disease resistance (Burrells et al., 2001a,b; Li and Gatlin, 2004; Sakai et al., 2001). Regarding the development of dietary nucleotides in fish nutrition research, the application of novel tools in molecular biology will be necessary to detect various aspects of metabolism and influences on
⁎ Corresponding author. Tel.: + 98 632 4234725; fax: + 98 632 4233322. E-mail address: [email protected] (S. Keyvanshokooh). 0044-8486/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.aquaculture.2011.10.013
physiological responses. Cutting edge technologies such as proteomics, genomics and metabolomics are probably the best approaches that offer a comprehensive method to study biochemical systems by expanding the level of investigation from single biomolecules to a wide range of molecules present in a cell or tissue at once, in terms of their presence and relative abundance, without a priori knowledge (Alves et al., 2010). Current annual production of farmed rainbow trout in Iran is approximately 50,000 tons. The recent accessibility to a large number of salmonid expressed sequences within public databases such as GenBank has made proteomic research on salmonids much more practical (Vilhelmsson et al., 2004). The present study was conducted to determine the effects of dietary nucleotides on muscle proteome of fingerling rainbow trout (Oncorhynchus mykiss). 2. Materials and methods 2.1. Fish and experimental conditions 675 rainbow trout fingerlings were obtained from a commercial rainbow trout farm in Lordegan, Iran. The fish were transported to the laboratory, acclimated to laboratory conditions and fed the basal experimental diet without dietary nucleotides for 2 weeks. At the end of the acclimation period, fish with an average weight of approximately 23 g were randomly selected and stocked in 15 1000-L tanks (triplicate groups per dietary treatment) at a density of 45 fish/tank.
80
S. Keyvanshokooh, A. Tahmasebi-Kohyani / Aquaculture 324–325 (2012) 79–84
The mean water quality parameters were maintained as follows: temperature 13.3 °C, dissolved oxygen 8.2 mg L − 1 and pH 7.7. 2.2. Feed and feeding Basal diet formulation and proximate composition analysis are shown in Table 1. The experimental diet was formulated to have 0% (basal), 0.05%, 0.1%, 0.15%, and 0.2% of “Optimun” supplements (Chemoforma, Augst, Switzerland). This product contained inosine monophosphate (IMP), adenosine monophosphate (AMP), guanosine monophosphate (GMP), uridine monophosphate (UMP), cytidine monophosphate (CMP) and ribonucleic acid (RNA). Dietary ingredients were thoroughly mixed in a mixer, made into pellets and air-dried at room temperature. The pellets were broken into small pieces, packed and stored in a freezer until used. Fish were fed by hand to apparent satiation five times per day (at hours: 8, 11, 14, 16 and 18) with one of the five experimental diets over 8 weeks. 2.3. Growth measurements At the end of feeding experiment, the fish were weighed from each tank. Percentage of body weight gain (WG) and feed efficiency (FE) were calculated for each group as follows: WG ¼ ½100 ðf inal body weight−initial body weight Þ=initial body weight
FE ¼ ½ðf inal body weight−initial body weight Þ=feed intake
Table 1 Ingredients composition of test diets fed by rainbow trout. Ingredient (% dry weight)
Basal diet
0.05%
0.10%
0.15%
0.20%
Fish meal a Wheat, white Fish oil a Soybean oil e Soybean meale Mineral premix d Vitamin premix c Vitamin C e Anti fungus e Di calcium phosphate Cellulose b Optimun g
43 18.65 3 3 26 1.5 1.5 0.1 0.25 1 2.0 0
43 18.65 3 3 26 1.5 1.5 0.1 0.25 1 1.95 0.05
43 18.65 3 3 26 1.5 1.5 0.1 0.25 1 1.90 0.10
43 18.65 3 3 26 1.5 1.5 0.1 0.25 1 1.85 0.15
43 18.65 3 3 26 1.5 1.5 0.1 0.25 1 1.80 0.20
Analyzed proximate composition (% dry weight) Crude protein 48.25 48.25 Crude lipid 18.86 18.86 h Carbohydrate 8.23 8.23 Ash 12.25 12.25 Dry matter % 87.59 87.59 Gross energy (Kj g− 1 diet) 20.24 20.24
48.25 18.86 8.23 12.25 87.59 20.24
48.25 18.86 8.23 12.25 87.59 20.24
48.25 18.86 8.23 12.25 87.59 20.24
f
a Pars Kilka Corporation., Iran, based on 100% dry weight included 72% crude protein, 16.2% crude lipid. b Merck Corporation, Germany. c Unit kg− 1 of mixture: Vitamin: retinol acetate (A), 1,200,000 IU; Cholecalciferol (D3), 400,000; DL-α-tocopheryl acetate (E), 30 IU; menadione sodium bisulfite (K3), 1200 mg; L-ascorbic acid (C), 5400 mg; D-biotin (H2), 200 mg; thiamin mononitrate (B1) ,200 mg; riboflavin (B2), 3600 mg; calcium d-pantothenate (B3), 7200 mg; niacinamide (B5), 9000 mg; pyridoxine hydrochloride (B6), 2400 mg; folic acid (B9), 600 mg; cyanocobalamin (B12), 4 mg; antioxidant 500 mg; Carrier up to 1 kg. d Unit kg− 1 of mixture: Mineral: Fe, 4500 mg; Cu, 500 mg; Co, 50 mg; Se, 50 mg; Zn, 6000 mg; Mn, 5000 mg; I, 150 mg; choline chloride, 150,000 mg; Carrier up to 1 kg. e Khorak-dam Abzian corporation, Iran. f Garmab shimi corporation, Iran. Contains: phosphor 17%, calcium 23.27%, moist 3%, fluorine 0.17%. g Chemoforma, Augst, Switzerland. h Carbohydrate calculated based on: 100 − (crude protein + crude lipid + ash).
2.4. Fish sampling and protein extraction Based on growth measurements (Table 2), fish fed diets with 0 and 0.2% NT were selected for proteomic analysis. Nine fish from each group (three fish per tank) were sacrificed. Muscle tissue samples were taken from behind the head and above the lateral line, within 5 min of slaughter, snap frozen in liquid nitrogen and stored at −80 °C until further analysis. In order to minimize the effects of individual variation, the muscle tissues of (each) three individuals were mixed before proteome extraction so that three pools were prepared for each experimental group. The tissue samples were washed in 40 mM Tris–HCl buffer (pH = 7), before being mechanically homogenized in cold lysis buffer containing 7 M urea, 2 M thiourea, 4% CHAPS, 50 mM DTT, 50 mM Tris (pH = 7), 0.2% carrier ampholyte, 1 mM PMSF, 0.25% RNase, and 1% DNase. Each sample was maintained for 1 h at room temperature for protein release. Cellular debris were removed by centrifugation at 12,000 ×g for 10 min, supernatants were collected and their protein content was determined according to the method of Bradford (Bradford, 1976) with BSA as standards. 2.5. Two dimensional gel electrophoresis 17 cm Immobilized pH gradient (IPG) strips with linear pH range of 3–10 (BioRad, USA) were passively rehydrated for at least 16 h with 1.2 mg protein in 300 μL of rehydration buffer containing 8 M urea, 4% CHAPS, 50 mM DTT, 0.2% ampholyte and bromophenol blue traces. Strips were focused at 20 °C with the following program: 20 min with a linear ramp (0–250 V), 5 h with a linear ramp (250– 10,000 V) and 50,000 Vh with a rapid ramp. After isoelectric focusing the strips were equilibrated in the first step with 6 M urea, 50 mM Tris–HCl pH 8.8, 20% glycerol, 2% SDS and 2% DTT for 20 min; and the second step with the same buffer but 2.5% iodoacetamide instead of DTT for another 20 min. Each IPG strip was then laid onto a 12% SDS-PAGE gel for second dimension electrophoresis. The running conditions were 16 mA/gel for 30 min and 24 mA/gel for approximately 5 h until the bromophenol blue front was 1 cm above the bottom of the gel. The gels were stained using colloidal Coomassie brilliant blue G-250 procedure (Candiano et al., 2004). To confirm the reproducibility of the results, three 2-DE runs were performed for each experimental condition. 2.6. Analysis of 2-D gels The gels were scanned using the Densitometer GS-800 scanner (BioRad, USA) and stored as TIF files. Spot detection and matching were performed using the PG200 software (Nonlinear Dynamics, UK). Each set of gel replicates was combined into average gels, which represented spots that were reproducibly present on both sets of the replicate gels. The software automatically selected the gel with greatest number of spots as the image for the reference gel and added unmatched spots from the other gels to this image to give a comprehensive reference gel
Table 2 Final weight, weight gain and feed efficiency (FE) of rainbow trout fed different levels of dietary nucleotides for 8 weeks. Experimental diets (%) 0 0.05 0.1 0.15 0.2
Final weight (g)
Weight gain (%)
Feed efficiency (FE)
46 ± 0.7a 46 ± 0.5a 58 ± 0.4b 66 ± 0.7c 67 ± 0.8c
98 ± 8a 99 ± 6a 151 ± 9b 187 ± 7c 192 ± 5c
0.62 ± 0.21a 0.73 ± 0.18 b 0.76 ± 0.24 b 0.85 ± 0.26 c 0.95 ± 0.17d
Values are mean±S.E.M. of three replicate groups. Mean values with different superscripts are significantly different from each other (significance level is defined as P b 0.05).
S. Keyvanshokooh, A. Tahmasebi-Kohyani / Aquaculture 324–325 (2012) 79–84
for matching spots on the different gels. The spots detected automatically by the software were visually inspected. Spot filtering and editing were performed manually to remove artifacts and to correct for spots that did not split correctly or were not detected by the software's automatic spot detection process. After the filtering and editing steps, the average gels were recreated, and the spots on the average gels were matched, using the comprehensive reference gel as a map to link the spots on different gels. The software subtracted a background value for each spot based on the entire surface area of the gel. Spot volumes were normalized, based on the total volume of all of the spots on each gel, and both of these operations were performed recursively every time spots were edited on any of the gels. Finally, normalized volume (NV) of the matched protein spots that were found to vary in expression between the diets were analyzed for statistical significance using Student's t-test, P b 0.05. Each spot that met this criterion was then visually verified to see if it was correctly detected, and the accuracy of the matching of the spots between the replicate sets was confirmed. Induction and repression factors (IF and RF) correspond to the ratio: NV nucleotide/NV controls. NV coefficients of variability (CV) were calculated for each spot between all the gels of the same condition, using the formula: CV = standard deviation × mean − 1 × 100. 2.7. In-gel trypsin digestion, MALDI-TOF/TOF MS and database searching Gel pieces were washed two times with 50% (v:v) aqueous acetonitrile containing 25 mM ammonium bicarbonate, then once with acetonitrile and dried in a vacuum concentrator for 20 min. Sequencing-grade, modified porcine trypsin (Promega) was dissolved in the 50 mM acetic
81
acid supplied by the manufacturer, then diluted 5-fold by adding 25 mM ammonium bicarbonate to give a final trypsin concentration of 0.02 μg/μL. Gel pieces were rehydrated by adding 10 μL of trypsin solution, and after 30 min enough 25 mM ammonium bicarbonate solution was added to cover the gel pieces. Digests were incubated overnight at 37 °C. A 1 μL aliquot of each peptide mixture was applied directly to the ground steel MALDI target plate, followed immediately by an equal volume of a freshly-prepared 5 mg/mL solution of 4-hydroxy-αcyano-cinnamic acid (Sigma) in 50% aqueous (v:v) acetonitrile containing 0.1%, trifluoroacetic acid (v:v). Positive-ion MALDI mass spectra were obtained using a Bruker ultraflex III in reflectron mode, equipped with a Nd:YAG smart beam laser. MS spectra were acquired over a mass range of m/z 800–4000. Final mass spectra were externally calibrated against an adjacent spot containing 6 peptides (des-Arg1-Bradykinin, 904.681; Angiotensin I, 1296.685; Glu1Fibrinopeptide B, 1750.677; ACTH (1–17 clip), 2093.086; ACTH (18–39 clip), 2465.198; ACTH (7–38 clip), 3657.929.). Monoisotopic masses were obtained using a SNAP averagine algorithm (C 4.9384, N 1.3577, O 1.4773, S 0.0417, H 7.7583) and a S/N threshold of 2. For each spot the ten strongest peaks of interest, with a S/N greater than 30, were selected for MS/MS fragmentation. Fragmentation was performed in LIFT mode without the introduction of a collision gas. The default calibration was used for MS/MS spectra, which were baseline-subtracted and smoothed (Savitsky-Golay, width 0.15 m/z, cycles 4); monoisotopic peak detection used a SNAP averagine algorithm (C 4.9384, N 1.3577, O 1.4773, S 0.0417, H 7.7583) with a minimum S/N of 6. Bruker flexAnalysis software (version 3.3) was used to perform the spectral processing and peak list generation for both the MS and MS/MS spectra.
Fig. 1. 2-DE map of muscle proteins of rainbow trout (Oncorhynchus mykiss), prepared by linear wide-range immobilized pH gradients (pH 3–10, 17 cm; BioRad, USA) in the first dimension and on 12% SDS-PAGE for the second dimension. Proteins were stained with colloidal Coomassie brilliant blue G-250. Labeled spots indicate proteins with significant altered expression profile after dietary nucleotides treatment (see Table 3).
82
S. Keyvanshokooh, A. Tahmasebi-Kohyani / Aquaculture 324–325 (2012) 79–84
Tandem mass spectral data were submitted to database searching using a locally-running copy of the Mascot program (Matrix Science Ltd., version 2.1), through the Bruker ProteinScape interface (version 2.1). Search criteria included: Enzyme, Trypsin; Fixed modifications, Carbamidomethyl (C); Variable modifications, Oxidation (M); Peptide tolerance, 250 ppm; MS/MS tolerance, 0.5 Da; Instrument, MALDITOF-TOF. 3. Results and discussion 3.1. Growth performance There was no fish mortality during the 8-week feeding trial among the five treatment groups. Fish fed 0.15–0.2% diets had highest WG, followed by fish fed the 0.1% diet, and lower in fish fed 0–0.05% diets. After 8 weeks of feeding, FEs of fish fed 0.05–0.2% diets were significantly higher than those of fish fed the NT-free basal diet (Table 2). There was an obvious trend of higher WG and FE with an increase in nucleotides in the diet, indicating that dietary supplementation of 0.15–0.2% nucleotides in diet exerts a positive effect on growth performance of rainbow trout fingerlings. Similarly, a growth-promoting effect was noted in Atlantic salmon (Salmo salar) fed nucleotides at level of 0.25% (Burrells et al., 2001b). Lin et al. (2009) also reported that dietary supplementation of 1.5 g mixed-NT kg− 1 exerted a positive effect on weight gain of grouper (Epinephelus malabaricus). Although the administration of dietary nucleotides has been reported to enhance the growth performance of cultured species such as tilapia (Ramadan et al., 1991), red drum (Li et al., 2007a) and white shrimp (Li et al., 2007b), the growthpromoting effect of nucleotides has not been observed in hybrid striped bass (Li and Gatlin, 2004) and juvenile red drum (Li et al., 2005). Based on current knowledge, it seems that growth enhancing effect of nucleotides may vary in different species and depends on feeding period, life stages and the type of nucleotide supplement (Li and Gatlin, 2006).
proliferation, development and differentiation. NDPK plays an important role in early embryonic development of Atlantic salmon (Murphy et al., 2000). NDPK is also important for the normal development of Drosophila. In shrimp, NDPK acts as a defense-related enzyme and plays roles in the anti-viral innate immune reaction (Zhao et al., 2007). The expression of NDPK was also up-regulated in hemolymph of scallop after bacterial challenge (Shi et al., 2008). Borda et al. (2003) presumed that an exogenous supply of nucleotides may promote growth of fish and crustaceans in early stages to meet their high rate of cell replication. Also, in the present study, the fish fed on 0.2% diet had higher levels of IgM, lysozyme and alternative complement activity (ACH50) (Tahmasebi-Kohyani et al., 2010). These results suggest that NDPK may play roles in the higher rate of cell proliferation and innate immune responses of fish fed dietary nucleotides. Adenylate kinase (AK) showed a decreased expression in fish fed nucleotides. AK is reported to play an important role in energy metabolism and in efficient energy transfer (Dzeja and Terzic, 2003). Ak interconverts Mg + 2 ATP (or Mg + 2 GTP) and free AMP with Mg + 2 ADP (or Mg + 2 GDP) and free ADP by catalyzing the addition of a phosphate to the β-position of AMP using a phosphate of the γposition of ATP (or GTP) as a phosphoryl donor, thereby resulting in the production of two molecules of ADP (or ADP and GDP) (Noda,
3.2. Proteomics The objective of this study was to examine the effects of dietary nucleotides on muscle proteome of rainbow trout. Muscles of control and treated groups were subjected to proteome analysis by 2-DE. Using the spot finding protocol of PG200 software, an average of 710 protein spots per gel were observed, with Mr ranging between 10 and 250 kDa and pI between 3.5 and 9.5. Most protein spots were located in the pI range between 5 and 8 (Fig. 1). Normalized volumes (vol. %) of the matched spots in replicates of basal and NT-fed samples were statistically analyzed by Student's ttest. Eight spots were significantly changed in abundance between the control and treated fish, and 6 of these were increased while the others were decreased in the NT-fed fish (Fig. 2). The differences in abundance between the two groups ranged from −2 to + 2.64-fold. All the spots were matched to the proteins of S. salar, and none of them could be matched to rainbow trout proteins since the genome of rainbow trout is not completely sequenced. Dietary NT caused differential expression of muscle metabolic proteins including glyceraldehyde-3-phosphate dehydrogenase, creatine kinase, adenylate kinase, nucleoside diphosphate kinase A, and triosephosphate isomerase. In addition to metabolic enzymes, troponin-T-1 as a structural protein was found to increase in abundance in the treated fish (Table 3). The expression of nucleoside diphosphate kinase (NDPK) was increased in fish fed dietary nucleotides. NDPK is a key metabolic enzyme that catalyzes the synthesis of non-adenine nucleoside triphosphate (NTP) by transferring the terminal phosphate between nucleoside diphosphate (NDP) and NTP (Shi et al., 2008). This enzyme is widely distributed and is believed to play a role in the general homeostasis of cellular nucleoside triphosphate pools. In addition to this metabolic role, a number of other functions have been assigned to NDPK such as
Fig. 2. Comparison of 2-DE spots with significant altered expression level (P b 0.05, student t-test) between control and NT-fed rainbow trout. The 2-D pattern of control is on the left and that of NT treated fish is on the right.
S. Keyvanshokooh, A. Tahmasebi-Kohyani / Aquaculture 324–325 (2012) 79–84
83
Table 3 Identification of differentially expressed proteins in the muscle tissue of rainbow trout fed dietary nucleotides (NT) for 8 weeks. Spot number
Protein name
1 2 3 4 5 6 7 8
Glyceraldehyde-3-phosphate dehydrogenase-1 Fast myotomal muscle troponin-T-1 Fast myotomal muscle troponin-T-1 Creatine kinase Adenylate kinase Nucleoside diphosphate kinase A Triosephosphate isomerase 1b Triosephosphate isomerase 1b
Accession number
Ms/ Ms score
Organism
gi|197632425 gi|197632601 gi|197632601 gi|197632231 gi|213511412 gi|213511196 gi|213515400 gi|213515400
47 125 113 96 107 106 336 500
Salmo salar S. salar S. salar S. salar S. salar S. salar S. salar S. salar
Expression
Overexpressed Overexpressed Overexpressed Overexpressed Underexpressed Overexpressed Underexpressed Overexpressed
IFa
2.64⁎ 2.60⁎ 2.06⁎ 2.52⁎ 1.7⁎ 1.55⁎
RFa
2⁎ 1.46⁎
Mean NV
CV (%)
NT/control
NT/control
0.127/0.048 0.138/0.053 0.066/0.032 0.043/0.017 0.013/0.026 0.041/0.024 0.097/0.142 0.502/0.323
15/23 8/21 20/16 5/9 23/23 16/13 21/10 14/17
⁎ P b 0.05. a The induction (IF) and repression (RF) factors are the ratios between normalized volumes in treated and control.
1973). The NDPK and adenylate kinase enzymes are both involved in the metabolism of nucleotides within the cell. AK belongs to the nucleoside monophosphate kinase class of enzymes, and differs from the NDPKs in its substrate specificity. While NDPK equilibrates nucleoside di- and triphosphates (Hooks et al., 1989), AK equilibrates adenylates and free Mg + 2 (Igamberdiev and Kleczkowski, 2003) in the reversible ATP-dependent phosphorylation of AMP to ADP and dAMP to dADP (Hooks et al., 1994). The decrease of AK in our study may be as a result of NDPK overexpression as it is shown that AK stimulated NDPK activity, whereas NDPK inhibited the activity of AK (Johansson et al., 2008). However, further studies have to be performed in order to understand how the interaction between NDPK and AK is regulated. The fish fed on nucleotide-supplemented diet exhibited an increased expression of glyceraldehyde-3-phosphate dehydrogenase (GAPDH). GAPDH is a key glycolytic enzyme that catalyzes the reversible conversion of glyceraldehyde-3-phosphate to 1,3-bisphosphoglycerate. Owing to its role in the glycolysis pathway, GAPDH has been routinely considered as a housekeeping gene. However, there is increasing evidence that GAPDH has gained several other new functions such as nuclear RNA export, DNA replication, DNA repair, membrane fusion and microtubule bundling (Sirover, 1999), as well as the apoptotic cascade (Chuang et al., 2005). The biochemical analysis of chicken muscle proteome dynamics under conditions of rapid growth has revealed drastic expression changes in various glycolytic enzymes, including GAPDH (Doherty et al., 2004; Teltathum and Mekchay, 2009). In analogy, the proteomic profiling of ovine muscle hypertrophy showed increased levels of GAPDH during muscle growth (Hamelin et al., 2006). In contrast, weight loss is accompanied by differential effects on numerous glycolytic enzymes, such as enolase, aldolase and GAPDH, as well as lactate dehydrogenase (Almeida et al., 2010a,b). The results of our study support the above outlined studies suggesting that GAPDH can be useful for the future establishment of a biomarker signature of muscle growth and weight gain in rainbow trout. Interestingly, two isoforms of triosephosphate isomerase (TPI) were detected in the present study, an acidic one (spot 7) and a less acidic one (spot 8). The TPI in spot 7 was down-regulated and the TPI in spot 8 was up-regulated. The proteomic analysis of chicken muscle under conditions of rapid growth has revealed increased levels of glycolytic enzymes including TPI (Doherty et al., 2004; Teltathum and Mekchay, 2009). Also, glycolytic enzymes such as TPI increased during muscle growth in sheep (Hamelin et al., 2006). Moreover, atrophying muscle showed decreased abundance of three successive enzymes in the intermediate steps of the glycolytic pathway, fructose-bisphosphate aldolase, TPI and GAPDH in rainbow trout (Salem et al., 2010). Although the abundance of both spots was affected by dietary supplementation of NT, the total TPI levels (spot 7+ spot 8) increased (Table 3), suggesting the role of glycolytic enzymes in better growth of fish fed dietary NT. Creatine kinase (CK) is an enzyme catalyzing the conversion of adenosine diphosphate (ADP) to adenosine triphosphate (ATP), consuming phosphocreatine and generating creatine. CK is typically found in tissues
with high energy demand like cardiac and skeletal muscle, brain, retina and primitive-type spermatozoa (Wallimann and Hemmer, 1994). The expression of CK was increased in fish fed dietary nucleotides. Reddish et al. (2008) reported the association of aldolase, enolase, pyruvate kinase, creatine kinase, and their fragments with body mass and length in Perca flavescens. Variability in expression levels of aldolase, enolase, and CK was also documented by other authors in association with development in zebrafish (Bosworth et al., 2005) and in trout (Morzel et al., 2006). In addition, Salem et al. (2010) reported the downregulation of CK in atrophying muscle. The over-expression of CK observed in this study could be related to higher growth rate in fish fed on NT-supplemented diet. Of the spots that were shown to increase with dietary NT, two were identified as the fast myotomal muscle troponin-T-1. In fish, muscle growth is commonly reported to occur by a combination of hyperplasia and hypertrophy, that is, through an increase in both myocyte number and size (Weatherley and Gill, 1985). Addis et al. (2010) related the expression of structural proteins including troponin with sea bream muscle growth. Structural proteins are the main constituents of sarcomere, the contractile unit of muscular fibers. The over-expression of both metabolic and structural proteins in fish fed NT may be related to the contribution of hyperplasia and hypertrophy to muscle growth in rainbow trout. In conclusion, we represented some changes in the muscle proteome of rainbow trout fed dietary nucleotides. The altered expression of both metabolic and structural proteins in fish fed NT may be related to higher growth rate in rainbow trout. These findings provide basic information to understand possible mechanisms of dietary NT contribution to better growth in fish. Acknowledgment This work was funded by grant P-1/4-12 from Khorramshahr University of Marine Science and Technology. We thank Dr. Behrouz Vaziri and Fatemeh Zandi (Pasteur Institute of Iran) for their technical and supportive assistance. We also thank Nemat Mahmoudi (Tarbiat Modares University) for his assistance. References Adamek, Z., Hamackova, J., Kouril, J., Vachta, R., Stibranyiova, I., 1996. Effect of Ascogen probiotics supplementation on farming success in rainbow trout (Oncorhynchus mykiss) and wels (Silurus glais) under conditions of intensive culture. Krmiva (Zagreb) 38, 11–20. Addis, M.F., Cappuccinelli, R., Tedde, V., Pagnozzi, D., Porcu, M.C., Bonaglini, E., Roggio, T., Uzzau, S., 2010. Proteomic analysis of muscle tissue from gilthead sea bream (Sparus aurata, L.) farmed in offshore floating cages. Aquaculture 309, 245–252. Almeida, A., Campos, A., Francisco, R., Van Harten, S., Cardoso, L., Coelho, A., 2010a. Proteomic investigation of the effects of weight loss in the gastrocnemius muscle of wild and NZW rabbits via 2D electrophoresis and MALDI TOF MS. Animal Genetics 41, 260–272. Almeida, A., Van Harten, S., Campos, A., Coelho, A., Cardoso, L., 2010b. The effect of weight loss on protein profiles of gastrocnemius muscle in rabbits: a study using
84
S. Keyvanshokooh, A. Tahmasebi-Kohyani / Aquaculture 324–325 (2012) 79–84
1D electrophoresis and peptide mass fingerprinting. Journal of Animal Physiology and Animal Nutrition 94, 174–185. Alves, R.N., Cordeiro, O., Silva, T.S., Richard, N., de Vareilles, M., Marino, G., Di Marco, P., Rodrigues, P.M., Conceição, L.E.C., 2010. Metabolic molecular indicators of chronic stress in gilthead seabream (Sparus aurata) using comparative proteomics. Aquaculture 299, 57–66. Borda, E., Martinez-Puig, D., Cordoba, X., 2003. Well-balanced nucleotide supply makes sense. Feed Mix 11, 24–26. Bosworth, I., Charles, A., Chou, C.W., Cole, R.B., Rees, B.B., 2005. Protein expression patterns in zebrafish skeletal muscle: initial characterization and the effects of hypoxic exposure. Proteomics 5, 1362–1371. Bradford, M.M., 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein–dye binding. Analytical Biochemistry 72, 248–254. Burrells, C., Williams, P., Forno, P., 2001a. Dietary nucleotides: a novel supplement in fish feeds: 1. Effects on resistance to disease in salmonids. Aquaculture 199, 159–169. Burrells, C., Williams, P., Southgate, P., Wadsworth, S., 2001b. Dietary nucleotides: a novel supplement in fish feeds: 2. Effects on vaccination, salt water transfer, growth rates and physiology of Atlantic salmon (Salmo salar L.). Aquaculture 199, 171–184. Candiano, G., Bruschi, M., Musante, L., Santucci, L., Ghiggeri, G.M., Carnemolla, B., Orecchia, P., Zardi, L., Righetti, P.G., 2004. Blue silver: A very sensitive colloidal Coomassie G 250 staining for proteome analysis. Electrophoresis 25, 1327–1333. Carver, J.D., Allan Walker, W., 1995. The role of nucleotides in human nutrition. The Journal of Nutritional Biochemistry 6, 58–72. Chuang, D.M., Hough, C., Senatorov, V.V., 2005. Glyceraldehyde-3-phosphate dehydrogenase, apoptosis, and neurodegenarative diseases. Annual Review of Pharmacology and Toxicology 45, 269–290. Doherty, M.K., McLean, L., Hayter, J.R., Pratt, J.M., Robertson, D.H.L., El Shafei, A., Gaskell, S.J., Beynon, R.J., 2004. The proteome of chicken skeletal muscle: changes in soluble protein expression during growth in a layer strain. Proteomics 4, 2082–2093. Dzeja, P.P., Terzic, A., 2003. Phosphotransfer networks and cellular energetics. The Journal of Experimental Biology 206, 2039. Gil, A., 2002. Modulation of the immune response mediated by dietary nucleotides. European Journal of Clinical Nutrition 56, S1. Hamelin, M., Sayd, T., Chambon, C., Bouix, J., Bibé, B., Milenkovic, D., Leveziel, H., Georges, M., Clop, A., Marinova, P., 2006. Proteomic analysis of ovine muscle hypertrophy. Journal of Animal Science 84, 3266. Hooks, M.A., Clark, R.A., Nieman, R.H., Roberts, J.K.M., 1989. Compartmentation of nucleotides in corn root tips studied by 31P-NMR and HPLC. Plant Physiology 89, 963. Hooks, M.A., Shearer, G.C., Roberts, J.K.M., 1994. Nucleotide availability in maize (Zea mays L.) root tips (estimation of free and protein-bound nucleotides using 31Pnuclear magnetic resonance and a novel protein–ligand-binding assay). Plant Physiology 104, 581. Igamberdiev, A.U., Kleczkowski, L.A., 2003. Membrane potential, adenylate levels and Mg2 + are interconnected via adenylate kinase equilibrium in plant cells. Biochimica et Biophysica Acta (BBA)-Bioenergetics 1607, 111–119. Johansson, M., Hammargren, J., Uppsäll, E., MacKenzie, A., Knorpp, C., 2008. The activities of nucleoside diphosphate kinase and adenylate kinase are influenced by their interaction. Plant Science 174, 192–199. Leonardi, M., Sandino, A., Klempau, A., 2003. Effect of a nucleotide-enriched diet on the immune system, plasma cortisol levels and resistance to infectious pancreatic necrosis (IPN) in juvenile rainbow trout (Oncorhynchus mykiss). Bulletin of the European Association of Fish Pathologists 23, 52–59. Li, P., Gatlin, D.M., 2004. Dietary brewers yeast and the prebiotic Grobiotic (TM) AE influence growth performance, immune responses and resistance of hybrid striped bass (Morone chrysops M. saxatilis) to Streptococcus iniae infection. Aquaculture 231, 445–456. Li, P., Gatlin III, D.M., 2006. Nucleotide nutrition in fish: current knowledge and future applications. Aquaculture 251, 141–152.
Li, P., Burr, G.S., Goff, J., Whiteman, K.W., Davis, K.B., Vega, R.R., Neill, W.H., Gatlin III, D.M., 2005. A preliminary study on the effects of dietary supplementation of brewers yeast and nucleotides, singularly or in combination, on juvenile red drum (Sciaenops ocellatus). Aquaculture Research 36, 1120–1127. Li, P., Gatlin III, D.M., Neill, W.H., 2007a. Dietary supplementation of a purified nucleotide mixture transiently enhanced growth and feed utilization of juvenile red drum, Sciaenops ocellatus. Journal of the World Aquaculture Society 38, 281–286. Li, P., Lawrence, A.L., Castille, F.L., Gatlin III, D.M., 2007b. Preliminary evaluation of a purified nucleotide mixture as a dietary supplement for Pacific white shrimp Litopenaeus vannamei (Boone). Aquaculture Research 38, 887–890. LIN, Y.H., Wang, H., SHIAU, S.Y., 2009. Dietary nucleotide supplementation enhances growth and immune responses of grouper, Epinephelus malabaricus. Aquaculture Nutrition 15, 117–122. Morzel, M., Chambon, C., Lefèvre, F., Paboeuf, G., Laville, E., 2006. Modifications of trout (Oncorhynchus mykiss) muscle proteins by preslaughter activity. Journal of Agricultural and Food Chemistry 54, 2997–3001. Murphy, M., Harte, T., McInerney, J., Smith, T.J., 2000. Molecular cloning of an Atlantic salmon nucleoside diphosphate kinase cDNA and its pattern of expression during embryogenesis. Gene 257, 139–148. Noda, L., 1973. 8 adenylate kinase. The Enzymes, 8, pp. 279–305. Ramadan, A., Atef, M., Afifi, N., 1991. Effect of the biogenic performance enhancer (Ascogen “S”) on growth rate of tilapia fish. Acta Veterinaria Scandinavica. Supplementum 87, S304–S306. Ramadan, A., Afifi, N.A., Moustafa, M., Samy, A., 1994. The effect of ascogen on the immune response of tilapia fish to Aeromonas hydrophila vaccine. Fish & Shellfish Immunology 4, 159–165. Reddish, J.M., St Pierre, N., Nichols, A., Green Church, K., Wick, M., 2008. Proteomic analysis of proteins associated with body mass and length in yellow perch, Perca flavescens. Proteomics 8, 2333–2343. Sakai, M., Taniguchi, K., Mamoto, K., Ogawa, H., Tabata, M., 2001. Immunostimulant effects of nucleotide isolated from yeast RNA on carp, Cyprinus carpio L. Journal of Fish Diseases 24, 433–438. Salem, M., Kenney, P.B., Rexroad III, C.E., Yao, J., 2010. Proteomic signature of muscle atrophy in rainbow trout. Journal of Proteomics 73, 778–789. Shi, X.Z., Zhao, X.F., Wang, J.X., 2008. Molecular cloning and analysis of function of nucleoside diphosphate kinase (NDPK) from the scallop Chlamys farreri. Biochemistry 73, 686–692. Sirover, M.A., 1999. New insights into an old protein: the functional diversity of mammalian glyceraldehyde-3-phosphate dehydrogenase. Biochimica et Biophysica Acta 1432, 159–184. Tahmasebi-Kohyani, A., Keyvanshokooh, S., Nematollahi, A., Mahmoudi, N., PashaZanoosi, H., 2010. Dietary administration of nucleotides to enhance growth, humoral immune responses, and disease resistance of the rainbow trout (Oncorhynchus mykiss) fingerlings. Fish & Shellfish Immunology 30, 189–193. Teltathum, T., Mekchay, S., 2009. Proteome changes in Thai indigenous chicken muscle during growth period. International Journal of Biological Sciences 5, 679. Vilhelmsson, O.T., Martin, S.A.M., Médale, F., Kaushik, S.J., Houlihan, D.F., 2004. Dietary plant-protein substitution affects hepatic metabolism in rainbow trout (Oncorhynchus mykiss). The British Journal of Nutrition 92, 71–80. Wallimann, T., Hemmer, W., 1994. Creatine kinase in non-muscle tissues and cells. Molecular and Cellular Biochemistry 133, 193–220. Weatherley, A., Gill, H., 1985. Dynamics of increase in muscle fibers in fishes in relation to size and growth. Cellular and Molecular Life Sciences 41, 353–354. Zhao, Z.Y., Yin, Z.X., Weng, S.P., Guan, H.J., Li, S.D., Xing, K., Chan, S.M., He, J.G., 2007. Profiling of differentially expressed genes in hepatopancreas of white spot syndrome virusresistant shrimp (Litopenaeus vannamei) by suppression subtractive hybridisation. Fish & Shellfish Immunology 22, 520–534.