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Functional changes in digestive enzymes and characterization of proteases of silver carp (♂) and bighead carp (♀) hybrid, during early ontogeny
Aquaculture 253 (2006) 694 – 702 www.elsevier.com/locate/aqua-online
Functional changes in digestive enzymes and characterization of proteases of silver carp (♂) and bighead carp (♀) hybrid, during early ontogeny Rina Chakrabarti a,⁎, Raja Mansingh Rathore a , Prabhat Mittal b , Sunil Kumar a a b
Aqua Research Lab, Department of Zoology, University of Delhi, Delhi 110 007, India Department of Statistics, Satyawati College, University of Delhi, Delhi 110 052, India
Received 31 May 2005; received in revised form 27 August 2005; accepted 31 August 2005
Abstract Study of digestive enzyme and partial characterization of proteases of silver carp (♂) and bighead carp (♀) hybrid during early ontogeny was performed. Specific amylase activity was observed in 4 DAH (0.07 ± 0.01 mg maltose mg protein− 1 min− 1) carp hybrid. Specific amylase activity showed polynomial relationship with the age of fish. Total protease, trypsin and chymotrypsin activities were 14.37 ± 2.21, 11.38 ± 1.67 and 2.83 ± 0.50 mUnits mg protein− 1 min− 1 in 4 DAH fish, respectively. Total protease activity showed exponential trend, whereas trypsin and chymotrypsin activities showed polynomial relationships with the increasing age of the fish. Lipase activity was 2.33 ± 0.18 mUnits in 4 DAH carp hybrid. Lipase activity showed polynomial trend with the increasing age of fish. In inhibition study, SBTI, PMSF, TLCK and TPCK inhibited the protease activity by 83.0% to 92.0%, 71.0% to 81.0%, 45.1% to 55.5% and 35.8% to 48.2%, respectively. SDS-PAGE showed the presence of various protein bands (20.9–69.4 kDa) in carp hybrid during ontogenic development. Substrate SDS-PAGE revealed the presence of several protease activity bands (19.1–73.7 kDa) in digestive tissue extract of fish during ontogenesis. Inhibition of protease activity bands in substrate SDS-PAGE with SBTI and PMSF revealed the abundance of serine proteases and inhibition of activity bands with TLCK and TPCK evidenced the presence of more than one isoform of trypsin and chymotrypsin in the digestive tissue extract of hybrid carp. © 2005 Elsevier B.V. All rights reserved. Keywords: Digestive enzymes; Proteases; Ontogeny; Inhibitors; Carp
1. Introduction The alarming increase of pollution level has become a menace for the freshwater biodiversities in the recent years. Amplifying demand for fish as food, poor ability of the fish larva to grow faster in environmental stress lead the aquaculturists to search for a desirable trait for ⁎ Corresponding author. Tel.: +91 11 27666496. E-mail address: [email protected] (R. Chakrabarti). 0044-8486/$ - see front matter © 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.aquaculture.2005.08.018
rapid growth, increased environmental fluctuation tolerance and higher production. Inter-specific breeding among fishes is a perfect tool to transfer desirable traits and is now been widely used in most of the world for a potential aquaculture (Bartley et al., 2001). Hybrid of microplanktophagous silver carp (Hypophthalmichthys molitrix) and macroplanktophagous bighead (Aristichthys nobilis) is widely accepted in aquaculture in Asia due to having the positive heterosis for growth rate. However, pure lines may be lost due to fertility of
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hybrids when parents and hybrids are grown together (Krasnai, 1987). This may be avoided by culture under control conditions. The process of embryonic and pre-larval development is associated with enhancement of two alternatives, synthesis and catabolism of proteins, which is accompanied by increase in the activities of trypsin and chymotrypsin-like proteases in Cyprinus carpio (Konovalov and Mestechkin, 1975). The rate of development is likely to determine the survival of fish with physiological and environmental changing status. Detailed knowledge of digestive physiology during the developmental stages of such fish is essential as the maximum growth rate of fish is partly contributed by digestive capacity, oxygen availability and their metabolic capacity required to support tissue protein synthesis (Blier et al., 1997). Age related changes in the diversity and quantity of digestive enzymes appear to represent evolutionary adaptations to the different natural diets and nutritional requirement of distinct developmental stages (Kumar et al., 2000). Fish must be competent at procuring and assimilating food before yolk sac absorption (Green and McCormick, 2001). The yolk sac of common carp is resorbed 2–5 days after hatching (depending on water temperature), whereas the activities of digestive enzymes are very low. The question remains which mechanisms maintain food assimilation at a level sufficient for intensive growth (II’ina and Turetskiy, 1987). For better survival of larvae, feeding must be initiated on digestible diets before or very soon after depletion of the endogenous energy sources, yolk and oil (Kim et al., 2001). The assessment of the presence and level of activity of certain enzymes may be used as a comparative indicator of the rate of development of the larvae, as well as their further survival rate (Ueberschar, 1993). The ratio of membrane and cavity digestion actually changes in fishes with age. It was found during the study of digestion in Ctenopharyngodon idella, that the ratio between amylase activity in hepatopancreas to its activity in mucosa increases with age: younger the fish, more significant the role of amylase associated with the mucosa (Buzinova, 1973). The development of larva to a fingerling relies on a proper development of digestive functions during larval life, and the maturation of digestive tract can be altered by diet composition (Hongming Ma et al., 2005). It has been suggested that growth of carp larvae on any artificial diet is not faster than on natural food. Dement’ev (1984) found deviation in type of enzyme activities found in silver carp fed with artificial diet as compared to younger fish fed with natural diet. It is well
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documented that at the early developmental stage fishes experience a crucial metabolic phase during the shifting of feeding. Considerable attention has been paid to evaluate the functional characteristics of the digestive enzymes that play an important role in the ontogenesis of larvae. Though substantial interest has been paid in recent years to bring out the new hybrid species in aquaculture for rapid growth and other desirable traits, information regarding mapping of the digestive physiology during ontogenesis of these hybrids is still unexplored. In the present study, we have tried to evaluate the fate of major functional digestive enzymes found in the gut of the hybrid fish during early developmental stages. Efforts have been made to characterize the digestive proteases. 2. Materials and methods 2.1. Fish species and culture Hybrids of silver carp Hypophthalmichthys molitrix (♀) × bighead Aristichthys nobilis (♂) (Class: Actinopterygii, Order: Cypriniformes, Family: Cyprinidae) were obtained from a commercial fish farm in India after 72 h (3 days old) of hatching (length 6.9 ± 0.1 mm; weight 1.32 ± 0.1 mg). Spawns were stocked at density of 45,000 m− 3 in 450 L tank having connection with recirculating system. Live food (Brachionus spp., Ceriodaphnia spp., Mesocyclops spp.) was provided to the fish ad libitum up to 14 days after hatching (DAH). From day-14 onwards mixed feeding with artificial diet (40% protein) was started at the rate of 4% of body weight. The experiment was continued for 30 days. Temperature and pH ranged from 28.1 to 32.4 °C and 7.5 to 8.9, respectively during the experimental period. Dissolved oxygen was maintained above 5 mg/L with help of aerators. 2.2. Sample preparation Fish were collected randomly from different tanks at 9 A.M. before morning feeding on every alternate day. Larvae were properly washed and were kept at −20 °C. Digestive system of individual fish was taken out on a glass platform maintained at 0 °C under dissecting microscope. 100 mg of gut tissue was collected for each sample and was homogenized with 1 ml pre-chilled distilled water. For 100 mg tissue the number of fish sacrificed was initially 315 ± 2.4 on day-4 and subsequently reduced to 24 ± 1.15 on day-34 as fish grew. Supernatant of the homogenate was obtained by centrifuging at 10,000 ×g at 4 °C for 10 min and stored
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at − 20 °C for further analysis. Three replicates were used for each assay. Total soluble protein was measured according to Bradford (1976). 2.3. Digestive enzyme assays Amylase was assayed by measuring the increase in reducing power of buffered starch solution with 3,5dinitro salicylic acid at 540 nm (Bernfeld, 1955). Specific amylase activity was expressed in terms of milligram of maltose liberated per milligram of protein per hour at 37 °C. Total protease activity was measured by using 1% azocasein as substrate in 50 mM Tris–HCl, pH 7.5 (Garcia-Carreno, 1992) and the change of absorbance was recorded at 366 nm. Protease activity was expressed in terms of mUnits mg protein− 1 min− 1. Trypsin and chymotrypsin activities were measured by taking N-α-benzoyl-DL-Arginine-p-nitroanilide (BAPNA) and Suc-Ala-Ala-Pro-Phe-p-nitroanalidine (SAPNA), as substrates, respectively. Change of absorbance was recorded under kinetic mode for 3 min at 410 nm (Erlanger et al., 1961). Activity units were expressed as change in absorbance mg protein− 1 min− 1 of the enzyme used in the assay. Activity units were calculated by the following equation: Activity units ðAbs410 =minÞ 1000 ml of reaction mixture ¼ 8800 mg protein in reaction mixture The molar extinction coefficient of para-nitroanalidine is €410 = 8800 cm2 mg− 1. Lipase activity was measured as described by Winkler and Stuckman (1979). The principle of the assay is the colorimetric estimation of para-nitrophenol (pNP) released due to enzymatic hydrolysis of para-nitrophenyl palmitate (pNPP) at 410 nm. One enzyme unit is defined as 1 nmol of p-nitrophenol enzymatically released from the substrate ml− 1 min− 1. The extinction coefficient of para-nitrophenol is €410 = 15,000 cm2 mg− 1.
phenylalanine chloromethylketone, TPCK (5 mM in methanol) were used as specific inhibitors for trypsin and chymotrypsin, respectively. Chelating agent Ethylenediamine-tetraacetate, EDTA (20 mM in distilled water) was used for inhibiting metal ion specific proteases. All these inhibitors were pre-incubated at 1 : 1 ratio for 1 h at room temperature. 2.5. Protein and enzyme composition assay Separation of proteins in the enzyme extracts was performed by using 12% SDS-PAGE (Laemmli, 1970). 20 μg of protein for each enzyme extract was loaded on to each well at controlled temperature of 4 °C. After electrophoresis the gels were stained with Coomassie brilliant blue for 2 h and then destained. The protease composition and evaluation of their classes were done by substrate SDS-PAGE (GarciaCarreno et al., 1993). Enzyme extract containing 5 mUnits of activity was loaded on to each well. After electrophoresis gel was immersed in 3% casein in 50 mM Tris–HCl, pH 7.5 for 30 min at 5 °C to allow the substrate to diffuse into the gel. Then the gel was incubated for 60 min at 25 °C. The gel was washed, stained and destained. Clear bands with blue background were indicated as protease activity bands. For protease class evaluation enzyme extract having 5 mUnits activity were incubated with different inhibitors like SBTI (250 μM), PMSF (100 mM), TLCK (10 mM), and EDTA (20 mM) prior to loading on to the wells. Then they are subjected to substrate SDS-PAGE. The bands were compared with activity bands without inhibition. 2.6. Statistical analysis Data of digestive enzyme activities were analyzed by using one-way ANOVA and appropriate regression model analysis. Statistical significance was accepted at P < 0.05 level. 3. Results
2.4. Protease inhibition assay 3.1. Enzyme activity The inhibition assay was conducted to evaluate the protease class by treating the enzymes with different specific inhibitors as described by Garcia-Carreno and Haard (1993). Phenylmethylsulfonylfluoride, PMSF (100 mM in 2-propanol) and Soybean trypsin inhibitor, SBTI (250 μm in distilled water) were used as serine class protease inhibitors. N α-p-tosyl-L-lysine chloromethylketone, TLCK (1 mM in HCL) and N-tosyl-L-
On day-4 the specific amylase activity was 0.078 ± 0.01 mg maltose mg protein− 1 h− 1. The enzyme activity increased significantly (P < 0.01) on day-8 compared to days 4 and 6. Whereas, the activity decreased (30%) significantly (P < 0.05) on day-12 than day-8 (Fig. 1a). The specific amylase activity then increased 22% between days 14 and 16 compared to 12th day. The
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b 0.2 0.18 0.16
y = 1E-08x 6 - 1E-06x 5 + 6E-05x 4 0.0011x3 + 0.0092x2 - 0.0221x + 0.0704 R2 = 0.603
0.14 0.12 0.1 0.08 0.06 0.04
Protease activity (mUnits/mg protein/min)
Specific amylase activity (mg maltose/mg protein/h)
a
1000 800
y = 12.063e 0. 12 73x R2 = 0.9478
600 400 200 0
2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34
2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34
Days after hatching 90 80 70 60 50 40 30 20 10 0
y = 0.0042x3 - 0.1846x 2 + 3.3739x + 2.8342 R2 = 0.9016
2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34
Days after hatching
d Chymotrypsin activity (mUnits/mg potein/min)
c Trypsin activity (mUnits/mg protein/min)
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600 y = 7E-06x6 + 0.0001x5 - 0.0501x 4 + 2.1316x3 - 33. 954x 2 + 222. 78x 500 481.17 2 400 R = 0.9426 300 200 100 0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34
Days after hatching
Lipase activity (mUnits)
e
Days after hatching
10 y = -5E-07x6 + 4E-05x5 - 0.0014x4 + 0.0195x 3 - 0.1205x2 + 0.4531x + 1.3737 8 R2 = 0.9396 6 4 2 0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34
Days after hatching Fig. 1. (a) Specific amylase activity show polynomial (6th degree), (b) protease activity shows exponential, (c) trypsin activity shows polynomial (3rd degree), (d) chymotrypsin activity shows polynomial (6th degree), and (e) lipase activity shows polynomial (6th degree) relationships with increasing age of silver carp × bighead hybrid larvae during ontogenesis. Each value represents mean ± SEM (n = 3).
enzyme activity ranged from 0.083 to 0.109 mg maltose mg protein− 1 min− 1 between day 18 and 34. The enzyme activity on day-34 was significantly (P < 0.05) higher (16%) than the activity found on day-32. The specific amylase activity showed a polynomial (6th degree) relationship with the increasing age of the hybrid fish. Total protease activity in 4 DAH larva was 14.37 ± 2.21 mUnits mg protein− 1 min− 1. The enzyme activity increased (35 to 120%) significantly (P < 0.01) on day8 compared to days 4 and 6. The protease activity on day-12 was also significantly (P < 0.05) higher than the
enzyme activity found in fish between days 8 and 10. But the enzyme activity decreased by 31% on day-14 compared to day-12. The enzyme activity increased by 25 to 31% in 18 DAH fish than the activity found in 14 and 16 DAH fish. The protease activity increased significantly (P < 0.05) between days 20 and 32 compared to the enzyme activity found in the respective previous days. On day-34 enzyme activity decreased (34%) significantly (P < 0.01) than the activity found on day-32. The enzyme activity showed exponential trend with the increasing age of the fish (Fig. 1b).
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Table 1 Percentage of inhibition of protease activity by different inhibitors in silver carp and bighead carp hybrid during ontogenesis % Inhibition Age of SBTI fish (DAH) 28 30 32 34
PMSF
TLCK
92.0 ± 0.7 80. 2 ± 6.8 53.6 ± 2.4 92.5 ± 1.7 77.2 ± 2.8 45.1 ± 5.5 83.7 ± 2.8 71.0 ± 6.1 48.9 ± 18.2 83.0 ± 1.8 81.0 ± 4.7 55.5 ± 3.0
TPCK
EDTA
45.6 ± 7.1 7.1 ± 1.1 48.2 ± 8.9 10.7 ± 2.8 47.9 ± 1.1 11.1 ± 5.4 35.8 ± 5.0 6.1 ± 1.7
Trypsin activity was 11.38 ± 1.6 mUnits mg protein− 1 min− 1 in 4 DAH fish. The activity increased significantly (P < 0.01) in 8 DAH larva than 4 DAH one. A significantly (P < 0.05) reduction (8 to 18%) in trypsin activity was observed between 10 and 18 days compared to the 8th day. On day-22 enzyme activity increased by 7 to 28% compared to the activity found on days 18 and 20. Then the enzyme activity decreased (13%) significantly (P < 0.05) on day-24 than the activity found on day-22. The trypsin activity increased significantly (P < 0.05) between days 26 and 32
compared to the activity found on respective previous days. On day-34, the enzyme activity decreased (17%) significantly (P < 0.05) than the activity found on day32. A polynomial (3rd degree) trend of trypsin activity with increasing age was observed in this hybrid fish (Fig. 1c). Chymotrypsin activity was 2.83 ± 0.5 mUnits mg protein− 1 min− 1 in 4 DAH fish. Significantly (P < 0.01) higher enzyme activity was found on day-10 compared to the activity found on day-4. On day-18 chymotrypsin activity increased (47 to 151%) significantly (P < 0.05) than the activity found between days 12 and 16. Enzyme activity on days 22 and 24 was significantly (P < 0.01) higher than day-20. But there was no significant (P > 0.05) difference in enzyme activity between days 22 and 24. A significant (P < 0.05) decrease (16%) in chymotrypsin activity was recorded on day-26 compared to day-24. The activity was significantly (P < 0.05) higher on day-32 compared to the other days of sampling. On day-34, the chymotrypsin activity decreased by 8% than day-32. Chymotrypsin activity showed a polynomial trend (6th degree) along with the age of the hybrid fish (Fig. 1d).
Fig. 2. (a) SDS-PAGE of digestive extract of silver carp × bighead larvae during ontogenesis. Lane M is molecular weight marker comprising, albumin, bovine (66,000); albumin, egg (45,000); glyceraldehyde-3-phosphate dehydrogenase (36,000); carbonic anhydrase, bovine (29,000); trypsinogen, bovine pancreas (24,000); trypsin inhibitor, soybean (20,100) and α-lactalbumin, bovine milk (14,200). Enzyme sample of different days was loaded on to each well. (b) Zymograms of enzyme samples of digestive extract of silver carp × bighead hybrid larvae during ontogenesis by Substrate SDS-PAGE. Lane M is molecular weight marker and other lanes correspond to samples of different days.
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In 4 DAH fish the lipase activity was 2.3 ± 0.1 mUnits. On day-8 lipase activity increased 35% than on day-4. Then the lipase activity decreased (8%) significantly (P < 0.05) on day 10 than the activity found on day-8. The activity then increased (44%) significantly (P < 0.05) on day-12 than the activity found on day-10. The lipase activity ranged from 3.06 to 4.25 mUnits between days 14 and 28. The enzyme activity increased significantly (P < 0.01) on days 30 and 32 to the respective previous days. A 14% decrease in activity was observed on day-34 compared to day-32 (Fig. 1e). Like amylase and chymotrypsin activities the lipase activity also showed a polynomial (6th degree) relationship with the age of fish. 3.2. Protease inhibition assay Enzyme samples of 28 DAH to 34 DAH fish treated with inhibitors like SBTI, PMSF, TLCK and TPCK showed 83.0% to 92.5%, 71.0% to 81.0%, 45.1% to 55.5% and 35.8% to 48.2% inhibition in the activity, respectively (Table 1). Whereas, enzyme samples treated with EDTA showed 6.1% to 11.1% inhibition of the activity. 3.3. SDS-PAGE and substrate SDS-PAGE Proteinograms of digestive extract of fish were studied by SDS-PAGE (Fig. 2a). Two bands with poor resolution (42.1 and 69.4 kDa) were found between day4 and day-8. One more band of 37.2 kDa appeared on day-10. Three more bands (53.4, 49.4 and 33.3 kDa) became visible on 16 DAH hybrid fish. Further, two low molecular weight bands of 23.9 and 27.1 kDa were visible on the 20th day followed by one more band of 20.9 kDa on day-24. Total 9 protein bands were visible between 24 and 34-day. Zymograms of various proteases of enzyme samples of the fish were observed by substrate SDS-PAGE (Fig. 2b). Two activity bands of proteases (73.7 and 43.6 kDa) were recorded between day-4 and day-10. One more zymogram of 58.1 kDa became visible from 12th day. One activity band of 35.6 kDa was apparent on day-20. Two additional bands of 24 and 19.1 kDa appeared on day-24. From the 24th day onwards, a total 6 activity bands ranging from 19.1 to 73.7 kDa were visible (Table. 2). Treatment of enzyme samples with various inhibitors reduced the number of bands (Table 2). Only one activity bands (73.7 kDa) was visible in SBTI treated enzyme samples. Other activity bands those appeared in the activity gel, were inhibited with SBTI. PMSF
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Table 2 Zymogram profiles of digestive extracts of silver carp × bighead hybrid larvae during ontogenesis Molecular weight of proteases DAH 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34
73.7 + + + + + + + + + + + + + + + +
58.1
43.6
35.6
24
19.1
+Is,p +Is,p +Is,p +Is,p +Is,p +Is,p +Is,p +Is,p
+Is,p,l,c +Is,p,l,c +Is,p,l,c +Is,p,l,c +Is,p,l,c +Is,p,l,c
+Is,p,c +Is,p,c +Is,p,c +Is,p,c +Is,p,c +Is,p,c
s,p
+Is,l,c +Is,l,c +Is,l,c +Is,l,c +Is,l,c +Is,l,c +Is,l,c +Is,l,c +Is,l,c +Is,l,c +Is,l,c +Is,l,c
+I +Is,p +Is,p +Is,p +Is,p +Is,p +Is,p +Is,p +Is,p +Is,p +Is,p +Is,p +Is,p +Is,p +Is,p +Is,p
+ = Activity band of protease. Is = Band Inhibited with SBTI. Ip = Band Inhibited with PMSF. Il = Band Inhibited with TLCK. Ic = Band Inhibited with TPCK.
inhibited four activity bands of 43.6, 35.6, 24 and 19.1 kDa. TLCK inhibited two trypsin-like enzyme activity bands in the gel having molecular weight of 58.1 and 24 kDa. TPCK treated samples showed inhibition of four chymotrypsin-like enzyme activities in the gel. Activity bands of 58.1, 43.6, 24 and 19.1 kDa were inhibited by TPCK. The protease activity bands those appeared in uninhibited samples were also found in EDTA treated samples of substrate SDS-PAGE. 4. Discussion Development and regulation of the digestive enzymes depends on the progressive changes in the digestive tract and the subsequent response to composition and amount of the available food. This is either genetically programmed or induced by the corresponding substrate in the food (Gruzdkov et al., 1986). In the present investigation all the enzymes studied were detected at the first feeding of the hybrid fish. Earlier studies (Chakrabarti and Sharma, 1997; Kumar and Chakrabarti, 1998; Kumar et al., 2000, Rathore et al., 2005) with Indian major carps Catla catla, Labeo rohita, and Cirrhinus mrigala evidenced the presence of amylase, protease, trypsin, chymotrypsin and lipase in all these species at the first feeding. However, high amylase activities between 8 and 10 DAH of the hybrid carp suggested the modulation of digestive system for
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carbohydrate metabolism in hybrid fish during early development. Tanaka (1973) suggested that dietary carbohydrates may fill the energy gap between endogenous and exogenous protein demand of the fish. Kim et al. (2001) found an increase in amylase activity followed by sharp decrease in unfed larval threadfins just before die off. This suggested the synthesis of amylase in early ontogenesis even in absence of food, and the carbohydrates are actively catalyzed during the time of organogenesis. The decrease in activity after the 10th and 16th day in hybrid fish may be possibly due to developmental changes in the gut morphology and increased protein level in the tissue. An increase in the specific amylase activity from 30-day onwards was evidenced in the present study. Silver carp, bighead carp and grass carp were reported to increase in their amylase activities from day-21 when they actively consume large phyto- and zooplankton (Volkova, 1999). Cahu et al. (2004) suggested that regulation of amylase is post-transcriptional in early larval stages of sea bass Dicetrarchus labrax (till day-25) and become transcriptional towards the end of larval period. The hybrid of sliver carp and bighead carp exhibited significant increase in protease activity from 8-day onwards in the present study and showed an exponential relationship with age. Kumar et al. (2000) documented a significant increase in protease activity from day-10 onwards in Catla catla fed with live food. The high activity of protease from 18-day onwards in the present study can be attributed to the adaptation of larva to digest greater protein content in the food. Similar results were also observed with Catla catla by Rathore et al. (2005). Sharma and Chakrabarti (2000) reported increase in proteolytic activity in common carp Cyprinus carpio with increase in their mass. Ershova et al. (2004) reported higher amount of proteases compared to carbohydrases in 12–18 day salmonid larvae. Pancreatic secretions like trypsin and chymotrypsin play a key role in digestive physiology of the carps as the stomach is absent. Both trypsin and chymotrypsin were detected on 4-day-old larva in the present study. Different trypsinogen expressions were reported in 5day-old winter flounder Pleuronectes americanus (Murray et al., 2004). Cahu et al. (2004) suggested that chain length of protein in the substrate in the food modulate trypsin transcription in sea bass larvae. The increase in the trypsin activity after the 24th day in the present study may be attributed to the transcriptional modulation for better protein digestion. It has been demonstrated that the exocrine part of the pancreas just begin to accumulate secretory granules at the early developmental stages of carp, i.e. the pancreas function
at a lower level, and secretion is activated after two weeks of development, whereas, the activity of alkaline proteases increases in the intestine (Ostroumova and Dement’eva, 1981). Chymotrypsin activity was low up to the first week after hatching compared to the later part of ontogeny. This may be because of delay in modulation of chymotrypsin and possibly associated with the regulation of trypsin and chymotrypsin in the gut of the fish at early development. The polynomial trend of the enzyme activity may imply the adaptation of the fish for shifting in the diet. Lipase activity was detected at the 4-day-old hybrid of silver carp × bighead carp. Major lipase in fishes appears to be non-specific and bile salt dependant (Gjellesvik et al., 1992). In Theragra chalogramma, two types of lipases, one related to yolk sac absorption and the other related to digestion of exogenous lipids were reported by Oozeki and Bailey (1995). The lipase activity showed polynomial relationship with the age of fish in the present study. Similar pattern of lipase activity was observed in Catla catla (Rathore et al., 2005). The significant increase in lipase activity from the fourth week in this study may be attributed to the adaptation of the larvae for better digestion and utilization of dietary lipids. The decrease in the proteolytic, trypsin, chymotrypsin and lipase activities on day-34 might be related to anatomical and physiological modifications before acquiring a constant activity towards adult mode of digestion (Cuvier-Peres and Kestemont, 2002). A wide range of proteins (17.4 to 69.4 kDa) was found in the enzyme samples of the hybrid fish in SDSPAGE. The significant increase in protease activity from day-20 in test tube analysis can be very well documented from the intensity of zymograms of proteases in substrate SDS-PAGE. The appearance of new activity bands at intervals attributed to the fate of protease activity in the test tube analysis as well. The decrease in band intensity relating to the biochemical assay of alkaline proteases was earlier documented in the enzyme extracts of white sea bream Diplodus sargus (Cara et al., 2003). Presence of serine proteases in the digestive tissue extract of carp hybrid was confirmed by the inhibition of the activity in test tube analysis. SBTI inhibited a major portion in both quantitative and substrate SDS-PAGE assay. This suggested the abundance of serine proteases in the digestive extract of hybrid fish. SBTI was inhibitorier in nature than PMSF. This was clear in the substrate SDS-PAGE analysis. Earlier works on intestinal extracts of juvenile piracanjuba, Brycon orbignyanus demonstrated inhibition of serine proteases by SBTI and PMSF (Garcia-Carreno et al., 2002). Jonas
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et al. (1983) reported a significance inhibition of protease activity by PMSF in Silurus glanis, Hypophthalmichthys molitrix and Cyprinus carpio, as PMSF selectively modifies the serine side chain located in the active centers of –OH type enzymes. Further, inhibition by specific TLCK and TPCK confirmed the presence of two trypsin-like and four chymotrypsin-like enzymes in the gut of the hybrid fish. Subsequent inhibition of the activity bands in the intestinal extract of Asian bony tongue Scleropages formosus by TLCK and TPCK was reported by Natalia et al. (2004). More than one isoform of trypsin and chymotrypsin in many fishes including Cyprinus carpio had been reported by a number of workers (Cohen et al., 1981; Simpson and Haard, 1984; Martinez et al., 1988; Kristiansson and Neilsen, 1992; Pivnenko, 2004). 6.1% to 11.1% inhibition of the metal ion specific activity was detected in the test tube assay in the present study. Inhibition of protease activity by EDTA suggested the presence of metal proteinases might be due to the microbial flora in the gut of carp hybrid. Ghosh et al. (2002) had reported the presence of Bacillus spp. plays a significant role in digestion of Catla catla and Labeo rohita. Alarcon et al. (1998) cited the dependence of divalent cations in the action of EDTA on the alkaline proteases of gilthead sea bream Sparus aurata. In conclusion, the elevated amylase activity can be attributed to the importance of carbohydrate metabolism at the first feeding of carp hybrid. The protease activity showed an exponential trend with the increasing age of fish. Trypsin, chymotrypsin and lipase activities showed polynomial trends during ontogeny of fish. Inhibition with SBTI and PMSF suggested the abundance of serine proteases in the digestive tissue extracts of carp hybrid. Two isoforms of trypsin and four isoforms of chymotrypsin were found at the early stages of development in silver carp × bighead carp hybrid. This information may be useful for the feed formulation of the carp larvae. Acknowledgement Authors are thankful to Department of Science and Technology (DST), Government of India, New Delhi for financial support. References Alarcon, F.J., Diaz, M., Moyano, F.J., Abellan, E., 1998. Characterization and functional properties of digestive proteases in two sparids; gilthead seabream (Sparaus aurata) and common dentex (Dentex dentex). Fish Physiol. Biochem. 19, 257–267.
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Bartley, D.M., Rana, K., Immink, A.J., 2001. The use of inter-specific hybrids in aquaculture and fisheries. Rev. Fish Biol. Fish. 10, 325–337. Bernfeld, P., 1955. Enzymes of carbohydrate metabolism. In: Colowick, S.P., Kaplan, N.O. (Eds.), Methods in Enzymology, vol. 1. Academic Press, New York, pp. 149–541. Blier, P.U., Pelletier, D., Dutil, J.D., 1997. Does aerobic capacity set a limit on fish growth rates? Rev. Fish. Sci. 5, 323–340. Bradford, M.M., 1976. A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein dye binding. Anal. Biochem. 72, 248–254. Buzinova, N.S., 1973. Amylase activity in amurs and carps. Ecological Physiology of Fishes (in Russian). Nauka Press, Moscow, pp. 48–50. Cahu, C., Ronnestad, I., Grangier, V., Zambonino-Infante, J.L., 2004. Expression and activities of pancreatic enzymes in developing sea bass larvae (Dicentrarchus labrax) in relation to intact and hydrolyzed dietary protein; involvement of cholecystokinin. Aquaculture 238, 295–308. Cara, J.B., Moyano, F.J., Cardenas, S., Fernandez-Diaz, C., Yufera, M., 2003. Assessment of digestive enzyme activities during larval development of white bream. J. Fish Biol. 63, 48–58. Chakrabarti, R., Sharma, J.G., 1997. Ontogenic changes of amylase and proteolytic enzyme activities of Indian major carp, Catla catla (Ham.) in relation to natural diet. Curr. Sci. 67, 932–934. Cohen, T., Gertle, A., Birk, J., 1981. Pancreatic proteolytic enzymes from carp Cyprinus carpio. Purification and physical properties of trypsin, chymotrypsin, elastase and carboxypeptidase. Comp. Biochem. Physiol. 69B, 639–646. Cuvier-Peres, A., Kestemont, P., 2002. Development of some digestive enzymes in Eurasian perch larvae Perca fluvialitis. Fish Physiol. Biochem. 24, 279–285. Dement’ev, M.C., 1984. Increasing the efficiency of culture of juvenile carps under fish farming. Authors Abstr. of Candidate Dissertation (in Russian). All-Union Pond Fisheries Research Institute, Moscow, p. 24. Erlanger, B.F., Kokowski, N., Cohen, W., 1961. The preparation and properties of two chromogenic substances of trypsin. Arch. Biochem. Biophys. 95, 271–278. Ershova, T.S., Volkova, I.V., Zaitsev, V.F., 2004. Specific features of digestive function development in larvae of some salmonid fish. Russ. J. Dev. Biol. 33, 233–237. Garcia-Carreno, F.L., 1992. The digestive proteases of langostilla (Pleuroncodes palanipes, Decapoda): their partial characterization and the effect of food on their composition. Comp. Biochem. Physiol. 103B, 575–578. Garcia-Carreno, F.L., Haard, N.F., 1993. Characterization of proteinase classes in langostilla (Pleuroncodes palanipes) extracts. J. Food Biochem. 17, 97–113. Garcia-Carreno, F.L., Dimes, L.E., Haard, N.F., 1993. Substrate–Gel electrophoresis for composition and molecular weight of proteinases or proteinaceous proteinase inhibitors. Anal. Biochem. 214, 65–69. Garcia-Carreno, F.L., Albuquerque-Cavalcanti, C., Navarrete del Toro, M., Zaniboni-Filho, E., 2002. Digestive proteinases of Brycon orbinyanus (Characidae, Teleostei): characteristics and effects of protein quality. Comp. Biochem. Physiol. 132B, 343–352. Ghosh, K., Sen, S.K., Ray, A.K., 2002. Characterization of Bacilli isolated from the gut of rohu, Labeo rohita fingerlings and its significance in digestion. J. Appl. Aquac. 12, 33–42. Gjellesvik, D.R., Lombardo, D., Walther, B.T., 1992. Pancreatic bile salt dependant lipase from cod (Gadus mohrua): purification and properties. Biochem. Biophys. Acta 1124, 123–134.
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Green, B.S., McCormick, M.I., 2001. Ontogeny of the digestive and feeding systems in the anemonefish Amphiprion melanopus. Environ. Biol. Fisches 61, 73–83. Gruzdkov, A.A., Zil’ber, Yu.D., Lezuitova, N.N., 1986. Physiology and biochemistry of enzyme adaptations. Pancreas, Membrannyi Gidroliz i Transport: Novye Dannye i Gipotezy (Membrane Hydrolysis and Transport: New Data and Hypotheses). Nauka, Leningrad, pp. 45–51. Hongming Ma, Cahu, C., Zambonino, J., Yu, H., Duan, Q., Le Gall, M., Mai, K., 2005. Activities of selected digestive enzymes during larval development of large yellow croaker (Pseudosciaena crocea). Aquaculture 245, 239–248. II’ina, I.D., Turetskiy, V.I., 1987. Development of digestive function in fishes. Vopr. Ihtiol. 27, 835–845 (in Russian). Jonas, E., Ragyanszki, M., Olah, J., Boross, L., 1983. Proteolytic digestive enzymes of carnivorous (Silurus glanis L.), herbivorous (Hypophthalmicthys molitrix), and omnivore (Cyprinus carpio L.) fishes. Aquaculture 30, 145–154. Kim, B.G., Divakaran, S., Brown, C.L., Ostrowski, A., 2001. Comparative digestive enzyme ontogeny in two marine larval fishes: Pacific threadfin (Polydactylus sexfilis) and bluefin trevally (Caranx melampygus). Fish Physiol. Biochem. 24, 225–241. Konovalov, Yu.D., Mestechkin, A.Ya., 1975. Activity of peptide hydrolases during embryonic development of common carp. Ontogenez 6, 201–205. Krasnai, Z.L., 1987. Interspecific hybridization of warm finfish. In: Tiews, K. (Ed.), Selection, Hybridization, and Genetic Engineering in Aquaculture, FAO, EIFAC and ICES, Rome, Italy and Copenhagen, Denmark, vol. 2, pp. 35–45. Kristiansson, M., Neilsen, H.H., 1992. Purification and characterization of chymotrypsin-like proteinase from pyloric caeca of Oncorhynchus mykiss. Comp. Biochem. Physiol. 101, 247–253. Kumar, S., Chakrabarti, R., 1998. Ontogenic development of amylase in three species of Indian major carps Catla catla, Labeo rohita and Cirrhinus mrigala, in relation to natural diet. Asian Fish. Sci. 10, 259–263. Kumar, S., Sharma, J.G., Chakrabarti, R., 2000. Quantitative estimation of proteolytic enzyme and structural study of anterior part of intestine of Indian major carp (Catla catla) larvae during ontogenesis. Curr. Sci. 79, 1007–1101. Laemmli, U.K., 1970. Cleavage of structural proteins during assembly of head of the bacteriophage T4. Nature (Lond.) 277, 680–685. Martinez, B., Ohlson, R.L., Serra, J.L., 1988. Purification and characterization of two trypsin-like enzymes from Anchovy. Comp. Biochem. Physiol. 91B, 202–214.
Murray, H.M., Perez-Casanova, J.C., Galant, J.W., Johnson, S.C., Douglas, S.E., 2004. Trypsinogen expression during the development of the exocrine pancreas in winter flounder (Pleuronectes americanus). Comp. Biochem. Physiol., Part A Mol. Integr. Physiol. 138, 53–59. Natalia, Y., Hashim, R., Ali, A., Chong, A., 2004. Characterization of digestive enzymes in a carnivorous ornamental fish, the Asian bony tongue Scleropages formosus (Osteoglossidae). Aquaculture 233, 305–320. Oozeki, Y., Bailey, K.M., 1995. Ontogenic development of digestive enzyme activities in larval walleye Pollock, Theragra chalcogramma. Mar. Biol. 122, 122–186. Ostroumova, I.N., Dement’eva, M.A., 1981. Beginning of the pancreatic function in the digestive process of carp larvae. J. Evol. Biochem. Physiol. 17, 302–305. Pivnenko, T.N., 2004. Activity, composition, and distribution of pancreatic serine proteinases in various taxonomic groups of hydrobionts. J. Evol. Biochem. Physiol. 40, 18–27. Rathore, R.M., Kumar, S., Chakrabarti, R., 2005. Digestive enzyme patterns and evaluation of protease classes in Catla catla (Family: Cyprinidae) during early developmental stages. Comp. Biochem. Physiol., B 142, 98–106. Sharma, J.G., Chakrabarti, R., 2000. Replacement of live-food with refrigerated-plankton food for Cyprinus carpio. Curr. Sci. 79, 214–219. Simpson, B.K., Haard, N.F., 1984. Trypsin from Greenland cod (Gadus ogac), isolation and comparative properties. Comp. Biochem. Physiol. 79B, 613–622. Tanaka, M., 1973. Studies on the structure and function of the digestive system of teleost larvae. Ph.D. Dissertation. Dept. of Fisheries, Faculty of Agriculture, Kyoto University. p. 136. Ueberschar, B., 1993. Measurement of proteolytic enzyme activity: significance and application in larval fish research. In: Walther, B.T., Fuhn, H.J. (Eds.), Physiological and Biochemical Aspects of Fish Development. Univ. of Bergen, Norway, pp. 233–239. Volkova, I.V., 1999. Activities of digestive enzymes in plant-eating fish at early phases of ontogenesis. Canad. Sci. (Biol.). Dissertation, Astrakhan. Winkler, U.K., Stuckman, M., 1979. Glycogen, Hyaluronate, and some other polysaccharides greatly enhance the formation of exolipase by Serratia marcescens. J. Bacteriol. 138, 663–670.
Functional changes in digestive enzymes and characterization of proteases of silver carp (♂) and bighead carp (♀) hybrid, during early ontogeny Rina Chakrabarti a,⁎, Raja Mansingh Rathore a , Prabhat Mittal b , Sunil Kumar a a b
Aqua Research Lab, Department of Zoology, University of Delhi, Delhi 110 007, India Department of Statistics, Satyawati College, University of Delhi, Delhi 110 052, India
Received 31 May 2005; received in revised form 27 August 2005; accepted 31 August 2005
Abstract Study of digestive enzyme and partial characterization of proteases of silver carp (♂) and bighead carp (♀) hybrid during early ontogeny was performed. Specific amylase activity was observed in 4 DAH (0.07 ± 0.01 mg maltose mg protein− 1 min− 1) carp hybrid. Specific amylase activity showed polynomial relationship with the age of fish. Total protease, trypsin and chymotrypsin activities were 14.37 ± 2.21, 11.38 ± 1.67 and 2.83 ± 0.50 mUnits mg protein− 1 min− 1 in 4 DAH fish, respectively. Total protease activity showed exponential trend, whereas trypsin and chymotrypsin activities showed polynomial relationships with the increasing age of the fish. Lipase activity was 2.33 ± 0.18 mUnits in 4 DAH carp hybrid. Lipase activity showed polynomial trend with the increasing age of fish. In inhibition study, SBTI, PMSF, TLCK and TPCK inhibited the protease activity by 83.0% to 92.0%, 71.0% to 81.0%, 45.1% to 55.5% and 35.8% to 48.2%, respectively. SDS-PAGE showed the presence of various protein bands (20.9–69.4 kDa) in carp hybrid during ontogenic development. Substrate SDS-PAGE revealed the presence of several protease activity bands (19.1–73.7 kDa) in digestive tissue extract of fish during ontogenesis. Inhibition of protease activity bands in substrate SDS-PAGE with SBTI and PMSF revealed the abundance of serine proteases and inhibition of activity bands with TLCK and TPCK evidenced the presence of more than one isoform of trypsin and chymotrypsin in the digestive tissue extract of hybrid carp. © 2005 Elsevier B.V. All rights reserved. Keywords: Digestive enzymes; Proteases; Ontogeny; Inhibitors; Carp
1. Introduction The alarming increase of pollution level has become a menace for the freshwater biodiversities in the recent years. Amplifying demand for fish as food, poor ability of the fish larva to grow faster in environmental stress lead the aquaculturists to search for a desirable trait for ⁎ Corresponding author. Tel.: +91 11 27666496. E-mail address: [email protected] (R. Chakrabarti). 0044-8486/$ - see front matter © 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.aquaculture.2005.08.018
rapid growth, increased environmental fluctuation tolerance and higher production. Inter-specific breeding among fishes is a perfect tool to transfer desirable traits and is now been widely used in most of the world for a potential aquaculture (Bartley et al., 2001). Hybrid of microplanktophagous silver carp (Hypophthalmichthys molitrix) and macroplanktophagous bighead (Aristichthys nobilis) is widely accepted in aquaculture in Asia due to having the positive heterosis for growth rate. However, pure lines may be lost due to fertility of
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hybrids when parents and hybrids are grown together (Krasnai, 1987). This may be avoided by culture under control conditions. The process of embryonic and pre-larval development is associated with enhancement of two alternatives, synthesis and catabolism of proteins, which is accompanied by increase in the activities of trypsin and chymotrypsin-like proteases in Cyprinus carpio (Konovalov and Mestechkin, 1975). The rate of development is likely to determine the survival of fish with physiological and environmental changing status. Detailed knowledge of digestive physiology during the developmental stages of such fish is essential as the maximum growth rate of fish is partly contributed by digestive capacity, oxygen availability and their metabolic capacity required to support tissue protein synthesis (Blier et al., 1997). Age related changes in the diversity and quantity of digestive enzymes appear to represent evolutionary adaptations to the different natural diets and nutritional requirement of distinct developmental stages (Kumar et al., 2000). Fish must be competent at procuring and assimilating food before yolk sac absorption (Green and McCormick, 2001). The yolk sac of common carp is resorbed 2–5 days after hatching (depending on water temperature), whereas the activities of digestive enzymes are very low. The question remains which mechanisms maintain food assimilation at a level sufficient for intensive growth (II’ina and Turetskiy, 1987). For better survival of larvae, feeding must be initiated on digestible diets before or very soon after depletion of the endogenous energy sources, yolk and oil (Kim et al., 2001). The assessment of the presence and level of activity of certain enzymes may be used as a comparative indicator of the rate of development of the larvae, as well as their further survival rate (Ueberschar, 1993). The ratio of membrane and cavity digestion actually changes in fishes with age. It was found during the study of digestion in Ctenopharyngodon idella, that the ratio between amylase activity in hepatopancreas to its activity in mucosa increases with age: younger the fish, more significant the role of amylase associated with the mucosa (Buzinova, 1973). The development of larva to a fingerling relies on a proper development of digestive functions during larval life, and the maturation of digestive tract can be altered by diet composition (Hongming Ma et al., 2005). It has been suggested that growth of carp larvae on any artificial diet is not faster than on natural food. Dement’ev (1984) found deviation in type of enzyme activities found in silver carp fed with artificial diet as compared to younger fish fed with natural diet. It is well
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documented that at the early developmental stage fishes experience a crucial metabolic phase during the shifting of feeding. Considerable attention has been paid to evaluate the functional characteristics of the digestive enzymes that play an important role in the ontogenesis of larvae. Though substantial interest has been paid in recent years to bring out the new hybrid species in aquaculture for rapid growth and other desirable traits, information regarding mapping of the digestive physiology during ontogenesis of these hybrids is still unexplored. In the present study, we have tried to evaluate the fate of major functional digestive enzymes found in the gut of the hybrid fish during early developmental stages. Efforts have been made to characterize the digestive proteases. 2. Materials and methods 2.1. Fish species and culture Hybrids of silver carp Hypophthalmichthys molitrix (♀) × bighead Aristichthys nobilis (♂) (Class: Actinopterygii, Order: Cypriniformes, Family: Cyprinidae) were obtained from a commercial fish farm in India after 72 h (3 days old) of hatching (length 6.9 ± 0.1 mm; weight 1.32 ± 0.1 mg). Spawns were stocked at density of 45,000 m− 3 in 450 L tank having connection with recirculating system. Live food (Brachionus spp., Ceriodaphnia spp., Mesocyclops spp.) was provided to the fish ad libitum up to 14 days after hatching (DAH). From day-14 onwards mixed feeding with artificial diet (40% protein) was started at the rate of 4% of body weight. The experiment was continued for 30 days. Temperature and pH ranged from 28.1 to 32.4 °C and 7.5 to 8.9, respectively during the experimental period. Dissolved oxygen was maintained above 5 mg/L with help of aerators. 2.2. Sample preparation Fish were collected randomly from different tanks at 9 A.M. before morning feeding on every alternate day. Larvae were properly washed and were kept at −20 °C. Digestive system of individual fish was taken out on a glass platform maintained at 0 °C under dissecting microscope. 100 mg of gut tissue was collected for each sample and was homogenized with 1 ml pre-chilled distilled water. For 100 mg tissue the number of fish sacrificed was initially 315 ± 2.4 on day-4 and subsequently reduced to 24 ± 1.15 on day-34 as fish grew. Supernatant of the homogenate was obtained by centrifuging at 10,000 ×g at 4 °C for 10 min and stored
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at − 20 °C for further analysis. Three replicates were used for each assay. Total soluble protein was measured according to Bradford (1976). 2.3. Digestive enzyme assays Amylase was assayed by measuring the increase in reducing power of buffered starch solution with 3,5dinitro salicylic acid at 540 nm (Bernfeld, 1955). Specific amylase activity was expressed in terms of milligram of maltose liberated per milligram of protein per hour at 37 °C. Total protease activity was measured by using 1% azocasein as substrate in 50 mM Tris–HCl, pH 7.5 (Garcia-Carreno, 1992) and the change of absorbance was recorded at 366 nm. Protease activity was expressed in terms of mUnits mg protein− 1 min− 1. Trypsin and chymotrypsin activities were measured by taking N-α-benzoyl-DL-Arginine-p-nitroanilide (BAPNA) and Suc-Ala-Ala-Pro-Phe-p-nitroanalidine (SAPNA), as substrates, respectively. Change of absorbance was recorded under kinetic mode for 3 min at 410 nm (Erlanger et al., 1961). Activity units were expressed as change in absorbance mg protein− 1 min− 1 of the enzyme used in the assay. Activity units were calculated by the following equation: Activity units ðAbs410 =minÞ 1000 ml of reaction mixture ¼ 8800 mg protein in reaction mixture The molar extinction coefficient of para-nitroanalidine is €410 = 8800 cm2 mg− 1. Lipase activity was measured as described by Winkler and Stuckman (1979). The principle of the assay is the colorimetric estimation of para-nitrophenol (pNP) released due to enzymatic hydrolysis of para-nitrophenyl palmitate (pNPP) at 410 nm. One enzyme unit is defined as 1 nmol of p-nitrophenol enzymatically released from the substrate ml− 1 min− 1. The extinction coefficient of para-nitrophenol is €410 = 15,000 cm2 mg− 1.
phenylalanine chloromethylketone, TPCK (5 mM in methanol) were used as specific inhibitors for trypsin and chymotrypsin, respectively. Chelating agent Ethylenediamine-tetraacetate, EDTA (20 mM in distilled water) was used for inhibiting metal ion specific proteases. All these inhibitors were pre-incubated at 1 : 1 ratio for 1 h at room temperature. 2.5. Protein and enzyme composition assay Separation of proteins in the enzyme extracts was performed by using 12% SDS-PAGE (Laemmli, 1970). 20 μg of protein for each enzyme extract was loaded on to each well at controlled temperature of 4 °C. After electrophoresis the gels were stained with Coomassie brilliant blue for 2 h and then destained. The protease composition and evaluation of their classes were done by substrate SDS-PAGE (GarciaCarreno et al., 1993). Enzyme extract containing 5 mUnits of activity was loaded on to each well. After electrophoresis gel was immersed in 3% casein in 50 mM Tris–HCl, pH 7.5 for 30 min at 5 °C to allow the substrate to diffuse into the gel. Then the gel was incubated for 60 min at 25 °C. The gel was washed, stained and destained. Clear bands with blue background were indicated as protease activity bands. For protease class evaluation enzyme extract having 5 mUnits activity were incubated with different inhibitors like SBTI (250 μM), PMSF (100 mM), TLCK (10 mM), and EDTA (20 mM) prior to loading on to the wells. Then they are subjected to substrate SDS-PAGE. The bands were compared with activity bands without inhibition. 2.6. Statistical analysis Data of digestive enzyme activities were analyzed by using one-way ANOVA and appropriate regression model analysis. Statistical significance was accepted at P < 0.05 level. 3. Results
2.4. Protease inhibition assay 3.1. Enzyme activity The inhibition assay was conducted to evaluate the protease class by treating the enzymes with different specific inhibitors as described by Garcia-Carreno and Haard (1993). Phenylmethylsulfonylfluoride, PMSF (100 mM in 2-propanol) and Soybean trypsin inhibitor, SBTI (250 μm in distilled water) were used as serine class protease inhibitors. N α-p-tosyl-L-lysine chloromethylketone, TLCK (1 mM in HCL) and N-tosyl-L-
On day-4 the specific amylase activity was 0.078 ± 0.01 mg maltose mg protein− 1 h− 1. The enzyme activity increased significantly (P < 0.01) on day-8 compared to days 4 and 6. Whereas, the activity decreased (30%) significantly (P < 0.05) on day-12 than day-8 (Fig. 1a). The specific amylase activity then increased 22% between days 14 and 16 compared to 12th day. The
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b 0.2 0.18 0.16
y = 1E-08x 6 - 1E-06x 5 + 6E-05x 4 0.0011x3 + 0.0092x2 - 0.0221x + 0.0704 R2 = 0.603
0.14 0.12 0.1 0.08 0.06 0.04
Protease activity (mUnits/mg protein/min)
Specific amylase activity (mg maltose/mg protein/h)
a
1000 800
y = 12.063e 0. 12 73x R2 = 0.9478
600 400 200 0
2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34
2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34
Days after hatching 90 80 70 60 50 40 30 20 10 0
y = 0.0042x3 - 0.1846x 2 + 3.3739x + 2.8342 R2 = 0.9016
2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34
Days after hatching
d Chymotrypsin activity (mUnits/mg potein/min)
c Trypsin activity (mUnits/mg protein/min)
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600 y = 7E-06x6 + 0.0001x5 - 0.0501x 4 + 2.1316x3 - 33. 954x 2 + 222. 78x 500 481.17 2 400 R = 0.9426 300 200 100 0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34
Days after hatching
Lipase activity (mUnits)
e
Days after hatching
10 y = -5E-07x6 + 4E-05x5 - 0.0014x4 + 0.0195x 3 - 0.1205x2 + 0.4531x + 1.3737 8 R2 = 0.9396 6 4 2 0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34
Days after hatching Fig. 1. (a) Specific amylase activity show polynomial (6th degree), (b) protease activity shows exponential, (c) trypsin activity shows polynomial (3rd degree), (d) chymotrypsin activity shows polynomial (6th degree), and (e) lipase activity shows polynomial (6th degree) relationships with increasing age of silver carp × bighead hybrid larvae during ontogenesis. Each value represents mean ± SEM (n = 3).
enzyme activity ranged from 0.083 to 0.109 mg maltose mg protein− 1 min− 1 between day 18 and 34. The enzyme activity on day-34 was significantly (P < 0.05) higher (16%) than the activity found on day-32. The specific amylase activity showed a polynomial (6th degree) relationship with the increasing age of the hybrid fish. Total protease activity in 4 DAH larva was 14.37 ± 2.21 mUnits mg protein− 1 min− 1. The enzyme activity increased (35 to 120%) significantly (P < 0.01) on day8 compared to days 4 and 6. The protease activity on day-12 was also significantly (P < 0.05) higher than the
enzyme activity found in fish between days 8 and 10. But the enzyme activity decreased by 31% on day-14 compared to day-12. The enzyme activity increased by 25 to 31% in 18 DAH fish than the activity found in 14 and 16 DAH fish. The protease activity increased significantly (P < 0.05) between days 20 and 32 compared to the enzyme activity found in the respective previous days. On day-34 enzyme activity decreased (34%) significantly (P < 0.01) than the activity found on day-32. The enzyme activity showed exponential trend with the increasing age of the fish (Fig. 1b).
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Table 1 Percentage of inhibition of protease activity by different inhibitors in silver carp and bighead carp hybrid during ontogenesis % Inhibition Age of SBTI fish (DAH) 28 30 32 34
PMSF
TLCK
92.0 ± 0.7 80. 2 ± 6.8 53.6 ± 2.4 92.5 ± 1.7 77.2 ± 2.8 45.1 ± 5.5 83.7 ± 2.8 71.0 ± 6.1 48.9 ± 18.2 83.0 ± 1.8 81.0 ± 4.7 55.5 ± 3.0
TPCK
EDTA
45.6 ± 7.1 7.1 ± 1.1 48.2 ± 8.9 10.7 ± 2.8 47.9 ± 1.1 11.1 ± 5.4 35.8 ± 5.0 6.1 ± 1.7
Trypsin activity was 11.38 ± 1.6 mUnits mg protein− 1 min− 1 in 4 DAH fish. The activity increased significantly (P < 0.01) in 8 DAH larva than 4 DAH one. A significantly (P < 0.05) reduction (8 to 18%) in trypsin activity was observed between 10 and 18 days compared to the 8th day. On day-22 enzyme activity increased by 7 to 28% compared to the activity found on days 18 and 20. Then the enzyme activity decreased (13%) significantly (P < 0.05) on day-24 than the activity found on day-22. The trypsin activity increased significantly (P < 0.05) between days 26 and 32
compared to the activity found on respective previous days. On day-34, the enzyme activity decreased (17%) significantly (P < 0.05) than the activity found on day32. A polynomial (3rd degree) trend of trypsin activity with increasing age was observed in this hybrid fish (Fig. 1c). Chymotrypsin activity was 2.83 ± 0.5 mUnits mg protein− 1 min− 1 in 4 DAH fish. Significantly (P < 0.01) higher enzyme activity was found on day-10 compared to the activity found on day-4. On day-18 chymotrypsin activity increased (47 to 151%) significantly (P < 0.05) than the activity found between days 12 and 16. Enzyme activity on days 22 and 24 was significantly (P < 0.01) higher than day-20. But there was no significant (P > 0.05) difference in enzyme activity between days 22 and 24. A significant (P < 0.05) decrease (16%) in chymotrypsin activity was recorded on day-26 compared to day-24. The activity was significantly (P < 0.05) higher on day-32 compared to the other days of sampling. On day-34, the chymotrypsin activity decreased by 8% than day-32. Chymotrypsin activity showed a polynomial trend (6th degree) along with the age of the hybrid fish (Fig. 1d).
Fig. 2. (a) SDS-PAGE of digestive extract of silver carp × bighead larvae during ontogenesis. Lane M is molecular weight marker comprising, albumin, bovine (66,000); albumin, egg (45,000); glyceraldehyde-3-phosphate dehydrogenase (36,000); carbonic anhydrase, bovine (29,000); trypsinogen, bovine pancreas (24,000); trypsin inhibitor, soybean (20,100) and α-lactalbumin, bovine milk (14,200). Enzyme sample of different days was loaded on to each well. (b) Zymograms of enzyme samples of digestive extract of silver carp × bighead hybrid larvae during ontogenesis by Substrate SDS-PAGE. Lane M is molecular weight marker and other lanes correspond to samples of different days.
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In 4 DAH fish the lipase activity was 2.3 ± 0.1 mUnits. On day-8 lipase activity increased 35% than on day-4. Then the lipase activity decreased (8%) significantly (P < 0.05) on day 10 than the activity found on day-8. The activity then increased (44%) significantly (P < 0.05) on day-12 than the activity found on day-10. The lipase activity ranged from 3.06 to 4.25 mUnits between days 14 and 28. The enzyme activity increased significantly (P < 0.01) on days 30 and 32 to the respective previous days. A 14% decrease in activity was observed on day-34 compared to day-32 (Fig. 1e). Like amylase and chymotrypsin activities the lipase activity also showed a polynomial (6th degree) relationship with the age of fish. 3.2. Protease inhibition assay Enzyme samples of 28 DAH to 34 DAH fish treated with inhibitors like SBTI, PMSF, TLCK and TPCK showed 83.0% to 92.5%, 71.0% to 81.0%, 45.1% to 55.5% and 35.8% to 48.2% inhibition in the activity, respectively (Table 1). Whereas, enzyme samples treated with EDTA showed 6.1% to 11.1% inhibition of the activity. 3.3. SDS-PAGE and substrate SDS-PAGE Proteinograms of digestive extract of fish were studied by SDS-PAGE (Fig. 2a). Two bands with poor resolution (42.1 and 69.4 kDa) were found between day4 and day-8. One more band of 37.2 kDa appeared on day-10. Three more bands (53.4, 49.4 and 33.3 kDa) became visible on 16 DAH hybrid fish. Further, two low molecular weight bands of 23.9 and 27.1 kDa were visible on the 20th day followed by one more band of 20.9 kDa on day-24. Total 9 protein bands were visible between 24 and 34-day. Zymograms of various proteases of enzyme samples of the fish were observed by substrate SDS-PAGE (Fig. 2b). Two activity bands of proteases (73.7 and 43.6 kDa) were recorded between day-4 and day-10. One more zymogram of 58.1 kDa became visible from 12th day. One activity band of 35.6 kDa was apparent on day-20. Two additional bands of 24 and 19.1 kDa appeared on day-24. From the 24th day onwards, a total 6 activity bands ranging from 19.1 to 73.7 kDa were visible (Table. 2). Treatment of enzyme samples with various inhibitors reduced the number of bands (Table 2). Only one activity bands (73.7 kDa) was visible in SBTI treated enzyme samples. Other activity bands those appeared in the activity gel, were inhibited with SBTI. PMSF
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Table 2 Zymogram profiles of digestive extracts of silver carp × bighead hybrid larvae during ontogenesis Molecular weight of proteases DAH 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34
73.7 + + + + + + + + + + + + + + + +
58.1
43.6
35.6
24
19.1
+Is,p +Is,p +Is,p +Is,p +Is,p +Is,p +Is,p +Is,p
+Is,p,l,c +Is,p,l,c +Is,p,l,c +Is,p,l,c +Is,p,l,c +Is,p,l,c
+Is,p,c +Is,p,c +Is,p,c +Is,p,c +Is,p,c +Is,p,c
s,p
+Is,l,c +Is,l,c +Is,l,c +Is,l,c +Is,l,c +Is,l,c +Is,l,c +Is,l,c +Is,l,c +Is,l,c +Is,l,c +Is,l,c
+I +Is,p +Is,p +Is,p +Is,p +Is,p +Is,p +Is,p +Is,p +Is,p +Is,p +Is,p +Is,p +Is,p +Is,p +Is,p
+ = Activity band of protease. Is = Band Inhibited with SBTI. Ip = Band Inhibited with PMSF. Il = Band Inhibited with TLCK. Ic = Band Inhibited with TPCK.
inhibited four activity bands of 43.6, 35.6, 24 and 19.1 kDa. TLCK inhibited two trypsin-like enzyme activity bands in the gel having molecular weight of 58.1 and 24 kDa. TPCK treated samples showed inhibition of four chymotrypsin-like enzyme activities in the gel. Activity bands of 58.1, 43.6, 24 and 19.1 kDa were inhibited by TPCK. The protease activity bands those appeared in uninhibited samples were also found in EDTA treated samples of substrate SDS-PAGE. 4. Discussion Development and regulation of the digestive enzymes depends on the progressive changes in the digestive tract and the subsequent response to composition and amount of the available food. This is either genetically programmed or induced by the corresponding substrate in the food (Gruzdkov et al., 1986). In the present investigation all the enzymes studied were detected at the first feeding of the hybrid fish. Earlier studies (Chakrabarti and Sharma, 1997; Kumar and Chakrabarti, 1998; Kumar et al., 2000, Rathore et al., 2005) with Indian major carps Catla catla, Labeo rohita, and Cirrhinus mrigala evidenced the presence of amylase, protease, trypsin, chymotrypsin and lipase in all these species at the first feeding. However, high amylase activities between 8 and 10 DAH of the hybrid carp suggested the modulation of digestive system for
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carbohydrate metabolism in hybrid fish during early development. Tanaka (1973) suggested that dietary carbohydrates may fill the energy gap between endogenous and exogenous protein demand of the fish. Kim et al. (2001) found an increase in amylase activity followed by sharp decrease in unfed larval threadfins just before die off. This suggested the synthesis of amylase in early ontogenesis even in absence of food, and the carbohydrates are actively catalyzed during the time of organogenesis. The decrease in activity after the 10th and 16th day in hybrid fish may be possibly due to developmental changes in the gut morphology and increased protein level in the tissue. An increase in the specific amylase activity from 30-day onwards was evidenced in the present study. Silver carp, bighead carp and grass carp were reported to increase in their amylase activities from day-21 when they actively consume large phyto- and zooplankton (Volkova, 1999). Cahu et al. (2004) suggested that regulation of amylase is post-transcriptional in early larval stages of sea bass Dicetrarchus labrax (till day-25) and become transcriptional towards the end of larval period. The hybrid of sliver carp and bighead carp exhibited significant increase in protease activity from 8-day onwards in the present study and showed an exponential relationship with age. Kumar et al. (2000) documented a significant increase in protease activity from day-10 onwards in Catla catla fed with live food. The high activity of protease from 18-day onwards in the present study can be attributed to the adaptation of larva to digest greater protein content in the food. Similar results were also observed with Catla catla by Rathore et al. (2005). Sharma and Chakrabarti (2000) reported increase in proteolytic activity in common carp Cyprinus carpio with increase in their mass. Ershova et al. (2004) reported higher amount of proteases compared to carbohydrases in 12–18 day salmonid larvae. Pancreatic secretions like trypsin and chymotrypsin play a key role in digestive physiology of the carps as the stomach is absent. Both trypsin and chymotrypsin were detected on 4-day-old larva in the present study. Different trypsinogen expressions were reported in 5day-old winter flounder Pleuronectes americanus (Murray et al., 2004). Cahu et al. (2004) suggested that chain length of protein in the substrate in the food modulate trypsin transcription in sea bass larvae. The increase in the trypsin activity after the 24th day in the present study may be attributed to the transcriptional modulation for better protein digestion. It has been demonstrated that the exocrine part of the pancreas just begin to accumulate secretory granules at the early developmental stages of carp, i.e. the pancreas function
at a lower level, and secretion is activated after two weeks of development, whereas, the activity of alkaline proteases increases in the intestine (Ostroumova and Dement’eva, 1981). Chymotrypsin activity was low up to the first week after hatching compared to the later part of ontogeny. This may be because of delay in modulation of chymotrypsin and possibly associated with the regulation of trypsin and chymotrypsin in the gut of the fish at early development. The polynomial trend of the enzyme activity may imply the adaptation of the fish for shifting in the diet. Lipase activity was detected at the 4-day-old hybrid of silver carp × bighead carp. Major lipase in fishes appears to be non-specific and bile salt dependant (Gjellesvik et al., 1992). In Theragra chalogramma, two types of lipases, one related to yolk sac absorption and the other related to digestion of exogenous lipids were reported by Oozeki and Bailey (1995). The lipase activity showed polynomial relationship with the age of fish in the present study. Similar pattern of lipase activity was observed in Catla catla (Rathore et al., 2005). The significant increase in lipase activity from the fourth week in this study may be attributed to the adaptation of the larvae for better digestion and utilization of dietary lipids. The decrease in the proteolytic, trypsin, chymotrypsin and lipase activities on day-34 might be related to anatomical and physiological modifications before acquiring a constant activity towards adult mode of digestion (Cuvier-Peres and Kestemont, 2002). A wide range of proteins (17.4 to 69.4 kDa) was found in the enzyme samples of the hybrid fish in SDSPAGE. The significant increase in protease activity from day-20 in test tube analysis can be very well documented from the intensity of zymograms of proteases in substrate SDS-PAGE. The appearance of new activity bands at intervals attributed to the fate of protease activity in the test tube analysis as well. The decrease in band intensity relating to the biochemical assay of alkaline proteases was earlier documented in the enzyme extracts of white sea bream Diplodus sargus (Cara et al., 2003). Presence of serine proteases in the digestive tissue extract of carp hybrid was confirmed by the inhibition of the activity in test tube analysis. SBTI inhibited a major portion in both quantitative and substrate SDS-PAGE assay. This suggested the abundance of serine proteases in the digestive extract of hybrid fish. SBTI was inhibitorier in nature than PMSF. This was clear in the substrate SDS-PAGE analysis. Earlier works on intestinal extracts of juvenile piracanjuba, Brycon orbignyanus demonstrated inhibition of serine proteases by SBTI and PMSF (Garcia-Carreno et al., 2002). Jonas
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et al. (1983) reported a significance inhibition of protease activity by PMSF in Silurus glanis, Hypophthalmichthys molitrix and Cyprinus carpio, as PMSF selectively modifies the serine side chain located in the active centers of –OH type enzymes. Further, inhibition by specific TLCK and TPCK confirmed the presence of two trypsin-like and four chymotrypsin-like enzymes in the gut of the hybrid fish. Subsequent inhibition of the activity bands in the intestinal extract of Asian bony tongue Scleropages formosus by TLCK and TPCK was reported by Natalia et al. (2004). More than one isoform of trypsin and chymotrypsin in many fishes including Cyprinus carpio had been reported by a number of workers (Cohen et al., 1981; Simpson and Haard, 1984; Martinez et al., 1988; Kristiansson and Neilsen, 1992; Pivnenko, 2004). 6.1% to 11.1% inhibition of the metal ion specific activity was detected in the test tube assay in the present study. Inhibition of protease activity by EDTA suggested the presence of metal proteinases might be due to the microbial flora in the gut of carp hybrid. Ghosh et al. (2002) had reported the presence of Bacillus spp. plays a significant role in digestion of Catla catla and Labeo rohita. Alarcon et al. (1998) cited the dependence of divalent cations in the action of EDTA on the alkaline proteases of gilthead sea bream Sparus aurata. In conclusion, the elevated amylase activity can be attributed to the importance of carbohydrate metabolism at the first feeding of carp hybrid. The protease activity showed an exponential trend with the increasing age of fish. Trypsin, chymotrypsin and lipase activities showed polynomial trends during ontogeny of fish. Inhibition with SBTI and PMSF suggested the abundance of serine proteases in the digestive tissue extracts of carp hybrid. Two isoforms of trypsin and four isoforms of chymotrypsin were found at the early stages of development in silver carp × bighead carp hybrid. This information may be useful for the feed formulation of the carp larvae. Acknowledgement Authors are thankful to Department of Science and Technology (DST), Government of India, New Delhi for financial support. References Alarcon, F.J., Diaz, M., Moyano, F.J., Abellan, E., 1998. Characterization and functional properties of digestive proteases in two sparids; gilthead seabream (Sparaus aurata) and common dentex (Dentex dentex). Fish Physiol. Biochem. 19, 257–267.
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