Different fish species have their own unique digestion and food assimilation properties due to differences in the structure of their digestive tracts and in their way of feeding.
Knowledge of the functional changes that are taking place in the digestive tract during food ingestion, digestion and assimilation is necessary to determine the ability of fish larvae to utilise a given diet (Segner et al. 1994).
Timeyko and Novokov (1987) found that the complexity of the morphological structure of the digestive tract is accompanied by periods of sharp increases in enzyme activity. The variations in digestive enzyme activity during larval development are indicative of the type and level of macronutrients that should be included in artificial feeds (Cahu and Zambonino-Infante 1995).
This study was undertaken to determine the activity of alkaline and acid type proteases, amylase, lipase, trypsin, chymotrypsin, leucine aminopeptidase, and alkaline and acid phosphatases during larval development of the grouper Epinephelus coioides .
Methods:
Samples for measuring digestive enzyme activity were collected (sampling time: 8:00–11:00 hours) at different larval stages: day 0, day 2, day 4, day 8, day 12, day 16, day 20, day 25, day 30, day 35, day 40, day 45, day 50, day 55 and day 60.
Whole larvae were used in the preparation of a crude enzyme extract from day 0 to day 30 and the mid-portion of the larvae was used from day 35 to day 60. Samples were freeze-dried, weighed and stored in a bio-freezer at–68°C prior to the preparation of crude enzyme extracts.
Freeze-dried larvae (70 mg/3.5 ml) were homogenised in 50 mM Tris-HCl buffer, pH 7.5, centrifuged (12,500 × G, 30 min at 4°C), filtered through a Sephadex G-25 M column (1 × 10 cm.), centrifuged (2000 × G, 5 min at 4°C) and then decanted. The supernatant (crude enzyme extract) was used for total protein and different enzyme assays.
Total protein was determined using the method of Lowry et al. (1951). Alkaline type protease activity was measured using 1% casein as substrate; and one unit of enzyme activity was defined as the amount of enzyme catalysing the formation of 1 µg of tyrosine per minute (modified method of Walter (1984)).
Acid type protease (pepsin) activity was determined using haemoglobin as a substrate; and one unit of pepsin activity expressed in tyrosine was equal to 0.001 of TCA soluble hydrolysis products per minute under standard conditions (Worthington Biochemical Corporation 1993). Amylase activity was quantified using soluble starch as a substrate; and one unit was defined as the amount of enzyme able to produce one micromole of reducing groups (calculated as maltose) per minute at 25 o C (Worthington Biochemical Corporation 1993).
Lipase activity was measured as the rate of hydrolysis of an olive oil emulsion that was determined by titration using a pH meter (Worthington Biochemical Corporation 1993). One unit of activity was equal to one micromole of acid produced per minute at 25°C under specified conditions. The activity of trypsin, chymotrypsin and leucine aminopeptidase was quantified according to methods described by Worthington Biochemical Corporation (1993).
Trypsin activity was equivalent to one micromole of N-p-Tosyl-L-arginine Methyl Ester (TAME) that was hydrolysed per minute (25°C; pH 8.1), chymotrypsin activity was equivalent to one micromole of N-Benzoyl-2-monophosphate-Na- Ca (BTEE) that was hydrolysed per minute (25°C; pH 7.8), and the activity of leucine aminopeptidase was equal to one micromole of leucinamide hydrolysed per minute (25°C; pH 8.5).
The activities of acid and alkaline phosphatases were determined at pH 4.8 and pH 9.8 respectively with nitrophenyl phosphate as the substrate (Bergmeyer 1974). The amount of 4-nitrophenol liberated per unit time in acidic solution was a measure of acid phosphatase activity, while the amount of 4-nitrophenol liberated per unit time in alkaline solution was a measure of alkaline phosphatase activity.
The total protein concentration of the newly hatched larvae (day 0) to day 2 was negligible (0.04 µg/larva). The concentration gradually increased with age of the larvae from 0.3 µg/ larva at day 12 to 84.8 µg/larva at day 60 (Fig. 1). Both alkaline and acid type protease activities (Fig. 2) were detected at early stages of development in grouper larvae. Alkaline type protease activity was identified in the newly hatched larvae (0.01 mU/larva) and gradually increased to a peak at day 50 (7334.9 mU/larva).
In contrast, acid type protease (pepsin) activity was not detected in the newly hatched larvae, but was detected at day 2 (2.2 U/larva). Its activity started to progress from day 12 (53.2 U/larva), which can be associated with the formation of the stomach. A two-fold increase in pepsin activity was observed from day 16 (53.2 U/larva) and every five days thereafter until day 40 (2706.7 U/larva).
The decrease in the activity of alkaline type protease from day 50 to day 60 can be linked to metamorphosis. The relationship between a marked decrease in the specific activity of alkaline type protease and metamorphosis was reported by Tanaka et al. (1996) in Japanese flounder, Paralichthys olivaceus, and also by Alliot et al. (1980) in Senegal sole.
Figure 3 shows that -amylase activity was detected in day 2 larvae (0.03 U/larvae) and a progressive increase was observed from day 16 (0.7 U/larva) until day 60 (141.9 U/larva). Early detection of -amylase activity has also been reported for other marine fish larvae and in all cases, the activity increased with age (Munilla- Moran et al. 1990). Moyano et al. (1996) observed that a marked increase in the activity of -amylase in sea bream was closely related to its feeding habits.
In this study, -amylase activity in grouper larvae increased with age and they may be capable of digesting carbohydrates at day 16. Also, the activity of lipase increased with age of grouper larvae (Fig. 4). The gradual increase in lipase activity (0.04–285 × 10 –5 U/ larva) can be related to the development of the pyloric caeca and intestine, which were fully developed at day 30.
Leucine aminopeptidase activity started to increase to an appreciable amount from day 16 (5.4 U/larva) until day 60 (447.3 U/larva) but was highest at day 40 (601.5 U/larva), which was the onset of metamorphosis in the grouper larvae (Fig. 5). The trypsin and chymotrypsin activity patterns are shown in Figure 6. Trypsin activity (9.0–16,407.5 mU/larva) seemed to be higher than chymotrypsin activity (1.3–10,368.7 mU/ larva) from day 8 until day 60.
However, there was a change in the pattern when chymotrypsin activity increased from 1964.6 mU/larva at day 40 to 2932.3 mU/larva at day 45 while trypsin activity decreased from 3130.6 mU/larva at day 40 to 709.5 mU/larva at day 50. As shown in Figure 7, the activity of acid phosphatase was increasing, which started from day 12 until day 60 (0.1–46.5 mU/larva), whereas alkaline phosphatase activity started from newly hatched larvae (day 0) to day 60 (0.03–207.2 mU/larva).
Both enzymes showed similar profiles, with higher values for alkaline phosphatase activity during metamorphosis. Moyano et al. (1996) also found that the activity of alkaline phosphatase was higher than that of acid phosphatase in gilthead seabream larvae.
Authors:
P.S. Eusebio, J.D. Toledo, R.E.P. Mamauag and M.J.G. Bernas
