Marine Biology (2004) 144: 863–869 DOI 10.1007/s00227-003-1255-9
R ES E AR C H A RT I C L E
M. Yu´fera Æ C. Ferna´ndez-Dı´ az Æ A. Vidaurreta J. B. Cara Æ F. J. Moyano
Gastrointestinal pH and development of the acid digestion in larvae and early juveniles of Sparus aurata (Pisces: Teleostei)
Received: 1 April 2003 / Accepted: 14 October 2003 / Published online: 26 November 2003 Springer-Verlag 2003
Abstract Changes in digestive pH and protease activity have been determined throughout the transition from larvae to the juvenile stage in Sparus aurata in rearing conditions (from 0.04 to 100 g wet weight). Measurements of pH have been taken in the stomach and different segments along the length of the intestine using a pH microelectrode. In starved fish, the gastric pH ranged between 6.0 and 8.0 approximately, except in juveniles of intermediate size (between 1.0 and 7.0 g wet weight), which exhibited a wider pH range of 2.0)8.0. Fed fish with digestive content showed, in general, lower pH values in the stomach. A progressive decrease was observed from a pH range of 5.5–8.0 in the youngest animals (0.04 g) to a pH range of 2.0)6.2 when juveniles were approaching 1.0 g wet weight. Above this weight, the gastric pH remained constant (between 2.0 and 6.0 approximately). The pH values in the intestine ranged between 6.7 and 8.4. They were similar in the different segments and weight classes examined, and there were no significant differences between fed and starved animals. Specific acid protease activity (units per milligram soluble protein) in fed animals increased from small (0.04–1.0 g) to intermediate juveniles (1.0 and 7.0 g), but then remained similar in larger juveniles. On the
Communicated by S.A. Poulet, Roscoff M. Yu´fera (&) Æ C. Ferna´ndez-Dı´ az Æ J. B. Cara Instituto de Ciencias Marinas de Andalucı´ a (CSIC), Apartado Oficial, 11520 Puerto Real, Ca´diz, Spain E-mail:
[email protected] Fax: +34-956-832612 A. Vidaurreta Cultivos Piscı´ colas Marinos SA, San Fernando, Ca´diz, Spain F. J. Moyano Dept. Biologı´ a aplicada, Escuela Polite´cnica Superior, Universidad de Almerı´ a, 04120 Almeria, Spain
contrary, specific alkaline protease activity in fed animals decreased from small to intermediate juveniles, and then remained at a similar level in larger juveniles. The results reflect a progressive transition during several months from alkaline digestion in larvae with undeveloped stomachs to the acid digestion in juveniles with fully developed stomachs. Full gastric capacity is developed in seabream juveniles of 1 g wet weight, which represents approximately 100 days post-hatching in cultured populations. Nevertheless, in the following 2.5 months, during which the intestine reaches the appropriate length, juveniles still show a transitional period in the regulatory mechanism of digestion, probably linked to the adaptation to a different feeding habit.
Introduction The transformation from larva to juvenile in fish implies a series of morphological, physiological and behavioural changes that vary among families and species. Metamorphosis in the sparids occurs during several weeks, in which the morphological features of juvenile fish appear progressively (Divanach et al. 1982; Koumoundouros et al. 1999). One of the most important events is the development of a completely functional, adult-style digestive system. The gilthead seabream (Sparus aurata) is a sparid with high commercial importance, inhabiting temperate waters in the north-eastern Atlantic Ocean and the Mediterranean Sea (Bauchot and Hureau 1986). Its rearing technology is well developed, and it is industrially produced in the Mediterranean countries. Seabream larvae hatch with a poorly developed digestive tract. Differentiation starts at the time of exogenous feeding, with the exception of the stomach, an organ that during the first weeks appears as a single enlargement in the oesophagus (Sarasquete et al. 1995). The pyloric caeca appears by 30 days post-hatching (dph) at temperatures close to 20C, and the first gastric glands can be detected by 45 dph (Domeneguini et al. 1998). These glands
864
secrete pepsinogen and hydrochloric acid, which decreases pH in the stomach and induces the conversion of pepsinogen to pepsin. Such changes affect both the way the food is processed within the digestive tract and the type and chemical composition of feeds that can be digested. During the first month, food is digested mainly by the action of alkaline proteases (Moyano et al. 1996), but, in wild populations, maturation of the digestive system allows the juveniles to change to a different prey spectrum more profitable for growth. In domesticated populations, the progressive appearance of the gastric function has been empirically considered the suitable period for weaning juveniles onto commercial feeds. At this point, juvenile status in this carnivorous fish has been reached, with a fully developed digestive tract including a stomach with gastric glands. Nevertheless, the presence of such glands does not determine complete functionality of the organ. It is also necessary that pepsin activity and the required acidification for its activity has taken place (Douglas et al. 1999). The aim of the present study was to advance the understanding of digestive processes, to examine the digestive conditions in the lumen of stomach and intestine during the transition from larva to juvenile, and to determine when acid digestion has been completely developed in the early juvenile stages of a species with high importance for aquaculture.
Materials and methods
pH values in the stomach dropped quickly after feeding in both groups, from values close to 7, and after 1 h the pH reached stable values (Fig. 1). Subsequently, pH determinations were taken between 1 and 2 h after feeding to standardise feeding-related pH changes. The fish used for pH and enzymatic measurements were grouped in three weight classes: 0.04–1.0 g, 1.0–7.0 g and 7.0– 95.0 g. These weight classes were determined a posteriori according to the results of gastric pH. We will refer to these classes as small, intermediate and large juveniles.
Enzyme-activity determinations For enzyme determination, the stomach and intestine were removed separately, opened and rinsed in distilled water. For each weight class, three samples were analysed, each prepared from pools of at least three animals. The samples were weighed and freeze-dried for later analysis. Enzyme extracts were prepared by homogenisation of the tissue sample (20 mg ml)1) in cold 100 mM Tris-HCl buffer+20 mM CaCl2, pH 8.0), followed by centrifugation (16,000 g, 30 min, 4C). Supernatants were stored at )20C for no more than 3 days until analysis. Concentration of soluble protein in extracts was determined by the Bradford method (Bradford 1976), using bovine serum albumin (1 mg ml)1) as a standard. Tissue extracts were assayed for the determination of acid and alkaline protease. Acid protease activity was measured using the method detailed by Anson (1938), modified by Dı´ az et al. (1998), using haemoglobin as substrate; 1 U of activity was defined as 1 lg of tyrosine released per minute. Alkaline protease activity was measured using azocasein as substrate (Sarath et al. 1989); 1 U of activity was defined as the amount of enzyme able to produce an increase of 1 U of absorbance per minute. The enzymatic analyses were performed on three samples in each range: 0.2–0.4 g, 2.0–4.0 g and 18.0–20.0 g, corresponding respectively to the three weight classes previously defined. Results are given as means (±SD).
Rearing conditions Sparus aurata L. were reared in captivity in a commercial hatchery according the following feeding schedule: rotifers (Brachionus plicatilis) and microalgae (Nanochloropsis gaditana), from mouth opening to 18 dph; Artemia nauplii from 15 to 50 dph; and commercial feeds from day 40 after hatching. After complete weaning the animals were routinely fed 12 h day)1 on a commercial diet (crude protein: 58%, crude fat: 13.5%, fibre: 10%). The rearing temperature was 19.5±1C. The photoperiod was 12 h light:12 h dark.
Data analysis Statistical differences in pH measured along the digestive tract were examined by a three-way analysis of variance (ANOVA), using feeding status, weight class and gut segment as factors, followed by Student–Newman–Keuls (SNK) multiple range test. Differences in enzymatic activities and the amount of soluble protein were examined by a two-way ANOVA, using weight class and feeding status as factors, followed by SNK multiple range test. Enzymeactivity data were log-transformed prior to analysis to reduce heteroscedasticity.
Digestive pH measurements Digestive pH was determined in juvenile fish with a wet weight ranging between 0.04 and 95 g (age: 50–300 dph approximately). Measurements were carried out throughout 2 years, in animals born from different egg batches. For pH determinations, the fish were anaesthetised with ethyl-4-aminobenzoate and then dissected to make the digestive tract accessible. Measurements were taken in still living animals using a pH microelectrode (WPI, Minicombo PH660). The tip of the microelectrode (660 lm diameter) was inserted into small slits made in the different sections of the digestive track: stomach, anterior intestine, medium intestine, posterior intestine and rectum. Two feeding conditions, fed and starved, were considered. Measurements in starved animals were always taken before the first morning feeding, and those animals still showing any gut content were not considered. The animals were starved for 24 h, with the exception of juveniles >40 g, which required 48 h to empty their gut completely. A preliminary test was performed with periodic gastric-pH determinations in the stomach during the first 2 h after feeding. This preliminary series was measured in three larvae with weights of 0.4–0.8 g and three juveniles of 7–8 g. The
Fig. 1 Sparus aurata. Decline in gastric pH (mean and SD) after ingestion, in larvae (0.4–0.8 g) and early juveniles (7–8 g)
865
Results The results of the pH determinations have been related to fish wet weight (WW). The correspondence with total length (TL) and approximate age is shown in Fig. 2. Wet weight increased as an exponential function of total body length (WW=0.0176TL2.922; r=0.985; P
