Fish Physiol Biochem DOI 10.1007/s10695-013-9884-5
Changes in digestive enzyme activities during larval development of leopard grouper (Mycteroperca rosacea) R. Martı´nez-Lagos • D. Tovar-Ramı´rez V. Gracia-Lo´pez • J. P. Lazo
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Received: 9 April 2013 / Accepted: 25 October 2013 Ó Springer Science+Business Media Dordrecht 2013
Abstract The leopard grouper is an endemic species of the Mexican Pacific with an important commercial fishery and good aquaculture potential. In order to assess the digestive capacity of this species during the larval period and aid in the formulation of adequate weaning diets, this study aimed to characterize the ontogeny of digestive enzymes during development of the digestive system. Digestive enzymes trypsin, chymotrypsin, acid protease, leucine–alanine peptidase, alkaline phosphatase, aminopeptidase N, lipase, amylase and maltase were quantified in larvae fed live prey and weaned onto a formulated microdiet at 31 days after hatching (DAH) and compared with fasting larvae. Enzyme activity for trypsin, lipase and amylase were detected before the opening of the mouth and the onset of exogenous feeding, indicating
R. Martı´nez-Lagos Centro Universitario Regional del Centro (CURC), Universidad Nacional Auto´noma de Honduras, Km. 1.5 carretera a Tegucigalpa, aldea de Tenguaje, Comayagua, Honduras D. Tovar-Ramı´rez (&) V. Gracia-Lo´pez Instituto Polite´cnico Nacional 195, Centro de Investigaciones Biolo´gicas del Noroeste (CIBNOR), Col. Playa Palo de Santa Rita Sur, 23096 La Paz, BCS, Mexico e-mail: [email protected] J. P. Lazo Centro de Investigacio´n Cientı´fica y de Educacio´n Superior de Ensenada Baja California, Km. 107 Carretera Tijuana, 22860 Ensenada, BC, Mexico
a precocious development of the digestive system that has been described in many fish species. The intracellular enzyme activity of leucine–alanine peptidase was high during the first days of development, with a tendency to decrease as larvae developed, reaching undetectable levels at the end of the experimental period. In contrast, activities of enzymes located in the intestinal brush border (i.e., aminopeptidase and alkaline phosphatase) were low at the start of exogenous feeding but progressively increased with larval development, indicating the gradual maturation of the digestive system. Based on our results, we conclude that leopard grouper larvae possess a functional digestive system at hatching and before the onset of exogenous feeding. The significant increase in the activity of trypsin, lipase, amylase and acid protease between 30 and 40 DAH suggests that larvae of this species can be successfully weaned onto microdiets during this period. Keywords Mycteroperca rosacea Digestive capacity Larval development Ontogeny
Introduction Optimization of the culture techniques for rearing marine fish larvae requires a thorough knowledge of the biology and physiology of the species of interest in order to adapt best aquaculture practices to increase
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growth and survival. One of the milestones of the larval period is the development of a juvenile-like digestive capacity during the first days of development to successfully acquire the proper nutrients for adequate growth and survival. With the aid of studies aiming to increase our knowledge with respect to larval nutrition of a species of interest, many new larval culture protocols have been developed, for example, for the turbot (Scophthalmus maximus) (Cousin et al. 1987; Tong et al. 2012), the European seabass (Dicentrarchus labrax) (Zambonino-Infante and Cahu 1994) and the California halibut (Paralichthys californicus) (Alvarez-Gonza´lez et al. 2006; Zacarı´as et al. 2006). In addition, digestive enzymes are considered to be reliable indicators of the nutritional condition of the individuals due to their species and age specificity, sensitivity and short latency (Lazo et al. 2011). Different digestive enzymes are used for this purpose, ranging from proteolytic pancreatic enzymes to intestinal brush border and cytosolic enzymes (Ueberscha¨r 1993; Cahu and Zambonino 2001; Cara et al. 2007). The leopard grouper (Mycteroperca rosacea) is an endemic species of Mexico, distributed from the southeast coast of Baja California (eastern central Pacific) to the Pacific coast of Jalisco, Mexico (Allen and Robertson 1998). This species has been identified as a good candidate for aquaculture because of its adaptability to captivity, resistance to handling and good market value. For this reason, previous work on breeding, farming (Gracia-Lo´pez et al 2004, 2005; Martı´nez-Lagos and Gracia-Lo´pez. 2009), nutrition and immunology (Reyes-Becerril et al. 2008) and natural history (Estrada-Godı´nez et al. 2011) have been performed. However, poor growth and survival during the larval period are still a bottleneck for the ´ lvarez-Lajonchere mass cultivation of this species (A and Herna´ndez-Molejo´n. 2001). Thus, the main objective of this study was to assess the digestive capacity of this species based on the characterization of the digestive enzymes during larval development.
Materials and methods Larval culture protocol Fertilized eggs were obtained from natural spawns of broodstock maintained in captivity (8 $, 2 #) at the
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Reproductive Biology Laboratory of the Centro de Investigaciones Biolo´gicas del Noroeste (CIBNOR) in La Paz, Baja California Sur, Mexico. Eggs were collected in a 300-lm mesh bag, counted volumetrically and distributed in 6–300-l tanks filled with seawater with a salinity of 35 % that had been filtered (1 lm) and sterilized (UV). Stocking density for the incubation period was 25 eggs l-1. After hatching, two experimental groups were established: one negative control using fasting larvae and one normally fed group. Digestive enzyme activities from fasted larvae were analyzed to assess the importance of enzymes, which exhibit activity before first feeding and are genetically controlled (Zambonino et al. 2008). Each treatment was evaluated in triplicate using three separate replicate tanks. Water temperature in the tanks was maintained throughout the experiment at 25 ± 1 °C using submersible heaters (300 W RENA, Annecy, France) in each tank. Central bubble aeration was applied to maintain the oxygen concentration C5.5 mg l-1. The photoperiod was set to 24 h light at a constant light intensity of 300 lux. The first live feed provided to the larvae consisted of rotifers (Brachionus plicatilis) without enrichment from 3 to 6 DAH. Rotifers had only been fed with the microalgae Nannochloropsis oculata. Rotifers were mesh screened to 75 lm and fed to larvae at a density of 10 rotifers ml-1 in the culture tank. After this period, rotifers were enriched (ER) with a commercial emulsion, Enrich HUFA (Salt Creek Inc., USA) and administered to the larvae 7–30 DAH. A second type of live food, Artemia nauplii (Artemia franciscana) (AN), was supplied 6–25 DAH at a density of 1–2 ml-1. Subsequently, larvae were fed with enriched Artemia metanauplii (EAN) 25–40 DAH at a density of 2–5 ml-1. Weaning onto a microdiet began 31 DAH using a commercial microparticulated feed (Caviar, Bernaqua, Olen, Belgium) (CD) with a particle size of 200–300 lm. Feed was supplied to the larval tanks once a day in the morning (Fig. 1). Cleaning of the tanks was performed daily using a siphon. Environmental parameters such as temperature and dissolved oxygen were daily monitored using a YSI model 55 sensor (Yellow Springs, USA); salinity was monitored using a Extech Instruments refractometer model RF 20 (Waltham, MA, USA), and light intensity was measured using a commercial light meter (Extech, Waltham, MA, USA).
Fish Physiol Biochem Fig. 1 Wet weight, total length (mean ± SD, n = 10) and type of food provided to leopard grouper larvae versus days after hatching (DAH). Feeding scheme during larval development: enriched rotifers (ER), Artemia nauplii (AN), enriched Artemia metanauplii (EAN) and microdiet (CD)
Larval sampling Digestive enzyme assays were performed using at least 500 mg wet weight of larval tissue, representing 3–2,400 larvae per sample, depending on their weight and age (Fig. 1). On each sampling date, larvae were weighed using a digital scale (OHAUS Adventure, NJ, USA). Larvae from the fasting treatment were only collected from 1 DAH up to 7 DAH, since 100 % of the larvae had died 8 DAH. Larvae from the live feed treatments were collected daily from 1 DAH to 7 DAH, and subsequent samples were collected 10, 15, 20, 25, 30, 35 and 40 DAH. Samples of viable fertilized eggs were also collected during the embryo (neurula) stage (4 h before hatching) to determine whether any of the enzymes quantified in this study were present prior to hatching. The samples were frozen at -80° C in an ultra-freezer until used for the enzyme assays. Biochemical analyses Larval samples were homogenized using a tissue homogenizer Model Pro-200 (Pro Scientific Inc., Oxford, CT, USA) with 4:1 v/w cold Tris-HCl buffer 50 mM, pH 7.5 (25 mg ml-1). The supernatant was obtained after centrifuging the homogenates at 12,000 g for 12 min at 5 °C; thereafter, the samples were frozen at -20 °C for subsequent enzymatic analyses. The soluble protein concentration was
determined using the method described by Bradford (1976) using bovine serum albumin as standard. Acid protease activity was assessed by the method described by Anson (1938) using 0.5 % hemoglobin as substrate in 0.1 mM glycine buffer/HCl, pH 2.0. The mixture was incubated at 37 °C and the reaction stopped by adding 0.5 ml of 20 % tri-chloro acetic acid (TCA). One unit of enzyme activity was defined as 1 lg of tyrosine released per minute using a molar extinction coefficient of 0.008 lg/ml cm-1 (calculated from a linear regression of different concentrations of tyrosine from 0 to 300 lg/ml and the absorbance at 280 nm). The activity of trypsin was determined at 25 °C according to the method described by Erlanger et al. (1961) using BAPNA (N-a-benzoyl-DL-arginine pnitroanilide) as substrate (10 mM in DMSO) at a concentration of 100 mM in 50 mM Tris-HCl pH 8.2 buffer containing 10 mM CaCl2. One unit was defined as 1 lM of p-nitroanilide released per minute using a molar extinction coefficient of 8,800 cm-1 M-1. Chymotrypsin activity was determined according to the method described by Asgeirsson and Bjarnson (1991) using 5 mM BTEE (benzoyl-tyrosine ethyl ester) as substrate (5 mM in DMSO) in 44.4 mM Tris buffer containing 55.5 mM CaCl2 pH 7.8. Enzyme activity was determined using a molar extinction coefficient for benzoyl-tyrosine of 964 cm-1 M-1 at 256 nm. Amylase activity was determined using 1 % soluble starch as substrate in 50 mM of Tris-HCl
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buffer, pH 7.5, according to the method described by Vega-Villasante et al. (1993). Sodium carbonate (2 N) and DNS reagent (di-nitrosalicylic acid) were added to increase color development of the reaction product. The reaction was stopped by boiling the reactants for 15 min. One unit of enzyme activity was defined as the quantity of enzyme required to increase 0.01 units of absorbance at 550 nm per minute. The bile-saltdependent lipase (BSD-lipase) activity was determined according to the method described by Versaw et al. (1989) using ß-naphtyl-caprylate (200 mM) as substrate and sodium taurocholate (100 mM) with a Tris–HCl buffer (50 mM, pH 7.2); the reaction was stopped with TCA (0.72 N). Fast blue (100 mM) and ethanol:ethyl acetate (1:1 v/v) were added to the reaction to develop color and clarify the solution. One unit of enzyme activity was defined as 1 lg naphtol released per minute at 540 nm. Alkaline phosphatase activity was determined according to the method described by (Bessey et al. 1946) using 4-nitrophenylphosphate as substrate in 30 mM NaCO3 buffer at pH 9.8. The extinction coefficient used for p-nitrophenol at 407 nm was 0.0183 lM-1 cm-1. A unit (U) of activity was defined as the amount of enzyme, which hydrolyzes 1 nmol of substrate in 1 min. The activity of aminopeptidase N was determined according to the method described by Maraux et al. (1973) using 0.1 M L-leucine p-nitroanilide as substrate in DMSO and 80 mM phosphate buffer at pH 7. One unit of enzyme activity is defined as 1 lg of nitroanilide released per minute using a molar extinction coefficient of 8,200 M-1 cm-1. The cytosolic (or intracellular) enzyme activity of leucine– alanine peptidase was determined according to the method described by Nicholson and Kim (1975) using 0.010 M L-leucyl-L-alanine as substrate and 50 mM Tris–HCl buffer at pH 8; the reaction was stopped using H2SO4. One unit of activity was defined as 1 nmol of hydrolyzed substrate released per min. Maltase activity was determined according to the method described by Dahlqvist (1970) using 56 mM of maltose as substrate and 500 mM of Tris–HCl buffer at pH 7. One unit of enzyme activity corresponded to the quantity of glucose released per minute. Specific activity (units/mg protein) of extracts were estimated using the following equations: (1) units per ml = (D abs) 9 reaction final volume (ml))/(MEC 9 time (min) 9 extract volume (ml); (2) units per mg of protein = (units/ml)/(mg of soluble protein). The
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increase in absorbance at a specific wavelength is represented by Dabs, while MEC corresponds to the molar extinction coefficient of the reaction product in ml lg-1 cm-1; (3) total activity units per larva = (units/ml)/no. of larvae/ml. All enzyme assays were performed in triplicate. Statistical analysis Mann–Whitney test was used to compare the enzyme activity of larvae. Since the distribution of data met the normality criteria, the Kruskal–Wallis test (P \ 0.05) was used to compare enzyme activity between the different days on which sampling took place. Values of the measured variables are expressed as mean ± standard deviation.
Results Larval growth Growth of leopard grouper larvae in terms of length (mm) and weight (mg) and the feeding protocol during larval development are shown in Fig. 1. Ontogeny of protease activity in fed larvae Trypsin activity was detected before the onset of exogenous feeding. Significant differences in enzymespecific and total activities were observed at 7 and 40 DAH (P \ 0.05). Total trypsin activity was low for the first 3 weeks of development, increasing at a slow but constant rate until 30 DHA. Subsequently, the activity significantly increased (P \ 0.05) to reach maximum values by the end of the experiment (Fig. 2a, b). Chymotrypsin and acid protease specific activities were detected before the onset of exogenous feeding and show similar patterns, reaching maximum specific activity levels by 7 DAH (P \ 0.05). Thereafter, a sharp decrease in activity was observed and remained at moderate and constant levels until the end of the experimental period, 40 DAH (Figs. 2c, 3a). However, total activity was low the first 2 weeks of development and began increasing by 20 DAH, and it continued to do so until the end of the experimental period (P \ 0.05) (Figs. 2d, 3b). Amylase and lipase activity showed a similar trend during development. Activity of these enzymes was
Fish Physiol Biochem
Fig. 2 Activity of digestive enzymes of fed leopard grouper larvae (mean ± SD, n = 3). Specific and total activities were determined for trypsin (a, b) and chymotrypsin (c, d). Fed larvae were provided different food: enriched rotifers (ER), Artemia
nauplii (AN), enriched Artemia metanauplii (EAN) and with microdiet (CD) during larval developmental. The values with astericks are significantly different (P \ 0.05)
detected at 1 DAH. Specific activity of these enzymes showed a slight variation the first 30 DAH, then increased till the end of the experiment, but only lipase activity showed significant differences (P \ 0.05) (Fig. 3a, c). Total activity for both enzymes remained low the first 4 weeks of development, but significantly increased after 30 DAH reaching maximum values by the end of the feeding trial (Fig. 3b, d). The specific activity of the intestinal enzymes aminopeptidase N and alkaline phosphatase resulted in a gradual but constant increase in activity until the end of the experiment (P \ 0.05) (Figs. 4c, 5a), concomitant with an increase in specific activity for trypsin, chymotrypsin and acid proteases (Figs. 2a, c, 4a). Total activities from both enzymes remained low until 20
DAH and then significantly increased to reach maximum values by the end of the feeding trial (P \ 0.05) (Figs. 4d, 5b). The intracellular enzyme leucine–alanine peptidase presented a different pattern of activity compared to the other proteases, with slight variations of activity through development and then a sharp decrease in activity from 30 DAH to 40 DAH, when no activity was detected with our assay. Although trends were observable during the experiment, data for this enzyme activity showed wide dispersion compared to most enzymes evaluated (P [ 0.05) (Fig. 5c, d). The specific activity of maltase was detected prior to hatching, increased 4 DAH and decreased to reach minimum levels by 8 DAH. Thereafter, specific
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Fig. 3 Activity of digestive enzymes of fed leopard grouper larvae (mean ± SD, n = 3). Specific and total activities were determined for amylase (a, b) and lipase (c, d). Fed larvae were provided different food: enriched rotifers (ER), Artemia nauplii
(AN), enriched Artemia metanauplii (EAN) and with microdiet (CD) during larval developmental. The values with asterisks are significantly different (P \ 0.05)
activity increased to reach a maximum level of 25 DAH, finally reaching intermediate values from 35 DAH to 40 DAH (Fig. 6a). Individual activity remained low the first 3 weeks of development and significantly increased by 20 DAH (Fig. 6b).
Discussion
Comparison of digestive activity between fasting and fed larvae Patterns observed for all digestive enzyme activities between fed and fasted larvae were surprisingly similar. No significant differences in the specific activity or total activity between fed and fasted larvae were observed for the first 7 DAH (data not show).
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Detailed knowledge of digestive physiology during larval development in fish species with good potential for aquaculture is essential for the production of healthy fingerlings. Changes in enzyme patterns in newly hatched larva have been extensively studied, and they have been correlated with both programmed genetic processes during the ontogeny of the digestive tract and/or changes in food administration, including both the quantity and quality of feeds. In the present study, the activities of trypsin and chymotrypsin were detected during the embryonic stage of the leopard grouper, at 4 h before hatching,
Fish Physiol Biochem
Fig. 4 Activity of digestive enzymes of fed leopard grouper larvae (mean ± SD, n = 3). Specific and total activities were determined for acid protease (a, b) and aminopeptidase N (c, d). Fed larvae were provided different food: enriched rotifers (ER),
Artemia nauplii (AN), enriched Artemia metanauplii (EAN) and with microdiet (CD) during larval developmental. The values with asterisks are significantly different (P \ 0.05)
indicating the importance of proteolytic activity during embryogenesis (cleavage of yolk proteins) and hatching (Sveinsdo´ttir et al. 2006; Civera-Cerecedo et al. 2004). The synthesis of these enzymes is considered to be genetically pre-programmed and has been observed for several species of fish larvae during embryogenesis, before exogenous feeding commenced and mouth opening (Cara et al. 2007, Cahu and Zambonino 2001; Alvarez et al. 2006; Zouiten et al. 2008). In the present study, the increase in enzyme activity coincided with both changes in food supplementation and ontogenic development associated with morphological changes. The slight increase of the main proteolytic enzymes, trypsin and chymotrypsin specific activity around 3
DAH may be associated with mouth opening in larvae ingesting exogenous food for the first time (Ribeiro et al. (1999); Lazo et al. (2000) and Alvarez-Gonza´lez et al. (2006). In addition, the sharp increase in trypsin and chymotrypsin specific activity of observed 7 DAH coincides with the initial supply of Artemia nauplii (from 6 DAH). Several studies have observed changes in enzyme activity in response to the quantity and quality of administered food, because this has been shown to stimulate digestive enzyme secretion in organisms (Cara et al. 2007; Pe´res et al. 1998). Furthermore, from a morphohistological point of view, the increase of trypsin activity may be related to further development of the exocrine pancreas during this
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Fig. 5 Activity of digestive enzymes of fed leopard grouper larvae (mean ± SD, n = 3). Specific and total activities were determined for alkaline phosphatase (a, b) and leucine–alanine peptidase (c, d). Fed larvae were provided different food:
enriched rotifers (ER), Artemia nauplii (AN), enriched Artemia metanauplii (EAN) and with microdiet (CD) during larval developmental. The values with asterisks are significantly different (P \ 0.05)
period of larval development (Gisbert et al. 2004). This was further corroborated with an increase in trypsin activity toward the end of the experiment, which is typically correlated with an increase in alkaline protein digestion, before the onset of acidic digestion in the stomach (Zambonino-Infante and Cahu 2001). In the present study, low trypsin-specific activity correlates well with not much growth for the first 10 DAH (Fig. 1). The low trypsin activity values observed during this period could have been affected by several factors such as the age of the larvae, the quality of spawns, and the food quality and availability. Similar results have been reported by Cara et al. (2007) for other species of fish larvae. However, some authors have shown that values of chymotrypsin activity had a higher correlation with nutritional status of the larvae,
for example, in red drum Sciaenops ocellatus (Applebaum and Holt 2003). Contrary to our expectations, the activity of acid protease was detected quite early in development (Fig. 4a). Activity of this enzyme followed a similar pattern to that observed for other proteases evaluated (trypsin and chymotrypsin). Nevertheless, acid protease activity observed early in development is probably not gastric pepsin, but rather some other acid enzyme present in the larva body such as the cathepsins, which are aspartic lysosomal proteolytic enzymes (EC 3.4.23.5). In fish, cathepsins are well distributed along different tissues such as the spleen, liver and muscle, but they are also expressed in other immune-related tissues such as head kidney, intestine and gills (Jia and Zhang 2009). In the present study, the acid protease
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Fig. 6 Activity of digestive enzymes of fed leopard grouper larvae (mean ± SD, n = 3). Specific and individual activities were determined for maltase (a, b). Fed larvae were provided different food: enriched rotifers (ER), Artemia nauplii (AN),
enriched Artemia metanauplii (EAN) and with microdiet (CD) during larval developmental. The values with asterisk are significantly different (P \ 0.05)
activity detected in embryonic eggs at 3 DAH could be related to enzymes involved in the digestion of the egg yolk proteins. In the case of the fasting larvae, these enzymes may be involved in the degradation of the intestinal tract and associated glands (Chen et al. 2007), as well as intracellular digestive processes, which include digestion or autolysis and cell death as part of the normal ontogenetic development (Mathews and van Holde 1998). In the present study, we detected a low acid protease activity up to 20 DAH; however, in other studies, this same enzyme was detected at 18 DAH in P. californicus (Alvarez-Gonza´lez et al. 2006), while Feng et al. (2008) detected pepsin activity even at hatching in larvae of Epinephelus coioides using the same technique used in this study. Thus, it is likely that other acid proteases are probably being detected since whole larval tissues were used for this assay. True gastric pepsin-like activity has been detected by molecular techniques only when the stomach becomes fully functional, for example, 30 DAH in Pagrus pagrus (Darias et al. 2007) and 41 DAH in Epinephelus coioides (Feng et al. 2008). Likewise, in studies where larvae dissection is performed to obtain enzymatic extracts, pepsin activity is detected in late larvae (just before metamorphosis) when gastric glands develop and a functional stomach is attained (Zacarı´as et al. 2006; Lazo et al.
2007). Zambonino-Infante and Cahu (2001) have reported that pepsin in marine fish is not detected early in development because marine fish lack a functional stomach in the first days of life, and HCl secreting glands are not functional (Govoni et al. 1986). For future studies, it is recommended to perform dissection of the digestive system to avoid the confounding effect of other enzymes present in the larval tissue and/or the use of molecular approaches to detect expression of the coding gene for pepsin by using specific primers. It is also important to corroborate pepsin activity with histological techniques to demonstrate the development of a functional stomach by the presence of gastric glands. We did not observe a significant increase in total acid protease activity until 20–30 DAH, which suggests a late development of a functional stomach in this species, which coincides with our histological studies, where the stomach starts to differentiate at 35 DAH and the gastric glands are visible at 40 DAH (results not shown). Unexpectedly in our study we did not find significant differences in enzyme activity patterns between fasting and fed larvae for the first 7 DAH. Although most studies report a higher digestive enzyme activity of fed larvae, our results could be explained by two different hypothesis: (1) Cannibalistic episodes among fasted larvae could explain the similarity in digestive
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enzyme activities between fed and starved larvae; this has been observed in previous experiments with the same fish species before the weaning period (GraciaLopez, personal communication). Previously, GraciaLo´pez et al. (2005) reported M. rosacea larval survival as low as 2 %, largely due to the cannibalistic behavior of the larvae. (2) At the time of sampling, the larvae for both treatments were not feeding and were in the similar ‘‘low-feeding’’ conditions. Thus, no significant differences were detected between the two experimental groups. Amylase-specific activity was detected as early as 1 DAH. Thereafter, the pattern observed is similar to that reported for other marine fish species (Moyano et al. 1996; Martı´nez et al. 1999). Despite the dispersion of the values and lack of statistical differences, amylase presents a constant specific activity for the first 5 DAH, suggesting M. rosacea’s ability to use carbohydrates at mouth opening; then it gradually decreases as the larvae develop. The decrease in amylase activity after hatching seems to be under genetic control, because amylase mRNA levels decreased independently of the dietary glucide concentration early in development for seabass larvae (Zambonino et al. 2008), leading some authors to hypothesize that this could be related to a low carbohydrate content of live prey (Cara et al. 2003). Fasting larvae showed low levels of amylase activity during the first days, and between 6 and 7 DAH, this activity decreased to undetectable levels with a pattern similar to that of the other enzymes (chymotrypsin, acid protease, lipase and maltase). The increase in amylase activity between 30 and 40 DAH is probably influenced by the carbohydrate content of the administered compound diet (Caviar, Bernaqua, 50–100 lm), which is known to contain carbohydrates derived from the yeast extract included in the formulation. Other studies have reported similar amylase patterns, which are thought to be influenced by the carbohydrates in the compound diets (Henning et al. 1994; Pe´res et al. 1998; Cara et al. 2003; Zouiten et al. 2008). Lipase activity was detected at 1 DAH and is typically associated with yolk lipid catabolism to provide energy for the developing larvae before exogenous feeding commences. Depending on the species of marine fish larvae, lipids may represent most of their energy sources during the endogenous feeding period (Sargent 1995; Rainuzzo et al. 1997); thus, lipase activity is essential for the proper larval development. At 6 DAH there is a slight, but not significant increase
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of lipase activity, and then it remains at constant values until the weaning period (30 DAH). The increase of lipase activity coincides with change of type of food (weaning to a compound diet), suggesting that the lipase activity may be strongly influenced by developmental stage and probably by the type of diet and lipid content (i.e., 15 % lipids). Our results are consistent with those reported for Sciaenops ocellatus (Holt et al. 1991) and Paralichthys californicus (Alvarez-Gonza´lez et al. 2006) whereby a strong capacity for lipid digestion is associated with the early stages of development. Morais et al. (2004) reported that the activity of this enzyme in Dicentrarchus labrax larvae is highly influenced by the type of lipids in the diet rather than the amount of lipids, since the response of the lipase is modulated mainly by the effect of the specific composition of fatty acids in the diet. Cahu et al. (1998) and Ribeiro et al. (1999) reported that the decrease of intracellular digestion (i.e., characterized by leu-ala peptidase) as well as the maturation of the brush border membrane of enterocytes (i.e., aminopeptidases, alkaline phosphatase) is typically considered the point of transformation to a juvenilelike mode of digestion. Aminopeptidase and alkaline phosphatase specific activities in our work were low early in development and increased as the larvae developed; in contrast, specific activity of the intracellular enzyme leucine–alanine peptidase remained at constant but moderate levels from 3 until 35 DAH (P [ 0.05) and then showed a decrease in activity to undetectable values, indicating a decrease in intracellular digestion in larvae as development of the digestive system progresses (Cahu and ZamboninoInfante 2001). It is important to acknowledge that intracellular digestion and cytosolic peptidase activity does not fully disappear during a fish life; in fact, higher activity has been detected in juveniles of Atherinopsids (Toledo et al. 2011) in contrast to the decrease in the activity of this enzyme with larval development described in most marine species (Zambonino and Cahu 1994). Zambonino and Cahu (2001) proposed the ratio between an intracellular enzyme (i.e., leu-ala peptidase) and a brush border enzyme (i.e., leucine– aminopeptidase) activities as an indicator of digestive system development in marine fish larvae, since the former has been identified in early pinocytotic processes (i.e., intracellular digestion) while the latter characterizes membrane digestion by enterocytes.
Fish Physiol Biochem
The detection of maltase is very common during larval development in fish, is a good indicator of an adequate differentiation and proliferation of intestinal cells, and has been used to assess the nutritional condition of many species (Zambonino and Cahu 2001). In our study, the pattern of maltase activity in both experimental groups showed a decrease from 5 to 7 DAH, which was probably caused by the lack of food and the use of glycogen stores to compensate for the high metabolic rate that is characteristic of fish during this larval stage. Despite the lack of significant differences, maltase-specific activity reached its maximum pick at 25 DAH and then decreased again to similar values as detected at 1 DAH. It remained constant until the end of the experiment (40 DAH), when the BBMs were well established. Finally, our results suggest that the leopard grouper larvae have a diverse and characteristic digestive enzyme activity, similar to that of other marine fish species. The information presented here can be used to formulate diets and weaning protocols in accordance with the digestive capacity of the larvae throughout development. Furthermore, we can reasonably conclude that the larvae of the leopard grouper possess a functional digestive system at the onset of exogenous feeding and that digestive capacity gradually increases as development progresses. The significant increase in the specific and total activities of pancreatic as well as intestinal enzymes (mainly brush border enzymes) and acidic proteases between 30 and 40 DAH suggests that the M. rosacea larvae can be successfully weaned onto microdiets around this age. Acknowledgments Thanks to Patricia Hinojosa Baltazar, Jorge Sandoval Soto and Francisco Encarnacio´n Ramı´rez for the technical support received and to the Ministry of Foreign Affairs of Mexico for the scholarship support provided to R.M. Special thanks go to our institution’s English-speaking editor and Miguel Co´rdoba for proofreading and editing help.
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´ lvarez-Lajonchere L, Herna´ndez-Molejo´n OG (2001) ProA duccio´n de peces estuarinos para un centro en Ame´rica Latina y El Caribe: Disen˜o, Operacio´n y Tecnologı´as. The World Aquaculture Society, Baton Rouge, p 424 Anson ML (1938) The estimation of pepsin, trypsin, papain and cathepsin with hemoglobin. J Gen Physiol 22:79–89 Applebaum SL, Holt AJ (2003) The digestive protease, chymotrypsin, as an indicator of nutritional condition in larval red drum (Sciaenops ocellatus). Mar Biol 142:1159–1167 ´ sgeirsson B, Bjarnasson JB (1991) Structural and kinetic A properties of chymotrypsin from atlantic cod (Gadus morhua). Comparison with bovine chymotrypsin. Comp Biochem Physiol 99:327–335 Bessey OA, Lowry SH, Brock MH (1946) A method for the rapid determination of alkaline phosphatase with five cubic milliliters of serum. J Biol Chem 164–321 Bradford MM (1976) A rapid and sensitive method for the quantization of microgram quantities of protein utilizing the principle of protein dye binding. Anal Biochem 72:248–254 Cahu CL, Zambonino-Infante J (2001) Substitution of live food by formulated diets in marine fish larvae. Aquaculture 200:161–180 Cahu CL, Zambonino-Infante J, Peres A, Quazuguel P, Le Gall MM (1998) Algal addition in sea bass (Dicentrarchus labrax) larvae rearing: effect on digestive enzymes. Aquaculture 161:479–489 Cara JB, Moyano FJ, Ca´rdenas S, Ferna´ndez-Dı´az C, Yu´fera M (2003) Assessment of digestive enzyme activities during larval development of white bream. J Fish Biol 63:48–58 Cara B, Moyano FJ, Zambonino JL, Fauvel C (2007) Trypsin and chymotrypsin as indicators of nutritional status of postweaned sea bass larvae. J Fish Biol 70:1798–1808 Chen BA, Qin JG, Carragher JF, Clarke SM, Kumar MS, Hutchinson WG (2007) Deleterious effects of food restrictions in yellowtail kingfish Seriola lalandi during early development. Aquaculture 271:326–335 Civera-Cerecedo R, Alvarez-Gonza´lez CA, Moyano-Lo´pez FJ (2004) Nutricio´n y alimentacio´n de larvas de peces marinos. In: Gonza´lez M, Cruz Suarez LE, Ricque Marie D, Nieto Lo´pez Mg, Villareal D, Scholz U (eds) Avances en Nutricio´n acuı´cola VII Memorias del VII simposium internacional de nutricio´n acuı´cola. Hermosillo, Sonora, Me´xico, pp 8–94 Cousin JC, Baudin-Laurencin F, Gabaudan J (1987) Ontogeny of enzymatic activities in fed and fasting turbot, Scophthalmus maximus L. J Fish Biol 30:15–33 Dahlqvist A (1970) Assay of intestinal disaccharidases. Enzymol Biol Clin 11:52–66 Darias M, Murray H, Gallant J, Douglas S, Yu´fera M, Martı´nezRodrı´guez G (2007) Ontogeny of pepsinogen and gastric proton pump expression in red porgy (Pagrus pagrus): determination of stomach functionality. Aquaculture 270:369–378 Erlanger B, Kolkowsky N, Cohen W (1961) The preparation and properties of two new chromogenic substrates of trypsin. Arch Biochem Biophys 95:271–278 Estrada-Godı´nez JA, Maldonado-Garcı´a M, Gracia-Lo´pez V, Carrillo M (2011) Ciclo reproductivo de la cabrilla sardinera Mycteroperca rosacea en la bahı´a de La Paz Me´xico. Ciencias Marinas 37(4):425–441
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development of gilthead seabream (Sparus aurata). Fish Physiol Biochem 15:121–130 Nicholson JA, Kim YS (1975) A one-step L-amino acid oxidase assay for intestinal peptide hydrolase activity. Anal Biochem 63:110–117 Pe´res A, Infante JL, Cahu C (1998) Dietary regulation of activities and mRNA levels of trypsin and amylase in sea bass (Dicentrarchus labrax) larvae. Fish Physiol Biochem 19:145–152 Rainuzzo JR, Reitan KI, Olsen Y (1997) The significance of lipids at early stages of marine fish: a review. Aquaculture 155:105–118 Reyes-Becerril M, Tovar-Ramı´rez D, Ascencio-Valle F, CiveraCerecedo R, Gracia-Lo´pez V, Barbosa-Solomieu V (2008) Effects of dietary live yeast Debaryomyces hansenii on the immune and antioxidant system in juvenile leopard grouper Mycteroperca rosacea exposed to stress. Aquaculture 280:39–44 Ribeiro L, Zambonino-Infante JL, Cahu C, Dinis MT (1999) Development of digestive enzymes in larvae of Solea senegalensis, Kaup 1858. Aquaculture 179:465–473 Sargent JR (1995) Origins and functions of egg lipids: Nutritional implications. In: Bromage NR, Roberts RJ (eds) Broodstock management and egg and larval quality. Blackwell, Oxford Sveinsdo´ttir S, Thorarensen H, Gudmundsdo´ttir A (2006) Involvement of trypsin and chymotrypsin activities in Atlantic cod (Gadus morhua) embryogenesis. Aquaculture 260:307–314 Toledo-Cuevas EM, Moyano-Lo´pez FJ, Tovar-Ramı´rez D, Alvarez-Gonza´lez CA, Stru¨ssmann CA, Martı´nez-Cha´vez CC, Martı´nez-Palacios CA (2011) Development of digestive biochemistry in the initial stages of three cultured atherinopsids. Aquac Res 42:776–786 Tong XH, Xu SH, Liu QH, Li J, Xia ZZ, Ma DY (2012) Digestive enzyme activities of turbot (Scophthalmus maximus, L.) during early developmental stages under culture condition. Fish Physiol Biochem.38:715–724 Ueberscha¨er B (1993) Measurement of proteolytic enzyme activity: significance and application in larval fish research. In: Walther BT, Fyhn HJ (eds) Physiological and biochemical aspects of fish development. Univ Bergen, Bergen, Norway Vega-Villasante F, Nolasco H, Civera R (1993) The digestive enzymes of the Pacific brown shrimp Penaeus californiensis. 1. Properties of amylase activity in the digestive tract. Comp Biochem Physiol 106:547–550 Versaw W, Cuppett SL, Winters DD, Williams LE (1989) An improved colorimetric assay for bacterial lipase in nonfat dry milk. J Food Sci 54:232–254 Zacarı´as M, Muguet JB, Lazo JP (2006) Proteolytic activity in California halibut larvae, (Paralichthys californicus). J World Aqua Soc 37:175–285 Zambonino JL, Gisbert E, Sarasquete C, Navarro I, Gutierrez J, Cahu C (2008) Ontogeny and physiology of the digestive system of marine fish larvae. In: feeding and digestive functions of fishes (Oxford & IBH Publishing Co. Pvt. Ltd) Zambonino-Infante JL, Cahu C (1994) Development and response to a diet change of some digestive enzymes in sea bass (Dicentrarchus labrax) larvae. Fish Physiol Biochem 12:399–408
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Changes in digestive enzyme activities during larval development of leopard grouper (Mycteroperca rosacea) R. Martı´nez-Lagos • D. Tovar-Ramı´rez V. Gracia-Lo´pez • J. P. Lazo
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Received: 9 April 2013 / Accepted: 25 October 2013 Ó Springer Science+Business Media Dordrecht 2013
Abstract The leopard grouper is an endemic species of the Mexican Pacific with an important commercial fishery and good aquaculture potential. In order to assess the digestive capacity of this species during the larval period and aid in the formulation of adequate weaning diets, this study aimed to characterize the ontogeny of digestive enzymes during development of the digestive system. Digestive enzymes trypsin, chymotrypsin, acid protease, leucine–alanine peptidase, alkaline phosphatase, aminopeptidase N, lipase, amylase and maltase were quantified in larvae fed live prey and weaned onto a formulated microdiet at 31 days after hatching (DAH) and compared with fasting larvae. Enzyme activity for trypsin, lipase and amylase were detected before the opening of the mouth and the onset of exogenous feeding, indicating
R. Martı´nez-Lagos Centro Universitario Regional del Centro (CURC), Universidad Nacional Auto´noma de Honduras, Km. 1.5 carretera a Tegucigalpa, aldea de Tenguaje, Comayagua, Honduras D. Tovar-Ramı´rez (&) V. Gracia-Lo´pez Instituto Polite´cnico Nacional 195, Centro de Investigaciones Biolo´gicas del Noroeste (CIBNOR), Col. Playa Palo de Santa Rita Sur, 23096 La Paz, BCS, Mexico e-mail: [email protected] J. P. Lazo Centro de Investigacio´n Cientı´fica y de Educacio´n Superior de Ensenada Baja California, Km. 107 Carretera Tijuana, 22860 Ensenada, BC, Mexico
a precocious development of the digestive system that has been described in many fish species. The intracellular enzyme activity of leucine–alanine peptidase was high during the first days of development, with a tendency to decrease as larvae developed, reaching undetectable levels at the end of the experimental period. In contrast, activities of enzymes located in the intestinal brush border (i.e., aminopeptidase and alkaline phosphatase) were low at the start of exogenous feeding but progressively increased with larval development, indicating the gradual maturation of the digestive system. Based on our results, we conclude that leopard grouper larvae possess a functional digestive system at hatching and before the onset of exogenous feeding. The significant increase in the activity of trypsin, lipase, amylase and acid protease between 30 and 40 DAH suggests that larvae of this species can be successfully weaned onto microdiets during this period. Keywords Mycteroperca rosacea Digestive capacity Larval development Ontogeny
Introduction Optimization of the culture techniques for rearing marine fish larvae requires a thorough knowledge of the biology and physiology of the species of interest in order to adapt best aquaculture practices to increase
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growth and survival. One of the milestones of the larval period is the development of a juvenile-like digestive capacity during the first days of development to successfully acquire the proper nutrients for adequate growth and survival. With the aid of studies aiming to increase our knowledge with respect to larval nutrition of a species of interest, many new larval culture protocols have been developed, for example, for the turbot (Scophthalmus maximus) (Cousin et al. 1987; Tong et al. 2012), the European seabass (Dicentrarchus labrax) (Zambonino-Infante and Cahu 1994) and the California halibut (Paralichthys californicus) (Alvarez-Gonza´lez et al. 2006; Zacarı´as et al. 2006). In addition, digestive enzymes are considered to be reliable indicators of the nutritional condition of the individuals due to their species and age specificity, sensitivity and short latency (Lazo et al. 2011). Different digestive enzymes are used for this purpose, ranging from proteolytic pancreatic enzymes to intestinal brush border and cytosolic enzymes (Ueberscha¨r 1993; Cahu and Zambonino 2001; Cara et al. 2007). The leopard grouper (Mycteroperca rosacea) is an endemic species of Mexico, distributed from the southeast coast of Baja California (eastern central Pacific) to the Pacific coast of Jalisco, Mexico (Allen and Robertson 1998). This species has been identified as a good candidate for aquaculture because of its adaptability to captivity, resistance to handling and good market value. For this reason, previous work on breeding, farming (Gracia-Lo´pez et al 2004, 2005; Martı´nez-Lagos and Gracia-Lo´pez. 2009), nutrition and immunology (Reyes-Becerril et al. 2008) and natural history (Estrada-Godı´nez et al. 2011) have been performed. However, poor growth and survival during the larval period are still a bottleneck for the ´ lvarez-Lajonchere mass cultivation of this species (A and Herna´ndez-Molejo´n. 2001). Thus, the main objective of this study was to assess the digestive capacity of this species based on the characterization of the digestive enzymes during larval development.
Materials and methods Larval culture protocol Fertilized eggs were obtained from natural spawns of broodstock maintained in captivity (8 $, 2 #) at the
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Reproductive Biology Laboratory of the Centro de Investigaciones Biolo´gicas del Noroeste (CIBNOR) in La Paz, Baja California Sur, Mexico. Eggs were collected in a 300-lm mesh bag, counted volumetrically and distributed in 6–300-l tanks filled with seawater with a salinity of 35 % that had been filtered (1 lm) and sterilized (UV). Stocking density for the incubation period was 25 eggs l-1. After hatching, two experimental groups were established: one negative control using fasting larvae and one normally fed group. Digestive enzyme activities from fasted larvae were analyzed to assess the importance of enzymes, which exhibit activity before first feeding and are genetically controlled (Zambonino et al. 2008). Each treatment was evaluated in triplicate using three separate replicate tanks. Water temperature in the tanks was maintained throughout the experiment at 25 ± 1 °C using submersible heaters (300 W RENA, Annecy, France) in each tank. Central bubble aeration was applied to maintain the oxygen concentration C5.5 mg l-1. The photoperiod was set to 24 h light at a constant light intensity of 300 lux. The first live feed provided to the larvae consisted of rotifers (Brachionus plicatilis) without enrichment from 3 to 6 DAH. Rotifers had only been fed with the microalgae Nannochloropsis oculata. Rotifers were mesh screened to 75 lm and fed to larvae at a density of 10 rotifers ml-1 in the culture tank. After this period, rotifers were enriched (ER) with a commercial emulsion, Enrich HUFA (Salt Creek Inc., USA) and administered to the larvae 7–30 DAH. A second type of live food, Artemia nauplii (Artemia franciscana) (AN), was supplied 6–25 DAH at a density of 1–2 ml-1. Subsequently, larvae were fed with enriched Artemia metanauplii (EAN) 25–40 DAH at a density of 2–5 ml-1. Weaning onto a microdiet began 31 DAH using a commercial microparticulated feed (Caviar, Bernaqua, Olen, Belgium) (CD) with a particle size of 200–300 lm. Feed was supplied to the larval tanks once a day in the morning (Fig. 1). Cleaning of the tanks was performed daily using a siphon. Environmental parameters such as temperature and dissolved oxygen were daily monitored using a YSI model 55 sensor (Yellow Springs, USA); salinity was monitored using a Extech Instruments refractometer model RF 20 (Waltham, MA, USA), and light intensity was measured using a commercial light meter (Extech, Waltham, MA, USA).
Fish Physiol Biochem Fig. 1 Wet weight, total length (mean ± SD, n = 10) and type of food provided to leopard grouper larvae versus days after hatching (DAH). Feeding scheme during larval development: enriched rotifers (ER), Artemia nauplii (AN), enriched Artemia metanauplii (EAN) and microdiet (CD)
Larval sampling Digestive enzyme assays were performed using at least 500 mg wet weight of larval tissue, representing 3–2,400 larvae per sample, depending on their weight and age (Fig. 1). On each sampling date, larvae were weighed using a digital scale (OHAUS Adventure, NJ, USA). Larvae from the fasting treatment were only collected from 1 DAH up to 7 DAH, since 100 % of the larvae had died 8 DAH. Larvae from the live feed treatments were collected daily from 1 DAH to 7 DAH, and subsequent samples were collected 10, 15, 20, 25, 30, 35 and 40 DAH. Samples of viable fertilized eggs were also collected during the embryo (neurula) stage (4 h before hatching) to determine whether any of the enzymes quantified in this study were present prior to hatching. The samples were frozen at -80° C in an ultra-freezer until used for the enzyme assays. Biochemical analyses Larval samples were homogenized using a tissue homogenizer Model Pro-200 (Pro Scientific Inc., Oxford, CT, USA) with 4:1 v/w cold Tris-HCl buffer 50 mM, pH 7.5 (25 mg ml-1). The supernatant was obtained after centrifuging the homogenates at 12,000 g for 12 min at 5 °C; thereafter, the samples were frozen at -20 °C for subsequent enzymatic analyses. The soluble protein concentration was
determined using the method described by Bradford (1976) using bovine serum albumin as standard. Acid protease activity was assessed by the method described by Anson (1938) using 0.5 % hemoglobin as substrate in 0.1 mM glycine buffer/HCl, pH 2.0. The mixture was incubated at 37 °C and the reaction stopped by adding 0.5 ml of 20 % tri-chloro acetic acid (TCA). One unit of enzyme activity was defined as 1 lg of tyrosine released per minute using a molar extinction coefficient of 0.008 lg/ml cm-1 (calculated from a linear regression of different concentrations of tyrosine from 0 to 300 lg/ml and the absorbance at 280 nm). The activity of trypsin was determined at 25 °C according to the method described by Erlanger et al. (1961) using BAPNA (N-a-benzoyl-DL-arginine pnitroanilide) as substrate (10 mM in DMSO) at a concentration of 100 mM in 50 mM Tris-HCl pH 8.2 buffer containing 10 mM CaCl2. One unit was defined as 1 lM of p-nitroanilide released per minute using a molar extinction coefficient of 8,800 cm-1 M-1. Chymotrypsin activity was determined according to the method described by Asgeirsson and Bjarnson (1991) using 5 mM BTEE (benzoyl-tyrosine ethyl ester) as substrate (5 mM in DMSO) in 44.4 mM Tris buffer containing 55.5 mM CaCl2 pH 7.8. Enzyme activity was determined using a molar extinction coefficient for benzoyl-tyrosine of 964 cm-1 M-1 at 256 nm. Amylase activity was determined using 1 % soluble starch as substrate in 50 mM of Tris-HCl
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buffer, pH 7.5, according to the method described by Vega-Villasante et al. (1993). Sodium carbonate (2 N) and DNS reagent (di-nitrosalicylic acid) were added to increase color development of the reaction product. The reaction was stopped by boiling the reactants for 15 min. One unit of enzyme activity was defined as the quantity of enzyme required to increase 0.01 units of absorbance at 550 nm per minute. The bile-saltdependent lipase (BSD-lipase) activity was determined according to the method described by Versaw et al. (1989) using ß-naphtyl-caprylate (200 mM) as substrate and sodium taurocholate (100 mM) with a Tris–HCl buffer (50 mM, pH 7.2); the reaction was stopped with TCA (0.72 N). Fast blue (100 mM) and ethanol:ethyl acetate (1:1 v/v) were added to the reaction to develop color and clarify the solution. One unit of enzyme activity was defined as 1 lg naphtol released per minute at 540 nm. Alkaline phosphatase activity was determined according to the method described by (Bessey et al. 1946) using 4-nitrophenylphosphate as substrate in 30 mM NaCO3 buffer at pH 9.8. The extinction coefficient used for p-nitrophenol at 407 nm was 0.0183 lM-1 cm-1. A unit (U) of activity was defined as the amount of enzyme, which hydrolyzes 1 nmol of substrate in 1 min. The activity of aminopeptidase N was determined according to the method described by Maraux et al. (1973) using 0.1 M L-leucine p-nitroanilide as substrate in DMSO and 80 mM phosphate buffer at pH 7. One unit of enzyme activity is defined as 1 lg of nitroanilide released per minute using a molar extinction coefficient of 8,200 M-1 cm-1. The cytosolic (or intracellular) enzyme activity of leucine– alanine peptidase was determined according to the method described by Nicholson and Kim (1975) using 0.010 M L-leucyl-L-alanine as substrate and 50 mM Tris–HCl buffer at pH 8; the reaction was stopped using H2SO4. One unit of activity was defined as 1 nmol of hydrolyzed substrate released per min. Maltase activity was determined according to the method described by Dahlqvist (1970) using 56 mM of maltose as substrate and 500 mM of Tris–HCl buffer at pH 7. One unit of enzyme activity corresponded to the quantity of glucose released per minute. Specific activity (units/mg protein) of extracts were estimated using the following equations: (1) units per ml = (D abs) 9 reaction final volume (ml))/(MEC 9 time (min) 9 extract volume (ml); (2) units per mg of protein = (units/ml)/(mg of soluble protein). The
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increase in absorbance at a specific wavelength is represented by Dabs, while MEC corresponds to the molar extinction coefficient of the reaction product in ml lg-1 cm-1; (3) total activity units per larva = (units/ml)/no. of larvae/ml. All enzyme assays were performed in triplicate. Statistical analysis Mann–Whitney test was used to compare the enzyme activity of larvae. Since the distribution of data met the normality criteria, the Kruskal–Wallis test (P \ 0.05) was used to compare enzyme activity between the different days on which sampling took place. Values of the measured variables are expressed as mean ± standard deviation.
Results Larval growth Growth of leopard grouper larvae in terms of length (mm) and weight (mg) and the feeding protocol during larval development are shown in Fig. 1. Ontogeny of protease activity in fed larvae Trypsin activity was detected before the onset of exogenous feeding. Significant differences in enzymespecific and total activities were observed at 7 and 40 DAH (P \ 0.05). Total trypsin activity was low for the first 3 weeks of development, increasing at a slow but constant rate until 30 DHA. Subsequently, the activity significantly increased (P \ 0.05) to reach maximum values by the end of the experiment (Fig. 2a, b). Chymotrypsin and acid protease specific activities were detected before the onset of exogenous feeding and show similar patterns, reaching maximum specific activity levels by 7 DAH (P \ 0.05). Thereafter, a sharp decrease in activity was observed and remained at moderate and constant levels until the end of the experimental period, 40 DAH (Figs. 2c, 3a). However, total activity was low the first 2 weeks of development and began increasing by 20 DAH, and it continued to do so until the end of the experimental period (P \ 0.05) (Figs. 2d, 3b). Amylase and lipase activity showed a similar trend during development. Activity of these enzymes was
Fish Physiol Biochem
Fig. 2 Activity of digestive enzymes of fed leopard grouper larvae (mean ± SD, n = 3). Specific and total activities were determined for trypsin (a, b) and chymotrypsin (c, d). Fed larvae were provided different food: enriched rotifers (ER), Artemia
nauplii (AN), enriched Artemia metanauplii (EAN) and with microdiet (CD) during larval developmental. The values with astericks are significantly different (P \ 0.05)
detected at 1 DAH. Specific activity of these enzymes showed a slight variation the first 30 DAH, then increased till the end of the experiment, but only lipase activity showed significant differences (P \ 0.05) (Fig. 3a, c). Total activity for both enzymes remained low the first 4 weeks of development, but significantly increased after 30 DAH reaching maximum values by the end of the feeding trial (Fig. 3b, d). The specific activity of the intestinal enzymes aminopeptidase N and alkaline phosphatase resulted in a gradual but constant increase in activity until the end of the experiment (P \ 0.05) (Figs. 4c, 5a), concomitant with an increase in specific activity for trypsin, chymotrypsin and acid proteases (Figs. 2a, c, 4a). Total activities from both enzymes remained low until 20
DAH and then significantly increased to reach maximum values by the end of the feeding trial (P \ 0.05) (Figs. 4d, 5b). The intracellular enzyme leucine–alanine peptidase presented a different pattern of activity compared to the other proteases, with slight variations of activity through development and then a sharp decrease in activity from 30 DAH to 40 DAH, when no activity was detected with our assay. Although trends were observable during the experiment, data for this enzyme activity showed wide dispersion compared to most enzymes evaluated (P [ 0.05) (Fig. 5c, d). The specific activity of maltase was detected prior to hatching, increased 4 DAH and decreased to reach minimum levels by 8 DAH. Thereafter, specific
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Fig. 3 Activity of digestive enzymes of fed leopard grouper larvae (mean ± SD, n = 3). Specific and total activities were determined for amylase (a, b) and lipase (c, d). Fed larvae were provided different food: enriched rotifers (ER), Artemia nauplii
(AN), enriched Artemia metanauplii (EAN) and with microdiet (CD) during larval developmental. The values with asterisks are significantly different (P \ 0.05)
activity increased to reach a maximum level of 25 DAH, finally reaching intermediate values from 35 DAH to 40 DAH (Fig. 6a). Individual activity remained low the first 3 weeks of development and significantly increased by 20 DAH (Fig. 6b).
Discussion
Comparison of digestive activity between fasting and fed larvae Patterns observed for all digestive enzyme activities between fed and fasted larvae were surprisingly similar. No significant differences in the specific activity or total activity between fed and fasted larvae were observed for the first 7 DAH (data not show).
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Detailed knowledge of digestive physiology during larval development in fish species with good potential for aquaculture is essential for the production of healthy fingerlings. Changes in enzyme patterns in newly hatched larva have been extensively studied, and they have been correlated with both programmed genetic processes during the ontogeny of the digestive tract and/or changes in food administration, including both the quantity and quality of feeds. In the present study, the activities of trypsin and chymotrypsin were detected during the embryonic stage of the leopard grouper, at 4 h before hatching,
Fish Physiol Biochem
Fig. 4 Activity of digestive enzymes of fed leopard grouper larvae (mean ± SD, n = 3). Specific and total activities were determined for acid protease (a, b) and aminopeptidase N (c, d). Fed larvae were provided different food: enriched rotifers (ER),
Artemia nauplii (AN), enriched Artemia metanauplii (EAN) and with microdiet (CD) during larval developmental. The values with asterisks are significantly different (P \ 0.05)
indicating the importance of proteolytic activity during embryogenesis (cleavage of yolk proteins) and hatching (Sveinsdo´ttir et al. 2006; Civera-Cerecedo et al. 2004). The synthesis of these enzymes is considered to be genetically pre-programmed and has been observed for several species of fish larvae during embryogenesis, before exogenous feeding commenced and mouth opening (Cara et al. 2007, Cahu and Zambonino 2001; Alvarez et al. 2006; Zouiten et al. 2008). In the present study, the increase in enzyme activity coincided with both changes in food supplementation and ontogenic development associated with morphological changes. The slight increase of the main proteolytic enzymes, trypsin and chymotrypsin specific activity around 3
DAH may be associated with mouth opening in larvae ingesting exogenous food for the first time (Ribeiro et al. (1999); Lazo et al. (2000) and Alvarez-Gonza´lez et al. (2006). In addition, the sharp increase in trypsin and chymotrypsin specific activity of observed 7 DAH coincides with the initial supply of Artemia nauplii (from 6 DAH). Several studies have observed changes in enzyme activity in response to the quantity and quality of administered food, because this has been shown to stimulate digestive enzyme secretion in organisms (Cara et al. 2007; Pe´res et al. 1998). Furthermore, from a morphohistological point of view, the increase of trypsin activity may be related to further development of the exocrine pancreas during this
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Fig. 5 Activity of digestive enzymes of fed leopard grouper larvae (mean ± SD, n = 3). Specific and total activities were determined for alkaline phosphatase (a, b) and leucine–alanine peptidase (c, d). Fed larvae were provided different food:
enriched rotifers (ER), Artemia nauplii (AN), enriched Artemia metanauplii (EAN) and with microdiet (CD) during larval developmental. The values with asterisks are significantly different (P \ 0.05)
period of larval development (Gisbert et al. 2004). This was further corroborated with an increase in trypsin activity toward the end of the experiment, which is typically correlated with an increase in alkaline protein digestion, before the onset of acidic digestion in the stomach (Zambonino-Infante and Cahu 2001). In the present study, low trypsin-specific activity correlates well with not much growth for the first 10 DAH (Fig. 1). The low trypsin activity values observed during this period could have been affected by several factors such as the age of the larvae, the quality of spawns, and the food quality and availability. Similar results have been reported by Cara et al. (2007) for other species of fish larvae. However, some authors have shown that values of chymotrypsin activity had a higher correlation with nutritional status of the larvae,
for example, in red drum Sciaenops ocellatus (Applebaum and Holt 2003). Contrary to our expectations, the activity of acid protease was detected quite early in development (Fig. 4a). Activity of this enzyme followed a similar pattern to that observed for other proteases evaluated (trypsin and chymotrypsin). Nevertheless, acid protease activity observed early in development is probably not gastric pepsin, but rather some other acid enzyme present in the larva body such as the cathepsins, which are aspartic lysosomal proteolytic enzymes (EC 3.4.23.5). In fish, cathepsins are well distributed along different tissues such as the spleen, liver and muscle, but they are also expressed in other immune-related tissues such as head kidney, intestine and gills (Jia and Zhang 2009). In the present study, the acid protease
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Fig. 6 Activity of digestive enzymes of fed leopard grouper larvae (mean ± SD, n = 3). Specific and individual activities were determined for maltase (a, b). Fed larvae were provided different food: enriched rotifers (ER), Artemia nauplii (AN),
enriched Artemia metanauplii (EAN) and with microdiet (CD) during larval developmental. The values with asterisk are significantly different (P \ 0.05)
activity detected in embryonic eggs at 3 DAH could be related to enzymes involved in the digestion of the egg yolk proteins. In the case of the fasting larvae, these enzymes may be involved in the degradation of the intestinal tract and associated glands (Chen et al. 2007), as well as intracellular digestive processes, which include digestion or autolysis and cell death as part of the normal ontogenetic development (Mathews and van Holde 1998). In the present study, we detected a low acid protease activity up to 20 DAH; however, in other studies, this same enzyme was detected at 18 DAH in P. californicus (Alvarez-Gonza´lez et al. 2006), while Feng et al. (2008) detected pepsin activity even at hatching in larvae of Epinephelus coioides using the same technique used in this study. Thus, it is likely that other acid proteases are probably being detected since whole larval tissues were used for this assay. True gastric pepsin-like activity has been detected by molecular techniques only when the stomach becomes fully functional, for example, 30 DAH in Pagrus pagrus (Darias et al. 2007) and 41 DAH in Epinephelus coioides (Feng et al. 2008). Likewise, in studies where larvae dissection is performed to obtain enzymatic extracts, pepsin activity is detected in late larvae (just before metamorphosis) when gastric glands develop and a functional stomach is attained (Zacarı´as et al. 2006; Lazo et al.
2007). Zambonino-Infante and Cahu (2001) have reported that pepsin in marine fish is not detected early in development because marine fish lack a functional stomach in the first days of life, and HCl secreting glands are not functional (Govoni et al. 1986). For future studies, it is recommended to perform dissection of the digestive system to avoid the confounding effect of other enzymes present in the larval tissue and/or the use of molecular approaches to detect expression of the coding gene for pepsin by using specific primers. It is also important to corroborate pepsin activity with histological techniques to demonstrate the development of a functional stomach by the presence of gastric glands. We did not observe a significant increase in total acid protease activity until 20–30 DAH, which suggests a late development of a functional stomach in this species, which coincides with our histological studies, where the stomach starts to differentiate at 35 DAH and the gastric glands are visible at 40 DAH (results not shown). Unexpectedly in our study we did not find significant differences in enzyme activity patterns between fasting and fed larvae for the first 7 DAH. Although most studies report a higher digestive enzyme activity of fed larvae, our results could be explained by two different hypothesis: (1) Cannibalistic episodes among fasted larvae could explain the similarity in digestive
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enzyme activities between fed and starved larvae; this has been observed in previous experiments with the same fish species before the weaning period (GraciaLopez, personal communication). Previously, GraciaLo´pez et al. (2005) reported M. rosacea larval survival as low as 2 %, largely due to the cannibalistic behavior of the larvae. (2) At the time of sampling, the larvae for both treatments were not feeding and were in the similar ‘‘low-feeding’’ conditions. Thus, no significant differences were detected between the two experimental groups. Amylase-specific activity was detected as early as 1 DAH. Thereafter, the pattern observed is similar to that reported for other marine fish species (Moyano et al. 1996; Martı´nez et al. 1999). Despite the dispersion of the values and lack of statistical differences, amylase presents a constant specific activity for the first 5 DAH, suggesting M. rosacea’s ability to use carbohydrates at mouth opening; then it gradually decreases as the larvae develop. The decrease in amylase activity after hatching seems to be under genetic control, because amylase mRNA levels decreased independently of the dietary glucide concentration early in development for seabass larvae (Zambonino et al. 2008), leading some authors to hypothesize that this could be related to a low carbohydrate content of live prey (Cara et al. 2003). Fasting larvae showed low levels of amylase activity during the first days, and between 6 and 7 DAH, this activity decreased to undetectable levels with a pattern similar to that of the other enzymes (chymotrypsin, acid protease, lipase and maltase). The increase in amylase activity between 30 and 40 DAH is probably influenced by the carbohydrate content of the administered compound diet (Caviar, Bernaqua, 50–100 lm), which is known to contain carbohydrates derived from the yeast extract included in the formulation. Other studies have reported similar amylase patterns, which are thought to be influenced by the carbohydrates in the compound diets (Henning et al. 1994; Pe´res et al. 1998; Cara et al. 2003; Zouiten et al. 2008). Lipase activity was detected at 1 DAH and is typically associated with yolk lipid catabolism to provide energy for the developing larvae before exogenous feeding commences. Depending on the species of marine fish larvae, lipids may represent most of their energy sources during the endogenous feeding period (Sargent 1995; Rainuzzo et al. 1997); thus, lipase activity is essential for the proper larval development. At 6 DAH there is a slight, but not significant increase
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of lipase activity, and then it remains at constant values until the weaning period (30 DAH). The increase of lipase activity coincides with change of type of food (weaning to a compound diet), suggesting that the lipase activity may be strongly influenced by developmental stage and probably by the type of diet and lipid content (i.e., 15 % lipids). Our results are consistent with those reported for Sciaenops ocellatus (Holt et al. 1991) and Paralichthys californicus (Alvarez-Gonza´lez et al. 2006) whereby a strong capacity for lipid digestion is associated with the early stages of development. Morais et al. (2004) reported that the activity of this enzyme in Dicentrarchus labrax larvae is highly influenced by the type of lipids in the diet rather than the amount of lipids, since the response of the lipase is modulated mainly by the effect of the specific composition of fatty acids in the diet. Cahu et al. (1998) and Ribeiro et al. (1999) reported that the decrease of intracellular digestion (i.e., characterized by leu-ala peptidase) as well as the maturation of the brush border membrane of enterocytes (i.e., aminopeptidases, alkaline phosphatase) is typically considered the point of transformation to a juvenilelike mode of digestion. Aminopeptidase and alkaline phosphatase specific activities in our work were low early in development and increased as the larvae developed; in contrast, specific activity of the intracellular enzyme leucine–alanine peptidase remained at constant but moderate levels from 3 until 35 DAH (P [ 0.05) and then showed a decrease in activity to undetectable values, indicating a decrease in intracellular digestion in larvae as development of the digestive system progresses (Cahu and ZamboninoInfante 2001). It is important to acknowledge that intracellular digestion and cytosolic peptidase activity does not fully disappear during a fish life; in fact, higher activity has been detected in juveniles of Atherinopsids (Toledo et al. 2011) in contrast to the decrease in the activity of this enzyme with larval development described in most marine species (Zambonino and Cahu 1994). Zambonino and Cahu (2001) proposed the ratio between an intracellular enzyme (i.e., leu-ala peptidase) and a brush border enzyme (i.e., leucine– aminopeptidase) activities as an indicator of digestive system development in marine fish larvae, since the former has been identified in early pinocytotic processes (i.e., intracellular digestion) while the latter characterizes membrane digestion by enterocytes.
Fish Physiol Biochem
The detection of maltase is very common during larval development in fish, is a good indicator of an adequate differentiation and proliferation of intestinal cells, and has been used to assess the nutritional condition of many species (Zambonino and Cahu 2001). In our study, the pattern of maltase activity in both experimental groups showed a decrease from 5 to 7 DAH, which was probably caused by the lack of food and the use of glycogen stores to compensate for the high metabolic rate that is characteristic of fish during this larval stage. Despite the lack of significant differences, maltase-specific activity reached its maximum pick at 25 DAH and then decreased again to similar values as detected at 1 DAH. It remained constant until the end of the experiment (40 DAH), when the BBMs were well established. Finally, our results suggest that the leopard grouper larvae have a diverse and characteristic digestive enzyme activity, similar to that of other marine fish species. The information presented here can be used to formulate diets and weaning protocols in accordance with the digestive capacity of the larvae throughout development. Furthermore, we can reasonably conclude that the larvae of the leopard grouper possess a functional digestive system at the onset of exogenous feeding and that digestive capacity gradually increases as development progresses. The significant increase in the specific and total activities of pancreatic as well as intestinal enzymes (mainly brush border enzymes) and acidic proteases between 30 and 40 DAH suggests that the M. rosacea larvae can be successfully weaned onto microdiets around this age. Acknowledgments Thanks to Patricia Hinojosa Baltazar, Jorge Sandoval Soto and Francisco Encarnacio´n Ramı´rez for the technical support received and to the Ministry of Foreign Affairs of Mexico for the scholarship support provided to R.M. Special thanks go to our institution’s English-speaking editor and Miguel Co´rdoba for proofreading and editing help.
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