Digestive enzyme activities during early ontogeny in ...

Digestive enzyme activities during early ontogeny in Common snook (Centropomus undecimalis) L. D. Jimenez-Martinez, C. A. AlvarezGonzález, D. Tovar-Ramírez, G. Gaxiola, A. Sanchez-Zamora, F. J. Moyano, F. J. Alarcón, G. Márquez-Couturier, et al. Fish Physiology and Biochemistry ISSN 0920-1742 Volume 38 Number 2 Fish Physiol Biochem (2012) 38:441-454 DOI 10.1007/s10695-011-9525-9

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Author's personal copy Fish Physiol Biochem (2012) 38:441–454 DOI 10.1007/s10695-011-9525-9

Digestive enzyme activities during early ontogeny in Common snook (Centropomus undecimalis) L. D. Jimenez-Martinez • C. A. Alvarez-Gonza´lez • D. Tovar-Ramı´rez • G. Gaxiola • A. Sanchez-Zamora • F. J. Moyano • F. J. Alarcoń • G. Ma´rquez-Couturier E. Gisbert • W. M. Contreras-Sańchez • N. Perales-Garcıá • L. Arias-Rodrı´guez • J. R. Indy • S. Pa´ramo-Delgadillo • I. G. Palomino-Albarrań

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Received: 19 December 2009 / Accepted: 3 June 2011 / Published online: 14 June 2011 Ó Springer Science+Business Media B.V. 2011

Abstract Common snook (Centropomus undecimalis) is one of the most important marine species under commercial exploitation in the Gulf of Mexico; for this reason, interest in developing its culture is a priority. However, larviculture remains as the main bottleneck for massive production. In this sense, our objective was to determine the changes of digestive enzymes activities using biochemical and electrophoretic techniques during 36 days of Common snook larviculture fed with live preys (microalgae, rotifers, and Artemia). During larviculture, all digestive enzymatic activities were detected with low values since yolk absorption, 2 days after hatching (dah) onwards. However, the maximum values for alkaline protease (6,500 U mg protein-1), trypsin (0.053 mU 9 10-3 mg protein-1), and Leucine aminopeptidase (1.4 9 10-3 mU mg protein-1) were detected at 12 dah; for chymotrypsin at 25 dah (3.8 9 10-3 mU mg protein-1), for carboxypeptidase

A (280 mU mg protein-1) and lipase at 36 dah (480 U mg protein-1), for a-amylase at 7 dah (1.5 U mg protein-1), for acid phosphatases at 34 dah (5.5 U mg protein-1), and finally for alkaline phosphatase at 25 dah (70 U mg protein-1). The alkaline protease zymogram showed two active bands, the first (26.3 kDa) at 25 dah onwards, and the second (51.6 kDa) at 36 dah. The acid protease zymogram showed two bands (RF = 0.32 and 0.51, respectively) at 34 dah. The digestive enzymatic ontogeny of C. undecimalis is very similar to other strictly marine carnivorous fish, and we suggest that weaning process should be started at 34 dah.

L. D. Jimenez-Martinez C. A. Alvarez-Gonza´lez (&) G. Ma´rquez-Couturier W. M. Contreras-Sańchez N. Perales-Garcıá L. Arias-Rodrı´guez J. R. Indy S. Pa´ramo-Delgadillo DACBIOL Laboratorio de Acuacultura, Universidad Jua´rez Autońoma de Tabasco, Carretera VillahermosaCa´rdenas km 0.5, 86139 Villahermosa, Tabasco, Mexico e-mail: [email protected]

F. J. Moyano F. J. Alarcoń Departamento de Biologıá Aplicada, Escuela Politećnica Superior, Universidad de Almerıá, 04120 La Canãda de San Urbano, Almerıá, Spain

G. Gaxiola A. Sanchez-Zamora I. G. Palomino-Albarrań Unidad Multidisciplinaria de Docencia e Investigacioń, Facultad de Ciencias, UNAM, Puerto de abrigo s/n, Sisal, Yucatań, Mexico

Keywords a-Amylase Centropomus undecimalis Common snook Lipase PAGE Phosphatase Protease

D. Tovar-Ramı´rez Centro de Investigaciones Biolo´gicas del Noroeste (CIBNOR), Mar Bermejo 195, Col. Playa Palo de Santa Rita, 23090 La Paz, B.C.S., Mexico E. Gisbert IRTA – Sant Carles de la Ra`pita, Crta. Poble Nou km 5.5, 43540 Sant Carles de la Rapita, Tarragona, Spain

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Fish Physiol Biochem (2012) 38:441–454

Introduction

Materials and methods

Marine fish culture in Mexico is a recent activity mainly based on some species with high economic value and adequate biological characteristics for their culture, such as spotted sand bass (Paralabrax maculatofasciatus), leopard grouper (Mycteroperca rosacea), bullseye puffer (Sphoeroides annulatus), rose snapper (Lutjanus guttatus), and more recently cobia (Rachycentrum canadum) (Avile´s-Quevedo et al. 1995; Gracia-Lo´pez et al. 2004; Komar et al. 2004; Ibarra-Castro and Duncan 2007; Holt et al. 2007). The fat snook (Centropomus parallelus) and the common snook (Centropomus undecimalis) of the family Centropomidae, a group with highly important commercial value in Mexico and the United States, have been widely studied to understand their basic biology and aiming for developing their culture (Cha´vez 1961; Stephen and Shafland 1982; Sherwood et al. 1993; Ramirez and Cerqueira 1994; Grier and Taylor 1998; Grier 2000; Cequeira and Bru¨gger 2001; Alvarez-Lajonche`re et al. 2002; Tarcisio et al. 2005; Gracia-Lo´pez et al. 2006; Wainwright et al. 2006; Yan˜es-Roca et al. 2009). However, for C. undecimalis, the bottleneck is still the massive fry production due to feeding problems when microalgae, rotifers, and Artemia nauplii are used. These live feeds are considered not adequate for fish larval culture (Versichelle et al. 1989; Garcıá-Ortega et al. 1998), resulting in low growth and survival. For this reason, many studies have been conducted to understand the digestive physiology during early ontogeny with many species, such as the seabream, Sparus aurata (Moyano et al. 1996), Siberian sturgeon, Acipenser baeri (Gisbert et al. 1999), white bream, Diplodus sargus (Cara et al. 2003), yellowtail amberjack, Seriola lalandi (Chen et al. 2006), common seabream, Pagrus pagrus (Darias et al. 2006), P. maculatofasciatus (Alvarez-Gonza´lez et al. 2008, 2010), and the orange-spotted grouper Epinephelus coioides (Shaozhen et al. 2008). These studies allow understanding the right moment to conduct early weaning using artificial diets (Brock et al. 1992; Zambonino-Infante and Cahu 1994; Ribeiro et al. 1999; Cara et al. 2003; Fabillo et al. 2004). In consequence, the objective of this work was to assess the development of digestive enzymes using biochemical and electrophoretical techniques during early ontogeny of C. undecimalis.

Rearing and sampling of larvae

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Twelve adult common snook C. undecimalis (4–5 kg per fish) were maintained under controlled conditions in four 13-m3 circular plastic tanks at the Unidad Multidisciplinaria de Docencia e Investigacioń (UMDI) from UNAM in Sisal, Merida, Mexico for 2 years. Spawning induction was done in adults using cholesterol implants with 150 lg of sGnRHa. fish-1 (Ovaplant, Syndel, Western Chemical, Fendale, WA, USA). A total of 1,10,000 embryos were obtained after 32 h after injection. After the embryos hatched, the yolk-sac larvae (3,250) were collected by siphoning and placed in a 400-l cylinder-conical tank with constant water exchange, and continuous aeration until the larvae absorbed the yolk (24 h later). Salinity (35.2 ± 1.1 ppt), dissolved oxygen (6.0 ± 0.3 mg l-1), and temperature (29.9 ± 1.1°C) were monitored daily. Larvae were fed four times per day (8:00, 12:00, 16:00, and 20:00 h), starting with green water culture, using the microalgae Nannochloropsis sp. (20 9 106 cells ml-1) and S-type rotifers Brachionus rotundiormis (R, 2–10 preys ml-1) from mouth opening (day 1 after hatching) until 10 dah. Rotifers were mixed with newly hatched Artemia nauplii (AN, INVE Aquaculture, Belgium, 2–10 preys ml-1) from day 10 until 25 dah and offered to the larvae. Finally, larvae were fed exclusively with lipidic enriched (SELCO, INVE Aquaculture, Belgium) Artemia meta-nauplii (EAMN, 2–15 preys ml-1) from day 25 up to 36 dah (end of the experiment). Nine samples of feed larvae were taken from one culture tank using a 500-lmdiameter mesh, in triplicate (the numbers in parentheses are the numbers of larvae sampled per replicate) on day 0 (embryos, 80), 1 (600), 3 (600), 5 (600), 7 (600), 12 (400), 25 (100), 34 (50), and 36 (30) after hatching. They were frozen with liquid nitrogen and stored at -80°C until analysis was done. For growth analysis, samples of 10 larvae were taken and fixed with buffered borate formalin solution (4%) to measure the total length for each larvae using a digital caliper (Neiko-HKMUD473, Neiko-HKMUD473, CA, USA). The individual wet weight (mg) of each larva was also recorded with an analytic balance (OHAUS-Phoenix GH-300, Pine Brook, NJ, USA; precision of 10-4 g) after elimination of water excess with filter paper.

Author's personal copy Fish Physiol Biochem (2012) 38:441–454

Biochemical analyses Sampled larvae were dissected individually to remove the head and tail, and the visceral bulks were homogenized as pool (30 mg ml-1) in cold 50 mmol l-1 Tris–HCl 20 mmol l-1 CaCl2 buffer, pH 7.5. The supernatant obtained after centrifugation (16,000g for 15 min at 5°C) was stored at -20°C until enzyme analysis. The concentration of soluble protein was determined by the Bradford (1976) method using bovine serum albumin as a standard. Total alkaline protease activity was measured using casein (0.5%) in 50 mmol l-1 Tris–HCl buffer, pH 9.0, following Kunitz’s (1947) method, modified by Walter (1984). Acid protease activity was evaluated according to Anson (1938) using 0.5% hemoglobin in 0.1 mmol l-1 glycine–HCl, pH 2.0. One unit of enzyme activity was defined as 1 lg tyrosine released per minute using a coefficient of molar extinction of 0.008 at 280 nm. Trypsin activity was assayed using BAPNA (N-a-benzoyl-DL-arginine 4-nitroanilide hydrochloride) as substrate according to Erlanger et al. (1961). Chymotrypsin activity in extracts was determined using SAAPNA (N-succinylala–ala-pro-phe p-nitroanilide) according to DelMar et al. (1979). Leucine aminopeptidase was determined using leucine p-nitroanilide (0.1 mmol l-1 in DMSO) as substrate, according to Maraux et al. (1973). For trypsin, chymotrypsin, and leucine aminopeptidase activities, one unit of enzyme activity was defined as 1 lmol p-nitroaniline released per minute using coefficients of molar extinction of 8.2 at 410 nm. Carboxypeptidase A activity was measured following the protocol of Folk and Schirmer (1963) using HPA (hippuryl-L-phenyl-alanine) as substrate dissolved in 25 mmol l-1 Tris–HCl, 10 mmol l-1 CaCl2 buffer, pH 7.8. One unit of enzyme activity was defined as 1 lmol of hydrolyzed hippuryl per minute using a coefficient of molar extinction of 0.36 at 254 nm. Determination of a-amylase activity was carried out following the Somoyi-Nelson procedure described by Robyt and Whelan (1968). One unit of activity was defined as the amount of enzyme able to produce 1 lg of maltose per minute at 600 nm. Lipase activity was quantified using b-naphthyl caprylate as substrate according to Versaw et al. (1989). One unit of activity was defined as 1 lg of naphthol released per minute

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using a molar extinction coefficient of 0.02 at 540 nm. Acid and alkaline phosphatases were assayed using 4-nitrophenyl phosphate in acid citrate buffer (pH 5.5) or glycine–NaOH buffer (pH 10.1) according to Bergmeyer (1974). One unit was defined as 1 lg of nitrophenyl released per minute using a molar extinction coefficient of 18.5 at 405 nm. All assays were performed by triplicate at 37°C. Digestive enzyme activities were expressed as U mg protein-1 and U larva-1 using the total number of larvae in each homogenized pooled sample. Electrophoretic analysis The analysis of the alkaline protease isoforms was done using sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE; 10% polyacrylamide) for each larval enzyme preparation in a Mini Protean II chamber (Bio-Rad) according to Laemmli (1970) using 8 9 10 9 0.075-cm gels. Zymograms of alkaline protease activities were obtained as described by Garcıá-Carrenõ et al. (1993). Electrophoresis was carried out during 60 min at a constant voltage of 100 V per gel at 5°C. After electrophoresis, the gels were washed and incubated for 30 min at 5°C in a 0.5% casein Hammerstein (Research Organics) solution at pH 9.0. The gels were then incubated for 90 min in the same solution at 25°C without agitation. Finally, the gels were washed and fixed in 12% trichloroacetic acid (Sigma–Aldrich) prior to staining with 0.1% Coomassie brilliant blue R-250 (Research Organics) in a solution of methanol-acetic acid (Sigma–Aldrich)water (50:20:50). Distaining was carried out in a solution of methanol-acetic acid–water (35:10:55). Clear zones, which indicated activity of alkaline proteases, were visible after 24 h. The acid protease activities in larval extracts were analyzed by neutral native polyacrylamide electrophoresis (Williams and Reisfeld 1964). All electrophoresis procedures were performed at a constant voltage and amperage (100 V and 64 mA). Acid protease isoforms were revealed according to the procedure of Dıáz-Lo´pez et al. (1998). Same quantity of protein (30 lg per well) was applied to carry out each electrophoresis. The gels were removed from the cell and soaked in 100 mmol l-1 HCl to reach pH 2.0 where the enzymes become active. After 15 min, the gel was soaked for 30 min at 4°C in a solution

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containing 0.25% hemoglobin in 100 mmol l-1 Glycine–HCl, pH 2.0, and then for 90 min in a fresh hemoglobin solution at 37°C. The gels were washed in distilled water and fixed for 15 min in a 12% trichloroacetic acid solution. When the clear areas of enzyme activity appeared, the gels were stained using Coomassie brilliant blue R-250 solution. Destaining was carried out as mentioned earlier. Clear zones revealed the activity of acid proteases within a few minutes although well-defined zones were obtained only after 2–4 h of staining. A low-range molecular weight marker (5 ll per well) containing phosphorylase b (97 kDa), bovine serum albumin (66 kDa), egg albumin (45 kDa), carbonic anhydrase (29 kDa), trypsinogen (24 kDa), and soybean trypsin inhibitor (20 kDa) was applied to each SDS–PAGE. The relative electromobility (Rf) was calculated for all zymograms (Igbokwe and Downe 1978), and the molecular weight (MW) of each band in the SDSzymograms (alkaline protease) was calculated by a linearly adjusted model between the Rf and the decimal logarithm of MW proteins using Quality One V. 4.6.5 (Hercules, CA) software program. Statistical analysis Larval growth was determined with an exponential model Y = aebX, with logarithm base 10 transformed data, and the model parameters were calculated by using the least-squares technique. A Kruskal–Wallis test was used to compare enzyme activity between ages for each activity. A nonparametric Nemenyi test was used when significant differences were detected. All tests were carried out with Statistica v7.0 (StatSoft, Tulsa, OK, USA) software.

Results Centropomus undecimalis larvae showed a typical daily growth rate (0.140 mg day-1) expressed by weight (Fig. 1a) and 0.078 mm day-1 expressed by length (Fig. 1b). For all specific and individual digestive enzyme activities, statistical differences were detected (P \ 0.05) when activities were compared between ages (Fig. 2a–l). Specific alkaline protease was detected from hatching (1 dah, 1,234.2 ± 0.5 U mg protein-1) and increased until 3461.5 ± 0.5 U mg protein-1 at 3

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Fig. 1 a Mean wet weight (lg larva-1 ± SD, n = 3 pooled larvae), and b total length (mm larvae-1 ± SD, n = 3 pooled larvae) of common snook larvae

dah, decreasing at 7 dah (393.4 ± 0.1 U mg protein-1) reaching the maximum value at 12 dah (6729.0 ± 0.3 U mg protein-1), to finally decreased (6729.5 ± 0.3 U mg protein-1) up to 36 dah (Fig. 2a). Specific acid protease activity was detected with low levels from hatching; it increased slightly at 7 dah (27.1 ± 0.8 U mg protein-1) remaining with statistically same values until 12 dah (19.2 ± 0.1 U mg protein-1), then increased rapidly at 34 dah (92.4 ± 6.0 U mg protein-1), reaching the maximum value at 36 dah (124.3 ± 0.8 U mg protein-1) (Fig. 2c). Specific trypsin activity showed low activity from hatching (0.03 ± 0.03 mU 9 10-3 mg protein-1), reaching two maximum peaks at 7 (0.05 ± 0.01 mU 9 10-3 mg protein-1) and 12 dah (0.05 ± 0.02 mU 9 10-3 mg protein-1), and then, it reduced rapidly at 34 and 36 dah (0.03 ± 0.04 and 0.03 ± 0.01 mU 9 10-3 mg protein-1, respectively) (Fig. 2e). Chymotrypsin-specific activity started with low values from hatching (1.0 ± 0.1 mU 9 10-3 mg protein-1) until day 5 after hatching (2.7 ± 0.3 mU 9 10-3 mg protein-1) and increased gradually, reaching the


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Fig. 2 Digestive enzyme activity during common snook larviculture (mean ± SD, n = 3 pooled larvae). a Specific alkaline protease and b individual alkaline protease activities, c specific acid protease and d individual acid protease activities, e specific trypsin and f individual trypsin activities, g specific chymotrypsin and h individual chymotrypsin activities, i specific carboxypeptidase A and j individual carboxypeptidase A activities, k specific leucine aminopeptidase and l individual leucine aminopeptidase activities. R rotifers, AN Artemia nauplii, EAMN Enriched Artemia metanauplii

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maximum activity at 25 dah (3.9 ± 0.2 mU 9 10-3 mg protein-1) and then decreasing rapidly from this day onwards (2.0 ± 1.0 mU 9 10-3 mg protein-1 at 36 dah) (Fig. 2g). Carboxypeptidase A-specific activity was low during the first days of larviculture around 25.1 ± 0.0 mU mg protein-1, and increased rapidly at days 7 (128.3 ± 0.3 mU mg protein-1) and 12 days after hatching (128.1 ± 0.2 mU mg protein-1) to reduce at 25 dah (47.8 ± 0.2 mU mg protein-1), and increased again at 34 and 36 dah (158.4 ± 0.1 and 278.2 ± 0.2 mU mg protein-1, respectively) (Fig. 2i). Specific Leucine aminopeptidase was first detected from hatching (1 dah, 0.23 ± 0.02 mU mg protein-1); it increased gradually until it reached the maximum peak of activity at 12 dah (1.4 ± 0.2 mU mg protein-1) and then decreased from this day up to the end of the larviculture at 36 dah (0.60 ± 0.03 mU mg protein-1) (Fig. 2k). For individual digestive proteases activities, a general pattern was observed with a gradual increase from the beginning of the larviculture to reach the maximum peak at 36 dah. For alkaline protease (Fig. 2b), the activity started from 20 to 50 mU larva-1, increasing to reach their maximum values at 34 and 36 dah (210 ± 3 and 225 ± 1 mU larva-1, respectively), for acid proteases (Fig. 2d) the lowest values varied from 0.1 to 0.25 mU larva-1 from hatching until 12 dah, reaching the highest values at 34 and 36 dah (3.5 ± 0.2 and 5.2 ± 0.8 mU larva-1, respectively), trypsin activity (Fig. 2f) had the lowest values from hatching until 12 dah (0.001–0.004 mU 9 10-3 larva-1), and reaching the highest values at 34 and 36 dah (0.1 ± 0.0 and 0.01 ± 0.00 mU 9 10-3 larva-1, respectively), finally, carboxypeptidase A (Fig. 2j) showed the lowest values from hatching until 25 dah (from 0.3 until 0.4 mU 9 10-3 larva-1), increasing the activity rapidly until the maximum values at 34 and 36 dah (5.9 ± 0.1 and 11.0 ± 0.04 mU 9 10-3 larva-1, respectively). Chymotrypsin individual activity showed a similar gradual increment as the specific activity from hatching until 25 dah (0.01–0.05 mU 9 10-3 larvae-1), and reaching the maximum peak at 34 dah (0.1 ± 0.0 mU 9 10-3 larva-1) and then decreased rapidly at 36 dah (0.1 ± 0.0 mU 9 10-3 larva-1) (Fig. 2h). Finally, the leucine aminopeptidase individual activity was different from the other enzyme patterns; it showed null activity from

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hatching up to day 5 after hatching, with a rapid increase at 7 dah (11.0 ± 0.1 mU 9 10-3 larva-1), reaching the maximum peak at 12 dah (14.5 ± 0.2 mU 9 10-3 larva-1); and then decreased at 25 dah (10.3 ± 0.2 mU 9 10-3 larva-1), and increasing again at 34 (13.2 ± 0.1 mU 9 10-3 larva-1) and 36 dah (12.5 ± 0.1 mU 9 10-3 larva-1) (Fig. 2i). Specific lipase activity showed slight increments at 3 (130.5 ± 0.3 U mg protein-1) and 7 dah (190.2 ± 0.3 U mg protein-1); afterward, the activity remained low (90–110 U mg protein-1) until 36 dah (480.4 ± 0.3 U mg protein-1) when it increased rapidly, being this day the peak with the maximum activity (Fig. 3a). The individual lipase activity gradually increased its activity from hatching (10 ± 1 mU 9 10-3 larva-1) until 12 dah (1.8 ± 0.1 mU larva-1), increasing its value (3.8 ± 0.9 mU larva-1) at 12 dah, reaching the maximum value at 34 dah (10.3 ± 0.12 mU larva-1), and finally decreasing rapidly at 36 dah (1.3 ± 0.1 mU larva-1) (Fig. 3b). Specific a-amylase activity was present from day 1 after hatching (0.2 ± 0.0 U mg protein-1); it increased from day 5 (0.9 ± 0.0 U mg protein-1) until day 12 after hatching (0.9 ± 0.0 U mg protein-1), reaching its maximum value at 7 dah (1.1 ± 0.0 U mg protein-1); the activity suddenly decreased from day 25 (0.1 ± 0.0 U mg protein- 1) until 34 dah (0.1 ± 0.0 U mg protein-1) and to increase slightly 36 dah (0.5 ± 0.1 U mg protein-1) (Fig. 3c). For the individual activity of a-amylase, two peaks were detected; the first one at 7 dah (8.0 ± 0.2 mU larva-1), with a gradual decrease on the activity until day 34 after hatching (2.1 ± 0.4 mU larva-1) and a rapid increase at 36 dah (18.2 ± 0.4 mU larva-1) being this the day of maximum activity (Fig. 3d). In the case of acid phosphatase-specific activity, low values were detected from hatching until day 12 (0.5 ± 0.1 U mg protein-1); however, from day 25 after hatching (3.8 ± 0.1 U mg protein-1) the activity increased, reaching the maximum peak at 34 dah (5.1 ± 0.1 U mg protein-1) (Fig. 3e). The individual acid phosphatase activity showed a similar tendency, being the maximum peaks obtained at 25 (50.5 ± 1.3 mU larva-1) and 36 dah (Fig. 3f). Finally, the specific and individual alkaline phosphatase activities (8–10 U mg protein-1 and 95–183 mU larva-1, respectively) had the same pattern with low values from


hatching until day 12 after hatching, increasing rapidly at 25 dah (78.1 ± 19.2 U mg protein-1 and 1,052.4 ± 11.8 mU larva-1, respectively) and decreasing on 34 (27.2 ± 14.8 U mg protein-1 and 253.6 ± 9.5 mU larva-1, respectively) and 36 dah (26.4 ± 9.5 U mg protein-1 and 322.6 ± 7.3 mU larva-1, respectively) (Fig. 3g, h). The zymogram for alkaline proteases showed only one band from 25 up to 36 dah (26.4 kDa), and the appearance of one additional active band of 51.6 kDa at 36 dah (Fig. 4a). The zymogram for acid proteases showed two active bands, the first band having a Rf of 0.32, and the second with a Rf of 0.51, both bands were observed at 34 and 36 dah (Fig. 4b).

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Discussion Growth Larval culture is considered the bottleneck for commercial production, especially in marine fish. For C. undecimalis, the growth rate showed an exponential value referred to wet weight and total length, which was slow during the first days of life, increasing rapidly after the day fifteen after hatching. Same growth has been observed in several species, such as Senegal sole (Solea senegalensis), California halibut (Paralichthys californicus), Atlantic cod (Gadus morhua), Atlantic halibut (Hippoglossus

Fig. 3 Digestive enzyme activity during common snook larviculture (mean ± SD, n = 3 pooled larvae). a Specific and b individual lipase activities, c specific and d individual a-amylase activities, e specific and f individual acid phosphatase activities, g specific and h individual alkaline phosphatase activities. R Rotifers, AN Artemia nauplii, EAMN Enriched Artemia metanauplii

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Fig. 4 Zymograms of acid (a) and alkaline digestive proteases (b) during larval development of common snook. Numbers at the top of the gel indicate the mean of days after hatching. The first well indicates LWMM (kDa): 97 Phosphorylase; 66 Bovine serum albumin; 45 Ovoalbumin; 29 Carbonic anhydrase; 24 Trypsinogen, 20 Trypsin soybean inhibitor

hippoglossus), and spotted sand bass (Paralabrax maculatofasciatus) (Ribeiro et al. 1999; AlvarezGonza´lez et al. 2006, 2008; Kva˚le et al. 2007), which has been related with the live food quality and quantity (mainly rotifers and Artemia nauplii), feeding frequency, weaning period, and morphophysiological changes that are related with digestion (enzymatic activity) and absorption of nutrients by enterocytes (Moyano et al. 1996). Alkaline proteases The trend observed for alkaline protease-, trypsin-, and chymotrypsin-specific activities during the first days of larviculture is similar to that of other carnivorous fishes like S. lalandi (Chen et al. 2006), P. californicus (Alvarez-Gonza´lez et al. 2006), red drum, Sciaenops ocellatus (Lazo et al. 2007), and P. maculatofasciatus (Alvarez-Gonza´lez et al. 2008a), which suggest that these enzymes play an important

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role in larval digestion, previous to the stomach functionality (Moyano et al. 1996). These changes in the alkaline enzymatic activity agree with Cahu and Zambonino-Infante (1994), who observed that pancreatic enzymes are responsible for most protein hydrolysis in the gut, which is initiated in the luminal zone, and then is continued in the parietal cell epithelium (microvilli), before the absorption of nutrients by enterocytes. These variations in the alkaline protease activities during C. undecimalis larviculture could be associated to morphophysiological changes and co-feeding process when rotifers were replaced by Artemia nauplii. These changes may increase the secretion of pancreatic zymogens, possibly influenced by the type and quantity of protein of the feed; also, the first feeding after yolk absorption may induce the activation of alkaline proteases such as chymotrypsin that occurred at 3 dah, which has been detected as the first alkaline exoprotease that acts in the digestive tract (Hjelmeland et al. 1983). In contrast, the level of trypsin in our species was detected early at low level and increased rapidly at 12 dah. This enzyme has also been early detected in Senegal sole (Solea senegalensis) and P. olivaceus larvae, even before the first feeding. Indeed, this enzyme is detected before the mouth opening, and it is related to the activation of other pancreatic zymogens and the protein hydrolysis in the lumen of S. senegalensis and P. olivaceus (Kurokawa and Suzuki 1996; Saénz et al. 2005). The differences between trypsin and chymotrypsin activities observed in C. undecimalis larvae could be considered as an indicator of nutritional status; for example, under normal conditions, the proportion of trypsin increases as a response to protein hydrolysis demand; however, if the larvae is reared with an inadequate diet or suffers a feeding restriction, this proportion decreases because relatively less trypsin is produced against a constant secretion of chymotrypsin (Moyano et al. 1996; Cara et al. 2003). For carboxipeptidase A in C. undecimalis larvae, low activity was detected from the embryos until 5 dah, increasing from 7 to 12 dah, decreasing at 25 dah, and reaching its maximum values at 34 and 36 dah. This enzyme has been primarily studied on adults of common carp, Cyprinus carpio (Cohen et al. 1981), amur catfish, Parasilurus asotus (Yoshinaka et al. 1985), S. maximus (Munilla-Morań and Stark 1990), and small-spotted catshark, Scyliorhinus


canicula (Hajjou et al. 1995), it is an important digestive parietal enzyme that depends on Zn (metallo-protease), and it is produced in the acinar pancreatic cells to hydrolyze peptides from carboxyl side (Vendrell et al. 2000). In P. olivaceus larvae has only been reported procarboxypetidase A as the major pancreatic enzyme, precursor for peptide digestion in the intestinal lumen and is synthesized at the first feeding (Srivastava et al. 2002). Leucine aminopeptidase activity was detected with low values in embryos, increasing between 2 and 12 dah, after that, decreasing was observed until the end of the larviculture. This pattern coincides with turbot, Scophthalmus maximus (Cousin et al. 1987) and European sea bass, Dicentrarchus labrax (Cahu and Zambonino-Infante 1997), where these parietal enzymes were localized on microvilli of the enterocyte as proenzymes and then activated by alkaline proteases (trypsin and chymotrypsin) to hydrolyze peptides from the N-terminal amino acid. Acid proteases (pepsin) In C. undecimalis larvae, the maximum activity of acidic protease was observed between 25 and 36 dah, similarly to what is observed in other marine fish larvae such as white bream (Diplodus sargus), common dentex (Dentex dentex), and redbanded seabream (Pagrus auriga) (Alarcon et al. 1998; Cara et al. 2003; Moyano et al. 2005). It has been reported that the appearance of pepsinogen like activity in the bastard halibut Paralichthys olivaceus larvae at 45 dah (Kurokawa and Suzuki 1996) indicates the starting of the juvenile stage and the settlement period. Detection of high peaks of acid protease activity (pepsin) indicates the presence of a functional stomach (gastric cells and acid chloride secretion) and could be taken, with other digestive enzymes, as an indicator of maturation of the digestive system (Ueberschaër 1993; Baglole et al. 1998; Kva˚le et al. 2007). In this sense, the change from an undifferentiated straight tube in larvae to a differentiated gastrointestinal tract, including the presence on gastric cells, allows to start the weaning period (Lazo et al. 2007). However, it is adequate to complement the information of enzymatic activity (biochemistry techniques) with the presence of gastric cells through histology, as was done with the D. dentex (Gisbert et al. 2009). On the other hand,

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low activity pepsin activity was detected during the first days of larviculture (from yolk-sac larvae until 12 dah) and is not necessarily from stomach origin, but it could be another type of hydrolases (cathepsines), which work at intracellular level under acidic conditions and could be detected when whole-body extracts are prepared (Moyano et al. 1996). Lipase Lipase activity was detected at 3 dah, increasing rapidly at 36 dah, in accordance to Green and McCormick (2001) who suggested that the presence of these digestive enzymes before hatching is strongly related with the absorption of nutritional components of the yolk sac. Specifically for lipase activity, diverse studies have been conducted through larval development of marine fish, where the activity shows two high peeks in a recurrent manner: the first one occurs at early days of life related to lipid hydrolysis of the yolk, and the second one when the digestive system maturation was reached (Oozeki and Baley 1995). However, these peeks could present fluctuations, which are strictly related to either changes in feed supplies and feed enrichment with lipid emulsifiers (Hoehne-Reitan et al. 2001), as it was observed in S. senegalensis larvae (Martıńez et al. 1999), in which two peeks of maximum activity were detected at 7 and 36 dah. It is admitted that lipid catabolism is performed primarily by esterase action hydrolyzing fatty acids as energy source in the first days of life, while true lipase is dependant on colipase and bile salts, acting over phospholipids and triacylglycerols (van Tilbeurgh et al. 1992); this enzyme is responsible for releasing highly polyunsaturated fatty acids and other more complex compounds, generally observed when maturation of the digestive system arrives (Ribeiro et al. 1999; Zambonino-Infante and Cahu 1999; Gawlicka et al. 2000; Sidell and Hazel 2002; Murray et al. 2003; Morais et al. 2005; Gisbert et al. 2009) Amylase Regarding a-amylase activity during ontogeny of C. undecimalis, maximum values were detected between 5 and 12 dah, and later decreased, in agreement with to earlier observations for other marine fish species, where the highest activity was reported before

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hatching and during the first days of life when the absorption of yolk-sac occurred and was followed by a subsequent reduction in activity (Cahu and Zambonino-Infante 1994; Zambonino-Infante and Cahu 2001). a-amylase activity and expression have been detected in fish fertilized eggs by biochemical or molecular approaches (Naz 2008; Darias et al. 2006) though its function has not been understood at early stages of fish development (Moyano et al. 1996; Pe´res et al. 1996; Martıńez et al. 1999; Buchet et al. 2000; Cuvier-Pe´res and Kestemont 2002). In larvae, it has been considered as a digestive system maturation indicator as it occurs in mammals with lactase (Zambonino-Infante and Cahu 1994). Fish larvae tend to maintain a-amylase at low levels of activity to utilize carbohydrates in the feed in carnivorous species (Munilla-Morań and Saborido-Rey 1996; Alvarez-Gonza´lez et al. 2008). Fange and Grove (1979), Ugolev et al. (1983), and Hidalgo et al. (1999) have proved that a-amylase activity tends to be higher with a progressive activity increasing in herbivorous and omnivorous fish when compared to carnivorous fish. Phosphatases On the other hand, parietal digestive enzymes, such as the brush border enzyme phosphatases, are very important because they are responsible for concluding digestion at intestinal epithelium level besides helping on hydrolyzed nutrient transport into the enterocytes (Harpaz and Uni 1999; Smith et al. 2000; Zambonino-Infante and Cahu 2001). Alvarez-Gonza´lez et al. (2008) reported that phosphatases have two main functions: the first one is to hydrolyze inorganic phosphate used for energy production, and the second one is to transport nutrients through cell membranes (absorption process). In this sense, Gawlicka et al. (2000) reported that when enterocytes reach their maximum hydrolysis and absorption capacity, phosphatase activity increases, which is related to a drop in leucine-alanine peptidase activity and maturation of enterocytes, as it has been detected in D. labrax larvae (Pe´res et al. 1997) and consequently determines the most adequate moment to carry on the substitution of live prey to artificial feeds (Ribeiro et al. 2002; Zambonino-Infante and Cahu 2007). In this manner, phosphatases act in the digestion process facilitating other enzyme actions,

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besides initiating the migration of nutrient processes from the cryptic region to the microvilli border to promote cell absorption (Copeland 1996). Zymogram analysis Zymogram of acid protease activity in C. undecimalis larvae allowed the detection of two isoforms (0.32 and 0.51 Rf’s) up to 34 dah, which is similar in juveniles as reported by Concha-Frıás (2008); this author found that the total acid protease activity was 86% inhibited using pepstatin A in the stomach of C. undecimalis juveniles. In this sense, pepsin activity detection through this technique has been reported for other species such as silk snapper, Lutjanus vivanus (26.1 kDa active fraction); grunt, Haemulon plumierii (24 kDa active fraction); spotted goatfish, Pseudupeneus maculatus (24.0 kDa active fraction); P. maculatofasciatus (one active fraction on 12 dah, 0.65 RF) (Rivera 2003; Rodrı´guez 2004; Souza et al. 2007; Alvarez-Gonza´lez et al. 2010) in agreement that these detected enzymes are similar to the swine pepsin A (35 kDa). However, the detection of a second acid protease isoform in C. undecimalis larvae could correspond not only to pepsin A, but also to pepsin C that was reported for Pacific bluefin tuna Thunnus orientalis juveniles using a molecular approach (Tanji et al. 2009). For alkaline protease, zymogram of C. undecimalis larvae showed only two isoforms, which apparently correspond to trypsin-like (51.6 kDa) detected at 36 dah, and chymotrypsin-like (26.4 kDa) detected at 34 and 36 dah. In this sense, an early detection using electrophoretical technique of these isoforms was not possible because of the low activity in whole-body homogenates until 25 dah, which was corroborated using biochemical technique. The molecular masses of active fractions found for C. undecimalis larvae were similar to those detected in wild juveniles of the same species (11 cm of total length) by Concha-Frıás (2008); however, this researcher found a third high molecular mass isoform ([75 kDa), which could correspond to an aminopeptidase. Our results are similar to those reported for other strictly carnivorous marine fish species such as the coho salmon (Oncorhynchus kisutch) and chinook salmon (Oncorhynchus tschawytscha) with two 22 kDa isoforms, Northern bluefin tuna (Thunnus thynnus) with three isoforms from 16.8 to 26.8 kDa and spotted sand bass (P. maculatofasciatus) with


two isoforms of 20.1–56.5 kDa, respectively. They are different from omnivorous fishes such as S. aurata (five active fractions ranging 24.5–90 kDa), D. dentex (eight fractions ranging from 24.5 to 69.5 kDa), pirapita´, Brycon orbignyanus (nine fractions ranging from 7 to 70 kDa), blue disk, Symphysodon aequifasciata (eight fractions ranging from 19.2 to76.5 kDa), and rohu, Labeo rohita (five fractions ranging from 20.9 to 69.4 kDa) (Dimes et al. 1994; Alarcon et al. 1998; Garcıá-Carrenõ et al. 2002; Chong et al. 2002; Essed et al. 2002; Chakrabarti et al. 2006; Alvarez-Gonza´lez et al. 2010). We can conclude that the low activities of alkaline protease, lipase, a-amylase, and phosphatase activity detected in C. undecimalis larvae at hatching and their increment throughout larviculture also the presence of only two isoforms of alkaline protease may be indicators of (1) genetic processes, specially while observing a slight specific activity in starving larvae during yolk absorption (for example, phospholipids hydrolysis); (2) a response to ingestion (feeding behavior), at the moment of adding live feeds (rotifers, Artemia and enriched Artemia metanauplii); and (3) progressive disappearance of the metabolic function in which a specific enzyme is involved (lactase in the case of mammals), or a relative increase in the soluble protein pool in the organism. Additionally, acidic protease activity, which starts at 25 dah reaching its maximum activity at 36 dah, allows us to consider C. undecimalis as a juvenile focusing on a digestive physiology point of view from 34 dah onwards, and let us situate this species as a strictly carnivorous fish larvae, also the weaning period for this species should be started after this age. Acknowledgments This work was made possible thanks to the Project ‘‘Estudio sobre la fisiologıá digestiva del robalo blanco Centropomus undecimalis’’ SEP-CONACyT (CB-20061-58931). We thank Claudia Durruty Lagunes and Jaime Sua´rez Bautista for their technical assistance. The Consejo Nacional de Ciencia y Tecnologıá (CONACYT) of Mexico provided a fellowship grant to the first author Luis Daniel Jimeńez-Martıńez.

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