Activity of digestive enzymes along the gut of juvenile red abalone, Haliotis rufescens, fed natural and balanced diets

Aquaculture 261 (2006) 615 – 625 www.elsevier.com/locate/aqua-online

Activity of digestive enzymes along the gut of juvenile red abalone, Haliotis rufescens, fed natural and balanced diets Zaul Garcia-Esquivel a,⁎, Horst Felbeck b a

Universidad Autónoma de Baja California, Instituto de Investigaciones Oceanológicas, Km. 107 carretera Tijuana-Ensenada, Ensenada, B.C., México b Scripps Institution of Oceanography, UCSD, 9565 Gilman Drive, La Jolla, CA 92109-0202, United States Accepted 18 August 2006

Abstract Two carbohydrases (cellulase, lysozyme), three proteases (trypsin, aminopeptidase and non-specific protease), a non-specific lipase, and semiquantitative tests of 19 digestive enzymes were assayed in different gut sections of juvenile red abalone, Haliotis rufescens, in order to identify the regions where digestion takes place and investigate the extent to which diet composition can modify the digestive capacity of abalone. The abalone were fed either fresh kelp (K) or balanced diets containing 25 or 38% crude protein for 6 months. Enzyme assays were carried out on different sections of the abalone's gut at the end of this period. On a weight-specific basis, the digestive gland was the site containing most of the enzymes. On a protein-specific basis, two main digestion regions were identified: the digestive gland-stomach region that is characterized by high activities of cellulase and lysozyme, chymotrypsin and protease, and the mouth-intestine region with a typically high activity of lipase and amino peptidase. Significant dietary effects were observed on the activity of enzymes, especially in the digestive gland. Abalone fed with 25 and 38% crude protein diets exhibited higher cellulase (39.8 ± 4.6 and 14.2 ± 0.8 mU mg− 1 protein, respectively) and lysozyme activities (88.0 ± 20.4 and 56.6 ± 15.7 U, respectively) than those fed with fresh kelp (5.5 ± 0.7 mU mg− 1 protein and 17.1± 1.8 U). In contrast, higher protease activity was found in kelp-fed organisms (234.1 ± 20.4 μg product/mg protein) than those fed the 25 and 38% crude protein diets (109.5 ± 20.7 and 119.5 ± 20.5 μg product/mg protein, respectively). Semiquantitative API ZYM assays resulted in no clear food-specific effects on the activity of carbohydrases, proteases, ester hydrolases or phosphohydrolases, yet organ-specific differences were conspicuous in various cases, and generally agreed with quantitative results. It is suggested that the increased carbohydrase activity exhibited by organisms fed the balanced diets resulted from a combination of an increased number of resident bacteria in the abalone's gut and facilitated contact between dietary substrates and digestive cells. The present results indicate that H. rufescens can adjust their enzyme levels in order to maximize the acquisition of dietary protein and carbohydrates. This characteristic can be advantageously used to search for suitable diets in abalone aquaculture. Published by Elsevier B.V. Keywords: Abalone; H. rufescens; Digestive enzymes; Juvenile abalone; Inert diet; Feed

1. Introduction

⁎ Corresponding author. Tel.: +52 646 174 4601; fax: +52 646 174 5303. E-mail address: [email protected] (Z. Garcia-Esquivel). 0044-8486/$ - see front matter. Published by Elsevier B.V. doi:10.1016/j.aquaculture.2006.08.022

Significant efforts have been recently applied to the study of nutritional aspects of abalone (e.g. Fleming et al., 1996; Takami and Kawamura, 2003), an economically important group whose fishery has been dramatically declining over the last decade (Gordon and Cook, 2004).

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The red abalone, Haliotis rufescens is the most abundant species in California (Rogers-Bennett et al., 2004), and its distribution extends to the Baja California coast in Mexico. H. rufescens typically live around kelp beds, and feeds on micro and macroalgae as its main source of food. This behavior has been used to culture organisms using natural kelp as the sole source of food (e.g. McBride, 1998; Zhang et al., 2004). Although abalone farming is primarily based on fresh algal foods, experimental evidence indicates that juvenile abalone can eat pelleted feed and grow at a higher rate when compared to kelp food (Viana et al., 1996; Lee, 2004). Pelleted feed has been used to quantify dietary requirements of protein and energy in abalone based on physiological budgets and ration-response experiments (reviewed by Fleming et al., 1996, see also GómezMontes et al., 2003). Parallel studies have also focused on the functional morphology of the abalone's gut (Cui et al., 2001) and early ontogenetic appearance of digestive enzymes (reviewed in Takami and Kawamura, 2003), in order to understand the digestive capacity of this group. The digestive potential of an organism not only depends on the composition of the diet, but also on the innate capacity of an organism to digest such a diet with endogenous and/or exogenous enzymes. Functional neuromorphological characteristics help explaining the feeding nature of gastropods (reviewed by Elliott and Susswein, 2002). Quantitative information on the activity of hydrolytic enzymes is also a useful tool for understanding the dietary preference (Boetius and Felbeck, 1995) and digestive capacity of an organism. For example, studies carried out on several lobster species indicate that carnivorous species typically exhibit high protease and low carbohydrase activity when compared to herviborous species, while omnivorous species show intermediate activity levels (Jones et al., 1997). Juvenile/adult stages of abalone typically exhibit significant activity of a range of proteases, including trypsin, chymotrypsin, non-specific protease and several aminopeptidases (Edwards and Condon, 2001; GarciaCarreño et al., 2003), thus suggesting that abalone have the capacity to digest diets with high protein content. Trypsin is expressed in abalone as early as the premetamorphic stage (Degnan et al., 1995), but quantitative assessment of protease activity is lacking in early postlarval stages. Significant activity of several carbohydrases was reported as early as the first month postsettlement in the Ezo abalone, Haliotis discus hannai (Takami et al., 1998), while activity of complex carbohydrases, lipases and proteases has been reported in several juvenile–adult species of abalone (e.g., Erasmus et al., 1997; see Picos-García et al., 2000, Table 1).

Table 1 Dietary treatments and ingredient composition (% dry weight) of experimental live diet (kelp) and balanced diets formulated with 38% crude protein (P38) and 25% crude protein (P25) Ingredients

P38

P25

Kelp

Fish meal Soybean protein isolateb Kelp mealc Menhaden fish oild Corn oile Modified corn starchf Common ingredientsg

24.70 15.22 22.05 2.69 0.38 26.10 14.51

14.10 8.70 16.70 3.61 0.51 47.5 14.51

– – – – – – –

Composition Dry matter (%) Crude protein (% dry wt) Ash (% dry wt) Energy (kcal/g dry wt)

94.91 ± 0.01 38.18 ± 0.12 20.28 ± 0.08 4.20 ± 0.06

94.98 ± 0.01 10.26 ± 0.11 25.46 ± 0.50 9.49 ± 0.07 15.99 ± 0.07 40.88 ± 7.93 4.09 ± 0.02 2.63 ± 0.01

a

a

Pre-cooked white fish (92% protein). ICN Biomedicals Inc. c Macrocystis pyrifera, kindly provided by Productos del Pacifico, S.A. de C.V. d Sigma cat. #F-8020. e Sigma cat. #C-8267. f Smart and Final®. g Vitamin mixture, 1.5 (ICN Biomedical, cat. # 904654); Stay C-35, 0.04 (DSM Nutritional Products Inc.); mineral mixture, 3.3 (ICN Biomedicals, Inc. cat.# 905455); D L-Methionine, 0.23 (Sigma, cat.# M-9500); benzoic acid, 0.23 (Sigma, cat.# B-3375); choline chloride, 0.11 (Sigma, cat.# C-7527); BTH, 0.09 (Sigma,# B-1378); αTocopherol, 0.01 (Sigma, cat # T-3634); acid fish silage from tuna viscera 3.0; gelatin (275 bloom, Duche). b

Recent feeding experiments with pelleted diets containing different protein/energy ratios showed that juvenile green abalone (H. fulgens) can grow faster with increasing levels of dietary fish/soybean protein than with fresh kelp (Gómez-Montes et al., 2003), thus suggesting that abalone possess certain digestive plasticity that can be modulated with diet. Garcia-Carreño et al. (2003) documented that various algal species and seagrass used as the sole source of food quantitatively affected the activity of digestive proteases in juvenile green abalone, H. fulgens. Detailed histological studies indicate that H. discus hannai contain five types of cells along the gut, including secretory cells that produce some proteases, lipases and carbohydrases (Cui et al., 2001), yet no comparable quantitative study exists in terms of the spatial distribution of digestive enzymes along the abalone's gut. With one exception (Edwards and Condon, 2001), most work on digestive enzymes of abalone has focused on the most conspicuous organ present in the abalone's gut, the digestive gland. It has been shown that carbohydrases are produced by both abalone and resident bacteria located in the gut (Erasmus et al., 1997; Kusumoto et al., 1997). It has also been

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demonstrated that abalone contain a range of proteases whose activity is quantitatively affected by the type of algal feed, but to date there are no comparative studies on the digestive response of abalone to dietary sources other than algae. The spectrum of hydrolytic enzymes present in an organism and the magnitude of their catalytic activity not only provide information on its feeding strategy (Boetius and Felbeck, 1995), but it is also indicative of the relative importance of each component of the diet (Johnston, 2003) and the plasticity of an organism when faced with changes in diet quality. In the present study, a two factor experimental design was used in juvenile red abalone, H. rufescens, in order to investigate the effect of diet composition (fresh kelp, low and high protein diets based on soybean/fish protein sources) on the digestive enzymes along the abalone's gut. Quantitative determination of the activity of digestive carbohydrases, lipases, and proteases can provide information on the degree of plasticity of the abalone's gut to different dietary conditions, while semi-quantitative screening of 19 enzymes can be used to gain a better understanding of the digestive potential of abalone. 2. Materials and methods 2.1. Organisms and experimental setup Abalone (H. rufescens) with an initial mean size of 4.2 ± 0.01 cm (mean ± SEM) were obtained from a local hatchery (The Cultured Abalone, Goleta, CA) and transported to the aquarium of Scripps Institution of Oceanography on November 2003, where they remained for two months under flow-through conditions with a mixture of cold and ambient water (average temperature 15.2 ± 0.1 °C), and feeding on fresh kelp (Macrocystis pyrifera). After this period, a group of 120 organisms (4.6 ± 0.02 cm) were separated from the tank, individually marked with water-proof paper tags, measured, and randomly distributed to growth chambers (10 organisms per chamber) located at a sea table. Growth chambers have been described elsewhere (GomezMontes et al., 2003). Briefly, they consisted of a black ABS pipe (20 cm diameter × 20 cm height) fitted with 1 mm mosquito mesh at 3 cm from the bottom. Chambers were held inside aerated 5l buckets and kept under flow-through conditions (300 ml min− 1) with constant air, and a photoperiod of ca. 12:12 light:dark. Three dietary treatments with four replicates consisting of fresh kelp (K), balanced low-protein diet (P25), and balanced high-protein diet (P38) were used in the experiment, for a total of 12 experimental buckets. High and low protein diets were prepared according to Gomez-

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Montes et al. (2003) using the ingredients listed in Table 1, while kelp was weekly collected from a near shore kelp bed, kept in a reservoir tank at ca. 12 °C, and harvested as needed. Food was daily provided in excess (ca. 4% of total abalone weight) throughout a six-month experimental period at 20:00 h. Any remaining feed was removed from the chambers at 08:00 h the following day. Buckets/chambers were cleaned daily with seawater and a soft cleaning pad. Monthly growth measurements were carried out throughout the experiment. Sampling, dissection, and evaluation of enzyme activities were carried out every week during the last month. 2.2. Sampling, dissection and preparation of crude extracts One organism per treatment was randomly collected every week during the last month of the experiment, except in the last week, where three organisms per treatment were collected. Organisms were measured, weighed, and transferred to a − 20 °C freezer for 20 to 30 min. After this period, organisms generally showed no tactile response and were considered dead. The shell was separated from the flesh, blot dried and weighed. The flesh was transferred to ice-cold seawater and the gut sections sequentially separated under a dissecting microscope according to the following order: digestive gland (DG), rectum (R), mouth-oesophagus NNN(M–O) (MO), intestine (I), and stomach (S). Fresh seawater was squeezed through each dissected organ to remove of any remaining food, and the excised tissue was then transferred to a cold Eppendorf microtube, where it remained at − 20 °C until ready for enzyme analyses. Gut sections were identified following the criteria of McLean (1970) and Erasmus et al. (1997). Between four and five replicates per treatment were used for quantitative assays of enzyme activity. Dissected sections of mouth-oesophagus, stomach, and digestive gland were individually assayed, while sections of rectum and intestine had to be pooled from at least three organisms in order to get enough material for quantitative assays. For this reason, data for MO, S and DG had four replicates, while those of R and I consisted of one and two replicates (respectively) of pooled tissues. Between three and six additional organisms per treatment were also dissected and used for semi-quantitative API ZYM enzyme assays during the last week of the experiment. 2.3. Quantitative assays Enzyme assays were carried during the same week that the abalone were dissected. About 200 mg tissue

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(wet wt) were excised with a scalpel, weighed, and manually ground in a glass Potter tissue homogenizer with 1.5 ml of ice-cold 10 mM Tris HCl, pH 7.5, until no tissue parts could be observed. The homogenate was transferred to 1.5 ml microcentrifuge tubes and spun for 10 min at 4 °C and 12,240 ×g in an IEC refrigerated microcentrifuge. The resulting supernatant was kept chilled and was used for protein quantitation and enzyme assays within 1 to 5 h of being prepared. Protein content of crude extracts was measured by the bincinchoninic acid method (SIGMA product code BCA-1 and B9643) using bovine serum albumin as standard. Lysozyme activity was determined according to the method of Weisner (1984), using a substrate of lyophilized Micrococcus luteus at a final concentration of 0.2 mg ml− 1. The reaction buffer consisted of fresh phosphate/NaCl/azide buffer (67 mM phosphate, 15.4 mM NaCl, 8 mM Na-azide, pH 6.3) and 50 to 100 μl of homogenate in a final volume of 1.525 ml. The decrease of absorbance in the reaction mixture was continuously followed at 546 nm for 3 min at room temperature (23 to 25 °C), and the activity of the enzyme is expressed in units mg− 1 protein. One unit is defined in the sense of Weisner (1984) as the amount of enzyme which causes a change in absorbance (ΔA) of 0.001 min− 1. Endocellulase activity was determined with the MEGAZYME kit (Megazyme Intl. Ireland Ltd, Ireland). Briefly, the reaction buffer consisted of 50–100 μl of homogenate and 25 mM Na-acetate (pH 4.5) in a final volume of 500 μl. The mixture was pre-equilibrated for 5 min at 40 °C and the reaction started with the addition of a tablet of Cellazyme C (azurine-crosslinked HE-cellulose). The reaction was stopped after 10 min with 2% Trizma base, and the slurry was passed through a Whatman No. 1 paper filter. The filtrate was read against a blank at 590 nm and the activity of cellulase is expressed in mU mg− 1 protein, using a standard curve of absorbance vs. mU. Non-specific lipase activity (EC 3.1.1.-) was determined according to Albro et al. (1985) and Ito et al. (2002). Briefly, the reaction mixture was prepared in a final volume of 1.1 ml and consisted of 100 mM Tris-HCl, pH 8.0, 10 mM deoxycholate, 6 Mm dithiotreitol (DTT) and 0.5 mM 4-p-nitrophenyl myristate. The stock solution of the latter was prepared according to Albro et al. (1985) using ethylene glycol monomethyl ether as a solvent. The increase in absorbance was continuously followed at 400 nm for 3 min to correct for non-lipase activity and reaction started with the addition of 50 to 100 μl of homogenate. Protease was assayed at 37 °C according to Head et al. (1984) with azocasein as substrate, and using

proper blanks to account for turbidity and background absorbance at 430 nm. Reaction mixture consisted of (final concentration) 400 mM Na-acetate, pH 5.5, 12.5 mM CaCl2, 125 mM NaCl, 1 mM EDTA and 0.4% azocasein. Stop solution was 1.5 mM trichloracetic acid. Azocasein digested with Streptomyces griseus protease was used to generate a calibration curve, and estimate protease activity in μg product h− 1 mg− 1 protein. Chymotrypsin was assayed in a total volume of 1.1 ml with the method of Garcia-Carreño et al. (2003), using a final concentration of 50 mM Tris-HCl /22 mM CaCl2 buffer, pH 7.5 and 0.1 mM SAPNA (succinylAla-Ala-Pro-Phe-p-nitroanilide). Activity was continuously recorded during 3 min at 410 nm and expressed in units mg− 1 protein, using a molar extinction coefficient of 8800 M− 1 cm− 1 for 4-nitroaniline. Aminopeptidase was assayed at 405 nm following the release of nitroaniline (NA) with the method of Boetius and Felbeck (1995). Reaction mixture consisted of 33.3 mM Tris-HCl, pH 8.01 mM, 1.67 mM MnCl2, 1 mM L-leucine nitroanilide-HCl, and 50 μl of tissue extract in a total volume of 250 μl. From the reaction solution 125 μl were withdrawn and immediately incubated for 15 min at 37 °C. After this period a 125 μl aliquot was withdrawn, mixed with one ml of distilled water, and the release of NA (nmol h− 1) was followed spectrophotometrically at 405 nm. Enzyme activity is expressed in mU mg− 1 protein, using nitroaniline as standard. 2.4. Api Zym tests Crude abalone tissues were homogenized with saline (0.85% NaCl in water) at ca. 1:10 w/v ratio. The extracts were further diluted to a protein content of 1 mg ml− 1, and 65 μl of the extract was added to each microcupule on the test trays. Nineteen digestive enzymes were tested semiquantitatively using the ApyZym system (bioMérieux Inc., Hazelwood, MI): acid phosphatase, naphthol-AS-BI-phosphohydrolase, α-galactosidase, βgalactosidase, β-glucoronidase, α-galactosidase, N-acetyl-β-glucosaminidase, α-mannosidase and α-fucosidase at pH 5.4; esterase (C4) at pH 6.5; esterase/lipase (C8), lipase, leucine arylamidase, valine arylamidase, cystine arylamidase and α-chymotrypsin at pH 7.5; alkaline phophatase and trypsin at pH 8.5. The trays were incubated for 4 h at 37–38 °C and the color developed by the reaction was revealed with the Zym A and Zym B reagents, following the recommended protocol. An arbitrary scale from 1 to 5 was used to score the intensity of the reaction for each enzyme tested.

Z. Garcia-Esquivel, H. Felbeck / Aquaculture 261 (2006) 615–625 Table 2 Initial and final (6 months) mean size and live weight (±SEM) of juvenile Haliotis fulgens, fed live kelp (kelp) or balanced diets with 38% (38P) or 25% (25P) crude protein content Treatment

Size (cm)

Weight (g)

Initial

Final

Initial

Final

38P 22P Kelp

4.62 ± 0.01 4.67 ± 0.05 4.65 ± 0.05

5.70 ± 0.06a 5.44 ± 0.02b 4.97 ± 0.10c

15.4 ± 0.18 16.1 ± 0.63 15.8 ± 0.75

30.9 ± 1.20a 26.7 ± 0.16b 19.7 ± 1.03c

Treatments with different superindices indicate significant differences at P b 0.05.

2.5. Statistics Organ, feed, and organ × gut interaction effects were tested with a two-way analysis of variance (two-way ANOVA) for each of the enzymes quantitatively assayed. When significant effects were found, a further Tukey test for multiple comparisons of means was carried out in order to identify those treatments with significant differences. All statistical tests were carried out in a personal computer using the statistical package SigmaStat 2.0 (Jandel Scientific, Chicago, IL).

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did not significantly affect the amount of soluble proteins. The weight-specific activity of enzymes followed the same trend of the proteins, as shown in the case of lipase (Fig. 2a). However, when expressed on a proteinspecific basis this trend was modified, especially in the digestive gland (Fig. 2b). Because the wet weight of the tissues was heterogeneous after blot-drying, it was decided to standardize the activity of enzymes on a protein-specific basis. Lipase activity was markedly different among different sections of the abalone gut (two-way ANOVA, P b 0.001), but it was not significantly affected by the type of food (two-way ANOVA, P = 0.814). Lipase activity was highest at the abalone's mouth (1.4 to 2.4 mU mg− 1 protein) and decreased towards the digestive gland and rectum, reaching values of b 80 nmol product mg− 1 protein in these tissues (Fig. 2b). The gradient was statistically significant in the organs tested, according to the following descendent order: M N S N DG (Tukey test, P b 0.05). The spatial distribution of cellulase and lysozyme activities showed an opposite distribution pattern to that of lipase (Fig. 3a, b), but only cellulase showed significant organ-dependent differences (two-way ANOVA,

3. Results Dietary effects were significantly reflected in the final size and weight of experimental abalone (ANOVA, P b 0.001), according to the following descending order: 38P N 22P N K (Table 2). The mean weight-specific content of soluble proteins (Fig. 1) was significantly influenced by the type of organ (two-way ANOVA, P b 0.001), with higher content in the digestive gland (100 to120 mg g− 1 wet tissue weight) than the rest of the organs (ca. 20 to 40 mg− 1 wet tissue weight). Feed type

Fig. 1. Weight-specific soluble protein content in different gut sections of juvenile red abalone, Haliotis fulgens, fed live kelp (kelp) or balanced diets with 38% or 25% crude protein content. MO = mouth– oesophagus, I = intestine, S = stomach, DG = digestive gland, R = rectum.

Fig. 2. Weight-specific (a) or protein-specific (b) lipase activity in different gut sections of juvenile red abalone, Haliotis fulgens, after being fed for six months with live kelp (Macrocystis pyrifera) or balanced diets with 38% or 25% crude protein content. MO = mouth–oesophagus, I = intestine, S = stomach, DG = digestive gland, R = rectum.

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in the kelp and P25 feed treatments confirmed (Tukey tests, P b 0.05) that protease activity was significantly higher at the DG (mean = 234.1 and 109.5 μg prod h− 1 mg− 1 protein, respectively) than the S–M sections (mean range 33.7–52.8 μg prod h− 1 mg− 1 protein). Chymotrypsin activity was generally low and exhibited the highest variability among all enzyme systems tested (Fig. 4b). Its activity was only

Fig. 3. Cellulase (a) and lysozyme activities in different gut sections of juvenile red abalone, Haliotis fulgens, after being fed for six months with live kelp (Macrocystis pyrifera) or balanced diets with 38% or 25% crude protein content. MO = mouth–oesophagus, I = intestine, S = stomach, DG = digestive gland, R = rectum.

P b 0.001), with highest activities in DG, followed by S and M (Fig. 3a). Feed effects were significant in both cellulase and lysozyme activities (two-way ANOVA, P b 0.001). Abalone fed kelp exhibited significantly lower cellulase and lysozyme activities (Tukey test, P b 0.05) than those fed the balanced diets. In addition, cellulase activity was also affected by the interaction between feed and gut section (two-way ANOVA, P b 0.001), such that its activity at the abalone's mouth was not significantly affected by the type of food, while its activity at the DG followed a significant gradient (Fig. 3a) according to the following order: P38N P25 N Kelp (Tukey test, P b 0.05). Specific lysozyme activity was ca. three orders of magnitude higher than cellulase activity (Fig. 3a, b). Two-way ANOVA tests indicated significant organ (P b 0.001), feed (P b 0.005) and interaction effects (P b 0.001) on the activity of non-specific protease. Overall, highest mean activity was detected in the DG of kelpfeeding abalone (Fig. 4a). Multiple comparisons of means within the digestive gland showed that abalone fed kelp exhibited the highest enzyme activity (Tukey test, P b 0.05). Likewise, multiple comparisons of means with-

Fig. 4. Protease (a), amino peptidase (b) and chymotrypsin (c) activities in different gut sections of juvenile red abalone, Haliotis fulgens, after being fed for six months with live kelp (Macrocystis pyrifera) or balanced diets with 38% or 25% crude protein content. MO = mouth–oesophagus, I = intestine, S = stomach, DG = digestive gland, R = rectum.

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Table 3 Results of API ZYM tests for glycosidases, carried out on homogenates of different gut sections of juvenile red abalone, Haliotis rufescens Enzyme

Glycosidases α-galactosidase β-galactosidase β-glucoronidase α-glucosidase β-glucosidase N-acetyl-βglucosaminidase α-mannosidase α-fucosidase

Mouth

Foregut

Stomach

Dig. Gland

K

22P

38P

K

22P

38P

K

22P

38P

K

22P

38P

0 4 3 0 3 3

0 3 5 0 3 4

0 3 4 0 3 3

0 4 5 3 2 5

0 4 4 1 0 5

0 4 4 2 1 5

1 4 5 3 1 4

1 4 5 2 1 4

0 4 4 2 + 5

2 5 5 2 2 4

1 5 5 2 3 5

1 5 5 2 3 5

2 5

2 5

2 5

3 5

2 4

3 5

3 4

3 4

3 4

4 5

4 5

4 5

Abalone had been feeding natural (kelp, K) or balanced diets prepared with 25% protein (P25) and 38% protein (P38). Homogenate concentration was adjusted to 1 mg protein in all cases. Numbers indicate the relative magnitude of enzyme activity. + = trace activity.

statistically affected by the spatial (organ) distribution (two-way ANOVA, P = 0.033), such that the stomachdigestive gland region exhibited significantly higher mean chymotrypsin activity (0.77–1.03 U mg − 1 protein) than the mouth (0.15 U mg− 1 protein) (Tukey test, P b 0.05). Trypsin activity was barely detected in the foregut and rectum (Fig. 4b). Finally, peptidase activity showed the same spatial trend described above for lipase, with high values at the beginning of the gut (mouth-foregut) and low values at the distal end (Fig. 4c). No significant food-dependent (two-way ANOVA, P = 0.463) or interaction effects (two-way ANOVA, P = 0.063) were observed in the activity of this enzyme, yet the spatial gradient of mean aminopeptidase activity was statistically significant (Tukey test, P b 0.05), with higher mean activity observed in M–OE (30.8 nmol min− 1 mg− 1 protein), followed by S (22.5 nmol min− 1 mg−1 protein), and DG (14.1 nmol min− 1 mg− 1 protein). Semiquantitative API ZYM assays resulted in no clear food-specific differences in the activity of carbohydrases (Table 3), proteases (Table 4), ester

hydrolases or phosphohydrolases (Table 5), yet organspecific differences were conspicuous in some cases. For example, α-galactosidase activity was restricted to stomach and digestive gland, β-glucosidase was highest at the mouth while α-glucosidase was absent from this site, and alkaline phosphatase decreased from the mouth towards the digestive gland. Overall, four carbohydrases (β-galactosidase, β-glucoronidase, N-acetyl-β-gulcosaminidase and α-fucosidase), one peptide hydrolase (leucine arylamidase) and two phosphohydrolases (acid phophatase and naphtol-AS-BI-phosphohydrolase) exhibited very high activities throughout the entire abalone's gut (Tables 3–5). In contrast, trypsin activity was not detected at all (Table 4), while low activities of lipase, esterase lipase and α-galactosidase were observed throughout the gut (Tables 3 and 5). The organspecific trend of enzyme activities observed with the API ZYM test appeared to be fairly consistent with the quantitative results described above. For example, esterase activity was higher at the mouth than the rest of the gut (Table 5), peptidase activities (leucine, valine and cystine arylamidase) were lowest in the digestive

Table 4 Results of API ZYM tests for peptide hydrolases, carried out on homogenates of different gut sections of juvenile red abalone, Haliotis rufescens Enzyme

Peptide hydrolases Leucine AP Valine AP Cystine AP Trypsin α-chymotrypsin

Mouth

Foregut

Stomach

Dig. Gland

K

22P

38P

K

22P

38P

K

22P

38P

K

22P

38P

5 5 3 0 0

5 5 3 0 0

5 5 3 0 0

5 4 3 0 0

5 4 2 0 0

5 4 3 0 0

5 4 3 0 2

5 4 3 0 2

5 4 3 0 1

4 3 2 0 0

4 3 2 0 0

4 2 2 0 0

Abalone had been feeding on natural (kelp, K) or balanced diets prepared with 25% protein (P25) and 38% protein (P38). Homogenate concentration was adjusted to 1 mg protein in all cases. Numbers indicate the relative magnitude of enzyme activity.

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Table 5 Results of API ZYM tests for ester and phospho hydrolases, carried out on homogenates of different gut sections of juvenile red abalone, Haliotis rufescens Enzyme

Mouth

Foregut

Stomach

Dig. Gland

K

22P

38P

K

22P

38P

K

22P

38P

K

22P

38P

Ester Hydrolases Esterase (C4) Esterase Lipase (C8) Lipase (C 14)

4 2 1

4 2 1

4 2 1

3 2 2

3 2 1

3 2 1

3 2 2

3 2 2

3 3 2

3 2 1

3 2 1

3 3 1

Phosphohydrolases Alkaline phosphatase Acid phosphatase Naphtol-AS-BI-phosphohydrolase

5 5 4

5 5 5

5 5 5

5 5 5

4 5 5

5 5 5

4 4 5

4 4 5

4 5 5

3 5 5

3 5 5

3 5 5

Abalone had been feeding on natural (kelp, K) or balanced diets prepared with 25% protein (P25) and 38% protein (P38). Homogenate concentration was adjusted to 1 mg protein in all cases. Numbers indicate the relative magnitude of enzyme activity.

gland, and α-chymotrypsin activity was only detected in the stomach (Table 4). 4. Discussion Both quantitative and semiquantitative (API ZYM) results obtained in the present study confirmed the herbivore nature of abalone, as revealed by the high specific activities of cellulase and lysozyme when compared to lipase, chymotrypsin and/or aminopeptidase. Previous studies also showed the presence of other complex carbohydrases in the digestive gland of abalone H. discus hannai and H. seibaldii (xylanase, carboxymethyl cellulase) and 13 other glycosidases (Nakagawa and Nagayama, 1988). The digestive gland appeared to be the most important site for storage/secretion of enzymes in juvenile red abalone, as shown by its high weight-specific protein content and enzyme activities. Similar findings have been reported for the green abalone, H. fulgens (Picos-García et al., 2000). This study evidenced the presence of two main digestion regions along the gut of juvenile H. rufescens: a) the stomach-digestive gland region, characterized by the presence of high amounts of complex carbohydrases (cellulose and lysozyme), and b) the mouth-intestine region, characterized by high specific activity of lipase and aminopeptidase. The stomach appeared to be the main site of chymotrypsin secretion, as evidenced by the APY ZYM strips and the higher specific activity, when compared to DG. In the latter case some activity values were barely different from zero, thus resulting in wide error bars (Fig. 4b). The possibility of cross-contamination between DG and S (this study) can be dismissed, as only the apical part of DG sections were used for the assays. Likewise, only stomach sections containing internal ridges and grooves

were used in the assays. In general, protease activity is reportedly low in haliotids. The magnitude of chymotrypsin activity found in the S–DG region of H. rufescens was comparable to that reported by GarciaCarreño et al. (2003) in the DG of kelp-fed H. fulgens (1.1 ± 0.3 U mg− 1 protein). Likewise, the activity levels of aminopeptidase (present study) were comparable to those of Leu-aminopeptidase (0.045 U mg− 1 protein) reported by the same authors. The high activity detected with the API ZYM strips for Leu, Val and Cys AP may be indicative of the high affinity of these enzymes for essential amino acids. Garcia-Carreño et al. (2003) also reported quantitatively higher activity of Arg-, Lys-and Met-aminopeptidase in the green abalone when compared to Ala-or Gly-aminopeptidases. Dietary effects were not as conspicuous as those of the different gut sections. Nevertheless, organisms exposed to balanced diets consistently exhibited lower protease and higher cellulase/lysozyme activities than those fed with kelp blades. The high carbohydrase activity most likely resulted from the inclusion of kelp meal in the balanced diets. The amount of powdered kelp present in the balanced diets (Table 1) coincides with the relative magnitude of lysozyme/cellulase activity recorded for these diets, being higher for the P25 diet and lower for the P38 diet (Fig. 3). Therefore, it is possible that finely ground kelp particles exerted a greater degree of stimulatory response than whole kelp blades for the production of endogenous carbohydrases, by facilitating a direct and rapid contact of the substrate with secretory cells lining the abalone gut. Alternatively, the high surface:volume ratio of the kelp powder promoted the growth of bacterial population already established in the stomach-digestive gland region of abalone. It is known that abalone's secretory type I gland cells contain proteinase, non-specific lipase,

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esterase and four types of polysaccharide-digesting enzymes (Cui et al., 2001). Recent molecular studies also demonstrated that abalone can produce endocellulase β-1,4-D-glucanase (EC 3.2.1.4) in the digestive gland (Susuki et al., 2003). Previous work also showed the presence of endogenous alginate lyase, CMCase, laminarinase, agarase and carrageenase in the in the digestive gland of abalone, and it was estimated that about 70-90% of bacterial polysaccharidase activity occurs extracellularly in this organ (Erasmus et al., 1997). It has been recognized that digestion of food may be reduced by a limited by colonization and penetration of cellulolytic microbes onto the exposed surface of feed particles (Beauchemin et al., 2004). It is also known that an increased amount of fiber stimulates the activity of cellulase and the growth of microflora in the gut of the fish Oreochromis mossambicus (Manju and Dhevendaran, 2002). Fresh kelp blades had to be torn down and chewed before attaining a size small enough as to increase its surface area and allow significant bacterial colonization. Taken together, these results reinforce the suggestion that finely ground kelp included in the diets stimulated the production of endogenous and/or exogenous carbohydrases. The stimulatory mechanisms suggested here need to be demonstrated in abalone, yet the high cellulase and lysozyme activities exhibited by abalone feeding of balanced diets was statistically supported in this study. These findings not only indicate that abalone can modulate their enzyme levels to optimize the utilization of dietary substrates, but it also shows for the first time that the non-specific immune response of abalone could also be enhanced by increasing the protein levels in the balanced diets. In this regard, it was evident that organisms fed the P25 and P38 diets exhibited higher lysozyme activity than those fed on low protein diets (kelp, ca. 10% protein). It has been previously shown that the non-specific immune response could be modulated by diet in the rainbow trout (Kiron et al., 1995), such that diets with high protein content (35 and 50%) resulted in increased lysozyme activity and C-reactive protein when compared to low-protein (10%) diets (Kiron et al., 1995). The stimulatory effect of kelp meal on the production of carbohydrases does not explain the high mean value of non-specific protease activity found in the digestive gland of kelp-fed organisms. Nevertheless, these findings are consistent with previous studies showing that phytotrophic lobster larvae exhibited lower amylase to protease ratio than omnivorous or carnivorous species (see review by Jones et al., 1997). Furthermore, when phytotrophic penaeid larvae were exposed to diets with plant-or animal-derived protein it was found that the

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former exhibited higher protease activity (Jones et al., 1997). Previous feeding experiments also showed that protease activity (acid protease, trypsin and chymotrypsin) was differentially affected by the type of algal or seagrass species consumed by the green abalone, H. fulgens (Garcia-Carreño et al., 2003). Together, these and the present studies suggest that protease activity was reduced in response to the high-protein content of the balanced diets, while increased activity resulted from the low protein content (ca. 10%) or low digestibility of kelp diets. In this regard, balanced diets contained ca. 38 and 25% crude protein, as compared to 10% protein of kelp. The final size attained by abalone followed the same ordinal pattern as the protein content of diets, thus suggesting that growth differences most likely resulted from differences in the amount of dietary protein. Simultaneous experiments carried out with H. rufescens (Garcia-Esquivel and Felbeck, unpubl. data) showed that the mean gut passage time is slightly shorter in kelpfed (23 h) than pelleted-fed organisms (18 h). Abalone however exhibited ca. 10% higher apparent dry matter digestibility of kelp diets when compared to balanced diets (Garcia-Esquivel and Felbeck, unpubl. data), thus suggesting that abalone can efficiently digest both types of diets. Previous work carried on H. midae also showed that the gut passage time of balanced diets ranged between 18–24 h (Britz et al., 1996). The use of API-ZYM strips proved to be practical for preliminary screening of the digestive potential of abalone. These results generally showed a good agreement with quantitative determinations of enzyme activities, even though API-ZYM strips were unable to clearly discriminate food-dependent effects. Strips revealed the presence of four carbohydrases with very high activity throughout the entire abalone's gut, as compared to one peptide hydrolase and two phosphohydrolases (Table 5), thus confirming the dominantly herbivorous nature of abalone. Chymotrypsin activity was only present in the stomach, while quantitative assays consistently showed high activity in the stomach–digestive gland region and minimal values in the rest of the gut. Both strips and quantitative assays identified the digestive gland as a site with comparatively lower peptidase activity in the gut. API ZYM tests indicated maximal lipase activity in the stomach–foregut region and minimal activity in the DG, while quantitative assays showed maximal activities in the mouth–stomach region and minimal in DG. The present study also showed high digestion of αglucosidase throughout the gut, except at the mouth. Conversely, most of the β-glucosidase activity occurred at the mouth region. It should be noted that seven glycosidases identified with the API ZYM strips in the

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present study are listed among the 13 glycosidases quantitatively determined by Kusumoto et al. (1997) in a cell line obtained from a parasitic/symbiont protist isolated from the from the digestive gland of the abalone H. midae (Kusumoto et al., 2000).Other studies have also shown that resident bacteria contribute to the hydrolysis of carbohydrates in the gut of the abalone H. midae (Erasmus et al., 1997). Therefore, it is possible that H. rufescens and H. midae contain the same type of endosymbionts in their gut. The API ZYM system has been previously used to screen the digestive capacity of other molluscan species, including the marine bivalves Lucinoma aequizonata (Boetius and Felbeck, 1995) and Crassostrea gigas (Luna-González et al., 2004), as well as the gastropod Aplysia punctata (Taïeb, 2001). Interestingly, enzyme profiles of apparently close groups such as A. punctata (Taïeb, 2001) and H. rufescens (this study) showed some differences under comparable conditions. For example, the API ZYM screening revealed much lower amino peptidase activity in the digestive gland of A. punctata than H. rufescens. There are also differences in the magnitude of activity of esterases and some glycosidases (a-glucosidase, trypsin, and n-acetyl-b-glucosaminidase). These differences may indicate specific adaptations to a certain food type that need to be revealed with specific studies. Interestingly, high α-fucosidase activity has been consistently reported in different gastropod species (Reglero and Cabezas, 1976; Nakagawa and Nagayama, 1988; Kusumoto et al., 2000; Taïeb, 2001), different bivalves (Boetius and Felbeck, 1995; Luna-González et al., 2004) and other deep-vent invertebrates (Boetius and Felbeck, 1995), therefore suggesting that this enzyme plays an important function in molluscs. L-fucose is known to be involved in glycoprotein metabolism and acts as a ligand in cell to cell recognition (Suzuki and Suzuki, 2001). It is presumed that α- L-fucosidase from H. gigantea acts on the substrate p-nitrophenyl α- L-fucoside (Reglero and Cabezas, 1976). Studies carried out on the polysaccharide fucoidan from Lessonia vadosa and other Laminarian species indicate that these molecules contain a backbone of L-fucose generally linked at position α-1,3 (Chandia et al., 2005 and refs. therein). Fucoidan comprises ca. 0.5 to 2% of the total dry weight of Macrocystis pyrifera (Cruz-Suárez et al., 2000). Because M. pyrifera was an important component of all three diets tested in the present study, it is assumed that dietary fucose was readily available for H. rufescens, and that in turn would explain the high activity of αfucosidase observed in all treatments. In summary, the present study confirms that the digestive system of the abalone H. rufescens is naturally

adapted to feed primarily on polysaccharide-rich diets. However this species could also optimize the utilization of feedstuffs with different dietary quality by adjusting their protease and carbohydrase levels. It was evident that low-protein diets (kelp) caused an increase of protease activity of H. rufescens, while the opposite was observed for protein-rich diets (P25 and P38). Likewise, the activity of cellulase and lysozyme were comparatively lower in abalone fed on carbohydrate-rich diets (kelp) when compared to those fed with P25 and P38. The most important digestive site for proteins and carbohydrates in abalone is the stomach-digestive gland region, yet the upper region (mouth–oesophagus) should also be considered in studies of lipid digestion. Despite the observed digestive plasticity of the abalone's gut, they grew faster when fed on balanced diets, thus suggesting that the amount of dietary protein defined the amount of substrate absorbed and deposited as tissue. The present qualitative and quantitative results confirm that balanced diets with different composition could potentially be used in order to find the most appropriate and economic feed for culturing abalone. Inert diets have demonstrated a promising option for rearing abalone under controlled conditions, yet further studies are still needed to optimize the composition of proteins and lipids in the diet, and to reduce the potential pollution hazard of balanced feeds. Acknowledgements This study was supported by a UC MEXUS-CONA CYT fellowship granted to ZGE, under the 2003–2004 Program for Sabbatical Residencies for Distinguished UC and Mexican Researchers. Thanks to Eddie Kisfaludy for providing fresh kelp throughout the experiment and his help in setting up the experimental system at the SIO aquarium. Marco A. González Gomez helped with the preparation of balanced diets. Thanks to Yoshihiro Fujiwara for his advice and for providing materials for the cellulase assay. Special thanks to John Wilson from DSM Nutritional Products Inc. for kindly donating the Stay C-35. References Albro, P.W., Hall, R.D., Corbett, J.T., Schroeder, J., 1985. Activation of non-specific lipase (EC 3.1.1.-) by bile salts. Biochim. Biophys. Acta 835, 477–490. Beauchemin, K.A., Colombatto, D., Morgavi, D.P., Yang, W.Z., Rode, L.M., 2004. Mode of action of exogenous degrading cell wall enzymes for ruminants. Can. J. Anim. Sci. 84, 13–22. Boetius, A., Felbeck, H., 1995. Digestive enzymes in marine invertebrates from hydrothermal vents and other reducing environments. Mar. Biol. 122, 105–113.

Z. Garcia-Esquivel, H. Felbeck / Aquaculture 261 (2006) 615–625 Britz, P.J., Hecht, T., Knauer, J., 1996. Gastric evacuation time and digestive enzyme activity in abalone Haliotis midae fed a formulated diet. S. Afr. J. Mar. Sci. 17, 297–303. Chandia, N.P., Matsuhiro, B., Ortiz, J.S., Mansilla, A., 2005. Carbohydrates from the sequential extraction of Lessonia vadosa (Phaeophyta). J. Chil. Chem. Soc. 50, 501–504. Cruz-Suárez, L.E., Ricque-Marie, D., Tapia-Salazar, M., GuajardoBarbosa, C., 2000. Uso de harina de kelp (Macrocystis pyrifera) en alimentos para camarón. In: Cruz-Suárez, L.E., Ricque-Marie, D., Tapia-Salazar, M., Olvera-Novoa, M.A., Civera-Cerecedo, R. (Eds.), Avances en Nutrición Acuícola V. Memorias del V Simposium Internacional de Nutrición Acuícola, 19–22 Noviembre, 2000, pp. 227–266. Mérida, Yucatán. Cui, L.-B., Liu, C.-L., Liu, X., Lu, Y.H., 2001. Structure and function of mucous epithelium of the intestine in Haliotis discus hannai. Acta Zool. Sin. 47, 324–338. Degnan, B.B., Groppe, J.C., Morse, D.E., 1995. Chymotrypsin, mRNA expression in digestive gland amoebocytes: cell specification occurs prior to metamorphosis and gut morphogenesis in the gastropod Haliotis rufescens. Roux's Arch. Dev. Biol. 205, 97–101. Edwards, S., Condon, C., 2001. Digestive protease characterization, localization and adaptation in backlip abalone (Haliotis rubra Leach). Aquac. Res. 32, 95–102. Elliott, C.J.H., Susswein, A.J., 2002. Comparative neuroethology of feeding control in molluscs. J. Exp. Biol. 205, 877–896. Erasmus, J.H., Cook, P.A., Coyne, V.E., 1997. The role of bacteria in the digestion of seaweed by the abalone Haliotis midae. Aquaculture 155, 377–386. Fleming, A.E., Van Barneveld, R.J., Hone, P.W., 1996. The development of artificial diets for abalone: a review and future directions. Aquaculture 140, 5–53. Garcia-Carreño, L.F., Navarrte del Toro, M.A., Serviere-Zaragoza, E., 2003. Digestive enzymes in juvenile green abalone, Haliotis fulgens, fed natural foods. Comp. Biochem. Physiol., B 134, 143–150. Gomez-Montes, L., Garcia-Esquivel, Z., D'Abramo, L.R., Shimada, A., Vasquez-Pelaez, C., Viana, M.T., 2003. Effect of dietary protein:energy ratio on intake, growth and metabolism of juvenile green abalone Haliotis fulgens. Aquaculture 220, 769–780. Gordon, H.R., Cook, P.A., 2004. Word abalone fisheries and aquaculture update: supply and market dynamics. J. Shellfish Res. 23, 935–939. Head, E.J.H., Wang, R., Conover, R.J., 1984. Comparison of diurnal feeding rhythms in Temora longicornis and Centropages hamatus with digestive enzyme activity. J. Plankton Res. 6, 543–550. Ito, M., Tchoua, U., Okamoto, M., Tojo, H., 2002. Purification and properties of a phospholipase A2/lipase preferring phosphatic acid, bis(monoacylglycerol) phosphate, and monoacylglycerol from rat testis. J. Biol. Chem. 277, 43674–43681. Johnston, D.J., 2003. Ontogenetic changes in digestive enzyme activity of the spiny lobster, Jasus edwarsii (Decapoda; Palinuridae). Mar. Biol. 143, 1071–1082. Jones, D.A., Kumlu, M., Le Vay, L., Fletcher, D.J., 1997. The digestive physiology of herbivorous, omnivorous and carnivorous crustacean larvae: a review. Aquaculture 155, 285–295. Kiron, V., Watanabe, T., Fukuda, H., Okamoto, N., Takeuchi, T., 1995. Protein nutrition and defence mechanisms in rainbow trout Oncorhynchus mykiss. Comp. Biochem. Physiol. IIIA, 351–359.

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Kusumoto, K., Shirahata, S., Katakuta, Y., Murakami, H., Kamei, Y., 1997. Establishment of an abalone digestive gland cell line secreting various glycosidases in protein-free culture. Cytotechnology 24, 169–175. Kusumoto, K., Shirahata, S., Kamei, Y., 2000. Purification and characterization of α-D-galactosidase produced by ADG cell line established from abalone digestive gland. Cytotechnology 33, 47–52. Lee, S.-M., 2004. Utilization of dietary protein, lipid and carbohydrate by abalone Haliotis discus hannai: a review. J. Shellfish Res. 23, 1027–1030. Luna-González, A., Maeda-Martínez, A.N., Ascencio-Valle, F., RoblesMungaray, M., 2004. Ontogenetic variations of hydrolytic enzymes in the Pacific oyster Crassostrea gigas. Fish Shellfish Immunol. 16, 287–294. Manju, K.G., Dhevendaran, K., 2002. Influence of Azolla spp. Incorporated feeds on the growth, conversion efficiency and gut flora of Oreochromis mossambicus (Peters). J. Aquac. Trop. 17, 221–230. McBride, S., 1998. Current status of abalone aquaculture in the Californias. J. Shellfish Res. 17, 593–600. McLean, N., 1970. Digestion of Haliotis rufescens Swainson (Gastropoda: Prosobranchia). J. Exp. Zool. 173, 303–318. Nakagawa, T., Nagayama, F., 1988. Distribution of glycosidase activities in marine invertebrates. J. Tokyo Univ. Fish. 75, 239–246. Picos-García, C., García-Carreño, F.L., Serviere-Zaragoza, E., 2000. Digestive proteases in juvenile Mexican green abalone, Haliotis fulgens. Aquaculture 181, 157–170. Reglero, A., Cabezas, J.A., 1976. Glycosidases of molluscs, purification and properties of α-L-fucosidase from Chamelea gallina L. Eur. J. Biochem. 66, 379–387. Rogers-Bennett, L., Allen, B.L., Davis, G.E., 2004. Measuring abalone (Haliotis spp.) recruitment in California to examine recruitment overfishing and recovery criteria. J. Shellfish Res. 23, 1201–1207. Susuki, K.-I., Ojima, T., Nishita, K., 2003. Purification and cDNA cloning of a cellulase from abalone Haliotis discus hannai. Eur. J. Biochem. 270, 771–778. Suzuki, M., Suzuki, A., 2001. Structural characterization of fucosecontaining oligosaccharides by high-performance liquid chromatography and matrix-assisted laser desorption/ionization time-offlight mass spectrometry. Biol. Chem. 382, 251–257. Taïeb, N., 2001. Distribution of digestive tubules and fine structure of digestive cells of Aplysia punctata (Cuvier, 1803). J. Molluscan Stud. 67, 169–182. Takami, H., Kawamura, T., 2003. Dietary changes in the abalone, Haliotis discus hannai, and relationship with the development of the digestive organ. JARQ 37, 89–98. Takami, H., Kawamura, T., Yamashita, Y., 1998. Development of polysacharide degradation activity in postlarval abalone Haliotis discus hannai. J. Shellfish Res. 17, 723–727. Viana, M.T., Lopez, L.M., García-Esquivel, Z., Mendez, E., 1996. The use of silage from fish and abalone viscera as an ingredient in abalone feed. Aquaculture 140, 87–98. Weisner, B., 1984. Lysozyme (Muramidase). In: Bergemeyer, H.U., Bergemeyer, J., Grassl, M. (Eds.), Methods of Enzymatic Analysis, vol. IV. Verlag Chemie, Weinheim, pp. 189–195. Zhang, G., Que, H., Liu, X., Xu, H., 2004. Abalone mariculture in China. J. Shellfish Res. 23, 947–950.