Effects of vitamins A and D on shell biomineralization of abalone Haliotis discus hannai, Ino.

ABSTRACT A 152-day feeding trial was conducted in a recirculating water system to investigate the effects of vitamins A (retinol) and D (cholecalciferol) on shell biomineralization of the abalone Haliotis discus hannai Ino. Triplicate groups of juvenile abalone (initial weight: 0.35 [+ or -] 0.03 g; initial shell length: 11.31 [+ or -] 0.25 mm) were fed to satiation daily with one of the 16 semipurified diets containing 0, 1 x [10.sup.3], 1 x [10.sup.5], 1 x [10.sup.6] IU/kg vitamin A and 0, 500, 1 x [10.sup.3], 5 x [10.sup.3] IU/kg vitamin D in a 4 x 4 factorial design. The results showed that vitamin D significantly increased the ratio of calcite/aragonite in the shell, in the case of deficient (0 IU/kg) or excessive (1 x [10.sup.6] IU/kg) dietary vitamin A supplementation. When dietary vitamin A supplementation ranged from 1 x [10.sup.3] to 1 X [10.sup.5] IU/kg, vitamin D significantly decreased the ratio of calcite/aragonite in the shell. The concentrations of Mg, Zn, and Sr in the shell were independent of the supplementation of these two vitamins. Meanwhile, the contents of Ca, P, and Cu in abalone shell increased with supplementation of the vitamins A and D, and significant interaction between vitamin A and D was observed on the concentrations of both Ca and Cu in shell. Vitamin A, instead of vitamin D, significantly increased Fe concentration in shell. Trends of the ratio of acidic/basic amino acids [(Asx + Glx)/(Lys + Arg + His)] in the shell soluble matrix protein (SMP) changing with dietary vitamin A and D were in agreement with those of the ratio of calcite/aragonite in the shell. The two vitamins did not change the shell ultrastructure as shown by scanning electron microscopy (SEM) and components of SMP expressed by SDS-PAGE.

KEY WORDS: Haliotis discus hannai, vitamin A, vitamin D, shell, biomineralization

INTRODUCTION

Vitamin A (retinol) is sequentially metabolized to retinaldehyde, retinoic acid for its functions in animals (Hofman & Eichele 1994, Saari 1994). All-trans-retinoic acid (ATRA) acts through a series of receptors, termed retinoic acid receptors (RAR) [alpha], [beta], and [gamma]. Meanwhile, 9-cis-retinoic acid (9-cis-RA) can bind another series of retinoid receptors, termed retinoid X receptors (RXR), and initiate transcription of RXR responsive genes in a ligand-dependent fashion (Rohde et al. 1999). Vitamin A is functionally related to vision, reproduction, and specific immune responses (Sporn & Roberts 1984, Dhur et al. 1991, Thompson et al. 1994). Furthermore, it induces the expression of genes associated with the terminal mineralization phase of chondrocyte maturation and promotes apatite deposition in the extracellular matrix (Iwamoto et al. 1993).

Vitamin D (cholecalciferol) requires two hydroxylations to be converted into its active hormonal form, 1[alpha],25-dihydroxyvitamin [D.sub.3] [1[alpha],25[(OH).sub.2][D.sub.3]]. It is activated by hydroxylation first in the liver to form 25-hydroxyvitamin [D.sub.3] [25(OH)[D.sub.3]], and then in the kidney to form 1[alpha],25[(OH).sub.2][D.sub.3] (Henry & Norman 1984, Norman & Henry 1993). 1[alpha],25[(OH).sub.2][D.sub.3] exerts its functions through 2 pathways: nongenomic pathway (reviewed in Revelli et al. 1998) and genomic pathway (reviewed in Jones et al. 1998). The genomic pathway requires binding of 1[alpha],25[(OH).sub.2][D.sub.3] to the nuclear vitamin D receptor (VDR), heterodimerization with a retinoid X receptor (RXR), and interaction with specific vitamin D-responsive element (VDRE) consensus sequences of target genes (Colnot et al. 1995, Haussler et al., 1998). Vitamin D plays an important role in calcium and phosphate homeostasis (DeLuca & Schnoes 1976). Recent studies have shown that vitamin D regulates the gene expression of proteins involved in the formation of mineralized tissues. 1[alpha],25[(OH).sub.2][D.sub.3] stimulates the expression of osteocalcin, which is a small extracelluar bone matrix protein with a capacity for specific Gla ([gamma]-carboxyglutamic acid)-dependent binding of [Ca.sup.2+] (Mahonen et al. 1990). Such function of 1[alpha],25[(OH).sub.2][D.sub.3] was further demonstrated by later studies (Luegmayr et al. 1998). In addition, 1[alpha],25[(OH).sub.2][D.sub.3] was found to be able to regulate the expression of a tooth-specific gene: amelogenin gene (Papagerakis et al. 1999). Amelogenins are the major (90%) matrix proteins for forming enamel (Sasaki & Shimokawa, 1995). Amelogenesis is a multistage process that generates a fully mineralized layer of enamel on the crowns of teeth (Smith & Nanci 1995). This process is controlled by ameloblasts, which secrete an extracellular protein matrix and provide a gel scaffold to support apatitic crystal growth (Fincham & Simmer 1997).

In contrast to bone and tooth, mollusc shell is mostly composed of calcium carbonate (CaC[O.sub.3]) (95% to 99% in weight), which is usually in the crystallographic form of calcite or (and) aragonite (Currey 1999). The shell contains a small amount of protein and polysaccharide (1% to 5% in weight). The organic material is responsible for the fracture toughness of the shell, which is 3000 times greater than pure crystalline aragonite (Weiner 1986). The organic matrix is also involved in the crystallization process: some parts may enhance, others may inhibit the crystal formation of calcium carbonate (Belcher et al. 1996, Weiss et al. 2000). It has been demonstrated that the soluble matrix proteins (SMP) play an important role in the control of crystal polymorphism phase switching and orientation (Belcher et al. 1996, Falini et al. 1996).

Shell biomineralization can be influenced by factors such as environmental (Kennedy et al. 1969, Almeida et al. 1998) and also the metabolic state of the organism (Wilkes & Crenshaw 1979). It was demonstrated in our previous studies that dietary Zn and P changed the character of mineralogy and the pattern of SMP in the shell of the abalone Haliotis discus hannai Ino (Zhang et al. 2002, Mai et al. 2003). Either vitamin A or D plays an important role in mineralization of bone and tooth as described earlier. There is no information available on the effects of vitamin A and D on mollusc shell biomineralization. There is potential for interaction between vitamin A and D signaling pathways, because RXR is needed for the two vitamins to exert their functions (Glass 1994, Carlberg 1995). Furthermore, such interaction influences bone mineralization in the rat (Rohde et al. 1999) and the performances of growth and metabolism in H. discus hannai (Zhang et al., unpublished data). The aim of this study is to investigate whether vitamin A and D have effects on shell biomineralization in H. discus hannai.

MATERIALS AND METHODS

Animals, Diets and Experimental Treatments

Abalone juveniles (initial weight: 0.35 [+ or -] 0.03 g; initial shell length: 11.31 [+ or -] 0.25 mm) used in this experiment were derived from a spawning at Huaxin Fisheries Co., Shandong, China.

Diets were formulated with purified ingredients. The composition and the proximate analysis of the diets are presented in Table 1. The basal diet contained 32.8 IU vitamin A/kg diet and 25.6 IU vitamin D/kg diet, analyzed by high-performance liquid chromatography (HPLC). Experimental diets with 4 levels of vitamin A (0, 1 x [10.sup.3], 1 x [10.sup.5] and 1 x [10.sup.6] IU/kg) and 4 levels of vitamin D (0, 500, 1 x [10.sup.3] and 5 x [10.sup.3] IU/kg) were prepared by adding appropriate quantities of all-trans-retinol acetate and cholecalciferol to the basal diet in a 4 x 4 factorial arrangement.

The growth experiment was conducted in a recirculating water system. Abalone were stocked at 30 animals for each rearing unit in a plastic basket (20 cm x 20 cm x 10 cm) per glass aquarium (45 cm x 25 cm x 35 cm). There were 16 treatments, and each treatment was conducted in three replicates. The feeding trial was run for 152 days.

Environmental Conditions of the Growth Experiment

During the 152-day growth experiment, water temperature was maintained at 18 [+ or -] 1[degrees]C, salinity 31-35, pH 7.8-8.0. Dissolved oxygen was not less than 6 mg/L, and there were negligible levels of free ammonia and nitrite. Mineral concentrations in the seawater of the rearing system were determined by inductively coupled plasma-atomic emission spectrophotometer (ICP-OES; VISTAMPX, VARIAN). Concentrations of Ca, P, Mg and Sr were 301.0, 0.5, 78.4 and 8.6 mg/L, and those of Cu, Zn, Fe, and Mn were 1.5, 8.2. 1.2 and 0.11 [micro]g/L.

Preparation of Shell Samples

At the termination of the feeding trial, animals were not fed for 3 days. All the abalone were removed from the baskets, weighted, measured and counted. Then, abalone from each replicate were immediately frozen (-70[degrees]C). Shells were separated from the soft body, and then were cleaned under tap water to remove residual dirt. Subsequently, shells were kept in 5% NaOH for 7 h. After being washed completely with deionized water, the samples were air-dried for 24 h for the following analyses.

Scanning Electron Microscopy

To examine variation in crystalline ultrastructure, shells of abalone fed the diets with different levels of vitamin A and D were analyzed by SEM. Vertical sections of shells for SEM were prepared by fracturing. SEM was carried out on a JEOL JSM-840 instrument.

Mineralogy and Chemical Compositions

Mineralogical composition of the shells was investigated by x-ray diffractometry on powdered samples (Cohen & Branch 1992). The fraction of each type of crystal (aragonite, calcite, and dolomite) was expressed as a percentage.

Concentrations of Ca, P, Mg, Fe, Zn, Cu, and Sr in shells were determined by ICP-OES. Preparation of the shells was according to the method of Carriker et al. (1980).

Isolation and Purification of the Soluble Matrix Protein

The cleaned shells were crushed into a fine powder, then suspended in 3,500 molecular weight cutoff dialysis membranes (Cole-Parmer Instrument Co., USA) and dialyzed against 5% acetic acid containing 0.01% (w/v) sodium azide for 72 h at room temperature. This was followed by exhaustive dialysis against ultra-pure water for 48 h to remove the acetic acid. The resultant dialysate was centrifuged at x10,000g for 30 min at 4[degrees]C. The supernatant containing protein SMP was collected and lyophilized for further analyses.

Vertical Polyacrylomide Gel Electrophoresis

Analysis of SMP was performed using the discontinuous buffer system. Electrophoresis of SDS-PAGE (18% separating gel and 4% stacking gel) was prepared with acrylamide:biacrylamide and run in Mini-PROTEAN II equipment (Bio-Rad, Hercules, CA, USA). Samples (20 [micro]L) were added to wells (1-cm high). All the stock solutions were prepared by the methods described in the instruction manual (Bio-Rad, Hercules, CA, USA). Gel thickness was established with 1-mm spacers. Molecular weight (MW) marker was obtained from Bio-Rad (catalog number 161-0304). Other electrophoretic chemicals were from Amersham-Pharmacia (New Territories, Hong Kong, People's Republic of China). Procedures were carried out at room temperature. The power condition was 150 V, constant voltage setting. The usual run time was approximately 90 min. The method in combination with silver nitrate and stains-all (4,5,4',5'- dibenzo -3,3'- diethyl-9-methylthiocarbocyanine bromide), in the present study, was accepted to stain the gels (Goldberg & Warner 1997).

Amino Acids Analyses

To have enough samples for amino acid analyses, SMP from the 3 replicates in a treatment were pooled together. Sample (1 mg) of SMP was hydrolyzed in vacuo using 6N HCl for 24 h at 110[degrees]C and derivitized with dabsyl chloride (Kamp 1990). Analyses of derivatized amino acids were performed by reversed-phase HPLC (RP-HPLC) on an ODS (octadecylsilane) Hypersil column (HP; 250 x 4 mm; 5 [micro]m). Eluant gradients with two solvent buffers were formed. The amount of acetonitrile (A) was gradually increased in 25 mM sodium acetate (pH 6.5) (B). Fifteen percent A was increased to 25% in 10 min, 40% in 20 min, 70% in 10 min, and held at 70% for 5 min before returning to 15% in 5 min. Tryptophan was lost during hydrolysis, and cystine coeluted with a reagent peak during amino acid separation. Therefore, tryptophan and cystine values are not reported. Values reported as Glx included both glutamate and glutamine, because glutamine was converted to glutamate during the analysis. Values for Asx included aspartate and asparagines, because asparagine was converted to aspartate.

Statistical Analysis

All percentage data were square-root arcsine transformed prior to analysis. Data were submitted to 2-way analysis of variance using the STATISTICA[TM] package. When significant differences (P < 0.05) were found, means were compared using the Tukey's test.

RESULTS

Scanning Electron Microscopy

There are no variations in shell ultrastructure of H. discus hannai fed the diets with different levels of vitamin A and D. Crystals in the shell could be divided into two layers from the outer to the inner shell surface: the prismatic layer and the nacreous layer. The former was composed of calcite. The latter was composed of tabular blocks of aragonite, and aragonitic tablets resemble a brick wall (Fig. 1). Growth line was found in the shells. It was continuous through the nacre (Fig. 2).

[FIGURES 1-2 OMITTED]

Mineralogy

Contents of aragonite, calcite, and dolomite in shell are given in Table 2. Aragonite was the dominant type of crystal. Although vitamin A significantly influenced contents of aragonite (91.90% to 96.39%) and calcite (1.72% to 5.25%), there was no obvious trend. Shell dolomite content (1.39% to 2.85%) was not significantly influenced by vitamin A.

In the case of hypo- or hyper-vitamin A supplement (0 or 1 x [10.sup.6] IU/kg), dietary vitamin D significantly increased shell calcite content. When dietary vitamin A supplements ranged from 1 x [10.sup.3] to 1 x [10.sup.5] IU/kg, vitamin D significantly decreased the content of calcite in shell. There were no significant effects of vitamin D on both aragonite and dolomite contents.

Significant interaction between vitamin A and D was found on contents of aragonite and calcite in the shell. When dietary vitamin A supplements ranged from 1 x [10.sup.3] to 1 x [10.sup.3] IU/kg, both vitamin A and D increased shell aragonite content, but decreased shell calcite content. Such significant interaction was not found with shell dolomite content.

Chemical Composition of Shells

Concentrations of Ca, P, Mg, Fe, Zn, Cu, and Sr in shell are given in Table 3. Vitamin A had significant effects on concentrations of Ca, P, Fe, and Cu. When dietary vitamin A levels ranged from 1 x [10.sup.3] to 1 x [10.sup.3] IU/kg it increased shell Ca concentration regardless of the dietary vitamin D supplements. Concentrations of the shell Fe in treatments with 1 x [10.sup.5] and 1 x [10.sup.6] IU/kg dietary vitamin A were significantly higher than those in treatments with 0 or 1 x [10.sup.3] IU/kg dietary vitamin A.

Vitamin D had no significant effects on concentrations of P, Mg, Fe, Zn, and Sr in the shell. When dietary vitamin A ranged from 1 x [10.sup.5] to 1 x [10.sup.6] IU/kg, vitamin D significantly increased shell Ca concentration. Vitamin D significantly elevated the shell Cu concentrations at all dietary vitamin A levels.

Interaction between vitamin A and D were found significant on concentrations of Ca and Cu in shell, not on other analyzed minerals. This interaction significantly increased concentrations of Ca and Cu in shell.

SDS-PAGE

The SDS-electrophoretical separation of the SMP gave five bands (Fig. 3). The gel was somewhat overstained to help visualize minor components. Preparations of SMP from the different shells of abalone fed the diets with different levels of vitamin A and D showed a similar staining profile. Four distinct bands were found at 36.6, 30.9, 24.5, and 19.7 kDa respectively. A heavy stained region was labeled a.

[FIGURE 3 OMITTED]

Amino Acid Compositions

Apparent amino acid contents of the soluble matrix protein (SMP) from the shell are given in Table 4. The dominant amino acids in shells were Asx, Ser, Gly, and Phe. Meanwhile, Pro and Met were not detected in shells. Vitamin D increased the ratio of acidic/basic amino acids [(Asx + Glx)/(Lys + Arg + His)], in the case of deficient (0 IU/kg) or excessive (1 x [10.sup.6] IU/kg) dietary vitamin A supplement. When dietary vitamin A supplements ranged from 1 x [10.sup.3] to 1 x [10.sup.5] IU/kg, vitamin D decreased those ratios in shell.

DISCUSSION

To our best knowledge, the present study is the first report on the effects of vitamin A and D on shell biomineralization in molluscs. The results showed that various levels of the two vitamins in diets did not change the ultrastructure or the soluble matrix protein (SMP) components in the shell of H. discus hannai. However, mineralogical and chemical compositions in shells were significantly influenced by dietary vitamin A and D. In previous study, it was also demonstrated that mineralogical and chemical compositions were very important to investigate the effects of dietary guaiacol and Zn on shell biomineralization in H. discus hannai (Mai & He 2000, Mai et al. 2003). It is suggested that changes in mineralogical and chemical compositions in the shell are responsive indicators to study the relationship between nutrients and shell biomineralization in abalone.

The role of vitamin A in bone mineralization has been well established. Low doses of vitamin A induce expression of the alkaline phosphatase (AKP), osteonectin, and osteopontin genes. As a result, massive hydroxyapatite is deposited. The mechanisms by which vitamin A induces changes in mineralization-related gene expression in chondrocytes is possibly the temporal expression of nuclear retinoic acid receptor (RAR) isoforms (lwamoto et al. 1993). Overdoses of vitamin A cause abnormalities in bone mineralization of larval flounder. These bone deformities were due to hypervitaminosis induced by excessive vitamin A that accelerated chondrocyte maturation (Dedi et al. 1995). In the present study, vitamin A significantly increased concentrations of Ca and P in the shell. Given that AKP plays an important role in metabolism of Ca and P (Norman et al. 1970, Birge & Avioli 1981), results mentioned earlier are suggested to relate to AKP activity in abalone. In our previous study, it was demonstrated that AKP activities in the viscera of abalone increased with dietary vitamin A levels (unpublished data). At present, it is difficult to identify whether vitamin A elevates expression of the AKP gene in abalone, because there are not enough direct data. Further study is needed to make the related mechanism clear.

Vitamin D can stimulate intestinal Ca and P intake and can lead to hypercalcemia and hyperphosphatemia in walking catfish Clarias batrachus and American eels Anguilla rostrata (Swarup & Srivastav 1982, Fenwick et al. 1984). Vitamin D did not significantly influence the concentrations of Ca and P in the bone of the blue tilapia Oreochromis aureus (O'Connell & Gatlin 1994). These findings indicate that the role of vitamin D in calcium metabolism of fish is not well established. In the present study, dietary vitamin D significantly increased concentrations of Ca and P in shell. These results are in agreement with those from a previous study (Zhou, pers. comm.). Given that AKP is involved in the metabolism of Ca and P (Norman et al. 1970, Birge & Avioli 1981), changes of AKP activity could be responsible for Ca and P concentrations in shell in the present study. It was demonstrated in our previous study that AKP activities were increased with dietary vitamin D (unpublished data). Vitamin D also elevated bone-specific AKP activity in vertebrates (Spiess et al. 1986). Combining these findings, it is suggested that vitamin D, as well as vitamin A, stimulate AKP activity, subsequently increasing the deposition of Ca and P in shell of H. discus hannai.

It has been demonstrated that the soluble matrix proteins (SMP) play important roles in the control of CaC[O.sub.3] crystal polymorphism phase switching and orientation in the mollusc shell (Belcher et al. 1996, Falini et al. 1996). Furthermore, aragonitic and calcitic SMP determine the nucleation and growth of aragonite and calcite, respectively (Belcher et al. 1996). Kawaguchi and Watabe (1993) reported high ratios of acidic/basic amino acids (50.1) in the calcitic prismatic layer soluble matrix (SM) of the American oyster Crassostrea virginica. Data calculated from some other studies shows that the ratio of acidic/basic amino acids (14.9) in calcitic shell of the Antarctic scallop Adamussium colbeki (Halloran & Donachy 1995) was greater than that (3.4) in aragonitic shell of the freshwater snail Biomphalaria glabrata (Marxen & Becker 1997). In the present study, trends of the ratio of calcite/aragonite in the shell, changing with different dietary vitamin A and D, were in agreement with those of the ratio of acidic/basic amino acids [(Asx + Glx)/(Lys + Arg + His)] in SMP. In our previous study, we found that ratios of acidic/basic amino acids, as well as ratios of calcite/ aragonite in the shell of H. discus hannai, were significantly increased with dietary zinc levels (Mai et al. 2003). Results from the present study, and previous studies on other molluscs discussed earlier, may imply that SMP from calcitic shell was more acidic than that from aragonitic shell. It is suggested that the most likely way for vitamin A and D to influence the ratio of calcite/aragonite is to change the concentrations of calcitic and aragonitic SMP in shell. It has been well established in vertebrates that the most important way for vitamin A and D to influence the mineralization of bone and tooth is to regulate the gene expression of proteins involved in the formation of these mineralized tissues (Mahonen et al. 1990, Iwamoto et al. 1993, Luegmayr et al. 1998, Papagerakis et al. 1999). It is still unclear if vitamin A and D regulate the gene expression of SMP. Further work will be necessary to better understand the relationship between the two vitamins and SMP, and to investigate the molecular mechanisms of shell biomineralization in H. discus hannai.

TABLE 1.

Composition of the basal diet.

                   Ingredient                       g/100 g dry wt.

Casein (vitamin-free, Sigma Chemical, St. Louis,
  MO, USA)                                                25.0
Gelatin (Sigma Chemical, St. Louis, MO, USA)               6.0
Dextrin (Shanghai Chemical Co.. Shanghai, China)          33.5
Sodium alginate (Shanghai Chemical Co.,
  Shanghai, China)                                        20.0
SO/MFO (Food grade) (a)                                    3.5
Choline chloride (Shanghai Chemical Co.,
  Shanghai, China)                                         0.5
Carboxymethylcellulose (Shanghai Chemical Co.,
  Shanghai, China)                                         5.0
Mineral mix (b)                                            4.5
Cholecalciferol and retinol-tree vitamin mix (c)           2.0

Proximate analysis (means of triplicate)
Crude protein (%)                                         29.0
Crude lipid (%)                                            3.3
Ash (%)                                                    9.6
Gross energy (kJ/g) (d)                                   18.8
Retinol (IU/kg)                                           32.8
Cholecalciferol (IU/kg)                                   25.6

(a) Soybean oil and menhaden fish oil (1:1) with 0.001% ethoxyquin.

(b) Mineral mix, each 1000 g of diet contained: NaCl, 0.4g;
MsS[O.sub.4] x 7[H.sub.2]O, 6.0g; Na[H.sub.2]P[O.sub.4] x 2[H.sub.2]O,
10.0g; K[H.sub.2]P[O.sub.4], 12.8g; Ca[([H.sub.2]P[O.sub.4]).sub.2] x
[H.sub.2]O, 8.0g; Fecitrate, 1.0g; Ca-lactate, 1.4g; ZnS[O.sub.4] x
7[H.sub.2]O, 141.2 mg; MnS[O.sub.4] x [H.sub.2]O x [H.sub.2]O,
64.8mg; CuS[O.sub.4]-5[H.sub.2]O, 12.4mg; Co[Cl.sub.2] x 6[H.sub.2]O,
0.4mg: KI[O.sub.3], 1.2 mg; [Na.sub.2]Se[O.sub.3], 0.4 mg.

(c) Cholecalciferol and retinol-free vitamin mix, each 1000 g of diet
contained: thiamin HCl, 120 mg: riboflavin, 100 mg; folic acid, 30 mg;
PABA, 400 mg: pyridoxine HCl, 40 mg: niacin, 800 mg: Ca pantothenate,
200 mg: inositol, 4000 mg; ascorbic acid, 4000 mg: biotin, 12 mg:
[B.sub.12], 0.18 mg: menadione, 80 mg; vitamin E, 450 mg: ethoxyquin
400 mg.

(d) Estimated with an XYR-1 bomb calorimeter.

TABLE 2. Contents of aragonite, calcite and dolomite in the shell of
Haliotis discus hannai fed the diets with different levels of
vitamin A (A) and D (D) for 152 days.

Dietary Supplement
                           Aragonite           Calcite
A (IU/kg)   D (IU/kg)         (%)                (%)

0                 0     [95.36.sup.ab]      [2.90.sup.ef]
                500     [94.39.sup.bcd]     [3.05.sup.ef]
              1,000     [93.66.sup.bcde]    [3.76.sup.bcde]
              5,000     [92.75.sup.de]      [4.59.sup.abcd]
1,000             0     [91.90.sup.e]       [5.25.sup.a]
                500     [92.79.sup.de]      [5.07.sup.a]
              1,000     [94.12.sup.bcd]     [3.38.sup.e]
              5,000     [94.17.sup.bcd]     [3.33.sup.e]
100.000           0     [93.77.sup.bcd]     [4.74.sup.ab]
                500     [94.13.sup.bcd]     [3.87.sup.bcd]
              1.000     [94.91.sup.abc]     [3.70.sup.bcd]
              5,000     [96.39.sup.a]       [1.72.sup.g]
1,000,000         0     [94.97.sup.abc]     [3.52.sup.de]
                500     [94.74.sup.abc]     [3.68.sup.bcde]
              1,000     [94.57.sup.abcd]    [3.89.sup.bcd]
              5.000     [93.39.sup.cde]     [4.68.sup.abc]
ANOVA
A                         0.0000             0.0001
D                         0.2955             0.0008
A x D                     0.0000             0.0000
Pooled s.e.               1.2222             0.9521

Dietary Supplement      Dolomite            C/A *
                          (%)               (%)
A (IU/kg)   D (IU/kg)
                          1.73           [3.04.sup.d]
0                 0       2.56           [3.23.sup.d]
                500       2.58           [4.01.sup.bcd]
              1,000       2.66           [4.95.sup.abc]
              5,000       2.85           [5.71.sup.a]
1,000             0       2.14           [5.47.sup.a]
                500       2.51           [3.59.sup.d]
              1,000       2.50           [3.54.sup.d]
              5,000       1.49           [5.05.sup.ab]
100.000           0       2.00           [4.11.sup,bcd]
                500       1.39           [3.89.sup.c]
              1.000       1.89           [1.79.sup.e]
              5,000       1.51           [3.71.sup.d]
1,000,000         0       1.57           [3.89.sup.c]
                500       1.54           [4.12.sup.bed]
              1,000       1.94           [5.01.sup.abc]
              5.000
ANOVA                     0.1158          0.0000
A                         0.7243          0.0040
D                         0.8459          0.0000
A x D                     0.7818          1.0257
Pooled s.e.

[sup.a-e] Means in the same column sharing a common superscript
letter were not significantly different (P > 0.05) as determined
by Tukey's test.

* C/A (%) = (calcite/aragonite) x 100.

TABLE 3.

Concentrations of Ca, P, Mg, Fe, Zn, Cu and Sr in the shell of Haliotis
discus hannai fed the diets with different levels of vitamin A (A) and
D (D) for 152 days

 Dietary Supplement
                               Ca                    P
A (IU/kg)     D (IU/kg)      (g/kg)               (mg/kg)

0                  0       [202.67.sup.c]      [80.06.sup.cd]
                 500       [196.33.sup.c]      [80.46.sup.cd]
               1,000       [200.67.sup.c]      [81.71.sup.bcd]
               5,000       [200.51.sup.c]      [82.30.sup.bcd]
1,000              0       [201.33.sup.c]      [80.12.sup.cd]
                 500       [201.67.sup.c]      [80.52.sup.cd]
               1,000       [199.67.sup.c]      [82.40.sup.bcd]
               5,000       [200.67.sup.c]      [82.64.sup.bcd]
100,000            0       [205.33.sup.bc]     [81.20.sup.cd]
                 500       [208.21.sup.bc]     [86.71.sup.abcd]
               1,000       [224.00.sup.abc]    [89.43.sup.ab]
               5,000       [243.03.sup.ab]     [91.87.sup.a]
1,000,000          0       [205.70.sup.bc]     [80.58.sup.cd]
                 500       [211.39.sup.bc]     [86.77.sup.abcd]
               1.000       [229.60.sup.abc]    [91.13.sup.a]
               5.000       [255.05.sup.a]      [91.99.sup.a]
ANOVA
A                             0.0000             0.0016
D                             0.0006             0.0597
A x D                         0.0206             0.5728
Pooled s.e.                  19.7782             5.8589

Dietary Supplement
                              Mg             Fe
A (IU/kg)     D (IU/kg)     (mg/kg)        (mg/kg)

0                  0       296.79       [405.67.sup.b]
                 500       298.79       [405.71.sup.b]
               1,000       300.00       [402.33.sup.b]
               5,000       295.88       [410.28.sup.b]
1,000              0       299.12       [402.36.sup.b]
                 500       297.75       [407.30.sup.b]
               1,000       308.65       [414.00.sup.b]
               5,000       302.20       [401.67.sup.b]
100,000            0       303.36       [539.22.sup.a]
                 500       304.94       [543.75.sup.a]
               1,000       300.63       [539.66.sup.a]
               5,000       307.07       [561.20.sup.a]
1,000,000          0       304.27       [562.35.sup.a]
                 500       299.26       [561.58.sup.a]
               1.000       304.54       [564.31.sup.a]
               5.000       312.26       [578.83.sup.a]
ANOVA
A                            0.7926        0.0000
D                            0.4639        0.5182
A x D                        0.9544        0.9348
Pooled s.e.                 10.6968       78.0254

Dietary Supplement
                              Zn              Cu
A (IU/kg)     D (IU/kg)     (mg/kg)        (mg/kg)

0                  0        53.37       [0.49.sup.c]
                 500        54.44       [0.50.sup.c]
               1,000        53.61       [0.51.sup.c]
               5,000        58.35       [0.52.sup.c]
1,000              0        54.68       [0.56.sup.c]
                 500        55.59       [0.56.sup.c]
               1,000        54.07       [0.55.sup.c]
               5,000        53.98       [0.50.sup.c]
100,000            0        53.07       [0.61.sup.c]
                 500        53.80       [0.66.sup.bc]
               1,000        52.02       [0.69.sup.bc]
               5,000        57.23       [1.04.sup.a]
1,000,000          0        57.59       [0.57.sup.c]
                 500        58.29       [0.62.sup.bc]
               1.000        59.05       [0.62.sup.bc]
               5.000        60.45       [0.83.sup.ab]
ANOVA
A                            0.2287      0.0000
D                            0.2767      0.0000
A x D                        0.9534      0.0002
Pooled s.e.                  4.1823      0.1520

Dietary Supplement
                              Sr
A (IU/kg)     D (IU/kg)     (g/kg)

0                  0         1.81
                 500         1.84
               1,000         1.90
               5,000         1.84
1,000              0         1.87
                 500         1.93
               1,000         1.86
               5,000         2.02
100,000            0         1.95
                 500         1.87
               1,000         2.03
               5,000         1.94
1,000,000          0         1.87
                 500         1.96
               1.000         1.97
               5.000         1.93
ANOVA
A                            0.6437
D                            0.8578
A x D                        0.9888
Pooled s.e.                  0.1846

[sup.a-d] Means in the same column sharing a common superscript letter
were not significantly different (P > 0.05) as determined by Tukey's
test.

TABLE 4.

Apparent amino acid contents of the soluble matrix protein (SMP) from
the shell of Haliotis discus hannai fed diets with different levels of
vitamin A (A) and D (D) for 152  days (a)

 Dietary Supplement                 Amino Acid (Mole %)

A (IU/kg)   D (IU/kg)    Asx    Glx    Ser    Thr    Gly    Ala

0                    0   30.5    4.3   10.2    0.6   20.4    0.9
                   500   30.8    5.0    9.5    0.6   20.6    0.8
                 1,000   30.3    4.6    9.5    0.7   21.0    0.8
                 5,000   31.0    4.7    9.2    0.4   20.7    0.6
1,000                0   30.6    4.0    9.7    0.6   19.6    0.5
                   500   30.9    4.8   10.0    0.8   20.0    0.8
                 1,000   31.0    4.3    9.2    0.6   20.8    1.0
                 5.000   31.0    4.1    9.1    0.7   20.9    0.6
100.000              0   31.5    4.8    9.2    0.6   22.8    0.6
                   500   30.2    5.0    9.2    0.6   20.0    0.7
                 1,000   30.8    4.3    9.8    0.6   20.4    0.7
                 5,000   30.7    4.0    9.0    0.6   19.6    0.9
1,000,000            0   30.2    5.1    9.6    0.7   20.7    0.8
                   500   30.0    5.0    9.3    0.6   21.5    0.7
                 1.000   30.1    4.8    9.4    0.6   21.0    0.7
                 5,000   30.8    4.7    9.4    0.6   20.4    0.7

 Dietary Supplement                    Amino Acid (Mole %)

A (IU/kg)   D (IU/kg)    Pro    Val    Arg    Met     Ile    Leu

0                    0    ND     3.8    3.5    ND     1.6    1.7
                   500    ND     4.6    3.0    ND     1.5    1.3
                 1,000    ND     4.2    3.2    ND     1.5    1.4
                 5,000    ND     5.3    3.5    ND     1.2    1.6
1,000                0    ND     6.4    3.6    ND     1.5    1.5
                   500    ND     3.2    3.8    ND     2.0    1.5
                 1,000    ND     3.5    3.5    ND     1.6    1.7
                 5.000    ND     4.7    3.5    ND     1.2    1.3
100.000              0    ND     2.9    3.7    ND     1.7    1.2
                   500    ND     4.0    4.0    ND     1.2    1.7
                 1,000    ND     3.9    3.8    ND     1.5    2.0
                 5,000    ND     6.1    3.4    ND     1.3    1.2
1,000,000            0    ND     4.3    3.0    ND     1.8    1.3
                   500    ND     5.2    3.0    ND     1.3    1.0
                 1.000    ND     5.1    3.6    ND     1.7    1.4
                 5,000    ND     5.1    3.6    ND     1.7    1.1

 Dietary Supplement                 Amino Acid (Mole %)

A (IU/kg)   D (IU/kg)    Phe    Lys    His    Tyr    A/B (b)

0                    0   11.6    7.5    1.5    2.3    2.8
                   500   11.3    7.3    1.5    2.4    3.0
                 1,000   11.5    7.1    1.4    2.2    3.0
                 5,000   11.7    7.0    1.2    2.1    3.1
1,000                0   11.2    7.0    0.8    2.3    3.0
                   500   11.5    7.2    1.2    2.7    2.9
                 1,000   11.4    7.1    1.5    2.5    2.9
                 5.000   11.7    7.2    1.4    2.3    2.9
100.000              0   11.6    7.1    1.2    2.1    3.0
                   500   11.5    7.5    1.5    2.6    2.7
                 1,000   11.2    7.2    1.5    2.3    2.8
                 5,000   11.4    7.1    1.6    2.2    2.9
1,000,000            0   11.6    7.3    1.6    2.0    3.0
                   500   11.8    7.0    1.3    2.1    3.1
                 1.000   11.2    7.2    1.2    2.0    2.9
                 5,000   11.7    6.9    1.2    2.0    3.0

(a) Because the amount of SMP from one replicate was not enough for
analysis of amino acid composition, SMP from 3 replicates in a
treatment were pooled as one sample for RP-HPLC.

(b) A/B, acidic/basic amino acids (Asx+Glx)/(Lys+Arg+His)]; ND,
not detected.

ACKNOWLEDGMENTS

This study was financially supported by grant No. 30200215 from the National Natural Science Foundation of China (NNSFC) and grant No. 2001AA628080 and No.2004AA628100 from the National High Technology Research and Development Program of China (863 Program).

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WENBING ZHANG, KANGSEN MAI, * WEI XU, QINGHUI AI, BEIPING TAN, ZHIGUO LIUFU, HONGMING MA

The Key Laboratory of Mariculture (Ministry of Education), Ocean University of China, Qingdao 266003, People's Republic of China

* Corresponding author. Fax: [phone]; E-mail: [e-mail]