Discus Hannai (haliotis Discus Hannai Ino,.) Shell Biomineralization Mechanism Of Nutrition

A series of experiments were conducted to investigate the effects of some nutrients, which related to metabolisms of vitamin D and minerals, on the growth, metabolism and shell biomineralization in abalone Haliotis discus hannai Ino. Relationships among nutrients, the growth and shell biomineralization of abalone were studied. The current studies include the followings: (1) Interaction between vitamin A and D on the survival, growth, body compositions, metabolisms of vitamin D and minerals, ultrastructure of shells, mineralogical and chemical compositions in shells, components of the soluble matrix proteins of shell (SMP), and amino acid compositions in SMP of H. discus hannai. (2) Effects of dietary zinc on the ultrastructure of shells, mineralogical and chemical compositions in shells, components of SMP, and amino acid compositions in SMP of H. discus hannai. (3) Interaction of dietary vitamin D and phosphorus on metabolisms of vitamin D and minerals, morphological charaters of calcite and aragonite on the "flat pearl"of H. discus hannai. The results are summarized as follows: (1) A 152-day growth experiment was conducted in a re-circulated water system to investigate the interaction between vitamin A (retinol) and D (cholecalciferol) on growth and metabolic responses in abalone Haliotis discus hannai Ino. Triplicate groups of juvenile abalone (initial weight: 0.35 ±0.03 g; initial shell length: 11.31 ±0.25 mm) were fed to satiation one of 16 semi-purified diets containing 0, 1×10³, 1×10⁵, 1×10⁶ IU/kg vitamin A and 0, 500, 1×10³, 5×10³ IU/kg vitamin D in a 4 ×4 factorial design. Abalone were weight and measured the shell length on the 76th day and the 152nd day, respectively. The total specific growth rate (SGR) during the 152 days, neither the SGR in the first 76 days nor in the second 76 days, was significantly (P<0.05) influenced by the interaction between vitamin A and D. Such interaction was not significant (P>0.05) in the daily increment in shell length (DISL) during the growth experiment. Dietary vitamin A and D significantly (P<0.05) stimulated viscera 25-hydroxyvitamin D3 [25(OH)D₃] and 1α,25-dihydroxyvitamin D₃ [1α,25(OH)₂D₃] contents in a cooperative fashion. Dietary vitamin A generally increased the alkaline phosphatase (AKP) activity in viscera except the excessive supplement (1×10⁶ IU/kg), which significantly (P<0.05) decreased AKP activity. Dietary vitamin D significantly (P<0.05) increased AKP activity. Content of P, not Ca and Mg in soft body increased with dietary vitamin D supplement. Although there was no interaction, both vitamin A and D elevated the soft body Zn content. Dietary vitamin A significantly (P<0.05) improved contents of lipid and retinol in soft body and viscera, respectively. Meanwhile, dietary vitamin D significantly (P<0.05) increased contents of ash and cholecalciferol in soft body and viscera, respectively. Based on these results, interaction between vitamin A and D was expressed in various manners as different indicators were considered, though there was potential antagonism mechanism at molecular level between the two fat-soluble vitamins. Vitamin D significantly (P<0.05) increased the ratio of calcite/aragonite in shell, in the case of deficient (0 IU/kg) or excessive (1×106 IU/kg) dietary vitamin A supplement. When dietary vitamin A supplements ranged from 1×103 to 1×105 IU/kg, vitamin D significantly (P<0.05) decreased the ratio of calcite/aragonite in shell. Effects of vitamin A or /and vitamin D on concentrations of Mg, Zn and Sr in shell were not significant (P>0.05). Interaction between vitamin A and D significantly (P<0.05) elevated the Ca concentration in shell. Although the interaction between vitamin A and D was not significant (P>0.05), both of the two vitamins significantly (P<0.05) increased the P concentration in shell. Vitamin A significantly (P<0.05) stimulated the Fe concentration in shell. However, effects of vitamin D on the shell Fe concentration were not significant (P>0.05). The ultrastructure and components of the soluble matrix protein (SMP) in shell were not obviously changed by vitamin A and D. (2) A study was conducted to investigate the relationship between dietary zinc and shell biomineralization in abalone Haliotis discus hannai Ino. Seven triplicate groups of juvenile abalone (mean weight: 0.74±0.01 g; mean shell length: 16.41±0.04 mm) were fed with one of seven semi-purified diets containing graded levels of dietary zinc (5.6, 10.7, 15.1, 25.2, 34.7, 45.3 and 84.6 mg/kg). After a 16-week rearing period, shells of abalone obviously consisted of two parts: the old shell (OS) and the newly grown shell (NS). Color of the OS kept brown as that of the abalone shell at the beginning of the experiment, but the NS's turned into green. For better understanding of the effects of dietary zinc on the shell deposition, NS was separated from OS. Mineralogical (aragonite, calcite and dolomite [CaMg(CO3)2]) and chemical (Zn, Fe, Mg, Cu and Sr) compositions in shells were analyzed. Meanwhile, SMP were extracted from both OS and NS followed by electrophoresis and amino acid composition analysis. The results showed that mineralogical and chemical compositions in OS were not significantly (P>0.05) influenced by dietary zinc. In NS, however, increasing dietary zinc significantly (P<0.05) decreased aragonite contents, but significantly (P<0.05) increased calcite contents. Concentrations of Zn and Fe inNS significantly (P<0.05) increased with dietary zinc. Compared with those in SMP of OS, two additional protein bands with molecular weights at 36.6 and 24.5 kDa, respectively, were found in the SMP of NS on the electrophoretogram. There's no difference in protein components among the seven NS. Nevertheless, patterns of amino acid compositions in NS changed with the dietary zinc content. The ratio of acidic/basic amino acids positively correlated with the ratio of calcite/aragonite in NS. (3) Metabolic responses of cholecalciferol (VD3) and minerals (Ca, P and Mg) in abalone Haliotis discus hannai Ino to dietary VD3 and phosphorus (P) were investigated. Based on a 2×2 factorial design, four casein-gelatin-based diets were formulated. The basal diet was supplemented with either 0 or 2000 IU VD3/kg diet and 0 or 10 g P/kg diet. The abalone was reared in P-free artificial seawater for 55 days. Results showed that dietary VD3 was hydroxylated to 25(OH)D3 and 1α,25(OH)2D3 in abalone, and subsequently raised the serum levels of these two VD3 metabolites. Dietary P deficiency elevated serum 1α,25(OH)2D3 level only when the dietary VD3 supplementation was sufficient. The supplementations of either dietary VD3 or P significantly (P<0.05) increased the levels of P in serum, mantle and hepatopancreas, and only the addition of VD3 significantly (P<0.05) raised the concentrations of Ca in serum and mantle. Interaction between dietary VD3 and P was only found significant (P<0.05) on the concentrations of P and Mg in mantle. The concentrations of Ca, P and Mg in muscle were not significantly (P>0.05) influenced by these dietary treatments. Hence, the metabolic responses in serum, muscle, mantle and hepatopancreas of abalone to dietary VD3 and P were in different manners. At the same time of rearing experiment mentioned above, glass coverslips (D = 6 mm; H = 0.2 mm) were inserted between the mantle and the inner surface of the shell of abalone. The experiment of the flat-pearl-culture lasted 40 days. Ultrastructures of normal shells and flat pearls were analyzed with scanning electron microscopy (SEM). Results showed that minearlizated structures of flat pearls in phosphorus-supplemented treatment were in line with those of normal shells. However, nucleation, growth and organization of CaCO3 crystal on flat pearls in phosphorus-deficiency treatment were significantly (P<0.05) changed. Arrangement and morphology of calcite and aragonite were irregular. It was suggested in present study that the flat-pearl-culture was able to simulate the normal formation of shell, and could be used to study the nutritional mechanism for shell biomineralization. It was demonstrated that dietary phosphorus was essential to shell biomineralization.