Comparison of the amino acid sequences of acorn barnacle lectins showing different inhibitory activities toward the crystal growth of calcium carbonate

ABSTRACT: The amino acid sequence of a D -galactose-binding lectin isolated from the hemolymph of the acorn barnacle Balanus rostratus was determined. The lectin (BRL) ( M _r 120K) is a multimeric protein whose subunit consists of 182 amino acids. The amino acid sequence was compared with those of multiple lectins (BRA-2, BRA-3) from Megabalanus rosa to explore the relationship between the structures and the inhibitory activity toward the crystal growth of calcium carbonate. Although BRL was 46% identical to BRA-2 and 15% identical to BRA-3, the lectin had no inhibitory activity, unlike BRA-2 and BRA-3. Both the number and the localization of acidic amino acid residues and their amide forms were different among them. Observations by scanning electron microscopy revealed modifications in the size and morphology of the calcium carbonate crystals grown in the presence of the lectins.     

Structure and possible function of <Emphasis Type="Italic">N</Emphasis>-glycans of an invertebrate C-type lectin from the acorn barnacle <Emphasis Type="Italic">megabalanus rosa</Emphasis>

ABSTRACT:   A C-type lectin (BRA-2) isolated from the acorn barnacle Megabalanus rosa , which was a glycoprotein having an N -linked sugar chain, was deglycosylated by N -glycopeptidase F. The structure of the released sugar chains was determined by a 2-D mapping method after derivatization with a fluorescent reagent, 2-aminopyridine, to be Manα1-6(Manα1-3)Manβ1-4GlcNAcβ1-4(Fucα1-6)GlcNAc and Manα1-6(GlcNAcβ1-2Manα1-3)Manβ1-4GlcNAcβ1-4(Fucα1-6)GlcNAc. The structures were confirmed by matrix-assisted laser desorption ionization mass spectrometry and a comparison with authentic sugar chains by high-pressure liquid chromatography. Various properties of BRA-2 were examined before and after deglycosylation. The susceptibility of BRA-2 to protease digestion was increased by deglycosylation. However, the inhibitory activity toward calcium carbonate crystallization as well as the hemagglutinating activity of deglycosylated BRA-2 was significantly decreased. These results suggest that the sugar chains of BRA-2 are important to both its structural stability and its function.     

Purification and characterization of a matrix shell protein from the shell of scallop <Emphasis Type="Italic">Patinopecten yessoensis</Emphasis>

ABSTRACT:   A new protein named MSP-SC (matrix shell protein from scallop) with a molecular weight of 14 kDa was isolated from the shell of a scallop, Patinopecten yessoensis , using gel filtration column chromatography, ion exchange column chromatography, and reverse phase C4 column chromatography. A comparison of the known protein sequences with the N-terminal sequence of MSP-SC showed that the protein sequence of MSP-SC was novel. Immunohistochemistry using a polyclonal antibody against MSP-SC showed that MSP-SC is expressed in the mantle pallial cell layer but not in the muscle tissue, and showed a punctate distribution along the horizontal calcified layer in the shell. The isolated MSP-SC inhibited the formation of calcium carbonate (CaCO₃) crystals in a dose-dependent manner. The CaCO₃ crystals grown in the presence of a lower concentration of MSP-SC were much larger and aggregated when compared with those formed in the absence of MSP-SC. In addition, the crystal had a radial and not cubical morphology. These results suggest that MSP-SC regulates the formation and the morphology of CaCO₃ crystals in the shell. Moreover, its ability to aggregate CaCO₃ crystals and its localization along the horizontal calcified layer in the shell suggest that MSP-SC may serve to connect the CaCO₃ layers in the scallop shell.     

Effects of acidity and a metabolic inhibitor on incorporation of calcium and inorganic carbon into endolymph and otoliths in salmon Oncorhynchus masou

ABSTRACT:   To investigate the mechanisms of calcium carbonate formation of teleost otoliths, using the double labeling technique of ⁴⁵Ca²⁺ and H¹⁴CO₃^–, the effects of tissue elimination, a metabolic inhibitor (cyanide) and acidity on the incorporation of calcium and inorganic carbon into endolymph and otoliths were examined. Incorporation of calcium and inorganic carbon into otoliths was decreased to 36% and 45%, respectively, by removing the sacculus. In contrast, cyanide inhibited incorporation of inorganic carbon and calcium into endolymph and otoliths in a concentration-dependent manner. Acidity in the incubation medium decreased the accumulation of inorganic carbon within the endolymph and otoliths. Calcium incorporation was not affected by variation of pH in the medium. These results indicate that calcium and bicarbonate are actively transported to endolymph via the sacculus, and it is suggested that the transport is involved in the oversaturation state for calcium carbonate in the endolymph and regulates subsequent calcification of otoliths.     

Effects of dietary guaiacol on shell biomineralization of juvenile abalone Haliotis discus hannai, Ino

A study was conducted to investigate the effects of guaiacol on shell biomineralization of abalone Haliotis discus hannai Ino. Seven semi-purified diets were formulated with graded levels of guaiacol (0, 5, 10, 50, 100, 500 and 1000 mg kg⁻¹). Abalone (initial weight: 0.29±0.01 g; initial shell length: 8.55±0.27 mm) were fed with these diets in a re-circulated water system for 152 days. The results showed that the contents of calcite in the shell increased significantly with dietary guaiacol levels, but the aragonite content decreased significantly. Dietary guaiacol significantly decreased the contents of calcium and strontium, but significantly increased the magnesium content in the shell. The effects of dietary guaiacol on the contents of phosphorus and zinc in shell were not significant. It was obvious that the ratio of potential acidic/basic amino acids [(Asx+Glx)/(Lys+Arg+His)] in the shell was elevated by dietary guaiacol. In conclusion, dietary guaiacol has effects on both the mineralogy and the amino acid composition in the shell. Furthermore, changes in mineralogy are related to the ratio of (Asx+Glx)/(Lys+Arg+His) in the shell.     

Phosphorus and calcium requirements in the diet of the American cichlid Cichlasoma urophthalmus (Günther)

An experiment was carried out with Cichlasoma urophthalmus (Günther) juveniles to determine the phosphorus requirement and its interaction with dietary calcium. Twelve isoenergetic and isoproteic diets were prepared using a basal artificial diet containing vitamin-free casein, dextrin, starch, corn oil, fish oil, vitamin mixture and a mineral mixture free of calcium and phosphorus. Calcium and phosphorus levels were determined in the casein. To the basal diets were added different concentrations of phosphorus as potassium monophosphate (0.5, 1.0, 1.5 and 2.5 g kg^–1) and calcium as calcium carbonate (0.5, 0.75, 1.0, 1.5, 2.0, 2.5, 3.0 and 4.0 g kg^–1). These concentrations resulted in varying Ca–P ratios (1:1, 1.33:1, 1.5:1, 1.6:1 and 2.0:1). Calcium and phosphorus concentrations in the water were 84 mg kg^–1 and 0.003 mg kg^–1, respectively. The diet with 0.5 g kg^–1 phosphorus resulted in deficiency signs such as reduced growth, high conversion ratio, high fat content and low bone mineralization. Increased levels of dietary calcium and phosphorus both gave improved growth and mineralization. Mineralization continued to increase with dietary phosphorus levels above that required for maximum growth. The optimum level of phosphorus in the diet was 1.5 g kg^–1, the optimum calcium level was 1.8 g kg^–1 and the optimum Ca–P ratio was 1.3. Carcass lipid levels were inversely related to dietary phosphorus.     

凡纳滨对虾C型凝集素（LvLc1）的特征和活性分析

C型凝集素是一种依赖于Ca~(2+)而发挥功能的糖蛋白,在一线的固有免疫防御过程中发挥着重要作用。围绕对虾C型凝集素开展深入研究,不仅可以丰富无脊椎动物固有免疫学内容,还有望将其开发为具有免疫增强效果的活性饵料,应用于对虾的健康养殖。本实验根据实验室前期转录组信息提示克隆获得了凡纳滨对虾一种新的C型凝集素基因(LvLc1,Gen Bank注册号:KY937940)。生物信息学分析显示LvLc1基因的开放阅读框全长891 bp,编码296个氨基酸,该基因编码的蛋白质含有一个保守的糖识别结构域(carbohydrate recognition domain,CRD),该结构域中具有潜在的半乳糖结合位点(QPD motif),进化发生分析显示LvLc1与来自节肢动物的甘露糖结合凝集素家族成员聚类在一起。对LvLc1基因的CRD结构域进行了原核重组表达与蛋白活性分析研究,结果显示:重组目的蛋白(rLvLc1)在Ca~(2+)存在的条件下,对多种病原菌(G~+、G~–和真菌)具有凝集作用,其凝集活性可被半乳糖、甘露糖、脂多糖等多种病原相关分子模式所抑制。研究表明,LvLc1作为C-型凝集素家族一个新成员,可能通过重要的模式识别受体作用,参与机体应答病原微生物侵染的防御过程。     

中国明对虾C 型凝集素基因(Fclectin)的重组表达及活性分析

研究拟通过分析对虾C型凝集素的活性特点,探讨其在对虾先天免疫应答过程中的潜在功能以及在养殖生产实践中的应用。实验利用原核表达系统对中国明对虾C-型凝集素基因的两个串联的糖识别结构域(carbohydrate recognition domain,CRD)进行了重组表达,并通过纯化复性获得了重组目的蛋白(rFclectin-CRD1和rFclectin-CRD2)。活性分析结果显示,重组目的蛋白对多种病原菌有凝集和抑制生长的作用,并且具有Ca2+依赖活性;其凝集活性可被半乳糖、肽聚糖、脂多糖等多种病原相关分子模式所抑制,研究结果证实,Fclectin是一种典型的C-型凝集素,它可能作为中国明对虾先天免疫中重要的模式识别受体,在一定程度上参与了机体应答病原微生物的防御过程。     

Effects of Reduced Carbonate Saturation State on Early Development in the Common Edible Sea Urchin Lytechinus variegatus: Implications for Land-Based Aquaculture

Land-based aquaculture facilities often utilize additional bicarbonate sources such as commercial sea salts that are designed to boost alkalinity in order to buffer seawater against reductions in pH. Despite these preventative measures, many facilities are likely to face occasional reductions in pH and corresponding reductions in carbonate saturation states due to the accumulation of metabolic waste products. We investigated the impact of reduced carbonate saturation states (Ω_Ca, Ω_Ar) on embryonic developmental rates, larval developmental rates, and echinoplutei skeletal morphometrics in the common edible sea urchin Lytechinus variegatus under high alkalinity conditions. Commercial artificial seawater was bubbled with a mixture of air and CO₂ gas to reduce the carbonate saturation state. Rates of embryonic and larval development were significantly delayed in both the low and extreme low carbonate saturation state groups relative to the control at a given time. Although symmetry of overall skeletal body lengths was not affected, allometric relationships were significantly different between treatment groups. Larvae reared under ambient conditions had significantly greater postoral arm and overall body lengths relative to body lengths than larvae grown under extreme low carbonate saturation state conditions, indicating that extreme changes in the carbonate system affected not only developmental rates but also larval skeletal shape. Reduced rates of embryonic development and delayed and altered larval skeletal growth are likely to negatively impact larval culturing of L. variegatus in land-based, intensive culture situations where calcite and aragonite saturation states are lowered by the accumulation of metabolic waste products.     

Digestive peptidases and proteinases in the midgut gland of the pink shrimp Farfantepenaeus paulensis (Crustacea, Decapoda, Penaeidae)

Proteases from the midgut gland of the Farfantepenaeus paulensis juveniles were assessed. Enzyme activity was determined using protease substrates and inhibitors. The effect of pH, temperature and calcium on proteolytic activity was assayed. Caseinolytic activity was analysed in substrate-sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). Trypsin, chymotrypsin and leucine aminopeptidase activity was detected. Proteolytic activity was strongly inhibited by the specific trypsin inhibitors. Tosyl-phenylalanine chloromethyl ketone inhibited 59.3% of chymotrypsin activity. The greatest trypsin-like activity occurred at pH 8.0 and 45 °C. Chymotrypsin-like activity reached maximal values at alkaline pH (7.2–9.0) and 55 °C. CaCl₂ did not increase trypsin-like activity, but rather inhibited it at concentrations of 30 (20%), 50 (30%) and 100 mM (50%). The substrate-SDS-PAGE zymogram revealed eight proteinase bands. Two possibly thermal-resistant (85 °C, 30 min) chymotrypsin isoforms were found, which were inhibited by phenyl-methyl-sulphonyl-fluoride. Aminopeptidase activity of enzyme extracts (Arg, Leu, Lys, Phe and Val) and the recommended concentrations of these essential amino acids in penaeid shrimp diets were positively correlated ( P <0.05). Beause protein digestion involves the combined action of different enzymes, adequate knowledge of shrimp digestion and enzyme characteristics is required for the assessment of the digestive potential of different feed sources and development of in vitro digestibility protocols.     

3种海产螺壳体相组成及结构特征分析

研究了3种不同产地的海螺—香螺、凤螺和芋螺壳的成份及相组成。结果表明:产自温带海域的香螺壳由方解石和文石共同构成,其不同部位组成相不同;产自热带海域的芋螺和凤螺均由单一的文石相组成。     

淡水养殖珍珠棱柱层的微结构形貌特征

通过x射线粉晶衍射（XRD）、场发射扫描电镜（FE—SEM）对不同颜色的淡水养殖珍珠的微结构进行较系统研究。结果表明,质优的珍珠其矿物相为文石,未见有方解石或球文石相,不同颜色的珍珠质层文石板片的厚度存在较大差异。质量较好的珍珠中接近珍珠核心区域存在棱柱层,该棱柱层由众多的纤维或条带状文石结晶体聚集而成,其中文石纤维或条带的定向近平行于珍珠层文石板片。此外,首次发现在淡水养殖珍珠的棱柱层中文石纤维与板片结晶体交替出现,同时初步建立了一种新的淡水养殖珍珠的内部结构模型。     

Purification and characterization of a lectin from the starfish Asterias amurensis

In this study, we describe a novel lectin (designated AMUL) from the Northern Pacific starfish Asterias amurensis. AMUL was purified from the coelomic fluid by affinity chromatography followed by size-exclusion chromatography. The native molecular mass of the lectin was found to be approximately 270 kDa by size-exclusion chromatography using a Superdex 200 column. AMUL showed distinct bands at approximately 14 or 18 kDa on SDS-PAGE under nonreducing or reducing conditions, respectively. The hemagglutination activity of AMUL against rabbit erythrocytes was strongly inhibited by the two sialic acids N-acetylneuraminic acid and N-glycolylneuraminic acid. The glycoproteins tested showed no inhibition of hemagglutination. The first 28 amino acid residues of AMUL were determined by automated Edman degradation, showing that AMUL has considerable sequence homology with C-type lectins from other echinoderms. These results show that AMUL is a novel echinoderm-derived sialic-acid-specific lectin that belongs to the C-type lectin superfamily.     

Pearl formation in the Japanese pearl oyster (Pinctada fucata) by CaCO3 polymorphs: Pearl quality‐specific biomineralization processes and their similarity to shell regeneration

Pearl oyster shell consists of two layers: a calcite prismatic layer (outer layer) and an aragonite nacreous layer (inner layer). Calcite and aragonite are CaCO₃ polymorphs, and their formations are controlled by shell‐forming tissue called mantle. Pearl sacs originating in the mantle form cultured pearls. Therefore, it has been widely accepted that pearl and shell are produced by the same process. However, this idea has been called into question by some recent mineralogical studies indicating microstructural and crystal‐polymorphic diversity in pearls. The pearl biomineralization process is still not fully understood in detail. Thus, in this study, we focused on the diversity of CaCO₃ polymorphism of non‐nacreous structures (NNSs) underlying the nacreous layer in pearl and regenerated shell, to reveal the biomineralization process of the Japanese pearl oyster (Pinctada fucata). Using Meigen's stain and scanning electron microscope‐energy dispersive X‐ray (SEM‐EDX), NNSs polymorphs in valuable and valueless pearls, in addition to regenerated shell, were compared. Aragonite was exclusively observed in the NNSs of valuable pearls, whereas calcite was dominant in those of valueless pearls. The same analysis of NNSs of regenerated shells was carried out. As in valueless pearls, almost all regenerated shell NNSs consisted of calcite, but one NNS was composed of aragonite. Accordingly, it seems that pearls are formed by the same biomineralization process as shell regeneration rather than shell formation.     

Larval calcification and growth of veligers to early pediveliger of the queen conch Strombus gigas in mesocosm and laboratory conditions

The goals of this study were to evaluate growth, development, and calcification process of veligers of Strombus gigas grown in natural conditions in mesocosm versus laboratory conditions. In this study, larvae bred in mesocosm conditions had a good growth rate (33.3±12.40 μm.day^?1) when fed with natural phytoplankton in natural flowing seawater versus a lower growth rate of 8.8±5.20 μm.day^?1 for larvae reared in laboratory conditions and fed with a monoalgal diet of Nannochloropsis oculata. Physicochemical parameters did not explain the difference on larval growth in both culture systems according to the principal component analysis done. Raman microspectrometry carried out on conch larvae grown in mesocosm and lab conditions allowed us to emphasize the biosynthesis of calcium carbonate species and their structure type evolution (amorphous, aragonite, or calcite structure) as a function of the breeding time by detecting and identifying in the spectra the characteristic Raman bands of CO₃ chemical groups and lattice vibrations. This analytical method seems to indicate that crystalline CaCO₃ structures are not detected in the recorded spectra on larvae reared in laboratory conditions during the early stages of the shell construction of 1 to 8 days. For this reason, light and current flow have also been considered in the discussion that can help to explain the differences found in this study. Contrarily, the Raman spectra acquired on larvae grown in mesocosms exhibit characteristic bands of aragonite (CO₃ double peak at 697–701 cm^?1 and lattice vibrations at 170 and 220 cm^?1) from the third day of breeding. The best shell growth and shell calcification pointed out in larvae grown in mesocosm compared to laboratory conditions are probably due to the nutrient amounts present in the food available in mesocosm.

     

Carbonate mineral filtrants with new surfaces reduce alkalinity in seawater and artificial seawater: Preliminary findings

Silica sand (silica), coral (aragonite), and oyster shell (calcite) were ground to similar particle sizes and placed in seawater and artificial seawater (GP2 Medium). Alkalinity and pH values of the artificial seawater decreased substantially over 24 h when in contact with coral and oyster shell; the effects in seawater were minor. Once alkalinity has been reduced, the maintenance of stable pH at values typical of seawater is made more difficult. The data, which are preliminary, have practical application.     

Chemical and Physical Properties of Shrimp Pond Bottom Soils in Ecuador

Chemical and physical analyses were conducted on bottom soil samples from 74 brackish-water ponds representing 40 shrimp farms in Ecuador. Most ponds had soils with pH > 6 and total carbon concentrations < 2.5%. Carbon was mostly in organic form, for the average concentration of carbonate carbon was 0.06%. The C: N ratio was 8 to 10 in soils with < 2.5% carbon. In ponds built in former mangrove areas, soil carbon was > 2.5% and C: N ratios were 25 to 30. Ponds soils in former mangrove areas also tended to be high in total sulfur and low in pH. Lack of correlation between carbon and sulfur in mangrove soils suggested that most of the sulfur was inorganic and presumably in sulfides. Soils containing above 0.4% free carbonate (as equivalent CaCO₃) had pH values > 7. Although carbonate concentration was a major factor controlling soil pH, calcium hardness of pond waters was strongly influenced by salinity (and calcium) in the water supply. Total phosphorus concentrations averaged 898 mg/kg, and dilute acid extractable phosphorus usually accounted for 25–35% of the total. Concentrations of major cations and minor elements varied greatly in soils and exhibited ranges of up to three orders of magnitude. Contrary to opinions of shrimp producers, many pond soils in Ecuador are not acidic and few soils have a high organic matter content. Proper use of soil and water testing could greatly improve the efficiency of liming and other soil management practices.     

Effect of dietary supplementation of calcium chloride on growth,survival, moulting frequency and body composition of narrow‐clawed crayfish,Astacus leptodactylus (Eschscholtz, 1823)

The effects of dietary calcium chloride (CaCl₂) concentration on the growth, survival, moulting and body composition of Astacus leptodactylus were studied. Diets were prepared using supplementation of 0 (control), 30, 60 and 120 g kg^?1 calcium chloride commercial trout larvae diet containing 50% protein and 12% lipid. Astacus leptodactylus larvae with an average total length of 22.0 ± 0.05 mm (TL) and weight of 0.53 ± 0.01 g obtained from eighteen ovigerous females collected from Seydisehir Sugla Dam lake in Konya, Turkey, were stocked in 0.2‐m² aquariums at a rate of 50 crayfish m^?2 and reared for 90 days. The experiments included four treatments (diets) with three replicates each. Calcium chloride exerted positive effects on growth, moulting frequency, feed efficiency and survival of narrow‐clawed crayfish. Crayfish fed with 60 g calcium kg^?1 calcium chloride‐supplemented diet exhibited the highest weight gain and specific growth rate (SGR) but the lowest survival rate (63.3%) (P < 0.05). There were no significant differences (P > 0.05) in mean moisture (803.5 g kg^?1) and protein content (177.0 g kg^?1) in crayfish tail meat. However, lipid values were between 3.8 and 11.6 g kg^?1 and significantly different among the diets (P < 0.05). Diet with 60 g kg^?1 calcium chloride is recommended for the best growth of freshwater crayfish.     

Roles of zinc and selenium in protecting sea cucumber Apostichopus japonicus coelomocytes against lipopolysaccharide‐induced oxidative stress in vitro

This study investigated the attenuate effects of zinc (Zn) and selenium (Se) on lipopolysaccharide (LPS)‐induced oxidative stress in sea cucumber Apostichopus japonicus coelomocytes in vitro. Coelomocytes were first treated with different concentrations of Zn (0.12, 0.48 and 1.2 mM) and Se (0.06, 0.24 and 0.6 mM) for 12 h and the optimal concentrations of Zn and Se as antioxidants for A. japonicus coelomocytes were selected based on antioxidant parameters including total antioxidant capacity (T‐AOC) and activities of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), glutathione‐S‐transferase (GST) and concentration of reduced glutathione (GSH). Next, coelomocytes were pretreated with 0 (control), 0.12 mM Zn or 0.24 mM Se for 12 h, and then were treated with LPS (100 μg mL^?1) for 6 h. Se completely inhibited LPS‐induced increase in superoxide anion () production and lipid peroxidation (LPO) and effectively prevented the LPS‐induced decreases of T‐AOC, activities of SOD, CAT, GPx and GST and concentration of GSH. Zn alleviated the LPS‐induced oxidative stress but the protective effects were not as effective as Se. The present work also proved that suboptimal amount of Zn and Se could impair the antioxidant system of A. japonicus coelomoytes. In conclusion, the present work demonstrated that both Zn and Se, especially Se, have the potentials to be the effective antioxidants for A. japonicus. Further work will be conducted for their optimal administration concentrations in vivo.