Shell Matrix Protein N38 of Pinctada fucata, Inducing Vaterite Formation, Extends the DING Protein to the Mollusca World.

In the animal kingdom, DING proteins were only found in Chordata and Aschelminthes. At present study, a potential DING protein, matrix protein N38, was isolated and purified from the shell of Pinctada fucata. Tandem mass spectrometry analysis revealed that 14 peptide segments matched between N38 and human phosphate-binding protein (HPBP). HPBP belongs to the DING protein family and has a "DINGGG-" sequence, which is considered a "signature" of HPBP. In this study, the mass spectrometry analysis results showed that N38 had a "DIDGGG-" sequence; this structure is a mutation from the "DINGGG-" structure, which is a distinctive feature of the DING protein family. The role of N38 during calcium carbonate formation was explored through the in vitro crystallization experiment. The results of scanning electron microscopy and Raman spectrum analysis indicated that N38 induced vaterite formation. These findings revealed that N38 might regulate and participate in the precise control of the crystal growth of the shell, providing new clues for biomineralization mechanisms in P. fucata and DING protein family studies. In addition, this study helped extend the research of DING protein to the Mollusca world.

Immobilization of Rhodopseudomonas palustris P1 on glass pumice to improve the removal of NH4 + -N and NO2 - -N from aquaculture pond water.

We conducted this research in order to investigate the potential of a new material called glass pumice for use as a microorganism immobilization carrier to improve aquaculture pond water quality. The pH adjustment capacity and the Rhodopseudomonas palustris P1 cell adsorption capacity of glass pumice were measured. The immobilized Rps. palustris P1 and the free sample were compared to determine which had an enhanced NH4 + -N and NO2 - -N removal efficiency. The results showed that glass pumice significantly affected the pH of the acid solution (P < 0.05); the pH increased from 3.0 ± 0.08 to 7.21 ± 0.13 in 12 H. Rps. palustris P1 adsorption to glass pumice was rapid and reached equilibrium within 60 Min. The Langmuir adsorption parameter data showed that glass pumice had a higher affinity for Rps. palustris P1 than SiO2 powder, with an adsorption capacity of 4.02 × 108  cells g-1 . The maximum NH4 + -N and NO2 - -N removal rates by immobilized Rps. palustris P1 were 134.82 ± 0.67% and 93.68 ± 0.14% higher than those of nonimmobilized P1, respectively. Based on the above results, we propose that glass pumice is potential as a microorganism carrier material in aquaculture water treatment.

Design and Application of a Solar Mobile Pond Aquaculture Water Quality-Regulation Machine Based in Bream Pond Aquaculture.

Bream pond aquaculture plays a very important role in China's aquaculture industry and is the main source of aquatic products. To regulate and control pond water quality and sediment, a movable solar pond aquaculture water quality regulation machine (SMWM) was designed and used. This machine is solar-powered and moves on water, and its primary components are a solar power supply device, a sediment lifting device, a mechanism for walking on the water's surface and a control system. The solar power supply device provides power for the machine, and the water walking mechanism drives the machine's motion on the water. The sediment lifting device orbits the main section of the machine and affects a large area of the pond. Tests of the machine's mechanical properties revealed that the minimum illumination necessary for the SMWM to function is 13,000 Lx and that its stable speed on the water is 0.02-0.03 m/s. For an illumination of 13,000-52,500 Lx, the sediment lifting device runs at 0.13-0.35 m/s, and its water delivery capacity is 110-208 m(3)/h. The sediment lifting device is able to fold away, and the angle of the suction chamber can be adjusted, making the machine work well in ponds at different water depths from 0.5 m to 2 m. The optimal distance from the sediment lifting device to the bottom of the pond is 10-15 cm. In addition, adjusting the length of the connecting rod and the direction of the traction rope allows the SMWM to work in a pond water area greater than 80%. The analysis of water quality in Wuchang bream (Parabramis pekinensis) and silver carp (Hypophthalmichthys molitrix) culture ponds using the SMWM resulted in decreased NH3(+)-N and available phosphorus concentrations and increased TP concentrations. The TN content and the amount of available phosphorus in the sediment were reduced. In addition, the fish production showed that the SMWM enhanced the yields of Wuchang bream and silver carp by more than 30% and 24%, respectively. These results indicate that the SMWM may be suitable for Wuchang bream pond aquaculture in China and that it can be used in pond aquaculture for regulating and controlling water quality.

Vertical Segregation and Phylogenetic Characterization of Ammonia-Oxidizing Bacteria and Archaea in the Sediment of a Freshwater Aquaculture Pond.

Pond aquaculture is the major freshwater aquaculture method in China. Ammonia-oxidizing communities inhabiting pond sediments play an important role in controlling culture water quality. However, the distribution and activities of ammonia-oxidizing microbial communities along sediment profiles are poorly understood in this specific environment. Vertical variations in the abundance, transcription, potential ammonia oxidizing rate, and community composition of ammonia-oxidizing bacteria (AOB) and ammonia-oxidizing archaea (AOA) in sediment samples (0-50 cm depth) collected from a freshwater aquaculture pond were investigated. The concentrations of the AOA amoA gene were higher than those of the AOB by an order of magnitude, which suggested that AOA, as opposed to AOB, were the numerically predominant ammonia-oxidizing organisms in the surface sediment. This could be attributed to the fact that AOA are more resistant to low levels of dissolved oxygen. However, the concentrations of the AOB amoA mRNA were higher than those of the AOA by 2.5- to 39.9-fold in surface sediments (0-10 cm depth), which suggests that the oxidation of ammonia was mainly performed by AOB in the surface sediments, and by AOA in the deeper sediments, where only AOA could be detected. Clone libraries of AOA and AOB amoA sequences indicated that the diversity of AOA and AOB decreased with increasing depth. The AOB community consisted of two groups: the Nitrosospira and Nitrosomonas clusters, and Nitrosomonas were predominant in the freshwater pond sediment. All AOA amoA gene sequences in the 0-2 cm deep sediment were grouped into the Nitrososphaera cluster, while other AOA sequences in deeper sediments (10-15 and 20-25 cm depths) were grouped into the Nitrosopumilus cluster.

Molecular cloning and mRNA expression of peroxiredoxin gene in black tiger shrimp (Penaeus monodon).

The techniques of homology cloning and anchored PCR were used to clone the peroxiredoxin (Prx) gene from black tiger shrimp (Penaeus monodon). The full length cDNA of black tiger shrimp Prx (PmPrx) contained a 5' untranslated region (UTR) of 51 bp, an ORF (open reading frame) of 582 bp encoding a polypeptide of 193 amino acids with an estimated molecular mass of 22.15 kDa and a 3' UTR of 948 bp. Sequence comparison showed that PmPrx shared higher identities with Prx IVs than that with other isoforms of Prx, indicating PmPrx was a member of the Prx IV family. A quantitative reverse transcriptase Real-Time PCR (qRT-PCR) assay was developed to assess the mRNA expression of PmPrx in different tissues and the temporal expression of PmPrx in the hepatopancreas challenged by lipopolyssacharide (LPS). Higher-level mRNA expression of PmPrx was detected in the tissues of hepatopancreas, gonad and heart. The expression of PmPrx in the hepatopancreas was up regulated after stimulated by LPS. The results indicated that PmPrx was a constitutive and inducible expressed protein and could be induced by LPS.

Calcineurin mediates the immune response of hemocytes through NF-kappaB signaling pathway in pearl oyster (Pinctada fucata).

Calcineurin (CN), a multifunctional protein, mediates the immune response through diverse signaling pathways in mammals, while the function of CN in the immune response of molluscan hemocytes still remains unclear. In the present study, we detected the distribution of CN in various tissues and the expression levels of Pf-CNA and Pf-CNB gene in hemocytes of Pinctada fucata. After the preparation of hemocyte monolayers, we checked the response of enzymatic activity of CN, the degradation level of IkappaBalpha, the activity of iNOS and the production of NO, and IL-2 to the challenge of lipopolysaccharide (LPS) and cyclosporin A (CsA). CN activity in hemocytes was very sensitive to both the stimulation of LPS and the inhibition of CsA. Most importantly, IkappaBalpha degradation in hemocytes was induced by LPS and attenuated by CsA. Consequently, the activity of iNOS was elevated and the production of NO was increased. Additionally, we found that the synthesis of IL-2 was increased by LPS but was apparently weakened by CsA. In vivo bacterial clearance experiments showed that CsA significantly decreased the ability of in vivo bacteria clearance in pearl oyster. All the results revealed, for the first time, that CN mediated the immune response of molluscan hemocytes via activating NF-kappaB signaling pathway.

Immunolocalization of matrix proteins in nacre lamellae and their in vivo effects on aragonitic tablet growth.

How matrix proteins precisely control the growth of nacre lamellae is an open question in biomineralization research. Using the antibodies against matrix proteins for immunolabeling and in vivo experiments, we investigate the structural and functional roles of EDTA-soluble matrix (SM) and EDTA-insoluble matrix (ISM) proteins in nacre biomineralization of the pearl oyster Pinctada fucata. Immunolabeling reveals that a SM protein, nacrein, distributes within aragonitic tablets and intertabular matrix. An ISM protein, which we named P43, has been specifically recognized by polyclonal antibodies raised against the recombinant protein of P. fucata bone morphogenetic protein 2 in immunoblot analysis. Immunolabeling indicates that P43 is localized to interlamellar sheet, and also embedded within aragonitic tablets. Although nacrein and P43 both distribute within aragonitic tablets, they function differently in aragonitic tablet growth. When nacrein is suppressed by the antibodies against it in vivo, crystal overgrowth occurs, indicating that this SM protein is a negative regulator in aragonitic tablet growth. When P43 is suppressed in vivo, the organo-mineral assemblage is disrupted, suggesting that P43 is a framework matrix. Taken together, SM and ISM proteins are indispensable factors for the growth of nacre lamellae, controlling crystal growth and constructing the framework of aragonitic tablets.

The inner-shell film: an immediate structure participating in pearl oyster shell formation.

In mollusks, the inner shell film is located in the shell-mantle zone and it is important in shell formation. In this study, we found that the film was composed of two individual films under certain states and some columnar structures were observed between the two individual films. The inner shell film was separated with the process of ethylenediaminetetraacetic acid (EDTA) treatment and the film proteins were extracted. Amino acid analysis showed that the film proteins may consist of shell framework proteins. The calcite crystallization experiment showed that the film proteins could inhibit the growth of calcite, while the CaCO(3) precipitation experiment showed that the film proteins could accelerate the rate of CaCO(3) precipitation. All these results suggested that the film plays an important role in shell formation. It may facilitate the aragonite formation by inhibiting the growth of calcite and accelerate the shell growth by promoting the precipitation of CaCO(3) crystals.

Characterization of calcium deposition and shell matrix protein secretion in primary mantle tissue culture from the marine pearl oyster Pinctada fucata.

In this study, we established and characterized a long-term primary mantle tissue culture from the marine pearl oyster Pinctada fucata for in vitro investigation of nacre biomineralization. In this culture system, the viability of mantle tissue cells lasted up to 2 months. The tissue cells were demonstrated to express nacre matrix proteins by RT-PCR, and a soluble shell matrix protein, nacrein, was detected in the culture medium by Western blot analysis. On the other hand, 15 days after initiating culture, a large amount of calcium deposits with major elements, including calcium, carbon, and oxygen, were generated in the mantle explants and cell outgrowth area. The quantity and size of calcium deposits increased with the prolonged cultivation, and their location and nanogranular structure suggested their biogenic origin. These calcium deposits specifically appeared in mantle tissue cultures, but not in heart tissue cultures. Taken together, these results demonstrate that the mantle tissue culture functions similarly to mantle cells in vivo. This study provides a reliable approach for the further investigation on nacre biomineralization at the cellular level.

Biomineralization: functions of calmodulin-like protein in the shell formation of pearl oyster.

Calmodulin-like protein (CaLP) was believed to be involved in the shell formation of pearl oyster. However, no further study of this protein was ever performed. In this study, the in vitro crystallization experiment showed that CaLP can modify the morphology of calcite. In addition, aragonite crystals can be induced in the mixture of CaLP and a nacre protein (at 16 kDa), which was detected and purified from the EDTA-soluble matrix of nacre. These results agreed with that of immunohistological staining in which CaLP was detected not only in the organic layer sandwiched between nacre (aragonite) and the prismatic layer (calcite), but also around the prisms of the prismatic layer. Take together, we concluded that (1) CaLP, as a component of the organic layer, can induce the nucleation of aragonite through binding with the 16-kDa protein, and (2) CaLP may regulate the growth of calcite in the prismatic layer.

A novel extrapallial fluid protein controls the morphology of nacre lamellae in the pearl oyster, Pinctada fucata.

Mollusk shell nacre is known for its superior mechanical properties and precisely controlled biomineralization process. However, the question of how mollusks control the morphology of nacre lamellae remains unresolved. Here, a novel 38-kDa extrapallial fluid (EPF) protein, named amorphous calcium carbonate-binding protein (ACCBP), may partially answer this question. Although sequence analysis indicated ACCBP is a member of the acetylcholine-binding protein family, it is actively involved in the shell mineralization process. In vitro, ACCBP can inhibit the growth of calcite and induce the formation of amorphous calcium carbonate. When ACCBP functions were restrained in vivo, the nacre lamellae grew in a screw-dislocation pattern, and low crystallinity CaCO(3) precipitated from the EPF. Crystal binding experiments further revealed that ACCBP could recognize different CaCO(3) crystal phases and crystal faces. With this capacity, ACCBP could modify the morphology of nacre lamellae by inhibiting the growth of undesired aragonite crystal faces and meanwhile maintain the stability of CaCO(3)-supersaturated body fluid by ceasing the nucleation and growth of calcite. Furthermore, the crystal growth inhibition capacity of ACCBP was proved to be directly related to its acetylcholine-binding site. Our results suggest that a "safeguard mechanism" of undesired crystal growth is necessary for shell microstructure formation.

A novel extracellular EF-hand protein involved in the shell formation of pearl oyster.

Mollusk shell formation is a complicated and highly controlled calcium metabolism process. Previous studies revealed that several EF-hand calcium-binding proteins actively participate in the regulation of shell mineralization. In this study, we cloned a full-length cDNA encoding a novel extracellular EF-hand calcium-binding protein (named EFCBP) from the pearl oyster, Pinctada fucata, according to the EF-hand motifs of calmodulin. Although it shares high similarity with the calmodulin family in its EF-hand signatures, EFCBP just has two EF-hand motifs and belongs to a new separate group from the other EF-hand proteins according to a phylogenetic analysis. EFCBP is specifically expressed in shell mineralization-related tissues, viz. the mantle, the gill, and the hemocytes. Moreover, its expression responds quickly only to the shell damage, but not to the damage of other tissues and the infection of the lipopolysaccharides from Escherichia coli. These results suggest that EFCBP might be an important regulator of shell formation. This finding may help better understand the functions of EF-hand proteins on the regulation of mollusk shell formation.

A novel matrix protein p10 from the nacre of pearl oyster (Pinctada fucata) and its effects on both CaCO3 crystal formation and mineralogenic cells.

A novel matrix protein, designated as p10 because of its apparent molecular mass of 10 kDa, was isolated from the nacreous layer of pearl oyster (Pinctada fucata) by reverse-phase high-performance liquid chromatography. In vitro crystallization experiments showed that p10 could accelerate the nucleation of calcium carbonate crystals and induce aragonite formation, suggesting that it might play a key role in nacre biomineralization. As nacre is known to contain osteogenic factors, two mineralogenic cell lines, MRC-5 fibroblasts and MC3T3-E1 preosteoblasts, were used to investigate the biological activity of p10. The results showed that p10 could increase alkaline phosphatase activity, an early marker of osteoblast differentiation, while the viability of MRC-5 and MC3T3-E1 remained unchanged after treatment of p10. Taken together, the findings led to identification of a novel matrix protein from the nacre of P. fucata that plays a role in both the mineral phase and in the differentiation of the cells involved in biomineralization.

cDNA cloning and characterization of a novel calmodulin-like protein from pearl oyster Pinctada fucata.

Calcium metabolism in oysters is a very complicated and highly controlled physiological and biochemical process. However, the regulation of calcium metabolism in oyster is poorly understood. Our previous study showed that calmodulin (CaM) seemed to play a regulatory role in the process of oyster calcium metabolism. In this study, a full-length cDNA encoding a novel calmodulin-like protein (CaLP) with a long C-terminal sequence was identified from pearl oyster Pinctada fucata, expressed in Escherichia coli and characterized in vitro. The oyster CaLP mRNA was expressed in all tissues tested, with the highest levels in the mantle that is a key organ involved in calcium secretion. In situ hybridization analysis reveals that CaLP mRNA is expressed strongly in the outer and inner epithelial cells of the inner fold, the outer epithelial cells of the middle fold, and the dorsal region of the mantle. The oyster CaLP protein, with four putative Ca(2+)-binding domains, is highly heat-stable and has a potentially high affinity for calcium. CaLP also displays typical Ca(2+)-dependent electrophoretic shift, Ca(2+)-binding activity and significant Ca(2+)-induced conformational changes. Ca(2+)-dependent affinity chromatography analysis demonstrated that oyster CaLP was able to interact with some different target proteins from those of oyster CaM in the mantle and the gill. In summary, our results have demonstrated that the oyster CaLP is a novel member of the CaM superfamily, and suggest that the oyster CaLP protein might play a different role from CaM in the regulation of oyster calcium metabolism.