Search

The bivalve hinge ligament is the hard tissue that functions to open and close shells. The ligament contains fibrous structures consisting of aragonite crystals surrounded by a dense organic matrix. This organic matrix may contribute to the formation of fibrous aragonite crystals, but the mechanism underlying this formation remains unclear. In this study, we identified a novel ligament-specific protein, Pinctada fucata tissue inhibitor of metalloproteinase (PfTIMP), from the fibrous organic matrix between aragonite crystals in the ligament using the amino acid sequence and cDNA cloning methods. PfTIMP consists of 143 amino acid residues and has a molecular weight of 13,580.4. To investigate the activity of PfTIMP, inhibition of matrix metalloproteinase (MMP) activity was measured. PfTIMP strongly inhibited human MMP13 and MMP9. Eight MMP homologs were identified from a P. fucata genomic database by BLAST search. To identify the specific MMP that may contribute to ligament formation, the expression level of each MMP was measured in the mantle isthmus, which secretes the ligament. The expression of MMP54089 increased after scratching of the ligament, while the expressions of other MMPs did not increase after doing the same operation. To identify the role of MMP54089 in forming the ligament structure, double stranded (ds) RNA targeting MMP54089 was injected into living P. fucata to suppress the function of MMP54089. Scanning electron microscopic images showed disordered growing surfaces of the ligament in individuals injected with MMP54089-specific dsRNA. These results suggest that PfTIMP and MMP54089 play important roles in the formation of the fibrous ligament structure.

The pearl oyster, Pinctada fucata, is cultured for pearl production in Japan. The shell of the pearl oyster consists of calcium carbonate and a small amount of organic matrix. Despite many studies of the shell matrix proteins, the mechanism by which calcium elements are transported from the mantle to the shell remains unclear. Investigating the molecular mechanism of calcium transportation, we prepared artificial seawater with a high concentration of calcium ions (10ASW) to induce calcification in the pearl oyster. When pearl oysters were cultured in 10ASW, unusual nanoparticles were precipitated on the surface of the nacreous layer. SDS-PAGE and 2D-PAGE analyses revealed that some calcium-sensing proteins (Sarcoplasmic Ca-binding Protein (Pf-SCP) and Pf-filamin A) might be related to the synthesis of these nanoparticles. The recombinant proteins of Pf-SCP can bind to calcium ions and accumulate nanoparticles of calcium carbonate crystals. However, transcriptomic analysis of the pearl oysters grown in 10ASW showed that the matrix protein genes in the shell did not differ before and after treatment with 10ASW. These results suggest that, despite increasing calcium transportation to the shell, treatment with a high concentration of calcium ions does not induce formation of the organic framework in the shell microstructure. These findings offer meaningful insights into the transportation of calcium elements from the mantle to the shell.

The bivalve hinge ligament is the hard tissue that functions to open and close shells. The ligament contains fibrous structures consisting of aragonite crystals surrounded by a dense organic matrix. This organic matrix may contribute to the formation of fibrous aragonite crystals, but the mechanism underlying this formation remains unclear. Recently, we showed that tissue inhibitor of metalloproteinase (TIMP) and matrix metalloproteinase (MMP) is related to the formation of the ligament in Pinctada fucata. BLAST search of genome database revealed that seven MMP genes are encoded in the genome of P. fucata. To identify the specific MMP that may contribute to ligament formation, the expression level of each MMP was measured in the mantle isthmus, which secretes the ligament. The expression of MMP54089 increased after scratching of the ligament, while the expressions of other MMPs did not increase after doing the same operation. To identify the role of MMP54089 in forming the ligament structure, double-stranded (ds) RNA targeting MMP54089 was injected into the living P. fucata to suppress the function of MMP54089. Scanning electron microscopic images showed disordered growing surfaces of the ligament in individuals injected with MMP54089-specific dsRNA. These results suggest that PfTIMP and MMP54089 play important roles in the formation of the fibrous ligament structure.

The effects of the removal of the oily surface film on initial swim bladder inflation and survival in cultured seven-band grouper Hyporthodus septemfasciatus were investigated during the larval stage. Additionally, the relationship between swim bladder inflation failure and malformations in later stages was examined. To explore promotion conditions for inflation, duplicate experiments were conducted under three different water surface conditions: removal of the oily surface film (ROF group), covering the water surface with an oil film (COF group), and sealing the water surface with liquid paraffin (SLP group). Larval swim bladder inflation was first observed 14 days after hatching (DAH) in the ROF group; thereafter, swim bladder inflation rates increased to 11.1% and 38.7% until the end of the experiments. This indicates that initial swim bladder inflation occurs at the pre-flexion stage in larval development. Conversely, bladder inflation was rarely observed in the COF or SLP groups, indicating the promotional effect of surface film removal on larval swim bladder inflation and suggesting that gulping atmospheric air is necessary for initial inflation. Although larval water surface death occurred frequently in the ROF group, no significant difference existed in any group between the survival rates at 10 DAH and at the end of the experiment. The incidence rate of spinal curvature and deformation of the centra was significantly higher in individuals without initial swim bladder inflation, and these malformations mainly occurred in the caudal vertebra. Therefore, the removal of the oily surface film promotes initial swim bladder inflation and reduces larval malformations.

The cause of abnormal musculature on both sides of the same myotome in artificially cultured seven-band grouper Epinephelus septemfasciatus (Thunberg) larvae remains unclear. We evaluated the effect of three factors: changes in temperature (between 22 and 24°C), disinfection with ozonated seawater (0.3 mg TROs L−1, 1 min), and hypoxia (22 or 14% saturation) during the early (20 h post-fertilization, hpf) and mid- (24 hpf) somite stages on trunk muscular development in larval seven-band grouper. The thermal regimes and disinfection with ozonated seawater had no effect on muscle development (P > 0.05, G-test). In contrast, exposure to hypoxia during both the early and mid-somite stages increased the incidence of abnormal musculature in the trunk (P

Since the 1990s, Akoya oyster disease (AOD) associated with reddish browning of the soft tissues and mortality of cultured pearl oyster, Pinctada fucata (Gould), has been occurring in the western part of Japan. Although transmission experiments suggested that the disease is infectious, the causative agent has not been identified. Previous studies suggested that a member of the Spirochaeta cause AOD, and Candidatus Maribrachyspira akoyae was proposed as the putative causative agent. However, difficulties in culturing the putative causal bacterium have hindered research on AOD. In this study, we developed a quantitative PCR assay to examine the spatial-temporal colonization patterns of Spirochaeta in pearl oysters. Spirochete genes were abundant in the mantle, adductor muscle, and hemolymph of oysters but scarce in the gill, crystalline style, hemocytes, heart, and digestive glands. Spirochete genes were detected in the hemolymph 2 weeks after infection, and their number increased with time and mortality. The Spirochaeta genes identified in the hemolymph of pearl oysters reared in culturing fields changed with variations in water temperature, and the number of Spirochaeta genes increased or decreased at approximately 20 °C. Hence, the bacterium was thought to grow at water temperatures above 20 °C. These results are consistent with those of infection tests and epidemiologic studies of the distribution and seasonal changes in AOD pathogens and support the hypothesis that a spirochete causes AOD.

The effects of rearing temperature (23–29 °C) during the larval and juvenile stage on survival, growth and skeletal malformations in the seven-band grouper Epinephelus septemfasciatus were investigated. The survival rate of juveniles 30–40 mm in total length emerging from eggs was higher at 25 and 26 °C (0.1–1.3 %) than at 23 °C or 27–29 °C (0.004–1.5 %). Growth (increase in total length) was accelerated at higher temperatures. The frequency of malformed individuals was lower at 25–27 °C (36.0–61.5 %) than at 23, 28 or 29 °C (65.3–76.9 %). Specific incidences of spinal curvature and centrum fusion or defects in juveniles were not related to rearing temperature. However, incidences of twisted or compressed vertebrae (6.5–64.0 %) were higher at higher temperatures, while the incidence of bifurcated neural spine was significantly higher at 23 °C (43.6–54.4 %) than at other temperatures (3.3–22.7 %). The incidence rate of spinal curvature (23.3 %) was significantly higher in juveniles with a deflated swim bladder, regardless of rearing temperature. The results of this study suggest that the optimum culture temperature for seven-band grouper is 25–26 °C, collectively considering the survival, growth and incidences of abnormalities. Our results also demonstrate the significance of identifying the conditions for swim bladder inflation to prevent spinal curvature in seven-band grouper.

This study examined the usage of a dry shipper for cryopreservation of Epinephelus septemfasciatus (Thunberg) spermatozoa. Milt was diluted 1:49 with 5% dimethyl sulfoxide plus 95% foetal bovine serum for cryopreservation. Computer-assisted sperm analysis was used to analyse sperm motility, while fertilization and hatching trials were conducted to gauge the applicability of the cryopreservation method for aquaculture. We showed that cooling rates of the dry shipper were stable for 14 days and could be manipulated by the use of different sized freezing straws and use of a simple polystyrene foam container (5 × 5 × 12 cm and 1 cm thickness on all sides with the upper layer exposed). Dry shipper cryopreserved spermatozoa had significantly lower post-thaw per cent motility and velocity than fresh sperm, but linearity of movement was unchanged. Fertilization and hatching rates were not significantly different at all tested sperm to egg ratios (3000:1–243000:1). The results indicated that 0.33 mL of milt when cryopreserved was sufficient to fertilize up to 450 g of oocytes. Application of this technology will help improve seed production in aquaculture and further develop breeding and genetics studies.

三重県南部のマダイ養殖漁場において，養殖強度（魚の収容密度，日間給餌率，生簀面積，生簀移動）と環境特性（水深，平均流速）が底質（酸揮発性硫化物［AVS］）に及ぼす影響を調査した。回帰分析で AVS に対する影響が確認された変数は，日間給餌率，生簀面積，生簀移動，水深および平均流速であった。特に平均流速は，有機物の堆積のしにくさと溶存酸素の供給のされやすさを反映し，AVS に対する影響が大きかった。底質改善の対策は，養殖強度と環境特性の両方を考慮して決定しなければならないことが明らかとなった。

The mollusk class, Bivalvia, plays an important role in the formation of calcium carbonate in the ocean. The bivalve hinge ligament is a hard but pliant tissue that resists the stress placed on the hinge during opening and closing. The ligament comprises a fine microstructure of fibrous aragonite crystals surrounded by a dense organic matrix. The matrix consists of organic fibers that are aligned with the fibrous aragonite crystals. Previous studies identified a tissue inhibitor of metalloproteinase (Pf-TIMP: Pinctada fucata-Tissue Inhibitor of Metalloproteinase) in the organic fibers of P. fucata ligaments. This enzyme exhibited strong inhibition of matrix metalloproteinase (MMP) activity in vitro, suggesting that MMPs also play a role in formation of the organic fibers of the ligament. Using transcriptome data, we identified MMP genes from the mantle isthmus, which is a soft tissue attached to the ligament. To investigate the function of MMP genes in vivo, we performed RNA interference experiments. The expression of MMP14973 and MMP07860 genes was inhibited after injection of each dsRNA. Cumulative injection of MMP07860 dsRNA induced aggregated aragonite fiber orientation, whereas the injection of MMP14973 showed minor effects. When the decalcified ligament was incubated in a solution saturated with calcium carbonate, aragonite fibers aligned along the surface. When the decalcified ligament was treated with recombinant human (hr) MMP-13, the precipitation of calcium carbonate was inhibited. To investigate general MMP functions in calcium carbonate crystallization in detail, we precipitated aragonite crystals in collagen gels treated with or without recombinant human (hr) MMP-1. Treatment with hrMMP-1 increased the interaction between collagen gels and calcium carbonate. These results imply that Pinctada fucata (Pf)-MMPs degrade extracellular matrices in the ligament to produce the fine organic fibers that regulate the orientation of fibrous aragonite crystals.

Mass mortality that is acompanied by reddish browning of the soft tissues has been occurring in cultured pearl oyster, Pinctada fucata martensii. The disease is called Akoya oyster disease (AOD). Although spreading pattern of the disease and transmission experiments suggest that the disease is infectious, the causative agent has not yet been identified. We used shotgun and 16S rRNA-based metagenomic analysis to identify genes that are present specifically in affected oysters. The genes found only in diseased oysters were mostly bacterial origin, suggesting that the causative agent was a bacterial pathogen. This hypothesis was supported by the inhibition of AOD development in naïve oysters injected with the hemolymph of diseased animals followed immediately with penicillin bath-administration. Further analyses of the hemolymph and mantle specifically and universally detected genes of bacteria that belong to phylum Spirochaetes in diseased pearl oysters but not in healthy oysters. By in situ hybridization or immunostaining, a Brachyspira-like bacterium was observed in the smears of hemolymph from affected oysters, but not from healthy oysters. Phylogenetic analysis using 16S rRNA sequences showed that the presumptive causative bacterium was outside of but most closely related to family Brachyspiraceae. We propose 'Candidatus Maribrachyspira akoyae' gen. nov, sp nov., for this bacterium.