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Mollusc shell color polymorphism is influenced by various factors. Pigments secreted in vivo by animals play a critical role in shell coloration. Among the different shell-color hues, orange pigmentation has been partially attributed to porphyrins. However, the detailed causal relationship between porphyrins and orange-shell phenotype in molluscs remains largely unexplored. The various strains of Pacific oyster (Crassostrea gigas) with different shell color provide useful models to study the molecular regulation of mollusc coloration. Accordingly, oysters with orange and gold-shells, exhibiting distinct porphyrin distributions, were selected for analysis of total metabolites and gene expression profile through mantle metabolomic and transcriptomic studies. Translocator protein (TspO) and protoporphyrin IX (PPIX) were identified as potential factors influencing oyster shell-color. The concentration of PPIX was measured using HPLC, while expression profiling of CgTspO was analyzed by qPCR, in situ hybridization, Western blotting, and immunofluorescence techniques. Moreover, the roles of CgTspO in regulating PPIX metabolism and affecting the orange-shell-coloration were investigated in vitro and in vivo. These studies indicate that PPIX and its associated metabolic protein, CgTspO may serve as new regulators of orange-shell-coloration in C. gigas. Data of this study offer new insights into oyster shell coloration and enhancing understandings of mollusc shell color polymorphism.

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BACKGROUND: Shell color formation is an important physiological process in bivalves, the molecular genetic basis has potential application in bivalve aquaculture, but there is still remaining unclear about this issue. The cystine/glutamate transporter (Slc7a11) and cystathionine beta-synthase (Cbs) are integral genes in pheomelanin synthesis pathway, which is vital to skin pigmentation. METHODS AND RESULTS: Here, the sequences of b (0, +) -type amino acid transporter 1 (B-aat1) and Cbs in Pacific oyster (Crassostrea gigas) (CgB-aat1, CgCbs) were characterized. Phylogenetically, the deduced amino acid sequences of CgB-aat1 and CgCbs both possessed conserved features. Genes were both ubiquitously expressed in six tested tissues with more abundant expression level in central mantle. Besides, the polyclonal antibodies of CgB-aat1, CgCbs, CgTyr, and CgTyrp2 were successfully prepared. Immunofluorescence analysis revealed that CgB-aat1 and CgCbs proteins were both expressed in gill rudiments of eyed-larvae and concentrated mainly in cytoplasm of epithelial cell and nerve axons in mantle. Additionally, after CgB-aat1 or CgCbs silencing, expressions at mRNA and protein levels of CgB-aat1 and CgCbs involved in pheomelanin synthesis were significantly suppressed, and CgTyr, CgTyrp1 and CgTyrp2 related to eumelanin synthesis were also down-regulated but no apparent differences, respectively. Moreover, micrographic examination found less brown-granules at mantle edge in CgB-aat1 interference group. CONCLUSION: These results implied that pheomelanin synthesis was possible induced by CgB-aat1-CgTyr-CgCbs axis, and it played an essential role on mantle pigmentation in the oysters. These findings provide the useful genetic knowledge and enrich the physiological information for the shell color formation in bivalve aquaculture.

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Porphyrins are a widespread group of pigments in nature which are believed to contribute to shell colors in mollusks. Previous studies have provided candidate genes for porphyrin shell coloration, however, the linkage analysis between functional genes and porphyrin pigmentation remains unclear in mollusks. RNA interference is a powerful molecular tool for analyzing the loss of functions of genes in vivo and alter gene expression. In this study, we used unicellular alga Platymonas subcordiformis and Nitzschia closterium f. minutissima as vectors to feed oysters with Escherichia coli strain HT115 engineered to express double-stranded RNAs targeting specific genes involved in porphyrin synthesis. A strain of Crassostrea gigas with orange shell was used to target key haem pathway genes expression using the aforementioned approach. We show here that feeding the oysters with E. coli, containing dsRNA targeting pigmentation genes, can cause changes in the color of the newly deposited shell. For example, the RNAi knockdown of CgALAS and CgPBGD resulted in the loss of uroporphyrin pigmentation from the shell due to the accumulation of the pigment in the oyster's mantle. The study probed the crucial role of ALAS and PBGD genes potential functions of uroporphyrin production and shell color pigmentation in C. gigas.

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Molluscs exhibit diverse shell colors. The molecular regulation of shell coloration is however not well understood. To investigate the connection of shell coloration with pigment synthesis, we analyzed the distribution of porphyrins, a widespread group of pigments in nature, in four Pacific oyster strains of different shell colors including black, orange, golden, and white. The porphyrin distribution was analyzed in oyster mantles and shells by fluorescence imaging and UV spectrophotometer. The results showed that red fluorescence emitted by porphyrins under the UV light was detected only on the nacre of the orange-shell strain and mantles of orange, black and white-shell strains. Extracts from newly deposit shell, nacre and mantle tissue from orange-shell specimens showed peaks in UV-vis spectra that are characteristic of porphyrins, but these were not observed for the other shell-color strains. In addition, genes of the haem synthetic pathway were isolated and characterized. Phylogenetic analysis of CgALAS, CgALAD, CgPBGD, CgUROS, and CgUROD provide further evidence for a conserved genetic pathway of haem synthesis during evolution. Differential expression of the haem genes expressed in mantle tissues support these findings and are consistent with porphyrins being produced by the orange strain only. Tissue in situ hybridization demonstrated the expression of these candidate genes at the outer fold of C. gigas mantles where shell is deposited. Our studies provide a better understanding of shell pigmentation in C. gigas and candidate genes for future mechanistic analysis of shell color formation in molluscs.

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Interleukin 1b (IL-1b) is a member of the cytokine family that serves as major mediators for early pro-inflammatory responses. During artificial breeding of Pacific cod (Gadus macrocephalus), outbreaks of NNV in the early development stages could lead to high mortality. Recent research reported that IL-1b participated in the host-virus interaction against NNV infection, however, no IL-1b gene has been identified from Pacific cod so far. In this study, we described the identification and characterization of a IL-1b from Pacific cod transcriptome at both transcript and protein level. Sequence alignment and phylogenetic analysis were conducted to determine and confirm their evolutionary relationship. Expression analysis revealed that IL-1b was expressed in all tested tissues and the western blot analysis confirmed the same expression profiles using purified recombinant IL-1b protein. In addition, significant up-regulation was detected in IL-1b transcripts during early development stages at 30dph and 50dph. After challenged with poly I:C, IL-1b was significantly induced after 24 h. Taken together, these results strongly suggested that IL-1b might play an essential role in innate defense mechanism of Pacific cod.

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(Ba0.4Sr0.6)TiO3 (BST) ceramics with various grain sizes (0.3-3.4 µm) were synthesized by the oxalate coprecipitation method and prepared by plasma activated sintering and conventional solid-state sintering process. The effect of grain boundary on the energy storage properties and the dielectric relaxation characteristics of BST paraelectric ceramics (Curie point ≈ -67°C) with various grain sizes were investigated. The dielectric breakdown strength (simplified as BDS) is obviously improved and then deteriorated with decreasing grain size, accounting for the energy density variation. The enhancement of interfacial polarization at grain boundary layers has a negative effect on the BDS, leading to the decreased values for samples with grain size smaller than 0.7 µm. In addition, the insulation effect of grain boundary barriers was discussed based on the complex impedance spectroscopy analysis, which was found to play a dominant role in controlling the BDS with coarser grain size. Among them, the sharply decreased BDS for BST with grain size of 1.8 µm was believed to be attributed to the combination of lower grain boundary density and higher interfacial polarization, due to the significant increase of oxygen vacancies at higher sintering temperature.