Identification and function analysis of BCL2 in immune response of Pteria penguin.

B-cell lymphoma/leukemia-2 (BCL2), an anti-apoptotic factor in the mitochondrial regulatory pathway of apoptosis, is critically important in immune defenses. In this study, a novel BCL2 gene was characterized from Pteria penguin (P. penguin). The PpBCL2 was 1482 bp long, containing an open reading frame (ORF) of 588 bp encoding 195 amino acids. Four highly conserved BCL-2 homology (BH) domains were found in PpBCL2. Amino acid alignment and phylogenetic tree showed that PpBCL2 had the highest similarity with BCL2 of Crassostrea gigas at 65.24 %. Tissue expression analysis showed that PpBCL2 had high constitutive expression in gill, digestive diverticulum and mantle, and was significantly increased 72 h of Vibrio parahaemolyticus (V. parahaemolyticus) challenge in these immune tissues. Furthermore, PpBCL2 silencing significantly inhibited antimicrobial activity of hemolymph supernatant by 1.4-fold, and significantly reduced the survival rate by 51.7 % at 72 h post infection in P. penguin. These data indicated that PpBCL2 played an important role in immune response of P. penguin against V. parahaemolyticus infection.

Creb2 involved in innate immunity by activating PpMitf-mediated melanogenesis in Pteria penguin.

cAMP response element binding protein 2 (CREB2) acts as an intracellular transcriptional factor and regulates many physiological processes, including melanogenesis and melanocyte differentiation. In our previous research, the Creb2 gene has been characterized from Pteria penguin (P. penguin), but its role and regulatory mechanism in P. penguin are still unclear. In this study, first, the function of PpCreb2 in melanogenesis and innate immunity were identified. PpCreb2 silencing significantly decreased the tyrosinase activity and melanin content, indicating PpCreb2 played an important role in melanogenesis. Meanwhile, PpCreb2 silencing visibly suppressed the antibacterial activity of hemolymph supernatant, indicating that PpCreb2 was involved in innate immunity of P. penguin. Second, the PpCreb2 was confirmed to perform immune function by regulating the melanogenesis. The decreased melanin oxidation product due to PpCreb2 silencing triggered the declining of antibacterial activity of hemolymph supernatant, which then could be rescued by adding exogenous melanin oxidation products. Third, the regulation pathway of PpCreb2 involved in innate immunity was analyzed. The promoter sequence analysis of PpMitf discovered 5 conserved cAMP response element (CRE), which were specifically recognized by basic Leucine zipper domain (bZIP) of upstream activation transcription factor. The luciferase activities analysis showed that PpCreb2 could activate the CRE in PpMitf promoter via highly conserved bZIP domain and regulate the expression of PpMitf, which further regulated the PpTyr expression. Therefore, the results collectively demonstrated that PpCreb2 participated in innate immunity by activating PpMitf-mediated melanogenesis in P. penguin.

Pax3 Gene Regulated Melanin Synthesis by Tyrosinase Pathway in Pteria penguin.

The paired-box 3 (Pax3) is a transcription factor and it plays an important part in melanin synthesis. In this study, a new Pax3 gene was identified from Pteria penguin (Röding, 1798) (P. penguin) by RACE-PCR (rapid-amplification of cDNA ends-polymerase chain reaction) and its effect on melanin synthesis was deliberated by RNA interference (RNAi). The cDNA of PpPax3 was 2250 bp long, containing an open reading fragment of 1365 bp encoding 455 amino acids. Amino acid alignment and phylogenetic tree showed PpPax3 shared the highest (69.2%) identity with Pax3 of Mizuhopecten yessoensis. Tissue expression profile showed that PpPax3 had the highest expression in mantle, a nacre-formation related tissue. The PpPax3 silencing significantly inhibited the expression of PpPax3, PpMitf, PpTyr and PpCdk2, genes involved in Tyr-mediated melanin synthesis, but had no effect on PpCreb2 and an increase effect on PpBcl2. Furthermore, the PpPax3 knockdown obviously decreased the tyrosinase activity, the total content of eumelanin and the proportion of PDCA (pyrrole-2,3-dicarboxylic acid) in eumelanin, consistent with influence of tyrosinase (Tyr) knockdown. These data indicated that PpPax3 played an important regulating role in melanin synthesis by Tyr pathway in P. penguin.

The structure and proteomic analysis of byssus in Pteria penguin: Insights into byssus evolution and formation.

Byssus is a unique external structure in sessile bivalves and is critical for settlement and metamorphosis. However, little is known about the stout byssus in Pteria penguin. We explored the byssus structure and proteins using scanning electron microscopy and proteomics, respectively. The results revealed that P. penguin byssus has a dense and highly aligned fiber inner core, and the outer cuticle contains protein granules embedded in the protein matrix. Proteomic analysis revealed 31 proteins in the byssus, among which 15 differentially expressed proteins were mainly enriched in the EGF/EGF-like and laminin EGF-like domains. Foot proteins were enriched in the EF-hand, immunoglobulin, and fibronectin domains. All these domains can participate in protein-protein and/or protein-metal interactions in the extracellular matrix (ECM), which, together with the seven types of ECM proteins detected in the byssus, supports the hypothesis that the byssus is derived from the ECM. We also found that in vitro acellular structures of the byssus and the shell shared commonalities in their formation processes. These results are useful for further understanding byssus evolution and the characterization of byssus-related proteins. SIGNIFICANCE: This manuscript investigates the structure and the origin of Pteria penguin byssus, given that byssus is vital to provide critical protection for reproduction and even against environmental stresses that affect survival. However, there is rare research on byssus protein composition. Hence, though scanning electron microscopy and proteomic analysis, we discovered that P. penguin byssus possesses the dense and highly aligned fiber inner core, and the outer cuticle has protein granules embedded in the protein matrix. Proteomic analysis showed that there were 31 proteins in the byssus, among which 15 proteins were mainly enriched in the EGF/EGF-like and laminin EGF-like domains. Foot proteins closely related to byssus formation were enriched in EF hand, immunoglobulin, and fibronectin domains. These domains are able to participate in protein-protein and/or protein-metal interactions in the extracellular matrix (ECM), which together with the seven types of ECM proteins detected in byssus support the hypothesis that byssus derive from the ECM. We also found in vitro acellular structures the byssus and the shell share commonalities in their formation processes. These results were useful for further understanding the byssus evolution and the characterization of the byssus-related proteins.

The mechanisms involved in byssogenesis in Pteria penguin under different temperatures.

Byssus is important for marine bivalves to adhere robustly to diverse substrates and resist environmental impacts. The winged pearl oyster, Pteria penguin, can reattach or not reattach to the same environment, which leaves the development and survival of the oyster population at risk. In this study, diverse methods were employed to evaluate the byssus quality and explore the mechanism of byssus secretion at different temperatures. The results demonstrated that oysters maintained their byssus properties at different temperatures through polyphenol oxidase (PPO) and reactive oxygen species (ROS) variation. They were both higher at 27 °C than at 21 °C. Furthermore, PPO activities of WB27 (31.78 U/g ± 1.50 U/g) were significantly higher than NB27, WB21, and NB21. Sectional observation revealed three types of vesicles, from which a novel vesicle might participate in byssogenesis as a putative metal storage particle. Moreover, cytoskeletal proteins may cooperate with cilia to transport byssal proteins, which then facilitate byssus formation under the regulation of upstream signals. Transcriptome analysis demonstrated that protein quality control, ubiquitin-mediated proteolysis, and cytoskeletal reorganization-related genes contributed to adaptation to temperature changes and byssus fabrication, and protection-related genes play a critical role in byssogenesis, byssus toughness, and durability. These results were utilized to create a byssogenesis mechanism model, to reveal the foot gland and vesicle types of P. penguin and provide new insights into adaptation to temperature changes and byssus fabrication in sessile bivalves.

Mitf Involved in Innate Immunity by Activating Tyrosinase-Mediated Melanin Synthesis in Pteria penguin.

The microphthalmia-associated transcription factor (MITF) is an important transcription factor that plays a key role in melanogenesis, cell proliferation, survival and immune defense in vertebrate. However, its function and function mechanism in bivalve are still rarely known. In this research, first, a Mitf gene was characterized from Pteria penguin (P. penguin). The PpMitf contained an open reading frame of 1,350 bp, encoding a peptide of 449 deduced amino acids with a highly conserved basic helix-loop-helix-leucine zipper (bHLH-LZ) domain. The PpMITF shared 55.7% identity with amino acid sequence of Crassostrea gigas (C. gigas). Tissue distribution analysis revealed that PpMitf was highly expressed in mantle and hemocytes, which were important tissues for color formation and innate immunity. Second, the functions of PpMitf in melanin synthesis and innate immunity were identified. The PpMitf silencing significantly decreased the tyrosinase activity and melanin content, indicating PpMitf involved in melanin synthesis of P. penguin. Meanwhile, the PpMitf silencing clearly down-regulated the expression of PpBcl2 (B cell lymphoma/leukemia-2 gene) and antibacterial activity of hemolymph supernatant, indicating that PpMitf involved in innate immunity of P. penguin. Third, the function mechanism of PpMitf in immunity was analyzed. The promoter sequence analysis of tyrosinase (Tyr) revealed two highly conserved E-box elements, which were specifically recognized by HLH-LZ of MITF. The luciferase activities analysis showed that Mitf could activate the E-box in Tyr promoter through highly conserved bHLH-LZ domain, and demonstrated that PpMitf involved in melanin synthesis and innate immunity by regulating tyrosinase expression. Finally, melanin from P. penguin, the final production of Mitf-Tyr-melanin pathway, was confirmed to have direct antibacterial activity. The results collectively demonstrated that PpMitf played a key role in innate immunity through activating tyrosinase-mediated melanin synthesis in P. penguin.

Identification of a tyrosinase gene and its functional analysis in melanin synthesis of Pteria penguin.

Tyrosinase is a key rate-limiting enzyme in melanin synthesis. In this study, a new tyrosinase gene (Tyr) was identified from Pteria penguin and its effect on melanin synthesis was deliberated by RNA interference (RNAi). The cDNA of PpTyr was 1728 bp long, containing a 5'untranslated region (UTR) of 11 bp, a 3'UTR of 295 bp, and an open reading fragment of 1422 bp encoding 473 amino acids. Amino acid alignment showed PpTyr had the highest (50%) identity to tyrosinase-like protein 1 from Pinctada fucata. Phylogenetic tree analysis classified PpTyr into α-subclass of type-3 copper protein. Tissue expression analysis indicated that PpTyr was highly expressed in mantle, a nacre formation related tissue. After PpTyr RNA interference, PpTyr mRNA was significantly inhibited by 71.0% (P < 0.05). For other melanin-related genes, PpCreb2 and PpPax3 expression showed no significant change, but PpBcl2 was obviously increased. By liquid chromatograph-tandem mass spectrometer (LC-MS/MS) analysis, the total content of PDCA (pyrrole-2, 3-dicarboxylic acid) and PTCA (pyrrole-2,3,5-tricarboxylic acid), as main markers for eumelanin, was sharply decreased by 66.6% after PpTyr RNAi (P < 0.05). The percentage of PDCA was also obviously decreased from 20.1% to 13.9%. This indicated that tyrosinase played a key role in melanin synthesis and color formation of P. penguin.

Characterization of transcriptome and identification of biomineralization genes in winged pearl oyster (Pteria penguin) mantle tissue.

The winged pearl oyster Pteria penguin is a commercially important marine pearl oyster species, with pearls that are quite different from those of other pearl oysters. Among such species, mantle tissue is the main organ responsible for shell and pearl formation, a biomineralization process that is regulated by a series of genes, most of which remain unknown. In this study, we sequenced and characterized the transcriptome of P. penguin mantle tissue using the HiSeq 2000 sequencing platform. A total of 93,204 unique transcripts were assembled from 51,580,076 quality reads, with a mean length of 608bp, and 40,974 unigenes were annotated. The sequence data enabled the identification of 79,702 potential single nucleotide polymorphism loci and 4345 putative simple sequence repeat loci. A total of 71 unique transcripts were identified homologous to known biomineralization genes, including mantle gene, nacrein, pearlin, pif, chitinase, and shematrin, of which only 3 were previously reported in P. penguin. qPCR analysis indicated that 10 randomly selected biomineralization genes were much more highly expressed in mantle tissue than in the other tissues. In addition, 30 unique sequences were identified as highly expressed, with FPKM values of >3000, and most of these were biomineralization-related genes, including shematrin family genes, a jacalin-related lectin synthesis gene, calponin-2, and paramyosin. These findings will be useful for future studies of biomineralization in P. penguin, as well as in other Pteria species.