Identification and comparative analysis of miRNA transcriptomes after allograft and xenograft transplantation in Pinctada fucata martensii.

Effective immune regulation after transplantation during pearl production is crucial for the cultivation of high-quality pearls. MicroRNAs (miRNAs) play an important role in a variety of physiological processes. To understand the regulatory rules of miRNAs after transplantation in Pinctada funcata martensii, we constructed 13 miRNA transcriptomes, including the control group (Con), allograft (Al), and xenograft (Xe) transplantation at six time points (6, 12, and 24 h and 3, 6, and 12 days), in which the xenografted mantle tissue was from Pinctada maxima. We identified 159 differentially expressed miRNAs (DEMs) and found that these DEMs showed high expression at 12 h, 24 h, and 3 days after transplantation. A total of 130 DEMs, such as Let-7, were present in the Al and Xe groups; miR-34 and 16 other DEMs were specifically present in the Al group; miR-216b and 13 other DEMs were specifically present in the Xe group. Compared with the Con group, the target genes of DEMs in the Al group were significantly enriched in protein complex, cytoskeleton, and macromolecular complex, and the Xe group was significantly enriched in ribonucleoside metabolic process, nucleoside binding, and cell division. Compared with the Al group, the target genes in the Xe group were significantly enriched in response to DNA damage stimulation. Overall, multiple pathways associated with cellular activity were enriched in higher numbers of genes in the Xe group than in the Al group. These findings enriched the information on immune regulatory mechanisms at the expression level of miRNAs in P. f. martensii after transplantation.

Globular C1q domain-containing protein from Pinctada fucata martensii participates in the immune defense process.

The globular C1q domain-containing (C1qDC) protein can recognize a variety of ligands, such as pathogen-associated molecular patterns, and plays an important role in the innate immune response. Our previous studies showed that a novel globular C1q domain-containing protein (PmC1qDC-1) is involved in the damage repair process of pearl oyster shells. However, the function of PmC1qDC-1 in pearl oyster innate immunity remains unknown. In the present study, the high-level structural analysis showed that PmC1qDC-1 was a spherical structure composed of 10 strands and was similar to the AiC1qDC-2 of bay scallop (Argopecten irradians). In situ hybridization indicated that PmC1qDC-1 had strong fluorescence signal in gills. Furthermore, the mRNA expression of PmC1qDC-1 was highly induced at 6-48 h in gill after lipopolysaccharide, peptidoglycan and polyinosinic-polycytidylic acid stimulation. Additionally, we obtained the recombinant protein of PmC1qDC-1 (rPmC1qDC-1) and found that rPmC1qDC-1 had antibacterial activity against Gram-negative (i.e., Pseudomonas aeruginosa, Vibrio parahaemolyticus, Escherichia coli, and Aeromonas hydrophila) and Gram-positive (i.e., Staphylococcus aureus and Bacillus subtilis) bacteria. These results indicated that PmC1qDC-1 might play an important role in the immune response against bacteria and viruses. This study provides clues for further studying the immune defense of Pinctada fucata martensii against pathogens and exploring the evolution of the classic pathway of complement system.

Polyamine Putrescine Regulates Oxidative Stress and Autophagy of Hemocytes Induced by Lipopolysaccharides in Pearl Oyster Pinctada fucata martensii.

The polyamine putrescine (Put) is a ubiquitous small cationic amine. It plays an essential role in controlling the innate immune response. However, little is known about its function in mollusks. In this study, the Put content was observed to increase in the serum of pearl oyster Pinctada fucata martensii after 6 and 24 h of lipopolysaccharide (LPS) stimulation. Activities of superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GSH-Px) increased, and nitric oxide synthase was downregulated in the Put group (i.e., combined treatment with Put and LPS) compared with that in the LPS group (i.e., combined treatment with phosphate-buffered saline and LPS). Furthermore, activities of alkaline phosphatase and acid phosphatase were inhibited after 6 h of LPS stimulation. The expression levels of the nuclear factor kappa B, IκB kinase, Janus kinase, and signal transducer and activator of transcription proteins genes were all significantly suppressed at 12 and 24 h in the Put group. Pseudomonas aeruginosa and Bacillus subtilis grew better after being incubated with the serum from the Put group than that from the LPS group. Additionally, the Put treatment remarkably inhibited the autophagy of hemocytes mediated by the AMP-activated protein kinase-mammalian target of rapamycin-Beclin-1 pathway. This study demonstrated that Put can effectively inhibit the inflammatory response induced by LPS in pearl oysters. These results provide useful information for further exploration of the immunoregulatory functions of polyamines in bivalves and contribute to the development of immunosuppressive agents.

Molecular Characterization of Complement Component 3 (C3) in the Pearl Oyster Pinctada fucata Improves Our Understanding of the Primitive Complement System in Bivalve.

As the central component in the complement system, complement component 3 (C3) plays essential roles in both the innate and adaptive immune responses. Here, a C3 gene (designated as pf-C3) was obtained from the pearl oyster Pinctada fucata by RT-PCR and rapid amplification of cDNA ends (RACE). The pf-C3 cDNA consists of 5,634 bp with an open reading frame (ORF) of 5,193 bp encoding a protein of 1,730 amino acids with a 19 residue signal peptide. The deduced pf-C3 protein possessed the characteristic structural features present in its homologs and contained the A2M_N_2, ANATO, A2M, A2M_comp, A2M_recep, and C345C domains, as well as the C3 convertase cleavage site, thioester motif, and conserved Cys, His, and Glu residues. Phylogenetic analysis revealed that pf-C3 is closely related to the C3s from other mollusks. Pf-C3 mRNA was expressed in all examined tissues including gill, digestive gland, adductor muscle, mantle and foot, while the highest expression was found in the digestive gland. Following the challenge with Vibrio alginolyticus, pf-C3 expression was significantly induced in hemocytes. Luciferase reporter assays indicated that pf-C3a could activate the NF-κB signal pathway in HEK293T cells. Further knockdown of pf-C3 by specific siRNA could significantly reduce the phagocytosis of V. alginolyticus by hemocytes in vitro. These results would help increase understanding of the function of C3 in the invertebrate immune system and therefore provide new insights into the roles of the primitive complement system in invertebrates.