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Copper/zinc superoxide dismutase (Cu/Zn-SOD) can effectively eliminate reactive oxygen species (ROS)，avoid damage from O 2 to the body, and maintain O 2 balance. In this study, multi-step high-performance liquid chromatography (HPLC), combined with Mass Spectrometry (MS), was used to isolate and identify Cu/Zn-SOD from the serum of Pinctada fucata martensii (P. f. martensii) and was designated as PmECSOD. With a length of 1864 bp and an open reading frame (ORF) of 1422 bp, the cDNA encodes a 473 amino acid protein. The PmECSOD transcript was detected in multiple tissues by quantitative real-time PCR (qRT-PCR), with its highest expression level being in the gills. Additionally, the temporal expression of PmECSOD mRNA in the hemolymph was highest at 48 h after in vivo stimulation with Escherichia coli and Micrococcus luteus. The results from this study provide a valuable base for further exploration of molluscan innate immunity and immune response., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

We have developed a black shell colored selected line observed to have higher survival ability. In this study, to understand its immune capacity, total carotenoid content (TCC) of the black shell colored line (BG) and the control group (CG) were compared. Survival and retention rates, immunity and antioxidant capacity of BG were compared relative to CG at different times after grafting operation. The results showed that BG had significantly larger TCC than CG (P < 0.05). BG had significantly higher survival and retention rates than CG on days 7, 30 and 360 after grafting (P < 0.05). On days 360, BG had significantly larger pearl thickness than CG (P < 0.05). BG exhibited increased ACP, AKP, SOD, CAT, TAOC and LZ activity than the CG on 0 h, 12 h, 1 d, 3 d, 5 d, 7 d and 30 d after grafting. BG had higher expression levels of Fascin, SOD, CDK-7, CDAP-1, IRAK-1, α2m, GST-1, TRAF-3 and Caspase-2 than CG. The results suggested that BG had higher immune competence and pearl production performances, which is promising to improve pearl quality and production., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

The mantle piece from the donor pearl oyster would be rejected by the immune system of recipient oyster in pearl culture practice, especially in the case that the donor and receptor are different species. Thus, investigation of the immune response of recipient oyster to grafted mantle pieces, particularly to xenografts, is of importance in creating xenograft transplantation technology for pearl culture industry. The humoral immune responses of P. fucata to allograft (mantle piece of P. fucata) and xenografts (mantle pieces of P. maxima and P. margaritifera, respectively) were studied in this paper. The oysters receiving no transplantations were served as the control group. The serum was collected from recipient P. fucata at 1 d, 2 d, 3 d, 4 d, 5 d, 7 d, 9 d, 11 d, 13 d, and 15 d, respectively after transplantation, and the serum antibacterial activity, lysozyme activity (LZM), alkaline phosphatase (AKP), acid phosphatase (ACP), total antioxidant capacity (TAC), and agglutination to rabbit red blood cells were investigated. The result indicated that serum of both the experimental groups and the control group can agglutinate rabbit red blood cells, with variation between groups and between time points, respectively. The antibacterial activity in the experimental group was significantly higher than that in the control group at 2-4 d, but lower at 5-11 d and returned back to normal at 15 d, with significant differences among experimental groups (P < 0.05). The LZM in the experimental group was significantly higher than that in the control group at 3-7 d, with significant differences in bacteriolytic activity among various groups (P < 0.05). Both the ACP and AKP activity levels in the experimental groups were higher than those in the control group at 2-9 d, with significant differences among various groups at 3-9 d (P < 0.05). The TAC level in the experimental groups was higher than that in the control group at 1-7 d, with significant differences among various groups at 4-7 d (P < 0.05). The above results indicated that all of the humoral immune factors investigated showed immune responses to both allografts and xenografts, with no specific to any of them. Thus, it is necessary to further screen immune rejection factors specific to xenografts, including cellular immune components., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Because of its capacity to rapidly convert superoxide to hydrogen peroxide, superoxide dismutase (SOD) is crucial in both intracellular signalling and regulation of oxidative stress. In this paper we report the cloning of a Cu/Zn SOD (designated as pfSOD) from the pearl oyster (Pinctada fucata) using rapid amplification of cDNA ends (RACE) PCR. The full-length cDNA of this Cu/Zn SOD contains an open reading frame (ORF) of 471 bp coding for 156 amino acids. No signal peptide was identified at the N-terminal amino acid sequence of Cu/Zn SOD indicating that this pfSOD encodes a cytoplasmic Cu/Zn SOD. This is supported by the presence of conserved amino acids required for binding copper and zinc. Semi-quantitative analysis in adult tissues showed that the pfSOD mRNA was abundantly expressed in haemocytes and gill and scarcely expressed in other tissues tested. After challenge with lipopolysaccharide (LPS), expression of pfSOD mRNA in haemocytes was increased, reaching the highest level at 8 h, then dropping to basal levels at 36 h. These results suggest that Cu/Zn SOD might be used as a bioindicator of the aquatic environmental pollution and cellular stress in pearl oyster., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Microsomal glutathione S-transferase (MGST) functions in cellular defense against xenobiotics and provides protection against the action of lipid hydroperoxides produced as a consequence of oxidative stress. In this study, a full-length cDNA encoding MGST3 (referred to as PmMGST3) was identified from the pearl oyster, Pinctada martensii by a combination of expressed sequence tag (EST) analysis and rapid amplification of cDNA ends (RACE). The full-length cDNA of PmMGST3 is 971 bp and contains a 5' UTR of 39 bp, a 3' UTR of 491 bp with a canonical polyadenylation signal sequence (AATAAA), and an open reading frame (ORF) of 447 bp encoding a polypeptide of 146 residues. The deduced polypeptide contains a conserved motif (FNCx(1)QRx(2)H) characteristic of the MGST3 subfamily. The PmMGST3 transcript could be detected in all tissues tested, with highest transcript level seen in hepatopancreas. Cadmium treatment significantly increased PmMGST3 mRNA levels in gill and hepatopancreas, while bacterial challenge initially depressed mRNA levels and then increased its level in haemocytes, gill and hepatopancreas in a time-dependent manner. In an assay using cumene hydroperoxide as a substrate, we demonstrated that PmMGST3 possesses glutathione-dependent peroxidase activity. These results suggest that PmMGST3 plays an important role in cellular defense against oxidative stress caused by cadmium and bacteria., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

This study assessed the effects of mechanical agitation, hypo-saline conditions, and exposure to the air on the Akoya pearl oyster, Pinctada imbricata, focusing specifically on the immunological activity of haemocytes. Both phagocytosis and phenoloxidase activity decreased significantly when oysters were exposed to all three stressors. Transient decreases were also evident in total haemocyte counts after mechanical stress and exposure to air, while significant increases in total haemocyte counts were evident after exposure to low salinity. Acid phosphatase activity increased significantly when oysters were exposed to air. The frequency of granulocytes in the haemolymph increased significantly when oysters were stressed by hypo-saline conditions, whilst the relative frequency of granulocytes did not differ significantly after mechanical agitation or exposure to air. The total protein content of haemolymph increased significantly when oysters were stressed by mechanical agitation and low salinity. These results suggest that fluctuations in environmental conditions affect circulating haemocytes and their cytochemistry, and that the different immunological parameters tested were influenced uniquely according to the type of stressor., (Crown Copyright © 2010. Published by Elsevier Ltd. All rights reserved.)

Cathepsin L is one of the crucial enzyme superfamilies and involved in the immune responses. In this study, a cDNA encoding cathepsin L cysteine protease was identified and characterized from pearl oyster Pinctada fucata (designated as poCL1). The poCL1 cDNA was 1160 bp long and consisted of a 5'-untranslated region (UTR) of 15 bp, a 3'-UTR of 149 bp with a polyadenylation signal (AATAAA) at 11 nucleotides upstream of the poly(A) tail, and an open reading frame (ORF) of 996 bp encoding a polypeptide of 331 amino acids, which contained a typical signal peptide sequence (Met(1)-Ala(16)), a prodomain (Thr(17)-Asp(113)), and a mature domain (Leu(114)-Val(331)). The preproprotein contained the oxyanion hole (Gln), the active triad formed by Cys, His and Asn, and the conserved ERFNIN, GNFD motifs, which is characteristic for cathepsin L proteases. Homology analysis revealed that the poCL1 shared 62.5-72.5% similarity and 42.9-56.0% identity to other known cathepsin L sequences. The phylogenetic tree showed that the poCL1 clustered with the invertebrate cathepsin L cysteine proteases and was closely related to Stichopus japonicus CL, Strongylocentrotus salar CL1 and Radix peregra CL. The mRNA expression of the poCL1 in blank group and bacterial challenge group could be detected in all studied tissues with the higher level in digestive gland. The expression level of poCL1 mRNA was significantly up-regulated at 4 h and 8 h, and then significantly down-regulated at 12 h and 24 h in digestive gland after Vibrio alginolyticus stimulation. These results provided important information for further exploring the roles of pearl oyster cathepsin L in the immune responses., (Copyright 2010 Elsevier Ltd. All rights reserved.)

The clip-domain serine proteases (SPs) are the essential components of extracellular signaling cascade in various biological processes, especially in embryonic development and the innate immune responses of invertebrate. Herein, we described the isolation and characterization of pearl oyster Pinctada fucata clip-domain SP gene (designated as poSP). The poSP cDNA was 1080 bp long and consisted of a 5'-untranslated region (UTR) of 13 bp, a 3'-UTR of 68 bp with a polyadenylation signal (AATAAA) at 22 nucleotides upstream of the poly(A) tail, and an open reading frame (ORF) of 999 bp encoding a polypeptide of 332 amino acids with an estimated molecular mass of 36.5 kDa and a theoretical isoelectric point of 7.3. A clip-domain and a trypsin-like serine protease domain were identified in the poSP using SMART analysis. Homology analysis of the deduced amino acid sequence of the poSP with other known SP sequences by MatGAT software revealed that the poSP shared 47.0-68.4% similarity to the other known SP sequences. The poSP mRNA was expressed in haemocytes, gonad, digestive gland and mantle, but not expressed in adductor muscle and gill. The poSP mRNA was up-regulated and increased nearly double-fold after LPS or Vibrio alginolyticus stimulation, respectively. These results suggested that the poSP was an inducible acute-phase protein that perhaps involved in the innate immune response of pearl oyster.

Inhibitor of NF-kappaB (IkappaB) is one important member of NF-kappaB signal pathway and plays a pivotal role in regulating the innate immune response of invertebrate. Herein, we described the isolation and characterization of pearl oyster Pinctada fucata IkappaB gene (designated as poIkappaB). The poIkappaB cDNA was 1975 bp long and consisted of a 5' untranslated region (UTR) of 73 bp, a 3' UTR of 807 bp with three RNA instability motifs (ATTTA) and a polyadenylation signal (AATAAA) at 13 nucleotides upstream of the poly (A) tail, and an open reading frame (ORF) of 1095 bp encoding a polypeptide of 364 amino acids with an estimated molecular mass of 40.11 kDa and theoretical isoelectric point of 4.61. A conserved degradation motif (DS(35)GFSS(39)) and six ankyrin repeats were identified in the poIkappaB by SMART analysis. Homology analysis of the deduced amino acid sequence of the poIkappaB with other known IkappaB sequences by MatGAT software revealed that the poIkappaB shared 23.5-63.3% similarities with other known IkappaB isoforms. The poIkappaB mRNA was constitutively expressed in all studied tissues with the most abundant mRNA in the haemocyte. The poIkappaB mRNA was up-regulated and increased 4.13- and 5.28-fold after LPS and Vibrio alginolyticus stimulation, respectively. These results suggested that the poIkappaB was a constitutive and inducible acute-phase protein that perhaps involved in the immune defense of pearl oyster.