The ferroptosis in haemocytes of Pacific oyster Crassostrea gigas upon erastin treatment.

Ferroptosis is an iron and oxidative dependent form of cell death usually mediated by redox related molecules in vertebrates. In the present study, a glutathione peroxidase 4 (GPX4) and a solute carrier family 7 member 11 (SLC7A11, xCT) homologues were identified from the oyster Crassostrea gigas (designed as CgGPX4 and CgxCT), which contained a GSHPx domain and an AA_permease domain, respectively. The mRNA transcripts of CgGPX4 and CgxCT were expressed in all the examined tissues, including gill, gonad, adductor muscle, labial palp, mantle, hepatopancreas and haemocytes, with the highest expression in haemocytes. After erastin treatment, the rate of cell malformation and cell death increased significantly in haemocytes, and the mitochondrial atrophy, crest loss and fracture were observed in haemocytes. While the amount of Fe2+ and Malondialdehyde (MDA) increased significantly, the mRNA expressions of CgGPX4, CgxCT and voltage-dependent anion channel 2 (CgVDAC2) in haemocytes decreased significantly after erastin treatment. These results indicated that erastin was able to induce the ferroptosis of oyster haemocytes.

BCL10 regulates the production of proinflammatory cytokines by activating MAPK-NF-κB/Rel signaling pathway in oysters.

B cell lymphoma/leukemia 10 (BCL10) is an important member of the caspase recruitment domain-containing (CARD) protein family, which plays crucial roles in mediating the host inflammatory response. In the present study, a BCL10 homologue was identified from Pacific oyster Crassostrea gigas (designed as CgBCL10). The full length cDNA of CgBCL10 was of 897 bp with an open reading frame of 522 bp encoding a polypeptide of 174 amino acids containing a classical CARD domain. The deduced amino acid sequence of CgBCL10 shared low similarity with the previously identified BCL10s from other species. In the phylogenetic tree, CgBCL10 was firstly clustered with CvBCL10 from Crassostrea virginica and then assigned into the branch of invertebrate BCL10s. The mRNA transcripts of CgBCL10 were highly expressed in gonad, gill, adductor muscle, and haemocytes. After Vibrio splendidus stimulation, the mRNA expression level of CgBCL10 in haemocytes increased significantly (p < 0.01) at 24, 72 and 96 h. In CgBCL10-RNAi oysters, the phosphorylation level of mitogen-activated protein kinases (MAPKs), nuclear translocation of NF-κB/Rel and activator protein-1 (AP-1) in haemocytes were inhibited, and the mRNA expressions of inflammatory cytokines including CgIL17-1, CgIL17-2, CgIL17-3, CgIL17-6 and CgTNF all decreased significantly (p < 0.01) at 12 h after V. splendidus stimulation. These results suggested that CgBCL10 regulated the expression of inflammatory cytokines by activating MAPK kinase, and nuclear translocation of NF-κB/Rel and AP-1 to defense pathogen.

Syk regulates the haemocyte autophagy through inducing the mRNA expressions of autophagy-related genes and the cleavage of CgLC3 in oyster antibacterial immunity.

Spleen tyrosine kinase (Syk) is reported to be involved in activating the autophagy. Recently, a homologue of Syk was identified from Pacific oyster Crassostrea gigas (defined as CgSyk). In the present study, the molecular characteristics of CgSyk and its regulation mechanism in autophagy were investigated in oyster C. gigas. The full-length cDNA of CgSyk was of 4566 bp with an open reading frame (ORF) of 1989 bp. CgSyk encoded a polypeptide of 662 amino acids, containing two Src homology 2 (SH2) domains and one tyrosine kinase catalytic (TyrKc) domain. The deduced amino acid sequence of CgSyk shared low similarity with the previously identified Syks from other species. In the phylogenetic tree, CgSyk was first clustered with Crassostrea virginica CvSyk, and then classified into a branch of invertebrate Syks. In CgSyk-RNAi oysters, the mRNA expressions of CgLC3, CgP62, CgBeclin-1 and CgATG5 in haemocytes decreased significantly at 12 h after Vibrio splendidus stimulation. At the same time, the abundance of CgLC3Ⅱ in haemocytes, and the autophagy rate of haemocytes in CgSyk-RNAi oysters decreased significantly at 12 h after V. splendidus stimulation. All the results collectively suggested that CgSyk regulated the autophagy through inducing the mRNA expressions of autophagy-related genes and the cleavage of CgLC3 to defend against bacterial invasion in oysters.

A autophagy related-like protein 16-1 promotes the formation of autophagosomes and autolysosomes in antibacterial immune response of Pacific oyster Crassostrea gigas.

Autophagy related 16-like (ATG16L) protein is a core autophagy protein, which promotes the extension of autophagosome membrane through microtubule-associated protein light chain 3 (LC3). In the present study, an ATG16L was identified from oyster Crassostrea gigas (defined as CgATG16L1). The full-length cDNA of CgATG16L1 was of 3184 bp with an open reading frame of 1650 bp that encoded a polypeptide of 549 amino acids. There was an ATG5-interacting motif (AFIM) domain, a coiled-coil (CC) domain and seven tryptophan-aspartic acid 40 (WD40) repeats in CgATG16L1. ATG16L1 mRNA was expressed in all the examined tissues with the highest expression in haemolymph (11.22-fold of that in hepatopancreas, p < 0.05). The mRNA expressions of CgATG16L1 in haemocytes increased significantly at 3, 6, 12, 24 and 72 h after lipopolysaccharide (LPS) stimulation, which were 81.15-fold, 24.95-fold, 6.02-fold, 3.90-fold and 5.97-fold (p < 0.05) of that in control group, respectively. The green positive signals of CgATG16L1 protein and the red positive signals of CgLC3 protein were dotted in the cytoplasm of agranulocytes, semi-granulocytes and granulocytes. The co-localization of CgATG16L1 and CgLC3 was observed in haemocytes after Vibrio splendidus stimulation. In CgATG16L1-RNAi oysters, the number of autophagosomes and autolysosomes in haemocytes was reduced. All these results suggested that CgATG16L1 participated in the bacteria-induced autophagy process in the haemocytes of oyster response to bacteria invasion.