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Identification of the autophagy pathway in a mollusk bivalve, Crassostrea gigas.

ABSTRACT: The Pacific oyster, Crassostrea gigas, is a mollusk bivalve commercially important as a food source. Pacific oysters are subjected to stress and diseases during culture. The autophagy pathway is involved in numerous cellular processes, including responses to starvation, cell death, and microorganism elimination. Autophagy also exists in C. gigas, and plays a role in the immune response against infections. Although this process is well-documented and conserved in most animals, it is still poorly understood in mollusks. To date, no study has provided a complete overview of the molecular mechanism of autophagy in mollusk bivalves. In this study, human and yeast ATG protein sequences and public databases (Uniprot and NCBI) were used to identify protein members of the C. gigas autophagy pathway. A total of 35 autophagy related proteins were found in the Pacific oyster. RACE-PCR was performed on several genes. Using molecular (real-time PCR) and protein-based (western blot and immunohistochemistry) approaches, the expression and localization of ATG12, ATG9, BECN1, MAP1LC3, MTOR, and SQSTM1, was investigated in different tissues of the Pacific oyster. Comparison with human and yeast counterparts demonstrated a high homology with the human autophagy pathway. The results also demonstrated that the key autophagy genes and their protein products were expressed in all the analyzed tissues of C. gigas. This study allows the characterization of the complete C. gigas autophagy pathway for the first time. Abbreviations: ATG: autophagy related; Atg1/ULK: unc-51 like autophagy activating kinase; ATG7: autophagy related 7; ATG9: autophagy related 9; ATG12: autophagy related 12; BECN1: beclin 1; BSA: bovine serum albumin; cDNA: complementary deoxyribonucleic acid; DNA: deoxyribonucleic acid; GABARAP: GABA type A receptor-associated protein; IHC: immunohistochemistry; MAP1LC3/LC3/Atg8: microtubule associated protein 1 light chain 3; MTOR: mechanistic target of rapamycin kinase; NCBI: national center for biotechnology information; ORF: open reading frame; PBS: phosphate-buffered saline; PCR: polymerase chain reaction; PtdIns3K: class III phosphatidylinositol 3-kinase; RACE-PCR: rapid amplification of cDNA-ends by polymerase chain reaction; RNA: ribonucleic acid; SQSTM1: sequestosome 1; Uniprot: universal protein resource; WIPI: WD repeat domain, phosphoinositide interacting.

SUBMITTER: Picot S

PROVIDER: S-EPMC7595595 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Background The Pacific oyster Crassostrea gigas (Mollusca, Lophotrochozoa) is an alternative and irregular protandrous hermaphrodite. Little is known about the genetic and phenotypic bases of sex determinism in oysters, and little more about the molecular pathways regulating reproduction. We have recently developed and validated a microarray containing 31,918 oligomers (Dheilly et al., 2011) representing the oyster transcriptome. The application of this microarray to the study of mollusks gametogenesis should provide a better understanding of the key factors involved in sex differentiation and the regulation of oyster reproduction. Results Expression of the 31,918 ESTs was studied in gonads of oysters cultured along the French Atlantic coasts over a yearly reproductive cycle. Principal component analysis and hierarchical clustering first showed a significant divergence in gene expression patterns of males and females beginning when gonial mitosis started. Early expressed male-specific genes included bindin and female-specific genes included foxL2, a pancreatic lipase related protein, cd63 and vitellogenin. ANOVA analysis of the data further revealed 2482 genes differentially expressed during the course of males and/or females gametogenesis. The expression of 434 genes could be localized in either germ cells or somatic cells of the gonad by comparing the transcriptome of gonads to the transcriptomes of striped oocytes and somatic tissues. Analysis of the annotated genes revealed conserved molecular mechanisms between mollusks and mammals. Genes involved in chromatin condensation, DNA replication and repair, mitosis and meiosis regulation, transcription, translation and apoptosis were expressed in both male and female gonads. Additional lists of genes more specifically involved in either spermatogenesis (meiotic phase, in spermatozoids and mature sperm formation and in flagella structure and movement) or oogenesis (female sex differentiation, transcriptional regulation, cell cycle regulation and oocytes maturation) were also generated. Conclusions Our study provides novel insight into spermatogenesis and oogenesis in an alternative hermaphrodite bivalve, the Pacific oyster C. gigas. We identified genes opening new perspectives for functional studies of the signaling pathways implicated in gonad differentiation and development.

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Identification of the autophagy pathway in a mollusk bivalve, Crassostrea gigas.

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