Comparative transcriptome analysis reveals molecular damage associated with cryopreservation in Crassostrea angulata D-larvae rather than to cryoprotectant exposure

Abstract

Background: The Portuguese oyster Crassostrea angulata, a bivalve of significant economic and ecological importance, has faced a decline in both production and natural populations due to pathologies, climate change, and anthropogenic factors. To safeguard its genetic diversity and improve reproductive management, cryopreservation emerges as a valuable strategy. However, the cryopreservation methodologies lead to some damage in structures and functions of the cells and tissues that can affect post-thaw quality. Transcriptomics may help to understand the molecular consequences related to cryopreservation steps and therefore to identify different freezability biomarkers. This study investigates the molecular damage induced by cryopreservation in C. angulata D-larvae, focusing on two critical steps: exposure to cryoprotectant solution and the freezing/thawing process.

Results: Expression analysis revealed 3 differentially expressed genes between larvae exposed to cryoprotectant solution and fresh larvae and 611 differentially expressed genes in cryopreserved larvae against fresh larvae. The most significantly enriched gene ontology terms were "carbohydrate metabolic process", "integral component of membrane" and "chitin binding" for biological processes, cellular components and molecular functions, respectively. Kyoto Encyclopedia of Genes and Genomes enrichment analysis identified the "neuroactive ligand receptor interaction", "endocytosis" and "spliceosome" as the most enriched pathways. RNA sequencing results were validate by quantitative RT-PCR, once both techniques presented the same gene expression tendency and a group of 11 genes were considered important molecular biomarkers to be used in further studies for the evaluation of cryodamage.

Conclusions: The current work provided valuable insights into the molecular repercussions of cryopreservation on D-larvae of Crassostrea angulata, revealing that the freezing process had a more pronounced impact on larval quality compared to any potential cryoprotectant-induced toxicity. Additionally, was identify 11 genes serving as biomarkers of freezability for D-larvae quality assessment. This research contributes to the development of more effective cryopreservation protocols and detection methods for cryodamage in this species.

Keywords: Cryodamage; Cryoprotectant exposure; D-larvae cryopreservation; Gene expression; Portuguese oyster; RNA-seq.

Fig. 1

Volcano plots representing the results of differential expression analysis between the two comparisons. A Cryoprotectant exposed larvae against fresh larvae. B Cryopreserved larvae against fresh larvae. C Venn diagram representing the distribution of DEGs between the two comparisons

Fig. 2

Gene ontology (GO) enrichment results for all differentially expressed genes (DEGs) in the comparison between cryopreserved larvae against fresh larvae

Fig. 3

Top 10 enriched pathways according to the results of the KEGG enrichment analysis for all differentially expressed genes in the comparison between cryopreserved larvae against fresh larvae

Fig. 4

Quantitative qPCR validation of RNA-seq results in terms of relative gene expression