Comparative transcriptome analysis reveals molecular damage associated with cryopreservation in Crassostrea angulata D-larvae rather than to cryoprotectant exposure
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. Expression analysis revealed 3 differentially expressed genes between larvae exposed to cryoprotectant solution and fresh larvae and 511 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. 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. Overall design: To characterize the transcriptional changes related with the different steps of cryopreservation, the whole transcriptome profile of fresh larvae diluted in FSW (fresh larvae), fresh larvae exposed to a CPAs (cryoprotectant exposed larvae) and post-thaw larvae (cryopreserved larvae) was compared. For this purpose, each D-larvae pool was exposed to the three following conditions, one control group and two treatments. As a control group, fresh larvae were concentrated in FSW as previously described. In the first treatment, larvae exposed to CPAs, the concentrated D-larvae were diluted with a 1:1 proportion in a CPAs consisting in a final concentration of 10% (v/v) dimethyl sulfoxide (DMSO) (Sigma-Aldrich), 1% (w/v) Polyvinylpyrrolidone (PVP)-40 40000 MW (Sigma-Aldrich) and 0.2 M Sucrose (Sigma-Aldrich) in milli-Q water, and incubated for 3 min of equilibrium time, at 4 ºC. After incubation, the pools of D-larvae were diluted in a 1:3 proportion in FSW to dissipate the CPAs. The second treatment intended to evaluate the effects of the freezing/thawing process. Following the same procedure as in the first treatment, the pooled larvae were incubated with the same CPAs. During the equilibrium time, the larvae pools diluted in the CPAs were loaded to 0.5 mL French straws (30,000 per straw) and maintained at 4 °C until finishes the 3 min equilibrium time. Subsequently, the straws were frozen in a programable biofreezer (Asymptote Grant EF600, United Kingdom) with the following freezing  protocol: 2.5 °C/min from 0 to -10 °C, hold for 5 min at -10 °C, 0.3 °C/min from -10 °C to -20 °C and 2.5 °C/min down to -35 °C, and finally, plunged into liquid nitrogen (LN) and stored in a LN container. After two months of storage, straws were thawed in a water bath set at 37 °C for 10 s. Afterward, a recovery bath was prepared, by diluting the content of each straw in 2 L of FSW during a period of incubation of 1 h at room temperature. This procedure allowed the removal of cryoprotectants. The post-thaw D-larvae were collected in a 30 µm mesh screen, washed and concentrated in 1.5 mL of FSW. Debris were eliminated, and the larvae quality was evaluated.