Anjos Catarina, Duarte Daniel, Fatsini Elvira, Matias Domitília, Cabrita Elsa
Centre of Marine Sciences-CCMAR/CIMAR.LA, University of Algarve, Faro, 8005-139, Portugal.
Portuguese Institute for Sea and Atmosphere-IPMA, Av. 5 de Outubro, Olhão, 8700-305, Portugal.
BMC Genomics. 2024 Jun 12;25(1):591. doi: 10.1186/s12864-024-10473-1.
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 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.
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.
葡萄牙牡蛎（Crassostrea angulata）是一种具有重要经济和生态意义的双壳贝类，由于病害、气候变化和人为因素，其产量和自然种群数量均有所下降。为了保护其遗传多样性并改善繁殖管理，冷冻保存成为一种有价值的策略。然而，冷冻保存方法会对细胞和组织的结构与功能造成一些损伤，进而影响解冻后的质量。转录组学有助于理解与冷冻保存步骤相关的分子后果，从而识别不同的可冻性生物标志物。本研究调查了冷冻保存对葡萄牙牡蛎D期幼虫造成的分子损伤，重点关注两个关键步骤：暴露于冷冻保护剂溶液以及冷冻/解冻过程。
表达分析显示，暴露于冷冻保护剂溶液的幼虫与新鲜幼虫之间有3个差异表达基因，冷冻保存幼虫与新鲜幼虫之间有611个差异表达基因。在生物学过程、细胞成分和分子功能方面，最显著富集的基因本体术语分别是“碳水化合物代谢过程”、“膜的 integral 成分”和“几丁质结合”。京都基因与基因组百科全书富集分析确定“神经活性配体受体相互作用”、“内吞作用”和“剪接体”为最富集的途径。RNA测序结果通过定量RT-PCR进行验证，因为两种技术呈现出相同的基因表达趋势，并且一组11个基因被认为是用于进一步研究冷冻损伤评估的重要分子生物标志物。
当前工作为冷冻保存对葡萄牙牡蛎D期幼虫的分子影响提供了有价值的见解，表明冷冻过程对幼虫质量的影响比任何潜在的冷冻保护剂诱导的毒性更为显著。此外，还鉴定出11个基因作为D期幼虫质量评估的可冻性生物标志物。本研究有助于开发更有效的冷冻保存方案以及该物种冷冻损伤的检测方法。
