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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 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.

Peer Review reports


The Portuguese oyster Crassostrea angulata, a bivalve of major economic value and widely distributed around the world [1], used to be an important resource for the European aquaculture industry until the 1970s. By this decade it suffered a great mortality mainly due to an iridovirus disease [2] and since then, not only its production but also its natural populations are endangered due to climate and anthropogenic factors [3]. To overcome this situation, there is a need to create tools and strategies to preserve this species natural banks and to improve its production [4].

Cryopreservation presents itself as a valuable strategy to secure the storage of important genetic lines of endangered species, preserving biodiversity, and to improve the management of a species reproduction [5]. Many studies show the possibility of cryopreserving both gametes and larvae of different invertebrates, including bivalve species such as oysters [6,7,8]. When compared to sperm storage, the main advantage of larvae cryopreservation is the immediate availability of a diploid organism upon thawing [9, 10]. However, there are some challenges on the cryopreservation of larvae when compared to gametes, such as the size and complexity of a multicellular organism [10, 11].

The improvement of cryopreservation methodologies is imperative to obtain high post-thaw larvae quality. For that purpose, the evaluation of the effect of cryopreservation steps, such as cryoprotectants solutions and freezing process, on the biological processes and pathways of the organism is essential for the establishment of a reliable protocol [12].

The most common quality parameters used to evaluate larval post-thaw quality are swimming activity, morphology, and survival [8]. Information is still scarce, but new technologies are finding their way to become valuable tools in this type of studies. From a molecular point of view, the use of techniques such as transcriptomic and proteomic analysis in cryobiology [13,14,15], may help to identify different freezability biomarkers and to understand the molecular consequences related to cryopreservation. This will be extremely important for a species such C. angulata, where cryopreservation tools can be applied for the creation of genebanks to secure species preservation.

The application of transcriptome analysis technologies such as next-generation sequencing (NGS) is becoming widespread, using tools such as RNA sequencing (RNA-seq) that allow gene identification and their respective expression [16, 17]. Gene expression have been successfully used in the investigation of sperm and larvae cryodamage in several species including bivalves [18,19,20,21]. There are studies in the use of RNA-seq to identify gene alterations in post-thaw sperm and embryos of different species [22,23,24]. In blue catfish (Ictalurus punctatus) cryopreserved sperm, authors identified an upregulation in genes related to sperm motility-related functions (cilium, motile cilium, and microtubule cytoskeleton) and amide (often used as a cryoprotectant in sperm preservation) biosynthesis pathway [23]. In kelp grouper cryopreserved larvae (Epinephelus moara) it was identified a downregulation of genes related to eye development, cranial nerve development, sensory light stimulation and neurotransmitter transport, suggesting an impairment of larvae central nervous system development [22]. However, information is still scarce regarding invertebrate larvae and the effects are only associated to the last step of cryopreservation not taking into account the possible toxicity of cryoprotectants during exposure. In a previous study performed by our group in cryopreserved C. angulata D-larvae it was identified that the use of different cryoprotectant solutions induced an increase in larvae abnormalities incidence and a reduction of larvae swimming velocity after thawing [25]. However, there is no information about more in-depth damage resulted from gene alterations that can compromise further survival and development. The use of transcriptomic tools would be useful to investigate the molecular damage induced by all steps of the cryopreservation process and to identify putative cryodamage biomarkers on oyster´s larvae. This can be achieved by the full screening of the alterations of larvae molecular networks and biological processes promoted by cryopreservation. Therefore, this comprehensive investigation would support the selection of the most successful cryopreservation methodologies and potentially the identification of new analytical methods for the detection of relevant cryodamage in this species.

The objective of this study was to characterize the molecular damage promoted by cryopreservation in C. angulata D-larvae, using transcriptomic tools in two critical cryopreservation steps namely exposure to cryoprotectant solution (CPAs) and freezing/thawing process. Additionally, this work aims to identify putative quality biomarkers to understand the potential impact in larval structures, biological, cellular and molecular functions.


Biological material

C. angulata breeders were acquired from Neptunpearl Lda. bivalve farm (Setúbal, Portugal) during their natural spawning period, between May and July. These individuals were transported to IPMA Experimental Station of Shellfish Production (Tavira, Portugal) and kept at 4 ºC for a maximum of 24 h. Prior to gametes collection, the oysters were wiped to removed debris and fouling organisms.

Gamete collection and D-larvae production

The oysters were opened, and their sex was determined by microscopic observation of gametes obtained by a small incision in the gonad. Each breeder was independently stripped to collect the gametes for posterior fertilization. Egg suspensions were filtered at 100 μm and retain in 20 μm mesh screen, and spermatozoa sieved at 20 μm, to reduce potential contaminations. Both gametes were maintained in filtered and UV- sterilized seawater (FSW), until the fertilization. The sperm motility and concentration were evaluated by computer-assisted sperm analysis (CASA) system (Proiser R + D S.L., Valencia, Spain), while the oocyte sphericity and concentration were confirmed after 20 min of contact with FSW through observation via light microscopy. Only males with motile spermatozoa and females with spherical oocytes were used. A total of three males and three females were crossed to produce each larvae pool. The fertilization was carried out with a spermatozoon to oocyte proportion of 1000:1. One hour after fertilization, the eggs were filtered at a 20 μm mesh screen, to remove the remaining sperm, and incubated at 21 ± 1 °C for 24 to 30 h until reaching the D-larvae stage. Each pool was incubated in a 250 L tank with FSW at a concentration of 100 eggs per mL, with slight aeration. The larvae were recovered at a 30 μm mesh screen and kept in FSW to determine their concentration. The total number of larvae in each pool was counted in triplicates under microscopic observation, using a Sedgewick Rafter counting chamber. Pooled D-larvae with no apparent malformations were concentrated (around 120,000 larvae per mL) in FSW and kept on ice (4 °C) for a maximum of 2 h. A total of three pools (n = 3) were obtained in this study.

Experimental 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) 40,000 MW (Sigma-Aldrich) and 0.2 M Sucrose (Sigma-Aldrich) in milli-Q water. Larvae were incubated in the cryoprotectant solution for 3 min (equilibrium time) at 4 ºC, since in a preliminary trial higher exposure time interfered with larval movement pattern. 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 using a cryoprotectant solution based on DMSO. DMSO was chosen to see any further implications at molecular level in Crassostrea angulata larval cryopreservation. 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) according to [9] 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 cryoprotectant dissipation. The post-thaw D-larvae were collected in a 30 μm mesh screen, washed and concentrated in 1.5 mL of FSW for further analyses.

Transcriptome analysis

RNA extraction, library preparation and sequencing

Whole transcriptome analysis was performed in triplicate (n = 3) for pools of the fresh larvae, cryoprotectant exposed larvae and cryopreserved larvae. For this purpose, pools of 30,000 D-larvae were centrifuged at 7,400 g for 5 min at 4 °C to remove the FSW. Larvae pellets were then resuspended in 1 mL of TRI Reagent® (Sigma-Aldrich) and stored at -80 °C, until the RNA extraction.

RNA was isolated using TRI Reagent® (Sigma-Aldrich), according to the manufacturer’s recommendations, and total RNA was posteriorly purified using the NucleoSpin® RNA II kit (Macherey-Nagel, Germany). One treatment of dsDNase was performed to avoid genomic DNA contamination. The concentration and purity of the total RNA samples were measured using a NanoDrop One spectrophotometer (Thermo Fisher Scientific, USA). The RNA integrity was assessed on a bioanalyzer in RIN value (RNA integrity number ≥ 8.6), assessed by Experion RNA StdSens analysis kit (BIO-RAD). A total of 9 RNA samples were stored at -80 °C, for further analyses.

Whole transcriptome sequencing and respective bioinformatic analysis were performed by the Lifesequencing S.L.-ADM company (Valencia, Spain).

A total of 9 libraries were prepared starting from 1 µg of total RNA using a TruSeq RNA Library Preparation Kit v2 of Illumina (Illumina, USA). To confirm the quality of these libraries (library size: 276–348 bp; concentration: 76–162 nM) a HS D5000 Kit of Agilent 4200 was used, in a TapeStation bioanalyzer (Agilent technologies, USA). The libraries were sequenced in the NovaSeq 6000 Illumina instrument generating paired-end 150 bases reads.

Reads processing, mapping, and annotation

The raw reads were filtered using the BBTols v38.75 software (Bushnell B.). The sequencing adapters, low quality sequences (< Q20) and short sequences (< 40 nucleotides) were removed. Additionally, the reads were checked for potential contamination of bacteria, eukaryote, or archaea rRNA with the SortMeRNA v2.1b program [26]. As C. angulata whole genome was not available [27], the clean reads were then aligned to the reference genome of Crassostrea gigas (RefSeq accession: GCF_000297895.1 from the NCBI database). The proteins were then functionally annotated with the gene ontology (GO) terms by Blast against the Bivalvia taxa (taxID 6544) proteins, using OmicsBox v1.2 program [28]. Due to the presence of many isoforms, the quantification of the expression was taken to the gene level, a step performed by the Salmon v1.1 software [29].

Differential expression analysis

Differences in expression were assessed using the DESeq2 v3.10 R package [30]. Counts were then filtered to remove the unexpressed genes and those with an expression lower than 5 counts in at least 3 samples. Thresholds were set for significant differential expression as False Discovery Rate (FDR) < 0.05 and │log2FC│> 1.5 (FC – Fold Change) for all the comparisons.

Gene set enrichment analysis (GSEA)

The functional enrichment analysis was carried out with all the genes and not only those with significant different expression. GO terms with 10–600 genes annotated and 1,000 permutations were the conditions chosen for the GSEA. The threshold defined for the results was a FDR of 0.05.

RT-qPCR confirmation

To confirm our RNA-seq data and define putative cryodamage markers in C. angulata cryopreservation, eleven differentially expressed genes (DEGs) were selected for quantitative RT-PCR (RT-qPCR). The genes were selected for their relation to the oyster growth (adgre3, dynein beta), structure-mantle and shell formation- (mp, fbn2, myob3b), oxidative stress (epx, hsp70) and immune response (bp10, muc19, socs5, Lectin). The 18 S gene was used as reference gene. The primers were designed using Primer-Blast (NCBI) (Table 1). Eight hundred nanograms of total RNA were reverse-transcribed to cDNA using a Maxima™ First Strand cDNA Synthesis Kit for RT-qPCR with dsDNase (Thermo Scientific™), following the manufacturer instructions. The qPCR reactions were conducted in duplicate on a CFX96 realtime PCR Detection System (Bio-Rad Laboratories, Hercules, CA) using a SsoFast™ EvaGreen® Supermix. In a total volume of 15 µL, the PCR reaction contained 7.5 µL of supermix, 0.75 µL of each 10 µM forward and reverse primers, and 3.75 µL of cDNA. The thermo cycling protocol used was as follow: 3 min at 95 ºC for an initial denaturation followed by 30 cycles of denaturation at 95 ºC for 30 s, annealing at 57 ºC for 30 s and extending at 72 ºC for 1 min. A final extension step was carried at 72 ºC for 5 min. To normalize the data, the expression of the reference gene in a pool of all samples was used. The relative expression of transcripts was evaluated by the 2ΔΔCt method [31].

Table 1 List of forward (FW) and reverse (RV) primers used for the different transcripts analyzed through quantitative RT-PCR


RNA sequencing and mapping

A pair end sequencing of 9 libraries was performed using a NovaSeq 6000 Illumina instrument. After a filtering step, 375,187,599 total clean reads were obtained. The average quality of the generated reads ranged from 35.50 to 36.18. The distribution of raw and clean reads among the different samples, as well as their mapping rate, are represented in the Table 2.

Table 2 RNA sequencing results for the reads counting and mapping to the reference genome (Crassostrea gigas) in each replicate of the three different treatments: Fresh (fresh larvae diluted in FSW), CPA exposure (fresh larvae exposed to CPAs) and Cryopreserved (post-thaw larvae)

Differential expression analysis

By using the DESeq2 R package, a total of 22,787 genes were identified among all the samples. In Fig. 1 is showed two volcano plots representing the differentially expressed genes in the two different comparisons: cryoprotectant exposed larvae against fresh larvae (Fig. 1A) and cryopreserved larvae against fresh larvae (Fig. 1B), along with a Venn diagram representing the distribution of DEGs between the two comparisons (Fig. 1C). For the first comparison, 3 DEGs were identified, 1 downregulated and 2 upregulated. Addressing the second comparison, a total of 611 DEGs were found, 378 of them were considered downregulated and the remaining 233 upregulated. Due to the low number of genes differentially expressed in the first condition (Fig. 1A) for further analysis only the second comparison was taken in account.

Fig. 1
figure 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

Enrichment analysis

For a further understanding of the biological meaning of the DEGs represented under cryopreservation of C. angulata larvae, a GO enrichment analysis was performed for all the DEGs, using the Database for Annotation, Visualization and Integrated Discovery (DAVID). The most significantly enriched GO terms in biological processes (BP), cellular components (CC) and molecular functions (MF) were “carbohydrate metabolic process” (GO:0005975), “integral component of membrane” (GO:0016021) and “chitin binding” (GO:0008061), respectively.

It is also important to mention the significant enrichment of the “extracellular region” (GO:0005576) and the “oxidoreductase activity, acting on paired donors, with incorporation or reduction of molecular oxygen, another compound as one donor, and incorporation of one atom of oxygen” (GO:0016716) (Fig. 2).

Fig. 2
figure 2

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

Moreover, a Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis for all the DEGs was performed, to assess the most enriched pathways. The 10 most enriched pathways from a total of 126 are represented in Fig. 3. The most enriched pathways were the “neuroactive ligand receptor interaction”, “endocytosis” and “spliceosome”. However, it is also important to notice the significant enrichment of protein regulation pathways such as “protein processing in endoplasmic reticulum” or “proteasome”, and other important developmental pathways as “ribosome” and “FoxO signaling pathway”.

Fig. 3
figure 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

RT-qPCR confirmation

Through RT-qPCR, the results from the RNA-seq analysis were confirmed using a set of 11 genes related to important mechanisms such as growth (adgre3, dynein beta), structure (mp, fbn2, myob3b), oxidative stress (epx, hsp70) and immune response (bp10, muc19, socs5, lectin). The comparison between the results of gene expression in both techniques presented the same tendency and for that reason the RNA-seq output was considered to be confirmed. More specifically there was a general upregulation of adgre3, socs5, hsp70 and myob3b regarding control group. Contrarily, dynein beta, bp10, muc19, lectin, epx, mp and fbn2 were downregulated in the two different methods. These results are shown in Fig. 4. Table 3 summarizes the expression results obtain using both methods for all the 11 selected genes, and their respective previously reported function.

Fig. 4
figure 4

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

Table 3 Functions of the differentially expressed genes selected as putative biomarkers of cryodamage. Relative expression and statistical significance are described


After a few studies in C. angulata to establish and improve freezing/thawing protocols, cryopreservation has been identified as a technique with the potential to preserve its genetic lines and enhance its reproduction management [18, 25, 32,33,34]. Although C. angulata, like other bivalves, had promising results in preserving sperm and larvae [18, 25, 32,33,34], cryopreserving larvae has proven to be challenging due to the multicellular organism’s size and complexity [25]. In bivalves, the evaluation of the post-thaw quality of larvae has been based on swimming activity, morphology, and survival [6, 8, 25]. To elucidate about the molecular changes that occur during the cryopreservation process and identifying the involved molecular mechanisms of cryodamage, the current study has analysed the alterations in gene expression of cryoprotectant exposed and cryopreserved C. angulata larvae using RNA sequencing.

Transcriptomic, functional and enrichment analysis

In the present study it was possible to identify a set of differentially expressed genes in both treatment comparisons. Assessing the comparison between cryoprotectant exposed larvae and fresh larvae, the number of DEGs was considered to be low (2 genes upregulated and 1 downregulated). Therefore, any possible toxic effect dealt by cryoprotectant exposure was not considered for further analysis. However, the number of DEGs detected when comparing cryopreserved larvae against fresh larvae, was considerably higher (233 genes upregulated and 378 downregulated). These results reveals that molecular damage is associated with freezing-thawing process during cryopreservation of Crassostrea angulata D-larvae rather than to cryoprotectant exposure.

Following a functional analysis, the relation of all DEGs to specific CC, MF and BP was evaluated through an enrichment analysis. Among these different gene ontologies (GO), the most significantly enriched were integral components of membrane (CC) and Chitin binding (MF). Chitin is the second most abundant natural polysaccharide and it is abundantly present in the shell matrix of oyster species such as Pinctata fucata martensii and C. gigas [35, 36]. Chitinase and Chitin synthases are vastly represented in the mantle, a very important tissue in shell formation and during early oyster larval development stages [37]. The results of the present study showed a down regulation of chitin synthase, chitin binding proteins and different isoforms of acidic mammalian chitinase in cryopreserved larvae, suggesting an impairment in shell formation and corroborating the data presented by Anjos et al. [25] in previous studies. Also, chitinases are known for their importance in immune response and by helping to maintain normal life cycle functions since, not only synthesis, but also degradation of chitin, are important in the development of chitin containing organisms [38]. Different DNA binding related molecular functions (RNA polymerase II transcription factor activity, sequence specific DNA binding; transcription factor activity, sequence-specific DNA binding; DNA binding) were also significantly enriched. An example of DNA binding functions are the changes occurring in DNA methylation during early development in C. gigas as reported by Riviere et al. [39]. Moreover, Zhao et al. [40] hypothesized that due to its connection to cell proliferation, DNA binding was a key element in stress response. This means that as a stress factor, the cryopreservation process may induce an impairment in the early development of C. angulata larvae.

Regarding the KEGG pathway enrichment analysis, the most significantly enriched pathway was the “neuroactive ligand-receptor interaction”. This pathway is made up of receptors situated on plasma membranes that are involved in signal transduction from the external environment into cells [41]. An analysis of the transcriptome of C. gigas and P.f. martensii at different stages of development revealed that neuroendocrine pathways such as this one, were implicated in important developmental functions like shell formation, settling, and metamorphosis [42]. Furthermore, a previous study by Lu et al. [43] reported a significant enrichment of this pathway upon induced stress in P.f. martensii. Both previous studies and the results of our study are a plausible evidence of the malformations observed by Anjos et al. [25].

The second two most significant enriched pathways were the endocytosis and the spliceosome. The first one is an imperative part of the membrane receptors activity and quantity control, regulating the signal transduction mediated by those receptors [44]. This pathway is also known to be related to cell proliferation and organism growth since it is connected to the degradation of epidermal growth factor receptors (EGFR) [45]. These receptors were referred to be directly corelated to the development and growth of the pearl oyster [46]. Also, Li et al. [47], proposed that endocytosis-induced processes such as signal transduction and plasma membrane proteins degradation, may regulate oyster growth by integrating endogenous signaling pathways and environmental input. These previous studies corroborate the results of the present study and therefore, it is hypothesized that cryopreservation may affect growth in C. angulata larvae.

Concerning “spliceosome”, as a key tool of genetic information processing, it is extremely important in the survival, adaptation and development of the organism [48]. Previous studies have already demonstrated an impairment of this process due to stress factors such as ocean acidification [49]. The same may be assumed by observing the functional analysis results of the present research, more specifically, the significant enrichment of processes like “DNA binding”.

In our study, there was also a significant enrichment of the “ribosome” pathway. This molecular organelle is responsible for the translational capacity of a cell and so, directly related to protein synthesis and functions such as cell growth, proliferation and apoptosis [50]. Moreover, other protein processing related pathways were significantly enriched, the “proteasome” and “protein processing in endoplasmic reticulum”, suggesting an increased amount of degraded proteins. Cryopreservation clearly induces a stress response in this organism and apparently leads to a development malfunction, mainly at the shell formation and membrane level.

Putative biomarkers of cryopreserved larval quality

To validate RNAseq data, a selection of 11 genes was done according to their relevance in certain functions. These genes were checked for their putative use as biomarkers of post-thaw larval quality. One of the functions affected by cryopreservation was the oxidative stress system. Heat shock proteins 70, hsp70, a subgroup of chaperones, are important to maintain the homeostasis of the cell and has the capacity of counteract apoptotic mechanisms, interviewing in cell processes such as cell movement and cytoskeleton stabilization [51, 52]. Our findings indicate that the expression levels of hsp70 were higher in post-thaw D-larvae than in fresh larvae (larvae dilute in FSW). This result is in line with data reported by Park et al. [53], where the expression levels of four apoptotic-related genes, including hsp70, were observed to be significantly elevated in cryopreserved bovine embryos. This expression may arise, as suggested by the authors, as a stress response and potentially compromised developmental ability. Overall, these proteins have the function of preventing thermal or oxidative stress, which is commonly associated with cryopreservation procedures.

Eosinophil peroxidase (epx) is an enzyme released from eosinophils granulocytes, that is essential to maintain the main function and homeostasis of eosinophils [54]. Eosinophils are made part of a group of cells known as hemocytes that play a crucial role in bivalve immune response to defend against different stressors, such as pathogens, temperature, acidification and pollution [55]. In the present work, the epx expression revealed that this gene was suppressed in D-larvae after freezing/thawing steps. Other studies, conducted in Anguilla japonica and C. gigas reported that when organisms were exposed to osmotic stress conditions, the levels of epx were also suppressed [56, 57], having been suggested by Zhao et al. [57] that this could serve as a protective tactic for mitigating cellular stress and lowering levels of reactive oxygen species (ROS). The observed suppression of epx expression in our study may be related to our experimental protocol that involved environmental fluctuations due to the freezing/thawing process, which must have induced thermal stress, leading to increased expression of hsp70 rather than epx.

The dynein beta chain flagellar outer arm, dyh4, belongs to the dynein protein family. Dynein proteins are divided into two main groups, which are cytoplasmic and axonemal dynein [58]. Cytoplasmic dynein is responsible for intracellular transport and cell mitosis [58, 59]. Axonemal dynein is a microtubule-based molecular motor that is in charge of the generation and regulation of the bending of cilia and flagella in eukaryotes [58, 60]. Dynein arms convert the chemical energy released upon ATP binding into mechanical force, producing the driving power for the organelles’ movement [60]. Cilia and flagella play important roles in bivalve sperm and larvae motility. The expression of dynein beta in thawed C. angulata D-larvae was lower than in the fresh larvae, suggesting some level of cilia impairment which may affect the larvae swimming performance and, in further larvae development stages, their feeding behavior and sensorial role. Unfortunately in the present study we could not follow larval development but this data corroborated previous findings reported by our group, where a significant lower velocity and motility was observed in thawed C. angulata D-larvae when compared with the control group [25]. Similar findings were reported by Suquet et al. [61] and Suneja et al. [62], where lower performances in swimming activity of C. gigas post-thaw D-larvae were reported. Suneja et al. [62] even explored the organogenesis of these larvae and identified development problems in the velum structure as a result of cryodamage, which suggests that modifications in the structure of the velum can lead to cilia impairment. This can affect the swimming and feeding behavior and compromise the performance of the larvae or even lead to their death at later stages. Apart from impairing structural modification associated with filtration and movement mechanisms, the downregulation of dynein motor proteins may also contribute to shell malformations, previously detected Anjos et al. [25] since as stated by De Wit et al. [63] these proteins have an important role as transporters of cellular components during the initial stages of shell formation in this species.

Myosin is the key muscle protein of thick filaments [64] and its function is to transform chemical energy in mechanical force that travels along actin filaments, resulting in the contraction of the muscle [65]. In bivalves, during larval development, the actin filaments or expression profiles of myosin heavy chain are mainly found in the velum retractor and adductor muscle, while in the adult phase in the adductor and mantle muscles [64, 66, 67]. Myosin heavy chain seems to have an important role in C. gigas embryo-larval developmental phases, especially in the stages of trochophore and D-larvae, due to the formation of the muscle structures during embryogenesis some associated with larval locomotion [64]. Thus, our results revealed that cryopreservation may have affected certain mechanisms in C. angulata D-larvae related to myosin protein production. This is evidenced by the higher gene expression of myob3b in the cryopreserved D-larvae compared to the control group, which may suggest a compensatory response to the cryopreservation process.

Fibrillin-2 is a protein encoded by the fbn2 gene that belongs to the fibrillin family [68]. In C. gigas, fibrillin is highly expressed in the mantle [69], a soft tissue layer that lines the inner shell and covers the visceral mass. This structure provides protection, contributes to shell formation, facilitates respiration and plays a crucial role in the oyster’s filter feeding mechanism [70, 71]. Our results showed that expression of the fbn2 transcript was suppressed after cryopreservation. This suggests that there may be an alteration at the level of the extracellular matrix of C. angulata larvae. This alteration is likely to have implications in the development and maintenance of tissues, potentially jeopardizing the subsequent larval developmental stages. David et al. [72] observed a down-regulation of the fibrillin gene in the mantle and gills of C. gigas after 24 days of hypoxia exposure. These results together with the ones obtained for mp, a specific mantle protein present in the epithelial cells, where expression was also suppressed, demonstrates that these alterations could compromise proper functioning, which can lead to alterations in the mantle tissues, shell formation and feeding capacity, especially during the early stages of development when structures are being formed. Some morphological alterations as mantle protuberance and reduction of body size related to the shell were detected in C. angulata post-thaw D-larvae in a prior work conducted by our group [25]. These morphological alterations can be related to mp gene suppression observed after freezing/thawing steps.

Suppressors of cytokine signaling (socs) are a class of inhibitory proteins that negatively regulate cytokine signal transduction [73, 74]. These inhibitory proteins play an essential role in several intercellular signal pathways that are engaged in the immune system [73, 75], growth and development of the organisms [76] being identified in several organs of C. gigas [75]. In our results, we identified the socs5 gene as being upregulated in cryopreserved larvae when compared with the fresh larvae. This result suggested that the cryopreservation protocol may have induced physical stress in the D-larvae of C. angulata, due to exposure to a combination of hyperosmotic solution (cryoprotectant solution) with temperature variations (freezing/thawing steps). These stress factors may have activated the production of cytokines, and to control an excessive response that can be harmful to the host, socs5 was activated. Similar results were obtained by De Zoysa et al. [77] with the socs2 gene being upregulated in Haliotis discus discus during thermal, low-salinity and hypoxic stress.

Blastula protease 10, bp10, involved in immune functions and embryonic development [78, 79] is an enzyme that belongs to the astacin metalloprotease family [80]. This gene has been used to evaluate the toxic effects of metals and other contaminants in Paracentrotus lividus embryos being upregulated or downregulated depending on the compound tested [79]. Our results revealed that cryopreservation suppressed the bp10 expression in the D-larvae of C. angulata when compared to the control. However, little is known about the bp10 gene in oysters and further studies will be needed to characterize and understand how variation in the expression of this metal binding protein is affected in cryopreserved larvae.

Lectins play a fundamental role in the self and nonself recognitions, innate immunity, reproduction and food capture and ingestion in bivalves [81,82,83,84]. Particularly in shell formation, these group of proteins are involved in the extracellular matrix agglutination [63]. In our study, the expression of lectins in C. angulata cryopreserved D-larvae was lower than in the fresh larvae. Since lectins can bind carbohydrates present in the surface of microalgae, they participate in the recognitions of food particles. Therefore, in our study changes in lectin gene expression may affect larvae’s digestive functions, impairing their ability to efficiently capture and process food. It is crucial to understand the implications of altered lectin gene expression in C. angulata thawed D-larvae as it can have consequences on their feeding selection and further digestive function and ultimately in larval developmental outcomes.


In this study, the analysis of differential gene expression revealed significant changes in genes associated with growth, structural development, oxidative stress response, and the immune system in cryopreserved C. angulata larvae, compared to fresh larvae. No effects were seen in larvae exposed to cryoprotectants. These findings underscore the importance of considering both molecular and physiological aspects in the development of reliable cryopreservation protocols and in the detection of relevant biomarkers of cryodamage in C. angulata D-larvae. Furthermore, they highlight the critical step in the cryopreservation process, revealing that gene expression is not affected by cryoprotectant exposure as it is by the freezing process itself. Importantly, this study emphasizes the significance of our findings for the shellfish sector, providing valuable insights for future research in cryobiology and support aquaculture and restocking programs.

Data availability

The datasets generated and analysed during the current study are available in the Gene Expression Omnibus (GEO) repository, with accession number GSE246924 to datasets.



Next-generation sequencing


RNA sequencing


Cryoprotectant solution


Filtered and UV- sterilized seawater


Computer-assisted sperm analysis


Dimethyl sulfoxide




Liquid nitrogen


Gene ontology


False Discovery Rate


Fold Change


Gene Set Enrichment Analysis


Differentially expressed genes


Quantitative RT-PCR


Database for Annotation, Visualization and Integrated Discovery


Biological processes


Cellular components


Molecular functions


Kyoto Encyclopedia of Genes and Genomes


Reactive oxygen species


Epidermal growth factor receptors


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The authors acknowledge F. Pinheiro for the support during experimental samplings, the technical team of IPMA (S. Joaquim, A. Rato, A. M. Ramos, A. B. Dias, and J. M. Teixeira) and bivalve farm Neptunpearl Lda. for maintaining and supplying the biological material, respectively.


This work was supported by the projects 0139_VENUS_5_E (Interreg POCTEP), ASSEMBLE + JRA2-H2020-INFRAIA-2016-2017 (No 730984), EBB-EAPA_501/2016 (Interreg Atlantic Area) and received national funds through FCT - Foundation for Science and Technology through projects CCMAR/Multi/04326/2022 and UID/Multi/04326/2019 and from the operational programs CRESC Algarve 2020 and COMPETE 2020 through project EMBRC, PT ALG-01-0145-FEDER-022121. CA and DD were supported by FCT fellowships (SFRH/BD/130910/2017 and COVID/BD/151914/2021) and Interreg POCTEP (0139_VENUS_5_E), respectively. EF was supported by FCT contract 2020.04181.CEECIND.

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E.C, designed the experimental work and acquired the funding. E.C., D.M. and E.F. supervised the experimental work. C.A. and D.D. collected the samples, performed the laboratory analyses, treated data and wrote the manuscript. C.A., D.M., E.F. and E.C. revised and edited the manuscript. All the authors contributed to the manuscript and approved the submitted manuscript.

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Correspondence to Elsa Cabrita.

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Anjos, C., Duarte, D., Fatsini, E. et al. Comparative transcriptome analysis reveals molecular damage associated with cryopreservation in Crassostrea angulata D-larvae rather than to cryoprotectant exposure. BMC Genomics 25, 591 (2024).

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