Increasing larval fitness prior to cryopreservation
The D-larvae of M. galloprovincialis from adult specimens in the Vigo estuary withstand a decrease in salinity of approximately 14.29% and an increase of 21.42% during the first 72 h of development (Fig. S1A-B-Fig. S2A-B). Based on the results of size and % normality, we can conclude that the optimum salinity range for larval development is between 30 and 42.5‰ (Fig. S1A-B). Previously, Hrs-Brenko12 and His et al.13 determined that the optimum salinity is 35‰. However, the effects of salinities higher than 35‰ were not analyzed, as presented in this work. The wide range of salinities determined to be suitable for larvae opens the possibility to use beneficial pre-freezing higher salinities10but this approach is not feasible at large aquaculture facilities unless they are situated in regions where the natural salinity is equally high. The cultures at various temperatures (from 12 to 25 °C) revealed that the optimal temperature range for the development of D-larvae at 72 h of life was between 16–20ºC (Fig. S2A-B), with 16 °C used as our control and the usual standard procedure. Our temperature range data disagrees with those obtained by Sánchez-Lazo & Martínez-Pita, 2012, 201214, who determined that the optimal temperature range for the growth of M. galloprovincialis larvae from the region of Galicia is between 20–24ºC. However, our data revealed a large decrease in the percentage of normality at 25 °C, which was not as evident in the size of the larvae, although there were significant differences with respect to the control at 16 °C. With these results, we decided to increase the incubation temperature standard from 16 °C to 20 °C for the next experiments to ensure high-quality larvae.
Mussel larvae, when they reach the D-larval stage (~ 48hpf at 18ªC), prior to that the trochophore larvae is lecytothrophic (lives of egg reserves)15,16. In our prior publications, the larvae were cryopreserved at 48 h and therefore not fed prior17. When our data revealed that older larvae were more resistant to cryopreservation, the protocols were modified to use 72 h larvae, and we continued without feeding, as a full stomach could be a source of internal ice formation during freezing in such complex larvae. The results of the cryopreservation of 72 h-old larvae after feeding (Fig. 2A) show significant differences in larval size between the controls, and these differences can also be observed between the cryopreserved larvae with and without food. The percentage of larval normality significantly differed between fed (84.3 ± 0.9%) and unfed larvae (74.4 ± 5.4%) in both the control and cryopreserved larvae. On the other hand, no significant differences were detected between the fed control larvae and the fed cryopreserved larvae, contrary to what happens if we compare the unfed cryopreserved larvae with their nonfeed control (Fig. 2B).
We succeeded in cryopreserving, for the first time, D-larvae of M. galloprovincialis 72 hpf after feeding. Moreover, all fed larvae, whether cryopreserved or not, were greater in size and percentage of normality than unfed larvae. In the case of the fed cryopreserved larvae, the average size was slightly larger (111 ± 1.64 μm) than that of those cryopreserved without food (107 ± 1.61 μm). In the case of % normality, the difference was much greater, ranging from 84.3%±0.9% in fed cryopreserved larvae to 74.4%±5.4% in nonfeed cryopreserved larvae. This implies that feeding the larvae improved their overall fitness, increasing their survival after thawing by approximately 10%.
Graph of the mean growth ± SD of cryopreserved larvae at 72 hpf (h post-fertilization) and cryopreserved unfed larvae at 72 hpf at 48 h post-thawing (A). For each parameter, 35 larvae were measured (n = 3). Graph of the percentage of larval normality ± SD of cryopreserved feed-fed larvae at 72 hpf and cryopreserved unfed larvae at 72 hpf 48 h post- thawing. (B) For each parameter, 100 larvae were examined (n = 3). Asterisks represent significant differences compared with the control without cryoprotectant; * indicates p ≤ 0.05. Images of cryopreserved fed (C,D) and unfed (E and F) larvae of M. galloprovincilis at 48 h post-thaw (hpt), stained with propidium iodide (red) and Hoechst (blue). Larvae cryopreserved after feeding, showing no damage and minimal variation between larval stages (C). Negative control (D). Unfed cryopreserved larvae showing localized damage in the ciliary veil area (thick arrow) (E) and some variation between larval stages (trochophore with slight damage) (F).
When the percentage of larval normality of our nonfeed cryopreserved larvae (74.4%) was compared with the current protocol, this percentage was close to that obtained by Heres, 20211 (77%± 4.31%). The survival of cryopreserved larvae after feeding increased by 7.3% (84.3 ± 0.96%).
To obtain the fittest larvae prior to starting the cryopreservation process, we decided to culture them at 35–37%, at 20 °C and with a microalgal diet.
Damage analysis of cryopreserved D-larvae 72 h after feeding
The results of the Hoechst and propidium iodide fluorescence dyes revealed small differences in the damage produced between fed and unfed cryopreserved larvae, which were located mainly in the ciliated part of the veil (Fig. 2E). Larvae cryopreserved after feeding do not present this damage (Fig. 2C), which appeared sporadically in unfed larvae (Fig. 2D as example of a clearly damaged larvae for comparison). In addition, for larvae that were fed prior to cryopreservation, the divergence between larval stages was minimal, whereas this high variability phenomenon was high in the case of unfed larvae, with trochophore larvae (a prior development stage that should have been overcome before 20 hpf) appearing sporadically (Fig. 2F). To date, no other records of larvae or organisms have been cryopreserved after feeding. Moreover, a few previous studies have analyzed the effects of pre-freezing diets after successful cryopreservation. Some works have used different pre-freezing diets on parental lines to modify the lipid composition of sperm, embryos, human T cells, etc18,19,20. However, in Guardieiro et al.19, the cryotolerance of beef heifer embryos was compromised when pre-freeze diets were used. The same is true for boar sperm fed DHA-enriched diets20. In contrast, in the case of Baynes & Scott, 198718, the use of pre-freeze diets increased viability and the post-thaw proliferation rate in human T cells. If we analyze our results together with those of the few previous studies, we can assume that it is not so much the use of lipid-enriched diets that is important but rather the diet itself.
The lower damage in fed larvae disproves the assumption that the full stomach could be a source of ice formation but also shows that larvae cryopreserved after feeding are greater in size and have higher survival rates. In contrast, the commonly reported developmental delays and lesions associated with cryopreservation11,21 are reduced. Our results suggest that feeding larvae before freezing significantly enhances both their survival and size, providing additional protection against cryopreservation, an important factor to consider when optimizing existing protocols.
Serial cryopreservation of larvae throughout the rearing cycle
By feeding larvae prior to cryopreservation and adding the prior acquired methodological knowledge, it was possible to study the success of cryopreserved larvae as their development progressed all the way to their settlement and transformation into juveniles. We successfully cryopreserved larvae at 24 hpf, 48 hpf, 72 hpf, 144 hpf, 10 dpf (10d), 15 dpf (15d) and 20 dpf (20d), as well as juveniles at 40 dpf (40d*) (Fig. 3). These achievements marked a significant milestone in cryopreservation research and paved the way for more intricate studies into long-term viability, growth patterns, and potential applications in aquaculture and conservation biology.
Graph of the mean growth ± SD of cryopreserved larvae and control larvae throughout larval development from 24 hpf (hours post-fertilization) until 40 dpf * (days post-fertilization) (40d * last modification protocol), 48 h post-thawing (A). For each parameter, 35 larvae were measured (n = 3). Graph of the percentage of larval normality ± SD of cryopreserved larvae and their controls throughout larval development from 24 hpf until 40 dpf * (40d* last modification protocol), 48 h post-thawing (B). For each parameter, 100 larvae were examined (n = 3). Asterisks represent significant differences compared with the control without cryoprotectant; * indicates p ≤ 0.05. Graph of % survival of cryopreserved larvae and their controls throughout larval development from 24 hpf to 40 dpf * (40d* last modification protocol), T1 (0 h post-thawing), T2 (24 h post-thawing) and T3 (48 h post-thawing), analyzed via the fluorescent live-dead dye (C). Images of control larvae obtained via light microscopy (D) and images of cryopreserved larvae after 72 h stained with propidium iodide and Hoechst, obtained via fluorescence microscopy, along different stages of larval development of M. galloprovincialis.
The size (Fig. 3A) and percentage of normality (Fig. 3B) of cryopreserved larvae after thawing throughout all developmental stages was smaller than that of the controls, similar to the work of Rusk et al., 202021 (Fig. 3A). However, these differences are small and seem to disappear as larval development progresses. It is possible that at times of intense development, changes in the cryopreservation process further accentuate the metabolic expenditure necessary for normal development. Moreover, cooling and thawing cryoinjuries could be more critical in each specific development stage. This results in slight delays in larval growth, but as indicated in Heres et al., 202211 and Paredes et al., 201522, this delay is minimized with the passage of days post-freezing.
Damage analysis of serial cryopreservation of larvae throughout the rearing cycle
Immediately after thawing (T1), larvae from all developmental stages were alive, with no dead or damaged individuals observed in the samples. However, survival analysis revealed a decline in survival percentage over time, as reflected in the 24-hour post-thaw survival data (T2). The percentage of larvae surviving 48 h post-thaw (T3) exceeded 60% in all cases, except for the 144hpf larvae. The low survival rates in these developmental stages (approximately 13%) may be due to different causes (Fig. 3C). The reduced survival of 144 hpf larvae could be attributed to the larvae being in a sensitive developmental phase during cryopreservation, such as during the formation of critical tissues or organs or the transition to a new larval stage. In our case, when the larvae were incubated at 20 °C, at 144 h, our larvae had already left the D-larvae stage and started to transform into umbonate at this time, and the gill septa and the rudiment of the foot began to form23. Therefore, damage to these structures or metabolic energy expenditure due to cryopreservation may be behind this decrease in the post-thaw survival rate.
However, it is important to note that the analysis was conducted without replicates, focusing solely on the total number of larvae present in 1 ml of each sample.
Using fluorescence staining (Fig. 3D), we identified common lesions of larvae and juveniles, which were predominantly localized in the digestive region. Although propidium iodide staining usually marks this region in general, even in controls, we occasionally observe small vesicles, some of which appeared to exhibit movement in the region; therefore, we suspect that these vesicles may have been remaining or living algae into which the dye may have penetrated when the cell membranes were compromised during the digestive process, i.e., half-digested microalgae. In any case, we cannot rule out the possibility that lethal or sublethal cryoinjury may occur in this region of the body, so in the future, it would be advisable to analyze this region in depth in a more specific way via histological, immunohistochemical or scanning microscopy techniques.
On the other hand, all the data presented from the size, survival and post-thaw damage analyses of 40d* juveniles are the result of the latest specialized modifications of the cryopreservation protocol. Details of the experiments performed to develop these modifications are provided in the following sections.
Strategies to improve survival in cryopreserved juvenile larvae
Initial attempts to cryopreserve 40-day-old juveniles (40 dpf) via the protocol developed by Heres (2021)24 were successful. However, analysis of survival rates over 48 h post-thaw revealed a significant decline, with survival decreasing to approximately 13% at 48 h (T3) (Fig. 4B). These juveniles, which are substantially larger (approximately 500 μm) than 72-hour D-larvae (approximately 100 μm) and possess fully developed organs and tissues such as adults, may require adjustments to the cryoprotectant concentrations. Specifically, the concentration used in the current protocol used for the larval stages (10% ethylene glycol [EG] + 0.4 M trehalose [TRE]) may be insufficient to provide adequate protection for larger, more complex individuals.
Dose‒response curve of 40-day-old M. galloprovincialis juveniles exposed to different concentrations of EG. For each parameter, 100 larvae were measured (n = 4) (A). Graph of % survival of cryopreserved juveniles at 40 days post-fertilization at three time points, T1 (0 h post-thawing), T2 (24 h post-thawing) and T3 (48 h post-thawing), analyzed via the fluorescent live-dead dye for two concentrations of cryoprotectant, EG10% or 12% with a TRE of 0.4 M, n = number of juveniles analyzed per treatment as present in 1 mL of sample (B). The thick arrow shows particulate material expelled by the juvenile after thawing. Images of cryopreserved juveniles fed with EG10% TRE 0.4 M and of cryopreserved juveniles fed EG10% TRE 0.4 M at three time points, (0 h post-thawing), T2 (24 h post-thawing) and T3 (48 h post-thawing), with propidium iodide and Hoechst dye (C).
The toxicity of ethylene glycol was studied through a dose‒response curve (Fig. 4A). The results revealed significant differences from the control at all EG concentrations. As the EG concentration increased, the survival rate decreased, reaching zero at the highest EG concentration (40% or 7.17 M EG). From the equation of this curve, we calculated the IC50 (half maximal inhibitory concentration or inhibitory concentration 50) of EG specifically for 40 dpf juveniles, which corresponds to 16.8% EG (3.03 M). Since our goal was for juvenile survival to be greater than 50%, we decided to increase the EG concentration from 10% (1.79 M) to 12% (2.15 M) for juveniles. With this adjustment, we aim to increase the level of cryoprotection of our juveniles without significantly increasing toxicity, which could compromise their survival.
The cryopreservation of 40 Dpf juveniles
The survival percentage results are shown on Fig. 4B. An increase of approximately 52% at time T3 (48 hpt, hours post-thaw) with respect to the juveniles cryopreserved with EG 10%+TRE 0.4 M TRE at this time. With this new cocktail, we increased the survival rate from approx. 13% to approx. 66% at 48 hpt. It is evident that at 24 hpt (T2), the % survival was greater with EG10%+TRE 0.4 M than with EG12%+TRE 0.4 M, however, at T3, the decrease in % survival was very evident with EG10%+TRE 0.4 M (Fig. 4). In 40 dpf juveniles (EG10%+TRE 0.04 M), a large amount of particulate matter was concentrated in the digestive tract region (thick arrow). These particles are expelled from the inside of the animal to the outside. Furthermore, these particles are stained with propidium iodide, which indicates that they are dead cells. We do not know the specific origin of these particles, but we suspect that they may be food remains (microalgae) or remains of some region of the digestive tract that is slowly disintegrating. However, once the 40 dpf* (EG12%+TRE 0.04 M) juveniles were cryopreserved with the new cocktail, the presence of this particulate material was practically nil (Fig. 4C). This allowed us to successfully cryopreserve 40 dpf juveniles (40d*), the % normality, length and survival data of which are discussed above (Fig. 3). On the other hand, no major differences were observed in % survival between treatments except for T3 (48 h post-thaw), when the increase in cryoprotection seemed to yield an increase in survival, as there were no replicates in this case, and no statistical analysis was performed at this specific point (due to the nature of the sample, as explained in Sect. 3.5).
Cryopreservation of 45 dpf juveniles
Ultimately, we cryopreserved 45-dpf juveniles with EG12%+TRE 0.4 M, and this time, we introduced a brief fasting period (96 h). We expected the introduction of fasting to help obtain a better survival rate. The size and percentage of normality data are very favorable at 48 h post-thawing (hpt) (Fig. 5A and B) and was not significantly different from that of the control. In this way, we managed to cryopreserve 45 dpf juveniles with a size of more than 1 mm (Fig. 5D) and maintained a normality percentage of more than 90%.
Damage analysis of 45 Dpf juveniles
The survival rate of 45 dpf juveniles decreases over time following cryopreservation. After the first 24 hpt (T1), the survival rate was approximately 63%, which decreased to approximately 11% by 72 hpt (T3) (Fig. 5C). Although this 72-hour survival rate may seem low, it is important to note that this is the first successful cryopreservation of juvenile mussels, which are 10 times larger than the size for which the current protocol was designed. Moreover, we are dealing with a fully developed organism that is indistinguishable from an adult, except for its size, and presents significant metabolic and systemic complexity (Fig. 5D). Thus, this represents the cryopreservation of an entire organism. In the images of Fig. 5C. no differences at the structural level were evident between the 45 dpf juveniles subjected to cryopreservation and the control juveniles. The main anatomical structures, such as the gills, foot, mantle edge and digestive region, appeared to remain intact in both treatments. In addition, no areas marked with propidium iodide were detected, suggesting the absence of cell damage in juveniles that survived the cryopreservation process during the first 72 hpt.
Graph of the mean growth ± SD of cryopreserved feed 45 hpf juvenile and control 45hpf juvenile 48 h post-thawing (A). For each parameter, 35 larvae were measured (n = 3). Graph of the percentage of larval normality ± SD of cryopreserved feed 45hpf juvenile and control 45 hpf juvenile 48 h post-thawing (B) For each parameter, 100 larvae were examined (n = 3). There were no significant differences (p ≤ 0.05) compared with the control without the cryoprotectant. Graph of % survival of cryopreserved juveniles at 45 dpf cryopreserved with EG 12%+TRE 0.4 M at three time points, T1 (24 h after thawing), T2 (48 h after thawing) and T3 (72 h after thawing), analyzed via the fluorescent live-dead dye EG 12%+TRE 0.4 M, n = number of juveniles analyzed per treatment as present in 1 mL of sample (C). Images of cryopreserved juveniles fed EG12% +TRE 0.4 M and of cryopreserved juveniles fed EG12%+TRE 0.4 M at three time points, T1 (24 hpt), T2 (48 hpt) and T3 (72 hpt), with propidium iodide (red, dead cells) and Hoechst (blue, active DNA) dye (D). The thin arrow points to the edge of the mantle, the thick arrow to the foot, the caret to the digestive tract and the asterisk to the gills.
After thawing (24 h), a high filtration rate can be observed in the gills, as well as some movement in the foot and mantle (Movies S1, S2). The byssus allows slight fixation, but this can be easily detached. At 48 h post-thawing, filtration and shrinkage of the mantle and foot were less evident than they were initially post-thawing. However, byssus formation is much clearer, and its binding power is more pronounced. We suspect that first, the filtration rate increases because juveniles try to eliminate the remains of the CPAs (Cryoprotectants Agents) from their organisms, also with the help of slight contractions of the mantle. Moreover, during the cryopreservation process, partial or total denaturation of the byssus proteins may have occurred because of the cold and exposure to the CPAs. On the other hand, the weakening of the byssus as an attachment structure to the substrate and to the rest of its congeners causes them to use the foot as a way of detecting the proximity of other juveniles in search of protection. Finally, the formation of new byssus strands and the increase in attachment strength indicate that the byssus gland is not damaged during cryopreservation process. In the future, it would be desirable to try to obtain hard evidence to validate this hypothesis quantitatively.
Owing to the technical complexity of juvenile analysis, it was not possible to perform replicates for a more detailed assessment of survival rates via this fluorescence technique. The fact that these organisms have byssus implies an extra difficulty. Naturally, the individuals of this species use the byssus structure to adhere to each other and to surfaces. This means that they appear in small aggregates of several individuals, making it very difficult to separate and work with a specific number of individuals in each straw. Despite this limitation, the preliminary data obtained were consistent across trials and provided valuable insights into the timing and location of lethal and sublethal cryoinjuries in larvae and juveniles. While the lack of replicates limits precise quantification, the consistency observed suggests that the results are representative of the effects of cryopreservation under the current experimental conditions. Moreover, this approach has proven useful as an exploratory tool to detect general patterns of cryodamage, offering critical insights into the processes that influence post-cryopreservation survival. For future studies, the optimization of this methodology is anticipated, along with the potential for additional replicates or alternative approaches to allow for more accurate survival quantification of organisms with a byssus that aggregates and attaches together.
To date, successful cryopreservation of Mytilus galloprovincialis larvae beyond 72 h post-fertilization (hpf) has not been achieved. However, we have not only surpassed this developmental stage but also successfully cryopreserved, for the first time, all larval stages of this species, including juvenile individuals. This makes us the first to cryopreserve all larval stages of any species globally and among the few studies to have successfully cryopreserved juveniles. These juveniles, both metabolically and in terms of tissue and organ complexity, are indistinguishable from adult individuals, except for their size.
Organismal cryopreservation studies are very novel, with few published examples, such as Caenorhabditis elegans25, which was cryopreserved via isochoric cryopreservation, and survival was tested for up to 24 h. Coral fragments from the species Porites compressa26 were successfully cryopreserved to a size of 1 cm. Other complex multicellular systems, such as embryos and larvae, can be equally important and comparable in size. Notable examples include the cryopreservation of Drosophila melanogaster embryos27, zebrafish Danio rerio embryos28, and shrimp larvae29, all of which are comparable in size to our 40 dpf larvae (more than 1 mm). These studies have successfully addressed the challenge posed by the low surface-to-volume ratio, which reduces the efflux of water and cryoprotectants. In the case of mussels, the animal body inside the shell has an elongated and hollow shape, making the penetration of cryoprotectants easier, as the surface-to-volume ratio is larger than the size suggests; on the other hand, their shells are impermeable to water and cryoprotectants, and mussels can open/close at will react to the stimuli of the environment (like when detecting a chemical in the water such as a cryoprotectant)30; therefore, this poses a completely unique set of challenges and advantages that have allowed us to cryopreserve mussel juveniles via slow cooling instead of vitrification.
Working with gregarious organisms poses the challenge of handling their naturally formed aggregates, which they use for protection. These juveniles attach to each other and to surfaces through the byssus, a protein-based structure critical for their survival. During the experiments, it was necessary to carefully detach the settled juveniles from the tanks, create aggregates of consistent numbers and sizes, and work quickly to prevent them from reaching the experimental surfaces. Although cryopreservation may cause byssus denaturation immediately after thawing, the integrity of the byssal gland allows juveniles to regenerate new byssus and restore its normal function.
This research lays an essential foundation for improving cryopreservation techniques and deepening our understanding of biological responses at both the cellular and systemic levels in whole organisms.
