Long-term study on survival and development of successive generations of Mytilus galloprovincialis cryopreserved larvae

Short-term experiments

Potential toxic and cryoprotection effects of different CPA combinations

Focusing on toxicity bioassays (Figs. 1A, 2A), although there were certain CPA combinations that yielded significant abnormality percentages compared to controls, in general the CPA combinations did not yield any significant toxic effect. The use of Milli-Q Water instead of FSW did not enhance normal larval development after CPA exposure, neither did the addition of PVP at the concentrations tested, even in combination with trehalose (TRE) (p > 0.05). In fact, the highest concentrations of PVP used in this experiment (9 and 12%) yielded significant abnormal development on exposed trochophores (Fig. 1A) (p < 0.05). The abnormality rates found in D-larvae developed from exposed 72 h-old D-stage did not show any dose–response relationship and only the CPA solutions containing 10% EG + 0.34% PVP with or without TRE showed significant differences with controls (Fig. 2A) (p < 0.05).

On the other hand, cryopreservation success (Figs. 1B, 2B) was significantly lower than control recovery after post-thawing incubation (p < 0.05). In general, those trials cryopreserved without PVP yielded significantly higher survival rates than the rest of the treatments regardless of the larval stage selected for cryopreservation (p < 0.05). The best survival rate from trochophore cryopreservation was 64 ± 9.66%, using 10% EG + 0.4 M TRE in Milli-Q Water (Fig. 1B). In the case of D-stage larvae, cryopreservation with 10% EG + 0.4 M TRE in FSW or Milli-Q water and 10% EG + 0.4 M TRE + 12% PVP did not show significant differences with survival of control larvae (p > 0.05). The treatment 10% EG + 0.4 M TRE in FSW had the highest survival percentage, 77 ± 4.31% (Fig. 2B).

Effect of different cooling and thawing rates

There was a significant decrease in survival rates of cryopreserved samples compared to control groups (80%) (p < 0.05). The cooling and thawing rate combination with the highest post-thaw survival percentage was cooling at − 1 °C/min after seeding (a.s.) and the use of a water bath at 35 °C for warming of the cryopreserved straws (Fig. 3), regardless the development stage selected. In this experiment, 63 ± 4.48% of normal D-larvae were obtained from trochophore cryopreservation and 71 ± 6.88% after cryopreservation using 72 h-old D-larvae. Slower cooling rates enhanced significantly lethal injuries and larval abnormalities. Meanwhile, the highest and the lowest thawing rates did not improve cryopreservation outcome (p > 0.05).

Effect of SUC solutions on post-thaw D-larvae at the CPA dilution step

In agreement with prior experiments, there was a significant decrease in survival rates of post-thaw cryopreserved larvae compared to controls, except when the cryopreserved 72 h-old D-larvae were added to the solution 6% SUC in FSW for CPA dilution. However, the CPA combinations did not significantly enhance the cryopreservation success and there was not any significant effect when using SUC solutions in Milli-Q water or FSW for dilution of CPA at both larval stages (Fig. 4).

The best post-thaw performance across short-term experiments was obtained when 72 h-old D-larvae were cryopreserved in 10% EG + 0.4 M TRE in FSW with 60 min for equilibration; followed by holding at 4 °C for 2 min, then cooling at − 1 °C/min to − 12 °C, holding for 2 min for seeding check, then cooling at − 1 °C/min to − 35 °C, and finally plunging into liquid nitrogen. The best thawing protocol was using a water bath at 35 °C, and once the ice was melted, the content of the straws was diluted in FSW at 1:1. The optimized protocol was selected to further long-term experiments.

Long-term experiments

Long-term experiment from cryopreserved F1 72 h-old D-larvae

Post-thaw larval parameters obtained during larval rearing and settlement are represented in Fig. 5. Regarding survival, there was a steep decline in larval density, especially between day 8 and 15 of culture, being more pronounced in cryopreserved samples. However, significant differences were not found between cryopreserved and control trials, excepting at day 22 (p < 0.05). The declining trend began to stabilize from day 16 onwards. At day 22, 5.24% of cryopreserved larvae developed into competent pediveliger larvae, representing 24% survival compared to the control group (Fig. 5A). Shell length was taken into account as an indicator of larval growth (Fig. 5B, Table 1). There was a significant difference in the size of of cryopreserved larvae on day 4 post-fertilization (control larvae: 116.47 ± 5.87 µm; cryopreserved larvae: 110.02 ± 5.51 µm, n = 35). This difference increased over the following 7 days of larval rearing until day 11, when shell length of cryopreserved larvae averaged 25% lower than that of controls (unfrozen larvae: 173.39 ± 22.30 µm; cryopreserved larvae: 129.71 ± 20.67 µm, n = 35). From day 12 onwards, these differences became less and no significant differences were found between larval size measurements at settlement (control larvae: 1775.03 ± 600.07 µm; cryopreserved larvae: 1600.99 ± 485.92 µm, n = 35) (p > 0.05).

Settlement analysis did not reveal significant differences when comparing successful percentage settlement values and shell sizes between treatments (p > 0.05). Settlement of mussel juveniles developed from cryopreserved larvae was 71.27 ± 19.10%, slightly higher than the 67.45 ± 31.65% of settled individuals (spat) from the control group (Fig. 5C). Focusing on the shell size of settled mussels, those from the control group reached a mean of 1755 ± 600.07 µm, whereas the cryopreserved group averaged 1595 ± 475.31 µm (Fig. 5D).

In this experiment, the growth rate of developed adult mussels cultured in rafts in Ria de Vigo, (NW Spain) were evaluated (Fig. 6). In general, there were no significant differences between treatments and the growth trend seemed to follow a linear behavior in both cryopreserved and control groups until the final samplings, when shell size approached a maximum of about 6 cm.

Long-term experiment from cryopreserved F2 72 h-old D-larvae

The short-term experiment with F2 larvae did not show significant differences when comparing the relative percentage of normal D-larvae (Fig. 7A) and larval development (Fig. 7B) after cryopreservation. Cryopreservation of larvae from adults developed from control larvae yielded higher relative normality rates, 90 ± 10.91%, than cryopreserved F2 from F1 adults developed from cryopreserved larvae, with 64 ± 4.46%. However, significant differences between pools were not found (p > 0.05). Comparing shell lengths measured at day 2 of incubation, cryopreserved larvae from cryopreserved adults averaged 108 ± 4.46 µm (median 108.34 µm), whereas cryopreserved larvae from control adults achieved 106 ± 6.05 µm (median 106.52 µm), without statistical significance between treatments (p > 0.05).

During the first days of larval rearing of cryopreserved F2 larvae, the survival rate declined until day 13, after which the trend stablished until metamorphosis into pediveliger larvae at day 20. Statistical analysis showed significant differences between cryopreserved trials and control groups (p < 0.05). At the settlement point, the treatment F1CRYO-F2CONTROL produced 48% of normal pediveliger larvae (percentage expressed as the proportion of juveniles produced from the initial subsample of competent larvae after 10 days of incubation for each treatment), followed by F1CONTROL-F2CONTROL with 11.92%; whereas F1CRYO-F2CRYO yielded 0.15% and F1CONTROL-F2CRYO, 0.12% (Fig. 7Aʹ). Larval growth during the first 20 days of culture was also significantly different between F2 cryopreserved and control groups (p < 0.05), although, this parameter seemed to stabilize over time and the shell size of cryopreserved larvae tended to become more similar to the shell size of the control larvae (Fig. 7Bʹ). Regarding the settlement success, there was not any significant difference between treatments when comparing the percentage of settled larvae (p > 0.05). The treatment F1CONTROL-F2CRYO achieved the highest rate, with 50.54 ± 29.39%, followed by F1CRYO-F2CRYO, with 35.41 ± 16.70%. Around 25% of pediveliger larvae from control groups were able to settle (Fig. 7Aʹʹ). The lengths of settled juveniles (spat) were significantly different only between F1CRYO-F2CRYO (471 ± 202 µm) and the rest of groups (around 388 µm) (p < 0.05) (Fig. 7Bʹʹ).

Discussion

New methodologies and good practices need to be developed for mollusk aquaculture to ensure a reliable supply of spat for aquaculture in order to meet increase demanded of the growing global population³. The implementation of breeding programs will be crucial for marine aquaculture, in the same way as it has already been successful for land agriculture. Selective breeding systems can be assisted by the application of cryopreservation tools, offering the possibility of storing interesting biological samples at high densities under minimal requirements compared to maintaining live stocks, able to survive and develop normally once they are thawed^20,22,38. In addition to several advantages mentioned in the Introduction, the establishment of biobanks could be the way to overcome the logistical issues of obtaining and maintaining living shellfish broodstock, as well as lowering costs and effort³⁵. For instance, shellfish hatchery requires daily a supply of diet composed of a mix of microalgal species in different proportions; continuous water flow and aeration are also needed, and the system must be efficient in terms of removing metabolites that can be deleterious. All of these activities can be time consuming, and expensive^39,40.

This is the first time a cryopreservation protocol for M. galloprovincialis larvae has been investigated in depth from fertilization to juvenile and adult production, and production of a second generation. Our work started with a very basic preliminary protocol described for trochophore larva by Paredes et al.²⁵, followed by studies on cryoprotectant toxicity⁴¹, cryopreservation of trochophore larvae and juvenile production³⁵. A similar long-term experiment was carried out by Suquet et al.¹, who evaluated the viability of progeny produced from cryopreserved oyster (Crassostrea gigas) larvae. However, the present research is pioneering in the field because we have studied the direct implementation of cryopreservation tools on aquaculture, focusing on the culture of the resulting spat in traditional mussel rafts in the natural environment and, in tandem, analysis of the potential long-term effects of cryopreservation on F2 progeny.

Improvements from our prior protocol for larvae were due to selection of the 72 h-old D-larval stage for cryopreservation, resulting not only in higher survival rates but also in increased quality of larval fitness. In addition, preference for the use of 72 h-old D-larvae is supported by our prior research that showed their higher tolerance to CPA toxicity and higher resistance to cryopreservation, compared with earlier development stages^35,41. The optimized protocol produced close to 75% of normal D-larvae at day 2 of incubation, compared with only 25% success obtained with cryopreserved trochophores in a 48 h short-term post-thaw fitness evaluation^25,36. Hence, by selecting older larval stages for cryopreservation, long-term outcomes could potentially be improved given the higher number of thawed normal D-larvae available for culture.

An explanation for the higher success when using an older stage larva, compared with trochophore larva, could be explained by Rusk⁴², who observed minor cell-specific damage and reduced neurogenesis in post-thawed trochophores. Other studies have explained larval mortality after thawing by higher oxidative stress from the production of reactive oxygen species (ROS) during the cryopreservation process^43,44,45. Trochophores have higher lipid contents compared with older development stages. These molecules are susceptible to oxidative stress, which may lead to ROS production and significant irreversible cell damages. Although the potential metabolic pathways blocked by ROS have not been identified yet, research suggests that those involved on larval development could be affected, resulting in cell death^32,46,47.

It is well known that selection of the optimal CPA and CPA concentration is species and cell specific. Hence, despite its detrimental effect on mussel development, dimethyl-sulfoxide (Me₂SO) has been considered the preferred CPA for mussel sperm cryopreservation^41,48, whereas research on mollusks has shown the success of using ethylene–glycol (EG) for larval cryopreservation^25,41,49. In order to improve the cryopreservation success, the addition of non-permeable CPAs has been investigated, based on their capacity to avoid potential harmful effects of permeable CPAs and diminish osmotic shock^26,50,51,52. The use of sugars, such as trehalose (TRE) is widely common, as well as polyvinylpyrrolidone (PVP)^{25,35,36,49,53}.

Liu et al.³⁶ recommended the use of PVP in combination with other CPAs to limit intracellular ice formation. However, the effect of PVP is unclear and they suggested further research focused on its toxicity to evaluate its potential harmful effect. The potential benefits of increasing PVP concentrations with 10% EG in combination or not with 0.4 M TRE were studied here, showing there was not any significant improvement.

On the other hand, the role of D-shell is unclear on CPA permeability. Further research on CPA permeability across tissues or complex cell aggregations and the role of barriers like the prodisoconch shell at the mussel D-stage could explain the differential responses of larval stages to cryopreservation. Moreover, 15 min of exposure seems to be enough time for osmotic equilibration to CPAs before slow cooling of mussel trochophores. However, research in our lab indicated⁵⁴ the improvement of the post-thaw survival and larval fitness when increasing the exposure time from 15 to 60 min when cryopreserving 72 h-old D-larvae.

Several authors reported benefits from the use of an extender to help CPA removal from cells, which could help the cells reach osmotic equilibrium, diminish potential osmotic damage after thawing. Some sugars have been applied for these purposes previously, especially when cryopreserving the trochophore stage^{25,36,49,53,55}. The final short-term experiment in this study tested the addition of increasing sucrose (SUC) concentrations in order to determine the optimal concentration to assist CPA removal. Despite the advantages reported in prior research, there was no enhancement of survival at any of the SUC tested concentrations.

This work has shown for the first time that cryopreserved mussel D-larvae can be reared to settlement and juveniles transferred to ropes and grown in the natural environment to produce a second generation. Long-term experiments suggested that cryopreservation does not compromise the development of larvae to adult stage. Adults that were cultured in traditional aquaculture systems showed similarities in growth rates; furthermore, histological analyses did not show any tissue alteration in cryopreserved individuals (data not shown). In addition, gametes of control and cryopreserved F1 had no differences in gamete quality, with oocytes with spherical shapes and high lipid droplet content and sperm with good motility and swimming in typical zig-zag trajectories. Moreover, larval rearing carried out with F2 larvae showed that cryopreservation of successive progenies did not affect production of mussel spat.

Differences F1 and F2 larval survival rates could have been due to difficulties in the maintenance of larval cultures, given the unusual circumstances of the Covid-19 pandemic, which limited the opportunities to maintain optimal conditions for larval development. In addition, the age of developed adults (F1) could negatively influence gamete quality and undermine cryopreservation success: adults selected for spawning events to obtain the initial F1 D-larvae were 9 months to 1 year old, averaging 7 cm. On their part, resulting adults cultured in rafts (F1) were 2 years old at the time of spawning, measuring 8 ± 0.56 cm (those developed from control larvae) and 8 ± 0.51 cm (adults developed from cryopreserved larvae) (Fig. 8). Despite the unavoidable difference in F1 and F2 parental age, there was no significant difference between survival and settlement of cryopreserved F2 larvae from F1 parents that had been cryopreserved or not.

Differences in size were found between control and cryopreserved larvae mainly during the first 22 days of larval rearing. A similar effect was observed in Refs.^25,35,49,53. Rusk et al.⁵³ also observed a lower activity in cryopreserved samples compared to controls in other parameters, including feeding consumption and delays in organogenesis. These differences could be explained by considering potential cryo-injuries that may have occurred during cryopreservation on organs or tissues, for example effects on the mantle and shell gland which are responsible for shell secretions. This explanation is supported by the number of abnormalities that were found in the shells of cryopreserved larvae, as described by Rodríguez-Riveiro et al³⁵ and Rusk et al.⁵³. This might mean that cryopreserved larvae grow slower than controls, due to a re-allocation of energy from growth to processes involved in tissue repair. Those larvae with lethal injuries or serious damage could die during through larval rearing, depending on the degree of abnormality. At the settlement, larvae were competent and able to develop normally into juveniles and grow at the same rate as juveniles developed from control larvae. In fact, this is supported by the settlement success of F1 and F2 (Fig. 7Aʹʹ) offspring, where the number of settled juveniles (spat) from cryopreserved larvae was higher than controls (p > 0.05). Further research is needed to improve our understanding of the mechanisms underlying our results. Focusing on the development of mussel juveniles cultured in rafts in natural environment, shell sizes were similar among treatments. The normal size of adult mussels ranges from 3 to 13 cm in natural conditions and they can reach 7 cm at the end of the first year when the environment is favorable⁵⁶. In this work, mussels reached 3 cm approximately 7 months after their transport to culture rafts and averaged 6–7 cm 1 year after. This observation indicates that cryopreservation does not have any detrimental effect on mussel growth, and they can reach commercial sizes as fast as individuals from non-cryopreserved larvae.

Conclusions

The current work describes an optimized cryopreservation protocol for 72 h-old D larvae of M. galloprovincialis based on a suitable development stage, cooling and thawing rates and CPA combinations tested. For the first time, mussel spat produced from cryopreserved larvae were able to develop into adults at the same growth rates as control individuals, be cultured in a natural environment, and even reach average commercial size at the same time as control mussels obtained from non-cryopreserved larvae. In addition, the viability of these produced adults is apparently unaffected by the cryopreservation process, with fecundity and gamete quality equivalent to control mussels, which is corroborated by subsequent work on F2 mussels. Moreover, cryopreservation of larvae from the F2 generation does not compromise larval development. This represents robust evidence of the suitability of this cryopreservation method for aquaculture or research purposes where animals must possess optimal health. Further research should be focused on factors involved in the cryopreservation process, such as membrane permeability parameters, to improve post-thaw success to provide higher survival rates from the outset and diminish cell injury which can lead to long-term lethal damages. In addition, it will also be important to determine the processes involved in the high mortality rates found at the beginning of culture and the delay in development of cryopreserved larvae needs to be undertaken to understand affected intracellular processes and metabolic pathways, particularly during the first hours after thawing. This work shows the high potential of cryopreservation to benefit the mussel industry and the suitability of this technique for selective breeding programs, one of the most promising ways to increase global production.

Source: Ecology - nature.com