Resumen de Mollusk cryopreservation and variability along development and species
The main objective of this doctoral thesis consists of the development of cryopreservation protocols for larval stages of marine mollusks, by carrying out a study of involved steps. This will optimize the method to obtain maximal survival rates and provide larvae with unaltered fitness by cryopreservation procedures. Therefore, developed cryopreservation protocols will be suitable for their implementation into research field, aquaculture sector and species conservation. Moreover, a comparative study of data collected from the work with related species could provide general trends about the response or behavior to cryopreservation, hence some standards could be formulated for the development of further cryopreservation protocols for larval stages of related species.
The application of cryopreservation techniques on areas of knowledge and aquaculture industry can offer several benefits, as well as the avoidance of severe problems. For example: • In research field, cryopreservation would enhance international interactions between institutions or researchers; it would provide the transport of unaltered biological material until handling. It would also allow greater flexibility in obtaining competent larvae on demand, without the need of sampling, thus avoiding the need to wait for administrative permits and the economic and personal effort they entail.
• In aquaculture industry, it would mean overcoming current global barriers that prevent the increase of the production of aquatic products, being able to establish a sustainable supply of mollusk seed, which is increasingly scarce in natural environment. This would make possible not to depend on the seasonal presence of spat -juvenile stage- marked by biological cycles of bivalve mollusks. In addition, it would enable the application of genetic techniques to perform selective breeding to generate families that grow faster or better in controlled culture conditions, have better organoleptic properties that make them more attractive or are preferred by the consumer in the market. And, in this way, make more efficient the good practices in mollusk aquaculture sector.
• In conservation of endangered species, the recruitment and restocking of species whose natural populations are in a danger situation could be significantly promoted, facilitating their monitoring and demographic control. Ultimately, it would be possible to conserve genomes of already extinct species, increasing possibilities for their study and application on research.
This doctoral thesis addressed the main aspects to be taken into account for the development of cryopreservation protocols for larval stages of the most important mollusk species from economic and cultural point of view of the Autonomous Community of Galicia, Spain and worldwide. The selected species were the Mediterranean mussel or Mytilus galloprovincialis, which was chosen as a model species; the slug carpet shell or Venerupis corrugata, the grooved carpet shell or Ruditapes decussatus and the Japanese clam or R. philippinarum. In addition, studies were also carried out with the GreenshellTM mussel or Perna canaliculus, endemic mussel from New Zealand.
Chapter I of this doctoral thesis introduces the reader to this field of science beginning with an explanation of cryobiology and cryopreservation concepts, followed by a brief summary of the main historical milestones that have allowed the development of this science until today. Next, the basic principles of cryopreservation are explained in order to provide the most important aspects to take into account that occur during the process and whose understanding is decisive in order to optimize each of the involved steps. This is followed by a review of research focused on the application of cryopreservation in marine mollusk species to date and a description of the biology of the target species and their economic importance.
Cryobiology is the branch of science that deals with the study of the relationship between organisms and low temperatures. The application of this knowledge to storage biological material at very low temperatures, generally below -140 °C, is known as cryopreservation. After its birth in the 1950s, the use of this technique has spread to all branches of knowledge due to its great potential. At present, this is only possible in the presence of Cryoprotecting Agents (CPAs), chemical compounds that prevent the organization of water molecules into ice crystals as temperature decreases, which are lethal for cells. In turn, these agents must be innocuous or with low toxicity. Therefore, when developing a cryopreservation protocol, a bioassay should be performed to select the one that offers low toxicity, but at the same time high cryoprotection by testing its capability in further cryopreservation experiments.
Cryopreservation involves the following steps: 1. The manner in which CPAs are added; 2. The time of exposure to CPAs prior to slow-cooling; 3. The cooling step until samples are immersed into liquid nitrogen; 4. Storage in liquid nitrogen; 5. Thawing step; 6. Dilution and CPA removal.
Throughout the whole process, osmotic changes occur by which cell volume varies due to solute movement between the external and internal medium according to cell membrane permeability, in an attempt to restore osmotic equilibrium. Consequently, during cryopreservation, the cell is partially dehydrated, which provide protection against the formation of intracellular ice crystals. Overall, the cell must deal with CPA toxicity, osmotic variations, changes in their volume and the presence of intra- and extracellular ice that can be lethal. The study of effects of each cryopreservation step allows the optimization of the protocol to obtain maximum survival rates.
Research applied to marine environment is scarce and focused on species of commercial interest. The most developed research line is sperm cryopreservation, which is widespread in fishes and mollusks, with reproducible and robust protocols. There is not any protocol described that successfully achieve oocyte preservation at the present and, nevertheless, cryopreservation of larval stages is scarce despite being the only way to preserve all the genetic information. Most of studies have examined factors and stages involved in cryopreservation process to maximize performance. To date, only Paniagua-Chávez et al., 1998 and Suquet et al., 2014 tested the ability to produce seed or adult individuals of Crassostrea virginica or C. gigas, respectively, from cryopreserved trials.
Chapter II contains a general explanation of the experimental design carried out during the development of this doctoral thesis.
Larvae of the target species were obtained by subjecting adult individuals to thermal shock cycles to induce spontaneous gamete spawning. Adults were collected from natural environment, generally from the Ria of Vigo or surrounding areas. In the case of GreenshellTM mussel (P. canaliculus), adults were selected from a broodstock previously conditioned to maximize the development and maturation of their gonadal tissue. Resulting embryos from in vitro fertilizations were incubated generally at a temperature of 18 ± 1 °C in filtered and UV-irradiated seawater to eliminate potential pathogens until their use.
Toxicity bioassays were carried out to study potential harmful effects of the most common CPAs used in marine cryopreservation on different developmental stages of selected species. The different solutions of CPAs tested in numerous bioassays were added in one step at 1:1 ratio. After exposure, samples were filtered and incubated until they were fixed with formaldehyde and analyzed.
For cryopreservation experiments, samples exposed to CPAs following the procedure described in toxicity bioassays were loaded into 0.25 mL straws and introduced into a cryochamber whereby the freezing rate is programmable. Regardless of the cooling rate used, once samples were at -35 °C, they were placed in liquid nitrogen for storage. A warm water bath was used for thawing.
Larvae sampled for in toxicity bioassays and those that were cryopreserved were incubated after experiments were completed at 18 ± 1 °C in filtered and UV-irradiated seawater. Incubation period was different according to the development stage, being until metamorphosis to stage D in the case of fertilized eggs and trochophore larvae; 48 h for D-larvae. The toxic effect of CPAs was determined by calculating the percentage of abnormal larvae developed after incubation. In the case of cryopreservation experiments, the percentage of normal larvae developed was taken into account. In both cases, these percentages were obtained after evaluating the state of 100 larvae of each replicate under optical microscope following previous works where anomalies and alterations in larval growth of bivalve mollusks have been described.
Chapter III includes studies focused on each step that takes place in cryopreservation process to identify and understand their effects on mollusk development stages in order to optimize them according to cellular requirements and thus achieve maximum survival rates, choosing the Mediterranean mussel (M. galloprovincialis) as model species. Short-term experiments were carried out with development stages in which the toxicity and cryoprotecting effect of different CPAs commonly used in marine cryopreservation were evaluated; different cooling and thawing rates, as well as the tolerance and resilience of different larval stages. This chapter also describes long-term experiments carried out with larval stages of model species in order to identify and understand potential long-term effects of cryopreservation, as well as the ability of the developed cryopreservation protocol to produce mussel seed and its degree of competence by monitoring its growth to commercial adult size. Research was subsequently focused on the viability of the next generation of larvae and the success of their cryopreservation long term.
Preliminary results with development stages of M. galloprovincialis are as follows: • Ethylene-glycol (EG) and Propylene-glycol (PG) were the permeable CPAs with the lowest toxicity, while dimethyl-sulfoxide (Me2SO) and Glycerol (GLY) showed a negative dose-dependent trend. The toxic effect varied among development stages according to age, independently of the chemical agent, being fertilized egg the most affected and 72 h-old D-larva the most tolerant. In fact, their development was not significantly compromised even at the highest concentrations of CPAs selected for this bioassay (up to 3 mol/L (M)). Post-thawing survival increased by selecting 72 h-old D-larva, up to 75-80%, starting from 50% resulting from cryopreserving trochophore stage. These findings were published in Cryobiology (Heres et al, 2019).
• Long-term experiment carried out with mussel trochophores cryopreserved using the preliminary protocol described in Paredes et al., 2013 produced less than 1% of pediveliger larvae at the end of larval rearing (20-22 days of rearing) and 64% of larvae compared to controls were able to settle. Moreover, a delay on larval growth was found which progressively diminished achieving minimal differences at the end of larval rearing. These results were published in Aquaculture (Rodríguez-Riveiro et al., 2019).
• Prolongation of equilibration time from 15 min to 60 min resulted in an improvement in larval fitness after cryopreservation of 72-h-old D larvae.
• Among all cooling and thawing rates tested on trochophore larvae and 72 h-old D-larvae, cooling at 1 °C/min and thawing using a 35 °C water bath yielded the highest survival rates.
• The addition of different dilutions of Sucrose (SUC), a sugar commonly used to enhance the sample dilution and progressive elimination of CPA molecules, thus diminishing their negative effects due to osmotic shock and/or prolonged exposure, did not improve the success of cryopreservation.
The final protocol was published in the book chapter Paredes et al., 2021, as described: CPA solution consists of 10% EG and 0.4 M TRE in filtered and UV-irradiated seawater (final concentrations). This is added in one step in a 1:1 ratio to 1 mL of seawater containing larvae, recommending 72 h-old D-stage. The resulting solution is loaded into 0.25 mL straws (IMV Technologies, France) and sealed with PVC powder. After 60 min of exposure at room temperature (18-20 °C), straws are deposited into a cryochamber (Cryologic, Ltd., Aus.), which is already at 4 °C. After 2 min at this temperature, samples are cooled at a constant rate of 1 °C/min to -12 °C. Cooling rate is then paused for 2 min, when seeding is performed to check whether nucleation process has already started and induce it otherwise. After this, cooling rate returns to 1 °C/min until -35 °C. After spending 2 min at this temperature, straws are directly immersed into liquid nitrogen. For thawing, straws are immersed into a water bath at 35 °C for 6 seconds, estimated time at which the ice inside the straw is melted. Cryopreserved samples are diluted with filtered UV-irradiated seawater at a 1:1 ratio. They are then ready for incubation.
• Long-term experiments carried out with 72 h-old D-stage to study the development of cryopreserved larvae indicated that during the first 2 weeks of incubation mortality rate is higher and more pronounced than in control group. From days 12 to 15 of larval rearing, the trend is stabilized in both groups. There is also a growth delay in cryopreserved samples, but the differences with controls are imperceptible at settlement point, at 20-22 days of larval rearing. The culture of juveniles developed from cryopreserved 72 h-old D-larvae allowed us to monitor their growth in rafts near Moaña (Galicia, Spain). The analysis showed no significant differences with control group. Percentage of pediveliger larvae depended on the larval stage selected for cryopreservation: starting from trochophore larvae, less than 1% of these larvae reached settlement, whereas with the first generation of 72 h old D larvae, over 20% of survival compared with control group was achieved. Cryopreservation of 72-h-old D larvae of the following generation also produced less than 1% of juveniles, a fact that has been justified by the advanced age of F1 adult mussels, which could have affected gamete quality and compromised the development of resulting larvae. This fact has occurred in other previous experiments and has also been reported in the field of marine cryopreservation (Paredes, 2014). In any case, settlement success was always higher in those cryopreserved treatments than in control group, except when trochophore larvae were selected for cryopreservation. These works are included in another research article recently submitted to Proceedings of the National Academy of Sciences of the United States of America (PNAS) (Heres et al., under review).
Chapter IV comprises research carried out with IceStartTM, a novel compound previously tested with encouraging results in cryopreservation of human cells. Its benefits are based on the capacity of this mineral agent to induce extracellular ice nucleation in homogeneous manner during cooling once the melting point of an aqueous solution has passed. In this way, the variability of spontaneous heterogeneous nucleation in the quality of cryopreserved samples is considerably reduced.
Initially, toxicity bioassays were carried out to test potential toxic effects of this product alone or in combination with a fixed combination of 10% EG and 0.4 M TRE in seawater on Mediterranean mussel trochophore larvae, 48 and 72 h-old D-larvae. In addition, toxicity was evaluated with different equilibration times. In parallel, cryopreservation experiments were performed where the effects of different equilibration times and the addition of 1µg/mL IceStartTM were tested. Finally, a last cryopreservation experiment was carried out with 72 h-old D-larvae cryopreserved in a solution of 10% EG and 0.4 M TRE in seawater and increasing concentrations of IceStartTM, from 1 to 32 µg/mL (final concentrations).
Findings are presented below: • Analyzing toxicity towards larval stages of Mediterranean mussel, IceStartTM had harmless effect acting on its own. There was not any decrease in toxicity when this compound was added to 10% EG + 0.4 M TRE solution. D-larva, especially at 72 h old, showed higher tolerance to CPA toxicity than the rest of larvae selected for the experiment.
• The increase of exposure to CPAs before cooling was beneficial, producing higher survival percentages than those obtained with shorter equilibration times. The effect was more evident when cryopreserving trochophore larvae, but in all larval stages selected, a considerable improvement of their health and fitness status under microscope was observed.
The following chapter (V) covers investigation on related mussel species and a comparative study with empirical data collected, with the aim of identifying general aspects of mollusk larval cryopreservation. Thus, standard basis could be established for the development of cryopreservation protocols for other phylogenetically related species in a more efficient way, emphasizing on those aspects that condition significantly the viability of cryopreserved larvae. The experimental design applied to Mediterranean mussels was taken into account to develop these experiments.
Toxicity bioassays were performed with the most used permeable CPAs (EG, PG, Me2SO and GLY) and developmental stages (fertilized egg, trochophore larvae, 48 and 72 h-old D-larvae) of three clam species (Venerupis corrugata, Ruditapes decussatus and R. philippinarum), followed by cryopreservation experiments with trochophore larvae, 48 and 72 h-old D-larvae to evaluate the effect of different CPA solutions and equilibration times.
Work carried out with GreenshellTM mussels (Perna canaliculus) was based on the protocol for D-larvae previously developed by Paredes et al., 2012 and Rusk, 2012, with aiming for improving its performance and thus being able to implement in mussel hatcheries. Experiments analyzed different CPA combinations mixing from 8% to 16% EG with 0.4 to 0.6 M TRE and 1% Polyvinylpyrrolidone (PVP), in seawater treated with ethylenediaminetetraacetic acid or milli-Q water (final concentrations). The effect of different equilibration times and the success of cryopreserving 48 or 72 h old D larvae were also evaluated.
Results of both investigations were: • The behavior of clam larval stages was similar among species and that found with Mediterranean mussel individuals: EG and PG were the least toxic CPAs, whereas ME2SO and GLY showed a dose-dependent trend with high mortality rates at high concentrations (2-3 M). Toxic effect also varied as a function of larval age, being more pronounced at fertilized egg. At this stage, mortality was close to 100% from concentrations between 1-1.5 M. The most tolerant stage was the 72 h-old D-larva. Cryopreservation experiments allowed discerning the optimal CPA for these species considering not only its low toxic effect but also its cryoprotective effect: in general, EG showed higher cryoprotection than PG. Survival rates varied according to developmental stage and species from 30 to 80%. The best results were with 72 h-old D-larvae of slug carpet shell and Japanese clam. Post-thawing parameters of the grooved carpet shell larvae were low as those belonging to respective control groups; therefore, this species may be even less tolerant to handling and in vitro conditions. The increase of equilibration time did not produce any apparent improvement as shown with the Mediterranean mussel.
This research conducted with three clam species provided sufficiently robust information to be published in Scientific Reports (Heres et al., 2021).
• The highest values of parameters analyzed in cryopreserved P. canaliculus larvae (survival, microalgae consumption, shell size and swimming activity) were obtained with 14% EG + 0.4 M TRE + 1% PVP prepared in milli-Q water; density between 50,000 to 150,000 larvae per straw (0.25 mL) and an equilibration time of 20 minutes. There were no significant differences between parameters collected from D-larvae cryopreserved at 48 or 72 h old. Survival was approximately 77% with respect to the control group.
The results of experiments with GreenshellTM mussel D-larvae were published in Cryobiology (Heres et al., 2020).
Chapter VI brings a general overview of findings achieved through the present PhD thesis and their implications in Cryobiology field. Finally, there are listed the main general conclusions and a proposal of future lines of research that could be of crucial importance for the impulse that cryopreservation of marine mollusks should take, as well as certain aspects that should be object of research to carry out a complete process of efficiency of this technique.
