Long term survival of cryopreserved mussel larvae (Mytilus galloprovinciallis)

Abstract

Due to the economic importance of the mussel Mytilus galloprovincialis in the Spanish aquaculture, there is a growing interest in developing alternative methods to ensure the supply of good-quality biological material and to minimize the uncertainty and variability that might be a source of economic risk for this sector. The aim of this work was to develop a successful cryopreservation 13 protocol for Mytilus galloprovincialis larvae and study long-term effects by assessing survival and 14 development post cryobanking. We evaluated the effect of previously studied cryoprotecting agent, 15Ethylene glycol (EG) for cryopreservation of trochophore larvae and D-veliger larvae 48h and 72h 16 old, following an existing preliminary protocol for M. galloprovincialis mussel trochophores. The 17 protocol selected for cryopreservation consisted on holding at 4ºC for 2 min, then cooling at 1ºC/min 18 to -12ºC, holding for 5 min, then cooling at -35ºC with a rate of 1ºC/min, then plunging into liquid 19 nitrogen for storage. Thawing took place by immersion in 35ºC water bath. With this protocol the initial percentage of short-term developed a D-larvae (48 hours incubation post-thaw) was 48.9 ± 7.6% with 10% EG + 0.2 M Trehalose (TRE). This initial test only studied the trochophore larvae until they reached the D-stage (48h old); in the present experiment experiments, we replicated this study but also carried out a long-term larval rearing with cryopreserved trochophore larvae in comparison to fresh larvae. This experiment allowed a comparative post-freezing analysis of both larval development and survival. Larval settlement was also characterized after 13 days. The data revealed that until settling, the survival of the cryopreserved larvae was slightly lower than the 2.8% reported in the larval rearing for Greenshell TM mussel. Over time, there was an initial difference in size of cryopreserved larvae when compared to controls, but from day 17 onwards the size between both types of larvae began to stabilize. Attending to settlement, we obtained a 64% of success of cryopreserved larvae respect to the control.

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

“…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 .…”

“…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 effort 35 . 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.…”

“…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 toxicity 41 , cryopreservation of trochophore larvae and juvenile production 35 . 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.…”

Genetic improvement technologies to support the sustainable growth of UK aquaculture

“…Transformative rearing technologies and husbandry regimes are integral to realizing the benefits of breeding programmes by ensuring optimal performances of the stocks. For example, cryopreservation of finfish milt (sperm) (Cabrita et al 2001;Yang et al 2018) and bivalve larvae (Suquet et al 2014;Rodriguez-Riveiro et al 2019) enables long-term storage of genetic material from optimal selection candidates. The high fecundity of aquatic species means that genetically improved stock can be disseminated from a single nucleus to a broad geographical and environmental range.…”

“…2014; Rodriguez‐Riveiro et al . 2019) enables long‐term storage of genetic material from optimal selection candidates. The high fecundity of aquatic species means that genetically improved stock can be disseminated from a single nucleus to a broad geographical and environmental range.…”