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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.

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