Cryopreservation of Platynereis dumerilii larvae

The marine annelid Platynereis dumerilii is a functional molecular model organism for developmental, evolutionary and chronobiological studies. Research on Platynereis is rapidly growing, and with it, the number of genetic variants that laboratories isolate or generate and must subsequently maintain and propagate. Therefore, there is an urgent need to alleviate the burden of live culture maintenance by developing cryopreservation techniques for this species. We report the first cryopreservation protocol for P. dumerilii larvae, which combined with a careful post-thawing culturing regime, allowed us to obtain animals that survived to adulthood and successfully reproduced. Our experiments show highest survival rate in 6-8 dayold larvae (dpf). Equilibration with cryoprotecting agents takes 1h in 5 % (v/v) Me2SO + 0.1 %(v/w) sucrose, followed by transfer to 0.25 ml straws. The protocol cools larvae at 2.5 °C/min from 20 °C to -35 °C using a programmable freezer, followed by a rapid transfer to liquid N₂. Larvae are thawed in a water bath at 18 °C. The post-thaw larvae feeding regime consisted of 50 % Tetraselmis + 50 % diatom strains mixture (Grammatophora marina and Nitzschia laevis). The maximum survival obtained with this protocol so far produced 34 % survival after ∼5 months, the average is 8.69 ± 13.08 % (140 days) with a large variability inter-individual batches.