Evaluation of Deep Eutectic Solvents for Cryopreservation of the Fish Pathogen Saprolegnia parasitica

Abstract

Saprolegnia parasitica (Oomycota) causes saprolegniosis and poses significant ecological and economic challenges in aquaculture. Experimental research on this pathogen is constrained by the lack of reliable long-term preservation methods, as routine maintenance by serial subculturing is labor-intensive and may result in genetic and phenotypic instability. Deep eutectic solvents (DESs), tunable low-melting mixtures, have recently gained attention as alternative cryoprotectants. However, their application has not been evaluated in oomycetes. Here, twelve glycerol-based two- and multicomponent DESs were assessed for cryopreservation of S. parasitica at −80 °C and compared with glycerol as a conventional cryoprotectant. Cryopreservation efficiency was assessed based on post-thaw survival and mycelial regeneration. Several two-component DESs, particularly glycerol-trehalose, supported 100% survival and high post-thaw mycelial regeneration, performing comparably to glycerol under the tested conditions. Shorter pre-incubation (30 min vs. 1 h and 3 h) and controlled-rate freezing (vs. direct freezing) significantly improved post-thaw growth. Although survival remained 100% under optimized conditions, extending storage from 7 to 32 days significantly reduced mycelial regeneration in the glycerol–trehalose treatment, indicating that survival alone, as done in existing literature, does not reflect physiological recovery. Overall, our results support the use of selected DESs as alternative cryoprotectants in oomycetes and contribute to the development of cryopreservation strategies for S. parasitica.