Cryopreservation of Steinernema carpocapsae and Heterorhabditis bacteriophora
A method for the cryopreservation of third-stage infective juveniles (IJ) of Steinernema carpocapsae and Heterorhabiditis bacteriophora was developed. Cryoprotection was achieved by incubating the nematodes in 22% glycerol (S. carpocapsae) or 14% glycerol (H. bacteriophora) for 24 hours, followed by 70% methanol at 0 C for 10 minutes. The viability of S. carpocapsae frozen in liquid nitrogen as 20 [mu]l volumes spread over cover slip glass was 80%. Survival of H. bacteriophora frozen on glass varied from 10 to 60% but was improved to 80% by replacing the glass with filter paper. Cryopreservation and storage of 1-ml aliqots of S. carpocapsae IJ resulted in 50% survival after 8 months; pathogenicity was retained and normal in vitro development took place. Trehalose and glycerol levels increased and glycogen levels decreased during incubation of S. carpocapsae IJ in glycerol. Normal levels of trehalose, glycerol and glycogen were restored during post freezing rehydration. Key words: cryopreservation, desiccation, entomopathogenic nematode, Heterorhabditis bacteriophora, nematode, Steinernema carpocapsae.