Cryopreservation of the entomogenous nematode parasite Steinernema feltiae (= Neoaplectana carpocapsae)

Abstract

Cryopreservation studies were conducted with the J1 juvenile and third stage infective juvenile (IJ) larval stages of the entomogenous parasitic nematode Steinernema feltiae (= Neoaplectana carpocapsae). The main parameters evaluated were (i) tolerance of the organisms to the cryoprotectants methanol, ethanediol, glycerol, and dimethyl sulfoxide; (ii) the incubation temperature and exposure period in cryoprotectant; and (iii) slow cooling (circa 1 °C min −1) vs rapid cooling (circa 5,100 °C min −1). The J1 stage was sensitive to all cryoprotectants at >20% ( v v ). This sensitivity increased at 22 and 37 °C. Exsheathed IJ stage organisms tolerated exposure to 50% ( v v ) ethanediol or Me 2SO and 60% ( v v ) methanol or glycerol. A proportion (3.4%) of J1s preincubated in 20% ( v v ) methanol for 10 min at 0 °C survived slow cooling to −35 °C followed by plunge into liquid nitrogen. Preincubation in 60% ( v v ) Me 2SO for 45 sec at 0 °C followed by rapid cooling yielded a higher proportion (12.3%) of surviving J1s. The infective IJ stage only survived rapid cooling. Preincubation for 20 min at 0 °C in either 60% ( v v ) methanol or 45% glycerol, followed by rapid cooling at 5,100 °C min −1, yielded 30–34% viable IJs following thawing. These larvae were capable of further growth and development in vitro. The results suggest that the organisms are vitrified during the rapid cooling step. For routine maintenance of strains of S. feltiae and related genera it is preferable to use the IJ stage.