Cryopreservation and low temperature storage of the pre-parasitic juveniles of Romanomermis culicivorax

Abstract

AbstractThe effects of low temperature storage and cryopreservation on the survival and infectivity of Romanomermis culicivorax were studied in the laboratory. When pre-parasitic juveniles of R. culicivorax were stored at-2 to 2°C, more than 90% survived for 9 days and 50% were motile for 13 days. The infective ability of the pre-parasitic juveniles for mosquitoes remained high (85%) after 5 days of cold storage and the infectivity was reduced only moderately (50 to 78%) after storage for 6 to 10 days. Various cryoprotectants were investigated to develop a cryopreservation procedure for the infectious pre-parasitic juveniles of R. culicivorax. After suspension in 1, 2.5 or 5% dimethylsulphoxide (DMSO), and cryopreservation with a two-step cooling sequence prior to storage in liquid nitrogen, approximately half of the pre-parasitic juveniles of R. culicivorax, cryopreserved in 2.5 and 5% DMSO, regained motility when thawed quickly after storage for 7 and 125 days. However, revived pre-parasitic juveniles were unable to infect mosquito larvae. Pre-parasitic juveniles treated with ethanediol, hydroxyethyl starch, and polyvinylpyrrolidone as cryoprotectants did not survive the cryopreservation procedure. Similar results were obtained with the pre-parasitic juveniles (PPJ) of Romanomermis yunanensis.