| Romanomermis culicicorax is an obligate parasite of mosquito larvae. Since its discovery in Luisana and so on, there has been considerable research on the use of the nematode for misquotes control. R. culicicorax has shown great potentials as an applied biological control agent.R. culicicorax can't be preserved for long time. In particular, the survival time of the infective pre-parasitic juveniles (PPJ) is relatively short. The vigor remains only 1 or 2 days and its application is limited. As to the freezing storage of the R culicicorax, only Song Jinzhang et al studied the pre-parasitic juveniles in liquid nitrogen in 2001.However, the revived pre-parasitic juveniles were unable to infect mosquito larvae.In order to research the croypreservation, application and the molecular mechanism antifreeze of the nematode, the effect of different frost-resisting agents, including DMSO, glycerol and trehalose, and concentrations on the survival rate, recovery motility and the infective capability of the pre-parasitic juveniles of R. culicivorax were studied. And the adults of R. culicivorax were studied also at low temperature. The ways were cooling with step-cooling way or non-step-cooling way, and then maintenance at -20~-10℃, -80~-70℃ or liquid nitrogen ,or -80~-70℃ after being cooled at different rates. The following are the results.The best one obtained among the frost-resisting agents was Dimethylsulphoxide (DMSO), The survival rates of the juveniles in 5% and 10%DMSO were 71.8 ±2.1 and 82.8 ±2. 9 respectively after 30 days at -80—-70 ℃. The results show the mixed frost-resisting agents composed of 5%DMSO and 5%trehalose or 10%trehalose is suitable to cryopreserve the juveniles. Storaged for 90 days, the survival rate is 78. 0±5. 8 and 64. 2 ±9. 2, and the recovery motilityis 39. 5 ±9.1 and 52. 5 ±14. 7 respectively.The juveniles are cooled with step-cooling way, and then put into liquid nitrogen. After 96 days, neither the survival rate nor the recovery motility is well with the same ways of recovery. Of all the frost-resisting agents, only trehalose can keep the juvenile bodies integrity.The survival rate and the vigor of the juveniles, cooled at 1.18°C per. minute rate at -20~-10°C, then maintained at -80~-70°C, prior to those cooled at 0.84°C and 0.92°C per. minute rate at -20~-10°C ; After equilibrated at 4-8°C,then put at 80~-70°C directly and storaged for 1 day,the effect of 10%DMSO is the best,in which the survival rate and the infective rate are 79.8 +5.3 ^ 89.1+4.9 respectively.The juveniles still had about 2.5% infective rate to the wigglers of culex after they were stored at -20~-10°C with 5%, 10%DMSO or 30% glycerol and at -80~ -70°C with 5%DMSO for one day. The survival rate of adults was 3.4% at -20—-10°C with 10%DMSO for one day.In conclusion? it hasn't been reported of adopting trehalose as impermeable frost-resisting agents in the Nematode field, even in the whole cryopreservation field. The results are well. The ways of step-cooling and immediately cooling are suitable for storage of R. culicicorax at the freezing temperature. The results have the value as the reference for cryopreserving nematodes, researching the mechanisms of freeze-tolerance in molecular biological field and cryopreserving other organism. The results also have certain value for controlling the insect with Nematode.
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