To begin, parasitize about 40 Lucilia sericata pupae onto emerging adult male and female Nasonia wasps mated for 1.5 days. Within 12 to 24 hours after parasitization, carefully open one end of the pupal shell using a sterile dissecting needle. Hold the other end of the shell and locate the wasp embryo.
Then transfer the embryo to a sterile cell strainer moistened with PBS. Once 20 to 30 embryos have been transferred, wash them with 1, 000 microliters of 10%sodium hypochlorite solution followed by sterile PBS and 70%ethanol. Finally, wash them three times with sterile PBS.
Next, place a polypropylene mesh sheet pre-wetted with PBS into a 24 well plate. Using a sterilized small brush, gently transfer the wasp embryos from the cell strainer to the polypropylene mesh sheet. For the wasp rearing, add 50 microliters of prepared NRM to each well with a sheet.
Then add one milliliter of sterile water between each well to maintain a humid environment for growth. The next day, transfer the polypropylene mesh containing the larva from one well to another well using alcohol disinfected tweezers, and add 50 microliters of equilibrated NRM. After nine to 11 days of feeding, over 80%of the larva should develop into white or yellow pupae.
then transfer the mesh to a clean well plate. In the present study, the survival rate of the germ-free wasps from larva to pupae was significantly improved compared to rearing germ-free wasps with NRM version three. Additionally, there was no difference in the generation period between the germ-free and conventionally reared wasps.
