Optimizing the Rearing Procedure of Germ-Free Wasps

Our research focuses on the study of insect-microbiota interactions, specifically investigating the impact of those symbiotic bacteria and exploring the dynamics of host-microbe relationships. Germ-free Nasonia is an essential tool for conducting this research. Current research utilizes technologies such as high-throughput sequencing, germ-free insect rearing, transcriptomics, proteomics, and advanced microscope.

This tool enable comprehensive analysis and virtualization of insect-microbe host interaction for advancing our understanding of this field. They optimized the procedure for preparing the Nasonia-rearing medium provides economic advantages and saves time and effort. Additionally, it effectively reduces the risk of NRM-contamination during the feeding process, greatly improving the efficiency of producing aseptic Nasonia vitripennis.

To begin, place commercially available Lucilia sericata pupae on a sheet of paper. Discard any underdeveloped larvae, dark old pupae, empty pupal shells, sawdust, or other impurities. Transfer young brownish red colored pupae to a beaker and immerse the pupae in deionized water.

Wrap the beaker with tinfoil or gauze and shake it to clean any surface impurities off the pupae. Then discard the water. Place the cleaned pupae in the filter tank of a garlic press and squeeze hard.

Then collect the pupae tissue fluid in a 50 millimeter sterile centrifuge tube and centrifuge it at 25, 000 G for 10 minutes at four degrees Celsius. Using a 22 gauge sterile needle, collect the protein layer into a new 50 millimeter polypropylene centrifuge tube and add commercial liquid Drosophila medium at an equal ratio to the protein extract. To remove the particles of different sizes from the mixture, filter it using a vacuum filtration system with filter paper of different pore sizes.

To prevent clogging, change the filter paper when the flow slows down. Once the filtrate is centrifuged, sterilize the supernatant through a 0.22 micrometer syringe filter and store the prepared Nasonia rearing medium, or NRM aliquots, at minus 20 degrees Celsius until further use. 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 1000 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 larvae 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 larvae should develop into white or yellow pupae, then transfer the mesh to a clean well plate. In the present study, the survival rates of the germ-free wasps from larvae to pupae was significantly improved compared to rearing germ-free wasps with NRMv3.

Additionally, there was no difference in the generation period between the germ-free and conventionally-reared wasps.