The overall goal of this procedure is to successfully perform a microinjection into nasonia vitripennis embryos in order to facilitate genomic manipulation. This method can help answer key questions related to nasonia vitripennis biology, such as in venom production and sex determination. The main advantage of using this technique is that it allows for direct manipulation into the wasp genome, and offers a wide variety of advanced genomic modifications.
Demonstrating the procedure will be Ming Li, a post-doc from our laboratory. To begin, set up several N.vitripennis colonies by placing 200 to 500 adults with a three to one ratio of females to males in bug dorm cages. Maintain the wasps at 25 plus or minus one degree Celsius with 30%relative humidity and a 12 to 12 light/dark cycle.
For two days, allow the mated females to feed on fresh S.bullata pupae, as well as small droplets of a one to ten volume to volume sucrose water solution. For the following two days, remove the blowfly pupae to deprive the female wasps of an oviposition site, making them very gravid. To keep S.bullota pupae hosts fresh, store them at 4 degrees Celsius immediately after obtaining them, and only remove them when needed.
Differentiate between young hosts that have a red-colored puparium, and older hosts that have a darker-colored puparium. Allow female wasps to parasitize into the young host by placing individual fresh S.bullata pupae into a foam stopper with a pupae-sized hole cut into the center. For maximum concentration of parasitization, and easier embryo collection, expose only about 0.2 centimeters of the anterior end of the host, which is rounded, compared to the posterior end, which is thicker, and contains a crater-like opening.
Place host pupae in this arrangement into the cage and wait roughly 45 minutes to allow wasps to parasitize them. Remove parasitized hosts by retrieving them by hand and gently tapping or blowing off any residual wasps. Replace them with fresh hosts approximately every 15 minutes to continue collecting early-staged eggs during injections.
Under a dissecting microscope, use forceps to carefully peel away the posterior end of the exterior puparium of a freshly parasitized host to expose the N.vitripennis eggs. Then, prepare an embryo alignment slide by using liquid adhesive to glue a cover slip onto a clean glass slide. With a wet, fine-tipped paintbrush, carefully brush off the embryos from the host, making sure not to damage the soft, pupal skin of the host.
Then, with the wet paintbrush, transfer approximately 20 embryos one by one onto the slide, immediately adjacent to the side of the cover slip. Orient the anterior end of the embryo against the cover slide's edge, so that the posterior ends of the embryo are pointed in the same direction. This will allow for higher precision of injecting into the same position among all embryos.
After pulling injection needles according to the text protocol, bevel the needle tip by slightly touching the tip to a diamond-abrasive plate at 25 degrees Celsius for approximately 10 seconds. Prepare the injection mixture consisting of genome modification reagents, and keep it on ice. Use a microloader tip to load the injection needle with two microliters of injection mixture.
Then, place the glass slide with the lined embryos onto the stage of a compound microscope. Carefully insert the needle into the posterior end of the embryo with a vertical angle of 25 to 35 degrees. Then, inject one to five picoliters of injection mixture and ensure that the embyro slightly swells with the injection.
If clogging occurs, try re-beveling the needles. The cytoplasm of N.vitripennis embryos is unusually viscous and sticky, which leads to frequent needle clogging. Inject approximately 20 to 40 eggs at a time, and then stop.
Transfer the injected eggs with a wet paintbrush by lightly touching the injected eggs and place them into a host. Then, continue injecting, using a fresh, newly laid batch of eggs. Ideally, this step is most effective when one person is collecting and lining up eggs, while another person is injecting genome modification components and transplanting injected embryos into host pupae.
After micro-injection, use a wet paintbrush to carefully place up to 40 injected G zero embryos one at a time onto a previously stung S.bullata pupae. Place the host pupae into a petri dish with damp filter paper and cotton balls and incubate them at 25 degrees Celsius with roughly 70%humidity until injected embryos hatch in about one to two days. Importantly, hosts can be left with a peeled-off puparium, and the N.vitripennis eggs will develop normally, so long as they are incubated in a humidified chamber to prevent desiccation.
After G zero embryos hatch, transfer the host pupae into a new petri dish and incubate them at 25 degrees Celsius and 70%humidity. Monitor the host pupae and injected G zero larvae daily. If the host pupae have a foul smell, transfer the injected larvae to new healthy host pupae.
For effective needle penetration and microinjection into N.vitripennis embryos, different micropipette pullers were tested with several types of capillary glass needles with filaments, including quartz, aluminosilicate, and borosilicate, to have a desired hypodermic-like long tip, effective for N.vitripennis embryo microinjection. The genome modification components used here were mixed guide RNAs and cas9 protein for crisper mediated genome editing. In this experiment, an sgRNA targeting the cinnabar gene was injected at varying amounts, along with cas9 protein, into N.vitripennis.
This table shows that the survival rates were dose-dependent, as increased concentration of sgRNA and cas9 protein led to decreased survival rates. While attempting this procedure, it is important to take care not to puncture nor desiccate the wasp embryos during manipulation or transfer by carefully using a moistened fine-tipped paintbrush or embryo pick. After watching this video, you should have a good understanding of how to retrieve, microinject, and transplant wasp embryos efficiently in order to successfully perform advanced genome modifications.
