RNA Interference in Aquatic Beetles as a Powerful Tool for Manipulating Gene Expression at Specific Developmental Time Points

Even as gene editing has become popular for studying the molecular genetics of non-model organisms, RNAi remains a powerful synergistic tool. The main advantage of this technique is that it can be applied to different developmental stages. In addition, partial knockdown can be used to study these whole genes.

The success of the procedure relies on the injection of DSR and having minimal harm. This takes practice, so don't be discouraged if it takes a few attempts to get it right. To prepare early stage embryos for micro injection, first pour boiling 20%agarose into a small Petri dish and place three one to two millimeter wide plastic tubes onto the surface of the liquid agarose before it solidifies.

Once the agarose has solidified use a pair of blunt forceps to remove the tubes and cover the resulting indentations with 500 microliters of autoclave distilled water. When the collection container is ready use a fine natural hair paint brush to transfer five eight hour old T.marmoratus embryo from the beetle nesting sites into a glass cavity dish filled with autoclave distilled water under a stereo microscope. Using the microscope and fine dissection forceps grasp the chorion of each embryo from both sides and rip the chorion open allowing the embryo to slide gently out of the tissue.

After removing both chorion layers from all the collected embryos use the paintbrush to carefully transfer the embryos from the glass cavity dish into the grooves of the prepared agarose plate. To inject early stage embryos with gene specific double stranded RNA first prepare microinjection needles on a needle puler and use the stereo microscope and find forceps to break each tip to a sharp edge. After throwing the purified stock double-stranded RNA of interest on ice, add one microliter of 10X injection buffer to the resulting solution and use double distilled water to bring the vinyl volume of the solution to 10 microliters.

Use a P10 pipette to backfill one of the prepared microinjection needles with the working double stranded RNA solution and load the needle into a microneedle holder connected to a microinjection system that utilizes pressure ejection technology. Turn on the microinjection system and the pressurized air supply and use the micro valve on the microinjection system to set the injection pressure to 15 pounds of pressure per square inch then use the time adjustment knob to set the injection duration to seconds. To calibrate the injection needle assess the volume of the fluid bubble that is formed at the tip of the needle when injecting into air for T.marmoratus embryo use an optimal bubble size of 100 to 200 microns.

The injection needle is of good quality if optimal bubbles can be achieved at 15 pounds of pressure per square inch with an injection duration of about three seconds. When the needle set up is ready align the needle on the agarose plate of embryos such that the needle approaches the embryos at a 45 to 60 degree angle. Use the micro manipulator to slowly move the microneedle until the tip is touching the surface of the middle of one embryo then carefully move the needle forward until it just pierces the surface of the embryo.

Once the needle is inside the embryo carefully press the micro injector injection button to deliver one to two nanoliters of double stranded RNA into the embryo. When all of the RNA has been delivered slowly retract the needle from the embryo without causing the embryo to rupture. Successfully injected embryos can be identified by the presence of a colored spot at the site of injection.

When all of the embryos have been injected carefully place the dish into a humidity chamber in a 25 degrees Celsius incubator set to a 14 hour light 10 hour dark cycle for four to five days. Monitor the embryo development progress daily under the stereo microscope to identify morphologically visible knockdown phenotypes. Using a high resolution digital camera to document the development process.

Remove any dead embryos and replenish the water on the agarose plate daily to avoid desiccation and the spread of microbial contamination to other surviving embryos during the incubation period. Before collecting the larva first add 60 to 70 degrees Celsius 2%liquid agarose to a small Petri dish. When the agarose has solidified use blunt forceps to make one shallow group per larva to be injected into the agarose and gently drain the water from the larva culture cups.

After anesthesia use soft ended forceps to carefully place the larva onto the immobilization plate with the necks and bodies in the grooves but the tail located above the agarose surface. When all of the larva had been placed, cover each lava with a thick layer of agarose that is still liquid but not dangerously hot and free any tales that were accidentally covered as necessary. for a double stranded RNA microinjection load a microneedle with the gene specific double stranded RNA working solution of interest as demonstrated.

Redrag the plunger of a manually controlled microinjection syringe and load the needle onto the microinjection system. Position the immobilized larva under the stereo microscope such that the injection site is aligned with the trajectory of the microinjection needle at a relatively flat angle. Use the micro manipulator to carefully move the needle tip into the larva and slowly and carefully apply pressure to the syringe, adjusting the injection pressure according to the movement of the colored injection fluid in the needle as necessary.

When all of the RNA has been injected use soft forceps to free the larva from the agarose and place the larva into a new container of room temperature water at 25 to 28 degrees Celsius and a 14 hour light 10 hour dark cycle. In this representative analysis three different genes were knocked down at a variety of different developmental stages of the sunburst diving beetle T.marmoratus. RNA interference is performed as demonstrated by injecting double-stranded RNA at a very early stage of embryogenesis.

Some of the embryos do not survive the process and turn necrotic. In these images they control individual and an individual with a slightly lighter eye color can be observed. In this individual, a more severe knockdown in which the more ventral lies of the cluster are completely unpigmented but the do allies still show some pigmentation can be observed.

Here an example of another individual with essentially complete pigment loss in all of the eyes is shown. Lac2 knocked down in larvae results in a less pigmented cuticle, lighter adult individuals with somewhat deformed wings likely due to the unusual softness of the tissue were also obtained. Be sure to look keenly to closely observe your injected animals on a daily basis and to immediately remove any dead individuals.

The power of this technique is that it can be applied to the genes of many different organisms. This technique has also been widely and successfully applied to evolutionary developmental biology.