The overall goal of this protocol is to deplete gene expression in the gut of cockroaches using RNA interference via oral ingestion of double-stranded RNA encapsulated in liposome carriers. This method can help answer key questions in the development of new pest control methods, such as can knocking down essential genes in the open field kill pests? The main advantage of this technique is that liposome protects the gene-specific double-stranded RNA to ensure its delivery to the target cell.
Demonstrating the procedure will be Jia-Hsin Huang, a postdoc, and Yun Liu, a technician from my laboratory. To begin, anesthetize the cockroaches on ice until they do not move for three minutes. Then, pick up an insect using the thumb and index finger to access its ventral side.
Next, use dissecting scissors to cut the tip of a coxa of a hind leg off between the coxa and the trochanter. Cutting elsewhere on the coxa is less efficient for the hemolymph collections. Now, position a 10-microliter micropipette at the incision, and squeeze the abdomen gently while drawing up the bleeding hemolymph.
Repeat this process until the hemolymph from five cockroaches has been collected into a single microcentrifuge tube. Next, briefly spin down the pooled hemolymph for 10 seconds. Then, combine 10 microliters of hemolymph with 50 microliters of 1x insect saline buffer.
Now, centrifuge the diluted hemolymph for 10 minutes to pull the hemocytes out of solution. Then, transfer the supernatant to a new tube. Lastly, quantify the total protein concentration using a microvolume UV-Vis spectrophotometer, and adjust the concentration of each sample to six milligrams of protein per microliter.
To collect midgut juice, first, anesthetize the cockroaches on ice. Then, transfer one to a dissection plate loaded with cold 1x insect saline buffer, and use insect pins to secure it ventral side up. Now, dissect the abdomen using fine tweezers.
Then, remove the entire gut, and transfer it to a fresh dish with 1x insect saline buffer. Next, isolate the midgut, which is the region between the crop and the Malpighian tubules. To do so, remove the front part of the animal and remove the hindgut.
Next, quickly transfer the midgut to a microcentrifuge tube with 100 microliters of insect saline buffer. Collect the midguts from six cockroaches into the same tube. Then, vortex the tube for 10 seconds.
Next, centrifuge the mixture for 10 minutes to separate out the hemocytes and gut tissues. Then, transfer the supernatant to a clean microcentrifuge tube, and adjust the concentration to six milligrams of protein per microliter. Double-stranded RNA lipoplexes must be used within an hour of preparation.
During their preparation, be sure to combine all of the diluted reagent with the diluted double-stranded RNA at the same time. Then, quickly vortex and incubate the mixture. Once ready, mix four microliters of the double-stranded RNA solution with 10 microliters of insect saline buffer as a control, or mix it with 10 microliters of extracted enzymes taken from hemolymph or midgut juice.
Next, make an enzyme-inhibited control by adding two microliters of EGTA. Otherwise, add two microliters of RNase-free water. Then, incubate the samples for an hour or longer at 25 degrees Celsius.
After the incubation, add 200 microliters of extraction reagent and 40 microliters of chloroform, and then vortex. Next, centrifuge the samples for 10 minutes. Next, transfer 150 microliters of each supernatant to a new tube, and precipitate the double-stranded RNA from each sample by adding 150 microliters of isopropanol and incubating the samples on ice for 15 minutes.
After the incubation, centrifuge the samples again and discard the supernatant. Then, wash the RNA pellets twice by adding 200 microliters of the 70%ethanol to each pellet and centrifuging the tube. After the second wash, dry the double-stranded RNA pellets using a centrifugal vacuum concentrator for three minutes.
Then, resuspend the pellets in 10 microliters of RNase-free water. Finally, check the integrity of the treated double-stranded RNA using a 1.5%agarose gel. Feed the cockroaches twice per day, one hour after lights on and one hour before lights off.
Do this for eight or 16 days without breaks. During this time, the cockroaches should be otherwise starved of water. For the feeding, have freshly prepared double-stranded RNA lipoplexes and naked double-stranded RNA available.
For the bolus, use with four microliters of double-stranded RNA lipoplexes or 250 nanograms of naked double-stranded RNA. To feed a cockroach, first, grab it by the wings using flexible forceps. Do not grab the body parts of a cockroach.
Then, slowly pipette the droplet of solution close to the mouthparts, and watch as the roach ingests the droplet. After the last feeding, provide water bottles to the roaches. Throughout the feeding period, assess the insects daily to check for mortality and remove any roaches that are no longer moving from the experiment.
Continuous oral administration of double-stranded tub lipoplexes was able to decrease tubulin expression in the midgut from 40%at day nine to 60%at day 17. By comparison, naked double-stranded RNA had no effect. This is most likely due to finding that exposure of the naked double-stranded RNA to midgut juice resulted in degradation within an hour, whereas the liposome-conjugated double-stranded RNA remained stable.
Not only did RNA levels decrease, but continuous feeding of the tubulin double-stranded RNA lipoplexes also resulted in significant lethality. This is unlikely to be due to the vector, as lipoplexes carrying double-stranded RNA to EGFP resulted in no lethality. While attempting this oral delivery system of RNA interference, it is important to perform a continuous feeding of double-stranded RNA for several days.
Once mastery, the feeding step can be done in two minutes per insect if it is performed properly. If needed, different formulation of liposome nanoparticles can be applied to improve the feeding procedure and RNAi efficiency. Ultimately, this technique has made it feasible for researchers in the field of the pest management to explore new control strategies using RNAi.
