Reproducible dsRNA Microinjection and Oviposition Bioassay in Mosquitoes and House Flies

This protocol describes a microinjection methodology that we have standardized and used for several years to deliver specific quantities of nucleic acids directly to the hemolymph of mosquitoes and house flies. This protocol results in minimal injection mortality and allows dose correlated measurements of fecundity.

This method can help answer key questions in the biopesticide field, such as how double strand RNA's impact gene expression, and fecundity in adult dipterans. The main advantage of this technique is that quantifiable doses of double strand RNA are delivered to the mosquito or fly, and resulting affects on multi-generational lethality can then be assessed. Though this method is presented for the mosquito, aedes aegypti, and the house fly, musca domestica, it can also be applied to other species of mosquitoes and flies.

Generally, individuals new to this method will struggle, because mastering the injection procedure itself is a time investment. Injecting each insect with the optimal needle tip size is critical to ensure delivery while minimizing injury and mortality. To begin, use forceps to carefully stage mosquitoes so they are eventually exposed on a microscope slide approximately half a centimeter apart.

To aid slide handling, leave a one to a one and half centimeter space at the left or right end of the slide. Then place the slide of staged insects at four degrees Celsius in a large petri dish. Set up a dissecting microscope and microinjector over a chill table.

After pulling glass capillaries to a fine tip, place the capillary needle into the microinjector and break the needle tip with forceps. Then rinse the needle by drawing up and expelling nuclease free water three times. Note that glass needle opening sizes vary between mosquitoes and house flies.

Next, prepare an injection solution with an appropriate concentration for mosquitoes. Add three micrograms per milliliter of Rhodamine B to the solution to aid in visualization. Use a pipette to transfer three to four microliters of solution to a clean surface, and draw it into the glass needle without taking in any air.

Depress the inject button repeatedly until the liquid begins to dispense from the needle. Use an ultra fine point marker to draw hash marks about one millimeter apart, starting from the liquid meniscus to the needle shank. Set the slide of mosquitoes under the needle and ensure that the field of view is wide enough to see the solution meniscus in the needle.

Align the needle with the middle one third of the mesocarape sternum. Brace the mosquito against the needle with forceps placed on the mosquito's opposite side, and gently puncture the cuticle with the needle tip. Gently slide the needle into the mosquito until the tip has passed through the midline.

Depress the inject button until the desired amount of liquid has been injected. If the meniscus does not move, slowly slide the mosquito or fly off the needle while watching for meniscus movement. If a portion of the injected solution beads out of the cuticle upon needle removal, or if the meniscus fails to move, discard the insect as the injection was not successful.

On the house fly, align the needle with the mesopleuron. Brace the fly against the needle with forceps placed on the fly's opposite side, and gently puncture the cuticle with the needle tip. Transfer the injected insects to clear 3.5 ounce holding cups in groups of ten to 15.

Then cover the cup with netting, and allow them to recover at room temperature. After the insects have recovered, invert the holding cup over a cotton ball soaked in 10%sucrose solution. First, fill a 12 inch artificial membrane with fresh blood, and heat it to 60 degrees Celsius in a hot water bath.

Dry the membrane with a paper towel, and lay it across the netting cap of the holding cup. After the mosquitoes feed, replace the cotton ball and allow the mosquitoes to rest for 24 hours. Next, construct oviposition cups by filling clear 3.5 ounce bio acetate cups with approximately 30 millimeters of deionized water.

Then place a piece of seed germination paper at the bottom of the cup. Cover the cup with netting, and cut a small slit in the netting cap. 24 hours after feeding, cut a small slit in the cap of the holding cup and transfer the females that successfully fed to oviposition cups.

Then seal the small slit in the oviposition cap with a 10%sucrose saturated cotton ball. Monitor the mosquitoes, and track daily mortality. After allowing the mosquitoes to oviposit for five to seven days, count the eggs under a dissecting microscope.

Demonstrating the procedure will be Dr.Christopher Geden, a USDA ARS Research Entomologist. Three days after injection, anesthetize the flies with carbon dioxide. Then transfer the flies to a clean cage with water and prepared fly diet.

Record mortality data, and remove dead flies daily. Next, mix 75%wheat bran with 25%pelleted live stock feed by weight to prepare the larval rearing medium. Add water to the mixture until 62%moisture is achieved.

Add the moistened wheat bran live stock feed mixture to a square of black cloth, and roll it into a ball. Use a rubber band to keep the cloth in place, and squeeze gently until the liquid medium seeps through. Place the ball in a 60 milliliter cup, and place it in the fly cage for five hours.

After this, rinse the eggs off of the ball, ensuring that the eggs are removed from under the folds of the cloth. Shake the eggs to disrupt any clusters, and transfer them to a graduated 20 milliliter centrifuge tube. Allow the eggs to settle, and note the volume of settled eggs in the tube.

Then add sufficient water to bring the volume to 20 times the volume of the settled eggs. Next, use a magnetic stir bar to mix the water and egg suspension. Finally, use a modified pipette tip to dispense 0.5 milliliters of the suspension onto a piece of pre-moistened black cloth in a series of lines.

In this protocol, female mosquitoes were micro-injected to evaluate gene expression. Injecting females with double strand RNA showed significant reduction in relative expression across multiple oviposition cycles. Average clutch sizes in mosquitoes in the first gonotrophic cycle were significantly reduced for both dsRPS6 and dsRPL26 treated groups.

An obvious dose effect was observed in clutch sizes when injecting dsRPS6 from one microgram to 50 nanograms in female mosquitoes. House flies were also injected with five micrograms of double strand RNA constructs. Similar to the mosquito observations, significant reduction in both specific transcript expression and clutch size was noted.

While attempting this procedure, it's important to remember to discard insects that were not successfully injected. Using this procedure, any double strand RNA construct or other biorational can be delivered to mosquitoes or flies. Don't forget that working with glass needles can be hazardous, and they should always be disposed of in appropriate sharps waste containers.