This video protocol demonstrates an effective technique to knock down a particular gene in an insect and conduct a novel bioassay to measure excretion rate. The technique we are gonna demonstrate today combines an RNAi mediated gene knockdown with an in vivo diuresis assay. It can be used to study diuresis in different a port species, and we actually developed it to study the function of aquaporin water channels in two different mosquito species.
The protocol I'll be demonstrating today is set up in two parts. The first demonstration illustrates how to construct a simple mosquito injection device and prepare and inject double stranded RNA into the thorax mosquitoes for RNA mediated gene knockdown. The second demonstration illustrates how to measure excretion rates in mosquitoes using an in vivo bioassay as the first step in order to perform RNAi mediated gene knockdown, specific doublet stranded RNAs against genes of interest and controlled doublet stranded RNAs are synthesized Additional equipment and materials that will be needed include an aspirator with collection vial for mosquito collection.
Fine tip forceps, CO2 fly pad hooked up to a CO2 gas bottle ethanol to clean the fly pad stereo microscope with light source needle puller if needed. Glass capillary needles with marked one millimeter increments, a micro injector to inject mosquitoes, A container to place mosquitoes in after the injection, and a food source for the mosquitoes after injection, such as 20%sucrose, soaked cotton ball or Water soaked raisins. A simple micro injector can be constructed by using scissors.
A metal needle, a glass capillary, a one milliliter syringe, one milliliter plastic pipette tip and tubing cut to about 40 centimeters in length. The tip of the syringe needle hub is cut at 0.2 milliliter scale mark and the rubber plunger head removed. A metal needle is used to punch a hole in the plunger head.
A glass capillary is placed in the hole and the head is placed back in the syringe. The tip of the syringe is then placed in one end of the tubing and a one milliliter plastic pipette tip placed in the other end. This will be used as the mouthpiece.
Alternatively, a 10 milliliter syringe can be used to produce the air pressure necessary for injection. Next place a glass capillary needle in the rubber plunger head hole and break the tip of the needle off so the width is big enough for liquid to flow through. Submerge the injection needle in the prepared D-S-R-N-A sample and draw the liquid sample into the injection needle by sucking the liquid up with a mouthpiece or a syringe.
Collect mosquitoes with a battery powered aspirator into a collection vial. Place a cap over the vial and place it on a clean CO2 pad to anesthetize mosquitoes. Alternatively, mosquitoes can be anesthetized on ice.
Discard all males before you begin the injection process. They can be manipulated on the CO2 pad using a feather or paintbrush. Grab the mosquitoes by the legs or wings with forceps to avoid injury.
When ready to inject the first mosquito, gently support one side of the thorax with the forceps inserting the tip of the needle into the other side of the thorax. It is better to inject into a thin portion of the cuticle and avoid pushing the needle too deeply into the thorax. Once the needle is in place, blow the liquid into the mosquito.
Double stranded. RNA disperses in the hemolymph and is taken up by the cells triggering an RNA eye mediated knockdown of the targeted jeans. After injection, mosquitoes are placed in a container for storage.
For example, a one pint wax line, cardboard cup with mesh covering secured with the cardboard lid. Once all the mosquitoes are placed into the container, the container should be placed in an environmentally controlled chamber for incubation, and the mosquito should also be provided with a food source such as 20%sucrose, soap, cotton balls placed on top of the mesh covering For generations. Insect diuresis is attractive researchers as a simple model system for study these days.
Techniques like RNAi can be used to study the the diuresis at a molecular level. The techniques demonstrated in this video should provide a a simple way to study diuresis in vivo. Female mosquitoes begin to excrete urine within two minutes after injection with PBS buffer, so it is important to take weight measurements before they start to excrete.
For the second part of this protocol, I would demonstrate how to perform an in vivo diuresis assay in Jedi mosquitoes. Additional materials and equipment that were not needed in knockdown protocol, but will be needed in this protocol, RPBS buffer solution and an analytical precision balance for taking weight measurements, the balance should be capable of a 10th milligram precision Before collection of the mosquitoes record, the weight of an empty collection vial with a cap using the analytical precision balance. This vial will be used for all subsequent measurements.
Take the initial weight measurement of five females by placing the vial containing the mosquitoes with the cap on the precision balance. Once you have anesthetized five female mosquitoes, you should line them up on the CO2 pad to allow for easier access with the micro injector. When the needle is inserted into the thorax, you'll blow PBS buffer into the mosquito.
After the injection, gently place the mosquitoes into the collection vial and cap it. Take the first weight measurement of the five injected mosquitoes by placing the field collection vial with the cap on the precision balance. The weight of the group of five mosquitoes can be calculated by taking the weight of the field collection vial with cap, and subtracting the weight of the empty collection vial with cap weight.
Measurements of the mosquito should be taken in 30 minute intervals, but this can be adjusted to shorter or longer intervals depending on excretion rates. After 30 minutes, collect the group of five mosquitoes with an aspirator into the same collection vial with cap. Then take the next weight measurement by placing the field collection vial with cap on the precision balance.
The diuresis assay was performed at different temperatures using two species of mosquito ADA aegypti and kyx kti. The excretion rate during the first hour after injection is given in percentage. The graph shows a strong increase in diuresis as temperature increases.
We also observed a difference in excretion rates between the Two mosquito species. There's a wide range of future Applications for the in vivo diuresis assay presented here. In addition to reverse genetics methods like RNAI, it can be used to study the effects of drugs on insect diuresis, for example, signal transduction inhibitors, et cetera.
It is also a powerful assay to study the process of xeno Biotic clearance in Target instance.
