RNA interference is one of the main tools used for reverse genetics in mosquitoes and oral delivery allows us to induce and maintain gene silencing in adult mosquitoes without the need for micro injection. The oral delivery of double strand RNA to adult mosquitoes is a low cost and versatile RNA interference method which significantly reduces work and time and effort to study gene function. This method could be used to study not only other target genes in Anopheles gambiae, but also in another anopheles of interest such as Anopheles albinamus.
To begin, grow a culture from a single bacterial colony of Escherichia coli strain HT115(DE3)containing the double stranded RNA expressing plasmid in five milliliters of lb containing ampicillin and tetracycline on a platform shaker for 12 hours at 37 degrees Celsius and 180 RPM. After 12 hours, take 0.5 milliliters of the overnight grown bacterial culture and make a one in 1000 dilution in 2X yeast tryptone media containing ampicillin and tetracycline. To induce the double stranded RNA production, add IPDG at the final concentration of 40 micromolar.
Then incubate the culture for two hours in shaking conditions as demonstrated. At the end of incubation when the optical density of culture reaches 0.4 at 600 nanometers, pellet the bacterial cells by centrification at 4, 000 times G for 10 minutes at four degrees Celsius and then wash the cells in one volume of PBS. After spinning the cells one more time, resuspend the cells in PBS and incubate at 70 degrees Celsius for one hour.
After heat killing the bacteria, make aliquots of 400 microliter volume and store them at 20 degrees Celsius until further use. Mix one 400 microliter aliquot of thaw heat killed bacteria expressing double stranded RNA with 1.6 milliliters of 12%sugar solution containing 0.2%methylparaben. Soak a small cotton ball in this solution and place it inside a cage containing five day old mosquitoes and ensure that the mosquitoes feed on this solution.
Change the cotton ball soaked in double stranded RNA sugar solution every other day for eight consecutive days, ensuring that the cage is maintained under constant conditions. To anesthetize the mosquitoes with cold, place the container on ice until mosquitoes stop moving, and then place the mosquitoes on a cold surface to isolate females for dissection. After spraying the mosquitoes with ethanol, place them on a glass surface with PBS.
With a pair of forceps, secure the mosquito head and pull the thorax very slowly allowing the salivary glands to be released into PBS. Once the salivary glands are dissected from 10 mosquitoes, pull the glands for RNA extraction. Upon completion of RNA extraction, suspend the RNA pellet in 30 microliters of RNAs free water.
Measure the absorbance and calculate the RNA concentration as described in the text manuscript. Using a commercial reverse transcription kit, synthesize cDNA from one microgram of RNA. Dilute the cDNA 10 times and set up the RTPCR reactions in triplicates for target and housekeeping genes as per the manufacturer's recommendations.
Amplify the CDNA with standard real time PCR conditions. To evaluate the ability to blood feed, place groups of 15 female mosquitoes treated with target and controlled double stranded RNA in small cages and starve them for four hours. Provide defibrillated cheap blood to the mosquitoes using a circulating water bath set to 37 degrees Celsius, glass mosquito feeders and peryfill membrane.
Observe, count and record the number of probing attempts to successfully acquire a blood meal from the first five females to become fully engorged in each group. After isolating fresh tissue and PBS has demonstrated previously, fix it in ice cold acetone for 90 seconds. Then rinse the tissue several times in PBS and incubate with primary antibodies overnight at four degrees Celsius with anti-serum diluted in PBS.
At the end of the incubation, wash the tissue several times with PBS. Add fluorescent secondary antibodies diluted in PBS, and incubate in the dark at room temperature for two hours. Add any counter stain 30 minutes before the end of the two hours incubation.
After two hours, wash the tissue three times with PBS, then mount the tissues in 100%glycerol on a standard microscope slide with a one millimeter thick cover slip and store at 20 degrees Celsius until imaging. The micro aray expression data showed the expression of all chosen targeted genes in adult salivary glands and the levels of AAPP and sage were particularly high. The double stranded RNA effectively reduced the abundance of fork head transcripts in the salivary gland.
The fork head double stranded RNA fed mosquitoes exhibited five times more feeding attempts than the control group or FEG double sex double stranded RNA fed mosquitoes to be completely engorged with blood. The levels of sage and CrebA staining were markedly reduced in all salivary gland lobes following fork head RNA interference compared to ant control RNA interference. When considering highly abundant saliva component proteins, levels of AAPP were reduced in all three salivary gland lobes following fork head RNA interference compared to control RNA interference treatment.
On the other hand, no changes in levels of mucin were observed. These data suggests that fork head contributes differently to the expression of different saliva protein genes. Reduced Rab11 fluorescence was observed in distal lateral lobes following fork head RNA interference treatment, however, increased Rab11 signal in the medial and proximal lateral lobes also occurred.
No discernible difference was observed in the Nile Red signal after fork head RNA interference compared to the control RNA interference treatment. some secretary machine reaction in a complex manner that differs between salivary gland lobes. This technique could allow researchers to silence genes for which expression can be reduced by a single injection of double strand RNA and to explore oral delivery of double strand RNA to use RNAi as a potential vector control method.
