This method can help to answer key questions in the field of mosquito-borne diseases, such as whether or not a mosquito species is a competant vector for a certain virus. The main advantage of this method is that a large amount of mosquitoes can be analyzed simultaneously, without the use of laboratory animals like mice. Generally, individuals new to this method will struggle because it needs fine motor skills that can only be acquired through practice.
Visual demonstration of this technique is critical, as the key steps are difficult to learn;they require experience with the handling of mosquito and viruses, and also fine motor skills. To begin, dilute the virus stock. Then, mix expired human blood with fructose, filtrated bovine serum, and the virus stock.
Freeze 140 microliters of bloodmeal mix for further analysis via TCID50. Next, put two 50-microliter droplets of the bloodmeal mix in a plastic vial, and allow the mosquitoes to feed for two hours. After anesthetizing the mosquitoes, count the fully-engorged individuals, and transfer them to a new vial containing a cotton pad soaked in fructose.
Then, maintain the mosquitoes at 27 degrees Celsius and 80%humidity for 14 or 21 days. Replenish the fructose-saturated cotton pads every 72 hours with 1.5 milliliters of fructose. First, seed 20, 000 Vero cells per well in a 96-well plate with supplemented growth medium.
To prepare the salivation device, place a squared glass plate onto the bench at a 30-degree angle. Then, adhere double-sided tape to the top of the glass plate. Next, roll modeling clay until it is 0.5 centimeters in diameter, and attach it to the plate horizontally one centimeter away from the double-sided tape.
Cut the tips off of the requisite number of 10-microliter filter tips, and fill each tip with 10 microliters of PBS. Then, place the tips on the modeling clay, and secure them with gentle pressure. Next, immobilize the mosquitoes by removing their legs and wings.
Fix the mosquitoes to the tape above the filter tips. Gently place the proboscis of each mosquito into a filter tip. After 30 minutes, remove the filter tips, and discard the clay and tape.
Transfer the collected saliva to reaction tubes containing 10 microliters of PBS. Mix the saliva and PBS gently, and transfer the mixture to the wells of a 96-well plate. Incubate the plate for seven days at 37 degrees Celsius with 5%carbon dioxide.
Using a light microscope, check the cells for the presence of cytopathic effect. Once a well is positive for cytopathic effect, use a pipette to harvest 140 microliters of the supernatant. Then, transfer the supernatant to a reaction tube.
Next, mix 140 microliters of the supernatant with 560 microliters of AVL buffer, and incubate for 10 minutes at room temperature. Finally, add 560 microliters of ethanol, and invert the sample to mix. Using this protocol, several mosquito species were tested for Zika-Virus infection and transmission under various climatic conditions.
Under 27-degree-Celsius incubation conditions all Aedes species displayed Zika-Virus-positive saliva. Conversely, none of the saliva samples from the Culex taxa contained evidence of the virus. Once mastered, this technique can be performed for 50 mosquitoes in one hour if performed properly.
After watching this video you should have a good understanding of how to perform forced-salivation experiments. Don't forget that working with infected mosquitoes can be very dangerous, so special safety measures for the handling of mosquitoes under BSA-3 conditions should always be taken while performing any experiment.
