The protocol allows us to detect the effect of treatment on the malaria susceptibility of African malaria vectors. For example, compounds aimed at blocking transmission from the human to the mosquito. This technique allows researchers to evaluate a large number of compounds under laboratory conditions, even before toxicity GATEs are available.
Midgut dissections can be challenging at first but it just takes some practice. Also, to be able to identify oocysts is not always easy. First, using a mouth aspirator, place 25 unfed female Anopheles gambiae mosquitoes into a 350 milliliter feeding cup.
Label the cups clearly to distinguish between control and treatment groups. Choose the number of cups per treatment according to the number of technical replicates included. Connect the glass feeder system to a water bath and maintain the temperature at 37 degrees Celsius.
Then, prepare the cow intestine or synthetic membrane by rinsing it in tap water and cutting it into pieces that are fitted for each feeder. Cover the bottom part of each feeder and fasten the membrane with an elastic band. For using the feeding cups as infection cups, first, keep the membrane on top of the nets of the cups.
Then place the infection cups underneath the feeders. Now, add one milliliter of gametocyte-infected blood to the feeders of the control cups, and gametocyte-infected blood with a test compound to the feeders of the treatment cups. Then leave the mosquitoes to feed for about 40 minutes.
After feeding, remove the feeders from the cups, rinse the feeders, and treat the excess blood with hypochlorite. Then, knock all the mosquitoes down on the ice for one to two minutes, and select the mosquitoes that have taken a blood meal by looking for swollen and red abdomens. Replace the sugar water that is given on alternate days to each infection cup with a 10%sugar water pad, and incubate the infection cups in a biosafety chamber for eight to 10 days.
Eight to 10 days after feeding, knock the infected mosquitoes down by placing them on ice. Then, transfer them to labeled tubes with 70%ethanol, keeping control in each treatment group in separate tubes. Keeping the control and test groups separate, transfer the mosquitoes to labeled Petri dishes lined with filter paper.
Add a drop of PBS on an appropriately-marked glass slide, and transfer a mosquito from the filter paper to the drop. Pin the thorax of the mosquito with dissecting needle. Then, remove the midgut by pulling the seventh abdominal segment with forceps, and transfer it to a droplet of 0.1%mercurochrome on a new microscope slide.
Leave the gut to stain for eight to 10 minutes. Then place a cover slip on the stained gut, and view it under bright field illumination at 20 to 40 times magnification. Record the number of oocysts per midgut for the control and each treatment group.
In this study, the compound MMV1581558 was found to significantly restrict the number of Plasmodium falciparum oocysts in the midgut of female Anopheles gambiae mosquitoes in comparison to the control. In the control group, the average prevalence of oocysts in the midgut was 89%with an average intensity of 9.5 oocysts per midgut. But in the MMV1581558 treated group, the average oocysts prevalence was 36%with an average intensity of only 1.5 oocysts per midgut.
Thus, MMV1581558 treatment resulted in 58%transmission blocking activity and 82%transmission reducing activity in all three biological replicates. It is critical to ensure that mosquitoes feed on a healthy gametocyte culture. If these are immature gametocytes, or not stage five gametocytes, the probability of getting an infection in the mosquito is limited or impossible.
The assay uses a visual morphological identification method, however, molecular quantification assays can be used. This is, however, costlier and might limit their use in resource-constrained laboratories. Having the ability to do this technique opens up new avenues to evaluate the change of malaria prevalence and intensity between almost anything in mosquito.
For example, comparing species vectorial capacity.
