The authors would like to acknowledgeProf. Lyn-Mari Birkholtz and Dr. Janette Reader from the&#160;Department of Biochemistry, Genetics and Microbiology, Institute for Sustainable Malaria Control, at the University of Pretoria, for culturing and supplying the gametocyte culture. The parasite strain was obtained from the latter department (not part of this publication). The Department of Science and Innovation (DSI) and the National Research Foundation (NRF); South African Research Chairs Initiative (UID 64763 to LK and UID 84627 to LMB); the NRF Communities of Practice (UID 110666 to LMB and LK); and the South African Medical Research Council Strategic Health Innovation Partnerships (SHIP) are also acknowledged for funds from the DSI.

Refer to Figure 1 for an illustration of the protocol. Ethical clearance was obtained from the University of Pretoria Health Sciences Ethics Committee (506/2018) for the withdrawal and use of human blood.
1. Gametocyte culture
NOTE: Prior to setting up the SMFA, a gametocyte culture was prepared at the University of Pretoria (see Reader et al.22 for the complete protocol).
<ol>
	<li>Prepare a g...

For this protocol to be executed successfully, attention should be given to each step, even though it might be a tedious and laborious process. One of the most important steps is to ensure that the gametocyte culture is of good quality and that it consists of mature gametocytes, with the correct male:female ratio, prior to starting the SMFA23,24. During the SMFA, it is also crucial to maintain the gametocyte culture at the correct temperature to prevent male game...

Malaria, known as one of the most destructive diseases worldwide, still poses a great threat to several countries-especially those within the African region-and contributes toward approximately 95% of cases worldwide1. This disease is caused by a protozoan parasite and, together with its Anopheles mosquito vector, these culprits can cause great harm to the human host2. More specifically, the falciparum species of the Plasmodium parasite genus is responsible for an estimated 99% of malaria cases in sub-Saharan Africa1. In addition to this, several major Anopheles mosquito vectors (including An. gambiae Giles, An. arabiensis Patton, An. coluzzii Coetzee &#38; Wilkerson sp.n., and An. funestus Giles) could be blamed for more than 95% of parasite transmission globally3,4,5,6,7,8. For the ideal parasite-vector companionship to be established, the mosquito vector should be susceptible to the parasite and be able to transmit it9. Furthermore, both the vector and parasite should overcome physical barriers to form the perfect infective combination-the mosquito vector should be able to sustain parasite development, and the parasite should have the ability to overcome the host&#39;s defense mechanisms10,11.
Gametocytes, the sexual stage of the P. falciparum parasite, play a crucial role in connecting the vector and parasite partners12. Sexual development takes place in vivo, and gametocytogenesis describes the process of the differentiation of mature gametocytes into motile male microgametes and female macrogametes13. Another process that takes place within the mosquito is exflagellation-the process during which the male gametocyte transforms into gametes and emerges from the red blood cells taken up during a blood meal11. The exflagellation process is further suggested to be enhanced by a favorable change in the environment of the mosquito midgut14. After exflagellation, a zygote is formed by the fusion of the male and female gametes13. From the zygote, a motile ookinete arises and moves from the blood meal to the epithelium of the mosquito midgut13. Here, the ookinete matures, and an oocyst is formed, which, in turn, produces motile sporozoites13,15. The sporozoites then migrate to the mosquito salivary glands and, as the mosquito takes a blood meal from its host, these sporozoites are injected into the host&#39;s bloodstream15.
Malaria control interventions, combining vector control strategies and the use of effective antimalarial drugs, have become crucial in combatting this disease15. With a rise in parasite and mosquito resistance, the urgency for the identification of novel antimalarial compounds is increasing16. Therefore, the in vivo evaluation of transmission-blocking compounds is important16. After the development of such effective transmission-blocking drugs, the SMFA has been used to assess whether these compounds inhibit the sexual development of P. falciparum in the Anopheles mosquito17,18,19. This assay has gained recognition since the 1970-1980s as the gold standard for evaluating transmission blocking20,21. This assay provides a cheaper alternative than other assays such as RT-qPCR, which requires specialized equipment. Furthermore, no patients are needed to execute the experiments. This assay also involves the provision of gametocyte-induced blood to female mosquitoes, which are then dissected to evaluate whether oocyst development is present21. This allows for gametocyte quantification and the detection of deformed oocysts because of the compounds22. For a compound to be classified as effective, the prevalence (the proportion of mosquitoes that harbor at least one oocyst in the midgut) and the number of oocysts (intensity) in the mosquito midgut must be evaluated to assess infection inhibition17,21,22.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Bovine intestine/</td>
			<td>Butchery</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Compound MMV1581558</td>
			<td>MMV</td>
			<td></td>
			<td>Pandemic response box</td>
		</tr>
		<tr>
			<td>Dissecting needles</td>
			<td>WRIM</td>
			<td></td>
			<td>Custom made</td>
		</tr>
		<tr>
			<td>falcon tube</td>
			<td>Lasec</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Glass feeders</td>
			<td>Glastechniek Peter Coelen B.V.</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Graphpad Prism (8.3.0)</td>
			<td>Graphpad</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Mercurochrome</td>
			<td>Merck (Sigma-Aldrich)</td>
			<td>129-16-8</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope slides</td>
			<td>Merch (Sigma-Aldrich)</td>
			<td>S8902</td>
			<td></td>
		</tr>
		<tr>
			<td>Parafilm</td>
			<td>Cleansafe</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>PBS tablets</td>
			<td>ThermoFisher Scientific</td>
			<td>BP2944</td>
			<td></td>
		</tr>
		<tr>
			<td>Perspex biosafety cabinet</td>
			<td>Wits University</td>
			<td></td>
			<td>Made by the contractors at Wits</td>
		</tr>
		<tr>
			<td>Plastic cups (350 mL)</td>
			<td>Plastic Land</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

standard membrane feeding assay for the detection of plasmodium falciparum infection in anopheles mosquito vectors

Malaria remains one of the most devastating diseases worldwide and, to date, the African region is still responsible for 94% of all cases worldwide. This parasitic disease requires a protozoan parasite, an Anopheles mosquito vector, and a vertebrate host. The Anopheles genus comprises more than 500 species, of which 60 are known as vectors of the parasite. The Plasmodium parasite genus consists of 250 species, and 48 of these are involved in disease transmission. Furthermore, the Plasmodium falciparum parasite has contributed toward an estimated 99.7% of malaria cases in sub-Saharan Africa in recent years. 
Gametocytes form part of the sexual stage of the parasite and are ingested by the female mosquito upon feeding on an infected human host. Further development of the parasite within the mosquito is enhanced by favorable environmental conditions in the midgut of the mosquito. Here, the fusion of the female and male gametes takes place, and the motile ookinetes originate. The ookinetes enter the midgut epithelium of the mosquito, and mature ookinetes form oocysts, which, in turn, produce motile sporozoites. These sporozoites migrate to the mosquito's salivary glands and are injected as a mosquito takes a blood meal. 
For drug discovery purposes, mosquitoes were artificially infected with gametocyte-infected blood in the standard membrane feeding assay (SMFA). To detect infection within the mosquito and/or to assess the efficacy of antimalarial compounds, the midguts of the female mosquitoes were removed post infection and were stained with mercurochrome. This method was used to enhance the visual detection of oocysts under the microscope for the accurate determination of oocyst prevalence and intensity.

The total number of control specimens dissected was 47, with an average to 89% prevalence and an intensity of 9.5 oocysts per midgut (Table 1, as published previously22). For the compound MMV1581558, the sample size reached a total of 42 specimens, with a 36% oocyst prevalence and an average intensity of 1.5&#160;oocysts. This shows a reduction in oocyst prevalence of 58% and a TRA of 82% across all three biological replicates (Table 1).
<p class="jove_content...

Watch this Scientific Journal Video about Standard Membrane Feeding Assay for the Detection of Plasmodium falciparum Infection in Anopheles Mosquito Vectors at JoVE.com

Standard Membrane Feeding Assay for the Detection of Plasmodium falciparum Infection in Anopheles Mosquito Vectors

The standard membrane feeding assay (SMFA) is regarded as the gold standard for the assessment and identification of potential antimalarial compounds. This artificial feeding system is used to infect mosquitoes to further evaluate the effects of such compounds on the intensity and prevalence of the Plasmodium falciparum parasite.

Standard Membrane Feeding Assay for the Detection of Plasmodium falciparum Infection in Anopheles Mosquito Vectors

Malaria remains one of the most devastating diseases worldwide and, to date, the African region is still responsible for 94% of all cases worldwide. This ...

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.

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2.5K Views.  University of the Witwatersrand. 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, u...