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

Summary

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.

Abstract

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.

Introduction

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 & Wilkerson sp.n., and An. funestus Giles) could be blamed for more than 95% of parasite transmission globally^3,4,5,6,7,8. 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's defense mechanisms^10,11.

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 sporozoites^13,15. 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'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 mosquito^17,18,19. This assay has gained recognition since the 1970-1980s as the gold standard for evaluating transmission blocking^20,21. 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 inhibition^17,21,22.

Protocol

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). Prepare a g…

Representative Results

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 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…

Discussion

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 SMFA^23,24. During the SMFA, it is also crucial to maintain the gametocyte culture at the correct temperature to prevent male game…

Materials

Bovine intestine/	Butchery
Compound MMV1581558	MMV		Pandemic response box
Dissecting needles	WRIM		Custom made
falcon tube	Lasec
Glass feeders	Glastechniek Peter Coelen B.V.
Graphpad Prism (8.3.0)	Graphpad
Mercurochrome	Merck (Sigma-Aldrich)	129-16-8
Microscope slides	Merch (Sigma-Aldrich)	S8902
Parafilm	Cleansafe
PBS tablets	ThermoFisher Scientific	BP2944
Perspex biosafety cabinet	Wits University		Made by the contractors at Wits
Plastic cups (350 mL)	Plastic Land