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 gametocyte culture that consists of stage V gametocytes from the NF54 parasite strain.</li>
	<li>Ensure that the gametocytemia of the culture is between 1.5% and 2.5%, with a 50% hematocrit in A+ male serum, to which fresh red blood cells are added.</li>
	<li>Separate the culture into different flasks and add 2 &#181;M of each compound for each respective treatment 48 h prior to conducting the SMFA. Leave the control group untreated.</li>
	<li>Assess the gametocyte culture shortly before conducting the SMFA to ensure the exflagellation of male gametes, with the presence of a 3:1 female:male ratio.</li>
</ol>
2. Artificial infection of mosquitoes through the SMFA
NOTE: Biosafety: infected mosquitoes should be housed in a biosafety level 2 (BSL2) facility with restricted access.
<ol>
	<li>Using a mouth aspirator, place 25 unfed female An. gambiae mosquitoes into a 350 mL feeding cup. Do the same for each treatment cup and label the cups clearly according to whether they are to be used as control or treatment groups. Choose the number of cups per treatment according to the number of technical replicates included. 
	NOTE: Colony mosquitoes between 5 and 7 days old are used in a typical transmission-blocking compound evaluation. Starving mosquitoes for 3-4 h or longer prior to blood feeding will facilitate the uptake of blood during SMFA.</li>
	<li>Connect the glass feeder system to the water bath and maintain the temperature at 37 &#176;C. 
	NOTE: The glass feeder consists of two arms, which are connected to the silicone tubing to which the water bath is connected (Figure 2). The hollow structure of the feeder allows water to circulate through and maintenance of the temperature of the blood.</li>
	<li>Prepare cow intestine (or synthetic membrane) by rinsing it in tap water and cut it into pieces that are fitted for each feeder. Cover each feeder and fasten the membrane with an elastic band. 
	NOTE: No ethical clearance was needed for the intestine, as it was bought from a local butchery, where it is sold to the public for food preparation.</li>
	<li>Place the infection cups underneath the feeders, with the membrane laying on top of the net of the cup.</li>
	<li>Add 1 mL of gametocyte-infected blood to the feeders of the control cups and gametocyte-infected blood with added compound to each corresponding compound feeder and cup.</li>
	<li>Leave the mosquitoes to feed for approximately 40 min with the feeders uncovered. 
	NOTE: Feedings take place under insectary conditions (25 &#176;C, 80% relative humidity) in the dark. The diameter of a feeder is approximately 13 mm.</li>
	<li>After feeding, remove the feeders from the cups, rinse the feeders, and treat the excess blood with hypochlorite.</li>
	<li>Remove the unfed mosquitoes from the cup by knocking all the mosquitoes down on ice (for 1-2 min) and separating the unfed mosquitoes from those that have taken a blood meal. Look for swollen and red abdomens (indicating blood) to distinguish the fed, fully engorged mosquitoes from the unfed ones (Figure 3).</li>
	<li>Place the infection cups in the biosafety chamber (Supplemental Figure S1) and provide each cup with a 10% sugar water pad, replacing the sugar water on alternate days for 8-10 days.</li>
</ol>
3. Preparation of infected mosquitoes
NOTE: This part of the protocol takes place within the BSL2 infection room. Only authorized, trained staff are permitted to enter the infection room where infected mosquitoes are housed. Mosquitoes are kept in modified cups that contain only one entry point, which automatically seals when the mouth aspirator is removed. These cups are placed inside a transparent, thermoplastic container to prevent escape. The container is located in the infection room behind a double-door system. All necessary protocols must be in place for accidental exposure to infected mosquitoes (Supplemental File S1). The protocols are country-specific and depend on the requirements of the institution.
<ol>
	<li>On days 8-10 post infection-feeding, knock the infected mosquitoes down by placing them on ice and transferring them to labeled tubes with 70% ethanol (keeping the mosquitoes of each control and treatment group separate).</li>
	<li>Ensure that all mosquitoes are dead before leaving the infection room.</li>
</ol>
4. Dissections of infected mosquitoes
NOTE: This part of the protocol is conducted in the laboratory.
<ol>
	<li>Transfer the mosquitoes to labeled Petri dishes lined with filter paper, keeping the control and test groups separate.</li>
	<li>Place a droplet of phosphate-buffered saline (PBS) on a microscope slide (marked according to the control/test group) and transfer an individual mosquito from the filter paper to the PBS.</li>
	<li>Remove the midgut from the immobilized, infected specimen by pinning the thorax of the mosquito with the dissecting needle whilst pulling the 7th abdominal segment with the forceps.</li>
	<li>With the gut being exposed and visible, look for the Malpighian tubules (Figure 4A,B) to distinguish the gut from the ovaries. Remove it from the PBS, transfer it to a droplet of 0.1% mercurochrome on a new microscope slide, and leave the gut to stain for 8-10 min.</li>
	<li>After staining, place a coverslip on the stained gut and view the gut under brightfield illumination at 20x-40x magnification (Figure 4C,D).</li>
	<li>Record the presence of and the number of oocysts per midgut for each control and treatment group (Supplemental File S2).</li>
	<li>Calculate the transmission-blocking activity using Equation (1): 
	%TBA &#160;<img alt="Equation 1" src="/files/ftp_upload/63546/63546eq01.jpg" style="margin: auto;" />&#160; &#160; (1) 
	where TBA = transmission-blocking activity (reduction in oocyst prevalence); p = oocyst prevalence; C = control; and T = treatment.</li>
	<li>Calculate the transmission-reducing activity using Equation (2): 
	%TRA = <img alt="Equation 2" src="/files/ftp_upload/63546/63546eq02.jpg" style="margin: auto;" />&#160; &#160; (2) 
	where TRA = transmission-reducing activity (reduction in oocyst intensity); I = oocyst intensity; C = control; and T = treatment. 
	NOTE: The TBA might not be significantly reduced, but a significant difference might be observed in the TRA and vice versa. This is dependent on the chemical material being evaluated.</li>
	<li>Perform statistical analysis using the non-parametric t-test (Mann-Whitney).</li>
</ol>

The authors have no conflicts of interest to disclose.

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 gametes from exflagellating before entering the mosquito. Another factor that plays a crucial role in establishing a successful artificial infection is to ensure that the female mosquitoes are keen to take a blood meal as the feeding behavior of mosquitoes can also greatly influence the outcome25. To ensure that only fully fed mosquitoes are dissected after the oocysts have developed, it is critical to focus on removing all unfed mosquitoes from each cup, as this could greatly influence the results. Midgut dissection is another important step to ensure that enough specimens are available for further analysis. Midguts have a tendency to tear, which can cause the content of the midgut to ooze out. Another important step in this protocol is the staining of the midguts, which is crucial for the detection and counting of the oocysts under the microscope.
Some limitations of the method include the small sample size of the mosquitoes, which can influence the accuracy and variance of TRA estimation20,26. This is mostly the result of a low feeding rate during the SMFA. As most mosquito colonies are maintained on animal blood, the provision of a gametocyte-infected human blood culture in the SMFA might contribute to the suboptimal feeding rate. This is also observed with the addition of certain compounds, which may act as repellants for the mosquitoes. Staining oocysts with mercurochrome has some drawbacks as it stains other structures within the midgut27. This causes poor visibility of the oocysts and makes counting oocysts challenging, affecting the results28. Conflicting results also arise when comparing those from the SFMA assay to alternative assays29,30. Furthermore, limitations such as time constraints and labor-intensive dissections restrict high-throughput processing when using this method31. The number of gametocytes ingested by the mosquitoes during this artificial infection process was also found to be low in some cases, possibly affecting oocyst intensity32,33,34.
Other alternative methods have also been developed, including bioluminescence assays, wherein parasite lines expressing firefly luciferase are used36. Although the latter method increases the throughput of samples, it also has its own restrictions, since such parasite lines could not be used to assess the transmission of infections that occur naturally or involve wild-type parasites37. Other methods that involve polymerase chain reaction (PCR) are also widely used. Although these methods enhance the sensitivity of oocyst detection, not all are fully quantitative38,39,40. The TaqMan qPCR assay is another promising method for the detection of P. falciparum oocysts, and this method was found to quantify much lower levels of parasitemia than microscopy41,42,43,44. This assay, however, requires expensive equipment and probes. Furthermore, possible changes in oocyst morphology due to the compounds cannot be visibly detected during qPCR. Compared to these methods, the SFMA method remains a valuable tool for infection detection and compound evaluation as most of the limitations of this method are relatively easy to overcome.

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><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).
Both the %TRA and %TBA for MMV1581558 were above 50%; thus, this compound could be regarded as a potential candidate for transmission-blocking and reduction. The statistical analysis for both intensity and prevalence showed a significant difference between the control group and MMV1581558 (p &#60; 0.0001) (Table 1 and Figure 5).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/63546/63546fig01.jpg" /> 
Figure 1: Illustration of the artificial infection system of Anopheles mosquitoes with Plasmodium falciparum gametocytes. Abbreviation: SMFA = standard membrane feeding assay. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63546/63546fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/63546/63546fig02.jpg" /> 
Figure 2: Membrane feeding system with water circulating through the glass feeders and silicone tubes to regulate the temperature of the gametocyte culture. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63546/63546fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/63546/63546fig03.jpg" /> 
Figure 3: Fully engorged Anopheles female after a blood meal. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63546/63546fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/63546/63546fig04.jpg" /> 
Figure 4: Midgut of Anopheles female. (A) With Malpighian tubules; (B) with ovaries; (C) with Plasmodium falciparum oocyst; and (D) uninfected midgut. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63546/63546fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/63546/63546fig5v2.jpg" /> 
Figure 5: Statistical summary of oocyst intensity between the control group (n = 47) and the compound MMV1581558 (n = 42). Error bars, &#177;SE; asterisks denote significant difference (ns P &#62; 0.05, ****P &#60; 0.0001). Abbreviation: DMSO = dimethyl sulfoxide. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63546/63546fig5v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Compound</td>
			<td>Number of reps.</td>
			<td>Sample size</td>
			<td>Av. Prevalence</td>
			<td>Av. Intensity/gut</td>
			<td>%TBA</td>
			<td>%TRA</td>
			<td>P-value Prevalence</td>
			<td>P-value Intensity</td>
		</tr>
		<tr>
			<td>Control</td>
			<td>3</td>
			<td>47</td>
			<td>89%</td>
			<td>9.5</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>MMV1581558</td>
			<td>3</td>
			<td>42</td>
			<td>36%</td>
			<td>1.5</td>
			<td>58%</td>
			<td>82%</td>
			<td>&#60;0.0001</td>
			<td>&#60;0.0001</td>
		</tr>
	</tbody>
</table>
Table 1: Dataset of an antimalarial compound, MMV1581558, evaluated during three biological replicates of the standard membrane feeding assay. For each control and test group, the sample size (n) is indicated, along with the average oocyst prevalence and intensity per midgut. The transmission-blocking activity and transmission-reducing activity are indicated. Abbreviations: TBA = transmission-blocking activity; TRA = transmission-reducing activity.
Supplemental Figure S1: Infected mosquitoes contained within a chamber in the infection room of the insectary. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63546/Supplemental Figure S1.pdf" target="_blank">Please click here to download this File.</a>
Supplemental File S1: Protocol for accidental exposure to infected mosquitoes. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63546/Supplemental File S1.pdf" target="_blank">Please click here to download this File.</a>
Supplemental File S2: Recording sheet for oocyst prevalence and intensity. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63546/Supplemental File S2.pdf" target="_blank">Please click here to download this File.</a>

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 ...

standard-membrane-feeding-assay-for-detection-plasmodium-falciparum

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

2.9K Views.  University of the Witwatersrand. Het protocol stelt ons in staat om het effect van de behandeling op de malariagevoeligheid van Afrikaanse malariavectoren te detecteren. Bijvoorbeeld verbindingen gericht op het blokkeren van de overdracht van de mens naar de mug. Deze techniek stelt onderzoekers in staat om een groot aantal verbindingen onder laboratoriumomstandigheden te evalueren, zelfs voordat toxiciteit GATE's beschikbaar zijn.Midgut-dissecties kunnen in het begin een uitdaging zijn, maar het vergt gewoon wat oefe...

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

Alphavirus Transducing System:  Tools for Visualizing Infection in Mosquito Vectors

Alphavirus transducing systems (ATSs) are important tools for expressing genes of interest (GOI) during infection. ATSs are derived from cDNA clones of mosquito-borne RNA viruses (genus Alphavirus; family Togaviridae). The Alphavirus genus contains about 30 different mosquito-borne virus species. Alphaviruses are enveloped viruses and contain single-stranded RNA genomes (~11.7 Kb). Alphaviruses transcribe a subgenomic mRNA that encodes the structural proteins of the virus required for encapsidation of the genome and maturation of the virus. Alphaviruses are usually highly lytic in vertebrate cells, but persistently infect susceptible mosquito cells with minimal cytopathology. These attributes make them excellent tools for gene expression in mosquito vectors. The most common ATSs in use are derived from Sindbis virus (SINV). The broad species tropism of SINV allows for infection of insect, avian, and mammalian cells8. However, ATSs have been derived from other alphaviruses as well9,10,20. Foreign gene expression is made possible by the insertion of an additional viral subgenomic RNA initiation site or promoter. ATSs in which an exogenous gene sequence is positioned 5' to the viral structural genes is used for stable protein expression in insects. ATSs, in which a gene sequence is positioned 3' to the structural genes, is used to trigger RNAi and silence expression of that gene in the insect.
ATSs have proven to be valuable tools for understanding vector-pathogen interactions, molecular details of viral replication and maintenance infectious cycles3,4,11,19,21. In particular, the expression of fluorescent and bioluminescent reporters has been instrumental tracking the viral infection in the vector and virus transmission5,14-16,18. Additionally, the vector immune response has been described using two strains of SINV engineered to express GFP2,9.
Here, we present a method for the production of SINV containing a fluorescent reporter (GFP) from the cDNA infectious clone. Infectious, full-length RNA is transcribed from the linearized cDNA clone. Infectious RNA is introduced into permissive target cells by electroporation. Transfected cells generate infectious virus particles expressing the GOI. Harvested virus is used to infect mosquitoes, as described here, or other host species (not shown herein). Vector competence is assessed by detecting fluorescence outside the midgut or by monitoring virus transmission7. Use of a fluorescent reporter as the GOI allows for convenient estimation of virus spread throughout a cell culture, for determination of rate of infection, dissemination in exposed mosquitoes, virus transmission from the mosquito and provides a rapid gauge of vector competence.

Methods for using alphavirus transducing systems to express fluorescent reporters in vitro and in adult mosquitoes are described. This technique may be adapted to express any protein of interest in lieu of or in addition to a reporter.

Alphavirus transducing systems (ATSs) are important tools for expressing genes of interest (GOI) during infection.  ATSs are derived from cDNA clones of ...

Protocol for Dengue Infections in Mosquitoes (A. aegypti) and Infection Phenotype Determination

The purpose of this procedure is to infect the Aedes mosquito with dengue virus in a laboratory condition and examine the infection level and dynamic of the virus in the mosquito tissues. This protocol is routinely used for studying mosquito-virus interactions, especially for identification of novel host factors that are able to determine vector competence. The entire experiment must be conducted in a BSL2 laboratory. Similar to Plasmodium falciparum infections, proper attire including gloves and lab coat must be worn at all times. After the experiment, all the materials that came in contact with the virus need to be treated with 75% ethanol and bleached before proceeding with normal washing. All other materials need to be autoclaved before discarding them.

Protocol for Dengue Infections in Mosquitoes A. aegypti and Infection Phenotype Determination

Once a gene is identified as potentially refractory for the dengue virus, it must be evaluated for it's role in preventing viral infections within the mosquito. This protocol illustrates how the extent of dengue infections of mosquitoes can be assayed. The techniques for growing up the virus in culture, membrane feeding mosquitoes human blood, and assaying viral titers in the mosquito midgut are demonstrated.

The purpose of this procedure is to infect the Aedes mosquito with dengue virus in a laboratory condition and examine the infection level and dynamic of ...

Biology

Protocol for Plasmodium falciparum Infections in Mosquitoes and Infection Phenotype Determination

Once a gene is identified as potentially refractory for malaria, it must be evaluated for its role in preventing Plasmodium infections within the mosquito.    This protocol illustrates how the extent of plasmodium infections of mosquitoes can be assayed.  The techniques for preparing the gametocyte culture, membrane feeding mosquitoes human blood, and assaying viral titers in the mosquito midgut are demonstrated.

Once a gene is identified as potentially refractory for malaria, it must be evaluated for its role in preventing Plasmodium infections within the mosquito. This protocol illustrates how the extent of plasmodium infections of mosquitoes can be assayed. The techniques for preparing the gametocyte culture, membrane feeding mosquitoes human blood, and assaying viral titers in the mosquito midgut are demonstrated.

Once a gene is identified as potentially refractory for malaria, it must be evaluated for its role in preventing Plasmodium infections within the ...

Phenotypic Analysis of Rodent Malaria Parasite Asexual and Sexual Blood Stages and Mosquito Stages

Recent advances in genetics and systems biology technologies have promoted our understanding of the biology of malaria parasites on the molecular level. However, effective malaria parasite targets for vaccine and chemotherapy development are still limited. This is largely due to the unavailability of relevant and practical in vivo infection models for human Plasmodium species, most notably for P.&#160;falciparum and P. vivax. Therefore, rodent malaria species have been extensively used as practical alternative in vivo models for malaria vaccine, drug targeting, immune response, and functional characterization studies of conserved Plasmodiumspp. genes. Indeed, rodent malaria models have proven to be invaluable, especially for exploring mosquito transmission and liver stage biology, and were indispensable for immunological studies. However, there are discrepancies in the methods used to evaluate the phenotypes of transgenic and wild-type asexual and sexual blood-stage parasites.&#160;Examples of these discrepancies are the choice of an intravenous vs. intraperitoneal infection of rodents with blood-stage parasites and the evaluation of male gamete exflagellation. Herein, we detail standardized experimental methods to evaluate the phenotypes of asexual and sexual blood stages in transgenic parasites expressing reporter-gene or wild-type rodent malaria parasite species. We also detail the methods to evaluate the phenotypes of malaria parasite mosquito stages (gametes, ookinetes, oocysts, and sporozoites) inside Anopheles mosquito vectors. These methods are detailed and simplified here for the lethal and non-lethal strains of P. berghei and P. yoelii but can also be applied with some adjustments to P. chabaudi and P. vinckei rodent malaria species.

Due to the striking similarities of the life cycle and biology of rodent malaria parasites to human malaria parasites, rodent malaria models have become indispensable for malaria research. Herein, we standardized some of the most important techniques used in the phenotypic analysis of wild-type and transgenic rodent malaria species.

Recent advances in genetics and systems biology technologies have promoted our understanding of the biology of malaria parasites on the molecular level. ...

Measuring Naturally Acquired Phagocytosis-Inducing Antibodies to Plasmodium falciparum Parasites by a Flow Cytometry-Based Assay

The protocol describes how to set up and run a flow cytometry-based phagocytosis assay of Plasmodium falciparum-infected erythrocytes (IEs) opsonized by naturally acquired IgG antibodies specific for VAR2CSA. VAR2CSA is the parasite antigen that mediates the selective sequestration of IEs in the placenta that can cause a severe form of malaria in pregnant women, called placental malaria (PM). Protection from PM is mediated by VAR2CSA-specific antibodies that are believed to function by inhibiting placental sequestration and/or by opsonizing IEs for phagocytosis. The assay employs late-stage-synchronized IEs that have been selected in vitro to express VAR2CSA, plasma/serum-antibodies from women with naturally acquired PM-specific immunity, and the phagocytic cell line THP-1. However, the protocol can easily be modified to assay the functionality of antibodies to any parasite antigen present on the IE surface, whether induced by natural exposure or by vaccination. The assay offers simple and high-throughput evaluation, with good reproducibility, of an important functional aspect of antibody-mediated immunity in malaria. It is, therefore, useful when evaluating clinical immunity to P. falciparum malaria, a major cause of morbidity and mortality in the tropics, particularly in sub-Saharan Africa.

Measuring Naturally Acquired Phagocytosis-Inducing Antibodies to Plasmodium falciparum Parasites by a Flow Cytometry-Based Assay

The overall goal of this protocol is to provide instruction on how to measure the capacity of antibodies present in sera or plasma of individuals, naturally exposed to Plasmodium falciparum infection, to opsonize and induce phagocytosis of the parasite-infected erythrocytes (IEs).

The protocol describes how to set up and run a flow cytometry-based phagocytosis assay of Plasmodium falciparum-infected erythrocytes (IEs) opsonized by ...

Generating Genetically Modified Plasmodium berghei Sporozoites

Malaria is a deadly disease caused by the parasite Plasmodium and is transmitted through the bite of female Anopheles mosquitoes. The sporozoite stage of Plasmodium deposited by mosquitoes in the skin of vertebrate hosts undergoes a phase of mandatory development in the liver before initiating clinical malaria. We know little about the biology of Plasmodium development in the liver; access to the sporozoite stage and the ability to genetically modify such sporozoites are critical tools for studying the nature of Plasmodium infection and the resulting immune response in the liver. Here, we present a comprehensive protocol for the generation of transgenic Plasmodium berghei sporozoites. We genetically modify blood-stage P. berghei and use this form to infect Anopheles mosquitoes when they take a blood meal. After the transgenic parasites undergo development in the mosquitoes, we isolate the sporozoite stage of the parasite from the mosquito salivary glands for in vivo and in vitro experimentation. We demonstrate the validity of the protocol by generating sporozoites of a novel strain of P. berghei expressing the green fluorescent protein (GFP) subunit 11 (GFP11), and show how it could be used to investigate the biology of liver-stage malaria.

Generating Genetically Modified Plasmodium berghei Sporozoites

Malaria is transmitted through inoculation of the sporozoite stage of Plasmodium by infected mosquitoes. Transgenic Plasmodium has allowed us to understand the biology of malaria better and has contributed directly to malaria vaccine development efforts. Here, we describe a streamlined methodology to generate transgenic Plasmodium berghei sporozoites.

Malaria is a deadly disease caused by the parasite Plasmodium and is transmitted through the bite of female Anopheles mosquitoes. The sporozoite stage of ...

A Multi-detection Assay for Malaria Transmitting Mosquitoes

The Anopheles gambiae species complex includes the major malaria transmitting mosquitoes in Africa. Because these species are of such medical importance, several traits are typically characterized using molecular assays to aid in epidemiological studies. These traits include species identification, insecticide resistance, parasite infection status, and host preference. Since populations of the Anopheles gambiae complex are morphologically indistinguishable, a polymerase chain reaction (PCR) is traditionally used to identify species. Once the species is known, several downstream assays are routinely performed to elucidate further characteristics. For instance, mutations known as KDR in a para gene confer resistance against DDT and pyrethroid insecticides. Additionally, enzyme-linked immunosorbent assays (ELISAs) or Plasmodium parasite DNA detection PCR assays are used to detect parasites present in mosquito tissues. Lastly, a combination of PCR and restriction enzyme digests can be used to elucidate host preference (e.g., human vs. animal blood) by screening the mosquito bloodmeal for host-specific DNA. We have developed a multi-detection assay (MDA) that combines all of the aforementioned assays into a single multiplex reaction genotyping 33SNPs for 96 or 384 samples at a time. Because the MDA includes multiple markers for species, Plasmodium detection, and host blood identification, the likelihood of generating false positives or negatives is greatly reduced from previous assays that include only one marker per trait. This robust and simple assay can detect these key mosquito traits cost-effectively and in a fraction of the time of existing assays.

Malaria transmitting mosquitoes have a number of epidemiologically important characteristics that can only be detected using molecular techniques. Utilizing a MALDI-TOF based SNP genotyping platform, we developed an assay for simultaneously detecting multiple key traits (species, insecticide resistance, parasite infection and host choice) of malaria vectors.

The Anopheles gambiae species complex includes the major malaria transmitting mosquitoes in Africa. Because these species are of such medical importance, ...

Plasmodium falciparum Gametocyte Culture and Mosquito Infection Through Artificial Membrane Feeding

Malaria remains one of the most important public health problems, causing significant morbidity and mortality. Malaria is a mosquito borne disease transmitted through an infectious bite from the female Anopheles mosquito. Malaria control will eventually rely on a multitude of approaches, which includes ways to block transmission to, through and from mosquitoes. To study mosquito stages of malaria parasites in the laboratory, we have optimized a protocol to culture highly infectious Plasmodium falciparum gametocytes, a parasite stage required for transmission from the human host to the mosquito vector. P. falciparum gametocytes mature through five morphologically distinct steps, which takes approximately 1-2 weeks. Gametocyte culture described in this protocol is completed in 15 days and are infectious to mosquitoes from days 15-18. These protocols were developed to maintain a continuous cycle of infection competent gametocytes and to maintain uninterrupted supply of mosquito stages of the parasite. Here, we describe the methodology of gametocyte culture and how to infect mosquitoes with these parasites using glass membrane feeders.

Plasmodium falciparum Gametocyte Culture and Mosquito Infection Through Artificial Membrane Feeding

Detailed investigations on mosquito stages of malaria parasites are critical to design effective transmission blocking strategies. This protocol demonstrates how to effectively culture infectious gametocytes and then feed these gametocytes to mosquitoes to generate mosquito stages of P. falciparum.

Malaria remains one of the most important public health problems, causing significant morbidity and mortality. Malaria is a mosquito borne disease ...

Detection of Plasmodium Sporozoites in Anopheles Mosquitoes using an Enzyme-linked Immunosorbent Assay

Plasmodium sporozoites are the infective stage of malaria parasites that infect humans. The sporozoites residing in the salivary glands of female Anopheles mosquitoes are transmitted to humans via mosquito bites during blood feeding. The presence of sporozoites in the mosquito salivary glands thus defines mosquito infectiousness. To determine whether an Anopheles mosquito carries Plasmodium sporozoites, the enzyme-linked immunosorbent assay (ELISA) method has been the standard tool to detect the Plasmodium circumsporozoite protein (CSP), the major surface protein of the sporozoites. In this method, the head along with the thorax of each mosquito is separated from the abdomen, homogenized, and subjected to a sandwich ELISA to detect the presence of CSP specific to Plasmodium falciparum and each of the two subtypes, VK210 and VK247, of Plasmodium vivax.This method has been used to study malaria transmission, including the seasonal dynamics of mosquito infection and the species of the major malaria vectors in the study sites.

Detection of Plasmodium Sporozoites in Anopheles Mosquitoes using an Enzyme-linked Immunosorbent Assay

This protocol describes a sandwich enzyme-linked immunosorbent assay to detect salivary gland sporozoites in mosquitoes. Using easily available monoclonal antibodies, the method enables cost-effective, high-throughput detection of mosquitoes carrying Plasmodium falciparum or Plasmodium vivax. The method is suitable for malaria transmission research, including vector surveys.

Plasmodium sporozoites are the infective stage of malaria parasites that infect humans. The sporozoites residing in the salivary glands of female ...

Mosquito Feeding Assay: A Method to Evaluate Test Compound Effects on Mortality and Fecundity of Adult Female Mosquitoes

Collect approximately 150 four- to five-day-old adult female mosquitoes with an aspirator, and transfer them to a separate cage. Remove the source of sugar 1 to 24 hours prior to the feeding assay.
Prepare an 80-millimolar stock solution of the testing compound in water. Then, serially dilute it with water to obtain working stock solutions of the desired concentrations.
To obtain desired testing concentrations, add 40 microliters of each dilution to 960 microliters of defibrinated rabbit blood in a 1.5-milliliter tube, and mix by pipetting. Place a membrane filter on a feeding unit, and seal it with a rubber ring.
Next, use a pipette to transfer 1 milliliter of the blood with testing solution through the delivery port located on the reverse side of the feeding unit. Attach the feeding unit to a heating unit. Then, swab the membrane surface gently with freshly prepared 10% lactic acid solution.
Place the feeding unit in the cage. Cover the cage with a dark cloth, and allow the mosquitoes to feed for one hour. After the mosquitoes feed, place the cage in a refrigerator at 4 degrees Celsius for five minutes to anesthetize the mosquitoes. Then, count and record the total number of mosquitoes in the cage.
By examining the abdomen, count and record the total number of fully- and partially-fed female mosquitoes. A minimum of 50 blood-fed mosquitoes should be obtained per dose. Remove any mosquitoes that have not fed. Next, transfer the cage to a growth chamber and use a score sheet to record the number of dead mosquitoes at the appropriate time points.
On day 3 post-blood feeding, place an egg cup in the cage for 72 hours. Use a dissecting microscope to count the total number of eggs produced per treatment.

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

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