Authors thank Bloomberg Philanthropies for financial support to Johns Hopkins Malaria Research Institute (JHMRI). This work would not have been possible without the expertise provided by JHMRI insect and parasitology core facilities.

Blood collections described below have been approved by the Institutional Review Board of Johns Hopkins University. P. falciparum is cultured in fresh RBCs under sterile conditions in a biosafety level 2 (BSL2) facility and caution is used to handle biological materials. After each step involving blood or blood products, every plasticware or glassware is rinsed with 10% bleach within the hood prior to proper disposal.
1. Reagents and preparation
<ol>
	<li>Parasite isolate P. falciparum&#160;NF54 (see Table of Materials) was used, which can produce infectious gametocytes for up to two months while in continuous culture. 
	NOTE: Not all culture adapted parasite lines produce gametocytes, and low passage NF54 isolates are most consistent.</li>
	<li>O+ Erythrocytes: Dilute whole blood by adding equal volume of RPMI 1640 media and centrifuge at 500 x g for 5 min at room temperature in a swing out rotor with deacceleration set at 0. Carefully remove the supernatant and buffy coat (a white band between plasma and packed erythrocytes, which contains most of white blood cells and platelets) and add equal volume of RPMI. Repeat the washing step twice and after the final wash add one pellet volume of RPMI to get 50% hematocrit for storage at 4 &#176;C. 
	NOTE: Gametocytes mature over a period of two weeks which makes it critical to begin cultures with fresh erythrocytes, typically erythrocytes drawn within a week works well.</li>
	<li>O+ Human Serum: Pool at-least 6 units of serum together to minimize the effect of normal variation in serum from different individuals. Sterilize pooled human serum using 0.2 &#181;m filtration flasks. Aliquot in portions of 50 mL or less based on the need and store at -20 &#176;C. 
	NOTE: Erythrocytes and serum needs to be from a compatible blood type for P. falciparum cultures.</li>
	<li>500x Hypoxanthine (100 mL) solution: Dissolve 0.5 g of hypoxanthine in 100 mL of 1 M NaOH. Filter sterilize and make 5-10 mL aliquots for storage. Stocks can be stored up to a year at -20 &#176;C and up to 2 weeks at 4 &#176;C.</li>
	<li>Sodium bicarbonate (Optional): Dissolve 7.5 g sodium bicarbonate in 100 mL of deionized, tissue culture grade water and filter sterilize with 0.2 &#181;m filter. 
	NOTE: This protocol uses the candle jar method to provide microaerophilic conditions for P. falciparum in vitro culture and does not require sodium bicarbonate. However, if parasites are cultured using a malaria gas mix (5% O2, 5% CO2 and 90% N2), it is important to supplement culture media with 0.2% sodium bicarbonate.</li>
	<li>Complete media: To prepare 500 mL of complete media, add 1 mL of hypoxanthine solution and 50 mL of pooled human serum to 500 mL of RPMI 1640. Add 15 mL of 7.5% sodium bicarbonate if using a malaria gas mixture. Store complete media at 4 &#176;C and discard if the color of complete media changes from orange to pink. To avoid waste, make enough complete media to be used within three days.</li>
	<li>Exflagellation media (Optional): Make exflagellation or ookinete media by dissolving 200 mg NaHCO3, 5 mg Hypoxanthine and 100 &#181;L of xanthurenic acid (from 100 mM stock in water) to 100 mL of incomplete media (RPMI 1640 with glutamine and HEPES). 
	NOTE: Exflagellation is the process of male gamete formation inside the midgut of female Anopheles mosquito few minutes after it takes blood meal infected with gametocytes. Male gametocytes of Plasmodium give rise to 8 male gametes after an exflagellation event. In vitro, this process occurs spontaneously when culture temperature is lowered to room temperature (RT) and can be observed in cultured mature gametocytes.</li>
	<li>N-Acetylglucosamine (Optional): Make 500 mM stock solution of N-acetylglucosamine in water or incomplete media, aliquot and store at -20 &#176;C.</li>
	<li>NaCl solutions: Dissolve 0.9 g, 1.6 g and 12 g NaCl in 100 mL of tissue culture grade deionized water to make 0.9%, 1.6% and 12% NaCl solutions. Filter sterilize with 0.22 &#181;m filter and store at 4 &#176;C.</li>
</ol>
2. P. falciparum asexual stage culture
<ol>
	<li>To begin parasite culture, remove a low passage frozen vial of P. falciparum NF54, from the liquid nitrogen tank and quickly thaw in water bath set at 37 &#176;C.</li>
	<li>Transfer contents (~1 mL) to a 50 mL sterile centrifuge tube and dropwise add 0.2 volume of prewarmed 12% NaCl, while gently shaking the tube to ensure even mixing. Incubate for 5 min at RT with intermittent gentle shaking.</li>
	<li>Add 9 volumes of 1.6% NaCl dropwise, while continuing gentle mixing. Centrifuge the contents at 500 x g for 5 min at RT, carefully discard the supernatant. Add 9 volumes of 0.9% NaCl dropwise, while making sure to consistently mix the parasite pellet. Centrifuge again at 500 x g for 5 min at RT. Remove the supernatant and resuspend parasites into 5 mL of complete media.</li>
	<li>Transfer contents to one well of a 6 well tissue culture plate and add 100-200 &#181;L of packed RBCs.</li>
	<li>Incubate parasite culture at 37 &#176;C in a candle jar or in a modular incubator chamber purged with special gas mix of 5% O2, 5% CO2 and 90% N2.</li>
	<li>Replace media every day and monitor the growth by making a thin blood smear by drawing a few microliters from settled RBC layer after the culture supernatant has been aspirated during regular media change.</li>
	<li>Use sterile glass Pasteur pipette to draw and then tap in the center of the glass slide to transfer a drop of culture to the slide. Place another glass slide in front of the of the drop and draw it back to contact the RBCs, quickly pushing it forward in one motion to make thin smear of RBC monolayer.</li>
	<li>Place the slide horizontally on a drying rack and let it air dry. Fix the blood smear by dropping absolute methanol onto the smear. Allow the fixed smear to dry completely and then carefully pour 10% Geimsa-stain freshly diluted in water, until blood smear is completely covered. Allow cells to stain for approximately 15 min.</li>
	<li>Wash off the excess stain by rinsing the slide under clean tap water and allow the slides to dry in vertical position.</li>
	<li>Determine parasitemia by viewing thin blood smear on a microscope using oil immersion 100x objective. Count the number of infected erythrocytes among a total of at-least 500 RBCs to determine percent parasitemia.</li>
</ol>
3. P. falciparum gametocyte culture
NOTE: Gametocyte cultures take two weeks to produce mature gametocytes infectious to mosquitoes. The steps of the gametocyte culture are outlined in Figure 1. P. falciparum isolates usually lose the ability to produce gametocyte after long term in vitro culture13. To ensure quality of gametocytes, culture should be initiated from low passage feeder culture, not more than 2 months old since thawing. Pre-warm media to 37 &#176;C and perform all the procedures on a slide warmer set at 38 &#176;C. Make sure gametocyte cultures are not out of incubator for extended period to minimize temperature fluctuations.
<ol>
	<li>Seed the gametocyte culture (day 0) using mixed asexual stage feeder culture at 0.3-1% parasitemia at 4% hematocrit.</li>
	<li>To set up a six well gametocyte culture, centrifuge 5 mL of feeder culture at 500 x g for 5 min at RT. Discard the supernatant and resuspend the pellet in 30 mL of complete media.</li>
	<li>Add 1.2 mL of packed RBCs, mix, and dispense 5 mL to each well of the 6 well plate and incubate in a candle jar at 37 &#176;C. 
	NOTE: Gametocyte culture set up described above is based on a mixed asexual stage feeder culture at 5% parasitemia.</li>
	<li>Change media daily for 15-18 days, without the addition of fresh blood, by carefully aspirating about 70-80% culture supernatant to avoid removing blood cells.</li>
	<li>Add 5 mL of fresh complete media to each well. While changing media, slowly add media using a serological pipette against the wall of the well to avoid disturbing the settled RBC layer. 
	NOTE: Because 1-2 mL of the culture medium will be left during media change, total volume of culture will be between 6-7 mL, after day 1 of gametocyte culture. While adapting this protocol, it is advisable to make blood smears every alternate day to make sure parasites are healthy. Making a blood smear can often deplete the number of cells in culture. To avoid this, draw a very small volume.</li>
	<li>To quantify mature gametocytemia, make blood smear between days 15-18 and count the number of mature gametocytes among total number of cells. 
	NOTE: Mature gametocytes can easily be identified with their classic crescent shape with smooth rounded ends (Figure 5B).</li>
	<li>Count a minimum of 1,000 RBCs and calculate the percentage of RBCs infected with mature gametocytes.</li>
	<li>To quantify exflagellation events, take 200 &#181;L of gametocyte culture and centrifuge at 500 x g for 5 min at RT in a pre-warmed tube. Resuspend the pellet in 20 &#181;L of exflagellation media and transfer to a glass slide with cover slip.</li>
	<li>After 15 min incubation at room temperature, begin counting exflagellation centers in phase contrast mode using 10x objective. Count exflagellation centers in at-least four fields to calculate exflagellation events. 
	NOTE: A detailed methodology of exflagellation assay has been described earlier by Delves et al10. More than 20 exflagellation events per field is considered suitable for membrane feeding assay.</li>
	<li>Gametocyte cultures typically have low levels of residual asexual stages. If the experiment requires pure gametocytes, treat the culture with 50 mM N-acetylglucosamine.</li>
	<li>Add 0.5 mL of N-acetylglucosamine from 10x stock solution per well (5 mL) for a minimum of 3 days while changing media to clear residual asexual stages. 
	NOTE: N-acetylglucosamine can block invasion of sexually committed merozoites too, treatment should be initiated only after day 7 of gametocyte culture.</li>
</ol>
4. Mosquito infection using standard membrane feeding assay (SMFA)
NOTE: Gametocytes grown in vitro can be fed to mosquitos using glass membrane feeders. Setting up of the blood feeding apparatus is shown in Figure 2. As described above, always maintain gametocytes at 37 &#176;C to avoid activation before they are ingested by mosquitoes. Prewarm plasticware, reagents and equipment&#39;s used with gametocyte culture to 37 &#176;C.
<ol>
	<li>Starve 3-7-day old female Anopheles mosquitoes by removing their sugar water for 6.5 h or overnight before membrane feeding.</li>
	<li>Transfer them using motorized or mouth operated aspirators to paper cups covered with double layer of fine mesh fabric. Alternatively, knock down mosquitoes in a cold room or refrigerator to transfer them to the paper cups. 
	NOTE: One-pint size cup can be used to feed up to 100 mosquitoes.</li>
	<li>Centrifuge washed blood at 500 x g for 5 min at RT and discard supernatant. Add an equal volume of freshly thawed human serum to reconstitute the whole blood. Transfer the blood and serum mix to the water bath set at 37 &#176;C for 30 min.</li>
	<li>Transfer gametocyte culture to pre-warmed 15 mL plastic tube and centrifuge at 600 x g for 5 min at 37 &#176;C. Carefully aspirate the culture supernatant and make thin blood smear to determine mature gametocytemia as described above.</li>
	<li>To make the final blood feed, dilute gametocyte pellet to a desired concentration using reconstituted whole blood. Keep the blood feed at 37 &#176;C until mosquitoes and glass feeders are ready. 
	NOTE: Mature gametocyte concentrations between 0.02 to 0.3% will provide consistent mosquito infectivity. However, it is advisable to optimize gametocytemia by feeding various concentrations to different cups of mosquitoes.</li>
	<li>Ensure that the glass membrane feeders have two openings, a narrow top opening to pipette the infected blood and a bottom opening for membrane attachment. Cut a paraffin film into squares, stretch to an even thickness and stick on to the opening of feeder to create sealed compartment for the blood feed.</li>
	<li>Attach membrane feeders to a circulatory water-bath by connecting tubing on each side to allow for the passage of warm water through the jacket around membrane feeders. 
	NOTE: Several glass feeders can be connected in series to accommodate for multiple feeding conditions.</li>
	<li>After all the feeders are connected to the water bath, turn it on and inspect for any leaks.</li>
	<li>Place feeders in the center of netting on mosquito cups with membrane-side down and secure feeders using clamps or tape. Make sure membrane feeders are not tilted to any side to allow even distribution of the blood feed and close contact of the entire feeder with the netting on cups, to allow mosquitoes access from inside the cup.</li>
	<li>Pipette about 200&#8211;1000 &#956;L of blood feed into membrane feeders. 
	NOTE: Volume of the blood feed will vary based on the number of mosquitoes and size of the membrane feeder. For 14 mm glass feeder and 50 mosquitoes use 200 &#956;L of blood feed.</li>
	<li>Make sure loading was done properly and blood feed was overlaid on the paraffin film.</li>
	<li>Allow mosquitoes to feed for about 30 min with intermittent monitoring. 
	NOTE: Gently blow mosquitoes with mouth to provide CO2 which will induce improved blood feeding.</li>
	<li>Remove unfed mosquitoes by knocking them down in a cold room. Visibly inspect mosquitoes for bulge and redness in the abdomen as a sign of fresh blood meal. Alternatively, use a mouth operated aspirator to selectively remove unfed mosquitoes.</li>
	<li>Discard unfed mosquitoes, after soaking them in 70% ethanol and put fed mosquitoes back into the mosquito cup.</li>
	<li>Double cage mosquito cups and transfer them to high containment incubator specified for mosquitoes infected with human malaria parasite P. falciparum.</li>
	<li>Place cotton pads soaked in 10% sucrose on the mosquito cups to provide them with sugar meal. Replace cotton pads every other day, until mosquitoes are dissected.</li>
</ol>
5. Mosquito mid-gut dissection and oocyst load quantification
NOTE: A schematic of midgut dissection is shown in Figure 3.
<ol>
	<li>7-8 days post blood feeding, transfer mosquito cups to 4 &#176;C for 10 min to knock down mosquitoes.</li>
	<li>Transfer mosquitoes to be dissected to a Petri dish on ice using fine-tipped forceps.</li>
	<li>Soak mosquitoes in 70% ethanol for 1-2 min to euthanize them. Add PBS to the Petri dish to wash off the ethanol after all the mosquitoes are euthanized. 
	NOTE: Before adding PBS make sure all the mosquitoes are dead.</li>
	<li>Mount a glass slide on dissecting microscope and pipette 100 &#956;L of PBS in the center. Carefully transfer one mosquito using forceps into the PBS and leave the rest of the mosquitoes in the Petri dish on ice.</li>
	<li>Using fine-tipped forceps, hold the third segment of the abdomen from the posterior end of the mosquito and with second forceps hold the junction between the thorax and abdomen. Gently pull the abdomen until the midgut is fully exposed.</li>
	<li>Discard the rest of the mosquito tissue and transfer the midgut to a clean slide containing a few drops of PBS.</li>
	<li>When the desired number of mosquitoes have been dissected, carefully remove the PBS using a pipette and stain the midgut with 0.2% mercurochrome for 2-5 min.</li>
	<li>Remove excess mercurochrome and line-up midguts on the slide so that they can be easily visualized under the light microscope.</li>
	<li>Place a cover slip and count oocysts on each midgut under 10x objective. Oocysts stain pink and are circular in shape (Figure 6C).</li>
</ol>
6. Mosquito salivary gland dissection and sporozoite load quantification
NOTE: A schematic of salivary gland dissection is shown in Figure 4.
<ol>
	<li>14-18 days post blood feeding, place mosquito cups at 4 &#176;C for 10 min. 
	NOTE: It is important to wait till mosquitoes stop moving.</li>
	<li>While waiting for mosquitoes to stop moving, prepare tools for dissection.</li>
	<li>Place a glass plate on a dissection microscope stage. Prepare 2 sets of 25 G needles on 1 mL syringes and a 9&#8221; Pasteur pipette and rubber bulb. Place 70% EtOH and dissection medium (HBSS, L-15 or PBS) in a 6 well plate.</li>
	<li>In the cold room, transfer anesthetized mosquitoes in a 6 well plate containing 70% EtOH. Gently move mosquitoes with forceps to make sure all mosquitoes are soaked, then transfer mosquitoes to the dissection medium by using forceps to wash off 70% EtOH.</li>
	<li>Move to the dissection room and place mosquitoes on a dissection microscope stage.</li>
	<li>Remove excess medium from mosquitoes, add new medium as necessary during dissection. Avoid mosquitoes from drying out yet too much liquid makes dissection more difficult.</li>
	<li>Using 2 syringes with 25 G needles, hold mosquito thorax and head, and gently pull the head upward to pull salivary glands from thorax.</li>
	<li>Disconnect salivary glands from head and thorax with a needle.</li>
	<li>Temporary place salivary glands in a separate droplet of medium on the glass plate.</li>
	<li>After dissecting 15-20 salivary glands, collect them in a low retention tube using the Pasteur pipette. 
	NOTE: Once salivary glands enter the wide part of the Pasteur pipette, they will stick to the glass and it is difficult to get them out.</li>
	<li>Pellet salivary glands by short pulse spin in a table-top centrifuge. Remove the dissection medium without disrupting the pellet and Add 100 &#181;L of new dissection medium.</li>
	<li>Grind salivary glands with a small homogenizer for 1 min to obtain sporozoites.</li>
	<li>Place 10 &#181;L of dissection medium containing sporozoites on a hemocytometer. 
	NOTE: Depending on the number of salivary glands, one may need to dilute the sporozoite solution to 1:10 to 1:50 with medium before counting.</li>
	<li>Count the number of sporozoites in two of the four quadrants and calculate number of sporozoites/mosquito:</li>
</ol>
<img alt="Equation 1" src="/files/ftp_upload/61426/61426equ01v3.jpg" />
<img alt="Equation 2" src="/files/ftp_upload/61426/61426equ02v3.jpg" />
<img alt="Equation 3" src="/files/ftp_upload/61426/61426equ03.jpg" />

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A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proteolytic+Cleavage+of+the+Plasmodium+falciparum+Circumsporozoite+Protein+Is+a+Target+of+Protective+Antibodies.">Proteolytic Cleavage of the Plasmodium falciparum Circumsporozoite Protein Is a Target of Protective Antibodies.</a> Journal of Infectious Diseases. 212 (7), 1111-1119 (2015).</li><li>Swearingen, K. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interrogating+the+Plasmodium+Sporozoite+Surface:+Identification+of+Surface-Exposed+Proteins+and+Demonstration+of+Glycosylation+on+CSP+and+TRAP+by+Mass+Spectrometry-Based+Proteomics.">Interrogating the Plasmodium Sporozoite Surface: Identification of Surface-Exposed Proteins and Demonstration of Glycosylation on CSP and TRAP by Mass Spectrometry-Based Proteomics.</a> PLoS Pathogens. 12 (4), 1005606 (2016).</li><li>Ifediba, T., Vanderberg, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Complete+in+vitro+maturation+of+Plasmodium+falciparum+gametocytes.">Complete in vitro maturation of Plasmodium falciparum gametocytes.</a> Nature. 294 (5839), 364-366 (1981).</li><li>Miura, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+inter-laboratory+comparison+of+standard+membrane-feeding+assays+for+evaluation+of+malaria+transmission-blocking+vaccines.">An inter-laboratory comparison of standard membrane-feeding assays for evaluation of malaria transmission-blocking vaccines.</a> Malaria Journal. 15, 463 (2016).</li><li>Miura, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Qualification+of+Standard+Membrane-Feeding+Assay+with+Plasmodium+falciparum+Malaria+and+Potential+Improvements+for+Future+Assays.">Qualification of Standard Membrane-Feeding Assay with Plasmodium falciparum Malaria and Potential Improvements for Future Assays.</a> PLoS One. 8 (3), 57909 (2013).</li></ol>

The Authors have nothing to disclose.

Methods described here have been successfully used at the Johns Hopkins Malaria Research Institute for more than 10 years15,16,17,18,19,20,21,22. Gametocytes produced using this protocol have been used for high throughput gametocytocidal assays<sup class="x...

Malaria is caused by Plasmodium parasites and is transmitted to their vertebrate hosts via infectious bite of female Anopheles mosquitoes. According to the 2019 World Health Organization (WHO) report, there were an estimated 405,000 deaths, from a total of 228 million cases of malaria1. Most of the malaria related deaths were concentrated in the African region, especially among children below five years of age. While the overall incidence rate of malaria has declined globally from 2010, in recent years decline has plateaued and additional control strategies are urgently needed to eliminate the disease.
Cyclic asexual blood stages of malaria parasites cause disease pathogenesis and a small subset of these differentiate into female and male gametocytes. Plasmodium falciparum&#160;gametocytes are unique in nature as they take 7-10 days to develop through five morphologically distinct stages. Immature gametocytes from stage I to IV are sequestered in bone marrow parenchyma and largely remain absent from peripheral circulation2,3,4,5. Erythrocytes infected with mature stage V gametocytes are released in the bloodstream and freely circulate to be taken up by mosquitoes. Once inside the mosquito midgut, gametocytes are activated, through a change in temperature and exposure to the midgut environment, transform into female and male gametes and begin development of the mosquito stages, which culminates with the infective stages of sporozoites in the mosquito salivary glands6,7.
Since Trager and Jenson8 described a standardized method to culture P. falciparum, studies on the asexual blood stages have greatly advanced. However, the lack of a reliable culture system for sexual stages has made it difficult to study P. falciparum gametocytes, transmission biology and mosquito stages. In recent years, several methods have been published which have aided laboratories in establishing gametocyte cultures9,10,11,12. This manuscript describes standardized and reliable protocol to culture P. falciparum gametocytes that can represent a valuable resource for the malaria research community. This method enables the robust production of mature and infectious gametocytes which along with a standardized mosquito feeding protocol, results in highly reliable mosquito infectivity. These methods were established to maintain uninterrupted supply of gametocytes, and mosquito stage parasites. In this manuscript, we describe a thorough gametocyte culture protocol (Figure 1), preparation of glass membrane feeders and infection of mosquitoes using these membrane feeders (Figure 2), dissection of midgut (Figure 3) and salivary gland of mosquitoes (Figure 4), and quantification of infection in mosquito after midgut and salivary gland dissection.

<table><tbody><tr><td>10% Sugar solution</td><td></td><td></td><td></td></tr><tr><td>10ml serological pipet</td><td>Falcon</td><td>357551</td><td></td></tr><tr><td>15 ml conical tube</td><td>Falcon</td><td>352096</td><td></td></tr><tr><td>1ml serological pipet</td><td>Falcon</td><td>357521</td><td></td></tr><tr><td>25 ml serological pipet</td><td>Falcon</td><td>357535</td><td></td></tr><tr><td>37&amp;deg;C Incubator</td><td></td><td></td><td></td></tr><tr><td>50 ml conical tube</td><td>Falcon</td><td>352070</td><td></td></tr><tr><td>5ml serological pipet</td><td>Falcon</td><td>357543</td><td></td></tr><tr><td>6 well tissue culture plates</td><td>Falcon</td><td>353046</td><td></td></tr><tr><td>70% Ethanol</td><td></td><td></td><td></td></tr><tr><td>9&quot; glass pipet</td><td>Fisherbrand</td><td>13-678-6B</td><td></td></tr><tr><td>Anopheles Mosquitoes</td><td>JHMRI, Insectary core</td><td></td><td>We use A. stephensi or A. gambiae (keele)</td></tr><tr><td>cell counter</td><td></td><td></td><td></td></tr><tr><td>Circulating water bath</td><td></td><td></td><td></td></tr><tr><td>fine tip forceps</td><td>Fisherbrand</td><td>12-000-122</td><td></td></tr><tr><td>Geimsa stain</td><td>Sigma</td><td>GS1L</td><td></td></tr><tr><td>Glass desiccator</td><td></td><td></td><td></td></tr><tr><td>Glass membrane feeder</td><td>Chemglass Life Sciences</td><td>CG183570</td><td></td></tr><tr><td>Glass slides</td><td>Fisherbrand</td><td>12-552-3</td><td></td></tr><tr><td>HBSS</td><td>Sigma</td><td>H6648</td><td></td></tr><tr><td>Human Blood O+</td><td>JHU</td><td></td><td>Wash RBCs three times with RPMI and refrigerate at 50% heamatocrit</td></tr><tr><td>Human Serum O+</td><td>Interstate blood bank</td><td></td><td>Pool at-least 6 units of serum from different donors and freeze down aliquots at -20&amp;deg;C.</td></tr><tr><td>Hypoxanthine</td><td>Sigma</td><td>H9337</td><td>Make 500x stock in 1M NaOH</td></tr><tr><td>Mercurochrome</td><td>Sigma</td><td>M7011</td><td>Prepare 1% stock solution in PBS that can be diluted to 0.1% when needed</td></tr><tr><td>Micro Pipette</td><td></td><td></td><td></td></tr><tr><td>Microscope</td><td>Olympus</td><td></td><td>Any microscope with 10x, 40x and 100x objective will work.</td></tr><tr><td>Mosquito cups</td><td>Neptune cups</td><td></td><td></td></tr><tr><td>N-acetylglucosamine</td><td>Sigma</td><td>A3286</td><td>Optional and needed only when pure gametocytes are required.</td></tr><tr><td>Netting</td><td></td><td></td><td>Make sure it can contain mosquitoes and allow blood feeding</td></tr><tr><td>Parafilm</td><td></td><td></td><td></td></tr><tr><td>PBS</td><td></td><td></td><td></td></tr><tr><td>Petri dish</td><td>Thermo Scientific</td><td>249964</td><td></td></tr><tr><td>Pipet tips</td><td></td><td></td><td></td></tr><tr><td>Pipetman</td><td></td><td></td><td></td></tr><tr><td>&lt;em&gt;Plasmodium falciparum&lt;/em&gt; NF54</td><td>BEI Resources</td><td>MRA-1000</td><td>Freeze down large numbers of early passage culture to make sure you have a constant supply</td></tr><tr><td>RPMI 1640</td><td>Corning</td><td>CV-041-CV</td><td>Media contains glutamine and HEPES</td></tr><tr><td>Slide warmer</td><td></td><td></td><td></td></tr><tr><td>Sodium bicarbonate</td><td>Sigma</td><td>S6297</td><td>Optional for media, add only when using malaria gas mix during culture incubation</td></tr><tr><td>water bath</td><td></td><td></td><td></td></tr><tr><td>Xanthurenic Acid</td><td>Sigma</td><td>D120804</td><td>For flagellation media</td></tr></tbody></table>

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.

Here we present results from a series of membrane feeds using P. falciparum NF54 gametocyte cultures generated using the protocol above (see (Figure 5). Gametocyte culture was initiated with approximately 0.5% mixed stage asexual culture on Day 0, which grew to a peak parasitemia of approximately 15% by Day 4 and Day 5. As shown in Figure 5A at this high parasitemia, the parasites are stressed and the asexual stage culture crashes. However, this stress ...

Watch this Scientific Journal Video about Plasmodium falciparum Gametocyte Culture and Mosquito Infection Through Artificial Membrane Feeding at JoVE.com

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.

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 ...

plasmodium-falciparum-gametocyte-culture-mosquito-infection-through

Research

JoVE Journal

Biology

7.4K Views.  Johns Hopkins University. 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.

Video: Plasmodium falciparum Gametocyte Culture and Mosquito Infection Through Artificial Membrane Feeding

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 ...

Immunology and Infection

Protocol for Production of a Genetic Cross of the Rodent Malaria Parasites

Variation in response to antimalarial drugs and in pathogenicity of malaria parasites is of biologic and medical importance. Linkage mapping has led to successful identification of genes or loci underlying various traits in malaria parasites of rodents1-3 and humans4-6. The malaria parasite Plasmodium yoelii is one of many malaria species isolated from wild African rodents and has been adapted to grow in laboratories. This species reproduces many of the biologic characteristics of the human malaria parasites; genetic markers such as microsatellite and amplified fragment length polymorphism (AFLP) markers have also been developed for the parasite7-9. Thus, genetic studies in rodent malaria parasites can be performed to complement research on Plasmodium falciparum. Here, we demonstrate the techniques for producing a genetic cross in P. yoelii that were first pioneered by Drs. David Walliker, Richard Carter, and colleagues at the University of Edinburgh10.

Genetic crosses in P. yoelii and other rodent malaria parasites are conducted by infecting mice Mus musculus with an inoculum containing gametocytes of two genetically distinct clones that differ in phenotypes of interest and by allowing mosquitoes to feed on the infected mice 4 days after infection. The presence of male and female gametocytes in the mouse blood is microscopically confirmed before feeding. Within 48 hrs after feeding, in the midgut of the mosquito, the haploid gametocytes differentiate into male and female gametes, fertilize, and form a diploid zygote (Fig. 1). During development of a zygote into an ookinete, meiosis appears to occur11. If the zygote is derived through cross-fertilization between gametes of the two genetically distinct parasites, genetic exchanges (chromosomal reassortment and cross-overs between the non-sister chromatids of a pair of homologous chromosomes; Fig. 2) may occur, resulting in recombination of genetic material at homologous loci. Each zygote undergoes two successive nuclear divisions, leading to four haploid nuclei. An ookinete further develops into an oocyst. Once the oocyst matures, thousands of sporozoites (the progeny of the cross) are formed and released into mosquito hemoceal. Sporozoites are harvested from the salivary glands and injected into a new murine host, where pre-erythrocytic and erythrocytic stage development takes place. Erythrocytic forms are cloned and classified with regard to the characters distinguishing the parental lines prior to genetic linkage mapping. Control infections of individual parental clones are performed in the same way as the production of a genetic cross.

Genetic crosses of rodent malaria parasites are performed by feeding two genetically distinct parasites to mosquitoes. Recombinant progeny are cloned from mouse blood after allowing mosquitoes to bite infected mice. This video shows how to produce genetic crosses of Plasmodium yoelii and is applicable to other rodent malaria parasites.

Variation in response to antimalarial drugs and in pathogenicity of malaria parasites is of biologic and medical importance. Linkage mapping has led to ...

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 ...

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 ...

An Experimental Model to Study Tuberculosis-Malaria Coinfection upon Natural Transmission of Mycobacterium tuberculosis and Plasmodium berghei

Coinfections naturally occur due to the geographic overlap of distinct types of pathogenic organisms. Concurrent infections most likely modulate the respective immune response to each single pathogen and may thereby affect pathogenesis and disease outcome. Coinfected patients may also respond differentially to anti-infective interventions. Coinfection between tuberculosis as caused by mycobacteria and the malaria parasite Plasmodium, both of which are coendemic in many parts of sub-Saharan Africa, has not been studied in detail. In order to approach the challenging but scientifically and clinically highly relevant question how malaria-tuberculosis coinfection modulate host immunity and the course of each disease, we established an experimental mouse model that allows us to dissect the elicited immune responses to both pathogens in the coinfected host. Of note, in order to most precisely mimic naturally acquired human infections, we perform experimental infections of mice with both pathogens by their natural routes of infection, i.e. aerosol and mosquito bite, respectively.

An Experimental Model to Study Tuberculosis-Malaria Coinfection upon Natural Transmission of Mycobacterium tuberculosis and Plasmodium berghei

Tuberculosis and malaria are two of the most prevalent infections in humans and major causes of morbidity and mortality in impoverished populations in the tropics. We established an experimental model system to study outcome of malaria-tuberculosis coinfection in mice after challenge with both pathogens via their natural route of infection.

Coinfections naturally occur due to the geographic overlap of distinct types of pathogenic organisms. Concurrent infections most likely modulate 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. ...

In Vivo Tracking of Edema Development and Microvascular Pathology in a Model of Experimental Cerebral Malaria Using Magnetic Resonance Imaging

Cerebral malaria is a sign of severe malarial disease and is often a harbinger of death. While aggressive management can be life-saving, the detection of cerebral malaria can be difficult. We present an experimental mouse model of cerebral malaria that shares multiple features of the human disease, including edema and microvascular pathology. Using magnetic resonance imaging (MRI), we can detect and track the blood-brain barrier disruption, edema development, and subsequent brain swelling. We describe multiple MRI techniques that can visualize these pertinent pathological changes. Thus, we show that MRI represents a valuable tool to visualize and track pathological changes, such as edema, brain swelling, and microvascular pathology, in vivo.

In Vivo Tracking of Edema Development and Microvascular Pathology in a Model of Experimental Cerebral Malaria Using Magnetic Resonance Imaging

We describe a mouse model of experimental cerebral malaria and show how inflammatory and microvascular pathology can be tracked in vivo using magnetic resonance imaging.

Cerebral malaria is a sign of severe malarial disease and is often a harbinger of death. While aggressive management can be life-saving, the detection of ...

Medicine

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.

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.

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 ...

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 ...

Identifying Kinases Rewiring in Malaria Parasite: A Chemical Genetics Approach

Understanding the Development of Compensatory Pathways in a Mutant Malaria Parasite Harbouring Hypomorphic Allele of Plant-Like Kinases

One of the mechanisms for subverting the effect of drugs by&#160;the malaria parasite is through rewiring of its transcriptome. The effect is more pronounced for target genes belonging to the multigene family. Plasmodium falciparum protein kinases belonging to the CDPK family are essential for blood stage development. As such, CDPKs are considered good targets for the development of anti-malarial compounds. The chemical genetics approach has been historically used to elucidate the function of protein kinases in higher eukaryotes. It requires the substitution of gatekeeper residue for another amino acid with a different side chain through genetic manipulation. Amino acid substitution at the gatekeeper position modulates the activity of a protein kinase and changes its susceptibility to a specific class of compounds known as bumped kinase inhibitors (BKIs) that help in the functional identification of the target gene. Here, we have exploited the chemical genetics approach to understand compensatory mechanisms evolved by a mutant parasite harboring a hypomorphic allele of cdpk1. Overall, our approach helps in identifying compensatory pathways that may be simultaneously targeted to prevent the development of drug resistance against individual kinases.

Author Spotlight: Identifying Compensatory Pathways in Malaria Parasites Containing Hypomorphic Allele of Essential Protein Kinases

Chemical genetics involves the substitution of a gatekeeper residue with an amino acid containing a different side chain at the target locus. Here, we have generated a mutant parasite containing a hypomorphic allele of cdpk1 and identified compensatory pathways adopted by the parasite in the mutant background.

Plasmodium falciparum Gametocyte Culture and Mosquito Infection Through Artificial Membrane Feeding

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