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

All animal experiments described here were conducted according to the approved protocols of the Institutional Animal Care and Use Committee (IACUC) of Tulane University and the animals ethics committee of Bezmialem Vakif University. All other experimental protocols and the use of recombinant DNA were conducted according to the approved protocols of the Institutional Biosafety Committee (IBC) of Tulane University.

1. Infection of Mice with Blood-stage Parasites for Parasitemia Analysis and Mosquito Infection Assays

1. Day -3: optional injection of phenylhydrazine into recipient mice
   1. Inject outbred SW or CD1 recipient mice (mice that will be used for mosquito infection and mosquito infection assays) intraperitoneally (IP) with 100 µL of phenylhydrazine (50 mg of phenylhydrazine in 5 mL of 1x phosphate-buffered saline (PBS)) using a 26 G needle syringe.
      NOTE: For robust mosquito infections of P. berghei and P. yoelii, phenylhydrazine is used to induce reticulocytosis in the recipient mice, which increases the parasitemia and, subsequently, the percentage of gametocytes, and significantly increases the exflagellation of male gametes (Figure 5), which will lead to a better mosquito stages infection for both P. berghei and P. yoelii strains^15,16,17,18.
2. Day -2: injection of frozen parasite stocks into donor mice
   1. Quickly thaw cryopreserved frozen stocks and immediately inject each donor mouse (typically two mice per genotype) IP with ~200 µL of stock using a 26 G needle syringe.
      NOTE: No more than five mice are housed per cage within controlled vivarium settings.
   2. Start checking parasitemia under a 100x microscope objective on Giemsa-stained thin blood smear after 24 h postinfection.Follow section 2.1 of the protocol for Giemsa-stained thin blood smears.
      NOTE: Due to the inability to precisely count the surviving viable blood stages after quickly thawing and injecting cryopreserved infected blood, donor mice receive the cryopreserved blood first. The infected erythrocytes of the donor mouse can be precisely quantified and used subsequently.
3. Day 0: bleeding of the donor mice
   1. Bleed the donor mice (see section 3.1) when their parasitemia is between 0.1% and 1%, which is usually obtained 2-3 days post-frozen-stock-infection in the case of P. yoelii 17X-NL and P. berghei ANKA strains.
   2. Use the facial vein puncture (to get between 200 and 500 µL), or terminally bleed the animals by heart puncture (to get between 600 µL and 1.2 mL) of infected blood. Terminally bleed the mice by heart puncture as described in detail in section 3.1.
      NOTE: Facial vein bleeding is not a routine method of obtaining blood as it is much more difficult to apply and is very distressful to mice, compared to terminal bleeding. The optimal parasitemia for the bleeding of donor mice is between 0.1%-1% because, at this parasitemia range, there are very few gametocytes (which cannot replicate to produce asexual blood stages) and there are less double- or triple-infected erythrocytes (which makes the quantification less accurate).
4. Day 0: quantification of infected blood to prepare doses of infected erythrocytes by serial dilutions
   1. Prepare serial dilutions of donor blood by adding 100 µL of blood to a microcentrifuge tube (dilution tube 1). Add 900 µL of RPMI medium to tube 1 and mix well by pipetting up and down with the same pipette tip, creating a 1:10 dilution.
   2. Take 100 µL of the diluted blood from tube 1 and add it to a new microcentrifuge tube (dilution tube 2). Add 900 µL of RPMI medium to tube 2 and mix well by pipetting up and down with the same pipette tip, creating a 1:100 dilution.
   3. Repeat steps 1.4.1 and 1.4.2 to make two more serial dilutions, creating 1:1,000 (dilution tube 3) and 1:10,000 dilutions (dilution tube 4).
   4. Add 12 µL of diluted blood from tube 3 and 12 µL of diluted blood from tube 4 to different sides of a hemocytometer and determine the average number of erythrocytes on each side of the hemocytometer. Multiply these numbers by 10. Multiply these numbers by the dilution factor, 1,000 or 10,000, respectively, to determine the number of erythrocytes in 1 µL of each dilution.
   5. Divide the desired number of infected erythrocytes to be injected by the number of erythrocytes in 1 µL of each dilution to determine the volume of diluted donor blood necessary for injection. Choose the dilution with the most appropriate volume for injection, add the volume to a new microcentrifuge tube, and complete it to 120 µL with RPMI medium.
5. Day 0: intravenous injection of the recipient mice
   1. Place the recipient mice under a heat red lamp to dilate their veins for injection. Load the parasite doses in a 27 G insulin syringe and place the recipient mouse in a restrainer.
   2. Inject 1 million or 10,000 infected erythrocytes intravenously (IV) using the 27 G insulin syringe in each recipient mouse for mosquito infection assays or for asexual and sexual blood-stage growth assays, respectively. Continue to maintain the mice under standard housing conditions.
      NOTE: An IP injection is an indirect method of introducing parasites into the bloodstream, as parasitized erythrocytes have to pass through the draining lymph nodes of the peritoneal cavity to reach the blood. This makes an IP injection an indirect delivery route for injecting a precise number of parasites into the bloodstream. Therefore, the IV route is preferred, as it delivers accurate parasite doses directly into the bloodstream, as shown in Figure 3 and described in the Representative Results.

2. Determination of the Blood-stage Parasite Load for Sexual and Asexual Stages

NOTE: In this section, standardized phenotypic evaluation methods of malaria parasite blood stages are listed. These methods are useful in the evaluation of novel antimalarial or vaccine candidates or even gene knockout on sexual and asexual stages development in the same experimental mice. Of note, P. chabaudi and P. vinckei are also very rational and important alternative options for these types of assays, especially in drug screening.

1. Determination of the parasitemia of asexual stages and male vs. female gametocytes by Giemsa-stained thin blood smears for parasite growth assays
   1. Using a 26 G or 27-G needle, prick the mouse's tail and collect the blood droplet on a microscope slide. Use the short edge of another microscope slide to quickly smear the blood across the slide.
   2. Fix the slide with 100% methanol for at least 1 min, only after the blood has completely dried on the slide. Stain in 10% Giemsa for 10-15 min. Wash the slide with single- or double-distilled water and allow it to dry.
   3. Read the slide using a 100x objective and oil immersion under a light microscope. For an accurate parasitemia measurement, check at least a total of 6,000 erythrocytes, which can be obtained by counting at least 30 or 40 microscopic grids with an average number of 200 or 150 erythrocytes (RBCs) per grid, respectively. Count the total number of RBCs in the first and last grids to determine the average number of red blood cells for all grids.
   4. Count the total number of infected erythrocytes per grid. Male and female gametocytes can be counted separately to determine gametocytemia but should also be included in the total parasitemia count.
   5. Determine the total parasitemia percentage by dividing the total number of parasitized erythrocytes by the total number of RBCs observed. Multiply this number by 100 to determine the parasitemia percentage.
   6. Determine the gametocytemia by dividing the total number of gametocytes counted by the total number of RBCs observed. Multiply this number by 100 to determine the gametocytemia percentage per mouse.
      NOTE: The only reliable method of differentiation between different asexual and sexual blood stages is the use of a microscope to evaluate Giemsa-stained thin blood smears, as each of the stages has different morphology, with the exception of immature gametocytes, which are mostly indistinguishable from asexual blood stages.
2. Determination of the blood-stage parasitemia by flow cytometry for fluorescent reporter protein-expressing WT-like parasite strains
   1. Label two microcentrifuge tubes for each mouse sample, designating one tube for a 1:1,000 dilution and the other for a 1:2,000 dilution. Add 1.5 µL of 1x heparin to the 1:1,000 dilution tube.
   2. Using a 26 G or 27 G needle, prick the mouse's tail and transfer 1.5 µL of blood to the tube containing 1.5 µL of 1x heparin (the 1:1,000 dilution tube). Mix the blood and the 1x heparin gently together by pipetting up and down.
   3. Add 1,497 µL of RPMI medium to the microcentrifuge tube, and gently pipette up and down to mix well.
   4. Transfer 500 µL of the mixture from the 1:1,000 dilution tube to the 1:2,000 dilution tube. Add 500 µL of RPMI medium to the 1:2,000 tube and gently mix by pipetting up and down.
   5. Follow the manufacturer's appropriate start-up protocol for the flow cytometer. Run the samples at a slow flow rate and set the detection for at least 20,000 events.
      NOTE: There is an alternative practical assay to measure parasitemia via tricolor FACS sorting, whether parasites express fluorescent markers or not³⁴. However, the assay described here offers an accurate measure of parasitemia in live parasites without the addition of any DNA-binding dyes and the use of FACS sorting.
3. Determination of the exflagellation rate of male gametes per microliter of infected mouse blood
   1. Prepare a 1:10 dilution microcentrifuge tube with 40 µL of incomplete ookinete medium (RPMI + sodium bicarbonate + hypoxanthine + xanthurenic acid) and 5 µL of 1x heparin.
   2. Using a 26 G or 27 G needle, prick the mouse's tail and transfer 5 µL of blood (using a micropipette) quickly to the tube containing the incomplete ookinete media + heparin. Mix gently and load 12 µL of the 1:10 blood dilution tube onto a hemocytometer and leave it at room temperature (20-24 °C).
   3. Start counting the exflagellation events 9-10 min after preparing the 1:10 dilution. Use a phase contrast light microscope with 40x magnification.
   4. Multiply the average number of exflagellation events counted by 10 and then multiply by the dilution factor (10) to determine the average number of exflagellation per microliter of infected blood.
      NOTE: This assay measures the number of male gamete exflagellation events in a 1 µL volume of blood, which can be quantified using a hemocytometer. An incomplete ookinete medium¹⁸ is mixed with the blood to closely mimic the conditions during which exflagellation occurs inside the mosquito midgut shortly after a blood meal.

3. Isolation and Processing of Blood-stage Parasites from Infected Erythrocytes and Frozen Stock Preparation

1. Terminal bleeding of infected rodents by heart puncture
   1. Prepare a 26 G needle syringe that contains approximately ≤20 µL of 1x heparin (200 units/mL) to collect the blood of the donor mouse by heart puncture.
   2. Use a slow flow of CO₂ to euthanize the mouse before bleeding. Alternatively, anesthetize the mouse with isoflurane (which has to be maintained before and during the skin incision to expose the heart).
   3. Lay the mouse on its back and pin down its appendages. Make a small incision in the skin near the base of the sternum by pulling up the skin with forceps and cutting it with scissors. Pull the skin apart at the site of the incision to expose the diaphragm and the top of the abdominal cavity.
   4. Hold the base of the rib cage with forceps and cut through the diaphragm to enter the thoracic cavity; be careful not to damage the heart or lungs. Pin back the rib cage to expose the heart.
   5. Gently puncture the heart and slowly pull up on the syringe plunger to collect ~1 mL of blood and transfer it to a microcentrifuge tube.
2. Preparation of frozen stocks from infected blood
   1. Bleed the mice (see section 3.1) with blood parasitemias that are between about 2% and 5%, with a significant amount of ring stages and not as many gametocytes.
   2. Mix the desired portion of blood with freezing solution (10% glycerol in Alsever's solution) in a 1:2 ratio and store in cryogenic vials. Freeze in liquid nitrogen.
3. Isolation and purification of blood-stage parasites for the extraction of DNA, RNA, and proteins
   1. Bleed mice (see section 3.1) with blood parasitemias that are higher than 0.5%.
   2. Prepare a 10 mL syringe by removing the plunger and inserting a small cotton swab. Pack down the cotton to the bottom of the syringe. Fill it with cellulose until the level of cellulose reaches the 3 mL mark but does not exceed the 4 mL mark on the syringe.
   3. Secure the syringe into a clip on a ring stand and place a waste collection tube below it. Add 5 mL of PBS to the syringe and allow it to flow through into the waste tube.
   4. Prepare frozen stocks if needed, using only 100-300 µL of blood. Add the rest of the infected blood (400-700 µL) to the column. Collect flow through with a waste tube until the droplets start to turn red. Once the droplets start to turn red, place a new 14-mL collection tube under the syringe to collect the flow through.
   5. Once the flow starts to slow, add PBS to the syringe and collect the flow through in the 15 mL tube until a total volume of 14 mL has been reached. Collect the remaining flow through with a waste tube.
   6. Centrifuge the 14 mL tube with blood/PBS mixture for 8 min at 250 x g with no brakes. Remove the supernatant and add 14 mL of chilled saponin (50 mg of saponin in 50 mL of PBS). To dislodge the pellet, invert the tube multiple times and vortex if necessary.
   7. Centrifuge the tube for 8 min at 1,217 x g with no brakes. Remove the supernatant, leaving about 0.5 mL of the solution above the pellet.
   8. Resuspend the pellet in the solution using a micropipette and transfer it to a microcentrifuge tube. Add 500 µL of PBS to the 14 mL tube to wash any residual parasites that are left in the tube. Add this to the same microcentrifuge tube.
   9. Centrifuge the tube for 2 min at 6,010 x g. Remove the supernatant and resuspend 1 mL of PBS. Centrifuge for 2 min at 9,391 x g.
   10. Proceed to extract genomic DNA, total RNA, and protein.
       1. For genomic DNA extraction, remove the supernatant and resuspend the pellet in 200 µL of PBS, and then follow any suitable protocol of gDNA extraction.
       2. For total RNA extraction, remove the supernatant, resuspend and vortex the pellet in 1 mL of any phenol- and guanidine isothiocyanate-based solution or any other suitable reagent and, then, follow any suitable protocol of total RNA extraction.
       3. For total protein extraction, remove the supernatant, resuspend the pellet in an appropriate volume of 6x sodium dodecyl sulfate (SDS)-loading dye or any other suitable reagent and, then, follow any suitable protocol of total protein processing.

4. Mosquito Infection Assays

NOTE: The mosquito is the primary host of the malaria parasites where sexual reproduction takes place. The infection of mice to transmit malaria parasites to mosquitoes is conducted by an IV injection of at least 1 million blood stages, followed by feeding an infected mouse (from each genotype that displays the highest male gamete exflagellation rate) to Anopheles mosquitoes in a cage at day 3 post-mouse-infection. The IV injection with 1 million blood-stage parasites in phenylhydrazine-treated mice will ensure the development of male and female gametocyte at a faster and higher rate. Mosquitoes infected with P. yoelii and P. berghei are incubated at 24 °C and 20-21 °C, respectively, to allow for the best possible mosquito stages development⁶.

1. Mosquito feeding and determination of the number of ookinetes per mosquito
   1. Starve adult female Anopheles stephensi or A. gambiae mosquitoes (4-7 days old) for 8-12 h prior to feeding. Allow adult mosquitoes to feed for at least 15 min on the infected anesthetized mice (injected with an appropriate dose of ketamine/xylazine) with the highest exflagellation rate (measured in section 2.3).
      NOTE: Ketamine/xylazine working solution is prepared by the 1:5 dilution of the stock solution in saline and IP injecting 100 µL per mouse. For a 10 mL stock solution, 1 mL of xylazine (100 mg/mL) is added to 9 mL of ketamine (100 mg/mL).
   2. Remove unfed female mosquitoes and male mosquitoes with a mouth aspirator.
   3. At 18-20 h postfeeding, collect 20-30 mosquitos and place them in the freezer for not less than 10 min to ensure death. Using a binocular dissection scope, dissect out 20-30 blood-filled midguts with two 26 G or 27 G needles or a needle and forceps in RPMI or PBS dissection medium and transfer the midguts to a microcentrifuge tube containing 200 µL of RPMI or PBS (For dissection method please see section 4.3).
   4. Centrifuge the collection tube for 1 min at 700 x g. Grind the pelleted midguts using a pestle. Repeat this step.
   5. Transfer 50 µL from the tube of ground midguts to a new microcentrifuge tube. Add 200 µL of RPMI or PBS to create a 1:5 dilution.
   6. Transfer 12 µL to a hemocytometer and incubate it at room temperature for 5 min to allow the contents to settle.
   7. Determine the average number of ookinetes by using a light microscope with a phase contrast and 40x magnification.
   8. Multiply the average number of mature ookinetes by 10 and then by the dilution factor of 5 to determine the average total number of ookinetes.
   9. Divide the average number of ookinetes by the number of blood-fed mosquitoes dissected to determine the number of ookinetes per blood-fed mosquito.
2. Determination of the number of early oocysts for fluorescent reporter protein-expressing WT-like parasite strains
   NOTE: The aim of this assay is to determine the number of live parasites expressing green fluorescent protein (GFP) that established a full infection of the mosquito midguts and formed early spherical oocysts. This assay determines if the ookinetes (their development estimated in the previous assay) completed their functions by the traversal through the mosquito midgut epithelial cells and the transformation to early oocysts on the basal lamina side of the midgut epithelia or not. This is another assay which makes great use of the parasites expressing GFP, as counting early oocysts at this stage will be nearly impossible without tedious immunostaining.
   1. Dissect the midguts (see section 4.1) of 20-30 blood-fed mosquitoes, at day 3 or 4 post-mosquito-feeding (pmf) for P. yoelii 17X-NL, and at day 6 or 7 pmf for P. berghei ANKA, with two 26 G or 27 G needles or a needle and forceps in RPMI or PBS dissection medium (For dissection method please see section 4.3).
   2. Spread 40-50 µL of dissection medium on the horizontal midline of the longer edge of the glass slide. Transfer the midguts to this line, one at a time, during dissection, and add more medium to the midguts, if needed, to avoid drying out.
   3. Place a coverslip gently on the dissected midguts, and seal it with nail polish.
   4. Using the 10x or 20x objective of the fluorescence microscope with the green fluorescence filter, count the number of early oocysts on each midgut, in at least 20 midguts.
3. Determination of the number of oocyst sporozoites per mosquito
   1. Dissect the midguts of at least 20-30 mosquitoes with two 26-G or 27-G needles or a needle and forceps in RPMI or PBS dissection medium and collect the dissected midguts in 200 µL of RPMI.
   2. Hold the lower abdomen segments firmly in place with one needle (or forceps) and  lightly push the thorax in an upward direction with the other needle at the junction between the thorax and the abdomen. Do carefully short pushes until the thorax separates from the abdomen. As soon as the separation starts the white midgut will appear stretched, and then the midgut will be cut from the esophagus end using the same needle that was used to separate the thorax. If still attached to the abdomen, the same needle can be used to pull the midgut away from the abdomen. Transfer the midguts of all mosquitoes to the collection tube by collecting them from the medium with a needle, one at a time.
   3. Centrifuge the collection tube for 1 min at 700x g to settle the midguts to the bottom of the collection tube and grind them with a pestle. Repeat this step.
   4. Transfer 12 µL to a hemocytometer and incubate it at room temperature for 5 min to allow its contents to settle.
   5. Count the oocyst sporozoites using a 40X objective of a light microscope. Use 1:5 and/or 1:10 dilutions if mosquito debris prevents the precise counting of the sporozoites or if the number of sporozoites is too high to count accurately.
   6. Calculate the average total number of oocyst sporozoites by multiplying the average number of sporozoites by 10 and, then, multiplying that number by the dilution factor, if any, and by the total volume (200 µL).
   7. Divide the average total number of sporozoites by the number of mosquitoes dissected to calculate the average number of oocyst sporozoites per mosquito.
      NOTE: A common mistake for measuring the success or productivity of mosquito stages infection is the counting of the number of oocysts at later stages of oocyst development. This is due to some oocysts being vacuolated at later time points of oocyst development, and others fail to develop sporozoites, even on the same midgut. The best and most reliable method to determine the productivity of mosquito stages infection is the counting of the average number of midgut oocyst sporozoites at days 10-12 post-mosquito-infection for P. yoelii and at days 12-14 post-mosquito-infection for P. berghei.
4. Determination of the number of salivary gland sporozoites per mosquito
   NOTE: The ultimate evaluation of the full completion of mosquito stages development is accomplished by estimating the number of sporozoites that invaded the salivary gland, which are the transmissible and infectious stages to vertebrates. The invasion of the salivary gland is established after the completion of oocyst sporozoite development, egress into the hemolymph, attachment to the basal lamina of the salivary gland acinar cells, and traversal of the acinar cells to reach the salivary glands ducts⁵. Therefore, this assay is also an evaluation of the success of all of these processes or not. Nonetheless, an estimation of hemolymph sporozoite numbers would allow the differentiation between defects in the egress from oocysts and defects in the invasion of salivary glands³⁵. The purification of salivary gland sporozoites allows conducting multiple functional assays on sporozoite motility and invasion phenotypes. The salivary gland sporozoites can also be used for the in vivo infection of mice and in immunization studies^36,37. Moreover, the most reproducible stages available to establish an in vitro liver stage invasion or a development assay is by the use of salivary gland sporozoites.
   1. Dissect the salivary glands of 50-100 female mosquitoes, at days 14-16 pmf for P. yoelii and at days 17-20 pmf for P. berghei, preferably with two 26 G or 27 G needles, in RPMI or DMEM medium kept on ice and supplemented with 5% fetal bovine serum (FBS) or/bovine serum albumin (BSA) for P. yoelii sporozoites or 3% FBS/BSA for P. berghei sporozoites. If the sporozoites are to be used in hepatoma in vitro assays, add 1% penicillin/streptomycin to the dissection medium. There are generally two methods for the dissection of the salivary glands. The first method is time-consuming but leads to the dissection of salivary glands with very little or no contaminating tissues. The second method is less time consuming but leads to the collection of a significant amount of contaminating tissues. The first method is detailed here.
   2. Hold the upper abdomen segments in place with the beveled side of one needle and carefully push the head very lightly (in very short pushes) at the junction between the head and thorax in an upward direction until the head is carefully separated from the thorax without tearing up the glassy salivary glands, the exposed salivary glands are then separated from the head with the same needle that pushed the head upward.
   3. The dissected salivary gland will be raised from the dissection medium using a short glass Pasteur pipette.
   4. When all salivary glands have been dissected and harvested, talent will place the tube containing the midguts into the centrifuge (1min, 700 x g).
   5. Count sporozoites using a hemocytometer under a light microscope set to Phase 2 contrast and 40x magnification. If the number of the mosquitos dissected ≥40, dilute the salivary glands to 1:5 or 1:10, depending on the expected infectivity yield (determined in earlier assays).
   6. Multiply the average number of sporozoites by 10 and the dilution factor (if any). Multiply by the total volume to determine the average number of salivary gland sporozoites. Divide by the number of dissected female mosquitoes to determine the average salivary gland sporozoites per mosquito.
      NOTE: The problem with the dissection of the salivary glands is their small size and glassy transparent appearance. Thus, it is very difficult to isolate the salivary glands and, therefore, they are usually dissected out as a lump sum with other tissues from the frontal part of the thorax, which is also important to protect the salivary glands from rupture during the collection.