EXPERIMENTAL PLASMODIUM FALCIPARUM INFECTIONS IN ANOPHELES
MOSQUITOES
Lindsey L. Garver, Luke Baton and GeorgeDimopoulos
W. Harry Feinstone Department of Molecular Microbiology and Immunology, Bloomberg
School of Public Health, Johns Hopkins University, 615N. Wolfe Street, Baltimore, MD 212052179, USA.
Introduction:
Transmission of the human malaria parasite, Plasmodium falciparum, is dependant on successful
development in an anopheline mosquito vector. In the mosquito, the parasite undergoes several
stage changes and meets considerable barriers to lifecycle progression that can be mediated by a
variety of mechanisms including the mosquito immune response. Elucidation of these barriers is
investigated using laboratory P. falciparum infections and phenotypic scoring based on counting
oocysts. Typically, experimental groups of mosquitoes are suspected to have an altered immune
response toward the parasite compared to the control group that has been differentially treated
with respect to gene expression of immune challenge. This can be due to RNAi gene silencing,
transgenic expression or other method. Both groups are fed on infected blood then midguts are
dissected and oocysts are visualized using light microscopy (Fig 1). A change in the number of
oocysts between groups suggests that the treatment of the experimental group is, for some
reason, affecting the ability of parasites to develop to that stage. Unlike P. berghei, P.
falciparum is not supported by a mouse model so infections in the laboratory require an artificial
membrane system. Such a system uses a water flow system to heat blood to room temperature
and a latex or parafilm membrane to contain the blood yet mimic human skin. This is the
simplest way to obtain infection phenotypes using a natural mosquito-malaria combination that is
relevant for applied research but can be performed in the lab. While we have focused on this
assay as a way to investigate mosquito immunity to P. falciparum, the same technique could also
be coupled to investigations of synthetic transmission blocking candidates, paratransgenic
candidates and any other experimental condition where effect on oocyst production would be a
useful phenotypic outcome to quantify. The pathogen used in this assay is transmissible to
humans if mishandled so proper caution must be taken at all times to ensure safety. Gloves and
protective clothing should be worn at all times, mosquitoes should always be contained and
contaminated surfaces should be treated with 10% bleach and/or discarded in biohazard
containers at the conclusion of experimental set up.
This method requires:
1) Anopheline mosquitoes (see Benedict 1997 for Anopheles gambiae rearing procedures) aged
at least 2 days and properly contained. Double containment (mosquitoes inside a cup inside a
cage) is preferable.
2) 1-pint cardboard cups with lids and mesh net covering to contain mosquitoes.
3) A culture of P. falciparum gametocytes with known gametocytemia.
4) Human serum and blood (O+)
5) Plastic 15 mL conical tubes
6) Tissue culture pipette tips
7) Pipetman
8) Sterile glass pasteur pipets
9) Water bath heated to 37oC
10) Centrifuge
11) Parafilm or other membrane
12) Circulating water bath
13) Glass membrane feeders
14) Rubber tubing to fit feeders (connects circulating water bath)
15) 10% bleach
16) Ice bucket
17) Glass petri dish and cover
18) Glass slides
19) Fine-tip forceps
20) Paper towels
21) 1X sterile PBS
22) 0.2% mercurochrome
23) 12-well glass slide
24) light microscope
Protocol:
A. Preparation of gametocyte culture:
1. Place fresh human blood and serum in a water bath and leave until 37oC is reached.
Maintain both at 37oC throughout the experiment.
2. Using a tissue culture hood and pipette, add 1 mL of warm human blood into an
Eppendorf tube. Centrifuge for 5 minutes at 2200 rpm. A pellet of red blood cells
(RBCs) with serum supernatant will be visible; remove and discard the serum. Add an
equal volume of new serum to the RBC pellet to wash. Centrifuge again, remove the
serum supernatant and wash again. Wash one more time and place in water bath until
you are ready to add the parasites.
3. Obtain a day 16 gametocyte culture that has been maintained on RPMI media.
Calculate the gametocytemia % by thin smear (from a drop of the culture) and Geimsa
stain, counting the number of gametes in a field of the parasite culture. It is usually in the
range of 2-4%. Use this percentage to calculate the volume of whole blood you need to
add to the parasites to reach a final % gametocytemia in the range of 0.3-1%. You may
choose a higher or lower final percentage based on the experiment and degree of
infection you’d like to achieve.
4. In the tissue culture hood and using a tissue culture grade pipettes, remove as much
RPMI from the gametocyte culture without removing or unsettling the parasites.
Carefully transfer the remaining media and parasites into a conical tube. Centrifuge for 5
minutes at 2200 rpm. Parasites and RBCs will pellet; remove and discard media
supernatant without unsettling parasites.
5. Add to the parasite pellet the volume of prepared whole blood that will give the
appropriate final gametocytemia (determined by your calculations). Mix gently though
thoroughly. It is this preparation that you will add to the feeding apparatus. Keep tube in
37oC water bath until you are ready to use.
B. Feeding mosquitoes :
1. While feeding, mosquitoes can be housed in small wire mesh cages or 1-pint waxlined cardboard cups covered with mesh netting secured by a rubber band or
cardboard lid supplied with cup. Use a battery-operated aspirator to transfer
mosquitoes from colony to either of these containers.
2. A circulation water bath can be prepared by connecting the necessary number of glass
feeders with rubber tubing in a series. Connect one end of the series to the circulator
of the water bath (the part that pushes water through the feeders) and the other end of
the series should empty the circulated water back into bath. Turn on the bath until
the water is about 37oC and the water is circulating through the feeders and back to
the bath. This is essential to attract the mosquitoes and keep the parasites alive.
3. Stretch parafilm or other membrane around the glass membrane feeder to mimic skin;
seal tightly to prevent leaks.
4. Place each feeder membrane-side down onto each cage or cup and secure feeders
using clamps or tape so that the membrane sits flush with the net. This ensures that
the mosquitoes can probe through the mesh to membrane and reach the blood from
inside the cage/cup.
5. Pipette parasite-blood meal (from step A) into the feeder, making sure the blood goes
through the feeder to contact the membrane. About 200 μl of blood per feeder should
be sufficient for up to 100 mosquitoes.
6. Allow mosquitoes to feed. (variable depending on experiment—for most
experiments, we allow them to feed for ~30 minutes, occasionally monitoring how
many have already taken a meal by looking at the blood content in the abdomen).
7. Once most feeding activity has stopped and a good proportion of mosquitoes have
visibly fed, remove feeders, turn off the water bath and dismantle the feeding
apparatus.
8.
Decontaminate everything that has contacted infected blood with 10% bleach and
rinse with water. Place disposable contaminated material (membranes, paper towels,
gloves, etc) into a small biohazard bag and deposit in a large biohazard receptacle.
9. Cold anesthetize mosquitoes by placing in refrigerator or cold room then transfer
them on a Petri dish embedded in an ice bucket. Sort mosquitoes, looking carefully at
the abdomens for any sign of a blood meal and discard those that did not feed. Some
mosquitoes will be fully engorged while others will show a narrow band of ingested
blood; all are acceptable as “fed” though you may want to record numbers of “fully
feds” and “partially feds”. Put the fed mosquitoes back into the cardboard cups.
Remember that these are Plasmodium infected mosquitoes so much care should be
taken to avoid escape.
10. Provide a sugar meal (10 % sucrose source) to mosquitoes and incubate under normal
insectary conditions for 7 days. Everyday the mosquitoes should be monitored for
mortality and sucrose should be replenished. We have seen improved survival when
we place a paper towel soaked in distilled water on top of the cages or cups to provide
extra moisture. These mosquitoes are P. falcipraum infected, so all care must be taken
so that no mosquitoes can escape from the cage/cup. We use a double layer of
protection by placing the cups/cages in to larger cages.
C. Dissecting mosquito midguts and estimating oocyst loads:
1. Aspirate mosquitoes (from step B) from cages/cups using a battery-operated aspirator and
cold anesthetize.
2. Transfer anesthetized mosquitoes to a glass Petri dish embedded in ice. Separate
mosquitoes out to a single layer and cover with a plastic Petri dish lid to avoid escape.
Mosquitoes that don’t fit on the dish should remain anesthetized and be contained
elsewhere.
3. Using fine-tipped forceps transfer a single mosquito from dish to 100 ul PBS contained
on a glass slide mounted under dissection microscope. Leave others covered in chilled
petri dish.
4. Use the forceps to lightly pinch mosquitoes at the thorax and the posterior abdomen. Pull
the body apart until midgut is exposed. Use forceps to remove excess tissue from gut
which will look like a white, sac-like body. Remove excess tissue from the gut. Mount
the gut in one well of a 12-well glass slide and immerse in 5-10 μl of 0.2%
mercurochrome. Squash the carcass and discard on damp paper towel. Continue for
other mosquitoes until 12-well slide is full. Cover the mounted guts with a glass
coverslip.
5. Examine guts under light microscope, looking for round oocysts that stain bright pink
against the clear/faint pink gut tissue. Count oocysts, record and continue to next sample.
Continue for all mosquitoes of all experimental treatments.
6. When finished, wipe slide clean with paper towel. Place paper towel with guts and paper
towel with carcasses are into small biohazard bag and put in trash. Gloves are worn
throughout procedure and also care taken not to let any infected mosquitoes escape.
Results and Discussion
This assay is an essential tool for anyone wishing to determine how an experimental treatment
affects the P. falciparum development in anopheline mosquitoes. Although the feeding
mechanism is artificial, the vector-parasite interaction is more applicable to natural infection
contexts than the rodent parasite model, P. berghei. This assay is limited in that the investigator
must have access to a successful P. falciparum gametocyte culture but is quite easy to carry out
if a culture can be obtained. P. falciparum infection has become particularly critical for research
in the immune response of mosquitoes since immunity to P. berghei and immunity to P.
falciparum are different (Cohuet, et al. 2006; Dong, et al. 2006). This is probably why more and
more investigators in this field are utilizing methods such as this one to score infection
phenotypes.
Benedict, M. (1997). Care and maintenance of anopheline mosquitoes. The molecular biology of
disease vectors: A methods manual. J. Crampton, C. Beard and C. Louis. London, Champman
and Hall: 3-12.
Cohuet, A., M. A. Osta, et al. (2006). "Anopheles and Plasmodium: from laboratory models to
natural systems in the field." EMBO Rep 7(12): 1285-9.
Dong, Y., R. Aguilar, et al. (2006). "Anopheles gambiae immune responses to human and rodent
Plasmodium parasite species." PLoS Pathog. 2(6): e52.
Figure 1: Midgut oocysts visualized by light microscopy. Using mercurochrome staining,
oocysts are visible in a midgut dissected from an infection mosquito at 7 days post-feeding.