Complete Development of Mosquito Phases of the Malaria Parasite in Vitro

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Science  25 Jan 2002:
Vol. 295, Issue 5555, pp. 677-679
DOI: 10.1126/science.1067159


Methods for reproducible in vitro development of the mosquito stages of malaria parasites to produce infective sporozoites have been elusive for over 40 years. We have cultured gametocytes ofPlasmodium berghei through to infectious sporozoites with efficiencies similar to those recorded in vivo and without the need for salivary gland invasion. Oocysts developed extracellularly in a system whose essential elements include co-cultured Drosophila S2 cells, basement membrane matrix, and insect tissue culture medium. Sporozoite production required the presence of para-aminobenzoic acid. The entire life cycle of P. berghei, a useful model malaria parasite, can now be achieved in vitro.

For over a century, a major objective of malaria control programs has been to block parasite transmission by mosquitoes. Such approaches would clearly benefit from a better understanding of parasite development within the vector, initiated when gametocytes are taken up in a blood meal. Fertilization of macrogametes within the mosquito midgut produces zygotes that transform into motile and invasive ookinetes. These penetrate and traverse the midgut epithelium and become sessile vegetative oocysts lying beneath the midgut basement lamina, each potentially producing 2 to 8000 sporozoites. Knowledge of the mosquito-related factors regulating these processes is improving (1–3), but it is difficult to determine the specific and separate effects of these factors in vivo. Early events associated with midgut invasion have recently been studied in vitro with the use of midgut preparations (4–6) or co-cultured mosquito cells (7), but these systems do not sustain long-term development or simulate oocyst interaction with the basal lamina and do not permit investigation of sporozoite differentiation.

Fertilization and ookinete development can be achieved in vitro for many malaria parasite species, including Plasmodium berghei, a parasite of rodents (8, 9). These culture systems have facilitated the study of ookinete molecules that may be targeted by antibodies induced by transmission-blocking vaccines or drugs (10, 11). After many pioneering attempts (12, 13), it is only recently that in vitro transformation of Plasmodium gallinaceum andPlasmodium falciparum ookinetes into oocysts and sporozoites has been achieved, but the numbers of oocysts produced are low and, more importantly, the infectivity of these sporozoites has not been demonstrated (14, 15). Here we confirm the need for a basement membrane–like substrate such as Matrigel, which may mimic the basal lamina of the mosquito midgut epithelium. In addition, co-culture with Drosophila melanogaster S2 cells is necessary for development, although the role of these insect cells is unclear.

We have based our work on the previously described P. gallinaceum culture system (15) and, where appropriate, substituted conditions that more nearly mimicked the mosquito environment or provided factors known to enhance oocyst growth in vivo. Thus, a culture system has been developed that consistently supports the transformation of large numbers of P. berghei ookinetes to extracellular oocysts and the production of infective sporozoites with efficiencies approaching those seen in vivo.

Plasmodium berghei ANKA (clone 2.34) ookinetes were produced in vitro (8, 9) and cultured to produce oocysts in eight-chamber slides (16). Previously, cultures of other malaria species used supplemented RPMI 1640 (15), a mammalian medium traditionally used to culture ookinetes. A comparison of oocysts growing extracellularly in RPMI 1640 and Schneider's medium (17), whose composition mirrors the high aminoacidaemia of mosquito hemolymph (18), demonstrated that Schneider's medium significantly improved oocyst yield [multiple analysis of variance (MANOVA) over time:F3,66 = 3.06, P = 0.03 (19)]. Therefore, a classic insect medium, Schneider's medium, was used in all subsequent investigations. Nutrition of oocysts may be better supported by this medium, or Schneider's medium may be more suitable for the co-cultured insect cells because growth ofDrosophila S2 cells is retarded in RPMI (20).

Extracellular oocyst development did not occur if chambers were not initially coated with Matrigel. Many ookinetes burrowed into the Matrigel matrix within hours and, within 1 to 2 days, transformed into oocysts within and on the surface of the matrix. Parasites not firmly attached to the matrix were probably removed during the repeated medium changes, which may account, in part, for the decline over time in oocyst number recovered from each chamber (19). We have previously observed that P. berghei ookinetes attach to plastic wells coated with the basal lamina components laminin, collagen IV, or fibronectin. Some ookinete-oocyst transformation occurs when bound to a laminin-collagen mixture (21). Ookinete proteins that may act as ligands for laminin (22) or collagen IV include the surface protein Pbs21 (23), Pgs28 (24), and a 215-kD protein, possibly PbCTRP (21, 25).

The culture of erythrocytic schizonts requires microaerobic conditions. Therefore, we compared the effect of air with low-oxygen gas phase on oocyst development (9). No significant effects on parasite development were observed due to gas phase when analyzed by days of interaction (MANOVA: F 3,42 = 0.5417, P = 0.66) (19). All subsequent incubations were performed in air. The hypothesis that fluctuations in midgut lumen pH normally associated with blood meal management (26) influence oocyst development was not upheld (9, 19), and oocyst differentiation in vitro was more efficient at a pH close to that of the hemocoele (pH 6.81 ± 0.08) (27) than the range of pH found in the gut during blood meal digestion (pH 7.68 to 8.03 inAnopheles stephensi) (26).

Pilot studies established that extracellular oocyst growth was markedly improved by the presence of DrosophilaS2 cells (19). We then asked whether oocyst development could be improved by co-culturing with cell lines from susceptible (Anopheles gambiae line, Sua 4.0, kindly supplied by H.-M. Müller, European Molecular Biology Laboratory, Heidelberg, Germany) or nonsusceptible mosquitoes [Mos20, from Aedes aegypti (28)]. All cells were added at a cell:ookinete ratio of 10:1. Cell type significantly affected the number of oocysts recovered throughout the culture period (MANOVA:F 3,28 = 33.51, P < 0.0001) (19). However, the Drosophila cell line supported significantly more oocysts than either mosquito cell line (Tukey's post hoc pairwise comparisons, P < 0.001 for all comparisons). In the absence of Matrigel, P. bergheiookinetes transform into early oocysts intracellularly in Aedes albopictus cells (29) and in A. gambiae4a-3A cells (7). The latter system supports intracellular oocyst development, including nuclear division and expression of circumsporozoite protein but not sporozoite formation (7). The role of co-cultured “feeder” cells in the sustained development of oocysts is unknown, but they could provide either soluble nutrients or unidentified intercellular matrix molecules (24) that may be involved in host-parasite signaling.

The development of P. berghei oocysts in the presence of S2 cells was very similar to that seen in the mosquito. Mosquitoes were fed on a P. berghei–infected mouse (8, 9), and groups of five female mosquitoes were dissected at various times post-infection. Comparisons of oocyst diameters in vivo and in vitro showed that growth proceeded at a similar rate in both conditions (19) and that maximum diameters of ∼40 μm were achieved by day 15. In contrast to reports of cultured P. gallinaceum oocysts (15), all cultured P. berghei oocysts were spherical and had a clearly defined capsule (Fig. 1D). Oocysts expressed Pbs21 until day 6 (Fig. 1B) and circumsporozoite protein was first detected 7 days after culture, as has been reported in vivo (30) (Fig. 1, E and H). Staining with 4′,6′-diamidino-2-phenylindole (DAPI) revealed several foci of nuclear material (Fig. 1, C, F, and I), indicative of the expected endomitosis.

Figure 1

In vitro sporogonic development ofP. berghei. (A through C) Day 3. Bars, 5 μm. (D through F) Day 9. Bar, 10 μm. (G through I) Day 15. Bars, 10 μm. (J and K) Day 25. Bars are as follows: (J), 4 μm, (K and L), 5 μm. (A, D, and G), light microscopy; (B, E, H, and K), fluorescence microscopy. Cell labeling was performed with reagents as follows: (B), Oocyst surface labeled with antibody 13.1 (antibody to Pbs21); (E, H, and K), parasite labeled with antibody 3D11 [antibody to circumsporozoite protein (CSP)] oocyst wall; (C, F, and I), oocyst nuclei (marked “N”) labeled with DAPI; (J), Giemsa-stained sporozoites (marked “Sp”) free on the Matrigel; (L), mouse erythrocytes infected with P. berghei 7 days after inoculation of sporozoites produced in culture. Oo, oocyst; Cap, oocyst wall.

Para-aminobenzoic acid (PABA), previously shown to support enhanced oocyst development in mosquitoes (31), was found to be essential for sporozoite differentiation in vivo. When added to Schneider's medium at a final concentration of 11 to 44 nM, oocysts containing sporozoites were visible by day 15. By day 20, the first sporozoites were naturally released from a small percentage of oocysts and were found adhering to Matrigel (Fig. 1, J and K). Parallel observations on parasite development in vivo showed that salivary gland invasion by sporozoites (and, therefore, prior oocyst rupture) was also first detected on day 20. Recovery of sporozoites adherent to the Matrigel increased with rising PABA concentrations to an optimum at 44 nM (19). In eight replicate experiments, culture medium removed from all wells every 2 to 3 days from day 20 onward contained large numbers of sporozoites.

Sporozoites were collected from 25- or 28-day-old cultures, washed, and resuspended in phosphate-buffered saline (PBS) and inoculated intravenously into Charles River Derived (C.D.) mice at doses of 4000 (25 day), 7000 (28 day), or 14,000 (28 day). Four of ten, one of three, and three of three mice became infected, respectively, demonstrating a patent parasitaemia by day 7 post-inoculation, a time consistent with the 6.3-day pre-patent period described after inoculation of salivary gland sporozoites (32,33) (Fig. 1L). This clearly demonstrates that a period of residence in the mosquito salivary glands is not essential to the onset of sporozoite infectivity, as suggested previously (33). One of these mice was then used as a blood meal source for A. stephensi mosquitoes, and 8 of 12 of the fully engorged mosquitoes developed oocyst infections on their midguts by day 10.

As described previously (34), ∼40% of the initial ookinete inoculum undergoes apoptosis and ∼68% of the survivors differentiated into oocysts by day 15. These efficiencies compare with 10 to 30% for cultured P. gallinaceum(15) and 77% for Plasmodium yoelii in vivo inA. stephensi (35). The ability to contrast parasite development in this culture system with the events of natural infection may enhance our ability to understand the regulation of parasite development in both susceptible and refractory mosquitoes.

We have described the successful culture of all the sporogonic stages of P. berghei in vitro. However, the partial success previously achieved with the avian parasite P. gallinaceum and the human parasite P. falciparum with the use of a comparable but less developed culture system (14, 15) gives every reason to expect our protocol to work with other strains and species ofPlasmodium. Whilst recognizing the potential applicability of this technique to a wide range of malaria species, we also appreciate that the possibility of culturing each and every stage of the life cycle of the rodent parasite P. berghei immediately opens up important new areas of investigation in this useful model species.

  • * Current address: Department of Parasitology, Biomedical Primate Research Centre, Lange Kleineg 139, Post Office Box 3306, 2280 GH Rijswijk, Netherlands.

  • To whom correspondence should be addressed. E-mail: h.hurd{at}


Summary of optimum culture conditions for sporogonic stages of P. berghei. Drosophila melanogaster S2 cells were incubated at 19° to 20°C in air on a layer of Matrigel in a ratio of 10:1 with ookinetes (36).

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