Species-specific escape of Plasmodium sporozoites from oocysts of avian, rodent, and human malarial parasites

Abstract

Background: Malaria is transmitted when an infected mosquito delivers Plasmodium sporozoites into a vertebrate host. There are many species of Plasmodium and, in general, the infection is host-specific. For example, Plasmodium gallinaceum is an avian parasite, while Plasmodium berghei infects mice. These two parasites have been extensively used as experimental models of malaria transmission. Plasmodium falciparum and Plasmodium vivax are the most important agents of human malaria, a life-threatening disease of global importance. To complete their life cycle, Plasmodium parasites must traverse the mosquito midgut and form an oocyst that will divide continuously. Mature oocysts release thousands of sporozoites into the mosquito haemolymph that must reach the salivary gland to infect a new vertebrate host. The current understanding of the biology of oocyst formation and sporozoite release is mostly based on experimental infections with P. berghei, and the conclusions are generalized to other Plasmodium species that infect humans without further morphological analyses.

Results: Here, it is described the microanatomy of sporozoite escape from oocysts of four Plasmodium species: the two laboratory models, P. gallinaceum and P. berghei, and the two main species that cause malaria in humans, P. vivax and P. falciparum. It was found that sporozoites have species-specific mechanisms of escape from the oocyst. The two model species of Plasmodium had a common mechanism, in which the oocyst wall breaks down before sporozoites emerge. In contrast, P. vivax and P. falciparum sporozoites show a dynamic escape mechanism from the oocyst via polarized propulsion.

Conclusions: This study demonstrated that Plasmodium species do not share a common mechanism of sporozoite escape, as previously thought, but show complex and species-specific mechanisms. In addition, the knowledge of this phenomenon in human Plasmodium can facilitate transmission-blocking studies and not those ones only based on the murine and avian models.

Keywords: Avian; Human; Mosquito vector; Murine; Oocyst; Plasmodium; Sporozoite escape.

Fig. 1

Plasmodium gallinaceum sporozoites escaping from oocysts. a and b Hundreds of rounded P. gallinaceum oocysts of similar size protruding from the external surface of the midgut (Mdgt) among the muscle fibers (Mfb) and tracheoles (Tch). Most oocysts form clusters of a few individuals (asterisks). All oocysts have a completely smooth surface. Magnification = ×100. c Two oocysts attached side by side to the midgut surface; one is completely smooth (S-ooc) and the other is flattened (F-ooc). Note the single hemocyte attached over the completely smooth oocyst wall (arrow). Magnification = ×1400. d One completely smooth oocyst (S-ooc) with a cracked wall (arrows) and a partly cracked oocyst (asterisks) showing hundreds of escaping sporozoites (Spz). Magnification = ×1700. e Thousands of clustered sporozoites (Spz) can be seen inside and escaping from a partly cracked oocyst (white asterisk). In the upper portion of the image, an empty half-shell of a broken oocyst can be seen, in which it is possible to observe details of the internal wall (In-wall). S-ooc = completely smooth oocyst. Magnification = ×1500. f Magnified view of the dashed area of Fig. 1d, showing the porous surface of the internal wall (In-wall) of the oocyst. Spz = clusters of escaping sporozoites. Magnification = ×4500

Fig. 2

Plasmodium berghei sporozoites escaping from oocysts. a P. berghei oocysts protruding among muscle fibers (Mfb) on the external surface of the midgut. The surfaces of the oocysts are partly smooth (white asterisks) and partly wrinkled (black asterisks), with the exception of a few flattened oocysts (F-ooc). Several oocysts have openings on their wrinkled surfaces, which appears as if the wall has peeled away, and sporozoites can be seen inside the hole (arrows). Muscle fibers = Mfb. Magnification = ×1000. b Small opening (arrowhead) in the oocyst (ooc) wall showing the orderly arrangement of several sporozoites (Spz) inside the oocyst. Note the wrinkled surface of the oocyst. Magnification = ×1900. c Oocyst (ooc) with a large opening, approximately half-size, showing a sponge-like part of the dissolving oocyst wall (D-wall). Note cluster of sporozoites (Spz) ready to escape from the oocyst. Magnification = ×1900. d A single oocyst (ooc) showing a large opening with a “cap” (asterisk) that appears to allow sporozoite escape. Note cluster of sporozoites (spz) attached to the internal side of the oocyst wall (arrows). Magnification = ×1700. e Magnified view of the dashed area from figure d, showing a large opening with several orderly arranged sporozoites (Spz) attached to the internal side of the oocyst wall (In-wall). Magnification = ×4300

Fig. 3

Plasmodium vivax sporozoites escaping from oocysts. a Low-power magnification of the external surface of the midgut showing protruding P. vivax oocysts (asterisks) of similar size. The rounded oocysts are arranged individually or in pairs protruding among muscle fibers (Mfb), trachea (Tra), and tracheoles (Tch) that rest on the external surface of the midgut. Magnification = ×200. b High-power magnification of a single sporozoite actively escaping by creating a hole (arrows) in the smooth oocyst (S-ooc) wall with its anterior end (asterisk). Note the rigid perpendicular shape of the escaping sporozoite (Spz). Magnification = ×8000. c and d Tens of grouped sporozoites (arrowheads) escaping from the lateral wall of an oocyst (S-ooc). Figure d is an enlarged image of the dashed area from figure c. Note the “rigid perpendicular shape” of the escaping sporozoites (Spz). Magnifications D = ×1400 and E = ×4000. e and f Free sporozoites (Spz) with the characteristic “comma-shape,” as seen in the mosquito hemocoel. Magnifications E = ×4100 and F = ×4300. g Empty oocyst (E-ooc) showing a hole through which the sporozoites escaped from the oocyst (asterisk). Magnification = ×1800. h Two side-by-side oocysts attached to the muscle fibers (Mfb) of the midgut. One smooth oocyst shows a completely stretched wall (S-ooc) and the other shows a flattened wall (F-ooc). Magnification = ×1300

Fig. 4

Plasmodium falciparum sporozoites escaping from oocysts. a P. falciparum oocysts, with numerous completely smooth oocysts (white asterisks) and wrinkled oocysts (black asterisks) protruding among muscle fibers (Mfb), trachea (Tra), and tracheoles (Tch) that rests on the external surface of the midgut. Magnification = ×350. b High-power magnification of the dashed area from figure a showing a group of four oocysts attached to the midgut. It is possible to observe surface details of two completely smooth oocysts (S-ooc) and two wrinkled oocysts (W-ooc). Magnification = ×800. c Enlarged image of a small portion of the initial process of a single sporozoite actively escaping from a completely smooth oocyst (S-ooc) by creating a hole (arrows). Note the rigid perpendicular shape of the escaping sporozoite and the anterior tip (large arrow) of the parasite (large arrow). Magnification = ×3000. d One completely smooth oocyst (S-ooc) with small broken areas (arrows) showing a group of twelve escaping sporozoites (Spz). Magnification = ×3500. e Flattened opened oocyst (Op-ooc) showing a lateral opening (asterisk) with a cluster of escaping sporozoites (Spz) that remain inside. Note hemocytes attached to the oocyst wall (arrows) and folded areas (asterisks) of the oocyst surface. Magnification = ×1300. f Enlarged image of the dashed area from figure e showing the oocyst opening and several escaping sporozoites (Spz). Note the “comma-shaped” sporozoites. Oocyst = Op-ooc. Magnification = ×6000. g Flattened empty oocyst (E-ooc) showing the lateral opening (asterisk). No sporozoites can be seen inside or around the oocyst opening. Magnification = ×1800