Background
Malaria remains a life-threatening disease that threatens approximately 3.4 billion people in 104 tropical countries, mainly in Africa, Asia, and South America, with an estimated 207 million cases and half a million deaths reported per year [
1]. This vector-borne disease is caused by protozoa of the genus
Plasmodium, of which
Plasmodium falciparum, endemic to Africa, is the most prevalent species, followed by
Plasmodium vivax in Asia and the Americas [
1]. Other
Plasmodium species infect other animal species, such as
Plasmodium gallinaceum and
Plasmodium berghei, responsible for avian and murine malaria, respectively [
2,
3]. Many experimental studies have used
P. berghei and
P. gallinaceum as laboratory models to investigate the interactions between the parasites and their vectors. These two
Plasmodium species are easily maintained in experimental animals, facilitating investigative research in laboratories [
4‐
8].
The
Plasmodium life cycle begins in a permissive vector when a female mosquito takes a blood meal from an infected vertebrate host that contains gametocytes, the stage of the parasite that can infect the invertebrate vector. Only a few minutes after the infective blood meal enters the midgut lumen of the susceptible mosquito, these gametocytes undergo activation to generate micro- and macro-gametes that fertilize to produce a diploid zygote. After DNA replication and the production of a 4N parasite, the zygote will differentiate into an ookinete over the next 18-24 h depending on the respective parasite species. Ookinetes are a motile form of the parasite that invade and pass through the midgut epithelium until they reach the midgut basal lamina towards the haemocoel of the mosquito. At this location, between the epithelial cells of the midgut and the basal lamina, the ookinete differentiates into a protruding rounded oocyst facing the mosquito haemocoel [
8‐
12]. The presence of well-developed protruding oocysts in the midgut wall is indicative of infection by
Plasmodium [
13‐
15], and is a reliable measurement to determine the infection rate and the susceptibility of a mosquito species to a particular
Plasmodium species. In the midgut wall, the oocysts progress to the asexual phase of multiplication known as sporogony, which is completed in approximately 1–2 weeks, the longest phase of the
Plasmodium life cycle in the mosquito vector. Ultimately, this biological process produces thousands of sporozoites, the final form of
Plasmodium in the vector. The sporozoites are motile sickle forms that escape from the oocysts into the mosquito hemocoel and invade the salivary gland. Once inside the salivary gland, the sporozoites are ready to be injected into a new vertebrate host via a mosquito bite, completing the
Plasmodium life cycle in the invertebrate vector [
16‐
18].
Completion of the
Plasmodium life cycle in the vector requires passage through several barriers inside and outside the midgut. One important and poorly studied barrier is the exit of sporozoites from the oocyst, a critical step that allows sporozoite release into the haemolymph and subsequent invasion of the mosquito salivary gland. Knowledge of the escape mechanism of various
Plasmodium species is largely unknown for the human malaria parasites, and only a few reports using the laboratory models have previously been published. Studies of the development of
P. berghei oocysts using a scanning electron microscope (SEM) showed a single small hole in the oocyst wall, inside which sporozoites could be seen [
19]. Sinden and Strong reported a torn oocyst from which several
P. falciparum sporozoites had been released [
20]. Meis and collaborators studying the sporogony of
P. falciparum and
P. berghei, reported some details of sporozoite escape and concluded that the two species showed similar mechanisms of escape, i.e., the oocysts burst and sporozoites were released into the hemocoel of the mosquito vector [
21]. Although published studies have provided some details, knowledge of sporozoite escape from the oocysts of distinct
Plasmodium species remains incomplete and is primarily based on
P. berghei, a classical murine malarial parasite used as an experimental model in several laboratories. Moreover, most of the studies on the molecular mechanism of oocyst formation and sporozoite escape have been done using murine
P. berghei mutant parasites, resulting in conclusions that have been generalized to human
Plasmodium species without further morphological study.
Understanding the mechanisms of sporozoite escape in various Plasmodium species as well as correlations with molecular findings, may contribute to our knowledge of the parasite life cycle in the mosquito vector. Scanning electron microscopy analysis of the external side of the dissected midguts of infected mosquitos is a valuable tool for studying sporozoite escape from oocysts and has not been well explored. Here, this study provides comprehensive insight into the microanatomy of the mechanism of sporozoite escape from oocysts in four species of Plasmodium: the two laboratory models, avian P. gallinaceum and rodent P. berghei, and the two primary causative agents of human malaria, P. vivax and P. falciparum. It was showed that sporozoite escape is not a common biological process, as previously thought, but the mechanism is complex and species-specific.
Discussion
The longest developmental stage of the
Plasmodium life cycle in the mosquito vector is sporogony, the process of formation of thousands of sporozoites. A single parasite invades the epithelium of the midgut of a mosquito vector and remains in the gut wall for several days. This single-celled protozoan remains outside the mosquito cells, and grows into a large-lobed syncytial nucleus by mitotic division, inside a structure named the oocyst, which forms mature sporozoites. These mature sporozoites escape from the oocysts into the mosquito cavity, after which they invade the salivary gland in preparation for injection in a new vector. The duration of this stage of the
Plasmodium life cycle varies according to the species, but usually lasts 8–14 days after the mosquito vector has ingested the infective blood meal [
7‐
9,
13,
27].
In this study, to examine the microanatomy of sporozoite escape from oocysts of the four Plasmodium species, 10–20 midgut sections were dissected daily from infected mosquito vectors, 6–16 days after the infective blood meal. The midgut samples were dissected, fixed, and processed in the same laboratory, following an identical rigorous protocol to facilitate comparative analyses. The microanatomical analyses presented here clearly and accurately show the ultrastructural aspects of the oocyst surfaces and the processes of sporozoite escape. Preliminary analyses revealed that in all Plasmodium species, the oocysts are rounded structures that protrude individually or in small groups from the exterior of the midgut wall of the mosquito vector. However, the oocysts of the four Plasmodium species differ in surface features of the external wall and in the process of sporozoite escape.
In the avian parasite
P. gallinaceum, the outer surfaces of all oocysts were completely smooth. During the process of sporozoite escape,
P. gallinaceum oocysts were cracked, suggestive of internal forces disrupting the oocyst wall from the inside. The broken oocysts were similar to broken eggs, exposing their internal surface, with subsequent release of large groups of sporozoites into the mosquito cavity. In contrast, all murine
P. berghei oocysts showed a hybrid surface, wrinkled on the top and smooth on the base. Compared to
P. gallinaceum, P. berghei sporozoites appear to have a less violent mechanism of escape from the oocysts. On the upper, wrinkled surface of the oocysts, a small part of the wall begins to decorticate, creating a small opening, followed by progressive dissolution of the oocyst wall. Then, the highly structured clusters of sporozoites detach from the internal oocyst wall. In the murine and avian species of
Plasmodium, the final steps of the sporozoite escape process, no empty oocysts were observed, distinct from the species of
Plasmodium that infect humans. Only one comparative study of
P. gallinaceum and
P. berghei oocysts has been published [
19]. In both
Plasmodium species, both completely smooth oocysts and rare, wrinkled oocysts were observed, which the authors considered matured oocysts or sample preparation artifacts. Although they only showed two images, they suggested these two
Plasmodium species have similar sporozoite escape mechanisms.
All
P. vivax oocysts showed similar completely smooth surfaces, and in this respect, they are morphologically similar to
P. gallinaceum oocysts. In contrast, two types of
P. falciparum oocysts were observed: completely smooth and wrinkled oocysts. These oocysts were randomly distributed, sometimes side-by-side, in the mosquito midgut at a 50:50 ratio. Previous studies found that infected
P. falciparum mosquitoes contained only wrinkled oocysts, but no escaping sporozoites were observed [
20,
21]. The authors suggested that the wrinkled surface was characteristic of mature oocysts. However, although we also observed two types of oocysts in
P. falciparum, sporozoites were only observed escaping from completely smooth oocysts, indicating that completely smooth oocysts contain mature sporozoites. The wrinkled oocysts may be immature oocysts or oocytes that cannot produce healthy, mature sporozoites.
The most noteworthy feature of the two human
Plasmodium species,
P. vivax and
P. falciparum, is the dynamic mechanism of sporozoite escape from oocysts, distinct from that of the laboratory model
Plasmodium species. Careful observation showed that the first signals of sporozoite escape are identical for the two human
Plasmodium species: escape begins with a single sporozoite, in a rigid perpendicular position, forcing an exit from through the oocyst wall. The rigid perpendicular sporozoite opens a tiny hole in the oocyst wall with its anterior end. The oocyst wall is composed of two layers; the internal layer is of
Plasmodium origin and the external thick layer that is derived from the basal lamina of the mosquito midgut [
28,
29]. Moreover, in addition to allowing for growth, the capsule must have an ordered structure to allow for precursors and nutrients that support parasite growth and differentiation to enter the oocyst and metabolites to exit it [
30,
31]. Subsequently, this tiny hole in the oocyst wall grows larger and allows other sporozoites to escape. Although this first step, with a single sporozoite making a tiny hole in the oocyst wall, is identical between the two species, the subsequent steps of sporozoite escape differ between
P. vivax and
P. falciparum. In
P. vivax, a small group of sporozoites continue, in the same rigid perpendicular position as the first, to actively move forward to enlarge the hole in the oocyst wall. In
P. falciparum oocysts, small groups of sporozoites escape, and individual sporozoites are flexible comma shapes, characteristic of random motion of the parasite [
32‐
34]. A geometrical model of malaria parasite migration demonstrated that sporozoites could be modeled as self-propelled individuals that can have curved or rigid structures for motion in distinct environments [
35]. This programmed rigidness and flexibility of the human
Plasmodium sporozoites appears to act distinctly in the two species of
Plasmodium, since it plays a role in opening the oocyst wall, allowing escape.
Molecular mechanisms related to oocyst formation and sporozoite escape have been demonstrated, mainly using mutants of murine
P. berghei, which infects rodents, but not in
Plasmodium species that infect humans. It is important to note that these analyses demonstrate that
P. berghei sporozoites escape from oocysts by a process that harms the oocyst wall. The circumsporozoite (CS) protein, secreted by sporozoites, covers the internal layer of the oocyst wall [
36,
37]. It was demonstrated in
P. berghei that the disruption or deletion of some regions of the CS protein affects the formation and maturation of sporozoites, escape from the oocyst, and subsequent progression of the
Plasmodium life cycle [
38,
39]. Likewise, several other gene deletions have been described that affect
P. berghei oocyst formation and consequent sporozoite escape: an oocyst-specific papain-like cysteine protease, known as the egress cysteine protease (ECP1), oocyst capsule protein (PbCAP380), fertilization gene (Pb GEX), lectin adhesive proteins (PbLAPs), protein kinases (PbCDLK), and nuclear forming-like protein (PbMISFIT) [
40‐
48]. The results showed that
P. berghei sporozoites are liberated from the oocyst by decortication and subsequent dissolution of the oocyst wall, which is consistent with a mechanism involving a proteolytic activity as has been proposed for
P. berghei [
42]. Thus, these findings indicate that proteins that act on the oocyst wall, rather than in the sporozoite, should be considered as target candidate molecules to stop transmission.
Analyses of the sporozoite escape processes in the
Plasmodium species that infect humans clearly showed the action of the actively protunding sporozoites is dissimilar from that of murine and avian
Plasmodium species.
Plasmodium belongs to the phylum Apicomplexa, which is well defined by polarized extracellular stages, which contain specialized secretory organelles named micronemes and rhoptries in their anterior edge. Proteins secreted by these organelles play essential roles in attachment and invasion of target cells, as well as gliding motility, locomotion, and morphological changes [
33,
49‐
52]. The main mode of active locomotion of the sporozoite is an actomyosin-dependent motility that is important for forward locomotion, and penetration and invasion of target cells [
53]. In addition, during sporozoite motility, TRAP may coordinate the formation of contact sites and the dissociation of these contact sites from the substrate, including involvement of actin filaments [
54,
55]. This raises the possibility that secretory proteins that are involved in the interplay of adhesion molecules and the invasion mechanism, well studied in invasion of host cells, can also play roles in the initial active stage that guides the escape of
P. vivax and
P. falciparum from the oocyst.
Careful comparative microanatomical analyses of midguts of mosquitos infected with four distinct
Plasmodium species allowed us to make novel observations of sporozoite escape from oocysts. The key findings of this study are the morphological features that reveal for first time the mechanisms of sporozoite escape from oocysts of four
Plasmodium species, including avian, murine, and human malarial parasites. Sporozoites of the four
Plasmodium species exit oocysts using different mechanisms. The avian
P. gallinaceum and murine
P. berghei have been used as experimental models in several laboratories for infection of vertebrates and mosquito vectors. Mice infected with
P. berghei have been used as laboratory models for human malaria [
56‐
58] and to investigate interaction of the parasite with vectors of human malaria such as
An. gambiae and
An. stephensi [
59‐
61]. It is important to state that the findings of the escape of
P. berghei and
P. falciparum sporozoites from oocysts were obtained from experimental infections of the same mosquito species, the
An. gambiae. This fact suggests that the distinct mechanisms of the sporozoite escape is not dependent of the
Anopheles species but is regulated by the
Plasmodium species. Nevertheless, it is noteworthy to consider that these
Plasmodium species differ in the oocyst microanatomical appearance and in the process of the sporozoite escape. Although the molecular mechanism that regulates sporozoite escape remains largely unknown, this study clearly indicates that
Plasmodium species do not share a common mechanism, as previously thought.
Authors’ contributions
ASO, RNP, LCP, KMMC, YTP, BC, RCS carried out the infection experiments of the mosquitoes, participated in the microscopy analysis and drafted the manuscript. LMV, APMD, NBR, MGVB, WMM, AMC participated in the analysis of the results and drafted the manuscript. MVGL, NFCS, MJL, CBM, participated in the design of the study, performed the analysis and helped to draft the manuscript. PFPP conceived the study, in its design and coordination and write the manuscript. All authors read and approved the final manuscript.