Background
Almost half of the world’s population is at risk of malaria infection caused by the intracellular parasite
Plasmodium. The World Health Organization (WHO) estimated 219 million cases for 2017, representing a 2 million case increase compared to 2016 [
1]. Two hosts are involved in the parasite’s biological cycle: the mosquito vector (where parasite sexual differentiation occurs) and the human host (where asexual replication prevails) [
2]. The parasite’s sexual phase (related to human-vector transmission) starts just after the female
Anopheles mosquito’s uptake of infected erythrocytes having parasite sexual forms (gametocytes) after biting an infected human. Sexual form fusion (macrogamete and microgamete) occurs inside the mosquito midgut; gametes then mate to form zygotes, which transform into ookinetes and embed themselves within insect midgut epithelial cells [
3,
4]. Physiological conditions occur simultaneously during the ookinete stage thereby facilitating parasite mobility and oocyst formation and maturation; ookinetes differentiate into oocysts and release large amounts of sporozoites into the haemocoel upon maturation which are responsible for mosquito vector-human transmission [
3,
5].
Gametocyte blockade is thus considered a good target for vaccine development because it is intended to stall the disease at human-vector transmission level [
6,
7]. Small quantities of gametocytes circulate in infected humans’ bloodstreams, being exposed to the host immune system; ookinetes could come into contact with the mosquito’s immune system that includes a complement-like immune response [
8‐
10]. The host’s immune response gradually controls
Plasmodium dispersal by reducing the amount of circulating gametocytes and limiting parasite development inside the mosquito vector, making this
Plasmodium sexual stage an important biological bottleneck [
11,
12]. However, even with such biological control, natural response against the parasite leaves asymptomatic human parasite carriers (i.e. an important cause of transmission) [
7,
8]. Parasite culture and the biological study of sexual-stage parasite antigens are important for identifying and selecting effective vaccine candidates to stop this never-ending infection cycle [
13].
It is known that infection susceptibility and disease transmission through
Anopheles is determined by the genetic characteristics of the mosquito and the parasite [
4]. Mosquito immune pressure generates genetic changes in
P. falciparum, thereby enabling its gradual adaptation to mosquitoes from different geographical regions [
11]. Parasite-vector interaction has been extensively followed up in African mosquitoes because of the ease of parasite isolate availability and the ease of establishing mosquito colonies to facilitate studying the mosquito’s immune response [
14‐
17]. Comparative analysis of parasite compatibility with mosquitoes from Africa and Southeast Asia has found parasite adaptive mechanisms regarding each
Anopheles species; a similar approach has been followed with the Latin-American
P. falciparum strain 7G8 and
Anopheles albimanus [
15]. Even so, knowledge is still lacking about the interaction of other Latin-American parasite strains with New World mosquitoes [
18]. This has mostly been due to difficulties in establishing
Anopheles colonies (from the New World, especially regarding epidemiologically important mosquito strains) and also the lack of parasite strains from this geographical area having characterized sexual differentiation capability [
18‐
21].
Plasmodium falciparum strains can be stimulated in vitro for gametocyte production; these sexual forms could be used in antibody invasion inhibition studies and testing candidate antigens for vaccine development [
22]. Some
P. falciparum strains (such as NF54) continuously cultured in vitro keep their sexual differentiation capability; however, many lose it because of spontaneous genetic mutations of sexual differentiation-related transcription factors, like the apetala 2-gametes (
ap2-
g) transcription factor [
23].
A group of Colombian
P. falciparum isolates were adapted to continuous in vitro culture more than 30 years ago; the
falciparum Colombia Bogotá 2 (FCB2) strain (an in vitro culture-adapted isolate from Colombia’s Eastern Plains) from that group was described as having sexual differentiation capability [
24]. This strain has been used for antigen analysis when developing an anti-malarial vaccine and in studies of the human immune response against the parasite [
25‐
27]. This strain has been maintained in in vitro continuous culture since then but it was not known if it conserved its sexual differentiation ability or whether sexual forms could evolve to mature forms and infect local
Anopheles species [
24].
The purpose of this study was thus to induce Colombian FCB2 strain gametocyte production and prove its infective ability by controlled female
Anopheles mosquito infection using an artificial feeding system involving parasitized erythrocytes. These FCB2 strain sexual forms (having mosquito infective capability) could thus be used in comparative studies with other
P. falciparum strains for evaluating antibodies produced against antigens, representing promising candidates for blocking malarial transmission [
28,
29]. This information increases knowledge about this specific Colombian parasite strain and provides another tool for developing anti-malarial drugs and vaccine candidates tackling parasite transmission.
Discussion
Malaria still remains a critical infectious disease because of the stalemate in controlling its progress since 2015 [
1]. This problem has mostly been associated with the appearance of parasite resistance to anti-malarial treatment and the mosquito’s resistance to currently available insecticides [
52,
53]. Asymptomatic patients (associated with silent host-vector transmission) are related to the disease’s epidemiological persistence thereby highlighting an increasing need for tools enabling the study of parasite transmissible forms [
54‐
57].
As the parasite’s sexual forms are directly related to host-vector transmission, the in vitro study of antigens blocking this parasite stage is important for attacking this infection. Most studies usually involve using
P. falciparum strains which have an already described differentiation capability [
43,
58,
59]. Many studies use the NF54 parasite strain because of the ease of gametocyte production; some approaches in Latin-America have used the 7G8 strain [
18,
19,
43]. Increasing the amount of characterized
P. falciparum strains from other geographical regions having sexual differentiation ability might upgrade variability analysis and provide a better response to the need for anti-malarial drugs and vaccines.
The
P. falciparum FCB2 strain was adapted from a severe malaria patient’s isolate and has been kept in in vitro culture for more than 30 years [
24]. Most in vitro cultured parasite strains lose their sexual differentiation ability because of mutations in genes associated with the proteins needed for it, such as
Pfap2-
g [
37]. This study has analysed the
P. falciparum Colombian FCB2 strain’s sexual differentiation capability by initially verifying
Pfap2g,
Pfs16,
Pfg27/25 and
Pfs25 gene expression used for detecting infected patients who could have gametocytes and female parasite sexual forms (Fig.
1) [
39‐
42,
60]. It is worth noting that the FCB2 strain has preserved its gametocyte production (although to a low degree: 0.2% gametocytes after 12 days culture) after more than 30 years of in vitro culture; it has conserved its characteristic phenotype during each gametocyte stage as seen in other sexually differentiated parasite strains (Fig.
2). These gametocytes were able to form zygotes and ookinetes and exflagellate after in vitro culture with low-temperature stimuli (Figs.
2,
3,
4).
The results highlighted the FCB2 strain’s differentiation ability and indicated its ability to infect mosquitoes. FCB2 strain gametocytes infected Colombian
An. albimanus and
An. stephensi using a controlled artificial mosquito feeding system; they differentiated into oocyst forms inside mosquito midgut, thereby confirming the conservation of the mosquitoes’ infection ability (Figs.
6 and
8). It is known that culture conditions influence gametocyte formation, i.e. erythrocyte percentage, hypoxanthine and glucose concentration [
22,
61,
62]. Serum also influences gametocyte production; gametocyte production and their infectivity become decreased when using serum replacement substances [
63]. Probably, such conserved FCB2 gametocyte production could have been associated with culture maintenance conditions, mostly related to parasite culture media always being supplemented with human plasma and this might have helped conserve this feature. Considering the implications of in vitro culture conditions regarding gametocytogenesis, it could be supposed that such conditions may also affect oocyst growth-associated genes, thereby causing impaired development of most of FCB2 oocysts. Genetic comparative analysis comparing high (e.g. NF54) and low (e.g. FCB2) oocyst-producing strains may help to resolve this question and also support the study of possible targets for anti-malarial drugs and vaccine development.
The FCB2 strain’s host geographical origin could also have influenced the amount of infected
An. albimanus females and could have been related to the high percentage of oocysts recorded in this study. Mosquito infection potential studies regarding some parasite strains from different regions worldwide have shown that malarial transmission success directly depends on the geographical origin of mosquitoes and parasites [
64‐
66]. However, further studies are needed (like the standard membrane-feeding assay using different strains) to confirm this hypothesis and confirming the compatibility between this strain and the geographic origin of
An. albimanus.
A mosquito’s parasite infectivity is related to the parasite’s genetic factors enabling mosquitoes to avoid host innate immune response resulting from coevolution of both organisms [
15]. Nevertheless, variation in mosquito infectivity has been reported when using
P. falciparum isolates from the same geographical area, e.g. African strain NF54 infected 90% of
Anopheles gambiae, Kenyan strain K39 infected 86% of
An. gambiae, whilst M24 infected only 6% of the same mosquito species [
15,
66]. Such huge difference regarding mosquito infection might be explained by variations in parasite strain infection susceptibility associated with mosquito immune response; specifically, increased
An. gambiae thioester-containing protein 1 (TEP1) has been shown to be involved in oocyst killing, whilst parasite polymorphism in the
Pfs47 gene has enabled evading mosquito immune response [
67].
Reports have shown that female
An. albimanus infection with the 7G8 Brazilian strain was 68% and average oocyst production was 2 oocysts; such production was small compared to the FCB2 strain studied here (56 oocysts by day 12) and highlighted differences regarding compatibility between South American parasite strains and mosquitoes from the same region [
11,
68]. Mosquito innate immune defence mechanisms may influence midgut epithelial ookinete invasion [
69,
70]; mosquito immune responses could thus be related to the aforementioned study’s findings. Gametocytaemia, mosquito midgut xanthurenic acid concentration, haemozoin concentration, temperature and other intrinsic mosquito characteristics also influenced FCB2 sporogony formation inside
An. albimanus midgut [
36,
71]; this might explain the large amount of these forms found on initial days post-feeding compared to the small amount of such forms which finally developed. Moreover, the
Anopheline late immune response against oocysts has been described in other mosquito species; haemocytes have been responsible for reduced parasite survival, using unknown mechanisms [
72]. Studying haemocyte cell defence response in
An. albimanus could be interesting for recognizing its cellular immunity.
Acknowledgements
The authors would like to thank Doctors Michael Delves and Diana Díaz for their help regarding parasite maintenance and gametocyte production and Doctor Ligia Inés Moncada for her helpful hints for mosquito breeding; Bei Resources for donating the P. falciparum 3D7 strain; the Instituto Nacional de Salud (INS) de Colombia for An. albimanus egg donation; Doctors Ana Catarina Alves, Henrique Silveira and João Pinto from the Instituto de Higiene e Medicina Tropical, Universidade Nova de Lisboa, Portugal, for donating An. stephensi eggs; the Instituto Distrital de Ciencia, Biotecnología e Innovación en Salud (IDCBIS) for donating the human plasma; Doctor Armando Moreno-Vranich for improving the quality of the images, Doctors Iván Darío Vélez and María Alejandra Vélez from PECET, Universidad de Antioquia, for helping us to restart mosquito infection experiments and Jason Garry for revising the manuscript.
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