Background
Plasmodium falciparum, the deadliest agent of human malaria, is exclusively transmitted by
Anopheles mosquitoes. In Africa, species belonging to the
Anopheles gambiae complex are responsible for a large proportion of malaria cases. This complex is composed of species morphologically identical but distinct in their distribution, ecology and contribution in malaria transmission. While
Anopheles merus, Anopheles melas, Anopheles bwambae and
Anopheles quadriannulatus have sporadic or null role in malaria transmission due to restricted geographical distribution and/or zoophily,
Anopheles gambiae s.s. and
Anopheles arabiensis are the most important in terms of epidemiology [
1,
2].
Anopheles gambiae s.s. itself was shown to be subdivided in incipient species, namely M and S molecular forms [
3], both vectors of malaria parasites [
4,
5]. Although these three taxa coexist in many zones, as in Senegal [
4], they have specific ecological niches and one species can be predominant on the others depending on the environmental conditions [
6,
7]. Especially at the larval stage, an habitat segregation has been demonstrated between M and S molecular forms [
8]. The biological differences between the incipient species most likely impact their vectorial capacity and their contribution in malaria transmission [
9]. A complete understanding of their role in malaria transmission would however not be possible without deciphering their relative susceptibility to the parasites.
The susceptibility of
Anopheles mosquitoes to
Plasmodium infection reflects the probability of successful parasite development from gamete fertilization to sporozoite production. During the sporogonic development steps in the mosquito midgut lumen, epithelium and haemolymph, parasites face a hostile environment, leading to a considerable reduction in the number of parasites reaching the oocyst stage [
10‐
12]. The mosquito susceptibility is the result of evolutionary process on both parasite and vector that maintains susceptible and refractoriness alleles in natural populations [
13]. Recently, a new cryptic sub-group inside
An. gambiae s.s., named 'Goundry', was identified [
14]. The exophilic behaviour of the taxa explains that it was not sampled before in dwellings. This new vector may have major importance in malaria transmission as it was found to be more susceptible to infection that the endophilic vectors. This finding highlighted that sibling species can have different levels of vector competence, which with regards to their vectorial capacity, will define their role in malaria transmission. Unfortunately, the study did not compare susceptibility of the already known species in the
An. gambiae complex.
The aim of the present study was to investigate the potential difference in susceptibility of the three malaria vectors of the An. gambiae complex in Senegal to infection by wild isolates of P. falciparum from the same area, using an in vitro model of infection.
Methods
Mosquito collection
Anopheles gambiae s.s. larvae were collected in five different breeding sites (minimum 100 larvae per site) in the village of Dielmo (13°43'N, 16°24'W). Larvae were raised until emergence; adults were fed on rabbit blood and 100 females (F0) randomly selected (20 from each collection site).
An. arabiensis larvae were sampled in one site in Dakar (14°72'N, 17°31'W). Larvae were also raised until emergence, fed on rabbit blood and 100 females (F0) randomly selected. Each F0 females was allowed to lays its eggs individually, before it was genotyped for the species and molecular forms by PCR-RFLP [
15]. According to the experiment, among the 100 selected
An. gambiae, 44 to 54 were molecular form M and 43 to 52 molecular form S (a few
An. arabiensis identified were discarded). All specimens sampled in Dakar were confirmed to be
An. arabiensis. The offspring of F0 females of the same taxa were then pooled and bred together in the same conditions for the three groups. Larvae were fed with Tetramin fish food. Pupae were collected and placed in 10-L plastic buckets, which were covered with mosquito gauze and provided with a cotton sleeve for easy access to 10% glucose on filter paper. Adults were maintained in a room at 27°C, 70% relative humidity and 12:12 h light/darkness, with a 30-mn dawn and dusk light regimen. In order to increase the proportion of mosquitoes accustomed to feeding on membrane, a selection of aggressive F1 and F2 females was performed. F3 females used for infection were all genotyped and species and molecular forms were confirmed.
Gametocyte carriers
Gametocyte carriers were detected by cross-sectional surveys in villages and schools, during the high transmission period from September to November 2006 in Hanene, (14°47'N, 16°55'W Thies region). Informed consent was obtained from adults or legal guardians of minors. Fingerprick blood was taken from each volunteer. The thick blood smears were stained with 10% Giemsa and examined microscopically with (100×) oil immersion lens for the presence of sexual and asexual parasites. Parasite density was estimated by counting against 1,000 white blood cells and converted to numbers of parasites per μL by assuming a standard white blood cell count of 8,000/μL. Symptomatic or non symptomatic individuals having an asexual density exceeding 1,000 parasites/mm3 were treated with artemisinin-based combination therapy according to national recommendations. Inclusion criteria of gametocyte carriers were: (1) age over 10 years; (2) a P. falciparum gametocyte density over 20/mm3 of blood; and (3) no anti-malarial treatment in the previous month. 6 mL of blood were drawn from the gametocyte carriers in a heparinised vacutainer tube. An insecticide-impregnated bed net was given to the participating individuals as compensation. This study was approved by the Senegalese National Ethical Committee.
Direct membrane feeding assay
Experimental infections were carried out by direct membrane feeding assays as described by Mulder
et al [
16]. Blood was rapidly distributed to three pools of three-days old females belonging to each taxa, through a warm-water (37°C) jacketed membrane feeder serially connected. Female mosquitoes were allowed to feed for 15 min before, partially fed and non-fed specimens were removed. Two batches of 50 mosquitoes of each taxa were randomly-selected from among fed females and maintained in the insectary under 10% sucrose diet for further analyses. The first batch of mosquitoes was dissected seven days later. Midguts were stained with 3% mercurochrome in PBS, and examined under light microscopy (40 × objective) for detection and quantification of oocysts. The percentage of oocyst-positive mosquitoes and number of oocysts were recorded. A second batch of mosquitoes were used to evaluated the presence of circumsporozoite protein (CSP) of
P. falciparum using an enzyme-linked immunosorbent assay (ELISA)[
17] performed on heads and thoraces 14 days after feeding. PCR RFLP [
15] were performed on the carcasses of dissected mosquitoes and the identification of molecular forms were confirmed.
Experiments were repeated four times with different samples of gametocyte-containing blood. Gametocytaemia was 413, 574, 597 and 866 gametocytes per μL, respectively for experiments 1, 2, 3 and 4.
Statistical analysis
Mortality rates were calculated after 7 and 14 days in each group. The number of oocyst was compared using non-parametric Kruskal-Wallis tests. Mortality rates, percentage of infected mosquito at oocyst and sporozoite stages were compared using Pearson Chi2 or Fisher exact test. Correlation between the number of oocyst and the gametocytemia was analysed using Spearman test. Statistical analyses were performed using Stata® 10.1. A P value of 0.05 or less was considered as significant.
Ethical approval
Experiments involving human subjects, population screenings as well as collection of blood samples, have been conducted in full accordance with ethical principles. Free and informed consent of the donors or their guardians was obtained at all times while community consent had been obtained beforehand. This study was approved by the Ethical National Comity of Senegal.
Discussion
This study is the first to evaluate the relative susceptibility of three major malaria vectors of the An. gambiae complex. It demonstrates that, in our experimental conditions, An. gambiae molecular form S is more susceptible than An. gambiae molecular form M and An. arabiensis to infection by Plasmodium falciparum at both oocyst and sporozoite stage.
Malaria transmission is known to depend on relationships that bind the pathogen, its invertebrate (vector) and vertebrate host (man). It is a complex phenomenon that implicates intrinsic factors associated with each actor as well as interactions between them [
13]. The present study focused on the intrinsic capacity of the anopheline vector to be infected. This is the reason why it was performed on an
in vitro model of mosquito infection that allowed a specific study of the ability of each vector to be infected [
16]. Most extrinsic factors and parameters depending on the parasite and on the human host were controlled. As a matter of fact, in each experiment performed, the same parasite isolate was present in the same blood sample. The impact of environmental factors was also controlled since all the feeding experiments were conducted in uniform laboratory conditions. The results demonstrate that, these experimental conditions, the rate of infection was higher in molecular form S than in M form and
An. arabiensis at both oocyst and sporozoite stages. The intensity of the infection was also higher in S form than M form and
An. arabiensis as shown by higher mean number of oocyst. Although variations in infection rate and intensity were observed in the different experiments (representing the variability of parasitological and human factors), a similar difference between taxa was detected. This indicates that species and molecular forms belonging to
An. gambiae complex did not display the same behaviour toward infection by
P. falciparum. The difference observed between molecular forms M and S of
An. gambiae and
An. arabiensis was only related to their intrinsic susceptibility to
P. falciparum infection. It is important to consider that although the method used for this study is the best available to evaluate susceptibility, it necessitates breeding mosquitoes in the insectary. Tested specimens belonged to the 3
rd generation and have, therefore, adapted to laboratory conditions. They may not be exactly representative of field populations.
During this study, mortality rate was different between species and forms. Although this parameter was not specifically studied, since all mosquitoes were potentially infected, mortality may have been influenced by the intensity of the infection.
Plasmodium infection has been shown to reduce vector survival in laboratory conditions [
18]. In this study,
An. gambiae S form mosquitoes which were the most infected were also the less surviving at both oocyst and sporozoite stage. Even though many parameters have to be taken into consideration when evaluating malaria transmission in the field, the most efficient vector will be the one mostly highly infected and longest lived. The divergence of these two parameters among
An. gambiae S and M forms could explain why dynamics studies of transmission have failed to demonstrated a difference between M and S forms [
4,
5]. The mechanisms responsible for parasite-induced mortality should be closely evaluated and taken into account in future anti-vectorial strategies.
The outcome of infection depends on the balance between the vector immune response, that aims to limit the infection [
19,
20], and the ability of the parasite to evade them [
13]. The observed difference in susceptibility level among anopheles species to
Plasmodium infection could be related to various levels of immune response of the vector. A variety of genes implicated in the immune response of the vector have been identified [
21,
22] and their allelic variants associated to relative refractoriness to infection [
23]. A study on the repartition of the allelic variant of these gens in the different taxa inside
An. gambiae complex could highlight their potential role in the difference susceptibility observed in this study. Beyond the M and S classification, the individual variation of susceptibility is a factor that should be investigated. The selection of resistant individuals may leads to refractory mechanisms at the molecular level, which could be exploited in the development of novel approaches to malaria control.
Susceptibility is known to increase with frequent and intense contact between parasite and vector because of evolution pressure of the parasite on the host [
13]. This co-adaptation is also known to be dependent on the environment. Therefore, it would be interesting to study the relative susceptibility of molecular form M and S of
An. gambiae from other areas or even their susceptibility to
Plasmodium strains from distant regions. Interestingly, a large range of oocyst number was observed in experiment 2 in
An. gambiae molecular form S. In the future, the relative variability of the susceptibility in the different taxa should be investigated.
Acknowledgements
We thank Charles Bouganali, Moussa Gueye and Moise Gnafouna for their technical assistance, the villagers in Hanene, Sian Clarke for the correction of the manuscript and Didier Fontenille and Isabelle Morlais for their help in the conception of the study.
Funding
This work was supported by the program Pal + of the French Ministry of Research and the Department Support and Formation of the south communities of the Research Institute for the Development (IRD).
Authors' contributions
MON, JFT, CS and CB have equally contributed to the design, acquisition, analysis and interpretation of data. LK, AG and CS contributed to the conception of study and to the analysis of entomological data. AC performed field activities and molecular biology study. OF designed the study protocol. MON, CM and AC drafted the manuscript. CS provided the scientific supervision in Dielmo and Hanene. All authors read and approved the final manuscript.