Christian Boudin

Immune response of Anopheles gambiae to the early sporogonic stages of the human malaria parasite Plasmodium falciparum

Deciphering molecular interactions between the malaria parasite and its mosquito vector is an emerging area of research that will be greatly facilitated by the recent sequencing of the genomes of Anopheles gambiae mosquito and of various Plasmodium species. So far, most such studies have focused on Plasmodium berghei, a parasite species that infects rodents and is more amenable to studies. Here, we analysed the expression pattern of nine An.gambiae genes involved in immune surveillance during development of the human malaria parasite P.falciparum in mosquitoes fed on parasite‐containing blood from patients in Cameroon. We found that P.falciparum ingestion triggers a midgut‐associated, as well as a systemic, response in the mosquito, with three genes, NOS, defensin and GNBP, being regulated by ingestion of gametocytes, the infectious stage of the parasite. Surprisingly, we found a different pattern of expression of these genes in the An.gambiae–P.berghei model. Therefore, differences in mosquito reaction against various Plasmodium species may exist, which stresses the need to validate the main conclusions suggested by the P.berghei–An.gambiae model in the P.falciparum–An.gambiae system.

Estimation of malaria transmission from humans to mosquitoes in two neighbouring villages in south Cameroon: evaluation and comparison of several indices

Malaria transmission from humans to mosquitoes was assessed in two neighbouring villages in a rural area near Yaoundé, Cameroon during high and low transmission seasons during 1998-2000, using several indices previously evaluated in different areas endemic for malaria but never directly compared. These indices were estimated from human parasitological data and mosquito infection rates and, for each individual, thick blood films were prepared at the same time as experimental infection of laboratory-bred mosquitoes. Among the 685 volunteers examined, the prevalence of Plasmodium falciparum gametocyte carriers was 16%, and 8% of individuals were able to infect mosquitoes. The percentage of mosquitoes that became infected by feeding on the infectious individuals was 21%. Children aged < 10 years contributed to about 75% of the infectious reservoir, although they constituted only 35% of the total population. Differences were found between the transmission seasons and the villages, and varied according to the index examined. Although there were more infectious individuals in one of the two villages, they were less infectious than those in the other village during the high transmission season. Comparative analysis of the transmission indices suggests the existence of functioning transmission-blocking immunity in one of the villages, which until now has been only hypothetically considered to play a role in malaria transmission in a natural setting. The epidemiological value of all the indices used and their accuracy in estimating the human infectious reservoir and its natural or induced variations are discussed.

Carboxypeptidases B of Anopheles gambiae as targets for a Plasmodium falciparum transmission-blocking vaccine.

ABSTRACT Anopheles gambiae is the major African vector of Plasmodium falciparum, the most deadly species of human malaria parasite and the most prevalent in Africa. Several strategies are being developed to limit the global impact of malaria via reducing transmission rates, among which are transmission-blocking vaccines (TBVs), which induce in the vertebrate host the production of antibodies that inhibit parasite development in the mosquito midgut. So far, the most promising components of a TBV are parasite-derived antigens, although targeting critical mosquito components might also successfully block development of the parasite in its vector. We previously identified A. gambiae genes whose expression was modified in P. falciparum-infected mosquitoes, including one midgut carboxypeptidase gene, cpbAg1. Here we show that P. falciparum up-regulates the expression of cpbAg1 and of a second midgut carboxypeptidase gene, cpbAg2, and that this up-regulation correlates with an increased carboxypeptidase B (CPB) activity at a time when parasites establish infection in the mosquito midgut. The addition of antibodies directed against CPBAg1 to a P. falciparum-containing blood meal inhibited CPB activity and blocked parasite development in the mosquito midgut. Furthermore, the development of the rodent parasite Plasmodium berghei was significantly reduced in mosquitoes fed on infected mice that had been immunized with recombinant CPBAg1. Lastly, mosquitoes fed on anti-CPBAg1 antibodies exhibited reduced reproductive capacity, a secondary effect of a CPB-based TBV that could likely contribute to reducing Plasmodium transmission. These results indicate that A. gambiae CPBs could constitute targets for a TBV that is based upon mosquito molecules.

The early sporogonic cycle of Plasmodium falciparum in laboratory‐infected Anopheles gambiae: an estimation of parasite efficacy

This study investigated the successive losses in the parasite densities of Plasmodium falciparum stages during the early sporogony in laboratory‐reared Anopheles gambiae infected by membrane feeding with blood from naturally infected gametocyte carriers (>50 gametocytes/mm3). The developmental stages of P. falciparum in the mosquito were studied from zygote to oocyst, by immunofluorescent method using monoclonal antibodies against the Pfs25 protein present on the surface of newly formed gametes. This method allows for assessment of the various sporogonic stages before, during and after passage of the midgut wall. Parasite densities were determined within the entire blood meal at 3 h (zygotes and macrogametes) and 24 h (ookinetes) post‐infection. At 48 h after the mosquito blood meal, midguts were checked for the presence of early oocysts. For the mid‐size oocysts count, classic microscopy examination was used at day 7 post‐infection. The parasite efficacy was estimated by following successive losses in parasite densities between different early stages of the sporogonic cycle in A. gambiae. Thirty‐seven experimental infections were realized with high gametocyte densities, ranging from 64 to 2392 gametocytes/mm3. All gametocyte carriers showed infection with round forms 100%; ookinetes were found in 91.9%. The prevalences of infections with oocysts were 48.6% at day 2 (young oocyst) and 37.8% at day 7 (mid‐size oocyst). The mean densities per mosquito for each parasite stage were 12.6 round forms, 5.5 ookinetes, 1.8 young oocyst and 2 mid‐size oocysts. Significant correlations were found between two consecutive parasite stages (round forms/ookinetes, ookinetes/young oocysts, young oocysts/mid‐size oocysts) and between round forms and mid‐size oocysts. The mean parasite density significantly decreased between round forms and ookinetes (yield Y1 = 41.6%) and between ookinetes and young oocysts (Y2 = 61.4%). By contrast, no significant decrease was observed between young oocysts and mid‐size oocysts (Y3 = 91.2%). The overall yield of the early sporogonic cycle (from round form to oocyst at day 7) was equal to 25.7%, indicating that almost 3/4 of the total parasites were lost during the early step of the sporogonic cycle, from 3 h post‐infection to day 7.

Comparison of artificial membrane feeding with direct skin feeding to estimate infectiousness of Plasmodium falciparum gametocyte carriers to mosquitoes

Human infectiousness to mosquitoes can be estimated by 2 tests: direct feeding on the skin and membrane feeding on venous blood. To validate the membrane feeding assay, the infectiousness of Plasmodium falciparum gametocyte carriers to Anopheles gambiae was estimated by these 2 methods in the same individuals in a rural area of Cameroon. Results from 37 experiments showed that direct feeding gave significantly higher infection rates than membrane feeding. We observed an average of 19.4% infected mosquitoes by direct feeding compared with 12.1% by membrane feeding, and a mean oocyst load of 5.63 by direct feeding compared with 2.65 by membrane feeding. However, there was a very good concordance between the 2 tests: 84.3% with the Kappa test on percentages of infected mosquitoes and 98.7% with the interclass correlation coefficient on oocyst loads. In addition, we found a good linear correlation between the 2 methods.

Stage-specific effects of host plasma factors on the early sporogony of autologous Plasmodium falciparum isolates within Anopheles gambiae

Quantitatively assessing the impact of naturally occurring transmission‐blocking (TB) immunity on malaria parasite sporogonic development may provide a useful interpretation of the underlying mechanisms. Here, we compare the effects of plasma derived from 23 naturally infected gametocyte carriers (OWN) with plasma from donors without previous malaria exposure (AB) on the early sporogonic development of Plasmodium falciparum in Anopheles gambiae. Reduced parasite development efficiency was associated with mosquitoes taking a blood meal mixed with the gametocyte carriers’ own plasma, whereas replacing autologous plasma with non‐immune resulted in the highest level of parasite survival. Seven days after an infective blood meal, 39.1% of the gametocyte carriers’ plasma tested showed TB activity as only a few macrogametocytes ingested along with immune plasma ended up as ookinetes but subsequent development was blocked in the presence of immune plasma. In other experiments (60.9%), the effective number of parasites declined dramatically from one developmental stage to the next, and resulted in an infection rate that was two‐fold lower in OWN than in AB infection group. These findings are in agreement with those in other reports and go further by quantitatively examining at which transition stages TB immunity exerts its action. The transitions from macrogametocytes to gamete/zygote and from gamete/zygote to ookinete were identified as main targets. However, the net contribution of host plasma factors to these interstage parasite reductions was low (5–20%), suggesting that irrespective of the host plasma factors, mosquito factors might also lower the survival level of parasites during the early sporogonic development.

Level and dynamics of malaria transmission and morbidity in an equatorial area of South Cameroon

We conducted parasitological and entomological malaria surveys among the population of Mengang district in southern Cameroon to analyse the relationship between malaria transmission intensity and malaria morbidity. We investigated two adjacent areas which differ 10‐fold in transmission intensity [annual entomological inoculation rate (EIR) 17 vs. 170], but have very similar Plasmodium falciparum malariometric profiles with parasite prevalences of 58 vs. 64%, high parasitaemia prevalences (> 1000 parasites/μl) of 15 vs. 16% and the same morbidity of 0.17–0.5 attacks/person/year. Plasmodium malariae prevalence was 14 vs. 16%. One possible explanation is that the similarity of the duration of the short and high transmission seasons in both areas is equally, if not more, significant for parasitological and clinical profiles as the annual EIR. We discuss the relationships between variations in transmission levels, parasitaemia and clinical incidence, and draw parallels to similar situations elsewhere.

Transcripts of the malaria vector Anopheles gambiae that are differentially regulated in the midgut upon exposure to invasive stages of Plasmodium falciparum

Understanding the interactions between the most deadly malaria parasite, Plasmodium falciparum, and its main vector, Anopheles gambiae, would be of great help in developing new malaria control strategies. The malaria parasite undergoes several developmental transitions in the mosquito midgut and suffers population losses to which mosquito factors presumably contribute. To identify such factors, we analysed An. gambiae midgut transcripts whose expression is regulated upon ingestion of invasive or non‐invasive forms of P. falciparum using a differential display approach. Sixteen cDNA were studied in detail; 12 represent novel genes of An. gambiae including a gene encoding profilin. Four transcripts were specifically regulated by P. falciparum gametocytes (invasive forms), whereas the others were regulated by either non‐invasive or both non‐invasive and invasive forms of the parasite. This differential regulation of some genes may reflect the adaptation of P. falciparum to its natural vector. These genes may be involved in the development of P. falciparum in An. gambiae or in the defence reaction of the mosquito midgut towards the parasite.

Dynamics of transmission of Plasmodium falciparum by Anopheles arabiensis and the molecular forms M and S of Anopheles gambiae in Dielmo, Senegal

BackgroundThe adaptation of Anopheles gambiae to humans and its environment involves an ongoing speciation process that can be best demonstrated by the existence of various chromosomal forms adapted to different environments and of two molecular forms known as incipient taxonomic units.MethodsThe aim of this study was to compare the epidemiologic role of Anopheles arabiens is and the molecular forms M and S of Anopheles gambiae in the transmission of Plasmodium in a rural areas of southern Senegal, Dielmo. The sampling of mosquitoes was carried out monthly between July and December 2004, during the rainy season, by human volunteers and pyrethrum spray catches.ResultsAnopheles arabiensis, An. gambiae M and S forms coexisted during the rainy season with a predominance of the M form in September and the peak of density being observed in August for the S form. Similar parity rates were observed in An. arabiensis [70.9%] (n = 86), An. gambiae M form [68.7%] (n = 64) and An. gambiae S form [81.1%] (n = 156). The circumsporozoite protein (CSP) rates were 2.82% (n = 177), 3.17% (n = 315) and 3.45% (n = 405), with the mean anthropophilic rates being 71.4% (n = 14), 86.3% (n = 22) and 91.6% (n = 24) respectively for An. arabiensis and An. gambiae M and S forms. No significant difference was observed either in host preference or in Plasmodium falciparum infection rates between sympatric M and S populations.ConclusionNo difference was observed either in host preference or in Plasmodium falciparum infection rates between sympatric M and S populations, but they present different dynamics of population. These variations are probably attributable to different breeding conditions.

Comparative susceptibility to Plasmodium falciparum of the molecular forms M and S of Anopheles gambiae and Anopheles arabiensis

BackgroundThe different taxa belonging to Anopheles gambiae complex display phenotypic differences that may impact their contribution to malaria transmission. More specifically, their susceptibility to infection, resulting from a co-evolution between parasite and vector, might be different. The aim of this study was to compare the susceptibility of M and S molecular forms of Anopheles gambiae and Anopheles arabiensis to infection by Plasmodium falciparum.MethodsF3 progenies of Anopheles gambiae s.l. collected in Senegal were infected, using direct membrane feeding, with P. falciparum gametocyte-containing blood sampled on volunteer patients. The presence of oocysts was determined by light microscopy after 7 days, and the presence of sporozoite by ELISA after 14 days. Mosquito species and molecular forms were identified by PCR.ResultsThe oocyst rate was significantly higher in the molecular S form (79.07%) than in the M form (57.81%, Fishers exact test p < 0.001) and in Anopheles arabiensis (55.38%, Fishers exact test vs. S group p < 0.001). Mean ± s.e.m. number of oocyst was greater in the An. gambiae S form (1.72 ± 0.26) than in the An. gambiae M form (0.64 ± 0.04, p < 0.0001) and in the An. arabiensis group (0.58 ± 0.04, vs. S group, p < 0.0001). Sporozoite rate was also higher in the molecular form S (83.52%) than in form M (50.98%, Fishers exact test p < 0.001) and Anopheles arabiensis 50.85%, Fishers exact test vs. S group p < 0.001).ConclusionInfected in the same experimental conditions, the molecular form S of An. gambiae is more susceptible to infection by P. falciparum than the molecular form M of An. gambiae and An. arabiensis.