PLoS Pathog.

The susceptibility of Anopheles mosquitoes to Plasmodium infections relies on complex interactions between the insect vector and the malaria parasite. A number of studies have shown that the mosquito innate immune responses play an important role in controlling the malaria infection and that the strength of parasite clearance is under genetic control, but little is known about the influence of environmental factors on the transmission success. We present here evidence that the composition of the vector gut microbiota is one of the major components that determine the outcome of mosquito infections. A. gambiae mosquitoes collected in natural breeding sites from Cameroon were experimentally challenged with a wild P. falciparum isolate, and their gut bacterial content was submitted for pyrosequencing analysis. The meta-taxogenomic approach revealed a broader richness of the midgut bacterial flora than previously described. Unexpectedly, the majority of bacterial species were found in only a small proportion of mosquitoes, and only 20 genera were shared by 80% of individuals. We show that observed differences in gut bacterial flora of adult mosquitoes is a result of breeding in distinct sites, suggesting that the native aquatic source where larvae were grown determines the composition of the midgut microbiota. Importantly, the abundance of Enterobacteriaceae in the mosquito midgut correlates significantly with the Plasmodium infection status. This striking relationship highlights the role of natural gut environment in parasite transmission. Deciphering microbe-pathogen interactions offers new perspectives to control disease transmission

Midgut microbiota of the malaria mosquito vector Anopheles gambiae and Interactions with plasmodium falciparum Infection

The susceptibility of Anopheles mosquitoes to Plasmodium infections relies on complex interactions between the insect vector and the malaria parasite. A number of studies have shown that the mosquito innate immune responses play an important role in controlling the malaria infection and that the strength of parasite clearance is under genetic control, but little is known about the influence of environmental factors on the transmission success. We present here evidence that the composition of the vector gut microbiota is one of the major components that determine the outcome of mosquito infections. A. gambiae mosquitoes collected in natural breeding sites from Cameroon were experimentally challenged with a wild P. falciparum isolate, and their gut bacterial content was submitted for pyrosequencing analysis. The meta-taxogenomic approach revealed a broader richness of the midgut bacterial flora than previously described. Unexpectedly, the majority of bacterial species were found in only a small proportion of mosquitoes, and only 20 genera were shared by 80% of individuals. We show that observed differences in gut bacterial flora of adult mosquitoes is a result of breeding in distinct sites, suggesting that the native aquatic source where larvae were grown determines the composition of the midgut microbiota. Importantly, the abundance of Enterobacteriaceae in the mosquito midgut correlates significantly with the Plasmodium infection status. This striking relationship highlights the role of natural gut environment in parasite transmission. Deciphering microbe-pathogen interactions offers new perspectives to control disease transmission.Institut de Recherche pour le Developpement (IRD); French Agence Nationale pour la Recherche [ANR-11-BSV7-009-01]; European Community [242095, 223601]info:eu-repo/semantics/publishedVersio

Comparative assessment of An. gambiae and An. stephensi mosquitoes to determine transmission-reducing activity of antibodies against P. falciparum sexual stage antigens.

BACKGROUND: With the increasing interest in vaccines to interrupt malaria transmission, there is a demand for harmonization of current methods to assess Plasmodium transmission in laboratory settings. Potential vaccine candidates are currently tested in the standard membrane feeding assay (SMFA) that commonly relies on Anopheles stephensi mosquitoes. Other mosquito species including Anopheles gambiae are the dominant malaria vectors for Plasmodium falciparum in sub-Saharan Africa. METHODS: Using human serum and monoclonal pre-fertilization (anti-Pfs48/45) and post-fertilization (anti-Pfs25) antibodies known to effectively inhibit sporogony, we directly compared SMFA based estimates of transmission-reducing activity (TRA) for An. stephensi and An. gambiae mosquitoes. RESULTS: In the absence of transmission-reducing antibodies, average numbers of oocysts were similar between An. gambiae and An. stephensi. Antibody-mediated TRA was strongly correlated between both mosquito species, and absolute TRA estimates for pre-fertilisation monoclonal antibodies (mAb) showed no significant difference between the two species. TRA estimates for IgG of naturally exposed individuals and partially effective concentrations of anti-Pfs25 mAb were higher for An. stephensi than for An. gambiae. CONCLUSION: Our findings support the use of An. stephensi in the SMFA for target prioritization. As a vaccine moves through product development, better estimates of TRA and transmission-blocking activity (TBA) may need to be obtained in epidemiologically relevant parasite-species combination

Composition of Anopheles coluzzii and Anopheles gambiae microbiota from larval to adult stages

During their immature life stages, malaria mosquitoes are exposed to a wide array of microbes and contaminants from the aquatic habitats. Although prior studies have suggested that environmental exposure shapes the microbial community structure in the adult mosquito, most reports have focused on laboratory-based experiments and on a single mosquito epithelium, the gut. In this study, we investigated the influence of the breeding site on the development of the Anopheles coluzzii and Anopheles gambiae microbiota in natural conditions. We characterized bacterial communities from aquatic habitats, at surface microlayer and subsurface water levels, to freshly emerge adult mosquitoes using multiplexed 16S rRNA gene pyrosequencing and we separately analyzed the microbiota associated with the different epithelia of adult individual, midguts, ovaries and salivary glands. We found that the distribution of bacterial communities in the aquatic habitats differed according to the depth of water collections. Inter-individual variation of bacterial composition was large in larvae guts but adult mosquitoes from a same breeding site shared quite similar microbiota. Although some differences in bacterial abundances were highlighted between the different epithelia of freshly emerged An. coluzzii and An. gambiae, an intriguing feature from our study is the particular similarity of the overall bacterial communities. Our results call for further investigations on the bacterial population dynamics in the different tissues to determine the distinctive characteristics of each microbiota during the mosquito lifespan and to identify specific interactions between certain key phyla or species and the insect life history traits

Anopheles gambiae salivary protein expression modulated by wild Plasmodium falciparum infection : highlighting of new antigenic peptides as candidates of An. gambiae bites

Background: Malaria is the major parasitic disease worldwide caused by Plasmodium infection. The objective of integrated malaria control programs is to decrease malaria transmission, which needs specific tools to be accurately assessed. In areas where the transmission is low or has been substantially reduced, new complementary tools have to be developed to improve surveillance. A recent approach, based on the human antibody response to Anopheles salivary proteins, has been shown to be efficient in evaluating human exposure to Anopheles bites. The aim of the present study was to identify new An. gambiae salivary proteins as potential candidate biomarkers of human exposure to P. falciparum-infective bites. Methods: Experimental infections of An. gambiae by wild P. falciparum were carried out in semi-field conditions. Then a proteomic approach, combining 2D-DIGE and mass spectrometry, was used to identify the overexpressed salivary proteins in infected salivary glands compared to uninfected An. gambiae controls. Subsequently, a peptide design of each potential candidate was performed in silico and their antigenicity was tested by an epitope-mapping technique using blood from individuals exposed to Anopheles bites. Results: Five salivary proteins (gSG6, gSG1b, TRIO, SG5 and long form D7) were overexpressed in the infected salivary glands. Eighteen peptides were designed from these proteins and were found antigenic in children exposed to the Anopheles bites. Moreover, the results showed that the presence of wild P. falciparum in salivary glands modulates the expression of several salivary proteins and also appeared to induce post-translational modifications. Conclusions: This study is, to our knowledge, the first that compares the sialome of An. gambiae both infected and not infected by wild P. falciparum, making it possible to mimic the natural conditions of infection. This is a first step toward a better understanding of the close interactions between the parasite and the salivary gland of mosquitoes. In addition, these results open the way to define biomarkers of infective bites of Anopheles, which could, in the future, improve the estimation of malaria transmission and the evaluation of malaria vector control tools

Prevalence of Plasmodium falciparum parasites resistant to sulfadoxine/pyrimethamine in pregnant women in Yaounde, Cameroon : emergence of highly resistant pfdhfr/pfdhps alleles

Objectives: To determine, 6 years after the adoption of intermittent preventive treatment of pregnant women with sulfadoxine/pyrimethamine (IPTp-SP) in Cameroon, (i) the polymorphism and prevalence of Plasmodium falciparum dihydrofolate reductase (pfdhfr) and dihydropteroate synthase (pfdhps) gene mutations associated with sulfadoxine/pyrimethamine resistance and (ii) the consequences of sulfadoxine/pyrimethamine use in the selection of pfdhfr/pfdhps alleles. Methods: pfdhfr and pfdhps genes from P. falciparum isolates collected in Yaounde (Cameroon) from pregnant women with symptomatic malaria before taking IPTp-SP [SP2 group (control) (n = 51)] or afterwards [SP+ group (n = 49)] were sequenced. Results: The pfdhfr N51I, C59R, S108N triple mutant had a prevalence close to 100% (96/100) and no mutations at codons 50 and 164 were detected in either of the groups. The most frequent pfdhps mutation was A437G with a prevalence of 76.5% (39/51) in the SP2 group, which was significantly higher in pregnant women who took sulfadoxine/pyrimethamine [95.9% (47/49)] (P = 0.012). Our study confirmed the presence of the pfdhps K540E mutation in Cameroon, but it remained rare. The prevalence of pfdhps A581G and A613S mutations had increased [5.9% (3/51) and 11.8% (6/51) in the control group, respectively] since the last studies in 2005. Surprisingly, the new pfdhps I431V mutation was detected, at a prevalence of 9.8% (5/51), and was found to be associated with other pfdhfr/pfdhps alleles to form an octuple N51I, C59R, S108N/I431V, S436A, A437G, A581G, A613S mutant. Conclusions: Significant changes were found in pfdhps polymorphism. In particular, we observed several parasites carrying eight mutations in pfdhfr/pfdhps genes, which are very susceptible to having a high level of resistance to sulfadoxine/pyrimethamine

Application of a qPCR assay in the investigation of susceptibility to malaria infection of the M and S molecular forms of An. gambiae s.s. in Cameroon

Plasmodium falciparum is the causative agent of malaria, a disease that kills almost one million persons each year, mainly in sub-Saharan Africa. P. falciparum is transmitted to the human host by the bite of an Anopheles female mosquito, and Anopheles gambiae sensus stricto is the most tremendous malaria vector in Africa, widespread throughout the afro-tropical belt. An. gambiae s.s. is subdivided into two distinct molecular forms, namely M and S forms. The two molecular forms are morphologically identical but they are distinct genetically, and differ by their distribution and their ecological preferences. The epidemiological importance of the two molecular forms in malaria transmission has been poorly investigated so far and gave distinct results in different areas. We have developed a real-time quantitative PCR (qPCR) assay, and used it to detect P. falciparum at the oocyst stage in wild An. gambiae s.s. mosquitoes experimentally infected with natural isolates of parasites. Mosquitoes were collected at immature stages in sympatric and allopatric breeding sites and further infected at the adult stage. We next measured the infection prevalence and intensity in female mosquitoes using the qPCR assay and correlated the infection success with the mosquito molecular forms. Our results revealed different prevalence of infection between the M and S molecular forms of An. gambiae s.s. in Cameroon, for both sympatric and allopatric populations of mosquitoes. However, no difference in the infection intensity was observed. Thus, the distribution of the molecular forms of An. gambiae s.s. may impact on the malaria epidemiology, and it will be important to monitor the efficiency of malaria control interventions on the two M and S forms

Impact of exposure to mosquito transmission-blocking antibodies on Plasmodium falciparum population genetic structure

Progress in malaria control has led to a significant reduction of the malaria burden. Interventions that interrupt transmission are now needed to achieve the elimination goal. Transmission-blocking vaccines (TBV) that aim to prevent mosquito infections represent promising tools and several vaccine candidates targeting different stages of the parasite's lifecycle are currently under development. A mosquito-midgut antigen, the anopheline alanyl aminopeptidase (AnAPN1) is one of the lead TBV candidates; antibodies against AnAPN1 prevent ookinete invasion. In this study, we explored the transmission dynamics of Plasmodium falciparum in mosquitoes fed with anti-AnAPN1 monoclonal antibodies(mAbs) vs. untreated controls, and investigated whether the parasite genetic content affects or is affected by antibody treatment. Exposure to anti-AnAPN1 mAbs was efficient at blocking parasite transmission and the effect was dose-dependent. Genetic analysis revealed a significant sib-mating within P. falciparum infra-populations infecting one host, as measured by the strong correlation between Wright's F-IS and multiplicity of infection. Treatments also resulted in significant decrease in F-IS as a by-product of drop in infra-population genetic diversity and concomitant increase of apparent panmictic genotyping proportions. Genetic differentiation analyses indicated that mosquitoes fed on a same donor randomly sampled blood-circulating gametocytes. We did not detect trace of selection, as the genetic differentiation between different donors did not decrease with increasing mAb concentration and was not significant between treatments for each gametocyte donor. Thus, there is apparently no specific genotype associated with the loss of diversity under mAb treatment. Finally, the anti-AnAPN1 mAbs were effective at reducing mosquito infection and a vaccine aiming at eliciting anti-AnAPN1 mAbs has a strong potential to decrease the burden of malaria in transmission- blocking interventions without any apparent selective pressure on the parasite population