Final Report Summary - MALVECBLOK (Population biology and molecular genetics of vectorial capacity in Anopheles gambiae: targeting reproductive behaviour and immunity( ))
Executive summary:
During the project period, MALVECBLOK implemented joint research efforts addressing fundamental topics of mosquito biology, and achieved the following objectives:
(i) The molecular bases of reproductive biology of the mosquito vector, and its effects on immunity and Plasmodium transmission. The major discoveries of this work package identified the key male and female molecules that shape post-mating responses and reproductive success in the malaria mosquitoes. We have characterized the composition of the mating plug and the key coagulation enzyme, transglutaminase, crucial for mosquito fertility. The specific roles of MAG secretions and the sperm cells in induction of post-mating responses in females were examined using sperm-less males that we established by silencing a key gene involved in gonadogenesis. These experiments demonstrated for the first time the pivotal role of male accessory glands in orchestrating female post-mating behaviour. In addition, these results brought a much needed proof of concept for vector control strategies based on sterile insect technique as spermless males efficiently engaged females into mating which, as expected, was fully sterile. Although no effect of mating on Plasmodium has been detected, our results revealed an unexpected connection between responses of females to mating and to blood feeding evidenced by mating-induced expression of a series of genes associated with a blood feeding.
(ii) The molecular mechanisms, which determine the mosquito immune status and regulate Plasmodium sporogony and transmission, both in laboratory settings and in natural populations. We have characterized the nature of TEP1/LRR complex and revealed its crucial role in defence against bacteria and malaria parasites. We uncovered that genetic diversity of P. falciparum enables parasites to survive immune responses in the vector; this discovery has important consequences for design of future vector control measures. Functional analysis of two major yolk proteins crucial for mosquito fertility revealed their role in TEP1 binding to parasites. Strikingly, expression of two major yolk proteins is negatively regulated by Rel1/Rel2 immune pathway, suggesting that activation of immune responses represses reproduction in the mosquitoes. One of the major achievements of MALVECBLOK is the discovery of TEP1 as a novel marker for evaluation of genetic structure of mosquito populations in the study sites. Established standardised protocols for TEP1 genotyping revealed that the allele conferring resistance to Plasmodium (TEP1R1) is massively spread in West Africa but is almost absent from Central and East Africa. Further experiments are needed to evaluate the contribution of TEP1R1 to the mosquito resistance to the parasites, but our results suggest that TEP1 can be used as a novel marker for population genetics studies of An. gambiae.
(iii) The role of genetic polymorphism in genes controlling reproduction and immunity on the structure of mosquito populations and malaria transmission in Africa. We characterized polymorphisms in reproductive genes and gene families, which reveal evolutionary relationships between subspecies of the An. gambiae complex. Some genes are under strong purifying selection, whereas others are subject for extensive variation with no signs of species-specific signatures. Unexpectedly, we found that polymorphism in TEP1 correlates with An. gambiae molecular forms, and performed massive TEP1 genotyping across West, Central and East Africa. Another key discovery of the consortium is the demonstration of the role of microbiota on the capacity of mosquitoes to transmit malaria. We have identified bacterial communities in the midguts of mosquitoes collected in breeding sites in Cameroon, and revealed a positive correlation between the presence of Enterobacteriaceae and Plasmodium development. Therefore we identified a new environmental factor that shapes malaria transmission in Africa.
In conclusion, during the project identified new potential targets for control of mosquito populations. Our results suggest that Plasmodium developed means to cope with the vector immune responses, especially in the areas of high transmission, and instruct the future studies on immuno-modulated mosquitoes. Special attention of the consortium was given to cutting-edge training opportunities and sharing of resources between the partners. The knowledge acquired in the project has a strong impact on European scientific competitiveness, and addresses societal issues in the endemic countries by recruitment of young researchers and by disseminating the study principles and knowledge of socio-economic impact of malaria in the research centres and, more importantly, among the local population involved in the study. As a result of the project, children of at least three villages in Africa benefit from regular medical surveillance and timely antimalarial treatment.
Project Context and Objectives:
This project is inspired by recent advances in the characterisation of An. gambiae genome, in the understanding of the differentiation/speciation processes ongoing within this species and in the development of powerful tools for functional gene analyses, and explores critical molecular mechanisms that underlie the vectorial capacity of the mosquito (i.e. its ability to support development of malaria parasites) with the ultimate purpose to block disease transmission. Europe has always been at the avant-garde of research in mosquito biology, however scientific exchanges between teams working in various aspects of the field, e.g. population biology, reproduction and immunity, remain fragmentary. This proposal integrates research groups from 3 European countries and scientists from 3 African malaria endemic countries with the main aim to increase our knowledge on two biological processes that crucially determine vectorial capacity, reproduction and immunity. The particular emphasis is on understanding the genetic bases of these two processes and the relationship between them, with the view to assess their role in malaria transmission and in shaping the structure of mosquito populations in Africa.
Accordingly, the Consortium draws together a critical mass of complementary expertise and resources which will be crucial to successfully implement joint research efforts addressing these fundamental topics of mosquito biology, organized in the following objectives:
(i)The analysis of the molecular bases of crucial aspects of reproductive biology of An. gambiae, such as mating and egg laying, and the role of reproduction in immunity and Plasmodium transmission. To this end, the Consortium will identify the key male (months 1-14, milestone 1.2) and female (months 1-42, milestone 1.1) factors, characterize male-female interactions (months 12-42, milestone 1.6) and signalling cascades shaping post-mating responses in An. gambiae (months 12-42, milestone 1.5). The project will considerably expand the fundamental knowledge of reproductive biology in Anopheles. In parallel, the relationship between reproductive biology and immunity in An. gambiae will be investigated. The effect of mating on the immune status will be examined both in the laboratory and field conditions (months 1-24, milestone 1.3).
(ii)The study of the molecular mechanisms which determine the An. gambiae immune status and regulate Plasmodium development and transmission, in both laboratory settings (months 1-42, milestones 2.1; 2.4) and natural populations (months 6-42, milestones 2.3; 2.5; 2.6; 3.3). Equally important, the trade-off on the immune status on the reproduction will be characterised (months 12-42, milestone 2.2).
(iii)The investigation of the role of genetic polymorphism in genes controlling reproduction and immunity on the structure of An. gambiae populations and malaria transmission in Africa (months 1-42, milestones 3.1; 3.2; 3.4; 3.6; 3.7).
Project Results:
WP1 Molecular mechanisms of vector reproduction and its effects on immunity and Plasmodium transmission
This work package has two main objectives:
1) the analysis of the molecular components shaping the reproductive success of male and female mosquitoes; and
2) the assessment of the effects of the reproductive status on female immunity and transmission of Plasmodium parasites.
Role of male-produced factors in inducing female post-mating responses (UNIPG)
In order to identify male factors transferred to the female during mating and likely to be responsible for the modulation of female behaviour after copulation, we have analyzed the composition of the mating plug. The mating plug is formed by coagulated male accessory gland (MAG) secretions, mainly consisting of proteins and lipids. We have performed mass spectrometry analysis of mating plugs dissected from freshly mated females, and have identified a number of male reproductive proteins transferred to females (Rogers et al. PLoS Biology 2009). Among these proteins is a MAG-specific transglutaminase (TGase), a cross-linking enzyme. In the frame of this project, we found that this transglutaminase is essential for the coagulation of male seminal secretions. Impairing expression of the TGase in the MAGs by RNA interference (RNAi), injecting dsRNA molecules into virgin males, prevents mating plug formation and transfer (D1.4). Moreover, females mated to males impaired in plug formation cannot store sperm properly, and are therefore sterile (M1.2). This major result was published in PLoS Biology and broadly discussed by the media worldwide. This is the first demonstration that meddling with mosquito mating can have applications for vector control.
Among other plug proteins are four Acps that may be triggering female post-mating responses. We have named AGAP009368 plugin, as this is the most abundant protein of the mating plug (Rogers et al., PLoS Biology 2009). The other three genes encoding Acps (AGAP009362, AGAP009370 and AGAP012830) have been silenced by injecting virgin males with the corresponding dsRNAs, however no obvious phenotype on female fertility, fecundity and receptivity to mating has been found yet.
We have developed polyclonal antibodies against all these male proteins (D1.3) and have studied their localization in the MAGs, plug and female reproductive tract. Our first results reveal non-overlapping expression patterns of these proteins, and we will continue our efforts to elucidate their functions.
The mating plug is formed by coagulated male accessory gland (MAG) secretions, mainly proteins and lipids. We have confirmed that a steroid hormone, 20 hydroxyecdysone (20E), is produced by the MAGs. Moreover, we have shown that 20E is transferred to females as part of the mating plug and that this male-derived hormone induces the expression of a number of reproductive genes in the atrium of virgin females, mimicking the transcriptional response that occurs in females after mating. We have also determined that one of the female genes, TED1 (see below), induced by the male-transferred 20E, interacts directly or indirectly with this hormone (M1.6). 20E transfer triggers a cascade of events that lead to increased egg development by the female. This is the first example of a male hormone that modulates female reproductive physiology (M1.2). We are targeting the biosynthesis of 20E in the MAGs by silencing the pathway of enzymes that are responsible for its synthesis. Our results show that we can reduce 20E levels in the MAGs by 50%, and that this reduction affects the female response after mating. These results are being written for publication (D1.8).
We have continued our functional analysis of MAG proteins. Specifically, we have focused on 3 enzymes, PAM, PHM and QPCT, which are involved in the synthesis of small peptide hormones. We have targeted QPCT by RNAi injecting virgin males with the corresponding dsRNAs, however no obvious phenotype on female fertility, fecundity and receptivity to mating has been found yet. We have developed polyclonal antibodies against all these male proteins (D1.3) and have studied their localization in the MAGs, plug and female reproductive tract, in collaboration with our partners from UNIROMA1.
Identification of female reproductive proteins and analysis of their interactions with male factors transferred during mating (UNIPG, UNIROMA1, CNRS)
We have functionally analysed a number of female genes regulated by mating (D1.1 M1.1). We have selected genes expressed in the spermatheca (sperm storage organ) and the atrium (where the mating plug is deposited and digested). Among the spermathecal genes, we have analyzed the heme peroxidase HPX15, whose expression is strongly upregulated 24h after mating. We have developed polyclonal antibodies against this protein and assessed that it is expressed predominantly in the secretory (glandular) cells surrounding the spermathecal capsule. RNAi knock down of HPX15 in virgin females has been successful at both transcript and protein levels, however no effects have been detected on fertility and fecundity of females (for these analyses we have developed specific protocols to assess the fertility and fecundity of females, D1.2).
Among the genes regulated by mating in the atrium, we have analyzed three genes encoding serine proteases, AGAP005194, AGAP005195 and AGAP005196 (the latter is also expressed in the spermatheca), strongly downregulated by mating. Antibodies raised against AGAP005194 have shown that this protein is closely linked to the mating plug surface, with a pattern resembling that of the major plug protein plugin, and its expression is not detectable at 24h post copulation. This suggests that AGAP005194, and possibly the other proteases, are involved in digestion of the mating plug, which is mostly completed at 24h post-mating.
To ascertain that our studies are relevant for natural populations, we have collected mating couples of field mosquitoes from natural swarms and examined the mating-regulated expression of a number of genes identified in our laboratory analyses. For this, UNIPG and UNIROMA1 performed a field trip to Burkina Faso in collaboration with IRSS. Mosquitoes in copula were collected in different villages from swarms of M and S molecular forms, believed to be incipient species in the An. gambiae complex. Quantitative PCR on the dissected tissues confirmed that most genes regulated in laboratory cages are also induced or repressed after mating in field populations. These results are very important for validating our candidate genes for further functional analyses.
We have continued to perform functional analyses on a number of female genes regulated by mating (D1.1 M1.1). We have selected genes expressed in the spermatheca (sperm storage organ) and the atrium (where the mating plug is deposited and digested). Among the spermathecal genes, we have analyzed the heme peroxidase HPX15, which is strongly upregulated by mating at 24h. We have developed polyclonal antibodies against this protein and assessed that it is expressed predominantly in the secretory cells (glandular) surrounding the spermathecal capsule. RNAi knock down of HPX15 in virgin females has been successful at both RNA and protein levels. Our initial studies had not identified any effect of HPX15 silencing on fertility, however a deeper analysis of the function of this gene has shown that HPX15 is important to preserve sperm function following multiple blood feedings. Fertility of egg batches after two blood feedings decreased significantly when HPX15 expression was reduced by RNAi injections (E. Teodori, F. Catteruccia, manuscript in preparation)(D1.8 M1.5). These results identify the oxidative stress derived from the blood meal as one of the risk factors for sperm function, and highlight the role HPX15 in preserving sperm viability after repeated blood meals.
We have performed a microarray analysis of spermathecae dissected from mated females and identified a signaling pathway comprising three G protein-coupled receptors (GPCRs) and downstream protein kinases that may play a crucial role for sperm viability and activation. We are currently studying the GPCRs activated by mating by RNAi silencing and by performing cell-based assays to establish the downstream signaling pathways. We have designed polyclonal antibodies to determine their exact localization in the spermatheca. We have chosen to focus on functional analyses of the spermatheca over proteomic analysis of the hemolymph (as originally planned) as the possibility of preventing sperm viability in the female has tremendous implications for reducing the fertility of mosquito populations in vector control programs.
Dissection of signalling cascades that regulate post-mating behaviour in females (UNIPG)
We identified a gene whose expression was strongly induced by mating and named it Matin (Mating induced). Although Matin does not feature any known functional domains, immunofluorescence experiments with polyclonal antibodies developed for the analysis have assessed that the protein is specifically expressed in the ampullae, a region in the female reproductive tract connecting the ovaries to the atrium. Inhibition of Matin expression by RNAi injections into virgin females strongly inhibits egg laying after mating and blood feeding, suggesting that this gene is involved in the signaling cascades initiated by mating and leading to oviposition.
We have generated transgenic lines that contain the promoter region of a mating-responsive gene controlling expression of a dtTomato or luciferase reporter genes for quantification of expression (D2.1). The promoter region of AGAP002620 (p2620), a gene expressed in the atrium and strongly regulated by mating, has been chosen for this purpose. An. gambiae embryos have been injected with the constructs p2620:luciferase and p2620::dsTomato following well established protocols and G1 individuals were screened for the occurrence of germline integration. The resulting homozygous transgenic line is currently being analysed to confirm the correlations between expression patterns of the endogene and the transgene.
We have further characterized PET1 (AGAP002620), the putative protein involved in egg laying trigger studied during the first reporting period. We have established that silencing of this gene does not affect egg laying as initially hypothesized, while it affects egg development. Mated females develop more eggs than virgin females after taking a blood meal. However, females in which PET1 was knocked-down by RNAi showed significant reduced fecundity compared to control females, and the overall number of eggs developed by dsPET1 females was decreased to levels similar to those obtained with virgin females. These results show that PET1 is a crucial gene in the cascade of events triggered by mating that contributes to egg development. For its role in egg development rather than egg laying, we have renamed this gene TED1 (trigger of egg development). Moreover, as discussed earlier, we have identified a male trigger of TED1. This is a steroid hormone, 20E, transferred to females during mating as part of the mating plug. Silencing of TED1 impairs the correct diffusion of 20E out of the female atrium into the hemolymph (M1.6) where 20E is needed to activate egg development though the induction of lipid transporters. These results are being prepared for publication (D1.8 M1.5). We are currently assessing whether TED1 silencing affects the development of Plasmodium parasites (D1.7 M1.4).
We have identified additional genes whose expression is triggered by mating in females at different time points in the spermatheca, atrium, ovaries, midgut, head and rest of the body (hemolymph, fat body, legs, Malpighian tubules and others). Besides providing an expanded collection of cDNAs from female tissues, these analyses are revealing additional candidates likely to be involved in modulating female post-mating behaviors (D1.8).
Moreover, we are characterizing a transgenic mating-responsive line FK that contains an expression cassette pAGAP002620:tdTomato. The FK line is being used to perform functional analyses of female candidate genes that may be regulating the mating-induced signaling cascades (D1.8 M1.5)
Effects of mating and post-mating responses on immune status and on sporogony and transmission of Plasmodium (UNIPG, CNRS, RUNMC)
The expression of a number of immune genes at different time points after mating has been studied in both laboratory and field-collected females. The expression of these genes (encoding antimicrobial peptides Cecropins, Gambicin and Defensin 1, antiparasitic genes like TEP1, LRIM1 and APL1) in the female reproductive tract and in the rest of the body has been assessed by quantitative RT-PCR. We found that expression of Gambicin is strongly downregulated in atrium and spermatheca after mating, while transcript levels of TEP1 are induced in the rest of the body. Moreover, in natural populations we found numerous polymorphisms associated with these genes (M1.3). The relevance of these polymorphisms in gene function is currently being assessed.
We also performed Plasmodium berghei infections and compared oocyst development in virgins versus mated females. For that, females were separated from males at the pupal stage and either mated or kept as virgin. At day 5 post-emergence virgin and mated females were fed an infectious P. berghei blood meal and oocyst numbers were compared in the two groups 10 days after infection. In this mouse model, no major difference was found in infection levels in the two groups, suggesting that the mated status does not directly affect development of the rodent malaria (M1.3).
We have confirmed that expression of the thioester-containing protein 1, TEP1, is induced in females after mating in the rest of the body. Moreover we have found a number of immune genes specifically induced by mating in the female head, indicating a strong immune response to yet undefined factors (possibly male peptides reaching the head or crossing the blood brain barrier). We have collected additional field samples from natural An. gambiae populations and found numerous polymorphisms associated with TEP1 (M1.3). In order to understand the relevance of these findings, we have collected in collaboration with our partners CNRS mosquito larvae from a number of different breeding sites (temporary and permanent) and adults from mating swarms to determine the microbiota of natural mosquito population. We are now determining whether there exists a correlation between different bacterial populations and specific TEP1 polymorphisms. Our hypothesis is that the latter may have been driven by selective pressures inflicted by a diverse range of microbes in An. gambiae populations adapted to different ecological habitats.
We have also generated male mosquitoes depleted of sperm cells and have determined that female mated to these sperm-less males do not exhibit any differences in their post-copulatory behaviors: these females lay (sterile) eggs and fail to re-mate, as normally observed in females mated to fertile males. These results have been recently published in Thailayil et al., PNAS 2011 (D1.5). Females mated to these spermless males are currently being tested for their ability to transmit malaria parasites. Preliminary data suggests that there is no difference in the number of parasites developing in these females compared to females mated to normal males (M1.4).
WP2 Molecular mechanisms that determine mosquito immune status and its effects on sporogony of Plasmodium and on mosquito reproduction
This work package has two objectives:
(i) to provide an integrated view of the molecular mechanisms which determine the mosquito immune status and regulate Plasmodium sporogony and transmission, in both laboratory settings and natural populations; and
(ii) to examine how the mosquito immune status affects reproductive capacity of mosquitoes.
Analysis of molecular mechanisms of TEP1-mediated Plasmodium recognition and killing in An. gambiae by biochemical and genetic approaches (CNRS, UNIPG, RUNMC)
Previous work demonstrated that two leucine-rich repeat proteins form a complex with TEP1 and that this complex is required to maintain the cleaved form of TEP1 in circulation. We performed biochemical analysis of the protein complex isolated by immuno-precipitation of the mosquito hemolymph and confirmed the presence of the two LRR proteins. However, no additional members could be identified, suggesting that the complex consists of these three proteins. Therefore, the complex can be studied in more detail in vitro. We asked which form of TEP1 is recruited into the complex, and in collaboration with Dr Baxter, University of Texas, USA, performed detailed characterization and solved X-ray structures of the two LRR proteins. Our data indicate that these proteins form a dimer, which is stabilized by a C-terminal coiled-coil domain interlinked by a single disulphide bridge. Our results suggest that these LRR proteins interact with a disarmed 'inactivated' form of TEP1 unable to form a covalent bond with the parasite surface. To confirm relevance of our observations in vitro for the mosquito biology in vivo, we used a recombinant TEP1/LRIM1/APL1 protein complex to rescue TEP1 knockdown phenotype in vivo. We developed a series of protocols to study immuno-induced and/or immuno-compromised mosquitoes (D2.3) and used bacterial infections to address this question. In this protocol, mosquitoes deprived of TEP1 succumb to bacterial infections within 24 h after challenge. We first demonstrated that depletion of the LRR proteins elicits a phenotype similar to dsTEP1, and then injected the recombinant complex together with bacteria 4 days after injection of dsRNA against TEP1, LRIM1 or APL1. Injection of the complex rescued survival of the mosquitoes confirming that it is fully functional in vivo, and further indicating that the complex does not target TEP1 to the surface of bacteria but has a different role. We are currently examining whether this complex associates with a bacterial protease to amplify TEP1 cleavage in the proximity of pathogens.
To perform structure to function analysis of TEP1, we expressed resistant and susceptible forms of the protein in a baculovirus system. In an in vitro system, the susceptible form of TEP1 displayed significantly shorter half-life than the resistant form, as evidenced by protein precipitation (M2.1). These results suggest that protein stability could underlie the more efficient binding of the resistant form to the parasites. This simple assay will be used to map residues that determine TEP1 stability by site-mutagenesis. Once important residues will be identified, we will confirm in vitro results in vivo by establishing transgenic mosquitoes. Currently, we are in the process of developing transgenic mosquitoes with a mutated TEP1 thioester site.
We have performed functional analysis of genes that are regulated by Rel1/Cactus cassette, and examined effects of knockdown of the top 10 candidates identified by microarrays on development of P. berghei within a mosquito. However no significant effect was observed. It is possible that other genes do not contribute to the response against rodent malaria parasites, suggesting that the phenotype could be totally explained by the contributions of TEP1, LRIM1 and APL1. These results are consistent with the biochemical data described above; therefore we are focusing our analysis on the TEP1/LRR complex. Interestingly, when the roles of these proteins were examined in experimental infections with P. falciparum in Cameroon, only TEP1 silencing increased parasite numbers, whereas depletion of the two LRRs had no effect. These results suggest that in human malaria infections, TEP1 must be activated by a distinct protease, and that rodent malaria parasites and bacteria share the same LRR-dependent TEP1 activation pathway. To confirm these results, we are performing P. falciparum infections in the laboratory settings with the aim to detect TEP1 on the surface of P. falciparum ookinetes.
A protocol has been developed to study immuno-induced and/or immuno-compromised mosquitoes (D2.3.). In this protocol, mosquitoes deprived of TEP1 succumb to bacterial infections within 24 h after challenge. We first demonstrated that depletion of the LRR proteins elicits a phenotype similar to dsTEP1. We extended this analysis to other candidate genes that are co-regulated with TEP1 according to the data of our microarray analyses. We have examined survival of mosquitoes silenced for genes encoding CLIP-domain serine proteases (catalytically active (B-type) or inactive (A or C type)). Out of 7 tested genes, silencing of CLIPA7 and CLIPA8 showed survival phenotypes similar to that of silencing TEP1, whereas no changes in the mosquito survival was detected for other genes. Interestingly, using this test we also examined whether fibrinogen 9 and cytochrome 450 collaborate with TEP1. As no effect of silencing of these genes on parasite development could be detected, only catalytically inactive CLIPA7 and CLIPA8 cooperate with TEP1. These results are in preparation for a publication.
In collaboration with Dr R. Baxter (Yale University, USA) we examined a series of recombinant TEP1 forms found in natural populations for their relative stability. We discovered that replacement of the thioester domain (TED) of TEP1 in resistant allele into susceptible was sufficient to reduce the half-life of the protein to the levels of the fully susceptible form. Using the developed assay, we are dissecting individual SNPs that are crucial for TEP1 stability and therefore, its function. These results are of high importance for the development of informative TEP1 genotyping protocols described in WP3.
RUNMC introduced Ngousso and resistant L3-5 strains of An. gambiae into the laboratory and performed P. falciparum infections with in vitro parasite cultures. Interestingly, significant differences were observed in the development of P. falciparum between Ngousso and G3 mosquitoes. Our results revealed that Ngousso are as susceptible to P. falciparum infections as An. stephensi, whereas a strong inhibition of the parasite development was detected in G3 and L3-5 mosquitoes. Silencing of TEP1 revealed that parasite killing in the G3 background is independent of TEP1. This result is unexpected as the immunofluorescence analysis using anti-TEP1 antibodies showed the presence of TEP1 on the surface of P. falciparum ookinetes. The intensity of TEP1 signal detected on the ookinetes was much stronger in the resistant strain than in Ngousso. Our results suggest that in spite of TEP1 binding to the parasites, some P. falciparum strains are insensitive to TEP1-mediated killing. Therefore, parasite genetic background is one of the factors that determine mosquito competence to control malaria infections.
Evaluation of environmental and physiological factors that affect mosquito immune status and Plasmodium sporogony in the laboratory and in the field (CNRS, RUNMC, IRD, MRTC, ICIPE)
The promoter sequences of immune-inducible genes were used to establish immune-responsive reporters (D2.1 and D2.6). We obtained 2 lines for Defensin 1 reporter, and 1 line for a 'universal' immune reporter containing Rel1- and Rel2-regulated sequences. We have characterized Defensin 1 reporter lines, that are activated by Gram-positive and Gram-negative bacteria but display no induction after infections with rodent malaria parasites. The 'universal' reporter line is currently under breeding and we should have first results shortly.
Establishment of experimental infections in Kenya and Mali were at the focus of the first reporting period. To this end, we organized an Ethical workshop in Cameroon, where these experiments are successfully running. MRTC and ICIPE now obtained Ethical clearance from national Committees and started experimental infections in Bamako and Mbita, respectively (D3.2). The study design involves identification of breeding sites with contrasting characteristics to examine the influence of these contrasting environmental factors on the capacity of mosquitoes to transmit malaria. First results in Mbita, Kenya, revealed significant effect of the breeding sites on the mosquito to develop of the parasites, this work is currently in progress.
RUNMC laboratory has successfully isolated 2 additional lines of P. falciparum from Malawi and Guinea. When these two P. falciparum strains were used for infections, one of them showed a strong susceptibility to TEP1-mediated killing. These results further highlight the importance of parasite genetic background as a determining factor in the mosquito - parasite interactions. Another factor that affects the efficiency of the mosquito responses is the genetic clonality of P. falciparum. Indeed, monoclonal infections produced high infection rates and were very sensitive to the wounding-induced immune responses (see below). In contrast, polyclonal infections were very low and resistant to the mosquito immune responses. Therefore, parasite genetic complexity is a crucial factor that shapes the outcome of Plasmodium infections in the field and should be taken into account for the development of new vector control strategies. These results of a collaborative work between IRD and CNRS were published in the International Journal for Parasitology (Nsango et al, 2012).
Modulation of the mosquito immune status and its effects on Plasmodium transmission (CNRS, RUNMC, IRD, MRTC, ICIPE)
Modulation of the mosquito immune status can be achieved by:
(i) genetic selection;
(ii) genetic manipulation by RNAi or by transgenesis.
(i) We have examined development of rodent malaria parasites in series of laboratory strains and observed a significant level of variation in outcomes of infection between these strains. For instance, the G3 strain is much more susceptible to infection than the Ngousso strain. Moreover, we performed selection on the Ngousso strain and obtained two lines which differ by 10-fold in the number of developing oocysts, suggesting that some mosquito factors regulate parasite development. Sequencing of TEP1 in these two lines revealed a single 'susceptible' allele shared by both lines, suggesting involvement of a different gene. We are currently isogenising these lines and will perform further genetic analysis to identify the gene(s) responsible for resistance to Plasmodium. These two lines will be also examined in infections with P. falciparum by RUNMC.
(ii) We have discovered that wounding induces a strong antiparasitic response against human but not rodent malaria parasites. To characterize molecular events underlying this effect, we used microarrays to compare mosquito transcriptomes before and 3h after wounding, and identified a number of candidate genes including components of the JNK pathway and TGases. Silencing of these genes rescued effect of wounding in the experimental infections in Cameroon, thereby confirming their involvement in the regulation of Plasmodium development. Our results suggest that injection of dsRNA can only be used to study wounding response against P. falciparum and that alternative methods of modulation of the mosquito immune status should be developed. These results are being submitted for publication. Therefore we now focus our efforts on the development of transgenic technology to assess gene function. We have developed a series of attP-containing lines for transgene integration and are in process of developing mosquito lines expressing the resistant allele of TEP1.
We have examined development of rodent and human malaria parasites in series of laboratory strains and observed a significant level of variation in outcomes of infection between these strains. For instance, the G3 strain is much more susceptible to P. berghei infection than the Ngousso strain, whereas G3 and L3-5 are more resistant to P. falciparum and are susceptible to P. berghei. Interestingly, the Ngousso colony is polymorphic for TEP1, and we are currently evaluating the contribution of TEP1 alleles to the resistance. We have discovered that wounding induces a strong antiparasitic response against human but not rodent malaria parasites. To characterize molecular events underlying this effect, we used microarrays to compare mosquito transcriptomes before and 3h after wounding, and identified a number of candidate genes including AP-1/Fos and TGases. Silencing of these genes rescued effect of wounding on P. falciparum development in the experimental infections in Cameroon. These results are being submitted for a joint publication between CNRS and IRD. We now focus our efforts on development of transgenic technology to assess gene function. We have developed a series of attP-containing lines for site-specific transgene integration and, as described above, are in process of developing mosquito lines expressing the resistant allele of TEP1. Recent studies from our and other laboratories revealed an important role of bacterial communities (or microbiote) in the mosquito-parasite interactions. To complement studies performed by IRD in the field populations, we are developing laboratory models of bacterial infections (as described above) to investigate this question. We will also extend these studies to P. falciparum infections in collaboration with RUNMC.
Analysis of reproductive success in An. gambiae mosquitoes with elevated or compromised immune status (UNIPG, CNRS)
Signal-independent activation of the Rel1 signalling cascade blocks development of rodent parasites but also abolishes egg development. Analysis of the genes regulated by Cactus pointed out to vitellogenin (Vg), one of the major yolk proteins in the mosquitoes. Indeed, we revealed that expression of this gene is negatively controlled by Rel1/Rel2 pathways, and in part explains Cactus knockdown phenotype. Silencing of vitellogenin and/or of lipophorin (Lp) decreased the number of surviving parasites indicating that these yolk proteins are important for Plasmodium development. In addition, only partial egg development was observed in dsVg mosquitoes, whereas ovaries remained undeveloped in dsLp females. The pro-parasitic effect of the yolk proteins required the function of TEP1, and TEP1 was more efficient in binding and killing of P. berghei in the absence of Vg and Lp. These results, published in PLoS Biology (Rono et al, 2010), for the first time establish a molecular link between reproduction and immunity and demonstrate that induction of immunity represses oogenesis in An. gambiae. Developed here tools will be used to study regulation of Lp and Vg expression after mating and effects of immune induction on mating efficiency. We will also examine the roles of the yolk proteins in infections with P. falciparum.
Careful immune-fluorescence analysis revealed the presence of TEP1 in the mosquito testes. Interestingly, TEP1-positive signal was observed inside the cyst stem cells, in the cystoblasts, and on the tails of the sperm cells. Functional analysis suggested that TEP1 is not important for mating but critical for maintaining male fertility, as progenies of the aged TEP1-depleted males are significantly less numerous as compared to control mosquitoes. Interestingly, a strong signal for TEP1 was detected on the sperm cells in the L3-5 mosquitoes that are resistant to P. berghei parasites. Experiments are now underway to examine whether allelic forms of TEP1 shape male fertility. The uncovered role of TEP1 in male fertility is crucial for understanding the mechanisms that drive malaria-resistance traits into vector populations.
We continued to examine the role of two yolk proteins in the antiparasitic responses of An. gambiae and in collaboration with RUNMC initiated analysis of Lp and Vg knockdowns on development of P. falciparum. Our results confirmed the protective role of yolk proteins in ookinete development, however in contrast to P. berghei, parasite killing in the absence of Lp and Vg is not mediated by TEP1. We also evaluated whether previously observed Lp-mediated effect on the oocyst growth affects ability of the mosquitoes to transmit Plasmodium. Our first results with P. berghei suggest a significant decrease in transmission efficiency. These experiments are being performed with P. falciparum isolates in collaboration with RUNMC.
WP3 Role of genetic polymorphism in genes controlling reproduction and immunity on structure of mosquito populations and malaria transmission in Africa.
This work package has two main objectives:
1) to examine genetic variability in genes controlling reproduction and immunity in natural An. gambiae populations, and
2) to evaluate the role of genetic differentiation in shaping the structure of mosquito populations, both in terms of the An. gambiae speciation and in ecological specialization, and in determining the vector competence to transmit malaria.
Analysis of genetic differentiation of genes involved in post-mating behaviour in An. gambiae (UNIPG, UNIROMA1, IRD, MRTC, ICIPE)
We developed PCR protocols to examine polymorphisms and rates of evolution in 13 MAG- and LRT-specific genes potentially involved in mating (based on results of WP1) and applied them on 5-10 specimens of An. gambiae s.s. M and S molecular forms, An. arabiensis, An. quadriannulatus, An. melas and An. merus. Genes encoding the principal components of the mating plug (e.g. plugin and TGase) were found to be conserved among taxa, whereas clusters of three MAG- and three LRT-specific genes showed high genetic divergence among sibling species of the complex, but not between M and S molecular forms. These two clusters were further investigated on a large collection of samples (either already available in UNIROMA1 or collected ad hoc by ICIPE and IRD) and were shown to evolve under selective pressures (D3.5; M3.2). Evidence of positive selective pressure and purifying selection maintaining lineage-specific products were found in the cluster of three paralogous MAG-specific genes (AGAP009369, AGAP009370, AGAP009370-copy). Adaptive evolution was detected in a number of codons of the three paralogous LRT-specific genes encoding for serine-proteases (AGAP005194, AGAP005195 expressed in the atrium and AGAP005196 detected in the spermatheca). Episodic selection was inferred in AGAP005196 along the branch leading to An. melas. The high level of replacement polymorphisms observed in all three proteases suggests that these duplicated genes might experience relaxed evolutionary constraints that could benefit a rapid diversification and eventually lead to a fixation of new advantageous variants. Two manuscripts on the obtained results are currently in preparation.
So far our analyses elucidated genes with species-specific but not molecular/chromosomal form-specific polymorphisms. These results did not allow us to develop standardized protocols for large-scale genotyping of the M and S forms. To overcome this roadblock, novel approaches are in progress to maximize the chances to identify molecular/chromosomal form-specific SNPs (D3.6 D3.7 and D3.9). On the other hand, absence of informative SNPs between M and S forms (also reported by other groups) may reflect as yet unknown biological phenomenon. As M/S hybrids are fertile in the laboratory conditions, it is possible that reproduction barriers have yet to be established between these two forms; in this case no informative polymorphisms for M/S will be detected in the reproductive genes.
The genetic characterization of samples collected in MALVECBLOK field sites and of other samples acquired by UNIROMA1 via other collaborators from Afrotropical malaria endemic areas and the development of a web-based database have been initiated in collaboration with CNRS (D3.4 expected delivery postponed to month 24).
We examined levels of polymorphism in genes with potential roles in post-mating responses identified in WP1 in samples collected by MALVECBLOK in malaria endemic countries and in other specimens available in UNIROMA1. To this end, we chose 8 genes expressed in the male accessory glands (MAGs) (AGAP009099 or TGase3, AGAP008276, AGAP008277, AGAP009368 or Plugin, AGAP009369, AGAP009370, AGAP009370c, and ACP1) and 4 genes expressed in the female low reproductive tract (LRT) (AGAP00885, AGAP005194, AGAP005195, AGAP005196).
A high level of conservation was observed in genes encoding the principal components of the mating plug Plugin and TGase3 suggesting strong evolutionary constrains exerted on these proteins.
In contrast, fixed genetic differences were found in a cluster of three genes expressed in the MAGs, whose products are delivered to female as components of the mating plug (AGAP009369, AGAP009370, AGAP009370c). Evolutionary analyses revealed signatures of purifying selection that acts to preserve the structure and function of these proteins. Fixed species-specific polymorphisms were found in at least one gene from each taxon along their geographical distribution (with the exception of An. gambiae s.s.). Notably, one fixed species-specific replacement in AGAP009370 coding region (Q to E amino acid change at the C-terminal region) was observed in An. arabiensis. This is the first report of an amino acid substitution that distinguishes An. arabiensis from An. gambiae s.s. which is present in an autosomal gene located outside the previously described characteristic chromosomal inversions.
We also detected signatures of adaptive evolution in the cluster of three genes encoding serine-proteases whose expression is inhibited in the female LRT after mating (AGAP005194, AGAP005195, AGAP005196). In all three genes fixed polymorphisms were detected in the regions encoding the specificity pockets that are known to mediate substrate recognition and/or binding. Furthermore, fixed amino acid replacements in An. melas and An. merus were detected on the exposed surfaces in the proximity of the protease active site in AGAP005195, suggesting potential species-specific differences in substrate recognition. The detected polymorphisms may have reinforced or directly contributed to the reproductive isolation among the species.
These results were published in BMC Evolutionary Biology, 2011, 11: 72; and in BMC Evolutionary Biology, 2011, 11: 292 (D3.5).
In other genes we detected patterns of incomplete lineage sorting due to a recent introgression event or sharing of ancestral polymorphisms among species/forms. Unfortunately, the observed evolutionary patterns were not helpful neither to reconstruct reliable phylogenetic relationships among members of the An. gambiae complex (D3.6) nor to develop standardized protocols for large-scale genotyping of An. gambiae s.s. chromosomal/molecular forms. We extended our analysis to 6 additional genes potentially involved in post-mating responses and previously reported by others to exhibit high levels of polymorphism (D3.9). However, in the large collected by MALVECBLOK all previously polymorphisms were randomly distributed among the samples and did not correlate with a particular chromosomal or molecular form.
Analysis of the expression of immune reporter genes in single specimens directly collected in the field to monitor their immune status in relation to their larval habitat (CNRS, IRD, MRTC)
We developed standardized protocols for collection of immature stages of An. gambiae from natural breeding sites and for subsequent membrane feeding of adult females on P. falciparum isolates. Procedures were discussed and approved by IRD, RUNMC, MRTC and ICIPE partners during a workshop set in Cameroon in June (D3.1).
Immature stages of An. gambiae were collected in multiple breeding sites (5 to 10) from 5 different locations in Cameroon (Mfou, Nolbission, Nkolondon, Obala and Mvan). Pools of 30 larvae (L4 stage) and 30 pupae were prepared directly in the field and qRT-PCR analyses were performed in Strasbourg. Examined breeding sites displayed a high level of heterogeneity in expression of mosquito antimicrobial genes (Defensin 1, Gambicin and TEP1) and no clear-cut correlation was established between expression patterns of the immune genes and mosquito resistance to Plasmodium in the field. A manuscript is in preparation (D3.8 expected delivery date postponed to month 28).
To characterize microbial diversity (D3.3) we collected additional pools of larvae and pupae in the same breeding sites. Midguts were dissected and 16S rDNA was analysed using TGGE and DGGE techniques. Only Gram-negative bacteria species were identified, suggesting that the mosquito midguts provide a thriving niche for this type of bacteria. Interestingly, we observed differences in the microbial flora in larval and pupal midguts from the same habitat, suggesting stage-specific proliferation of microbes (M3.3). For instance, Thorsellia anophelis was only found in larval midguts. No significant differences in bacterial loads were detected between M and S molecular forms. To reveal correlations between mosquito genotype, microbiota composition and Plasmodium genotype, we sequenced mosquito/parasite/microbiota genomes after experimental infections with the field parasites using the Illumina platform. Moreover, we compared the complexity of microbiota in the mosquito midgut between P. falciparum infected and non-infected mosquitoes by sequencing DNA extracted from the midguts using the 454 GS Flex Titanium sequencer. Analyses of data are in progress. These results should: i) provide a detailed description of microflora in selected larval habitats in Cameroon, and ii) determine whether exposure to bacterial communities in natural populations of mosquitoes modulates their susceptibility to P. falciparum.
We reported high levels of variability in expression of antimicrobial peptide genes in larvae collected from breeding sites at different geographical locations in Cameroon. These results indicated that larval habitats shape the mosquito immune status. Using SOPs for collection of immature stages of An. gambiae from natural breeding sites and for membrane feeding of females on P. falciparum isolates (D3.1) we examined whether exposure to distinct bacterial communities modulates mosquito susceptibility to P. falciparum. We first selected breeding sites where larvae showed distinct patterns of AMP expression and collected larval stages for further culturing in the laboratory. Eclosed females were exposed to P. falciparum infection, their midguts were dissected 8 days after the infectious blood meal and analysed for parasite loads. We then determined the microbiota of individual midguts using new generation sequencing approaches (454 pyrosequencing). Laboratory mosquitoes were used as a control. The microbial diversity was analysed according to the origin of the mosquitoes. After having obtained a snapshot of bacterial communities, we looked to see whether there existed a correlation between the bacterial content and the malaria infection status by comparing midgut microbiota of P. falciparum-infected and non-infected mosquitoes.
Significant differences were observed in microbiota between the laboratory mosquitoes and the mosquitoes collected in natural habitats. The diversity of bacterial species was very limited in the laboratory mosquitoes. For mosquitoes from natural habitats, the gut microbiota displayed a large inter-individual variability with a dominant few taxa. Interestingly, differences in the bacterial composition of the mosquito midguts correlated with the breeding sites. Our results suggest that the environmental conditions at larval stages shape the midgut microbiota in adults.
A significant positive correlation was detected between the relative abundance of Enterobacteriaceae bacteria in the mosquito midgut and P. falciparum infection levels. These results suggest that the midgut microbiota is an important factor that shapes vector competence, and that Enterobacteriaceae family of Gram-negative bacteria benefit P. falciparum development. These results were published in PLoS Pathogens (2012) (D3.8).
Contribution of genetic polymorphism to the immune status in the African populations of An. gambiae (IRD, CNRS, MRTC, ICIPE)
Experimental infections were established in Kenya using standardized MALVECBLOK protocols (Del. 3.2). Field- and lab-based experiments are being conducted in Cameroon, Mali and Kenya to investigate vector-host-parasite interactions using natural isolates of P. falciparum. Mbita laboratory An. gambiae S-form colony was used in Kenya. Larvae were bred in soils obtained from different larval breeding sites: significant differences were observed in infection rates in adult females that emerged. Analysis of microbial flora from mosquito guts and these breeding environments (soil and water) is in progress. Natural populations of mosquitoes were infected with field isolates of P. falciparum in Cameroon: preliminary results showed that although the M molecular form harbored higher infection rates, in sympatric conditions the M and S molecular forms were equal in transmitting P. falciparum.
Sequencing of mosquitoes to investigate putative correlation between TEP1 alleles and the mosquito resistance to P. falciparum is in progress at IRD-Montpellier and CNRS-Strasbourg (M3.1) and we expect that the sequencing will:
i) identify new TEP1 alleles from field populations (M3.6) and
ii) reveal informative polymorphisms linked to the mosquito infection status (D3.11; M3.7).
A standardized protocol for TEP1 sequencing and characterization in field mosquitoes has been established and distributed to all partners (D3.7). Silencing of TEP1 interacting proteins has been conducted in Cameroon and analysis of results is in progress.
We investigated the genetic polymorphism of TEP1 in Cameroon, Mali and Kenya by collecting immature stages of mosquitoes from a series of breeding sites. The TEP1 gene was first sequenced in individual mosquitoes and compared with the sequences of TEP1 alleles identified in the laboratory colonies. Unexpected the same 4 major allelic classes were identified in African natural populations: S1, S2, R2, and R1. Based on the sequence analysis and on results obtained in WP2, we developed a PCR-based RFLP protocol for TEP1 genotyping (D3.11). A SOP for TEP1 genotyping was developed and used in Cameroon, Mali and Kenya (D3.7). Our analysis revealed TEP1 allelic distribution in the countries across the African continent. Importantly, we observed that alleles were unevenly distributed among An. gambiae molecular forms. A manuscript is currently in preparation, which describes TEP1 polymorphism in the different malaria endemic areas (D3.12).
In Cameroon, IRD collected more than 2000 larvae in the field breeding sites and cultured them to imago in the laboratory. The eclosed 920 females were infected with field isolates of P. falciparum. Midguts were dissected eight days later to determine mosquito infection status by quantitative RT-PCR, whereas the carcasses were used for genotyping (M&S form and TEP1). The R1 allele was very rarely found (approximately 1 in 1000). Allelic frequencies of TEP1 R2 and S2 differed between M and S forms, where the S2 allele was more frequent in the M form and the R2 allele - in the S form. The majority of mosquitoes had S1 allele. Meta-analysis of 920 experiments indicated a higher prevalence of infection for the M form in both allopatric and sympatric M and S populations. But no difference in the infection intensity was detected. Statistical analysis also did not reveal any correlation between TEP1 genotypes and prevalence of infection. This is not surprising as only R1 allele, which is almost absent from Cameroonian samples, correlates with resistance to infections with P. berghei. Nonetheless, the M molecular form of An. gambiae has a higher frequency of the S2 allele and is more susceptible to P. falciparum infection, which is indicative of genetic association between TEP1 polymorphism and malaria infection.
The genetic characterization of samples collected in MALVECBLOK field sites is summarized in a database (Del. 3.4).
Potential Impact:
MALVECBLOK project focused on the major threat to global human health: malaria, the cause of death of more than a million people each year, and of illness of hundred millions individuals in tropical and subtropical countries. The pluri-disciplinary and complementary efforts of five European research teams, located in four different member states and one African ICPC country, were linked to those of two sub-Saharan groups, located in two disease-endemic ICPC countries.
The Consortium focused on the following topics, all related to the biology of the major malaria vector An. gambiae:
- reproductive biology
- immunity (defence strategies of the mosquito against the parasite)
- genetic structure of natural mosquito populations in Africa.
The major impact of the project on the global health issue of malaria is represented by the development of new concepts for innovative vector control measures. By promoting international cooperation with scientists of three disease-endemic countries, this Consortium elucidated the molecular events involved in reproduction and immunity of the mosquito vector and examined their effects on the development of the malaria parasite, both in laboratory and field conditions. The knowledge acquired in this project had a strong impact on European scientific competitiveness, as evidenced by a series of publications in the international peer reviewed journals.
Scientific issues: innovative ideas and findings
Fully exploiting the recent advances in the characterisation of the An. gambiae genome and functional gene analysis, MALVECBLOK comprehensively elucidated molecular events involved in reproductive and immune functions of the mosquito vector. This was achieved through the integration of expertise from different disciplines contributed by the teams involved in the project. The three topics covered by the project (reproductive biology and immunity and genetic structure of mosquito populations in Africa), were studied using complementary approaches and a variety of standard operational procedures. The obtained findings were published in scientific prestigious international scientific journals such as PNAS, PLoS Biology, PLoS Pathogens, Inter J Parasitol, BMC Evol Biol and presented at numerous prestigious international conferences.
In this bi-directional North-South collaboration, the fieldwork carried out in Africa allowed scientific validation of experimental results obtained in the European laboratories, and vice versa, African field data from the different African countries gave rise to new hypotheses which have been then tested in Europe. This collaborative project created strong links between European and ICPC research teams, and benefit the conception of highly innovative ideas and findings that improved the knowledge on crucial biological processes of the malaria mosquito. The MALVECBLOK project promoted the exchange of good practice and standardisation of fieldwork scientific procedures and protocols. Young researchers had excellent opportunities for training in their everyday scientific work, visits to the partners laboratories and through their participation at Consortium meetings at international Congresses.
By the integration of its complementary research skills, this collaborative research project strongly contributed to the development of a dynamic and successful European Research Environment. Based on the knowledge generated, it proposed new approaches for prevention and management of malaria.
European Competitiveness
The complementary skills of the MALVECBLOK partners led to identification of novel key molecules underlying reproduction biology and immunity of An. gambiae mosquitoes. The complementary expertise available in the Consortium was instrumental for understanding the bases of the transmission of the disease. The knowledge and tools provided by this project is now ready to be exploited for the design of novel active compounds, either inhibiting the reproduction of the insect, or stimulating the insect immune system for parasite killing.
For example, the identified in the WP1 crucial role of transglutaminase in male fertility could be exploited to prevent efficient female insemination. Currently, chemical formulations are being designed to block the activity of this enzyme.
On the other hand, identification of genetic structuring of mosquito populations relative to TEP1 genotype revealed by MALVECBLOK, is important for the development of future diagnostic tools to gauge susceptibility of local vectors to Plasmodium.
Finally, identification of the molecules that are required for both mosquito reproduction and Plasmodium development offers new interesting targets for the design of novel insect-specific to manipulate vector fertility and immunity at the population level, a topic that has great public health and economic relevance.
The findings and tools developed by MALVECBLOK are therefore expected to boost the innovative capacity of sustainable EU health-related industries and business.
European added value
The work undertaken by MALVECBLOK would not be possible at a regional or national level. A total of 3 different member states (comprising 5 Participants) and 3 ICPC contributed their complementary expertise in order to implement an international trans-disciplinary project. The scientific excellence skills of the 5 European partners have been essential for the success at the intellectual and infrastructural level.
- partner 1 (CNRS, FR) : mosquito immunity (RNAi silencing, cell biology, analysis of immune phenotypes, rodent parasite cultures, mosquito breeding colonies)
- partner 3 (RUNMC, NL) : immunology, biology of Plasmodia spp. (experimental membrane infections)
- partner 4 (INIROMA1, IT) : population biology of An. gambiae s.s. (cytology, genotyping, experience in fieldwork including sample collection)
- partner 5 (IRD, FR, tight collaboration with OCEAC in Cameroon) : P. falciparum transmission in mosquito vectors (functional analysis and immune gene polymorphisms, genetic analysis, experience in the fieldwork and sample collection, mosquito breeding colonies)
- partner 6 (MRTC, Mali): mosquito population biology, antiviral responses (mosquito breeding colonies, experimental membrane infections, experience in fieldwork and sample collection, fully equipped outstations)
- partner 7 (ICIPE, Kenya): mosquito population biology, strong parasitological background (mosquito breeding colonies, fully equipped outstations)
- partner 8 (UNIPG, IT) : reproduction biology of An. gambiae (transgenesis, analysis of post-mating phenotypes, mosquito breeding colonies).
International cooperation with ICPC countries was undertaken in order to share, generate and use knowledge through novel and consolidated research partnerships, on the basis of mutual interest and mutual benefit. In order to study and understand the geographical variations in local mosquito and parasite populations, the fieldwork in the malaria-endemic area was performed in three different sub-Saharan countries covering West, Central and East Africa.
List of Websites:
http://www.malvecblok.org
During the project period, MALVECBLOK implemented joint research efforts addressing fundamental topics of mosquito biology, and achieved the following objectives:
(i) The molecular bases of reproductive biology of the mosquito vector, and its effects on immunity and Plasmodium transmission. The major discoveries of this work package identified the key male and female molecules that shape post-mating responses and reproductive success in the malaria mosquitoes. We have characterized the composition of the mating plug and the key coagulation enzyme, transglutaminase, crucial for mosquito fertility. The specific roles of MAG secretions and the sperm cells in induction of post-mating responses in females were examined using sperm-less males that we established by silencing a key gene involved in gonadogenesis. These experiments demonstrated for the first time the pivotal role of male accessory glands in orchestrating female post-mating behaviour. In addition, these results brought a much needed proof of concept for vector control strategies based on sterile insect technique as spermless males efficiently engaged females into mating which, as expected, was fully sterile. Although no effect of mating on Plasmodium has been detected, our results revealed an unexpected connection between responses of females to mating and to blood feeding evidenced by mating-induced expression of a series of genes associated with a blood feeding.
(ii) The molecular mechanisms, which determine the mosquito immune status and regulate Plasmodium sporogony and transmission, both in laboratory settings and in natural populations. We have characterized the nature of TEP1/LRR complex and revealed its crucial role in defence against bacteria and malaria parasites. We uncovered that genetic diversity of P. falciparum enables parasites to survive immune responses in the vector; this discovery has important consequences for design of future vector control measures. Functional analysis of two major yolk proteins crucial for mosquito fertility revealed their role in TEP1 binding to parasites. Strikingly, expression of two major yolk proteins is negatively regulated by Rel1/Rel2 immune pathway, suggesting that activation of immune responses represses reproduction in the mosquitoes. One of the major achievements of MALVECBLOK is the discovery of TEP1 as a novel marker for evaluation of genetic structure of mosquito populations in the study sites. Established standardised protocols for TEP1 genotyping revealed that the allele conferring resistance to Plasmodium (TEP1R1) is massively spread in West Africa but is almost absent from Central and East Africa. Further experiments are needed to evaluate the contribution of TEP1R1 to the mosquito resistance to the parasites, but our results suggest that TEP1 can be used as a novel marker for population genetics studies of An. gambiae.
(iii) The role of genetic polymorphism in genes controlling reproduction and immunity on the structure of mosquito populations and malaria transmission in Africa. We characterized polymorphisms in reproductive genes and gene families, which reveal evolutionary relationships between subspecies of the An. gambiae complex. Some genes are under strong purifying selection, whereas others are subject for extensive variation with no signs of species-specific signatures. Unexpectedly, we found that polymorphism in TEP1 correlates with An. gambiae molecular forms, and performed massive TEP1 genotyping across West, Central and East Africa. Another key discovery of the consortium is the demonstration of the role of microbiota on the capacity of mosquitoes to transmit malaria. We have identified bacterial communities in the midguts of mosquitoes collected in breeding sites in Cameroon, and revealed a positive correlation between the presence of Enterobacteriaceae and Plasmodium development. Therefore we identified a new environmental factor that shapes malaria transmission in Africa.
In conclusion, during the project identified new potential targets for control of mosquito populations. Our results suggest that Plasmodium developed means to cope with the vector immune responses, especially in the areas of high transmission, and instruct the future studies on immuno-modulated mosquitoes. Special attention of the consortium was given to cutting-edge training opportunities and sharing of resources between the partners. The knowledge acquired in the project has a strong impact on European scientific competitiveness, and addresses societal issues in the endemic countries by recruitment of young researchers and by disseminating the study principles and knowledge of socio-economic impact of malaria in the research centres and, more importantly, among the local population involved in the study. As a result of the project, children of at least three villages in Africa benefit from regular medical surveillance and timely antimalarial treatment.
Project Context and Objectives:
This project is inspired by recent advances in the characterisation of An. gambiae genome, in the understanding of the differentiation/speciation processes ongoing within this species and in the development of powerful tools for functional gene analyses, and explores critical molecular mechanisms that underlie the vectorial capacity of the mosquito (i.e. its ability to support development of malaria parasites) with the ultimate purpose to block disease transmission. Europe has always been at the avant-garde of research in mosquito biology, however scientific exchanges between teams working in various aspects of the field, e.g. population biology, reproduction and immunity, remain fragmentary. This proposal integrates research groups from 3 European countries and scientists from 3 African malaria endemic countries with the main aim to increase our knowledge on two biological processes that crucially determine vectorial capacity, reproduction and immunity. The particular emphasis is on understanding the genetic bases of these two processes and the relationship between them, with the view to assess their role in malaria transmission and in shaping the structure of mosquito populations in Africa.
Accordingly, the Consortium draws together a critical mass of complementary expertise and resources which will be crucial to successfully implement joint research efforts addressing these fundamental topics of mosquito biology, organized in the following objectives:
(i)The analysis of the molecular bases of crucial aspects of reproductive biology of An. gambiae, such as mating and egg laying, and the role of reproduction in immunity and Plasmodium transmission. To this end, the Consortium will identify the key male (months 1-14, milestone 1.2) and female (months 1-42, milestone 1.1) factors, characterize male-female interactions (months 12-42, milestone 1.6) and signalling cascades shaping post-mating responses in An. gambiae (months 12-42, milestone 1.5). The project will considerably expand the fundamental knowledge of reproductive biology in Anopheles. In parallel, the relationship between reproductive biology and immunity in An. gambiae will be investigated. The effect of mating on the immune status will be examined both in the laboratory and field conditions (months 1-24, milestone 1.3).
(ii)The study of the molecular mechanisms which determine the An. gambiae immune status and regulate Plasmodium development and transmission, in both laboratory settings (months 1-42, milestones 2.1; 2.4) and natural populations (months 6-42, milestones 2.3; 2.5; 2.6; 3.3). Equally important, the trade-off on the immune status on the reproduction will be characterised (months 12-42, milestone 2.2).
(iii)The investigation of the role of genetic polymorphism in genes controlling reproduction and immunity on the structure of An. gambiae populations and malaria transmission in Africa (months 1-42, milestones 3.1; 3.2; 3.4; 3.6; 3.7).
Project Results:
WP1 Molecular mechanisms of vector reproduction and its effects on immunity and Plasmodium transmission
This work package has two main objectives:
1) the analysis of the molecular components shaping the reproductive success of male and female mosquitoes; and
2) the assessment of the effects of the reproductive status on female immunity and transmission of Plasmodium parasites.
Role of male-produced factors in inducing female post-mating responses (UNIPG)
In order to identify male factors transferred to the female during mating and likely to be responsible for the modulation of female behaviour after copulation, we have analyzed the composition of the mating plug. The mating plug is formed by coagulated male accessory gland (MAG) secretions, mainly consisting of proteins and lipids. We have performed mass spectrometry analysis of mating plugs dissected from freshly mated females, and have identified a number of male reproductive proteins transferred to females (Rogers et al. PLoS Biology 2009). Among these proteins is a MAG-specific transglutaminase (TGase), a cross-linking enzyme. In the frame of this project, we found that this transglutaminase is essential for the coagulation of male seminal secretions. Impairing expression of the TGase in the MAGs by RNA interference (RNAi), injecting dsRNA molecules into virgin males, prevents mating plug formation and transfer (D1.4). Moreover, females mated to males impaired in plug formation cannot store sperm properly, and are therefore sterile (M1.2). This major result was published in PLoS Biology and broadly discussed by the media worldwide. This is the first demonstration that meddling with mosquito mating can have applications for vector control.
Among other plug proteins are four Acps that may be triggering female post-mating responses. We have named AGAP009368 plugin, as this is the most abundant protein of the mating plug (Rogers et al., PLoS Biology 2009). The other three genes encoding Acps (AGAP009362, AGAP009370 and AGAP012830) have been silenced by injecting virgin males with the corresponding dsRNAs, however no obvious phenotype on female fertility, fecundity and receptivity to mating has been found yet.
We have developed polyclonal antibodies against all these male proteins (D1.3) and have studied their localization in the MAGs, plug and female reproductive tract. Our first results reveal non-overlapping expression patterns of these proteins, and we will continue our efforts to elucidate their functions.
The mating plug is formed by coagulated male accessory gland (MAG) secretions, mainly proteins and lipids. We have confirmed that a steroid hormone, 20 hydroxyecdysone (20E), is produced by the MAGs. Moreover, we have shown that 20E is transferred to females as part of the mating plug and that this male-derived hormone induces the expression of a number of reproductive genes in the atrium of virgin females, mimicking the transcriptional response that occurs in females after mating. We have also determined that one of the female genes, TED1 (see below), induced by the male-transferred 20E, interacts directly or indirectly with this hormone (M1.6). 20E transfer triggers a cascade of events that lead to increased egg development by the female. This is the first example of a male hormone that modulates female reproductive physiology (M1.2). We are targeting the biosynthesis of 20E in the MAGs by silencing the pathway of enzymes that are responsible for its synthesis. Our results show that we can reduce 20E levels in the MAGs by 50%, and that this reduction affects the female response after mating. These results are being written for publication (D1.8).
We have continued our functional analysis of MAG proteins. Specifically, we have focused on 3 enzymes, PAM, PHM and QPCT, which are involved in the synthesis of small peptide hormones. We have targeted QPCT by RNAi injecting virgin males with the corresponding dsRNAs, however no obvious phenotype on female fertility, fecundity and receptivity to mating has been found yet. We have developed polyclonal antibodies against all these male proteins (D1.3) and have studied their localization in the MAGs, plug and female reproductive tract, in collaboration with our partners from UNIROMA1.
Identification of female reproductive proteins and analysis of their interactions with male factors transferred during mating (UNIPG, UNIROMA1, CNRS)
We have functionally analysed a number of female genes regulated by mating (D1.1 M1.1). We have selected genes expressed in the spermatheca (sperm storage organ) and the atrium (where the mating plug is deposited and digested). Among the spermathecal genes, we have analyzed the heme peroxidase HPX15, whose expression is strongly upregulated 24h after mating. We have developed polyclonal antibodies against this protein and assessed that it is expressed predominantly in the secretory (glandular) cells surrounding the spermathecal capsule. RNAi knock down of HPX15 in virgin females has been successful at both transcript and protein levels, however no effects have been detected on fertility and fecundity of females (for these analyses we have developed specific protocols to assess the fertility and fecundity of females, D1.2).
Among the genes regulated by mating in the atrium, we have analyzed three genes encoding serine proteases, AGAP005194, AGAP005195 and AGAP005196 (the latter is also expressed in the spermatheca), strongly downregulated by mating. Antibodies raised against AGAP005194 have shown that this protein is closely linked to the mating plug surface, with a pattern resembling that of the major plug protein plugin, and its expression is not detectable at 24h post copulation. This suggests that AGAP005194, and possibly the other proteases, are involved in digestion of the mating plug, which is mostly completed at 24h post-mating.
To ascertain that our studies are relevant for natural populations, we have collected mating couples of field mosquitoes from natural swarms and examined the mating-regulated expression of a number of genes identified in our laboratory analyses. For this, UNIPG and UNIROMA1 performed a field trip to Burkina Faso in collaboration with IRSS. Mosquitoes in copula were collected in different villages from swarms of M and S molecular forms, believed to be incipient species in the An. gambiae complex. Quantitative PCR on the dissected tissues confirmed that most genes regulated in laboratory cages are also induced or repressed after mating in field populations. These results are very important for validating our candidate genes for further functional analyses.
We have continued to perform functional analyses on a number of female genes regulated by mating (D1.1 M1.1). We have selected genes expressed in the spermatheca (sperm storage organ) and the atrium (where the mating plug is deposited and digested). Among the spermathecal genes, we have analyzed the heme peroxidase HPX15, which is strongly upregulated by mating at 24h. We have developed polyclonal antibodies against this protein and assessed that it is expressed predominantly in the secretory cells (glandular) surrounding the spermathecal capsule. RNAi knock down of HPX15 in virgin females has been successful at both RNA and protein levels. Our initial studies had not identified any effect of HPX15 silencing on fertility, however a deeper analysis of the function of this gene has shown that HPX15 is important to preserve sperm function following multiple blood feedings. Fertility of egg batches after two blood feedings decreased significantly when HPX15 expression was reduced by RNAi injections (E. Teodori, F. Catteruccia, manuscript in preparation)(D1.8 M1.5). These results identify the oxidative stress derived from the blood meal as one of the risk factors for sperm function, and highlight the role HPX15 in preserving sperm viability after repeated blood meals.
We have performed a microarray analysis of spermathecae dissected from mated females and identified a signaling pathway comprising three G protein-coupled receptors (GPCRs) and downstream protein kinases that may play a crucial role for sperm viability and activation. We are currently studying the GPCRs activated by mating by RNAi silencing and by performing cell-based assays to establish the downstream signaling pathways. We have designed polyclonal antibodies to determine their exact localization in the spermatheca. We have chosen to focus on functional analyses of the spermatheca over proteomic analysis of the hemolymph (as originally planned) as the possibility of preventing sperm viability in the female has tremendous implications for reducing the fertility of mosquito populations in vector control programs.
Dissection of signalling cascades that regulate post-mating behaviour in females (UNIPG)
We identified a gene whose expression was strongly induced by mating and named it Matin (Mating induced). Although Matin does not feature any known functional domains, immunofluorescence experiments with polyclonal antibodies developed for the analysis have assessed that the protein is specifically expressed in the ampullae, a region in the female reproductive tract connecting the ovaries to the atrium. Inhibition of Matin expression by RNAi injections into virgin females strongly inhibits egg laying after mating and blood feeding, suggesting that this gene is involved in the signaling cascades initiated by mating and leading to oviposition.
We have generated transgenic lines that contain the promoter region of a mating-responsive gene controlling expression of a dtTomato or luciferase reporter genes for quantification of expression (D2.1). The promoter region of AGAP002620 (p2620), a gene expressed in the atrium and strongly regulated by mating, has been chosen for this purpose. An. gambiae embryos have been injected with the constructs p2620:luciferase and p2620::dsTomato following well established protocols and G1 individuals were screened for the occurrence of germline integration. The resulting homozygous transgenic line is currently being analysed to confirm the correlations between expression patterns of the endogene and the transgene.
We have further characterized PET1 (AGAP002620), the putative protein involved in egg laying trigger studied during the first reporting period. We have established that silencing of this gene does not affect egg laying as initially hypothesized, while it affects egg development. Mated females develop more eggs than virgin females after taking a blood meal. However, females in which PET1 was knocked-down by RNAi showed significant reduced fecundity compared to control females, and the overall number of eggs developed by dsPET1 females was decreased to levels similar to those obtained with virgin females. These results show that PET1 is a crucial gene in the cascade of events triggered by mating that contributes to egg development. For its role in egg development rather than egg laying, we have renamed this gene TED1 (trigger of egg development). Moreover, as discussed earlier, we have identified a male trigger of TED1. This is a steroid hormone, 20E, transferred to females during mating as part of the mating plug. Silencing of TED1 impairs the correct diffusion of 20E out of the female atrium into the hemolymph (M1.6) where 20E is needed to activate egg development though the induction of lipid transporters. These results are being prepared for publication (D1.8 M1.5). We are currently assessing whether TED1 silencing affects the development of Plasmodium parasites (D1.7 M1.4).
We have identified additional genes whose expression is triggered by mating in females at different time points in the spermatheca, atrium, ovaries, midgut, head and rest of the body (hemolymph, fat body, legs, Malpighian tubules and others). Besides providing an expanded collection of cDNAs from female tissues, these analyses are revealing additional candidates likely to be involved in modulating female post-mating behaviors (D1.8).
Moreover, we are characterizing a transgenic mating-responsive line FK that contains an expression cassette pAGAP002620:tdTomato. The FK line is being used to perform functional analyses of female candidate genes that may be regulating the mating-induced signaling cascades (D1.8 M1.5)
Effects of mating and post-mating responses on immune status and on sporogony and transmission of Plasmodium (UNIPG, CNRS, RUNMC)
The expression of a number of immune genes at different time points after mating has been studied in both laboratory and field-collected females. The expression of these genes (encoding antimicrobial peptides Cecropins, Gambicin and Defensin 1, antiparasitic genes like TEP1, LRIM1 and APL1) in the female reproductive tract and in the rest of the body has been assessed by quantitative RT-PCR. We found that expression of Gambicin is strongly downregulated in atrium and spermatheca after mating, while transcript levels of TEP1 are induced in the rest of the body. Moreover, in natural populations we found numerous polymorphisms associated with these genes (M1.3). The relevance of these polymorphisms in gene function is currently being assessed.
We also performed Plasmodium berghei infections and compared oocyst development in virgins versus mated females. For that, females were separated from males at the pupal stage and either mated or kept as virgin. At day 5 post-emergence virgin and mated females were fed an infectious P. berghei blood meal and oocyst numbers were compared in the two groups 10 days after infection. In this mouse model, no major difference was found in infection levels in the two groups, suggesting that the mated status does not directly affect development of the rodent malaria (M1.3).
We have confirmed that expression of the thioester-containing protein 1, TEP1, is induced in females after mating in the rest of the body. Moreover we have found a number of immune genes specifically induced by mating in the female head, indicating a strong immune response to yet undefined factors (possibly male peptides reaching the head or crossing the blood brain barrier). We have collected additional field samples from natural An. gambiae populations and found numerous polymorphisms associated with TEP1 (M1.3). In order to understand the relevance of these findings, we have collected in collaboration with our partners CNRS mosquito larvae from a number of different breeding sites (temporary and permanent) and adults from mating swarms to determine the microbiota of natural mosquito population. We are now determining whether there exists a correlation between different bacterial populations and specific TEP1 polymorphisms. Our hypothesis is that the latter may have been driven by selective pressures inflicted by a diverse range of microbes in An. gambiae populations adapted to different ecological habitats.
We have also generated male mosquitoes depleted of sperm cells and have determined that female mated to these sperm-less males do not exhibit any differences in their post-copulatory behaviors: these females lay (sterile) eggs and fail to re-mate, as normally observed in females mated to fertile males. These results have been recently published in Thailayil et al., PNAS 2011 (D1.5). Females mated to these spermless males are currently being tested for their ability to transmit malaria parasites. Preliminary data suggests that there is no difference in the number of parasites developing in these females compared to females mated to normal males (M1.4).
WP2 Molecular mechanisms that determine mosquito immune status and its effects on sporogony of Plasmodium and on mosquito reproduction
This work package has two objectives:
(i) to provide an integrated view of the molecular mechanisms which determine the mosquito immune status and regulate Plasmodium sporogony and transmission, in both laboratory settings and natural populations; and
(ii) to examine how the mosquito immune status affects reproductive capacity of mosquitoes.
Analysis of molecular mechanisms of TEP1-mediated Plasmodium recognition and killing in An. gambiae by biochemical and genetic approaches (CNRS, UNIPG, RUNMC)
Previous work demonstrated that two leucine-rich repeat proteins form a complex with TEP1 and that this complex is required to maintain the cleaved form of TEP1 in circulation. We performed biochemical analysis of the protein complex isolated by immuno-precipitation of the mosquito hemolymph and confirmed the presence of the two LRR proteins. However, no additional members could be identified, suggesting that the complex consists of these three proteins. Therefore, the complex can be studied in more detail in vitro. We asked which form of TEP1 is recruited into the complex, and in collaboration with Dr Baxter, University of Texas, USA, performed detailed characterization and solved X-ray structures of the two LRR proteins. Our data indicate that these proteins form a dimer, which is stabilized by a C-terminal coiled-coil domain interlinked by a single disulphide bridge. Our results suggest that these LRR proteins interact with a disarmed 'inactivated' form of TEP1 unable to form a covalent bond with the parasite surface. To confirm relevance of our observations in vitro for the mosquito biology in vivo, we used a recombinant TEP1/LRIM1/APL1 protein complex to rescue TEP1 knockdown phenotype in vivo. We developed a series of protocols to study immuno-induced and/or immuno-compromised mosquitoes (D2.3) and used bacterial infections to address this question. In this protocol, mosquitoes deprived of TEP1 succumb to bacterial infections within 24 h after challenge. We first demonstrated that depletion of the LRR proteins elicits a phenotype similar to dsTEP1, and then injected the recombinant complex together with bacteria 4 days after injection of dsRNA against TEP1, LRIM1 or APL1. Injection of the complex rescued survival of the mosquitoes confirming that it is fully functional in vivo, and further indicating that the complex does not target TEP1 to the surface of bacteria but has a different role. We are currently examining whether this complex associates with a bacterial protease to amplify TEP1 cleavage in the proximity of pathogens.
To perform structure to function analysis of TEP1, we expressed resistant and susceptible forms of the protein in a baculovirus system. In an in vitro system, the susceptible form of TEP1 displayed significantly shorter half-life than the resistant form, as evidenced by protein precipitation (M2.1). These results suggest that protein stability could underlie the more efficient binding of the resistant form to the parasites. This simple assay will be used to map residues that determine TEP1 stability by site-mutagenesis. Once important residues will be identified, we will confirm in vitro results in vivo by establishing transgenic mosquitoes. Currently, we are in the process of developing transgenic mosquitoes with a mutated TEP1 thioester site.
We have performed functional analysis of genes that are regulated by Rel1/Cactus cassette, and examined effects of knockdown of the top 10 candidates identified by microarrays on development of P. berghei within a mosquito. However no significant effect was observed. It is possible that other genes do not contribute to the response against rodent malaria parasites, suggesting that the phenotype could be totally explained by the contributions of TEP1, LRIM1 and APL1. These results are consistent with the biochemical data described above; therefore we are focusing our analysis on the TEP1/LRR complex. Interestingly, when the roles of these proteins were examined in experimental infections with P. falciparum in Cameroon, only TEP1 silencing increased parasite numbers, whereas depletion of the two LRRs had no effect. These results suggest that in human malaria infections, TEP1 must be activated by a distinct protease, and that rodent malaria parasites and bacteria share the same LRR-dependent TEP1 activation pathway. To confirm these results, we are performing P. falciparum infections in the laboratory settings with the aim to detect TEP1 on the surface of P. falciparum ookinetes.
A protocol has been developed to study immuno-induced and/or immuno-compromised mosquitoes (D2.3.). In this protocol, mosquitoes deprived of TEP1 succumb to bacterial infections within 24 h after challenge. We first demonstrated that depletion of the LRR proteins elicits a phenotype similar to dsTEP1. We extended this analysis to other candidate genes that are co-regulated with TEP1 according to the data of our microarray analyses. We have examined survival of mosquitoes silenced for genes encoding CLIP-domain serine proteases (catalytically active (B-type) or inactive (A or C type)). Out of 7 tested genes, silencing of CLIPA7 and CLIPA8 showed survival phenotypes similar to that of silencing TEP1, whereas no changes in the mosquito survival was detected for other genes. Interestingly, using this test we also examined whether fibrinogen 9 and cytochrome 450 collaborate with TEP1. As no effect of silencing of these genes on parasite development could be detected, only catalytically inactive CLIPA7 and CLIPA8 cooperate with TEP1. These results are in preparation for a publication.
In collaboration with Dr R. Baxter (Yale University, USA) we examined a series of recombinant TEP1 forms found in natural populations for their relative stability. We discovered that replacement of the thioester domain (TED) of TEP1 in resistant allele into susceptible was sufficient to reduce the half-life of the protein to the levels of the fully susceptible form. Using the developed assay, we are dissecting individual SNPs that are crucial for TEP1 stability and therefore, its function. These results are of high importance for the development of informative TEP1 genotyping protocols described in WP3.
RUNMC introduced Ngousso and resistant L3-5 strains of An. gambiae into the laboratory and performed P. falciparum infections with in vitro parasite cultures. Interestingly, significant differences were observed in the development of P. falciparum between Ngousso and G3 mosquitoes. Our results revealed that Ngousso are as susceptible to P. falciparum infections as An. stephensi, whereas a strong inhibition of the parasite development was detected in G3 and L3-5 mosquitoes. Silencing of TEP1 revealed that parasite killing in the G3 background is independent of TEP1. This result is unexpected as the immunofluorescence analysis using anti-TEP1 antibodies showed the presence of TEP1 on the surface of P. falciparum ookinetes. The intensity of TEP1 signal detected on the ookinetes was much stronger in the resistant strain than in Ngousso. Our results suggest that in spite of TEP1 binding to the parasites, some P. falciparum strains are insensitive to TEP1-mediated killing. Therefore, parasite genetic background is one of the factors that determine mosquito competence to control malaria infections.
Evaluation of environmental and physiological factors that affect mosquito immune status and Plasmodium sporogony in the laboratory and in the field (CNRS, RUNMC, IRD, MRTC, ICIPE)
The promoter sequences of immune-inducible genes were used to establish immune-responsive reporters (D2.1 and D2.6). We obtained 2 lines for Defensin 1 reporter, and 1 line for a 'universal' immune reporter containing Rel1- and Rel2-regulated sequences. We have characterized Defensin 1 reporter lines, that are activated by Gram-positive and Gram-negative bacteria but display no induction after infections with rodent malaria parasites. The 'universal' reporter line is currently under breeding and we should have first results shortly.
Establishment of experimental infections in Kenya and Mali were at the focus of the first reporting period. To this end, we organized an Ethical workshop in Cameroon, where these experiments are successfully running. MRTC and ICIPE now obtained Ethical clearance from national Committees and started experimental infections in Bamako and Mbita, respectively (D3.2). The study design involves identification of breeding sites with contrasting characteristics to examine the influence of these contrasting environmental factors on the capacity of mosquitoes to transmit malaria. First results in Mbita, Kenya, revealed significant effect of the breeding sites on the mosquito to develop of the parasites, this work is currently in progress.
RUNMC laboratory has successfully isolated 2 additional lines of P. falciparum from Malawi and Guinea. When these two P. falciparum strains were used for infections, one of them showed a strong susceptibility to TEP1-mediated killing. These results further highlight the importance of parasite genetic background as a determining factor in the mosquito - parasite interactions. Another factor that affects the efficiency of the mosquito responses is the genetic clonality of P. falciparum. Indeed, monoclonal infections produced high infection rates and were very sensitive to the wounding-induced immune responses (see below). In contrast, polyclonal infections were very low and resistant to the mosquito immune responses. Therefore, parasite genetic complexity is a crucial factor that shapes the outcome of Plasmodium infections in the field and should be taken into account for the development of new vector control strategies. These results of a collaborative work between IRD and CNRS were published in the International Journal for Parasitology (Nsango et al, 2012).
Modulation of the mosquito immune status and its effects on Plasmodium transmission (CNRS, RUNMC, IRD, MRTC, ICIPE)
Modulation of the mosquito immune status can be achieved by:
(i) genetic selection;
(ii) genetic manipulation by RNAi or by transgenesis.
(i) We have examined development of rodent malaria parasites in series of laboratory strains and observed a significant level of variation in outcomes of infection between these strains. For instance, the G3 strain is much more susceptible to infection than the Ngousso strain. Moreover, we performed selection on the Ngousso strain and obtained two lines which differ by 10-fold in the number of developing oocysts, suggesting that some mosquito factors regulate parasite development. Sequencing of TEP1 in these two lines revealed a single 'susceptible' allele shared by both lines, suggesting involvement of a different gene. We are currently isogenising these lines and will perform further genetic analysis to identify the gene(s) responsible for resistance to Plasmodium. These two lines will be also examined in infections with P. falciparum by RUNMC.
(ii) We have discovered that wounding induces a strong antiparasitic response against human but not rodent malaria parasites. To characterize molecular events underlying this effect, we used microarrays to compare mosquito transcriptomes before and 3h after wounding, and identified a number of candidate genes including components of the JNK pathway and TGases. Silencing of these genes rescued effect of wounding in the experimental infections in Cameroon, thereby confirming their involvement in the regulation of Plasmodium development. Our results suggest that injection of dsRNA can only be used to study wounding response against P. falciparum and that alternative methods of modulation of the mosquito immune status should be developed. These results are being submitted for publication. Therefore we now focus our efforts on the development of transgenic technology to assess gene function. We have developed a series of attP-containing lines for transgene integration and are in process of developing mosquito lines expressing the resistant allele of TEP1.
We have examined development of rodent and human malaria parasites in series of laboratory strains and observed a significant level of variation in outcomes of infection between these strains. For instance, the G3 strain is much more susceptible to P. berghei infection than the Ngousso strain, whereas G3 and L3-5 are more resistant to P. falciparum and are susceptible to P. berghei. Interestingly, the Ngousso colony is polymorphic for TEP1, and we are currently evaluating the contribution of TEP1 alleles to the resistance. We have discovered that wounding induces a strong antiparasitic response against human but not rodent malaria parasites. To characterize molecular events underlying this effect, we used microarrays to compare mosquito transcriptomes before and 3h after wounding, and identified a number of candidate genes including AP-1/Fos and TGases. Silencing of these genes rescued effect of wounding on P. falciparum development in the experimental infections in Cameroon. These results are being submitted for a joint publication between CNRS and IRD. We now focus our efforts on development of transgenic technology to assess gene function. We have developed a series of attP-containing lines for site-specific transgene integration and, as described above, are in process of developing mosquito lines expressing the resistant allele of TEP1. Recent studies from our and other laboratories revealed an important role of bacterial communities (or microbiote) in the mosquito-parasite interactions. To complement studies performed by IRD in the field populations, we are developing laboratory models of bacterial infections (as described above) to investigate this question. We will also extend these studies to P. falciparum infections in collaboration with RUNMC.
Analysis of reproductive success in An. gambiae mosquitoes with elevated or compromised immune status (UNIPG, CNRS)
Signal-independent activation of the Rel1 signalling cascade blocks development of rodent parasites but also abolishes egg development. Analysis of the genes regulated by Cactus pointed out to vitellogenin (Vg), one of the major yolk proteins in the mosquitoes. Indeed, we revealed that expression of this gene is negatively controlled by Rel1/Rel2 pathways, and in part explains Cactus knockdown phenotype. Silencing of vitellogenin and/or of lipophorin (Lp) decreased the number of surviving parasites indicating that these yolk proteins are important for Plasmodium development. In addition, only partial egg development was observed in dsVg mosquitoes, whereas ovaries remained undeveloped in dsLp females. The pro-parasitic effect of the yolk proteins required the function of TEP1, and TEP1 was more efficient in binding and killing of P. berghei in the absence of Vg and Lp. These results, published in PLoS Biology (Rono et al, 2010), for the first time establish a molecular link between reproduction and immunity and demonstrate that induction of immunity represses oogenesis in An. gambiae. Developed here tools will be used to study regulation of Lp and Vg expression after mating and effects of immune induction on mating efficiency. We will also examine the roles of the yolk proteins in infections with P. falciparum.
Careful immune-fluorescence analysis revealed the presence of TEP1 in the mosquito testes. Interestingly, TEP1-positive signal was observed inside the cyst stem cells, in the cystoblasts, and on the tails of the sperm cells. Functional analysis suggested that TEP1 is not important for mating but critical for maintaining male fertility, as progenies of the aged TEP1-depleted males are significantly less numerous as compared to control mosquitoes. Interestingly, a strong signal for TEP1 was detected on the sperm cells in the L3-5 mosquitoes that are resistant to P. berghei parasites. Experiments are now underway to examine whether allelic forms of TEP1 shape male fertility. The uncovered role of TEP1 in male fertility is crucial for understanding the mechanisms that drive malaria-resistance traits into vector populations.
We continued to examine the role of two yolk proteins in the antiparasitic responses of An. gambiae and in collaboration with RUNMC initiated analysis of Lp and Vg knockdowns on development of P. falciparum. Our results confirmed the protective role of yolk proteins in ookinete development, however in contrast to P. berghei, parasite killing in the absence of Lp and Vg is not mediated by TEP1. We also evaluated whether previously observed Lp-mediated effect on the oocyst growth affects ability of the mosquitoes to transmit Plasmodium. Our first results with P. berghei suggest a significant decrease in transmission efficiency. These experiments are being performed with P. falciparum isolates in collaboration with RUNMC.
WP3 Role of genetic polymorphism in genes controlling reproduction and immunity on structure of mosquito populations and malaria transmission in Africa.
This work package has two main objectives:
1) to examine genetic variability in genes controlling reproduction and immunity in natural An. gambiae populations, and
2) to evaluate the role of genetic differentiation in shaping the structure of mosquito populations, both in terms of the An. gambiae speciation and in ecological specialization, and in determining the vector competence to transmit malaria.
Analysis of genetic differentiation of genes involved in post-mating behaviour in An. gambiae (UNIPG, UNIROMA1, IRD, MRTC, ICIPE)
We developed PCR protocols to examine polymorphisms and rates of evolution in 13 MAG- and LRT-specific genes potentially involved in mating (based on results of WP1) and applied them on 5-10 specimens of An. gambiae s.s. M and S molecular forms, An. arabiensis, An. quadriannulatus, An. melas and An. merus. Genes encoding the principal components of the mating plug (e.g. plugin and TGase) were found to be conserved among taxa, whereas clusters of three MAG- and three LRT-specific genes showed high genetic divergence among sibling species of the complex, but not between M and S molecular forms. These two clusters were further investigated on a large collection of samples (either already available in UNIROMA1 or collected ad hoc by ICIPE and IRD) and were shown to evolve under selective pressures (D3.5; M3.2). Evidence of positive selective pressure and purifying selection maintaining lineage-specific products were found in the cluster of three paralogous MAG-specific genes (AGAP009369, AGAP009370, AGAP009370-copy). Adaptive evolution was detected in a number of codons of the three paralogous LRT-specific genes encoding for serine-proteases (AGAP005194, AGAP005195 expressed in the atrium and AGAP005196 detected in the spermatheca). Episodic selection was inferred in AGAP005196 along the branch leading to An. melas. The high level of replacement polymorphisms observed in all three proteases suggests that these duplicated genes might experience relaxed evolutionary constraints that could benefit a rapid diversification and eventually lead to a fixation of new advantageous variants. Two manuscripts on the obtained results are currently in preparation.
So far our analyses elucidated genes with species-specific but not molecular/chromosomal form-specific polymorphisms. These results did not allow us to develop standardized protocols for large-scale genotyping of the M and S forms. To overcome this roadblock, novel approaches are in progress to maximize the chances to identify molecular/chromosomal form-specific SNPs (D3.6 D3.7 and D3.9). On the other hand, absence of informative SNPs between M and S forms (also reported by other groups) may reflect as yet unknown biological phenomenon. As M/S hybrids are fertile in the laboratory conditions, it is possible that reproduction barriers have yet to be established between these two forms; in this case no informative polymorphisms for M/S will be detected in the reproductive genes.
The genetic characterization of samples collected in MALVECBLOK field sites and of other samples acquired by UNIROMA1 via other collaborators from Afrotropical malaria endemic areas and the development of a web-based database have been initiated in collaboration with CNRS (D3.4 expected delivery postponed to month 24).
We examined levels of polymorphism in genes with potential roles in post-mating responses identified in WP1 in samples collected by MALVECBLOK in malaria endemic countries and in other specimens available in UNIROMA1. To this end, we chose 8 genes expressed in the male accessory glands (MAGs) (AGAP009099 or TGase3, AGAP008276, AGAP008277, AGAP009368 or Plugin, AGAP009369, AGAP009370, AGAP009370c, and ACP1) and 4 genes expressed in the female low reproductive tract (LRT) (AGAP00885, AGAP005194, AGAP005195, AGAP005196).
A high level of conservation was observed in genes encoding the principal components of the mating plug Plugin and TGase3 suggesting strong evolutionary constrains exerted on these proteins.
In contrast, fixed genetic differences were found in a cluster of three genes expressed in the MAGs, whose products are delivered to female as components of the mating plug (AGAP009369, AGAP009370, AGAP009370c). Evolutionary analyses revealed signatures of purifying selection that acts to preserve the structure and function of these proteins. Fixed species-specific polymorphisms were found in at least one gene from each taxon along their geographical distribution (with the exception of An. gambiae s.s.). Notably, one fixed species-specific replacement in AGAP009370 coding region (Q to E amino acid change at the C-terminal region) was observed in An. arabiensis. This is the first report of an amino acid substitution that distinguishes An. arabiensis from An. gambiae s.s. which is present in an autosomal gene located outside the previously described characteristic chromosomal inversions.
We also detected signatures of adaptive evolution in the cluster of three genes encoding serine-proteases whose expression is inhibited in the female LRT after mating (AGAP005194, AGAP005195, AGAP005196). In all three genes fixed polymorphisms were detected in the regions encoding the specificity pockets that are known to mediate substrate recognition and/or binding. Furthermore, fixed amino acid replacements in An. melas and An. merus were detected on the exposed surfaces in the proximity of the protease active site in AGAP005195, suggesting potential species-specific differences in substrate recognition. The detected polymorphisms may have reinforced or directly contributed to the reproductive isolation among the species.
These results were published in BMC Evolutionary Biology, 2011, 11: 72; and in BMC Evolutionary Biology, 2011, 11: 292 (D3.5).
In other genes we detected patterns of incomplete lineage sorting due to a recent introgression event or sharing of ancestral polymorphisms among species/forms. Unfortunately, the observed evolutionary patterns were not helpful neither to reconstruct reliable phylogenetic relationships among members of the An. gambiae complex (D3.6) nor to develop standardized protocols for large-scale genotyping of An. gambiae s.s. chromosomal/molecular forms. We extended our analysis to 6 additional genes potentially involved in post-mating responses and previously reported by others to exhibit high levels of polymorphism (D3.9). However, in the large collected by MALVECBLOK all previously polymorphisms were randomly distributed among the samples and did not correlate with a particular chromosomal or molecular form.
Analysis of the expression of immune reporter genes in single specimens directly collected in the field to monitor their immune status in relation to their larval habitat (CNRS, IRD, MRTC)
We developed standardized protocols for collection of immature stages of An. gambiae from natural breeding sites and for subsequent membrane feeding of adult females on P. falciparum isolates. Procedures were discussed and approved by IRD, RUNMC, MRTC and ICIPE partners during a workshop set in Cameroon in June (D3.1).
Immature stages of An. gambiae were collected in multiple breeding sites (5 to 10) from 5 different locations in Cameroon (Mfou, Nolbission, Nkolondon, Obala and Mvan). Pools of 30 larvae (L4 stage) and 30 pupae were prepared directly in the field and qRT-PCR analyses were performed in Strasbourg. Examined breeding sites displayed a high level of heterogeneity in expression of mosquito antimicrobial genes (Defensin 1, Gambicin and TEP1) and no clear-cut correlation was established between expression patterns of the immune genes and mosquito resistance to Plasmodium in the field. A manuscript is in preparation (D3.8 expected delivery date postponed to month 28).
To characterize microbial diversity (D3.3) we collected additional pools of larvae and pupae in the same breeding sites. Midguts were dissected and 16S rDNA was analysed using TGGE and DGGE techniques. Only Gram-negative bacteria species were identified, suggesting that the mosquito midguts provide a thriving niche for this type of bacteria. Interestingly, we observed differences in the microbial flora in larval and pupal midguts from the same habitat, suggesting stage-specific proliferation of microbes (M3.3). For instance, Thorsellia anophelis was only found in larval midguts. No significant differences in bacterial loads were detected between M and S molecular forms. To reveal correlations between mosquito genotype, microbiota composition and Plasmodium genotype, we sequenced mosquito/parasite/microbiota genomes after experimental infections with the field parasites using the Illumina platform. Moreover, we compared the complexity of microbiota in the mosquito midgut between P. falciparum infected and non-infected mosquitoes by sequencing DNA extracted from the midguts using the 454 GS Flex Titanium sequencer. Analyses of data are in progress. These results should: i) provide a detailed description of microflora in selected larval habitats in Cameroon, and ii) determine whether exposure to bacterial communities in natural populations of mosquitoes modulates their susceptibility to P. falciparum.
We reported high levels of variability in expression of antimicrobial peptide genes in larvae collected from breeding sites at different geographical locations in Cameroon. These results indicated that larval habitats shape the mosquito immune status. Using SOPs for collection of immature stages of An. gambiae from natural breeding sites and for membrane feeding of females on P. falciparum isolates (D3.1) we examined whether exposure to distinct bacterial communities modulates mosquito susceptibility to P. falciparum. We first selected breeding sites where larvae showed distinct patterns of AMP expression and collected larval stages for further culturing in the laboratory. Eclosed females were exposed to P. falciparum infection, their midguts were dissected 8 days after the infectious blood meal and analysed for parasite loads. We then determined the microbiota of individual midguts using new generation sequencing approaches (454 pyrosequencing). Laboratory mosquitoes were used as a control. The microbial diversity was analysed according to the origin of the mosquitoes. After having obtained a snapshot of bacterial communities, we looked to see whether there existed a correlation between the bacterial content and the malaria infection status by comparing midgut microbiota of P. falciparum-infected and non-infected mosquitoes.
Significant differences were observed in microbiota between the laboratory mosquitoes and the mosquitoes collected in natural habitats. The diversity of bacterial species was very limited in the laboratory mosquitoes. For mosquitoes from natural habitats, the gut microbiota displayed a large inter-individual variability with a dominant few taxa. Interestingly, differences in the bacterial composition of the mosquito midguts correlated with the breeding sites. Our results suggest that the environmental conditions at larval stages shape the midgut microbiota in adults.
A significant positive correlation was detected between the relative abundance of Enterobacteriaceae bacteria in the mosquito midgut and P. falciparum infection levels. These results suggest that the midgut microbiota is an important factor that shapes vector competence, and that Enterobacteriaceae family of Gram-negative bacteria benefit P. falciparum development. These results were published in PLoS Pathogens (2012) (D3.8).
Contribution of genetic polymorphism to the immune status in the African populations of An. gambiae (IRD, CNRS, MRTC, ICIPE)
Experimental infections were established in Kenya using standardized MALVECBLOK protocols (Del. 3.2). Field- and lab-based experiments are being conducted in Cameroon, Mali and Kenya to investigate vector-host-parasite interactions using natural isolates of P. falciparum. Mbita laboratory An. gambiae S-form colony was used in Kenya. Larvae were bred in soils obtained from different larval breeding sites: significant differences were observed in infection rates in adult females that emerged. Analysis of microbial flora from mosquito guts and these breeding environments (soil and water) is in progress. Natural populations of mosquitoes were infected with field isolates of P. falciparum in Cameroon: preliminary results showed that although the M molecular form harbored higher infection rates, in sympatric conditions the M and S molecular forms were equal in transmitting P. falciparum.
Sequencing of mosquitoes to investigate putative correlation between TEP1 alleles and the mosquito resistance to P. falciparum is in progress at IRD-Montpellier and CNRS-Strasbourg (M3.1) and we expect that the sequencing will:
i) identify new TEP1 alleles from field populations (M3.6) and
ii) reveal informative polymorphisms linked to the mosquito infection status (D3.11; M3.7).
A standardized protocol for TEP1 sequencing and characterization in field mosquitoes has been established and distributed to all partners (D3.7). Silencing of TEP1 interacting proteins has been conducted in Cameroon and analysis of results is in progress.
We investigated the genetic polymorphism of TEP1 in Cameroon, Mali and Kenya by collecting immature stages of mosquitoes from a series of breeding sites. The TEP1 gene was first sequenced in individual mosquitoes and compared with the sequences of TEP1 alleles identified in the laboratory colonies. Unexpected the same 4 major allelic classes were identified in African natural populations: S1, S2, R2, and R1. Based on the sequence analysis and on results obtained in WP2, we developed a PCR-based RFLP protocol for TEP1 genotyping (D3.11). A SOP for TEP1 genotyping was developed and used in Cameroon, Mali and Kenya (D3.7). Our analysis revealed TEP1 allelic distribution in the countries across the African continent. Importantly, we observed that alleles were unevenly distributed among An. gambiae molecular forms. A manuscript is currently in preparation, which describes TEP1 polymorphism in the different malaria endemic areas (D3.12).
In Cameroon, IRD collected more than 2000 larvae in the field breeding sites and cultured them to imago in the laboratory. The eclosed 920 females were infected with field isolates of P. falciparum. Midguts were dissected eight days later to determine mosquito infection status by quantitative RT-PCR, whereas the carcasses were used for genotyping (M&S form and TEP1). The R1 allele was very rarely found (approximately 1 in 1000). Allelic frequencies of TEP1 R2 and S2 differed between M and S forms, where the S2 allele was more frequent in the M form and the R2 allele - in the S form. The majority of mosquitoes had S1 allele. Meta-analysis of 920 experiments indicated a higher prevalence of infection for the M form in both allopatric and sympatric M and S populations. But no difference in the infection intensity was detected. Statistical analysis also did not reveal any correlation between TEP1 genotypes and prevalence of infection. This is not surprising as only R1 allele, which is almost absent from Cameroonian samples, correlates with resistance to infections with P. berghei. Nonetheless, the M molecular form of An. gambiae has a higher frequency of the S2 allele and is more susceptible to P. falciparum infection, which is indicative of genetic association between TEP1 polymorphism and malaria infection.
The genetic characterization of samples collected in MALVECBLOK field sites is summarized in a database (Del. 3.4).
Potential Impact:
MALVECBLOK project focused on the major threat to global human health: malaria, the cause of death of more than a million people each year, and of illness of hundred millions individuals in tropical and subtropical countries. The pluri-disciplinary and complementary efforts of five European research teams, located in four different member states and one African ICPC country, were linked to those of two sub-Saharan groups, located in two disease-endemic ICPC countries.
The Consortium focused on the following topics, all related to the biology of the major malaria vector An. gambiae:
- reproductive biology
- immunity (defence strategies of the mosquito against the parasite)
- genetic structure of natural mosquito populations in Africa.
The major impact of the project on the global health issue of malaria is represented by the development of new concepts for innovative vector control measures. By promoting international cooperation with scientists of three disease-endemic countries, this Consortium elucidated the molecular events involved in reproduction and immunity of the mosquito vector and examined their effects on the development of the malaria parasite, both in laboratory and field conditions. The knowledge acquired in this project had a strong impact on European scientific competitiveness, as evidenced by a series of publications in the international peer reviewed journals.
Scientific issues: innovative ideas and findings
Fully exploiting the recent advances in the characterisation of the An. gambiae genome and functional gene analysis, MALVECBLOK comprehensively elucidated molecular events involved in reproductive and immune functions of the mosquito vector. This was achieved through the integration of expertise from different disciplines contributed by the teams involved in the project. The three topics covered by the project (reproductive biology and immunity and genetic structure of mosquito populations in Africa), were studied using complementary approaches and a variety of standard operational procedures. The obtained findings were published in scientific prestigious international scientific journals such as PNAS, PLoS Biology, PLoS Pathogens, Inter J Parasitol, BMC Evol Biol and presented at numerous prestigious international conferences.
In this bi-directional North-South collaboration, the fieldwork carried out in Africa allowed scientific validation of experimental results obtained in the European laboratories, and vice versa, African field data from the different African countries gave rise to new hypotheses which have been then tested in Europe. This collaborative project created strong links between European and ICPC research teams, and benefit the conception of highly innovative ideas and findings that improved the knowledge on crucial biological processes of the malaria mosquito. The MALVECBLOK project promoted the exchange of good practice and standardisation of fieldwork scientific procedures and protocols. Young researchers had excellent opportunities for training in their everyday scientific work, visits to the partners laboratories and through their participation at Consortium meetings at international Congresses.
By the integration of its complementary research skills, this collaborative research project strongly contributed to the development of a dynamic and successful European Research Environment. Based on the knowledge generated, it proposed new approaches for prevention and management of malaria.
European Competitiveness
The complementary skills of the MALVECBLOK partners led to identification of novel key molecules underlying reproduction biology and immunity of An. gambiae mosquitoes. The complementary expertise available in the Consortium was instrumental for understanding the bases of the transmission of the disease. The knowledge and tools provided by this project is now ready to be exploited for the design of novel active compounds, either inhibiting the reproduction of the insect, or stimulating the insect immune system for parasite killing.
For example, the identified in the WP1 crucial role of transglutaminase in male fertility could be exploited to prevent efficient female insemination. Currently, chemical formulations are being designed to block the activity of this enzyme.
On the other hand, identification of genetic structuring of mosquito populations relative to TEP1 genotype revealed by MALVECBLOK, is important for the development of future diagnostic tools to gauge susceptibility of local vectors to Plasmodium.
Finally, identification of the molecules that are required for both mosquito reproduction and Plasmodium development offers new interesting targets for the design of novel insect-specific to manipulate vector fertility and immunity at the population level, a topic that has great public health and economic relevance.
The findings and tools developed by MALVECBLOK are therefore expected to boost the innovative capacity of sustainable EU health-related industries and business.
European added value
The work undertaken by MALVECBLOK would not be possible at a regional or national level. A total of 3 different member states (comprising 5 Participants) and 3 ICPC contributed their complementary expertise in order to implement an international trans-disciplinary project. The scientific excellence skills of the 5 European partners have been essential for the success at the intellectual and infrastructural level.
- partner 1 (CNRS, FR) : mosquito immunity (RNAi silencing, cell biology, analysis of immune phenotypes, rodent parasite cultures, mosquito breeding colonies)
- partner 3 (RUNMC, NL) : immunology, biology of Plasmodia spp. (experimental membrane infections)
- partner 4 (INIROMA1, IT) : population biology of An. gambiae s.s. (cytology, genotyping, experience in fieldwork including sample collection)
- partner 5 (IRD, FR, tight collaboration with OCEAC in Cameroon) : P. falciparum transmission in mosquito vectors (functional analysis and immune gene polymorphisms, genetic analysis, experience in the fieldwork and sample collection, mosquito breeding colonies)
- partner 6 (MRTC, Mali): mosquito population biology, antiviral responses (mosquito breeding colonies, experimental membrane infections, experience in fieldwork and sample collection, fully equipped outstations)
- partner 7 (ICIPE, Kenya): mosquito population biology, strong parasitological background (mosquito breeding colonies, fully equipped outstations)
- partner 8 (UNIPG, IT) : reproduction biology of An. gambiae (transgenesis, analysis of post-mating phenotypes, mosquito breeding colonies).
International cooperation with ICPC countries was undertaken in order to share, generate and use knowledge through novel and consolidated research partnerships, on the basis of mutual interest and mutual benefit. In order to study and understand the geographical variations in local mosquito and parasite populations, the fieldwork in the malaria-endemic area was performed in three different sub-Saharan countries covering West, Central and East Africa.
List of Websites:
http://www.malvecblok.org