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1.  Differential antibody response to the Anopheles gambiae gSG6 and cE5 salivary proteins in individuals naturally exposed to bites of malaria vectors 
Parasites & Vectors  2014;7(1):549.
Background
Mosquito saliva plays crucial roles in blood feeding but also evokes in hosts an anti-saliva antibody response. The IgG response to the Anopheles gambiae salivary protein gSG6 was previously shown to be a reliable indicator of human exposure to Afrotropical malaria vectors. We analyzed here the humoral response to the salivary anti-thrombin cE5 in a group of individuals from a malaria hyperendemic area of Burkina Faso.
Methods
ELISA was used to measure the anti-cE5 IgG, IgG1 and IgG4 antibody levels in plasma samples collected in the village of Barkoumbilen (Burkina Faso) among individuals of the Rimaibé ethnic group. Anti-gSG6 IgG levels were also determined for comparison. Anopheles vector density in the study area was evaluated by indoor pyrethrum spray catches.
Results
The cE5 protein was highly immunogenic and triggered in exposed individuals a relatively long-lasting antibody response, as shown by its unchanged persistence after a few months of absent or very low exposure (dry season). In addition cE5 did not induce immune tolerance, as previously suggested for the gSG6 antigen. Finally, IgG subclass analysis suggested that exposed individuals may mount a Th1-type immune response against the cE5 protein.
Conclusions
The anti-cE5 IgG response is shown here to be a sensitive indicator of human exposure to anopheline vectors and to represent an additional tool for malaria epidemiological studies. It may be especially useful in conditions of low vector density, to monitor transiently exposed individuals (i.e. travellers/workers/soldiers spending a few months in tropical Africa) and to evaluate the impact of insecticide treated nets on vector control. Moreover, the gSG6 and cE5 salivary proteins were shown to trigger in exposed individuals a strikingly different immune response with (i) gSG6 evoking a short-lived IgG response, characterized by high IgG4 levels and most likely induction of immune tolerance, and (ii) cE5 eliciting a longer-living IgG response, dominated by anti-cE5 IgG1 antibodies and not inducing tolerance mechanisms. We believe that these two antigens may represent useful reagents to further investigate the so far overlooked role of Anopheles saliva and salivary proteins in host early immune response to Plasmodium parasites.
Electronic supplementary material
The online version of this article (doi:10.1186/s13071-014-0549-8) contains supplementary material, which is available to authorized users.
doi:10.1186/s13071-014-0549-8
PMCID: PMC4253619  PMID: 25428638
Anopheles gambiae; Salivary proteins; Immune response; IgG; IgG1; IgG4; Marker of exposure; Plasmodium transmission; Malaria epidemiology
2.  IgG1 and IgG4 Antibody Responses to the Anopheles gambiae Salivary Protein gSG6 in the Sympatric Ethnic Groups Mossi and Fulani in a Malaria Hyperhendemic Area of Burkina Faso 
PLoS ONE  2014;9(4):e96130.
Human antibody response to the Anopheles gambiae salivary protein gSG6 has recently emerged as a potentially useful tool for malaria epidemiological studies and for the evaluation of vector control interventions. However, the current understanding of the host immune response to mosquito salivary proteins and of the possible crosstalk with early response to Plasmodium parasites is still very limited. We report here the analysis of IgG1 and IgG4 subclasses among anti-gSG6 IgG responders belonging to Mossi and Fulani from Burkina Faso, two ethnic groups which are known for their differential humoral response to parasite antigens and for their different susceptibility to malaria. The IgG1 antibody response against the gSG6 protein was comparable in the two groups. On the contrary, IgG4 titers were significantly higher in the Fulani where, in addition, anti-gSG6 IgG4 antibodies appeared in younger children and the ratio IgG4/IgG1 stayed relatively stable throughout adulthood. Both gSG6-specific IgG1 and IgG4 antibodies showed a tendency to decrease with age whereas, as expected, the IgG response to the Plasmodium circumsporozoite protein (CSP) exhibited an opposite trend in the same individuals. These observations are in line with the idea that the An. gambiae gSG6 salivary protein induces immune tolerance, especially after intense and prolonged exposure as is the case for the area under study, suggesting that gSG6 may trigger in exposed individuals a Th2-oriented immune response.
doi:10.1371/journal.pone.0096130
PMCID: PMC3997568  PMID: 24760038
3.  Discovery and characterization of two Nimrod superfamily members in Anopheles gambiae 
Pathogens and Global Health  2013;107(8):463-474.
Anti-bacterial proteins in mosquitoes are known to play an important modulatory role on immune responses to infections with human pathogens including malaria parasites. In this study we characterized two members of the Anopheles gambiae Nimrod superfamily, namely AgNimB2 and AgEater. We confirm that current annotation of the An. gambiae genome incorrectly identifies AgNimB2 and AgEater as a single gene, AGAP009762. Through in silico and experimental approaches, it has been shown that AgNimB2 is a secreted protein that mediates phagocytosis of Staphylococcus aureus but not of Escherichia coli bacteria. We also reveal that this function does not involve a direct interaction of AgNimB2 with S. aureus. Therefore, AgNimB2 may act downstream of complement-like pathway activation, first requiring bacterial opsonization. In addition, it has been shown that AgNimB2 has an anti-Plasmodium effect. Conversely, AgEater is a membrane-bound protein that either functions redundantly or is dispensable for phagocytosis of E. coli or S. aureus. Our study provides insights into the role of members of the complex Nimrod superfamily in An. gambiae, the most important African vector of human malaria.
doi:10.1179/204777213X13867543472674
PMCID: PMC4073527  PMID: 24428830
Anopheles gambiae; Innate immunity; Microbiota; Phagocytosis; Nimrod superfamily; NIM repeats; EGF-like repeats
4.  The Anopheles gambiae cE5, a tight- and fast-binding thrombin inhibitor with post-transcriptionally regulated salivary-restricted expression 
Mosquito saliva carries a large number of factors with anti-hemostatic, anti-inflammatory and immuno-modulatory activities. The cE5 protein was initially identified during an Anopheles gambiae salivary gland transcriptome study and later shown to share sequence similarity with anophelin, a thrombin inhibitor from the saliva of the New World mosquito Anopheles albimanus. The cE5 gene was found to encode different mRNA isoforms coexisting in several tissues of both male and female mosquitoes, a highly unusual profile for a gene potentially encoding an anti-thrombin and involved in blood feeding. Expression of the cE5 protein and assessment of its activity and inhibitory properties showed that it is a highly specific and tight-binding thrombin inhibitor, which differs from the An. albimanus orthologue for the fast-binding kinetics. Despite the widespread occurrence of cE5 transcripts in different mosquito tissues the corresponding protein was only found in female salivary glands, where it undergoes post-translational modification. Therefore, tissue-specific restriction of the An. gambiae cE5 is not achieved by transcriptional control, as common for mosquito salivary genes involved in hematophagy, but by post-trascriptional gene regulatory mechanisms. Our observations provide a paradigm of post-transcriptional regulation as key determinant of tissue specificity for a protein from an important disease vector and point out that transcriptomic data should be interpreted with caution in the absence of concomitant proteomic support.
doi:10.1016/j.ibmb.2012.04.008
PMCID: PMC3416949  PMID: 22617725
Anopheles; salivary protein; anti-thrombin; anophelin; hematophagy; post-transcriptional regulation
5.  The Anopheles gambiae salivary protein gSG6: an anopheline-specific protein with a blood-feeding role 
The Anopheles gambiae salivary gland protein 6 (gSG6) is a small protein specifically found in the salivary glands of adult female mosquitoes. We report here the expression of a recombinant form of the protein and we show that in vivo gSG6 is expressed in distal-lateral lobes and is secreted with the saliva while the female mosquito probes for feeding. Injection of gSG6 dsRNA into adult An. gambiae females results in decreased gSG6 protein levels, increased probing time and reduced blood feeding ability. gSG6 orthologs have been found so far only in the salivary glands of Anopheles stephensi and Anopheles funestus, both members of the Cellia subgenus. We report here the gSG6 sequence from five additional anophelines, four species of the An. gambiae complex and Anopheles freeborni, a member of the subgenus Anopheles. We conclude that gSG6 plays some essential blood feeding role and was recruited in the anopheline subfamily most probably after the separation of the lineage which gave origin to Cellia and Anopheles subgenera.
doi:10.1016/j.ibmb.2009.04.006
PMCID: PMC3740408  PMID: 19442731
Anopheles gambiae; gSG6; salivary glands; saliva; blood feeding
6.  Comprehensive Genetic Dissection of the Hemocyte Immune Response in the Malaria Mosquito Anopheles gambiae 
PLoS Pathogens  2013;9(1):e1003145.
Reverse genetics in the mosquito Anopheles gambiae by RNAi mediated gene silencing has led in recent years to an advanced understanding of the mosquito immune response against infections with bacteria and malaria parasites. We developed RNAi screens in An. gambiae hemocyte-like cells using a library of double-stranded RNAs targeting 109 genes expressed highly or specifically in mosquito hemocytes to identify novel regulators of the hemocyte immune response. Assays included phagocytosis of bacterial bioparticles, expression of the antimicrobial peptide CEC1, and basal and induced expression of the mosquito complement factor LRIM1. A cell viability screen was also carried out to assess dsRNA cytotoxicity and to identify genes involved in cell growth and survival. Our results identify 22 novel immune regulators, including proteins putatively involved in phagosome assembly and maturation (Ca2+ channel, v-ATPase and cyclin-dependent protein kinase), pattern recognition (fibrinogen-domain lectins and Nimrod), immune modulation (peptidase and serine protease homolog), immune signaling (Eiger and LPS-induced factor), cell adhesion and communication (Laminin B1 and Ninjurin) and immune homeostasis (Lipophorin receptor). The development of robust functional cell-based assays paves the way for genome-wide functional screens to study the mosquito immune response to infections with human pathogens.
Author Summary
The mosquito immune system relies on innate humoral and cellular reactions to fight infections, including those by malaria parasites that must pass through mosquitoes before they can infect humans. Therefore, a detailed molecular understanding of these reactions could assist the design of new ways to control the spread of malaria and other mosquito-borne diseases. Here we use a technique to silence in mosquito cultured cells genes that are highly and/or specifically expressed in mosquito hemocytes, the equivalent of human white blood cells, as a means to investigate their function in reactions of the mosquito immune system. Our study identifies several novel regulators of immune reactions including phagocytosis, the engulfment and subsequent destruction of bacteria and other pathogens by hemocytes, the production of antimicrobial peptides, which directly kill or inhibit the proliferation of microbes, and the basal and induced production of an important complement regulator. Complement is a robust reaction of mosquitoes against malaria parasites and bacteria through phagocytosis, lysis or melanization (the enclosure of pathogens in a melanin capsule). We also reveal intriguing molecular connections between these reactions such as phagocytosis and regulation of complement. Our study provides novel insights into mosquito immune system and its reactions against infections.
doi:10.1371/journal.ppat.1003145
PMCID: PMC3561300  PMID: 23382679
7.  Humoral Response to the Anopheles gambiae Salivary Protein gSG6: A Serological Indicator of Exposure to Afrotropical Malaria Vectors 
PLoS ONE  2011;6(3):e17980.
Salivary proteins injected by blood feeding arthropods into their hosts evoke a saliva-specific humoral response which can be useful to evaluate exposure to bites of disease vectors. However, saliva of hematophagous arthropods is a complex cocktail of bioactive factors and its use in immunoassays can be misleading because of potential cross-reactivity to other antigens. Toward the development of a serological marker of exposure to Afrotropical malaria vectors we expressed the Anopheles gambiae gSG6, a small anopheline-specific salivary protein, and we measured the anti-gSG6 IgG response in individuals from a malaria hyperendemic area of Burkina Faso, West Africa. The gSG6 protein was immunogenic and anti-gSG6 IgG levels and/or prevalence increased in exposed individuals during the malaria transmission/rainy season. Moreover, this response dropped during the intervening low transmission/dry season, suggesting it is sensitive enough to detect variation in vector density. Members of the Fulani ethnic group showed higher anti-gSG6 IgG response as compared to Mossi, a result consistent with the stronger immune reactivity reported in this group. Remarkably, anti-gSG6 IgG levels among responders were high in children and gradually declined with age. This unusual pattern, opposite to the one observed with Plasmodium antigens, is compatible with a progressive desensitization to mosquito saliva and may be linked to the continued exposure to bites of anopheline mosquitoes. Overall, the humoral anti-gSG6 IgG response appears a reliable serological indicator of exposure to bites of the main African malaria vectors (An. gambiae, Anopheles arabiensis and, possibly, Anopheles funestus) and it may be exploited for malaria epidemiological studies, development of risk maps and evaluation of anti-vector measures. In addition, the gSG6 protein may represent a powerful model system to get a deeper understanding of molecular and cellular mechanisms underlying the immune tolerance and progressive desensitization to insect salivary allergens.
doi:10.1371/journal.pone.0017980
PMCID: PMC3060095  PMID: 21437289
8.  Genomic organization and splicing evolution of the doublesex gene, a Drosophila regulator of sexual differentiation, in the dengue and yellow fever mosquito Aedes aegypti 
Background
In the model system Drosophila melanogaster, doublesex (dsx) is the double-switch gene at the bottom of the somatic sex determination cascade that determines the differentiation of sexually dimorphic traits. Homologues of dsx are functionally conserved in various dipteran species, including the malaria vector Anopheles gambiae. They show a striking conservation of sex-specific regulation, based on alternative splicing, and of the encoded sex-specific proteins, which are transcriptional regulators of downstream terminal genes that influence sexual differentiation of cells, tissues and organs.
Results
In this work, we report on the molecular characterization of the dsx homologue in the dengue and yellow fever vector Aedes aegypti (Aeadsx). Aeadsx produces sex-specific transcripts by alternative splicing, which encode isoforms with a high degree of identity to Anopheles gambiae and Drosophila melanogaster homologues. Interestingly, Aeadsx produces an additional novel female-specific splicing variant. Genomic comparative analyses between the Aedes and Anopheles dsx genes revealed a partial conservation of the exon organization and extensive divergence in the intron lengths. An expression analysis showed that Aeadsx transcripts were present from early stages of development and that sex-specific regulation starts at least from late larval stages. The analysis of the female-specific untranslated region (UTR) led to the identification of putative regulatory cis-elements potentially involved in the sex-specific splicing regulation. The Aedes dsx sex-specific splicing regulation seems to be more complex with the respect of other dipteran species, suggesting slightly novel evolutionary trajectories for its regulation and hence for the recruitment of upstream splicing regulators.
Conclusions
This study led to uncover the molecular evolution of Aedes aegypti dsx splicing regulation with the respect of the more closely related Culicidae Anopheles gambiae orthologue. In Aedes aegypti, the dsx gene is sex-specifically regulated and encodes two female-specific and one male-specific isoforms, all sharing a doublesex/mab-3 (DM) domain-containing N-terminus and different C-termini. The sex-specific regulation is based on a combination of exon skipping, 5' alternative splice site choice and, most likely, alternative polyadenylation. Interestingly, when the Aeadsx gene is compared to the Anopheles dsx ortholog, there are differences in the in silico predicted default and regulated sex-specific splicing events, which suggests that the upstream regulators either are different or act in a slightly different manner. Furthermore, this study is a premise for the future development of transgenic sexing strains in mosquitoes useful for sterile insect technique (SIT) programs.
doi:10.1186/1471-2148-11-41
PMCID: PMC3045327  PMID: 21310052
9.  Analysis of apyrase 5' upstream region validates improved Anopheles gambiae transformation technique 
BMC Research Notes  2009;2:24.
Background
Genetic transformation of the malaria mosquito Anopheles gambiae has been successfully achieved in recent years, and represents a potentially powerful tool for researchers. Tissue-, stage- and sex-specific promoters are essential requirements to support the development of new applications for the transformation technique and potential malaria control strategies. During the Plasmodium lifecycle in the invertebrate host, four major mosquito cell types are involved in interactions with the parasite: hemocytes and fat body cells, which provide humoral and cellular components of the innate immune response, midgut and salivary glands representing the epithelial barriers traversed by the parasite during its lifecycle in the mosquito.
Findings
We have analyzed the upstream regulatory sequence of the An. gambiae salivary gland-specific apyrase (AgApy) gene in transgenic An. gambiae using a piggyBac transposable element vector marked by a 3xP3 promoter:DsRed gene fusion. Efficient germ-line transformation in An. gambiae mosquitoes was obtained and several integration events in at least three different G0 families were detected. LacZ reporter gene expression was analyzed in three transgenic lines/groups, and in only one group was tissue-specific expression restricted to salivary glands.
Conclusion
Our data describe an efficient genetic transformation of An. gambiae embryos. However, expression from the selected region of the AgApy promoter is weak and position effects may mask tissue- and stage- specific activity in transgenic mosquitoes.
doi:10.1186/1756-0500-2-24
PMCID: PMC2669092  PMID: 19284522
10.  An annotated catalogue of salivary gland transcripts in the adult female mosquito, Ædes ægypti* 
BMC Genomics  2007;8:6.
Background
Saliva of blood-sucking arthropods contains a cocktail of antihemostatic agents and immunomodulators that help blood feeding. Mosquitoes additionally feed on sugar meals and have specialized regions of their glands containing glycosidases and antimicrobials that might help control bacterial growth in the ingested meals. To expand our knowledge on the salivary cocktail of Ædes ægypti, a vector of dengue and yellow fevers, we analyzed a set of 4,232 expressed sequence tags from cDNA libraries of adult female mosquitoes.
Results
A nonredundant catalogue of 614 transcripts (573 of which are novel) is described, including 136 coding for proteins of a putative secretory nature. Additionally, a two-dimensional gel electrophoresis of salivary gland (SG) homogenates followed by tryptic digestion of selected protein bands and MS/MS analysis revealed the expression of 24 proteins. Analysis of tissue-specific transcription of a subset of these genes revealed at least 31 genes whose expression is specific or enriched in female SG, whereas 24 additional genes were expressed in female SG and in males but not in other female tissues. Most of the 55 proteins coded by these SG transcripts have no known function and represent high-priority candidates for expression and functional analysis as antihemostatic or antimicrobial agents. An unexpected finding is the occurrence of four protein families specific to SG that were probably a product of horizontal transfer from prokaryotic organisms to mosquitoes.
Conclusion
Overall, this paper contributes to the novel identification of 573 new transcripts, or near 3% of the Æ. ægypti proteome assuming a 20,000-protein set, and to the best-described sialome of any blood-feeding insect.
doi:10.1186/1471-2164-8-6
PMCID: PMC1790711  PMID: 17204158

Results 1-10 (10)