Peptides with Antimicrobial Activity in the Saliva of the Malaria Vector Anopheles coluzzii.

Mosquito saliva plays a crucial physiological role in both sugar and blood feeding by helping sugar digestion and exerting antihemostatic functions. During meal acquisition, mosquitoes are exposed to the internalization of external microbes. Since mosquitoes reingest significant amounts of saliva during feeding, we hypothesized that salivary antimicrobial components may participate in the protection of mouthparts, the crop, and the gut by inhibiting bacterial growth. To identify novel potential antimicrobials from mosquito saliva, we selected 11 candidates from Anopheles coluzzii salivary transcriptomic datasets and obtained them either using a cell-free transcription/translation expression system or, when feasible, via chemical synthesis. Hyp6.2 and hyp13, which were predicted to be produced as propeptides and cleaved in shorter mature forms, showed the most interesting results in bacterial growth inhibition assays. Hyp6.2 (putative mature form, 35 amino acid residues) significantly inhibited the growth of Gram-positive (Staphylococcus aureus) and Gram-negative (Escherichia coli and Serratia marcescens) bacteria. Hyp13 (short form, 19 amino acid residues) dose-dependently inhibited E. coli and S. marcescens growth, inducing membrane disruption in both Gram-positive and Gram-negative bacteria as indicated with scanning electron microscopy. In conclusion, we identified two A. coluzzii salivary peptides inhibiting Gram-positive and Gram-negative bacteria growth and possibly contributing to the protection of mosquito mouthparts and digestive tracts from microbial infection during and/or after feeding.

An insight into the sialotranscriptome and virome of Amazonian anophelines.

BACKGROUND: Saliva of mosquitoes contains anti-platelet, anti-clotting, vasodilatory, anti-complement and anti-inflammatory substances that help the blood feeding process. The salivary polypeptides are at a fast pace of evolution possibly due to their relative lack of structural constraint and possibly also by positive selection on their genes leading to evasion of host immune pressure. RESULTS: In this study, we used deep mRNA sequence to uncover for the first time the sialomes of four Amazonian anophelines species (Anopheles braziliensis, A. marajorara, A. nuneztovari and A. triannulatus) and extend the knowledge of the A. darlingi sialome. Two libraries were generated from A. darlingi mosquitoes, sampled from two localities separated ~ 1100 km apart. A total of 60,016 sequences were submitted to GenBank, which will help discovery of novel pharmacologically active polypeptides and the design of specific immunological markers of mosquito exposure. Additionally, in these analyses we identified and characterized novel phasmaviruses and anpheviruses associated to the sialomes of A. triannulatus, A. marajorara and A. darlingi species. CONCLUSIONS: Besides their pharmacological properties, which may be exploited for the development of new drugs (e.g. anti-thrombotics), salivary proteins of blood feeding arthropods may be turned into tools to prevent and/or better control vector borne diseases; for example, through the development of vaccines or biomarkers to evaluate human exposure to vector bites. The sialotranscriptome study reported here provided novel data on four New World anopheline species and allowed to extend our knowledge on the salivary repertoire of A. darlingi. Additionally, we discovered novel viruses following analysis of the transcriptomes, a procedure that should become standard within future RNAseq studies.

Human exposure to Anopheles farauti bites in the Solomon Islands is not associated with IgG antibody response to the gSG6 salivary protein of Anopheles gambiae.

BACKGROUND: Mosquito saliva elicits immune responses in humans following mosquito blood feeding. Detection of human antibodies recognizing the Anopheles gambiae salivary gland protein 6 (gSG6) or the gSG6-P1 peptide in residents of Africa, South America and Southeast Asia suggested the potential for these antibodies to serve as a universal marker to estimate human biting rates. Validating the utility of this approach requires concurrent comparisons of anopheline biting rates with antibodies to the gSG6 protein to determine the sensitivity and specificity of the assay for monitoring changes in vector populations. This study investigated whether seroprevalence of anti-gSG6 antibodies in humans reflected the relative exposure to Anopheles farauti bites in the Solomon Islands as estimated from sympatric human landing catches. METHODS: Human biting rates by An. farauti were estimated by landing catches at 10 sampling sites in each of 4 villages during the wet and dry seasons. Human serum samples from these same villages were also collected during the wet and dry seasons and analysed for antibody recognition of the gSG6 antigen by the Luminex xMAP© platform. Antibody titres and prevalence were compared to HLCs at the sampling sites nearest to participants' residences for utility of anti-gSG6 antibodies to estimate human exposure to anopheline bites. RESULTS: In this study in the Solomon Islands only 11% of people had very high anti-gSG6 antibody titres, while other individuals did not recognize gSG6 despite nightly exposures of up to 190 bites by An. farauti. Despite clear spatial differences in the human biting rates within and among villages, associations between anti-gSG6 antibody titres and biting rates were not found. CONCLUSIONS: Few studies to date have concurrently measured anopheline biting rates and the prevalence of human antibodies to gSG6. The lack of association between anti-gSG6 antibody titres and concurrently measured human biting rates suggests that the assay for human anti-gSG6 antibodies lacks sufficient sensitivity to be a biomarker of An. farauti exposure at an epidemiologically relevant scale. These findings imply that an improvement in the sensitivity of serology to monitor changes in anopheline biting exposure may require the use of saliva antigens from local anophelines, and this may be especially true for species more distantly related to the African malaria vector An. gambiae.

Functional analyses yield detailed insight into the mechanism of thrombin inhibition by the antihemostatic salivary protein cE5 from Anopheles gambiae.

Saliva of blood-feeding arthropods carries several antihemostatic compounds whose physiological role is to facilitate successful acquisition of blood. The identification of novel natural anticoagulants and the understanding of their mechanism of action may offer opportunities for designing new antithrombotics disrupting blood clotting. We report here an in-depth structural and functional analysis of the anophelin family member cE5, a salivary protein from the major African malaria vector Anopheles gambiae that specifically, tightly, and quickly binds and inhibits thrombin. Using calorimetry, functional assays, and complementary structural techniques, we show that the central region of the protein, encompassing amino acids Asp-31-Arg-62, is the region mainly responsible for α-thrombin binding and inhibition. As previously reported for the Anopheles albimanus orthologue anophelin, cE5 binds both thrombin exosite I with segment Glu-35-Asp-47 and the catalytic site with the region Pro-49-Arg-56, which includes the highly conserved DPGR tetrapeptide. Moreover, the N-terminal Ala-1-Ser-30 region of cE5 (which includes an RGD tripeptide) and the additional C-terminal serine-rich Asn-63-Glu-82 region (absent in orthologues from anophelines of the New World species A. albimanus and Anopheles darlingi) also played some functionally relevant role. Indeed, we observed decreased thrombin binding and inhibitory properties even when using the central cE5 fragment (Asp-31-Arg-62) alone. In summary, these results shed additional light on the mechanism of thrombin binding and inhibition by this family of salivary anticoagulants from anopheline mosquitoes.

Anopheline salivary protein genes and gene families: an evolutionary overview after the whole genome sequence of sixteen Anopheles species.

BACKGROUND: Mosquito saliva is a complex cocktail whose pharmacological properties play an essential role in blood feeding by counteracting host physiological response to tissue injury. Moreover, vector borne pathogens are transmitted to vertebrates and exposed to their immune system in the context of mosquito saliva which, in virtue of its immunomodulatory properties, can modify the local environment at the feeding site and eventually affect pathogen transmission. In addition, the host antibody response to salivary proteins may be used to assess human exposure to mosquito vectors. Even though the role of quite a few mosquito salivary proteins has been clarified in the last decade, we still completely ignore the physiological role of many of them as well as the extent of their involvement in the complex interactions taking place between the mosquito vectors, the pathogens they transmit and the vertebrate host. The recent release of the genomes of 16 Anopheles species offered the opportunity to get insights into function and evolution of salivary protein families in anopheline mosquitoes. RESULTS: Orthologues of fifty three Anopheles gambiae salivary proteins were retrieved and annotated from 18 additional anopheline species belonging to the three subgenera Cellia, Anopheles, and Nyssorhynchus. Our analysis included 824 full-length salivary proteins from 24 different families and allowed the identification of 79 novel salivary genes and re-annotation of 379 wrong predictions. The comparative, structural and phylogenetic analyses yielded an unprecedented view of the anopheline salivary repertoires and of their evolution over 100 million years of anopheline radiation shedding light on mechanisms and evolutionary forces that contributed shaping the anopheline sialomes. CONCLUSIONS: We provide here a comprehensive description, classification and evolutionary overview of the main anopheline salivary protein families and identify two novel candidate markers of human exposure to malaria vectors worldwide. This anopheline sialome catalogue, which is easily accessible as hyperlinked spreadsheet, is expected to be useful to the vector biology community and to improve the capacity to gain a deeper understanding of mosquito salivary proteins facilitating their possible exploitation for epidemiological and/or pathogen-vector-host interaction studies.

Deciphering the olfactory repertoire of the tiger mosquito Aedes albopictus.

BACKGROUND: The Asian tiger mosquito Aedes albopictus is a highly invasive species and competent vector of several arboviruses (e.g. dengue, chikungunya, Zika) and parasites (e.g. dirofilaria) of public health importance. Compared to other mosquito species, Ae. albopictus females exhibit a generalist host seeking as well as a very aggressive biting behaviour that are responsible for its high degree of nuisance. Several complex mosquito behaviours such as host seeking, feeding, mating or oviposition rely on olfactory stimuli that target a range of sensory neurons localized mainly on specialized head appendages such as antennae, maxillary palps and the mouthparts. RESULTS: With the aim to describe the Ae. albopictus olfactory repertoire we have used RNA-seq to reveal the transcriptome profiles of female antennae and maxillary palps. Male heads and whole female bodies were employed as reference for differential expression analysis. The relative transcript abundance within each tissue (TPM, transcripts per kilobase per million) and the pairwise differential abundance in the different tissues (fold change values and false discovery rates) were evaluated. Contigs upregulated in the antennae (620) and maxillary palps (268) were identified and relative GO and PFAM enrichment profiles analysed. Chemosensory genes were described: overall, 77 odorant binding proteins (OBP), 82 odorant receptors (OR), 60 ionotropic receptors (IR) and 30 gustatory receptors (GR) were identified by comparative genomics and transcriptomics. In addition, orthologs of genes expressed in the female/male maxillary palps and/or antennae and involved in thermosensation (e.g. pyrexia and arrestin1), mechanosensation (e.g. piezo and painless) and neuromodulation were classified. CONCLUSIONS: We provide here the first detailed transcriptome of the main Ae. albopictus sensory appendages, i.e. antennae and maxillary palps. A deeper knowledge of the olfactory repertoire of the tiger mosquito will help to better understand its biology and may pave the way to design new attractants/repellents.

MicroRNAs and other small RNAs in Aedes aegypti saliva and salivary glands following chikungunya virus infection.

Mosquito saliva facilitates blood feeding through the anti-haemostatic, anti-inflammatory and immunomodulatory properties of its proteins. However, the potential contribution of non-coding RNAs to host manipulation is still poorly understood. We analysed small RNAs from Aedes aegypti saliva and salivary glands and show here that chikungunya virus-infection triggers both the siRNA and piRNA antiviral pathways with limited effects on miRNA expression profiles. Saliva appears enriched in specific miRNA subsets and its miRNA content is well conserved among mosquitoes and ticks, clearly pointing to a non-random sorting and occurrence. Finally, we provide evidence that miRNAs from Ae. aegypti saliva may target human immune and inflammatory pathways, as indicated by prediction analysis and searching for experimentally validated targets of identical human miRNAs. Overall, we believe these observations convincingly support a scenario where both proteins and miRNAs from mosquito saliva are injected into vertebrates during blood feeding and contribute to the complex vector-host-pathogen interactions.

Human IgG responses to the Aedes albopictus 34k2 salivary protein: analyses in Réunion Island and Bolivia confirm its suitability as marker of host exposure to the tiger mosquito.

BACKGROUND: The rapid worldwide spreading of Aedes aegypti and Aedes albopictus is expanding the risk of arboviral diseases transmission, pointing out the urgent need to improve monitoring and control of mosquito vector populations. Assessment of human-vector contact, currently estimated by classical entomological methods, is crucial to guide planning and implementation of control measures and evaluate transmission risk. Antibody responses to mosquito genus-specific salivary proteins are emerging as a convenient complementary tool for assessing host exposure to vectors. We previously showed that IgG responses to the Ae. albopictus 34k2 salivary protein (al34k2) allow detection of seasonal and geographic variation of human exposure to the tiger mosquito in two temperate areas of Northeast Italy. The main aim of this study was to confirm and extend these promising findings to tropical areas with ongoing arboviral transmission. METHODS: IgG responses to al34k2 and to the Ae. aegypti orthologous protein ae34k2 were measured by ELISA in cohorts of subjects only exposed to Ae. albopictus (Réunion Island), only exposed to Ae. aegypti (Bolivia) or unexposed to both these vectors (North of France). RESULTS AND CONCLUSION: Anti-al34k2 IgG levels were significantly higher in sera of individuals from Réunion Island than in unexposed controls, indicating that al34k2 may be a convenient and reliable proxy for whole saliva or salivary gland extracts as an indicator of human exposure to Ae. albopictus. Bolivian subjects, exposed to bites of Ae. aegypti, carried in their sera IgG recognizing the Ae. albopictus al34k2 protein, suggesting that this salivary antigen can also detect, even though with low sensitivity, human exposure to Ae. aegypti. On the contrary, due to the high background observed in unexposed controls, the recombinant ae34k2 appeared not suitable for the evaluation of human exposure to Aedes mosquitoes. Overall, this study confirmed the suitability of anti-al34k2 IgG responses as a specific biomarker of human exposure to Ae. albopictus and, to a certain extent, to Ae. aegypti. Immunoassays based on al34k2 are expected to be especially effective in areas where Ae. albopictus is the main arboviral vector but may also be useful in areas where Ae. albopictus and Ae. aegypti coexist.

Biochemical and structural analysis of a cytosolic sulfotransferase of the malaria vector Anopheles gambiae overexpressed in the reproductive tissues.

The temporary or permanent chemical modification of biomolecules is a crucial aspect in the physiology of all living species. However, while some modules are well characterised also in insects, others did not receive the same attention. This holds true for sulfo-conjugation that is catalysed by cytosolic sulfotransferases (SULT), a central component of the metabolism of endogenous low molecular weight molecules and xenobiotics. In particular, limited information is available about the functional roles of the mosquito predicted enzymes annotated as SULTs in genomic databases. The herein described research is the first example of a biochemical and structural study of a SULT of a mosquito species, in general, and of the malaria vector Anopheles gambiae in particular. We confirmed that the AGAP001425 transcript displays a peculiar expression pattern that is suggestive of a possible involvement in modulating the mosquito reproductive tissues physiology, a fact that could raise attention on the enzyme as a potential target for insect-containment strategies. The crystal structures of the enzyme in alternative ligand-bound states revealed elements distinguishing AgSULT-001425 from other characterized SULTs, including a peculiar conformational plasticity of a discrete region that shields the catalytic cleft and that could play a main role in the dynamics of the reaction and in the substrate selectivity of the enzyme. Along with further in vitro biochemical studies, our structural investigations could provide a framework for the discovery of small-molecule inhibitors to assess the effect of interfering with AgSULT-001425-mediated catalysis at the organismal level.

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.

Wide cross-reactivity between Anopheles gambiae and Anopheles funestus SG6 salivary proteins supports exploitation of gSG6 as a marker of human exposure to major malaria vectors in tropical Africa.

BACKGROUND: The Anopheles gambiae gSG6 is an anopheline-specific salivary protein which helps female mosquitoes to efficiently feed on blood. Besides its role in haematophagy, gSG6 is immunogenic and elicits in exposed individuals an IgG response, which may be used as indicator of exposure to the main African malaria vector A. gambiae. However, malaria transmission in tropical Africa is sustained by three main vectors (A. gambiae, Anopheles arabiensis and Anopheles funestus) and a general marker, reflecting exposure to at least these three species, would be especially valuable. The SG6 protein is highly conserved within the A. gambiae species complex whereas the A. funestus homologue, fSG6, is more divergent (80% identity with gSG6). The aim of this study was to evaluate cross-reactivity of human sera to gSG6 and fSG6. METHODS: The A. funestus SG6 protein was expressed/purified and the humoral response to gSG6, fSG6 and a combination of the two antigens was compared in a population from a malaria hyperendemic area of Burkina Faso where both vectors were present, although with a large A. gambiae prevalence (>75%). Sera collected at the beginning and at the end of the high transmission/rainy season, as well as during the following low transmission/dry season, were analysed. RESULTS: According to previous observations, both anti-SG6 IgG level and prevalence decreased during the low transmission/dry season and showed a typical age-dependent pattern. No significant difference in the response to the two antigens was found, although their combined use yielded in most cases higher IgG level. CONCLUSIONS: Comparative analysis of gSG6 and fSG6 immunogenicity to humans suggests the occurrence of a wide cross-reactivity, even though the two proteins carry species-specific epitopes. This study supports the use of gSG6 as reliable indicator of exposure to the three main African malaria vectors, a marker which may be useful to monitor malaria transmission and evaluate vector control measures, especially in conditions of low malaria transmission and/or reduced vector density. The Anopheles stephensi SG6 protein also shares 80% identity with gSG6, suggesting the attractive possibility that the A. gambiae protein may also be useful to assess human exposure to several Asian malaria vectors.

Effects of Local and Systemic Immune Challenges on the Expression of Selected Salivary Genes in the Malaria Mosquito Anopheles coluzzii.

Salivary glands play a crucial tripartite role in mosquito physiology. First, they secrete factors that greatly facilitate both sugar and blood meal acquisition. Second, the transmission of pathogens (parasites, bacteria and viruses) to the vertebrate host requires both the recognition and invasion of the salivary glands. Third, they produce immune factors that both protect the organ from invading pathogens and are also able to exert their activity in the crop and the midgut when saliva is re-ingested during feeding. Studies on mosquito sialomes have revealed the presence of several female and/or male salivary gland-specific or enriched genes whose function is completely unknown so far. We focused our attention on these orphan genes, and we selected, according to sequence and structural features, a shortlist of 11 candidates with potential antimicrobial properties. Afterwards, using qPCR, we investigated their expression profile at 5 and 24 h after an infectious sugar meal (local challenge) or thoracic microinjection (systemic challenge) of Gram-negative (Escherichia coli, EC) or Gram-positive (Staphylococcus aureus, SA) bacteria. We observed a general increase in the transcript abundance of our salivary candidates between 5 and 24 h after local challenge. Moreover, transcriptional modulation was determined by the nature of the stimulus, with salivary gland-enriched genes (especially hyp15 upon SA stimulus) upregulated shortly after the local challenge and later after the systemic challenge. Overall, this work provides one of the first contributions to the understanding of the immune role of mosquito salivary glands. Further characterization of salivary candidates whose expression is modulated by immune challenge may help in the identification of possible novel antimicrobial peptides.

IgG Antibody Responses to the Aedes albopictus 34k2 Salivary Protein as Novel Candidate Marker of Human Exposure to the Tiger Mosquito.

Mosquitoes of the Aedes genus transmit arboviruses of great importance to human health as dengue, chikungunya, Zika and yellow fever. The tiger mosquito Aedes albopictus can play an important role as arboviral vector, especially when Aedes aegypti is absent or present at low levels. Remarkably, the rapid worldwide spreading of the tiger mosquito is expanding the risk of arboviral transmission also to temperate areas, and the autochthonous cases of chikungunya, dengue and Zika in Europe emphasize the need for improved monitoring and control. Proteomic and transcriptomic studies on blood feeding arthropod salivary proteins paved the way toward the exploitation of genus-specific mosquito salivary proteins for the development of novel tools to evaluate human exposure to mosquito bites. We previously found that the culicine-specific 34k2 salivary protein from Ae. albopictus (al34k2) evokes specific IgG responses in experimentally exposed mice, and provided preliminary evidence of its immunogenicity to humans. In this study we measured IgG responses to al34k2 and to Ae. albopictus salivary gland protein extracts (SGE) in individuals naturally exposed to the tiger mosquito. Sera were collected in two areas of Northeast Italy (Padova and Belluno) during two different time periods: at the end of the low- and shortly after the high-density mosquito seasons. Anti-SGE and anti-al34k2 IgG levels increased after the summer period of exposure to mosquito bites and were higher in Padova as compared to Belluno. An age-dependent decrease of anti-saliva IgG responses was found especially in Padova, an area with at least 25 years history of Ae. albopictus colonization. Moreover, a weak correlation between anti-saliva IgG levels and individual perception of mosquito bites by study participants was found. Finally, determination of anti-al34k2 IgG1 and IgG4 levels indicated a large predominance of IgG1 antibodies. Overall, this study provides a convincing indication that antibody responses to al34k2 may be regarded as a reliable candidate marker to detect temporal and/or spatial variation of human exposure to Ae. albopictus; a serological tool of this kind may prove useful both for epidemiological studies and to estimate the effectiveness of anti-vectorial measures.

MicroRNAs from saliva of anopheline mosquitoes mimic human endogenous miRNAs and may contribute to vector-host-pathogen interactions.

During blood feeding haematophagous arthropods inject into their hosts a cocktail of salivary proteins whose main role is to counteract host haemostasis, inflammation and immunity. However, animal body fluids are known to also carry miRNAs. To get insights into saliva and salivary gland miRNA repertoires of the African malaria vector Anopheles coluzzii we used small RNA-Seq and identified 214 miRNAs, including tissue-enriched, sex-biased and putative novel anopheline miRNAs. Noteworthy, miRNAs were asymmetrically distributed between saliva and salivary glands, suggesting that selected miRNAs may be preferentially directed toward mosquito saliva. The evolutionary conservation of a subset of saliva miRNAs in Anopheles and Aedes mosquitoes, and in the tick Ixodes ricinus, supports the idea of a non-random occurrence pointing to their possible physiological role in blood feeding by arthropods. Strikingly, eleven of the most abundant An. coluzzi saliva miRNAs mimicked human miRNAs. Prediction analysis and search for experimentally validated targets indicated that miRNAs from An. coluzzii saliva may act on host mRNAs involved in immune and inflammatory responses. Overall, this study raises the intriguing hypothesis that miRNAs injected into vertebrates with vector saliva may contribute to host manipulation with possible implication for vector-host interaction and pathogen transmission.

Analysis in a murine model points to IgG responses against the 34k2 salivary proteins from Aedes albopictus and Aedes aegypti as novel promising candidate markers of host exposure to Aedes mosquitoes.

BACKGROUND: Aedes mosquitoes are vectors of arboviral diseases of great relevance for public health. The recent outbreaks of dengue, Zika, chikungunya and the rapid worldwide spreading of Aedes albopictus emphasize the need for improvement of vector surveillance and control. Host antibody response to mosquito salivary antigens is emerging as a relevant additional tool to directly assess vector-host contact, monitor efficacy of control interventions and evaluate risk of arboviral transmission. METHODOLOGY/PRINCIPAL FINDINGS: Groups of four BALB/c mice were immunized by exposure to bites of either Aedes albopictus or Aedes aegypti. The 34k2 salivary proteins from Ae. albopictus (al34k2) and Ae. aegypti (ae34k2) were expressed in recombinant form and Ae. albopictus salivary peptides were designed through B-cell epitopes prediction software. IgG responses to salivary gland extracts, peptides, al34k2 and ae34k2 were measured in exposed mice. Both al34k2 and ae34k2, with some individual and antigen-specific variation, elicited a clearly detectable antibody response in immunized mice. Remarkably, the two orthologous proteins showed very low level of immune cross-reactivity, suggesting they may eventually be developed as species-specific markers of host exposure. The al34k2 immunogenicity and the limited immune cross-reactivity to ae34k2 were confirmed in a single human donor hyperimmune to Ae. albopictus saliva. CONCLUSIONS/SIGNIFICANCE: Our study shows that exposure to bites of Ae. albopictus or Ae. aegypti evokes in mice species-specific IgG responses to al34k2 or ae34k2, respectively. Deeper understanding of duration of antibody response and validation in natural conditions of human exposure to Aedes mosquitoes are certainly needed. However, our findings point to the al34k2 salivary protein as a promising potential candidate for the development of immunoassays to evaluate human exposure to Ae. albopictus. This would be a step forward in the establishment of a serological toolbox for the simultaneous assessment of human exposure to Aedes vectors and the pathogens they transmit.

Purification and biochemical characterization of a recombinant Anopheles gambiae tryptophan 2,3-dioxygenase expressed in Escherichia coli.

In the malaria vector Anopheles gambiae, tryptophan 2,3-dioxygenase (TDO) is the only enzyme able to initiate l-tryptophan degradation through the kynurenine pathway. TDO converts l-tryptophan to N-formylkynurenine by catalyzing the heme-dependent oxidative opening of the substrate indole ring. Despite the central role exerted by kynurenines in the physiology of living organisms, only a few insect TDOs have been subjected to biochemical characterization in vitro. We performed a RT-PCR-based analysis of the tissue distribution of TDO mRNA in A. gambiae that revealed a ubiquitous expression of the gene, thus further underlining the importance of the enzyme in the mosquito biology. We developed an expression/purification procedure yielding pure and active recombinant A. gambiae TDO. Spectral analyses showed that the enzyme was purified in its heme-ferric form that was subsequently used to determining the Michaelis-Menten constants of the TDO catalyzed reaction in the presence of reducing agents. The screening of a number of compounds as potential TDO modulators showed that several kynurenines and other Tryptophan-derived molecules interfere with the enzyme activity in vitro. Our study could contribute to understanding TDO regulation in vivo and to the identification of inhibitors to be used to alter Tryptophan homeostasis in the malaria vector.

Saliva of hematophagous insects: a multifaceted toolkit.

Transcriptomic, proteomic and genomic studies significantly improved our understanding of the complexity of blood feeding insect saliva providing unparalleled evolutionary insights. Salivary genes appeared to be under strong selective pressure with gene duplication and functional diversification being a powerful driver in the evolution of novel salivary genes/functions. The first insect salivary proteins responsible for complement inhibition were identified and a widespread mechanism of action shared by unrelated salivary protein families was recognized and named kratagonism. microRNAs were for the first time described in the saliva of a few blood feeding arthropods raising intriguing questions on their possible contribution to vertebrate host manipulation and pathogen transmission and further emphasizing how much we still have to learn on blood feeding insect saliva.

An annotated catalogue of salivary gland transcripts in the adult female mosquito, Aedes aegypti.

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 AEdes aegypti, 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 AE. aegypti proteome assuming a 20,000-protein set, and to the best-described sialome of any blood-feeding insect.

An insight into the sialome of the adult female mosquito Aedes albopictus.

To gain insight into the molecular repertoire of the adult female salivary glands of the tiger mosquito Aedes albopictus, we performed transcriptome and proteome analysis. cDNA clones were sequenced and assembled in clusters of related sequences and the corresponding genes assigned to one of three categories: housekeeping (H; 31%), secreted (S; 34%), or unknown (U; 35%) function. Among the putative secreted factors are proteins known to be widely distributed in the saliva of blood-sucking Diptera, such as D7 and antigen 5 family members, as well as proteins that are mosquito- or culicine-specific, i.e., the 30-kDa allergen or the 62-kDa and 34-kDa families, respectively. Expression of 15 of these salivary proteins was confirmed by Edman degradation. Tissue and sex specificity of selected transcripts were evaluated by RT-PCR and identified at least 32 genes whose expression is restricted or enriched in the female salivary glands of Ae. albopictus, whereas 17 additional genes were expressed in female glands and adult males but not in other tissues of adult females. For approximately one third of the genes analyzed, involvement in blood-feeding, sugar digestion, immune response, or other more generic physiological roles can be postulated; however, no functions can be suggested for the remaining sequences, which therefore likely represent either novel functions or novel molecules recruited during the evolution of hematophagy. Supplemental spreadsheets with hyperlinks to all sequences used in this manuscript are hyperlinked throughout the text and can be found at http://www.ncbi.nlm.nih.gov/projects/omes/#salivarytranscriptomes.

Spatial repellents and malaria transmission in an endemic area of Cambodia with high mosquito net usage.

BACKGROUND: The spread of artemisinin resistant malaria from SE Asia to the rest of the world remains a threat that will only be ended by eliminating malaria from the region. Novel control approaches are required to mitigate this threat. Spatial repellents (SR) are one such approach. We therefore conducted a multiple cross-over experiment from April 2013 - April 2014, in which all houses in one of two villages in Mondolkiri Province, Cambodia were alternately supplied with an emanator of the spatial repellent metofluthrin per 30 m3 of protected area to cover all potential peridomestic areas where people might spend their time before sleeping. Emanators were replaced every month for a three-month period. MATERIAL AND METHODS: Mosquito densities were simultaneously monitored in each village for two weeks every month using six CDC light-traps/night run from 18.00 to 07.00 hrs inside bedrooms and malaria prevalence, seroconversion and gSG6 protein rates assessed from prevalence surveys. After emanators were installed in the first village they were installed in the second village for a further three-month period and following that were again used in the initial village for a further three months. Surveys were undertaken before the initial installation of the emanators and at each cross-over point. RESULTS: Anopheles dirus densities were highest in houses closest to the forest. Transmission rates were low even before the application of the emanators. Perhaps due to the low levels of malaria transmission in Mondolkiri no significant relationships were found in Plasmodium cases or seroconversion rates between villages, surveys or by intervention. Adult males, who might spend more time unprotected in the forest at night, appeared to be at greater risk of becoming infected with P. falciparum malaria as compared to women or young children. CONCLUSION: At the malaria transmission levels present in Mondolkiri the metofluthrin emanators evaluated had no observable effect on malaria prevalence. This may be due to confounding by low prevalence rates.