The global expansion of Aedes albopictus from Southeast Asia to various regions worldwide poses a significant public health concern due to its role as a vector for several pathogens, including chikungunya virus (CHIKV), which infects over one million people annually. In this study, aimed at understanding the molecular interactions between Ae. albopictus and CHIKV, we analyzed by RNA sequencing CHIKV-infected and uninfected control mosquitoes. We focused our attention on key mosquito organs at one- and five-days post-blood meal ingestion, which correspond to viral entry and dissemination, and found specific transcriptional changes involving various pathways during the CHIKV lifecycle. The mosquito midgut plays a crucial role in the early stages, when the virus enters along with human blood components, encounters the resident microbiota, interacts with the developing peritrophic matrix, and counteracts the mosquito's digestive enzymes. We found that RNA interference (RNAi) was predominantly activated in the midgut during the initial virus invasion. Additionally, several key enzymes involved in autophagy and ubiquitination were also more abundant in infected midguts compared to controls. At later time points, after viral dissemination into the hemocoel, key immune responses are triggered in the hemolymph and, accordingly, immune mechanisms such as the activation of leucine-rich repeats (LRRs) proteins, secretion of antimicrobial peptides (e.g., holotricin), and melanization (mediated by phenoloxidase, PO) were the most prominent. RNA-seq results were validated by RT-qPCR on selected candidates in different tissues and a catalogue of Ae. albopictus immune genes (891 contigs) grouped into 24 different immune and immune-related families was compiled. This study explores the molecular interactions between Ae. albopictus and CHIKV across developmental stages, providing key insights into arbovirus transmission dynamics and mosquito vector competence.

Mosquito-borne arboviral diseases represent a growing threat and serious worldwide concern for public health authorities. Host immunoglobulin G (IgG) responses to mosquito salivary antigens emerged as a useful additional tool to evaluate human–vector contact, which is crucial for transmission risk assessment and planning vector control interventions. We previously reported that IgG responses to the Aedes albopictus 34k2 salivary protein (al34k2) are suitable, although with some limitations, to reveal variation of human exposure to the tiger mosquito. In this study we evaluated the Ae. albopictus Ag5-3 (alAg5-3), an Antigen 5 family member specifically and abundantly expressed in the saliva of adult females. IgG responses to recombinant alAg5-3, as well as to a combination of alAg5-3 and al34k2, were measured in a set of sera previously collected from healthy human blood donors before and after the summer season of exposure to mosquito bites. Surveys were conducted in two districts of Northeast Italy, Padua and Belluno, with different density and history of colonization by the tiger mosquito Ae. albopictus. A preliminary pilot study, performed on a small subset of individuals from Padua, indicated that alAg5-3 was more immunogenic than al34k2 and may be suitable to detect variations of exposure to Ae. albopictus. Analysis of the whole set of 523 sera showed that anti-alAg5-3 IgG levels significantly increased, in both study areas, after the summer period of high mosquito density. However, differences between the two study sites were only found when a mixture of the two antigens, alAg5-3 and al34k2, was used. IgG responses to alAg5-3 represent a novel appropriate marker to evaluate seasonal variation of human exposure to Ae. albopictus and, because of its higher sensitivity, it appears preferable to al34k2, especially for longitudinal studies in conditions of low-to-moderate mosquito density. However, the combination of both antigens may be a better surrogate of Ae. albopictus saliva since it allows the detection of both temporal and spatial variations of exposure to Ae. albopictus bites. The high conservation of the Ag5-3 protein among Aedes species suggests it may be exploited to also reveal exposure to Aedes aegypti and perhaps to other Aedes species.

Mosquito saliva is a powerful cocktail of bioactive molecules that help blood feeding and, in virtue of their immunomodulatory properties, may modify the local environment at the bite site facilitating transmission and establishment of mosquito-borne pathogens. We describe here some basic experimental procedures for studying and characterizing mosquito salivary components. These include mosquito salivary gland dissection, preparation of salivary gland protein extracts, and collection of mosquito saliva, both on a small scale from individual mosquitoes and on a larger scale by artificial membrane feeding from hundreds of caged mosquitoes at the same time.

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.

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.

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. 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). 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.

Anisakids are widespread marine parasites of medical, veterinary and economic relevance. They infect marine natural hosts but humans can accidentally acquire the fish-borne zoonosis anisakiasis by ingesting infected raw fishes or mollusks. Among the several species described, Anisakis pegreffii is one of the main etiological agent of the disease, in particular in the Mediterranean area. Despite the growing evidence of miRNAs involvement in host-parasite interplay, and the emerging role of exosomal microvesicles in shuttling them between different cell types (and sometime across species), no information on miRNAs from any Anisakis species is presently available. In this study we isolated extracellular vesicles (EVs) released by Anisakis pegreffii infective third-stage larvae (L3) and analyzed by RNA-seq small RNAs from both L3 and EVs. We showed by nanoparticle tracking analysis that L3 release in culture medium particles of size compatible with the one of extracellular vesicles. A catalogue of 156 miRNAs from A. pegreffii was compiled by sequence comparison to evolutionary close species and miRNA prediction software. Using differential expression analysis, we identified a small number of highly abundant miRNAs in larvae and extracellular vesicles fractions whose potential biological relevance may deserve future investigation. Finally, A. pegreffii miRNAs were compared to those described in other parasitic helminths and predicted targets among human genes were searched, suggesting their potential involvement during infection.

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.

Mosquitoes of theAedesgenus transmit arboviruses of great importance to human health as dengue, chikungunya, Zika and yellow fever. The tiger mosquitoAedes albopictuscan play an important role as arboviral vector, especially whenAedes aegyptiis 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 fromAe. 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 toAe. albopictussalivary 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 ofAe. albopictuscolonization. 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 toAe. albopictus; a serological tool of this kind may prove useful both for epidemiological studies and to estimate the effectiveness of anti-vectorial measures.

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.

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. 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. 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. 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.

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.

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. 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. 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.

A RGD motif was identified in the N-terminal region of cE5, a potent salivary thrombin inhibitor from the African malaria vector Anopheles gambiae. A peptide (APQ30) encompassing the first 30 amino acids residues of the protein and including the RGD tripeptide was tested in cell adhesion assays and found to inhibit alpha(v)beta(3) and alpha(v)beta(5) mediated adhesion. A shorter peptide (APQ16), strongly conserved among members of the A. gambiae species complex and including only the first 16 residues, retained adhesion inhibitory properties, however with enhanced specificity toward avb5. In addition, migration and invasion assays showed its capacity to inhibit the invasiveness of the malignant cell lines HepG2 and MDA-MB231. Altogether our data point to APQ16 as a new promising candidate as theranostic agent.

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.

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.

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.

Background: Seroepidemiology can provide evidence for temporal changes in malaria transmission and is an important tool to evaluate the effectiveness of control interventions. During the early 2000s, Vanuatu experienced an acute increase in malaria incidence due to a lapse in funding for vector control. After the distribution of subsidised insecticide-treated nets (ITNs) resumed in 2003, malaria incidence decreased in the subsequent years. This study was conducted to find the serological evidence supporting the impact of ITN on exposure to Anopheles vector bites and parasite prevalence.Methods: On Ambae Island, blood samples were collected from 231 and 282 individuals in 2003 and 2007, respectively. Parasite prevalence was determined by microscopy. Antibodies to three Plasmodium falciparum (PfSE, PfMSP-1(19), and PfAMA-1) and three Plasmodium vivax (PvSE, PvMSP-1(19), and PvAMA-1) antigens, as well as the Anopheles-specific salivary antigen gSG6, were detected by ELISA. Age-specific seroprevalence was analysed using a reverse catalytic modelling approach to estimate seroconversion rates (SCRs).Results: Parasite rate decreased significantly (P < 0.001) from 19.0% in 2003 to 3.2% in 2007, with a shift from P. falciparum predominance to P. falciparum-P. vivax co-dominance. Significant (P < 0.001) decreases were observed in seroprevalence to all three P. falciparum antigens but only two of three P. vivax antigens (except PvAMA-1; P = 0.153), consistent with the more pronounced decrease in P. falciparum prevalence. Seroprevalence to gSG6 also decreased significantly (P < 0.001), suggesting that reduced exposure to vector bites was important to the decrease in parasite prevalence between 2003 and 2007. Analyses of age-specific seroprevalence showed a three-fold decrease in P. falciparum transmission, but the evidence for the decrease in P. vivax transmission was less clear.Conclusions: Serological markers pointed to the effectiveness of ITNs in reducing malaria prevalence on Ambae Island between 2003 and 2007. The recombinant gSG6 antigen originally developed to indicate exposure to the Afrotropical vector An. gambiae may be used in the Pacific to complement the traditional measure of entomological inoculation rate (EIR).

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 -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.