The mosquito taste system and disease control.

Mosquitoes are a widely diverse group of organisms, comprising ∼3,500 species that live in an enormous range of habitats. Some species are vectors of diseases that afflict hundreds of millions of people each year. Although understanding of mosquito olfaction has progressed dramatically in recent years, mosquito taste remains greatly understudied. Since taste is essential to feeding, egg laying, and mating decisions in insects, improved understanding of taste in mosquitoes could provide new mechanistic insight into many aspects of their behavior. We provide a guide to current knowledge in the field, and we suggest a wealth of opportunities for research that are now enabled by recent scientific and technological advances. We also propose means by which taste might be exploited in new strategies for mosquito control, which may be urgently needed as the geographical ranges of vector species increase with climate change.

Experimental host and vector ranges of the emerging maize yellow mosaic polerovirus.

Maize yellow mosaic virus (MaYMV) is an emerging polerovirus that has been detected in maize, other cereal crops and weedy grass species in Asia, Africa, and the Americas. Disease symptoms in maize include prominent leaf tip reddening and stunting. Infection by MaYMV has been reported to reduce plant growth and yields by 10-30% in some instances. In this study, an experimental host range for MaYMV among agronomically important cereal crops and common grasses was established. Additional aphid species were assessed as potential vectors for MaYMV and their transmission efficiencies were determined. Here we report oats, foxtail millet, barley, and rye as new experimental cereal crop hosts of MaYMV in addition to confirming the previously reported hosts of corn, sorghum, wheat, and broom millet. Four of the nine other grass species evaluated were also identified as suitable experimental hosts for MaYMV: ryegrass, switchgrass, green foxtail, and sand love grass. Interestingly, no visible symptoms were present in any of the infected hosts besides the susceptible maize control. Vector range studies identified the greenbug aphid, Schizaphis graminum, as a new vector of MaYMV, though transmission efficiency was lower than the previously reported Rhopalosiphum maidis vector and similar to the other known aphid vector, R. padi. Given MaYMV's global ubiquity, ability to evade detection, and broad host range, further characterization of yield impacts and identification of viable control strategies are desirable.

Knockdown of E1- and E2-ubiquitin enzymes triggers defective chorion biogenesis and modulation of autophagy-related genes in the follicle cells of the vector Rhodnius prolixus.

In insects, the last stage of oogenesis is the process where the chorion layers (eggshell) are synthesized and deposited on the surface of the oocytes by the follicle cells. Protein homeostasis is determined by the fine-tuning of translation and degradation pathways, and the ubiquitin-proteasome system is one of the major degradative routes in eukaryotic cells. The conjugation of ubiquitin to targeted substrates is mediated by the ordered action of E1-activating, E2-conjugating, and E3-ligase enzymes, which covalently link ubiquitin to degradation-targeted proteins delivering them to the proteolytic complex proteasome. Here, we found that the mRNAs encoding polyubiquitin (pUbq), E1, and E2 enzymes are highly expressed in the ovaries of the insect vector of Chagas Disease Rhodnius prolixus. RNAi silencing of pUbq was lethal whereas the silencing of E1 and E2 enzymes resulted in drastic decreases in oviposition and embryo viability. Eggs produced by the E1- and E2-silenced insects presented particular phenotypes of altered chorion ultrastructure observed by high-resolution scanning electron microscopy as well as readings for dityrosine cross-linking and X-ray elemental microanalysis, suggesting a disruption in the secretory routes responsible for the chorion biogenesis. In addition, the ovaries from silenced insects presented altered levels of autophagy-related genes as well as a tendency of upregulation in ER chaperones, indicating a disturbance in the general biosynthetic-secretory pathway. Altogether, we found that E1 and E2 enzymes are essential for chorion biogenesis and that their silencing triggers the modulation of autophagy genes suggesting a coordinated function of both pathways for the progression of choriogenesis.

Transcriptome profiling reveals sex-specific gene expressions in pupal and adult stages of the mosquito Culex pipiens.

Understanding the development process of male and female mosquitoes provides important basic information for sterile insect release programmes and is important for improving other vector control strategies. However, little is known about the molecular mechanisms that distinguish male from female-specific developmental processes in this species. We used IlluminaRNA-seq to identify sex-specific genes during pupal and adult stages. One hundred and forty-seven genes were expressed only in pupal males, 56 genes were expressed in adult males and another 82 genes were commonly expressed in both male samples. In addition, 26 genes were expressed only in the pupal females, 163 genes were found in the adult females and only one gene was expressed in both female samples. A further quantitative real-time PCR validation of selected genes from the RNA sequencing (RNA-seq) analysis confirmed upregulation of those genes in a sex-specific manner, including: fibrinogen and fibronectin, a zinc finger protein, phospholipase A(2) and a serine protein for female pupae; venom allergen 3, a perlecan, testis-specific serine/threonine-protein kinase 1, testis-specific serine/threonine-protein kinase 6 and cytochrome c-2 for male pupae; a salivary protein, D7 protein precursor, trypsin 7 precursor, D7 protein and nanos for female adults; and tetraspanin F139, cytosol aminopeptidase, testis-specific serine/threonine-protein kinase 1, a testis-specific serine/threonine-protein kinase 6 and a C-type lectin for male adults. These findings provide insight into the development and physiology of Culex mosquitoes, which will help in the development of more effective control methods for these disease vectors.

Introduction of two new programming tools in Bengali and measurement of their reception among high-school students in Purba Bardhaman, India with the prototypic inclusion of a vector-biology module.

We introduce two new software tools, Bongojontro and Bongojontro Baksobandi, aimed at reducing the barriers to programming for native speakers of Bengali, the fifth most spoken language in the world. The highlights of these software include programming in the native language of Bengali, simpler construction of programs which is friendly for beginners, and the possibility of creating and using modules which can be used to incorporate a level of abstraction that can be helpful for users of different technical skills and roles. We introduced the software to students of two semirural schools in Purba Bardhamman, West Bengal, India. The participants were a section of class XI students of age group 16-17 from both the schools. 40 students provided the full data in the succeeding survey, with 2 providing incomplete data. Among those who participated in the survey, it was found that the reception was overwhelmingly positive, with mean score greater than 6(out of 7) in 12 out of 15 survey questions. The scores were especially high on the usage of their native language on the software and its easy workflow. However, the mean "ease of learning" score was a bit low (4.45/7) compared to the other high ratings. The prototypic vector-biology module, which was a part of Bongojontro Baksobandi, also received very favorable reviews. Further work along these lines using the software and its modules seems to be a promising avenue for useful research and inclusive development in education and information technologies.

Identification of new Anopheles gambiae transcriptional enhancers using a cross-species prediction approach.

The success of transgenic mosquito vector control approaches relies on well-targeted gene expression, requiring the identification and characterization of a diverse set of mosquito promoters and transcriptional enhancers. However, few enhancers have been characterized in Anopheles gambiae to date. Here, we employ the SCRMshaw method we previously developed to predict enhancers in the A. gambiae genome, preferentially targeting vector-relevant tissues such as the salivary glands, midgut and nervous system. We demonstrate a high overall success rate, with at least 8 of 11 (73%) tested sequences validating as enhancers in an in vivo xenotransgenic assay. Four tested sequences drive expression in either the salivary gland or the midgut, making them directly useful for probing the biology of these infection-relevant tissues. The success of our study suggests that computational enhancer prediction should serve as an effective means for identifying A. gambiae enhancers with activity in tissues involved in malaria propagation and transmission.

Deficiency of ULK1/ATG1 in the follicle cells disturbs ER homeostasis and causes defective chorion deposition in the vector Rhodnius prolixus.

In insects, synthesis and deposition of the chorion (eggshell) are performed by the professional secretory follicle cells (FCs) that surround the oocytes in the course of oogenesis. Here, we found that ULK1/ATG1, an autophagy-related protein, is highly expressed in the FCs of the Chagas-Disease vector Rhodnius prolixus, and that parental RNAi silencing of ULK1/ATG1 results in oocytes with abnormal chorion ultrastructure and FCs presenting expanded rough ER membranes as well as increased expression of the ER chaperone BiP3, both indicatives of ER stress. Silencing of LC3/ATG8, another essential autophagy protein, did not replicate the ULK1/ATG1 phenotypes, whereas silencing of SEC16A, a known partner of the noncanonical ULK1/ATG1 function in the ER exit sites phenocopied the silencing of ULK1/ATG1. Our findings point to a cooperated function of ULK1/ATG1 and SEC16A in the FCs to complete choriogenesis and provide additional in vivo phenotype-based evidence to the literature of the role of ULK1/ATG1 in the ER in a professional secretory cell.

Vector-borne plant pathogens modify top-down and bottom-up effects on insect herbivores.

Ecological theory predicts that host-plant traits affect herbivore population growth rates, which in turn modulates predator-prey interactions. However, while vector-borne plant pathogens often alter traits of both host plants and vectors, a few studies have assessed how pathogens may act as interaction modifiers within tri-trophic food webs. By applying a food web motif framework, we assessed how a vector-borne plant pathogen (Pea-enation mosaic virus, PEMV) modified both bottom-up (plant-herbivore) and top-down (predator-prey) interactions. Specifically, we assessed trophic interactions with PEMV-infectious Acyrthosiphon pisum (pea aphid) vectors compared to non-infectious aphids in a factorial experiment that manipulated predator and plant communities. We show that PEMV altered bi-trophic relationships, whereby on certain plant species, PEMV reduced vector performance but also increased their susceptibility to predators. However, on other plant species, PEMV weakened top-down control or increased vector performance. Our results suggest that vector-borne plant pathogens are important interaction modifiers for plant-herbivore-predator dynamics: host-plant response to viruses can decrease herbivore abundance by reducing herbivore performance, but also increase herbivore abundance by weakening top-down control. Broadly speaking, trophic interactions that regulate herbivore outbreaks appear to be modified for herbivores actively transmitting viruses to host plants. Consequently, management and monitoring of outbreaking herbivores should consider the infection status of focal populations.

Discovery of Novel Thrips Vector Proteins That Bind to the Viral Attachment Protein of the Plant Bunyavirus Tomato Spotted Wilt Virus.

The plant-pathogenic virus tomato spotted wilt virus (TSWV) encodes a structural glycoprotein (GN) that, like with other bunyavirus/vector interactions, serves a role in viral attachment and possibly in entry into arthropod vector host cells. It is well documented that Frankliniella occidentalis is one of nine competent thrips vectors of TSWV transmission to plant hosts. However, the insect molecules that interact with viral proteins, such as GN, during infection and dissemination in thrips vector tissues are unknown. The goals of this project were to identify TSWV-interacting proteins (TIPs) that interact directly with TSWV GN and to localize the expression of these proteins in relation to virus in thrips tissues of principal importance along the route of dissemination. We report here the identification of six TIPs from first-instar larvae (L1), the most acquisition-efficient developmental stage of the thrips vector. Sequence analyses of these TIPs revealed homology to proteins associated with the infection cycle of other vector-borne viruses. Immunolocalization of the TIPs in L1 revealed robust expression in the midgut and salivary glands of F. occidentalis, the tissues most important during virus infection, replication, and plant inoculation. The TIPs and GN interactions were validated using protein-protein interaction assays. Two of the thrips proteins, endocuticle structural glycoprotein and cyclophilin, were found to be consistent interactors with GN These newly discovered thrips protein-GN interactions are important for a better understanding of the transmission mechanism of persistent propagative plant viruses by their vectors, as well as for developing new strategies of insect pest management and virus resistance in plants.IMPORTANCE Thrips-transmitted viruses cause devastating losses to numerous food crops worldwide. For negative-sense RNA viruses that infect plants, the arthropod serves as a host as well by supporting virus replication in specific tissues and organs of the vector. The goal of this work was to identify thrips proteins that bind directly to the viral attachment protein and thus may play a role in the infection cycle in the insect. Using the model plant bunyavirus tomato spotted wilt virus (TSWV), and the most efficient thrips vector, we identified and validated six TSWV-interacting proteins from Frankliniella occidentalis first-instar larvae. Two proteins, an endocuticle structural glycoprotein and cyclophilin, were able to interact directly with the TSWV attachment protein, GN, in insect cells. The TSWV GN-interacting proteins provide new targets for disrupting the viral disease cycle in the arthropod vector and could be putative determinants of vector competence.

Tissue Tropisms and Transstadial Transmission of a Rickettsia Endosymbiont in the Highland Midge, Culicoides impunctatus (Diptera: Ceratopogonidae).

Rickettsia is a genus of intracellular bacteria which can manipulate host reproduction and alter sensitivity to natural enemy attack in a diverse range of arthropods. The maintenance of Rickettsia endosymbionts in insect populations can be achieved through both vertical and horizontal transmission routes. For example, the presence of the symbiont in the follicle cells and salivary glands of Bemisia whiteflies allows Belli group Rickettsia transmission via the germ line and plants, respectively. However, the transmission routes of other Rickettsia bacteria, such as those in the Torix group of the genus, remain underexplored. Through fluorescence in situ hybridization (FISH) and transmission electron microscopy (TEM) screening, this study describes the pattern of Torix Rickettsia tissue tropisms in the highland midge, Culicoides impunctatus (Diptera: Ceratopogonidae). Of note is the high intensity of infection of the ovarian suspensory ligament, suggestive of a novel germ line targeting strategy. Additionally, localization of the symbiont in tissues of several developmental stages suggests transstadial transmission is a major route for ensuring maintenance of Rickettsia within C. impunctatus populations. Aside from providing insights into transmission strategies, the presence of Rickettsia bacteria in the fat body of larvae indicates potential host fitness and vector capacity impacts to be investigated in the future.IMPORTANCE Microbial symbionts of disease vectors have garnered recent attention due to their ability to alter vectorial capacity. Their consideration as a means of arbovirus control depends on symbiont vertical transmission, which leads to spread of the bacteria through a population. Previous work has identified a Rickettsia symbiont present in several species of biting midges (Culicoides spp.), which transmit bluetongue and Schmallenberg arboviruses. However, symbiont transmission strategies and host effects remain underexplored. In this study, we describe the presence of Rickettsia in the ovarian suspensory ligament of Culicoides impunctatus Infection of this organ suggests the connective tissue surrounding developing eggs is important for ensuring vertical transmission of the symbiont in midges and possibly other insects. Additionally, our results indicate Rickettsia localization in the fat body of Culicoides impunctatus As the arboviruses spread by midges often replicate in the fat body, this location implies possible symbiont-virus interactions to be further investigated.

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.

An insight into the sialome, mialome and virome of the horn fly, Haematobia irritans.

BACKGROUND: The horn fly (Haematobia irritans) is an obligate blood feeder that causes considerable economic losses in livestock industries worldwide. The control of this cattle pest is mainly based on insecticides; unfortunately, in many regions, horn flies have developed resistance. Vaccines or biological control have been proposed as alternative control methods, but the available information about the biology or physiology of this parasite is rather scarce. RESULTS: We present a comprehensive description of the salivary and midgut transcriptomes of the horn fly (Haematobia irritans), using deep sequencing achieved by the Illumina protocol, as well as exploring the virome of this fly. Comparison of the two transcriptomes allow for identification of uniquely salivary or uniquely midgut transcripts, as identified by statistically differential transcript expression at a level of 16 x or more. In addition, we provide genomic highlights and phylogenetic insights of Haematobia irritans Nora virus and present evidence of a novel densovirus, both associated to midgut libraries of H. irritans. CONCLUSIONS: We provide a catalog of protein sequences associated with the salivary glands and midgut of the horn fly that will be useful for vaccine design. Additionally, we discover two midgut-associated viruses that infect these flies in nature. Future studies should address the prevalence, biological effects and life cycles of these viruses, which could eventually lead to translational work oriented to the control of this economically important cattle pest.

The Cat Flea (Ctenocephalides felis) Immune Deficiency Signaling Pathway Regulates Rickettsia typhi Infection.

Rickettsia species are obligate intracellular bacteria with both conserved and lineage-specific strategies for invading and surviving within eukaryotic cells. One variable component of Rickettsia biology involves arthropod vectors: for instance, typhus group rickettsiae are principally vectored by insects (i.e., lice and fleas), whereas spotted fever group rickettsiae are exclusively vectored by ticks. For flea-borne Rickettsia typhi, the etiological agent of murine typhus, research on vertebrate host biology is facilitated using cell lines and animal models. However, due to the lack of any stable flea cell line or a published flea genome sequence, little is known regarding R. typhi biology in flea vectors that, importantly, do not suffer lethality due to R. typhi infection. To address if fleas combat rickettsial infection, we characterized the cat flea (Ctenocephalides felis) innate immune response to R. typhi Initially, we determined that R. typhi infects Drosophila cells and increases antimicrobial peptide (AMP) gene expression, indicating immune pathway activation. While bioinformatics analysis of the C. felis transcriptome identified homologs to all of the Drosophila immune deficiency (IMD) and Toll pathway components, an AMP gene expression profile in Drosophila cells indicated IMD pathway activation upon rickettsial infection. Accordingly, we assessed R. typhi-mediated flea IMD pathway activation in vivo using small interfering RNA (siRNA)-mediated knockdown. Knockdown of Relish and Imd increased R. typhi infection levels, implicating the IMD pathway as a critical regulator of R. typhi burden in C. felis These data suggest that targeting the IMD pathway could minimize the spread of R. typhi, and potentially other human pathogens, vectored by fleas.

Diaphorina citri Nymphs Are Resistant to Morphological Changes Induced by "Candidatus Liberibacter asiaticus" in Midgut Epithelial Cells.

"Candidatus Liberibacter asiaticus" is the causative bacterium associated with citrus greening disease. "Ca Liberibacter asiaticus" is transmitted by Diaphorina citri more efficiently when it is acquired by nymphs rather than adults. Why this occurs is not known. We compared midguts of D. citri insects reared on healthy or "Ca Liberibacter asiaticus"-infected citrus trees using quantitative PCR, confocal microscopy, and mitochondrial superoxide staining for evidence of oxidative stress. Consistent with its classification as propagative, "Ca Liberibacter asiaticus" titers were higher in adults than in nymphs. Our previous work showed that adult D. citri insects have basal levels of karyorrhexis (fragmentation of the nucleus) in midgut epithelial cells, which is increased in severity and frequency in response to "Ca Liberibacter asiaticus." Here, we show that nymphs exhibit lower levels of early-stage karyorrhexis than adults and are refractory to the induction of advanced karyorrhexis by "Ca Liberibacter asiaticus" in the midgut epithelium. MitoSox Red staining showed that guts of infected adults, particularly males, experienced oxidative stress in response to "Ca Liberibacter asiaticus." A positive correlation between the titers of "Ca Liberibacter asiaticus" and the Wolbachia endosymbiont was observed in adult and nymph midguts, suggesting an interplay between these bacteria during development. We hypothesize that the resistance of the nymph midgut to late-stage karyorrhexis through as yet unknown molecular mechanisms benefits "Ca Liberibacter asiaticus" for efficient invasion of midgut epithelial cells, which may be a factor explaining the developmental dependency of "Ca Liberibacter asiaticus" acquisition by the vector.

Variable Inhibition of Zika Virus Replication by Different Wolbachia Strains in Mosquito Cell Cultures.

Mosquito-borne arboviruses are a major source of human disease. One strategy to reduce arbovirus disease is to reduce the mosquito's ability to transmit virus. Mosquito infection with the bacterial endosymbiont Wolbachia pipientis wMel is a novel strategy to reduce Aedes mosquito competency for flavivirus infection. However, experiments investigating cyclic environmental temperatures have shown a reduction in maternal transmission of wMel, potentially weakening the integration of this strain into a mosquito population relative to that of other Wolbachia strains. Consequently, it is important to investigate additional Wolbachia strains. All Zika virus (ZIKV) suppression studies are limited to the wMel Wolbachia strain. Here we show ZIKV inhibition by two different Wolbachia strains: wAlbB (isolated from Aedes albopictus mosquitoes) and wStri (isolated from the planthopper Laodelphax striatellus) in mosquito cells. Wolbachia strain wStri inhibited ZIKV most effectively. Single-cycle infection experiments showed that ZIKV RNA replication and nonstructural protein 5 translation were reduced below the limits of detection in wStri-containing cells, demonstrating early inhibition of virus replication. ZIKV replication was rescued when Wolbachia was inhibited with a bacteriostatic antibiotic. We observed a partial rescue of ZIKV growth when Wolbachia-infected cells were supplemented with cholesterol-lipid concentrate, suggesting competition for nutrients as one of the possible mechanisms of Wolbachia inhibition of ZIKV. Our data show that wAlbB and wStri infection causes inhibition of ZIKV, making them attractive candidates for further in vitro mechanistic and in vivo studies and future vector-centered approaches to limit ZIKV infection and spread.IMPORTANCE Zika virus (ZIKV) has swiftly spread throughout most of the Western Hemisphere. This is due in large part to its replication in and spread by a mosquito vector host. There is an urgent need for approaches that limit ZIKV replication in mosquitoes. One exciting approach for this is to use a bacterial endosymbiont called Wolbachia that can populate mosquito cells and inhibit ZIKV replication. Here we show that two different strains of Wolbachia, wAlbB and wStri, are effective at repressing ZIKV in mosquito cell lines. Repression of virus growth is through the inhibition of an early stage of infection and requires actively replicating Wolbachia Our findings further the understanding of Wolbachia viral inhibition and provide novel tools that can be used in an effort to limit ZIKV replication in the mosquito vector, thereby interrupting the transmission and spread of the virus.

Use of chemical probes to explore the toxicological potential of the K+/Cl- cotransporter (KCC) as a novel insecticide target to control the primary vector of dengue and Zika virus, Aedes aegypti.

The majority of commercialized insecticides target the insect nervous system and therefore, neural proteins are well-validated targets for insecticide development. Considering that only a few neural targets are exploited for insecticidal action and the development of insecticide resistance has reduced the efficacy of current insecticidal classes, we sought to test the toxicological potential of the potassium-chloride cotransporter (KCC). In mammals, KCC proteins have seminal roles in shaping GABAergic signaling and inhibitory neurotransmission, thus ion transport through KCC is critical for proper neurotransmission. Therefore, we hypothesized that mosquito KCC represents a putative insecticide target site and that pharmacological inhibition of KCC constructs in Aedes aegypti will be lethal. To test this hypothesis, we developed a robust, cell-based fluorescence assay that enables in vitro characterization of small-molecules against Ae. aegypti KCC and performed a proof-of-concept study employing well characterized mammalian KCC modulators to determine the toxicological potential of Ae. aegypti KCC. The selective inhibitor of mammalian KCC, termed VU0463271, was found to be a potent inhibitor Ae. aegypti KCC and microinjection induced lethality in a concentration-dependent manner to susceptible and pyrethroid resistant strains. Importantly, an analog of VU0463271 was shown to be >40-fold less potent and did not induce toxicity, suggesting that the observed physiological effects and mortality are likely due to KCC inhibition. This proof-of-concept study suggests that Ae. aegypti KCC represents a putative target site for mosquitocide design that can mitigate the current mechanisms of insecticide resistance.

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.

Role of inward rectifier potassium channels in salivary gland function and sugar feeding of the fruit fly, Drosophila melanogaster.

The arthropod salivary gland is of critical importance for horizontal transmission of pathogens, yet a detailed understanding of the ion conductance pathways responsible for saliva production and excretion is lacking. A superfamily of potassium ion channels, known as inward rectifying potassium (Kir) channels, is overexpressed in the Drosophila salivary gland by 32-fold when compared to the whole body mRNA transcripts. Therefore, we aimed to test the hypothesis that pharmacological and genetic depletion of salivary gland specific Kir channels alters the efficiency of the gland and reduced feeding capabilities using the fruit fly Drosophila melanogaster as a model organism that could predict similar effects in arthropod disease vectors. Exposure to VU041, a selective Kir channel blocker, reduced the volume of sucrose consumption by up to 3.2-fold and was found to be concentration-dependent with an EC50 of 68µM. Importantly, the inactive analog, VU937, was shown to not influence feeding, suggesting the reduction in feeding observed with VU041 is due to Kir channel inhibition. Next, we performed a salivary gland specific knockdown of Kir1 to assess the role of these channels specifically in the salivary gland. The genetically depleted fruit flies had a reduction in total volume ingested and an increase in the time spent feeding, both suggestive of a reduction in salivary gland function. Furthermore, a compensatory mechanism appears to be present at day 1 of RNAi-treated fruit flies, and is likely to be the Na+-K+-2Cl- cotransporter and/or Na+-K+-ATPase pumps that serve to supplement the inward flow of K+ ions, which highlights the functional redundancy in control of ion flux in the salivary glands. These findings suggest that Kir channels likely provide, at least in part, a principal potassium conductance pathway in the Drosophila salivary gland that is required for sucrose feeding.

A Deep Insight Into the Sialotranscriptome of the Chagas Disease Vector, Panstrongylus megistus (Hemiptera: Heteroptera).

Saliva of blood-sucking arthropods contains a complex cocktail of pharmacologically active compounds that assists feeding by counteracting their hosts' hemostatic and inflammatory reactions. Panstrongylus megistus (Burmeister) is an important vector of Chagas disease in South America, but despite its importance there is only one salivary protein sequence publicly deposited in GenBank. In the present work, we used Illumina technology to disclose and publicly deposit 3,703 coding sequences obtained from the assembly of >70 million reads. These sequences should assist proteomic experiments aimed at identifying pharmacologically active proteins and immunological markers of vector exposure. A supplemental file of the transcriptome and deducted protein sequences can be obtained from http://exon.niaid.nih.gov/transcriptome/P_megistus/Pmeg-web.xlsx.

The role of mites in the transmission and maintenance of Hantaan virus (Hantavirus: Bunyaviridae).

This review examines the evidence indicating a role for parasitic mites in the transmission and maintenance of Hantaan virus in nature. The available data, much of it from recent studies in China, indicate that both trombiculid and gamasid mites are naturally infected with Hantaan virus and that infected mites can transmit the virus by bite to laboratory mice and transovarially (vertically) through eggs to their offspring. Collectively, these findings challenge the current paradigm of hantavirus transmission, namely, that rodents serve as the reservoir of human pathogenic hantaviruses in nature and that humans are infected with these viruses by inhalation of aerosols of infectious rodent excreta. Further research is needed to confirm the mite-hantavirus association and to determine if parasitic mites are in fact the major source and principal vectors of human pathogenic hantaviruses, such as Hantaan. If the mite hypothesis is correct, then it will significantly alter current concepts about the epidemiology, prevention, and control of human hantavirus infection.