Annotated Differentially Expressed Salivary Proteins of Susceptible and Insecticide-Resistant Mosquitoes of Anopheles stephensi

Vector control is one of the major global strategies for control of malaria. However, the major obstacle for vector control is the development of multiple resistances to organochlorine, organophosphorus insecticides and pyrethroids that are currently being used in public health for spraying and in bednets. Salivary glands of vectors are the first target organ for human-vector contact during biting and parasite-vector contact prior to parasite development in the mosquito midguts. The salivary glands secrete anti-haemostatic, anti-inflammatory biologically active molecules to facilitate blood feeding from the host and also inadvertently inject malaria parasites into the vertebrate host. The Anopheles stephensi mosquito, an urban vector of malaria to both human and rodent species has been identified as a reference laboratory model to study mosquito—parasite interactions. In this study, we adopted a conventional proteomic approach of 2D-electrophoresis coupled with MALDI-TOF mass spectrometry and bioinformatics to identify putative differentially expressed annotated functional salivary proteins between An. stephensi susceptible and multiresistant strains with same genetic background. Our results show 2D gel profile and MALDI-TOF comparisons that identified 31 differentially expressed putative modulated proteins in deltamethrin/DDT resistant strains of An. stephensi. Among these 15 proteins were found to be upregulated and 16 proteins were downregulated. Our studies interpret that An. stephensi (multiresistant) caused an upregulated expression of proteins and enzymes like cytochrome 450, short chain dehyrdogenase reductase, phosphodiesterase etc that may have an impact in insecticide resistance and xenobiotic detoxification. Our study elucidates a proteomic response of salivary glands differentially regulated proteins in response to insecticide resistance development which include structural, redox and regulatory enzymes of several pathways. These identified proteins may play a role in regulating mosquito biting behavior patterns and may have implications in the development of malaria parasites in resistant mosquitoes during parasite transmission.     

Inhibition of Malaria Infection in Transgenic Anopheline Mosquitoes Lacking Salivary Gland Cells

Malaria is an important global public health challenge, and is transmitted by anopheline mosquitoes during blood feeding. Mosquito vector control is one of the most effective methods to control malaria, and population replacement with genetically engineered mosquitoes to block its transmission is expected to become a new vector control strategy. The salivary glands are an effective target tissue for the expression of molecules that kill or inactivate malaria parasites. Moreover, salivary gland cells express a large number of molecules that facilitate blood feeding and parasite transmission to hosts. In the present study, we adapted a functional deficiency system in specific tissues by inducing cell death using the mouse Bcl-2-associated X protein (Bax) to the Asian malaria vector mosquito, Anopheles stephensi. We applied this technique to salivary gland cells, and produced a transgenic strain containing extremely low amounts of saliva. Although probing times for feeding on mice were longer in transgenic mosquitoes than in wild-type mosquitoes, transgenic mosquitoes still successfully ingested blood. Transgenic mosquitoes also exhibited a significant reduction in oocyst formation in the midgut in a rodent malaria model. These results indicate that mosquito saliva plays an important role in malaria infection in the midgut of anopheline mosquitoes. The dysfunction in the salivary glands enabled the inhibition of malaria transmission from hosts to mosquito midguts. Therefore, salivary components have potential in the development of new drugs or genetically engineered mosquitoes for malaria control.     

Differential protein expression in the midgut of Culex quinquefasciatus mosquitoes induced by the insecticide temephos

Mosquitoes are vectors for pathogens of malaria, lymphatic filariasis, dengue, chikungunya, yellow fever and Japanese encephalitis. Culex quinquefasciatus Say, 1823 (Diptera: Culicidae) is a known vector of lymphatic filariasis. Its control in Brazil has been managed using the organophosphate temephos. Studies examining the proteins of Cx. quinquefasciatus that are differentially expressed in response to temephos further understanding of the modes of action of the insecticide and may potentially identify resistance factors in the mosquito. In the present study, a comparative proteomic analysis, using 2‐dimensional electrophoresis coupled with matrix‐assisted laser desorption/ionization (MALDI) time of flight (TOF)/TOF mass spectrometry, and bioinformatics analyses were performed to identify midgut proteins in Cx. quinquefasciatus larvae that were differentially expressed in response to exposure to temephos relative to those in untreated controls. A total of 91 protein spots were differentially expressed; 40 were upregulated and 51 were downregulated by temephos. A total of 22 proteins, predominantly upregulated, were identified as known to play a role in the immune response, whereas the downregulated proteins were involved in energy and protein catabolism. This is the first proteome study of the midgut of Cx. quinquefasciatus and it provides insights into the molecular mechanisms of insecticide‐induced responses in the mosquito.     

Comparative proteomics reveals mechanisms that underlie insecticide resistance in Culex pipiens pallens Coquillett

Mosquito control based on chemical insecticides is considered as an important element of the current global strategies for the control of mosquito-borne diseases. Unfortunately, the development of insecticide resistance of important vector mosquito species jeopardizes the effectiveness of insecticide-based mosquito control. In contrast to target site resistance, other mechanisms are far from being fully understood. Global protein profiles among cypermethrin-resistant, propoxur-resistant, dimethyl-dichloro-vinyl-phosphate-resistant and susceptible strain of Culex pipiens pallens were obtained and proteomic differences were evaluated by using isobaric tags for relative and absolute quantification labeling coupled with liquid chromatography/tandem mass spectrometric analysis. A susceptible strain of Culex pipiens pallens showed elevated resistance levels after 25 generations of insecticide selection, through iTRAQ data analysis detected 2,502 proteins, of which 1,513 were differentially expressed in insecticide-selected strains compared to the susceptible strain. Finally, midgut differential protein expression profiles were analyzed, and 62 proteins were selected for verification of differential expression using iTRAQ and parallel reaction monitoring strategy, respectively. iTRAQ profiles of adaptation selection to three insecticide strains combined with midgut profiles revealed that multiple insecticide resistance mechanisms operate simultaneously in resistant insects of Culex pipiens pallens. Significant molecular resources were developed for Culex pipiens pallens, potential candidates were involved in metabolic resistance and reducing penetration or sequestering insecticide. Future research that is targeted towards RNA interference of the identified metabolic targets, such as cuticular proteins, cytochrome P450s, glutathione S-transferases and ribosomal proteins proteins and biological pathways (drug metabolism—cytochrome P450, metabolism of xenobiotics by cytochrome P450, oxidative phosphorylation, ribosome) could lay the foundation for a better understanding of the genetic basis of insecticide resistance in Culex pipiens pallens.     

Different mutation patterns of atovaquone resistance to Plasmodium falciparum in vitro and in vivo : rapid detection of codon 268 polymorphisms in the cytochrome b as potential in vivo resistance marker

Background

Anopheles gambiae is the main vector of Plasmodium falciparum in Africa. The mosquito midgut constitutes a barrier that the parasite must cross if it is to develop and be transmitted. Despite the central role of the mosquito midgut in the host/parasite interaction, little is known about its protein composition. Characterisation of An. gambiae midgut proteins may identify the proteins that render An. gambiae receptive to the malaria parasite.

Methods

We carried out two-dimensional gel electrophoresis of An. gambiae midgut proteins and compared protein profiles for midguts from males, sugar-fed females and females fed on human blood.

Results

Very few differences were detected between male and female mosquitoes for the approximately 375 silver-stained proteins. Male midguts contained ten proteins not detected in sugar-fed or blood-fed females, which are therefore probably involved in male-specific functions; conversely, female midguts contained twenty-three proteins absent from male midguts. Eight of these proteins were specific to sugar-fed females, and another ten, to blood-fed females.

Conclusion

Mass spectrometry analysis of the proteins found only in blood-fed female midguts, together with data from the recent sequencing of the An. gambiae genome, should make it possible to determine the role of these proteins in blood digestion or parasite receptivity.     

Aedes aegypti SNAP and a calcium transporter ATPase influence dengue virus dissemination

Dengue virus (DENV) is a flavivirus that causes marked human morbidity and mortality worldwide, and is transmitted to humans by Aedes aegypti mosquitoes. Habitat expansion of Aedes, mainly due to climate change and increasing overlap between urban and wild habitats, places nearly half of the world’s population at risk for DENV infection. After a bloodmeal from a DENV-infected host, the virus enters the mosquito midgut. Next, the virus migrates to, and replicates in, other tissues, like salivary glands. Successful viral transmission occurs when the infected mosquito takes another blood meal on a susceptible host and DENV is released from the salivary gland via saliva into the skin. During viral dissemination in the mosquito and transmission to a new mammalian host, DENV interacts with a variety of vector proteins, which are uniquely important during each phase of the viral cycle. Our study focuses on the interaction between DENV particles and protein components in the A. aegypti vector. We performed a mass spectrometry assay where we identified a set of A. aegypti salivary gland proteins which potentially interact with the DENV virion. Using dsRNA to silence gene expression, we analyzed the role of these proteins in viral infectivity. Two of these candidates, a synaptosomal-associated protein (AeSNAP) and a calcium transporter ATPase (ATPase) appear to play a role in viral replication both in vitro and in vivo, observing a ubiquitous expression of these proteins in the mosquito. These findings suggest that AeSNAP plays a protective role during DENV infection of mosquitoes and that ATPase protein is required for DENV during amplification within the vector.     

Blood Feeding and Plasmodium Infection Alters the miRNome of Anopheles stephensi

Blood feeding is an integral process required for physiological functions and propagation of the malaria vector Anopheles. During blood feeding, presence of the malaria parasite, Plasmodium in the blood induces several host effector molecules including microRNAs which play important roles in the development and maturation of the parasite within the mosquito. The present study was undertaken to elucidate the dynamic expression of miRNAs during gonotrophic cycle and parasite development in Anopheles stephensi. Using next generation sequencing technology, we identified 126 miRNAs of which 17 were novel miRNAs. The miRNAs were further validated by northern hybridization and cloning. Blood feeding and parasitized blood feeding in the mosquitoes revealed regulation of 13 and 16 miRNAs respectively. Expression profiling of these miRNAs revealed that significant miRNAs were down-regulated upon parasitized blood feeding with a repertoire of miRNAs showing stage specific up-regulation. Expression profiles of significantly modulated miRNAs were further validated by real time PCR. Target prediction of regulated miRNAs revealed overlapping targeting by different miRNAs. These targets included several metabolic pathways including metabolic, redox homeostasis and protein processing machinery components. Our analysis revealed tight regulation of specific miRNAs post blood feeding and parasite infection in An. stephensi. Such regulated expression suggests possible role of these miRNAs during gonotrophic cycle in mosquito. Another set of miRNAs were also significantly regulated at 42 h and 5 days post infection indicating parasite stage-specific role of host miRNAs. This study will result in better understanding of the role of miRNAs during gonotrophic cycle and parasite development in mosquito and can probably facilitate in devising novel malaria control strategies at vector level.     

Identification of Proteasome Subunit Beta Type 6 (PSMB6) Associated with Deltamethrin Resistance in Mosquitoes by Proteomic and Bioassay Analyses

Deltamethrin (DM) insecticides are currently being promoted worldwide for mosquito control, because of the high efficacy, low mammalian toxicity and less environmental impact. Widespread and improper use of insecticides induced resistance, which has become a major obstacle for the insect-borne disease management. Resistance development is a complex and dynamic process involving many genes. To better understand the possible molecular mechanisms involved in DM resistance, a proteomic approach was employed for screening of differentially expressed proteins in DM-susceptible and -resistant mosquito cells. Twenty-seven differentially expressed proteins were identified by two-dimensional electrophoresis (2-DE) and mass spectrometry (MS). Four members of the ubiquitin-proteasome system were significantly elevated in DM-resistant cells, suggesting that the ubiquitin-proteasome pathway may play an important role in DM resistance. Proteasome subunit beta type 6 (PSMB6) is a member of 20S proteasomal subunit family, which forms the proteolytic core of 26S proteasome. We used pharmaceutical inhibitor and molecular approaches to study the contributions of PSMB6 in DM resistance: the proteasome inhibitor MG-132 and bortezomib were used to suppress the proteasomal activity and siRNA was designed to block the function of PSMB6. The results revealed that both MG-132 and bortezomib increased the susceptibility in DM-resistant cells and resistance larvae. Moreover, PSMB6 knockdown decreased cellular viability under DM treatment. Taken together, our study indicated that PSMB6 is associated with DM resistance in mosquitoes and that proteasome inhibitors such as MG-132 or bortezomib are suitable for use as a DM synergist for vector control.     

An α‐amylase is a novel receptor for Bacillus thuringiensis ssp. israelensis Cry4Ba and Cry11Aa toxins in the malaria vector mosquito Anopheles albimanus (Diptera: Culicidae)

Bacillus thuringiensis ssp. israelensis (Bti) produces four Cry toxins (Cry4Aa, Cry4Ba, Cry10Aa and Cry11Aa), and two Cyt proteins (Cyt1Aa and Cyt2Ba), toxic to mosquito‐larvae of the genus Aedes, Anopheles and Culex, important human disease vectors that transmit dengue virus, malaria and filarial parasites respectively. Previous work showed that Bti is highly toxic to Anopheles albimanus, the main vector for transmission of malaria in Mexico. In this work, we analysed the toxicity of isolated Cry proteins of Bti and identified an An. albimanus midgut protein as a putative Cry4Ba and Cry11Aa receptor molecule. Biossays showed that Cry4Ba and Cry11Aa of Bti are toxic to An. albimanus larvae. Ligand blot assays indicated that a 70 kDa glycosylphosphatidylinositol‐anchored protein present in midgut brush border membrane vesicles of An. albimanus interacts with Cry4Ba and Cry11Aa toxins. This protein was identified as an α‐amylase by mass spectrometry and enzymatic activity assays. The cDNA that codes for the α‐amylase was cloned by means of 5′‐ and 3′‐RACE experiments. Recombinant α‐amylase expressed in Escherichia coli specifically binds Cry4Ba and Cry11Aa toxins.     

In vivo germination and host specificity of Nosema algerae in mosquitoes.

Of five species of mosquitoes tested, Anopheles stephensi and A. albimanus were the most susceptible, A. quadrimaculatus and Culex pipiens were relatively unsusceptible and A. atroparvus was almost completely refractory to Nosema algerae. Spore germination percentages differed in the larval midguts of the five species of mosquito but not in the order which would explain the susceptibility differences. Neither midgut pH difference nor differences in passage rate through the midgut were sufficient to explain the observed differences in spore germination percentages.     

Alpha-COPI coatomer protein is required for rough endoplasmic reticulum whorl formation in mosquito midgut epithelial cells

Background

One of the early events in midgut epithelial cells of Aedes aegypti mosquitoes is the dynamic reorganization of rough endoplasmic reticulum (RER) whorl structures coincident with the onset of blood meal digestion. Based on our previous studies showing that feeding on an amino acid meal induces TOR signaling in Ae. aegypti, we used proteomics and RNAi to functionally identify midgut epithelial cell proteins that contribute to RER whorl formation.

Methodology/Principal Findings

Adult female Ae. aegypti mosquitoes were maintained on sugar alone (unfed), or fed an amino acid meal, and then midgut epithelial cells were analyzed by electron microscopy and protein biochemistry. The size and number of RER whorls in midgut epithelial cells were found to decrease significantly after feeding, and several KDEL-containing proteins were shown to have altered expression levels. LC-MS/MS mass spectrometry was used to analyze midgut microsomal proteins isolated from unfed and amino acid fed mosquitoes, and of the 127 proteins identified, 8 were chosen as candidate whorl forming proteins. Three candidate proteins were COPI coatomer subunits (alpha, beta, beta''), all of which appeared to be present at higher levels in microsomal fractions from unfed mosquitoes. Using RNAi to knockdown alpha-COPI expression, electron microscopy revealed that both the size and number of RER whorls were dramatically reduced in unfed mosquitoes, and moreover, that extended regions of swollen RER were prevalent in fed mosquitoes. Lastly, while a deficiency in alpha-COPI had no effect on early trypsin protein synthesis or secretion 3 hr post blood meal (PBM), expression of late phase proteases at 24 hr PBM was completely blocked.

Conclusions

alpha-COPI was found to be required for the formation of RER whorls in midgut epithelial cells of unfed Aa. aegypti mosquitoes, as well as for the expression of late phase midgut proteases.     

Epoxide hydrolase activities and epoxy fatty acids in the mosquito Culex quinquefasciatus

Culex mosquitoes have emerged as important model organisms for mosquito biology, and are disease vectors for multiple mosquito-borne pathogens, including West Nile virus. We characterized epoxide hydrolase activities in the mosquito Culex quinquefasciatus, which suggested multiple forms of epoxide hydrolases were present. We found EH activities on epoxy eicosatrienoic acids (EETs). EETs and other eicosanoids are well-established lipid signaling molecules in vertebrates. We showed EETs can be synthesized in vitro from arachidonic acids by mosquito lysate, and EETs were also detected in vivo both in larvae and adult mosquitoes by LC-MS/MS. The EH activities on EETs can be induced by blood feeding, and the highest activity was observed in the midgut of female mosquitoes. The enzyme activities on EETs can be inhibited by urea-based inhibitors designed for mammalian soluble epoxide hydrolases (sEH). The sEH inhibitors have been shown to play diverse biological roles in mammalian systems, and they can be useful tools to study the function of EETs in mosquitoes. Besides juvenile hormone metabolism and detoxification, insect epoxide hydrolases may also play a role in regulating lipid signaling molecules, such as EETs and other epoxy fatty acids, synthesized in vivo or obtained from blood feeding by female mosquitoes.     

Anopheles Midgut Epithelium Evades Human Complement Activity by Capturing Factor H from the Blood Meal

Hematophagous vectors strictly require ingesting blood from their hosts to complete their life cycles. Exposure of the alimentary canal of these vectors to the host immune effectors necessitates efficient counteractive measures by hematophagous vectors. The Anopheles mosquito transmitting the malaria parasite is an example of hematophagous vectors that within seconds can ingest human blood double its weight. The innate immune defense mechanisms, like the complement system, in the human blood should thereby immediately react against foreign cells in the mosquito midgut. A prerequisite for complement activation is that the target cells lack complement regulators on their surfaces. In this work, we analyzed whether human complement is active in the mosquito midgut, and how the mosquito midgut cells protect themselves against complement attack. We found that complement remained active for a considerable time and was able to kill microbes within the mosquito midgut. However, the Anopheles mosquito midgut cells were not injured. These cells were found to protect themselves by capturing factor H, the main soluble inhibitor of the alternative complement pathway. Factor H inhibited complement on the midgut cells by promoting inactivation of C3b to iC3b and preventing the activity of the alternative pathway amplification C3 convertase enzyme. An interference of the FH regulatory activity by monoclonal antibodies, carried to the midgut via blood, resulted in increased mosquito mortality and reduced fecundity. By using a ligand blotting assay, a putative mosquito midgut FH receptor could be detected. Thereby, we have identified a novel mechanism whereby mosquitoes can tolerate human blood.     

Suppressing Dengue-2 Infection by Chemical Inhibition of Aedes aegypti Host Factors

Dengue virus host factors (DENV HFs) that are essential for the completion of the infection cycle in the mosquito vector and vertebrate host represent potent targets for transmission blocking. Here we investigated whether known mammalian DENV HF inhibitors could influence virus infection in the arthropod vector A. aegypti. We evaluated the potency of bafilomycin (BAF; inhibitor of vacuolar H+-ATPase (vATPase)), mycophenolic acid (MPA; inhibitor of inosine-5′-monophosphate dehydrogenase (IMPDH)), castanospermine (CAS; inhibitor of glucosidase), and deoxynojirimycin (DNJ; inhibitor of glucosidase) in blocking DENV infection of the mosquito midgut, using various treatment methods that included direct injection, ingestion by sugar feeding or blood feeding, and silencing of target genes by RNA interference (RNAi). Injection of BAF (5 µM) and MPA (25 µM) prior to feeding on virus-infected blood inhibited DENV titers in the midgut at 7 days post-infection by 56% and 60%, and in the salivary gland at 14 days post-infection by 90% and 83%, respectively, while treatment of mosquitoes with CAS or DNJ did not affect susceptibility to the virus. Ingestion of BAF and MPA through a sugar meal or together with an infectious blood meal also resulted in various degrees of virus inhibition. RNAi-mediated silencing of several vATPase subunit genes and the IMPDH gene resulted in a reduced DENV infection, thereby indicating that BAF- and MPA-mediated virus inhibition in adult mosquitoes most likely occurred through the inhibition of these DENV HFs. The route and timing of BAF and MPA administration was essential, and treatment after exposure to the virus diminished the antiviral effect of these compounds. Here we provide proof-of-principle that chemical inhibition or RNAi-mediated depletion of the DENV HFs vATPase and IMPDH can be used to suppress DENV infection of adult A. aegypti mosquitoes, which may translate to a reduction in DENV transmission.     

Midgut Microbiota of the Malaria Mosquito Vector Anopheles gambiae and Interactions with Plasmodium falciparum Infection

The susceptibility of Anopheles mosquitoes to Plasmodium infections relies on complex interactions between the insect vector and the malaria parasite. A number of studies have shown that the mosquito innate immune responses play an important role in controlling the malaria infection and that the strength of parasite clearance is under genetic control, but little is known about the influence of environmental factors on the transmission success. We present here evidence that the composition of the vector gut microbiota is one of the major components that determine the outcome of mosquito infections. A. gambiae mosquitoes collected in natural breeding sites from Cameroon were experimentally challenged with a wild P. falciparum isolate, and their gut bacterial content was submitted for pyrosequencing analysis. The meta-taxogenomic approach revealed a broader richness of the midgut bacterial flora than previously described. Unexpectedly, the majority of bacterial species were found in only a small proportion of mosquitoes, and only 20 genera were shared by 80% of individuals. We show that observed differences in gut bacterial flora of adult mosquitoes is a result of breeding in distinct sites, suggesting that the native aquatic source where larvae were grown determines the composition of the midgut microbiota. Importantly, the abundance of Enterobacteriaceae in the mosquito midgut correlates significantly with the Plasmodium infection status. This striking relationship highlights the role of natural gut environment in parasite transmission. Deciphering microbe-pathogen interactions offers new perspectives to control disease transmission.