The dual roles of Armigeres subalbatus prophenoloxidase V in parasite melanization and egg chorion melanization in the mosquito Ar. subalbatus

Phenoloxidases (POs) play key roles in various physiological functions in insects, e.g., cuticular sclerotization, wound healing, egg tanning, cuticle formation and melanotic encapsulaction of pathogens. Previously, we identified five POs, designated As-pro-PO I–V, from the mosquito Armigeres subalbatus and demonstrated that the functions of As-pro-PO I, II and III, were associated with filarial parasite melanization, blood feeding and cuticle formation, respectively. In the present study, we delineate the dual functions of As-pro-PO V. We found that the level of As-pro-PO V mRNA in mosquitoes was significantly increased after microfilaria challenge or blood feeding, and decreased to normal level after oviposition. Knockdown of As-pro-PO V by dsRNA resulted in significant decreases in the degree of microfilaria melanization, egg chronic melanization rates and egg hatching rates in Ar. subalbatus. Further transfection and electrophoretic mobility-shift assays verified the As-pro-PO V gene might regulated by both AP-1, a putative immune-related regulatory element and CdxA, a developmental regulatory element. The binding of AP-1 and CdxA motif with mosquito nuclear extracts was significantly enhanced after microfilaria challenge and blood-feeding in Ar. subalbatus, respectively. These results indicate that As-pro-PO V is a critical enzyme that is required for both an effective melanization immune response and egg chorion melanization in this mosquito.     

Hemolytic activity is mediated by the endogenous lectin in the mosquito hemolymph serum

Although cytolysis of invading organisms is an innate form of immunity used by invertebrates, so far the underlying mechanism remains less explored. The pupal hemolymph of the mosquito Armigeres subalbatus induces an activity that causes hemolysis of human red blood cells (HRBC). This hemolytic activity was inhibited by sialic acid (N-acetylneuraminic acid) and serine protease inhibitors. We purified the sialic acid-specific lectin(s) from the pupal hemolymph using formaldehyde-fixed HRBC and determined the sequence of the amino-terminal 19 amino acid residues. A polyclonal antibody produced against this N-terminal peptide clearly inhibited the hemolytic activity of the hemolymph in vitro, thus suggesting that the hemolysis of HRBC is caused by the lectin present in the mosquito hemolymph. We suggest that mosquitoes possess a cytolysis system.     

Plasmodium yoelii-Infected A. stephensi Inefficiently Transmit Malaria Compared to Intravenous Route

It was recently reported that when mosquitoes infected with P. berghei sporozoites feed on mice, they deposit approximately 100–300 sporozoites in the dermis. When we inoculate P. yoelii (Py) sporozoites intravenously (IV) into BALB/c mice, the 50% infectious dose (ID₅₀) is often less than 3 sporozoites, indicating that essentially all Py sporozoites in salivary glands are infectious. Thus, it should only take the bite of one infected mosquito to infect 100% of mice. In human subjects, it takes the bite of at least 5 P. falciparum-infected mosquitoes to achieve 100% blood stage infection. Exposure to 1–2 infected mosquitoes only leads to blood stage infection in approximately 50% of subjects. If mosquitoes carrying Py sporozoites inoculate 100–300 sporozoites per bite, and 1 to 2 mosquito bites achieve 50% blood stage infection rates, then this would suggest that the majority of sporozoites inoculated by mosquitoes into the dermis are not responsible for a productive infection, or that a significant number of sporozoite-infected mosquitoes do not inoculate any sporozoites. The objective of this study was to determine if this is the case. We therefore studied the infectivity to mice of the bites of 1, 2, 4, or 5–8 Py-infected mosquitoes. The bite of one Py sporozoite-infected mosquito caused blood stage infection in 41.4% (12/29) of mice, two bites infected 66.7% (22/33), four bites infected 75% (18/24), and five to eight bites infected 100% (21/21). These findings demonstrate that inoculation of sporozoites by mosquito bite is much less efficient than IV inoculation of Py sporozoites by needle and syringe. Such data may have implications for determining the best route and dose of administration to humans of our attenuated P. falciparum sporozoite vaccine, the scientific basis of which is immunity by bites from irradiated infected mosquitoes, and suggest that the challenge is to develop a method of administration that approximates IV inoculation, not one that mimics mosquito bite.     

Linking Mosquito Infestation to Resident Socioeconomic Status, Knowledge, and Source Reduction Practices in Suburban Washington, DC

Eliminating water-holding containers where mosquitoes oviposit and develop (source reduction) can help manage urban disease-vector mosquitoes. Source reduction requires residents to be knowledgeable of effective practices and motivated to implement them. We tested relationships between demographics, resident knowledge, attitudes, and practices (KAP), and mosquito infestation by administering larval mosquito surveys and KAP questionnaires in Washington, DC. Respondents who reported practicing source reduction had lower numbers of pupae-positive containers and Culex pipiens-positive containers, but not Aedes albopictus-positive containers or water-holding containers, in their yards. When controlling for numbers of water-holding containers in statistical models, residents who reported source reduction had lower numbers of A. albopictus-positive containers in addition to numbers of pupae-positive containers and C. pipiens-positive containers. These results suggest that while active container reduction may be effective at reducing C. pipiens and overall pupal production, it may be offset by other resident activities that add containers to yards, and that source reduction that involves mosquito habitat management without outright container removal can also be effective at reducing A. albopictus. Source reduction was related to respondent knowledge of mosquitoes and, in particular, specific knowledge of mosquito development, which both varied with demographics alongside respondent motivation to control mosquitoes. Respondents from high socioeconomic status households reported greater knowledge but lower motivation than respondents from middle and low socioeconomic-status households. We conclude that mosquito-related education will help promote community-based container management as part of integrated mosquito management programs, particularly in middle and low socioeconomic status neighborhoods with lower knowledge and high motivation.     

Coordinated changes in JH biosynthesis and JH hemolymph titers in Aedes aegypti mosquitoes

Juvenile hormone III (JH) is synthesized by the corpora allata (CA) and plays a key role in mosquito development and reproduction. A decrease in JH titer during the last instar larvae allows pupation and metamorphosis to proceed. As the anti-metamorphic role of JH comes to an end, the CA of the late pupa once again synthesizes JH, which plays an essential role in orchestrating reproductive maturation. In spite of the importance of Aedes aegypti as a vector, a detailed study of the changes of JH hemolymph titers during the gonotrophic cycle has never been performed. In the present studies, using a high performance liquid chromatography coupled to a fluorescent detector (HPLC–FD) method, we measured changes in JH levels in the hemolymph of female mosquitoes during the pupal and adult stages. Our results revealed tightly concomitant changes in JH biosynthesis and JH hemolymph titers during the gonotrophic cycle of female mosquito. Feeding high sugar diets resulted in an increase of JH titers, and mating also modified JH titers in hemolymph. In addition these studies confirmed that JH titer in mosquitoes is fundamentally determined by the rate of biosynthesis in the CA.     

Cloning and blood‐meal dependent induction pattern of apolipophorin‐III from Anopheles sinensis

Apolipophorin‐III is known to play a role in transporting lipids in insects, and much attention has been paid to lepidopteran insects' apolipophorin. Thus, we were interested in examining the effects of blood‐meal on the expression pattern of apolipophorin‐III in mosquitoes. This led us to clone and partially characterize the full‐length cDNA of apoLp‐III (AnsiApoLp‐III) from Anopheles sinensis. Analysis of AnsiApoLp‐III cDNA shows that the 728‐bp sequence has a 582‐bp protein‐coding region with 94 bp of putative 5′ untranslated region and 152 bp of 3′ untranslated region. The deduced amino acid sequence begins with a methionine codon at position 95 and extends to position 674, encompassing a polypeptide of 193 amino acids. AnsiApoLp‐III has the highest identity (63%) to Culex quinquefasciatus apoLp‐III. Temporal expression pattern analysis shows that although AnsiApoLp‐III was expressed at all developmental stages, it was highly detected at egg and adult stages in the female mosquitoes. In addition, we found out that AnsiApoLp‐III was induced in An. sinensis adult females after uptaking a blood‐meal. Spatial expression patterns of AnsiApoLp‐III shows that AnsiApoLp‐III mRNA was strongly induced at day 1 and gradually decreased from day 1 to day 4 in the ovaries. Most interestingly, AnsiApoLp‐III mRNA in the Malpighian tubule was strongly induced at day 1, decreased during days 1–3, and then became elevated again at day 4. These data suggest that blood‐meal influences AnsiApoLp‐III mRNA induction in ovaries and Malpighian tubules. It remains to further elucidate the biological roles of AnsiApoLp‐III in these organs.     

Host Immune Response to Mosquito-Transmitted Chikungunya Virus Differs from That Elicited by Needle Inoculated Virus

Background

Mosquito-borne diseases are a worldwide public health threat. Mosquitoes transmit viruses or parasites during feeding, along with salivary proteins that modulate host responses to facilitate both blood feeding and pathogen transmission. Understanding these earliest events in mosquito transmission of arboviruses by mosquitoes is essential for development and assessment of rational vaccine and treatment strategies. In this report, we compared host immune responses to chikungunya virus (CHIKV) transmission by (1) mosquito bite, or (2) by needle inoculation.

Methods and Findings

Differential cytokine expression was measured using quantitative real-time RT-PCR, at sites of uninfected mosquito bites, CHIKV-infected mosquito bites, and needle-inoculated CHIKV. Both uninfected and CHIKV infected mosquitoes polarized host cytokine response to a T_H2 profile. Compared to uninfected mosquito bites, expression of IL-4 induced by CHIKV-infected mosquitoes were 150 fold and 527.1 fold higher at 3 hours post feeding (hpf) and 6 hpf, respectively. A significant suppression of T_H1 cytokines and TLR-3 was also observed. These significant differences may result from variation in the composition of uninfected and CHIKV-infected mosquito saliva. Needle injected CHIKV induced a robust interferon-γ, no detectable IL-4, and a significant up-regulation of TLR-3.

Conclusions

This report describes the first analysis of cutaneous cytokines in mice bitten by CHIKV–infected mosquitoes. Our data demonstrate contrasting immune activation in the response to CHIKV infection by mosquito bite or needle inoculation. The significant role of mosquito saliva in these earliest events of CHIKV transmission and infection are highlighted.     

Malaria Mosquitoes Host-Locate and Feed upon Caterpillars

Adult female mosquitoes need blood to develop their eggs and both sexes use nectar and honeydew as carbohydrate resources for flight, survival and to enhance reproduction. However, there are also a few reports in the literature of mosquitoes feeding on haemolymph of soft-bodied insects such as caterpillars. The frequency and significance of this entomophagous behavior is not well understood, but is thought to be a vestige of ancestral feeding behavior or an opportunistic behavior that has evolved over time. In our current paper we investigated the extent to which the malaria mosquito, Anopheles stephensi, is attracted to, and can successfully feed on, larvae of two common moth species, Manduca sexta and Heliothis subflexa. Using y-tube olfactometer assays we found that female An. stephensi readily flew upwind to and landed on the caterpillars of both moth species. The nature of the volatile cues used in host location remains unclear but respirometer studies suggest a possible role of CO₂. Laboratory cage assays further showed that the female mosquitoes were able to actively feed on moth larvae and gain sufficient nutritional benefit to influence survival. The extent to which such an opportunistic behavior occurs in the field has yet to be explored but our results suggest that this haemolymph feeding behavior could play a role in malaria mosquito life history and could provide a novel mechanism for horizontal transmission of pathogens and other micro-organisms between hosts.     

Wolbachia Enhances West Nile Virus (WNV) Infection in the Mosquito Culex tarsalis

Novel strategies are required to control mosquitoes and the pathogens they transmit. One attractive approach involves maternally inherited endosymbiotic Wolbachia bacteria. After artificial infection with Wolbachia, many mosquitoes become refractory to infection and transmission of diverse pathogens. We evaluated the effects of Wolbachia (wAlbB strain) on infection, dissemination and transmission of West Nile virus (WNV) in the naturally uninfected mosquito Culex tarsalis, which is an important WNV vector in North America. After inoculation into adult female mosquitoes, Wolbachia reached high titers and disseminated widely to numerous tissues including the head, thoracic flight muscles, fat body and ovarian follicles. Contrary to other systems, Wolbachia did not inhibit WNV in this mosquito. Rather, WNV infection rate was significantly higher in Wolbachia-infected mosquitoes compared to controls. Quantitative PCR of selected innate immune genes indicated that REL1 (the activator of the antiviral Toll immune pathway) was down regulated in Wolbachia-infected relative to control mosquitoes. This is the first observation of Wolbachia-induced enhancement of a human pathogen in mosquitoes, suggesting that caution should be applied before releasing Wolbachia-infected insects as part of a vector-borne disease control program.     

The entomopathogenic fungus Beauveria bassiana activate toll and JAK-STAT pathway-controlled effector genes and anti-dengue activity in Aedes aegypti

Here we investigated the effect of Beauveria bassiana infection on the survival of Aedes aegypti mosquitoes and the modulation of their susceptibility to dengue virus infection. Application of B. bassiana caused a reduction in the life span of A. aegypti and hindered dengue virus replication in the mosquito midgut. Fungus infection induced the expression a variety anti-microbial and dengue virus restriction factor genes. Transient reverse genetic analyses showed that the JAK-STAT pathway is implicated anti-fungal defense of Aedes mosquitoes. Our data suggest that this B. bassiana-mediated anti-dengue activity is likely to be at least partly indirectly mediated through the activation of the mosquito’s anti-dengue Toll and JAK-STAT pathways.     

Molecular Epidemiology of Japanese Encephalitis Virus in Mosquitoes in Taiwan during 2005–2012

Japanese encephalitis (JE) is a mosquito-borne zoonotic disease caused by the Japanese encephalitis virus (JEV). Pigs and water birds are the main amplifying and maintenance hosts of the virus. In this study, we conducted a JEV survey in mosquitoes captured in pig farms and water bird wetland habitats in Taiwan during 2005 to 2012. A total of 102,633 mosquitoes were collected. Culex tritaeniorhynchus was the most common mosquito species found in the pig farms and wetlands. Among the 26 mosquito species collected, 11 tested positive for JEV by RT-PCR, including Cx. tritaeniorhynchus, Cx. annulus, Anopheles sinensis, Armigeres subalbatus, and Cx. fuscocephala. Among those testing positive, Cx. tritaeniorhynchus was the predominant vector species for the transmission of JEV genotypes I and III in Taiwan. The JEV infection rate was significantly higher in the mosquitoes from the pig farms than those from the wetlands. A phylogenetic analysis of the JEV envelope gene sequences isolated from the captured mosquitoes demonstrated that the predominant JEV genotype has shifted from genotype III to genotype I (GI), providing evidence for transmission cycle maintenance and multiple introductions of the GI strains in Taiwan during 2008 to 2012. This study demonstrates the intense JEV transmission activity in Taiwan, highlights the importance of JE vaccination for controlling the epidemic, and provides valuable information for the assessment of the vaccine''s efficacy.     

RNAi Trigger Delivery into Anopheles gambiae Pupae

RNA interference (RNAi), a naturally occurring phenomenon in eukaryotic organisms, is an extremely valuable tool that can be utilized in the laboratory for functional genomic studies. The ability to knockdown individual genes selectively via this reverse genetic technique has allowed many researchers to rapidly uncover the biological roles of numerous genes within many organisms, by evaluation of loss-of-function phenotypes. In the major human malaria vector Anopheles gambiae, the predominant method used to reduce the function of targeted genes involves injection of double-stranded (dsRNA) into the hemocoel of the adult mosquito. While this method has been successful, gene knockdown in adults excludes the functional assessment of genes that are expressed and potentially play roles during pre-adult stages, as well as genes that are expressed in limited numbers of cells in adult mosquitoes. We describe a method for the injection of Serine Protease Inhibitor 2 (SRPN2) dsRNA during the early pupal stage and validate SRPN2 protein knockdown by observing decreased target protein levels and the formation of melanotic pseudo-tumors in SRPN2 knockdown adult mosquitoes. This evident phenotype has been described previously for adult stage knockdown of SRPN2 function, and we have recapitulated this adult phenotype by SRPN2 knockdown initiated during pupal development. When used in conjunction with a dye-labeled dsRNA solution, this technique enables easy visualization by simple light microscopy of injection quality and distribution of dsRNA in the hemocoel.     

Phenoloxidase Activity Acts as a Mosquito Innate Immune Response against Infection with Semliki Forest Virus

Several components of the mosquito immune system including the RNA interference (RNAi), JAK/STAT, Toll and IMD pathways have previously been implicated in controlling arbovirus infections. In contrast, the role of the phenoloxidase (PO) cascade in mosquito antiviral immunity is unknown. Here we show that conditioned medium from the Aedes albopictus-derived U4.4 cell line contains a functional PO cascade, which is activated by the bacterium Escherichia coli and the arbovirus Semliki Forest virus (SFV) (Togaviridae; Alphavirus). Production of recombinant SFV expressing the PO cascade inhibitor Egf1.0 blocked PO activity in U4.4 cell- conditioned medium, which resulted in enhanced spread of SFV. Infection of adult female Aedes aegypti by feeding mosquitoes a bloodmeal containing Egf1.0-expressing SFV increased virus replication and mosquito mortality. Collectively, these results suggest the PO cascade of mosquitoes plays an important role in immune defence against arboviruses.     

A novel herbal product based on Piper betle and Sphaeranthus indicus essential oils: Toxicity,repellent activity and impact on detoxifying enzymes GST and CYP450 of Aedes aegypti Liston (Diptera: Culicidae)

Fresh leaves of Piper betle Linn. (Piperales: Piperaceae) and Sphaeranthus indicus Linn. (Asterales: Asteraceae), commonly known as betel leaves and East Indian globe thistle, respectively, were harvested and steam distilled for the extraction of P. betle and S. indicus crude volatile oils (Pb-CVO and Si-CVO, respectively). LC₅₀ calculated on 3^rd instar larvae of dengue mosquito Aedes aegypti Linn. (Diptera: Culicidae) was 42.17?ppm for PbSi-CVO (i.e., herbal formulation based on the EOs of P. betle and S. indicus). The larval and pupal duration were significantly longer post-treatment with 100?ppm of PbSi-CVO, if compared to control. We observed that PbSi-CVO significantly altered the detoxifying enzymes GST and CYP450 compared to the expression of control. Sub-lethal concentrations of PbSi-CVO showed strong repellent properties against dengue mosquitoes, without adverse reactions on the volunteers experiencing the repellent assays. Lastly, the adulticidal activity of PbSi-CVO was studied. Overall, our study outlined that this herbal product represents a promising candidate for the development of botanical based adulticidal agents.     

The Effect of Pyriproxyfen as a “Population Growth Regulator” against Aedes albopictus under Semi-Field Conditions

An insect growth regulator, pyriproxyfen, has been used for the control of a range of pest insects, including mosquitoes. Pyriproxyfen is effective in inhibiting adult emergence and sterilizing adult females. The Asian tiger mosquito, Aedes albopictus (Skuse), is an important vector of dengue and chikungunya, and is expanding its distribution throughout Europe and the Americas. In the present study, we evaluated the impact of pyriproxyfen-treated bed nets on population growth of Ae. albopictus under semi-field conditions, using 6 small microcosms. We created microcosms containing breeding sites to simulate the natural ecosystem of vector mosquito and installing miniature bed net treated with 350 mg/m² pyriproxyfen in Experiment I and 35 mg/m² in Experiment II. For each experiment, we also established microcosms installing untreated polyethylene net (untreated control). The installing nets were provided with artificially torn holes, to simulate damage and allow mosquitoes to penetrate. We released 100 pairs of Ae. albopictus into each microcosm, and allowed them to feed on a mouse under the bed nets at approximately 1-week intervals. In comparison with the untreated control microcosms, the number of eggs laid by the released adults in the pyriproxyfen-treated microcosms was significantly lower in both Experiment I and II. Moreover, egg hatchability was significantly suppressed and pupal mortality was increased. Our results indicate that tarsal contact with pyriproxyfen has been shown to suppress egg production and hatchability in adult females and the auto-dissemination of pyriproxyfen into larval breeding sites by adult mosquitoes, through contact with pyriproxyfen-treated polyethylene bed nets, may suppress the mosquito population density.     

Molecular and functional characterization of Anopheles gambiae inward rectifier potassium (Kir1) channels: A novel role in egg production

Inward rectifier potassium (Kir) channels play essential roles in regulating diverse physiological processes. Although Kir channels are encoded in mosquito genomes, their functions remain largely unknown. In this study, we identified the members of the Anopheles gambiae Kir gene family and began to investigate their function. Notably, we sequenced the A. gambiae Kir1 (AgKir1) gene and showed that it encodes all the canonical features of a Kir channel: an ion pore that is composed of a pore helix and a selectivity filter, two transmembrane domains that flank the ion pore, and the so-called G-loop. Heterologous expression of AgKir1 in Xenopus oocytes revealed that this gene encodes a functional, barium-sensitive Kir channel. Quantitative RT-PCR experiments then showed that relative AgKir1 mRNA levels are highest in the pupal stage, and that AgKir1 mRNA is enriched in the adult ovaries. Gene silencing of AgKir1 by RNA interference did not affect the survival of female mosquitoes following a blood meal, but decreased their egg output. These data provide evidence for a new role of Kir channels in mosquito fecundity, and further validates them as promising molecular targets for the development of a new class of mosquitocides to be used in vector control.     

Insecticidal Activity and Expression of Cytochrome P450 Family 4 Genes in Aedes albopictus After Exposure to Pyrethroid Mosquito Coils

Mosquito coils are insecticides commonly used for protection against mosquitoes due to their toxic effects on mosquito populations. These effects on mosquitoes could induce the expression of metabolic enzymes in exposed populations as a counteractive measure. Cytochrome P450 family 4 (CYP4) are metabolic enzymes associated with a wide range of biological activities including insecticide resistance. In this study, the efficacies of three commercial mosquito coils with different pyrethroid active ingredients were assessed and their potential to induce the expression of CYP4 genes in Aedes albopictus analyzed by real-time quantitative PCR. Coils containing 0.3 % d-allethrin and 0.005 % metofluthrin exacted profound toxic effects on Ae. albopictus, inducing high mortalities (≥90 %) compared to the 0.2 % d-allethrin reference coil. CYP4H42 and CYP4H43 expressions were significantly higher in 0.3 % d-allethrin treated mosquitoes compared to the other treated populations. Short-term (KT₅₀) exposure to mosquito coils induced significantly higher expression of both genes in 0.005 % metofluthrin exposed mosquitoes. These results suggest the evaluated products provided better protection than the reference coil; however, they also induced the expression of metabolic genes which could impact negatively on personal protection against mosquito.