Performance of MALDI-TOF Mass Spectrometry to Determine the Sex of Mosquitoes and Identify Specific Colonies from French Polynesia.

Mosquitoes are the main arthropod vectors of human pathogens. The current methods for mosquito identification include morphological and molecular methods. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), now routinely used for bacterial identification, has recently emerged in the field of entomology. The aim of this study was to use MALDI-TOF MS to identify mosquito colonies from French Polynesia. Five hundred specimens from French Polynesia belonging to three species, Aedes aegypti, Aedes polynesiensis, and Culex quinquefasciatus, were included in the study. Testing the legs of these mosquitoes by MALDI-TOF MS revealed a 100% correct identification of all specimens at the species level. The MALDI-TOF MS profiles obtained allowed differentiation of male from female mosquitoes and the specific identification of female mosquito colonies of the same species but different geographic origin.

ADP binding by the Culex quinquefasciatus mosquito D7 salivary protein enhances blood feeding on mammals.

During blood-feeding, mosquito saliva is injected into the skin to facilitate blood meal acquisition. D7 proteins are among the most abundant components of the mosquito saliva. Here we report the ligand binding specificity and physiological relevance of two D7 long proteins from Culex quinquefasciatus mosquito, the vector of filaria parasites or West Nile viruses. CxD7L2 binds biogenic amines and eicosanoids. CxD7L1 exhibits high affinity for ADP and ATP, a binding capacity not reported in any D7. We solve the crystal structure of CxD7L1 in complex with ADP to 1.97 Å resolution. The binding pocket lies between the two protein domains, whereas all known D7s bind ligands either within the N- or the C-terminal domains. We demonstrate that these proteins inhibit hemostasis in ex vivo and in vivo experiments. Our results suggest that the ADP-binding function acquired by CxD7L1 evolved to enhance blood-feeding in mammals, where ADP plays a key role in platelet aggregation.

Sugar Feeding Patterns for Aedes aegypti and Culex quinquefasciatus (Diptera: Culicidae) Mosquitoes in South Texas.

Effective mosquito surveillance and management depend on a thorough understanding of the biology and feeding patterns unique to species and sex. Given that a propensity to sugar feed is necessary for some mosquito surveillance and newer control strategies, we sought to document the amount of total sugar in wild Aedes aegypti (L.) and Culex quinquefasciatus (Say) captured from five different locations in the Lower Rio Grande Valley (LRGV) of South Texas over 2 yr. We used Biogents Sentinel 2 (BGS2) traps in year 1 and aspirators, BGS2, and CDC resting traps in years 2 and 3 to collect adult mosquitoes. The hot anthrone test was used to quantify total sugar content in each mosquito. Additionally, the cold and hot anthrone tests were used to distinguish fructose content from total sugars for mosquitoes captured in 2019. Overall, Ae. aegypti females had significantly lower total sugar content than Ae. aegypti males as well as both sexes of Cx. quinquefasciatus. However, the percentage of Ae. aegypti positive for fructose consumption was four to eightfold higher than Ae. aegypti previously reported in other regions. The difference between locations was significant for males of both species, but not for females. Seasonality and trapping method also revealed significant differences in sugar content of captured mosquitoes. Our results reinforce that sugar feeding in female Ae. aegypti is less than Cx. quinquefasciatus, although not absent. This study provides necessary data to evaluate the potential effectiveness of sugar baits in surveillance and control of both Ae. aegypti and Cx. quinquefasciatus mosquitoes.

An Improved Multiplex Polymerase Chain Reaction (PCR) Assay for the Identification of Mosquito (Diptera: Culicidae) Blood Meals.

The analysis of vertebrate blood meals serves as an integral component of vector incrimination studies where feeding preferences and host associations influence vector-borne disease transmission. Diagnostic polymerase chain reaction (PCR)-based techniques have been widely used to determine host associations, yet applications for Culex (Diptera: Culicidae), which feed primarily on bird populations, have been limited by multistep PCR techniques that approach each potential host species singly. As a result, we have developed a multiplexed primer set targeting mitochondrial cytochrome b sequences that can distinguish human, bird, and mammalian host blood meals in a single PCR reaction, an improvement over previous analyses relying on single primers or other multiplex primer approaches through the inclusion of avian primers. To validate this new methodology, we demonstrate its application on blood samples as well as field-collected Culex samples. Although designed for applications with mosquito vectors, this multiplex PCR assay is not mosquito-specific, and should serve as a valuable tool for identifying the blood meals of other blood-feeding arthropods, contributing greatly to the study of vector-borne disease.

Efficiency of Tinospora crispa against Culex quinquefasciatus larva.

Tinospora crispa stem aqueous extractions for various time durations were determined regarding their total phenolic content and their larvicidal abilities. The results revealed that the total phenolic content in 1-, 3-, 5-, 10-, and 24-h extracts were 8.26, 8.43, 13.57, 12.52, and 12.43 mg/g gallic acid equivalent, respectively. The 5-h extract of T. crispa was evaluated against Culex quinquefasciatus mosquito larva in concentrations 3.125, 6.25, 12.5, and 25 mg/l, by determining the lethal concentration (LC) within 24 h and by histopathological analysis. The 24-h LC50 and LC90 values were 16.95 and 30.12 mg/l, respectively. The histopathological lesions after exposure to 50% of the 24-h LC50 were observed primarily in the midgut of the larva. The lesions observed were for the example epithelial cells lifting from the basement membrane, cell elongation protruding into the lumen, brush border disrupting with absent microvilli, and vesicle appearance. The present study indicated that the aqueous extract of this herb may have a suitable property for a larvicidal natural product and may replace harmful chemical pesticides.

Use of MALDI-TOF MS for the Identification of Chad Mosquitoes and the Origin of Their Blood Meal.

Matrix-assisted desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) is a clinical microbiology tool for the systematic identification of microorganisms. It has recently been presented as an innovative tool for the rapid and accurate identification of mosquitoes and their blood meal. To evaluate the capacity of this tool to identify mosquitoes collected in a tropical environment and preserved with silica gel, we analyzed 188 mosquitoes of different species collected in Chad, which were preserved with silica gel for 2 months. The MALDI-TOF MS analysis correctly identified 96% of the mosquitoes and 37.5% of their blood meals. Using MALDI-TOF MS and molecular biology, eight mosquito species were identified, including Anopheles gambiae s.l., Anopheles rufipes, Culex quinquefasciatus, Culex neavei, Culex pipiens, Culex perexiguus, Culex rima, and Culex watti. Blood meal identification revealed that mosquitoes fed mainly on humans, birds, and cows. Matrix-assisted desorption/ionization time-of-flight mass spectrometry appears to be a promising, fast, and reliable tool to identify mosquitoes and the origin of their blood meal for samples stored with silica gel.

Mosquito-larvicidal binary toxin receptor protein (Cqm1): crystallization and X-ray crystallographic analysis.

Cqm1 from Culex quinquefasciatus has been identified as the receptor for Lysinibacillus sphaericus binary toxin (BinAB). It is an amylomaltase that is presented on the epithelial membrane in the larval midgut through a glycosyl-phosphatidylinositol anchor. The active core of this protein (residues 23-560) was overexpressed in Escherichia coli, purified and successfully crystallized by the sitting-drop vapor-diffusion method using D-arabinose and CaCl2 as additives, as identified using high-throughput differential scanning fluorimetry analysis. X-ray diffraction data were collected to a resolution of 2.8 Šusing a laboratory X-ray source. The crystals had the symmetry of space group P212121, with unit-cell parameters a = 191.3, b = 205.3, c = 59.0 Šand with four monomers in the asymmetric unit. Structure refinement is in progress. This is the first structure report for a binary toxin receptor and for a member of the GH13_17 subfamily in the CAZy database.

Evaluation of resting traps to examine the behaviour and ecology of mosquito vectors in an area of rapidly changing land use in Sabah, Malaysian Borneo.

BACKGROUND: Widespread deforestation occurring in the tropics is hypothesized to impact the transmission of vector-borne diseases (VBD). Predicting how environmental changes will impact VBD transmission is dependent on understanding the ecology and behaviour of potential vector species outside of domestic settings. However there are few reliable sampling tools for measuring the habitat preference and host choice of mosquito vectors; with almost none suitable for sampling recently blood-fed, resting mosquitoes. This study evaluated the use of two mosquito traps: the resting bucket (RB) and sticky resting bucket (SRB) traps relative to CDC backpack aspiration (CDC) for sampling mosquitoes resting in a range of habitats representing a gradient of deforestation. Eight habitats were selected for sampling around two villages in Kudat District, Malaysian Borneo, to reflect the range of habitats available to mosquitoes in and around human dwellings, and nearby forest habitats where reservoir hosts are present: secondary forest (edge, interior and canopy); plantations (palm and rubber); and human settlements (inside, under and around houses). RESULTS: Over 31 days, 2243 mosquitoes were collected in 5748 discrete collections. Nine mosquito genera were sampled with Aedes and Culex species being present in all habitats and most abundant. RB and CDC backpack aspiration were most efficient for sampling Culex whereas CDC backpack aspiration and SRB were most efficient for Aedes. Most Aedes identified to species level were Ae. albopictus (91%), with their abundance being highest in forest edge habitats. In contrast, Culex were most abundant under houses. Most blood-fed mosquitoes (76%) were found in human settlements; with humans and chickens being the only blood source. CONCLUSIONS: RB and SRB traps proved capable of sampling mosquitoes resting in all sampled habitats. However, sampling efficiency was generally low (c.0.1 per trap per day), necessitating traps to be deployed in high numbers for mosquito detection. None of the traps were effective for sampling zoonotic malaria vectors; however, SRB collected relatively higher numbers of the dengue vector Ae. albopictus. The higher abundance of mosquitoes in forest edge habitats indicates the potential value of these traps for investigating sylvatic dengue transmission. This study has demonstrated the merits in application of simple resting traps for characterising mosquito vector resting behaviour outside of the home.

Conglomeration of novel Culex quinquefasciatus salivary proteins to contrive multi-epitope subunit vaccine against infections caused by blood imbibing transmitter.

The southern house vector, Culex quinquefasciatus is the paramount cause of Japanese encephalitis, West Nile fever and Lymphatic Filariasis, which is globally affecting the worldwide population. Many attempts were made by researchers with different perceptions to discover regimen against these aforementioned ailments but the output was not that effectual. Consequently, there is an imminent need to develop very effective and potential treatment against these perilous diseases. Employing immunoinformatic approaches, we have designed the multi-epitope subunit vaccine by exploring salivary proteins of Culex quinquefasciatus, which possess both antigenic and potent immunogenic behaviour. The immunogenic epitopes from the immune cells (B-cell, CTL, and HTL) were predicted and linked together with the help of linkers. Apart from this, at the N-terminal of the construct, an adjuvant was added in order to enhance the immunogenicity of the vaccine. The physiological parameters, antigenicity and allergenicity were also evaluated for the designed vaccine construct. Molecular docking between ligand (vaccine construct) and TLR-4 receptor was performed. Molecular dynamics simulation of the docked complex was performed to identify the stability, patterns, macromolecules interactions and their behaviour. Finally, to ensure the translation and gene expression efficiency of designed construct, insilico restriction cloning was executed into suitable expression vector pET28a.

Rapid identification of medically important mosquitoes by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

BACKGROUND: Accurate and rapid identification of dipteran vectors is integral for entomological surveys and is a vital component of control programs for mosquito-borne diseases. Conventionally, morphological features are used for mosquito identification, which suffer from biological and geographical variations and lack of standardization. We used matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) for protein profiling of mosquito species from North India with the aim of creating a MALDI-TOF MS database and evaluating it. METHODS: Mosquito larvae were collected from different rural and urban areas and reared to adult stages. The adult mosquitoes of four medically important genera, Anopheles, Aedes, Culex and Armigerus, were morphologically identified to the species level and confirmed by ITS2-specific PCR sequencing. The cephalothoraces of the adult specimens were subjected to MALDI-TOF analysis and the signature peak spectra were selected for creation of database, which was then evaluated to identify 60 blinded mosquito specimens. RESULTS: Reproducible MALDI-TOF MS spectra spanning over 2-14 kDa m/z range were produced for nine mosquito species: Anopheles (An. stephensi, An. culicifacies and An. annularis); Aedes (Ae. aegypti and Ae. albopictus); Culex (Cx. quinquefasciatus, Cx. vishnui and Cx. tritaenorhynchus); and Armigerus (Ar. subalbatus). Genus- and species-specific peaks were identified to create the database and a score of > 1.8 was used to denote reliable identification. The average numbers of peaks obtained were 55-60 for Anopheles, 80-100 for Aedes, 30-60 for Culex and 45-50 peaks for Armigeres species. Of the 60 coded samples, 58 (96.67%) were correctly identified by MALDI-TOF MS with a score > 1.8, while there were two unreliable identifications (both Cx. quinquefasciatus with scores < 1.8). CONCLUSIONS: MALDI-TOF MS appears to be a pragmatic technique for accurate and rapid identification of mosquito species. The database needs to be expanded to include species from different geographical regions and also different life-cycle stages to fully harness the technique for entomological surveillance programs.

Using MALDI-TOF MS to identify mosquitoes collected in Mali and their blood meals.

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) has been recently described as an innovative and effective tool for identifying arthropods and mosquito blood meal sources. To test this approach in the context of an entomological survey in the field, mosquitoes were collected from five ecologically distinct areas of Mali. We successfully analysed the blood meals from 651 mosquito abdomens crushed on Whatman filter paper (WFPs) in the field using MALDI-TOF MS. The legs of 826 mosquitoes were then submitted for MALDI-TOF MS analysis in order to identify the different mosquito species. Eight mosquito species were identified, including Anopheles gambiae Giles, Anopheles coluzzii, Anopheles arabiensis, Culex quinquefasciatus, Culex neavei, Culex perexiguus, Aedes aegypti and Aedes fowleri in Mali. The field mosquitoes for which MALDI-TOF MS did not provide successful identification were not previously available in our database. These specimens were subsequently molecularly identified. The WFP blood meal sources found in this study were matched against human blood (n = 619), chicken blood (n = 9), cow blood (n = 9), donkey blood (n = 6), dog blood (n = 5) and sheep blood (n = 3). This study reinforces the fact that MALDI-TOF MS is a promising tool for entomological surveys.

Eco-friendly and cost-effective Ag nanocrystals fabricated using the leaf extract of Habenaria plantaginea: toxicity on six mosquito vectors and four non-target species.

Recently, the biofabrication of metal nanoparticles has gained wide interest owing to its inherent features such as swift, simplicity, eco-friendliness, and cheaper costs. Different green-reducing agents led to the production of nanoparticles with varying toxicity on insects. In the current study, silver nanoparticles (AgNPs) were successfully synthesized using Habenaria plantaginea leaf extract. Ag nanoparticles were studied by UV-Vis spectroscopy (UV-Vis), X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FTIR), atomic force microscopy (AFM), scanning electron microscopy (SEM) coupled with energy-dispersive spectroscopy (EDS), and transmission electron microscopy (TEM). H. plantaginea extract and AgNPs were tested for mosquito larvicidal activity on Anopheles stephensi, Aedes aegypti, Culex quinquefasciatus, An. subpictus, Ae. albopictus, and Cx. tritaeniorhynchus. LC50 values were 102.51, 111.99, 123.47, 123.96, 136.56, 149.42 µg/ml and 12.23, 13.38, 14.78, 14.37, 15.39, 16.89 µg/ml, respectively. Moreover, H. plantaginea aqueous extract and AgNPs were tested against the non-target species Anisops bouvieri, Diplonychus indicus, Poecilia reticulata, and Gambusia affinis obtaining LC50 values ranging from 831.82 to 36,212.67 µg/ml. Overall, this study showed the effectiveness of H. plantaginea-fabricated nanoparticles on a wide range of important mosquito vectors, highlighting their scarce toxicity on four natural enemies predating mosquito larvae and pupae.

Curzerene, trans-ß-elemenone, and γ-elemene as effective larvicides against Anopheles subpictus, Aedes albopictus, and Culex tritaeniorhynchus: toxicity on non-target aquatic predators.

A wide number of studies dealing with mosquito control focus on toxicity screenings of whole plant essential oils, while limited efforts shed light on main molecules responsible of toxicity, as well as their mechanisms of action on non-target organisms. In this study, GC-MS shed light on main essential oil components extracted from leaves of the Suriname cherry Eugenia uniflora, i.e., curzerene (35.7%), trans-ß-elemenone (11.5%), and γ-elemene (13.6%), testing them on Anopheles subpictus, Aedes albopictus, and Culex tritaeniorhynchus larvae. Non-target toxicity experiments were carried out on four species of aquatic larvivorous organisms, including fishes, backswimmers, and waterbugs. The essential oil from E. uniflora leaves tested on An. subpictus, Ae. Albopictus, and Cx. tritaeniorhynchus showed LC50 of 31.08, 33.50, and 36.35 µg/ml, respectively. Curzerene, trans-ß-elemenone, and γ-elemene were extremely toxic to An. subpictus (LC50 = 4.14, 6.13, and 10.53 µg/ml), Ae. albopictus (LC50 = 4.57, 6.74, and 11.29 µg/ml), and Cx. tritaeniorhynchus (LC50 = 5.01, 7.32, and 12.18 µg/ml). The essential oil from E. uniflora leaves, curzerene, trans-ß-elemenone, and γ-elemene showed low toxicity to larvivorous fishes, backswimmers, and waterbugs, with LC50 ranging from 303.77 to 6765.56 µg/ml. Predator safety factor (PSF) ranged from 55.72 to 273.45. Overall, we believe that curzerene isolated from the essential oil from E. uniflora leaves can represent an ideal molecule to formulate novel mosquito larvicides, due to its extremely low LC50 on all tested mosquito vectors (4.14-5.01 µg/ml), which far encompasses most of the botanical pesticides tested till now. Notably, the above-mentioned LC50 did not damage the four aquatic predators tested in this study.

Cry11Aa Interacts with the ATP-Binding Protein from Culex quinquefasciatus To Improve the Toxicity.

Cry11Aa displays high toxicity to the larvae of several mosquito species, including Aedes, Culex, and Anopheles. To study its binding characterization against Culex quinquefasciatus, Cry11Aa was purified and western blot results showed that Cry11Aa could bind successfully to the brush border membrane vesicles. To identify Cry11Aa-binding proteins in C. quinquefasciatus, a biotin-based protein pull-down experiment was performed and seven Cry11Aa-binding proteins were isolated from the midgut of C. quinquefasciatus larvae. Analysis of liquid chromatography-tandem mass spectrometry showed that one of the Cry11Aa-binding proteins is the ATP-binding domain 1 family member B. To investigate its binding property and effect on the toxicity of Cry11Aa, western blot, far-western blot, enzyme-linked immunosorbent assay, and bioassays of Cry11Aa in the presence and absence of the recombinant ATP-binding protein were performed. Our results showed that the ATP-binding protein interacted with Cry11Aa and increased the toxicity of Cry11Aa against C. quinquefasciatus. Our study suggests that midgut proteins other than the toxin receptors may modulate the toxicity of Cry toxins against mosquitoes.

Pharmacological potential of Bidens pilosa L. and determination of bioactive compounds using UHPLC-QqQLIT-MS/MS and GC/MS.

BACKGROUND: Research of natural products from traditionally used medicinal plants to fight against the human ailments is fetching attention of researchers worldwide. Bidens pilosa Linn. var. Radiata (Asteraceae) is well known for its folkloric medicinal use against various diseases from many decades. Mizoram, North East India, has high plant diversity and the use of this plant as herbal medicine is deep rooted in the local tribes. The present study was executed to understand the pharmacological potential of B. pilosa leaves extract. METHODS: The antimicrobial potential was determined using agar well diffusion and broth microdilution method against bacterial and yeast pathogens. Cytotoxicity was evaluated using MTT and apoptotic DNA fragmentation assays. Further, the antioxidant ability of the extract was analysed using DPPH and ABTS free radical scavenging assay. Mosquitocidal activity was evaluated against third in-star larvae of C. quinquefasciatus using dose response and time response larvicidal bioassay. Additionally, the major phenolic and volatile compounds were determined using UHPLC-QqQLIT-MS/MS and GC/MS respectively. RESULTS: We found that the extract showed highest antimicrobial activity against E. coli (MIC 80 µg/mL and IC50 110.04 µg/mL) and showed significant cytotoxicity against human epidermoid carcinoma (KB-3-1) cells with IC50 values of 99.56 µg/mL among the tested cancer cell lines. The IC50 values for scavenging DPPH and ABTS was 80.45 µg/mL and 171.6 µg/mL respectively. The extract also showed the high phenolics (72 µg GAE/mg extract) and flavonoids (123.3 µg Quercetin /mg extract). Lastly, five bioactive and six volatile compounds were detected using UHPLC-QqQLIT-MS/MS and GC-MS respectively which may be responsible for the plant's bioactivities. An anticancerous compound, Paclitaxel was detected and quantified for the first time from B. pilosa leaves extract, which further showed the anticancerous potential of the tested extract. CONCLUSION: On the basis of the present investigation, we propose that the leaf extract of B. pilosa might be a good candidate for the search of efficient environment friendly natural bioactive agent and pharmaceutically important compounds.

mosGCTL-7, a C-Type Lectin Protein, Mediates Japanese Encephalitis Virus Infection in Mosquitoes.

Japanese encephalitis virus (JEV) is an arthropod-borne flavivirus prevalent in Asia and the Western Pacific and is the leading cause of viral encephalitis. JEV is maintained in a transmission cycle between mosquitoes and vertebrate hosts, but the molecular mechanisms by which the mosquito vector participates in transmission are unclear. We investigated the expression of all C-type lectins during JEV infection in Aedes aegypti The C-type lectin mosquito galactose-specific C-type lectin 7 (mosGCTL-7) (VectorBase accession no. AAEL002524) was significantly upregulated by JEV infection and facilitated infection in vivo and in vitro mosGCTL-7 bound to the N-glycan at N154 on the JEV envelope protein. This recognition of viral N-glycan by mosGCTL-7 is required for JEV infection, and we found that this interaction was Ca2+ dependent. After mosGCTL-7 bound to the glycan, mosPTP-1 bound to mosGCTL-7, promoting JEV entry. The viral burden in vivo and in vitro was significantly decreased by mosPTP-1 double-stranded RNA (dsRNA) treatment, and infection was abolished by anti-mosGCTL-7 antibodies. Our results indicate that the mosGCTL-7/mosPTP-1 pathway plays a key role in JEV infection in mosquitoes. An improved understanding of the mechanisms underlying flavivirus infection in mosquitoes will provide further opportunities for developing new strategies to control viral dissemination in nature.IMPORTANCE Japanese encephalitis virus is a mosquito-borne flavivirus and is the primary cause of viral encephalitis in the Asia-Pacific region. Twenty-four countries in the WHO Southeast Asia and Western Pacific regions have endemic JEV transmission, which exposes >3 billion people to the risks of infection, although JEV primarily affects children. C-type lectins are host factors that play a role in flavivirus infection in humans, swine, and other mammals. In this study, we investigated C-type lectin functions in JEV-infected Aedes aegypti and Culex pipiens pallens mosquitoes and cultured cells. JEV infection changed the expression of almost all C-type lectins in vivo and in vitro, and mosGCTL-7 bound to the JEV envelope protein via an N-glycan at N154. Cell surface mosPTP-1 interacted with the mosGCTL-7-JEV complex to facilitate virus infection in vivo and in vitro Our findings provide further opportunities for developing new strategies to control arbovirus dissemination in nature.

A novel anticoagulant protein with antithrombotic properties from the mosquito Culex pipiens pallens.

A newly discovered protein, named 'CPP protein', which possesses antithrombotic and anticoagulant properties, was purified from the salivary gland of the mosquito Culex pipiens pallens. CPP protein was found to have an estimated molecular mass of 21.7kDa, and to be active at 60°C, and pH 7.0. The anticoagulation activity of CPP protein was strongly inhibited by calcium ions. CPP protein inhibited fibrin clot formation and platelet activation, and it degraded blood clots. It also inhibited the enzymatic activities of activated factor X and thrombin. In addition, CPP protein prolonged the activated partial thromboplastin time and prothrombin time. CPP protein demonstrated antithrombotic effect in two mouse models, a thrombin-induced acute thromboembolism model, and an ex vivo coagulation model. CPP protein at a dose of 20mg/kg was devoid of hemorrhagic activity. These results suggest that CPP protein could have potential as a therapeutic agent for thrombosis, due to its antithrombotic properties and lack of hemorrhagic activity.

The mitochondrial genomes of Culex tritaeniorhynchus and Culex pipiens pallens (Diptera: Culicidae) and comparison analysis with two other Culex species.

BACKGROUND: Culex tritaeniorhynchus and Culex pipiens pallens are the major vectors of the Japanese encephalitis virus and Wuchereria bancrofti, the causative agent of filariasis. The knowledge of mitochondrial genomes has been widely useful for the studies on molecular evolution, phylogenetics and population genetics. METHODS: In this study, we sequenced and annotated the mitochondrial (mt) genomes of Cx. tritaeniorhynchus and Cx. p. pallens, and performed a comparative analysis including four known mt genomes of species of the subgenus Culex (Culex). The phylogenetic relationships of Cx. tritaeniorhynchus, Cx. p. pallens and four known Culex mt genome sequences were reconstructed by maximum likelihood based on concatenated protein-coding gene sequences. RESULTS: Culex tritaeniorhynchus and Cx. p. pallens mt genomes are 14,844 bp and 15,617 bp long, both consists of 13 PCGs, 22 tRNAs, 2 rRNAs and 1 CR (not sequenced for Cx. tritaeniorhynchus). The initiation and termination codons of PCGs are ATN and TAA, respectively, except for COI starting with TCG, and COI and COII terminated with T. tRNAs have the typical clover-leaf secondary structures except for trnS ((AGN)) that is lacking the DHU stem. 16S rRNA and 12S rRNA secondary structures were drawn for the first time for mosquito mt genomes. The control region of Cx. p. pallens mt genome is 747 bp long and with four tandem repeat structures. Phylogenetic analyses demonstrated that the mt genome of Cx. tritaeniorhynchus was significantly separated from the remaining five mt genomes of Culex spp. Culex p. pipiens, Cx. p. pallens and Cx. p. quinquefasciatus formed a monophyletic clade with Cx. p. quinquefasciatus linked in the middle of the clade, and Cx. p. pallens should have the same taxonomic level as Culex p. pipiens and Cx. p. quinquefasciatus. CONCLUSIONS: The mt genomes of Cx. tritaeniorhynchus and Cx. p. pallens share the same gene composition and order with those of two other Culex species. Culex p. pallens of the Pipiens complex should have the same taxonomic level as Culex p. pipiens and Cx. p. quinquefasciatus investigated. We enriched the Culex mt genome data and provided a reference basis for further Culex mt genome sequencing and analyses.

Interaction of Lysinibacillus sphaericus Cry48Aa/Cry49Aa toxin with midgut brush-border membrane fractions from Culex quinquefasciatus larvae.

The Cry48Aa/Cry49Aa mosquitocidal toxin from Lysinibacillus sphaericus was uniquely composed of a three-domain (Cry) toxin and binary (Bin) toxin-like protein, with high toxicity against Culex spp. However, its mode of action against the target mosquitoes is still unknown. In this study, Cry48Aa, Cry49Aa and its N- and C-terminal truncated proteins were expressed and purified, and the binding affinities of the purified proteins with midgut brush-border membrane fractions (BBMFs) from Culex quin-quefasciatus larvae were performed. The results showed that both Cry48Aa and Cry49Aa have specific and high binding affinity to BBMFs, with dissociation constants of 9.5 ± 1.8 and 25.4 ± 3.8 nM, respectively. Competition assays demonstrated that Cry49Aa C-terminal derivatives were able to bind to the BBMFs, whereas Far-Western dot blot analysis revealed that its N-terminal constructs interacted with Cry48Aa. Nevertheless, larvicidal activity was almost lost when Cry49Aa truncated proteins, either individually or in pairs, combined with Cry48Aa. It is concluded that Cry49Aa is responsible for receptor binding and interaction with Cry48Aa and plays an important role in the mechanism of action of these two-component toxins.

Physical features and chitin content of eggs from the mosquito vectors Aedes aegypti, Anopheles aquasalis and Culex quinquefasciatus: Connection with distinct levels of resistance to desiccation.

Mosquito eggs are laid in water but freshly laid eggs are susceptible to dehydration, if their surroundings dry out at the first hours of development. During embryogenesis of different mosquito vectors the serosal cuticle, an extracellular matrix, is produced; it wraps the whole embryo and becomes part of the eggshell. This cuticle is an essential component of the egg resistance to desiccation (ERD). However, ERD is variable among species, sustaining egg viability for different periods of time. While Aedes aegypti eggs can survive for months in a dry environment (high ERD), those of Anopheles aquasalis and Culex quinquefasciatus in the same condition last, respectively, for one day (medium ERD) or a few hours (low ERD). Resistance to desiccation is determined by the rate of water loss, dehydration tolerance and total amount of water of a given organism. The ERD variability observed among mosquitoes probably derives from diverse traits. We quantified several attributes of whole eggs, potentially correlated with the rate of water loss: length, width, area, volume, area/volume ratio and weight. In addition, some eggshell aspects were also evaluated, such as absolute and relative weight, weight/area relationship (herein called surface density) and chitin content. Presence of chitin specifically in the serosal cuticle as well as aspects of endochorion external surface were also investigated. Three features could be related to differences on ERD levels: chitin content, directly related to ERD, the increase in the egg volume during embryogenesis and the eggshell surface density, which were both inversely related to ERD. Although data suggest that the amount of chitin in the eggshell is relevant for egg impermeability, the participation of other yet unidentified eggshell attributes must be considered in order to account for the differences in the ERD levels observed among Ae. aegypti, An. aquasalis and Cx. quinquefasciatus.