Salivary factor LTRIN from Aedes aegypti facilitates the transmission of Zika virus by interfering with the lymphotoxin-ß receptor.

Pathogens have co-evolved with mosquitoes to optimize transmission to hosts. Mosquito salivary-gland extract is known to modulate host immune responses and facilitate pathogen transmission, but the underlying molecular mechanisms of this have remained unknown. In this study, we identified and characterized a prominent 15-kilodalton protein, LTRIN, obtained from the salivary glands of the mosquito Aedes aegypti. LTRIN expression was upregulated in blood-fed mosquitoes, and LTRIN facilitated the transmission of Zika virus (ZIKV) and exacerbated its pathogenicity by interfering with signaling through the lymphotoxin-ß receptor (LTßR). Mechanically, LTRIN bound to LTßR and 'preferentially' inhibited signaling via the transcription factor NF-κB and the production of inflammatory cytokines by interfering with the dimerization of LTßR during infection with ZIKV. Furthermore, treatment with antibody to LTRIN inhibited mosquito-mediated infection with ZIKV, and abolishing LTßR potentiated the infectivity of ZIKV both in vitro and in vivo. This study provides deeper insight into the transmission of mosquito-borne diseases in nature and supports the therapeutic potential of inhibiting the action of LTRIN to disrupt ZIKV transmission.

Modulation of acyl-carnitines, the broad mechanism behind Wolbachia-mediated inhibition of medically important flaviviruses in Aedes aegypti.

Wolbachia-infected mosquitoes are refractory to flavivirus infections, but the role of lipids in Wolbachia-mediated virus blocking remains to be elucidated. Here, we use liquid chromatography mass spectrometry to provide a comprehensive picture of the lipidome of Aedes aegypti (Aag2) cells infected with Wolbachia only, either dengue or Zika virus only, and Wolbachia-infected Aag2 cells superinfected with either dengue or Zika virus. This approach identifies a class of lipids, acyl-carnitines, as being down-regulated during Wolbachia infection. Furthermore, treatment with an acyl-carnitine inhibitor assigns a crucial role for acyl-carnitines in the replication of dengue and Zika viruses. In contrast, depletion of acyl-carnitines increases Wolbachia density while addition of commercially available acyl-carnitines impairs Wolbachia production. Finally, we show an increase in flavivirus infection of Wolbachia-infected cells with the addition of acyl-carnitines. This study uncovers a previously unknown role for acyl-carnitines in this tripartite interaction that suggests an important and broad mechanism that underpins Wolbachia-mediated pathogen blocking.

Molecular characteristics of odorant-binding protein 1 in Anopheles maculipennis.

BACKGROUND: Anopheles maculipennis complex, the historic vector of malaria, causes serious medical problems worldwide and exhibits different behaviours. Studying the odorant-binding proteins (OBPs), which influence the chemosensory system and behavioural responses, is essential to understanding the population structure and developing effective control measures against this vector. The present study was designed to identify and analyse the obp1 gene in An. maculipennis. METHODS: Adults of An. maculipennis sensu stricto were collected in Zanjan Province, northwest of Iran, and gDNAs of female mosquitoes were extracted. Fragments of An. maculipennis obp1 (Amacobp1) gene were amplified using degenerate and specific primers, and some of amplicons were selected for sequencing. RESULTS: Analysis of amplified products identified that the sequence of Amacobp1 gene was 1341 bp long. This gene contains three exons (5', internal, and 3'of 160, 256, and 18 bp, respectively) and encodes 144 amino acids. The sizes of introns I and II in deduced gene are 268 and 358 nucleotides, respectively. The amino acid sequence in the C-terminal of AmacOBP1 is similar to that of major malaria vector Anopheles species. However, its N-terminal has a specific signal peptide with 19 amino acids. This peptide is conserved in different studied populations, and its sequence of amino acids shows the most variation among anopheline species. CONCLUSIONS: Degenerate primers in this study are suggested for studying obp1 gene in Anopheles species. Amacobp1 gene is proposed as a molecular marker for the detection of intraspecific ecotypes and diagnosis of different species within Maculipennis Group. Moreover, the N-terminal of AmacOBP1 peptide is recommended as a molecular marker to identify the Amacobp1 expression patterns in different chemosensory organs for assessing the molecular mechanisms and developing novel behavioural disturbance agents to control An. maculipennis.

Identification of mixed and successive blood meals of mosquitoes using MALDI-TOF MS protein profiling.

BACKGROUND: The accurate and rapid identification of mosquito blood meals is critical to study the interactions between vectors and vertebrate hosts and, subsequently, to develop vector control strategies. Recently, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) profiling has been shown to be a reliable and effective tool for identifying single blood meals from mosquitoes. METHODS: In this study, we developed MALDI-TOF MS profiling protocols to identify Anopheles gambiae Giles, Anopheles coluzzii and Aedes albopictus mosquitoes' mixed blood meals and the last of successive blood meals. The mosquitoes were either successively artificially fed with distinct host bloods or engorged with mixed bloods from distinct vertebrate hosts, such as humans, sheep and dogs. RESULTS: Blind test analyses revealed a correct identification of mixed blood meals from mosquitoes using MALDI-TOF MS profiling. The 353 MS spectra from mixed blood meals were identified using log score values >1.8. All MS spectra (n = 244) obtained from mosquitoes' successive blood meals were reproducible and specific to the last blood meal, suggesting that the previous blood meals do not have an impact on the identification of the last one. CONCLUSION: MALDI-TOF MS profiling approach appears to be an effective and robust technique to identify the last and mixed blood meals during medical entomological surveys.

Mutant cycle analysis identifies a ligand interaction site in an odorant receptor of the malaria vector Anopheles gambiae.

Lack of information about the structure of insect odorant receptors (ORs) hinders the development of more effective repellants to control disease-transmitting insects. Mutagenesis and functional analyses using agonists to map the odorant-binding sites of these receptors have been limited because mutations distant from an agonist-binding site can alter agonist sensitivity. Here we use mutant cycle analysis, an approach for exploring the energetics of protein-protein or protein-ligand interactions, with inhibitors, to identify a component of the odorant-binding site of an OR from the malaria vector, Anopheles gambiae The closely related odorant-specificity subunits Agam/Or15 and Agam/Or13 were each co-expressed with Agam/Orco (odorant receptor co-receptor subunit) in Xenopus oocytes and assayed by two-electrode voltage clamp electrophysiology. We identified (-)-fenchone as a competitive inhibitor with different potencies at the two receptors and used this difference to screen a panel of 37 Agam/Or15 mutants, surveying all positions that differ between Agam/Or15 and Agam/Or13 in the transmembrane and extracellular regions, identifying position 195 as a determinant of (-)-fenchone sensitivity. Inhibition by (-)-fenchone and six structurally related inhibitors of Agam/Or15 receptors containing each of four different hydrophobic residues at position 195 served as input data for mutant cycle analysis. Several mutant cycles, calculated from the inhibition of two receptors by each of two ligands, yielded coupling energies of ≥1 kcal/mol, indicating a close, physical interaction between the ligand and residue 195 of Agam/Or15. This approach should be useful in further expanding our knowledge of odorant-binding site structures in ORs of disease vector insects.

Multiple Salivary Proteins from Aedes aegypti Mosquito Bind to the Zika Virus Envelope Protein.

Aedes aegypti mosquitoes are important vectors of several debilitating and deadly arthropod-borne (arbo) viruses, including Yellow Fever virus, Dengue virus, West Nile virus and Zika virus (ZIKV). Arbovirus transmission occurs when an infected mosquito probes the host's skin in search of a blood meal. Salivary proteins from mosquitoes help to acquire blood and have also been shown to enhance pathogen transmission in vivo and in vitro. Here, we evaluated the interaction of mosquito salivary proteins with ZIKV by surface plasmon resonance and enzyme-linked immunosorbent assay. We found that three salivary proteins AAEL000793, AAEL007420, and AAEL006347 bind to the envelope protein of ZIKV with nanomolar affinities. Similar results were obtained using virus-like particles in binding assays. These interactions have no effect on viral replication in cultured endothelial cells and keratinocytes. Additionally, we found detectable antibody levels in ZIKV and DENV serum samples against the recombinant proteins that interact with ZIKV. These results highlight complex interactions between viruses, salivary proteins and antibodies that could be present during viral transmissions.

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.

Mosquito vector proteins homologous to α1-3 galactosyl transferases of tick vectors in the context of protective immunity against malaria and hypersensitivity to vector bites.

BACKGROUND: An epitope, Galα1-3Galß1-4GlcNAc-R, termed α-gal, is present in glycoconjugates of New World monkeys (platyrrhines) and other mammals but not in hominoids and Old World monkeys (catarrhines). The difference is due to the inactivation of α1-3 galactosyl transferase (α1-3 GT) genes in catarrhines. Natural antibodies to α-gal are therefore developed in catarrhines but not platyrrhines and other mammals. Hypersensitivity reactions are commonly elicited by mosquito and tick vector bites. IgE antibodies against α-gal cause food allergy to red meat in persons who have been exposed to tick bites. Three enzymes synthesising the terminal α1-3-linked galactose in α-gal, that are homologous to mammalian α and ß1-4 GTs but not mammalian α1-3 GTs, were recently identified in the tick vector Ixodes scapularis. IgG and IgM antibodies to α-gal are reported to protect against malaria because mosquito-derived sporozoites of malaria parasites express α-gal on their surface. This article explores the possibility that the α-gal in sporozoites are acquired from glycoconjugates synthesised by mosquitoes rather than through de novo synthesis by sporozoites. METHODS: The presence of proteins homologous to the three identified tick α1-3 GTs and mammalian α1-3 GTs in two important mosquito vectors, Aedes aegypti and Anopheles gambiae, as well as Plasmodium malaria parasites, was investigated by BLASTp analysis to help clarify the source of the α-gal on sporozoite surfaces. RESULTS: Anopheles gambiae and Ae. aegypti possessed several different proteins homologous to the three I. scapularis proteins with α1-3 GT activity, but not mammalian α1-3 GTs. The putative mosquito α1-3 GTs possessed conserved protein domains characteristic of glycosyl transferases. However, the genus Plasmodium lacked proteins homologous to the three I. scapularis proteins with α1-3 GT activity and mammalian α1-3 GTs. CONCLUSIONS: The putative α1-3 GTs identified in the two mosquito vectors may synthesise glycoconjugates containing α-gal that can be transferred to sporozoite surfaces before they are inoculated into skin during blood feeding. The findings merit further investigation because of their implications for immunity against malaria, hypersensitivity to mosquito bites, primate evolution, and proposals for immunisation against α-gal.

Classification of Mosquitoes with Infrared Spectroscopy and Partial Least Squares-Discriminant Analysis.

Mosquito-borne diseases are responsible for considerable morbidity and mortality globally. Given the absence of effective vaccines for most arthropod-borne viruses, mosquito control efforts remain the dominant method of disease prevention. Ideal control efforts begin with entomologic surveillance in order to determine the abundance, identity, and infection status of pathogen-vectoring mosquito populations. Traditionally, much of the surveillance work involves morphological species identification by trained entomologists. Limited operational funding and lack of specialized training is a known barrier to surveillance and effective control efforts for many operational mosquito control personnel. Therefore, there is a need for surveillance workflow improvements and rapid mosquito identification methods. Herein, is presented a proof of concept study in which infrared spectroscopy coupled with partial least squares-discriminant analysis was explored as a means of automatically classifying mosquitoes at the species level. The developed method resulted in greater than 94% accuracy for four mosquitoes of public health relevance: Aedes aegypti, Aedes albopictus, Aedes japonicus, and Aedes triseriatus.

Ability of near-infrared spectroscopy and chemometrics to predict the age of mosquitoes reared under different conditions.

BACKGROUND: Practical, field-ready age-grading tools for mosquito vectors of disease are urgently needed because of the impact that daily survival has on vectorial capacity. Previous studies have shown that near-infrared spectroscopy (NIRS), in combination with chemometrics and predictive modeling, can forecast the age of laboratory-reared mosquitoes with moderate to high accuracy. It remains unclear whether the technique has utility for identifying shifts in the age structure of wild-caught mosquitoes. Here we investigate whether models derived from the laboratory strain of mosquitoes can be used to predict the age of mosquitoes grown from pupae collected in the field. METHODS: NIRS data from adult female Aedes albopictus mosquitoes reared in the laboratory (2, 5, 8, 12 and 15 days-old) were analysed against spectra from mosquitoes emerging from wild-caught pupae (1, 7 and 14 days-old). Different partial least squares (PLS) regression methods trained on spectra from laboratory mosquitoes were evaluated on their ability to predict the age of mosquitoes from more natural environments. RESULTS: Models trained on spectra from laboratory-reared material were able to predict the age of other laboratory-reared mosquitoes with moderate accuracy and successfully differentiated all day 2 and 15 mosquitoes. Models derived with laboratory mosquitoes could not differentiate between field-derived age groups, with age predictions relatively indistinguishable for day 1-14. Pre-processing of spectral data and improving the PLS regression framework to avoid overfitting can increase accuracy, but predictions of mosquitoes reared in different environments remained poor. Principal components analysis confirms substantial spectral variations between laboratory and field-derived mosquitoes despite both originating from the same island population. CONCLUSIONS: Models trained on laboratory mosquitoes were able to predict ages of laboratory mosquitoes with good sensitivity and specificity though they were unable to predict age of field-derived mosquitoes. This study suggests that laboratory-reared mosquitoes do not capture enough environmental variation to accurately predict the age of the same species reared under different conditions. Further research is needed to explore alternative pre-processing methods and machine learning techniques, and to understand factors that affect absorbance in mosquitoes before field application using NIRS.

Protocols for Testing the Toxicity of Novel Insecticidal Chemistries to Mosquitoes.

New classes of insecticides with novel modes of action are needed to control insecticide resistant populations of mosquitoes that transmit diseases such as Zika, dengue and malaria. Assays for rapid, high-throughput analyses of unformulated novel chemistries against mosquito larvae and adults are presented. We describe protocols for single point-dose and dose response assays to evaluate the toxicity of small molecule chemistries to the Aedes aegypti vector of Zika, dengue and yellow fever, the malaria vector, Anopheles gambiae and the northern house mosquito, Culex quinquefasciatus, on contact and via ingestion. As an example, we evaluated the toxicity of amitriptyline, a small molecule antagonist of G protein-coupled receptors, via larval, adult topical and adult blood-feeding assay. The protocols provide a starting point to investigate insecticide potential. Results are discussed in the context of additional experiments to explore product applications and mechanisms for delivery.

Odorant ligands for the CO2 receptor in two Anopheles vectors of malaria.

Exhaled CO2 is an important host-seeking cue for Anopheles mosquitoes, which is detected by a highly conserved heteromeric receptor consisting of three 7-transmembrane proteins Gr22, Gr23, and Gr24. The CO2 receptor neuron has been shown to also respond sensitively to a variety of odorants in Aedes aegypti. The detection of CO2 is important for upwind navigation and for enhancing the attraction to body heat as well as to skin odorants. The orthologs of the CO2 receptor proteins are present in malaria-transmitting mosquitoes like Anopheles coluzzii and Anopheles sinensis. Activators and inhibitors of the CO2-neuron were tested on the maxillary palps in these two species by single-sensillum electrophysiology. The electrophysiological testing of three prolonged-activator odorants identified originally in Aedes aegypti also showed varying ability to reduce the CO2-ellicited increase in spikes. These findings provide a foundation for comparing the functional conservation with the evolutionary conservation of an important class of odorant receptor. The identification of a suite of natural odorants that can be used to modify the CO2-detection pathway may also contribute to odor-blends that can alter the behavior of these disease transmitting mosquitoes.

Effects of La Crosse virus infection on the host-seeking behavior and levels of two neurotransmitters in Aedes triseriatus.

BACKGROUND: La Crosse virus (LACV) infection has been shown to manipulate the blood-feeding behaviors of its main vector, Aedes triseriatus. Here, we investigated the effects of virus infection on serotonin and dopamine and their potential roles in host-seeking. In mosquitoes, serotonin depletion has been shown to interfere with blood-feeding but not host-seeking. Dopamine depletion does not affect either blood-feeding or host-seeking; elevations of dopamine, however, has been shown to inhibit host-seeking. The purpose of this study was to determine the effects of LACV infection on the host-seeking behavior of and neurotransmitter levels in Ae. triseriatus. METHODS: Host-seeking behavior was evaluated using a uni-port olfactometer and a membrane feeder assay. Levels of serotonin and dopamine in infected and control mosquito heads were measured using HPLC-ED. RESULTS: Infection with LACV significantly inhibited the activation and attraction of Ae. triseriatus females to a host. A higher proportion of uninfected Ae. triseriatus females were activated by the presence of a host compared to infected mosquitoes and more uninfected mosquitoes were full responders (95.7%) compared to infected ones (91.1%). However, infection with LACV did not significantly affect the landing, probing, or blood-feeding rates of female mosquitoes. LACV-infected mosquitoes had lower serotonin levels than controls (104.5 vs 138.3 pg/head) while the dopamine levels were not affected by infection status (282.3 vs 237 pg/head). CONCLUSIONS: Our work suggests that virus-induced reduction of serotonin is related to previously reported blood-feeding alterations in LACV-infected mosquitoes and could lead to enhanced transmission and increased vectorial capacity. In addition, some aspects of host-seeking were inhibited by virus infection.

Monitoring the Age of Mosquito Populations Using Near-Infrared Spectroscopy.

Mosquito control with bednets, residual sprays or fumigation remains the most effective tool for preventing vector-borne diseases such as malaria, dengue and Zika, though there are no widely used entomological methods for directly assessing its efficacy. Mosquito age is the most informative metric for evaluating interventions that kill adult mosquitoes but there is no simple or reliable way of measuring it in the field. Near-Infrared Spectroscopy (NIRS) has been shown to be a promising, high-throughput method that can estimate the age of mosquitoes. Currently the ability of NIRS to measure mosquito age is biased, and has relatively high individual mosquito measurement error, though its capacity to rigorously monitor mosquito populations in the field has never been assessed. In this study, we use machine learning methods from the chemometric literature to generate more accurate, unbiased estimates of individual mosquito age. These unbiased estimates produce precise population-level measurements, which are relatively insensitive to further increases in NIRS accuracy when feasible numbers of mosquitoes are sampled. The utility of NIRS to directly measure the impact of pyrethroid resistance on mosquito control is illustrated, showing how the technology has potential as a highly valuable tool for directly assessing the efficacy of mosquito control interventions.

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.

Accurate identification of Anopheles gambiae Giles trophic preferences by MALDI-TOF MS.

The determination of the trophic preferences of the Anopheles gambiae Giles (Diptera: Culicidae) is a decisive parameter for the monitoring and the prevention of malaria risk transmission. Currently, arthropod blood feeding sources are identified using immunological or molecular biology traditional techniques. Despite the effectiveness of these methods, they present several limitations, and notably, they are time-consuming and costly techniques. A recent study demonstrated that MALDI-TOF MS could be a useful tool for the identification of blood meal origins in freshly engorged mosquitoes. However, the limited number of blood vertebrate species tested to date, did not allow an assessment of the efficiency of MALDI-TOF MS in distinguishing blood MS spectra among close host species, such as humans versus primates. Therefore, in the present study, blood from ten distinct vertebrate host species, including four domestic species, four wild species, and two primates, was selected to control the reliability of MALDI-TOF MS based identification. Host blood species-specific MS profiles, up to 24h post-feeding in engorged Anopheles abdomens, were confirmed. Blind tests underlined the high specificity of MS spectra for the recognition of each host species, preventing misidentification. Nevertheless, an accurate analysis of the results from MS spectra queried against the MS database revealed that the reliability of identification is directly linked to the comprehensiveness of the MS reference database. Finally, the rapidity, the low-cost reagents, the simplicity of data analysis, and the accuracy of the tool for blood origin determination, make this proteomic strategy a promising complementary method for the elucidation of host/vector interactions.

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.

High efficacy of (Z)-γ-bisabolene from the essential oil of Galinsoga parviflora (Asteraceae) as larvicide and oviposition deterrent against six mosquito vectors.

The eco-friendly management of mosquitoes with novel and effective larvicides and oviposition deterrents is a crucial challenge to prevent outbreaks of mosquito-borne diseases. However, most of the herbal formulations tested in these years showed LC50 values higher of 40 ppm, and significant oviposition deterrent activity only when tested at relatively higher doses (> 50 µg/ml). Herein, we studied the chemical composition of the Galinsoga parviflora essential oil (EO). This plant is an annual herb native to South America naturalized all over the world. We tested the EO larvicidal and oviposition deterrent action on 6 mosquito species. Totally 37 compounds were identified in the EO of G. parviflora by GC and GC-MS analyses. The major constituent was (Z)-γ-bisabolene (38.9%). The G. parviflora EO and (Z)-γ-bisabolene showed acute toxicity on An. stephensi (LC50 = 31.04 and 2.04 µg/ml, respectively), Ae. aegypti (LC50 = 34.22 and 2.26 µg/ml, respectively), Cx. quinquefasciatus (LC50 = 37.10 and 2.47 µg/ml, respectively), An. subpictus (LC50 = 40.97 and 4.09 µg/ml, respectively), Ae. albopictus (LC50 = 45.55 and 4.50 µg/ml, respectively) and Cx. tritaeniorhynchus (LC50 = 49.56 and 4.87 µg/ml, respectively) larvae. Furthermore, the oviposition deterrent potential of the G. parviflora EO and (Z)-γ-bisabolene was studied on six mosquito vectors, showing that 25 µg/ml of (Z)-γ-bisabolene led to an Oviposition Activity Index lower of - 0.79 in all tested mosquito vectors. Overall, all larvicidal LC50 values estimated for (Z)-γ-bisabolene were lower than 5 µg/ml. This result far encompasses current evidences of toxicity reported for the large majority of botanical products currently tested against mosquito young instars, allowing us to propose this compound as an highly effective mosquito larvicide and oviposition deterrent.

Fabrication of highly effective mosquito nanolarvicides using an Asian plant of ethno-pharmacological interest, Priyangu (Aglaia elaeagnoidea): toxicity on non-target mosquito natural enemies.

Mosquitoes threaten the lives of humans, livestock, pets and wildlife around the globe, due to their ability to vector devastating diseases. Aglaia elaeagnoidea, commonly known as Priyangu, is widely employed in Asian traditional medicine and pest control. Medicinal activities include anti-inflammatory, analgesic, anticancer, and anesthetic actions. Flavaglines, six cyclopenta[b]benzofurans, a cyclopenta[bc]benzopyran, a benzo[b]oxepine, and an aromatic butyrolactone showed antifungal properties, and aglaroxin A and rocaglamide were effective to control moth pests. Here, we determined the larvicidal action of A. elaeagnoidea leaf aqueous extract. Furthermore, we focused on Priyangu-mediated synthesis of Ag nanoparticles toxic to Culex quinquefasciatus, Aedes aegypti and Anopheles stephensi. The plant extract and the nanolarvicide were tested on three mosquito vectors, following the WHO protocol, as well as on three non-target mosquito predators. Priyangu-synthesized Ag nanoparticles were characterized by spectroscopic (UV, FTIR, XRD, and EDX) and microscopic (AFM, SEM, and TEM) analyses. Priyangu extract toxicity was moderate on Cx. quinquefasciatus (LC50 246.43; LC90 462.09 µg/mL), Ae. aegypti (LC50 229.79; LC90 442.71 µg/mL), and An. stephensi (LC50 207.06; LC90 408.46 µg/mL), respectively, while Priyangu-synthesized Ag nanoparticles were highly toxic to Cx. quinquefasciatus (LC50 24.91; LC90 45.96 µg/mL), Ae. aegypti (LC50 22.80; LC90 43.23 µg/mL), and An. stephensi (LC50 20.66; LC90 39.94 µg/mL), respectively. Priyangu extract and Ag nanoparticles were found safer to non-target larvivorous fishes, backswimmers, and waterbugs, with LC50 ranging from 1247 to 37,254.45 µg/mL, if compared to target pests. Overall, the current research represents a modern approach integrating traditional botanical pesticides and nanotechnology to the control of larval populations of mosquito vectors, with negligible toxicity against non-target including larvivorous fishes, backswimmers, and waterbugs.

Structure of the G119S Mutant Acetylcholinesterase of the Malaria Vector Anopheles gambiae Reveals Basis of Insecticide Resistance.

Malaria is a devastating disease in sub-Saharan Africa and is transmitted by the mosquito Anopheles gambiae. While indoor residual spraying of anticholinesterase insecticides has been useful in controlling the spread of malaria, widespread application of these compounds has led to the rise of an insecticide-resistant mosquito strain that harbors a G119S mutation in the nervous system target enzyme acetylcholinesterase. We demonstrate the atomic basis of insecticide resistance through structure determination of the G119S mutant acetylcholinesterase of An. gambiae in the ligand-free state and bound to a potent difluoromethyl ketone inhibitor. These structures reveal specific features within the active-site gorge distinct from human acetylcholinesterase, including an open channel at the base of the gorge, and provide a means for improving species selectivity in the rational design of improved insecticides for malaria vector control.