Transcriptomic analysis and molecular docking reveal genes involved in the response of Aedes aegypti larvae to an essential oil extracted from Eucalyptus.

BACKGROUND: Aedes aegypti (L.) is an urban mosquito, vector of several arboviruses that cause severe diseases in hundreds of million people each year. The resistance to synthetic insecticides developed by Ae. aegypti populations worldwide has contributed to failures in vector control campaigns, increasing the impact of arbovirus diseases. In this context, plant-derived essential oils with larvicidal activity could be an attractive alternative for vector control. However, the mode of action and the detoxificant response of mosquitoes to plant derived compounds have not been established, impairing the optimization of their use. METHODS AND FINDINGS: Here we compare gene expression in Ae. aegypti larvae after 14 hrs of exposure to Eucalyptus camaldulensis essential oil with a control group exposed to vehicle (acetone) for the same lapse, by using RNA-Seq. We found differentially expressed genes encoding for cuticle proteins, fatty-acid synthesis, membrane transporters and detoxificant related gene families (i.e. heat shock proteins, cytochromes P450, glutathione transferases, UDP-glycosyltransferases and ABC transporters). Finally, our RNA-Seq and molecular docking results provide evidence pointing to a central involvement of chemosensory proteins in the detoxificant response in mosquitoes. CONCLUSIONS AND SIGNIFICANCE: Our work contributes to the understanding of the physiological response of Ae. aegypti larvae to an intoxication with a natural toxic distilled from Eucalyptus leafs. The results suggest an involvement of most of the gene families associated to detoxification of xenobiotics in insects. Noteworthy, this work provides important information regarding the implication of chemosensory proteins in the detoxification of a natural larvicide. Understanding the mode of detoxification of Eucalyptus distilled compounds could contribute to their implementation as a tool in mosquito control.

Development of cellulose nanocrystal-stabilized Pickering emulsions of massoia and nutmeg essential oils for the control of Aedes albopictus.

We investigated the larvicidal potential of 10 plant essential oils (EOs) against the Asian tiger mosquito Aedes albopictus. Among the EOs, larvicidal activity against Ae. albopictus was strongest in those derived from massoia (Massoia aromatica) and nutmeg (Myristica fragrans). Larvicidal activities of massoia and nutmeg EOs against Ae. albopictus were 95.0% and 85.0% at 50 µg/mL, respectively. A total of 4 and 14 compounds were identified from massoia and nutmeg, respectively, and two massoia lactones, C10 and C12, were isolated from massoia EO. Among the identified compounds, benzyl salicylate, terpinolene, C12 massoia lactone, sabinene, benzyl benzoate, methyl eugenol, and C10 massoia lactone exhibited the strong larvicidal activity. Cellulose nanocrystal (CNC)-stabilized Pickering emulsions of massoia and nutmeg EOs were developed to overcome the insolubility of EOs in water. CNC/massoia and CNC/nutmeg emulsions were stable for at least 10 days, and larvicidal activities of CNC/massoia PE and CNC/nutmeg were higher than those of crude massoia and nutmeg EOs. This study presents a CNC-stabilized PE, a suitable formulation for EOs, as a potential larvicide against Ae. albopictus.

A natural agonist of mosquito TRPA1 from the medicinal plant Cinnamosma fragrans that is toxic, antifeedant, and repellent to the yellow fever mosquito Aedes aegypti.

Plants produce various secondary metabolites that offer a potential source of novel insecticides and repellents for the control of mosquito vectors. Plants of the genus Cinnamosma are endemic to, and widely-distributed throughout, the island of Madagascar. The barks of these species are commonly used in traditional medicines for treating a wide range of maladies. The therapeutic nature of the bark is thought to be associated with its enrichment of pungent drimane sesquiterpenes, which elicit antifeedant and toxic effects in some insects. Here we test the hypothesis that a bark extract of Cinnamosma fragrans (CINEX) and its major drimane sesquiterpenes are insecticidal, antifeedant, and repellent to Aedes aegypti, the principal mosquito vector of chikungunya, dengue, yellow fever, and Zika viruses. We demonstrate that CINEX is 1) toxic to larval and adult female mosquitoes, and 2) antifeedant and repellent to adult female mosquitoes. Moreover, we show that cinnamodial (CDIAL), a sesquiterpene dialdehyde isolated from CINEX, duplicates these bioactivities and exhibits similar toxic potency against pyrethroid-susceptible and -resistant strains of Ae. aegypti. Importantly, we show that CDIAL is an agonist of heterologously-expressed mosquito Transient Receptor Potential A1 (TRPA1) channels, and the antifeedant activity of CDIAL is dampened in a TRPA1-deficient strain of Ae. aegypti (TRPA1-/-). Intriguingly, TRPA1-/- mosquitoes do not exhibit toxic resistance to CDIAL. The data indicate that modulation of TRPA1 is required for the sensory detection and avoidance of CDIAL by mosquitoes, but not for inducing the molecule's toxicity. Our study suggests that CDIAL may serve as a novel chemical platform for the development of natural product-based insecticides and repellents for controlling mosquito vectors.

Positional stable isotope tracer analysis reveals carbon routes during ammonia metabolism of Aedes aegypti mosquitoes.

In Aedes aegypti females, the ammonia released during blood meal digestion is partially metabolized to facilitate the disposal of excess nitrogen. In this study, we used low- and high-resolution liquid chromatography-mass spectrometry (LC/MS) techniques to investigate the role of glucose during ammonia detoxification. Mosquitoes were fed a blood meal supplemented with [1,2-13C2]glucose, and downstream metabolites were measured for 24 h. Quantification of [13C] amino acids in the entire mosquito body was conducted without sample derivatization using selected reaction monitoring of mass transitions that are indicative of the structural position of [13C] atom incorporation. Identification of unlabeled and [13C] isotopologs of 43 compounds, including amino acids, amino acid derivatives, and organic acids, was performed by high-resolution LC/MS techniques. Blood-fed mosquitoes synthesized [13C] metabolites in mainly 2 carbon positions from [1,2-13C2]glucose. [13C2]Ala and [13C2]Pro were the most abundant and rapidly labeled amino acids synthesized. Additional [13C] amino acids, [13C] amino acid derivatives, and [13C] organic acids in 1 or 2 carbon positions were also identified. Two kinetic routes were proposed based on the incorporation of a [13C] atom at position 1 in specific amino acids. Our findings provide evidence that glucose is used for ammonia detoxification and [13C] uric acid synthesis through multiple metabolic pathways, uncovering a metabolic link at the carbon atomic level in ammonia metabolism of A. aegypti-Horvath, T. D., Dagan, S., Lorenzi, P. L., Hawke, D. H., Scaraffia, P. Y. Positional stable isotope tracer analysis reveals carbon routes during ammonia metabolism of Aedes aegypti mosquitoes.

Acetylcholinesterase inhibitory activity of stigmasterol & hexacosanol is responsible for larvicidal and repellent properties of Chromolaena odorata.

BACKGROUND: Chromolaena odorata, has been traditionally known for its insect repellent property. Aim of this study was to determine larvicidal tendency of C. odorata on Culex quinquefasciatus and isolate compounds responsible for this activity and to determine the mechanism of action of these compounds. METHODS: C. odorata plant extract was screened for mosquito larvicidal activity. The extract was fractionated using chromatography and the bioactive fraction showing larvicidal activity was identified. The chemical nature of the compounds in the bioactive fraction was determined using NMR and Mass spectrometry. RESULTS: We identified phytosterols and alkanols to be the compounds regulating larvicidal activity in the bioactive fraction of the plant extract. Stigmasterol and 1-hexacosanol were identified to be the chief orchestrators of larvicidal activity and their mode of action has been observed to be neurotoxicity. At a molecular level both stigmasterol and 1-hexacosanol were found to be inhibiting acetylcholinesterase activity in C. quinquefasciatus & A. aegypti. The acetylcholinesterase inhibitory effect was validated in vitro using recombinant acetylcholinesterase and ex vivo in larval homogenates of Culex and Aedes. Electrophysiological studies using electroantennography have shown enhanced neural response to these compounds. CONCLUSIONS: Neurotoxic effect of C. odorata derived stigmasterol and 1-hexacosanol, exerted through acetylcholinesterase inhibition was responsible for the mortality of C. quinquefasciatus, A. aegypti &Chironomus riparius. EAG studies pointed out hyper-excitability of the olfactory system by these compounds. GENERAL SIGNIFICANCE: These compounds are natural agents for mosquito control that can be used in vector control as larvicidal compounds, pending further investigations.

Plant essential oils affect the toxicities of carbaryl and permethrin against Aedes aegypti (Diptera: Culicidae).

ABSTRACT Phytochemicals have been considered as alternatives for conventional pesticides because of their low mammalian toxicity and environmental safety. They usually display less potent insecticidal effects than synthetic compounds, but may express as yet unknown modes of action. In the current study, we evaluated 14 plant essential oils for their toxicities and synergistic effects with carbaryl and permethrin against fourth instars of Aedes aegypti (L.) as well as 5-7-d-old adults. Six essential oils showed significant synergistic effects with carbaryl at 10-50 mg/liter, but paradoxically all of them decreased the toxicity of permethrin against Ae. aegypti larvae. None showed toxicity or synergistic effects on Ae. aegypti adults, at doses up to 2,000 ng/ insect. The six essential oils displaying synergistic effects in Ae. aegypti larvae inhibited the in vitro activities of cytochrome P450 monooxygenases and carboxylesterases in the low milligram per liter range. The data indicated that cytochrome P450 monooxygenases and carboxylesterase were probably targets for these natural synergists. Thus, the mechanism of synergism was most likely inhibition of metabolism and not interacting target site effects.

A cell-based screening platform identifies novel mosquitocidal toxins.

Pesticides currently in widespread use often lack species specificity and also become less effective as resistance emerges. Consequently, there is a pressing need to develop novel agents that are narrowly targeted and safe to humans. A cell-based screening platform was designed to discover compounds that are lethal to mosquito (Anopheles and Aedes) cells but show little or no activity against other insect (Drosophila) or human cell lines. Mosquito-specific, aqueous-stable cytotoxins were recovered at rare frequencies. Three of these were profiled for structure-activity relationships and also assessed in whole-animal toxicity assays. In at least one test case, species-specific cytotoxicity seen in culture effectively translated to the whole-animal level, with potent toxicity against Anopheles yet none against Drosophila. Therefore, this initiative has the potential to advance novel mosquitocidal agents and, in a broader sense, could establish a versatile platform for developing customized pesticides that selectively target other disease vectors as well.

Cloning and functional characterization of inward-rectifying potassium (Kir) channels from Malpighian tubules of the mosquito Aedes aegypti.

Inward-rectifying K(+) (Kir) channels play critical physiological roles in a variety of vertebrate cells/tissues, including the regulation of membrane potential in nerve and muscle, and the transepithelial transport of ions in osmoregulatory epithelia, such as kidneys and gills. It remains to be determined whether Kir channels play similar physiological roles in insects. In the present study, we sought to 1) clone the cDNAs of Kir channel subunits expressed in the renal (Malpighian) tubules of the mosquito Aedes aegypti, and 2) characterize the electrophysiological properties of the cloned Kir subunits when expressed heterologously in oocytes of Xenopus laevis. Here, we reveal that three Kir subunits are expressed abundantly in Aedes Malpighian tubules (AeKir1, AeKir2B, and AeKir3); each of their full-length cDNAs was cloned. Heterologous expression of the AeKir1 or the AeKir2B subunits in Xenopus oocytes elicits inward-rectifying K(+) currents that are blocked by barium. Relative to the AeKir2B-expressing oocytes, the AeKir1-expressing oocytes 1) produce larger macroscopic currents, and 2) exhibit a modulation of their conductive properties by extracellular Na(+). Attempts to functionally characterize the AeKir3 subunit in Xenopus oocytes were unsuccessful. Lastly, we show that in isolated Aedes Malpighian tubules, the cation permeability sequence of the basolateral membrane of principal cells (Tl(+) > K(+) > Rb(+) > NH(4)(+)) is consistent with the presence of functional Kir channels. We conclude that in Aedes Malpighian tubules, Kir channels contribute to the majority of the barium-sensitive transepithelial transport of K(+).

The antioxidant role of xanthurenic acid in the Aedes aegypti midgut during digestion of a blood meal.

In the midgut of the mosquito Aedes aegypti, a vector of dengue and yellow fever, an intense release of heme and iron takes place during the digestion of a blood meal. Here, we demonstrated via chromatography, light absorption and mass spectrometry that xanthurenic acid (XA), a product of the oxidative metabolism of tryptophan, is produced in the digestive apparatus after the ingestion of a blood meal and reaches milimolar levels after 24 h, the period of maximal digestive activity. XA formation does not occur in the White Eye (WE) strain, which lacks kynurenine hydroxylase and accumulates kynurenic acid. The formation of XA can be diminished by feeding the insect with 3,4-dimethoxy-N-[4-(3-nitrophenyl)thiazol-2-yl] benzenesulfonamide (Ro-61-8048), an inhibitor of XA biosynthesis. Moreover, XA inhibits the phospholipid oxidation induced by heme or iron. A major fraction of this antioxidant activity is due to the capacity of XA to bind both heme and iron, which occurs at a slightly alkaline pH (7.5-8.0), a condition found in the insect midgut. The midgut epithelial cells of the WE mosquito has a marked increase in occurrence of cell death, which is reversed to levels similar to the wild type mosquitoes by feeding the insects with blood supplemented with XA, confirming the protective role of this molecule. Collectively, these results suggest a new role for XA as a heme and iron chelator that provides protection as an antioxidant and may help these animals adapt to a blood feeding habit.

Differential ammonia metabolism in Aedes aegypti fat body and midgut tissues.

In order to understand at the tissue level how Aedes aegypti copes with toxic ammonia concentrations that result from the rapid metabolism of blood meal proteins, we investigated the incorporation of (15)N from (15)NH(4)Cl into amino acids using an in vitro tissue culture system. Fat body or midgut tissues from female mosquitoes were incubated in an Aedes saline solution supplemented with glucose and (15)NH(4)Cl for 10-40min. The media were then mixed with deuterium-labeled amino acids, dried and derivatized. The (15)N-labeled and unlabeled amino acids in each sample were quantified by mass spectrometry techniques. The results demonstrate that both tissues efficiently incorporate ammonia into amino acids, however, the specific metabolic pathways are distinct. In the fat body, the (15)N from (15)NH(4)Cl is first incorporated into the amide side chain of Gln and then into the amino group of Gln, Glu, Ala and Pro. This process mainly occurs via the glutamine synthetase (GS) and glutamate synthase (GltS) pathway. In contrast, (15)N in midgut is first incorporated into the amino group of Glu and Ala, and then into the amide side chain of Gln. Interestingly, our data show that the GS/GltS pathway is not functional in the midgut. Instead, midgut cells detoxify ammonia by glutamate dehydrogenase, alanine aminotransferase and GS. These data provide new insights into ammonia metabolism in A. aegypti mosquitoes.

Sensitivity of Aedes aegypti adults (Diptera: Culicidae) to the vapors of Eucalyptus essential oils.

Vapors of essential oils extracted from various species of Eucalyptus (E. gunnii, E. tereticornis, E. grandis, E. camaldulensis, E. dunnii, E. cinerea, E. saligna, E. sideroxylon, E. globulus ssp. globulus, E. globulus ssp. maidenii, E. viminalis and the hybrids E. grandisxE. tereticornis and E. grandisxE. camaldulensis) and their major components were found to be toxic to Aedes aegypti adults, the yellow fever mosquito. An aliquot of each oil was placed in a cylindrical test chamber and the number of knocked-down mosquitoes was recorded as function of time. Knockdown time 50% was then calculated. Results showed that E. viminalis had the fastest knockdown time at of 4.2 min, on the same order as dichlorvos, a standard knockdown agent. A correlation was observed between the content of 1,8-cineole in the Eucalyptus essential oils and the corresponding toxic effect. The correlation between KT(50) values and calculated vapor pressures of the essential oil components showed that the fumigant activity of simple organic compounds in insects is correlated with their volatility.

Identification of two sterol carrier protein-2 like genes in the yellow fever mosquito, Aedes aegypti.

Two genes encoding sterol carrier protein-2 like proteins are identified from fourth instar cDNAs of the yellow fever mosquito, Aedes aegypti. The predicted AeSCP-2like1 (AeSCP-2L1) and AeSCP-2like2 (AeSCP-2L2) proteins are small, acidic and lacking the peroxisomal targeting sequence at the C-termini. Purified recombinant AeSCP-2L1 and -2L2 bind to cholesterol with a Kd of 5.4 x 10(-6) M and 2.6 x 10(-6) M, respectively. The Kd values of AeSCP-2L1 and -2L2 to palmitic acid are 3.7 x 10(-7) M and 2.6 x 10(-7) M, respectively. Both genes are expressed predominantly in gut tissues. The transcripts of the AeSCP-2L1 gene are only detected in larval stages, whereas AeSCP-2L2 is expressed in larval and adult stages. AeSCP-2L2 transcription increases within 5 h after a bloodmeal and stays at high levels during vitellogenesis. In in vitro larval gut tissue cultures, AeSCP-2L1 transcripts were increased in the presence of juvenile hormone III, whereas AeSCP-2L2 mRNA levels increased in the presence 20-hydroxylecdysone. The results suggest that transcription of AeSCP-2L1 and -2L2 genes are regulated differently through the mosquito life cycle.

Derris (Lonchocarpus) urucu (Leguminosae) extract modifies the peritrophic matrix structure of Aedes aegypti (Diptera:Culicidae).

Aqueous suspension of ethanol extracts of Derris (Lonchocarpus) urucu (Leguminosae), collected in the state of Amazonas, Brazil, were tested for larvicidal activity against the mosquito Aedes aegypti (Diptera:Culicidae). The aim of this study was to observe the alterations of peritrophic matrix in Ae. aegypti larvae treated with an aqueous suspension of D. urucu extract. Different concentrations of D. urucu root extract were tested against fourth instar larvae. One hundred percent mortality was observed at 150 microg/ml (LC(50) 17.6 microg/ml) 24 h following treatment. In response to D. urucu feeding, larvae excreted a large amount of amorphous feces, while control larvae did not produce feces during the assay period. Ultrastructural studies showed tha larvae fed with 150 microg/ml of D. urucu extract for 4 h have an imperfect peritrophic matrix and extensive damage of the midgut epithelium. Data indicate a protective role for the peritrophic matrix. The structural modification of the peritrophic matrix is intrinsically associated with larval mortality.

Inhibitory potential of neem (Azadirachta indica Juss) leaves on dengue virus type-2 replication.

In the present study we report in vitro and in vivo inhibitory potential of crude aqueous extract of neem leaves and pure neem compound (Azadirachtin) on the replication of Dengue virus type-2. In vitro antiviral activity of aqueous neem leaves extract assessed in C(6/36) (cloned cells of larvae of Aedes albopictus) cells employing virus inhibition assay showed inhibition in dose dependent manner. The aqueous extract of neem leaves at its maximum non-toxic concentration of 1.897 mg/ml completely inhibited 100-10,000 TCID(50) of virus as indicated by the absence of cytopathic effects. The in vivo protection studies with neem leaves extract at its maximum non-toxic concentrations 120-30 mg/ml resulted in inhibition of the virus replication as confirmed by the absence of Dengue related clinical symptoms in suckling mice and absence of virus specific 511 bp amplicon in RT-PCR. The pure neem i.e. Azadirachtin did not reveal any inhibition on Dengue virus type-2 replication in both in vitro and in vivo systems.

Requirement of co-factors for the ligand-mediated activity of the insect ecdysteroid receptor in yeast.

In insects, a steroid hormone 20-hydroxyecdysone has an important role in regulating critical events such as development and reproduction. The action of 20-hydroxyecdysone is mediated by its binding to the ecdysteroid receptor (EcR), which requires a heterodimeric partner, ultraspiracle protein (USP), a homologue of the retinoid X receptor (RXR). The EcR-USP heterodimer represents a functional receptor complex capable of initiating transcription of early genes. Our goal was to establish a ligand-dependent transactivation system in yeast utilizing an insect EcR-USP heterodimer. This has been achieved using mosquito Aedes aegypti AaEcR-USP. Expression of AaEcR alone, but not USP, resulted in constitutive transcription of the ecdysone reporter gene coupled with the Drosophila heat shock protein-27 ecdysone response elements. Removal of the N-terminal A/B domain of AaEcR abolished its constitutive transcription. Constitutive transcription was also eliminated in the presence of its heterodimeric partner, AaUSPa, AaUSPb or mammalian RXR. This suggests that the A/B domain is essential for the EcR ligand-independent transactivation and its interaction with the yeast transcription complex. A ligand-mediated transactivation of Aa(Delta A/B)EcR-USP or Aa(Delta A/B)EcR-RXR heterodimers in response to an ecdysteroid agonist RH-5992 was observed only in the presence of GRIP1, a mouse co-activator. In the presence of a co-repressor, SMRT, Aa(Delta A/B)EcR-USP heterodimer exhibited a ligand-dependent repression activity. In addition, ligand-dependent transactivation systems for spruce budworm and fruit fly ecdysone receptors were also reported. This is the first report establishing the requirements of co-factors for a highly efficient ligand-dependent function of the insect EcR-USP in yeast. These findings open a way to study insect EcR-USP structure and function and to identify ligands that are specific for a certain group of insects, such as mosquitoes.

Biosynthesis of Aedes aegypti lipophorin and gene expression of its apolipoproteins.

The biosynthesis of lipophorin of the yellow fever mosquito, Aedes aegypti, was investigated. Fat bodies were incubated in vitro with radiolabeled methionine and cysteine, and radiolabeled proteins secreted into the medium were analyzed by density gradient ultracentrifugation, SDS-PAGE and fluorography. Lipophorin was synthesized in the fat body and secreted into the medium. Its density was 1.114 g/ml, similar to that of lipophorin circulating in hemolymph. Three peptides of a tryptic digest of apolipophorin II were sequenced and degenerate oligonucleotide primers were designed based on the amino acid sequences. With these primers, a cDNA product of 1.2 kb was amplified by RT-PCR using as template RNA extracted from adult female mosquitoes 24 h after ingestion of a blood meal. This cDNA was cloned, sequenced and used as a probe for Northern blot analysis, which revealed that the apoproteins of lipophorin were coded for by a single mRNA of approximately 10 kb. The expression of the apolipophorins was induced by blood feeding. From the data presented we concluded that Aedes aegypti lipophorin is synthesized in the fat body and that the expression of its apolipophorins is induced by blood feeding.

A decrease in cysteine levels causes the glutathione deficiency of aging in the mosquito.

Our previous results indicated that a glutathione (GSH) deficiency is a determinant of the aging process in many tissues and organisms. Correction of this deficiency in the aging mosquito by feeding the cysteine (Cys) precursor magnesium thiazolidine carboxylic acid (MgTc) suggested that the cause could be a lack of Cys. Adult mosquitoes (Aedes aegypti) were fed either a control diet or a diet supplemented with MgTC and then were analyzed for their Cys, cystine, GSH, and glutathione disulfide contents with our HPLC method. The life span profile of Cys levels paralleled that of GSH in the control group with high levels in the young that decreased during maturity and aging. Cystine and glutathione disulfide were undetectable. The causal relationship between the Cys and the GSH deficiencies was shown in the MgTC-supplemented group with an 83% increase in Cys and a 39% increase in GSH relative to control values. Further the conversion steps of MgTC to Cys and then to GSH were verified by use of buthionine sulfoximine. These results demonstrate that a Cys deficiency occurs in the aging mosquito and is the cause of the GSH deficiency.