Development of monoclonal antibodies for monitoring Aedes atropalpus vitellogenesis

Monoclonal antibodies to a mixture of Aedes atropalpus and A. aegypti soluble yolk proteins were produced by hybridomas between the fusion of P3X63.653 myeloma cells and splenocytes of immunized BALB/c mice. Ascites fluid collected from mice innoculated with cloned hybridoma cells contained high specificity and affinity to the soluble yolk proteins of both Aedes species. Seven different hybridoma lines produced antibodies with specificity to both A. atropalpus and A. aegypti and one cell line produced antibodies monospecific to A. aegypti soluble yolk proteins. Monoclonal antibodies specific to A. atropalpus vitellin and vitellogenin were characterized by a combination of gel electrophoresis, western blotting and immunohistochemical staining. An indirect double antibody sandwich enzyme-linked immunosorbent assay was developed using a mixture of the seven hybridoma antibodies to A. atropalpus vitellin for monitoring vitellogenin levels in individual mosquito haemolymph samples. With this procedure, the peak period of vitellogenin synthesis in A. atropalpus was found to be 18 to 30 h after adult eclosion.     

Biosynthesis,purification, and characterization of Aedes aegypti vitellin and vitellogenin

Vitellin, the major egg yolk protein, and vitellogenin, the hemolymph precursor of egg yolk protein, have been purified to apparent homogeneity from the mosquito Aedes aegypti. The purification procedure included chromatography on ion exchange, hydrophobic, and gel filtration columns. Vitellin and vitellogenin have a similar molecular weight (M_r 300,000) on gel filtration columns. However, the molecular weights of vitellin and vitellogenin, as determined from SDS electrophoresis, were 393,000 and 337,000, respectively. Vitellin in sodium dodecyl sulfate released six subunits of molecular weight 116,000, 83,000, 75,000, 54,000, 36,000, and 29,000, whereas vitellogenin released only three subunits (155,000, 120,000, and 62,000). The average molecular weights of vitellin and vitellogenin after gel filtration and SDS electrophoresis were 346,000 and 318,000, respectively. Vitellin has a high content of aspartic acid and glutamic acid, and a low content of histidine, methionine, cysteine, and tryptophan. Vitellin also contains 0.9% mol of glucosamine and no galactosamine. The isoelectric points of vitellin and vitellogenin are at pH 6.4 and 6.3, respectively. Aedes aegypti fat bodies incubated for short intervals in tissue culture medium in the presence of [³H]valine showed incorporation by radio-immunoprecipitation and SDS electrophoresis into three primary vitellogenin polypeptides of molecular weights (± SEM) 156,000 ± 4,000, 114,000 ± 5,000, and 62,000 ± 400 inside the fat body and 162,000 ± 3,000, 118,200 ± 2,000, and 63,000 ± 300 in the medium. These results suggest that the molecular weight of vitellogenin synthesized inside the fat body (M_r 332,000) remains unchanged when secreted into the hemolymph (M_r 343,000). The three vitellogenin subunits are processed by the ovary into six subunits which are then deposited in the yolk granules as vitellin.     

A preparation method for Aedes aegypti and Aedes atropalpus chorions for protein characterization

This investigation presents a method of preparation and comparison of the fully-formed eggshells (chorions) of the aedine mosquitoes, Aedes aegypti and Aedes atropalpus, by a gentle lysis-washing protocol. Light and scanning electron micrographs of the prepared chorions display a two-layered structure in the characteristically sculptured endochorion and the tubercle-patterned exochorion. SDS-gel electrophoresis of solubilized whole ovaries and prepared chorions, stained for protein and carbohydrate, show significant differences between the two species mainly in the chorion electrophoresis patterns.     

Inter-species cross-reactivity of the prothoracicotropic hormone of Manduca sexta and egg-development neurosecretory hormone of Aedes aegypti

The cross-reactivities of the big and small forms of prothoracicotropic hormone (PTTH) from pupal brains of Manduca sexta and egg-development neurosecretory hormone (EDNH) from heads of adult Aedes aegypti were examined for PTTH by the in vitro Manduca prothoracic gland assay and for EDNH by the in vitro and in vivo Aedes ovary assays. The synthesis of ecdysone by both larval and pupal prothoracic glands of Manduca was increased in a dose-dependent manner by crude extracts of Aedes aegypti heads, reaching a maximum of approx. 3- and 2-fold, respectively. Gel filtration chromatography of the Aedes head extract revealed a peak of EDNH activity with an apparent mol. wt of 11 kD. This partially purified EDNH did not possess prothoracicotropic activity in the in vitro prothoracic gland assay, nor did any other fractions from the gel filtration column. Similarly, partially purified big and small PTTH did not activate Aedes atropalpus ovaries to synthesize ecdysone in vitro, nor did they cause ovarian maturation in vivo. Thus, it appears that the structural differences between PTTH and EDNH are sufficient enough to prevent functional cross-reactivity. The apparent discrepancy in the results obtained with the crude and partially purified EDNH and PTTHs raises questions about the reliability of bioassays for screening the presence and cross-reactivity of peptide neurohormones in crude extracts.     

Effects of Amino Acid Deletion on the Antiplasmodial Activity of Angiotensin II

Malaria is an infectious disease for which effective treatment and prevention strategies remain limited. Our group recently reported that angiotensin II (AII) presents antiplasmodial activity and inhibits the development of Plasmodium gallinaceum in Aedes aegypti. However, details concerning role of each amino acid residue in the antiplasmodial activity of the peptide and information about the minimal structure responsible for this activity remain unknown. In this work, we investigated the effects of specific deletions (i.e., mono-, di-, tri- and tetra-deletions) of AII amino acids on the antiplasmodial activity of this molecule. The peptides were synthesized on solid phase method using the t-Boc strategy, purified using high performance liquid chromatography and characterized using mass spectrometry. The lytic activity of the peptides was assessed in vitro using mature sporozoites extracted from the salivary glands of infected Aedes aegypti mosquitoes. The results demonstrate that all of the deletions reduced antiplasmodial activity compared to native AII and that active analogs tend to adopt β-turn conformations; however, the deletion of bulky hydrophobic residues causes greater reductions of bioactivity than the deletion of hydrophilic residues. Corroborating previous studies, we observed that analog extremities are susceptible to changes and can be carefully modified without compromising the activity of this compound. This research contributes to our understanding of the role of each AII amino acid residue in activity against Plasmodium gallinaceum and identifies two short analogs with similar antiplasmodial activity to AII. These analogs may be candidates for additional antimalarial assays because they are inexpensive and easy to synthesize.     

Isolation and primary structure of neuropeptides from the mosquito,Aedes aegypti,immunoreactive to FMRFamide antiserum

Two novel neuropeptides, Aea-HP-I and II, have been isolated from a head extract of the mosquito, Aedes aegypti; they were detected by a FMRFamide radioimmunoassay. The peptides were purified by gel filtration, ion exchange chromatography, and reversed-phase high performance liquid chromatography. Amino acid composition and sequence analysis, combined with enzymatic digestion, established the primary structure of Aea-HP-I as pGlu-Arg-Pro-Hyp-Ser-Leu-Lys-Thr-Arg-Phe-NH₂ and Aea-HP-II as Thr-Arg-Phe-NH₂. Aea-HP-I was synthesized, and chromatographic properties of the synthetic peptide were the same as those of the native peptide, thus confirming the structural analysis. The peptide has three unusual residues: an amino-terminal pGlu, a Hyp in the fourth position, and a carboxyl-terminal amide. The Pro-Hyp sequence occurs in toxin peptides from the venoms of cone snails and wasps and in bradykinin analogues. Although the functions of Aea-HP-I and II have not been determined, the peptides have the same RFa sequence at the carboxyl-terminal as Lem-SK-I and II (leucosulfakinins) and Lem-MS (leucomyosuppressin) in cockroaches and FMRFamide-related peptides in molluscs.     

Isolation and characterization of highly purified mosquito oostatic hormone

Oostatic hormone, the hormone that inhibits vitellogenesis in mosquitoes, was purified 7,000-fold with a recovery of 70% from the ovaries of the mosquito Aedes aegypti. The purification procedure included heat treatment and chromatography on ion exchange and gel filtration columns. The hormone is a small peptidelike molecule of molecular weight 2,200 at pH 4.5, which aggregates into larger molecular species of trimer and octamer at pH 7.0 as determined by gel filtration. The hormone is positively charged at pH 7.8 and has a low Rf at pH 9.4 on disc gel electrophoresis. Injection of purified oostatic hormone (9 ng) into female mosquitoes inhibited yolk deposition and vitellogenin synthesis. Activity of the oostatic hormone in the mosquito ovary increased rapidly following blood feeding and reached a maximum after 48 h. Oostatic hormone of A. aegypti injected into autogenous Aedes taeniorhynchus inhibited egg development. Repeated injections of dilute oostatic hormone at 24 h intervals partially arrested egg development, resulting in 60% reduction in the number of eggs laid. This hormone does not block release of egg development neurosecretory hormone (EDNH) from the mosquito brain but rather appears to act on the ovary.     

Toxicité du champignon entomopathogèneCordyceps militaris pour des larves de culicides

Différents métabolites excrétés lors de la croissancein vitro deCordyceps militaris (L.)Link ont un effet toxique marqué sur les larves deCulex pipiens et d'Aedes atropalpus mais plus faible sur celles d'A. aegypti. Ces produits toxiques partiellement thermostables entraînent, suivant leur concentration, la mortalité d'une population entière deC. pipiens pipiens et d'A. atropalpus tandis qu'une proportion élevée deC. pipiens pipiens et d'A. atropalpus tandis qu'une proportion élevée des larves d'A. aegypti semble résistante à leur action. Nous pensons pouvoir utiliser ces résultats dans la recherche d'un synergisme entre ces métabolites toxiques et d'autres microorganismes ou insecticides dans un programme de lutte intégrée contre les larves de moustiques.     

Evolution of glutathione S-transferase subunits in culicidae and related nematocera: Electrophoretic and immunological evidence for conserved enzyme structure and expression

The antigenic relatedness and molecular weights of glutathione S-transferase (GST) protein subunits expressed by representative members of the Dipteran suborder Nematocera were analyzed by immunoblotting and affinity chromatography. Ten species within the genus Aedes expressed two subunits which cross-reacted with an antiserum developed against the GST-1b isozyme purified from Aedes aegypti. One of these two subunits showed no discernible molecular weight variability within the ten Aedes species. Its molecular weight (25,900 Da) was identical to the GST-1a subunit found in A. aegypti. The molecular weight of the other subunits varied among the ten species, but in all cases was larger than 25,900 Da. In ten species within the genus Anopheles (considered to be among the more primitive genera in the Culicidae), only one immunologically related GST subunit was expressed. Its molecular weight was also identical to the GST-1a subunit found in the Aedes species. Members of genera considered phylogenetically intermediate between Anopheles and Aedes had either one or two cross-reacting subunits. Representative species of families closely related to the Culicidae (the Chaoboridae, Chironomidae, Simuliidae, and Cecidomyiidae) also expressed immunologically related GST subunits. GST isozymes were partially purified from Anopheles freeborni and A aegypti adults, larvae, and ovaries using S-hexylglutathione affinity chromatography. Adult and larval Anopheles freeborni expressed not only the immunologically related subunit, but an additional, immunologically unrelated subunit. In fully developed ovaries of both species, however, only subunits immunologically related to GST-1b were expressed. These results suggest that there is an immunologically conserved domain shared among specific GST isozymes within the Nemotocea. The conserved nature of these GST subunits, their correlation with oocyte development, and their localization within ovary tissue, suggests that they share a conserved in vivo function during oocyte maturation. In addition, the conserved antibody binding domain within these subunits apparently has been duplicated in several species of Culicidae.     

Purification of the toxic protein from Bacillus thuringiensis serotype 10 isolate demonstrating a preferential larvicidal activity to the mosquito

The protein demonstrating larvicidal activity to the mosquito Aedes aegypti was purified from the alkali extract of the spore-parasporal inclusion complex of the isolate, 73-E-10-2, belonging to Bacillus thuringiensis serotype 10. By Sepharose CL-4B gel filtration and DEAE-cellulose column chromatography, a toxic protein was obtained, and its homogeneity was confirmed by Sephadex G-150 gel filtration and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The molecular weight of the toxic protein was 67,000, when estimated by SDS-PAGE. The LC₅₀ of the toxic protein against 4-day-old larvae of A. aegypti was 16.8 μg/ml. There was no serological relationship between the toxic protein from the isolate 73-E-10-2 and that (M_r 67,000) from the type strain of B. thuringiensis subsp. israelensis.     

Mosquitoes rely on their gut microbiota for development

Field studies indicate adult mosquitoes (Culicidae) host low diversity communities of bacteria that vary greatly among individuals and species. In contrast, it remains unclear how adult mosquitoes acquire their microbiome, what influences community structure, and whether the microbiome is important for survival. Here, we used pyrosequencing of 16S rRNA to characterize the bacterial communities of three mosquito species reared under identical conditions. Two of these species, Aedes aegypti and Anopheles gambiae, are anautogenous and must blood‐feed to produce eggs, while one, Georgecraigius atropalpus, is autogenous and produces eggs without blood feeding. Each mosquito species contained a low diversity community comprised primarily of aerobic bacteria acquired from the aquatic habitat in which larvae developed. Our results suggested that the communities in Ae. aegypti and An. gambiae larvae share more similarities with one another than with G. atropalpus. Studies with Ae. aegypti also strongly suggested that adults transstadially acquired several members of the larval bacterial community, but only four genera of bacteria present in blood fed females were detected on eggs. Functional assays showed that axenic larvae of each species failed to develop beyond the first instar. Experiments with Ae. aegypti indicated several members of the microbial community and Escherichia coli successfully colonized axenic larvae and rescued development. Overall, our results provide new insights about the acquisition and structure of bacterial communities in mosquitoes. They also indicate that three mosquito species spanning the breadth of the Culicidae depend on their gut microbiome for development.     

Ovary ecdysteroidogenic hormone functions independently of the insulin receptor in the yellow fever mosquito,Aedes aegypti

Most mosquito species must feed on the blood of a vertebrate host to produce eggs. In the yellow fever mosquito, Aedes aegypti, blood feeding triggers medial neurosecretory cells in the brain to release insulin-like peptides (ILPs) and ovary ecdysteroidogenic hormone (OEH). Theses hormones thereafter directly induce the ovaries to produce ecdysteroid hormone (ECD), which activates the synthesis of yolk proteins in the fat body for uptake by oocytes. ILP3 stimulates ECD production by binding to the mosquito insulin receptor (MIR). In contrast, little is known about the mode of action of OEH, which is a member of a neuropeptide family called neuroparsin. Here we report that OEH is the only neuroparsin family member present in the Ae. aegypti genome and that other mosquitoes also encode only one neuroparsin gene. Immunoblotting experiments suggested that the full-length form of the peptide, which we call long OEH (lOEH), is processed into short OEH (sOEH). The importance of processing, however, remained unclear because a recombinant form of lOEH (rlOEH) and synthetic sOEH exhibited very similar biological activity. A series of experiments indicated that neither rlOEH nor sOEH bound to ILP3 or the MIR. Signaling studies further showed that ILP3 activated the MIR but rlOEH did not, yet both neuropeptides activated Akt, which is a marker for insulin pathway signaling. Our results also indicated that activation of TOR signaling in the ovaries required co-stimulation by amino acids and either ILP3 or rlOEH. Overall, we conclude that OEH activates the insulin signaling pathway independently of the MIR, and that insulin and TOR signaling in the ovaries is coupled.     

Insecticidal peptides from the theraposid spider Brachypelma albiceps: An NMR-based model of Ba2

Soluble venom and purified fractions of the theraposid spider Brachypelma albiceps were screened for insecticidal peptides based on toxicity to crickets. Two insecticidal peptides, named Ba1 and Ba2, were obtained after the soluble venom was separated by high performance liquid chromatography and cation exchange chromatography. The two insecticidal peptides contain 39 amino acid residues and three disulfide bonds, and based on their amino acid sequence, they are highly identical to the insecticidal peptides from the theraposid spiders Aphonopelma sp. from the USA and Haplopelma huwenum from China indicating a relationship among these genera. Although Ba1 and Ba2 were not able to modify currents in insect and vertebrate cloned voltage-gated sodium ion channels, they have noteworthy insecticidal activities compared to classical arachnid insecticidal toxins indicating that they might target unknown receptors in insect species. The most abundant insecticidal peptide Ba2 was submitted to NMR spectroscopy to determine its 3-D structure; a remarkable characteristic of Ba2 is a cluster of basic residues, which might be important for receptor recognition.     

Isolation and characterization of mosquito cell membrane glycoproteins

Plasma membranes have been purified from an established cell line, Mos 20A of Aedes aegypti, and analysed for glycoprotein and polypeptide constituents by isoelectric focusing and sodium dodecyl sulphate polyacrylamide gel electrophoresis. A major glycoprotein of molecular weight 110 000 carrying binding sites for concanavalin A and soybean agglutinin has been purified to homogeneity. Although located on the cell surface, the 110 kdalton glycoprotein is not labelled by lactoperoxidase-catalysed radioactive iodination of whole cells. Analysis indicates the presence of N-glycans, containing on average nine mannose residues, and the N-acetylglucosaminyl-β1,4-N-acetylglucosamine sequence. In addition, O-glycosidically linked N-acetylgalactosamine residues are present.     

Purification and expression of glutathione S-transferase from a Malaysian population of Aedes albopictus (Diptera: Culicidae)

Glutathione S-transferase (GST) from the 4^th instar larvae of the dengue vector Aedes albopictus was purified by glutathione-agarose affinity chromatography and characterised using SDS-PAGE. The expression of the purified enzyme in the life stages and insecticide treated populations of Ae. albopictus as well as its cross-reactivity with larval GST of two dipteran species Aedes aegypti and Batrocera papayae were observed using western blotting. The purified GST had a specific activity of 196.0 ± 11 μmol/min/mg with a purification fold and yield of 28 and 69%, respectively. The SDS-PAGE analysis of the purified GST depicted a single band size of 23 kDa. The GST was expressed in all the larval and adult stages of Ae. albopictus with the exception of the pupal stage. However, the expression level in the adult stage was visibly reduced as compared to the larval stages. Western blotting analysis showed no cross-reactivity with the GST of Ae. aegypti (4^th instar) and B. papayae (3^rd instar) larvae. The expression of this enzyme was not inducible by exposure to the insecticides dichlorodiphenyltrichloroethane (1.25 mg/L) and malathion (0.3125 mg/L).     

Defense reactions of mosquitoes to filarial worms: comparative studies on the response of three different mosquitoes to inoculated Brugia pahangi and Dirofilaria immitis microfilariae

The melanization response of Aedes trivittatus and the Rockefeller (RKF) and black-eyed Liverpool (LVP) strains of Aedes aegypti against intrathoracically inoculated Dirofilaria immitis and Brugia pahangi microfilariae (mff) was investigated. All mff of either species were melanized in A. trivittatus following Day 2 postinoculation, and the response of this species was significantly more rapid and effective than either strain of A. aegypti. The refractory RKF strain had a significantly greater response against both D. immitis and B. pahangi than the highly susceptible LVP strain, but data suggest that the increased responsiveness was due to a physiologic incompatibility in RKF A. aegypti, thereby resulting in a greater mortality and subsequent melanization of inoculated mff. Inoculation of large numbers of mff overloaded the defense capabilities of A. aegypti (LVP), but not those of A. trivittatus. The melanization response against D. immitis mff was effectively reduced for up to 4 days in A. aegypti (LVP), but for only 1 day in A. trivittatus, when mosquitoes were maintained on a 0.3 m sucrose diet containing from 0.1 to 1.0% (w/v) phenylthiourea.     

Isolation and partial sequencing of a pancreatic polypeptide-like neuropeptide from the liver fluke,Fasciola hepatica

1. A pancreatic polypeptide (PP)-immunoreactive neuropeptide has been isolated and partially sequenced from the liver fluke, Fasciola hepatica.2. Gel permeation chromatography of an acid ethanol extract of cattle flukes showed that the peptide is similar in size to mammalian (bovine) PP.3. The Fasciola peptide was purified to homogeneity by means of reverse-phase HPLC, employing different column chemistries.4. The purified peptide was sequenced using automated gas-phase Edman degradation and the first 24 amino acid residues determined.     

Evaluation of the Activity of the Essential Oil from an Ornamental Flower against Aedes aegypti: Electrophysiology,Molecular Dynamics and Behavioral Assays

Dengue fever has spread worldwide and affects millions of people every year in tropical and subtropical regions of Africa, Asia, Europe and America. Since there is no effective vaccine against the dengue virus, prevention of disease transmission depends entirely on regulating the vector (Aedes aegypti) or interrupting human-vector contact. The aim of this study was to assess the oviposition deterrent activity of essential oils of three cultivars of torch ginger (Etlingera elatior, Zingiberaceae) against the dengue mosquito. Analysis of the oils by gas chromatography (GC)—mass spectrometry revealed the presence of 43 constituents, of which α-pinene, dodecanal and n-dodecanol were the major components in all cultivars. Solutions containing 100 ppm of the oils exhibited oviposition deterrent activities against gravid Ae. aegypti females. GC analysis with electroantennographic detection indicated that the oil constituents n-decanol, 2-undecanone, undecanal, dodecanal, trans-caryophyllene, (E)-β-farnesene, α-humulene, n-dodecanol, isodaucene and dodecanoic acid were able to trigger antennal depolarization in Ae. aegypti females. Bioassays confirmed that solutions containing 50 ppm of n-dodecanol or dodecanal exhibited oviposition deterrent activities, while a solution containing the alcohol and aldehyde in admixture at concentrations representative of the oil presented an activity similar to that of the 100 ppm oil solution. Docking and molecular dynamics simulations verified that the interaction energies of the long-chain oil components and Ae. aegypti odorant binding protein 1 were quite favorable, indicating that the protein is a possible oviposition deterrent receptor in the antenna of Ae. aegypti.     

Purification and partial characterization of mosquito egg development neurosecretory hormone: Evidence for gonadotropic and steroidogenic effects

Egg development neurosecretory hormone (EDNH), a hormone that stimulates vitellogenesis in mosquitoes, was purified 10,000-fold from the mosquito Aedes aegypti. The purification procedure included chromatography on hydrophobic, ion exchange, and gel filtration columns, and preparative electrophoresis to give an almost homogeneous preparation. The hormone is a polypeptide monomer of molecular weight of 18,700 ± 500 as determined from SDS electrophoresis and gel filtration chromatography. Using lowpressure chromatography throughout the purification procedure, the hormone was recovered at a high yield (39%). The amount of EDNH in a mosquito is about 0.6-1.6 ng, corresponding to 32–85 fmol. Injection of purified EDNH into female mosquitoes resulted in the conversion of [¹⁴C]cholesterol into labeled ecdysone and 20-hydroxyecdysone which were separated and identified using thin-layer chromatography and high-performance liquid chromatography separation procedures. Egg development and vitellogenin synthesis were also induced when EDNH was injected into several mosquito species, indicating that the hormone is not species-specific. This report is the first to show that a purified preparation of EDNH has both steroidogenic and a gonadotropic effects on female mosquitoes.