Characterization of N-linked oligosaccharides in chorion peroxidase of Aedes aegypti mosquito

A peroxidase is present in the chorion of Aedes aegypti eggs and catalyzes chorion protein cross-linking during chorion hardening, which is critical for egg survival in the environment. The unique chorion peroxidase (CPO) is a glycoprotein. This study deals with the N-glycosylation site, structures, and profile of CPO-associated oligosaccharides using mass spectrometric techniques and enzymatic digestion. CPO was isolated from chorion by solubilization and several chromatographic methods. Mono-saccharide composition was analyzed by HPLC with fluorescent detection. Our data revealed that carbohydrate (D-mannose, N-acetyl D-glucosamine, D-arabinose, N-acetyl D-galactosamine, and L-fucose) accounted for 2.24% of the CPO molecular weight. A single N-glycosylation site (Asn328-Cys- Thr) was identified by tryptic peptide mapping and de novo sequencing of native and PNGase A-deglycosylated CPO using matrix-assisted laser/desorption/ionization time-of-flight mass spectrometry (MALDI/TOF/MS) and liquid chromatography/tandem mass spectrometry (LC/MS/MS). The Asn328 was proven to be a major fully glycosylated site. Potential tryptic glycopeptides and profile were first assessed by MALDI/TOF/MS and then by precursor ion scanning during LC/MS/MS. The structures of N-linked oligosaccharides were elucidated from the MS/MS spectra of glycopeptides and exoglycosidase sequencing of PNGase A-released oligosaccharides. These CPO-associated oligosaccharides had dominant Man3GlcNAc2 and Man3 (Fuc) GlcNAc2 and high mannose-type structures (Man(4-8)GlcNAc2). The truncated structures, Man2GlcNAc2 and Man2 (Fuc) GlcNAc2, were also identified. Comparison of CPO activity and Stokes radius between native and deglycosylated CPO suggests that the N-linked oligosaccharides influence the enzyme activity by stabilizing its folded state.     

Multiple functions of an odorant-binding protein in the mosquito Aedes aegypti

To prevent spreading of deadly diseases, populations of mosquitoes can be controlled by interfering with their chemical communication system. Odorant-binding proteins, recently shown to be required for olfaction, represent interesting targets for such purpose. Here we describe the ligand-binding properties and the unusual tissue expression of odorant-binding protein 22 from the repertoire of Aedes aegypti. Best ligands are molecules with two aromatic rings connected by a short rigid chain. The protein is expressed not only in sensory organs, such as the antennae and proboscis, but also in the male reproductive apparatus and transferred to the spermathecs of females. This suggests an additional function for this protein as pheromone carrier, analogously to vertebrates’ urinary and salivary proteins as well as some insect chemosensory proteins. Antiserum against odorant-binding protein 22 also stained the edges and sensilla of spiracles, indicating a third, still unknown, role for this protein.     

Natural vertical transmission of dengue viruses by Aedes aegypti in Bolivia

The natural transmission of dengue virus from an infected female mosquito to its progeny, namely the vertical transmission, was researched in wild caught Aedes aegypti during an important outbreak in the town of Santa Cruz de la Sierra, Bolivia. Mosquitoes were collected at the preimaginal stages (eggs, larvae and pupae) then reared up to adult stage for viral detection using molecular methods. Dengue virus serotypes 1 and 3 were found to be co-circulating with significant higher prevalence in male than in female mosquitoes. Of the 97 pools of Ae. aegypti (n = 635 male and 748 female specimens) screened, 14 pools, collected in February-May in 2007, were found positive for dengue virus infection: five DEN-1 and nine DEN-3. The average true infection rate (TIR) and minimum infection rate (MIR) were respectively 1.08% and 1.01%. These observations suggest that vertical transmission of dengue virus may be detected in vectors at the peak of an outbreak as well as several months before an epidemic occurs in human population.     

Assessment of diet choice by the yellow fever mosquito Aedes aegypti

To investigate feeding‐related decisions in Aedes aegypti (L.), adults are presented with simple diets of paired gustatory stimuli conveying information concerning energy content, nutrient richness, osmotic balance and food toxicity in a two‐diet matrix assay. Assessment of mosquito gut contents indicates that both sexes accept single sugar diets in a dose‐dependent manner. When presented with a choice between two different yet equimolar sugar solutions, more individuals of both sexes accept the disaccharides, sucrose and trehalose, than the monosacharrides, fructose and glucose. The combination of pyranose and furanose sugars in solution, either physically associated (as in sucrose) or present as monomers (as glucose and fructose), is accepted over solutions containing a single sugar moiety. Using the two‐diet matrix assay, mosquito diet‐choice is also tested between two equimolar sucrose ‘driver’ solutions in which one is presented with various concentrations of another potential feeding cue ‘test’ compound (i.e. each of the 20 naturally‐occurring amino acids, sodium chloride, quinine or caffeine). Diet‐choice between the ‘driver’ sucrose‐only solution and the solution of the ‘driver’ sucrose containing a ‘test’ amino acid is influenced by sex, amino acid concentration and sucrose concentration. There is also an example of synergism between the diet components, leucine and sucrose. Mosquitoes demonstrate a dose‐dependent acceptance of sucrose‐only diets over sodium chloride‐containing sucrose when presented together. Interestingly, the sucrose‐only diet is accepted by more mosquitoes than all concentrations of the saline‐containing sucrose diets except those approximately isotonic to mosquito haemolymph, at which concentration mosquitoes show no clear choice between the diets. More individuals of both sexes accept sucrose‐only diets than the diets of caffeine‐containing sucrose in a dose‐dependent manner. Only females, however, respond to quinine‐containing sucrose diets and modulate this behaviour in relation to the energetic reward: more females imbibed quinine‐containing sucrose at the higher sucrose concentration (1 m ). A systematic characterization of diet selection behaviour of A. aegypti is presented for 27 putative feeding cues potentially involved in nectar/honeydew feeding. This study will be used as a basis from which to investigate further the mosquito's assessment of food quality and ultimately host choice.     

Time and site of assembly of the peritrophic membrane of the mosquito Aedes aegypti

Summary We determined the time and site of secretion of the precursors of the peritrophic membrane (PM) in Aedes aegypti and when the structure is assembled. The fine structure of the developing membrane of blood-feed females was described, and the pattern of secretion of injected tritiated glucosamine analyzed autoradiographically. Immediately following blood feeding, ingested red cells rapidly become compressed, such that the surrounding plasma is extruded to the margin of the midgut contents. Thereby, ingested fluids form a narrow margin separating the blood mass from the midgut epithelium. By electron microscopy, the PM first becomes evident at about 4 to 8 h after blood is ingested, and the membrane attains mature texture by 12 h. The compacted mass of ingested erythrocytes seems to serve as a template for the forming structure. In contrast, tritiated glucosamine, injected into freshly engorged mosquitoes, begins to concentrate on the midgut microvilli by 2 h after feeding. By 8 h the label assumes the layered appearance that characterizes the fine structure of the mature membrane. In contrast to the prevailing concept that the PM of mosquitoes first assumes texture anteriorly immediately after blood is ingested, we find that this potential barrier to pathogen development forms no earlier than 4 h after feeding and that it is formed from precursors secreted along the entire length of the epithelium overlying the food mass.     

Worldwide patterns of genetic differentiation imply multiple 'domestications' of Aedes aegypti, a major vector of human diseases

Understanding the processes by which species colonize and adapt to human habitats is particularly important in the case of disease-vectoring arthropods. The mosquito species Aedes aegypti, a major vector of dengue and yellow fever viruses, probably originated as a wild, zoophilic species in sub-Saharan Africa, where some populations still breed in tree holes in forested habitats. Many populations of the species, however, have evolved to thrive in human habitats and to bite humans. This includes some populations within Africa as well as almost all those outside Africa. It is not clear whether all domestic populations are genetically related and represent a single 'domestication' event, or whether association with human habitats has developed multiple times independently within the species. To test the hypotheses above, we screened 24 worldwide population samples of Ae. aegypti at 12 polymorphic microsatellite loci. We identified two distinct genetic clusters: one included all domestic populations outside of Africa and the other included both domestic and forest populations within Africa. This suggests that human association in Africa occurred independently from that in domestic populations across the rest of the world. Additionally, measures of genetic diversity support Ae. aegypti in Africa as the ancestral form of the species. Individuals from domestic populations outside Africa can reliably be assigned back to their population of origin, which will help determine the origins of new introductions of Ae. aegypti.     

Evidence of two lineages of the dengue vector Aedes aegypti in the Brazilian Amazon, based on mitochondrial DNA ND4 gene sequences

Genetic variation was estimated in ten samples populations of Aedes aegypti from the Brazilian Amazon, by using a 380 bp fragment of the mitochocondrial NADH dehydrogenase subunit 4 (ND4) gene. A total of 123 individuals were analyzed, whereby 13 haplotypes were found. Mean genetic diversity was slightly high (h = 0.666 ± 0.029; π = 0.0115 ± 0.0010). Two AMOVA analyses indicated that most of the variation (~70%-72%) occurred within populations. The variation found among and between populations within the groups disclosed lower, but even so, highly significant values. F(ST) values were not significant in most of the comparisons, except for the samples from Pacaraima and Rio Branco. The isolation by distance (IBD) model was not significant (r = 0.2880; p = 0.097) when the samples from Pacaraima and Rio Branco were excluded from the analyses, this indicating that genetic distance is not related to geographic distance. This result may be explained either by passive dispersal patterns (via human migrations and commercial exchange) or be due to the recent expansion of this mosquito in the Brazilian Amazon. Phylogenetic relationship analysis showed two genetically distinct groups (lineages) within the Brazilian Amazon, each sharing haplotypes with populations from West Africa and Asia.     

The cost of immunity in the yellow fever mosquito, Aedes aegypti depends on immune activation

Although host immunity offers the obvious benefit of reducing parasite infection, it is often traded-off with other fitness components. We investigated whether the cost of an immune response in the yellow fever mosquito, Aedes aegypti, is modulated by the antigen that activates the melanization immune response. Thus, one of three different novel antigens were injected into the mosquito's thorax--either a glass bead, a negatively charged (C-25) Sephadex bead, or a neutral (G-25) Sephadex bead--and fecundity and bead melanization were observed. Glass beads are immunologically inert and were therefore used as an inoculation control. The fecundity of mosquitoes inoculated with these beads did not differ from the fecundity of mosquitoes that did not melanize negatively charged or neutral beads. The ability of A. aegypti to melanize negatively charged Sephadex beads was associated with reduced fecundity, showing a clear cost of immunity. In contrast, melanization of the neutral beads was quite strong but had no effect on fecundity. Thus, the cost of what appeared to be the same immune response--melanization of a bead--depended on the type of bead that stimulated the immune system. Such differences might help to explain variation of immune efficacy against different parasites in natural populations.     

Lipid metabolism during sequential gonotrophic cycles in large and small female Aedes aegypti

The lipid metabolism was investigated during six gonotrophic cycles of Aedes aegypti. Females of constant body size were analyzed for their total lipid content: large females with a body size of 41.06 (wing length cubed) and small females with 15.63. Their lipid contents at eclosion were compared to lipid values after two days of sugar-feeding, shortly before a blood meal, after oviposition, of their total egg batches, and again before the next blood meal, with intermittent access to sugar for two days for six gonotrophic cycles.Large females transferred most of their pre-blood meal lipid into the ovaries. Their low lipid content after oviposition was restored by synthesis from intermittent sugar meals. After the third gonotrophic cycle, they withheld more and more of the resynthesized lipid in their fat body, thus gradually reducing their fecundity. Since blood consumption was not altered significantly during these six cycles, age-related reduction of fecundity was clearly caused by limitations of yolk lipid.Small females transferred a considerably smaller, but constant segment of sugar-derived lipids to the ovaries. In both size classes, lipid content per oocyte was constant throughout all cycles with 9 mcal/oocyte in large and 7 mcal/oocyte in small females. Total fecundity reached 450 eggs in large and 280 eggs in small females. Large females that were maintained on water without sucrose took large blood meals from which part of the yolk lipid was synthesized. Extrapolations suggest that only one or two additional gonotrophic cycles would be possible without additional carbohydrate sources, despite lipogenesis from blood protein.     

Geographic and developmental variation in expression of the circadian rhythm gene, timeless, in the pitcher-plant mosquito, Wyeomyia smithii

Expression of the circadian rhythm gene timeless was investigated in the pitcher-plant mosquito, Wyeomyia smithii (Coq.), and was found to vary with time of day, instar of diapause, and latitude of origin. The temporal pattern of timeless expression differed between the two diapausing instars and was significantly higher in southern (38-40 degrees N) than in northern (50 degrees N) populations, when diapausing instar was held constant. Expression of timeless is therefore both developmentally and evolutionarily variable. This result provides the first example of a latitudinal difference in the expression of timeless, suggesting that, along with evidence from other insects, timeless has the potential to affect photoperiodic response and its adaptive evolution in temperate seasonal environments.     

Isolation, primary culture and morphological characterization of oenocytes from Aedes aegypti pupae

Oenocytes are ectodermic cells that participate in a number of critical physiological roles such as detoxification and lipid storage and metabolism in insects. In light of the lack of information on oenocytes from Aedes aegypti and the potential role of these cells in the biology of this major yellow fever and dengue vector, we developed a protocol to purify and maintain Ae. aegypti pupa oenocytes in primary culture. Ae. aegypti oenocytes were cultured as clustered and as isolated ovoid cells with a smooth surface. Our results demonstrate that these cells remain viable in cell culture for at least two months. We also investigated their morphology in vivo and in vitro using light, confocal, scanning and transmission electron microscopes. This work is the first successful attempt in isolating and maintaining Ae. aegypti oenocytes in culture, and a significant step towards understanding the role of this cell type in this important disease vector. The purification and the development of primary cultures of insect oenocytes will allow future studies of their metabolism in producing and secreting compounds.     

Infection sites of the entomopathogenic fungus Beauveria bassiana in the larvae of the mosquito Aedes aegypti

A series of experiments were conducted to determine the infection sites of the entomopathogenic fungus Beauveria bassiana after ingestion by the larvae of Aedes aegypti. The timing of the host death in relation to larval molting, the principle sites of fungal infection and development in host tissues were studied. Fungal conidiospores (CS) and blastospores (BS) were used separately for treatment of mosquito larvae. Although in most instances CS germinated and developed within the host, in others there was a premature abortion of the fungal development cycle. On the other hand, BS ingested by the larvae showed differences in the fungal development stages in the larval tissues. While the two primary infection sites were the head and the anal region, the most preferred site for fungal development was the larval gut. No more than two cycles of fungal development can occur in the host. Although both CS and BS are effective as larvicides, BS is far more pathogenic.

---

Résumé Une série d'expériences a été menée afin de déterminer les emplacements d'infection du fongus entomopathogénique Beauveria bassiana (souche GK 2016) lequel avait été ingesté des larves d'Aedes aegypti. On a étudié le moment de la mort des hôtes en relation des mues des larves, les emplacements principaux où se conduit l'infection fongale et le développement du fongus dans des tissus divers d'hôte. On a utilisé les conidiospores (CS) et les blastospores (BS) séparément pour traitement des larves des moustiques. Bien que dans la plupart des cas CS ait germiné et se soit développé chez l'hôte, en d'autres cas, CS a germiné mais n'avait pas produit du BS évidemment grâce à l'avortement prématuré du cycle de développement des fongus chez l'hôte. Le BS qui avait été ingesté dans les larves présentait des stages différents de développement du fongus dans les tissus des larves. Quoique les deux emplacements placements principaux étaient la tête et la région anale, l'emplacement préféré pour le développement du fongus était l'intestin des larves. Nous montrons qu'il n'y a plus de deux cycles de développement du fongus chez l'hôte. Bien que CS et BS soient efficaces comme larvicides, BS est de loin plus pathogène.

     

Mass Production of Genetically Modified Aedes aegypti for Field Releases in Brazil

New techniques and methods are being sought to try to win the battle against mosquitoes. Recent advances in molecular techniques have led to the development of new and innovative methods of mosquito control based around the Sterile Insect Technique (SIT)^1-3. A control method known as RIDL (Release of Insects carrying a Dominant Lethal)⁴, is based around SIT, but uses genetic methods to remove the need for radiation-sterilization^5-8. A RIDL strain of Ae. aegypti was successfully tested in the field in Grand Cayman^9,10; further field use is planned or in progress in other countries around the world.Mass rearing of insects has been established in several insect species and to levels of billions a week. However, in mosquitoes, rearing has generally been performed on a much smaller scale, with most large scale rearing being performed in the 1970s and 80s. For a RIDL program it is desirable to release as few females as possible as they bite and transmit disease. In a mass rearing program there are several stages to produce the males to be released: egg production, rearing eggs until pupation, and then sorting males from females before release. These males are then used for a RIDL control program, released as either pupae or adults^11,12.To suppress a mosquito population using RIDL a large number of high quality male adults need to be reared^13,14. The following describes the methods for the mass rearing of OX513A, a RIDL strain of Ae. aegypti ⁸, for release and covers the techniques required for the production of eggs and mass rearing RIDL males for a control program.