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.     

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.     

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.

     

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.     

Toxicity of the blue-green alga Oscillatoria agardhii to the mosquito Aedes aegypti and the shrimp Artemia salina

The cyanobacterium Oscillatoria agardhii 27, which does not produce mammalian neuro- or hepatotoxins, was highly toxic to the larval stages of the yellow fever mosquito Aedes aegypti: its 24-h LC₅₀ values against fourth-and second-instar larvae of A. aegypti were 8.7 and 6.1 g live cells/ml, respectively. The toxin was water-soluble and was partially purified but the chemical nature of the toxic compound(s) is still unknown. Aqueous solutions were also toxic to the newborn larvae of the brine shrimp, Artemia salina, used for the bioassay. The toxic activity of these solutions decreased markedly on heating to 90°C for 15 min.J. Kiviranta is with the Department of Pharmacy, P.O. Box 15, FIN-00014 University of Helsinki, Helsinki, Finland; A. Abdel-Hameed is usually with the Department of Microbiology, Faculty of Pharmacy, Cairo University, Kasr-El-Aini Street, Cairo, Egypt, but is presently with the Department of Applied Chemistry and Microbiology, P.O. Box 27, Viikki, Building B, FIN-00014 University of Helsinki, Helsinki, Finland.     

Differentiation of Aedes aegypti and Aedes albopictus (Diptera: Culicidae) with egg surface morphology and morphometrics using scanning electron microscopy

Aedes aegypti and Aedes albopictus are potential arboviral vectors leading to high human fatality worldwide. Efforts in the present study were made to differentiate the eggs of A. aegypti and A. albopictus morphologically and morphometrically using scanning electron microscopy (SEM). Morphometrically, these species’ eggs were 48.48% significantly different of the 33 attributes including egg dimensions, micropylar apparatus, dimensions and density of outer chorionic cells (OCCs), tubercles and width of exochorionic network. In comparison to A. aegypti eggs, A. albopictus eggs were significantly smaller and more tapered at the posterior end; however, the micropylar disc of A. aegypti was wider and had incomplete circular sectors whereas it was a narrower polygon without sectors in A. albopictus. These species were also significantly different with regards to OCC which enclose both large central and small peripheral tubercles. Specifically, the exochorionic networks in A. aegypti were interwoven, reticulated and extensively wide whereas they were narrow, prominent and solid-wall-like in A. albopictus. This feature may strengthen A. albopictus eggs against desiccation, when they are laid in containers. The morphometrical and morphological analysis of the egg’s attributes of A. aegypti and A. albopictus may be helpful in understanding egg biology as well as in species confirmation.     

Some consequences of selection for fast and slow recovery from the larval alarm reaction in Aedes aegypti

Summary Replicated divergent selection based upon the time taken to recover from the larval alarm reaction in the mosquito Aedes aegypti resulted in lines which recovered faster and slower than the control lines. Estimates of the realized heritability were consistent, ranging from 0.21 to 0.24 in the fast replicates and 0.19 to 0.20 in the slow replicates. After 11 generations of selection an apparent change in the fitness was examined using an application of the path analysis. The relevance of the findings to natural selection is also discussed.     

Effect of body size and sugar meals on oviposition of the yellow fever mosquito,Aedes aegypti (Diptera: Culicidae)

The effects of dietary sugar and body size on the oviposition of Ae. aegypti were studied under laboratory conditions. In female mosquitoes provided with sugar, the start of maximum fecundity was significantly delayed and the oviposition period was longer than in females provided with water. The peak of oviposition was also delayed in sugar‐fed females. Large females oviposited more eggs per day than small females at maximum fecundity and during eight days of observations. Large females also visited significantly more water‐containing cups in their cages per day than small females at maximum fecundity. During the eight days of observations, large females and sugar‐fed females visited more water‐containing cups in their cages than water‐fed small females. Both large females and sugar‐fed females oviposited their eggs at sites higher above the water line than water‐fed small females. These results suggested that large and sugar‐fed female Ae. aegypti mosquitoes had more energy reserves and oviposited their eggs at higher sites, which would lead to a time lag in hatching.     

Laboratory evalution of a translocation double heterozygote for genetic control of Aedes aegypti

Summary Two pure translocation homozygote stocks, T₁/T₁ and T₃/T₃, were used to produce a double translocation heterozygote system designated T₁/T₃, employing T₁/T₁ as the male and T₃/T₃ as the female parent. The double heterozygote showed 73 % sterility when mated to wild females. Tests on mating competitiveness, recombination frequency in the differential segment, insemination rate and inheritance of sterility after release, for four generations in laboratory cages, have been carried out to evaluate the efficiency of this strain as an agent for a population control programme.     

In-silico homology modeling of three isoforms of insect defensins from the dengue vector mosquito, Aedes aegypti (Linn., 1762)

Dengue is a serious public health problem in tropical and subtropical countries. It is caused by any of the four serologically distinct dengue viruses, namely DENV1–4. The viruses are transmitted by Aedes mosquitoes. Understanding various defence mechanisms of insects has become a prime area of research worldwide. In insects, the first line of defence against invading pathogens includes cellular mechanisms and a battery of antimicrobial peptides such as defensins, cecropins etc. Defensins—cationic, cysteine-rich peptides consisting of ∼40 amino acids with broad-spectrum activity against Gram-positive bacteria—have been reported from a wide range of organisms. In the dengue vector mosquito, Aedes aegypti, three isoforms of defensins are reported to be expressed in a spatial and temporal fashion. This report presents the three-dimensional structures of the three isoforms of Ae. aegypti defensins predicted by comparative modeling. Prediction was done with Modeller 9v1 and the structures validated through a series of tests. The best results of the prediction study are presented, and may help lead to the discovery of new synthetic peptides or derivatives of defensins that could be useful in the control of vector-borne diseases.     

Ultrastructural changes in the fat body of adult female Aedes aegypti in relationship to vitellogenin synthesis

Summary The ultrastructure of the fat body of Aedes aegypti was followed from emergence through a blood meal. Changes in the volume of protein granules and lipid droplets were also examined. The relationship of these events to the known endocrinology of vitellogenin synthesis in mosquitoes is discussed.Massachusetts Experiment Station Paper No. 2130. Supported by the Massachusetts Agricultural Experiment Station, Project n° 356, and by NIH grant n° AI-11909     

Mosquito trypsin: immunocytochemical localization in the midgut of blood-fed Aedes aegypti (L.)

Summary A polyclonal antibody was raised against trypsin purified from the midgut of blood-fed Aedes aegypti. Using this antibody and our modification of the peroxidase-antiperoxidase immunocytochemical reaction, strong activity was found in the lumen of the midgut at the light-microscopical level. The activity was localized mainly in the posterior part of the distensible, abdominal midgut, along the periphery of the blood bolus and within the peritrophic membrane. Immunoreactivity appeared 8 h after the blood meal and was most prominent around 24 h, coinciding with our previous spectrophotometric determinations of trypsin.At the electron-microscopical level, secretory granules, immunocytochemically labelled with anti-trypsin antibody and protein A-colloidal gold, were first detected about 12 h after the blood meal. At 18 h, the secretory pathway could be followed immunocytochemically from the formation of granules in the Golgi complex until their release by exocytosis in the midgut lumen. By 24 h, there was a reduction in secretory granules, and large lysosomes appeared.The process of secretion described for this mosquito is comparable to similar events in vertebrate secretory systems and the presence of an intracellular trypsinogen is suggested.     

Identification and linkage relationships of three hexokinase genes in Aedes aegypti

Four regions of hexokinase activity are detected by starch gel electrophoresis of adult Aedes aegypti. Three of the regions, Hk-2, Hk-3, and Hk-4, are produced by three tightly linked loci, located on the third chromosome 7.25 map units from the locus fuzzy. The three loci show developmental differences as well as differences in substrate specificity.This work was supported by grants from the National Institutes of Health, AI 05118-02 and AI 12016.     

Natural skip oviposition of the mosquito Aedes aegypti indicated by codominant genetic markers

This study examines the use of codominant restriction fragment length polymorphism (RFLP) markers to estimate the number of sibling families found within and among oviposition sites used by the mosquito Aedes aegypti (L) (Diptera: Culicidae). Estimates were made using pairwise relatedness (rxy) calculations based on alleles shared between individuals. Genotypes for eight laboratory mosquito families were determined at six RFLP loci and the observed allele frequencies were used to generate simulated distributions of rxy from full-sibling and unrelated pairs of individuals. The midpoint (mp) between the means of the pairwise rxy distributions was used to discriminate full-sibling families from unrelated families. Clusters of individuals with rxy values higher than the mp value were grouped as putative sibling families. This method was tested by calculating actual rxy for all pairwise comparisons of the known laboratory full-sibling and paternal half-sibling families, followed by upgma cluster analysis to group sibling families. The technique was then used for sibling estimations on wild caught mosquitoes collected at three locations in Trinidad, West Indies. From field populations, 35 families were estimated among 122 individuals tested with an average of 6.2 families per container. Members of 19 predicted families clustered as groups across multiple containers, providing molecular evidence for skip-oviposition behaviour in Ae. aegypti females, whereby individual females oviposit in more than one container.     

Effect of four chlorophytes on larval survival, development and adult body size of the mosquito Aedes aegypti

The effect of four microalgal chlorophytes, Chlorella vulgaris,Scenedesmus quadricauda, Chlorococcum sp. and Ankistrodesmus convolutus, on the survival, larval development and adultbody size of the mosquito Aedes aegypti was investigated. The percentage mortality of larvae fed with C. vulgaris, Chlorococcum sp.or S. quadricauda after six days was 100%, 84% and 88%,respectively. Delayed pupation and body size reduction of the mosquitoesfed with C. vulgaris, Chlorococcum sp. and S. quadricaudawere observed. In contrast, larvae fed with A. convolutus werebigger than those fed with normal insectory feed. The study showed thatC. vulgaris, S. quadricauda and Chlorococcum sp. have potentialto be used as larvicidal agents.     

The Harmonic Convergence of Fathers Predicts the Mating Success of Sons in Aedes aegypti

During courtship males often communicate information about their fitness to females. The matching of harmonic components of flight tone in male-female pairs of flying mosquitoes, or harmonic convergence, was recently described. This behaviour occurs prior to mating and has been suggested to function in mate selection. We investigated the hypothesis that harmonic convergence is a component of mosquito courtship. A key prediction of this hypothesis is that harmonic convergence should provide information to potential mates about fitness benefits. We measured the effect of harmonic convergence behaviour on the direct and indirect benefits obtained by females. We found that the sons of pairs that converged at harmonic frequencies prior to mating had increased mating success and that these offspring were themselves more likely to converge prior to mating. These results suggest that males may be able to signal information about their genetic quality to females prior to mating and that this signal may be heritable. These findings are important for our understanding of mosquito behaviour and have applications in the control of mosquito-borne disease. This study also contributes to the study of male-female interactions and signal coevolution.     

Indigenous fish species for the control of Aedes aegypti in water storage tanks in Southern México

Because of inadequate supply of water, inhabitants of five villagesclose to Tapachula, Chiapas, México, store water in cement tanksthat support large populations of Aedes aegypti. Biologicalcontrol using indigenous fish species were studied to control A. aegypti larvae in thosecontainers, since other organisms used as biological control agents areexpensive and unfamiliar to inhabitants of those towns. Other measures(chemical or physical control) are expensive and time consuming. Fiveindigenous fish species, Lepisosteus tropicus (Gill)(Lepisosteiformes: Lepisosteidae), Astyanax fasciatus (Cuvier)(Cypriniformes: Characinidae), Brycon guatemalensis (Regan)(Cypriniformes: Characinidae), Ictalurus meridionalis(Günther) (Cypriniformes: Ictaluridae) and Poecilia sphenopsValenciennes (Cyprinodontiformes: Poeciliidae), currently used asmosquito control agents in the area were tested. Container indexes (ameasure of disease transmission potential) in the tested area werealways zero during the year of the study, independent of towns and fishspecies; this was significantly (P < 0.05) different from containerindexes prior to the test as well as from controls without fish. Nosignificant (P > 0.05) differences were recorded in the efficiency ofthe tested fish species feeding on A. aegypti larvae. Our resultsshow that all tested fish species can be considered as good biologicalagents for controlling A. aegypti larvae in Southern Mexico.     

Identification of immune-related protein kinases from mosquitoes (Aedes aegypti)

Protein kinases are known to be involved in signal transduction for numerous physiological events. However, little is known about the roles of protein kinases in insect immunity. A fragment around 150 bp was amplified by polymerase chain reaction using cDNA templates from bacterial inoculated mosquitoes and primers corresponding to the conserved domain of protein kinases. Based on sequence analysis, 11 groups of protein kinases were characterized including 3 nonreceptor tyrosine kinases, 3 receptor tyrosine kinases, 3 serine/threonine kinases, and 2 novel protein kinases. The most abundant kinase obtained in this study reveals a high degree of similarity to human cholinesterase-related cell division controller (CHED) protein kinase. The expression of this mosquito CHED-like kinase is not detectable in normal female mosquitoes, but induced only after bacterial inoculation and trauma. A mosquito protein kinase was demonstrated to share homology with a plant Tousled gene, but has not yet been characterized in the animal system. In addition, analysis of the sequences of several protein kinases cloned from mosquitoes suggests that they might be involved in the regulation of cellular or humoral immunity.     

Ultrastructural changes in the muscles,midgut, hemopoietic organ,imaginal discs and Malpighian tubules of the mosquito Aedes aegypti larvae infected by the fungus Coelomomyces stegomyiae

Fungi belonging to the genus Coelomomyces can infect mosquito larvae and develop within the larval hemocoel. To examine fungal development, Aedesaegypti larvae infected with Coelomomyces stegomyiae Keilin were fixed, embedded and sectioned for both light and electron microscopy. While fungal hyphae of C. stegomyiae did not invade cells other than the cuticular epithelial cells, they did penetrate a number of tissues including muscles, midgut, hemopoietic organ, imaginal discs, and Malpighian tubules. This revised version was published online in July 2006 with corrections to the Cover Date.