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.     

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.     

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.     

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.     

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.     

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.     

Defense reactions by larvae of Aedes aegypti during infection by the aquatic fungus Lagenidium giganteum (Oomycete)

Summary The adherence of zoospores of Lagenidium giganteum to the cuticle of mosquito larvae is the initial step in the infection process. Subsequently, a germ tube penetrates the integument, inducing a rapid melanization of the injured cuticle and epidermis. After entering the hemocoel the developing hyphae are occasionally encapsulated locally. This process is slow (6 to 12 h postincubation) and most frequently cell-free, although it can be mediated by circulating hemocytes. Sporadic hemocyte mediation of the humoral encapsulation process in larval stages of Culicidae adds a previously unreported dimension to this unusual type of defense reaction. The defense reactions of larvae of Aedes aegypti were ineffective against observed infection by Lagenidium giganteum.     

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.     

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.     

Ultrastructure and morphology of midgut visceral muscle in early pupal Aedes aegypti mosquitoes

These studies focus on the pupal Aedes aegypti midgut muscularis for the first 26 h following larval-pupal transition. The midgut muscularis of Ae. aegypti pupae during this first half of the pupal stadium is a grid of both circularly and longitudinally oriented muscle bands, arranged in a manner resembling that of the larvae. While many muscle bands exhibit signs of degeneration during the time period studied, not all bands degrade, nor is this degradation simultaneous. Band deterioration involves destruction of internal elements while the muscle fiber plasma membrane remains intact. Deterioration of contractile elements may involve proteosome-like structures and associated enzymes. Many features of the larval muscularis including cruciform cells, bifurcating circular bands, and bifurcating longitudinal bands of muscle are retained during the time period investigated. Neuromuscular junctions along some muscle bands are retained through at least 16 h into the pupal stadium. The selective nature of muscle fiber degradation, coupled with the retention of larval features and neural input, may allow for limited functionality of the muscularis during metamorphosis. Evidence of sexual dimorphism in the midgut muscularis of male and female Ae. aegypti pupae was not observed during the time period studied.     

Proteome of Aedes aegypti larvae in response to infection by the intracellular parasite Vavraia culicis

We report on the modification of the Aedes aegypti larval proteome following infection by the microsporidian parasite Vavraia culicis. Mosquito larvae were sampled at 5 and 15 days of age to compare the effects of infection when the parasite was in two different developmental stages. Modifications of the host proteome due to the stress of infection were distinguished from those of a more general nature by treatments involving hypoxia. We found that the major reaction to stress was the suppression of particular protein spots. Older (15 days) larvae reacted more strongly to infection by V. culicis (46% of the total number of spots affected; 17% for 5 days larvae), while the strongest reaction of younger (5 days) larvae was to hypoxia for pH range 5-8 and to combined effects of infection and hypoxia for pH range 3-6. MALDI-TOF results indicate that proteins induced or suppressed by infection are involved directly or indirectly in defense against microorganisms. Finally, our MALDI-TOF results suggest that A. aegypti larvae try to control or clear V. culicis infection and also that V. culicis probably impairs the immune defense of this host via arginases-NOS competition.     

Organization, ultrastructure, and development of midgut visceral muscle in larval Aedes aegypti

The midgut muscularis of larvae of the mosquito Aedes aegypti takes the form of a grid of longitudinal and circular muscle bands. The longitudinal and circular bands overlap at near right angles at many areas of intersection. The longitudinal bands run the length of the midgut. However, some bands of circular muscle, located in the anterior midgut, pass only partway around the gut. An unusual feature was observed at some regions where longitudinal and circular bands of muscle intersect: filaments oriented at near right angles to one another were present in the same membrane-bound fiber. These cruciform regions send contractile elements into both circular and longitudinal bands. The muscularis was fixed in a contracted state, so most of the sarcomeres are represented by complete overlap of myosin and lighter staining actin filaments. Features characteristic of supercontracting muscle, including perforated Z-lines, were seen in sarcomeres of circular muscle bands. Small invaginations resembling transverse tubules were present but a sarcoplasmic reticulum was not observed. While occasional cells that may be neurons or neurosecretory cells were observed, a network that might serve to coordinate the segmentation and peristaltic movement of the muscularis was not apparent.     

The role of the Aedes aegypti Epsilon glutathione transferases in conferring resistance to DDT and pyrethroid insecticides

The Epsilon glutathione transferase (GST) class in the dengue vector Aedes aegypti consists of eight sequentially arranged genes spanning 53,645 bp on super contig 1.291, which maps to chromosome 2. One Epsilon GST, GSTE2, has previously been implicated in conferring resistance to DDT. The amino acid sequence of GSTE2 in an insecticide susceptible and a DDT resistant strain differs at five residues two of which occur in the putative DDT binding site. Characterization of the respective recombinant enzymes revealed that both variants have comparable DDT dehydrochlorinase activity although the isoform from the resistant strain has higher affinity for the insecticide. GSTe2 and two additional Epsilon GST genes, GSTe5 and GSTe7, are expressed at elevated levels in the resistant population and the recombinant homodimer GSTE5-5 also exhibits low levels of DDT dehydrochlorinase activity. Partial silencing of either GSTe7 or GSTe2 by RNA interference resulted in an increased susceptibility to the pyrethroid, deltamethrin suggesting that these GST enzymes may also play a role in resistance to pyrethroid insecticides.     

Additional morphological and physiological heterogeneity within the midgut of larval Aedes aegypti (Diptera: Culicidae) revealed by histology, electrophysiology, and effects of Bacillus thuringiensis endotoxin

Analysis of larval Aedes aegypti midgut using scanning electron microscopy, nuclear and mitochondrial dyes, response to Bacillus thuringiensis israelensis CryIVB toxin, and electrophysiology is described. The anterior ventriculus ("stomach") region is found to have much lower mitochondrial densities than other midgut regions. The transitional region is distinguished by apical surface architecture, and by region-specific effects of CryIVB endotoxin. In this region CryIVB causes holes ranging from 1.0 to 7.0 microm in diameter (mean 3.3+/-0.53 microm, N=12), blisters 16.9+/-1.54 microm in diameter (N=10), and separation of adjacent cells. The holes are not consistent with damage due to the colloid osmotic lysis model of delta-endotoxin activity. The posterior ventriculus possesses a distinctive cellular architecture consisting of hemispherical, domed apical membranes surrounded by deep clefts. Functional and morphological heterogeneity is revealed within the posterior ventriculus, with the anterior end dominating the electrical profile of isolated, perfused preparations and showing the greatest response to serotonin. Hyperpolarization of the transepithelial potential by serotonin occurred in conjunction with a decrease in the space constant lambda, ruling out closure of ion channels as the mechanism of action of serotonin.     

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.     

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.     

Susceptibility of Aedes aegypti and Aedes albopictus larvae to Ascogregarina culicis and Ascogregarina taiwanensis (Apicomplexa: Lecudinidae) from Florida

The susceptibility of Aedes aegypti to Ascogregarina culicis and Aedes albopictus to Ascogregarina taiwanensis was examined with mosquito and parasite strains from Tampa, FL. When each host was bioassayed with its natural gregarine, the infection intensity indicated that Ae. aegypti was 59% more susceptible to A. culicis (87 gamonts/larva) than Ae. albopictus to A. taiwanensis (47 gamonts/larva). Infections in single and mixed host populations exposed to 100 oocysts/larva of one and both parasites demonstrated that Ae. aegypti harbors higher A. culicis gamont loads than Ae. albopictus of A. taiwanensis. In dual gregarine exposures of single host populations, the A. culicis infection intensity in Ae. aegypti was reduced by approximately 50%. A. taiwanensis exhibited the same capability of infecting Ae. albopictus in single and dual exposures. In mixed host populations there were no cross infections, but A. taiwanensis in Ae. albopictus produced an infection intensity of approximately 70% lower than that of A. culicis in Ae. aegypti.     

Estimation of the number of full sibling families at an oviposition site using RAPD-PCR markers: applications to the mosquito Aedes aegypti

There are many species in which groups of individuals encountered in the field are known to consist of mixtures of full-sibling families. We describe a statistical technique, based on the use of random amplified polymorphic DNA-polymerase chain reaction (RAPD-PCR) markers, that allows for the estimation of the number of families contained in these groups. We test the technique on full-sibling families of the mosquito Aedes aegypti, a species that distributes its eggs among several locations. Mixtures of 10 families with 15 individuals per family were analyzed using 40 RAPD-PCR loci amplified by 5 primers. Our analysis accurately estimated the number of families. The technique was accurate when the number of families was small or when family sizes were small and variable.