Egg chorion tanning in Aedes aegypti mosquito

The biochemical pathway of egg chorion tanning in the mosquito, Aedes aegypti, is described and compared with chorion protein crosslinking in Drosophila and silkmoths and the biochemical pathways of cuticular tanning in insects. Phenol oxidase, dopa decarboxylase and tyrosine are critical components involved in egg chorion tanning in A. aegypti. Tanning of the mosquito egg chorion is initiated following activation of phenol oxidase, which then catalyzes the hydroxylation of tyrosine to dopa and further oxidizes dopa and dopamine to their respective o-quinones. Because intramolecular cyclization is much slower in dopaminequinone than dopaquinone, the chance to react with external nucleophiles to participate in protein crosslinking reactions also is much greater in dopaminequinone than dopaquinone. This might partly explain the necessity for the involvement of dopa decarboxylase in mosquito chorion tanning. Intramolecular cyclization of dopaquinone and dopaminequinone to form dopachrome and dopaminechrome, respectively, the structural rearrangement of these aminochromes to produce 5,6-dihydroxyindole, and the subsequent oxidation of 5,6-dihydroxyindole by phenol oxidase also lead to melanin formation during egg chorion tanning.     

Isolation of trypsin isozymes from the mosquito Aedes aegypti (L.)

Trypsin has been isolated from midgut homogenates of blood-fed females of Aedes aegypti by a simple two-step purification procedure: ion-exchange chromatography followed by affinity chromatography. The resulting mosquito trypsin contains a number of isozymes, among which 5 major SDS-PAGE bands are recognized with molecular weights of 26.7, 28.5, 29.7, 31.0 and 32.0 kdaltons, as are some minor bands above and below this range. The isozymic pattern is comparable to that in crude homogenates. Isoelectric focussing of purified trypsin however, revealed over 20 tryptic isozymes, demonstrating that several isozymes segregate into subforms. A high correlation between TAME-active fractions and their DFP equivalent was demonstrated by using ³H-labelled DFP as a marker for trypsin on native acrylamide gels.The purification factor and the specific activities are discussed with respect to the unusual amounts of protein of dietary origin present in the midgut homogenates. Interference of blood-borne coagulation factors of a tryptic nature is unlikely.     

Distention-mediated egg maturation in the mosquito,Aedes aegypti

Abdominal distention accelerates the release of a factor from the head of blood-fed Aedes aegypti mosquitoes. The critical period during which the head is required for oögenesis following blood ingestion is approx 6 h with a 5 μl meal, but small blood meals of 1 μl require the head to be present for significantly longer. Increasing the abdominal distention by supplementing the 1 μl meal with saline results in a critical period similar to that with 5 μl of blood. The information from the distended abdomen appears to travel via the ventral nerve cord. Transection of the ventral nerve cord prevents oögenesis from occurring after small blood meals, but not with larger blood volumes. Topical application of 100 pg of juvenile hormone III can substitute for the distention message.     

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.     

Population structure of the mosquito Aedes aegypti (Stegomyia aegypti) in Pakistan

Eleven microsatellite markers were used to determine the genetic population structure and spread of Aedes aegypti (Stegomyia aegypti) (Diptera: Culicidae) in Pakistan using mosquitoes collected from 13 different cities. There is a single genetic cluster of Ae. aegypti in Pakistan with a pattern of isolation by distance within the population. The low level of isolation by distance suggests the long‐range passive dispersal of this mosquito, which may be facilitated by the tyre trade in Pakistan. A decrease in genetic diversity from south to north suggests a recent spread of this mosquito from Karachi. A strong negative correlation between genetic distance and the quality of road connections shows that populations in cities connected by better road networks are less differentiated, which suggests the human‐aided passive dispersal of Ae. aegypti in Pakistan. Dispersal on a large spatial scale may facilitate the strategy of introducing transgenic Ae. aegypti or intracellular bacteria such as Wolbachia to control the spread of dengue disease in Pakistan, but it also emphasizes the need for simple measures to control container breeding sites.     

Lethal genes on the sex chromosomes concealed in a population of the mosquito Aedes aegypti L.

A population has been examined in which an overall parity between the sexes hides considerable between-family variation in sex ratio. A proportion of families show highly distorted sex ratios, with either an excess of females or an excess of males. Distorted sex ratios are invariably associated with mortality in the immature stages at a level appropriate to the action of recessive lethal genes. It has been shown that 26% of M-bearing (Y) chromosomes and at least 24% of m-bearing (X) chromosomes carry a recessive lethal gene.Two such genes have been investigated. l kills males and, in a cross between two heterozygotes, gives rise to a sex ratio close to 2:1 (excess families). k kills females and, in a cross between two heterozygotes, gives rise to a sex ratio close to 1:2 (excess families). Selection for excess or excess did not increase the level of sex ratio distortion.No crossing over occurs between k and the M/m locus whereas l shows 5–10% recombination with M/m. A test for allelism confirmed that l and k are not allelic. The penetrance of k is complete whereas l shows somewhat less than full penetrance. The penetrance of l has been improved by selection.The high frequency of lethals remained in the population during the two year period of study. There was evidence for heterosis preserving this frequency, the heterozygotes living longer and producing more progeny. However lethals were no longer to be found after four further years of laboratory culture.     

FLP-mediated recombination in the vector mosquito, Aedes aegypti.

The activity of a yeast recombinase, FLP, on specific target DNA sequences, FRT, has been demonstrated in embryos of the vector mosquito, Aedes aegypti. In a series of experiments, plasmids containing the FLP recombinase under control of a heterologous heat-shock gene promoter were co-injected with target plasmids containing FRT sites into preblastoderm stage mosquito embryos. FLP-mediated recombination was detected between (i) tandem repeats of FRT sites leading to the excision of specific DNA sequences and (ii) FRT sites located on separate plasmids resulting in the formation of heterodimeric or higher order multimeric plasmids. In addition to FRT sites originally isolated from the yeast 2 microns plasmid, a number of synthetic FRT sites were also used. The synthetic sites were fully functional as target sites for recombination and gave results similar to those derived from the yeast 2 microns plasmid. This successful demonstration of yeast FLP recombinase activity in the mosquito embryo suggests a possible future application of this system in establishing transformed lines of mosquitoes for use in vector control strategies and basic studies.     

Genomic analysis of detoxification genes in the mosquito Aedes aegypti

Annotation of the recently determined genome sequence of the major dengue vector, Aedes aegypti, reveals an abundance of detoxification genes. Here, we report the presence of 235 members of the cytochrome P450, glutathione transferase and carboxy/cholinesterase families in Ae. aegypti. This gene count represents an increase of 58% and 36% compared with the fruitfly, Drosophila melanogaster, and the malaria mosquito, Anopheles gambiae, respectively. The expansion is not uniform within the gene families. Secure orthologs can be found across the insect species for enzymes that have presumed or proven biosynthetic or housekeeping roles. In contrast, subsets of these gene families that are associated with general xenobiotic detoxification, in particular the CYP6, CYP9 and alpha esterase families, have expanded in Ae. aegypti. In order to identify detoxification genes associated with resistance to insecticides we constructed an array containing unique oligonucleotide probes for these genes and compared their expression level in insecticide resistant and susceptible strains. Several candidate genes were identified with the majority belonging to two gene families, the CYP9 P450s and the Epsilon GSTs. This 'Ae. aegypti Detox Chip' will facilitate the implementation of insecticide resistance management strategies for arboviral control programmes.     

The mosquito Aedes aegypti (L.): evidence for three new proteinases

The effect of inhibitors was studied on the proteolytic activity of crude extracts of the mosquito Aedes aegypti (L.), prepared 25 h after blood intake. This activity is only partially inhibited by the lima bean trypsin inhibitor (LBTI) or by EDTA. Experiments with mixed inhibitors are used to indicate that, apart from the well characterized trypsin-like enzymes, at least three other proteinases are present. These are an LBTI-resistant serine proteinase, a metal chelator-sensitive proteinase, and a proteinase which can only be inhibited by the ovomucoid inhibitor. These newly described proteinases are inactive against several model substrates developed for mammalian enzymes. They are partially separable by ion-exchange chromatography.     

A cytogenetic analysis of meiotic drive in the mosquito,Aedes aegypti (L.)

Meiotic drive in Aedes aegypti (L.) is shown by a Giemsa C-banding technique to be associated with preferential isochromatid breakage of the X chromosome during male meiosis. These breaks remain open at least until anaphase-I and, since the range of cells affected is proportional to the sensitivity of the X chromosome to the Distorter gene, it is argued that they are directly related to the decreased number of spermatozoa found in distorting males. This reduction is considered to be attributable to the degeneration of more X- than Y-bearing spermatids but it is probable that some non-functional X-bearing spermatozoa are also produced. Chromosome breakage is almost completely confined to four sites, two adjacent to the centromere, one just proximal to the intercalary band and another about the centre of the unbanded arm. Although the first three of these lie within a region in which crossing-over does not take place, fragmentation occurs more frequently in a chiasmate arm than in one devoid of chromatid exchange.     

Chemicals isolated from Justicia adhatoda Linn reduce fitness of the mosquito,Aedes aegypti L

Extracts from Justicia adhatoda L. (Acanthaceae) strongly reduced the fitness of the mosquito, Aedes aegypti Linn. The methanolic extracts inhibited several enzymes responsible for protecting insects from oxidative and other damage, including glutathione‐S‐transferase, superoxide dismutase, cytochrome P450, and α‐ and β‐esterases. They increased repellency (maximum repellency at 100 ppm) in host‐seeking adult females using the “arm‐in cage assay.” Histopathological examination showed the extracts led to serious midgut cell damage. Justicia adhatoda extracts led to reduced fecundity and oviposition of gravid females compared to controls. The extracts led to substantially reduced A. aegypti survival. We infer that the extracts have potential to reduce pathogen transmission by suppressing population growth of A. aegypti, and possibly other mosquito species.     

Activation of vitellogenin synthesis in the mosquito Aedes aegypti by ecdysone

Injected β-ecdysone was found to induce the synthesis of yolk protein (vitellogenin) in adult female Aedes aegypti without a blood meal. After injection of 5 μg ecdysone per mosquito, vitellogenin constituted 80 per cent of the total protein secreted by explanted fat body, a proportion comparable to that produced by fat body from blood-fed females. Moreover, the time course of induction of vitellogenin synthesis in ecdysone-injected mosquitoes was similar to that triggered by a blood meal. Response to ecdysone is dosedependent: 0·5 μg per female was required to stimulate synthesis to 50 per cent of the level found 18 hr after a blood meal. Ecdysone was effective in decapitated or ovariectomized mosquitoes, and also when applied directly to fat body preparations in vitro. Thus it appears that ecdysone acts directly on the fat body to induce specific protein synthesis, as does the vitellogenin stimulating hormone (VSH) from the ovary of blood-fed mosquitoes. These results suggest that ecdysone can replace VSH in inducing vitellogenin synthesis in the unfed mosquito.     

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.