Linkage of the genes for DDT and dieldrin resistance in larvae of the mosquito Aedes aegypti.

The DDT resistance gene RDDT1, and the dieldrin resistance gene Rd1 have been mapped on linkage group II with respect to visible markers, in the mosquito Aedes argypti L. The best interpretation of the data gives the order wa - Rdl - ds RDDT1 - s - y but was - Rdl -ds - y - s - RDDT1 is also possible. h is very loosely linked with RDDT1. The length of the linkage group has been considerably extended from previous studies.     

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.     

A genetical study of DDT resistance in the mosquito Aedes aegypti.

Crosses have been carried out to determine the relationship between adult DDT resistance and the three linkage groups of the mosquito Aedes aegypti. Two linkage groups were implicated in the control of DDT resistance. In the Bangkok-HR strain resistance derived mainly from linkage group III, probably with the maor effect from the gene R-DDT2. When resistance was transferred into a susceptible background, by outcrossing Bangkok-HR to strain 64 and reselecting, resistance in the resulting Bangkok-MR strain came from both linkage groups II and III.     

A salivary gland-specific, maltase-like gene of the vector mosquito, Aedes aegypti

Genomic and cDNA clones of a gene expressed specifically in the salivary glands of adult Aedes aegypti have been isolated and sequenced. This gene encodes an abundant mRNA that is transcribed throughout the male salivary gland but only in the cells of the proximal lateral lobes of the female gland. The deduced protein has many basic amino acids, several possible sites for N-glycosylation, and displays striking similarities with the products of a yeast maltase gene and three previously unidentified genes from Drosophila melanogaster. We propose the name 'Maltase-like I' (MalI) to designate this gene. The presumed function of this gene product is to assist the mosquito in its sugar-feeding capabilities. The mosquito and fruitfly genes have similar structural features 5' to the protein coding regions, indicating that these genes may share common control mechanisms.     

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.     

Pathology of mosquito iridescent virus of Aedes taeniorhynchus in cell cultures of Aedes aegypti.

An electron microscopical study was conducted on the pathology of the mosquito iridescent virus (MIV) of Aedes taeniorhynchus in monolayer cultures of Aedes aegypti cells. The sequence of events in the pathology, from the initiation of attachment through maturation and release, is presented.MIV attaches to cells and is taken up by the process of viropexis (phagocytosis) within 15 min after inoculation. Intact virions are released into the cytoplasm at 30–60 min by disruption of the phagocytic vesicles. Discrete foci of replication (viroplasm) develop in the cytoplasm within 1 day after infection. Progeny virus is assembled in the viroplasm within 2 days after infection and later appears at the cell surface, where it acquires an envelope from the plasma membrane upon budding from the cell. Virus does not accumulate to form aggregates in the cytoplasm; instead, it buds from the cell after assembly.     

Bioassay of mosquito iridescent virus of Aedes taeniorhynchus in cell cultures of Aedes aegypti.

A bioassay of mosquito iridescent virus (MIV) of Aedes taeniorhynchus was developed using cell cultures of Aedes aegypti. The dilution end point technique was based on the occurrence of cytopathic effects which were optimum at 31°C. Peleg's A. aegypti cell line was more sensitive and reliable than Singh's A. aegypti cell line for infectivity titration of the “R” and “T” strains of MIV. The highest tissue culture infectivity dose 50s (TCID₅₀) were elicited by virion:cell ratios of approximately 10. TCID₅₀ titers were significantly reduced by virus neutralization with either homologous or heterologous antiserum to either RMIV or TMIV. The virus propagated in either cell line was not infectious to A. taeniorhynchus larvae, or to the respective cells from which the virus was produced. All plaque assay attempts were unsuccessful.     

Studies on prophenoloxidase activation in the mosquito Aedes aegypti L

This study, the first of its kind in a mosquito vector species, demonstrates the feasibility of studying prophenoloxidase activation in an insect containing not more than a few microliters of hemolymph. Mosquito phenoloxidase was found to be in an inactive proenzyme form, prophenoloxidase. Mosquito prophenoloxidase required bivalent cation for its activation; Ca2+ was found to be the most efficient for activation. Concomitant amidase activity was also observed prior to phenoloxidase activity. Through Western blotting, using a cross-reactive silkworm antiprophenoloxidase antibody, our results strongly suggest that mosquito prophenoloxidase activation resulted from limited proteolysis. Protease inhibitor studies reinforced this contention showing the involvement of (a) serine protease(s) with trypsin-like activity in the activation of mosquito prophenoloxidase.     

Evidence for hormonal control of diuresis after a blood meal in the mosquito Aedes aegypti

In previous studies we have presented evidence for the role of peptides, isolated from heads of the mosquito Aedes aegypti, in stimulating fluid secretion by isolated Malpighian tubules. In the present study we conducted experiments to investigate whether these peptides are involved in hormone-mediated diuresis after a blood meal. In vivo experiments showed that the head was required to maintain diuresis after the blood meal. Whereas feeding on blood triggered a prompt diuresis in the intact mosquito, subsequent decapitation caused a gradual, not an abrupt, decline in urine excretion rate. Hemolymph collected from mosquitoes fed blood significantly stimulated fluid secretion in vitro by isolated Malpighian tubules, whereas hemolymph from unfed or blood-fed decapitated mosquitoes did not. These results indicate that a diuretic factor was released into the hemolymph after a blood meal. This factor was not present in the hemolymph of decapitated females. We identified the head as a source of diuretic factors. Peptides isolated from a head extract by high-performance liquid chromatography, when injected into the hemocoel of blood-fed decapitated mosquitoes, triggered diuresis in vivo and also stimulated fluid secretion in isolated Malpighian tubules. These studies support the hypothesis that the head is a storage site for diuretic peptides that may be released after a blood meal to control diuresis.     

Population genetic structure of the dengue mosquito Aedes aegypti in Venezuela

The mosquito Aedes aegypti is the main vector of dengue in Venezuela. The genetic structure of this vector was investigated in 24 samples collected from eight geographic regions separated by up to 1160 km. We examined the distribution of a 359-basepair region of the NADH dehydrogenase subunit 4 mitochondrial gene among 1144 Ae. aegypti from eight collections. This gene was amplified by the polymerase chain reaction and tested for variation using single strand conformation polymorphism analysis. Seven haplotypes were detected throughout Venezuela and these were sorted into two clades. Significant differentiation was detected among collections and these were genetically isolated by distance.