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.     

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.     

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.     

Amplified fragment length polymorphism mapping of quantitative trait loci for malaria parasite susceptibility in the yellow fever mosquito Aedes aegypti

The yellow fever mosquito Aedes aegypti has been the subject of extensive genetic research due to its medical importance and the ease with which it can be manipulated in the laboratory. A molecular genetic linkage map was constructed using 148 amplified fragment length polymorphism (AFLP) and six single-strand conformation polymorphism (SSCP) markers. Eighteen AFLP primer combinations were used to genotype two reciprocal F2 segregating populations. Each primer combination generated an average of 8.2 AFLP markers eligible for linkage mapping. The length of the integrated map was 180.9 cM, giving an average marker resolution of 1.2 cM. Composite interval mapping revealed a total of six QTL significantly affecting Plasmodium susceptibility in the two reciprocal crosses of Ae. aegypti. Two common QTL on linkage group 2 were identified in both crosses that had similar effects on the phenotype, and four QTL were unique to each cross. In one cross, the four main QTL accounted for 64% of the total phenotypic variance, and digenic epistasis explained 11.8% of the variance. In the second cross, the four main QTL explained 66% of the variance, and digenic epistasis accounted for 16% of the variance. The actions of these QTL were either dominance or underdominance. Our results indicated that at least three new QTL were mapped on chromosomes 1 and 3. The polygenic nature of susceptibility to P. gallinaceum and epistasis are important factors for significant variation within or among mosquito strains. The new map provides additional information useful for further genetic investigation, such as identification of new genes and positional cloning.     

Isolation and identification of three RFamide-immunoreactive peptides from the mosquito Aedes aegypti

Three RFamide-immunoreactive peptides were isolated from headless Aedes aegypti mosquitoes. Their structures were identified by Edman degradation or a combination of amino acid composition analysis and mass spectrometry to be: Aedes head peptide 1 [23], [Pro4]-Aedes head peptide (i.e., pGlu-Arg-Pro-Pro-Ser-Leu-Lys-Thr- Arg-Phe-amide), and Leu-Lys-Thr-Arg-Phe-amide which have been named Aedes head peptides 3 and 4, respectively.     

Microsatellite isolation and linkage group identification in the yellow fever mosquito Aedes aegypti

Microsatellites have proved to be very useful as genetic markers, as they seem to be ubiquitous and randomly distributed throughout most eukaryote genomes. However, our laboratories and others have determined that this paradigm does not necessarily apply to the yellow fever mosquito Aedes aegypti. We report the isolation and identification of microsatellite sequences from multiple genomic libraries for A. aegypti. We identified 6 single-copy simple microsatellites from 3 plasmid libraries enriched for (GA)(n), (AAT)(n), and (TAGA)(n) motifs from A. aegypti. In addition, we identified 5 single-copy microsatellites from an A. aegypti cosmid library. Genetic map positions were determined for 8 microsatellite loci. These markers greatly increase the number of microsatellite markers available for A. aegypti and provide additional tools for studying genetic variability of mosquito populations. Additionally, most A. aegypti microsatellites are closely associated with repetitive elements that likely accounts for the limited success in developing an extensive panel of microsatellite marker loci.     

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.     

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.     

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.     

Annotation and expression profiling of apoptosis-related genes in the yellow fever mosquito, Aedes aegypti

Apoptosis has been extensively studied in Drosophila by both biochemical and genetic approaches, but there is a lack of knowledge about the mechanisms of apoptosis regulation in other insects. In mosquitoes, apoptosis occurs during Plasmodium and arbovirus infection in the midgut, suggesting that apoptosis plays a role in mosquito innate immunity. We searched the Aedes aegypti genome for apoptosis-related genes using Drosophila and Anopheles gambiae protein sequences as queries. In this study we have identified eleven caspases, three inhibitor of apoptosis (IAP) proteins, a previously unreported IAP antagonist, and orthologs of Drosophila Ark, Dnr1, and BG4 (also called dFadd). While most of these genes have been previously annotated, we have improved the annotation of several of them, and we also report the discovery of four previously unannotated apoptosis-related genes. We examined the developmental expression profile of these genes in Ae. aegypti larvae, pupae and adults, and we also studied the function of a novel IAP antagonist, IMP. Expression of IMP in mosquito cells caused apoptosis, indicating that it is a functional pro-death protein. Further characterization of these genes will help elucidate the molecular mechanisms of apoptosis regulation in Ae. aegypti.     

The genetic basis of resistance and sensitivity to the meiotic drive gene D in the mosquito Aedes aegypti L.

A study has been made on the genetic basis of meiotic drive at the Distorter (D) locus which, in coupling with the male-determining gene (or region) M on the Y chromosome, causes production of excess male progeny. Its effect is regulated by the sensitivity/resistance of the X chromosome. This study demonstrates that there are two major loci controlling resistance/sensitivity to MD: (1) the m gene (or region) on the X chromosome (allelic with M) which may be either m ^R or m ^S (resistant or sensitive), (2) the t (tolerance) gene (or genes) which recombines with m and, if present, largely counteracts the effect of m ^S . There is also evidence that MD itself is capable of limited adaptation.The conclusions were derived from using MD males of the T30 or ACCRA strains (from Trinidad and Ghana respectively). The work involved the use of the CHIPEI and RED strains with sensitive X chromosomes, the latter also carrying the t (tolerance) gene which is linked to re (red eye) and m (the sex-determining locus or region) but recombines with both. The implications of these findings for using MD as a method of population control are discussed.     

Comparative genome analysis of the yellow fever mosquito Aedes aegypti with Drosophila melanogaster and the malaria vector mosquito Anopheles gambiae

An in silico comparative genomics approach was used to identify putative orthologs to genetically mapped genes from the mosquito, Aedes aegypti, in the Drosophila melanogaster and Anopheles gambiae genome databases. Comparative chromosome positions of 73 D. melanogaster orthologs indicated significant deviations from a random distribution across each of the five A. aegypti chromosomal regions, suggesting that some ancestral chromosome elements have been conserved. However, the two genomes also reflect extensive reshuffling within and between chromosomal regions. Comparative chromosome positions of A. gambiae orthologs indicate unequivocally that A. aegypti chromosome regions share extensive homology to the five A. gambiae chromosome arms. Whole-arm or near-whole-arm homology was contradicted with only two genes among the 75 A. aegypti genes for which orthologs to A. gambiae were identified. The two genomes contain large conserved chromosome segments that generally correspond to break/fusion events and a reciprocal translocation with extensive paracentric inversions evident within. Only very tightly linked genes are likely to retain conserved linear orders within chromosome segments. The D. melanogaster and A. gambiae genome databases therefore offer limited potential for comparative positional gene determinations among even closely related dipterans, indicating the necessity for additional genome sequencing projects with other dipteran species.     

Building a better mosquito: identifying the genes enabling malaria and dengue fever resistance in A. gambiae and A. aegypti mosquitoes

In this interview, George Dimopoulos focuses on the physiological mechanisms used by mosquitoes to combat Plasmodium falciparum and dengue virus infections. Explanation is given for how key refractory genes, those genes conferring resistance to vector pathogens, are identified in the mosquito and how this knowledge can be used to generate transgenic mosquitoes that are unable to carry the malaria parasite or dengue virus.