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.     

Linkage of a gene for DDT-resistance in adults of the mosquito Aedes aegypti.

The gene RDDT2, which gives resistance to DDT in the imago of Aedes aegypti L. has been mapped on linkage group III with respect to six visible markers. The best interpretation of the order of the genes is:- blp - blt - co - fz - wi -RDDT2 - min, but the order blp - blt - co - fz -RDDT2 - wi - min is also possible.     

The effect of DDT and rearing temperature on pecten spine number in larvae of the mosquito Aedes aegypti L.

Pecten spine number has been studied in larvae of Aedes aegypti exposed during development to low temperatures or to DDT at the fourth instar, both of which caused substantial mortality. But neither contingency caused stabilising selection for spine number, in contrast to what was observed in a previous study when larvae were exposed to scum on the surface of the rearing medium. — Two larval colour morphs, yellow and brown, did not differ in spine number.     

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.     

Inheritance of DDT dehydrochlorination and of a mechanism restricting uptake of DDT in the mosquito Aedes aegypti

Crosses and backcrosses were made between the T8 dichlorodiphenyltrichloroethane (DDT) resistant strain and NS susceptible strain. Each generation was tested for resistance, for internal levels of DDT and dichlorodiphenyldichloroethylene (DDE) (thus "DDT uptake" (DDT + DDE) and percentage DDT dehydrochlorination in vivo), and for DDT dehydrochlorination in vitro, both at the larval and adult stages. The patterns of inheritance of uptake and dehydrochlorination were different. At both life stages, dehydrochlorination (both in vivo and in vitro) was intermediate in the F1, reverting to or exceeding the parental strains in the backcrosses except in adult tests on the backcrosses to the susceptible strain where it remained intermediate. Uptake increased very substantially in the F1 compared with either parental strain and was also high in the backcrosses. This was interpreted as being due to the disruption of an uptake-restricting mechanism in T8 brought about by outcrossing. Larval resistance in the various generations was correlated significantly with dehydrochlorination, both in vivo and in vitro but bore no clear relation to uptake. Resistance in adults was found not to be correlated significantly with either.     

Targeted identification of markers linked to malaria and filarioid nematode parasite resistance genes in the mosquito Aedes aegypti.

Quantitative trait loci (QTL) have been identified for competence of the mosquito Aedes aegypti to transmit the avian malaria parasite Plasmodium gallinaceum and the human filarial parasite Brugia malayi. Efforts towards the map-based cloning of the associated genes are limited by the availability of genetic markers for fine-scale mapping of the QTL positions. Two F2 mosquito populations were subjected to bulked segregant analysis to identify random amplified polymorphic DNA (RAPD)-PCR fragments linked with the major QTL determining susceptibility to both parasites. Individual mosquitoes for the bulks were selected on the basis of their genotypes at restriction fragment length polymorphism (RFLP) loci tightly linked with the QTL. Pool-positive RAPD fragments were cloned and evaluated as RFLP markers. Of the 62 RAPD/RFLP fragments examined, 10 represented low-copy number sequences. Five of these clones were linked with the major QTL for P. gallinaceum susceptibility (pgs1), of which one clone mapped within the flanking markers that define the QTL interval. The remaining five clones were linked with the major QTL for B. malayi susceptibility (fsb1), and again one clone mapped within the flanking markers that define the QTL interval. In addition, nine RAPD/RFLP fragments were isolated that seem to be of non-mosquito origin.     

The genetic basis of pyrethroid and DDT resistance inter-relationships in Aedes aegypti I. Isolation of DDT and pyrethroid resistance factors

DDT resistance in adults of the BKPM3 strain of Aedes aegypti, homozygous for the pyrethroid resistance gene R ^py , was found to be due to factors on both chromosomes II and III. No evidence was found to suggest the presence of factors conferring resistance to permethrin on chromosome II and consequently that DDT resistance factor(s) on this chromosome are related to permethrin resistance. Further confirmation that R ^py is the major gene controlling pyrethroid resistance was obtained and also evidence that it is closely linked or allelic to the chromosome III DDT resistance factor.     

Limonoids as larvicidal components against mosquito larvae (Aedes aegypti Linn.)

The study focussed on four limonoids (calodendrolide, harrisonin, pedonin and pyroangolensolide) as larvicidal components against mosquito 2nd instar larvae of the species Aedes aegypti Linn. senso stricto. Since pyroangolensolide is close to calodendrolide in structure, it was synthesized through reduction of calodendrolide with chromium(II) chloride in acetone. Harrisonin and pedonin were extracted with cold methanol from the root bark of Harrisonia abyssinica while calodendrolide was extracted with the same solvent from the root bark of Calodendrum capense. The structure of pyroangolensolide was elucidated using physical and spectroscopic techniques. 25, 50, 75, and 100 microM of each compound were tested against the mosquito larvae. Calodendrolide was the most toxic since 100% mortality was registered at all concentrations, while pyroangolensolide showed 100% mortality up to 50 microM and for contents of 25 microM, a mortality of 70% was registered. As a result of this toxicity, lower concentrations (5, 10 and 15 microM) were tested for both calodendrolide and pyroangolensolide. Toxicity of harrisonin and pedonin was lower. The relative toxicity was in the order: calodendrolide > pyroangolensolide > harrisonin > pedonin with LC50 values of 13.2, 16.6, 28.1 and 59.2 microM, respectively.     

The effect of larval selection on adult resistance to DDT in two strains of the mosquito Aedes aegypti (L).

Evidence is presented which confirms the influence of linkage group II on adult DDT-resistance in one strain of Aedes aegypti (Bangkok-MR) but not in another strain (Bangkok-HR).     

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.     

Bioactivity of selected plant essential oils against the yellow fever mosquito Aedes aegypti larvae

The bioactivity of 14 essential oils from five plants has been studied using the brine shrimp lethality test and the Aedes aegypti larvicidal assay. All essential oils screened had LC50 values smaller than 200 microg/ml, showing significant lethality against brine shrimp. In addition, nine of the 14 essential oils tested showed toxicity against the fourth-instar A. aegypti larvae in 24 h (LC50<100 microg/ml). Of these, the leaf and bark essential oils of Cryptomeria japonica demonstrated high larvicidal activity, the most active being the leaf essential oil of C. japonica, with a LC50=37.6 microg/ml (LC90=71.9 microg/ml), followed by the bark essential oil of C. japonica also showing high activity against A. aegypti larvae, with a LC50=48.1 microg/ml (LC90=130.3 microg/ml). The results obtained from this study suggest that the leaf and bark essential oils of C. japonica are promising as larvicides against A. aegypti larvae and could be useful in the search for new natural larvicidal compounds.     

Effect of selection for dichlorodiphenyltrichloroethane (DDT) resistance on the uptake and breakdown of DDT in Aedes aegypti L

Aedes aegypti larvae and adults were selected to high levels of resistance with dichlorodiphenyltrichloroethane (DDT) along separate lines. The larval-selected line showed three responses associated with larval resistance: increased detoxication of DDT by dehydrochlorination to dichlorodiphenyldichloroethane DDE (demonstrated both in vivo and in vitro), increased tolerance to unmetabolised ("residual") DDT and, a reduction in uptake of DDT. Larval selection caused very little change in adult resistance or the uptake of DDT by adults, but there was an increase in dehydrochlorination. In the adult-selected line dehydrochlorination was increased by selection and was significantly correlated with resistance.     

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.

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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.

     

Characterisation of DDT and pyrethroid resistance in Trinidad and Tobago populations of Aedes aegypti

Insecticide resistance is an important factor in the effectiveness of Aedes aegypti control and the related spread of dengue. The objectives of this study were to investigate the status of the organochlorine dichlorodiphenyltrichloroethane (DDT) and pyrethroid (permethrin and deltamethrin) resistance in Trinidad and Tobago populations of Ae. aegypti and the underlying biochemical mechanisms. Nine populations of Ae. aegypti larvae from Trinidad and Tobago were assayed to DDT and PYs using the Centers for Disease Control and Prevention (CDC) time-mortality-based bioassay method. A diagnostic dosage (DD) was established for each insecticide using the CAREC reference susceptible Ae. aegypti strain and a resistance threshold (RT), time in which 98-100% mortality was observed in the CAREC strain, was calculated for each insecticide. Mosquitoes which survived the DD and RT were considered as resistant, and the resistance status of each population was categorised based on the WHO criteria with mortality <80% indicative of resistance. Biochemical assays were conducted to determine the activities of α and β esterases, mixed function oxidases (MFO) and glutathione-S-transferases (GST) enzymes which are involved in resistance of mosquitoes to DDT and PYs. Enzymatic activity levels in each population were compared with those obtained for the CAREC susceptible strain, and significant differences were determined by Kruskal-Wallis and Tukey's non-parametric tests (P<0.05). The established DDs were 0.01 mg l(-1), 0.2 mg l(-1) and 1.0 mg l(-1) for deltamethrin, permethrin and DDT, respectively; and the RTs for deltamethrin, permethrin and DDT were 30, 75 and 120 min, respectively. All Ae. aegypti populations were resistant to DDT (<80% mortality); two strains were incipiently resistant to deltamethrin and three to permethrin (80-98% mortality). Biochemical assays revealed elevated levels of α-esterase and MFO enzymes in all Ae. aegypti populations. All, except three populations, showed increased levels of β-esterases; and all populations, except Curepe, demonstrated elevated GST levels.Metabolic detoxification of enzymes is correlated with the manifestation of DDT and PY resistance in Trinidad and Tobago populations of Ae. aegypti. The presence of this resistance also suggests that knock down (kdr)-type resistance may be involved, hence the need for further investigations. This information can contribute to the development of an insecticide resistance surveillance programme and improvement of resistance management strategies aimed at combatting the spread of dengue in Trinidad and Tobago.