Mating behaviour in the mosquito, Anopheles gambiae s.1.save

ABSTRACT. Mating behaviour in the Anopheles gambiae complex was studied in the laboratory, using an infra-red TV system to make observations in the 'dark'. Maximum fertilization occurred when both sexes were at least 2 days old. In LD 12:12 most mating took place in the first hour of the dark phase. At this time, the males had erect fibrillae on the antennae and showed the maximum response to an artificial female flight tone, approaching, landing-on and attempting to clasp the sound source. The cycle of fibrillar erection and of responsiveness to sound continued in constant dark. Males did not appear to discriminate on contact, between their own and other species. Previously mated females actively resisted copulation, but, when tethered, were unable to prevent the deposition of further internal mating plugs. Free-flying, previously inseminated, females prevented the males from depositing internal plugs, but not from ejaculating and depositing external plugs. The differences between A.gambiae and the better known Stegomyia mosquitoes appear to be related to their different habits, A.gambiae males being less likely to encounter females of other species or inseminated females of their own species at the restricted time and place of mating.     

Oogenesis in the malaria mosquito Anopheles gambiae

Summary Oogenesis has been followed with the electron microscope in 2 strains of the malaria mosquito Anopheles gambiae, from the emergence of the adult (oocytes at leptonema) till shortly before the oocytes are ready for oviposition. After pachynema the chromosomes form a karyosphere and a fibrous capsule develops around it. Work on other mosquitoes suggests that the capsule may be related to the synaptonemal complexes. Both Anopheles strains contain at some time an extrachromosomal (not DNA-containing) body comparable to the karyosphere in size. Clusters of granules are present at the surface of the nucleolus and free in the nucleoplasm. Tentative results indicate that they may contain DNA. During oogenesis the nucleolus becomes very large, mainly because of proliferation of the nucleolonema. Towards the end of oocyte development the nucleus assumes the large canoe-shape also seen in Aedes and Culex. Nucleolonema traverse the entire nucleus, and modified granular clusters are found throughout.     

Thioredoxin reductase from the malaria mosquito Anopheles gambiae.

The mosquito, Anopheles gambiae, is an important vector of Plasmodium falciparum malaria. Full genome analysis revealed that, as in Drosophila melanogaster, the enzyme glutathione reductase is absent in A. gambiae and functionally substituted by the thioredoxin system. The key enzyme of this system is thioredoxin reductase-1, a homodimeric FAD-containing protein of 55.3 kDa per subunit, which catalyses the reaction NADPH + H+ + thioredoxin disulfide-->NADP+ + thioredoxin dithiol. The A. gambiae trxr gene is located on chromosome X as a single copy; it represents three splice variants coding for two cytosolic and one mitochondrial variant. The predominant isoform, A. gambiae thioredoxin reductase-1, was recombinantly expressed in Escherichia coli and functionally compared with the wild-type enzyme isolated in a final yield of 1.4 U.ml(-1) of packed insect cells. In redox titrations, the substrate A. gambiae thioredoxin-1 (Km=8.5 microm, kcat=15.4 s(-1) at pH 7.4 and 25 degrees C) was unable to oxidize NADPH-reduced A. gambiae thioredoxin reductase-1 to the fully oxidized state. This indicates that, in contrast to other disulfide reductases, A. gambiae thioredoxin reductase-1 oscillates during catalysis between the four-electron reduced state and a two-electron reduced state. The thioredoxin reductases of the malaria system were compared. A. gambiae thioredoxin reductase-1 shares 52% and 45% sequence identity with its orthologues from humans and P. falciparum, respectively. A major difference among the three enzymes is the structure of the C-terminal redox centre, reflected in the varying resistance of catalytic intermediates to autoxidation. The relevant sequences of this centre are Thr-Cys-Cys-SerOH in A. gambiae thioredoxin reductase, Gly-Cys-selenocysteine-GlyOH in human thioredoxin reductase, and Cys-X-X-X-X-Cys-GlyOH in the P. falciparum enzyme. These differences offer an interesting approach to the design of species-specific inhibitors. Notably, A. gambiae thioredoxin reductase-1 is not a selenoenzyme but instead contains a highly unusual redox-active Cys-Cys sequence.     

Insecticide susceptibility levels of Anopheles gambiae s.l. in the area of Ouagadougou, Burkina Faso

Tests to evaluate the susceptibility level in Anopheles gambiae s.1. from Ouagadougou and two nearby villages have been carried out. Anopheles gambiae s.1. larvae from Ouagadougou showed complete susceptibility to organophosphates and carbamates, and adults showed low-level resistance to DDT. Nine percent survival of adult An. gambiae s.1. to one-hour exposure of 4% DDT was observed in samples from Zagtouli village while in those from Koubri village, where dieldrin also was tested, resistance to both organochlorine insecticides was detected.     

Movement of Anopheles gambiae s.l. malaria vectors between villages in The Gambia

ovement of mosquitoes belonging to the Anopheles gambiae complex (mixed wild populations of An.arabiensis, An.gambiae and An.melas ) between three neighbouring rural villages in The Gambia was investigated by mark-release-recapture. A total of 12,872 mosquitoes were collected in bednets, marked with a magenta fluorescent powder and released over a 15-day period in one of the villages. A further 15,507 mosquitoes were collected in exit traps, marked with a yellow powder and released over the same period. Mosquitoes were captured daily in all three villages using pyrethrum spray catches, as well as bednet and exit trap catches. The catching period extended for 6 days after the last day of release.
Of the mosquitoes released, 372 (1.3%) were recaptured 2–21 days later. Of these recaptures, 272 were caught in the release village, and 98 were caught in other villages situated 1–1.4 km away. The 'movement index' between villages was calculated as 17.2% (12.2–22.4% confidence limits) for mosquitoes released after feeding and 20.1% (14.7–25.3%) for those released unfed.
These results suggest that movement of mosquitoes between neighbouring villages in The Gambia seriously affects the entomological evaluation of pyrethroid-impregnated bednet programmes in areas where treated and untreated villages are interspersed.     

Chromosome end elongation by recombination in the mosquito Anopheles gambiae.

One of the functions of telomeres is to counteract the terminal nucleotide loss associated with DNA replication. While the vast majority of eukaryotic organisms maintain their chromosome ends via telomerase, an enzyme system that generates short, tandem repeats on the ends of chromosomes, other mechanisms such as the transposition of retrotransposons or recombination can also be used in some species. Chromosome end regression and extension were studied in a medically important mosquito, the malaria vector Anopheles gambiae, to determine how this dipteran insect maintains its chromosome ends. The insertion of a transgenic pUChsneo plasmid at the left end of chromosome 2 provided a unique marker for measuring the dynamics of the 2L telomere over a period of about 3 years. The terminal length was relatively uniform in the 1993 population with the chromosomes ending within the white gene sequence of the inserted transgene. Cloned terminal chromosome fragments did not end in short repeat sequences that could have been synthesized by telomerase. By late 1995, the chromosome ends had become heterogeneous: some had further shortened while other chromosomes had been elongated by regenerating part of the integrated pUChsneo plasmid. A model is presented for extension of the 2L chromosome by recombination between homologous 2L chromosome ends by using the partial plasmid duplication generated during its original integration. It is postulated that this mechanism is also important in wild-type telomere elongation.     

An eclosion timing mechanism in the mosquito Anopheles gambiae

The circadian control of adult emergence was studied in Anopheles gambiae. In contrast to the situation reported for other mosquitoes, the timing of adult eclosion can be modified by the light regime. Comparison of the timing of pupal—adult ecdysis in groups of individuals pupating at the same time and then kept either in 12 h light alternating with 12 h dark (LD 12:12) or constant light, at temperatures from 22 to 34·5°C, showed that the timing can be modified by the light regime. In LD, eclosion due to take place during the middle and later part of the light phase was delayed, giving a peak near light-off; the maximum delay was of the order of 4–5 h at both 22 and 34·5°C. This effect appears to be mediated by a temperature-compensated timing mechanism. Experiments with different light regimes indicated that the time cue is a previous change from light to dark. When this was given to late fourth stage larvae it affected the timing of adult eclosion without affecting the time of pupation.     

Evolution of the dorsal-ventral patterning network in the mosquito, Anopheles gambiae

The dorsal-ventral patterning of the Drosophila embryo is controlled by a well-defined gene regulation network. We wish to understand how changes in this network produce evolutionary diversity in insect gastrulation. The present study focuses on the dorsal ectoderm in two highly divergent dipterans, the fruitfly Drosophila melanogaster and the mosquito Anopheles gambiae. In D. melanogaster, the dorsal midline of the dorsal ectoderm forms a single extra-embryonic membrane, the amnioserosa. In A. gambiae, an expanded domain forms two distinct extra-embryonic tissues, the amnion and serosa. The analysis of approximately 20 different dorsal-ventral patterning genes suggests that the initial specification of the mesoderm and ventral neurogenic ectoderm is highly conserved in flies and mosquitoes. By contrast, there are numerous differences in the expression profiles of genes active in the dorsal ectoderm. Most notably, the subdivision of the extra-embryonic domain into separate amnion and serosa lineages in A. gambiae correlates with novel patterns of gene expression for several segmentation repressors. Moreover, the expanded amnion and serosa anlage correlates with a broader domain of Dpp signaling as compared with the D. melanogaster embryo. Evidence is presented that this expanded signaling is due to altered expression of the sog gene.     

Multimodal pyrethroid resistance in malaria vectors, Anopheles gambiae s.s., Anopheles arabiensis, and Anopheles funestus s.s. in western Kenya

Anopheles gambiae s.s., Anopheles arabiensis, and Anopheles funestus s.s. are the most important species for malaria transmission. Pyrethroid resistance of these vector mosquitoes is one of the main obstacles against effective vector control. The objective of the present study was to monitor the pyrethroid susceptibility in the 3 major malaria vectors in a highly malaria endemic area in western Kenya and to elucidate the mechanisms of pyrethroid resistance in these species. Gembe East and West, Mbita Division, and 4 main western islands in the Suba district of the Nyanza province in western Kenya were used as the study area. Larval and adult collection and bioassay were conducted, as well as the detection of point mutation in the voltage-gated sodium channel (1014L) by using direct DNA sequencing. A high level of pyrethroid resistance caused by the high frequency of point mutations (L1014S) was detected in An. gambiae s.s. In contrast, P450-related pyrethroid resistance seemed to be widespread in both An. arabiensis and An. funestus s.s. Not a single L1014S mutation was detected in these 2 species. A lack of cross-resistance between DDT and permethrin was also found in An. arabiensis and An. funestus s.s., while An. gambiae s.s. was resistant to both insecticides. It is noteworthy that the above species in the same area are found to be resistant to pyrethroids by their unique resistance mechanisms. Furthermore, it is interesting that 2 different resistance mechanisms have developed in the 2 sibling species in the same area individually. The cross resistance between permethrin and DDT in An. gambiae s.s. may be attributed to the high frequency of kdr mutation, which might be selected by the frequent exposure to ITNs. Similarly, the metabolic pyrethroid resistance in An. arabiensis and An. funestus s.s. is thought to develop without strong selection by DDT.     

DDT resistance in Anopheles gambiae declines with mosquito age

Adult Anopheles gambiae Giles mosquitoes from Zanzibar were tested on a standard discriminating dose of DDT which reliably kills susceptible mosquitoes. Adults from wild-caught larvae reared in the laboratory, and from the F1 progeny of wild-caught adults, showed less than 5% test mortality when newly-emerged, but mortality rose with age to over 90% when they were 12-14 days old. Wild-caught mixed-age adults showed an intermediate mortality rate of 25%, close to the rate predicted from laboratory results for a fully resistant population with an age-structure typical of this species in natural conditions. It is inferred that older, genetically resistant insects may be scored as susceptible, so that routine susceptibility tests with wild-caught adults underestimate the frequency of resistance. In Zanzibar, such tests probably helped to persuade spraying authorities to continue spraying DDT in spite of resistance. Resistance which is restricted to younger insects may nonetheless reduce the effectiveness of spraying.     

Conserved boundary elements from the Hox complex of mosquito,Anopheles gambiae

The conservation of hox genes as well as their genomic organization across the phyla suggests that this system of anterior–posterior axis formation arose early during evolution and has come under strong selection pressure. Studies in the split Hox cluster of Drosophila have shown that proper expression of hox genes is dependent on chromatin domain boundaries that prevent inappropriate interactions among different types of cis-regulatory elements. To investigate whether boundary function and their role in regulation of hox genes is conserved in insects with intact Hox clusters, we used an algorithm to locate potential boundary elements in the Hox complex of mosquito, Anopheles gambiae. Several potential boundary elements were identified that could be tested for their functional conservation. Comparative analysis revealed that like Drosophila, the bithorax region in A. gambiae contains an extensive array of boundaries and enhancers organized into domains. We analysed a subset of candidate boundary elements and show that they function as enhancer blockers in Drosophila. The functional conservation of boundary elements from mosquito in fly suggests that regulation of hox genes involving chromatin domain boundaries is an evolutionary conserved mechanism and points to an important role of such elements in key developmentally regulated loci.     

Dispersion of Anopheles gambiae s.l. in an urban zone of Ouagadougou (Burkina Faso)

Dispersion of Anopheles gambiae s.l. from larval breeding places was examined in the town of Ougadougou, Burkina Faso. A suitable study area was chosen, characterized by high concentration of larval breeding places along a water reservoir. Mosquito density per room was calculated from the results of pyrethrum spray catches carried out on a series of seven groups of collecting stations along a transect from 100 to 1050 m from the water reservoir area. High density and uniform distribution of houses and human host in the urban area appear associated with a remarkably low degree of dispersion since most of An. gambiae s.l. were collected within 200 m (males) or 300 m (females) from the breeding sites. Malaria transmission is consequently very focal in these conditions.     

Spectrum of metabolic-based resistance to DDT and pyrethroids in Anopheles gambiae s.l. populations from Cameroon.

Some populations of Anopheles gambiae s.l. from Cameroon were reported to develop resistance to DDT or pyrethroids but were free of the kdr mutation "Leucine-Phenylalanine" (Leu-Phe). This study reports on the metabolic activity of non-specific esterases (NSEs), mixed function oxidases (MFOs), and glutathione S-transferases (GSTs), three enzyme systems commonly involved in insecticide resistance. Biochemical assays were performed in DDT or pyrethroid-resistant populations of An. gambiae s.l. from Douala, Mbalmayo, Pitoa, and Simatou neighborhoods. Enzyme activity was compared to the Kisumu-susceptible reference strain using the Mann-Whitney test. Most of the tested samples had elevated NSE activity (P < 0.02). The Douala sample evenly displayed elevated GST activity (P < 0.001), while high MFO level was recorded in the Pitoa sample (P < 0.001). MFO or GST levels were sometimes lower or similar to that of the Kisumu strain. These results suggest metabolic detoxification is a major DDT or pyrethroid resistance mechanism and emphasize the need for further investigations on An. gambiae s.l. resistance mechanisms in Cameroon.     

Spatial and temporal distribution of Anopheles gambiae s.l. (Diptera: Culicidae) in two Tanzanian villages: implication for designing mosquito sampling routines

This paper describes the spatial and temporal distribution of Anopheles gambiae s.l. Giles in two Tanzanian villages based on data collected from a five-month intensive mosquito sampling programme and analysed using Taylor's power law. The degree of spatial aggregation of female A. gambiae in each village was similar to its corresponding temporal aggregation, indicating that in designing sampling routines for estimating the abundance of mosquitoes, sampling effort should be allocated equally to houses (spatial) and nights (temporal). The analysis also showed that for a given amount of sampling effort, estimates of village-level mosquito abundance are more precise when sampling is carried out in randomly selected houses, than when the same houses are used on each sampling occasion. Also, the precision of estimating parous rates does not depend on whether mosquito sampling is carried out in the same or a random selection of houses. The implications of these findings for designing sampling routines for entomological evaluation of vector control trials are discussed.     

Multilevel analyses of genetic differentiation in Anopheles gambiae s.s. reveal patterns of gene flow important for malaria-fighting mosquito projects

Malaria control projects based on the introduction and spread of transgenes into mosquito populations depend on the extent of isolation between those populations. On the basis of the distribution of paracentric inversions, Anopheles gambiae has been subdivided into five subspecific chromosomal forms. Estimating gene flow between and within these forms of An. gambiae presents a number of challenges. We compared patterns of genetic divergence (F(ST)) between sympatric populations of the Bamako and Mopti forms at five sites. We used microsatellite loci within the j inversion on chromosome 2, which is fixed in the Bamako form but absent in the Mopti form, and microsatellites on chromosome 3, a region void of inversions. Estimates of genetic diversity and F(ST)'s suggest genetic exchanges between forms for the third chromosome but little for the j inversion. These results suggest a role for the inversion in speciation. Extensive gene flow within forms among sites resulted in populations clustering according to form despite substantial gene flow between forms. These patterns underscore the low levels of current gene flow between chromosomal forms in this area of sympatry. Introducing refractoriness genes in areas of the genome void of inversions may facilitate their spread within forms but their passage between forms may prove more difficult than previously thought.     

Reduced susceptibility to pyrethroid insecticide treated nets by the malaria vector Anopheles gambiae s.l. in western Uganda

Background

Cloning of parasites by limiting dilution is an essential and rate-limiting step in many aspects of malaria research including genomic and genetic manipulation studies. The standard Giemsa-stained blood smears to detect parasites is time-consuming, whereas the more sensitive parasite lactate dehydrogenase assay involves multiple steps and requires fresh reagents. A simple PCR-based method was therefore tested for parasite detection that can be adapted to high throughput studies.

Methods

Approximately 1 μL of packed erythrocytes from each well of a microtiter cloning plate was directly used as template DNA for a PCR reaction with primers for the parasite 18s rRNA gene. Positive wells containing parasites were identified after rapid separation of PCR products by gel electrophoresis.

Results

The PCR-based method can consistently detect a parasitaemia as low as 0.0005%, which is equivalent to 30 parasite genomes in a single well of a 96-well plate. Parasite clones were easily detected from cloning plates using this method and a comparison of PCR results with Giemsa-stained blood smears showed that PCR not only detected all the positive wells identified in smears, but also detected wells not identified otherwise, thereby confirming its sensitivity.

Conclusion

The PCR-based method reported here is a simple, sensitive and efficient method for detecting parasite clones in culture. This method requires very little manual labor and can be completely automated for high throughput studies. The method is sensitive enough to detect parasites a week before they can be seen in Giemsa smears and is highly effective in identifying slow growing parasite clones.