A standard cytogenetic photomap for the mosquito Anopheles stephensi (Diptera: Culicidae): application for physical mapping

To facilitate physical genome mapping, we have developed a new cytogenetic photomap for Anopheles stephensi (Liston) (Diptera: Culicidae), an important malaria vector in Asia. The high-resolution images of the ovarian polytene chromosomes have been straightened and divided by numbered divisions and lettered subdivisions. The exact chromosomal locations of eight DNA probes have been determined by fluorescent in situ hybridization. Using the DNA sequences, we have established correspondence between chromosomal arms among An. stephensi, Anopheles gambiae (Patton), and Anopheles funestus (Giles). The results support previous cytogenetic observations of arm translocations taking place during diversification of the species. To make the cytogenetic map useful for population genetics studies, we have indicated the chromosomal positions for the breakpoints of 19 polymorphic inversions.     

High malaria transmission intensity due to Anopheles funestus (Diptera: Culicidae) in a village of savannah-forest transition area in Cameroon

An entomological survey was conducted on vectors of malaria in a village of the forest-savannah transition area in Cameroon from February 1999 to October 2000. A total of 2,050 anopheline mosquitoes belonging to eight species were caught 1) after landing on human volunteers, 2) by using pyrethrum spray collections in human dwellings, and 3) in resting sites outdoors. Anopheles funestus Giles was the most abundant species (accounting for 91% of anophelines caught) followed by Anopheles gambiae Giles (7%). Applying polymerase chain reaction led to the identification of all specimens of the An. funestus group as An. funestus sensu stricto and mosquitoes from the An. gambiae complex were mostly An. gambiae sensu stricto of the S molecular form. Malaria transmission was perennial with an entomological inoculation rate estimated at 172 infective bites per person during the period of study. An. funestus was responsible for 88% of the total malaria transmission, with a Plasmodium falciparum circumsporozoite rate of 6.8% and an anthropophilic rate of 99.3%. These results confirm that in high agricultural activity areas, An. funestus can be, by far, the major malaria vector.     

Estimating dispersal and survival of Anopheles gambiae and Anopheles funestus along the Kenyan coast by using mark-release-recapture methods

Mark-release-recapture (MRR) experiments were conducted with emerging Anopheles gambiae s.l. and Anophelesfunestus Giles at Jaribuni and Mtepeni in Kilifi, along the Kenyan Coast. Of 739 and 1246 Anopheles released at Jaribuni and Mtepeni, 24.6 and 4.33% were recaptured, respectively. The daily survival probability was 0.96 for An. funestus and 0.95 for An. gambiae in Jaribuni and 0.83 and 0.95, respectively, in Mtepeni. The maximum flight distance recorded was 661 m. The high survival probability of An. gambiae and An. funestus estimated accounts for the continuous transmission of malaria along the Kenyan coast. This study also shows that the release of young, emergent female Anopheles improves the recapture rates and may be a better approach to MRR studies.     

Observations on the life cycle of Coelomomyces indicus (Blastocladiales: Coelomomycetaceae) in anopheline mosquitoes from the Philippines and Thailand

The water mold Coelomomyces indicus Iyengar is a widespread pathogen of anopheline mosquitoes in Asia and Africa, and it infects the copepod Microcyclops varicans Sars as its crustacean alternate host. This was determined by direct observation of field-infected copepods, selective meiospore encystment on M. varicans, and experimental infections of the copepod with zoospores from both thick and thin-walled meiosporangia. The physiological conditions governing germination of the 2 sporangial types were determined. The gametothallus in the copepod displays a light yellow pigmentation at maturity, and gametogenesis in both field and experimentally infected copepods occurs just at night fall, or 24 h after dark induction. In vivo culture was attained with the mosquito host Anopheles culicifacies Giles. Attempts to infect Anopheles stephensi Liston and Anopheles gambiae Giles, reported hosts of C. indicus, were unsuccessful.     

Anopheles funestus (Diptera: Culicidae) in a humid savannah area of western Burkina Faso: bionomics, insecticide resistance status, and role in malaria transmission

An entomological survey was carried out in three humid savannah sites of western Burkina Faso (Bama, Lena, and Soumousso) to (1) update the taxonomy of the Anopheles funestus Giles group, (2) examine the role of each species in malaria transmission, (3) characterize the insecticide resistance status of this malaria vector, and (4) determine the distribution of An. funestus chromosomal forms in these areas. Polymerase chain reaction identification of the members showed the occurrence of An. leesoni Evans in Lena and An. rivulorum-like in Soumousso in addition to An. funestus s.s. Malaria transmission was ensured mainly by An. funestus s.s. both in Soumousso and Lena and by An. gambiae s.s. Giles in Bama, the rice-growing area. The insecticide resistance status performed only on An. funestus indicated that this mosquito was susceptible to pyrethroids irrespective of the study area, but it was resistant to dieldrin. Furthermore, the occurrence of the two chromosomal forms of An. funestus, namely, Kiribina and Folonzo, seemed to follow ecological setups where Kiribina predominated in the irrigated area and Folonzo was more frequent in classic savannah. This study revealed that the problematic of An. funestus taxonomy was closer to that of An. gambiae requiring more structured studies to understand its genetic ecology.     

Effect of human circumsporozoite antibodies in Plasmodium-infected Anopheles (Diptera: Culicidae)

Human circumsporozoite (CS) antibodies to Plasmodium falciparum were detected in blood meals from 45.0% of 1,547 field-collected Anopheles gambiae Giles sensu lato and Anopheles funestus Giles from western Kenya. Possible effects on malaria infections within the Anopheles host were investigated. Circumsporozoite antibodies were detected in blood meals up to 36 h after feeding. Antibodies crossing the midgut were detected experimentally in hemolymph from 4 to 36 h after feeding; human IgG also was present in hemolymph from fully gravid field-collected Anopheles. Ingestion of high-titer human CS antibodies or 2A10 monoclonal antibody to P. falciparum sporozoites by P. falciparum-infected An. gambiae, 10 d after feeding on an infected human, had no effect on o?cyst maturation, sporozoite rates, or sporozoite loads. Contact between CS antibodies and sporozoites in the hemocoel did not block sporozoite invasion of salivary glands. Human IgG antibodies were detected by an indirect fluorescent antibody technique on salivary gland sporozoites from 83.3% of 114 field-collected Anopheles. In 65.4% of 26 infections, antibodies persisted on sporozoites for at least three days. Thus, a high proportion of naturally infected An. gambiae s.l. and An. funestus in western Kenya transmit sporozoites that are bound with human IgG acquired during previous blood meals. The infectivity of such sporozoites needs to be determined in relation to natural transmission and to the potential use of malaria sporozoite vaccines.     

Attraction of Anopheles (Diptera: culicidae) to volatile chemicals in Western Kenya

Anopheles gambiae s.l. and Anopheles funestus Giles are the primary vectors of malaria in East Africa. Identification of host-location olfactory cues may increase trap sensitivity for vector control and surveillance programs. Solid-state army miniature light traps were operated near sleeping humans in huts at night without lights and augmented with the potential attractants: L-lactic acid, Limburger cheese volatiles, hexanoic acid, and carbon dioxide. Mosquito response varied between species and gender. Female An. funestus exhibited a greater response to traps baited with L-lactic acid in combination with carbon dioxide than carbon dioxide alone in two different experiments.     

Detection of knockdown resistance mutations in Anopheles sacharovi (Diptera: Culicidae) and genetic distance with Anopheles gambiae (Diptera: Culicidae) using cDNA sequencing of the voltage-gated sodium channel gene

The knockdown resistance (kdr) mutation in the voltage-gated sodium channel gene (VGSCG), an important resistance mechanism against pyrethroids, was studied in Anopheles sacharovi Favre. It was found that the specific primers Agd1 and Agd2 used for polymerase chain reaction (PCR) amplification of Anopheles gambiae Giles VGSCG also amplified this genomic region in An. sacharovi. Comparison of the IIs4-IIs6 domain segments of the gene indicated 70% nucleotides common to both species and a genetic distance of 0.255 between them. Four different samples of pyrethroid-resistant An. sacharovi produced three types of amino acid, serine (TCG),leucine (TTG),and phenylalanine (TTT) at the kdr mutation point, whereas only two kdr mutations, leucine to phenylalanine and leucine to serine, occur in An. gambiae.     

Effect of Plasmodium falciparum on the survival of naturally infected afrotropical Anopheles (Diptera: Culicidae)

The effect of the malarial parasite, Plasmodium falciparum Welch, on the daily survival rates and longevity of Anopheles gambiae Giles sensu lato and Anopheles funestus Giles was determined for wild-caught, naturally infected females from western Kenya. Mosquitoes were collected inside houses and held in cages until death, after which they were assayed for P. falciparum circumsporozoite protein by an enzyme-linked immunosorbent assay (ELISA). Survival rates of field populations determined by parity rates were significantly higher than survival rates estimated by regression for Anopheles dying in cages. Overall, An. gambiae s.l. had a significantly higher daily mortality rate (means = 17.5%) than An. funestrus (means = 13.2%). P. falciparum ELISA infection rates, which were higher for An. gambiae s.l. (means = 19.8%; n = 1,221) than for An. funestus (means = 11.9%; n = 1,128), did not increase as a function of time for caged Anopheles. For An. gambiae s.l., there was a significant negative correlation between holding time and P. falciparum ELISA absorbance, suggesting that detectable circumsporozoite protein and perhaps the number of sporozoites may decrease with time in the vector. In western Kenya, an area where Anopheles populations often have extremely high malaria infection rates. Plasmodium infections did not reduce vector survivorship.     

A proteogenomic analysis of Anopheles gambiae using high-resolution Fourier transform mass spectrometry

Anopheles gambiae is a major mosquito vector responsible for malaria transmission, whose genome sequence was reported in 2002. Genome annotation is a continuing effort, and many of the approximately 13,000 genes listed in VectorBase for Anopheles gambiae are predictions that have still not been validated by any other method. To identify protein-coding genes of An. gambiae based on its genomic sequence, we carried out a deep proteomic analysis using high-resolution Fourier transform mass spectrometry for both precursor and fragment ions. Based on peptide evidence, we were able to support or correct more than 6000 gene annotations including 80 novel gene structures and about 500 translational start sites. An additional validation by RT-PCR and cDNA sequencing was successfully performed for 105 selected genes. Our proteogenomic analysis led to the identification of 2682 genome search-specific peptides. Numerous cases of encoded proteins were documented in regions annotated as intergenic, introns, or untranslated regions. Using a database created to contain potential splice sites, we also identified 35 novel splice junctions. This is a first report to annotate the An. gambiae genome using high-accuracy mass spectrometry data as a complementary technology for genome annotation.     

Comparative sensitivity of four Anopheles (Diptera: Culicidae) to five repellents

Five repellents were evaluated against Anopheles albimanus Wiedemann An. freeborni Aitken, An. gambiae Giles, and An. stephensi Liston, using a "free choice," dose-response testing procedure on rabbits. Overall, deet was the most effective repellent based on effective dosages (ED50's and ED95's), except against An. albimanus. An. albimanus was significantly more tolerant to deet compared with dimethyl phthalate or ethyl hexanediol. Permethrin was effective against all four anopheline species at the ED50 levels; however, the ED95 values were higher compared with the other repellents. There were significant differences in sensitivity of some of the four species to all the repellents tested, except dimethyl phthalate and indalone. The variability in the response of these anopheline species to repellents confirm that the extrapolation of repellent data from one anopheline species to another is unreliable and that comprehensive tests are needed for each species.     

Insecticide susceptibility status of Anopheles gambiae s.l. (Diptera: Culicidae) in the Republic of Cameroon

A large-scale survey of Anopheles gambiae Giles, 1902 susceptibility to DDT, dieldrin, permethrin, and deltamethrin was conducted in the Republic of Cameroon. 15 field populations from various geographical areas were tested using World Health Organization test kits for adult mosquitoes. The laboratory Kisumu susceptible reference strain was tested as a control. Results showed that dieldrin and DDT resistance was still present in some populations, and indicated permethrin or deltamethrin resistance. Within the Anopheles gambiae complex, resistant individuals belonged to An. gambiae s.s. and An. arabiensis species. Both M and S molecular forms of An. gambiae s.s. were found resistant. In most of resistant populations, the knockdown times were 2-5-folds increased. However, none of the surviving mosquitoes was positive to the kdr "Leu-Phe" mutation using polymerase chain reaction (PCR) diagnostic test. These results likely suggested involvement of other resistance mechanism(s), such as enzyme detoxification or kdr "Leu-Ser" mutation. Researches on An. gambiae s.l. resistance should be promoted in Cameroon, to improve malaria vector control programs and to implement resistance management strategies.     

Reassociation kinetics of Anopheles gambiae (Diptera: Culicidae) DNA.

The renaturation kinetics of nuclear DNA from the G3 colony of Anopheles gambiae Giles was studied to estimate the genome size and to determine the proportion of repeated sequences. An. gambiae has a haploid DNA content of 0.27 picograms or 2.6 x 10(8) basepairs. Analysis of reassociation kinetics indicated that the genome is composed of 61% single-copy and 33% repetitive sequences, with 6% foldback sequences.     

Relationship between kdr mutation and resistance to pyrethroid and DDT insecticides in natural populations of Anopheles gambiae

The spread of insecticide resistance genes in Anopheles gambiae Giles sensu stricto threatens to compromise vector-based malaria control programs. Two mutations at the same locus in the voltage-gated sodium channel gene are known to confer knockdown resistance (kdr) to pyrethroids and DDT. Kdr-e involves a leucine-serine substitution, and it was until recently thought to be restricted to East Africa, whereas kdr-w, which involves a leucine-phenylalanine substitution, is associated with resistance in West Africa. In this study, we analyze the frequency and relationship between the kdr genotypes and resistance to type I and type II pyrethroids and DDT by using WHO test kits in both the Forest-M and S molecular forms of An. gambiae in Cameroon. Both kdr-w and kdr-e polymorphisms were found in sympatric An. gambiae, and in many cases in the same mosquito. Kdr-e and kdr-w were detected in both forms, but they were predominant in the S form. Both kdr-e and kdr-w were closely associated with resistance to DDT and weakly associated with resistance to type II pyrethroids. Kdr-w conferred greater resistance to permethrin than kdr-e. We also describe a modified diagnostic designed to detect both resistant alleles simultaneously.     

High malaria transmission intensity in a village close to Yaounde, the capital city of Cameroon

A 2-yr longitudinal malaria study was undertaken in a suburb of Yaounde, the capital city of Cameroon, in the village of Simbock, approximately 2 km from the city limits. This study allowed assessment of malaria transmission intensity and dynamics in this region before implementation of pyrethroid impregnated bed nets through the national vector control program. Anophelines were captured on human volunteers by pyrethrum spray collections and in resting sites outdoors. Malaria vectors were Anopheles funestus Giles, Anopheles gambiae s.s. Giles (M and S forms), Anopheles moucheti Evans, and Anopheles nili Theobald. An. moucheti was the most abundant mosquito captured during the study, accounting for >54% of total anophelines caught. The annual Plasmodium falciparum Welch entomological inoculation rates measured by enzyme-linked immunosorbent assay were 277 infected bites per human for the first year and 368 for the second year. An. gambiae s.s., An. funestus, An. moucheti, and An. nili were responsible for 23.8%, 26.8%, 39.2%, and 10.2% of malaria transmission, respectively. Malaria transmission is perennial throughout the year. All these vectors were highly anthropophagous because only two out of 566 mosquitoes blood-meal tested were not taken on humans.     

Spatial distribution patterns of malaria vectors and sample size determination in spatially heterogeneous environments: a case study in the west Kenyan highland

The current study examined temporal and spatial distribution patterns of anopheline malaria vectors in a highland site and determined the number of houses to be sampled to achieve the targeted precision level. Adult mosquito sampling was conducted seasonally in May and August 2002 in a 3 by 3-km2 area, and in November 2002 and February 2003 in an expanded 4 by 4-km2 area in Kakamega District, western Kenya. Anopheles gambiae Giles was the predominant malaria vector species, constituting 84.6% of the specimens, whereas Anopheles funestus Giles constituted 15.4% of the vector populations. An. gambiae abundance increased by six- to eight-fold in the long rainy season over the dry seasons, but An. funestus abundance peaked 3 mo after the long rainy season. For both species, the coefficient of variation was larger than 1, suggesting that the distribution of mosquito adults was aggregated. Mosquito clustering occurred in houses <400 m from a valley bottom. The negative binomial distribution was accepted in one sample period (August 2002) for An. gambiae and in two sampling periods (May and August 2002) for An. funestus. Taylor's power law analyses indicated that An. gambiae distribution was more aggregated in the wet seasons than in the dry seasons, whereas the degree of aggregation of An. funestus was similar in all four seasons. The minimum number of houses required to estimate anopheline female abundance within the commonly acceptable precision level (0.2) should be 17 houses per km2 for An. gambiae and 42 houses per km2 for An. funestus. The potential factors causing aggregated anopheline mosquito distribution are discussed.     

Monoclonal antibody MG96 completely blocks Plasmodium yoelii development in Anopheles stephensi

In spite of research efforts to develop vaccines against the causative agent of human malaria, Plasmodium falciparum, effective control remains elusive. The predominant vaccine strategy focuses on targeting parasite blood stages in the vertebrate host. An alternative approach has been the development of transmission-blocking vaccines (TBVs). TBVs target antigens on parasite sexual stages that persist within the insect vector, anopheline mosquitoes, or target mosquito midgut proteins that are presumed to mediate parasite development. By blocking parasite development within the insect vector, TBVs effectively disrupt transmission and the resultant cascade of secondary infections. Using a mosquito midgut-specific mouse monoclonal antibody (MG96), we have partially characterized membrane-bound midgut glycoproteins in Anopheles gambiae and Anopheles stephensi. These proteins are present on the microvilli of midgut epithelial cells in both blood-fed and unfed mosquitoes, suggesting that the expression of the protein is not induced as a result of blood feeding. MG96 exhibits a dose-dependent blocking effect against Plasmodium yoelii development in An. stephensi. We achieved 100% blocking of parasite development in the mosquito midgut. Preliminary deglycosylation assays indicate that the epitope recognized by MG96 is a complex oligosaccharide. Future investigation of the carbohydrate epitope as well as gene identification should provide valuable insight into the possible mechanisms of ookinete attachment and invasion of mosquito midgut epithelial cells.     

Method for in situ hybridization to polytene chromosomes from ovarian nurse cells of Anopheles gambiae (Diptera: Culicidae)

A procedure for in situ hybridization to polytene chromosomes from the ovarian nurse cells of Anopheles gambiae Giles has been developed. This procedure involves a modification of established methods for Drosophila larval salivary gland polytene chromosomes. Treatment of chromosome squashes with xylene followed by slow rehydration provides required resolution of chromosome banding patterns, possibly because fatty contaminants are removed from ovarian nurse cell preparations. Using this procedure, unique DNA sequences from a genomic library of An. gambiae have been mapped on adult mosquito polytene chromosomes. The ability to locate genetic markers on chromosomes will allow correlation of physical and genetic maps. Such maps will facilitate identification of genetic loci and expand our knowledge of the genomic organization of An. gambiae.     

Spatial and habitat distribution of Anopheles gambiae and Anopheles arabiensis (Diptera: Culicidae) in Banambani village, Mali

We studied the larval distribution and composition of Anopheles arabiensis Patton, An. gambiae s.s. Giles, and its forms, among local habitats; and their association with the adults between these habitats in Banambani village, Mali during the mid-rainy seasons of 1997-1999. For species and form identification we used polymerase chain reaction (PCR) and PCR-restriction fragment-length polymorphism (RFLP). Differences among species in the distribution of larvae were observed in 1998, but not in 1997 or 1999, although they were on the borderline of statistical significance. Differences among the M and S molecular forms were statistically significant in 1999 when rainfall was high, but not in the two prior, drier sampling periods. Combining all information into the Fisher multiple comparisons test, there were statistically significant differences between species and molecular forms during the 3-yr study period. Hybrid larvae between the M and S forms were observed (0.57%), the first such observation to our knowledge. In spite of differences among larval distribution, no differences of adult species composition were observed among habitats. Factors that influence the distributions of An. gambiae larval populations are discussed.     

Innate preference for host-odor blends modulates degree of anthropophagy of Anopheles gambiae sensu lato (Diptera: Culicidae).

In field studies, Anopheles gambiae Giles sensu stricto obtains most blood meals from humans, whereas Anopheles quadriannulatus Theobald bites predominantly bovids. We investigated whether host odors modulate the host preference of these mosquito species. In a dual-choice olfactometer, mosquitoes were given a choice between clean air and putative host-specific odor blends. An. gambiae chose 'human odor' over clean air and clean air over cow odor.' Although Anopheles quadriannulatus did not choose cow odor over clean air, it chose clean air over human odor. Cheese odor, which attracted An. gambiae, did not result in higher trap catch of An. quadriannulatus. We conclude that the degree of anthropophagy of An. gambiae s.l. has an innate olfactory basis.