Quantitation of malaria sporozoites in the salivary glands of wild Afrotropical Anopheles

The number of malaria sporozoites in the salivary glands was determined microscopically for 1137 wild, naturally infected Anopheles from western Kenya. Infective Anopheles gambiae Giles sensu lato (n = 874) contained a geometric mean (GM) of 962 sporozoites and An.funestus Giles (n = 263) contained 812. No significant differences were detected in geometric mean numbers of sporozoites between species, collection techniques or sites. Of the infective An.gambiae, 1.7% (15/874) contained more than 41,830 sporozoites, the maximum observed for An.funestus. Microscopic techniques were found to be more sensitive than enzyme-linked immunosorbent assays (ELISA) for detecting low-grade sporozoite infections in salivary glands. Salivary gland sporozoites from 83.6% of the 1137 gland infections were identified by ELISA as either Plasmodium falciparum Welch (n = 910), P.ovale Stephens (n = 7), P.malariae Grassi & Feletti (n = 3) or mixed (n = 30). The 187 gland infections which could not be identified by ELISA contained significantly fewer sporozoites (GM = 242) than those which could be identified (GM = 1200).     

ELISA absorbance cut-off method affects malaria sporozoite rate determination in wild Afrotropical Anopheles

ABSTRACT. Malaria sporozoite infection rates in a mixed species group of 244 Anopheles gambiae Giles sensu lato and 115 An. funestus Giles wild female mosquitoes were compared using three methods to determine cutoff absorbance values for positivity of a Plasmodium falciparum Welch enzyme-linked immunosorbent assay (ELISA). Positive controls were based on P. falciparum circumsporozoite protein. As negative controls, four wild male Anopheles were included on each microtitre plate; tests were repeated on four consecutive days for each plate.
Infection rates were estimated at 13.1–22.8% using the mean absorbance value of negative controls plus three standard deviations, 11.7–12.8% using double the mean and 12.5–13.6% using the fixed cut-off value of 0.20 (allowing for 20% variation in negative control absorbance values).
Observed agreement for positivity or negativity among samples tested four times was 98.6% for the 2× mean method, 97.2% for the fixed cut-off 0.20 value, but only 82.7% for the mean +3 SD method. It was concluded that the 2× mean cut-off method is most reliable for field studies. P. falciparum sporozoite rates of 12.2% in An. funestus and 11.9% in An. gambiae s. l . were thus determined on the basis of the 2× mean cut-off method.
This comparative evaluation demonstrates that vector infectivity rates can be seriously over-estimated from sporozoite ELISA tests, by as much as 87% in one case considered here, depending on the absorbance cut-off method applied for negative controls.     

Isolation of leishmanial parasites from a wild caught Anopheles gambiae mosquito in Kenya

A total of 232 mosquitoes were collected and dissected for leishmanial parasites in the Baringo District, Kenya. Anopheles gambiae sensu lato comprised 90.9% of the sample. One female A. gambiae was found to be infected with leishmanial promastigotes. The parasites when injected into Balb C mice caused skin lesions characterized by heavy amastigote infections. The average size of the parasite was: body length, 11.7 ± 0.19 μm; width, 1.3 ± 0.04 μm; flagellum length, 15.5 ± 0.28 μm.     

Anopheles nili as the main vector of human malaria in villages of southern Cameroon

In villages near Sanaga river, in the forest zone of south Cameroon, Anopheles nili Theobald is the main species of mosquito regularly found biting man inside houses. Its densities are related to the flow level of the river. It is also the main malaria vector in terms of intensity and seasonal duration of transmission. The yearly malaria inoculation rate due to An. nili alone is 104 infective bites/man, varying between 3 infective bites/man in October and 20 in March. Anopheles gambiae Giles is a less important vector in the area but reached its peak in the dry season, when the Sanaga river is at its lowest level. These observations underline the influence of local ecology on malaria transmission and the need for entomological studies in each situation.     

Efficacy of permethrin-impregnated curtains for malaria vector control

Preliminary results obtained by the use of permethrin-impregnated curtains against the Afrotropical malaria vectors Anopheles gambiae Giles s.l. and An.funestus Giles are reported and discussed. Field trials were carried out in villages near Ouagadougou, Burkina Faso. Houses were provided with curtains made from 100% cotton netting, impregnated with permethrin at the dose of 1 g a.i./m2, to cover the doorway, the window(s) and the space under the eaves. Entomological data collected during the period 1985-86 showed residual permethrin activity for about a year, and almost complete prevention of indoor-resting mosquitoes. Increased exit-rate and mortality-rate of house-entering malaria vectors were also obtained. Utilization of this malaria vector control method in primary health care programmes is advocated.     

Exposure of Gambian children to Anopheles gambiae malaria vectors in an irrigated rice production area

Abstract. Variation in exposure of children to malaria vectors of the Anopheles gambiae complex was recorded in a Gambian village situated near an irrigated area of rice cultivation. Observations were made in 1987 and 1988 during two dry seasons, when pumped water was used to grow rice, and two rainy seasons, when rice was produced using a combination of irrigated and rainfed paddies. Routine collections of mosquitoes were made from under bednets. Most of these specimens were assumed to have fed on the occupants of the net and thus represented a crude measure of exposure to malaria. Most nets in the village were in good condition, but even these were a poor defence against blood-seeking mosquitoes. Two annual peaks in the numbers of An. gambiae s.l. corresponded with the irrigation of rice paddies in the dry and wet seasons. When there were few vectors in the village the frequency distribution of mosquitoes caught under nets was described best by a Poisson process. When high numbers were present the daily distributions were over-dispersed and fitted a negative binomial model. The spatial distribution of mosquitoes varied between dry and wet seasons and was related to the predominant wind direction at night, suggesting that wind assisted the dispersal of mosquitoes from their breeding sites. For individual children in the rainy season, increased exposure to malaria vectors was associated with living adjacent to a mosquito breeding site, being resident in larger compounds, having open eaves in the house, a store-room adjacent to the bedroom, the absence of a ceiling in the bedroom, the absence of wood smoke indoors and leaving the bednet untucked at night. In the dry season a high level of exposure was associated with living close to a mosquito breeding site, having an unfenced compound, sleeping in a room without a ceiling and using insecticide aerosols. These observations demonstrate that within a village there are systematic and persistent differences in the level of exposure to malaria parasites experienced by individual children.     

Anopheles arabiensis and An. funestus are equally important vectors of malaria in Matola coastal suburb of Maputo, southern Mozambique

Transmission characteristics of malaria were studied in Matola, a coastal suburb of Maputo, the capital City, in southern Mozambique, from November 1994 to April 1996. The local climate alternates between cool dry season (May-October) and hot rainy season (November-April) with mean annual rainfall 650-850 mm. Saltmarsh and freshwater pools provide mosquito breeding sites in Matola. Malaria prevalence reached approximately 60% among people living nearest to the main breeding sites of the vectors. Plasmodium falciparum caused 97% of malaria cases, others being P. malariae and P. ovale. Potential malaria vector mosquitoes (Diptera: Culicidae) collected at Matola during daytime indoor-resting (n = 1021) and on human bait at night (n = 5893) comprised 12% Anopheles coustani Laveran (93% biting outdoors), 46% An. funestus Giles (68% biting indoors) and 42% An. gambiae Giles sensu lato (60% biting outdoors). All 215 specimens of An. gambiae s.l. identified genetically were An. arabiensis Patton. Anopheles funestus populations remained stable throughout the year, whereas densities of the An. gambiae complex fluctuated considerably, with An. arabiensis peaking during the rainy season. No concomitant rise in malaria incidence was observed. Human landing indices of An. funestus and An. arabiensis averaged 1.8 and 3.8 per man-night, respectively. Overall Plasmodium sporozoite rates were 2.42+/-1.24% in 2181 An. funestus and 1.11+/-1.25% in 1689 An. arabiensis dissected and examined microscopically. Mean daily survival rates were 0.79 for both vector species. Estimated infective bites/person/year were 15 An. funestus and 12 An. arabiensis. Biting rates were greatest at 2100-24.00 hours for An. funestus (68% endophagic) and 21.00-03.00 hours for An. arabiensis (40% endophagic). The entomological inoculation rate (EIR) declined sharply over very short distances (50% per 90m) away from breeding-sites of the vectors. Consequently, P. falciparum prevalence among Matola residents was halved 350 m within the town. Implications for the protective effectiveness of a 'cordon sanitaire' by residual house-spraying and/or the use of insecticide-treated bednets are discussed.     

Probing behaviour and sporozoite delivery by Anopheles stephensi infected with Plasmodium berghei

We observed that Plasmodium berghei sporozoite-infected Anopheles stephensi was not impaired in its ability to locate blood on a host. When probing rats, infected mosquitoes took as long as non-infected mosquitoes to locate blood. Contrary to previous suggestions, infective mosquitoes delivered sporozoites into mineral oil even after extensively probing a vertebrate host. We observed that, in mosquitoes having probed a host, both the mean number of sporozoites ejected over 3 min into oil (35.9 v. 31.7 sporozoites) and the proportion of mosquitoes delivering sporozoites (60% v. 50%) were similar to mosquitoes not having probed. We then developed a model of sporozoite delivery, taking into account observations that sporozoites are clumped in the lumen of the glands as well as upon delivery, and that output is uneven and inconsistent. We conclude that clumping optimizes transmission, if a threshold of infection exists and the mean number of sporozoites per clump is greater than the threshold.     

Impact of permethrin-treated bednets on malaria transmission by the Anopheles gambiae complex in The Gambia

Malaria vector mosquitoes belonging to the Anopheles gambiae complex were studied in four hamlets in The Gambia. All inhabitants were given bednets treated either with a placebo (milk) in two hamlets or with the pyrethroid insecticide permethrin (500 mg/m2) in two other hamlets. Malaria transmission occurred mainly during a few weeks of the rainy season, in September and October 1987. The indoor resting densities of mosquitoes in permethrin-treated hamlets were reduced, and we estimated over 90% reduction in biting on man by An. gambiae Giles sensu stricto in these hamlets. No mosquitoes were found under permethrin-treated bednets compared with eighty-one recovered from placebo-treated bednets. Mosquitoes exited more readily from rooms where permethrin-treated bednets were used than from rooms with placebo-treated nets. The annual mean probability that a child would receive an infective bite was estimated to be 0.09 in hamlets with insecticide-treated bednets, compared with 1.9 where placebo-treated bednets were used. Permethrin-treated bednets are therefore recommended as a means of effectively reducing the risk of exposure to malaria transmission, particularly in areas of low seasonal transmission.     

The impact of permethrin-impregnated bednets on malaria vectors of the Kenyan coast

Abstract. The effects of introducing permethrin-impregnated bednets on local populations of the malaria vector mosquitoes Anopheles funestus and the An.gambiae complex was monitored during a randomized controlled trial at Kilifi on the Kenyan coast. Pyrethrum spray collections inside 762 households were conducted between May 1994 and April 1995 after the introduction of bednets in half of the study area. All-night human bait collections were performed in two zones (one control and one intervention) for two nights each month during the same period. PCR identifications of An.gambiae sensu lato showed that proportions of sibling species were An.gambiae sensu stricto > An.merus > An.arabiensis.
Indoor-resting densities of An.gambiae s.l. and the proportion of engorged females decreased significantly in intervention zones as compared to control zones. However, the human blood index and Plasmodium falciparum sporozoite rate remained unaffected. Also vector parous rates were unaltered by the intervention, implying that survival rates of malaria vectors were not affected. The human-biting density of An.gambiae s.l. , the predominant vector, was consistently higher in the intervention zone compared to the control zone, but showed 8% reduction compared to pre-intervention biting rates - versus 94% increase in the control zone.
Bioassay, susceptibility and high-performance liquid chromatography results all indicated that the permethrin content applied to the nets was sufficient to maintain high mortality of susceptible vectors throughout the trial. Increased rates of early outdoor-biting, as opposed to indoor-biting later during the night, were behavioural or vector composition changes associated with this intervention, which would require further monitoring during control programmes employing insecticide-treated bednets.     

Malaria transmission potential of Anopheles mosquitoes in the Mwea-Tebere irrigation scheme, Kenya

1. Anopheles arabiensis Patton and An. funestus Giles were identified as vectors of Plasmodium falciparum malaria in the Mwea-Tebere irrigation scheme, Kenya. An. arabiensis was the only member of the An. gambiae complex identified from chromosome characteristics. Other Anopheles species found included An. pharoensis Theobald, An. rufipes Gough and An. coustani Laveran. Survival rates per gonotrophic cycle for An. arabiensis averaged 0.37 during the short rains (October-November), 0.49 during the dry season (February) and 0.78 during the long rains (May-June). Vectorial capacities were correspondingly low due to low survival rates and a high degree of zoophily. The average duration of infective life for P. falciparum was 0.2 days for both An. arabiensis and An. funestus. In contrast, entomological inoculation rates were comparatively high: 6-8 infective bites/man/month. An. pharoensis averaged 110 bites/man/night during the short rains; 1/999 (0.1%) was positive by ELISA for P. falciparum circumsporozoite antigen, but the ELISA evidence is not conclusive for vector incrimination. In correspondence with clinical observations, the transmission of P. malariae and P. ovale is unlikely due to the low vector survival rates. The observed anomaly between low vectorial capacities and high entomological inoculation rates demonstrates the importance of accurately estimating vector sporozoite rates to monitor unstable malaria transmission in irrigated areas.     

Gut content identification of larvae of the Anopheles gambiae complex in western Kenya using a barcoding approach

Although larvae feeding and food source are vital to the development, survival and population regulation of African malaria vectors, the prey organisms of Anopheles gambiae larvae in the natural environment have not been well studied. This study used a molecular barcoding approach to investigate the natural diets of Anopheles gambiae s.l. larvae in western Kenya. Gut contents from third- and fourth-instar larvae from natural habitats were dissected and DNA was extracted. The 18S ribosomal DNA gene was amplified, the resulting clones were screened using a restriction fragment length polymorphism method and nonmosquito clones were sequenced. Homology search and phylogenetic analyses were then conducted using the sequences of non-mosquito clones to identify the putative microorganisms ingested. The phylogenetic analyses clustered ingested microorganisms in four clades, including two clades of green algae (Chlorophyta, Chlorophyceae Class, Chlamydomonadales and Chlorococcales families), one fungal clade, and one unknown eukaryote clade. In parallel, using the same approach, an analysis of the biodiversity present in the larval habitats was carried out. This present study demonstrated the feasibility of the barcoding approach to infer the natural diets of Anopheles gambiae larvae. Our analysis suggests that despite the wide range of microorganisms available in natural habitats, mosquito larvae fed on specific groups of algae. The novel tools developed from this study can be used to improve our understanding of the larval ecology of African malaria vectors and to facilitate the development of new mosquito control tools.     

Modulation of Anopheles gambiae Epsilon glutathione transferase activity by plant natural products in vitro

Elevated glutathione transferase (GST) E2 activity is associated with DDT resistance in the mosquito Anopheles gambiae. The search for chemomodulators that inhibit the function of AgGSTE2 would enhance the insecticidal activity of DDT. Therefore, we examined the interaction of novel natural plant products with heterologously expressed An. gambiae GSTE 2 in vitro. Five of the ten compounds, epiphyllocoumarin (Tral-1), knipholone anthrone, isofuranonaphthoquinones (Mr 13/2, Mr13/4) and the polyprenylated benzophenone (GG1) were shown to be potent inhibitors of AgGSTE2 with IC₅₀ values of 1.5 μM, 3.5 μM, 4 μM, 4.3 μM and 4.8 μM respectively. Non-competitive inhibition was obtained for Tral 1 and GG1 with regards to GSH (K_i of 0.24 μM and 0.14 μM respectively). Competitive inhibition for Tral1 was obtained with CDNB (K_i = 0.4 μM) whilst GG1 produced mixed type of inhibition. The K_i and K_i' for GSH for Tral-1 and GG1 were 0.2 μM and 0.1 μM respectively. These results suggest that the novel natural plant products, particularly Tral-1, represent potent AgGSTE2 in vitro inhibitors.     

Mitochondrial and ribosomal internal transcribed spacer (ITS2) diversity of the African malaria vector Anopheles funestus

The pattern of sequence variation in the mitochondrial DNA cytochrome b gene (cyt-b) and ribosomal DNA internal transcribed spacer 2 (ITS2) was examined in Anopheles funestus from Senegal and Burkina Faso in West Africa and Kenya in East Africa. From both West African countries, samples included individuals hypothesized to represent reproductively isolated taxa based upon different karyotypes and behaviours. Analysis of the cyt-b data revealed high haplotypic diversity (86%) and an average pairwise difference per site of 0.42%. Sequence variation was not partitioned by geographical origin or karyotype class. The most common haplotype was sampled across Africa (approximately 6000 km). Analysis of the ITS2 data revealed one of the longest spacers yet found in anophelines (approximately 704 bp). In common with other anopheline ITS2 sequences, this one had microsatellites and frequent runs of individual nucleotides. Also in common with data from other anopheline ITS2 studies, the An. funestus sequences were almost monomorphic, with only two rare polymorphisms detected. The results from both markers are congruent and do not support the hypothesis of reproductively isolated chromosomal taxa within An. funestus. Whether the lack of support by mitochondrial DNA (mtDNA) and ribosomal DNA (rDNA) sequences is a result of the recent origin of the presumptive taxa, or of the absence of barriers to gene flow, remains to be elucidated, using more rapidly evolving markers such as microsatellites.     

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.