Deep sequencing reveals extensive variation in the gut microbiota of wild mosquitoes from Kenya.

The mosquito midgut is a hostile environment that vector-borne parasites must survive to be transmitted. Commensal bacteria in the midgut can reduce the ability of mosquitoes to transmit disease, either by having direct anti-parasite effects or by stimulating basal immune responses of the insect host. As different bacteria have different effects on parasite development, the composition of the bacterial community in the mosquito gut is likely to affect the probability of disease transmission. We investigated the diversity of mosquito gut bacteria in the field using 454 pyrosequencing of 16S rRNA to build up a comprehensive picture of the diversity of gut bacteria in eight mosquito species in this population. We found that mosquito gut typically has a very simple gut microbiota that is dominated by a single bacterial taxon. Although different mosquito species share remarkably similar gut bacteria, individuals in a population are extremely variable and can have little overlap in the bacterial taxa present in their guts. This may be an important factor in causing differences in disease transmission rates within mosquito populations.

Biological cost of tolerance to heavy metals in the mosquito Anopheles gambiae.

The global rate of heavy metal pollution is rapidly increasing in various habitats. Anopheles malaria vector species (Diptera: Culicidae) appear to tolerate many aquatic habitats with metal pollutants, despite their normal proclivity for 'clean' water (i.e. low levels of organic matter). Investigations were conducted to establish whether there are biological costs for tolerance to heavy metals in Anopheles gambiae Giles sensu stricto and to assess the potential impact of heavy metal pollution on mosquito ecology. Anopheles gambiae s.s. were selected for cadmium, copper or lead tolerance through chronic exposure of immature stages to solutions of the metals for three successive generations. Biological costs were assessed in the fourth generation by horizontal life table analysis. Tolerance in larvae to cadmium (as cadmium chloride, CdCl(2)), copper [as copper II nitrate hydrate, Cu(NO(3))(2) 2.5 H(2)O] and lead [as lead II nitrate, Pb(NO(3))(2)], monitored by changes in LC(50) concentrations of the metals, changed from 6.07 microg/L, 12.42 microg/L and 493.32 microg/L to 4.45 microg/L, 25.02 microg/L and 516.69 microg/L, respectively, after three generations of exposure. The metal-selected strains had a significantly lower magnitude of egg viability, larval and pupal survivorship, adult emergence, fecundity and net reproductive rate than the control strain. The population doubling times were significantly longer and the instantaneous birth rates lower in most metal-selected strains relative to the control strain. Our results suggest that although An. gambiae s.s. displays the potential to develop tolerance to heavy metals, particularly copper, this may occur at a significant biological cost, which can adversely affect its ecological fitness.

Development and survival of Anopheles gambiae eggs in drying soil: influence of the rate of drying, egg age, and soil type.

Little is known about the contribution made by the egg stage of African malaria vectors to the rapid rise in adult populations following the onset of seasonal rains. To examine this issue, we evaluated the viability of Anopheles gambiae eggs in drying soil in the laboratory. Survival data were collected from field-caught mosquitoes kept in sandy loam soil and laboratory-reared colonies kept in sandy loam soil and black cotton soil. Under high, medium, and low soil-moisture regimes, egg viability declined sharply with increased duration of drying. Eggs remained viable in drying sandy loam soil for 1, 5, and 10 days, but not after 15 or 20 days. The most dramatic decline in hatching success occurred between drying days 1 (78-83% hatch) and 5 (20-23% hatch). In contrast, eggs reared in high-moisture black cotton soil remained viable for up to 15 days. Furthermore, after 5 drying days, high-, medium-, and low-moisture soils averaged 59, 47, and 31% hatching success, respectively. We recovered unhatched eggs from sandy loam soils to examine the developmental status of the embryos. A majority of the unhatched eggs that were recovered from days 15 and 20 in sandy loam soils contained fully developed late-stage embryos. Thus, unhatched eggs completed embryonic development but probably died before receiving an appropriate hatching stimulus. Our results suggest that the absolute moisture content of the soil does not alone determine hatching success of anopheline eggs. Rather, soil moisture, together with the rate of drying, physiological factors associated with the age of the egg, and the type of soil in which the egg rests likely influence survival.

Efficacy of Bacillus thuringiensis israelensis, Bacillus sphaericus and temephos for managing Anopheles larvae in Eritrea.

We evaluated the larvicidal activity of the granular formulation of Bacillus thuringiensis israelensis (Bti) serotype H-14 (Vectobac G, 200 ITU/mg) and Bacillus sphaericus (Bsph) serotype H5a5b (Vectolex CG, 670 Bs ITU/mg) against Anopheles arabiensis and other mosquitoes in breeding habitats in 3 sites, Gash-Barka, Anseba, and Debub zones, in Eritrea. The primary objective was to determine the optimal application rate and duration of effect for Bti and Bsph in representative larval habitats as compared with the organophosphate temephos. The biolarvicides were tested at 100% (high) and 50% (low) of the maximum recommended application rate. Temephos was applied at a rate of 100 ml/ha. At least 4 replicate experiments with Vectobac G (5.6 and 11.2 kg/ha), Vectolex CG (11.2 and 22.4 kg/ha) were conducted in each study site. All 3 larvicides caused significant mortality of the main malaria vector species, An. arabiensis, and other mosquito species (Anopheles cinereus, Anopheles pretoriensis, Culex quinquefasciatus). The larvicidal activity for Bti and Bsph was variable depending upon breeding habitat, mosquito species, and general ecology of the area. Both biopesticides had a similar duration of activity (2-3 wk) and were generally as effective as temephos for these time periods. In some cases, the high and low application rates for Bti and Bsph produced equivalent control over 2-3 wk. The 2 Bacillus biopesticides were less effective in habitats with high algal content and in fast flowing streams primarily because of the inability to penetrate algal mats and dilution effect, respectively. The results show that application of the 2 biolarvicides bimonthly to streambed pools, rain pools, and similar habitats would maintain control of the anopheline mosquito population.

Extensive multiple test centre evaluation of the VecTest malaria antigen panel assay.

To determine which species and populations of Anopheles transmit malaria in any given situation, immunological assays for malaria sporozoite antigen can replace traditional microscopical examination of freshly dissected Anopheles. We developed a wicking assay for use with mosquitoes that identifies the presence or absence of specific peptide epitopes of circumsporozoite (CS) protein of Plasmodium falciparum and two strains of Plasmodium vivax (variants 210 and 247). The resulting assay (VecTest Malaria) is a rapid, one-step procedure using a 'dipstick' test strip capable of detecting and distinguishing between P. falciparum and P. vivax infections in mosquitoes. The objective of the present study was to test the efficacy, sensitivity, stability and field-user acceptability of this wicking dipstick assay. In collaboration with 16 test centres world-wide, we evaluated more than 40 000 units of this assay, comparing it to the standard CS ELISA. The 'VecTest Malaria' was found to show 92% sensitivity and 98.1% specificity, with 97.8% accuracy overall. In accelerated storage tests, the dipsticks remained stable for > 15 weeks in dry conditions up to 45 degrees C and in humid conditions up to 37 degrees C. Evidently, this quick and easy dipstick test performs at an acceptable level of reliability and offers practical advantages for field workers needing to make rapid surveys of malaria vectors.

Linear and spatial organization of polytene chromosomes of the African malaria mosquito Anopheles funestus.

Anopheles funestus Giles is one of the major malaria vectors in Africa, but little is known about its genetics. Lack of a cytogenetic map characterized by regions has hindered the progress of genetic research with this important species. This study developed a cytogenetic map of An. funestus using ovarian nurse cell polytene chromosomes. We demonstrate an important application with the cytogenetic map for characterizing various chromosomal inversions for specimens collected from coastal Kenya. The linear and spatial organization of An. funestus polytene chromosomes was compared with the best-studied malaria mosquito, An. gambiae Giles. Comparisons of chromosome morphology between the two species have revealed that the most extensive chromosomal rearrangement occurs in pericentromeric heterochromatin of autosomes. Differences in pericentromeric heterochromatin types correlate with nuclear organization differences between An. funestus and An. gambiae. Attachments of chromosomes to the nuclear envelope strongly depend on the presence of diffusive beta-heterochromatin. Thus, An. funestus and An. gambiae exhibit species-specific characteristics in chromosome-linear and -spatial organizations.

Sporozoite loads of naturally infected Anopheles in Kilifi District, Kenya.

The number of salivary gland malaria sporozoites (sporozoite load) was determined by hemacytometer counts for 2,055 field-collected Anopheles mosquitoes from Kilifi District, Kenya. Of 48 gland-positive Anopheles gambiae s.l., sporozoite loads ranged from 125 to 79,875, with a geometric mean of 1,743 sporozoites per infected mosquito. About half of the infected mosquitoes had sporozoite loads < 1,000. Following hemacytometer examination of salivary gland samples, the same samples were subsequently tested for Plasmodium falciparum circumsporozoite (CS) protein by enzyme-linked immunosorbent assay (ELISA). The confirmation by ELISA of CS protein in 89.6% (43/48) of the salivary gland-positive samples compared to only 1.4% (28/2,007) of the dissection-negative mosquitoes indicated that dissection methods with hemacytometer counts of sporozoites were adequate for detecting even low numbers of sporozoites in field-collected mosquitoes. Detection of 17 or fewer sporozoites in blood meals of 7 freshly bloodfed An. gambiae s.l. provides a further indication that the actual number of sporozoites transmitted during bloodfeeding may be quite low.

Resting behaviour, ecology and genetics of malaria vectors in large scale agricultural areas of Western Kenya.

In Kenya indoor and outdoor resting densities of Anopheles arabiensis and Anopheles funestus at the Ahero rice irrigation scheme, and Anopheles gambiae s.s., An. arabiensis and An. funestus at the Miwani sugar belt were assessed for 13 months by pyrethrum spray collections in houses and granaries. The vector's house leaving behaviour was evaluated with exit traps and it was noted that early exophily (i.e., deliberate) was not detected in any of the vectors. Assortative indoor/outdoor resting behaviour was studied by a capture-mark-release-recapture method and showed that in An. arabiensis both indoor and outdoor resting traits were present in the same individuals. Samples of half-gravid female An. gambiae s.l. were chromosomally identified either as Anopheles gambiae s.s. or An. arabiensis and in a subsample chromosomal inversions were read. Anopheles gambiae s.s. and An. arabiensis had the 2Rb inversion but in addition the 2La inversion was found in An. gambiae s.s. and this is an indication of low chromosomal variation. At Ahero An. arabiensis was most abundant when the rice crop was immature and An. funestus when the crop was mature. This succession of vectors facilitated the transmission of malaria throughout the year. At Miwani, An. gambiae s.l. population peaked during the long rains but the proportion of An. arabiensis was highest during the dry season. The indoor resting density of males of the three vector species was less than half of the females.

Origin of blood meals in indoor and outdoor resting malaria vectors in western Kenya.

Blood meals were obtained from indoor and outdoor resting malaria vectors in three villages of western Kenya and tested by sandwich ELISA to determine host preferences and their human blood index (HBI). Anopheles gambiae s.s. collected indoors at Kisian village had a HBI of 0.97 while that of Anopheles arabiensis collected at Ahero was 0.23. However, the HBI of A. arabiensis varied depending on the availability of outdoor resting shelters. Most female A. arabiensis (98.9%) collected outdoors in granaries at Ahero had fed on cattle. Indoor-collected female Anopheles funestus had mainly fed on people (93.0%), but taken at least some of their blood (20.2%) from cattle. Although small numbers of A. arabiensis fed on sheep or goats and birds, none of the female A. gambiae s.s. and A. funestus tested had fed on these hosts. The absence of human-fed A. arabiensis in outdoor shelters indicated that exiting after feeding, a behaviour pattern that mitigates indoor insecticidal spraying, is not prevalent in this species in western Kenya.

Confirmation that Plasmodium falciparum has aperiodic infectivity to Anopheles gambiae.

In preparation for field studies of transmission-blocking malaria vaccines, a study was carried out to determine whether P. falciparum infections obtained in An. gambiae blood-fed at 16.00 hours were quantitatively similar to infections obtained at 23.00 hours. Using a group of children aged 5-12 years from villages at Ahero, near Kisumu in Kenya, 71/74 (96%) of whom were found to be positive for P.falciparum parasitaemia, one batch of fifty colony-bred An.gambiae females were fed on volunteers at 16.00 hours and another batch at 23.00 hours. No statistically significant differences were found in the proportions of mosquitoes becoming infected, the numbers of children infecting mosquitoes or the mean numbers of malaria oocysts developing in mosquitoes blood-fed at the different times. Because mosquito infections obtained by day (16.00 hours) are equivalent in quantity to those obtained at night (23.00 hours), experimental infections can be carried out in the afternoon, when it is most convenient, rather than during the night.

Plasmodium falciparum sporozoite and entomological inoculation rates at the Ahero rice irrigation scheme and the Miwani sugar-belt in western Kenya.

Anopheles arabiensis and An. funestus were collected by pyrethrum spray sheet collections in houses and by human-bait catches at a village in western Kenya adjacent to the Ahero rice irrigation scheme; and using the same methods, An. gambiae s.l. and An. funestus were collected at Miwani, a village in the sugar-cane belt. Plasmodium falciparum sporozoite rates were determined by ELISA. At Ahero the mean sporozoite rates were 1.1% and 4.3% in An. arabiensis and An. funestus, respectively, while at Miwani the rates were 6.0% in An. gambiae s.l. and 4.3% in An. funestus. Entomolgoical inoculation rates (EIR) were derived from both human-bait collections (IR-HBC) and by the proportion of human blood-fed females caught resting indoors (IR-HBF). The IR-HBF appeared to be a more realistic index of EIR. At Ahero and Miwani people were exposed to an average of 416 and 91 infective bites/person/year, respectively. The main vectors were An. funestus at Ahero and An. gambiae s.l. at Miwani. In view of the intense and perennial malaria transmission at Ahero, vector control by insecticides should be considered, while at Miwani, where transmission is seasonal, permethrin-impregnated bed nets could be an alternative to indoor spraying. These measures must be augmented with availability of effective antimalarials.