Pyrethroid resistance in Anopheles gambiae s.s. and Anopheles arabiensis in western Kenya: phenotypic, metabolic and target site characterizations of three populations.

Field and laboratory investigations revealed phenotypic, target site and metabolic resistance to permethrin in an Anopheles gambiae s.s. (Diptera: Culicidae) population in Bungoma District, a region in western Kenya in which malaria is endemic and rates of ownership of insecticide-treated bednets are high. The sensitivity of individual An. gambiae s.l. females as indicated in assays using World Health Organization (WHO) test kits demonstrated reduced mortality in response to permethrin, deltamethrin and bendiocarb. Estimated time to knock-down of 50% (KDT50 ) of the test population in Centers for Disease Control (CDC) bottle bioassays was significantly lengthened for the three insecticides compared with that in a susceptible control strain. Anopheles arabiensis from all three sites showed higher mortality to all three insecticides in the WHO susceptibility assays compared with the CDC bottle assays, in which they showed less sensitivity and longer KDT50 than the reference strain for permethrin and deltamethrin. Microplate assays revealed elevated activity of ß-esterases and oxidases, but not glutathione-S-transferase, in An. gambiae s.s. survivors exposed to permethrin in bottle bioassays compared with knocked down and unexposed individuals. No An. arabiensis showed elevated enzyme activity. The 1014S kdr allele was fixed in the Bungoma An. gambiae s.s. population and absent from An. arabiensis, whereas the 1014F kdr allele was absent from all samples of both species. Insecticide resistance could compromise vector control in Bungoma and could spread to other areas as coverage with longlasting insecticide-treated bednets increases.

A supervised land cover classification of a western Kenya lowland endemic for human malaria: associations of land cover with larval Anopheles habitats.

BACKGROUND: A supervised land cover classification was developed from very high resolution IKONOS satellite data and extensive ground truth sampling of a ca. 10 sq km malaria-endemic lowland in western Kenya. The classification was then applied to an investigation of distribution of larval Anopheles habitats. The hypothesis was that the distribution and abundance of aquatic habitats of larvae of various species of mosquitoes in the genus Anopheles is associated with identifiable landscape features. RESULTS AND DISCUSSION: The classification resulted in 7 distinguishable land cover types, each with a distinguishable vegetation pattern, was highly accurate (89%, Kappa statistic = 0.86), and had a low rate of omission and commission errors. A total of 1,198 habitats and 19,776 Anopheles larvae of 9 species were quantified in samples from a rainy season, and 184 habitats and 582 larvae from a dry season. Anopheles gambiae s.l. was the dominant species complex (51% of total) and A. arabiensis the dominant species. Agricultural land covers (mature maize fields, newly cultivated fields, and pastured grasslands) were positively associated with presence of larval habitats, and were located relatively close to stream channels; whilst nonagricultural land covers (short shrubs, medium shrubs, tall shrubs, and bare soil around residences) were negatively associated with presence of larval habitats and were more distant from stream channels. Number of larval habitats declined exponentially with distance from streams. IKONOS imagery was not useful in direct detection of larval habitats because they were small and turbid (resembling bare soil), but was useful in localization of them through statistical associations with specific land covers. CONCLUSION: A supervised classification of land cover types in rural, lowland, western Kenya revealed a largely human-modified and fragmented landscape consisting of agricultural and domestic land uses. Within it, larval habitats of Anopheles vectors of human malaria were associated with certain land cover types, of largely agricultural origin, and close to streams. Knowledge of these associations can inform malaria control to gather information on potential larval habitats more efficiently than by field survey and can do so over large areas.

Sampling outdoor, resting Anopheles gambiae and other mosquitoes (Diptera: Culicidae) in western Kenya with clay pots.

Clay pots were analyzed as devices for sampling the outdoor resting fraction of Anopheles gambiae Giles (Diptera: Culicidae) and other mosquito species in a rural, western Kenya. Clay pots (Anopheles gambiae resting pots, herein AgREPOTs), outdoor pit shelters, indoor pyrethrum spray collections (PSC), and Colombian curtain exit traps were compared in collections done biweekly for nine intervals from April to June 2005 in 20 housing compounds. Of 10,517 mosquitoes sampled, 4,668 An. gambiae s.l. were sampled in total of which 63% were An. gambiae s.s. (46% female) and 37% were An. arabiensis (66% female). The clay pots were useful and practical for sampling both sexes of An. gambiae s.l. Additionally, 617 An. funestus (58% female) and 5,232 Culex spp. (males and females together) were collected. Temporal changes in abundance of An. gambiae s.l. were similarly revealed by all four sampling methods, indicating that the clay pots could be used as devices to quantify variation in mosquito population density. Dispersion patterns of the different species and sexes fit well the negative binomial distribution, indicating that the mosquitoes were aggregated in distribution. Aside from providing a useful sampling tool, the AgREPOT also may be useful as a delivery vehicle for insecticides or pathogens to males and females that enter and rest in them.

Impact of sustained use of insecticide-treated bednets on malaria vector species distribution and culicine mosquitoes.

Insecticide-treated bednets (ITNs) significantly reduce malaria vector populations. Susceptibility to ITNs differs by vector species, and culicine mosquitoes have not been shown to be significantly affected by the use of ITNs. We examined the impact of 2-4 yr of ITN use on malaria vector species distribution and culicine mosquitoes. Routine entomological surveillance was conducted in adjacent areas with and without ITNs from November 1999 to January 2002. Use of ITNs reduced the proportion of Anopheles gambiae Giles relative to Anopheles arabiensis Giles. The number of culicines per house was significantly lower in the ITN area than in the neighboring area. Changes in the An. gambiae sibling species distribution may help to explain apparent mosquito behavioral changes attributed to ITNs. Reductions in culicines by ITNs may have implications for community perceptions of ITN effectiveness and for control of other diseases such as lymphatic filariasis.

Genetic and morphological variation in three snow pool Aedes mosquito species of the subgenus Ochlerotatus (Diptera: Culicidae).

Thirteen Aedes hexodontus Dyar populations from throughout the western United States were examined for genetic and morphological variation. Analysis of allozyme frequencies at 16 loci revealed a pattern of genetic variation that formed a north-south cline across Washington, Oregon, and California in the number of alleles per locus, the percent of polymorphic loci, and the frequency of one allele of aconitase-1. Comparison of the genetic profile of Ae. hexodontus populations to two other widely distributed members of the punctor subgroup, Aedes punctor (Kirby) and Aedes abserratus (Felt & Young), revealed only one diagnostic locus for all three species. Seven loci exhibited frequency differences among species but were not diagnostic. Morphological characters also exhibited little variation within and among the three species. The adult females differed only in the scaling of the probasisternum. This area was extensively scaled in 91% of Ae. hexodontus specimens but bare or only lightly scaled in Ae. puntor and Ae. abserratus. No other differences were observed in the adult females or the male genitalia in any of the three species. The larvae of Ae. abserratus could be separated by the single-branched seta 2-X. Six larval characters differed between Ae. hexodontus and Ae. punctor but the ranges of each character overlapped and none were diagnostic. These comparisons indicated that Ae. hexodontus is a single species, at least in the southern part of its range. Also, genetic and morphological comparison of the three species within the punctor subgroup attested to the close relationship hypothesized for these mosquitoes.

Genetic and morphological characterization of the Aedes (Ochlerotatus) dorsalis (Diptera: Culicidae) group in North America.

An examination of the electrophoretically detectable variation among the North American members of the Aedes (Ochlerotatus) dorsalis group revealed large genetic differences among all 4 species. At least 9 of 18 loci examined (50%) were diagnostic for each species pair. However, morphological variation observed among species was low. Only Aedes canadensis (Theobald) was separated readily from the other members of this group [Aedes dorsalis (Meigen), Aedes melanimon Dyar and Aedes campestris Dyar & Knab] in all life stages. Characters traditionally used to separate the remaining 3 species were less reliable. In the adult female, Ae. melanimon may be distinguished from Ae. campestris by the scaling patterns of the wings and abdomen, but Ae. dorsalis could not be distinguished reliably by these characters. Adults of Ae. dorsalis may be separated reliably from those of Ae. campestris and Ae. melanimon only by the length of the subapical tooth relative to the length of the tarsal claw. Ae. melanimon was identified in the larval stage by the short mesothoracic hair 1. Eight larval characters differed between Ae. dorsalis and Ae. campestris. However, the ranges of these characters overlapped and no character was truly diagnostic. Genetic variation within species was low as measured by average heterozygosity and Nei's genetic distance coefficients. No allozymes were diagnostic for coastal and inland populations of Ae. dorsalis, and the pattern of genetic differentiation within this species did not correspond to the geographic location of the populations examined. Therefore, the genetic data did not support the hypothesis that Ae. dorsalis represents a complex of 2 or more cryptic species.

Characteristics of larval anopheline (Diptera: Culicidae) habitats in Western Kenya.

A longitudinal survey of mosquito larval habitats was carried out in Asembo Bay, western Kenya, during the rainy season of 1998. All pools of standing water along a 700-m transect were sampled twice per week. For each habitat, eight environmental variables were recorded and a sample of anopheline larvae was collected for identification. In total, 1,751 Anopheles gambiae s.l. and 2,784 Anopheles funestus Giles were identified. Identification of An. gambiae s.l. by polymerase chain reaction (PCR) indicated that 240 (14.7%) were An. gambiae Giles and 858 (52.4%) were An. arabiensis Patton; PCR failed to identify 539 (32.9%) specimens. Repeated measures logistic regression analysis indicated that An. gambiae and An. arabiensis larvae were associated with small, temporary habitats with algae and little or no aquatic vegetation. Anopheles funestus larvae were associated with larger, semipermanent bodies of water containing aquatic vegetation and algae. Direct comparison of habitat characteristics associated with either An. gambiae or An. arabiensis revealed that algae were associated more commonly with habitats containing An. gambiae; no other differences were detected. Chi-square analysis indicated that these species were collected from the same habitat more frequently than would be expected by chance alone. Together, these results indicate that An. gambiae and An. arabiensis have similar requirements for the larval environment and that, at least in western Kenya, they do not segregate into separate habitats.

Temporal and spatial genetic variation within and among populations of the mosquito Culex tarsalis (Diptera: Culicidae) from California.

The genetic structure of 11 populations of Culex tarsalis Coquillett from California and 1 population from Nevada was examined at 18 loci using polyacrylamide gel electrophoresis. Six populations from northern and southern California were sampled repeatedly to determine if the genetic structure of Cx. tarsalis changes seasonally. Significant differences in allele frequencies at 13 different loci were seen in 3 populations over time as determined by contingency chi-square tests. Nei's genetic distance coefficients among different sampling dates was consistently < 0.025. The number of alleles per locus in these populations ranged from 1.6 to 2.7, whereas the average heterozygosity ranged from 0.086 to 0.228. No single locus was found to vary in a consistent pattern within all populations that were sampled repeatedly. These results indicate that Cx. tarsalis populations are genetically stable over time and that temporal variation is due to fluctuations in population size or immigration of genetically distinct individuals. In contrast, Cx. tarsalis did exhibit some differences in genetic structure that were related to geographical features including the Sierra Nevada and the Tehachapi Mountains of southern California. Genetically differentiated populations occurred in Nevada, southern and northeastern California, and the Central Valley of California. Little differentiation was observed among populations located in the Central Valley of California and those located at high elevations in the Sierra Nevada. Thus, in the populations examined, mountain ranges or arid conditions that limit the number of larval development sites appeared to be important barriers to the dispersal of Cx. tarsalis.

Effect of permethrin-impregnated nets on exiting behavior, blood feeding success, and time of feeding of malaria mosquitoes (Diptera: Culicidae) in western Kenya.

The impact of permethrin-treated bednets on the feeding and house entering/exiting behavior of malaria vectors was assessed in two studies in western Kenya. In one study, matched pairs of houses were allocated randomly to receive bednets or no bednets. Exiting mosquitoes were collected in Colombian curtains hung around half of each house; indoor resting mosquitoes were collected by pyrethrum spray catches. The number of Anopheles gambiae Giles and An. arabiensis Patton estimated to have entered the houses was unaffected by the presence of bednets; Anopheles funestus Giles was less likely to enter a house if bednets were present. Anopheles gambiae and An. funestus were less likely to obtain a blood meal and significantly more likely to exit houses when bednets were present. No difference was detected in An. arabiensis rates of blood feeding and exiting. In a second experiment, hourly night biting collections were done on 13 nights during the rainy season to assess whether village-wide use of permethrin-treated bednets caused a shift in the time of biting of malaria vectors. A statistically significant shift was detected in the biting times of An. gambiae s.l., although the observed differences were small. No change was observed in the hourly distribution of An. funestus biting. Our study demonstrated that, at least in the short-term, bednets reduced human-vector contact and blood feeding success but did not lead to changes in the biting times of the malaria vectors in western Kenya.

Genetic differences among Anopheles vestitipennis subpopulations collected using different methods in Chiapas state, southern México.

Biting activity and population genetic studies of the malaria vector Anopheles vestitipennis were conducted in southern México. Three subpopulations were collected from 2 villages; 2 subpopulations were from the same village, one on human bait and one with an animal-baited trap; the third was collected from a cattle corral in the 2nd village (280 km away SSE). The anthropophilic subpopulation had steady activity with 61% of bites occurring before midnight, significantly different from those of the 2 zoophilic subpopulations, which had 78-82% of bites before midnight and 2 biting peaks, one at 1900-2100 h and the other at 0400-0500 h. Isozyme analysis (13 enzymes) of these subpopulations indicated that differences between the 2 sympatric subpopulations (D = 0.07), collected using 2 different methods, were greater than that between the 2 allopatric ones (D = 0.03). These studies suggest the existence of 2 genetically different subpopulations of An. vestitipennis with specific host preferences.

The distribution of species of the Aedes increpitus complex in the western United States.

Maps of the California and Oregon distribution of members of the Aedes increpitus complex (Aedes clivis Lanzaro and Eldridge, Aedes increpitus Dyar, and Aedes washinoi Lanzaro and Eldridge) are presented that are based on collections reported by Lanzaro and Eldridge (1992) and new collections from various sites, many in the Central Valley of California. Analysis of individually reared specimens by polyacrylamide gel electrophoresis and conventional morphological methods confirmed the diagnostic value of isozymes for these species and of larval head hairs for distinguishing Ae. clivis from other members of the complex. Other larval characters and pupal hairs did not appear to have diagnostic value. An additional site was discovered where apparent hybrids between Ae. washinoi and Ae. increpitus occur, and a single possible hybrid between Ae. washinoi and Ae. clivis was found at a site where these species had previously been reported to occur sympatrically.

Ribosomal DNA internal transcribed spacer (ITS2) sequences differentiate Anopheles funestus and An. rivulorum, and uncover a cryptic taxon.

Differentiation among the closely related Afrotropical species comprising the Funestus Group is difficult by traditional taxonomic measures. Anopheles rivulorum is the second most abundant and widespread species in the Funestus Group, and is occasionally collected indoors along with the dominant member and major malaria vector, An. funestus. The prospect of misidentification of An. rivulorum as An. funestus prompted the development of a rapid, polymerase chain reaction (PCR)-based method for identifying these two species. The ribosomal internal transcribed spacer 2 (ITS2) was amplified from thirty-five specimens of An. rivulorum collected from the extremes of its range: Eastern Africa (Kenya), Southern Africa (South Africa) and Western Africa (Burkina Faso). The ITS2 region of An. rivulorum ( approximately 380 bp) is sufficiently different in size from the ITS2 of An. funestus ( approximately 700 bp) that these species can be distinguished by agarose gel electrophoresis of PCR products without further manipulation. Comparison of the An. rivulorum and An. funestus ITS2 nucleotide sequences revealed such extensive divergence that meaningful alignment was impossible, except for a 25 bp island near the 5' end. Intraspecific sequence comparisons revealed no variation among An. rivulorum individuals collected from the same country. However, sequence divergence was 2% between specimens from South Africa and Kenya, and nearly tenfold higher ( approximately 19%) between specimens from Burkina Faso and either South Africa or Kenya, an unprecedented level of intraspecific ITS2 divergence in Anopheles. Taken together, these data suggest that the Burkina Faso sample is not An. rivulorum, but rather a cryptic taxon within the Funestus Group.