Molecular Characterization Reveals Diverse and Unknown Malaria Vectors in the Western Kenyan Highlands.

The success of mosquito-based malaria control is dependent upon susceptible bionomic traits in local malaria vectors. It is crucial to have accurate and reliable methods to determine mosquito species composition in areas subject to malaria. An unexpectedly diverse set of Anopheles species was collected in the western Kenyan highlands, including unidentified and potentially new species carrying the malaria parasite Plasmodium falciparum. This study identified 2,340 anopheline specimens using both ribosomal DNA internal transcribed spacer region 2 and mitochondrial DNA cytochrome oxidase subunit 1 loci. Seventeen distinct sequence groups were identified. Of these, only eight could be molecularly identified through comparison to published and voucher sequences. Of the unidentified species, four were found to carry P. falciparum by circumsporozoite enzyme-linked immunosorbent assay and polymerase chain reaction, the most abundant of which had infection rates comparable to a primary vector in the area, Anopheles funestus. High-quality adult specimens of these unidentified species could not be matched to museum voucher specimens or conclusively identified using multiple keys, suggesting that they may have not been previously described. These unidentified vectors were captured outdoors. Diverse and unknown species have been incriminated in malaria transmission in the western Kenya highlands using molecular identification of unusual morphological variants of field specimens. This study demonstrates the value of using molecular methods to compliment vector identifications and highlights the need for accurate characterization of mosquito species and their associated behaviors for effective malaria control

Ecological limitations on aquatic mosquito predator colonization in the urban environment

Urban malaria cases are becoming common in Africa as more people move into cities and industrialization proceeds. While many species of Anopheles mosquitoes vector malaria in rural areas, only a few are found within cities. The success of anthropophilic species in cities, such as members of the An. gambiae complex, may be explained by limitations on colonization by predator species in urban environments. Habitats that are temporal or structurally simple have lower predator survivorship in a variety of ecosystems, but these have not been investigated previously in an urban area. Areas within and around the Kenyan coastal town of Malindi were previously sampled for the presence of standing water using a geographic sampling strategy with probability proportional to size sampling of planned well-drained, unplanned poorly-drained, planned poorly-drained, unplanned well-drained, and peri-urban locations. Standing aquatic habitats in these areas were reassessed. During monthly sampling, presence/absence of mosquitoes and predator taxa were noted, as were ecological habitat variables: structural complexity and presence of water. Lambda statistics were calculated to associate predator guilds, habitat types, location variables, and ecological variables. All predator guilds found in habitats were strongly associated with habitat type, as were the structural complexity and temporal nature of the habitats. Types of habitat were heterogeneously distributed throughout Malindi, with swimming pools as a common habitat type in planned urban areas and tire track pools a common habitat type in peri-urban areas of Malindi. Predator colonization of aquatic habitats in Malindi was strongly influenced by habitat type, and not associated with location characteristics. Ecological variables were affected by the type of habitats, which are co-associated with planning and drainage in Malindi. While habitat types are distributed heterogeneously within Malindi, habitats with low predation pressure are available for mosquito colonization in both urban and peri-urban areas. The temporal, peri-urban tire track pools and the structural simplicity of urban swimming pools may discourage predator colonization, thereby increasing the probability of malaria vectors in these areas of Malindi. Future studies should evaluate habitats for use in malaria surveillance and experimentally test the effects of structural complexity and temporal nature of urban habitats on the densities of mosquito larvae and their aquatic predators

Novel vectors of malaria parasites in the western highlands of Kenya.

: TO THE EDITOR: The primary malaria control techniques, indoor application of residual insecticides and insecticide-treated bed nets, are used on the basis of previously assumed key characteristics of behaviors of vectors of malaria parasites, i.e., resting and feeding indoors (1). Any deviation from the typical activities of a species related to exophagy (feeding outdoors) and exophily (living and resting outdoors) (2) or to population replacement, followed by increased outdoor biting or resting (3), may undermine malaria control efforts. Identification of mosquitoes that transmit malaria parasites has, for the most part, relied on the use of outdated morphologic keys (4,5) and, more recently, species-diagnostic PCR (6). Cryptic species or subpopulations that exhibit divergent behaviors (7) may be responsible for maintaining malaria parasite transmission, and without adequate discriminatory techniques, these vectors may be misidentified and their key behavioral differences overlooked

Heavy metals in mosquito larval habitats in urban Kisumu and Malindi, Kenya, and their impact

Concentrations and distribution of cadmium, chromium, copper, iron, lead, manganese and zinc in mosquito larval habitats in urban Kisumu and Malindi, Kenya and their effect on the presence of Anopheles gambiae, Aedes aegypti, Culex quinquefasciatus and Anopheles funestus larvae were investigated. Manganese and iron were the most prevalent heavy metals in water of larval habitats in urban Kisumu and Malindi, respectively. Iron was the most prevalent heavy metal in bottom sediments in larval habitats in both cities. The highest concentrations of all heavy metals, except cadmium and iron, were recorded in the poorly planned–well drained stratum in the two cities. All heavy metals were more concentrated in human-made than in natural larval habitats. Copper was positively associated with the presence of Ae. aegypti, and lead was associated with the presence of An. gambiae and Ae. aegypti in urban Kisumu. Absence of significant correlation between the other metals and mosquito species in both cities, despite relatively high concentrations, suggest that the local larval populations, including key malaria vectors have adapted to the detected levels of these metals

Efficacy and persistence of long-lasting microbial larvicides against malaria vectors in western Kenya highlands.

BackgroundChemical-based malaria vector control interventions are threatened by the development of insecticide resistance and changes in the behavior of the vectors, and thus require the development of alternative control methods. Bacterial-based larvicides have the potential to target both insecticide resistant and outdoor-biting mosquitoes and are safe to use in the environment. However, the currently available microbial larvicide formulations have a short duration of activity requiring frequent re-applications which increase the cost of control interventions. This study was designed to evaluate the efficacy and duration of activity of two long-lasting formulations of Bacillus thuringiensis var. israelensis (Bti) and Bacillus sphaericus (Bs) (LL3 and FourStar®) under field conditions in western Kenya highlands.MethodsThree sites were selected for this study in the highlands of western Kenya. In each site, one hundred anopheline larval habitats were selected and assigned to one of three arms: (i) LL3; (ii) FourStar®; and (iii) untreated control larval habitats. Four types of larval habitats were surveyed: abandoned gold mines, drainage canals, fish ponds and non-fish ponds. The habitats were sampled for mosquito larvae by using a standard dipping technique and collected larvae were recorded according to the larval stages of the different Anopheles species. The larvicides were applied at manufacturers' recommended dosage of 1 briquette per 100 square feet. Both treatment and control habitats were sampled for mosquito larvae immediately before treatment (day 0), and then at 24 hours, 3 days and weekly post-treatment for 5 months.ResultsOverall larval density in treatment habitats was significantly reduced after application of the two microbial larvicides as compared to the control habitats. Post-intervention reduction in anopheline larval density by LL3 was 65, 71 and 84% for 1 day, 2 weeks and 4 weeks, respectively. FourStar® reduced anopheline larval density by 60, 66 and 80% for 1 day, 2 weeks and 4 weeks, respectively. Comparisons between the treatments reveal that LL3 and FourStar® were similar in efficacy. A higher reduction in Anopheles larval density was observed in the abandoned goldmines, while drainage canals had the lowest reduction.ConclusionsBoth LL3 and FourStar® long-lasting microbial larvicides were effective in reducing immature stages of An. gambiae complex and An. funestus group species, with significant reductions lasting for three months post-application

Efficacy and persistence of long-lasting microbial larvicides against malaria vectors in western Kenya highlands

Abstract Background Chemical-based malaria vector control interventions are threatened by the development of insecticide resistance and changes in the behavior of the vectors, and thus require the development of alternative control methods. Bacterial-based larvicides have the potential to target both insecticide resistant and outdoor-biting mosquitoes and are safe to use in the environment. However, the currently available microbial larvicide formulations have a short duration of activity requiring frequent re-applications which increase the cost of control interventions. This study was designed to evaluate the efficacy and duration of activity of two long-lasting formulations of Bacillus thuringiensis var. israelensis (Bti) and Bacillus sphaericus (Bs) (LL3 and FourStar®) under field conditions in western Kenya highlands. Methods Three sites were selected for this study in the highlands of western Kenya. In each site, one hundred anopheline larval habitats were selected and assigned to one of three arms: (i) LL3; (ii) FourStar®; and (iii) untreated control larval habitats. Four types of larval habitats were surveyed: abandoned gold mines, drainage canals, fish ponds and non-fish ponds. The habitats were sampled for mosquito larvae by using a standard dipping technique and collected larvae were recorded according to the larval stages of the different Anopheles species. The larvicides were applied at manufacturers’ recommended dosage of 1 briquette per 100 square feet. Both treatment and control habitats were sampled for mosquito larvae immediately before treatment (day 0), and then at 24 hours, 3 days and weekly post-treatment for 5 months. Results Overall larval density in treatment habitats was significantly reduced after application of the two microbial larvicides as compared to the control habitats. Post-intervention reduction in anopheline larval density by LL3 was 65, 71 and 84% for 1 day, 2 weeks and 4 weeks, respectively. FourStar® reduced anopheline larval density by 60, 66 and 80% for 1 day, 2 weeks and 4 weeks, respectively. Comparisons between the treatments reveal that LL3 and FourStar® were similar in efficacy. A higher reduction in Anopheles larval density was observed in the abandoned goldmines, while drainage canals had the lowest reduction. Conclusions Both LL3 and FourStar® long-lasting microbial larvicides were effective in reducing immature stages of An. gambiae complex and An. funestus group species, with significant reductions lasting for three months post-application

Microbial larvicides for mosquito control: Impact of long lasting formulations of Bacillus thuringiensis var. israelensis and Bacillus sphaericus on non-target organisms in western Kenya highlands.

The microbial larvicides Bacillus thuringiensis var. israelensis and Bacillus sphaericus have been used extensively for mosquito control and have been found to be effective and safe to non-target organisms cohabiting with mosquito larvae. Recently developed long lasting microbial larvicides (LLML), although evading the previous challenge of short duration of activity, increase the risk of persistence of toxins in the treated larval habitats. This study monitored the impact of LLML FourStar® and LL3 on non-target organisms cohabiting with mosquito larvae in an operational study to control malaria vectors in western Kenya highlands. A total of 300 larval habitats were selected in three highland villages. The habitats were first monitored for 5 weeks to collect baseline data on non-target organisms cohabiting with mosquito larvae and then randomized into two treatment arms (respective FourStar® and LL3) and one control arm. Non-target organisms were sampled weekly for 5 months after treatment to assess the impact of LLML intervention. Before treatment, the mean density of all non-target organisms combined in the control, LL3 and FourStar® treated habitats was 1.42, 1.39 and 1.49 individuals per habitat per sampling occasion, respectively. Following treatment, this density remained fairly unchanged for 21 weeks at which time it was 1.82, 2.11, and 2.05 for the respective control, LL3 and FourStar® treated habitats. Statistical analysis revealed that LL3 and FourStar® did not significantly alter abundance, richness or diversity of the 11 taxa studied, when comparing the intervention and control larval habitats. However, both FourStar® and LL3 significantly reduced the density of malaria vectors. In conclusion, one round of label rate application of FourStar® or LL3 in natural larval habitats did not alter richness, abundance or diversity of the monitored aquatic non-target organisms cohabiting with mosquito larvae to an ecologically significant level

Microbial larvicides for mosquito control: Impact of long lasting formulations of Bacillus thuringiensis var. israelensis and Bacillus sphaericus on non-target organisms in western Kenya highlands.

The microbial larvicides Bacillus thuringiensis var. israelensis and Bacillus sphaericus have been used extensively for mosquito control and have been found to be effective and safe to non-target organisms cohabiting with mosquito larvae. Recently developed long lasting microbial larvicides (LLML), although evading the previous challenge of short duration of activity, increase the risk of persistence of toxins in the treated larval habitats. This study monitored the impact of LLML FourStar® and LL3 on non-target organisms cohabiting with mosquito larvae in an operational study to control malaria vectors in western Kenya highlands. A total of 300 larval habitats were selected in three highland villages. The habitats were first monitored for 5 weeks to collect baseline data on non-target organisms cohabiting with mosquito larvae and then randomized into two treatment arms (respective FourStar® and LL3) and one control arm. Non-target organisms were sampled weekly for 5 months after treatment to assess the impact of LLML intervention. Before treatment, the mean density of all non-target organisms combined in the control, LL3 and FourStar® treated habitats was 1.42, 1.39 and 1.49 individuals per habitat per sampling occasion, respectively. Following treatment, this density remained fairly unchanged for 21 weeks at which time it was 1.82, 2.11, and 2.05 for the respective control, LL3 and FourStar® treated habitats. Statistical analysis revealed that LL3 and FourStar® did not significantly alter abundance, richness or diversity of the 11 taxa studied, when comparing the intervention and control larval habitats. However, both FourStar® and LL3 significantly reduced the density of malaria vectors. In conclusion, one round of label rate application of FourStar® or LL3 in natural larval habitats did not alter richness, abundance or diversity of the monitored aquatic non-target organisms cohabiting with mosquito larvae to an ecologically significant level