Background Hypoendemic malaria transmission in western Kenya highlands is not expected to lead to rapid acquisition of immunity to malaria. However, the asymptomatic subpopulation may play a significant role as an infection reservoir that should be considered in malaria control programs. Determination of spatio-temporal dynamics of asymptomatic subpopulations provides an opportunity to estimate the epidemiological importance of this group to malaria transmission. Methods Monthly parasitological surveys were undertaken on a cohort of 246 children for 12 months. Plasmodium falciparum infection prevalence was analyzed by both microscopy and PCR, and infection durations were determined. Results Infection prevalence and duration (1–12 months) decreased with age and altitude. Prevalence among age groups 5–9 and 10–14 years was high (34.4% and 34.1%, respectively), but significantly lower in older children (9.1%). Prevalence decreased from (52.4%) at ~1,430 m to 23.3% at 1,580 m. Conclusions Prevalence of asymptomatic P. falciparum infections was high, with PCR detecting a significantly higher number of infections than microscopy. Our results are consistent with gradual acquisition of immunity with age upon repeated infection, and also show that malaria transmission risk is highly heterogeneous in the highland area. The results provide strong support for targeted control.

Anopheles funestus Giles is one of the major African malaria vectors. It has previously been implicated in a major outbreak of malaria in KwaZulu/Natal, South Africa, during the period 1996 to 2000. The re-emergence of this vector was associated with monooxygenase-based resistance to pyrethroid insecticides. We have identified a gene from the monooxygenase CYP6 family, CYP6P9, which is over expressed in a pyrethroid resistant strain originating from Mozambique. Quantitative Real-Time PCR shows that this gene is highly over expressed in the egg and adult stages of the resistant strain relative to the susceptible strain but the larval stages showed almost no difference in expression between strains. This gene is genetically linked to a major locus associated with pyrethroid resistance in this A. funestus population.

Genetically-modified mosquitoes that are unable to transmit pathogens offer opportunities for controlling vector-borne diseases such as malaria and dengue. Site-specific gene recombination technologies are advantageous in the development of these insects because anti-pathogen effector genes can be inserted at integration sites in the genome that cause the least alteration in mosquito fitness. Here we describe Anopheles stephensi transgenic lines containing φC31 attP "docking" sites linked to a fluorescent marker gene. Chromosomal insertion sites were determined and lifetable parameters were assessed for transgenic mosquitoes of each line. No significant differences in fitness between the transgenic and non-transgenic mosquitoes were detected in this study. These transgenic lines are suitable for future site-specific integrations of anti-parasite transgenes into the attP sites.

While genome and transcriptome sequencing has revealed a large number and diversity of Anopheles gambiae predicted proteins, identifying their functions and biosynthetic pathways remains challenging. Applied mass spectrometry based proteomics in conjunction with mosquito genome and transcriptome databases were used to identify 44 proteins as putative components of the eggshell. Among the identified molecules are two vitelline membrane proteins and a group of seven putative chorion proteins. Enzymes with peroxidase, laccase and phenoloxidase activities, likely involved in cross-linking reactions that stabilize the eggshell structure, also were identified. Seven odorant binding proteins were found in association with the mosquito eggshell, although their role has yet to be demonstrated. This analysis fills a considerable gap of knowledge about proteins that build the eggshell of anopheline mosquitoes.

Human travel to malaria endemic lowlands from epidemic highlands has been shown to increase the risk of malaria infections in the highlands. In order to gain insight on the impact of human travel, we examined prevalence, genetic variability and population genetic structure of Plasmodium falciparum in asymptomatic children from one highland site and three surrounding malaria endemic lowland sites in western Kenya, using multilocus microsatellite genotyping. We further analyzed the frequencies of mutations at the genes conferring resistance to chloroquine and sulphadoxinepyrimethamine. We found a significant decrease in malaria prevalence in the highland site from 2006 to 2007, one year after the introduction of the artemisinin-based combination therapy as first-line treatment for uncomplicated malaria and the scale-up of insecticide treated bed nets. Population genetic diversity, measured by the number of observed and effective microsatellite alleles and Nei's unbiased genetic diversity, was high and comparable for both highland and lowland populations. Analysis of molecular variance did not detect a significant genetic structure across highland and lowland regions. Similarly, mutations at key antimalarial -resistance codons of the pfcrt, pfmdr1, pfdhfr and pfdhps genes were found at comparable high frequencies in all four sites. High level of gene flow and lack of significant genetic structure in malaria parasites between highland and lowland areas suggest the importance of human travel in shaping parasite population structure.

Pyrethroid resistance has been demonstrated in populations of Anopheles funestus from South Africa and southern Mozambique. Resistance is associated with elevated P450 monooxygenase enzymes. In this study, degenerate primers based on conserved regions of Anopheles gambiae P450 CYP4, 6 and 9 families were used to amplify genomic and cDNA templates from A. funestus. A total of 12 CYP4, 12 CYP6 and 7 CYP9 partial genes have been isolated and sequenced. BLAST results revealed that A. funestus P450s generally have a high sequence identity to A. gambiae with above 75% identity at the amino acid level. The exception is CYP9J14. The A. gambiae P450 showing highest identity to CYP9J14 exhibits only 55% identity suggesting that CYP9J14 may have arisen from a recent duplication event. Molecular phylogenetic analysis based on amino acid sequences also supported this hypothesis. Intron positions, but not size, were highly conserved between the two species. The high level of orthology that exists in the P450 gene families of these two species may facilitate the prediction of individual P450 protein function.

BackgroundThe Kenyan highlands were malaria-free before the 1910s, but a series of malaria epidemics have occurred in the highlands of western Kenya since the 1980s. Longitudinal studies of the genetic structure, complexity, infection dynamics, and duration of naturally acquired Plasmodium falciparum infections are needed to facilitate a comprehensive understanding of malaria epidemiology in the complex Kenyan highland eco-epidemiological systems where malaria recently expanded, as well as the evaluation of control measures.MethodsWe followed a cohort of 246 children residing in 3 villages at altitudes 1430 - 1580 m in western Kenya. Monthly parasitological surveys were undertaken for one year, yielding 866 P. falciparum isolates that were analyzed using 10 microsatellite markers.ResultsInfection complexity and genetic diversity were high (HE = 0.787-0.816), with ≥83% of infections harboring more than one parasite clone. Diversity remained high even during the low malaria transmission season. There was no significant difference between levels of genetic diversity and population structure between high and low transmission seasons. Infection turn-over rate was high, with the average infection duration of single parasite genotypes being 1.11 months, and the longest genotype persistence was 3 months.ConclusionsThese data demonstrate that despite the relatively recent spread of malaria to the highlands, parasite populations seem to have stabilized with no evidence of bottlenecks between seasons, while the ability of residents to clear or control infections indicates presence of effective anti-plasmodial immune mechanisms.

A knowledge gap in mosquito functional genetic analysis is the dearth of characterized regulatory regions that can target tissue specific transgene expression. To broaden the tools available, a promoter region of the Anopheles gambiaeα-tubulin1b gene has been assayed following fusion to the green fluorescent protein (GFP) reporter gene and stable transformation of An. gambiae. In eight transgenic lines, the Angtub α1b regulatory region directed a core profile of tissue specific expression in the head, chordotonal organs, ventral nerve cord and testes. This profile overlaps those seen for α2-tubulin expression in Drosophila melanogaster and Bombyx mori. In addition, widespread position dependant expression was observed in other specific tissues that were unique to each line. For example, in different lines, expression was observed in larval and adult muscles, fatbody, cuticle and midgut secretory cells. The majority of genomic transgene insertions were mapped to within 10 kb of a gene, suggesting that the Angtub α1b basal promoter is particularly sensitive to enhancers and may be suitable to form the basis of a sensitive enhancer trapping construct, in combination with a binary expression system such as Gal4-UAS.