Distribution of a Knockdown Resistance Mutation (L1014S) in Anopheles gambiae s.s. and Anopheles arabiensis in Western and Southern Kenya

In Kenya, insecticide-treated mosquito nets (ITNs) distributed to pregnant women and children under 5 years old through various programs have resulted in a significant reduction in malaria deaths. All of the World Health Organization-recommended insecticides for mosquito nets are pyrethroids, and vector mosquito resistance to these insecticides is one of the major obstacles to an effective malaria control program. Anopheles gambiae s.s. and Anopheles arabiensis are major malaria vectors that are widely distributed in Kenya. Two point mutations in the voltage-gated sodium channel (L1014F and L1014S) are associated with knockdown resistance (kdr) to DDT and pyrethroids in An. gambiae s.s. While the same point mutations have been reported to be rare in An. arabiensis, some evidence of metabolic resistance has been reported in this species. In order to determine the distribution of the point mutation L1014S in An. gambiae s.s. and An. arabiensis in southern and western Kenya, we collected larvae and screened for the mutation by DNA sequencing. We found high allelic and homozygous frequencies of the L1014S mutation in An. gambiae s.s. The L1014S mutation was also widely distributed in An. arabiensis, although the allelic frequency was lower than in An. gambiae s.s. The same intron sequence (length: 57 base) found in both species indicated that the mutation was introgressed by hybridization. The allelic frequency of L1014S was higher in both species in western regions, demonstrating the strong selection pressure imposed by long-lasting insecticide-treated nets (LLITN)/ITN on the An. gambiae s.s. and An. arabiensis populations in those areas. The present contribution of the L1014S mutation to pyrethroid resistance in An. arabiensis may be negligible. However, the homozygous frequency could increase with continuing selection pressure due to expanded LLITN coverage in the future

Radiosensitizing potential of the selective cyclooygenase-2 (COX-2) inhibitor meloxicam on human glioma cells

The COX-2 protein is frequently overexpressed in human malignant gliomas. This expression has been associated with their aggressive growth characteristics and poor prognosis for patients. Targeting the COX-2 pathway might improve glioma therapy. In this study, the effects of the selective COX-2 inhibitor meloxicam alone and in combination with irradiation were investigated on human glioma cells in vitro. A panel of three glioma cell lines (D384, U87 and U251) was used in the experiments from which U87 cells expressed constitutive COX-2. The response to meloxicam and irradiation (dose-range of 0–6 Gy) was determined by the clonogenic assay, cell proliferation was evaluated by growth analysis and cell cycle distribution by FACS. 24–72 h exposure to 250–750 μM meloxicam resulted in a time and dose dependent growth inhibition with an almost complete inhibition after 24 h for all cell lines. Exposure to 750 μM meloxicam for 24 h increased the fraction of cells in the radiosensitive G2/M cell cycle phase in D384 (18–27%) and U251 (17–41%) cells. 750 μM meloxicam resulted in radiosensitization of D384 (DMF:2.19) and U87 (DMF:1.25) cells, but not U251 cells (DMF:1.08). The selective COX-2 inhibitor meloxicam exerted COX-2 independent growth inhibition and radiosensitization of human glioma cells

Genetic population structure of Anopheles gambiae in Equatorial Guinea

BACKGROUND: Patterns of genetic structure among mosquito vector populations in islands have received particular attention as these are considered potentially suitable sites for experimental trials on transgenic-based malaria control strategies. In this study, levels of genetic differentiation have been estimated between populations of Anopheles gambiae s.s. from the islands of Bioko and Annobón, and from continental Equatorial Guinea (EG) and Gabon. METHODS: Genotyping of 11 microsatellite loci located in chromosome 3 was performed in three island samples (two in Bioko and one in Annobón) and three mainland samples (two in EG and one in Gabon). Four samples belonged to the M molecular form and two to the S-form. Microsatellite data was used to estimate genetic diversity parameters, perform demographic equilibrium tests and analyse population differentiation. RESULTS: High levels of genetic differentiation were found between the more geographically remote island of Annobón and the continent, contrasting with the shallow differentiation between Bioko island, closest to mainland, and continental localities. In Bioko, differentiation between M and S forms was higher than that observed between island and mainland samples of the same molecular form. CONCLUSION: The observed patterns of population structure seem to be governed by the presence of both physical (the ocean) and biological (the M-S form discontinuity) barriers to gene flow. The significant degree of genetic isolation between M and S forms detected by microsatellite loci located outside the "genomic islands" of speciation identified in A. gambiae s.s. further supports the hypothesis of on-going incipient speciation within this species. The implications of these findings regarding vector control strategies are discussed

Multiple Origins of Knockdown Resistance Mutations in the Afrotropical Mosquito Vector Anopheles gambiae

How often insecticide resistance mutations arise in natural insect populations is a fundamental question for understanding the evolution of resistance and also for modeling its spread. Moreover, the development of resistance is regarded as a favored model to study the molecular evolution of adaptive traits. In the malaria vector Anopheles gambiae two point mutations (L1014F and L1014S) in the voltage-gated sodium channel gene, that confer knockdown resistance (kdr) to DDT and pyrethroid insecticides, have been described. In order to determine whether resistance alleles result from single or multiple mutation events, genotyping of the kdr locus and partial sequencing of the upstream intron-1 was performed on a total of 288 A. gambiae S-form collected from 28 localities in 15 countries. Knockdown resistance alleles were found to be widespread in West Africa with co-occurrence of both 1014S and 1014F in West-Central localities. Differences in intron-1 haplotype composition suggest that kdr alleles may have arisen from at least four independent mutation events. Neutrality tests provided evidence for a selective sweep acting on this genomic region, particularly in West Africa. The frequency and distribution of these kdr haplotypes varied geographically, being influenced by an interplay between different mutational occurrences, gene flow and local selection. This has important practical implications for the management and sustainability of malaria vector control programs

Patterns of Reproductive Isolation in Toads

Understanding the general features of speciation is an important goal in evolutionary biology, and despite significant progress, several unresolved questions remain. We analyzed an extensive comparative dataset consisting of more than 1900 crosses between 92 species of toads to infer patterns of reproductive isolation. This unique dataset provides an opportunity to examine the strength of reproductive isolation, the development and sex ratios of hybrid offspring, patterns of fertility and infertility, and polyploidization in hybrids all in the context of genetic divergence between parental species. We found that the strength of intrinsic postzygotic isolation increases with genetic divergence, but relatively high levels of divergence are necessary before reproductive isolation is complete in toads. Fertilization rates were not correlated to genetic divergence, but hatching success, the number of larvae produced, and the percentage of tadpoles reaching metamorphosis were all inversely related with genetic divergence. Hybrids between species with lower levels of divergence developed to metamorphosis, while hybrids with higher levels of divergence stopped developing in gastrula and larval stages. Sex ratios of hybrid offspring were biased towards males in 70% of crosses and biased towards females in 30% of crosses. Hybrid females from crosses between closely related species were completely fertile, while approximately half (53%) of hybrid males were sterile, with sterility predicted by genetic divergence. The degree of abnormal ploidy in hybrids was positively related to genetic divergence between parental species, but surprisingly, polyploidization had no effect on patterns of asymmetrical inviability. We discuss explanations for these patterns, including the role of Haldane's rule in toads and anurans in general, and suggest mechanisms generating patterns of reproductive isolation in anurans

Inferring selection in the Anopheles gambiae species complex: an example from immune-related serine protease inhibitors

<p>Abstract</p> <p>Background</p> <p>Mosquitoes of the <it>Anopheles gambiae </it>species complex are the primary vectors of human malaria in sub-Saharan Africa. Many host genes have been shown to affect <it>Plasmodium </it>development in the mosquito, and so are expected to engage in an evolutionary arms race with the pathogen. However, there is little conclusive evidence that any of these mosquito genes evolve rapidly, or show other signatures of adaptive evolution.</p> <p>Methods</p> <p>Three serine protease inhibitors have previously been identified as candidate immune system genes mediating mosquito-Plasmodium interaction, and serine protease inhibitors have been identified as hot-spots of adaptive evolution in other taxa. Population-genetic tests for selection, including a recent multi-gene extension of the McDonald-Kreitman test, were applied to 16 serine protease inhibitors and 16 other genes sampled from the <it>An. gambiae </it>species complex in both East and West Africa.</p> <p>Results</p> <p>Serine protease inhibitors were found to show a marginally significant trend towards higher levels of amino acid diversity than other genes, and display extensive genetic structuring associated with the 2La chromosomal inversion. However, although serpins are candidate targets for strong parasite-mediated selection, no evidence was found for rapid adaptive evolution in these genes.</p> <p>Conclusion</p> <p>It is well known that phylogenetic and population history in the <it>An. gambiae </it>complex can present special problems for the application of standard population-genetic tests for selection, and this may explain the failure of this study to detect selection acting on serine protease inhibitors. The pitfalls of uncritically applying these tests in this species complex are highlighted, and the future prospects for detecting selection acting on the <it>An. gambiae </it>genome are discussed.</p

Ecological and genetic relationships of the Forest-M form among chromosomal and molecular forms of the malaria vector Anopheles gambiae sensu stricto

<p>Abstract</p> <p>Background</p> <p><it>Anopheles gambiae sensu stricto</it>, one of the principal vectors of malaria, has been divided into two subspecific groups, known as the M and S molecular forms. Recent studies suggest that the M form found in Cameroon is genetically distinct from the M form found in Mali and elsewhere in West Africa, suggesting further subdivision within that form.</p> <p>Methods</p> <p>Chromosomal, microsatellite and geographic/ecological evidence are synthesized to identify sources of genetic polymorphism among chromosomal and molecular forms of the malaria vector <it>Anopheles gambiae s.s</it>.</p> <p>Results</p> <p>Cytogenetically the Forest M form is characterized as carrying the standard chromosome arrangement for six major chromosomal inversions, namely 2La, 2Rj, 2Rb, 2Rc, 2Rd, and 2Ru. Bayesian clustering analysis based on molecular form and chromosome inversion polymorphisms as well as microsatellites describe the Forest M form as a distinct population relative to the West African M form (Mopti-M form) and the S form. The Forest-M form was the most highly diverged of the <it>An. gambiae s.s</it>. groups based on microsatellite markers. The prevalence of the Forest M form was highly correlated with precipitation, suggesting that this form prefers much wetter environments than the Mopti-M form.</p> <p>Conclusion</p> <p>Chromosome inversions, microsatellite allele frequencies and habitat preference all indicate that the Forest M form of <it>An. gambiae </it>is genetically distinct from the other recognized forms within the taxon <it>Anopheles gambiae sensu stricto</it>. Since this study covers limited regions of Cameroon, the possibility of gene flow between the Forest-M form and Mopti-M form cannot be rejected. However, association studies of important phenotypes, such as insecticide resistance and refractoriness against malaria parasites, should take into consideration this complex population structure.</p

Differential Plasmodium falciparum infection of Anopheles gambiae s.s. molecular and chromosomal forms in Mali

BACKGROUND: Anopheles gambiae sensu stricto (s.s.) is a primary vector of Plasmodium falciparum in sub-Saharan Africa. Although some physiological differences among molecular and chromosomal forms of this species have been demonstrated, the relative susceptibility to malaria parasite infection among them has not been unequivocally shown. The objective of this study was to investigate P. falciparum circumsporozoite protein infection (CSP) positivity among An. gambiae s.s. chromosomal and molecular forms. METHODS: Wild An. gambiae from two sites Kela (n = 464) and Sidarebougou (n = 266) in Mali were screened for the presence of P. falciparum CSP using an enzyme-linked immunosorbent assay (ELISA). Samples were then identified to molecular form using multiple PCR diagnostics (n = 713) and chromosomal form using chromosomal karyotyping (n = 419). RESULTS: Of 730 An. gambiae sensu lato (s.l.) mosquitoes, 89 (12.2%) were CSP ELISA positive. The percentage of positive mosquitoes varied by site: 52 (11.2%) in Kela and 37 (13.9%) in Sidarebougou. Eighty-seven of the positive mosquitoes were identified to molecular form and they consisted of nine Anopheles arabiensis (21.4%), 46 S (10.9%), 31 M (12.8%), and one MS hybrid (14.3%). Sixty of the positive mosquitoes were identified to chromosomal form and they consisted of five An. arabiensis (20.0%), 21 Savanna (15.1%), 21 Mopti (30.4%), 11 Bamako (9.2%), and two hybrids (20.0%). DISCUSSION: In this collection, the prevalence of P. falciparum infection in the M form was equivalent to infection in the S form (no molecular form differential infection). There was a significant differential infection by chromosomal form such that, P. falciparum infection was more prevalent in the Mopti chromosomal forms than in the Bamako or Savanna forms; the Mopti form was also the most underrepresented in the collection. Continued research on the differential P. falciparum infection of An. gambiae s.s. chromosomal and molecular forms may suggest that Plasmodium – An. gambiae interactions play a role in malaria transmission

Molecular and Behavioral Differentiation among Brazilian Populations of Lutzomyia longipalpis (Diptera: Psychodidae: Phlebotominae)

Lutzomyia longipalpis is the main vector of visceral leishmaniasis in the Americas. There is strong evidence that L. longipalpis is a species complex, but there is still no consensus regarding the number of species occurring in Brazil. We combined molecular and behavioral analyses of a number of L. longipalpis populations in order to help clarify this question. This approach has allowed us to identify two main groups of populations in Brazil. One group probably represents a single species distributed mainly throughout the coastal regions of North and Northeast Brazil and whose males produce the same type of copulation song and pheromone. The second group is more heterogeneous, probably represented by a number of incipient species with different levels of genetic divergence among the siblings that produce different combinations of copulation songs and pheromones. The high level of complexity observed raises important questions concerning the epidemiological consequences of this incipient speciation process