Improving the population genetics toolbox for the study of the African malaria vector Anopheles nili: microsatellite mapping to chromosomes

<p>Abstract</p> <p>Background</p> <p><it>Anopheles nili </it>is a major vector of malaria in the humid savannas and forested areas of sub-Saharan Africa. Understanding the population genetic structure and evolutionary dynamics of this species is important for the development of an adequate and targeted malaria control strategy in Africa. Chromosomal inversions and microsatellite markers are commonly used for studying the population structure of malaria mosquitoes. Physical mapping of these markers onto the chromosomes further improves the toolbox, and allows inference on the demographic and evolutionary history of the target species.</p> <p>Results</p> <p>Availability of polytene chromosomes allowed us to develop a map of microsatellite markers and to study polymorphism of chromosomal inversions. Nine microsatellite markers were mapped to unique locations on all five chromosomal arms of <it>An. nili </it>using fluorescent <it>in situ </it>hybridization (FISH). Probes were obtained from 300-483 bp-long inserts of plasmid clones and from 506-559 bp-long fragments amplified with primers designed using the <it>An. nili </it>genome assembly generated on an Illumina platform. Two additional loci were assigned to specific chromosome arms of <it>An. nili </it>based on <it>in silico </it>sequence similarity and chromosome synteny with <it>Anopheles gambiae</it>. Three microsatellites were mapped inside or in the vicinity of the polymorphic chromosomal inversions <it>2Rb </it>and <it>2Rc</it>. A statistically significant departure from Hardy-Weinberg equilibrium, due to a deficit in heterozygotes at the <it>2Rb </it>inversion, and highly significant linkage disequilibrium between the two inversions, were detected in natural <it>An. nili </it>populations collected from Burkina Faso.</p> <p>Conclusions</p> <p>Our study demonstrated that next-generation sequencing can be used to improve FISH for microsatellite mapping in species with no reference genome sequence. Physical mapping of microsatellite markers in <it>An. nili </it>showed that their cytological locations spanned the entire five-arm complement, allowing genome-wide inferences. The knowledge about polymorphic inversions and chromosomal locations of microsatellite markers has been useful for explaining differences in genetic variability across loci and significant differentiation observed among natural populations of <it>An. nili</it>.</p

Organisation et evolution des genes des immunoglobulines et des recepteurs T gamma/delta humains

Available from INIST (FR), Document Supply Service, under shelf-number : TD 79349 / INIST-CNRS - Institut de l'Information Scientifique et TechniqueSIGLEFRFranc

Wolbachia prevalence, diversity, and ability to induce cytoplasmic incompatibility in mosquitoes

International audienc

Improving the antigen affinity of an antibody Fv-fragment by protein design.

International audienceThe affinity of an antibody for its ligand 2-phenyloxazolone was improved by protein design. For the design two-dimensional nuclear magnetic resonance spectroscopy, protein engineering and molecular modelling were used in an interactive scheme. Initially the binding site was localized with the help of transferred nuclear Overhauser enhancement signals from two, site specifically assigned tyrosine side-chains in the complementarity-determining regions of the antibody to the ligand 4-glycyl-2-phenyloxazolone. On their basis the hapten was placed into a model of the Fv-fragment built according to the principles of canonical antibody structures. From the model, unfavourable contacts between hapten and an aspartyl side-chain in complementarity-determining region 3 of the heavy chain were predicted. Substitution of the aspartyl residue by alanine resulted in a threefold increase in affinity of the antibody Fv-fragment for two hapten derivatives when compared with the wild-type. Nuclear magnetic resonance analysis of the improved Fv-fragment revealed an interaction between the alpha-carbon proton of alanyl residue with the ligand, which was not seen for the aspartyl residue. This interaction is not entirely in accordance with the model, which predicts an interaction between the side-chain of this residue and the hapten. However, it shows that by combined use of nuclear magnetic resonance analysis and molecular modelling, a residue that is critical for antigen binding was identified, whose mutation allowed the design of an improved antibody combining site

Identification of spider-mite species and their endosymbionts using multiplex PCR

International audienceSpider mites of the genus Tetranychidae are severe crop pests. In the Mediterranean a few species coexist, but they are difficult to identify based on morphological characters. Additionally, spider mites often harbour several species of endosymbiotic bacteria, which may affect the biology of their hosts. Here, we propose novel, cost-effective, multiplex diagnostic methods allowing a quick identification of spider-mite species as well as of the endosymbionts they carry. First, we developed, and successfully multiplexed in a single PCR, primers to identify Tetranychus urticae, T. evansi and T. ludeni, some of the most common tetranychids found in southwest Europe. Moreover, we demonstrated that this method allows detecting multiple species in a single pool, even at low frequencies (up to 1/100), and can be used on entire mites without DNA extraction. Second, we developed another set of primers to detect spider-mite endosymbionts, namely Wolbachia, Cardinium and Rickettsia in a multiplex PCR, along with a generalist spider-mite primer to control for potential failure of DNA amplification in each PCR. Overall, our method represents a simple, cost-effective and reliable method to identify spider-mite species and their symbionts in natural field populations, as well as to detect contaminations in laboratory rearings. This method may easily be extended to other species

Direct Selection of EGF Mutants Displayed on Filamentous Phage Using Cells Overexpressing EGF Receptor

International audienceUnderstanding receptor-ligand interactions, and the signal transduction pathways they activate, is of great interest for the discovery of novel antagonists and agonists. In this report we describe a rapid and efficient procedure to evaluate the importance of several different epidermal growth factor (EGF) residues for the binding and activation of its receptor (EGFR). We constructed an EGF mutant library randomized at positions 13, 15 and 16 and expressed them on filamentous phages. Phage display is a powerful system, allowing rapid isolation of binding mutants. Since many of the most pharmacologically interesting receptors cannot be produced in a soluble form, we developed a technique to rapidly select receptor-binding molecules directly on cells. A luciferase assay, simple to perform, was then used to test their biological transduction activity and to rapidly detect mutants of interest. Analysis of the resulting sequences revealed that the wild-type amino acids at positions 13, 15 and 16 are optimized for binding and activity. EGF mutants with agonist properties were also isolated and tolerated substitutions were identified

Relating fitness to long-term environmental variations in natura

International audienceQuantifying links between ecological processes and adaptation dynamics in natura remains a crucial challenge. Many studies have documented the strength, form and direction of selection, and its variations in space and time, but only a few managed to link these variations to their proximal causes. This step is, however, crucial, if we are to understand how the variation in selective pressure affects adaptive allele dynamics in natural settings. We used data from a long-term survey (about 30 years) monitoring the adaptation to insecticides of Culex pipiens mosquitoes in Montpellier area (France), focusing on three resistance alleles of the Ester locus. We used a population genetics model taking temporal and spatial variations in selective pressure into account, to assess the quantitative relationships between variations in the proximal agent of selection (amounts of insecticide sprayed) and the fitness of resistance alleles. The response to variations in selective pressure was fast, and the alleles displayed different fitness-to-environment relationships: the analyses revealed that even slight changes in insecticide doses could induce changes in the strength and direction of selection, thus changing the fitness ranking of the adaptive alleles. They also revealed that selective pressures other than the insecticides used for mosquito control affected the resistance allele dynamics. These fitness-to-environment relationships, fast responses and continuous evolution limit our ability to predict the outcome of adaptive allele dynamics in a changing environment, but they clearly contribute to the maintenance of polymorphism in natural populations. Our study also emphasizes the necessity of long-term surveys in evolutionary ecology

ORIGINAL PAPER Polymorphism, haplotype composition, and selection in the Mhc-DRB of wild baboons

Abstract General patterns of organization in the major histocompatibility complex (MHC) have been successfully explained by the model of birth-and-death evolution, but understanding why certain MHC genes are maintained together into specific haplotypes remains challenging. The haplotype configurations of the functionally important class II DR region have been described in few primates and display important interspecific variability with respect to the extent of allelic variation, the number of loci and/or combinations of loci present. Understanding the evolutionary mechanisms driving such variation is conditional upon characterizing haplotypes in new species and identifying the selective pressures acting on haplotypes. This study explores the variability of haplotype configurations in the Mhc-DRB region (exon 2) for the first time in wild non-human primates, chacma baboons (Papio ursinus). Paur-DRB haplotypes were characterized through segregation studies and linkage disequilibrium. 23 Paur-DRB sequences and 15 haplotype configurations were identified in 199 animals. The Paur-DRB exon 2 is shown to be subjected to intense positive selection and frequent recombination. An approach recently developed for human vaccine studies was used to classify Paur-DRB sequences into supertypes, based on the physico-chemical properties of amino acids that are posi-tively selected, thus most probably involved in antigen recognition. Sequences grouped into the same supertype (thus presumably sharing antigen-binding affinities) are non-randomly distributed within haplotypes, leading to an increased individual diversity of supertypes. Our results suggest that selection favoring haplotypes with complemen-tary sets of DRB supertypes shapes functionally tuned haplotypes in this natural baboon population

Wolbachia Diversity And Cytoplasmic Incompatibility Patterns In Culex Pipiens Populations In Turkey

Background Wolbachia are maternally transmitted bacteria that can manipulate their hosts’ reproduction causing cytoplasmic incompatibility (CI). CI is a sperm-egg incompatibility resulting in embryonic death. Due to this sterilising effect on mosquitoes, Wolbachia are considered for vector control strategies. Important vectors for arboviruses, filarial nematodes and avian malaria, mosquitoes of Culex pipiens complex are suitable for Wolbachia-based vector control. They are infected with Wolbachia wPip strains belonging to five genetically distinct groups (wPip-I to V) within the Wolbachia B supergroup. CI properties of wPip strongly correlate with this genetic diversity: mosquitoes infected with wPip strains from a different wPip group are more likely to be incompatible with each other. Turkey is a critical spot for vector-borne diseases due to its unique geographical position as a natural bridge between Asia, Europe and Africa. However, general wPip diversity, distribution and CI patterns in natural Cx. pipiens (s.l.) populations in the region are unknown. In this study, we first identified wPip diversity in Turkish Cx. pipiens (s.l.) populations, by assigning them to one of the five groups within wPip (wPip-Ito V). We further investigated CI properties between different wPip strains from this region. Results We showed a wPip fixation in Cx. pipiens (s.l.) populations in Turkey by analysing 753 samples from 59 sampling sites. Three wPip groups were detected in the region: wPip-I, wPip-II and wPip-IV. The most dominant group was wPip-II. While wPip-IV was restricted to only two locations, wPip-I and wPip-II had wider distributions. Individuals infected with wPip-II were found co-existing with individuals infected with wPip-I or wPip-IV in some sampling sites. Two mosquito isofemale lines harbouring either a wPip-I or a wPip-II strain were established from a population in northwestern Turkey. Reciprocal crosses between these lines showed that they were fully compatible with each other but bidirectionally incompatible with wPip-IV Istanbul infected line. Conclusion Our findings reveal a high diversity of wPip and CI properties in Cx. pipiens (s.l.) populations in Turkey. Knowledge on naturally occurring CI patterns caused by wPip diversity in Turkey might be useful for Cx. pipiens (s.l.) control in the region.PubMedWoSScopu