Analysis of expression in the Anopheles gambiae developing testes reveals rapidly evolving lineage-specific genes in mosquitoes

<p>Abstract</p> <p>Background</p> <p>Male mosquitoes do not feed on blood and are not involved in delivery of pathogens to humans. Consequently, they are seldom the subjects of research, which results in a very poor understanding of their biology. To gain insights into male developmental processes we sought to identify genes transcribed exclusively in the reproductive tissues of male <it>Anopheles gambiae </it>pupae.</p> <p>Results</p> <p>Using a cDNA subtraction strategy, five male-specifically or highly male-biased expressed genes were isolated, four of which remain unannotated in the <it>An. gambiae </it>genome. Spatial and temporal expression patterns suggest that each of these genes is involved in the mid-late stages of spermatogenesis. Their sequences are rapidly evolving; however, two genes possess clear homologs in a wide range of taxa and one of these probably acts in a sperm motility control mechanism conserved in many organisms, including humans. The other three genes have no match to sequences from non-mosquito taxa, thus can be regarded as orphans. RNA <it>in situ </it>hybridization demonstrated that one of the orphans is transcribed in spermatids, which suggests its involvement in sperm maturation. Two other orphans have unknown functions. Expression analysis of orthologs of all five genes indicated that male-biased transcription was not conserved in the majority of cases in <it>Aedes </it>and <it>Culex</it>.</p> <p>Conclusion</p> <p>Discovery of testis-expressed orphan genes in mosquitoes opens new prospects for the development of innovative control methods. The orphan encoded proteins may represent unique targets of selective anti-mosquito sterilizing agents that will not affect non-target organisms.</p

Update of the Anopheles gambiae PEST genome assembly

BACKGROUND: The genome of Anopheles gambiae, the major vector of malaria, was sequenced and assembled in 2002. This initial genome assembly and analysis made available to the scientific community was complicated by the presence of assembly issues, such as scaffolds with no chromosomal location, no sequence data for the Y chromosome, haplotype polymorphisms resulting in two different genome assemblies in limited regions and contaminating bacterial DNA. RESULTS: Polytene chromosome in situ hybridization with cDNA clones was used to place 15 unmapped scaffolds (sizes totaling 5.34 Mbp) in the pericentromeric regions of the chromosomes and oriented a further 9 scaffolds. Additional analysis by in situ hybridization of bacterial artificial chromosome (BAC) clones placed 1.32 Mbp (5 scaffolds) in the physical gaps between scaffolds on euchromatic parts of the chromosomes. The Y chromosome sequence information (0.18 Mbp) remains highly incomplete and fragmented among 55 short scaffolds. Analysis of BAC end sequences showed that 22 inter-scaffold gaps were spanned by BAC clones. Unmapped scaffolds were also aligned to the chromosome assemblies in silico, identifying regions totaling 8.18 Mbp (144 scaffolds) that are probably represented in the genome project by two alternative assemblies. An additional 3.53 Mbp of alternative assembly was identified within mapped scaffolds. Scaffolds comprising 1.97 Mbp (679 small scaffolds) were identified as probably derived from contaminating bacterial DNA. In total, about 33% of previously unmapped sequences were placed on the chromosomes. CONCLUSION: This study has used new approaches to improve the physical map and assembly of the A. gambiae genome

Complete mtDNA genomes of Anopheles darlingi and an approach to anopheline divergence time

Abstract Background The complete sequences of the mitochondrial genomes (mtDNA) of members of the northern and southern genotypes of Anopheles (Nyssorhynchus) darlingi were used for comparative studies to estimate the time to the most recent common ancestor for modern anophelines, to evaluate differentiation within this taxon, and to seek evidence of incipient speciation. Methods The mtDNAs were sequenced from mosquitoes from Belize and Brazil and comparative analyses of structure and base composition, among others, were performed. A maximum likelihood approach linked with phylogenetic information was employed to detect evidence of selection and a Bayesian approach was used to date the split between the subgenus Nyssorhynchus and other Anopheles subgenera. Results The comparison of mtDNA sequences within the Anopheles darlingi taxon does not provide sufficient resolution to establish different units of speciation within the species. In addition, no evidence of positive selection in any protein-coding gene of the mtDNA was detected, and purifying selection likely is the basis for this lack of diversity. Bayesian analysis supports the conclusion that the most recent ancestor of Nyssorhynchus and Anopheles+Cellia was extant ~94 million years ago. Conclusion Analyses of mtDNA genomes of Anopheles darlingi do not provide support for speciation in the taxon. The dates estimated for divergence among the anopheline groups tested is in agreement with the geological split of western Gondwana (95 mya), and provides additional support for explaining the absence of Cellia in the New World, and Nyssorhynchus in the Afro-Eurasian continents

Effects of stable ectopic expression of the primary sex determination gene Yob in the mosquito Anopheles gambiae

Abstract Background Mosquito-borne diseases, such as malaria, are controlled primarily by suppressing mosquito vector populations using insecticides. The current control programmes are seriously threatened by the emergence and rapid spread of resistance to approved insecticides. Genetic approaches proposed to complement the existing control efforts may be a more sustainable solution to mosquito control. All such approaches would rely on releases of modified male mosquitoes, because released females would contribute to biting and pathogen transmission. However, no sufficiently large-scale methods for sex separation in mosquitoes exist. Results Here we exploited the female embryo-killing property of the sex determining gene Yob from the African malaria mosquito, Anopheles gambiae, to evaluate the feasibility of creating transgenic An. gambiae sexing strains with a male-only phenotype. We generated An. gambiae lines with Yob expression, in both sexes, controlled by the vas2 promoter. Penetrance of the female-lethal phenotype was highly dependent on the location of the transgenic construct within the genome. A strong male bias was observed in one of the lines. All the females that survived to adulthood in that line possessed masculinized head appendages and terminal abdominal segments. They did not feed on blood, lacked host-seeking behavior, and thus were effectively sterile. Males, however, were not affected by Yob overexpression. Conclusions Our study demonstrates that ectopic expression of Yob results in a recovery of viable, fertile males, and in death, or otherwise strongly deleterious effects, in females. This result shows potential for generation of transgenic sexing strains of Anopheles gambiae with a conditional male-only phenotype

Satellite DNA From the Y Chromosome of the Malaria Vector Anopheles gambiae

Satellite DNA is an enigmatic component of genomic DNA with unclear function that has been regarded as “junk.” Yet, persistence of these tandem highly repetitive sequences in heterochromatic regions of most eukaryotic chromosomes attests to their importance in the genome. We explored the Anopheles gambiae genome for the presence of satellite repeats and identified 12 novel satellite DNA families. Certain families were found in close juxtaposition within the genome. Six satellites, falling into two evolutionarily linked groups, were investigated in detail. Four of them were experimentally confirmed to be linked to the Y chromosome, whereas their relatives occupy centromeric regions of either the X chromosome or the autosomes. A complex evolutionary pattern was revealed among the AgY477-like satellites, suggesting their rapid turnover in the A. gambiae complex and, potentially, recombination between sex chromosomes. The substitution pattern suggested rolling circle replication as an array expansion mechanism in the Y-linked 53-bp satellite families. Despite residing in different portions of the genome, the 53-bp satellites share the same monomer lengths, apparently maintained by molecular drive or structural constraints. Potential functional centromeric DNA structures, consisting of twofold dyad symmetries flanked by a common sequence motif, have been identified in both satellite groups

Correspondence

Acquisition of genetic information through horizontal gene transfer (HGT) is an important evolutionary process by which micro-organisms gain novel phenotypic characteristics. In pathogenic bacteria, for example, it facilitates maintenance and enhancement of virulence and spread of drug resistance. In the genus Mycobacterium, to which several primary human pathogens belong, HGT has not been clearly demonstrated. The few existing reports suggesting this process are based on circumstantial evidence of similarity of sequences found in distantly related species. Here, direct evidence of HGT between strains of Mycobacterium avium representing two different serotypes is presented. Conflicting evolutionary histories of genes encoding elements of the glycopeptidolipid (GPL) biosynthesis pathway led to an analysis of the GPL cluster genomic sequences from four Mycobacterium avium strains. The sequence of M. avium strain 2151 appeared to be a mosaic consisting of three regions having alternating identities to eitherM. avium strains 724 or 104. Maximum-likelihood estimation of two breakpoints allowed a~4100 bp region horizontally transferred into the strain 2151 genome to be pinpointed with confidence. The maintenance of sequence continuity at both breakpoints and the lack of insertional elements at these sites strongly suggest that the integration of foreign DNA occurred by homologous recombination. To our knowledge, this is the first report to demonstrate naturally occurring homologous recombination inMycobacterium. This previously undiscovered mechanism of genetic exchange may have major implications for the understanding of Mycobacterium pathogenesis

Culicoides vectors of bluetongue virus in Chester Zoo.

On four nights in June 2008, light traps were operated for Culicoides biting midges, the vector species for bluetongue virus (BTV), at five sites in Chester Zoo in north-west England. Over 35,000 Culicoides midges, of 25 species, were captured, including high densities inside animal enclosures. Over 94 per cent of all the Culicoides trapped were females of the Obsoletus group, which is implicated as the vector of BTV serotype 8 in northern Europe. The mean catch of this group per trap per night was over 1500, suggesting a potential risk of BTV transmission if the virus is introduced to Chester Zoo in the animals or midges in the summer

Analysis of the evolutionary forces shaping mitochondrial genomes of a Neotropical malaria vector complex

Many vectors of human malaria belong to complexes of morphologically indistinguishable cryptic species. Here we report the analysis of the newly sequenced complete mitochondrial DNA molecules from six recognized or putative species of one such group, the Neotropical Anopheles albitarsis complex. The molecular evolution of these genomes had been driven by purifying selection, particularly strongly acting on the RNA genes. Directional mutation pressure associated with the strand-asynchronous asymmetric mtDNA replication mechanism may have shaped a pronounced DNA strand asymmetry in the nucleotide composition in these and other Anopheles species. The distribution of sequence polymorphism, coupled with the conflicting phylogenetic trees inferred from the mitochondrial DNA and from the published white gene fragment sequences, indicates that the evolution of the complex may have involved ancient mtDNA introgression. Six protein coding genes (nad5, nad4, cox3, atp6, cox1 and nad2) have high levels of sequence divergence and are likely informative for population genetics studies. Finally, the extent of the mitochondrial DNA variation within the complex supports the notion that the complex consists of a larger number of species than until recently believed