Pilot Anopheles gambiae full-length cDNA study: sequencing and initial characterization of 35,575 clones

We describe the preliminary analysis of over 35,000 clones from a full-length enriched cDNA library from the malaria mosquito vector Anopheles gambiae. The clones define nearly 3,700 genes, of which around 2,600 significantly improve current gene definitions. An additional 17% of the genes were not previously annotated, suggesting that an equal percentage may be missing from the current Anopheles genome annotation

Anopheles gambiae genome reannotation through synthesis of ab initio and comparative gene prediction algorithms

BACKGROUND: Complete genome annotation is a necessary tool as Anopheles gambiae researchers probe the biology of this potent malaria vector. RESULTS: We reannotate the A. gambiae genome by synthesizing comparative and ab initio sets of predicted coding sequences (CDSs) into a single set using an exon-gene-union algorithm followed by an open-reading-frame-selection algorithm. The reannotation predicts 20,970 CDSs supported by at least two lines of evidence, and it lowers the proportion of CDSs lacking start and/or stop codons to only approximately 4%. The reannotated CDS set includes a set of 4,681 novel CDSs not represented in the Ensembl annotation but with EST support, and another set of 4,031 Ensembl-supported genes that undergo major structural and, therefore, probably functional changes in the reannotated set. The quality and accuracy of the reannotation was assessed by comparison with end sequences from 20,249 full-length cDNA clones, and evaluation of mass spectrometry peptide hit rates from an A. gambiae shotgun proteomic dataset confirms that the reannotated CDSs offer a high quality protein database for proteomics. We provide a functional proteomics annotation, ReAnoXcel, obtained by analysis of the new CDSs through the AnoXcel pipeline, which allows functional comparisons of the CDS sets within the same bioinformatic platform. CDS data are available for download. CONCLUSION: Comprehensive A. gambiae genome reannotation is achieved through a combination of comparative and ab initio gene prediction algorithms

Identification and Characterization of Two Novel RNA Viruses from Anopheles gambiae Species Complex Mosquitoes

Mosquitoes of the Anopheles gambiae complex display strong preference for human blood-meals and are major malaria vectors in Africa. However, their interaction with viruses or role in arbovirus transmission during epidemics has been little examined, with the exception of O'nyong-nyong virus, closely related to Chikungunya virus. Deep-sequencing has revealed different RNA viruses in natural insect viromes, but none have been previously described in the Anopheles gambiae species complex. Here, we describe two novel insect RNA viruses, a Dicistrovirus and a Cypovirus, found in laboratory colonies of An. gambiae taxa using small-RNA deep sequencing. Sequence analysis was done with Metavisitor, an open-source bioinformatic pipeline for virus discovery and de novo genome assembly. Wild-collected Anopheles from Senegal and Cambodia were positive for the Dicistrovirus and Cypovirus, displaying high sequence identity to the laboratory-derived virus. Thus, the Dicistrovirus (Anopheles C virus, AnCV) and Cypovirus (Anopheles Cypovirus, AnCPV) are components of the natural virome of at least some anopheline species. Their possible influence on mosquito immunity or transmission of other pathogens is unknown. These natural viruses could be developed as models for the study of Anopheles-RNA virus interactions in low security laboratory settings, in an analogous manner to the use of rodent malaria parasites for studies of mosquito anti-parasite immunity

Mycobacterial genomics

The small size of their genomes made bacterial ideal model organisms for the emerging field of genomics. Elucidating the genome sequences of mycobacteria was particularly attractive owing to the difficulties inherent in their manipulation. The slow growth rate, clumping, and requirement for category III containment make manipulation of Mycobacterium tuberculosis-complex strains laborious. M. leprae presents even greater problems as it has resisted all attempts at axenic culture. Availability of genome sequence data promised to accelerate our knowledge of the fundamental biology of these organisms, and to offer clues to the basis for their virulence, tropism and persistence in the host. This article will focus on what the genome sequences of M. tuberculosis and M. leprae have taught us about these pathogens, and how comparative genomics has exposed some of the fundamental differences between the species

Composition and genetics of malaria vector populations in the Central African Republic

International audienceBACKGROUND:In many African countries malaria has declined sharply due to a synergy of actions marked by the introduction of vector control strategies, but the disease remains a leading cause of morbidity and mortality in Central African Republic (CAR). An entomological study was initiated with the aim to characterize the malaria vectors in Bangui, the capital of CAR, and determine their vector competence.METHODS:A cross-sectional entomological study was conducted in 15 sites of the district of Bangui, the capital of CAR, in September-October 2013 and a second collection was done in four of those sites between November and December 2013. Mosquitoes were collected by human landing catch (HLC) indoors and outdoors and by pyrethrum spray catch of indoor-resting mosquitoes. Mosquitoes were analysed for species and multiple other attributes, including the presence of Plasmodium falciparum circumsporozoite protein or DNA, blood meal source, 2La inversion karyotype, and the L1014F kdr insecticide resistance mutation.RESULTS:Overall, 1292 anophelines were analysed, revealing a predominance of Anopheles gambiae and Anopheles funestus, with a small fraction of Anopheles coluzzii. Molecular typing of the An. gambiae complex species showed that An. gambiae was predominant (95.7 %) as compared to An. coluzzii (2.1 %), and Anopheles arabiensis was not present. In some areas the involvement of secondary vectors, such as Anopheles coustani, expands the risk of infection. By HLC sampling, An. funestus displayed a stronger endophilic preference than mosquitoes from the An. gambiae sister taxa, with a mean indoor-capture rate of 54.3 % and 67.58 % for An. gambiae sister taxa and An. funestus, respectively. Human biting rates were measured overall for each of the species with 28 or 29 bites/person/night, respectively. Both vectors displayed a strong human feeding preference as determined by blood meal source, which was not different between the different sampling sites. An. coustani appears to be highly exophilic, with 92 % of HLC samples captured outdoors. The mean CSP rate in head-thorax sections of all Anopheles was 5.09 %, and was higher in An. gambiae s.l. (7.4 %) than in An. funestus (3.3 %). CSP-positive An. coustani were also detected in outdoor HLC samples. In the mosquitoes of the An. gambiae sister taxa the kdr-w mutant allele was nearly fixed, with 92.3 % resistant homozygotes, and no susceptible homozygotes detected.CONCLUSIONS:This study collected data on anopheline populations in CAR, behaviour of vectors and transmission levels. Further studies should investigate the biting behaviour and susceptibility status of the anophelines to different insecticides to allow the establishment of appropriate vector control based on practical entomological knowledge

Genetics and immunity of Anopheles response to the entomopathogenic fungus Metarhizium anisopliae overlap with immunity to Plasmodium

International audienceEntomopathogenic fungi have been explored as a potential biopesticide to counteract the insecticide resistance issue in mosquitoes. However, little is known about the possibility that genetic resistance to fungal biopesticides could evolve in mosquito populations. Here, we detected an important genetic component underlying Anopheles coluzzii survival after exposure to the entomopathogenic fungus Metarhizium anisopliae . A familiality study detected variation for survival among wild mosquito isofemale pedigrees, and genetic mapping identified two loci that significantly influence mosquito survival after fungus exposure. One locus overlaps with a previously reported locus for Anopheles susceptibility to the human malaria parasite Plasmodium falciparum . Candidate gene studies revealed that two LRR proteins encoded by APL1C and LRIM1 genes in this newly mapped locus are required for protection of female A. coluzzii from M. anisopliae , as is the complement-like factor Tep1. These results indicate that natural Anopheles populations already segregate frequent genetic variation for differential mosquito survival after fungal challenge and suggest a similarity in Anopheles protective responses against fungus and Plasmodium . However, this immune similarity raises the possibility that fungus-resistant mosquitoes could also display enhanced resistance to Plasmodium , suggesting an advantage of selecting for fungus resistance in vector populations to promote naturally diminished malaria vector competence

Bacterial Artificial Chromosome-Based Comparative Genomic Analysis Identifies Mycobacterium microti as a Natural ESAT-6 Deletion Mutant

Mycobacterium microti is a member of the Mycobacterium tuberculosis complex that causes tuberculosis in voles. Most strains of M. microti are harmless for humans, and some have been successfully used as live tuberculosis vaccines. In an attempt to identify putative virulence factors of the tubercle bacilli, genes that are absent from the avirulent M. microti but present in human pathogen M. tuberculosis or Mycobacterium bovis were searched for. A minimal set of 50 bacterial artificial chromosome (BAC) clones that covers almost all of the genome of M. microti OV254 was constructed, and individual BACs were compared to the corresponding BACs from M. bovis AF2122/97 and M. tuberculosis H37Rv. Comparison of pulsed-field gel-separated DNA digests of BAC clones led to the identification of 10 regions of difference (RD) between M. microti OV254 and M. tuberculosis. A 14-kb chromosomal region (RD1(mic)) that partly overlaps the RD1 deletion in the BCG vaccine strain was missing from the genomes of all nine tested M. microti strains. This region covers 13 genes, Rv3864 to Rv3876, in M. tuberculosis, including those encoding the potent ESAT-6 and CFP-10 antigens. In contrast, RD5(mic), a region that contains three phospholipase C genes (plcA to -C), was missing from only the vole isolates and was present in M. microti strains isolated from humans. Apart from RD1(mic) and RD5(mic) other M. microti-specific deleted regions have been identified (MiD1 to MiD3). Deletion of MiD1 has removed parts of the direct repeat region in M. microti and thus contributes to the characteristic spoligotype of M. microti strains

Molecular characterization and genetic authentication assay for Anopheles ‘hemocyte-like’ cell lines 4a-3A and 4a-3B

International audienceBackground: Anopheles cell lines are used in a variety of ways to better understand the major vectors of malaria in sub-Saharan Africa. Despite this, commonly used cell lines are not well characterized, and no tools are available for cell line identification and authentication. Methods: Utilizing whole genome sequencing, genomes of 4a-3A and 4a-3B 'hemocyte-like' cell lines were characterized for insertions and deletions (indels) and SNP variation. Genomic locations of distinguishing sequence variation and species origin of the cell lines were also examined. Unique indels were targeted to develop a PCR-based cell line authentication assay. Mitotic chromosomes were examined to survey the cytogenetic landscape for chromosome structure and copy number in the cell lines. Results: The 4a-3A and 4a-3B cell lines are female in origin and primarily of Anopheles coluzzii ancestry. Cytogenetic analysis indicates that the two cell lines are essentially diploid, with some relatively minor chromosome structural rearrangements. Whole-genome sequence was generated, and analysis indicated that SNPs and indels which differentiate the cell lines are clustered on the 2R chromosome in the regions of the 2Rb, 2Rc and 2Ru chromosomal inversions. A PCR-based authentication assay was developed to fingerprint three indels unique to each cell line. The assay distinguishes between 4a-3A and 4a-3B cells and also uniquely identifies two additional An. coluzzii cell lines tested, Ag55 and Sua4.0. The assay has the specificity to distinguish four cell lines and also has the sensitivity to detect cellular contamination within a sample of cultured cells. Conclusions: Genomic characterization of the 4a-3A and 4a-3B Anopheles cell lines was used to develop a simple diagnostic assay that can distinguish these cell lines within and across research laboratories. A cytogenetic survey indicated that the 4a-3A and Sua4.0 cell lines carry essentially normal diploid chromosomes, which makes them amenable to CRISPR/Cas9 genome editing. The presented simple authentication assay, coupled with screening for mycoplasma, will allow validation of the integrity of experimental resources and will promote greater experimental reproducibility of results