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Female Anopheles gambiae antennae: increased transcript accumulation of the mosquito-specific odorant-binding-protein OBP2
Seth A Hoffman, Lakshminarayanan Aravind, Soundarapandian Velmurugan
Parasites & Vectors , 2012, DOI: 10.1186/1756-3305-5-27
Abstract: Our initial focus is on odorant binding proteins with differential transcript accumulation between female and male mosquitoes. We report that the odorant binding protein, OBP2 (AGAP003306), had increased expression in the antennae of female vs. male Anopheles gambiae sensu stricto (G3 strain). The increased expression in antennae of females of this gene by quantitative RT-PCR was 4.2 to 32.3 fold in three independent biological replicates and two technical replicate experiments using A. gambiae from two different laboratories. OBP2 is a member of the vast OBP superfamily of insect odorant binding proteins and belongs to the predominantly dipteran clade that includes the Culex oviposition kairomone-binding OBP1. Phylogenetic analysis indicates that its orthologs are present across culicid mosquitoes and are likely to play a conserved role in recognizing a molecule that might be critical for female behavior.OBP2 has increased mRNA transcript accumulation in the antennae of female as compared to male A. gambiae. This molecule and related molecules may play an important role in female mosquito feeding and breeding behavior. This finding may be a step toward providing a foundation for understanding mosquito olfactory requirements and developing control strategies based on reducing mosquito feeding and breeding success.Factors that influence mosquito fitness, especially host seeking and mate finding are complex and modulated by multiple cues, of which olfactory cues are most important [1-4]. Detection of odor molecules requires odorant binding proteins (OBPs) that are abundant in antennal chemosensilla [5,6]. OBPs are low molecular weight soluble proteins that bind and transport odor molecules from sensillae to G-protein-coupled receptors in olfactory sensory neurons [6]. The finding of receptor AgamOBP1 binding to its ligand indole demonstrated the significance of OBPs in odor recognition [7]. Understanding olfactory function could lead to development of malaria control
Genome annotation of Anopheles gambiae using mass spectrometry-derived data
Dário E Kalume, Suraj Peri, Raghunath Reddy, Jun Zhong, Mobolaji Okulate, Nirbhay Kumar, Akhilesh Pandey
BMC Genomics , 2005, DOI: 10.1186/1471-2164-6-128
Abstract: We searched 3,967 mass spectra from 16 LC-MS/MS runs of Anopheles gambiae salivary gland homogenates against the Anopheles gambiae genome database. This allowed us to validate 23 known transcripts and 50 novel transcripts. In addition, a novel gene was identified on the basis of peptides that matched a genomic region where no gene was known and no transcript had been predicted. The amino termini of proteins encoded by two predicted transcripts were confirmed based on N-terminally acetylated peptides sequenced by tandem mass spectrometry. Finally, six sequence polymorphisms could be annotated based on experimentally obtained peptide sequences.The peptide sequences from this study were mapped onto the genomic sequence using the distributed annotation system available at Ensembl and can be visualized in the context of all other existing annotations. The strategy described in this paper can be used to correct and confirm genome annotations and permit discovery of novel proteins in a high-throughput manner by mass spectrometry.The recent completion of Anopheles gambiae genome sequence [1] provided an architectural scaffold for mapping, identifying, selecting, and exploiting malaria insect vector genes for future studies. An. gambiae genome consists of 3 pairs of chromosome, designated as 2R/2L, 3R/3L and X. The Y chromosome is yet to be completely sequenced and assembled because of the high number of transposable element fragments. Thus far, approximately 85% of the genome has been assembled with the total genome size being 278 Mbp. About 15,189 genes are annotated in the An. gambiae genome, of which 11,757 are derived from prediction programs [2]. Currently, there are approximately 700 known An. gambiae proteins that are annotated in the databases. The annotation of the An. gambiae genome sequence has been an ongoing process since it was completed in 2002 [1]. The assembled genome is publicly available through NCBI (National Center for Biotechnology Information) and EBI
The Anopheles gambiae Odorant Binding Protein 1 (AgamOBP1) Mediates Indole Recognition in the Antennae of Female Mosquitoes  [PDF]
Harald Biessmann,Evi Andronopoulou,Max R. Biessmann,Vassilis Douris,Spiros D. Dimitratos,Elias Eliopoulos,Patrick M. Guerin,Kostas Iatrou,Robin W. Justice,Thomas Kr?ber,Osvaldo Marinotti,Panagiota Tsitoura,Daniel F. Woods,Marika F. Walter
PLOS ONE , 2012, DOI: 10.1371/journal.pone.0009471
Abstract: Haematophagous insects are frequently carriers of parasitic diseases, including malaria. The mosquito Anopheles gambiae is the major vector of malaria in sub-Saharan Africa and is thus responsible for thousands of deaths daily. Although the role of olfaction in A. gambiae host detection has been demonstrated, little is known about the combinations of ligands and odorant binding proteins (OBPs) that can produce specific odor-related responses in vivo. We identified a ligand, indole, for an A. gambiae odorant binding protein, AgamOBP1, modeled the interaction in silico and confirmed the interaction using biochemical assays. RNAi-mediated gene silencing coupled with electrophysiological analyses confirmed that AgamOBP1 binds indole in A. gambiae and that the antennal receptor cells do not respond to indole in the absence of AgamOBP1. This case represents the first documented instance of a specific A. gambiae OBP–ligand pairing combination, demonstrates the significance of OBPs in odor recognition, and can be expanded to the identification of other ligands for OBPs of Anopheles and other medically important insects.
Identification of Cryptic Anopheles Mosquito Species by Molecular Protein Profiling  [PDF]
Pie Müller, Valentin Pflüger, Matthias Wittwer, Dominik Ziegler, Fabrice Chandre, Frédéric Simard, Christian Lengeler
PLOS ONE , 2013, DOI: 10.1371/journal.pone.0057486
Abstract: Vector control is the mainstay of malaria control programmes. Successful vector control profoundly relies on accurate information on the target mosquito populations in order to choose the most appropriate intervention for a given mosquito species and to monitor its impact. An impediment to identify mosquito species is the existence of morphologically identical sibling species that play different roles in the transmission of pathogens and parasites. Currently PCR diagnostics are used to distinguish between sibling species. PCR based methods are, however, expensive, time-consuming and their development requires a priori DNA sequence information. Here, we evaluated an inexpensive molecular proteomics approach for Anopheles species: matrix assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). MALDI-TOF MS is a well developed protein profiling tool for the identification of microorganisms but so far has received little attention as a diagnostic tool in entomology. We measured MS spectra from specimens of 32 laboratory colonies and 2 field populations representing 12 Anopheles species including the A. gambiae species complex. An important step in the study was the advancement and implementation of a bioinformatics approach improving the resolution over previously applied cluster analysis. Borrowing tools for linear discriminant analysis from genomics, MALDI-TOF MS accurately identified taxonomically closely related mosquito species, including the separation between the M and S molecular forms of A. gambiae sensu stricto. The approach also classifies specimens from different laboratory colonies; hence proving also very promising for its use in colony authentication as part of quality assurance in laboratory studies. While being exceptionally accurate and robust, MALDI-TOF MS has several advantages over other typing methods, including simple sample preparation and short processing time. As the method does not require DNA sequence information, data can also be reviewed at any later stage for diagnostic or functional patterns without the need for re-designing and re-processing biological material.
Matrix-Assisted Laser Desorption Ionization - Time of Flight Mass Spectrometry: An Emerging Tool for the Rapid Identification of Mosquito Vectors  [PDF]
Amina Yssouf, Cristina Socolovschi, Christophe Flaudrops, Mamadou Ousmane Ndiath, Seynabou Sougoufara, Jean-Sebastien Dehecq, Guillaume Lacour, Jean-Michel Berenger, Cheikh Sadibou Sokhna, Didier Raoult, Philippe Parola
PLOS ONE , 2013, DOI: 10.1371/journal.pone.0072380
Abstract: Background The identification of mosquito vectors is typically based on morphological characteristics using morphological keys of determination, which requires entomological expertise and training. The use of protein profiling by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS), which is increasingly being used for the routine identification of bacteria, has recently emerged for arthropod identification. Methods To investigate the usefulness of MALDI-TOF-MS as a mosquito identification tool, we tested protein extracts made from mosquito legs to create a database of reference spectra. The database included a total of 129 laboratory-reared and field-caught mosquito specimens consisting of 20 species, including 4 Aedes spp., 9 Anopheles spp., 4 Culex spp., Lutzia tigripes, Orthopodomyia reunionensis and Mansonia uniformis. For the validation study, blind tests were performed with 76 specimens consisting of 1 to 4 individuals per species. A cluster analysis was carried out using the MALDI-Biotyper and some spectra from all mosquito species tested. Results Biomarker mass sets containing 22 and 43 masses have been detected from 100 specimens of the Anopheles, Aedes and Culex species. By carrying out 3 blind tests, we achieved the identification of mosquito vectors at the species level, including the differentiation of An. gambiae complex, which is possible using MALDI-TOF-MS with 1.8 as the cut-off identification score. A cluster analysis performed with all available mosquito species showed that MALDI-Biotyper can distinguish between specimens at the subspecies level, as demonstrated for An gambiae M and S, but this method cannot yet be considered a reliable tool for the phylogenetic study of mosquito species. Conclusions We confirmed that even without any specific expertise, MALDI-TOF-MS profiling of mosquito leg protein extracts can be used for the rapid identification of mosquito vectors. Therefore, MALDI-TOF-MS is an alternative, efficient and inexpensive tool that can accurately identify mosquitoes collected in the field during entomological surveys.
A Proteomic Investigation of Soluble Olfactory Proteins in Anopheles gambiae  [PDF]
Guido Mastrobuoni, Huili Qiao, Immacolata Iovinella, Simona Sagona, Alberto Niccolini, Francesca Boscaro, Beniamino Caputo, Marta R. Orejuela, Alessandra della Torre, Stefan Kempa, Antonio Felicioli, Paolo Pelosi, Gloriano Moneti, Francesca Romana Dani
PLOS ONE , 2013, DOI: 10.1371/journal.pone.0075162
Abstract: Odorant-binding proteins (OBPs) and chemosensory proteins (CSPs) are small soluble polypeptides that bind semiochemicals in the lymph of insect chemosensilla. In the genome of Anopheles gambiae, 66 genes encode OBPs and 8 encode CSPs. Here we monitored their expression through classical proteomics (2D gel-MS analysis) and a shotgun approach. The latter method proved much more sensitive and therefore more suitable for tiny biological samples as mosquitoes antennae and eggs. Females express a larger number and higher quantities of OBPs in their antennae than males (24 vs 19). OBP9 is the most abundant in the antennae of both sexes, as well as in larvae, pupae and eggs. Of the 8 CSPs, 4 were detected in antennae, while SAP3 was the only one expressed in larvae. Our proteomic results are in fairly good agreement with data of RNA expression reported in the literature, except for OBP4 and OBP5, that we could not identify in our analysis, nor could we detect in Western Blot experiments. The relatively limited number of soluble olfactory proteins expressed at relatively high levels in mosquitoes makes further studies on the coding of chemical messages at the OBP level more accessible, providing for few specific targets. Identification of such proteins in Anopheles gambiae might facilitate future studies on host finding behavior in this important disease vector.
An expression map for Anopheles gambiae
Robert M MacCallum, Seth N Redmond, George K Christophides
BMC Genomics , 2011, DOI: 10.1186/1471-2164-12-620
Abstract: We have clustered microarray-based gene-averaged expression values, available from VectorBase, for 10194 genes over 93 experimental conditions using a self-organizing map. Map regions corresponding to known biological events, such as egg production, are revealed. Many individual gene clusters (nodes) on the map are highly enriched in biological and molecular functions, such as protein synthesis, protein degradation and DNA replication. Gene families, such as odorant binding proteins, can be classified into distinct functional groups based on their expression and evolutionary history. Immunity-related genes are non-randomly distributed in several distinct regions on the map, and are generally distant from genes with house-keeping roles. Each immunity-rich region appears to represent a distinct biological context for pathogen recognition and clearance (e.g. the humoral and gut epithelial responses). Several immunity gene families, such as peptidoglycan recognition proteins (PGRPs) and defensins, appear to be specialised for these distinct roles, while three genes with physically interacting protein products (LRIM1/APL1C/TEP1) are found in close proximity.The map provides the first genome-scale, multi-experiment overview of gene expression in A. gambiae and should also be useful at the gene-level for investigating potential interactions. A web interface is available through the VectorBase website http://www.vectorbase.org/ webcite. It is regularly updated as new experimental data becomes available.Genome sequencing [1] and gene expression microarray technologies have, in recent years, enabled systems-level research into the malaria-transmitting mosquito Anopheles gambiae. By measuring transcript levels with respect to biological events, such as blood feeding, development, parasite infection and mating, one can identify genes that are likely to be involved in the underlying processes. However, due to the wealth of information produced by individual experiments and the n
Demasculinization of the Anopheles gambiae X chromosome
Kalle Magnusson, Gareth J Lycett, Antonio M Mendes, Amy Lynd, Philippos-Aris Papathanos, Andrea Crisanti, Nikolai Windbichler
BMC Evolutionary Biology , 2012, DOI: 10.1186/1471-2148-12-69
Abstract: We performed a meta-analysis of sex-biased gene expression in Anopheles gambiae which provides evidence for a general underrepresentation of male-biased genes on the X-chromosome that increased in significance with the observed degree of sex-bias. A phylogenomic comparison between Drosophila melanogaster, Aedes aegypti and Culex quinquefasciatus also indicates that the Anopheles X chromosome strongly disfavours the evolutionary conservation of male-biased expression and that novel male-biased genes are more likely to arise on autosomes. Finally, we demonstrate experimentally that transgenes situated on the Anopheles gambiae X chromosome are transcriptionally silenced in the male germline.The data presented here support the hypothesis that the observed demasculinization of the Anopheles X chromosome is driven by X-chromosome inactivation in the male germline and by sexual antagonism. The demasculinization appears to be the consequence of a loss of male-biased expression, rather than a failure in the establishment or the extinction of male-biased genes.
Gene Expression-Based Biomarkers for Anopheles gambiae Age Grading  [PDF]
Mei-Hui Wang, Osvaldo Marinotti, Daibin Zhong, Anthony A. James, Edward Walker, Tom Guda, Eliningaya J. Kweka, John Githure, Guiyun Yan
PLOS ONE , 2013, DOI: 10.1371/journal.pone.0069439
Abstract: Information on population age structure of mosquitoes under natural conditions is fundamental to the understanding of vectorial capacity and crucial for assessing the impact of vector control measures on malaria transmission. Transcriptional profiling has been proposed as a method for predicting mosquito age for Aedes and Anopheles mosquitoes, however, whether this new method is adequate for natural conditions is unknown. This study tests the applicability of transcriptional profiling for age-grading of Anopheles gambiae, the most important malaria vector in Africa. The transcript abundance of two An. gambiae genes, AGAP009551 and AGAP011615, was measured during aging under laboratory and field conditions in three mosquito strains. Age-dependent monotonic changes in transcript levels were observed in all strains evaluated. These genes were validated as age-grading biomarkers using the mark, release and recapture (MRR) method. The MRR method determined a good correspondence between actual and predicted age, and thus demonstrated the value of age classifications derived from the transcriptional profiling of these two genes. The technique was used to establish the age structure of mosquito populations from two malaria-endemic areas in western Kenya. The population age structure determined by the transcriptional profiling method was consistent with that based on mosquito parity. This study demonstrates that the transcription profiling method based on two genes is valuable for age determination of natural mosquitoes, providing a new approach for determining a key life history trait of malaria vectors.
Comparison of K+-channel genes within the genomes of Anopheles gambiae and Drosophila melanogaster
Thomas J McCormack
Genome Biology , 2003, DOI: 10.1186/gb-2003-4-9-r58
Abstract: This study identifies at least eight voltage-gated K+-channel genes in Anopheles, as well as three Slo-family, three Eag-family and six inward rectifier K+-channel genes. The genomic organization of K+-channel genes from Drosophila and Anopheles is well conserved. The sequence identity of the most similar K+-channel gene products between these two species ranges from 42% to 98%, with a mean value of 85%. Although most K+-channel genes in Drosophila and Anopheles are present in a 1:1 ratio, Anopheles has more genes in three K+-channel types, namely KQT, Kv3, and inward rectifier channels. Microsynteny between the genes flanking K+-channel genes in Drosophila and Anopheles was seldom observed; however, most of the K+-channel genes are indeed located at positions which a previous genome-wide comparison has designated as homologous chromosomal regions.The Anopheles genome encodes more voltage-gated and inward rectifier K+-channel genes than that of Drosophila. Despite the conservation of intron-exon boundaries, orthologs of genes flanking K+-channel genes in Drosophila are generally not found adjacent to the Anopheles K+-channel orthologs, suggesting that extensive translocation of genes has occurred since the divergence of these two organisms.The rapid rate of sequence acquisition has revolutionized molecular biology. The sequencing of entire genomes, in addition to new computer-based search tools has allowed us to identify and analyze large sets of data very rapidly. The acceleration of data acquisition, in fields such as whole-genome sequence determination and genome-wide gene-expression profiling, has opened the door for the study of model organisms and organisms of importance to the study of medicine and disease states by allowing for the analysis of the entirety of genetic information in a given organism. The recent completion of the sequencing of the Anopheles gambiae genome provides us with the entire genetic makeup of this organism. Furthermore, the completion
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