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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.
Exploring Proteins in Anopheles gambiae Male and Female Antennae through MALDI Mass Spectrometry Profiling  [PDF]
Francesca R. Dani, Simona Francese, Guido Mastrobuoni, Antonio Felicioli, Beniamino Caputo, Frederic Simard, Giuseppe Pieraccini, Gloriano Moneti, Mario Coluzzi, Alessandra della Torre, Stefano Turillazzi
PLOS ONE , 2008, DOI: 10.1371/journal.pone.0002822
Abstract: MALDI profiling and imaging mass spectrometry (IMS) are novel techniques for direct analysis of peptides and small proteins in biological tissues. In this work we applied them to the study of Anopheles gambiae antennae, with the aim of analysing expression of soluble proteins involved in olfaction perireceptor events. MALDI spectra obtained by direct profiling on single antennae and by the analysis of extracts, showed similar profiles, although spectra obtained through profiling had a richer ion population and higher signal to noise ratio. Male and female antennae showed distinct protein profiles. MALDI imaging experiments were also performed and differences were observed in the localization of some proteins. Two proteins were identified through high resolution measurement and top-down MS/MS experiments. A 8 kDa protein only present in the male antennae matched with an unannotated sequence of the An. gambiae genome, while the presence of odorant binding protein 9 (OBP-9) was confirmed through experiments of 2-DE, followed by MS and MS/MS analysis of digested spots. This work shows that MALDI MS profiling is a technique suitable for the analysis of proteins of small and medium MW in insect appendices, and allows obtaining data for several specimens which can be investigated for differences between groups. Proteins of interest can be identified through other complementary MS approaches.
Identification and expression profiling of putative odorant-binding proteins in the malaria mosquitoes, Anopheles gambiae and A. arabiensis
Zhengxi Li,Jing-Jiang Zhou,Zuorui Shen,Field Lin
Science China Life Sciences , 2004, DOI: 10.1360/03yc0232
Abstract: Olfaction plays a major role in host-seeking behaviour of mosquitoes. An informatics-based genome-wide analysis of odorant-binding protein (OBP) homologues is undertaken, and 32 putative OBP genes in total in the whole genome sequences of Anopheles gambiae are identified. Tissue-specific expression patterns of all A. gambiae OBP candidates are determined by semi-quantitative Reverse Transcription (RT)-PCR using mosquito actin gene as internal expression control standard. The results showed that 20 OBP candidates had strong expression in mosquito olfactory tissues (female antennae), which indicate that OBPs may play an important role in regulating mosquito olfactory behaviours. Species-specific expression patterns of all putative anopheline OBPs are also studied in two of the most important malaria vectors in A. gambiae complex, i.e. A. gambiae and A. arabiensis, which found 12 of the putative OBP genes examined displayed species-differential expression patterns. The cumulative relative expression intensity of the OBPs in A. arabiensis antennae was higher than that in A. gambiae (the ratio is 1441.45:1314.12), which might be due to their different host preference behaviour. While A. gambiae is a highly anthropophilic mosquito, A. arabiensis is more opportunistic (varying from anthropophilic to zoophilic). So the latter should need more OBPs to support its host selection preference. Identification of mosquito OBPs and verification of their tissue- and species-specific expression patterns represent the first step towards further molecular analysis of mosquito olfactory mechanism, such as recombinant expression and ligand identification.
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.
Genome Analysis and Expression Patterns of Odorant-Binding Proteins from the Southern House Mosquito Culex pipiens quinquefasciatus  [PDF]
Julien Pelletier, Walter S. Leal
PLOS ONE , 2009, DOI: 10.1371/journal.pone.0006237
Abstract: Olfactory-based behaviors in mosquitoes are mediated by odorant-binding proteins (OBPs). They form a multigenic family involved in the peripheral events in insect olfaction, specifically the transport of odorants to membrane-bound odorant receptors. OBPs contribute to the remarkable sensitivity of the insect's olfactory system and may be involved in the selective transport of odorants. We have employed a combination of bioinformatics and molecular approaches to identify and characterize members of the “classic” OBP family in the Southern House mosquito Culex pipiens quinquefasciatus ( = Cx. quinquefasciatus), a vector of pathogens causing several human diseases. By taking advantage of the recently released genome sequences, we have identified fifty-three putative Cx. quinquefasciatus OBP genes by Blast searches. As a first step towards their molecular characterization, expression patterns by RT-PCR revealed thirteen genes that were detected exclusively and abundantly in chemosensory tissues. No clear differences were observed in the transcripts levels of olfactory-specific OBPs between antennae of both sexes using semi-quantitative RT-PCR. Phylogenetic and comparative analysis revealed orthologous of Cx. quinquefasciatus OBPs in Anopheles gambiae and Aedes aegypti. The identification of fifty-three putative OBP genes in Cx. quinquefasciatus highlights the diversity of this family. Tissue-specificity study suggests the existence of different functional classes within the mosquito OBP family. Most genes were detected in chemosensory as well as non chemosensory tissues indicating that they might be encapsulins, but not necessarily olfactory proteins. On the other hand, thirteen “true” OBP genes were detected exclusively in olfactory tissues and might be involved specifically in the detection of “key” semiochemicals. Interestingly, in Cx. quinquefasciatus olfactory-specific OBPs belong exclusively to four distinct phylogenetic groups which are particularly well conserved among three mosquito species.
Expression and Membrane Topology of Anopheles gambiae Odorant Receptors in Lepidopteran Insect Cells  [PDF]
Panagiota Tsitoura,Evi Andronopoulou,Daniela Tsikou,Adamantia Agalou,Maria P. Papakonstantinou,Georgia A. Kotzia,Vassiliki Labropoulou,Luc Swevers,Zafiroula Georgoussi,Kostas Iatrou
PLOS ONE , 2012, DOI: 10.1371/journal.pone.0015428
Abstract: A lepidopteran insect cell-based expression system has been employed to express three Anopheles gambiae odorant receptors (ORs), OR1 and OR2, which respond to components of human sweat, and OR7, the ortholog of Drosophila's OR83b, the heteromerization partner of all functional ORs in that system. With the aid of epitope tagging and specific antibodies, efficient expression of all ORs was demonstrated and intrinsic properties of the proteins were revealed. Moreover, analysis of the orientation of OR1 and OR2 on the cellular plasma membrane through the use of a novel ‘topology screen’ assay and FACS analysis demonstrates that, as was recently reported for the ORs in Drosophila melanogaster, mosquito ORs also have a topology different than their mammalian counterparts with their N-terminal ends located in the cytoplasm and their C-terminal ends facing outside the cell. These results set the stage for the production of mosquito ORs in quantities that should permit their detailed biochemical and structural characterization and the exploration of their functional properties.
Identification and Expression Profile Analysis of Odorant Binding Proteins in the Oriental Fruit Fly Bactrocera dorsalis  [PDF]
Weiwei Zheng,Wei Peng,Chipan Zhu,Qun Zhang,Giuseppe Saccone,Hongyu Zhang
International Journal of Molecular Sciences , 2013, DOI: 10.3390/ijms140714936
Abstract: Olfaction is crucial in many insects for critical behaviors, including those regulating survival and reproduction. Insect odorant-binding proteins (OBPs) function in the first step of the olfactory system and play an essential role in the perception of odorants, such as pheromones and host chemicals. The oriental fruit fly, Bactrocera dorsalis, is a destructive fruit-eating pest, due to its wide host range of up to 250 different types of fruits and vegetables, and this fly causes severe economic damage to the fruit and vegetable industry. However, OBP genes have not been largely identified in B. dorsalis. Based on our previously constructed B. dorsalis cDNA library, ten OBP genes were identified in B. dorsalis for the first time. A phylogenetic tree was generated to show the relationships among the 10 OBPs of B. dorsalis to OBP sequences of two other Dipteran species, including Drosophila melanogaster and the mosquito Anopheles gambiae. The expression profiles of the ten OBPs in different tissues (heads, thoraxes, abdomens, legs, wings, male antennae and female antenna) of the mated adults were analyzed by real-time PCR. The results showed that nine of them are highly expressed in the antenna of both sexes, except BdorOBP7. Four OBPs ( BdorOBP1, BdorOBP4, BdorOBP8, and BdorOBP10) are also enriched in the abdomen, and BdorOBP7 is specifically expressed in leg, indicating that it may function in other biological processes. This work will provide insight into the roles of OBPs in chemoreception and help develop new pest-control strategies.
The Co-Expression Pattern of Odorant Binding Proteins and Olfactory Receptors Identify Distinct Trichoid Sensilla on the Antenna of the Malaria Mosquito Anopheles gambiae  [PDF]
Anna Schultze, Pablo Pregitzer, Marika F. Walter, Daniel F. Woods, Osvaldo Marinotti, Heinz Breer, Jürgen Krieger
PLOS ONE , 2013, DOI: 10.1371/journal.pone.0069412
Abstract: The initial steps of odorant recognition in the insect olfactory system involve odorant binding proteins (OBPs) and odorant receptors (ORs). While large families of OBPs have been identified in the malaria vector A. gambiae, little is known about their expression pattern in the numerous sensory hairs of the female antenna. We applied whole mount fluorescence in Situ hybridization (WM-FISH) and fluorescence immunohistochemistry (WM-FIHC) to investigate the sensilla co-expression of eight A. gambiae OBPs (AgOBPs), most notably AgOBP1 and AgOBP4, which all have abundant transcripts in female antenna. WM-FISH analysis of female antennae using AgOBP-specific probes revealed marked differences in the number of cells expressing each various AgOBPs. Testing combinations of AgOBP probes in two-color WM-FISH resulted in distinct cellular labeling patterns, indicating a combinatorial expression of AgOBPs and revealing distinct AgOBP requirements for various functional sensilla types. WM-FIHC with antisera to AgOBP1 and AgOBP4 confirmed expression of the respective proteins by support cells and demonstrated a location of OBPs within sensilla trichodea. Based on the finding that AgOBP1 and AgOBP4 as well as the receptor type AgOR2 are involved in the recognition of indole, experiments were performed to explore if the AgOBP-types and AgOR2 are co-expressed in distinct olfactory sensilla. Applying two-color WM-FISH with AgOBP-specific probes and probes specific for AgOR2 revealed a close association of support cells bearing transcripts for AgOBP1 and AgOBP4 and neurons with a transcript for the receptor AgOR2. Moreover, combined WM-FISH/-FIHC approaches using an AgOR2-specific riboprobe and AgOBP-specific antisera revealed the expression of the “ligand-matched” AgOBP1, AgOBP4 and AgOR2 to single trichoid hairs. This result substantiates the notion that a specific response to indole is mediated by an interplay of the proteins.
An Odorant Receptor from the Southern House Mosquito Culex pipiens quinquefasciatus Sensitive to Oviposition Attractants  [PDF]
Julien Pelletier,David T. Hughes,Charles W. Luetje,Walter S. Leal
PLOS ONE , 2012, DOI: 10.1371/journal.pone.0010090
Abstract: Insect odorant receptors (ORs) are heteromers comprised of highly variable odorant-binding subunits associated with one conserved co-receptor. They are potential molecular targets for the development of novel mosquito attractants and repellents. ORs have been identified in the malaria mosquito, Anopheles gambiae, and in the yellow fever mosquito, Aedes aegypti. However, they are still unknown in the Southern house mosquito, Culex quinquefasciatus, which transmits pathogens that cause human diseases throughout the world, including West Nile Virus in the United States.
Antennal expression pattern of two olfactory receptors and an odorant binding protein implicated in host odor detection by the malaria vector Anopheles gambiae
Danuta Schymura, Maike Forstner, Anna Schultze, Thomas Kr?ber, Luc Swevers, Kostas Iatrou, Jürgen Krieger
International Journal of Biological Sciences , 2010,
Abstract: Odor-detection in the malaria mosquito Anopheles gambiae involves large families of diverse proteins, including multiple odorant binding proteins (AgOBPs) and olfactory receptors (AgORs). The receptors AgOR1 and AgOR2, as well as the binding protein AgOBP1, have been implicated in the recognition of human host odors. In this study, we have explored the expression of these olfactory proteins, as well as the ubiquitous odorant receptor heteromerization partner AgOR7, in the thirteen flagellomeres (segments) of female and male antenna. Expressing cells were visualized by adapting a whole mount fluorescence in situ hybridization method. In female mosquitoes, AgOR1-expressing olfactory receptor neurons (ORNs) were almost exclusively segregated in segments 3 to 9, whereas AgOR2-expressing ORNs were distributed over flagellomeres 2 to 13. Different individuals comprised a similar number of cells expressing a distinct AgOR type, although their antennal topography and number per flagellomere varied. AgOBP1-expressing support cells were present in segments 3 to 13 of the female antenna, with increasing numbers towards the distal end. In male mosquitoes, total numbers of AgOR- and AgOBP1-expressing cells were much lower. While AgOR2-expressing cells were found on both terminal flagellomeres, AgOR1 cells were restricted to the most distal segment. High densities of AgOBP1-expressing cells were identified in segment 13, whereas segment 12 comprised very few. Altogether, the results demonstrate that both sexes express the two olfactory receptor types as well as the binding protein AgOBP1 but there is a significant sexual dimorphism concerning the number and distribution of these cells. This may suggest gender-specific differences in the ability to detect distinct odorants, specifically human host-derived volatiles.
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