Patterns of nucleotide polymorphism and divergence in the odorant-binding protein genes OS-E and OS-F: analysis in the melanogaster species subgroup of Drosophila

The Olfactory Specific-E and -F genes (OS-E and OS-F) belong to the odorant-binding protein gene family, which includes the general odorant-binding proteins and the pheromone-binding proteins. In Drosophila melanogaster, these genes are arranged in tandem in a genomic region near the centromere of chromosome arm 3R. We examined the pattern of DNA sequence variation in an approximately 7-kb genomic region encompassing the two OS genes in four species of the melanogaster subgroup of Drosophila and in a population sample of D. melanogaster. We found that both the OS-E and the OS-F gene are present in all surveyed species. Nucleotide divergence estimates would support that the two genes are functional, although they diverge in their functional constraint. The pattern of nucleotide variation in D. melanogaster also differed between genes. Variation in the OS-E gene region exhibited an unusual and distinctive pattern: (i) a relatively high number of fixed amino acid replacements in the encoded protein and (ii) a peak of nucleotide polymorphism around the OS-E gene. These results are unlikely under the neutral model and suggest the action of natural selection in the evolution of the two odorant-binding protein genes.     

Three odorant-binding proteins are co-expressed in sensilla trichodea of Drosophila melanogaster

Odorant-binding proteins (OBPs) are small soluble proteins present in the aqueous medium surrounding olfactory receptor neurones. In this study we examine the expression patterns of three Drosophila OBPs (LUSH=OBP76a, OS-E=OBP83b and OS-F=OBP83a), using post-embedding immunocytochemistry. All three OBPs are co-expressed in sensilla trichodea whereas sensilla intermedia show co-expression of OS-E and OS-F only, but not of LUSH. Thus, it is confirmed that an individual sensillum can contain more than one OBP, even if it comprises only a single receptor neurone, such as the subtype T-1. In s. trichodea of lush⁻ mutants, expression of OS-E and OS-F is not impaired. No other sensillum type on antenna or maxillary palp (e.g. sensilla basiconica, sensilla coeloconica) expresses LUSH, OS-E or OS-F. Within the s. trichodea the three OBPs show the same labelling pattern: the extracellular sensillum lymph in the hair lumen and the sensillum-lymph cavities are heavily labelled. Intracellularly, the three OBPs are co-localised in a variety of dense granules in all auxiliary cells, and also in the receptor neurones. Immunocytochemical data from antennal sections of flies where lush gene expression has been tagged with the reporter gene lacZ suggest that LUSH is synthesised only in the trichogen and the thecogen cells. Thus, LUSH OBP is produced and secreted by two auxiliary cells, whereas its turnover and decomposition does not appear to be restricted to these auxiliary cells but may also occur in the tormogen and receptor cells. The immunocytochemical results are discussed with respect to current concepts of the function of odorant-binding proteins.     

Odorant binding protein diversity and distribution among the insect orders, as indicated by LAP, an OBP-related protein of the true bug Lygus lineolaris (Hemiptera, Heteroptera)

Insect odorant binding proteins (OBPs) are thought to deliver odors to olfactory receptors, and thus may be the first biochemical step in odor reception capable of some level of odor discrimination. OBPs have been identified from numerous species of several insect orders, including Lepidoptera, Diptera, Coleoptera and Hymenoptera; all are holometabolous insects belonging to the monophyletic division of insects known as the Endopterygota. Recently, an antennal protein with OBP-like properties was identified from Lygus lineolaris, a hemipteran insect representing the Hemipteroid Assemblage, a sister division to the Endopterygota. The full length sequence of Lygus antennal protein (LAP) is presented in this report. In situ hybridization analysis revealed LAP expression in cell clusters associating with olfactory sensilla; expression was adult-specific, initiating in developing adult tissue during the transitional period that precedes the actual adult molt. Sequence analysis confirmed that LAP is homologous with the OBP-related protein family, and most similar to the OS-E and OS-F proteins of Drosophila, the ABPX proteins of Lepidoptera and the OBPRP proteins of the Coleoptera. Assuming that the OBP-related proteins represent one homologous family, the identification of LAP significantly expands the phylogenetic depth of that family and its underlying role in odor detection to encompass all members of the Endopterygota and Hemipteroid Assemblage, which comprise >90% of all insect species.     

Unusual pattern of nucleotide sequence variation at the OS-E and OS-F genomic regions of Drosophila simulans

Nucleotide variation at the genomic region encompassing the odorant-binding protein genes OS-E and OS-F (OS region) was surveyed in two populations of Drosophila simulans, one from Europe and the other from Africa. We found that the European population shows an atypical and large haplotype structure, which extends throughout the approximately 5-kb surveyed genomic region. This structure is depicted by two major haplotype groups segregating at intermediate frequency in the sample, one haplogroup with nearly no variation, and the other at levels more typical for this species. This pattern of variation was incompatible with neutral predictions for a population at a stationary equilibrium. Nevertheless, neutrality tests contrasting polymorphism and divergence data fail to detect any departure from the standard neutral model in this species, whereas they confirm the non-neutral behavior previously observed at the OS-E gene in D. melanogaster. Although positive Darwinian selection may have been responsible for the observed unusual nucleotide variation structure, coalescent simulation results do not allow rejecting the hypothesis that the pattern was generated by a recent bottleneck in the history of European populations of D. simulans.     

The Lepidoptera Odorant Binding Protein gene family: Gene gain and loss within the GOBP/PBP complex of moths and butterflies

Butterflies and moths differ significantly in their daily activities: butterflies are diurnal while moths are largely nocturnal or crepuscular. This life history difference is presumably reflected in their sensory biology, and especially the balance between the use of chemical versus visual signals. Odorant Binding Proteins (OBP) are a class of insect proteins, at least some of which are thought to orchestrate the transfer of odor molecules within an olfactory sensillum (olfactory organ), between the air and odor receptor proteins (ORs) on the olfactory neurons. A Lepidoptera specific subclass of OBPs are the GOBPs and PBPs; these were the first OBPs studied and have well documented associations with olfactory sensilla. We have used the available genomes of two moths, Manduca sexta and Bombyx mori, and two butterflies, Danaus plexippus and Heliconius melpomene, to characterize the GOBP/PBP genes, attempting to identify gene orthologs and document specific gene gain and loss. First, we identified the full repertoire of OBPs in the M. sexta genome, and compared these with the full repertoire of OBPs from the other three lepidopteran genomes, the OBPs of Drosophila melanogaster and select OBPs from other Lepidoptera. We also evaluated the tissue specific expression of the M. sexta OBPs using an available RNAseq databases. In the four lepidopteran species, GOBP2 and all PBPs reside in single gene clusters; in two species GOBP1 is documented to be nearby, about 100 kb from the cluster; all GOBP/PBP genes share a common gene structure indicating a common origin. As such, the GOBP/PBP genes form a gene complex. Our findings suggest that (1) the lepidopteran GOBP/PBP complex is a monophyletic lineage with origins deep within Lepidoptera phylogeny, (2) within this lineage PBP gene evolution is much more dynamic than GOBP gene evolution, and (3) butterflies may have lost a PBP gene that plays an important role in moth pheromone detection, correlating with a shift from olfactory (moth) to visual (butterfly) communication, at least regarding long distance mate recognition. These findings will be clarified by additional lepidopteran genomic data, but the observation that moths and butterflies share most of the PBP/GOBP genes suggests that they also share common chemosensory-based behavioral pathways.     

Identification and expression of odorant-binding proteins of the malaria-carrying mosquitoes Anopheles gambiae and Anopheles arabiensis

Host preference and blood feeding are restricted to female mosquitoes. Olfaction plays a major role in host-seeking behaviour, which is likely to be associated with a subset of mosquito olfactory genes. Proteins involved in olfaction include the odorant receptors (ORs) and the odorant-binding proteins (OBPs). OBPs are thought to function as a carrier within insect antennae for transporting odours to the olfactory receptors. Here we report the annotation of 32 genes encoding putative OBPs in the malaria mosquito Anopheles gambiae and their tissue-specific expression in two mosquito species of the Anopheles complex; a highly anthropophilic species An. gambiae sensu stricto and an opportunistic, but more zoophilic species, An. arabiensis. RT-PCR shows that some of the genes are expressed mainly in head tissue and a subset of these show highest expression in female heads. One of the genes (agCP1588) which has not been identified as an OBP, has a high similarity (40%) to the Drosophila pheromone-binding protein 4 (PBPRP4) and is only expressed in heads of both An. gambiae and An. arabiensis, and at higher levels in female heads. Two genes (agCP3071 and agCP15554) are expressed only in female heads and agC15554 also shows higher expression levels in An. gambiae. The expression profiles of the genes in the two members of the Anopheles complex provides the first step towards further molecular analysis of the mosquito olfactory apparatus.     

Novel odorant-binding proteins expressed in the taste tissue of the fly

A taste tissue cDNA library of the fleshfly Boettcherisca peregrina was screened with a subtracted cDNA probe enriched with taste-receptor-tissue-specific cDNA. Seven genes were identified with sequence similarity to insect odorant-binding protein (OBP) genes. The predicted amino acid sequences of the genes contain the putative signal peptide sequence at the N-terminal and most of them conserve the six cysteines common to known insect OBPs. These genes show a high degree of sequence divergence with approximately 20% amino acid identity. The most striking feature was that all seven of these genes are expressed mainly in the taste tissues, such as the labellum and tarsus, unlike the known insect OBP genes expressed in olfactory tissue. The predicted amino acid sequences had the highest degree of sequence similarity to the Drosophila melanogaster OBPs named pheromone binding protein-related proteins (PBPRPs). These gene products are here referred to as gustatory PBP-related proteins (GPBPRPs) 1-7. Homologous GPBPRP genes were found also in D. melanogaster by database search and are shown to be expressed in Drosophila taste tissues.     

Evolution of gene expression in the Drosophila olfactory system

Host plant shifts by phytophagous insects play a key role in insect evolution and plant ecology. Such shifts often involve major behavioral changes as the insects must acquire an attraction and/or lose the repulsion to the new host plant's odor and taste. The evolution of chemotactic behavior may be due, in part, to gene expression changes in the peripheral sensory system. To test this hypothesis, we compared gene expression in the olfactory organs of Drosophila sechellia, a narrow ecological specialist that feeds on the fruit of Morinda citrifolia, with its close relatives Drosophila simulans and Drosophila melanogaster, which feed on a wide variety of decaying plant matter. Using whole-genome microarrays and quantitative polymerase chain reaction, we surveyed the entire repertoire of Drosophila odorant receptors (ORs) and odorant-binding proteins (OBPs) expressed in the antennae. We found that the evolution of OR and OBP expression was accelerated in D. sechellia compared both with the genome average in that species and with the rate of OR and OBP evolution in the other species. However, some of the gene expression changes that correlate with D. sechellia's increased sensitivity to Morinda odorants may predate its divergence from D. simulans. Interspecific divergence of olfactory gene expression cannot be fully explained by changes in the relative abundance of different sensilla as some ORs and OBPs have evolved independently of other genes expressed in the same sensilla. A number of OR and OBP genes are upregulated in D. sechellia compared with its generalist relatives. These genes include Or22a, which likely responds to a key odorant of M. citrifolia, and several genes that are yet to be characterized in detail. Increased expression of these genes in D. sechellia may have contributed to the evolution of its unique chemotactic behavior.     

The SNMP/CD36 gene family in Diptera, Hymenoptera and Coleoptera: Drosophila melanogaster, D. pseudoobscura, Anopheles gambiae, Aedes aegypti, Apis mellifera, and Tribolium castaneum

Sensory neuron membrane proteins (SNMPs) are membrane bound proteins initially identified in olfactory receptor neurons of Lepidoptera and are thought to play a role in odor detection; SNMPs belong to a larger gene family characterized by the human protein CD36. We have identified 12-14 candidate SNMP/CD36 homologs from each of the genomes of Drosophila melanogaster, D. pseudoobscura, Anopheles gambiae and Aedes aegypti (Diptera), eight candidate homologs from Apis mellifera (Hymenoptera), and 15 from Tribolium castaneum (Coleoptera). Analysis (sequence similarity and intron locations) suggests that the insect SNMP/CD36 genes fall into three major groups. Group 1 includes the previously characterized D. melanogaster emp (epithelial membrane protein). Group 2 includes the previously characterized D. melanogaster croquemort, ninaD, santa maria, and peste. Group 3 genes include the SNMPs, which fall into two subgroups referred to as SNMP1 and SNMP2. D. melanogaster SNMP1 (CG7000) shares both significant sequence similarity and five of its six intron insertion sites with the lepidopteran Bombyx mori SNMP1. The topological conservation of this gene family within the three major holometabolous lineages indicates that it predates the coleopteran and hymenoptera/dipera/lepidoptera split 300+ million years ago. The current state of knowledge of the characterized insect members of this gene family is discussed.     

A cluster of candidate odorant receptors from the malaria vector mosquito,Anopheles gambiae

Olfaction is critical to the host preference selection behavior of many disease-transmitting insects, including the mosquito Anopheles gambiae sensu stricto (hereafter A. gambiae), one of the major vectors for human malaria. In order to more fully understand the molecular biology of olfaction in this insect, we have previously identified several members member of a family of candidate odorant receptor proteins from A. gambiae (AgORs). Here we report the cloning and characterization of an additional AgOR gene, denoted as AgOr5, which shows significant similarity to putative odorant receptors in A. gambiae and Drosophila melanogaster and which is selectively expressed in olfactory organs. AgOr5 is tightly clustered within the A. gambiae genome to two other highly homologous candidate odorant receptors, suggesting that these genes are derived from a common ancestor. Analysis of the developmental expression within members of this AgOR gene cluster reveals considerable variation between these AgORs as compared to candidate odorant receptors from D. melanogaster.     

Functional dissection of Odorant binding protein genes in Drosophila melanogaster

Most organisms rely on olfaction for survival and reproduction. The olfactory system of Drosophila melanogaster is one of the best characterized chemosensory systems and serves as a prototype for understanding insect olfaction. Olfaction in Drosophila is mediated by multigene families of odorant receptors and odorant binding proteins (OBPs). Although molecular response profiles of odorant receptors have been well documented, the contributions of OBPs to olfactory behavior remain largely unknown. Here, we used RNAi-mediated suppression of Obp gene expression and measurements of behavioral responses to 16 ecologically relevant odorants to systematically dissect the functions of 17 OBPs. We quantified the effectiveness of RNAi-mediated suppression by quantitative real-time polymerase chain reaction and used a proteomic liquid chromatography and tandem mass spectrometry procedure to show target-specific suppression of OBPs expressed in the antennae. Flies in which expression of a specific OBP is suppressed often show altered behavioral responses to more than one, but not all, odorants, in a sex-dependent manner. Similarly, responses to a specific odorant are frequently affected by suppression of expression of multiple, but not all, OBPs. These results show that OBPs are essential for mediating olfactory behavioral responses and suggest that OBP-dependent odorant recognition is combinatorial.     

Odorant-binding-protein subfamilies associate with distinct classes of olfactory receptor neurons in insects

The olfactory receptors of terrestrial animals exist in an aqueous environment, yet detect odorants that are primarily hydrophobic. The aqueous solubility of hydrophobic odorants is thought to be greatly enhanced via odorant binding proteins (OBP) which exist in the extracellular fluid surrounding the odorant receptors. We have isolated and partially sequenced 14 candidate OBPs from six insect (moth) species. All 14 represent a single homologous family based on conserved sequence domains. The 14 proteins can be divided into three subfamilies based on differences in tissue specific expression and similarities in amino acid sequences. All 14 proteins are specifically expressed in antennal olfactory tissue. Subfamily I represents previously described pheromone binding proteins (PBP), which are male-specific, associate with pheromone-sensitive neurons, and are highly variable in their sequences when compared among species. Subfamilies II and III are expressed in both male and female antennae, appear to associate with general-odorant-sensitive neurons, and are highly conserved when compared among species. The properties of the subfamily II and III proteins suggest these are general-odorant binding proteins (GOBP). The properties of the respective insect OBP subfamilies suggest that they have different odorant binding specificities. The association of different insect OBP subfamilies with distinct classes of olfactory neurons having different odorant specificities suggests that OBPs can act as selective signal filters, peripheral to the actual receptor proteins.     

Unique Features of Odorant-Binding Proteins of the Parasitoid Wasp Nasonia vitripennis Revealed by Genome Annotation and Comparative Analyses

Insects are the most diverse group of animals on the planet, comprising over 90% of all metazoan life forms, and have adapted to a wide diversity of ecosystems in nearly all environments. They have evolved highly sensitive chemical senses that are central to their interaction with their environment and to communication between individuals. Understanding the molecular bases of insect olfaction is therefore of great importance from both a basic and applied perspective. Odorant binding proteins (OBPs) are some of most abundant proteins found in insect olfactory organs, where they are the first component of the olfactory transduction cascade, carrying odorant molecules to the olfactory receptors. We carried out a search for OBPs in the genome of the parasitoid wasp Nasonia vitripennis and identified 90 sequences encoding putative OBPs. This is the largest OBP family so far reported in insects. We report unique features of the N. vitripennis OBPs, including the presence and evolutionary origin of a new subfamily of double-domain OBPs (consisting of two concatenated OBP domains), the loss of conserved cysteine residues and the expression of pseudogenes. This study also demonstrates the extremely dynamic evolution of the insect OBP family: (i) the number of different OBPs can vary greatly between species; (ii) the sequences are highly diverse, sometimes as a result of positive selection pressure with even the canonical cysteines being lost; (iii) new lineage specific domain arrangements can arise, such as the double domain OBP subfamily of wasps and mosquitoes.     

An overview of odorant-binding protein functions in insect peripheral olfactory reception

Insect olfactory perception involves many aspects of insect life, and can directly or indirectly evoke either individual or group behaviors. Insect olfactory receptors and odorant-binding proteins (OBPs) are considered to be crucial to insect-specific and -sensitive olfaction. Although the mechanisms of interaction between OBPs or OBP/ligand complex with olfactory receptors are still not well understood, it has been shown that many OBPs contribute to insect olfactory perception at various levels. Some of these are numerous and divergent members in OBP family; expression in the olfactory organ at high concentration; a variety of combinational patterns between different OBPs and ligands, but exclusive affinity for one OBP to specific binding ligands; complicated interactions between OBP/ligand complex and transmembrane proteins (olfactory receptors or sensory neuron membrane proteins). First, we review OBPs' ligand-binding property based on OBP structural research and ligand-binding test; then, we review current progress around the points cited above to show the role of such proteins in insect olfactory signal transmission; finally, we discuss applications based on insect OBP research.     

The molecular logic of olfaction in Drosophila

Drosophila fruit flies display robust olfactory-driven behaviors with an olfactory system far simpler than that of vertebrates. Endowed with 1300 olfactory receptor neurons, these insects are able to recognize and discriminate between a large number of distinct odorants. Candidate odorant receptor molecules were identified by complimentary approaches of differential cloning and genome analysis. The Drosophila odorant receptor (DOR) genes encode a novel family of proteins with seven predicted membrane-spanning domains, unrelated to vertebrate or nematode chemosensory receptors. There are on the order of 60 or more members of this gene family in the Drosophila genome, far fewer than the hundreds to thousands of receptors found in vertebrates or nematodes. DOR genes are selectively expressed in small subsets of olfactory neurons, in expression domains that are spatially conserved between individuals, bilaterally symmetric and not sexually dimorphic. Double in situ RNA hybridization with a number of pairwise combinations of DOR genes fails to reveal any overlap in gene expression, suggesting that each olfactory neuron expresses one or a small number of receptor genes and is therefore functionally distinct. How is activation of such a subpopulation of olfactory receptor neurons in the periphery sensed by the brain? In the mouse, all neurons expressing a given receptor project with precision to two of 1800 olfactory bulb glomeruli, creating a spatial map of odor quality in the brain. We have employed DOR promoter transgenes that recapitulate expression of endogenous receptor to visualize the projections of individual populations of receptor neurons to subsets of the 43 glomeruli in the Drosophila antennal lobe. The results suggest functional conservation in the logic of olfactory discrimination from insects to mammals.     

昆虫气味结合蛋白的研究进展

摘要: 昆虫主要依赖其复杂且灵敏的化学感受系统来识别并区分外界环境中的各种化学信号。嗅觉是负责嗅觉信号传导的感官方式,能够引起昆虫觅食、产卵、交配和躲避天敌等对生存和繁殖至关重要的行为反应。在嗅觉感知过程中,气味结合蛋白(odorant binding proteins, OBPs)最先与外界脂溶性化学物质相互作用,并将其转运至化学受体神经元上,激活树突膜表面分布的嗅觉受体(olfactory receptors, ORs),是嗅觉系统正常运行的必需蛋白。近年来,随着高通量测序和分子生物学技术的快速发展,越来越多的昆虫OBPs相继得以鉴定并开展功能研究。昆虫OBPs是一类可溶性的小分子蛋白,一般由6个α-螺旋构成一个稳定、紧密的疏水性结合腔,其构象变化因昆虫种类和配体结构不同而有所差异。OBPs的分布不受限于嗅觉器官,还在口器、足、中肠、腺体等非嗅觉组织中表达,具有嗅觉识别、味觉感受、营养物质转运、信息素合成与释放、组织发育与分化等生理功能。OBPs行使以上功能的共同特性为结合和溶解包括信息素组分、普通气味分子和非挥发性物质等的疏水性小分子物质。昆虫OBPs的稳定性和多功能性暗示其可广泛应用于害虫防治、生物传感器、分析化学、生态学等多个领域。本文对过去20多年来昆虫OBPs的相关研究进行综述,为进一步深入开展OBPs的功能研究提供理论参考。