Functions Of Odorant-binding Proteins Research Articles

Odorant Binding Proteins Facilitate the Gas-Phase Uptake of Odorants Through the Nasal Mucus.

Mammalian odorant binding proteins (OBPs) have long been suggested to transport hydrophobic odorant molecules through the aqueous environment of the nasal mucus. While the function of OBPs as odorant transporters is supported by their hydrophobic beta-barrel structure, no rationale has been provided on why and how these proteins facilitate the uptake of odorants from the gas phase. Here, a multi-scale computational approach validated through available high-resolution spectroscopy experiments reveals that the conformational space explored by carvone inside the binding cavity of porcine OBP (pOBP) is much closer to the gas than the aqueous phase, and that pOBP effectively manages to transport odorants by lowering the free energy barrier of odorant uptake. Understanding such perireceptor events is crucial to fully unravel the molecular processes underlying the olfactory senseand move towards the development of protein-based biomimetic sensor units that can serve as artificial noses.

Identification of odorant-binding proteins and functional analysis of antenna-specific BhorOBP28 in Batocera horsfieldi (Hope).

The important wood-boring pest Batocera horsfieldi has evolved a sensitive olfactory system to locate host plants. Odorant-binding proteins (OBPs) are thought to play key roles in olfactory recognition. Therefore, exploring the physiological function of OBPs could facilitate a better understanding of insect chemical communications. In this research, 36 BhorOBPs genes were identified via transcriptome sequencing of adults' antennae from B. horsfieldi, and most BhorOBPs were predominantly expressed in chemosensory body parts. Through fluorescence competitive binding and fluorescence quenching assays, the antenna-specific BhorOBP28 was investigated and displayed strong binding affinities forming stable complexes with five volatiles, including (+)-α-Pinene, (+)-Limonene, β-Pinene, (-)-Limonene, and (+)-Longifolene, which could also elicit conformation changes when they were interacting with BhorOBP28. Batocera horsfieldi females exhibited a preference for (-)-Limonene, and a repellent response to (+)-Longifolene. Feeding dsOBP19 produced by a bacteria-expressed system with a newly constructed vector could lead to the knockdown of BhorOBP28, and could further impair B. horsfieldi attraction to (-)-Limonene and repellent activity of (+)-Longifolene. The analysis of site-directed mutagenesis revealed that Leu7, Leu72, and Phe121 play a vital role in selectively binding properties of BhorOBP28. By modeling the molecular mechanism of olfactory recognition, these results demonstrate that BhorOBP28 is involved in the chemoreception of B. horsfieldi. The bacterial-expressed dsRNA delivery system gains new insights into potential population management strategies. Through the olfactory process concluded that discovering novel behavioral regulation and environmentally friendly control options for B. horsfieldi in the future. © 2024 Society of Chemical Industry.

Odorant Binding Protein Expressed in Legs Enhances Malathion Tolerance in Bactrocera dorsalis (Hendel).

Bactrocera dorsalis is a highly invasive species and is one of the most destructive agricultural pests worldwide. Organophosphorus insecticides have been widely and chronically used to control it, leading to the escalating development of resistance. Recently, odorant binding proteins (OBPs) have been found to play a role in reducing insecticide susceptibility. In this study, we used RT-qPCR to measure the expression levels of four highly expressed OBP genes in the legs of B. dorsalis at different developmental stages and observed the effect of malathion exposure on their expression patterns. The results showed that OBP28a-2 had a high expression level in 5 day old adults of B. dorsalis, and its expression increased after exposure to malathion. By CRISPR/Cas9 mutagenesis, we generated OBP28a-2-/- null mutants and found that they were more susceptible to malathion than wild-type adults. Furthermore, in vitro direct affinity assays confirmed that OBP28a-2 has a strong affinity for malathion, suggesting that it plays a role in reducing the susceptibility of B. dorsalis to malathion. Our findings enriched our understanding of the function of OBPs. The results highlighted the potential role of OBPs as buffering proteins that help insects survive exposure to insecticides.

An OBP gene highly expressed in non-chemosensory tissues affects the phototaxis and reproduction of Spodoptera frugiperda.

Insect odorant binding proteins (OBPs) were initially regarded as carriers of the odorants involved in chemosensation. However, it had been observed that a growing number of OBP genes exhibited broad expression patterns beyond chemosensory tissues. Here, an OBP gene (OBP31) was found to be highly expressed in the larval ventral nerve cord, adult brain and male reproductive organ of Spodoptera frugiperda. An OBP31 knockout strain (OBP31-/- ) was generated by CRISPR/Cas9 mutagenesis. For OBP31-/- , the larvae needed longer time to pupate, but there was no difference in the pupal weight between OBP31-/- and wild type (WT). OBP31-/- larvae showed stronger phototaxis than the WT larvae, indicating the importance of OBP31 in light perception. For mating rhythm of adults, OBP31-/- moths displayed an earlier second mating peak. In the cross-pairing of OBP31-/- and WT moths, the mating duration was longer, and hatchability was lower in OBP31-/- group and OBP31+/- ♂ group than that in the WT group. These results suggested that OBP31 played a vital role in larval light perception and male reproductive process and could provide valuable insights into understanding the biological functions of OBPs that were not specific in chemosensory tissues.

NlugOBP8 in Nilaparvata lugens Involved in the Perception of Two Terpenoid Compounds from Rice Plant

Odorant binding proteins (OBPs) play an important role in insect peripheral olfactory systems and exploring the physiological function of OBPs could facilitate the understanding of insects' chemical communication. Here, the functional analysis of an antenna-based NlugOBP8 from brown planthopper (BPH) Nilaparvata lugens (Stål) was performed both in vitro and in vivo. Recombinant NlugOBP8 exhibited strong binding affinity to 13 out of 26 rice plant volatiles and could form a stable complex with 9 of them according to the fluorescence binding and fluorescence quenching experiments. Circular dichroism spectra demonstrated that six volatiles could give rise to significant conformational change of recombinant NlugOBP8. H-tube olfactometer bioassay confirmed that BPHs were significantly attracted by nerolidol and significantly repelled by linalool, caryophyllene oxide, and terpinolene, respectively. Antennae of dsNlugOBP8-injected BPHs exhibited significantly lower electrophysiological response to linalool and caryophyllene oxide. Moreover, the repellent responses of BPHs to these two volatiles were also impaired upon silencing NlugOBP8. These data suggest that NlugOBP8 is involved in recognizing linalool and caryophyllene oxide and provide additional target for the sustainable control of BPHs.

Identification of Odorant-Binding and Chemosensory Protein Genes in Mythimna separata Adult Brains Using Transcriptome Analyses.

Large numbers of chemosensory genes have been identified in the peripheral sensory organs of the pest Mythimna separata (Walker) to increase our understanding of chemoreception-related molecular mechanisms and to identify molecular targets for pest control. Chemosensory-related genes are expressed in various tissues, including non-sensory organs, and they play diverse roles. To better understand the functions of chemosensory-related genes in non-sensory organs, transcriptomic analyses of M. separata brains were performed. In total, 29 odorant-binding proteins (OBPs) and 16 chemosensory proteins (CSPs) putative genes were identified in the transcriptomic data set. The further examination of sex- and tissue-specific expression using RT-PCR suggested that eight OBPs (OBP5, -7, -11, -13, -16, -18, -21, and -24) and eight CSPs (CSP2–4, -8, CSP10–12, and -15) genes were expressed in the brain. Furthermore, bands representing most OBPs and CSPs could be detected in antennae, except for a few that underwent sex-biased expression in abdomens, legs, or wings. An RT-qPCR analysis of the expression profiles of six OBPs (OBP3–5, -9, -10, and -16) and two CSPs (CSP3 and CSP4) in different tissues and sexes indicated that OBP16 was highly expressed in male brain, and CSP3 and CSP4 were female-biased and highly expressed in brain. The expression levels of OBP5 and OBP10 in brain were not significantly different between the sexes. The findings expand our current understanding of the expression patterns of OBPs and CSPs in M. separata sensory and non-sensory tissues. These results provide valuable reference data for exploring novel functions of OBPs and CSPs in M. separata and may help in developing effective biological control strategies for managing this pest by exploring novel molecular targets.

The molecular identification, odor binding characterization, and immunolocalization of odorant-binding proteins in Liriomyza trifolii

The Liriomyza trifolii is a highly invasive polyphagia pest. Understanding the physiological functions of odorant binding proteins (OBPs) in the chemical communication of L. trifolii can lead to effective pest management strategies. Seven full-length OBPs were identified by transcriptome screening of L. trifolii adults. Bioinformatics analyses classified the seven OBPs into two subfamilies (six classic OBPs, one minus-C OBP). The analysis of their expression in different development stages revealed that LtriOBP5 was highly expressed in the larval stage, LtriOBP4 in the pupa stage, and LtriOBP1, 2, 3, 6, 7 in the adult stage; the expression levels were higher in male adults than in females. The analysis of different tissues showed high expression of LtriOBP1, 3, 6, 7 in the antennae, which were selected for in vitro purification. To explore the ligand compounds of OBPs, fluorescence competitive binding experiments were performed. Immunofluorescence localization revealed that LtriOBP1, 3, 6, 7 showed strong binding abilities to plant volatiles and were located in the antennae, implying that LtriOBP1, 3, 6, 7 may play key roles in olfaction, such as host location. LtriOBP6 and LtriOBP7 had strong binding abilities to specific herbivore-induced plant volatiles, suggesting LtriOBP6 and LtriOBP7 may also play critical roles in chemoreception. This study provides preliminary exploration of the olfactory perception mechanism of L. trifolii, which can be used as a basis to design insect behavior regulators and develop highly effective insecticides using mixture of ligands and known pesticides.

BarH1 regulates odorant-binding proteins expression and olfactory perception of Monochamus alternatus Hope

Insect odorant-binding proteins (OBPs) are a class of small soluble proteins that can be found in various tissues wherein binding and transport of small molecules are required. Thus, OBPs are not only involved in typical olfactory function by specific activities with odorants but also participate in other physiological processes in non-chemosensory tissues. To better understand the complex biological functions of OBPs, it is necessary to study the transcriptional regulation of their expression patterns. In this paper, an apparent gradient expression pattern of Obp19, that was highly and specifically expressed in antennae and played an essential role in the detection of camphene, was defined in the antennae of the Japanese pine sawyer. Further, the transcription factor BarH1, that also presented gradient expression pattern in antennae, was found to regulate expression of Obp19 directly through binding to its upstream DNA sequence. The condition of BarH1 gene silence, the gene expression levels of Obp19 significantly decreased. At the same time, additional olfactory genes also were regulated and thus influence camphene reception. These findings provide us an opportunity to incorporate Obps in the gene regulatory networks of insects, which contribute to a better understanding of the multiplicity and diversity of OBPs and the olfactory mediated behaviors.

A Detailed Spatial Expression Analysis of Wing Phenotypes Reveals Novel Patterns of Odorant Binding Proteins in the Soybean Aphid, Aphis glycines.

The wide range of insect niches has led to a rapid expansion of chemosensory gene families as well as their relatively independent evolution and a high variation. Previous studies have revealed some functions for odorant-binding proteins (OBPs) in processes beyond olfaction, such as gustation and reproduction. In this study, a comparative transcriptomic analysis strategy was applied for the soybean aphid, Aphis glycines, focusing on various functional tissues and organs of winged aphids, including the antenna, head, leg, wing, thorax, cauda, and cornicle. Detailed spatial OBP expression patterns in winged and wingless parthenogenetic aphids were detected by RT-qPCR. Twelve OBPs were identified, and three new OBPs in A. glycines are first reported. All OBPs showed comparatively higher expression in sensory organs and tissues, such as the antenna, head, or leg. Additionally, we found some novel expression patterns for aphid OBPs (Beckendorf et al., 2008). Five OBPs exhibited high-expression levels in the cauda and four in the cornicle (Biasio et al., 2015). Three genes (OBP2/3/15) were highly expressed in the wing (Calvello et al., 2003). Two (OBP3/15) were significantly more highly expressed in the wingless thorax than in the winged thorax with the wings removed, and these transcripts were significantly enriched in the removed wings. More details regarding OBP spatial expression were revealed under our strategy. These findings supported the existence of carrier transport functions other than for foreign chemicals and therefore broader ligand ranges of aphid OBPs. It is important for understanding how insect OBPs function in chemical perception as well as their other potential physiological functions.

Antennal Enriched Odorant Binding Proteins Are Required for Odor Communication in Glossina f. fuscipes.

Olfaction is orchestrated at different stages and involves various proteins at each step. For example, odorant-binding proteins (OBPs) are soluble proteins found in sensillum lymph that might encounter odorants before reaching the odorant receptors. In tsetse flies, the function of OBPs in olfaction is less understood. Here, we investigated the role of OBPs in Glossina fuscipes fuscipes olfaction, the main vector of sleeping sickness, using multidisciplinary approaches. Our tissue expression study demonstrated that GffLush was conserved in legs and antenna in both sexes, whereas GffObp44 and GffObp69 were expressed in the legs but absent in the antenna. GffObp99 was absent in the female antenna but expressed in the male antenna. Short odorant exposure induced a fast alteration in the transcription of OBP genes. Furthermore, we successfully silenced a specific OBP expressed in the antenna via dsRNAi feeding to decipher its function. We found that silencing OBPs that interact with 1-octen-3-ol significantly abolished flies’ attraction to 1-octen-3-ol, a known attractant for tsetse fly. However, OBPs that demonstrated a weak interaction with 1-octen-3-ol did not affect the behavioral response, even though it was successfully silenced. Thus, OBPs’ selective interaction with ligands, their expression in the antenna and their significant impact on behavior when silenced demonstrated their direct involvement in olfaction.

Odorant Binding Protein C17 Contributes to the Response to Artemisia vulgaris Oil in Tribolium castaneum.

The red flour beetle, Tribolium castaneum (T. castaneum), generates great financial losses to the grain storage and food processing industries. Previous studies have shown that essential oil (EO) from Artemisia vulgaris (A. vulgaris) has strong contact toxicity to larvae of the beetle, and odorant-binding proteins (OBPs) contribute to the defense of larvae against A. vulgaris. However, the functions of OBPs in insects defending against plant oil is still not clear. Here, expression of one OBP gene, TcOBPC17, was significantly induced 12–72 h after EO exposure. Furthermore, compared to the control group, RNA interference (RNAi) against TcOBPC17 resulted in a higher mortality rate after EO treatment, which suggests that TcOBPC17 involves in the defense against EO and induces a declining sensitivity to EO. In addition, the tissue expression profile analysis revealed that the expression of TcOBPC17 was more abundant in the metabolic detoxification organs of the head, fat body, epidermis, and hemolymph than in other larval tissue. The expression profile of developmental stages showed that TcOBPC17 had a higher level in early and late adult stages than in other developmental stages. Taken together, these results suggest that TcOBPC17 could participate in the sequestration process of exogenous toxicants in T. castaneum larvae.

Odorant-Binding Proteins Contribute to the Defense of the Red Flour Beetle, Tribolium castaneum, Against Essential Oil of Artemisia vulgaris.

The function of odorant-binding proteins (OBPs) in insect chemodetection has been extensively studied. However, the role of OBPs in the defense of insects against exogenous toxic substances remains elusive. The red flour beetle, Tribolium castaneum, a major pest of stored grains, causes serious economic losses for the agricultural grain and food processing industries. Here, biochemical analysis showed that essential oil (EO) from Artemisia vulgaris, a traditional Chinese medicine, has a strong contact killing effect against larvae of the red flour beetle. Furthermore, one OBP gene, TcOBPC11, was significantly induced after exposure to EO. RNA interference (RNAi) against TcOBPC11 led to higher mortality compared with the controls after EO treatment, suggesting that this OBP gene is associated with defense of the beetle against EO and leads to a decrease in sensitivity to the EO. Tissue expression profiling showed that expression of TcOBPC11 was higher in the fat body, Malpighian tubule, and hemolymph than in other larval tissues, and was mainly expressed in epidermis, fat body, and antennae from the early adult. The developmental expression profile revealed that expression of TcOBPC11 was higher in late larval stages and adult stages than in other developmental stages. These data indicate that TcOBPC11 may be involved in sequestration of exogenous toxicants in the larvae of T. castaneum. Our results provide a theoretical basis for the degradation mechanism of exogenous toxicants and identify potential novel targets for controlling the beetle.

Functional Analysis of Two Odorant-Binding Proteins, MaltOBP9 and MaltOBP10, in Monochamus alternatus Hope.

Odorant-binding proteins (OBPs) are important for the perception of chemical signals by insects. Effective pest management strategies can be developed by understanding the host location mechanism and the physiological functions of OBPs in olfactory detection. In this study, we cloned two OBPs from Monochamus alternatus, where MaltOBP9 was highly expressed in multiple insect tissues and MaltOBP10 was highly expressed in the female antenna according to the results of qRT-PCR. The recombinant proteins were successfully purified in vitro. Immunocytochemistry indicated the high expression of MaltOBP9 and MaltOBP10 in the sensillum lymph of sensilla basiconica, sensilla trichodea, sensilla auricillica, and sensilla chaetica, thereby demonstrating their broad participation in semiochemical detection. Both proteins were localized in the inner cavity of mechanoreceptors and they exhibited broad binding abilities with volatiles from pine bark according to fluorescence competitive binding assays. Due to its broad binding ability and distribution, MaltOBP9 may be involved in various physiological processes as well as olfactory detection. MaltOBP10 appears to play a role in the fundamental olfactory recognition process of female adults according to its broad binding ability. These findings suggest that OBPs may have various physiological functions in insects, thereby providing novel insights into the olfactory receptive mechanism.

An Antennae-Specific Odorant-Binding Protein Is Involved in Bactrocera dorsalis Olfaction

Insect antennae are important olfactory organs that house high concentrations of odorant-binding proteins (OBPs) in the sensillum lymph. Previous studies in other insects have shown that OBPs play important roles in transporting odorants and enhancing the sensitivity of the olfactory system. However, the functions of OBPs in the oriental fruit fly, Bactrocera dorsalis, especially those specifically expressed in antennae, have not been fully elucidated. In this study, cDNA libraries were constructed from both the male and female antennal transcriptome, and twenty OBPs were identified in total. The expression profiles of these OBPs were examined in the adult antenna, head, thorax, leg, and abdomen of both sexes. Seven of the identified OBP genes had significantly higher expression in both the male and female antennae than in other tissues, while the transcript levels of the remaining OBPs varied across different tissues. Regarding the function of antenna-specific OBPs, we targeted Bdorsobp2 as a representative for further RNA interference (RNAi) and identified via electrophysiology a decrease in detection of a potential species-specific a potent attractant, methyl eugenol. Moreover, subsequent behavioral assay data showed that the behavioral response of B. dorsalis toward this odorant decreased when Bdorobp2 was silenced with injection of double-stranded RNA (dsRNA). Combined, these results support our initial hypothesis that antennae-specific OBPs are of critical importance for insect odorant detection, sensitivity, and behavior.

Chemical structure of semiochemicals and key binding sites together determine the olfactory functional modes of odorant-binding protein 2 in Eastern honey bee, Apis cerana

Insects can exhibit flexible olfaction that is sensitive to complex natural chemical environments. Odorant-binding proteins (OBPs) in insects' antennal chemosensilla can act as transporters of plant volatiles and pheromones across the sensillar lymph. Although the physiological functions of OBPs have been widely reported, it is still unclear how OBP binds to ligands with various structures in detail. Here, we further investigated the ligand-binding modes and characteristics of AcerOBP2 from the Eastern honey bee (Apis cerana). The results showed that, as a specific protein distributed below the base of chemosensilla on the antennal surface, AcerOBP2 was strongly bound with the candidate floral volatiles and bee pheromones. By docking analysis and site-directed mutagenesis, four different binding modes were found in the five AcerOBP2 mutants between six ligands. Two key amino acids, Ser123 and Lys51, play a key role in AcerOBP2 binding to odors, depending on the presence or absence of hydrogen bonds. In addition, the binding modes depend on their chemical structures and the binding poses of the diverse ligands. These results not only further prompted the functional basis of the relationship between the chemical structures of odorants and bee OBPs, but also revealed the complexity of the flexible behavioral modes of odor binding in insect olfactory systems.

Expression profiles and ligand-binding properties of two odorant-binding proteins from Grapholita molesta (Busck) (Lepidoptera: Tortricidae)

Abstract The ligand-binding function of odorant binding proteins (OBPs) that are expressed exclusively or enriched in antennae are well-studied, whilst the ligand binding properties of relatively low expressed OBPs in insect antennae are still unclear. Here, two low expressed GmolOBPs (namely GmolOBP12 and GmolOBP16) were cloned based on the antennal transcriptome of Grapholita molesta, and then their expression profiles and binding properties were investigated via real-time quantitative PCR (qRT-PCR) and fluorescence binding assays, respectively. Multiple sequence alignment and phylogenetic tree analyses displayed that both of GmolOBP12 and GmolOBP16 possessed the typical six-cysteine motifs unique to the classic OBPs subfamily and were classified as such. Although the abundance of transcripts of GmolOBP12 and GmolOBP16 were relatively low compared to that of many other OBPs in the antennal transcriptome of G. molesta, qRT-PCR results indicated that the transcript levels of GmolOBP12 and GmolOBP16 in antennae were significantly higher than those in other tissues. GmolOBP12 displayed higher expression level in male antennae than in female antennae, while the transcript level of GmolOBP16 in antennae was similar for both sexes. Both recombinant GmolOBP12 and GmolOBP16 exhibited strong binding affinities to the sex pheromone (Z)-8-dodecenyl alcohol and host-plant volatile pear ester. Besides, rGmolOBP12 showed outstanding binding affinities to (E)-2-hexenal, benzaldehyde, 1-hexanol, and (Z)-3-hexenyl acetate with Ki values of 4.21, 6.81, 4.68 and 4.68 μM, respectively. rGmolOBP16 had moderate binding abilities with hexanal, decanal, 1-hexanol, methyl myristate and benzonitrile. We speculated that GmolOBP12 may have dual functions in recognition of green leaf volatiles and sex pheromone components and GmolOBP16 may participate in the detection of host-plant volatiles in chemoreception.

Enhancement of the Olfactory Response by Lipocalin Cp-Lip1 in Newt Olfactory Receptor Cells: An Electrophysiological Study.

Previously, we have detected the expression of 2 lipocalin genes (lp1 and lp2) in the olfactory epithelium of the Japanese newt Cynops pyrrhogaster. Recombinant proteins of these genes (Cp-Lip1 and Cp-Lip2, respectively) exhibited high affinities to various odorants, suggesting that they work like the odorant-binding proteins (OBPs). However, the physiological functions of OBP generally remain inconclusive. Here, we examined the effect of Cp-Lip1 on the electrophysiological responses of newt olfactory receptor cells. We observed that the electro-olfactogram induced by the vapor of an odorant with high affinity to Cp-Lip1 appeared to increase in amplitude when a tiny drop of Cp-Lip1 solution was dispersed over the olfactory epithelium. However, the analysis was difficult because of possible interference by intrinsic components in the nasal mucus. We subsequently adopted a mucus-free condition by using suction electrode recordings from isolated olfactory cells, in which impulses were generated by puffs of odorant solution. When various concentration (0-5 µM) of Cp-Lip1 was mixed with the stimulus solution of odorants highly affinitive to Cp-Lip1, the impulse frequency increased in a concentration-dependent manner. The increase by Cp-Lip1 was seen more evidently at lower concentration ranges of stimulus odorants. These results strongly suggest that Cp-Lip1 broadens the sensitivity of the olfactory cells toward the lower concentration of odorants, by which animals can detect very low concentration of odorants.

Sensilla Morphology and Complex Expression Pattern of Odorant Binding Proteins in the Vetch Aphid Megoura viciae (Hemiptera: Aphididae).

Chemoreception in insects is mediated by several components interacting at different levels and including odorant-binding proteins (OBPs). Although recent studies demonstrate that the function of OBPs cannot be restricted to an exclusively olfactory role, and that OBPs have been found also in organs generally not related to chemoreception, their feature of binding molecules remains undisputed. Studying the vetch aphid Megoura viciae (Buckton), we used a transcriptomic approach to identify ten OBPs in the antennae and we examined the ultrastructural morphology of sensilla and their distribution on the antennae, legs, mouthparts and cauda of wingless and winged adults by scanning electron microscopy (SEM). Three types of sensilla, trichoid, coeloconic and placoid, differently localized and distributed on antennae, mouthparts, legs and cauda, were described. The expression analysis of the ten OBPs was performed by RT-qPCR in the antennae and other body parts of the wingless adults and at different developmental stages and morphs. Five of the ten OBPs (MvicOBP1, MvicOBP3, MvicOBP6, MvicOBP7, and MvicOBP8), whose antibodies were already available, were selected for experiments of whole-mount immunolocalization on antennae, mouthparts, cornicles and cauda of adult aphids. Most of the ten OBPs were more expressed in antennae than in other body parts; MvicOBP1, MvicOBP3, MvicOBP6, MvicOBP7 were also immunolocalized in the sensilla on the antennae, suggesting a possible involvement of these proteins in chemoreception. MvicOBP6, MvicOBP7, MvicOBP8, MvicOBP9 were highly expressed in the heads and three of them (MvicOBP6, MvicOBP7, MvicOBP8) were immunolocalized in the sensilla on the mouthparts, supporting the hypothesis that also mouthparts may be involved in chemoreception. MvicOBP2, MvicOBP3, MvicOBP5, MvicOBP8 were highly expressed in the cornicles-cauda and two of them (MvicOBP3, MvicOBP8) were immunolocalized in cornicles and in cauda, suggesting a possible new function not related to chemoreception. Moreover, the response of M. viciae to different components of the alarm pheromone was assessed by behavioral assays on wingless adult morph; (-)-α-pinene and (+)-limonene were found to be the components mainly eliciting an alarm response. Taken together, our results represent a road map for subsequent in-depth analyses of the OBPs involved in several physiological functions in M. viciae, including chemoreception.

Molecular and functional characterization of three odorant binding proteins from the oriental fruit moth Grapholita molesta (Busck) (Lepidoptera: Tortricide).

Odorant binding proteins (OBPs) act in recognizing odor molecules and their most well-studied functions are transporting odors across the sensillum lymph to olfactory receptor neurons within the insect antennal sensillum. The adults of Grapholita molesta highly depend on olfactory cues in locating host plants and selecting oviposition sites, in which OBPs play an important role in perceiving and recognizing host plant volatiles. Exploring the physiological function of OBPs could facilitate our understanding of their importance in insects' chemical communication. In this study, three OBP genes were cloned and named GmolOBP4, GmolOBP5, and GmolOBP10. Quantitative real-time PCR results indicated that GmolOBP4 and GmolOBP10 were predominantly expressed in adult antennae and GmolOBP5 was expressed in multiple tissues, including head, legs, and wings in addition to antennae. The binding affinities of the three recombinant GmolOBPs (rGmolOBPs) with four sex pheromone components and twenty-nine host plant volatiles were measured using 1-N-Phenyl-naphthylamine as a fluorescence probe. The three rGmolOBPs exhibited specific binding properties to potential ligands, GmolOBP4 and GmolOBP10 bound to minor sex pheromone components, such as (Z)-8-dodecenyl alcohol and dodecanol, respectively. rGmolOBP4 showed intermediate binding ability with hexanal, benzyl alcohol, and pear ester, rGmolOBP5 had a weak affinity for benzaldehyde, pear ester and, methyl jasmonate, and rGmolOBP10 showed strong binding capacity toward hexanol, decanol, and α-ocimene. We speculate that the GmolOBP4 and GmolOBP10 have dual functions in perception and recognition of host plant volatiles and sex pheromone components, while GmolOBP5 may serve other function(s).

Screening behaviorally active compounds based on fluorescence quenching in combination with binding mechanism analyses of SspOBP7, an odorant binding protein from Sclerodermus sp.

Reverse chemical ecology approaches based on the recognition and transport function of odorant binding proteins (OBPs) have been used to screen behaviorally active compounds of insects. In the first place, behaviorally active compounds from Sclerodermus sp., an important ectoparasite of Monochamus alternatus Hope, were screened by SspOBP7. The Fluorescence quenching assays revealed that only six of 19 ligands that had binding affinities in fluorescence competition-binding assays formed complexes with SspOBP7. Pursuing this further, two non-polar ligands, terpinolene and (+)-α-longipinene showed strong attractant activities for Sclerodermus sp. The pH change could lead to conformational transition of SspOBP7 from one state to another, which results in low binding affinities at low pH. Finally, a mutational analysis of the SspOBP7 binding cavity proved that changing the cavity had a greater effect on non-polar ligands, and the specific recognition of ligands by SspOBP7 might depend mainly on the appropriate shapes of the cavity and ligands. The most obvious finding to emerge from this work is that the use of fluorescence quenching to study the binding mechanism of OBPs could aid reverse chemical ecology approaches by narrowing the scope of candidate behaviorally active compounds.