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1.  Characterization of an Antennal Carboxylesterase from the Pest Moth Spodoptera littoralis Degrading a Host Plant Odorant 
PLoS ONE  2010;5(11):e15026.
Carboxyl/cholinesterases (CCEs) are highly diversified in insects. These enzymes have a broad range of proposed functions, in neuro/developmental processes, dietary detoxification, insecticide resistance or hormone/pheromone degradation. As few functional data are available on purified or recombinant CCEs, the physiological role of most of these enzymes is unknown. Concerning their role in olfaction, only two CCEs able to metabolize sex pheromones have been functionally characterized in insects. These enzymes are only expressed in the male antennae, and secreted into the lumen of the pheromone-sensitive sensilla. CCEs able to hydrolyze other odorants than sex pheromones, such as plant volatiles, have not been identified.
In Spodoptera littoralis, a major crop pest, a diversity of antennal CCEs has been previously identified. We have employed here a combination of molecular biology, biochemistry and electrophysiology approaches to functionally characterize an intracellular CCE, SlCXE10, whose predominant expression in the olfactory sensilla suggested a role in olfaction. A recombinant protein was produced using the baculovirus system and we tested its catabolic properties towards a plant volatile and the sex pheromone components.
We showed that SlCXE10 could efficiently hydrolyze a green leaf volatile and to a lesser extent the sex pheromone components. The transcript level in male antennae was also strongly induced by exposure to this plant odorant. In antennae, SlCXE10 expression was associated with sensilla responding to the sex pheromones and to plant odours. These results suggest that a CCE-based intracellular metabolism of odorants could occur in insect antennae, in addition to the extracellular metabolism occurring within the sensillar lumen. This is the first functional characterization of an Odorant-Degrading Enzyme active towards a host plant volatile.
PMCID: PMC2993938  PMID: 21124773
2.  Plant odorants interfere with detection of sex pheromone signals by male Heliothis virescens 
In many insects, mate finding relies on female-released sex pheromones, which have to be deciphered by the male olfactory system within an odorous background of plant volatiles present in the environment of a calling female. With respect to pheromone-mediated mate localization, plant odorants may be neutral, favorable, or disturbing. Here we examined the impact of plant odorants on detection and coding of the major sex pheromone component, (Z)-11-hexadecenal (Z11-16:Ald) in the noctuid moth Heliothis virescens. By in vivo imaging the activity in the male antennal lobe (AL), we monitored the interference at the level of olfactory sensory neurons (OSN) to illuminate mixture interactions. The results show that stimulating the male antenna with Z11-16:Ald and distinct plant-related odorants simultaneously suppressed pheromone-evoked activity in the region of the macroglomerular complex (MGC), where Z11-16:Ald-specific OSNs terminate. Based on our previous findings that antennal detection of Z11-16:Ald involves an interplay of the pheromone binding protein (PBP) HvirPBP2 and the pheromone receptor (PR) HR13, we asked if the plant odorants may interfere with any of the elements involved in pheromone detection. Using a competitive fluorescence binding assay, we found that the plant odorants neither bind to HvirPBP2 nor affect the binding of Z11-16:Ald to the protein. However, imaging experiments analyzing a cell line that expressed the receptor HR13 revealed that plant odorants significantly inhibited the Z11-16:Ald-evoked calcium responses. Together the results indicate that plant odorants can interfere with the signaling process of the major sex pheromone component at the receptor level. Consequently, it can be assumed that plant odorants in the environment may reduce the firing activity of pheromone-specific OSNs in H. virescens and thus affect mate localization.
PMCID: PMC3465774  PMID: 23060749
pheromone detection; antennal lobe; pheromone receptor; pheromone binding protein; olfaction
3.  A carboxylesterase, Esterase-6, modulates sensory physiological and behavioral response dynamics to pheromone in Drosophila 
BMC Biology  2012;10:56.
Insects respond to the spatial and temporal dynamics of a pheromone plume, which implies not only a strong response to 'odor on', but also to 'odor off'. This requires mechanisms geared toward a fast signal termination. Several mechanisms may contribute to signal termination, among which odorant-degrading enzymes. These enzymes putatively play a role in signal dynamics by a rapid inactivation of odorants in the vicinity of the sensory receptors, although direct in vivo experimental evidences are lacking. Here we verified the role of an extracellular carboxylesterase, esterase-6 (Est-6), in the sensory physiological and behavioral dynamics of Drosophila melanogaster response to its pheromone, cis-vaccenyl acetate (cVA). Est-6 was previously linked to post-mating effects in the reproductive system of females. As Est-6 is also known to hydrolyze cVA in vitro and is expressed in the main olfactory organ, the antenna, we tested here its role in olfaction as a putative odorant-degrading enzyme.
We first confirm that Est-6 is highly expressed in olfactory sensilla, including cVA-sensitive sensilla, and we show that expression is likely associated with non-neuronal cells. Our electrophysiological approaches show that the dynamics of olfactory receptor neuron (ORN) responses is strongly influenced by Est-6, as in Est-6° null mutants (lacking the Est-6 gene) cVA-sensitive ORN showed increased firing rate and prolonged activity in response to cVA. Est-6° mutant males had a lower threshold of behavioral response to cVA, as revealed by the analysis of two cVA-induced behaviors. In particular, mutant males exhibited a strong decrease of male-male courtship, in association with a delay in courtship initiation.
Our study presents evidence that Est-6 plays a role in the physiological and behavioral dynamics of sex pheromone response in Drosophila males and supports a role of Est-6 as an odorant-degrading enzyme (ODE) in male antennae. Our results also expand the role of Est-6 in Drosophila biology, from reproduction to olfaction, and highlight the role of ODEs in insect olfaction.
PMCID: PMC3414785  PMID: 22715942
carboxylesterase; esterase 6; olfaction; pheromone; signal termination
4.  Antennal carboxylesterases in a moth, structural and functional diversity 
Pheromone-degrading enzymes (PDEs) are supposed to be involved in the signal inactivation step within the olfactory sensilla of insects by quickly degrading pheromone molecules. Because esters are widespread insect pheromone components, PDEs belonging to the carboxylesterase (CCE) family have been the most studied. However, only two CCEs were both identified at the molecular level and functionally characterized as PDEs until recently. In the pest moth Spodoptera littoralis, we have identified an unsuspected diversity of antennal CCEs, with a total number of 30 genes. Two CCEs, enriched in antennae and belonging to distinct clades, were shown to present different substrate specificities toward pheromone and plant compounds. A same CCE was also shown to efficiently degrade both pheromone and plant components. Our results suggest that the structural evolution of antennal CCEs reflects their functional diversity and that a complex set of CCE-mediated reactions take place is the olfactory organs of moths.
PMCID: PMC3419116  PMID: 22896794
lepidoptera; noctuidae; olfaction; carboxylesterase; odorant-degrading enzymes; pheromone
5.  Identification and Characterization of an Antennae-Specific Aldehyde Oxidase from the Navel Orangeworm 
PLoS ONE  2013;8(6):e67794.
Antennae-specific odorant-degrading enzymes (ODEs) are postulated to inactivate odorant molecules after they convey their signal. Different classes of insect ODEs are specific to esters, alcohols, and aldehydes – the major functional groups of female-produced, hydrophobic sex pheromones from moth species. Esterases that rapidly inactive acetate and other esters have been well-studied, but less is known about aldehyde oxidases (AOXs). Here we report cloning of an aldehyde oxidase, AtraAOX2, from the antennae of the navel orangeworm (NOW), Amyelois transitella, and the first activity characterization of a recombinant insect AOX. AtraAOX2 gene spans 3,813 bp and encodes a protein with 1,270 amino acid residues. AtraAOX2 cDNA was expressed in baculovirus-infected insect Sf21 cells as a ≈280 kDa homodimer with 140 kDa subunits. Recombinant AtraAOX2 degraded Z11Z13–16Ald and plant volatile aldehydes as substrates. However, as expected for aldehyde oxidases, recombinant AtraAOX2 did not show specificity for Z11Z13–16Ald, the main constituent of the sex pheromone, but showed high activity for plant volatile aldehydes. Our data suggest AtraAOX2 might be involved in degradation of a diversity of aldehydes including sex pheromones, plant-derived semiochemicals, and chemical cues for oviposition sites. Additionally, AtraAOX2 could protect the insect's olfactory system from xenobiotics, including pesticides that might reach the sensillar lymph surrounding the olfactory receptor neurons.
PMCID: PMC3691121  PMID: 23826341
6.  Moth Sex Pheromone Receptors and Deceitful Parapheromones 
PLoS ONE  2012;7(7):e41653.
The insect's olfactory system is so selective that male moths, for example, can discriminate female-produced sex pheromones from compounds with minimal structural modifications. Yet, there is an exception for this “lock-and-key” tight selectivity. Formate analogs can be used as replacement for less chemically stable, long-chain aldehyde pheromones, because male moths respond physiologically and behaviorally to these parapheromones. However, it remained hitherto unknown how formate analogs interact with aldehyde-sensitive odorant receptors (ORs). Neuronal responses to semiochemicals were investigated with single sensillum recordings. Odorant receptors (ORs) were cloned using degenerate primers, and tested with the Xenopus oocyte expression system. Quality, relative quantity, and purity of samples were evaluated by gas chromatography and gas chromatography-mass spectrometry. We identified olfactory receptor neurons (ORNs) housed in trichoid sensilla on the antennae of male navel orangeworm that responded equally to the main constituent of the sex pheromone, (11Z,13Z)-hexadecadienal (Z11Z13-16Ald), and its formate analog, (9Z,11Z)-tetradecen-1-yl formate (Z9Z11-14OFor). We cloned an odorant receptor co-receptor (Orco) and aldehyde-sensitive ORs from the navel orangeworm, one of which (AtraOR1) was expressed specifically in male antennae. AtraOR1•AtraOrco-expressing oocytes responded mainly to Z11Z13-16Ald, with moderate sensitivity to another component of the sex pheromone, (11Z,13Z)-hexadecadien-1-ol. Surprisingly, this receptor was more sensitive to the related formate than to the natural sex pheromone. A pheromone receptor from Heliothis virescens, HR13 ( = HvirOR13) showed a similar profile, with stronger responses elicited by a formate analog than to the natural sex pheromone, (11Z)-hexadecenal thus suggesting this might be a common feature of moth pheromone receptors.
PMCID: PMC3401280  PMID: 22911835
7.  Differential Interactions of Sex Pheromone and Plant Odour in the Olfactory Pathway of a Male Moth 
PLoS ONE  2012;7(3):e33159.
Most animals rely on olfaction to find sexual partners, food or a habitat. The olfactory system faces the challenge of extracting meaningful information from a noisy odorous environment. In most moth species, males respond to sex pheromone emitted by females in an environment with abundant plant volatiles. Plant odours could either facilitate the localization of females (females calling on host plants), mask the female pheromone or they could be neutral without any effect on the pheromone. Here we studied how mixtures of a behaviourally-attractive floral odour, heptanal, and the sex pheromone are encoded at different levels of the olfactory pathway in males of the noctuid moth Agrotis ipsilon. In addition, we asked how interactions between the two odorants change as a function of the males' mating status. We investigated mixture detection in both the pheromone-specific and in the general odorant pathway. We used a) recordings from individual sensilla to study responses of olfactory receptor neurons, b) in vivo calcium imaging with a bath-applied dye to characterize the global input response in the primary olfactory centre, the antennal lobe and c) intracellular recordings of antennal lobe output neurons, projection neurons, in virgin and newly-mated males. Our results show that heptanal reduces pheromone sensitivity at the peripheral and central olfactory level independently of the mating status. Contrarily, heptanal-responding olfactory receptor neurons are not influenced by pheromone in a mixture, although some post-mating modulation occurs at the input of the sexually isomorphic ordinary glomeruli, where general odours are processed within the antennal lobe. The results are discussed in the context of mate localization.
PMCID: PMC3299628  PMID: 22427979
8.  A Comparison of the Olfactory Gene Repertoires of Adults and Larvae in the Noctuid Moth Spodoptera littoralis 
PLoS ONE  2013;8(4):e60263.
To better understand the olfactory mechanisms in a lepidopteran pest model species, the cotton leafworm Spodoptera littoralis, we have recently established a partial transcriptome from adult antennae. Here, we completed this transcriptome using next generation sequencing technologies, namely 454 and Illumina, on both adult antennae and larval tissues, including caterpillar antennae and maxillary palps. All sequences were assembled in 77,643 contigs. Their analysis greatly enriched the repertoire of chemosensory genes in this species, with a total of 57 candidate odorant-binding and chemosensory proteins, 47 olfactory receptors, 6 gustatory receptors and 17 ionotropic receptors. Using RT-PCR, we conducted the first exhaustive comparison of olfactory gene expression between larvae and adults in a lepidopteran species. All the 127 candidate olfactory genes were profiled for expression in male and female adult antennae and in caterpillar antennae and maxillary palps. We found that caterpillars expressed a smaller set of olfactory genes than adults, with a large overlap between these two developmental stages. Two binding proteins appeared to be larvae-specific and two others were adult-specific. Interestingly, comparison between caterpillar antennae and maxillary palps revealed numerous organ-specific transcripts, suggesting the complementary involvement of these two organs in larval chemosensory detection. Adult males and females shared the same set of olfactory transcripts, except two male-specific candidate pheromone receptors, two male-specific and two female-specific odorant-binding proteins. This study identified transcripts that may be important for sex-specific or developmental stage-specific chemosensory behaviors.
PMCID: PMC3614943  PMID: 23565215
9.  Changes in Odor Background Affect the Locomotory Response to Pheromone in Moths 
PLoS ONE  2013;8(1):e52897.
Many animals rely on chemical cues to recognize and locate a resource, and they must extract the relevant information from a complex and changing odor environment. For example, in moths, finding a mate is mediated by a sex pheromone, which is detected in a rich environment of volatile plant compounds. Here, we investigated the effects of a volatile plant background on the walking response of male Spodoptera littoralis to the female pheromone. Males were stimulated by combining pheromone with one of three plant compounds, and their walking paths were recorded with a locomotion compensator and analyzed. We found that the addition of certain volatile plant compounds disturbed the orientation toward the sex pheromone. The effect on locomotion was correlated with the capacity of the plant compound to antagonize pheromone detection by olfactory receptor neurons, suggesting a masking effect of the background over the pheromone signal. Moths were more sensitive to changes in background compared to a constant background, suggesting that a background odor also acts as a distracting stimulus. Our experiments show that the effects of odorant background on insect responses to chemical signals are complex and cannot be explained by a single mechanism.
PMCID: PMC3534683  PMID: 23301000
10.  Identification of pheromone components and their binding affinity to the odorant binding protein CcapOBP83a-2 of the Mediterranean fruit fly, Ceratitis capitata 
The Mediterranean fruit fly (or medfly), Ceratitis capitata (Wiedemann; Diptera: Tephritidae), is a serious pest of agriculture worldwide, displaying a very wide larval host range with more than 250 different species of fruit and vegetables. Olfaction plays a key role in the invasive potential of this species. Unfortunately, the pheromone communication system of the medfly is complex and still not well established. In this study, we report the isolation of chemicals emitted by sexually mature individuals during the “calling” period and the electrophysiological responses that these compounds elicit on the antennae of male and female flies. Fifteen compounds with electrophysiological activity were isolated and identified in male emissions by gas chromatography coupled to electroantennography (GC–EAG). Within the group of 15 identified compounds, 11 elicited a response in antennae of both sexes, whilst 4 elicited a response only in female antennae. The binding affinity of these compounds, plus 4 additional compounds known to be behaviourally active from other studies, was measured using C. capitata OBP, CcapOBP83a-2. This OBP has a high homology to Drosophila melanogaster OBPs OS-E and OS-F, which are associated with trichoid sensilla and co-expressed with the well-studied Drosophila pheromone binding protein LUSH. The results provide evidence of involvement of CcapOBP83a-2 in the medfly's odorant perception and its wider specificity for (E,E)-α-farnesene, one of the five major compounds in medfly male pheromone emission. This represents the first step in the clarification of the C. capitata and pheromone reception pathway, and a starting point for further studies aimed towards the creation of new powerful attractants or repellents applicable in the actual control strategies.
Graphical abstract
•The pheromone blend produced by medfly males during the calling period is investigated using air entrainment and GC–EAG.•Out of 15 chemicals, 11 elicited antennal responses in both sexes and 4 elicited the response only in female antennae.•The first medfly odorant binding protein CcapOBP83a-2 has been expressed and purified (87).•CcapOBP83a-2 showed binding affinity for (E,E)-α-farnesene, one of the components of medfly pheromone.•The ability of CcapOBP83a-2 to bind other electrophysiologically active compounds shows its broad binding specificity.
PMCID: PMC4003389  PMID: 24607850
Medfly; Ceratitis capitata; Olfaction; Odorant binding protein; Pheromone binding protein; Pheromone; Binding studies; Protein expression; Electroantennography; GC–EAG; Fluorescence displacement; OBP, Odorant binding protein; PBP, Pheromone binding protein; CSP, Chemosensory protein; OR, Odorant receptor; EAG, Electroantennography; GC–EAG, Gas chromatography coupled to electroantennography; SIT, Sterile Insect Technique
11.  Identification of genes expressed in the sex pheromone gland of the black cutworm Agrotis ipsilon with putative roles in sex pheromone biosynthesis and transport 
BMC Genomics  2013;14:636.
One of the challenges in insect chemical ecology is to understand how insect pheromones are synthesised, detected and degraded. Genome wide survey by comparative sequencing and gene specific expression profiling provide rich resources for this challenge. A. ipsilon is a destructive pest of many crops and further characterization of the genes involved in pheromone biosynthesis and transport could offer potential targets for disruption of their chemical communication and for crop protection.
Here we report 454 next-generation sequencing of the A. ipsilon pheromone gland transcriptome, identification and expression profiling of genes putatively involved in pheromone production, transport and degradation. A total of 23473 unigenes were obtained from the transcriptome analysis, 86% of which were A. ipsilon specific. 42 transcripts encoded enzymes putatively involved in pheromone biosynthesis, of which 15 were specifically, or mainly, expressed in the pheromone glands at 5 to 120-fold higher levels than in the body. Two transcripts encoding for a fatty acid synthase and a desaturase were highly abundant in the transcriptome and expressed more than 40-fold higher in the glands than in the body. The transcripts encoding for 2 acetyl-CoA carboxylases, 1 fatty acid synthase, 2 desaturases, 3 acyl-CoA reductases, 2 alcohol oxidases, 2 aldehyde reductases and 3 acetyltransferases were expressed at a significantly higher level in the pheromone glands than in the body. 17 esterase transcripts were not gland-specific and 7 of these were expressed highly in the antennae. Seven transcripts encoding odorant binding proteins (OBPs) and 8 encoding chemosensory proteins (CSPs) were identified. Two CSP transcripts (AipsCSP2, AipsCSP8) were highly abundant in the pheromone gland transcriptome and this was confirmed by qRT-PCR. One OBP (AipsOBP6) were pheromone gland-enriched and three OBPs (AipsOBP1, AipsOBP2 and AipsOBP4) were antennal-enriched. Based on these studies we proposed possible A. ipsilon biosynthesis pathways for major and minor sex pheromone components.
Our study identified genes potentially involved in sex pheromone biosynthesis and transport in A. ipsilon. The identified genes are likely to play essential roles in sex pheromone production, transport and degradation and could serve as targets to interfere with pheromone release. The identification of highly expressed CSPs and OBPs in the pheromone gland suggests that they may play a role in the binding, transport and release of sex pheromones during sex pheromone production in A. ipsilon and other Lepidoptera insects.
PMCID: PMC3849270  PMID: 24053512
12.  A Single Sex Pheromone Receptor Determines Chemical Response Specificity of Sexual Behavior in the Silkmoth Bombyx mori 
PLoS Genetics  2011;7(6):e1002115.
In insects and other animals, intraspecific communication between individuals of the opposite sex is mediated in part by chemical signals called sex pheromones. In most moth species, male moths rely heavily on species-specific sex pheromones emitted by female moths to identify and orient towards an appropriate mating partner among a large number of sympatric insect species. The silkmoth, Bombyx mori, utilizes the simplest possible pheromone system, in which a single pheromone component, (E, Z)-10,12-hexadecadienol (bombykol), is sufficient to elicit full sexual behavior. We have previously shown that the sex pheromone receptor BmOR1 mediates specific detection of bombykol in the antennae of male silkmoths. However, it is unclear whether the sex pheromone receptor is the minimally sufficient determination factor that triggers initiation of orientation behavior towards a potential mate. Using transgenic silkmoths expressing the sex pheromone receptor PxOR1 of the diamondback moth Plutella xylostella in BmOR1-expressing neurons, we show that the selectivity of the sex pheromone receptor determines the chemical response specificity of sexual behavior in the silkmoth. Bombykol receptor neurons expressing PxOR1 responded to its specific ligand, (Z)-11-hexadecenal (Z11-16:Ald), in a dose-dependent manner. Male moths expressing PxOR1 exhibited typical pheromone orientation behavior and copulation attempts in response to Z11-16:Ald and to females of P. xylostella. Transformation of the bombykol receptor neurons had no effect on their projections in the antennal lobe. These results indicate that activation of bombykol receptor neurons alone is sufficient to trigger full sexual behavior. Thus, a single gene defines behavioral selectivity in sex pheromone communication in the silkmoth. Our findings show that a single molecular determinant can not only function as a modulator of behavior but also as an all-or-nothing initiator of a complex species-specific behavioral sequence.
Author Summary
Like many animal species, moths use chemical signals called sex pheromones to communicate with conspecific individuals of the opposite sex in the context of reproduction. Typically, male moths depend on sex pheromones emitted by conspecific females to identify and locate their mates. Therefore, the behavioral preference of male moths to conspecific pheromones is a critical factor for successful reproduction. Sex pheromone receptor proteins expressed in specialized antennal olfactory receptor neurons reportedly play a central role in sex pheromone discrimination. However, the causal relationship between sex pheromone receptor specificity and behavioral preference remains to be proven. We have addressed this question in a genetically tractable moth species, the silkmoth (Bombyx mori), because this species possesses the simplest possible pheromone system in which a single pheromone substance, bombykol, elicits full sexual behavior. Using transgenic silkmoths expressing a sex pheromone receptor from another moth species, we revealed that solely the chemical specificity of the odorant receptors in bombykol receptor neurons determines the behavioral preference in male silkmoths. Our results show that the initiation of a complex programmed sexual behavior can depend on the properties of a single pheromone receptor gene expressed in a population of olfactory receptor neurons.
PMCID: PMC3128102  PMID: 21738481
13.  Olfactory Proteins Mediating Chemical Communication in the Navel Orangeworm Moth, Amyelois transitella 
PLoS ONE  2009;4(9):e7235.
The navel orangeworm, Amyelois transitella Walker (Lepidoptera: Pyralidae), is the most serious insect pest of almonds and pistachios in California for which environmentally friendly alternative methods of control — like pheromone-based approaches — are highly desirable. Some constituents of the sex pheromone are unstable and could be replaced with parapheromones, which may be designed on the basis of molecular interaction of pheromones and pheromone-detecting olfactory proteins.
By analyzing extracts from olfactory and non-olfactory tissues, we identified putative olfactory proteins, obtained their N-terminal amino acid sequences by Edman degradation, and used degenerate primers to clone the corresponding cDNAs by SMART RACE. Additionally, we used degenerate primers based on conserved sequences of known proteins to fish out other candidate olfactory genes. We expressed the gene encoding a newly identified pheromone-binding protein, which was analyzed by circular dichroism, fluorescence, and nuclear magnetic resonance, and used in a binding assay to assess affinity to pheromone components.
We have cloned nine cDNAs encoding olfactory proteins from the navel orangeworm, including two pheromone-binding proteins, two general odorant-binding proteins, one chemosensory protein, one glutathione S-transferase, one antennal binding protein X, one sensory neuron membrane protein, and one odorant receptor. Of these, AtraPBP1 is highly enriched in male antennae. Fluorescence, CD and NMR studies suggest a dramatic pH-dependent conformational change, with high affinity to pheromone constituents at neutral pH and no binding at low pH.
PMCID: PMC2749207  PMID: 19789654
14.  Requirement for Drosophila SNMP1 for Rapid Activation and Termination of Pheromone-Induced Activity 
PLoS Genetics  2014;10(9):e1004600.
Pheromones are used for conspecific communication by many animals. In Drosophila, the volatile male-specific pheromone 11-cis vaccenyl acetate (cVA) supplies an important signal for gender recognition. Sensing of cVA by the olfactory system depends on multiple components, including an olfactory receptor (OR67d), the co-receptor ORCO, and an odorant binding protein (LUSH). In addition, a CD36 related protein, sensory neuron membrane protein 1 (SNMP1) is also involved in cVA detection. Loss of SNMP1 has been reported to eliminate cVA responsiveness, and to greatly increase spontaneous activity of OR67d-expressing olfactory receptor neurons (ORNs). Here, we found the snmp11 mutation did not abolish cVA responsiveness or cause high spontaneous activity. The cVA responses in snmp1 mutants displayed a delayed onset, and took longer to reach peak activity than wild-type. Most strikingly, loss of SNMP1 caused a dramatic delay in signal termination. The profound impairment in signal inactivation accounted for the previously reported “spontaneous activity,” which represented continuous activation following transient exposure to environmental cVA. We introduced the silk moth receptor (BmOR1) in OR67d ORNs of snmp11 flies and found that the ORNs showed slow activation and deactivation kinetics in response to the BmOR1 ligand (bombykol). We expressed the bombykol receptor complex in Xenopus oocytes in the presence or absence of the silk moth SNMP1 (BmSNMP) and found that addition of BmSNMP accelerated receptor activation and deactivation. Our results thus clarify SNMP1 as an important player required for the rapid kinetics of the pheromone response in insects.
Author Summary
Pheromones are chemicals produced and released by animals for social communication with other members of their species. For example, male fruit flies produce a volatile pheromone that is sensed by both males and females, and which functions in gender recognition. This volatile male pheromone, called 11-cis vaccenyl acetate, is detected by olfactory neurons housed in hair-like appendages on the insect antenna. To effectively sense the pheromone, especially during navigation, the olfactory neurons must respond rapidly, and then quickly inactivate after the stimulation ceases. We found that a CD36-related protein referred to as sensory neuron membrane protein 1 (SNMP1) was required by olfactory neurons for the rapid on and off responses to 11-cis vaccenyl acetate. Loss of SNMP1 reduced the initial sensitivity to the pheromone, and then caused a strikingly slower termination of the response after removal of the pheromone. Our findings demonstrate that SNMP1 is a critical player that allows olfactory neurons to achieve sensitive and rapid on and off responses to a pheromone that is critical for social interactions in insects.
PMCID: PMC4177743  PMID: 25255106
15.  Molecular Characterization and Differential Expression of Olfactory Genes in the Antennae of the Black Cutworm Moth Agrotis ipsilon 
PLoS ONE  2014;9(8):e103420.
Insects use their sensitive and selective olfactory system to detect outside chemical odorants, such as female sex pheromones and host plant volatiles. Several groups of olfactory proteins participate in the odorant detection process, including odorant binding proteins (OBPs), chemosensory proteins (CSPs), odorant receptors (ORs), ionotropic receptors (IRs) and sensory neuron membrane proteins (SNMPs). The identification and functional characterization of these olfactory proteins will enhance our knowledge of the molecular basis of insect chemoreception. In this study, we report the identification and differential expression profiles of these olfactory genes in the black cutworm moth Agrotis ipsilon. In total, 33 OBPs, 12 CSPs, 42 ORs, 24 IRs, 2 SNMPs and 1 gustatory receptor (GR) were annotated from the A. ipsilon antennal transcriptomes, and further RT-PCR and RT-qPCR revealed that 22 OBPs, 3 CSPs, 35 ORs, 14 IRs and the 2 SNMPs are uniquely or primarily expressed in the male and female antennae. Furthermore, one OBP (AipsOBP6) and one CSP (AipsCSP2) were exclusively expressed in the female sex pheromone gland. These antennae-enriched OBPs, CSPs, ORs, IRs and SNMPs were suggested to be responsible for pheromone and general odorant detection and thus could be meaningful target genes for us to study their biological functions in vivo and in vitro.
PMCID: PMC4118888  PMID: 25083706
16.  Functional Characterizations of Chemosensory Proteins of the Alfalfa Plant Bug Adelphocoris lineolatus Indicate Their Involvement in Host Recognition 
PLoS ONE  2012;7(8):e42871.
Insect chemosensory proteins (CSPs) have been proposed to capture and transport hydrophobic chemicals from air to olfactory receptors in the lymph of antennal chemosensilla. They may represent a new class of soluble carrier protein involved in insect chemoreception. However, their specific functional roles in insect chemoreception have not been fully elucidated. In this study, we report for the first time three novel CSP genes (AlinCSP1-3) of the alfalfa plant bug Adelphocoris lineolatus (Goeze) by screening the antennal cDNA library. The qRT-PCR examinations of the transcript levels revealed that all three genes (AlinCSP1-3) are mainly expressed in the antennae. Interestingly, these CSP genes AlinCSP1-3 are also highly expressed in the 5th instar nymphs, suggesting a proposed function of these CSP proteins (AlinCSP1-3) in the olfactory reception and in maintaining particular life activities into the adult stage. Using bacterial expression system, the three CSP proteins were expressed and purified. For the first time we characterized the types of sensilla in the antennae of the plant bug using scanning electron microscopy (SEM). Immunocytochemistry analysis indicated that the CSP proteins were expressed in the pheromone-sensitive sensilla trichodea and general odorant-sensitive sensilla basiconica, providing further evidence of their involvement in chemoreception. The antennal activity of 55 host-related semiochemicals and sex pheromone compounds in the host location and mate selection behavior of A. lineolatus was investigated using electroantennogram (EAG), and the binding affinities of these chemicals to the three CSPs (AlinCSP1-3) were measured using fluorescent binding assays. The results showed several host-related semiochemicals, (Z)-3-hexen-1-ol, (E)-2-hexen-1-al and valeraldehyde, have a high binding affinity with AlinCSP1-3 and can elicit significant high EAG responses of A. lineolatus antennae. Our studies indicate the three antennae-biased CSPs may mediate host recognition in the alfalfa plant bug A. lineolatus.
PMCID: PMC3416781  PMID: 22900060
17.  Functional Analysis of General Odorant Binding Protein 2 from the Meadow Moth, Loxostege sticticalis L. (Lepidoptera: Pyralidae) 
PLoS ONE  2012;7(3):e33589.
Odorant binding proteins play a crucial role in transporting semiochemicals across the sensillum lymph to olfactory receptors within the insect antennal sensilla. In this study, the general odorant binding protein 2 gene was cloned from the antennae of Loxostege sticticalis, using reverse transcription PCR and rapid amplification of cDNA ends. Recombinant LstiGOBP2 was expressed in Escherichia coli and purified by Ni ion affinity chromatography. Real-time PCR assays indicated that LstiGOBP2 mRNA is expressed mainly in adult antennae, with expression levels differing with developmental age. Ligand-binding experiments using N-phenyl-naphthylamine (1-NPN) as a fluorescent probe demonstrated that the LstiGOBP2 protein has binding affinity to a broad range of odorants. Most importantly, trans-11-tetradecen-1-yl acetate, the pheromone component of Loxostege sticticalis, and trans-2-hexenal and cis-3-hexen-1-ol, the most abundant plant volatiles in essential oils extracted from host plants, had high binding affinities to LstiGOBP2 and elicited strong electrophysiological responses from the antennae of adults.
PMCID: PMC3316592  PMID: 22479417
18.  Molecular Characterization, Expression Pattern, and Ligand-Binding Property of Three Odorant Binding Protein Genes from Dendrolimus tabulaeformis 
Journal of Chemical Ecology  2014;40(4):396-406.
Odorant binding proteins (OBPs) play important roles in insect olfactory processes. The Chinese pine caterpillar moth, Dendrolimus tabulaeformis (Lepidoptera, Lasiocampidae) is a serious economic pest in China, and the pheromones of this species have been identified to monitor their presence. However, the molecular mechanisms by which D. tabulaeformis perceive pheromones and host volatiles remain unknown. In this study, we identified and characterized three new OBPs, including one pheromone binding protein (PBP1) and two general odor binding proteins (GOBPs), from antennal cDNA of D. tabulaeformis. The deduced amino acid sequences of DtabPBP1, DtabGOBP1, and DtabGOBP2 revealed mature proteins of 140, 147, and 140 amino acids, respectively. Each has six cysteine residues in conserved positions relative to other known OBPs. Amino-acid alignments indicated that the two GOBPs are more conserved (DtabGOBP1 is 52.9–67.4 % identical to orthologs from other Lepidoptera, and DtabGOBP2 is 55.2–81.8 % identical) than the PBP (32.5–46.0 %). Real-time PCR indicated tissue- and sex-specific expression patterns of the three genes. DtabPBP1 was mainly expressed in the antennae of males, whereas female antennae had only 1.09 % the expression in male antennae. Both DtabGOBP1 and DtabGOBP2 were more highly expressed in antennae than in other tissues, while DtabGOBP1 was more abundant in male antennae and DtabGOBP2 in female antennae. In addition, the binding specificities of the three proteins were investigated, and all three OBPs exhibited high binding affinities for the pheromone component (5Z,7E)-5,7-dodecadien-1-yl propionate (Z5,E7-12:OPr). This suggests a role in binding pheromone for GOBPs, as well as PBP1, in D. tabulaeformis.
Electronic supplementary material
The online version of this article (doi:10.1007/s10886-014-0412-6) contains supplementary material, which is available to authorized users.
PMCID: PMC4008786  PMID: 24728949
Pheromone binding proteins; General odorant binding proteins; Real-time PCR; Fluorescence competitive binding assay; Forest insect; Economic pest; Lepidoptera
19.  Molecular and neural mechanisms of sex pheromone reception and processing in the silkmoth Bombyx mori 
Male moths locate their mates using species-specific sex pheromones emitted by conspecific females. One striking feature of sex pheromone recognition in males is the high degree of specificity and sensitivity at all levels, from the primary sensory processes to behavior. The silkmoth Bombyx mori is an excellent model insect in which to decipher the underlying mechanisms of sex pheromone recognition due to its simple sex pheromone communication system, where a single pheromone component, bombykol, elicits the full sexual behavior of male moths. Various technical advancements that cover all levels of analysis from molecular to behavioral also allow the systematic analysis of pheromone recognition mechanisms. Sex pheromone signals are detected by pheromone receptors expressed in olfactory receptor neurons in the pheromone-sensitive sensilla trichodea on male antennae. The signals are transmitted to the first olfactory processing center, the antennal lobe (AL), and then are processed further in the higher centers (mushroom body and lateral protocerebrum) to elicit orientation behavior toward females. In recent years, significant progress has been made elucidating the molecular mechanisms underlying the detection of sex pheromones. In addition, extensive studies of the AL and higher centers have provided insights into the neural basis of pheromone processing in the silkmoth brain. This review describes these latest advances, and discusses what these advances have revealed about the mechanisms underlying the specific and sensitive recognition of sex pheromones in the silkmoth.
PMCID: PMC3978319  PMID: 24744736
insect; silkmoth; olfaction; sex pheromone; pheromone-source searching behavior
20.  Sex-Linked Pheromone Receptor Genes of the European Corn Borer, Ostrinia nubilalis, Are in Tandem Arrays 
PLoS ONE  2011;6(4):e18843.
Tuning of the olfactory system of male moths to conspecific female sex pheromones is crucial for correct species recognition; however, little is known about the genetic changes that drive speciation in this system. Moths of the genus Ostrinia are good models to elucidate this question, since significant differences in pheromone blends are observed within and among species. Odorant receptors (ORs) play a critical role in recognition of female sex pheromones; eight types of OR genes expressed in male antennae were previously reported in Ostrinia moths.
Methodology/Principal Findings
We screened an O. nubilalis bacterial artificial chromosome (BAC) library by PCR, and constructed three contigs from isolated clones containing the reported OR genes. Fluorescence in situ hybridization (FISH) analysis using these clones as probes demonstrated that the largest contig, which contained eight OR genes, was located on the Z chromosome; two others harboring two and one OR genes were found on two autosomes. Sequence determination of BAC clones revealed the Z-linked OR genes were closely related and tandemly arrayed; moreover, four of them shared 181-bp direct repeats spanning exon 7 and intron 7.
This is the first report of tandemly arrayed sex pheromone receptor genes in Lepidoptera. The localization of an OR gene cluster on the Z chromosome agrees with previous findings for a Z-linked locus responsible for O. nubilalis male behavioral response to sex pheromone. The 181-bp direct repeats might enhance gene duplications by unequal crossovers. An autosomal locus responsible for male response to sex pheromone in Heliothis virescens and H. subflexa was recently reported to contain at least four OR genes. Taken together, these findings support the hypothesis that generation of additional copies of OR genes can increase the potential for male moths to acquire altered specificity for pheromone components, and accordingly, facilitate differentiation of sex pheromones.
PMCID: PMC3081303  PMID: 21526121
21.  Glomerular interactions in olfactory processing channels of the antennal lobes 
An open question in olfactory coding is the extent of interglomerular connectivity: do olfactory glomeruli and their neurons regulate the odorant responses of neurons innervating other glomeruli? In the olfactory system of the moth Manduca sexta, the response properties of different types of antennal olfactory receptor cells are known. Likewise, a subset of antennal lobe glomeruli has been functionally characterized and the olfactory tuning of their innervating neurons identified. This provides a unique opportunity to determine functional interactions between glomeruli of known input, specifically, (1) glomeruli processing plant odors and (2) glomeruli activated by antennal stimulation with pheromone components of conspecific females. Several studies describe reciprocal inhibitory effects between different types of pheromone-responsive projection neurons suggesting lateral inhibitory interactions between pheromone component-selective glomerular neural circuits. Furthermore, antennal lobe projection neurons that respond to host plant volatiles and innervate single, ordinary glomeruli are inhibited during antennal stimulation with the female’s sex pheromone. The studies demonstrate the existence of lateral inhibitory effects in response to behaviorally significant odorant stimuli and irrespective of glomerular location in the antennal lobe. Inhibitory interactions are present within and between olfactory subsystems (pheromonal and non-pheromonal subsystems), potentially to enhance contrast and strengthen odorant discrimination.
PMCID: PMC4066976  PMID: 23893248
Electrophysiology; Glomerulus; Neural coding; Olfaction; Pheromone
22.  A General Odorant Background Affects the Coding of Pheromone Stimulus Intermittency in Specialist Olfactory Receptor Neurones 
PLoS ONE  2011;6(10):e26443.
In nature the aerial trace of pheromone used by male moths to find a female appears as a train of discontinuous pulses separated by gaps among a complex odorant background constituted of plant volatiles. We investigated the effect of such background odor on behavior and coding of temporal parameters of pheromone pulse trains in the pheromone olfactory receptor neurons of Spodoptera littoralis. Effects of linalool background were tested by measuring walking behavior towards a source of pheromone. While velocity and orientation index did drop when linalool was turned on, both parameters recovered back to pre-background values after 40 s with linalool still present. Photo-ionization detector was used to characterize pulse delivery by our stimulator. The photo-ionization detector signal reached 71% of maximum amplitude at 50 ms pulses and followed the stimulus period at repetition rates up to 10 pulses/s. However, at high pulse rates the concentration of the odorant did not return to base level during inter-pulse intervals. Linalool decreased the intensity and shortened the response of receptor neurons to pulses. High contrast (>10 dB) in firing rate between pulses and inter-pulse intervals was observed for 1 and 4 pulses/s, both with and without background. Significantly more neurons followed the 4 pulses/s pattern when delivered over linalool; at the same time the information content was preserved almost to the control values. Rapid recovery of behavior shows that change of perceived intensity is more important than absolute stimulus intensity. While decreasing the response intensity, background odor preserved the temporal parameters of the specific signal.
PMCID: PMC3196569  PMID: 22028879
23.  An RNA-Seq Screen of the Drosophila Antenna Identifies a Transporter Necessary for Ammonia Detection 
PLoS Genetics  2014;10(11):e1004810.
Many insect vectors of disease detect their hosts through olfactory cues, and thus it is of great interest to understand better how odors are encoded. However, little is known about the molecular underpinnings that support the unique function of coeloconic sensilla, an ancient and conserved class of sensilla that detect amines and acids, including components of human odor that are cues for many insect vectors. Here, we generate antennal transcriptome databases both for wild type Drosophila and for a mutant that lacks coeloconic sensilla. We use these resources to identify genes whose expression is highly enriched in coeloconic sensilla, including many genes not previously implicated in olfaction. Among them, we identify an ammonium transporter gene that is essential for ammonia responses in a class of coeloconic olfactory receptor neurons (ORNs), but is not required for responses to other odorants. Surprisingly, the transporter is not expressed in ORNs, but rather in neighboring auxiliary cells. Thus, our data reveal an unexpected non-cell autonomous role for a component that is essential to the olfactory response to ammonia. The defective response observed in a Drosophila mutant of this gene is rescued by its Anopheles ortholog, and orthologs are found in virtually all insect species examined, suggesting that its role is conserved. Taken together, our results provide a quantitative analysis of gene expression in the primary olfactory organ of Drosophila, identify molecular components of an ancient class of olfactory sensilla, and reveal that auxiliary cells, and not simply ORNs, play an essential role in the coding of an odor that is a critical host cue for many insect vectors of human disease.
Author Summary
Olfaction underlies the attraction of insect pests and vectors of disease to their plant and human hosts. In the genetic model insect Drosophila, the neuronal basis of odor coding has been extensively analyzed in the antenna, its major olfactory organ, but the molecular basis of odor coding has not. Additionally, there has been little analysis of any olfactory cells other than neurons. We have undertaken a comprehensive and quantitative analysis of gene expression in the Drosophila antenna. This analysis revealed a surprisingly broad dynamic range of odor receptor and odor binding protein expression, and unexpected expression of taste receptor genes. Further analysis identified 250 genes that are expressed at reduced levels in a mutant lacking an evolutionarily ancient class of sensilla, antennal hairs housing neurons that respond to human odors. One of these genes, a transporter, is expressed in non-neuronal cells but is essential to the response of a neuron to ammonia, a key cue for insect vectors of disease. A mutation in this transporter can be rescued by its mosquito homolog. While many studies of sensory coding consider the neural circuit in isolation, our analysis reveals an essential role for an auxiliary cell.
PMCID: PMC4238959  PMID: 25412082
24.  An Expressed Sequence Tag collection from the male antennae of the Noctuid moth Spodoptera littoralis: a resource for olfactory and pheromone detection research 
BMC Genomics  2011;12:86.
Nocturnal insects such as moths are ideal models to study the molecular bases of olfaction that they use, among examples, for the detection of mating partners and host plants. Knowing how an odour generates a neuronal signal in insect antennae is crucial for understanding the physiological bases of olfaction, and also could lead to the identification of original targets for the development of olfactory-based control strategies against herbivorous moth pests. Here, we describe an Expressed Sequence Tag (EST) project to characterize the antennal transcriptome of the noctuid pest model, Spodoptera littoralis, and to identify candidate genes involved in odour/pheromone detection.
By targeting cDNAs from male antennae, we biased gene discovery towards genes potentially involved in male olfaction, including pheromone reception. A total of 20760 ESTs were obtained from a normalized library and were assembled in 9033 unigenes. 6530 were annotated based on BLAST analyses and gene prediction software identified 6738 ORFs. The unigenes were compared to the Bombyx mori proteome and to ESTs derived from Lepidoptera transcriptome projects. We identified a large number of candidate genes involved in odour and pheromone detection and turnover, including 31 candidate chemosensory receptor genes, but also genes potentially involved in olfactory modulation.
Our project has generated a large collection of antennal transcripts from a Lepidoptera. The normalization process, allowing enrichment in low abundant genes, proved to be particularly relevant to identify chemosensory receptors in a species for which no genomic data are available. Our results also suggest that olfactory modulation can take place at the level of the antennae itself. These EST resources will be invaluable for exploring the mechanisms of olfaction and pheromone detection in S. littoralis, and for ultimately identifying original targets to fight against moth herbivorous pests.
PMCID: PMC3045336  PMID: 21276261
25.  Sex Pheromone Evolution Is Associated with Differential Regulation of the Same Desaturase Gene in Two Genera of Leafroller Moths 
PLoS Genetics  2012;8(1):e1002489.
Chemical signals are prevalent in sexual communication systems. Mate recognition has been extensively studied within the Lepidoptera, where the production and recognition of species-specific sex pheromone signals are typically the defining character. While the specific blend of compounds that makes up the sex pheromones of many species has been characterized, the molecular mechanisms underpinning the evolution of pheromone-based mate recognition systems remain largely unknown. We have focused on two sets of sibling species within the leafroller moth genera Ctenopseustis and Planotortrix that have rapidly evolved the use of distinct sex pheromone blends. The compounds within these blends differ almost exclusively in the relative position of double bonds that are introduced by desaturase enzymes. Of the six desaturase orthologs isolated from all four species, functional analyses in yeast and gene expression in pheromone glands implicate three in pheromone biosynthesis, two Δ9-desaturases, and a Δ10-desaturase, while the remaining three desaturases include a Δ6-desaturase, a terminal desaturase, and a non-functional desaturase. Comparative quantitative real-time PCR reveals that the Δ10-desaturase is differentially expressed in the pheromone glands of the two sets of sibling species, consistent with differences in the pheromone blend in both species pairs. In the pheromone glands of species that utilize (Z)-8-tetradecenyl acetate as sex pheromone component (Ctenopseustis obliquana and Planotortrix octo), the expression levels of the Δ10-desaturase are significantly higher than in the pheromone glands of their respective sibling species (C. herana and P. excessana). Our results demonstrate that interspecific sex pheromone differences are associated with differential regulation of the same desaturase gene in two genera of moths. We suggest that differential gene regulation among members of a multigene family may be an important mechanism of molecular innovation in sex pheromone evolution and speciation.
Author Summary
Chemical signals are prevalent in sexual communication systems, especially within the Lepidoptera where sex pheromones are typically one of the defining characteristics of species. We have isolated six desaturases from two groups of sibling species of leafroller moths belonging to the genera Ctenopseustis and Planotortrix. Functional analyses in yeast and quantitative RT–PCR indicate that three of the desaturases are involved in the biosynthesis of sex pheromone components in these species. One of three enzymes is a Δ10-desaturase that is differentially expressed in the pheromone glands of the two sets of sibling species, consistent with differences in the pheromone blend in both species pairs. In the pheromone glands of species that utilize (Z)-8-tetradecenyl acetate as sex pheromone component (C. obliquana and P. octo), the expression levels of the Δ10-desaturase are significantly higher than pheromone gland expression levels in their sibling species (C. herana and P. excessana). Our results demonstrate that interspecific sex pheromone differences are associated with differential regulation of the same desaturase gene in these two genera of moths. Based on these findings differential gene regulation among members of a multigene family may be an important mechanism of molecular innovation in sex pheromone evolution and speciation.
PMCID: PMC3266893  PMID: 22291612

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